WO2010043700A1 - Procédé de clarification à une densité de cellules supérieure - Google Patents

Procédé de clarification à une densité de cellules supérieure Download PDF

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Publication number
WO2010043700A1
WO2010043700A1 PCT/EP2009/063565 EP2009063565W WO2010043700A1 WO 2010043700 A1 WO2010043700 A1 WO 2010043700A1 EP 2009063565 W EP2009063565 W EP 2009063565W WO 2010043700 A1 WO2010043700 A1 WO 2010043700A1
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WO
WIPO (PCT)
Prior art keywords
cell
cells
desired biological
supernatant layer
broth
Prior art date
Application number
PCT/EP2009/063565
Other languages
English (en)
Inventor
Grigorios Zarbis-Papastoitsis
Michael Christopher Kuczewski
Emily Belcher Schirmer
Original Assignee
Dsm Ip Assets B.V.
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 Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Priority to CN2009801405074A priority Critical patent/CN102186875A/zh
Priority to CA2739501A priority patent/CA2739501A1/fr
Priority to JP2011531501A priority patent/JP2012505863A/ja
Priority to EP09820285A priority patent/EP2346891A1/fr
Priority to AU2009305343A priority patent/AU2009305343A1/en
Publication of WO2010043700A1 publication Critical patent/WO2010043700A1/fr
Priority to US13/083,538 priority patent/US20110184154A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Definitions

  • the present invention relates to a method for the clarification of a cell broth and to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance.
  • Fermentative production of biological substances delivers a complex cell broth from which the biological substances should be isolated and purified by a great number of steps.
  • solid material such as the cells and cell debris is to be separated from the cell broth fluid - a step called clarification.
  • the biological substances are present extracellularly and will thus be present in the cell broth fluid.
  • clarification methods used to-date include centrifugation, filtration (such as microfiltration, depth filtration and filtration through absolute pore size membranes) and expanded bed chromatography. Flocculation may be employed in order to enhance any of these clarification methods, in particular in combination with filtration.
  • Known flocculation agents for this purpose can range from simple electrolytes to synthetic poly-electrolytes (such as DEAE dextran, acryl-based polymers, polyethylene amine) or inorganic materials (such as diatomaceous earth or perlites).
  • synthetic poly-electrolytes such as DEAE dextran, acryl-based polymers, polyethylene amine
  • inorganic materials such as diatomaceous earth or perlites.
  • flocculation agents are, amongst others, that they may bind the desired biological substances of interest, that they may inactivate the desired biological substances of interest, that the flocculation process takes too long and/or that the flocculation agent may be hard or expensive to prepare in the high quality needed for medical use.
  • the flocculent needs to be removed from the final purified molecule a process that often requires expensive and time consuming analytical assays to verify its removal.
  • the method according to the present invention does not have any of these disadvantages.
  • An objective of the present invention is to provide a cost-effective method for the clarification of a cell broth harvested from a bioreactor and which contains mammalian cells as well as secreted desired biological substances.
  • a further objective of the present invention is to provide a method for the clarification of a cell broth containing mammalian cells at a higher cell density.
  • the present invention relates to a method for the clarification of a cell broth containing mammalian cells and culture medium as well as secreted desired biological substances having an overall positive charge in the cell broth wherein the cells are at an initial cell density of at least 15x10 6 cells/ml by the following steps: a. contacting the cell broth with particulate anion exchange material, b. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, and c. separating the resulting cell pellet from the supernatant layer.
  • cell broth means a cell culture inoculated with intact mammalian cells, and which may further contain culture medium as defined below, as well as secreted biological substances.
  • culture medium as defined below, as well as secreted biological substances.
  • the so diluted material is still covered by the term cell broth.
  • secreted biological substances biological substances which upon the production thereof by the mammalian cells are predominantly released (actively or passively) into the culture medium.
  • desired biological substance is intentionally being produced making use of the mammalian cells.
  • all positive charge of the secreted desired biological substances is meant here that the electrostatic contribution of positive and negatively charged ionogenic groups on the biological substance under the solvent conditions in the cell broth results in a net positive charge.
  • the overall charge of a biological substance is based on the pK a of the acidic and basic residues and the pH of the solution - in this case the pH of the cell broth.
  • the pi the pH where the net charge is zero
  • the substance must be higher than the pH of the cell broth.
  • culture medium is meant here the extracellular environment of the cells, which contains the nutrients and other constituents supporting the growth and production of cells, but may also contain waste products or host cell proteins (HCP) or material from lysed cells.
  • HCP host cell proteins
  • the composition of the culture medium may vary in time during the course of the culturing of cells and at the stage of clarification may be depleted of one or more of the original constituents.
  • contacting is meant here introduction of anion exchange material to cell broth and settling of cells (e.g. under gravity or with centrifugation).
  • anion exchange material is meant here particulate weak or strong anion exchange chromatography media.
  • the anion exchange material generally comprises a carrier, which may be organic material or inorganic material or a mixture of organic and inorganic material. Suitable organic materials are agarose based media and metacrylate. Suitable inorganic materials are silica, ceramics and metals.
  • the particles preferably may have a size of between 15 and 150 ⁇ m. More preferably their size is between 15 and 70 ⁇ m. The particles may have a density suitable for effecting relatively rapid sedimentation of the cells from the cell broth, but not too high as it was observed that too dense particles did not effect the sedimentation.
  • equate incubation time is meant here the time in which the precipitation of the cells results in a distinct cell pellet volume and a supernatant layer.
  • separating is meant here any method to remove the supernatant from the cell pellet, such as by decanting or drawing out the supernatant or e.g. by draining the pellet from the vessel through a port at the bottom.
  • the "supernatant layer” is the liquid overlying volume as a result of the settling.
  • the supernatant layer may (and generally will) still contain cells, be it at a cell density significantly lower than the initial cell density.
  • the initial cell density for the process of the present invention is at least 15x10 6 cells/ ml, more preferably at least 80x10 6 cells/ml.
  • the process according to the present invention may be carried out with cell densities up to 175x10 6 cells/ml, more preferably up to 130x10 6 cells/ml.
  • the cell density can be measured using a cell counter such as
  • Vi-CELLTM (with the trypan blue exclusion method) but other suitable methods include cytometry, packed cell volume determination, or Coulter counters (with the Electrical Sensing Zone Method).
  • the initial cell density is above 130x10 6 cells/ml it is advisable to first dilute the cell broth.
  • a cell broth with an initial cell density above 100x10 6 cells/ml be first diluted. Dilution preferably may be done to a cell density of not more than 80x10 6 cells/ml.
  • the cell broth may be diluted with a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, i.e. an isotonic solution such as PBS.
  • the present invention relates to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance having an overall positive charge in the cell broth wherein the cells in the cell broth are mammalian cells at an initial cell density of at least 15x10 6 cells/ml by a. contacting the cell broth with particulate anion exchange material b. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. extracting the secreted desired biological substances from the supernatant layer.
  • recovery is meant here obtaining the desired product essentially free from by-products and waste.
  • the present invention further relates to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance as described above and wherein the cells in the cell broth are mammalian cells at an initial cell density of at least 15x10 6 cells/ml, wherein the resulting cell pellet is further processed by e. re-suspending the resulting cell pellet, f. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, g. separating the resulting cell pellet from the supernatant layer and h. extracting the secreted desired biological substances from the supernatant layer.
  • step e. through h. of the above process are repeated one or more times.
  • the resulting cell pellet is re-suspended in a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, such as an aqueous (preferably isotonic) salt solution, more preferably in PBS.
  • aqueous (preferably isotonic) salt solution preferably in PBS.
  • the supernatant layers are collected and the secreted desired biological substance is extracted from the pooled supernatants.
  • mammalian cells include CHO (Chinese Hamster Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells, for example HEK-293 cells, human lymphoblastoid cells, E1 immortalized HER cells, mouse cells, for example NSO cells. More preferably, E1 immortalized HER cells are used, most preferably PER.C6 cells.
  • the cells in the process of the present invention are E1 -immortalized HER cells, more preferably PER.C6 cells (see U.S.
  • Patent 5,994,1208 the content of which is incorporated by reference here.
  • PER.C6 cells are exemplified by cells as deposited under ECACC No. 96022940 (see, e.g., U.S. Patent 5,994,128, EP 0833934 B1 , the contents of which are incorporated by reference here).
  • the cell broth for clarification may be obtained by any cell culturing method suitable for attaining a cell density of the mammalian cells of at least 15x10 6 cells/ml. Suitable methods in this respect are described in e.g. WO2005095578, WO2004099396 and WO2008006494. The contents thereof are incorporated herein by reference.
  • Biological substances which may be produced by the cells according to the present invention (for example by expressing a (recombinant) gene coding therefore) are for example viruses or (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood proteins such as proteins from the blood coagulation cascade, multifunctional proteins such as for instance erythropoietin, virus or bacterial proteins for instance for use in vaccines; immunoglobulins such as for example IgG or IgM, and the like.
  • a protein, more preferably an immunoglobulin or a part thereof is produced by the cells.
  • the biological substances such as proteins or vaccines produced by the cells can be used as an active ingredient in a pharmaceutical preparation.
  • the terms 'product' and 'biological substance' are interchangeable.
  • Suitable methods for extracting the secreted desired biological substances from the supernatant layer are for example filtration (such as depth filtration, microfiltration, ultrafiltration, diafiltration), chromatography (such as size exclusion chromatography, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography), aqueous two-phase extraction, precipitation or centrifugation.
  • the desired biological substance can be extracted very efficiently by cation exchange chromatography.
  • affinity chromatography in particular protein A chromatography, and cation exchange chromatography are especially suitable separation methods.
  • Si-PEI Bakerbond Wide-Pore PEI (PolyEthylenelmine) Prep LC Packing grafted silica beads (JT Baker)
  • Streamline DEAE diethylaminoethyl grafted agarose beads (GE Healthcare)
  • PrA HPLC Analytical Protein A High Pressure Liquid Chromatography.
  • PER.C6 ® cells at various cell densities and prepared according to the procedure outlined in WO2008006494.
  • the PER.C6 ® cells produced an antibody.
  • the concentration of antibody in the supernatant layers was measured by PrA HPLC. The concentrations were corrected for biomass when necessary, i.e. when the cell density is extremely high, the cells contribute significantly to the working volume.
  • Example 1 Clarification with low cell density. Different amounts of Si-PEI were added to individual vials containing
  • Si-PEI greatly reduced the time needed for the cells to settle. Also, the addition of Si-PEI resulted in a more compact pellet in comparison with the control (no Si-PEI added).
  • Si-PEI Different amounts of Si-PEI were added to individual vials containing 5 ml of cell culture.
  • X 1 63.5x10 6 cells/ml. The cells were allowed to settle for 30 minutes. Adding 5% (vol) of Si-PEI settled 87% of the cells. Adding 10% (vol) of
  • Si-PEI settled 89% of the cells. Adding 20% (vol) of Si-PEI settled 85% of the cells. In each case the resulting cell density was below 10x10 6 cells/ml which is a suitable feed for depth filtration. Product recovery was 97%.
  • the addition of Si-PEI greatly reduced the time needed for the cells to settle. Also, the addition of Si-PEI resulted in a more compact pellet in comparison with the control (no Si-PEI added).
  • Example 3 Clarification with high cell density. 10% (vol) of Si-PEI was added to 345 ml of cell culture broth.
  • Xt 123x10 6 cells/ml. Due to the high cell density two hours of settling were allowed. After these two hours the cell density in the resulting supernatant was 13.6x10 6 cells/ml. The pellet volume was 53% of the total volume (93% for the control where no Si-PEI was added). The supernatant was decanted and the pellet was washed twice with isotonic PBS with 1 hour of settling after each wash. Product recovery was 93% after the two washes. The total process time was 4 hours. After pooling the supernatants, the final process volume was 600 ml and the cell density was 9.9x10 6 cells/ml. Example 4 Maximizing recovery of product by repeated washings
  • X t 78x10 6 or 120x10 6 cells/ml.
  • Example 5 Clarification with high cell density and with various anion exchange materials 10 % (vol) of the anion exchange materials Si-PEI, DEAE Hyper D or
  • the cells were allowed to settle for 2 hours.
  • Figure 2a shows the cell densities in the supernatants as a function of time for each of the anion exchange materials as well as the control where no anion exchange material was added.
  • Figure 2b shows the supernatant volume as a function of time.
  • the addition of anion exchange material resulted in a more compact pellet, i.e. increased supernatant volume.
  • the total process time was 4 hours. After pooling the supernatants, the final process volume was 48 ml (4.8 x increase) and the cell density was
  • An cell culture harvest with initial cell density of 175 ⁇ 10 6 cells/ml_ was diluted to ⁇ 75 ⁇ 10 6 cells/mL with PBS(lnitial volume of 1.7 L). Following dilution Si-PEI chromatography media were added to the harvest (0.1 L of Si-PEI resin per L of diluted harvest). The cells were allowed to settle for ⁇ 60 minutes. The product containing supernatant was decanted and the settled cells were washed twice with PBS. The initial supernatant was pooled together with the two washes to maximize product recovery ( ⁇ 95%). The combined pool contains less than 5 ⁇ 10 6 cells/ml, and the HCP content is reduced by 59%.
  • the product recovered after the Si-PEI settling is further purified by depth filtration.
  • Depth filtration consist of a primary filter (typically 10 or 5 ⁇ m pore size) used for further reduction of the cell mass, followed by a secondary filter (typically 3 or 1 ⁇ m pore size) that removes smaller particles and prepares the clarified harvest for sterile filtration typically through a gradient 0.8/0.2 ⁇ m filter.
  • the depth filtration train can be Millipore Millistak+HC filters containing media such as DOHC (primary) followed by XOHC (secondary) or CUNO ZetaPlus filters containing such as 10M02 (primary) followed by 60ZA05A (secondary). In either case the clarified harvest is further filtered through 0.8/0.2 Dm filters (Supor, Pall).
  • the monoclonal antibody (product) is immobilized on the resin at a capacity of > 95 g/L of chromatography media.
  • the conditions used for immobilizing the antibody are slightly acidic (pH ⁇ 5.3) and conductivity of ⁇ 4.5 mS/cm. After binding the antibody is washed with equilibration buffer and finally eluted with a buffer step containing 100 mM sodium chloride. An additional reduction in HCP content (78%) is obtained by this step.
  • the eluted antibody can be further purified by a combination of chromatography and filtration techniques until the required purity specifications are met.
  • the overall reduction in Host Cell Proteins from the cell culture harvest through the CEX step is summarized below:

Abstract

La présente invention porte sur un procédé pour la clarification d'un bouillon de culture cellulaire contenant des cellules de mammifère ainsi que des substances biologiques secrétées souhaitées ayant une charge globale positive dans le bouillon de culture cellulaire, les cellules étant présentes à une densité de cellules élevée, par la mise en contact du bouillon de culture cellulaire avec une matière d'échange d'anions, l'étape consistant à laisser un temps d'incubation adéquat pour permettre la formation d'un culot de cellules et d'une couche de surnageant et la séparation du culot de cellules ainsi obtenu et de la couche de surnageant. La présente invention porte en outre sur un procédé pour la récupération de substances biologiques sécrétées souhaitées à partir d'un bouillon de culture cellulaire contenant des cellules de mammifère à une densité de cellules élevée produisant la substance biologique sécrétée souhaitée ayant une charge globale positive dans le bouillon de culture cellulaire par la mise en œuvre des étapes ci-dessus, suivies par l'extraction des substances biologiques sécrétées souhaitées à partir de la couche de surnageant.
PCT/EP2009/063565 2008-10-17 2009-10-16 Procédé de clarification à une densité de cellules supérieure WO2010043700A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN2009801405074A CN102186875A (zh) 2008-10-17 2009-10-16 较高细胞密度的澄清方法
CA2739501A CA2739501A1 (fr) 2008-10-17 2009-10-16 Procede de clarification a une densite de cellules superieure
JP2011531501A JP2012505863A (ja) 2008-10-17 2009-10-16 高密度細胞における清澄化方法
EP09820285A EP2346891A1 (fr) 2008-10-17 2009-10-16 Procédé de clarification à une densité de cellules supérieure
AU2009305343A AU2009305343A1 (en) 2008-10-17 2009-10-16 Clarification process at higher cell density
US13/083,538 US20110184154A1 (en) 2008-10-17 2011-04-09 Cell broth clarification and host cell protein removal

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10632008P 2008-10-17 2008-10-17
US61/106,320 2008-10-17
EP08167506 2008-10-24
EP08167506.8 2008-10-24

Related Child Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/063566 Continuation-In-Part WO2010043701A1 (fr) 2008-10-17 2009-10-16 Procédé de clarification

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WO2010043700A1 true WO2010043700A1 (fr) 2010-04-22

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PCT/EP2009/063565 WO2010043700A1 (fr) 2008-10-17 2009-10-16 Procédé de clarification à une densité de cellules supérieure

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EP (1) EP2346891A1 (fr)
JP (1) JP2012505863A (fr)
KR (1) KR20110085980A (fr)
CN (1) CN102186875A (fr)
AU (1) AU2009305343A1 (fr)
CA (1) CA2739501A1 (fr)
WO (1) WO2010043700A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012030512A1 (fr) 2010-09-03 2012-03-08 Percivia Llc. Procédé de purification de protéine en flux
US10421986B2 (en) 2013-09-30 2019-09-24 Janssen Vaccines & Prevention B.V. Method for the clarification of high-density crude cell culture harvest
US10695744B2 (en) 2015-06-05 2020-06-30 W. R. Grace & Co.-Conn. Adsorbent biprocessing clarification agents and methods of making and using the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030170810A1 (en) * 2001-09-18 2003-09-11 Masoud Vedadi Methods and apparatuses for purification
WO2007123961A2 (fr) * 2006-04-20 2007-11-01 Wyeth Procédés de purification pour isolation de virus purifié de la stomatite vésiculaire dans une culture cellulaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030170810A1 (en) * 2001-09-18 2003-09-11 Masoud Vedadi Methods and apparatuses for purification
WO2007123961A2 (fr) * 2006-04-20 2007-11-01 Wyeth Procédés de purification pour isolation de virus purifié de la stomatite vésiculaire dans une culture cellulaire

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012030512A1 (fr) 2010-09-03 2012-03-08 Percivia Llc. Procédé de purification de protéine en flux
US10421986B2 (en) 2013-09-30 2019-09-24 Janssen Vaccines & Prevention B.V. Method for the clarification of high-density crude cell culture harvest
US10695744B2 (en) 2015-06-05 2020-06-30 W. R. Grace & Co.-Conn. Adsorbent biprocessing clarification agents and methods of making and using the same

Also Published As

Publication number Publication date
AU2009305343A1 (en) 2010-04-22
KR20110085980A (ko) 2011-07-27
JP2012505863A (ja) 2012-03-08
CA2739501A1 (fr) 2010-04-22
EP2346891A1 (fr) 2011-07-27
CN102186875A (zh) 2011-09-14

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