EP3504328A1 - Modification de protéines de cellules hôtes - Google Patents

Modification de protéines de cellules hôtes

Info

Publication number
EP3504328A1
EP3504328A1 EP17798018.2A EP17798018A EP3504328A1 EP 3504328 A1 EP3504328 A1 EP 3504328A1 EP 17798018 A EP17798018 A EP 17798018A EP 3504328 A1 EP3504328 A1 EP 3504328A1
Authority
EP
European Patent Office
Prior art keywords
protein
seq
modification
host cell
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17798018.2A
Other languages
German (de)
English (en)
Inventor
Gang Chen
Ning Li
Michael Goren
Darya Burakov
Chen Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regeneron Pharmaceuticals Inc
Original Assignee
Regeneron Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Publication of EP3504328A1 publication Critical patent/EP3504328A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01013—Sterol esterase (3.1.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01034—Lipoprotein lipase (3.1.1.34)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01023—Ceramidase (3.5.1.23)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature

Definitions

  • the invention provides for cells and methods for expression and purification of recombinant proteins in eukaryotic cells.
  • the invention includes methods and compositions for expression of proteins in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, that employ downregulating gene expression of endogenous proteins in order to control production of such unwanted "sticky" host cell proteins.
  • the invention includes polynucleotides and modified cells that facilitate purification of an exogenous recombinant protein of interest.
  • the methods of the invention efficiently target host cell proteins in the Chinese hamster cellular genome in order to facilitate enhanced and stable expression of recombinant proteins expressed by the modified cells.
  • HCPs host cell proteins
  • LC/MS Advanced liquid chromatography/mass spectrometry
  • HCPs Changes in cell culture conditions of eukaryotic cells has been shown to impact the purity and stability of manufactured proteins, as seen by the increased quantity of HCPs of CHO cells upon downstream bioprocessing alterations (Tait, et al, 2013, Biotechnol Prog 29(3):688-696).
  • the detrimental effect of leftover HCPs in any product may affect the overall quality or quantity, or both the quality and quantity of the product.
  • HCPs if present even at low levels in a therapeutic product, may induce an undesired immune response which causes concern for patient safety and efficacy of the drug product (Singh SK. 201 1 .
  • the invention provides a recombinant host cell, wherein the cell is modified to decrease the expression levels of two or more fatty acid hydrolases (FAHs) relative to the expression levels of FAH in an unmodified cell.
  • FHs fatty acid hydrolases
  • the invention provides a recombinant host cell, wherein the cell is modified to have no expression of two or more target FAHs.
  • one of said two or more target FAHs is an esterase.
  • the esterase is a lipase.
  • the lipase is: (1 ) a
  • one of said two or more target FAHs is an amidase.
  • the amidase is a fatty acid acylase.
  • the fatty acid acylase is an acid ceramidase.
  • a gene of interest is exogenously added to the recombinant host cell.
  • the exogenously added gene encodes a protein of interest (POI), for example the POI is selected form the group consisting of antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein.
  • POI protein of interest
  • the invention relates to a composition comprising one or more proteins of interest (POIs) obtainable by a method according to the invention.
  • the protein may be a recombinant protein and may be e.g. selected form the group comprising antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein, or any combinations thereof.
  • the invention relates to a composition comprising one or more proteins of interest (POIs), wherein the adverse enzyme activity is ⁇ 50%, 40%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, 30%, 29%, 28%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.05%, or ⁇ 0.05% active relative to the adverse enzyme activity of a wild-type production system.
  • POIs proteins of interest
  • the phrase "adverse enzyme activity" refers to any enzyme and its action upon the resulting composition as a whole, wherein the action results in metabolism of any protein components which metabolites reduces the shelf-life of the composition or results in the formation of subvisible particles (SVPs) above prescribed regulations.
  • the protein may be a recombinant protein and may e.g. be selected from the group consisting of antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein or any combinations thereof.
  • the enzymes and their activities may be, e.g., various esterases, hydrolases, lipases, phospholipases, ceramidases and the likes, or any combinations thereof.
  • Adverse enzyme activity may be measured using a functional assay ⁇ e.g., polysorbate fatty acid hydrolysis assay), or a structural assay ⁇ e.g., nano LC-MS of peptide fragments, liquid chromatography-tandem mass spectrometry (LC-MS/MS) or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) or the like (deZafra et al, 2015, Biotechnol Bioeng;1 12(1 1 ):2284- 91 )).
  • a functional assay e.g., polysorbate fatty acid hydrolysis assay
  • a structural assay ⁇ e.g., nano LC-MS of peptide fragments, liquid chromatography-tandem mass spectrometry (LC-MS/MS) or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-
  • the invention provides a cell comprising a nonfunctional PLBD2 protein and one or more additional nonfunctional fatty acid hydrolases (FAH).
  • FAH nonfunctional fatty acid hydrolases
  • nonfunctional FAH is a nonfunctional lipoprotein lipase (LPL), lysosomal acid lipase (LI PA), acid ceramidase (ACE), platelet-activating factor acetylhydrolase IB subunit gamma (PAFAHG), phosphoinositide phospholipase C fragment (PIPLCf), phosphoinositide phospholipase C (PIPLC), liver carboxylesterase 1 (LCE), isoamyl acetate-hydrolyzing esterase 1 -like (IAH1 ), group XV phospholipase A2 (LPLA2), carboxylic ester hydrolase (CEH), and/or arylsulfatase A (ASA).
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • PAFAHG platelet-activating factor acetylhydrolase IB subunit gamma
  • the invention provides making a cell by FAH target disruption.
  • the method comprises a site-specific nuclease for disrupting or editing the cell genome at a target site or sequence.
  • the FAH target site is (1 ) a PLBD2 target site, (2) an LPL target site, (3) an LI PA target site, (4) an ACE target site, (5) a PAFAHG site, (6) a PIPLCf or PIPLC site, (7) an LCE site, (8) an IAH1 site, (9) an LPLA2 site, (9) an CEH site, and /or (10) an ASA site.
  • the PLBD2 target site comprises a position within SEQ ID NO:33, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:33, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:33, within exon 1 of SEQ ID NO:33, within exon 2 of SEQ ID NO:33, or within exon 3 of SEQ ID NO:33.
  • the PLBD2 target site comprises a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-230, 190-210, 200-220, 210-230, 220-240, 230-250, 240-260, and 250-270 of SEQ ID NO:33.
  • the target site at a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 is selected from the group consisting of nucleotides spanning positions numbered 37-56, 44-56, 33-62, 40-69, 1 10-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the PLBD2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:33 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:33 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPL target site comprises a position within SEQ ID NO:61 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:61 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO:61 , within exon 1 of SEQ ID NO:61 , within exon 2 of SEQ ID NO:61 , or within exon 3 of SEQ ID NO:61.
  • the LPL target site comprises a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430, 420-440
  • the target site at a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 is selected from the group consisting of nucleotides spanning positions numbered 465-484, 558-577, and 593-612 of SEQ ID NO:61.
  • the LPL target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:61 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:61 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LIPA target site comprises a position within SEQ ID NO:69, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:69, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:69, within exon 1 of SEQ ID NO:69, within exon 2 of SEQ ID NO:69, or within exon 3 of SEQ ID NO:69.
  • the LIPA target site comprises a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350 and 340-360 of SEQ ID NO:69.
  • the target site at a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 is selected from the group consisting of nucleotides spanning positions numbered 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the LIPA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:69 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:69 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ACE target site comprises a position within SEQ ID NO:77, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:77, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:77, within exon 1 of SEQ ID NO:77, within exon 2 of SEQ ID NO:77, within exon 3 of SEQ ID NO:77, or within exon 4 of SEQ ID NO:77.
  • the ACE target site comprises a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370 and 360-380 of SEQ ID NO:77.
  • the target site at a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 is selected from the group consisting of nucleotides spanning positions numbered 135-154, 237-256, and 332-351 of SEQ ID NO:77.
  • the ACE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:77 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:77 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 1 17, 100 nucleotides
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:1 17 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:1 17 is selected from the group consisting of nucleotides spanning positions numbered 101-120, 1 1 1-130, 121 -140, 131-150, 141-160, and 150-169 of SEQ ID NO:
  • the PAFAHG target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 17 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 17 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PIPLC target site comprises a position within SEQ ID NO:1 18, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 1 18, 100 nucleotides
  • the PIPLC target site comprises a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:1 18 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:1 18 is selected from the group consisting of nucleotides spanning positions numbered 39-188, 39-58, 49-68, 59-78, 69-88, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 149-178, and 159-188 of SEQ ID NO:1 18.
  • the PIPLC target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 18 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 18 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LCE target site comprises a position within SEQ ID NO:1 19, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 1 19, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 1 19, within exon 1 of SEQ ID NO: 1 19, within exon 2 of SEQ ID NO: 1 19, within exon 3 of SEQ ID NO: 1 19, within exon 4 of SEQ ID NO: 1 19, or within exon 5 of SEQ ID NO:1 19.
  • the LCE target site comprises a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:1 19 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:1 19 is selected from the group consisting of nucleotides spanning positions numbered 89-140, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, and 139-158 of SEQ ID NO:1 19.
  • the LCE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 19 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 19 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the IAH1 target site comprises a position within SEQ ID NO:120, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 120, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 120, within exon 1 of SEQ ID NO: 120, within exon 2 of SEQ ID NO:120, within exon 3 of SEQ ID NO:120, or within exon 4 of SEQ ID NO:120.
  • the IAH1 target site comprises a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO: 120 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430
  • the target site at a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO: 120 is selected from the group consisting of nucleotides spanning positions numbered 104-325, 104-123, 1 14-133, 124-143, 134-153, 144-163, 154-173, 164-183, 174-193, 184-203, 194-213, 204-223, 214-233, 224-243, 234-253, 244-263, 254-273, 264-283, 274-293, 284-303, 294-313, 304-323, and 314-333 of SEQ ID NO:120.
  • the IAH1 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO: 120 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:120 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 121 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 121 , within exon 1 of SEQ ID NO: 121 , within exon 2 of SEQ ID NO: 121 , within exon 3 of SEQ ID NO: 121 , or within exon 4 of SEQ ID NO: 121.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 is selected from the group consisting of nucleotides spanning positions numbered 69-195, 69-88, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, and 179-198 of SEQ ID NO:121.
  • the LPLA2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:121 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:121 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the CEH target site comprises a position within SEQ ID NO:122, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 122, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 122, within exon 1 of SEQ ID NO: 122, within exon 2 of SEQ ID NO: 122, within exon 3 of SEQ ID NO: 122, within exon 4 of SEQ ID NO: 122, or within exon 5 of SEQ ID NO: 122.
  • the CEH target site comprises a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO: 122 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430
  • the target site at a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO: 122 is selected from the group consisting of nucleotides spanning positions numbered 79-186, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, and 169-188 of SEQ ID NO:122.
  • the CEH target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:122 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:122 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ASA target site comprises a position within SEQ ID NO:123, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 123, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 123, within exon 1 of SEQ ID NO: 123, within exon 2 of SEQ ID NO: 123, within exon 3 of SEQ ID NO: 123, or within exon 4 of SEQ ID NO: 123.
  • the ASA target site comprises a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO: 123 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430
  • the target site at a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO: 123 is selected from the group consisting of nucleotides spanning positions numbered 79-296, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, 179-198 , 189-208 , 199-218, 209-228, 219-238, 229-248, 239-258, 249-268, 259-278, 269-288, and 279-298 of SEQ ID NO:123.
  • the ASA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:123 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:123 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the cell further integrates an exogenous nucleic acid sequence.
  • the cell is capable of producing an exogenous protein of interest.
  • the altered protein resulting from a disrupted gene does not bind to the protein of interest produced by the cell.
  • an isolated Chinese hamster ovary (CHO) cell comprising an engineered nucleic acid sequence comprising a variant of the PLBD2 gene (such as a variant of SEQ ID NO:33).
  • the PLBD2 gene comprises GACAGTCACG TGGCCCGACT GAGGCACGCG , nucleotides 1-30 of SEQ ID NO:33 (SEQ ID NO: 44).
  • the PLBD2 gene is engineered to disrupt expression of the open reading frame.
  • the invention provides an isolated CHO cell comprising (a) a disrupted PLBD2 gene comprising GACAGTCACG TGGCCCGACT GAGGCACGCG (SEQ ID NO: 44, also nucleotides 1- 30 of SEQ ID NO:33), (b) a disrupted esterase gene comprising a nucleotide encoding any one of the amino acid sequences in Table 2, or (c) a protein fragment of Table 2 expressed by a disrupted PLBD2 gene; and an exogenous nucleic acid sequence comprising a gene of interest.
  • the CHO cell that comprises an engineered nucleic acid sequence comprising a variant of the PLBD2 gene also comprises variants of one or more of the genes encoding (1 ) LPL (variant of SEQ ID NO:61 ), (2) LIPA (SEQ ID NO:69), (3) ACE (SEQ ID NO:77), (4) PAFAHG (SEQ ID NO:1 17), (5) PIPLC (SEQ ID NO:1 18), (6) LCE (SEQ ID NO:1 19), (7) IAH1 (SEQ ID NO:120), (8) LPLA2 (SEQ ID NO:121 ), (9) CEH (SEQ ID NO:122), and/or (10) ASA (SEQ ID NO:123).
  • a method of producing a protein of interest using a recombinant host cell wherein the host cell is modified to decrease the expression levels of esterase relative to the expression levels of esterase in an unmodified cell.
  • the method comprises the modified host cell having decreased esterase expression and an exogenous nucleic acid sequence comprising a gene of interest (GOI).
  • GOI gene of interest
  • the exogenous nucleic acid sequence comprises one or more genes of interest.
  • the one or more genes of interest are selected from the group consisting of a first GOI, a second GOI and a third GOI.
  • the invention provides expression systems comprising the recombinant host cell comprising modified or nonfunctional esterase.
  • the cell comprises a GOI operably linked to a promoter capable of driving expression of the GOI, wherein the promoter comprises a eukaryotic promoter that can be regulated by an activator or inhibitor.
  • the eukaryotic promoter is operably linked to a prokaryotic operator, and the eukaryotic cell optionally further comprises a prokaryotic repressor protein.
  • one or more selectable markers are expressed by the modified host cell.
  • the genes of interest and/or the one or more selectable markers are operably linked to a promoter, wherein the promoter may be the same or different.
  • the promoter comprises a eukaryotic promoter (such as, for example, a CMV promoter or an SV40 late promoter), optionally controlled by a prokaryotic operator (such as, for example, a tet operator).
  • the cell further comprises a gene encoding a prokaryotic repressor (such as, for example, a tet repressor).
  • a CHO host cell comprising recombinase recognition sites.
  • the recombinase recognition sites are selected from a LoxP site, a Lox5 1 1 site, a Lox2272 site, Lox2372, Lox5171 , and a frt site.
  • the cell further comprises a gene capable of expressing a recombinase.
  • the recombinase is a Cre recombinase.
  • the selectable marker gene is a drug resistance gene.
  • the drug resistance gene is a neomycin resistance gene or a hygromycin resistance gene.
  • the second and third selectable marker genes encode two different fluorescent proteins.
  • the two different fluorescent proteins are selected from the group consisting of Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (mKate).
  • the first, second, and third promoters are the same. In another embodiment, the first, second, and third promoters are different from each other. In another embodiment, the first promoter is different from the second and third promoters, and the second and third promoters are the same. In more embodiments, the first promoter is an SV40 late promoter, and the second and third promoters are each a human CMV promoter. In other embodiments, the first and second promoters are operably linked to a prokaryotic operator.
  • the host cell line has an exogenously added gene encoding a recombinase integrated into its genome, operably linked to a promoter.
  • the recombinase is Cre recombinase.
  • the host cell has a gene encoding a regulatory protein integrated into its genome, operably linked to a promoter.
  • the regulatory protein is a tet repressor protein.
  • the first GOI and the second GOI encode a light chain, or fragment thereof, of an antibody or a heavy chain, or fragment thereof, of an antibody.
  • the first GOI encodes a light chain of an antibody and the second GOI encodes a heavy chain of an antibody.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a first light chain, or fragment thereof, a second light chain, or fragment thereof and a heavy chain, or fragment thereof.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a light chain, or fragment thereof, a first heavy chain, or fragment thereof and a second heavy chain, or fragment thereof.
  • a method for making a protein of interest comprising (a) introducing into a CHO host cell a gene of interest (GOI), wherein the GOI integrates into a specific locus such as a locus described in US Patent No. 7771997B2, issued August 10, 2010 or other stable integration and/or expression-enhancing locus; (b) culturing the cell of (a) under conditions that allow expression of the GOI; and (c) recovering the protein of interest.
  • the protein of interest is selected from the group consisting of a subunit of an immunoglobulin, or fragment thereof, and a receptor, or ligand-binding fragment thereof.
  • the protein of interest is selected from the group consisting of an antibody light chain, or antigen-binding fragment thereof, and an antibody heavy chain, or antigen-binding fragment thereof.
  • the CHO host cell genome comprises further modifications, and comprises one or more recombinase recognition sites as described above, and the GOI is introduced into a specific locus through the action of a recombinase that recognizes the
  • the GOI is introduced into the cell employing a targeting vector for recombinase-mediated cassette exchange (RMCE) when the CHO host cell genome comprises at least one exogenous recognition sequence within a specific locus.
  • RMCE recombinase-mediated cassette exchange
  • the GOI is introduced into the cell employing a targeting vector for homologous recombination, and wherein the targeting vector comprises a 5' homology arm homologous to a sequence present in the specific locus, a GOI, and a 3' homology arm
  • the targeting vector further comprises two, three, four, or five or more genes of interest.
  • one or more of the genes of interest are operably linked to a promoter.
  • a method for modifying a CHO cell genome to integrate an exogenous nucleic acid sequence comprising the step of introducing into the cell a vehicle comprising an exogenous nucleic acid sequence wherein the exogenous nucleic acid integrates within a locus of the genome.
  • the invention provides a process for manufacturing a stable protein formulation comprising the steps of: (a) extracting a protein fraction from the modified host cell of the invention having decreased or ablated expression of esterase, (b) contacting the protein fraction comprising a protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (c) collecting the protein of interest from the media, wherein a reduced level of the esterase activity is associated with the protein fraction collected at step (c), thus providing a stable protein formulation.
  • PA protein A affinity
  • CEX cation exchange
  • AEX anion exchange
  • the invention provides a process for reducing esterase activity in a protein formulation comprising the steps of: (a) modifying a host cell to decrease or ablate expression of esterase, (b) transfecting the host cell with a protein of interest, (c) extracting a protein fraction from the modified host cell, (c) contacting the protein fraction comprising the protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (d) collecting the protein of interest from the media, and (e) combining the protein of interest with a fatty acid ester, and optionally a buffer and thermal stabilizer, thus providing a protein formulation essentially free of detectable esterase activity.
  • the protein formulation is essentially free of PLBD2 protein or PLBD2 activity.
  • a method for modifying a CHO cell genome to express a therapeutic agent comprising a vehicle for introducing, into the genome, an exogenous nucleic acid comprising a sequence for expression of the therapeutic agent, wherein the vehicle comprises a 5' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33, a nucleic acid encoding the therapeutic agent, and a 3' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33.
  • the invention provides a modified CHO host cell comprising a modified CHO genome wherein the CHO genome is modified by disruption of target sequence within a nucleotide sequence at least 90% identical to SEQ ID NO: 33.
  • the modified CHO host cell further comprises another FAH target sequence disruption.
  • the another FAH target sequence is within (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:61 , (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:69 and/or (3) a nucleotide sequence at least 90% identical to SEQ ID NO:77.
  • the invention provides a modified eukaryotic host cell comprising a modified eukaryotic genome wherein the eukaryotic genome is modified at a target sequence in a coding region of the target gene by a site-specific nuclease.
  • the site-specific nuclease comprises a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, or an RNA-guided DNA endonuclease.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • TAL effector domain fusion protein or an RNA-guided DNA endonuclease.
  • the invention also provides methods of making such a modified eukaryotic host cell.
  • the target sequence can be placed in the indicated orientation as in SEQ ID NO:33, 61 , 69, 77, 1 17, 1 18, 1 19, 120, 121 , 122, or 123; or in the reverse of the orientation of SEQ ID NO:33, 61 , 69, 77, 1 17, 1 18, 1 19, 120, 121 , 122, or 123.
  • Fig. 1 depicts the results of Taqman® quantitative polymerase chain reaction (qPCR) experiments to detect genomic (gDNA) or transcripts (mRNA) of the modified clones.
  • Primers and probes were designed to flank the sequences predicted as subject to targeted disruption within exon 1 , either starting at nucleotide 37 (sgRNAI ) or starting at nucleotide 44 (sgRNA2) of SEQ ID NO:33.
  • Relative amount of amplicons from clones targeted by either sgRNAI or sgRNA2 are graphed (i.e., relative to amplicons derived from the negative control transfection clones which were subject to no sgRNA or unmatched sgRNA).
  • Clone 1 for example, has relatively no amplified gDNA nor mRNA per qPCR of the targeted exon 1 region. Clone 1 and several other clones were selected for follow up analysis. The vertical axes represent the relative amount of template containing sgRNAI sequence (upper panel) or sgRNA2 sequence (lower panel).
  • FIG. 2A and Fig. 2B illustrate the results of further PCR analysis of a Clone 1 cells population compared to wild type Chinese hamster overy (CHO) cells.
  • Fig. 2A shows a PCR amplicon from Clone 1 that is shorter in length (bp) compared to the amplicon from genomic DNA of wild type cells.
  • Fig. 2B shows a PCR amplicon from Clone 1 that is shorter in length (bp) compared to the amplicon from mRNA of wild type cells. Sequencing confirmed an 1 1 bp deletion in the PLBD2 gene of Clone 1 .
  • the vertical axes represent the size of the PCR fragments in base-pairs (bp).
  • Fig. 3 illustrates the relative protein titer of monoclonal antibody 1 (mAbl )-expressing Clone 1 cells (RS001 ) or mAb1 -expressing wild type CHO cells (RS0WT) subject to the same fed-batch culture conditions for 12 days. Samples of conditioned medium were extracted for each culture, and the Protein A binding fraction was quantified at Day 2, 4, 6, 9 and 12.
  • Fig. 4 shows the results of RS001 or RSOWT cells following production culture and protein purification using either Protein A (PA) alone, or PA and anion exchange (AEX) chromatography. PA-purified mAb1 from RS001 and RSOWT was analyzed for lipase abundance using trypsin digest mass spectrometry. As such, trypsin digests of RS001 - and RSOWT-produced mAb1 were injected into a reverse phase liquid chromatography column coupled to a triple quadrupole mass
  • spectrometer set to monitor a specific PLBD2 product fragment (as in Table 2).
  • Control reactions containing reference samples of mAb1 (with no endogenous PLBD2) spiked with varying amounts of recombinant PLBD2 were also analyzed and plotted. The signals detected in the experiments were compared to the control reactions to determine concentration of PLBD2.
  • mAb1 produced from Clone 1 shows no detectable amounts of PLBD2 when purified with PA alone.
  • Fig. 5 is a line plot depicting percent cell viability as a function of time in days. Open circles (-0-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A -) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 6 is a line plot depicting protein production (titer) in grams per liter as a function of time in days. Open circles (-o-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A-) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 7 is a line plot depicting viable cell counts in cells per milliliter as a function of time in days. Open circles (-o-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A-) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 8 shows a formatted alignment of LPL knock out constructs clone 19, clone 20, clone 21 , and clone 22, represented by SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161 , and SEQ ID NO: 162, respectively.
  • the partial wildtype LPL sequence is represented by SEQ ID NO: 158.
  • exogenously added gene refers to any DNA sequence or gene not present within the genome of the cell as found in nature.
  • an "exogenously added gene” within a CHO genome can be a gene from any other species (e.g., a human gene), a chimeric gene (e.g., human/mouse), or a hamster gene not found in nature within the particular CHO locus in which the gene is inserted (i.e., a hamster gene from another locus in the hamster genome), or any other gene not found in nature to exist within a CHO locus of interest.
  • Percent identity when describing an esterase, e.g., a hydrolase protein, such as SEQ ID NO:32, 34, 35, 37, 62,70, 74, 75, 76, 78, 82, 83, 84; 124, 125, 126, 127, 128, 129, and 130; or gene, such as SEQ ID NO:33, 61 , 69, 77, 1 17, 1 18, 1 19, 120, 121 , 122, and 123 includes homologous sequences that display the recited identity along regions of contiguous homology, but the presence of gaps, deletions, or insertions that have no homolog in the compared sequence are not taken into account in calculating percent identity.
  • a hydrolase protein such as SEQ ID NO:32, 34, 35, 37, 62,70, 74, 75, 76, 78, 82, 83, 84; 124, 125, 126, 127, 128, 129, and 130; or gene, such as SEQ ID NO
  • a "percent identity" determination between, e.g., SEQ ID NO:32 with a species homolog would not include a comparison of sequences where the species homolog has no homologous sequence to compare in an alignment (i.e., SEQ ID NO:32 compared to a fragment thereof, or the species homolog has a gap or deletion, as the case may be). Thus, “percent identity” does not include penalties for gaps, deletions, and insertions.
  • Target sites are the sites within the genomic sequence selected for cleavage or break by a nuclease.
  • the DNA break is normally repaired by the non-homologous end-joining (NHEJ) DNA repair pathway.
  • NHEJ non-homologous end-joining
  • InDels insertions or deletions
  • ORF open reading frame
  • Targeted insertion refers to gene targeting methods employed to direct insertion or integration of a gene or nucleic acid sequence to a specific location on the genome, i.e., to direct the DNA to a specific site between two nucleotides in a contiguous polynucleotide chain. Targeted insertion may also be performed to introduce a small number of nucleotides or to introduce an entire gene cassette, which includes multiple genes, regulatory elements, and/or nucleic acid sequences. "Insertion” and “integration” are used interchangeably.
  • Recognition site or “recognition sequence” is a specific DNA sequence recognized by a nuclease or other enzyme to bind and direct site-specific cleavage of the DNA backbone.
  • Endonucleases cleave DNA within a DNA molecule.
  • Recognition sites are also referred to in the art as recognition target sites.
  • Polysorbates are fatty acid esters of sorbitan or iso-sorbide (polyoxyethylene sorbitan or iso- sorbide mono- or di- esters).
  • the polyoxyethylene serves as the hydrophilic head group and the fatty acid as the lipophilic hydrophobic tail.
  • the effectiveness as a surfactant of the polysorbate depends upon the amphiphilic nature of the molecule with both hydrophilic head and hydrophobic tail present (in a single molecule).
  • SVP subvisible particle
  • SVPs may attribute to immunogenicity. Regulatory authorities like the United States Food and Drug Administration (USFDA) provide limitations on the number of subvisible particles (SVPs) allowed in a pharmaceutical formulation. United States Pharmacopeia (USP) publishes standards for strength, purity and quality of drugs and drug ingredients, as well as food ingredients and dietary
  • USP 31 monograph ⁇ 788> sets the limit for number of particles allowed in parenteral formulations.
  • USP 31 monograph ⁇ 788> is available at http://www.uspnf.com/official- text/revision-bulletins/particulate-matter-iniections: and as Revision Bulletin Official July 1 , 2012, ⁇ 788> Particulate Matter in Injections, The United States Pharmacopeial Convention.
  • the limit is set at no more than 25 particles of at least 10 microns per ml_, and no more than 3 particles of at least 25 microns per ml_.
  • the limit is set at no more than 6,000 particles of at least 10 microns per container, and no more than 600 particles of at least 25 microns per container.
  • the term "stability” refers to the retention of an acceptable degree of physical structure (colloidal, nature), chemical structure or biological function of the biologically active agent (e.g., biotherapeutic or other protein produced in a cell-based bioprocess) over time during storage (a.k.a. "shelf-life"), during processing, or after administration and while in vivo.
  • the biologically active agent may be stable even though it does not maintain 100% of its structure or function after storage or administration for a defined amount of time.
  • the biologically active agent and formulation containing the biologically active agent may be regarded as "stable".
  • Stability can be measured, inter alia, by determining the percentage of native molecule that remains in the formulation after storage or administration for a defined amount of time at a defined temperature.
  • the percentage of native molecule can be determined by, inter alia, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]), such that native means non-aggregated and non-degraded.
  • SE-HPLC size exclusion high performance liquid chromatography
  • At least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the biologically active agent can be detected in the formulation after a defined amount of time at a defined temperature or under physiological conditions after administration.
  • the defined amount of time after which stability is measured can be about 14 days, about 28 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 1 1 months, about 12 months, about 18 months, about 24 months, or more.
  • the temperature at which the formulation containing the biologically active agent may be kept when assessing stability can be any temperature from about -80°C to about 45°C, e.g., storage at about - 80°C, about -30°C, about -20°C, about 0°C, about 4°-8°C, about 5°C, about 25°C, about 35°C, about 37°C or other physiological temperatures, or about 45°C.
  • the biologically active agent may be deemed stable if after 3 months under physiological conditions, greater than about 75%, 80%, 85% or 90% of native molecule is detected in the soluble fraction by SE-HPLC or other size exclusion or size determination method.
  • “Physiological temperature” includes the body temperature of any vertebrate.
  • physiological temperature of humans is about 37°C.
  • physiological temperature is between about 25°C and about 45°C.
  • physiological temperature is about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, and about 45°C.
  • Stability can be measured, inter alia, by determining the percentage of biologically active agent, such as a protein, that forms an aggregate (i.e., high molecular weight species) after a defined amount of time at a defined temperature, wherein stability is inversely proportional to the percent high molecular weight (HMW) species that is formed of the biologically active agent (protein).
  • the percentage of HMW species of the biologically active agent can be determined by, inter alia, size exclusion chromatography, as described above.
  • a pharmaceutical formulation containing the biologically active agent may also be deemed stable if after three months at physiological conditions less than about 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1 % of the biologically active agent is detected in a HMW form.
  • Stability can be measured, inter alia, by determining the percentage of a biologically active agent, such as a protein, that is degraded or otherwise is found as a low molecular weight (LMW) species after a defined amount of time at a defined temperature. Stability is inversely proportional to the percent LMW species that is formed in the soluble fraction. The percentage of LMW species of the biologically active agent in the soluble fraction can be determined by, inter alia, size exclusion chromatography, as described above.
  • LMW low molecular weight
  • a pharmaceutical formulation may also be deemed stable if after three months under storage conditions less than about 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1 % of the biologically active agent is detected in a LMW form.
  • compositions prepared in host cells as described herein are envisioned to comply with standards set forth by multiple regulatory authorities and ICH guidelines.
  • the composition complies if tested for subvisible particles and the test results in the average number of particles present in the units tested does not exceed 12 per milliliter equal to or greater than 10 um in size, and does not exceed 2 per milliliter equal to or greater than 25 um.
  • Various test for microscopic particles in solution are well-known in the art, including but not limited to tests recommended in ICH Guideline Q4B Annex 3(R1 ), dated 27 September 2010 (Evaluation And Recommendation Of Pharmacopoeial Texts For Use In The lch Regions On Test For Particulate Contamination: Sub-Visible Particles General Chapter).
  • HCPs proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins. HCPs are generally process-related impurities during biologies production. The amount of residual HCPs in drug product is generally considered a critical quality attribute (CQA), due to their potential to affect product safety and efficacy. Regulatory authorities require a product sponsor to monitor the removal of HCPs in drug product during bioprocess development. A sensitive assay e.g., immunoassay, capable of detecting a wide range of protein impurities is generally utilized. This testing can include verification at commercial scale in accordance with regional regulations and may be done at the time of submission of a marketing approval application.
  • CQA critical quality attribute
  • ICH Specifications (Q6A and Q6B, section 2.3), if a drug substance or drug product does not contain any impurity in the specific formulation, i.e. if efficient control or removal to acceptable levels is demonstrated by suitable studies, further testing may be reduced or eliminated upon approval by the regulatory authorities.
  • compositions prepared in host cells as described herein are envisioned to comply with standards set forth by multiple regulatory authorities and ICH guidelines.
  • the composition prepared by the host cells described herein comprise less than 100 ng/mg (ppm), less than about 90 ppm, less than about 80 ppm, less than about 70 ppm, less than about 60 ppm, less than about 50 ppm, less than about 40 ppm, less than about 30 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or 0 ppm of the target host cell protein, i.e. fatty acid hydrolase.
  • fatty acid hydrolase or “FAH” refers to any hydrolytic enzyme that cleaves at a carbonyl group creating a carboxylic acid product in which the carboxylic acid comprises an R- group that is lipophilic or otherwise hydrophobic.
  • the carboxylic acid product is a fatty acid.
  • Esterases and “fatty acid acylases/amidases” are included as subgenera of fatty acid hydrolase.
  • Lipases are a subgenus of esterases that cleave lipids (fats, waxes, sterols, glycerides and phospholipids.
  • Phospholipases are a subgenus of lipases that cleave
  • phospholipids phospholipids. Esterases cleave fatty acid esters into fatty acids and alcohols. Lipases include PLBD2, LPL and LIPA. "Ceramidase” is a subgenus of fatty acid acylase that cleaves ceramide and releases a fatty acid and sphingosine, which is an amino alcohol. Examples of ceramidases include acid ceramidase, neutral ceramidase, alkaline ceramidase 1 , alkaline ceramidase 2 and alkaline ceramidase 3.
  • Protein A-binding fraction refers to the fraction of cell lysate from cultured cells expressing a protein of interest which binds to a Protein A affinity format. It is well understood in the art that Protein A affinity chromatography, such as Protein A chromatography medium, such as resins, beads, columns and the like, are utilized to capture Fc-containing proteins due to their affinity to Protein A.
  • Protein A affinity chromatography such as Protein A chromatography medium, such as resins, beads, columns and the like
  • Phospholipase B-like 2 refers to the homologs of a phospholipase gene known as NCBI RefSeq. XM_003510812.2 (SEQ ID NO:33) or protein known as NCBI RefSeq. XP_003510860.1 (SEQ ID NO:32), and further described herein.
  • PLBD2 is also referred to in the art as putative phospholipase B-like 2 (PLBL2), 76 kDa protein, LAMA-like protein 2, PLB homolog 2, lamina ancestor homolog 2, mannose-6-phosphate protein associated protein p76, p76, phospholipase B-like 2 32 kDa form, phospholipase B-like 2 45 kDa form, or Lysosomal 66.3 kDa protein.
  • Lipoprotein lipase is a glycosylated homodimer secreted by parenchymal cells and associated with endothelial cells of the capillary lumen.
  • Exemplary LPL proteins include Chinese hamster LPL (SEQ ID NO:62), mouse LPL (SEQ ID NO:66), ), rat LPL (SEQ ID NO:67) and human LPL (SEQ ID NO:68).
  • Mouse LPL is 92% identical to Chinese hamster LPL.
  • Rat LPL is 92% identical to Chinese hamster LPL.
  • Human LPL is 88% identical to Chinese hamster LPL.
  • Chinese hamster LPL is encoded by a polynucleotide sequence of SEQ ID NO:61 .
  • Lysosomal acid lipase also known as lysosomal lipase, lipase A, lysosomal acid and cholesterol esterase is an intracellular lipase that functions in the lysosome.
  • LI PA reversibly catalyzes cholesteryl ester bond formation and cleavage.
  • LI PA is also a glycosylated homodimer.
  • Exemplary LIPA proteins include Chinese hamster LIPA (SEQ ID NO:70), mouse LIPA (SEQ ID NO:74), ), rat LIPA (SEQ ID NO:75) and human LIPA (SEQ ID NO:76).
  • Mouse LIPA is 72% identical to Chinese hamster LIPA.
  • Rat LIPA is 75% identical to Chinese hamster LIPA.
  • Human LIPA is 74% identical to Chinese hamster LIPA.
  • Chinese hamster LIPA is encoded by a polynucleotide sequence of SEQ ID NO:69.
  • Acid ceramidase also known as ASAH1 , AC, ACDase, ASAH, PHP, PHP32, SMAPME, N-acylsphingosine amidohydrolase (acid ceramidase) 1 is an acylase that cleaves ceramide to produce fatty acid and sphingosine. It is a heterodimer comprising a non-glycosylated alpha subunit and a glycosylated beta subunit. Acid ceramidase has an acid pH optimum. The lipid accumulation disease, Farber Lipogranulomatosis, is associated with a deficiency in acid ceramidase activity.
  • Exemplary acid ceramidases include Chinese hamster ACE (SEQ ID NO:78), mouse ACE (SEQ ID NO:82), ), rat ACE (SEQ ID NO:83) and human ACE (SEQ ID NO:84).
  • Mouse ACE is 87% identical to Chinese hamster ACE.
  • Rat ACE is 89% identical to Chinese hamster ACE.
  • Human ACE is 83% identical to Chinese hamster ACE.
  • Chinese hamster ACE is encoded by a polynucleotide sequence of SEQ ID NO:77.
  • Platelet-activating factor acetylhydrolase IB subunit gamma (PAFAHG), also known as PAFAH1 B3, PAFAHG, and platelet activating factor acetylhydrolase 1 b catalytic subunit 3 is one of the catalytic subunits along with beta of the cytosolic tetrameric platelet-activating factor
  • acetylhydrolase IB belongs to the phospholipase A2 family and catalyzes the hydrolysis of the acyl group at position 2 of glycerol in bioactive phospholipids (see Stafforini et al., Journal of Biological Chemistry, 272:17895-17898, July 1997).
  • Chinese hamster PAFAHG (SEQ ID NO:124) is 98% identical to both rat and mouse PAFAHG, and 96% identical to human PAFAHG.
  • Chinese hamster PAFAHG is encoded by a polynucleotide sequence of SEQ ID NO:1 17.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO: 125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO: 125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO: 125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO: 125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO: 125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Additional fatty acid hydrolases that can serve as targets for deletion, either individually, or in combination with one of more additional fatty acid hydrolases are listed in Table 1. Any of the following hydrolases, or their equivalents, listed in Table A may be the target protein in the creation of a knockout cell line, where removal of the host cell protein is necessitated due to contamination in the preparation of a biopharmaceutical product.
  • cell or “cell line” includes any cell that is suitable for expressing a recombinant nucleic acid sequence.
  • Cells include those of prokaryotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g. , strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g.,S. cerevisiae, S. pombe, P. partoris, P.
  • the cell is a human, monkey, ape, hamster, rat or mouse cell.
  • the cell is eukaryotic and is selected from the following cells: CHO (e.g.,CHO K1 , DXB-1 1 CHO, Veggie-CHO), COS (e.g.,COS-7), retinal cells, Vero, CV1 , kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21 ), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1 , U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT cell, tumor cell, and a cell line derived from an aforementioned cell.
  • the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6® cell).
  • the invention is based at least in part on a recombinant host cell and cell expression system thereof that decreases expression of two or more an endogenous host cell fatty acid hydrolases (FAHs), decreases the enzymatic function or binding ability of two or more endogenous host cell FAHs, or lacks detectable expression of two or more FAHs.
  • FHs endogenous host cell fatty acid hydrolases
  • the inventors discovered that the disruption of genes encoding at least two FAHs allows for the optimized and efficient production and purification of biopharmaceutical products expressed in such expression systems.
  • the invention may be employed in several ways, such as 1 ) utilizing gene editing tools to totally knockout FAH expression, whereas no measurable full-length FAH enzyme is expressed in the cell due to disruption of the gene encoding the FAH; 2) utilizing gene editing tools to eliminate or reduce enzymatic activity, whereas the FAH protein is expressed but rendered nonfunctional due to disruptions in its gene; and 3) utilizing gene editing tools to eliminate or reduce the ability of an endogenous host cell FAH to bind exogenous recombinant protein produced by the cell.
  • FAH activity was determined in protein fractions of certain antibody-producing cells. Several particular fatty acid hydrolases were determined as contaminants in these protein fractions, including three carboxylic esterases (a.k.a.
  • esterases phospholipase B-like (PLBD2), lipoprotein lipase (LPL) and lysosomal acid lipase (LIPA), and acid ceramidase, a carboxylic amidase (an acylase).
  • PLBD2 phospholipase B-like
  • LPL lipoprotein lipase
  • LIPA lysosomal acid lipase
  • acid ceramidase a carboxylic amidase (an acylase).
  • Gene editing target sites were identified in hamster PLBD2, LPL, LIPA and acid ceramidase (ACE) genes that enable targeted disruption of those genes in a hamster cell (i.e., CHO) genome.
  • ACE acid ceramidase
  • An optimized host cell comprising a combination of genetic modifications that affect the expression of genes encoding (1 ) PLBD2 and LPL; (2) PLBD2 and LIPA; (3) PLBD2 and ACE; (4) LPL and LIPA; (5) LPL and ACE; (6) LIPA and ACE; (7) PLBD2, LPL and LIPA; (8) PLBD2, LPL and ACE; (9) PLBD2, LIPA and ACE; (10) LPL, LIPA and ACE, (1 1 ) PLBD2, LPL, LIPA and ACE, (12) PLBD2 and PIPLC, (13) PLBD2 and CEH, (14) PLBD2 and PAFAHG, (15) PLBD2 and LCE, (16) PLBD2 and ASA, (17) PLBD2 and IAH1 , (18) PLBD2 and LPLA2, (19) LPL and PIPLC, (20) LPL and CEH, (21 ) LPL and PAFAHG, (22) LPL and LCE, (23)
  • Such a cell is envisioned to reduce the burden of certain purification steps, thereby reducing time and cost, while increasing production yield. Also, the formulated protein is expected to have improved stability due to the reduced hydrolase burden.
  • the invention is also based on the specific targeting of an exogenous gene to the integration site.
  • the methods of the invention allow efficient modification of the cell genome, thus producing a modified or recombinant host cell useful as a cell expression system for the bioprocessing of therapeutic or other commercial protein products.
  • the methods of the invention employ cellular genome gene editing strategies for the alteration of particular genes of interest that otherwise may diminish or contaminate the quality of recombinant protein formulations, or require multiple purification steps.
  • compositions of the invention can also be included in expression constructs for example, in expression vectors for cloning and engineering new cell lines. These cell lines comprise the modifications described herein, and further modifications for optimal incorporation of expression constructs for the purpose of protein expression are envisioned.
  • Expression vectors comprising polynucleotides can be used to express proteins of interest transiently, or can be integrated into the cellular genome by random or targeted recombination such as, for example, homologous recombination or recombination mediated by recombinases that recognize specific recombination sites (e.g., Cre-lox-mediated recombination).
  • Target sites for disruption or insertion of DNA are typically identified with the maximum effect of the gene disruption or insertion in mind.
  • target sequences may be chosen near the N-terminus of the coding region of the gene of interest whereas a DNA break is introduced within the first or second exon of the gene.
  • Introns non-coding regions
  • the changes introduced by these modifications are permanent to the genomic DNA of the organism.
  • GOI expressible gene of interest
  • any other desirable elements such as, e.g., promoters, enhancers, markers, operators, ribosome binding sites (e.g., internal ribosome entry sites), etc. are also employed.
  • the resulting recombinant cell line conveniently provides more efficient downstream bioprocess methods with respect to expressible exogenous genes of interest (GOIs), since purification steps for exogenous proteins of interest are eliminated due to the absence of the contaminant host cell protein. Eliminating or refining purification procedures also results in higher amounts (titer) of the recovered protein of interest.
  • GOIs expressible exogenous genes of interest
  • Applicants have discovered enzymatic activities associated with the destabilization of polysorbates (including polysorbate 20 and polysorbate 80) and/or enzymatic activities co-purifying with highly concentrated, multimerized or aggregated protein. Those activities were found to be associated with one or more fatty acid hydrolases (FAHs).
  • FAHs fatty acid hydrolases
  • One such FAH was identified from the peptide sequences listed in Table 2.
  • a BLAST search of those peptide sequences revealed identity with a putative phospholipase B-like 2 (PLBD2, also referred to as PLBL2).
  • PLBD2 is highly conserved in hamster (SEQ ID NO:32), mice (SEQ ID NO:34), rat (SEQ ID NO:35), human (SEQ ID NO:36), and bovine (SEQ ID NO:37).
  • the applicants discovered that PLBD2 which co-purifies under certain processes with some classes of proteins-of-interest manufactured in a mammalian cell line, has enzymatic activity responsible for the hydrolysis of polysorbate 20 and 80.
  • Other FAH species, of which PLBD2 is an example may contribute to polysorbate instability or persist as hydrophobic "sticky" proteins that bind protein multimers or aggregates during purification and ultimate formulation, depending upon the particular protein-of-interest and/or background of the host cell.
  • IgGs formulated with polysorbate 20 did not form particles and the putative esterase did not hydrolyze the polysorbate 20.
  • the author reported that the putative lipase associated with the IgG did not affect saturated C12 fatty acid (i.e., laurate) (Id at 7.)
  • LPL lipoprotein lipase
  • LIPA lysosomal acid lipase
  • ACE acid ceramidase
  • LPL is a triacylglycerol/diacylglycerol hydrolase of the carboxylic ester hydrolase (esterase) family (see Hide ef al., "Structure and evolution of the lipase superfamily," J Lipid Res. 1992 Feb; 33(2): 167-78).
  • LIPA is a sterol esterase and synthase that acts on esters of sterols and long-chain fatty acids (see Dubland and Francis, “Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism,” Front. Cell Dev. Biol. 2015 Feb 2; 3(3): 1-1 1 ).
  • Acid ceramidase is not a carboxylic ester hydrolase, but rather an amide hydrolase that cleaves fatty acids from ceramide at the amide bond (carboxylic amide hydrolase) (see Park and Schuchman, "Acid ceramidase and human disease,” Biochim. Biophys. Acta. 2006 Dec; 1758(12): 2133-8).
  • Phospholipases are a family of esterase enzymes that catalyze the cleavage of phospholipids. Each phospholipase subclass has different substrate specificity based on its target cleavage site.
  • Phospholipase B was identified as related to a group of prokaryotic and eukaryotic lipase proteins by virtue of the presence of a highly conserved amino acid sequence motif, Gly-Asp-Ser-Leu (GDSL) (Upton, C, and Buckley, JT. A new family of lipolytic enzymes? Trends Biochem Sci. 1995; 20:178-179).
  • phospholipase B is also classified with known GDSL(S) hydrolases, and has little sequence homology to true lipases, differentiating itself structurally from phospholipases by having a serine-containing motif closer to the N-terminus than other lipases.
  • phospholipase B-like proteins are also classified as N-terminal nucleophile (Ntn) hydrolases.
  • Ntn N-terminal nucleophile hydrolases.
  • PLB-like proteins such as phospholipase B-like protein 1 (PLBD1 ) and phospholipase B-like protein 2 (PLBD2), also have amidase activity, similar to other Ntn hydrolases (Repo, H. et al, Proteins 2014; 82:300-31 1 ).
  • Lipoprotein lipases have also been demonstrated to cleave carboxylic ester bonds of polysorbate 20 and 80; and to associate with some monoclonal antibodies during production (see N. Levy, "Host cell protein impurities and protein-protein interactions in downstream purification of monoclonal antibodies," Dissertation submitted to the Faculty of the University of Delaware, Summer 2014, UMI 3642330, Published by ProQuest LLC, 2014).
  • Cell-cultures of a CHO-K1 RNAi knock-down of LPL revealed diminished polysorbate esterase activity. The effect of such a knockdown on overall cell viability or the production of useful titers of ectopic protein has not been investigated.
  • Knockout of a host cell gene such as an FAH, more particularly one or more of phospholipase B-like protein 2, lipoprotein lipase, lysosomal acid lipase and acid ceramidase may be accomplished in several ways. Rendering the FAH encoding gene nonfunctional, or reducing the functional activity of the target FAH protein may be done by introducing point mutations in the FAH genomic sequence, particularly in the exons (coding regions).
  • nucleic acid sequences of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 were identified and sequences upstream and downstream of the target site (i.e., homologous arms) may be utilized to integrate an expression cassette comprising a mutated gene by homologous recombination. Further gene editing tools are described herein in accordance with the invention.
  • Cell lines devoid of multiple FAH activities are useful for the production of therapeutic proteins to be purified and stored long term, and such cell lines solve problems associated with long term storage of pharmaceutical compositions in a formulation containing a fatty acid ester surfactant by maintaining protein stability and reducing subvisible particle (SVP) formation (see also PCT International Application No. PCT/US 15/54600 filed October 8, 2015, which is hereby incorporated in its entirety into the specification).
  • SVP subvisible particle
  • Assays to detect FAH activity include polysorbate degradation measurements. Unpurified protein supernatants or fractions from CHO cells, and supernatant at each step or sequence of steps when subjected to sequential purification steps, is tested for stability of polysorbate, such as polysorbate 20 or 80. The measurement of percent intact polysorbate reported is inversely proportional to the amount of contaminating FAH activity. Other measurements for detection of FAH activity or presence of FAH in a protein sample are known in the art. Detection of FAH protein ⁇ e.g., lipase, phospholipase, PLBD2, LPL, LIPA, acylase, ACE) may be done by trypsin digest mass spectrometry.
  • micrometers in diameter may be counted in the protein formulation in order to detect esterase or other FAH activity.
  • glycerophospho[ 3 H]choline formation from phosphatidyl[3H]choline following incubation of phosphatidyl[ 3 H]choline and protein supernatant may be determined by thin-layer chromatography (following similar protocols according to Kanoh, H. et al. 1991 Comp Biochem Physiol 102B(2):367- 369).
  • SEQ ID NO:32 disclosed herein was identified from proteins expressed in CHO cells. Other mammalian species (such as, for example, humans, rats, mice), were found to have high homology to the identified FAH. Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques. In addition, variants of PLBD2, can then be tested for FAH activity as described herein. DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:32, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical
  • compositions and are candidates for targeted disruption in the engineered cell line are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:32 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian PLBD2 sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 3 identifies exemplary mammalian PLBD2 proteins and their degree of homology.
  • the targeted disruption of SEQ ID NO:33 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 33-62, 37-56, 40-69, 44-63, 1 10-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 33-62, 37-56, 40-69, 44-63, 1 10-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the PLBD2 nucleic acid sequence is at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:33 or target sequence thereof.
  • SEQ ID NO:62 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LPL can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:62, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:62 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian LPL sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 4 identifies exemplary mammalian LPL proteins and their degree of homology.
  • the targeted disruption of SEQ ID NO:61 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400- 420, 410-430, 420-440, 430-450, 440-460
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410- 430, 420-440, 430-450, 440-460, 450-470
  • the LPL nucleic acid sequence is at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:61 or target sequence thereof.
  • SEQ ID NO:70 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LIPA can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:70, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:70 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian LIPA sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 5 identifies exemplary mammalian LIPA proteins and their degree of homology.
  • the targeted disruption of SEQ ID NO:69 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the LIPA nucleic acid sequence is at least about 70% identical, at least about 71 % identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, or at least about 79% identical, at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:69 or target sequence thereof.
  • SEQ ID NO:78 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LPL can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:78, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:78 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • ACE mammalian acid ceramidase
  • the targeted disruption of SEQ ID NO:77 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 135-154, 237-256, and 332-351 of SEQ ID NO:77.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 350-370, 360-380, 135-154, 237-256, and 332-351 of SEQ I D NO:77.
  • the ACE nucleic acid sequence is at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:77 or target sequence thereof.
  • Cell populations expressing enhanced levels of a protein of interest can be developed using the cell lines and methods provided herein.
  • the isolated commercial protein, protein supernatant or fraction thereof, produced by the cells of the invention have no detectable esterase or esterase activity.
  • Cell pools further modified with exogenous sequence(s) integrated within the genome of the modified cells of the invention are expected to be stable over time, and can be treated as stable cell lines for most purposes. Recombination steps can also be delayed until later in the process of development of the cell lines of the invention.
  • Methods for genetically engineering a host cell genome in a particular location may be achieved in several ways. Genetic editing techniques were used to modify a nucleic acid sequence in a eukaryotic cell, wherein the nucleic acid sequence is an endogenous sequence normally found in such cells and expressing a contaminant host cell protein. Clonal expansion is necessary to ensure that the cell progeny will share the identical genotypic and phenotypic characteristics of the engineered cell line.
  • native cells are modified by a homologous recombination technique to integrate a nonfunctional or mutated target nucleic acid sequence encoding a host cell protein, such as a variant of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and/or SEQ ID NO:77.
  • One such method of editing the CHO PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and ASA genomic sequences involves the use of guide RNAs and a type II Cas enzyme to specifically target an exon of PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and/or ASA.
  • RNAs directed to particular exons of CHO PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and ASA have been employed (Table 7) in a site-specific nuclease editing method as described herein.
  • Other methods of targeted genome editing, for example nucleases, recombination-based methods, or RNA interference, to modify the FAH genes may be employed for the targeted disruption of the CHO genome.
  • the engineered mammalian host cell comprises one or more disruptions within gene sequences selected from the group consisting of nucleotides 37-63 of SEQ ID NO:33, nucleotides 465-612 of SEQ ID NO:61 , nucleotides 180-295 of SEQ ID NO:69, nucleotides 135-351 of SEQ ID NO:77, nucleotides 249-388 of SEQ ID NO:1 17, nucleotides 1624-2157 of SEQ ID NO:163, nucleotides 372-1399 of SEQ ID NO:1 18, nucleotides 1 155-1600 of SEQ ID NO:1 19, nucleotides 423-615 of SEQ ID NO:120, nucleotides 753-1 141 of SEQ ID NO:121 , nucleotides 31 1 -581 of SEQ ID NO:164, nucleotides 1 155-1443 of SEQ ID NO:122, and nucle
  • methods and compositions for knockout or downregulation of a nucleic acid molecule encoding an ortholog of a host cell FAH protein having at least 80% identity to SEQ ID NO:33 (PLBD2), SEQ ID NO:61 (LPL), SEQ ID NO:69 (LIPA) and/or SEQ ID NO:77 (ACE); or antibody-binding variant thereof, are via homologous recombination.
  • a nucleic acid molecule encoding an FAH protein (or any protein of interest in general) can be targeted by homologous recombination or by using site-specific nuclease methods that specifically target sequences at the FAH-expressing site of the host cell genome.
  • homologous polynucleotide molecules i.e., homologous arms
  • a transgene can be introduced during this exchange if the transgene is flanked by homologous genomic sequences.
  • a recombinase recognition site can also be introduced into the host cell genome at the integration sites.
  • Homologous recombination in eukaryotic cells can be facilitated by introducing a break in the chromosomal DNA at the integration site.
  • Model systems have demonstrated that the frequency of homologous recombination during gene targeting increases if a double-strand break is introduced within the chromosomal target sequence. This may be accomplished by targeting certain nucleases to the specific site of integration. DNA-binding proteins that recognize DNA sequences at the target gene are known in the art. Gene targeting vectors are also employed to facilitate homologous recombination.
  • NHEJ non-homologous end-joining
  • ZFNs zinc finger nucleases
  • Some embodiments can utilize ZFNs with a combination of individual zinc finger domains targeting multiple target sequences.
  • ZFN methods to target disruption of two or more FAH genes are also embodied by the invention.
  • TAL effector nucleases may also be employed for site-specific genome editing.
  • TAL effector protein DNA-binding domain is typically utilized in combination with a non-specific cleavage domain of a restriction nuclease, such as Fokl.
  • a fusion protein comprising a TAL effector protein DNA-binding domain and a restriction nuclease cleavage domain is employed to recognize and cleave DNA at a target sequence within an exon of the gene encoding the target host cell protein, for example an esterase, such as a phospholipase B-like 2 protein(or other mammalian phospholipase), a lipoprotein lipase and a lysosomal acid lipase, and/or a fatty acylase, such as acid ceramidase (or other mammalian ceramidase).
  • an esterase such as a phospholipase B-like 2 protein(or other mammalian phospholipase), a lipoprotein lipase and a lysosomal acid lipase, and/or a fatty acylase, such as acid ceramidase (or other mammalian ceramidase).
  • Targeted disruption or insertion of exogenous sequences into a specific exon of the CHO protein encoded by SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and/or SEQ ID NO:77 may be done by employing a TALE nuclease (TALEN) targeted to locations within exon 1 , exon 2, exon 3, etc. of the fatty acid hydrolase genomic DNA (see Tables 6 and 7).
  • TALE nuclease TALEN
  • the TALEN target cleavage site within the gene sequences may be selected based on ZiFit.partners.org (ZiFit Targeter Version 4.2) and then TALENs are designed based on known methods (Boch J et al., 2009 Science 326:1509-1512; Bogdanove, A. J. & Voytas, D. F. 201 1 Science 333, 1843-1846; Miller, J. C. et al., 201 1 Nat Biotechnol 29, 143-148).
  • TALEN methods to target disruption of two or more of the PLBD2 gene ⁇ e.g., exon 1 or exon 2), LPL gene ⁇ e.g., exon 2, 3 or 4), LI PA gene ⁇ e.g., exon 1 or 2) and ACE gene ⁇ e.g., exon 1 , 3 or 4) are also embodied by the invention.
  • RNA-guided endonucleases are programmable genome engineering tools that were developed from bacterial adaptive immune machinery.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated
  • the protein Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection.
  • RGENs consist of components (Cas9 and tracrRNA) and a target- specific CRISPR RNA (crRNA).
  • CRISPR-Cas9 methods to target disruption of two or more of the PLBD2 gene e.g., exon 1 or exon 2
  • LPL gene e.g., exon 2, 3 or 4
  • LIPA gene e.g., exon 1 or 2
  • ACE gene e.g., exon 1 , 3 or 4
  • BuD-derived nucleases with precise DNA-binding specificities (Stella, S. et al. Acta Cryst. 2014, D70, 2042-2052).
  • a single residue-to-nucleotide code guides the BuDN to the specific DNA target within polynucleotide of interest ⁇ e.g., SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and or SEQ ID NO:77).
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding PLBD2, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003614971 .1 , at: Exon 1 within nucleotides (nt) 175367 to 175644 (SEQ ID NO:47); Exon 2 within nt 168958 to 169051 (SEQ ID NO:48); Exon 3 within nt 166451 to166609 (SEQ ID NO:49); Exon 4 within nt 164966 to 165066 (SEQ ID NO:50); Exon 5 within nt 164564 to164778 (SEQ ID NO:51 ); Exon 6 within nt 162682 to162779 (SEQ ID NO:52); Exon 7 within nt 160036 to160196 (SEQ ID NO:53); Exon 8 within nt 159733 to 159828 (S
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding LPL, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003613760.1 , at: Exon 1 within nucleotides (nt) 1257424 to 1257507 (SEQ ID NO:85); Exon 2 within nt 1266450 to 1266610 (SEQ ID NO:86); Exon 3 within nt 1270069 1270248 (SEQ ID NO:87); Exon 4 within nt 1271770 to 1271881 (SEQ ID NO:88); Exon 5 within nt 12283518 12283751 (SEQ ID NO:89); Exon 6 within nt 123715 1273957 (SEQ ID NO:90); Exon 7 within nt 1276672 1276792 (SEQ ID NO:91 ); Exon 8 within nt 1278328 to 12
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding LIPA, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003614200.1 , at: Exon 1 within nucleotides (nt) 985778 to 985674 (SEQ ID NO:94); Exon 2 within nt 984375 to 984258 (SEQ ID NO:95); Exon 3 within nt 970771 970573 (SEQ ID NO:96); Exon 4 within nt 969327 to 969218 (SEQ ID NO:97); Exon 5 within nt 968139 968003 (SEQ ID NO:98); Exon 6 within nt 961871 to 961725 (SEQ ID NO:99); Exon 7 within nt 960826 to 960755 (SEQ ID NO:100); Exon 8 within nt 95
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding ACE, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003613654.1 , at: Exon 1 within nucleotides (nt) 1378167 to 1378244 (SEQ ID NO: 103); Exon 2 within nt 1393746 to 1393792 (SEQ ID NO: 104); Exon 3 within nt 1398208 to 1398298 (SEQ ID NO:105); Exon 4 within nt 1399171 to 1399257 (SEQ ID NO: 106); Exon 5 within nt 1402147 to 1402225 (SEQ ID NO: 107); Exon 6 within nt 1404854 to 1404928 (SEQ ID NO:108); Exon 7 within nt 1405714 to 1405759 (SEQ ID NO:109); Exon 8 within nt 140
  • Precise genome modification methods are chosen based on the tools available compatible with unique target sequences within SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 so that disruption of the cell phenotype is avoided.
  • any protein of interest suitable for expression in prokaryotic or eukaryotic cells can be used in the engineered host cell systems provided.
  • the protein of interest includes, but is not limited to, an antibody or antigen-binding fragment thereof, a chimeric antibody or antigen- binding fragment thereof, an ScFv or fragment thereof, an Fc-fusion protein or fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, or an extracellular domain of a cell surface receptor or a fragment thereof.
  • Proteins of interest may be simple polypeptides consisting of a single subunit, or complex multisubunit proteins comprising two or more subunits.
  • the protein of interest may be a biopharmaceutical product, food additive or preservative, or any protein product subject to purification and quality standards.
  • the protein product is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody.
  • the antibody is an lgG1 antibody.
  • the antibody is an lgG2 antibody.
  • the antibody is an lgG4 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG4 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG1 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG1/lgG4 antibody. [00172] In some embodiments, the antibody is selected from the group consisting of an anti- Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1 ), an anti-Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580A1 ), an anti-DII4 antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2 antibody as described in U.S. Pat. No.
  • an anti- Angiopoetin-Like 3 antibody e.g. an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356
  • an anti-platelet derived growth factor receptor antibody e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827
  • an anti-Erb3 antibody an anti- Prolactin
  • Receptor antibody e.g. anti-PRLR antibody as described in U.S. Pat. No. 9,302,015
  • an anti- Complement 5 antibody e.g. an anti-C5 antibody as described in U.S. Pat. Appln. Pub. No
  • an anti-TNF antibody an anti-epidermal growth factor receptor antibody (e.g. an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvlll antibody as described in U.S. Pat. Appln. Pub. No. US2015/0259423A1 ), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g. an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1 ), an anti-Growth And Differentiation Factor-8 antibody (e.g.
  • an anti-GDF8 antibody also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871 ,209 or 9,260,515)
  • an anti-Glucagon Receptor e.g. anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1
  • an anti-VEGF antibody e.g. anti-IL1 R antibody
  • an interleukin 4 receptor antibody e.g an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271681 A1 or U.S. Pat Nos. 8,735,095 or
  • an anti-interleukin 6 receptor antibody e.g. an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody, an anti-IL3 antibody, an anti-l L4 antibody, an anti-IL5 antibody, an anti-l L6 antibody, an anti-l L7 antibody
  • an anti-interleukin 33 e.g. anti- IL33 antibody as described in U.S. Pat. Appln. Pub. Nos.
  • an anti-Respiratory syncytial virus antibody e.g. anti- RSV antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271653A1
  • an anti-Cluster of differentiation 3 e.g. an anti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos.
  • an anti- Cluster of differentiation 20 e.g. an anti-CD20 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1 , and in U.S. Pat. No. 7,879,984
  • an anti-CD19 antibody, an anti-CD28 antibody, an anti- Cluster of Differentiation-48 e.g. anti-CD48 antibody as described in U.S. Pat. No. 9,228,014
  • an anti-Fel d1 antibody e.g. as described in U.S. Pat. No.
  • an anti-Middle East Respiratory Syndrome virus e.g. an anti-MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015/0337029A1
  • an anti-Ebola virus antibody e.g. as described in U.S. Pat. Appln. Pub. No. US2016/0215040
  • an anti-Zika virus antibody e.g. an anti- Lymphocyte Activation Gene 3 antibody, or an anti-CD223 antibody
  • an anti-Nerve Growth Factor antibody e.g. an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 and U.S. Pat. Nos.
  • the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos.
  • the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.
  • the protein of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein).
  • an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety.
  • the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG.
  • the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands.
  • an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the 11-1 R1 extracellular region fused to Fc of hlgG1 ; see U.S. Pat. No. 6,927,004, which is herein incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hlgG1 ; see U.S. Pat. Nos.
  • IL-1 trap e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the 11-1 R1 extracellular region fused to Fc of hlgG1 ; see U.S. Pat. No
  • an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.
  • the host cells used in the methods of the invention are eukaryotic host cells including, for example, Chinese hamster ovary (CHO) cells, human cells, rat cells and mouse cells.
  • the invention provides a cell comprising a disrupted nucleic acid sequence fragment of SEQ ID NO:33 and at least one more of SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77.
  • the invention includes an engineered mammalian host cell further transfected with an expression vector comprising an exogenous gene of interest, such gene encoding the
  • biopharmaceutical product While any mammalian cell may be used, in one particular embodiment the host cell is a CHO cell.
  • Transfected host cells include cells that have been transfected with expression vectors that comprise a sequence encoding a protein or polypeptide. Expressed proteins will preferably be secreted into the culture medium for use in the invention, depending on the nucleic acid sequence selected, but may be retained in the cell or deposited in the cell membrane. Various mammalian cell culture systems can be employed to express recombinant proteins. Other cell lines developed for specific selection or amplification schemes will also be useful with the methods and
  • compositions provided herein provided that at least two genes encoding a different fatty acid hydrolase (FAH) having at least 80% homology to at least two of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 have been downregulated, knocked out or otherwise disrupted in accordance with the invention.
  • An embodied cell line is the CHO cell line designated K1 .
  • the host cell line may be pre-adapted to bioreactor medium in the appropriate case.
  • transfection protocols are known in the art, and are reviewed in Kaufman (1988) Meth. Enzymology 185:537.
  • the transfection protocol chosen will depend on the host cell type and the nature of the GOI, and can be chosen based upon routine experimentation. The basic requirements of any such protocol are first to introduce DNA encoding the protein of interest into a suitable host cell, and then to identify and isolate host cells which have incorporated the
  • heterologous DNA in a relatively stable, expressible manner.
  • Electroporation can also be used to introduce DNA directly into the cytoplasm of a host cell, for example, as described by Potter ef al. (Proc. Natl. Acad. Sci. USA 81 :7161 , 1988) or Shigekawa ef al. (BioTechniques 6:742, 1988). Unlike protoplast fusion, electroporation does not require the selection marker and the GOI to be on the same plasmid.
  • reagents useful for introducing heterologous DNA into a mammalian cell have been described, such as LipofectinTM Reagent and LipofectamineTM Reagent (Gibco BRL, Gaithersburg, Md.). Both of these commercially available reagents are used to form lipid-nucleic acid complexes (or liposomes) which, when applied to cultured cells, facilitate uptake of the nucleic acid into the cells.
  • Methods for amplifying the GOI are also desirable for expression of the recombinant protein of interest, and typically involves the use of a selection marker (reviewed in Kaufman supra). Resistance to cytotoxic drugs is the characteristic most frequently used as a selection marker, and can be the result of either a dominant trait ⁇ e.g., can be used independent of host cell type) or a recessive trait ⁇ e.g., useful in particular host cell types that are deficient in whatever activity is being selected for).
  • amplifiable markers are suitable for use in the cell lines of the invention and may be introduced by expression vectors and techniques well known in the art (e.g., as described in Sambrook, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).
  • Useful selectable markers and other tools for gene amplification such as regulatory elements, described previously or known in the art, can also be included in the nucleic acid constructs used to transfect mammalian cells.
  • the transfection protocol chosen and the elements selected for use therein will depend on the type of host cell used. Those of skill in the art are aware of numerous different protocols and host cells in order to adapt the invention for a particular use, and can select an appropriate system for expression of a desired protein, based on the
  • the invention relates to the following items:
  • a recombinant host cell comprising a modification in two or more genes encoding two or more fatty acid hydrolases (FAH).
  • the recombinant host cell according to item 1 wherein the two or more FAHs are selected from the group consisting of phospholipase B-like 2 (PLBD2) protein, lipoprotein lipase (LPL), lysosomal acid lipase (LI PA) and acid ceramidase (ACE).
  • PLBD2 phospholipase B-like 2
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • [00191] a modification in a coding sequence of a polynucleotide encoding the PLBD2 protein, wherein the modification decreases the expression level of the PLBD2 protein relative to the expression level of a PLBD2 protein in a cell lacking the modification;
  • [00194] a modification in a coding sequence of a polynucleotide encoding the PLBD2 protein, wherein the modification decreases the expression level of the PLBD2 protein relative to the expression level of a PLBD2 protein in a cell lacking the modification;
  • PLBD2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
  • the LPL protein comprises an amino acid sequence at least 80% identical to SEQ ID NO:62.
  • LPL protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68.
  • LI PA protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.
  • ACE protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.
  • the recombinant host cell according to any of previous items further comprising a polynucleotide encoding an exogenous protein of interest.
  • the exogenous protein of interest is selected from the group consisting of an antibody heavy chain, an antibody light chain, an antigen-binding fragment, an antigen-binding protein, and an Fc-fusion protein.
  • a method of producing a recombinant protein comprising the steps of: (a) obtaining a sample comprising a recombinant protein and a plurality of host cell proteins from a host cell that is modified to produce reduced levels of fatty acid hydrolase compared to an unmodified cell; and (b) subjecting the sample to at least one purification step to remove at least one host cell protein.
  • a. does not comprise a detectable amount of a phospholipase B-like 2 (PLBD2) protein
  • b a modification in a coding sequence of a polynucleotide encoding a fatty acid hydrolase selected from the group consisting of lipoprotein lipase (LPL), lysosomeal acid lipase (LI PA), acid ceramidase (ACE), or a combination thereof.
  • LPL lipoprotein lipase
  • LI PA lysosomeal acid lipase
  • ACE acid ceramidase
  • a process for reducing fatty acid hydrolase activity in a protein formulation comprising the steps of: (a) modifying a host cell to decrease or ablate the expression of phospholipase B-like 2 (PLBD2) protein; (b) modifying the host cell to decrease or ablate the expression of lipoprotein lipase (LPL), lysosomal acid lipase (LI PA) and/or acid ceramidase (ACE); (c) transfecting the host cell with a polynucleotide encoding a protein of interest; (d) extracting a protein fraction comprising the protein of interest from the host cell; (e) contacting the protein fraction with a chromatography media selected from the group consisting of protein A affinity (PA) media, cation exchange (CEX) media, and anion exchange (AEX) media; (f) collecting the protein of interest from the media; and (g) combining the protein of interest with a fatty acid ester, and optionally a buffer and thermal stabilizer.
  • PA protein A
  • step of modifying a host cell to decrease or ablate the expression of phospholipase B-like 2 (PLBD2) protein comprises inserting or deleting at least one nucleotide within exon 1 of a polynucleotide encoding the PLBD2 protein.
  • step of modifying the host cell to decrease or ablate the expression of lipoprotein lipase comprises inserting or deleting at least one nucleotide within exon 2, exon 3 or exon 4 of a polynucleotide encoding the LPL protein.
  • step of modifying the host cell to decrease or ablate the expression of acid ceramidase comprises inserting or deleting at least one nucleotide within exon 1 , exon 3 or exon 4 of a polynucleotide encoding the ACE protein.
  • composition comprising one or more recombinant proteins obtainable by the method according to any of items 31-36 or the process according to any of items 37-45.
  • composition obtainable according to any of items 46-47, wherein the stable composition is characterized by one or more of:
  • composition obtainable according to any of items 46-48, wherein the adverse enzyme activity is the activity of one or more of esterases, hydrolases, lipases, phospholipases, ceramidases.
  • composition comprising one or more recombinant proteins, wherein the composition is stable.
  • composition according to item 50, wherein the stable composition is characterized by one or more of:
  • composition according to any of items 50-51 , wherein the adverse enzyme activity is the activity of one or more of esterases, hydrolases, lipases, phospholipases, ceramidases.
  • a Type II CRISPR/Cas system which requires at least 20 nucleotides (nt) of homology between a chimeric RNA (i.e., guide RNA) and its genomic target was used.
  • Guide RNA sequences were designed for specific targeting of an exon within the CHO phospholipase B-like 2 (PLBD2) nucleic acid (SEQ ID NO:33) and are considered unique (to minimize off-target effects in the genome).
  • sgRNA small guide RNAs
  • the sgRNA expression plasmid (System Biosciences, CAS940A-1 ) contains a human H1 promoter that drives expression of the small guide RNA and the tracrRNA following the sgRNA.
  • Immortalized Chinese hamster ovary (CHO) cells were transfected with the plasmid encoding Cas9- H1 enzyme followed by one of the sgRNA sequences, for instance sgRNAI (SEQ ID NO:45) or sgRNA2 (SEQ ID NO:46), designed to target the first exon of CHO PLBD2.
  • sgRNAI and sgRNA2 were predicted to generate a double strand break (DSB) at or around nucleotides 53 and 59 of SEQ ID NO:33, respectively.
  • a DSB was therefore predicted to occur approx. 23 or 29 nucleotides downstream of the PLBD2 start codon. (Note that nucleotides 1-30 of SEQ ID NO:33 encode a signal peptide.)
  • a negative control transfection was performed where the parental CHO line was transfected with the plasmid encoding Cas9-H1 enzyme without a proceeding sgRNA, or an sgRNA encoding a gene sequence not present in the CHO genome.
  • Genomic DNA gDNA
  • messenger RNA mRNA
  • qPCR primers and probes were designed to overlap with the sgRNA sequence used for the double strand break targeting event, in order to detect disruption of the genomic DNA and its transcription.
  • the relative abundance of PLBD2 gene or transcript in the candidate clones was determined using relative qPCR method, where the clones derived from the negative control transfection were used as a calibrator. See Figure 1.
  • the qPCR primers and probes were designed to detect sequences either in the sgRNAI or sgRNA2 position in PLBD2 exon 1 .
  • gDNA and RNA isolated from clone 1 failed to support qPCR amplification of PLBD2 exon 1 in either sgRNAI or sgRNA2 regions, but amplification of the housekeeping gene, GAPDH, was detected. Based on this data, clone 1 was identified as a potential knock out of PLBD2 in which both genomic alleles of PLBD2 of exonl were disrupted. It is noted that amplification of genomic DNA and mRNA was not detected in Clone 8 using primers overlapping with sgRNA2, however, sgRNAI primers/probes detected genomic DNA above control values. Clone 8, and others were further analyzed in order to understand the performance of the site- directed nuclease method.
  • the inventors also unexpectedly identified Clone 8 as a PLBD2 knockout despite the fact that genomic DNA fragments were identified by qPCR primers overlapping with the sgRNAI sequence.
  • the identification of a clone that has no detectable phospholipase activity or no detectable phospholipase protein was technically challenging and time-consuming.
  • Site-directed nuclease techniques may provide an ease-of-use, however, careful screening and elimination of false positives is necessary and still there may be unpredictable outcomes with regard to the identity of a single clone having two disrupted alleles.
  • Clone 1 and the wild type control host cell line were transfected with plasmids encoding the light and heavy chains of mAb1 , a fully human IgG, in the presence of Cre recombinase to facilitate recombination mediated cassette exchange (RMCE) into EESYR locus (US Patent No. 7771997B2, issued August 10, 2010).
  • the transfected cultures were selected for 1 1 days in serum-free medium containing 400 ug/mL hygromycin. Cells that underwent RMCE, were isolated by flow cytometry.
  • PLBD2 knock out clone 1 and the wild type host cell line produced equivalent observed
  • the clone 1 derived isogenic cell line expressing mAb1 was designated RS001
  • the mAb1 expressing cell line originated from the PLBD2 wild type host was designated RS0WT.
  • Polysorbate 20 or polysorbate 80 degradation was measured to detect putative esterase activity in the supernatants of PLBD2 mutants. Unpurified protein supernatant from CHO cells, and supernatant taken at each step or sequence of steps when subjected to sequential purification steps, was tested for stability of polysorbate. The percent intact polysorbate reported was inversely proportional to the amount of contaminant esterase activity. Unpurified protein supernatant from CHO cells, and supernatant at each step or sequence of steps when subjected to sequential purification steps, was tested in assays measuring polysorbate degradation. The relative levels of intact polysorbate reported is inversely proportional to levels of contaminant esterase activity.
  • Monoclonal antibody was produced in an unmodified CHO cell and purified by different processes according to Table 1 1 , and the esterase activity measured by percent intact polysorbate 20, as in Table 9.
  • HIC Hydrophobic interaction chromatography
  • Example 4 Esterase protein abundance and activity detection in mAbl purified from modified compared to unmodified CHO cells.
  • mAbl was produced from RS001 and RSOWT and purified from the conditioned media using either PA alone, or PA and AEX chromatography
  • the PA-purified mAB1 from RS001 and RSOWT were analyzed for lipase abundance using trypsin digest mass spectrometry.
  • the trypsin digests of RS001 and RSOWT mAbl were injected into a reverse phase liquid chromatography column coupled to a triple quadrupole mass spectrometer set to monitor a specific product ion fragmented from SEQ ID NO:32.
  • a hydrophobic interaction column (Phenyl Sepharose® High Performance [GE Healthcare, Little Chalfont, Buckinghamshire, UK]) was used to generate a "HIC strip” fraction containing a protein of interest (i.e., mAb2) and associated host cell proteins.
  • the column was first equilibrated with two column volumes (CV) of buffer containing 40 mM Tris, 200 mM citrate at pH8.0.
  • the monoclonal antibody-containing load material from an anion exchange pool (“Q pool”) was adjusted to 200 mM sodium citrate, pH8.0, then loaded onto the column at a loading amount of 20-40 grams of protein per liter of the phenyl sepharose resin.
  • the column was washed with six CVs of 40 mM Tris, 200 mM citrate, at pH8.0. Then the column was stripped with three CVs of reverse osmosis deionized water. The stripped fraction was collected for subsequent analysis.
  • a 50 ⁇ g aliquot of the mAb2-containing HIC strip sample was denatured and reduced in a solution containing 5 mM acetic acid and 5 mM tris(2-carboxyethyl)phosphine-HCI by heating at 80°C for 10 minutes.
  • the sample was then diluted in 50 mM Tris-HCI buffer (pH 8.0) and alkylated with 1 .5 mM iodoacetamide (IAA) and digested with trypsin (modified, sequencing grade from Promega, Madison, Wl) with an enzyme to substrate ratio of 1 :20 (w/w) at 37°C in the dark for three hours.
  • the digestion was then stopped by addition of 10% trifluoroacetic acid (TFA).
  • Peptides were separated using a linear gradient from 1 % mobile phase B (0.1 % FA in acetonitrile) to 7% mobile phase B for the first 5 minutes, followed by a second linear increase from 7% to 27% mobile phase B over the next 1 10 minutes, and another subsequent linear increase from 27%-40% in 10 minutes and a final increase to 90% in 5 minutes. The gradient was held at 90% for 20 minutes.
  • a Thermo Q ExactiveTM Plus mass spectrometer (Thermo Scientific, Waltham, MA) was used for peptide mass analyses, with high-energy collisional dissociation (HCD) employed for peptide fragmentation for MS/MS experiments.
  • Thermo XcaliburTM version 2.2.42
  • Proteome Discoverer version 1.4
  • Thermo Scientific, Waltham, MA was also used to perform the peptide identification using both Mascot and Sequent search engines.
  • the peptide spectra from the Proteome Discovery was manually examined to confirm the spectral assignment and protein identification.
  • Lipoprotein lipase (UniProtein ID: P06858), lysosomal acid lipase (UniProtein ID: P38571 ) and acid ceramidase (UniProtein ID: Q13510) were identified as potential active fatty acid hydrolases by more than three unique peptides per protein in the mAb2-containing HIC strip fraction.
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) were transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the lipoprotein lipase (LPL) gene (SEQ ID NO:61 ): sgRNA3 (SEQ ID NO:63), sgRNA4 (SEQ ID NO:64) or sgRNA5 (SEQ ID NO:65) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette was used in some
  • Cre recombinase was co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures were selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin. Cells that underwent RMCE were isolated by flow cytometry.
  • the desired knock out genotype was confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the LPL knock-out loci is designated as "clone 9".
  • the resultant clone containing both the PLBD2 and LPL knock-out loci is designated as "clone 10".
  • the wild-type host cell, Clone 1 (PLBD2-KO), Clone 9 (LPL-KO) and Clone 10 (LPL+PLBD2 KO) were cultured in 2L bioreactors under fed-batch conditions at 36.5°C (pH 6.9-7.4) with dissolved oxygen.
  • Cells were inoculated in the production bioreactor (i.e. transferred from a seed train culture (N-1 ) at a cell titer of 5.0 x 10 6 - 7.0 x 10 6 cells/mL) and protein production was induced by the addition of doxycycline.
  • Cells were cultured for 14 days in chemically defined media supplemented with nutrient feeds as needed during the batch culture up to day 12.
  • FIG. 8 depicts an alignment of LPL-KO clones 19-22 (SEQ ID NOs:159-162, respectively) compared to a partial Chinese hamster LPL sequence (SEQ ID NO:158) showing the gaps in the respective clone sequences.
  • LPL-KO clones (as well as any other fatty acid hydrolase knock-out clone), plasmids encoding for Cas9 nuclease, eYFP and site-specific sgRNA were stably integrated into the CHO genome using Lipofectamine-based transfection protocol followed by selection for neomycin resistant cells. Seventeen days post transfection, the YFP positive cells were enriched by flow cytometry prior to single cell-sorting. After a 21 -day expansion, mRNA was isolated and the single cell clones were analyzed by qPCR for the presence of gene disruption.
  • 1 , 2 or 3 different sgRNA expression cassettes may be placed in the same plasmid.
  • Some constructs are manufactured using at least 2 sgRNA cassettes per lipase in a single plasmid.
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) are transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the lysosomal acid lipase (LIPA) gene (SEQ ID NO:69): sgRNA6 (SEQ ID NO:71 ), sgRNA7 (SEQ ID NO:72) or sgRNA8 (SEQ ID NO:73) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the LI PA knock-out or knock-down loci is designated as "clone 1 1 ".
  • the resultant clone containing both the PLBD2 and LPL knock-out or knock-down loci is designated as "clone 12".
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) are transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the acid ceramidase (ACE) gene (SEQ ID NO:77): sgRNA9 (SEQ ID NO:79), sgRNAI O (SEQ ID NO:80) or sgRNA1 1 (SEQ ID NO:81 ) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the ACE knock-out or knock-down loci is designated as "clone 13".
  • the resultant clone containing both the PLBD2 and LPL knock-out or knock-down loci is designated as "clone 14".
  • Clone 9 in which the LPL gene and is modified
  • Clone 10 in which both the LPL gene and the PLBD2 gene are modified
  • plasmids encodes the Cas9 protein
  • the other plasmid encodes the guide RNA designed to target the lysosomal acid lipase (LIPA) gene (SEQ ID NO:69): sgRNA6 (SEQ ID NO:71 ), sgRNA7 (SEQ ID NO:72) and/or sgRNA8 (SEQ ID NO:73) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co- transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing the LIPA modified locus and the LPL modified locus designated as "clone 15".
  • the resultant clone containing the PLBD2, LPL and LIPA modified loci is designated as "clone 16".
  • LIPA Lipase
  • ACE Acid Ceramidase
  • Clone 15 in which the LPL and LIPA genes and are modified
  • Clone 16 in which the PLBD2, LPL and LIPA genes are modified
  • a pair of plasmids One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the acid ceramidase (ACE) gene (SEQ ID NO:77): sgRNA9 (SEQ ID NO:79), sgRNAI O (SEQ ID NO:80) or sgRNA1 1 (SEQ ID NO:81 ) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co- transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing the LIPA modified locus, the LPL modified locus and the ACE modified locus is designated as "clone 17".
  • the resultant clone containing the PLBD2 modified locus, the LIPA modified locus, the LPL modified locus and the ACE modified locus is designated as "clone 18".
  • the invention provides for cells and methods for expression and purification of recombinant proteins in eukaryotic cells.
  • the invention includes methods and compositions for expression of proteins in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, that employ downregulating gene expression of endogenous proteins in order to control production of such unwanted "sticky" host cell proteins.
  • the invention includes polynucleotides and modified cells that facilitate purification of an exogenous recombinant protein of interest.
  • the methods of the invention efficiently target host cell proteins in the Chinese hamster cellular genome in order to facilitate enhanced and stable expression of recombinant proteins expressed by the modified cells.
  • HCPs host cell proteins
  • LC/MS Advanced liquid chromatography/mass spectrometry
  • HCPs Changes in cell culture conditions of eukaryotic cells has been shown to impact the purity and stability of manufactured proteins, as seen by the increased quantity of HCPs of CHO cells upon downstream bioprocessing alterations (Tait, et al, 2013, Biotechnol Prog 29(3):688-696).
  • the detrimental effect of leftover HCPs in any product may affect the overall quality or quantity, or both the quality and quantity of the product.
  • HCPs if present even at low levels in a therapeutic product, may induce an undesired immune response which causes concern for patient safety and efficacy of the drug product (Singh SK. 201 1.
  • the invention provides a recombinant host cell, wherein the cell is modified to decrease the expression levels of two or more fatty acid hydrolases (FAHs) relative to the expression levels of FAH in an unmodified cell.
  • FHs fatty acid hydrolases
  • the invention provides a recombinant host cell, wherein the cell is modified to have no expression of two or more target FAHs.
  • one of said two or more target FAHs is an esterase.
  • the esterase is a lipase.
  • the lipase is: (1 ) a phospholipase, such as a phospholipase B-like protein or a phospholipase B-like 2 protein, (2) a lipoprotein lipase or (3) a lysosomal acid lipase.
  • one of said two or more target FAHs is an amidase.
  • the amidase is a fatty acid acylase.
  • the fatty acid acylase is an acid ceramidase.
  • a gene of interest is exogenously added to the recombinant host cell.
  • the exogenously added gene encodes a protein of interest (POI), for example the POI is selected form the group consisting of antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein.
  • POI protein of interest
  • the invention relates to a composition comprising one or more proteins of interest (POIs) obtainable by a method according to the invention.
  • the protein may be a recombinant protein and may be e.g. selected form the group comprising antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein, or any combinations thereof.
  • the invention relates to a composition comprising one or more proteins of interest (POIs), wherein the adverse enzyme activity is ⁇ 50%, 40%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, 30%, 29%, 28%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.05%, or ⁇ 0.05% active relative to the adverse enzyme activity of a wild-type production system.
  • POIs proteins of interest
  • the phrase "adverse enzyme activity" refers to any enzyme and its action upon the resulting composition as a whole, wherein the action results in metabolism of any protein components which metabolites reduces the shelf-life of the composition or results in the formation of subvisible particles (SVPs) above prescribed regulations.
  • the protein may be a recombinant protein and may e.g. be selected from the group comprising antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein or any combinations thereof.
  • the enzymes and their activities may be, e.g., various esterases, hydrolases, lipases, phospholipases, ceramidases and the likes, or any combinations thereof.
  • Adverse enzyme activity may be measured using a functional assay ⁇ e.g., polysorbate fatty acid hydrolysis assay), or a structural assay ⁇ e.g., nano LC-MS of peptide fragments, or the like).
  • the invention provides a cell comprising a nonfunctional PLBD2 protein and one or more additional nonfunctional fatty acid hydrolases (FAH).
  • FAH nonfunctional fatty acid hydrolases
  • nonfunctional FAH is a nonfunctional lipoprotein lipase (LPL), lysosomal acid lipase (LI PA), acid ceramidase (ACE), platelet-activating factor acetylhydrolase IB subunit gamma (PAFAHG), phosphoinositide phospholipase C fragment (PIPLCf), phosphoinositide phospholipase C (PIPLC), liver carboxylesterase 1 (LCE), isoamyl acetate-hydrolyzing esterase 1 -like (IAH1 ), group XV phospholipase A2 (LPLA2), carboxylic ester hydrolase (CEH), and/or arylsulfatase A (ASA).
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • PAFAHG platelet-activating factor acetylhydrolase IB subunit gamma
  • the invention provides making a cell by FAH target disruption.
  • the method comprises a site-specific nuclease for disrupting or editing the cell genome at a target site or sequence.
  • the FAH target site is (1 ) a PLBD2 target site, (2) an LPL target site, (3) an LI PA target site, (4) an ACE target site, (5) a PAFAHG site, (6) a PIPLCf or PIPLC site, (7) an LCE site, (8) an IAH1 site, (9) an LPLA2 site, (9) an CEH site, and /or (10) an ASA site.
  • the PLBD2 target site comprises a position within SEQ ID NO:33, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:33, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:33, within exon 1 of SEQ ID NO:33, within exon 2 of SEQ ID NO:33, or within exon 3 of SEQ ID NO:33.
  • the PLBD2 target site comprises a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-230, 190-210, 200-220, 210-230, 220-240, 230-250, 240-260, and 250-270 of SEQ ID NO:33.
  • the target site at a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 is selected from the group consisting of nucleotides spanning positions numbered 37-56, 44-56, 33-62, 40-69, 110-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the PLBD2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:33 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:33 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPL target site comprises a position within SEQ ID NO:61 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:61 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO:61 , within exon 1 of SEQ ID NO:61 , within exon 2 of SEQ ID NO:61 , or within exon 3 of SEQ ID NO:61.
  • the LPL target site comprises a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430, 420-440
  • the target site at a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 is selected from the group consisting of nucleotides spanning positions numbered 465-484, 558-577, and 593-612 of SEQ ID NO:61.
  • the LPL target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:61 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:61 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LIPA target site comprises a position within SEQ ID NO:69, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:69, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:69, within exon 1 of SEQ ID NO:69, within exon 2 of SEQ ID NO:69, or within exon 3 of SEQ ID NO:69.
  • the LIPA target site comprises a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350 and 340-360 of SEQ ID NO:69.
  • the target site at a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 is selected from the group consisting of nucleotides spanning positions numbered 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the LIPA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:69 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:69 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ACE target site comprises a position within SEQ ID NO:77, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:77, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:77, within exon 1 of SEQ ID NO:77, within exon 2 of SEQ ID NO:77, within exon 3 of SEQ ID NO:77, or within exon 4 of SEQ ID NO:77.
  • the ACE target site comprises a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370 and 360-380 of SEQ ID NO:77.
  • the target site at a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 is selected from the group consisting of nucleotides spanning positions numbered 135-154, 237-256, and 332-351 of SEQ ID NO:77.
  • the ACE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:77 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:77 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:117, 100 nucleotides
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:1 17 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:117 is selected from the group consisting of nucleotides spanning positions numbered 101-120, 1 1 1-130, 121 -140, 131-150, 141-160, and 150-169 of SEQ ID NO:1 17.
  • the PAFAHG target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 17 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 17 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PIPLC target site comprises a position within SEQ ID NO:1 18, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:118, 100 nucleotides
  • SEQ ID NO:1 downstream of the 5-prime end of SEQ ID NO: 118, within exon 1 of SEQ ID NO:1 18, within exon 2 of SEQ ID NO:118, within exon 3 of SEQ ID NO:118, within exon 4 of SEQ ID NO:1 18, within exon 5 of SEQ ID NO:1 18, within exon 6 of SEQ ID NO:1 18, within exon 7 of SEQ ID NO:1 18, within exon 8 of SEQ ID NO: 1 18, within exon 9 of SEQ ID NO: 1 18, within exon 10 of SEQ ID NO: 1 18, within exon 1 1 of SEQ ID NO:1 18, within exon 12 of SEQ ID NO:1 18, or within exon 13 of SEQ ID NO:1 18.
  • the PIPLC target site comprises a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:1 18 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:118 is selected from the group consisting of nucleotides spanning positions numbered 39-188, 39-58, 49-68, 59-78, 69-88, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 149-178, and 159-188 of SEQ ID NO:118.
  • the PIPLC target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 18 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 18 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LCE target site comprises a position within SEQ ID NO:1 19, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 1 19, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:1 19, within exon 1 of SEQ ID NO:1 19, within exon 2 of SEQ ID NO:1 19, within exon 3 of SEQ ID NO:119, within exon 4 of SEQ ID NO:1 19, or within exon 5 of SEQ ID NO:1 19.
  • the LCE target site comprises a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:1 19 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:119 is selected from the group consisting of nucleotides spanning positions numbered 89-140, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, and 139-158 of SEQ ID NO:1 19.
  • the LCE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 19 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 19 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the IAH1 target site comprises a position within SEQ ID NO:120, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 120, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:120, within exon 1 of SEQ ID NO:120, within exon 2 of SEQ ID NO:120, within exon 3 of SEQ ID NO:120, or within exon 4 of SEQ ID NO:120.
  • the IAH1 target site comprises a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO:120 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430
  • the target site at a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO:120 is selected from the group consisting of nucleotides spanning positions numbered 104-325, 104-123, 1 14-133, 124-143, 134-153, 144-163, 154-173, 164-183, 174-193, 184-203, 194-213, 204-223, 214-233, 224-243, 234-253, 244-263, 254-273, 264-283, 274-293, 284-303, 294-313, 304-323, and 314-333 of SEQ ID NO:120.
  • the IAH1 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO: 120 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:120 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 121 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 121 , within exon 1 of SEQ ID NO: 121 , within exon 2 of SEQ ID NO: 121 , within exon 3 of SEQ ID NO: 121 , or within exon 4 of SEQ ID NO: 121.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 is selected from the group consisting of nucleotides spanning positions numbered 69-195, 69-88, 79-98, 89-108, 99-118, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, and 179-198 of SEQ ID NO:121.
  • the LPLA2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO: 121 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:121 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • a standard method such as mass spectrometry
  • the CEH target site comprises a position within SEQ ID NO:122, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 122, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 122, within exon 1 of SEQ ID NO: 122, within exon 2 of SEQ ID NO: 122, within exon 3 of SEQ ID NO: 122, within exon 4 of SEQ ID NO: 122, or within exon 5 of SEQ ID NO: 122.
  • the CEH target site comprises a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO:122 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430,
  • the target site at a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO:122 is selected from the group consisting of nucleotides spanning positions numbered 79-186, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, and 169-188 of SEQ ID NO:122.
  • the CEH target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:122 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO: 122 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ASA target site comprises a position within SEQ ID NO:123, within 100 nucleotides upstream of the 5 prime end of SEQ ID NO: 123 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 123, within exon 1 of SEQ ID NO: 123, within exon 2 of SEQ ID NO: 123, within exon 3 of SEQ ID NO: 123, or within exon 4 of SEQ ID NO: 123.
  • the ASA target site comprises a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO:123 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430,
  • the target site at a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO:123 is selected from the group consisting of nucleotides spanning positions numbered 79-296, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, 179-198 , 189-208 , 199-218, 209-228, 219-238, 229-248, 239-258, 249-268, 259-278, 269-288, and 279-298 of SEQ ID NO:123.
  • the ASA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:123 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO: 123 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the cell further integrates an exogenous nucleic acid sequence.
  • the cell is capable of producing an exogenous protein of interest.
  • the altered protein resulting from a disrupted gene does not bind to the protein of interest produced by the cell.
  • an isolated Chinese hamster ovary (CHO) cell comprising an engineered nucleic acid sequence comprising a variant of the PLBD2 gene (such as a variant of SEQ ID NO:33).
  • the PLBD2 gene comprises GACAGTCACG TGGCCCGACT GAGGCACGCG , nucleotides 1-30 of SEQ ID NO:33 (SEQ ID NO: 44).
  • the PLBD2 gene is engineered to disrupt expression of the open reading frame.
  • the invention provides an isolated CHO cell comprising (a) a disrupted PLBD2 gene comprising GACAGTCACG TGGCCCGACT GAGGCACGCG (SEQ ID NO: 44, also nucleotides 1- 30 of SEQ ID NO:33), (b) a disrupted esterase gene comprising a nucleotide encoding any one of the amino acid sequences in Table 2, or (c) a protein fragment of Table 2 expressed by a disrupted PLBD2 gene; and an exogenous nucleic acid sequence comprising a gene of interest.
  • the CHO cell that comprises an engineered nucleic acid sequence comprising a variant of the PLBD2 gene also comprises variants of one or more of the genes encoding (1 ) LPL (variant of SEQ ID NO:61 ), (2) LIPA (SEQ ID NO:69), (3) ACE (SEQ ID NO:77), (4) PAFAHG (SEQ ID NO:1 17), (5) PIPLC (SEQ ID NO:1 18), (6) LCE (SEQ ID NO:1 19), (7) IAH1 (SEQ ID NO:120), (8) LPLA2 (SEQ ID NO:121 ), (9) CEH (SEQ ID NO:122), and/or (10) ASA (SEQ ID NO:123).
  • a method of producing a protein of interest using a recombinant host cell wherein the host cell is modified to decrease the expression levels of esterase relative to the expression levels of esterase in an unmodified cell.
  • the method comprises the modified host cell having decreased esterase expression and an exogenous nucleic acid sequence comprising a gene of interest (GOI).
  • GOI gene of interest
  • the exogenous nucleic acid sequence comprises one or more genes of interest.
  • the one or more genes of interest are selected from the group consisting of a first GOI, a second GOI and a third GOI.
  • the invention provides expression systems comprising the recombinant host cell comprising modified or nonfunctional esterase.
  • the cell comprises a GOI operably linked to a promoter capable of driving expression of the GOI, wherein the promoter comprises a eukaryotic promoter that can be regulated by an activator or inhibitor.
  • the eukaryotic promoter is operably linked to a prokaryotic operator, and the eukaryotic cell optionally further comprises a prokaryotic repressor protein.
  • one or more selectable markers are expressed by the modified host cell.
  • the genes of interest and/or the one or more selectable markers are operably linked to a promoter, wherein the promoter may be the same or different.
  • the promoter comprises a eukaryotic promoter (such as, for example, a CMV promoter or an SV40 late promoter), optionally controlled by a prokaryotic operator (such as, for example, a tet operator).
  • the cell further comprises a gene encoding a prokaryotic repressor (such as, for example, a tet repressor).
  • a CHO host cell comprising recombinase recognition sites.
  • the recombinase recognition sites are selected from a LoxP site, a /_ox51 1 site, a Lox2272 site, Lox2372, Lox5171 , and a frt site.
  • the cell further comprises a gene capable of expressing a recombinase.
  • the recombinase is a Cre recombinase.
  • the selectable marker gene is a drug resistance gene.
  • the drug resistance gene is a neomycin resistance gene or a hygromycin resistance gene.
  • the second and third selectable marker genes encode two different fluorescent proteins.
  • the two different fluorescent proteins are selected from the group consisting of Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (mKate).
  • the first, second, and third promoters are the same. In another embodiment, the first, second, and third promoters are different from each other. In another embodiment, the first promoter is different from the second and third promoters, and the second and third promoters are the same. In more embodiments, the first promoter is an SV40 late promoter, and the second and third promoters are each a human CMV promoter. In other embodiments, the first and second promoters are operably linked to a prokaryotic operator.
  • the host cell line has an exogenously added gene encoding a recombinase integrated into its genome, operably linked to a promoter.
  • the recombinase is Cre recombinase.
  • the host cell has a gene encoding a regulatory protein integrated into its genome, operably linked to a promoter.
  • the regulatory protein is a tet repressor protein.
  • the first GOI and the second GOI encode a light chain, or fragment thereof, of an antibody or a heavy chain, or fragment thereof, of an antibody.
  • the first GOI encodes a light chain of an antibody and the second GOI encodes a heavy chain of an antibody.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a first light chain, or fragment thereof, a second light chain, or fragment thereof and a heavy chain, or fragment thereof.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a light chain, or fragment thereof, a first heavy chain, or fragment thereof and a second heavy chain, or fragment thereof.
  • a method for making a protein of interest comprising (a) introducing into a CHO host cell a gene of interest (GOI), wherein the GOI integrates into a specific locus such as a locus described in US Patent No. 7771997B2, issued August 10, 2010 or other stable integration and/or expression-enhancing locus; (b) culturing the cell of (a) under conditions that allow expression of the GOI; and (c) recovering the protein of interest.
  • the protein of interest is selected from the group consisting of a subunit of an immunoglobulin, or fragment thereof, and a receptor, or ligand-binding fragment thereof.
  • the protein of interest is selected from the group consisting of an antibody light chain, or antigen-binding fragment thereof, and an antibody heavy chain, or antigen-binding fragment thereof.
  • the CHO host cell genome comprises further modifications, and comprises one or more recombinase recognition sites as described above, and the GOI is introduced into a specific locus through the action of a recombinase that recognizes the
  • the GOI is introduced into the cell employing a targeting vector for recombinase-mediated cassette exchange (RMCE) when the CHO host cell genome comprises at least one exogenous recognition sequence within a specific locus.
  • RMCE recombinase-mediated cassette exchange
  • the GOI is introduced into the cell employing a targeting vector for homologous recombination, and wherein the targeting vector comprises a 5' homology arm homologous to a sequence present in the specific locus, a GOI, and a 3' homology arm
  • the targeting vector further comprises two, three, four, or five or more genes of interest.
  • one or more of the genes of interest are operably linked to a promoter.
  • a method for modifying a CHO cell genome to integrate an exogenous nucleic acid sequence comprising the step of introducing into the cell a vehicle comprising an exogenous nucleic acid sequence wherein the exogenous nucleic acid integrates within a locus of the genome.
  • the invention provides a process for manufacturing a stable protein formulation comprising the steps of: (a) extracting a protein fraction from the modified host cell of the invention having decreased or ablated expression of esterase, (b) contacting the protein fraction comprising a protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (c) collecting the protein of interest from the media, wherein a reduced level of the esterase activity is associated with the protein fraction collected at step (c), thus providing a stable protein formulation.
  • PA protein A affinity
  • CEX cation exchange
  • AEX anion exchange
  • the invention provides a process for reducing esterase activity in a protein formulation comprising the steps of: (a) modifying a host cell to decrease or ablate expression of esterase, (b) transfecting the host cell with a protein of interest, (c) extracting a protein fraction from the modified host cell, (c) contacting the protein fraction comprising the protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (d) collecting the protein of interest from the media, and (e) combining the protein of interest with a fatty acid ester, and optionally a buffer and thermal stabilizer, thus providing a protein formulation essentially free of detectable esterase activity.
  • the protein formulation is essentially free of PLBD2 protein or PLBD2 activity.
  • a method for modifying a CHO cell genome to express a therapeutic agent comprising a vehicle for introducing, into the genome, an exogenous nucleic acid comprising a sequence for expression of the therapeutic agent, wherein the vehicle comprises a 5' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33, a nucleic acid encoding the therapeutic agent, and a 3' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33.
  • the invention provides a modified CHO host cell comprising a modified CHO genome wherein the CHO genome is modified by disruption of target sequence within a nucleotide sequence at least 90% identical to SEQ ID NO: 33.
  • the modified CHO host cell further comprises another FAH target sequence disruption.
  • the another FAH target sequence is within (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:61 , (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:69 and/or (3) a nucleotide sequence at least 90% identical to SEQ ID NO:77.
  • the invention provides a modified eukaryotic host cell comprising a modified eukaryotic genome wherein the eukaryotic genome is modified at a target sequence in a coding region of the target gene by a site-specific nuclease.
  • the site-specific nuclease comprises a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, or an RNA-guided DNA endonuclease.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • TAL effector domain fusion protein or an RNA-guided DNA endonuclease.
  • the invention also provides methods of making such a modified eukaryotic host cell.
  • the target sequence can be placed in the indicated orientation as in SEQ ID NO:33, 61 , 69, 77, 1 17, 118, 119, 120, 121 , 122, or 123; or in the reverse of the orientation of SEQ ID NO:33, 61 , 69, 77, 1 17, 1 18, 119, 120, 121 , 122, or 123.
  • Fig. 1 depicts the results of Taqman® quantitative polymerase chain reaction (qPCR) experiments to detect genomic (gDNA) or transcripts (mRNA) of the modified clones.
  • Primers and probes were designed to flank the sequences predicted as subject to targeted disruption within exon 1 , either starting at nucleotide 37 (sgRNAI ) or starting at nucleotide 44 (sgRNA2) of SEQ ID NO:33.
  • Relative amount of amplicons from clones targeted by either sgRNAI or sgRNA2 are graphed (i.e., relative to amplicons derived from the negative control transfection clones which were subject to no sgRNA or unmatched sgRNA).
  • Clone 1 for example, has relatively no amplified gDNA nor mRNA per qPCR of the targeted exon 1 region. Clone 1 and several other clones were selected for follow up analysis. The vertical axes represent the relative amount of template containing sgRNAI sequence (upper panel) or sgRNA2 sequence (lower panel).
  • FIG. 2A and Fig. 2B illustrate the results of further PCR analysis of a Clone 1 cells population compared to wild type Chinese hamster overy (CHO) cells.
  • Fig. 2A shows a PCR amplicon from Clone 1 that is shorter in length (bp) compared to the amplicon from genomic DNA of wild type cells.
  • Fig. 2B shows a PCR amplicon from Clone 1 that is shorter in length (bp) compared to the amplicon from mRNA of wild type cells. Sequencing confirmed an 1 1 bp deletion in the PLBD2 gene of Clone 1 .
  • the vertical axes represent the size of the PCR fragments in base-pairs (bp).
  • Fig. 3 illustrates the relative protein titer of monoclonal antibody 1 (mAbl )-expressing Clone 1 cells (RS001 ) or mAb1 -expressing wild type CHO cells (RS0WT) subject to the same fed-batch culture conditions for 12 days. Samples of conditioned medium were extracted for each culture, and the Protein A binding fraction was quantified at Day 2, 4, 6, 9 and 12.
  • Fig. 4 shows the results of RS001 or RS0WT cells following production culture and protein purification using either Protein A (PA) alone, or PA and anion exchange (AEX) chromatography.
  • PA-purified mAb1 from RS001 and RS0WT was analyzed for lipase abundance using trypsin digest mass spectrometry.
  • trypsin digests of RS001 - and RSOWT-produced mAb1 were injected into a reverse phase liquid chromatography column coupled to a triple quadrupole mass spectrometer set to monitor a specific PLBD2 product fragment (as in Table 2).
  • Control reactions containing reference samples of mAb1 (with no endogenous PLBD2) spiked with varying amounts of recombinant PLBD2 were also analyzed and plotted. The signals detected in the experiments were compared to the control reactions to determine concentration of PLBD2.
  • mAb1 produced from Clone 1 shows no detectable amounts of PLBD2 when purified with PA alone.
  • Fig. 5 is a line plot depicting percent cell viability as a function of time in days. Open circles (-0-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A-) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 6 is a line plot depicting protein production (titer) in grams per liter as a function of time in days. Open circles (-o-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A-) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 7 is a line plot depicting viable cell counts in cells per milliliter as a function of time in days. Open circles (-o-) represent wildtype cells. Filled diamonds (- ⁇ -) represent PLBD2-KO cells. Filled squares (- ⁇ -) represent LPL-KO cells. Filled triangles (-A-) represent the double knock-out LPL-KO / PLBD2KO.
  • Fig. 8 shows a formatted alignment of LPL knock out constructs clone 19, clone 20, clone 21 , and clone 22, represented by SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161 , and SEQ ID NO:162, respectively.
  • the partial wildtype LPL sequence is represented by SEQ ID NO:158.
  • exogenously added gene refers to any DNA sequence or gene not present within the genome of the cell as found in nature.
  • an "exogenously added gene” within a CHO genome can be a gene from any other species (e.g., a human gene), a chimeric gene (e.g., human/mouse), or a hamster gene not found in nature within the particular CHO locus in which the gene is inserted (i.e., a hamster gene from another locus in the hamster genome), or any other gene not found in nature to exist within a CHO locus of interest.
  • Percent identity when describing an esterase, e.g., a hydrolase protein, such as SEQ ID NO:32, 34, 35, 37, 62,70, 74, 75, 76, 78, 82, 83, 84; 124, 125, 126, 127, 128, 129, and 130; or gene, such as SEQ ID NO:33, 61 , 69, 77, 1 17, 118, 119, 120, 121 , 122, and 123 includes homologous sequences that display the recited identity along regions of contiguous homology, but the presence of gaps, deletions, or insertions that have no homolog in the compared sequence are not taken into account in calculating percent identity.
  • a hydrolase protein such as SEQ ID NO:32, 34, 35, 37, 62,70, 74, 75, 76, 78, 82, 83, 84; 124, 125, 126, 127, 128, 129, and 130; or gene, such as SEQ
  • a "percent identity" determination between, e.g., SEQ ID NO:32 with a species homolog would not include a comparison of sequences where the species homolog has no homologous sequence to compare in an alignment (i.e., SEQ ID NO:32 compared to a fragment thereof, or the species homolog has a gap or deletion, as the case may be). Thus, “percent identity” does not include penalties for gaps, deletions, and insertions.
  • “Targeted disruption” of a gene or nucleic acid sequence refers to gene targeting methods that direct cleavage or breaks (such as double stranded breaks) in genomic DNA and thus cause a modification to the coding sequence of such gene or nucleic acid sequence.
  • Gene target sites are the sites selected for cleavage or break by a nuclease.
  • the DNA break is normally repaired by the non-homologous end-joining (NHEJ) DNA repair pathway.
  • NHEJ non-homologous end-joining
  • InDels insertions or deletions
  • a small number of nucleotides are either inserted or deleted at random at the site of the break and these InDels may shift or disrupt the open reading frame (ORF) of the target gene. Shifts in the ORF may cause significant changes in the resulting amino acid sequence downstream of the DNA break, or may introduce a premature stop codon, therefore the expressed protein, if any, is rendered nonfunctional or subject to degradation.
  • Targeted insertion refers to gene targeting methods employed to direct insertion or integration of a gene or nucleic acid sequence to a specific location on the genome, i.e., to direct the DNA to a specific site between two nucleotides in a contiguous polynucleotide chain. Targeted insertion may also be performed to introduce a small number of nucleotides or to introduce an entire gene cassette, which includes multiple genes, regulatory elements, and/or nucleic acid sequences. "Insertion” and “integration” are used interchangeably.
  • Recognition site or “recognition sequence” is a specific DNA sequence recognized by a nuclease or other enzyme to bind and direct site-specific cleavage of the DNA backbone.
  • Endonucleases cleave DNA within a DNA molecule.
  • Recognition sites are also referred to in the art as recognition target sites.
  • Polysorbates are fatty acid esters of sorbitan or iso-sorbide (polyoxyethylene sorbitan or iso- sorbide mono- or di- esters).
  • the polyoxyethylene serves as the hydrophilic head group and the fatty acid as the lipophilic hydrophobic tail.
  • the effectiveness as a surfactant of the polysorbate depends upon the amphiphilic nature of the molecule with both hydrophilic head and hydrophobic tail present (in a single molecule).
  • SVP subvisible particle
  • SVPs may attribute to immunogenicity. Regulatory authorities like the United States Food and Drug Administration (USFDA) provide limitations on the number of subvisible particles (SVPs) allowed in a pharmaceutical formulation. United States Pharmacopeia (USP) publishes standards for strength, purity and quality of drugs and drug ingredients, as well as food ingredients and dietary
  • USP 31 monograph ⁇ 788> sets the limit for number of particles allowed in parenteral formulations.
  • USP 31 monograph ⁇ 788> is available at http://www.uspnf.com/official- text/revision-bulletins/particulate-matter-injections; and as Revision Bulletin Official July 1 , 2012, ⁇ 788> Particulate Matter in Injections, The United States Pharmacopeial Convention.
  • the limit is set at no more than 25 particles of at least 10 microns per ml_, and no more than 3 particles of at least 25 microns per ml_.
  • the limit is set at no more than 6,000 particles of at least 10 microns per container, and no more than 600 particles of at least 25 microns per container.
  • the term "stability” refers to the retention of an acceptable degree of physical structure (colloidal, nature), chemical structure or biological function of the biologically active agent (e.g., biotherapeutic or other protein produced in a cell-based bioprocess) over time during storage (a.k.a. "shelf-life"), during processing, or after administration and while in vivo.
  • the biologically active agent may be stable even though it does not maintain 100% of its structure or function after storage or administration for a defined amount of time.
  • the biologically active agent and formulation containing the biologically active agent may be regarded as "stable".
  • Stability can be measured, inter alia, by determining the percentage of native molecule that remains in the formulation after storage or administration for a defined amount of time at a defined temperature.
  • the percentage of native molecule can be determined by, inter alia, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]), such that native means non-aggregated and non-degraded.
  • SE-HPLC size exclusion high performance liquid chromatography
  • At least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the biologically active agent can be detected in the formulation after a defined amount of time at a defined temperature or under physiological conditions after administration.
  • the defined amount of time after which stability is measured can be about 14 days, about 28 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 1 1 months, about 12 months, about 18 months, about 24 months, or more.
  • the temperature at which the formulation containing the biologically active agent may be kept when assessing stability can be any temperature from about -80°C to about 45°C, e.g., storage at about - 80°C, about -30°C, about -20°C, about 0°C, about 4°-8°C, about 5°C, about 25°C, about 35°C, about 37°C or other physiological temperatures, or about 45°C.
  • the biologically active agent may be deemed stable if after 3 months under physiological conditions, greater than about 75%, 80%, 85% or 90% of native molecule is detected in the soluble fraction by SE-HPLC or other size exclusion or size determination method.
  • “Physiological temperature” includes the body temperature of any vertebrate.
  • physiological temperature of humans is about 37°C.
  • physiological temperature is between about 25°C and about 45°C.
  • physiological temperature is about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, and about 45°C.
  • Stability can be measured, inter alia, by determining the percentage of biologically active agent, such as a protein, that forms an aggregate (i.e., high molecular weight species) after a defined amount of time at a defined temperature, wherein stability is inversely proportional to the percent high molecular weight (HMW) species that is formed of the biologically active agent (protein).
  • the percentage of HMW species of the biologically active agent can be determined by, inter alia, size exclusion chromatography, as described above.
  • a pharmaceutical formulation containing the biologically active agent may also be deemed stable if after three months at physiological conditions less than about 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1% of the biologically active agent is detected in a HMW form.
  • Stability can be measured, inter alia, by determining the percentage of a biologically active agent, such as a protein, that is degraded or otherwise is found as a low molecular weight (LMW) species after a defined amount of time at a defined temperature. Stability is inversely proportional to the percent LMW species that is formed in the soluble fraction. The percentage of LMW species of the biologically active agent in the soluble fraction can be determined by, inter alia, size exclusion chromatography, as described above.
  • LMW low molecular weight
  • a pharmaceutical formulation may also be deemed stable if after three months under storage conditions less than about 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1% of the biologically active agent is detected in a LMW form.
  • compositions prepared in host cells as described herein are envisioned to comply with standards set forth by multiple regulatory authorities and ICH guidelines.
  • the composition complies if tested for subvisible particles and the test results in the average number of particles present in the units tested does not exceed 12 per milliliter equal to or greater than 10 um in size, and does not exceed 2 per milliliter equal to or greater than 25 um.
  • Various test for microscopic particles in solution are well-known in the art, including but not limited to tests recommended in ICH Guideline Q4B Annex 3(R1 ), dated 27 September 2010 (Evaluation And Recommendation Of Pharmacopoeial Texts For Use In The lch Regions On Test For Particulate Contamination: Sub-Visible Particles General Chapter).
  • HCPs proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins. HCPs are generally process-related impurities during biologies production. The amount of residual HCPs in drug product is generally considered a critical quality attribute (CQA), due to their potential to affect product safety and efficacy. Regulatory authorities require a product sponsor to monitor the removal of HCPs in drug product during bioprocess development. A sensitive assay e.g., immunoassay, capable of detecting a wide range of protein impurities is generally utilized. This testing can include verification at commercial scale in accordance with regional regulations and may be done at the time of submission of a marketing approval application.
  • CQA critical quality attribute
  • ICH Specifications (Q6A and Q6B, section 2.3), if a drug substance or drug product does not contain any impurity in the specific formulation, i.e. if efficient control or removal to acceptable levels is demonstrated by suitable studies, further testing may be reduced or eliminated upon approval by the regulatory authorities.
  • compositions prepared in host cells as described herein are envisioned to comply with standards set forth by multiple regulatory authorities and ICH guidelines.
  • the composition prepared by the host cells described herein comprise less than 100 ng/mg (ppm), less than about 90 ppm, less than about 80 ppm, less than about 70 ppm, less than about 60 ppm, less than about 50 ppm, less than about 40 ppm, less than about 30 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm, or 0 ppm of the target host cell protein, i.e. fatty acid hydrolase.
  • fatty acid hydrolase or “FAH” refers to any hydrolytic enzyme that cleaves at a carbonyl group creating a carboxylic acid product in which the carboxylic acid comprises an R- group that is lipophilic or otherwise hydrophobic.
  • the carboxylic acid product is a fatty acid.
  • Esterases and “fatty acid acylases/amidases” are included as subgenera of fatty acid hydrolase.
  • Lipases are a subgenus of esterases that cleave lipids (fats, waxes, sterols, glycerides and phospholipids.
  • Phospholipases are a subgenus of lipases that cleave
  • phospholipids phospholipids. Esterases cleave fatty acid esters into fatty acids and alcohols. Lipases include PLBD2, LPL and LIPA. "Ceramidase” is a subgenus of fatty acid acylase that cleaves ceramide and releases a fatty acid and sphingosine, which is an amino alcohol. Examples of ceramidases include acid ceramidase, neutral ceramidase, alkaline ceramidase 1 , alkaline ceramidase 2 and alkaline ceramidase 3.
  • Protein A-binding fraction refers to the fraction of cell lysate from cultured cells expressing a protein of interest which binds to a Protein A affinity format. It is well understood in the art that Protein A affinity chromatography, such as Protein A chromatography medium, such as resins, beads, columns and the like, are utilized to capture Fc-containing proteins due to their affinity to Protein A.
  • Protein A affinity chromatography such as Protein A chromatography medium, such as resins, beads, columns and the like
  • Phospholipase B-like 2 refers to the homologs of a phospholipase gene known as NCBI RefSeq. XM_003510812.2 (SEQ ID NO:33) or protein known as NCBI RefSeq.
  • PLBD2 is also referred to in the art as putative phospholipase B-like 2 (PLBL2), 76 kDa protein, LAMA-like protein 2, PLB homolog 2, lamina ancestor homolog 2, mannose-6-phosphate protein associated protein p76, p76, phospholipase B-like 2 32 kDa form, phospholipase B-like 2 45 kDa form, or Lysosomal 66.3 kDa protein.
  • Lipoprotein lipase is a glycosylated homodimer secreted by parenchymal cells and associated with endothelial cells of the capillary lumen.
  • Exemplary LPL proteins include Chinese hamster LPL (SEQ ID NO:62), mouse LPL (SEQ ID NO:66), ), rat LPL (SEQ ID NO:67) and human LPL (SEQ ID NO:68).
  • Mouse LPL is 92% identical to Chinese hamster LPL.
  • Rat LPL is 92% identical to Chinese hamster LPL.
  • Human LPL is 88% identical to Chinese hamster LPL.
  • Chinese hamster LPL is encoded by a polynucleotide sequence of SEQ ID NO:61 .
  • Lysosomal acid lipase also known as lysosomal lipase, lipase A, lysosomal acid and cholesterol esterase is an intracellular lipase that functions in the lysosome.
  • LIPA reversibly catalyzes cholesteryl ester bond formation and cleavage.
  • LIPA is also a glycosylated homodimer.
  • Exemplary LIPA proteins include Chinese hamster LIPA (SEQ ID NO:70), mouse LIPA (SEQ ID NO:74), ), rat LIPA (SEQ ID NO:75) and human LIPA (SEQ ID NO:76).
  • Mouse LIPA is 72% identical to Chinese hamster LIPA.
  • Rat LIPA is 75% identical to Chinese hamster LIPA.
  • Human LIPA is 74% identical to Chinese hamster LIPA.
  • Chinese hamster LIPA is encoded by a polynucleotide sequence of SEQ ID NO:69.
  • Acid ceramidase also known as ASAH1 , AC, ACDase, ASAH, PHP, PHP32, SMAPME, N-acylsphingosine amidohydrolase (acid ceramidase) 1 is an acylase that cleaves ceramide to produce fatty acid and sphingosine. It is a heterodimer comprising a non-glycosylated alpha subunit and a glycosylated beta subunit. Acid ceramidase has an acid pH optimum. The lipid accumulation disease, Farber Lipogranulomatosis, is associated with a deficiency in acid ceramidase activity.
  • Exemplary acid ceramidases include Chinese hamster ACE (SEQ ID NO:78), mouse ACE (SEQ ID NO:82), ), rat ACE (SEQ ID NO:83) and human ACE (SEQ ID NO:84).
  • Mouse ACE is 87% identical to Chinese hamster ACE.
  • Rat ACE is 89% identical to Chinese hamster ACE.
  • Human ACE is 83% identical to Chinese hamster ACE.
  • Chinese hamster ACE is encoded by a polynucleotide sequence of SEQ ID NO:77.
  • Platelet-activating factor acetylhydrolase IB subunit gamma (PAFAHG), also known as PAFAH1 B3, PAFAHG, and platelet activating factor acetylhydrolase 1 b catalytic subunit 3 is one of the catalytic subunits along with beta of the cytosolic tetrameric platelet-activating factor acetylhydrolase IB.
  • PAFAHG belongs to the phospholipase A2 family and catalyzes the hydrolysis of the acyl group at position 2 of glycerol in bioactive phospholipids (see Stafforini et al., Journal of Biological Chemistry, 272:17895-17898, July 1997).
  • Chinese hamster PAFAHG (SEQ ID NO:124) is 98% identical to both rat and mouse PAFAHG, and 96% identical to human PAFAHG.
  • Chinese hamster PAFAHG is encoded by a polynucleotide sequence of SEQ ID NO:1 17.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO:125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO:125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO:125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • phosphodiesterase monophosphatidylinositol phosphodiesterase, phosphatidylinositol phospholipase C, PI-PIPLC, and 1-phosphatidyl-D-myo-inositol-4,5-bisphosphate
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO:125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Phosphoinositide phospholipase C also known as triphosphoinositide
  • inositoltrisphosphohydrolase is member of the phospholipase C superfamily of phophodiesterases that participate in phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism and calcium-dependent lipid signaling.
  • PIPLC hydrolyzes PIP2 at the inner membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG).
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Chinese hamster PIPLC (SEQ ID NO:125) is 96% identical to rat PIPLC, 95% identical to mouse PIPLC, and 92% identical to human PIPLC.
  • Chinese hamster PIPLC is encoded by a polynucleotide sequence of SEQ ID NO:1 18.
  • Additional fatty acid hydrolases that can serve as targets for deletion, either individually, or in combination with one of more additional fatty acid hydrolases are listed in Table 1. Any of the following hydrolases, or their equivalents, listed in Table A may be the target protein in the creation of a knockout cell line, where removal of the host cell protein is necessitated due to contamination in the preparation of a biopharmaceutical product.
  • cell or “cell line” includes any cell that is suitable for expressing a recombinant nucleic acid sequence.
  • Cells include those of prokaryotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g.,S. cerevisiae, S. pombe, P. partoris, P.
  • the cell is a human, monkey, ape, hamster, rat or mouse cell.
  • the cell is eukaryotic and is selected from the following cells: CHO (e.g.,CHO K1 , DXB-1 1 CHO, Veggie-CHO), COS (e.g.,COS-7), retinal cells, Vero, CV1 , kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21 ), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1 , U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT cell, tumor cell, and a cell line derived from an aforementioned cell.
  • the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g.,a PER.C6® cell).
  • the invention is based at least in part on a recombinant host cell and cell expression system thereof that decreases expression of two or more an endogenous host cell fatty acid hydrolases (FAHs), decreases the enzymatic function or binding ability of two or more endogenous host cell FAHs, or lacks detectable expression of two or more FAHs.
  • FHs endogenous host cell fatty acid hydrolases
  • the inventors discovered that the disruption of genes encoding at least two FAHs allows for the optimized and efficient production and purification of biopharmaceutical products expressed in such expression systems.
  • the invention may be employed in several ways, such as 1 ) utilizing gene editing tools to totally knockout FAH expression, whereas no measurable full-length FAH enzyme is expressed in the cell due to disruption of the gene encoding the FAH; 2) utilizing gene editing tools to eliminate or reduce enzymatic activity, whereas the FAH protein is expressed but rendered nonfunctional due to disruptions in its gene; and 3) utilizing gene editing tools to eliminate or reduce the ability of an endogenous host cell FAH to bind exogenous recombinant protein produced by the cell.
  • FAH activity was determined in protein fractions of certain antibody-producing cells. Several particular fatty acid hydrolases were determined as contaminants in these protein fractions, including three carboxylic esterases (a.k.a.
  • esterases phospholipase B-like (PLBD2), lipoprotein lipase (LPL) and lysosomal acid lipase (LIPA), and acid ceramidase, a carboxylic amidase (an acylase).
  • PLBD2 phospholipase B-like
  • LPL lipoprotein lipase
  • LIPA lysosomal acid lipase
  • acid ceramidase a carboxylic amidase (an acylase).
  • Gene editing target sites were identified in hamster PLBD2, LPL, LIPA and acid ceramidase (ACE) genes that enable targeted disruption of those genes in a hamster cell ⁇ i.e., CHO) genome.
  • ACE acid ceramidase
  • An optimized host cell comprising a combination of genetic modifications that affect the expression of genes encoding (1 ) PLBD2 and LPL; (2) PLBD2 and LIPA; (3) PLBD2 and ACE; (4) LPL and LIPA; (5) LPL and ACE; (6) LIPA and ACE; (7) PLBD2, LPL and LIPA; (8) PLBD2, LPL and ACE; (9) PLBD2, LIPA and ACE; (10) LPL, LIPA and ACE, (1 1 ) PLBD2, LPL, LIPA and ACE, (12) PLBD2 and PIPLC, (13) PLBD2 and CEH, (14) PLBD2 and PAFAHG, (15) PLBD2 and LCE, (16) PLBD2 and ASA, (17) PLBD2 and IAH1 , (18) PLBD2 and LPLA2, (19) LPL and PIPLC, (20) LPL and CEH, (21 ) LPL and PAFAHG, (22) LPL and LCE, (23)
  • Such a cell is envisioned to reduce the burden of certain purification steps, thereby reducing time and cost, while increasing production yield. Also, the formulated protein is expected to have improved stability due to the reduced hydrolase burden.
  • the invention is also based on the specific targeting of an exogenous gene to the integration site.
  • the methods of the invention allow efficient modification of the cell genome, thus producing a modified or recombinant host cell useful as a cell expression system for the
  • the methods of the invention employ cellular genome gene editing strategies for the alteration of particular genes of interest that otherwise may diminish or contaminate the quality of recombinant protein formulations, or require multiple purification steps.
  • compositions of the invention can also be included in expression constructs for example, in expression vectors for cloning and engineering new cell lines. These cell lines comprise the modifications described herein, and further modifications for optimal incorporation of expression constructs for the purpose of protein expression are envisioned.
  • Expression vectors comprising polynucleotides can be used to express proteins of interest transiently, or can be integrated into the cellular genome by random or targeted recombination such as, for example, homologous recombination or recombination mediated by recombinases that recognize specific recombination sites (e.g., Cre-lox-mediated recombination).
  • Target sites for disruption or insertion of DNA are typically identified with the maximum effect of the gene disruption or insertion in mind.
  • target sequences may be chosen near the N-terminus of the coding region of the gene of interest whereas a DNA break is introduced within the first or second exon of the gene.
  • Introns non-coding regions
  • the changes introduced by these modifications are permanent to the genomic DNA of the organism.
  • RNA editing protocols were employed to render a nonfunctional versions of two or more of those genes.
  • the one of those genes encodes PLBD2 (e.g., SEQ ID NO:33).
  • protocols known in the art for introducing an expressible gene of interest (GOI), such as a multi- subunit antibody, along with any other desirable elements such as, e.g., promoters, enhancers, markers, operators, ribosome binding sites (e.g., internal ribosome entry sites), efc. are also employed.
  • the resulting recombinant cell line conveniently provides more efficient downstream bioprocess methods with respect to expressible exogenous genes of interest (GOIs), since purification steps for exogenous proteins of interest are eliminated due to the absence of the contaminant host cell protein. Eliminating or refining purification procedures also results in higher amounts (titer) of the recovered protein of interest.
  • GOIs expressible exogenous genes of interest
  • Applicants have discovered enzymatic activities associated with the destabilization of polysorbates (including polysorbate 20 and polysorbate 80) and/or enzymatic activities co-purifying with highly concentrated, multimerized or aggregated protein. Those activities were found to be associated with one or more fatty acid hydrolases (FAHs).
  • FAHs fatty acid hydrolases
  • One such FAH was identified from the peptide sequences listed in Table 2.
  • a BLAST search of those peptide sequences revealed identity with a putative phospholipase B-like 2 (PLBD2, also referred to as PLBL2).
  • PLBD2 is highly conserved in hamster (SEQ ID NO:32), mice (SEQ ID NO:34), rat (SEQ ID NO:35), human (SEQ ID NO:36), and bovine (SEQ ID NO:37).
  • the applicants discovered that PLBD2 which co-purifies under certain processes with some classes of proteins-of-interest manufactured in a mammalian cell line, has enzymatic activity responsible for the hydrolysis of polysorbate 20 and 80.
  • Other FAH species, of which PLBD2 is an example may contribute to polysorbate instability or persist as hydrophobic "sticky" proteins that bind protein multimers or aggregates during purification and ultimate formulation, depending upon the particular protein-of-interest and/or background of the host cell.
  • IgGs formulated with polysorbate 20 did not form particles and the putative esterase did not hydrolyze the polysorbate 20.
  • the author reported that the putative lipase associated with the IgG did not affect saturated C 12 fatty acid ⁇ i.e., laurate) (Id at 7.)
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • LPL is a triacylglycerol/diacylglycerol hydrolase of the carboxylic ester hydrolase (esterase) family (see Hide et al., "Structure and evolution of the lipase superfamily," J Lipid Res. 1992 Feb; 33(2): 167-78).
  • LI PA is a sterol esterase and synthase that acts on esters of sterols and long-chain fatty acids (see Dubland and Francis, “Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism,” Front. Cell Dev. Biol. 2015 Feb 2; 3(3): 1-11 ).
  • Acid ceramidase is not a carboxylic ester hydrolase, but rather an amide hydrolase that cleaves fatty acids from ceramide at the amide bond (carboxylic amide hydrolase) (see Park and Schuchman, "Acid ceramidase and human disease,” Biochim. Biophys. Acta. 2006 Dec; 1758(12): 2133-8).
  • Phospholipases are a family of esterase enzymes that catalyze the cleavage of phospholipids. Each phospholipase subclass has different substrate specificity based on its target cleavage site.
  • Phospholipase B was identified as related to a group of prokaryotic and eukaryotic lipase proteins by virtue of the presence of a highly conserved amino acid sequence motif, Gly-Asp-Ser-Leu (GDSL) (Upton, C, and Buckley, JT. A new family of lipolytic enzymes? Trends Biochem Sci. 1995; 20:178-179).
  • phospholipase B is also classified with known GDSL(S) hydrolases, and has little sequence homology to true lipases, differentiating itself structurally from phospholipases by having a serine-containing motif closer to the N-terminus than other lipases.
  • phospholipase B-like proteins are also classified as N-terminal nucleophile (Ntn) hydrolases.
  • Ntn N-terminal nucleophile hydrolases.
  • PLB-like proteins such as phospholipase B-like protein 1 (PLBD1 ) and phospholipase B-like protein 2 (PLBD2), also have amidase activity, similar to other Ntn hydrolases (Repo, H. et al, Proteins 2014; 82:300-31 1 ).
  • Lipoprotein lipases have also been demonstrated to cleave carboxylic ester bonds of polysorbate 20 and 80; and to associate with some monoclonal antibodies during production (see N. Levy, "Host cell protein impurities and protein-protein interactions in downstream purification of monoclonal antibodies," Dissertation submitted to the Faculty of the University of Delaware, Summer 2014, UMI 3642330, Published by ProQuest LLC, 2014).
  • Cell-cultures of a CHO-K1 RNAi knock-down of LPL revealed diminished polysorbate esterase activity. The effect of such a knockdown on overall cell viability or the production of useful titers of ectopic protein has not been investigated.
  • Knockout of a host cell gene such as an FAH, more particularly one or more of phospholipase B-like protein 2, lipoprotein lipase, lysosomal acid lipase and acid ceramidase may be accomplished in several ways. Rendering the FAH encoding gene nonfunctional, or reducing the functional activity of the target FAH protein may be done by introducing point mutations in the FAH genomic sequence, particularly in the exons (coding regions).
  • nucleic acid sequences of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 were identified and sequences upstream and downstream of the target site ⁇ i.e., homologous arms) may be utilized to integrate an expression cassette comprising a mutated gene by homologous recombination. Further gene editing tools are described herein in accordance with the invention.
  • Cell lines devoid of multiple FAH activities are useful for the production of therapeutic proteins to be purified and stored long term, and such cell lines solve problems associated with long term storage of pharmaceutical compositions in a formulation containing a fatty acid ester surfactant by maintaining protein stability and reducing subvisible particle (SVP) formation (see also PCT International Application No. PCT/US 15/54600 filed October 8, 2015, which is hereby incorporated in its entirety into the specification).
  • SVP subvisible particle
  • Assays to detect FAH activity include polysorbate degradation measurements. Unpurified protein supernatants or fractions from CHO cells, and supernatant at each step or sequence of steps when subjected to sequential purification steps, is tested for stability of polysorbate, such as polysorbate 20 or 80. The measurement of percent intact polysorbate reported is inversely proportional to the amount of contaminating FAH activity. Other measurements for detection of FAH activity or presence of FAH in a protein sample are known in the art. Detection of FAH protein (e.g., lipase, phospholipase, PLBD2, LPL, LIPA, acylase, ACE) may be done by trypsin digest mass spectrometry.
  • Detection of FAH protein e.g., lipase, phospholipase, PLBD2, LPL, LIPA, acylase, ACE
  • micrometers in diameter may be counted in the protein formulation in order to detect esterase or other FAH activity.
  • glycerophospho[ 3 H]choline formation from phosphatidyl[3H]choline following incubation of phosphatidyl[ 3 H]choline and protein supernatant may be determined by thin-layer chromatography (following similar protocols according to Kanoh, H. et al. 1991 Comp Biochem Physiol 102B(2):367- 369).
  • SEQ ID NO:32 disclosed herein was identified from proteins expressed in CHO cells. Other mammalian species (such as, for example, humans, rats, mice), were found to have high homology to the identified FAH. Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques. In addition, variants of PLBD2, can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:32, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:32 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian PLBD2 sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 3 identifies exemplary mammalian PLBD2 proteins and their degree of homology.
  • the targeted disruption of SEQ ID NO:33 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 33-62, 37-56, 40-69, 44-63, 1 10-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 33-62, 37-56, 40-69, 44-63, 1 10-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the PLBD2 nucleic acid sequence is at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:33 or target sequence thereof.
  • SEQ ID NO:62 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LPL can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:62, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:62 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian LPL sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 4 identifies exemplary mammalian LPL proteins and their degree of homology. TABLE 4: Amino acid identity of LPL homologs
  • the targeted disruption of SEQ ID NO:61 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400- 420, 410-430, 420-440, 430-450, 440-460
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410- 430, 420-440, 430-450, 440-460, 450-470
  • the LPL nucleic acid sequence is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:61 or target sequence thereof.
  • SEQ ID NO:70 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LI PA can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:70, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:70 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • the mammalian LIPA sequences are conserved among hamster, human, mouse and rat genomes.
  • Table 5 identifies exemplary mammalian LIPA proteins and their degree of homology.
  • the targeted disruption of SEQ ID NO:69 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the LIPA nucleic acid sequence is at least about 70% identical, at least about 71% identical, at least about 72% identical, at least about 73% identical, at least about 74% identical, at least about 75% identical, at least about 76% identical, at least about 77% identical, at least about 78% identical, or at least about 79% identical, at least about 80% identical, at least about 81 % identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:69 or target sequence thereof.
  • SEQ ID NO:78 disclosed herein was identified from proteins expressed in CHO cells.
  • Other mammalian species such as, for example, humans, rats, mice
  • Homologous sequences may also be found in cell lines derived from other tissue types of Cricetulus griseus, or other homologous species, and can be identified and isolated by techniques that are well-known in the art. For example, one may identify other homologous sequences by cross-species hybridization or PCR-based techniques.
  • variants of LPL can then be tested for FAH activity as described herein.
  • DNAs that encode proteins at least about 80% identical in amino acid identity to SEQ ID NO:78, or variants thereof, and having FAH activity are expected to exhibit their FAH activity on biopharmaceutical compositions and are candidates for targeted disruption in the engineered cell line. Accordingly, homologs of SEQ ID NO:78 or variants thereof, and the cells expressing such homologs are also encompassed by embodiments of the invention.
  • ACE mammalian acid ceramidase
  • the targeted disruption of SEQ ID NO:77 is directed to the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130- 150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200- 220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300- 320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 135-154, 237-256, and 332-351 of SEQ ID NO:77.
  • the target sequence is wholly or partially within the region selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 1 10-140, 120-140, 130-150, 140- 160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210- 230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310- 330, 320-340, 330-350, 340-360, 350-370, 360-380, 135-154, 237-256, and 332-351 of SEQ I D NO:77.
  • the ACE nucleic acid sequence is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, or at least about 89% identical, at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the sequence of SEQ ID NO:77 or target sequence thereof.
  • Cell populations expressing enhanced levels of a protein of interest can be developed using the cell lines and methods provided herein.
  • the isolated commercial protein, protein supernatant or fraction thereof, produced by the cells of the invention have no detectable esterase or esterase activity.
  • Cell pools further modified with exogenous sequence(s) integrated within the genome of the modified cells of the invention are expected to be stable over time, and can be treated as stable cell lines for most purposes. Recombination steps can also be delayed until later in the process of development of the cell lines of the invention.
  • Methods for genetically engineering a host cell genome in a particular location may be achieved in several ways. Genetic editing techniques were used to modify a nucleic acid sequence in a eukaryotic cell, wherein the nucleic acid sequence is an endogenous sequence normally found in such cells and expressing a contaminant host cell protein. Clonal expansion is necessary to ensure that the cell progeny will share the identical genotypic and phenotypic characteristics of the engineered cell line.
  • native cells are modified by a homologous recombination technique to integrate a nonfunctional or mutated target nucleic acid sequence encoding a host cell protein, such as a variant of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and/or SEQ ID NO:77.
  • One such method of editing the CHO PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and ASA genomic sequences involves the use of guide RNAs and a type II Cas enzyme to specifically target an exon of PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and/or ASA.
  • RNAs directed to particular exons of CHO PLBD2, LPL, LIPA, ACE, PAFAHG, PIPLC, LCE, IAH1 , LPLA2, CEH, and ASA have been employed (Table 7) in a site-specific nuclease editing method as described herein.
  • Other methods of targeted genome editing, for example nucleases, recombination-based methods, or RNA interference, to modify the FAH genes may be employed for the targeted disruption of the CHO genome.
  • the engineered mammalian host cell comprises one or more disruptions within gene sequences selected from the group consisting of nucleotides 37-63 of SEQ ID NO:33, nucleotides 465-612 of SEQ ID NO:61 , nucleotides 180-295 of SEQ ID NO:69, nucleotides 135-351 of SEQ ID NO:77, nucleotides 249-388 of SEQ ID NO:1 17, nucleotides 1624-2157 of SEQ ID NO:163, nucleotides 372-1399 of SEQ ID NO:1 18, nucleotides 1 155-1600 of SEQ ID NO:1 19, nucleotides 423-615 of SEQ ID NO:120, nucleotides 753-1 141 of SEQ ID NO:121 , nucleotides 311 -581 of SEQ ID NO:164, nucleotides 1 155-1443 of SEQ ID NO:122, and nucle
  • methods and compositions for knockout or downregulation of a nucleic acid molecule encoding an ortholog of a host cell FAH protein having at least 80% identity to SEQ ID NO:33 (PLBD2), SEQ ID NO:61 (LPL), SEQ ID NO:69 (LIPA) and/or SEQ ID NO:77 (ACE); or antibody-binding variant thereof, are via homologous recombination.
  • a nucleic acid molecule encoding an FAH protein (or any protein of interest in general) can be targeted by homologous recombination or by using site-specific nuclease methods that specifically target sequences at the FAH-expressing site of the host cell genome.
  • homologous polynucleotide molecules ⁇ i.e., homologous arms
  • a transgene can be introduced during this exchange if the transgene is flanked by homologous genomic sequences.
  • a recombinase recognition site can also be introduced into the host cell genome at the integration sites.
  • Homologous recombination in eukaryotic cells can be facilitated by introducing a break in the chromosomal DNA at the integration site.
  • Model systems have demonstrated that the frequency of homologous recombination during gene targeting increases if a double-strand break is introduced within the chromosomal target sequence. This may be accomplished by targeting certain nucleases to the specific site of integration. DNA-binding proteins that recognize DNA sequences at the target gene are known in the art. Gene targeting vectors are also employed to facilitate homologous recombination.
  • NHEJ non-homologous end-joining
  • ZFNs zinc finger nucleases
  • Some embodiments can utilize ZFNs with a combination of individual zinc finger domains targeting multiple target sequences.
  • ZFN methods to target disruption of two or more FAH genes are also embodied by the invention.
  • TAL effector nucleases may also be employed for site-specific genome editing.
  • TAL effector protein DNA-binding domain is typically utilized in combination with a non-specific cleavage domain of a restriction nuclease, such as Fokl.
  • a fusion protein comprising a TAL effector protein DNA-binding domain and a restriction nuclease cleavage domain is employed to recognize and cleave DNA at a target sequence within an exon of the gene encoding the target host cell protein, for example an esterase, such as a phospholipase B-like 2 protein(or other mammalian phospholipase), a lipoprotein lipase and a lysosomal acid lipase, and/or a fatty acylase, such as acid ceramidase (or other mammalian ceramidase).
  • an esterase such as a phospholipase B-like 2 protein(or other mammalian phospholipase), a lipoprotein lipase and a lysosomal acid lipase, and/or a fatty acylase, such as acid ceramidase (or other mammalian ceramidase).
  • Targeted disruption or insertion of exogenous sequences into a specific exon of the CHO protein encoded by SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and/or SEQ ID NO:77 may be done by employing a TALE nuclease (TALEN) targeted to locations within exon 1 , exon 2, exon 3, etc. of the fatty acid hydrolase genomic DNA (see Tables 6 and 7).
  • TALE nuclease TALEN
  • the TALEN target cleavage site within the gene sequences may be selected based on ZiFit.partners.org (ZiFit Targeter Version 4.2) and then TALENs are designed based on known methods (Boch J et al., 2009 Science 326:1509-1512; Bogdanove, A. J. & Voytas, D. F. 201 1 Science 333, 1843-1846; Miller, J. C. et al., 201 1 Nat Biotechnol 29, 143-148).
  • TALEN methods to target disruption of two or more of the PLBD2 gene ⁇ e.g., exon 1 or exon 2), LPL gene ⁇ e.g., exon 2, 3 or 4), LI PA gene ⁇ e.g., exon 1 or 2) and ACE gene ⁇ e.g., exon 1 , 3 or 4) are also embodied by the invention.
  • RNA-guided endonucleases are programmable genome engineering tools that were developed from bacterial adaptive immune machinery.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated
  • the protein Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection.
  • RGENs consist of components (Cas9 and tracrRNA) and a target- specific CRISPR RNA (crRNA).
  • CRISPR-Cas9 methods to target disruption of two or more of the PLBD2 gene e.g., exon 1 or exon 2
  • LPL gene e.g., exon 2, 3 or 4
  • LIPA gene e.g., exon 1 or 2
  • ACE gene e.g., exon 1 , 3 or 4
  • BuD-derived nucleases with precise DNA-binding specificities (Stella, S. et al. Acta Cryst. 2014, D70, 2042-2052).
  • a single residue-to-nucleotide code guides the BuDN to the specific DNA target within polynucleotide of interest ⁇ e.g., SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and or SEQ ID NO:77).
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding PLBD2, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003614971 .1 , at: Exon 1 within nucleotides (nt) 175367 to 175644 (SEQ ID NO:47); Exon 2 within nt 168958 to 169051 (SEQ ID NO:48); Exon 3 within nt 166451 to166609 (SEQ ID NO:49); Exon 4 within nt 164966 to 165066 (SEQ ID NO:50); Exon 5 within nt 164564 to164778 (SEQ ID NO:51 ); Exon 6 within nt 162682 to162779 (SEQ ID NO:52); Exon 7 within nt 160036 to160196 (SEQ ID NO:53); Exon 8 within nt 159733 to 159828 (S
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding LPL, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003613760.1 , at: Exon 1 within nucleotides (nt) 1257424 to 1257507 (SEQ ID NO:85); Exon 2 within nt 1266450 to 1266610 (SEQ ID NO:86); Exon 3 within nt 1270069 1270248 (SEQ ID NO:87); Exon 4 within nt 1271770 to 1271881 (SEQ ID NO:88); Exon 5 within nt 12283518 12283751 (SEQ ID NO:89); Exon 6 within nt 123715 1273957 (SEQ ID NO:90); Exon 7 within nt 1276672 1276792 (SEQ ID NO:91 ); Exon 8 within nt 1278328 to 12
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding LI PA, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003614200.1 , at: Exon 1 within nucleotides (nt) 985778 to 985674 (SEQ ID NO:94); Exon 2 within nt 984375 to 984258 (SEQ ID NO:95); Exon 3 within nt 970771 970573 (SEQ ID NO:96); Exon 4 within nt 969327 to 969218 (SEQ ID NO:97); Exon 5 within nt 968139 968003 (SEQ ID NO:98); Exon 6 within nt 961871 to 961725 (SEQ ID NO:99); Exon 7 within nt 960826 to 960755 (SEQ ID NO:100); Exon 8 within nt 95
  • Sequence-specific endonucleases may be directed to a target sequence at any one of the exons encoding ACE, for example in the CHO-K1 genome, NCBI Reference Sequence: NW_003613654.1 , at: Exon 1 within nucleotides (nt) 1378167 to 1378244 (SEQ ID NO: 103); Exon 2 within nt 1393746 to 1393792 (SEQ ID NO: 104); Exon 3 within nt 1398208 to 1398298 (SEQ ID NO:105); Exon 4 within nt 1399171 to 1399257 (SEQ ID NO: 106); Exon 5 within nt 1402147 to 1402225 (SEQ ID NO:107); Exon 6 within nt 1404854 to 1404928 (SEQ ID NO:108); Exon 7 within nt 1405714 to 1405759 (SEQ ID NO:109); Exon 8 within nt 14067
  • Precise genome modification methods are chosen based on the tools available compatible with unique target sequences within SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 so that disruption of the cell phenotype is avoided.
  • any protein of interest suitable for expression in prokaryotic or eukaryotic cells can be used in the engineered host cell systems provided.
  • the protein of interest includes, but is not limited to, an antibody or antigen-binding fragment thereof, a chimeric antibody or antigen- binding fragment thereof, an ScFv or fragment thereof, an Fc-fusion protein or fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, or an extracellular domain of a cell surface receptor or a fragment thereof.
  • Proteins of interest may be simple polypeptides consisting of a single subunit, or complex multisubunit proteins comprising two or more subunits.
  • the protein of interest may be a biopharmaceutical product, food additive or preservative, or any protein product subject to purification and quality standards.
  • the protein product is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody.
  • the antibody is an lgG1 antibody.
  • the antibody is an lgG2 antibody.
  • the antibody is an lgG4 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG4 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG1 antibody. In one embodiment, the antibody is a chimeric lgG2/lgG1/lgG4 antibody.
  • the antibody is selected from the group consisting of an anti- Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1 ), an anti-Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580A1 ), an anti-DII4 antibody, an anti-Angiopoetin-2 antibody (e.g. an anti-ANG2 antibody as described in U.S. Pat. No.
  • an anti- Programmed Cell Death 1 antibody e.g. an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1
  • an anti-Programmed Cell Death Ligand-1 e.g. an anti-PD-L1 antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580
  • an anti- Angiopoetin-Like 3 antibody e.g. an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356
  • an anti-platelet derived growth factor receptor antibody e.g. an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827
  • an anti-Erb3 antibody e.g. an anti- Prolactin Receptor antibody
  • an anti- Complement 5 antibody e.g. an anti-C5 antibody as described in U.S. Pat. Appln. Pub. No
  • an anti-TNF antibody an anti-epidermal growth factor receptor antibody (e.g. an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti-EGFRvlll antibody as described in U.S. Pat. Appln. Pub. No. US2015/0259423A1 ), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g. an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. Appln. Pub. No. US2014/0044730A1 ), an anti-Growth And Differentiation Factor-8 antibody (e.g.
  • an anti-GDF8 antibody also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871 ,209 or 9,260,515)
  • an anti-Glucagon Receptor e.g. anti-GCGR antibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 or US2016/0075778A1
  • an anti-VEGF antibody e.g. anti-VEGF antibody
  • an anti-l L1 R antibody e.g an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271681 A1 or U.S. Pat Nos. 8,735,095 or
  • an anti-interleukin 6 receptor antibody e.g. an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880
  • an anti-IL1 antibody e.g. an anti-IL2 antibody, an anti-IL3 antibody, an anti-l L4 antibody, an anti-IL5 antibody, an anti-l L6 antibody, an anti-l L7 antibody
  • an anti-interleukin 33 e.g. anti- IL33 antibody as described in U.S. Pat. Appln. Pub. Nos.
  • an anti-Respiratory syncytial virus antibody e.g. anti- RSV antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271653A1
  • an anti-Cluster of differentiation 3 e.g. an anti-CD3 antibody, as described in U.S. Pat. Appln. Pub. Nos.
  • an anti- Cluster of differentiation 20 e.g. an anti-CD20 antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1 , and in U.S. Pat. No. 7,879,984
  • an anti-CD19 antibody, an anti-CD28 antibody, an anti- Cluster of Differentiation-48 e.g. anti-CD48 antibody as described in U.S. Pat. No. 9,228,014
  • an anti-Fel d1 antibody e.g. as described in U.S. Pat. No.
  • an anti-Middle East Respiratory Syndrome virus e.g. an anti-MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015/0337029A1
  • an anti-Ebola virus antibody e.g. as described in U.S. Pat. Appln. Pub. No. US2016/0215040
  • an anti-Zika virus antibody e.g. an anti- Lymphocyte Activation Gene 3 antibody, or an anti-CD223 antibody
  • an anti-Nerve Growth Factor antibody e.g. an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 and U.S. Pat. Nos.
  • the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos.
  • the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.
  • the protein of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein).
  • an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety.
  • the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG.
  • the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands.
  • an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the 11-1 R1 extracellular region fused to Fc of hlgG1 ; see U.S. Pat. No. 6,927,004, which is herein incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hlgG1 ; see U.S. Pat. Nos.
  • IL-1 trap e.g., rilonacept, which contains the IL-1 RAcP ligand binding region fused to the 11-1 R1 extracellular region fused to Fc of hlgG1 ; see U.S. Pat. No
  • an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.
  • the host cells used in the methods of the invention are eukaryotic host cells including, for example, Chinese hamster ovary (CHO) cells, human cells, rat cells and mouse cells.
  • the invention provides a cell comprising a disrupted nucleic acid sequence fragment of SEQ ID NO:33 and at least one more of SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77.
  • the invention includes an engineered mammalian host cell further transfected with an expression vector comprising an exogenous gene of interest, such gene encoding the
  • biopharmaceutical product While any mammalian cell may be used, in one particular embodiment the host cell is a CHO cell.
  • Transfected host cells include cells that have been transfected with expression vectors that comprise a sequence encoding a protein or polypeptide. Expressed proteins will preferably be secreted into the culture medium for use in the invention, depending on the nucleic acid sequence selected, but may be retained in the cell or deposited in the cell membrane. Various mammalian cell culture systems can be employed to express recombinant proteins.
  • cell lines developed for specific selection or amplification schemes will also be useful with the methods and compositions provided herein, provided that at least two genes encoding a different fatty acid hydrolase (FAH) having at least 80% homology to at least two of SEQ ID NO:33, SEQ ID NO:61 , SEQ ID NO:69 and SEQ ID NO:77 have been downregulated, knocked out or otherwise disrupted in accordance with the invention.
  • An embodied cell line is the CHO cell line designated K1 .
  • the host cell line may be pre-adapted to bioreactor medium in the appropriate case.
  • transfection protocols are known in the art, and are reviewed in Kaufman (1988) Meth. Enzymology 185:537.
  • the transfection protocol chosen will depend on the host cell type and the nature of the GOI, and can be chosen based upon routine experimentation. The basic requirements of any such protocol are first to introduce DNA encoding the protein of interest into a suitable host cell, and then to identify and isolate host cells which have incorporated the
  • heterologous DNA in a relatively stable, expressible manner.
  • Electroporation can also be used to introduce DNA directly into the cytoplasm of a host cell, for example, as described by Potter et al. (Proc. Natl. Acad. Sci. USA 81 :7161 , 1988) or Shigekawa et al. (BioTechniques 6:742, 1988). Unlike protoplast fusion, electroporation does not require the selection marker and the GOI to be on the same plasmid.
  • reagents useful for introducing heterologous DNA into a mammalian cell have been described, such as LipofectinTM Reagent and LipofectamineTM Reagent (Gibco BRL, Gaithersburg, Md.). Both of these commercially available reagents are used to form lipid-nucleic acid complexes (or liposomes) which, when applied to cultured cells, facilitate uptake of the nucleic acid into the cells.
  • Methods for amplifying the GOI are also desirable for expression of the recombinant protein of interest, and typically involves the use of a selection marker (reviewed in Kaufman supra). Resistance to cytotoxic drugs is the characteristic most frequently used as a selection marker, and can be the result of either a dominant trait (e.g., can be used independent of host cell type) or a recessive trait (e.g., useful in particular host cell types that are deficient in whatever activity is being selected for).
  • a dominant trait e.g., can be used independent of host cell type
  • a recessive trait e.g., useful in particular host cell types that are deficient in whatever activity is being selected for.
  • amplifiable markers are suitable for use in the cell lines of the invention and may be introduced by expression vectors and techniques well known in the art (e.g., as described in Sambrook, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).
  • Useful selectable markers and other tools for gene amplification such as regulatory elements, described previously or known in the art, can also be included in the nucleic acid constructs used to transfect mammalian cells.
  • the transfection protocol chosen and the elements selected for use therein will depend on the type of host cell used. Those of skill in the art are aware of numerous different protocols and host cells in order to adapt the invention for a particular use, and can select an appropriate system for expression of a desired protein, based on the
  • the invention relates to the following items:
  • a recombinant host cell comprising a modification in two or more genes encoding two or more fatty acid hydrolases (FAH).
  • the recombinant host cell according to item 1 wherein the two or more FAHs are selected from the group consisting of phospholipase B-like 2 (PLBD2) protein, lipoprotein lipase (LPL), lysosomal acid lipase (LI PA) and acid ceramidase (ACE).
  • PLBD2 phospholipase B-like 2
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • [00191] a. a modification in a coding sequence of a polynucleotide encoding the PLBD2 protein, wherein the modification decreases the expression level of the PLBD2 protein relative to the expression level of a PLBD2 protein in a cell lacking the modification; and [00192] b. a modification in a coding sequence of a polynucleotide encoding the LI PA, wherein the modification decreases the expression level of the LI PA relative to the expression level of LI PA in a cell lacking the modification.
  • [00194] a modification in a coding sequence of a polynucleotide encoding the PLBD2 protein, wherein the modification decreases the expression level of the PLBD2 protein relative to the expression level of a PLBD2 protein in a cell lacking the modification;
  • [00202] a. a modification in a coding sequence of a polynucleotide encoding the LPL, wherein the modification decreases the expression level of the LPL relative to the expression level of LPL in a cell lacking the modification; and [00203] b. a modification in a coding sequence of a polynucleotide encoding the ACE protein, wherein the modification decreases the expression level of the ACE protein relative to the expression level of a ACE protein in a cell lacking the modification.
  • PLBD2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
  • LPL protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68.
  • LI PA protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.
  • ACE protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.
  • exogenous protein of interest is selected from the group consisting of an antibody heavy chain, an antibody light chain, an antigen-binding fragment, an antigen-binding protein, and an Fc-fusion protein.
  • a method of producing a recombinant protein comprising the steps of: (a) obtaining a sample comprising a recombinant protein and a plurality of host cell proteins from a host cell that is modified to produce reduced levels of fatty acid hydrolase compared to an unmodified cell; and (b) subjecting the sample to at least one purification step to remove at least one host cell protein.
  • a. does not comprise a detectable amount of a phospholipase B-like 2 (PLBD2) protein
  • b a modification in a coding sequence of a polynucleotide encoding a fatty acid hydrolase selected from the group consisting of lipoprotein lipase (LPL), lysosomeal acid lipase (LI PA), acid ceramidase (ACE), or a combination thereof.
  • LPL lipoprotein lipase
  • LI PA lysosomeal acid lipase
  • ACE acid ceramidase
  • a process for reducing fatty acid hydrolase activity in a protein formulation comprising the steps of: (a) modifying a host cell to decrease or ablate the expression of phospholipase B-like 2 (PLBD2) protein; (b) modifying the host cell to decrease or ablate the expression of lipoprotein lipase (LPL), lysosomal acid lipase (LI PA) and/or acid ceramidase (ACE); (c) transfecting the host cell with a polynucleotide encoding a protein of interest; (d) extracting a protein fraction comprising the protein of interest from the host cell; (e) contacting the protein fraction with a chromatography media selected from the group consisting of protein A affinity (PA) media, cation exchange (CEX) media, and anion exchange (AEX) media; (f) collecting the protein of interest from the media; and (g) combining the protein of interest with a fatty acid ester, and optionally a buffer and thermal stabilizer.
  • PA protein A
  • step of modifying a host cell to decrease or ablate the expression of phospholipase B-like 2 (PLBD2) protein comprises inserting or deleting at least one nucleotide within exon 1 of a polynucleotide encoding the PLBD2 protein.
  • step of modifying the host cell to decrease or ablate the expression of acid ceramidase comprises inserting or deleting at least one nucleotide within exon 1 , exon 3 or exon 4 of a polynucleotide encoding the ACE protein.
  • composition comprising one or more recombinant proteins obtainable by the method according to any of items 31-36 or the process according to any of items 37-45.
  • composition obtainable according to any of items 46-47, wherein the stable composition is characterized by one or more of:
  • composition obtainable according to any of items 46-48, wherein the adverse enzyme activity is the activity of one or more of esterases, hydrolases, lipases, phospholipases, ceramidases.
  • composition comprising one or more recombinant proteins, wherein the composition is stable.
  • stable composition is characterized by one or more of:
  • composition according to any of items 50-51 , wherein the adverse enzyme activity is the activity of one or more of esterases, hydrolases, lipases, phospholipases, ceramidases.
  • a Type II CRISPR/Cas system which requires at least 20 nucleotides (nt) of homology between a chimeric RNA (i.e., guide RNA) and its genomic target was used.
  • Guide RNA sequences were designed for specific targeting of an exon within the CHO phospholipase B-like 2 (PLBD2) nucleic acid (SEQ ID NO:33) and are considered unique (to minimize off-target effects in the genome).
  • sgRNA small guide RNAs
  • the sgRNA expression plasmid (System Biosciences, CAS940A-1 ) contains a human H1 promoter that drives expression of the small guide RNA and the tracrRNA following the sgRNA.
  • Immortalized Chinese hamster ovary (CHO) cells were transfected with the plasmid encoding Cas9- H1 enzyme followed by one of the sgRNA sequences, for instance sgRNAI (SEQ ID NO:45) or sgRNA2 (SEQ ID NO:46), designed to target the first exon of CHO PLBD2.
  • sgRNAI and sgRNA2 were predicted to generate a double strand break (DSB) at or around nucleotides 53 and 59 of SEQ ID NO:33, respectively.
  • a DSB was therefore predicted to occur approx. 23 or 29 nucleotides downstream of the PLBD2 start codon. (Note that nucleotides 1-30 of SEQ ID NO:33 encode a signal peptide.)
  • a negative control transfection was performed where the parental CHO line was transfected with the plasmid encoding Cas9-H1 enzyme without a proceeding sgRNA, or an sgRNA encoding a gene sequence not present in the CHO genome.
  • Genomic DNA gDNA
  • messenger RNA mRNA
  • qPCR primers and probes were designed to overlap with the sgRNA sequence used for the double strand break targeting event, in order to detect disruption of the genomic DNA and its transcription.
  • the relative abundance of PLBD2 gene or transcript in the candidate clones was determined using relative qPCR method, where the clones derived from the negative control transfection were used as a calibrator. See Figure 1.
  • the qPCR primers and probes were designed to detect sequences either in the sgRNAI or sgRNA2 position in PLBD2 exon 1 .
  • gDNA and RNA isolated from clone 1 failed to support qPCR amplification of PLBD2 exon 1 in either sgRNAI or sgRNA2 regions, but amplification of the housekeeping gene, GAPDH, was detected. Based on this data, clone 1 was identified as a potential knock out of PLBD2 in which both genomic alleles of PLBD2 of exonl were disrupted. It is noted that amplification of genomic DNA and mRNA was not detected in Clone 8 using primers overlapping with sgRNA2, however, sgRNAI primers/probes detected genomic DNA above control values. Clone 8, and others were further analyzed in order to understand the performance of the site- directed nuclease method.
  • the inventors also unexpectedly identified Clone 8 as a PLBD2 knockout despite the fact that genomic DNA fragments were identified by qPCR primers overlapping with the sgRNAI sequence.
  • the identification of a clone that has no detectable phospholipase activity or no detectable phospholipase protein was technically challenging and time-consuming.
  • Site-directed nuclease techniques may provide an ease-of-use, however, careful screening and elimination of false positives is necessary and still there may be unpredictable outcomes with regard to the identity of a single clone having two disrupted alleles.
  • Clone 1 and the wild type control host cell line were transfected with plasmids encoding the light and heavy chains of mAbl , a fully human IgG, in the presence of Cre recombinase to facilitate recombination mediated cassette exchange (RMCE) into EESYR locus (US Patent No. 7771997B2, issued August 10, 2010).
  • the transfected cultures were selected for 1 1 days in serum-free medium containing 400 ug/mL hygromycin. Cells that underwent RMCE, were isolated by flow cytometry.
  • PLBD2 knock out clone 1 and the wild type host cell line produced equivalent observed
  • the clone 1 derived isogenic cell line expressing mAbl was designated RS001
  • the mAbl expressing cell line originated from the PLBD2 wild type host was designated RS0WT.
  • Polysorbate 20 or polysorbate 80 degradation was measured to detect putative esterase activity in the supernatants of PLBD2 mutants. Unpurified protein supernatant from CHO cells, and supernatant taken at each step or sequence of steps when subjected to sequential purification steps, was tested for stability of polysorbate. The percent intact polysorbate reported was inversely proportional to the amount of contaminant esterase activity. Unpurified protein supernatant from CHO cells, and supernatant at each step or sequence of steps when subjected to sequential purification steps, was tested in assays measuring polysorbate degradation. The relative levels of intact polysorbate reported is inversely proportional to levels of contaminant esterase activity.
  • Monoclonal antibody was produced in an unmodified CHO cell and purified by different processes according to Table 1 1 , and the esterase activity measured by percent intact polysorbate 20, as in Table 9.
  • HIC Hydrophobic interaction chromatography
  • Example 4 Esterase protein abundance and activity detection in mAbl purified from modified compared to unmodified CHO cells.
  • mAbl was produced from RS001 and RSOWT and purified from the conditioned media using either PA alone, or PA and AEX chromatography
  • the PA-purified mAB1 from RS001 and RSOWT were analyzed for lipase abundance using trypsin digest mass spectrometry.
  • the trypsin digests of RS001 and RSOWT mAbl were injected into a reverse phase liquid chromatography column coupled to a triple quadrupole mass spectrometer set to monitor a specific product ion fragmented from SEQ ID NO:32.
  • a hydrophobic interaction column (Phenyl Sepharose® High Performance [GE Healthcare, Little Chalfont, Buckinghamshire, UK]) was used to generate a "HIC strip” fraction containing a protein of interest ⁇ i.e., mAb2) and associated host cell proteins.
  • the column was first equilibrated with two column volumes (CV) of buffer containing 40 mM Tris, 200 mM citrate at pH8.0.
  • the monoclonal antibody-containing load material from an anion exchange pool (“Q pool”) was adjusted to 200 mM sodium citrate, pH8.0, then loaded onto the column at a loading amount of 20-40 grams of protein per liter of the phenyl sepharose resin.
  • the column was washed with six CVs of 40 mM Tris, 200 mM citrate, at pH8.0. Then the column was stripped with three CVs of reverse osmosis deionized water. The stripped fraction was collected for subsequent analysis.
  • a 50 pg aliquot of the mAb2-containing HIC strip sample was denatured and reduced in a solution containing 5 mM acetic acid and 5 mM ir/ ' s(2-carboxyethyl)phosphine-HCI by heating at 80°C for 10 minutes.
  • the sample was then diluted in 50 mM Tris-HCI buffer (pH 8.0) and alkylated with 1 .5 mM iodoacetamide (IAA) and digested with trypsin (modified, sequencing grade from Promega, Madison, Wl) with an enzyme to substrate ratio of 1 :20 (w/w) at 37°C in the dark for three hours.
  • the digestion was then stopped by addition of 10% trifluoroacetic acid (TFA).
  • Peptides were separated using a linear gradient from 1 % mobile phase B (0.1 % FA in acetonitrile) to 7% mobile phase B for the first 5 minutes, followed by a second linear increase from 7% to 27% mobile phase B over the next 1 10 minutes, and another subsequent linear increase from 27%-40% in 10 minutes and a final increase to 90% in 5 minutes. The gradient was held at 90% for 20 minutes.
  • a Thermo Q ExactiveTM Plus mass spectrometer (Thermo Scientific, Waltham, MA) was used for peptide mass analyses, with high-energy collisional dissociation (HCD) employed for peptide fragmentation for MS/MS experiments.
  • Thermo XcaliburTM version 2.2.42
  • Proteome Discoverer version 1.4
  • Thermo Scientific, Waltham, MA was also used to perform the peptide identification using both Mascot and Sequent search engines.
  • the peptide spectra from the Proteome Discovery was manually examined to confirm the spectral assignment and protein identification.
  • Lipoprotein lipase (UniProtein ID: P06858), lysosomal acid lipase (UniProtein ID: P38571 ) and acid ceramidase (UniProtein ID: Q13510) were identified as potential active fatty acid hydrolases by more than three unique peptides per protein in the mAb2-containing HIC strip fraction.
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) were transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the lipoprotein lipase (LPL) gene (SEQ ID NO:61 ): sgRNA3 (SEQ ID NO:63), sgRNA4 (SEQ ID NO:64) or sgRNA5 (SEQ ID NO:65) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette was used in some
  • Cre recombinase was co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/0115502A1 , published April 28, 2016).
  • the transfected cultures were selected for 1 1 days in serum-free medium containing 400 ⁇ g mL hygromycin. Cells that underwent RMCE were isolated by flow cytometry.
  • the desired knock out genotype was confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the LPL knock-out or knock-down loci is designated as "clone 9".
  • the resultant clone containing both the PLBD2 and LPL knock-out or knock-down loci is designated as "clone 10".
  • FIG. 8 depicts an alignment of LPL-KO clones 19-22 (SEQ ID NOs:159-162, respectively) compared to a partial Chinese hamster LPL sequence (SEQ ID NO:158) showing the gaps in the respective clone sequences.
  • LPL-KO clones (as well as any other fatty acid hydrolase knock-out clone), plasmids encoding for Cas9 nuclease, eYFP and site-specific sgRNA were stably integrated into the CHO genome using Lipofectamine-based transfection protocol followed by selection for neomycin resistant cells. Seventeen days post transfection, the YFP positive cells were enriched by flow cytometry prior to single cell-sorting. After a 21 -day expansion, mRNA was isolated and the single cell clones were analyzed by qPCR for the presence of gene disruption.
  • 1 , 2 or 3 different sgRNA expression cassettes may be placed in the same plasmid.
  • Some constructs are manufactured using at least 2 sgRNA cassettes per lipase in a single plasmid.
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) are transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the lysosomal acid lipase (LIPA) gene (SEQ ID NO:69): sgRNA6 (SEQ ID NO:71 ), sgRNA7 (SEQ ID NO:72) or sgRNA8 (SEQ ID NO:73) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent
  • the transfected cultures are selected for 11 days in serum-free medium containing 400 pg/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the LIPA knock-out or knock-down loci is designated as "clone 1 1 ".
  • the resultant clone containing both the PLBD2 and LPL knock-out or knock-down loci is designated as "clone 12".
  • Clone 1 in which PLBD2 gene was knocked out, and a wild type host cell line (EESYR®) are transfected with a pair of plasmids.
  • One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the acid ceramidase (ACE) gene (SEQ ID NO:77): sgRNA9 (SEQ ID NO:79), sgRNAI O (SEQ ID NO:80) or sgRNA11 (SEQ ID NO:81 ) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co-transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/0115502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 pg/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing only the ACE knock-out or knock-down loci is designated as "clone 13".
  • the resultant clone containing both the PLBD2 and LPL knock-out or knock-down loci is designated as "clone 14".
  • Clone 9 in which the LPL gene and is modified
  • Clone 10 in which both the LPL gene and the PLBD2 gene are modified
  • plasmids encodes the Cas9 protein
  • the other plasmid encodes the guide RNA designed to target the lysosomal acid lipase (LIPA) gene (SEQ ID NO:69): sgRNA6 (SEQ ID NO:71 ), sgRNA7 (SEQ ID NO:72) and/or sgRNA8 (SEQ ID NO:73) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co- transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing the LIPA modified locus and the LPL modified locus designated as "clone 15".
  • the resultant clone containing the PLBD2, LPL and LIPA modified loci is designated as "clone 16".
  • Example 10 Targeted Disruption of a Lipoprotein Lipase (LPL) Gene, a Lysosomal Acid Lipase (LIPA) Gene and an Acid Ceramidase (ACE) Gene
  • Clone 15 in which the LPL and LIPA genes and are modified
  • Clone 16 in which the PLBD2, LPL and LIPA genes are modified
  • a pair of plasmids One of the plasmids encodes the Cas9 protein and the other plasmid encodes the guide RNA designed to target the acid ceramidase (ACE) gene (SEQ ID NO:77): sgRNA9 (SEQ ID NO:79), sgRNAI O (SEQ ID NO:80) or sgRNA1 1 (SEQ ID NO:81 ) (Table 7).
  • Tandem RNA polymerase III promoters such as H1 and/or U6 in any order can be used.
  • a single plasmid encoding both the Cas9 and the sgRNA expression cassette is used in some experiments. In other experiments, two separate plasmids are used, which confers flexibility for combining Cas9 with different sgRNAs.
  • Cre recombinase is co- transfected along with the Cas9 and the sgRNA plasmid to facilitate recombination mediated cassette exchange (RMCE) into the EESYR® locus (US Patent No. 7771997B2, issued August 10, 2010) or the EESYR® II locus (US Patent Application Publication No. 2016/01 15502A1 , published April 28, 2016).
  • the transfected cultures are selected for 1 1 days in serum-free medium containing 400 ⁇ g/mL hygromycin.
  • Cells that undergo RMCE are isolated by flow cytometry.
  • the desired knock out genotype is confirmed by standard molecular biology methods such as qPCR and sequencing.
  • the resultant clone containing the LIPA modified locus, the LPL modified locus and the ACE modified locus is designated as "clone 17".
  • the resultant clone containing the PLBD2 modified locus, the LIPA modified locus, the LPL modified locus and the ACE modified locus is designated as "clone 18".
  • the invention provides for cells and methods for expression and purification of recombinant proteins in eukaryotic cells.
  • the invention includes methods and compositions for expression of proteins in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, that employ downregulating gene expression of endogenous proteins in order to control production of such unwanted "sticky" host cell proteins.
  • the invention includes polynucleotides and modified cells that facilitate purification of an exogenous recombinant protein of interest.
  • the methods of the invention efficiently target host cell proteins in the Chinese hamster cellular genome in order to facilitate enhanced and stable expression of recombinant proteins expressed by the modified cells.
  • HCPs host cell proteins
  • LC/MS Advanced liquid chromatography/mass spectrometry
  • HCPs Changes in cell culture conditions of eukaryotic cells has been shown to impact the purity and stability of manufactured proteins, as seen by the increased quantity of HCPs of CHO cells upon downstream bioprocessing alterations (Tait, et al, 2013, Biotechnol Prog 29(3):688-696).
  • the detrimental effect of leftover HCPs in any product may affect the overall quality or quantity, or both the quality and quantity of the product.
  • HCPs if present even at low levels in a therapeutic product, may induce an undesired immune response which causes concern for patient safety and efficacy of the drug product (Singh SK. 201 1.
  • the invention provides a recombinant host cell, wherein the cell is modified to decrease the expression levels of two or more fatty acid hydrolases (FAHs) relative to the expression levels of FAH in an unmodified cell.
  • FHs fatty acid hydrolases
  • the invention provides a recombinant host cell, wherein the cell is modified to have no expression of two or more target FAHs.
  • one of said two or more target FAHs is an esterase.
  • the esterase is a lipase.
  • the lipase is: (1 ) a phospholipase, such as a phospholipase B-like protein or a phospholipase B-like 2 protein, (2) a lipoprotein lipase or (3) a lysosomal acid lipase.
  • one of said two or more target FAHs is an amidase.
  • the amidase is a fatty acid acylase.
  • the fatty acid acylase is an acid ceramidase.
  • a gene of interest is exogenously added to the recombinant host cell.
  • the exogenously added gene encodes a protein of interest (POI), for example the POI is selected form the group consisting of antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein.
  • POI protein of interest
  • the invention relates to a composition comprising one or more proteins of interest (POIs) obtainable by a method according to the invention.
  • the protein may be a recombinant protein and may be e.g. selected form the group comprising antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein, or any combinations thereof.
  • the invention relates to a composition comprising one or more proteins of interest (POIs), wherein the adverse enzyme activity is ⁇ 50%, 40%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, 30%, 29%, 28%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.05%, or ⁇ 0.05% active relative to the adverse enzyme activity of a wild-type production system.
  • POIs proteins of interest
  • the phrase "adverse enzyme activity" refers to any enzyme and its action upon the resulting composition as a whole, wherein the action results in metabolism of any protein components which metabolites reduces the shelf-life of the composition or results in the formation of subvisible particles (SVPs) above prescribed regulations.
  • the protein may be a recombinant protein and may e.g. be selected from the group comprising antibody heavy chain, antibody light chain, antigen-binding fragment, and Fc-fusion protein or any combinations thereof.
  • the enzymes and their activities may be, e.g., various esterases, hydrolases, lipases, phospholipases, ceramidases and the likes, or any combinations thereof.
  • Adverse enzyme activity may be measured using a functional assay ⁇ e.g., polysorbate fatty acid hydrolysis assay), or a structural assay ⁇ e.g., nano LC-MS of peptide fragments, or the like).
  • the invention provides a cell comprising a nonfunctional PLBD2 protein and one or more additional nonfunctional fatty acid hydrolases (FAH).
  • FAH nonfunctional fatty acid hydrolases
  • nonfunctional FAH is a nonfunctional lipoprotein lipase (LPL), lysosomal acid lipase (LI PA), acid ceramidase (ACE), platelet-activating factor acetylhydrolase IB subunit gamma (PAFAHG), phosphoinositide phospholipase C fragment (PIPLCf), phosphoinositide phospholipase C (PIPLC), liver carboxylesterase 1 (LCE), isoamyl acetate-hydrolyzing esterase 1 -like (IAH1 ), group XV phospholipase A2 (LPLA2), carboxylic ester hydrolase (CEH), and/or arylsulfatase A (ASA).
  • LPL lipoprotein lipase
  • LI PA lysosomal acid lipase
  • ACE acid ceramidase
  • PAFAHG platelet-activating factor acetylhydrolase IB subunit gamma
  • the invention provides making a cell by FAH target disruption.
  • the method comprises a site-specific nuclease for disrupting or editing the cell genome at a target site or sequence.
  • the FAH target site is (1 ) a PLBD2 target site, (2) an LPL target site, (3) an LI PA target site, (4) an ACE target site, (5) a PAFAHG site, (6) a PIPLCf or PIPLC site, (7) an LCE site, (8) an IAH1 site, (9) an LPLA2 site, (9) an CEH site, and /or (10) an ASA site.
  • the PLBD2 target site comprises a position within SEQ ID NO:33, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:33, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:33, within exon 1 of SEQ ID NO:33, within exon 2 of SEQ ID NO:33, or within exon 3 of SEQ ID NO:33.
  • the PLBD2 target site comprises a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-230, 190-210, 200-220, 210-230, 220-240, 230-250, 240-260, and 250-270 of SEQ ID NO:33.
  • the target site at a position within SEQ ID NO:33 or adjacent to a position within SEQ ID NO:33 is selected from the group consisting of nucleotides spanning positions numbered 37-56, 44-56, 33-62, 40-69, 110-139, 198-227, 182-21 1 , and 242-271 of SEQ ID NO:33.
  • the PLBD2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:33 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:33 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPL target site comprises a position within SEQ ID NO:61 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:61 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO:61 , within exon 1 of SEQ ID NO:61 , within exon 2 of SEQ ID NO:61 , or within exon 3 of SEQ ID NO:61.
  • the LPL target site comprises a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430, 420-440
  • the target site at a position within SEQ ID NO:61 or adjacent to a position within SEQ ID NO:61 is selected from the group consisting of nucleotides spanning positions numbered 465-484, 558-577, and 593-612 of SEQ ID NO:61.
  • the LPL target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:61 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:61 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LIPA target site comprises a position within SEQ ID NO:69, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:69, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:69, within exon 1 of SEQ ID NO:69, within exon 2 of SEQ ID NO:69, or within exon 3 of SEQ ID NO:69.
  • the LIPA target site comprises a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350 and 340-360 of SEQ ID NO:69.
  • the target site at a position within SEQ ID NO:69 or adjacent to a position within SEQ ID NO:69 is selected from the group consisting of nucleotides spanning positions numbered 180-199, 239-258, and 276-295 of SEQ ID NO:69.
  • the LIPA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:69 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:69 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ACE target site comprises a position within SEQ ID NO:77, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:77, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:77, within exon 1 of SEQ ID NO:77, within exon 2 of SEQ ID NO:77, within exon 3 of SEQ ID NO:77, or within exon 4 of SEQ ID NO:77.
  • the ACE target site comprises a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 110-140, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370 and 360-380 of SEQ ID NO:77.
  • the target site at a position within SEQ ID NO:77 or adjacent to a position within SEQ ID NO:77 is selected from the group consisting of nucleotides spanning positions numbered 135-154, 237-256, and 332-351 of SEQ ID NO:77.
  • the ACE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:77 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:77 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:117, 100 nucleotides
  • the PAFAHG target site comprises a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:1 17 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 17 or adjacent to a position within SEQ ID NO:117 is selected from the group consisting of nucleotides spanning positions numbered 101-120, 1 1 1-130, 121 -140, 131-150, 141-160, and 150-169 of SEQ ID NO:1 17.
  • the PAFAHG target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 17 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 17 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the PIPLC target site comprises a position within SEQ ID NO:1 18, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO:118, 100 nucleotides
  • SEQ ID NO:1 downstream of the 5-prime end of SEQ ID NO: 118, within exon 1 of SEQ ID NO:1 18, within exon 2 of SEQ ID NO:118, within exon 3 of SEQ ID NO:118, within exon 4 of SEQ ID NO:1 18, within exon 5 of SEQ ID NO:1 18, within exon 6 of SEQ ID NO:1 18, within exon 7 of SEQ ID NO:1 18, within exon 8 of SEQ ID NO: 1 18, within exon 9 of SEQ ID NO: 1 18, within exon 10 of SEQ ID NO: 1 18, within exon 1 1 of SEQ ID NO:1 18, within exon 12 of SEQ ID NO:1 18, or within exon 13 of SEQ ID NO:1 18.
  • the PIPLC target site comprises a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:1 18 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410
  • the target site at a position within SEQ ID NO:1 18 or adjacent to a position within SEQ ID NO:118 is selected from the group consisting of nucleotides spanning positions numbered 39-188, 39-58, 49-68, 59-78, 69-88, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 149-178, and 159-188 of SEQ ID NO:118.
  • the PIPLC target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 18 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 18 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LCE target site comprises a position within SEQ ID NO:1 19, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 1 19, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:1 19, within exon 1 of SEQ ID NO:1 19, within exon 2 of SEQ ID NO:1 19, within exon 3 of SEQ ID NO:119, within exon 4 of SEQ ID NO:1 19, or within exon 5 of SEQ ID NO:1 19.
  • the LCE target site comprises a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:1 19 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:1 19 or adjacent to a position within SEQ ID NO:119 is selected from the group consisting of nucleotides spanning positions numbered 89-140, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, and 139-158 of SEQ ID NO:1 19.
  • the LCE target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:1 19 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:1 19 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the IAH1 target site comprises a position within SEQ ID NO:120, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 120, 100 nucleotides downstream of the 5-prime end of SEQ ID NO:120, within exon 1 of SEQ ID NO:120, within exon 2 of SEQ ID NO:120, within exon 3 of SEQ ID NO:120, or within exon 4 of SEQ ID NO:120.
  • the IAH1 target site comprises a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO:120 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430
  • the target site at a position within SEQ ID NO:120 or adjacent to a position within SEQ ID NO:120 is selected from the group consisting of nucleotides spanning positions numbered 104-325, 104-123, 1 14-133, 124-143, 134-153, 144-163, 154-173, 164-183, 174-193, 184-203, 194-213, 204-223, 214-233, 224-243, 234-253, 244-263, 254-273, 264-283, 274-293, 284-303, 294-313, 304-323, and 314-333 of SEQ ID NO:120.
  • the IAH1 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO: 120 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:120 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 , within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 121 , 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 121 , within exon 1 of SEQ ID NO: 121 , within exon 2 of SEQ ID NO: 121 , within exon 3 of SEQ ID NO: 121 , or within exon 4 of SEQ ID NO: 121.
  • the LPLA2 target site comprises a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 selected from the group consisting of nucleotides spanning positions numbered -20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-
  • the target site at a position within SEQ ID NO:121 or adjacent to a position within SEQ ID NO:121 is selected from the group consisting of nucleotides spanning positions numbered 69-195, 69-88, 79-98, 89-108, 99-118, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, and 179-198 of SEQ ID NO:121.
  • the LPLA2 target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO: 121 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO:121 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the CEH target site comprises a position within SEQ ID NO:122, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 122, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 122, within exon 1 of SEQ ID NO: 122, within exon 2 of SEQ ID NO: 122, within exon 3 of SEQ ID NO: 122, within exon 4 of SEQ ID NO: 122, or within exon 5 of SEQ ID NO: 122.
  • the CEH target site comprises a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO:122 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430,
  • the target site at a position within SEQ ID NO:122 or adjacent to a position within SEQ ID NO:122 is selected from the group consisting of nucleotides spanning positions numbered 79-186, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, and 169-188 of SEQ ID NO:122.
  • the CEH target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:122 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO: 122 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell ⁇ i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the ASA target site comprises a position within SEQ ID NO:123, within 100 nucleotides upstream of the 5-prime end of SEQ ID NO: 123, 100 nucleotides downstream of the 5-prime end of SEQ ID NO: 123, within exon 1 of SEQ ID NO: 123, within exon 2 of SEQ ID NO: 123, within exon 3 of SEQ ID NO: 123, or within exon 4 of SEQ ID NO: 123.
  • the ASA target site comprises a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO:123 selected from the group consisting of nucleotides spanning positions numbered 1-20, 10-30, 20-40, 30-50, 30-60, 30-70, 40-60, 40-70, 50-70, 60-80, 70-90, 80-100, 90-1 10, 100-120, 1 10-130, 120-140, 130-150, 140-160, 150-170, 160-180, 160-180, 170-190, 180-200, 180-220, 190-210, 190-230, 200-220, 210-230, 220-240, 230-250, 240-260, 250-270, 260-280, 270-290, 280-300, 290- 310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430,
  • the target site at a position within SEQ ID NO:123 or adjacent to a position within SEQ ID NO:123 is selected from the group consisting of nucleotides spanning positions numbered 79-296, 79-98, 89-108, 99-1 18, 109-128, 1 19-138, 129-148, 139-158, 149-168, 159-178, 169-188, 179-198 , 189-208 , 199-218, 209-228, 219-238, 229-248, 239-258, 249-268, 259-278, 269-288, and 279-298 of SEQ ID NO:123.
  • the ASA target site is partially or fully within or encompassed by the nucleotide positions of SEQ ID NO:123 provided herein, and disrupting or editing the cell genome at the target site or sequence may consist of deleting or inserting one or more nucleotides within the nucleotide positions of SEQ ID NO: 123 provided herein, whereas disrupting or editing alters a subsequent transcript as compared to that transcribed from a wild-type cell (i.e., a cell free of genomic disruption or gene editing).
  • the subsequent transcript of an altered gene results in a frameshift of the translated protein.
  • the subsequent transcript of an altered gene results in an altered protein that is subject to degradation, is not detectable by a standard method such as mass spectrometry, or has no detectable activity.
  • the targeted disruption or editing occurs on both alleles of the gene.
  • the cell further integrates an exogenous nucleic acid sequence.
  • the cell is capable of producing an exogenous protein of interest.
  • the altered protein resulting from a disrupted gene does not bind to the protein of interest produced by the cell.
  • an isolated Chinese hamster ovary (CHO) cell comprising an engineered nucleic acid sequence comprising a variant of the PLBD2 gene (such as a variant of SEQ ID NO:33).
  • the PLBD2 gene comprises GACAGTCACG TGGCCCGACT GAGGCACGCG , nucleotides 1-30 of SEQ ID NO:33 (SEQ ID NO: 44).
  • the PLBD2 gene is engineered to disrupt expression of the open reading frame.
  • the invention provides an isolated CHO cell comprising (a) a disrupted PLBD2 gene comprising GACAGTCACG TGGCCCGACT GAGGCACGCG (SEQ ID NO: 44, also nucleotides 1- 30 of SEQ ID NO:33), (b) a disrupted esterase gene comprising a nucleotide encoding any one of the amino acid sequences in Table 2, or (c) a protein fragment of Table 2 expressed by a disrupted PLBD2 gene; and an exogenous nucleic acid sequence comprising a gene of interest.
  • the CHO cell that comprises an engineered nucleic acid sequence comprising a variant of the PLBD2 gene also comprises variants of one or more of the genes encoding (1 ) LPL (variant of SEQ ID NO:61 ), (2) LIPA (SEQ ID NO:69), (3) ACE (SEQ ID NO:77), (4) PAFAHG (SEQ ID NO:1 17), (5) PIPLC (SEQ ID NO:1 18), (6) LCE (SEQ ID NO:1 19), (7) IAH1 (SEQ ID NO:120), (8) LPLA2 (SEQ ID NO:121 ), (9) CEH (SEQ ID NO:122), and/or (10) ASA (SEQ ID NO:123).
  • a method of producing a protein of interest using a recombinant host cell wherein the host cell is modified to decrease the expression levels of esterase relative to the expression levels of esterase in an unmodified cell.
  • the method comprises the modified host cell having decreased esterase expression and an exogenous nucleic acid sequence comprising a gene of interest (GOI).
  • GOI gene of interest
  • the exogenous nucleic acid sequence comprises one or more genes of interest.
  • the one or more genes of interest are selected from the group consisting of a first GOI, a second GOI and a third GOI.
  • the invention provides expression systems comprising the recombinant host cell comprising modified or nonfunctional esterase.
  • the cell comprises a GOI operably linked to a promoter capable of driving expression of the GOI, wherein the promoter comprises a eukaryotic promoter that can be regulated by an activator or inhibitor.
  • the eukaryotic promoter is operably linked to a prokaryotic operator, and the eukaryotic cell optionally further comprises a prokaryotic repressor protein.
  • one or more selectable markers are expressed by the modified host cell.
  • the genes of interest and/or the one or more selectable markers are operably linked to a promoter, wherein the promoter may be the same or different.
  • the promoter comprises a eukaryotic promoter (such as, for example, a CMV promoter or an SV40 late promoter), optionally controlled by a prokaryotic operator (such as, for example, a tet operator).
  • the cell further comprises a gene encoding a prokaryotic repressor (such as, for example, a tet repressor).
  • a CHO host cell comprising recombinase recognition sites.
  • the recombinase recognition sites are selected from a LoxP site, a /_ox51 1 site, a Lox2272 site, Lox2372, Lox5171 , and a frt site.
  • the cell further comprises a gene capable of expressing a recombinase.
  • the recombinase is a Cre recombinase.
  • the selectable marker gene is a drug resistance gene.
  • the drug resistance gene is a neomycin resistance gene or a hygromycin resistance gene.
  • the second and third selectable marker genes encode two different fluorescent proteins.
  • the two different fluorescent proteins are selected from the group consisting of Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (mKate).
  • the first, second, and third promoters are the same. In another embodiment, the first, second, and third promoters are different from each other. In another embodiment, the first promoter is different from the second and third promoters, and the second and third promoters are the same. In more embodiments, the first promoter is an SV40 late promoter, and the second and third promoters are each a human CMV promoter. In other embodiments, the first and second promoters are operably linked to a prokaryotic operator.
  • the host cell line has an exogenously added gene encoding a recombinase integrated into its genome, operably linked to a promoter.
  • the recombinase is Cre recombinase.
  • the host cell has a gene encoding a regulatory protein integrated into its genome, operably linked to a promoter.
  • the regulatory protein is a tet repressor protein.
  • the first GOI and the second GOI encode a light chain, or fragment thereof, of an antibody or a heavy chain, or fragment thereof, of an antibody.
  • the first GOI encodes a light chain of an antibody and the second GOI encodes a heavy chain of an antibody.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a first light chain, or fragment thereof, a second light chain, or fragment thereof and a heavy chain, or fragment thereof.
  • the first, second and third GOI encode a polypeptide selected from the group consisting of a light chain, or fragment thereof, a first heavy chain, or fragment thereof and a second heavy chain, or fragment thereof.
  • a method for making a protein of interest comprising (a) introducing into a CHO host cell a gene of interest (GOI), wherein the GOI integrates into a specific locus such as a locus described in US Patent No. 7771997B2, issued August 10, 2010 or other stable integration and/or expression-enhancing locus; (b) culturing the cell of (a) under conditions that allow expression of the GOI; and (c) recovering the protein of interest.
  • the protein of interest is selected from the group consisting of a subunit of an immunoglobulin, or fragment thereof, and a receptor, or ligand-binding fragment thereof.
  • the protein of interest is selected from the group consisting of an antibody light chain, or antigen-binding fragment thereof, and an antibody heavy chain, or antigen-binding fragment thereof.
  • the CHO host cell genome comprises further modifications, and comprises one or more recombinase recognition sites as described above, and the GOI is introduced into a specific locus through the action of a recombinase that recognizes the
  • the GOI is introduced into the cell employing a targeting vector for recombinase-mediated cassette exchange (RMCE) when the CHO host cell genome comprises at least one exogenous recognition sequence within a specific locus.
  • RMCE recombinase-mediated cassette exchange
  • the GOI is introduced into the cell employing a targeting vector for homologous recombination, and wherein the targeting vector comprises a 5' homology arm homologous to a sequence present in the specific locus, a GOI, and a 3' homology arm
  • the targeting vector further comprises two, three, four, or five or more genes of interest.
  • one or more of the genes of interest are operably linked to a promoter.
  • a method for modifying a CHO cell genome to integrate an exogenous nucleic acid sequence comprising the step of introducing into the cell a vehicle comprising an exogenous nucleic acid sequence wherein the exogenous nucleic acid integrates within a locus of the genome.
  • the invention provides a process for manufacturing a stable protein formulation comprising the steps of: (a) extracting a protein fraction from the modified host cell of the invention having decreased or ablated expression of esterase, (b) contacting the protein fraction comprising a protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (c) collecting the protein of interest from the media, wherein a reduced level of the esterase activity is associated with the protein fraction collected at step (c), thus providing a stable protein formulation.
  • PA protein A affinity
  • CEX cation exchange
  • AEX anion exchange
  • the invention provides a process for reducing esterase activity in a protein formulation comprising the steps of: (a) modifying a host cell to decrease or ablate expression of esterase, (b) transfecting the host cell with a protein of interest, (c) extracting a protein fraction from the modified host cell, (c) contacting the protein fraction comprising the protein of interest with a column selected from the group consisting of protein A affinity (PA), cation exchange (CEX) and anion exchange (AEX) chromatography, (d) collecting the protein of interest from the media, and (e) combining the protein of interest with a fatty acid ester, and optionally a buffer and thermal stabilizer, thus providing a protein formulation essentially free of detectable esterase activity.
  • the protein formulation is essentially free of PLBD2 protein or PLBD2 activity.
  • a method for modifying a CHO cell genome to express a therapeutic agent comprising a vehicle for introducing, into the genome, an exogenous nucleic acid comprising a sequence for expression of the therapeutic agent, wherein the vehicle comprises a 5' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33, a nucleic acid encoding the therapeutic agent, and a 3' homology arm homologous to a sequence present in the nucleotide sequence of SEQ ID NO:33.
  • the invention provides a modified CHO host cell comprising a modified CHO genome wherein the CHO genome is modified by disruption of target sequence within a nucleotide sequence at least 90% identical to SEQ ID NO: 33.
  • the modified CHO host cell further comprises another FAH target sequence disruption.
  • the another FAH target sequence is within (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:61 , (1 ) a nucleotide sequence at least 90% identical to SEQ ID NO:69 and/or (3) a nucleotide sequence at least 90% identical to SEQ ID NO:77.
  • the invention provides a modified eukaryotic host cell comprising a modified eukaryotic genome wherein the eukaryotic genome is modified at a target sequence in a coding region of the target gene by a site-specific nuclease.
  • the site-specific nuclease comprises a zinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-like effector nuclease (TALEN), a TAL effector domain fusion protein, or an RNA-guided DNA endonuclease.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • TAL effector domain fusion protein or an RNA-guided DNA endonuclease.
  • the invention also provides methods of making such a modified eukaryotic host cell.
  • the target sequence can be placed in the indicated orientation as in SEQ ID NO:33, 61 , 69, 77, 1 17, 118, 119, 120, 121 , 122, or 123; or in the reverse of the orientation of SEQ ID NO:33, 61 , 69, 77, 1 17, 1 18, 119, 120, 121 , 122, or 123.

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Abstract

L'invention concerne des compositions et des procédés pour l'obtention de lignées cellulaires modifiées et de systèmes d'expression qui permettent l'expression de protéines recombinantes dans des cellules eucaryotes et leur facilité d'isolement. L'invention concerne également des systèmes d'expression cellulaire pouvant exprimer une protéine d'intérêt sensiblement sans protéine de cellule hôte liée.
EP17798018.2A 2016-08-24 2017-08-24 Modification de protéines de cellules hôtes Withdrawn EP3504328A1 (fr)

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Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200141472A (ko) * 2018-05-04 2020-12-18 시그마-알드리치 컴퍼니., 엘엘씨 변형된 숙주 세포 단백질 프로파일을 갖는 공작된 세포
EP3902412A4 (fr) 2018-10-17 2022-08-31 Perfect Day, Inc. Composants recombinants et compositions destinées à être utilisées dans des produits alimentaires
CA3140713A1 (fr) * 2019-05-21 2020-11-26 Regeneron Pharmaceuticals, Inc. Procedes d'identification et de quantification de proteine de cellule hote
KR20220034169A (ko) * 2019-07-10 2022-03-17 리제너론 파아마슈티컬스, 인크. 감소된 수준의 숙주 세포 단백질을 포함하는 방법 및 조성물
CN114555792A (zh) * 2019-10-15 2022-05-27 伊莱利利公司 重组改造的、脂肪酶/酯酶缺陷型哺乳动物细胞系
EP4127153A2 (fr) * 2020-03-26 2023-02-08 Genentech, Inc. Cellules de mammifère modifiées
CN115989416A (zh) * 2020-08-31 2023-04-18 基因泰克公司 用于评估生物制药开发过程中聚山梨醇酯降解风险的高通量、基于荧光的酯酶活性测定法
BR112023004206A2 (pt) * 2020-09-08 2023-04-11 Regeneron Pharma Método para detectar proteínas de célula hospedeira em anticorpos terapêuticos combinando digestão de tripsina, gradientes de cromatografia e espectrometria de massa boxcar
WO2024033465A1 (fr) * 2022-08-10 2024-02-15 Boehringer Ingelheim International Gmbh Miarn artificiels ciblant de multiples hydrolases

Family Cites Families (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7087411B2 (en) 1999-06-08 2006-08-08 Regeneron Pharmaceuticals, Inc. Fusion protein capable of binding VEGF
US6927004B2 (en) 2002-03-08 2005-08-09 Asml Netherlands B.V. Mask for use in lithography, method of making a mask, lithographic apparatus, and device manufacturing method
DK2374818T3 (da) 2006-06-02 2013-01-21 Regeneron Pharma Højaffinitetsantistoffer mod human IL-6-receptor
US7608693B2 (en) 2006-10-02 2009-10-27 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human IL-4 receptor
US7771997B2 (en) 2007-06-04 2010-08-10 Regeneron Pharmaceuticals, Inc. Enhanced expression and stability regions
CA2695199C (fr) 2007-07-31 2016-07-19 Regeneron Pharmaceuticals, Inc. Anticorps humains en cd20 humain et leur procede d'utilisation
US8309088B2 (en) 2007-08-10 2012-11-13 Regeneron Pharmaceuticals, Inc. Method of treating osteoarthritis with an antibody to NGF
JO3672B1 (ar) 2008-12-15 2020-08-27 Regeneron Pharma أجسام مضادة بشرية عالية التفاعل الكيماوي بالنسبة لإنزيم سبتيليسين كنفرتيز بروبروتين / كيكسين نوع 9 (pcsk9).
JO3417B1 (ar) 2010-01-08 2019-10-20 Regeneron Pharma الصيغ المستقرة التي تحتوي على الأجسام المضادة لمضاد مستقبل( interleukin-6 (il-6r
JO3340B1 (ar) 2010-05-26 2019-03-13 Regeneron Pharma مضادات حيوية لـعامل تمايز النمو 8 البشري
JOP20190250A1 (ar) 2010-07-14 2017-06-16 Regeneron Pharma صيغ مستقرة تحتوي على الأجسام المضادة لمضاد عامل نمو الأعصاب
AR083044A1 (es) 2010-09-27 2013-01-30 Regeneron Pharma Anticuerpos anti-cd48 y usos de los mismos
EP3733711A1 (fr) 2010-10-06 2020-11-04 Regeneron Pharmaceuticals, Inc. Formulations stabilisées contenant des anticorps anti-récepteur de l'interleukine-4 (il-4r)
JO3756B1 (ar) 2010-11-23 2021-01-31 Regeneron Pharma اجسام مضادة بشرية لمستقبلات الجلوكاجون
JO3412B1 (ar) 2011-06-17 2019-10-20 Regeneron Pharma أجسام مضادة ل angptl3 واستخداماتها
AU2012339722B2 (en) 2011-11-14 2017-09-14 Regeneron Pharmaceuticals, Inc. Compositions and methods for increasing muscle mass and muscle strength by specifically antagonizing GDF8 and/or Activin A
WO2013112438A1 (fr) 2012-01-23 2013-08-01 Regeneron Pharmaceuticals, Inc. Formulations stabilisées contenant des anticorps anti-ang2
JO3820B1 (ar) 2012-05-03 2021-01-31 Regeneron Pharma أجسام مضادة بشرية لـ fel d1وطرق لاستخدامها
TWI641619B (zh) 2012-06-25 2018-11-21 美商再生元醫藥公司 抗－ｅｇｆｒ抗體及其用途
US9540449B2 (en) 2012-08-13 2017-01-10 Regeneron Pharmaceuticals, Inc. Anti-PCSK9 antibodies with pH-dependent binding characteristics
JOP20200236A1 (ar) 2012-09-21 2017-06-16 Regeneron Pharma الأجسام المضادة لمضاد cd3 وجزيئات ربط الأنتيجين ثنائية التحديد التي تربط cd3 وcd20 واستخداماتها
JO3405B1 (ar) 2013-01-09 2019-10-20 Regeneron Pharma الأجسام المضادة لمضاد مستقبل عامل النمو المشتق من الصفائح الدموية - بيتا واستخداماتها
JO3532B1 (ar) 2013-03-13 2020-07-05 Regeneron Pharma الأجسام المضادة لمضاد انترلوكين-33 واستعمالاتها
TWI659968B (zh) 2013-03-14 2019-05-21 再生元醫藥公司 針對呼吸道融合病毒ｆ蛋白質的人類抗體及其使用方法
US9637535B2 (en) 2013-03-15 2017-05-02 Regeneron Pharmaceuticals, Inc. IL-33 antagonists and uses thereof
TWI641620B (zh) 2013-08-21 2018-11-21 再生元醫藥公司 抗－ｐｒｌｒ抗體及其用途
WO2015095568A1 (fr) * 2013-12-18 2015-06-25 Kelvin Lee Réduction de l'activité d'une lipase dans des formulations de produits
TWI681969B (zh) 2014-01-23 2020-01-11 美商再生元醫藥公司 針對ｐｄ-１的人類抗體
TWI680138B (zh) 2014-01-23 2019-12-21 美商再生元醫藥公司 抗ｐｄ-ｌ１之人類抗體
JP6632984B2 (ja) 2014-03-11 2020-01-22 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗ＥＧＦＲｖＩＩＩ抗体およびその使用
TWI701042B (zh) 2014-03-19 2020-08-11 美商再生元醫藥公司 用於腫瘤治療之方法及抗體組成物
EP3636073B1 (fr) 2014-05-05 2023-11-15 Regeneron Pharmaceuticals, Inc. Animaux c5 et c3 humanisés
JO3701B1 (ar) 2014-05-23 2021-01-31 Regeneron Pharma مضادات حيوية بشرية لمتلازمة الشرق الأوسط التنفسية - بروتين كورونا فيروس الشوكي
EP3194441A1 (fr) 2014-09-16 2017-07-26 Regeneron Pharmaceuticals, Inc. Anticorps anti-glucagon et leurs utilisations
TW201628649A (zh) * 2014-10-09 2016-08-16 再生元醫藥公司 減少醫藥調配物中微可見顆粒之方法
BR112017008022A2 (pt) 2014-10-23 2018-02-20 Regeneron Pharmaceuticals, Inc. célula, polinucleotídeo, veículo, e, métodos para modificação de um genoma de células cho e para preparação de uma proteína de interesse.
TWI710573B (zh) 2015-01-26 2020-11-21 美商再生元醫藥公司 抗伊波拉病毒醣蛋白之人類抗體
TW201702380A (zh) * 2015-02-27 2017-01-16 再生元醫藥公司 宿主細胞蛋白質修飾

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