WO2017115773A1 - Fc領域含有ポリペプチドの精製を効率化するための方法 - Google Patents
Fc領域含有ポリペプチドの精製を効率化するための方法 Download PDFInfo
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- WO2017115773A1 WO2017115773A1 PCT/JP2016/088820 JP2016088820W WO2017115773A1 WO 2017115773 A1 WO2017115773 A1 WO 2017115773A1 JP 2016088820 W JP2016088820 W JP 2016088820W WO 2017115773 A1 WO2017115773 A1 WO 2017115773A1
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- 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
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/528—CH4 domain
Definitions
- the present invention relates to a method for increasing the dynamic binding capacity of an Fc region-containing polypeptide to a protein A resin in protein A column chromatography, a purification method using the method, and the like.
- protein A resin has been improved, and more antibodies can be bound to protein A resin. Accordingly, the efficiency of antibody purification has been improved.
- GE Healthcare's rProtein A sepharoseGEFast Flow which is a representative first generation protein A resin, has a typical antibody binding capacity of 15-20 / g / L resin, whereas its second generation protein
- the binding capacity of about 30 g / L resin is generally recognized in Mab Select SuRe, which is an A-resin and is currently the world's most used.
- the latter can cope with a linear flow velocity about 1.5-2 times higher than the former, and protein A purification of antibody molecules can be performed more efficiently.
- Bispecific antibodies have two types of heavy chains due to their ability to recognize two different types of antigens. Therefore, the culture supernatant containing a bispecific antibody includes not only a bispecific antibody containing two types of H chains but also an antibody having only one type of H chain. In order to separate these from bispecific antibodies, Fc region mutants having altered binding power to protein A resin are used (Patent Documents 1 and 2). There was concern that protein A purification of bispecific antibodies would become inefficient due to the effects of such molecular modifications.
- An object of the present invention is to provide a method for more efficiently purifying an Fc region-containing polypeptide, particularly a bispecific antibody, using a protein A resin column.
- the present inventors have determined that the Fc region has a binding force to the protein A resin of the first polypeptide chain of the Fc region and the second polypeptide chain of the Fc region. It was found that by using different Fc regions, the dynamic binding capacity of the antibody increases and the purification efficiency of the antibody increases, and the present invention has been completed.
- the present invention provides the following.
- [1] A method for increasing the dynamic binding capacity of an Fc region-containing polypeptide to a protein A resin in protein A column chromatography.
- [2] preparing a first polypeptide chain and a second polypeptide chain having different binding forces to the resin as the first polypeptide chain and the second polypeptide of the Fc region, [1].
- [3] A first polypeptide chain that binds to the resin is prepared as a first polypeptide chain of the Fc region, and the second polypeptide chain of the Fc region does not bind to the resin or
- the Fc region of the Fc region-containing polypeptide is bound to the resin, while the first polypeptide chain of the Fc region binds to the resin, or the second polypeptide chain of the Fc region does not bind to the resin, or The method according to any one of [1] to [3], comprising a step of modifying the Fc region so that the binding (to the resin is weaker than that of the first polypeptide chain).
- the first polypeptide chain of the Fc region contains CH3 of IgG1, IgG2 or IgG4, and the second polypeptide chain of the Fc region contains CH3 of IgG3, any one of [1] to [4] The method of crab.
- the EU numbering 435th amino acid of the first polypeptide chain of the Fc region is His, and the EU numbering 435th amino acid of the second polypeptide chain is Arg, [1] to [5] The method according to any one. [7] The method according to any one of [1] to [6], wherein the increase in the binding capacity is 5 g / L resin or more. [8] The method according to any one of [1] to [7], wherein the increased dynamic binding capacity is 45 g / L resin or more. [9] The method according to any one of [1] to [8], wherein the Fc region-containing polypeptide is an antibody. [10] The method according to [9], wherein the antibody is a bispecific antibody.
- [11] A method for purifying an Fc region-containing polypeptide using the method according to [1] to [10].
- An Fc region-containing polypeptide purified by the method according to [11].
- a method for producing an Fc region-containing polypeptide using protein A resin comprising the following steps: (A) preparing a first polypeptide chain and a second polypeptide chain of the Fc region having different binding forces to the resin, (B) In protein A column chromatography, the dynamic binding capacity of the Fc region-containing polypeptide in step (a) to protein A resin is substantially the same as the binding force to the resin in protein A column chromatography.
- a first polypeptide chain that binds to the resin is prepared as the first polypeptide chain of the Fc region, and the second polypeptide chain of the Fc region is added to the resin as the second polypeptide chain.
- the method according to [15], which is a step of preparing a second polypeptide chain that does not bind or has a weak bond (to the resin as compared to the first polypeptide chain) [17]
- the step (a) However, the Fc region of the Fc region-containing polypeptide to be purified binds to the resin, while the first polypeptide chain of the Fc region binds to the resin, and the second polypeptide chain of the Fc region binds to the resin.
- the sample according to the step (c) is capable of binding to both a polypeptide containing the first polypeptide chain of the Fc region and a polypeptide containing the second polypeptide chain of the Fc region.
- the purification method according to [11], wherein the Fc region-containing polypeptide is an antibody.
- the purification method according to [19], wherein the antibody is a bispecific antibody.
- the present invention provides a method for more efficiently purifying Fc region-containing polypeptides, particularly bispecific antibodies, using protein A resin.
- the Fc region-containing polypeptide used in the present invention only needs to contain the Fc region of an antibody, and includes a polypeptide in which the Fc region and another polypeptide are fused, such as an antibody.
- the polypeptide in the present invention usually refers to peptides and proteins having a length of about 10 amino acids or more.
- it is usually a biological polypeptide, but is not particularly limited, and may be, for example, a polypeptide comprising an artificially designed sequence.
- any of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. may be sufficient.
- Fc region usually refers to a region in an antibody molecule comprising two polypeptide chains comprising a hinge region or a part thereof, a CH2 domain, and a CH3 domain, but is not particularly limited to this, and the hinge region Or some of them may not be included.
- the Fc region of the human IgG class is Kabat EU numbering, which means, for example, from the 226th cysteine to the C terminus, or from the 230th proline to the C terminus, but is not limited thereto.
- the human CH2 domain means the 231st to 340th in the Kabat EU numbering
- the human CH3 domain means the 341th to 447th in the Kabat EU numbering, but is not limited thereto.
- the Fc region can be suitably obtained by re-elution of the fraction adsorbed to the protein A resin after partial digestion of IgG1, IgG2, IgG3, Fc region-containing monoclonal antibodies, etc. with a protease such as pepsin. .
- protease such as pepsin.
- Such proteolytic enzymes are not particularly limited as long as full-length antibodies can be digested so that Fab and F (ab ') 2 can be produced in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin, papain, etc. can be illustrated.
- the Fc region include human IgG type Fc.
- any of IgG1, IgG2, IgG3, and IgG4 isotypes may be used.
- the Fc region of the present invention includes the first polypeptide chain and the second polypeptide chain.
- One embodiment of the present invention is a method for increasing the dynamic binding capacity of an Fc region-containing polypeptide to a protein A resin in protein A column chromatography.
- the first polypeptide chain and the second polypeptide chain contained in the Fc region preferably have different binding forces to the protein A resin, for example, bind to the protein A resin as the first polypeptide chain.
- a polypeptide chain that binds weakly or does not bind to protein A resin as compared to the first polypeptide chain can be used as the second polypeptide chain.
- a polypeptide chain containing CH3 of IgG1, IgG2, or IgG4 can be used as such a first polypeptide chain, and a polypeptide chain containing CH3 of IgG3 can be used as a second polypeptide chain .
- IgG1, IG2, IgG3, and IgG4 may be natural types or may contain mutations within a range that can achieve the object of the present invention.
- a polypeptide chain in which EU numbering 435 is His (H) can be used as the first polypeptide chain, and EU numbering 435 is Arg (R) as the second polypeptide chain.
- a polypeptide chain can be used.
- EU polypeptide chain in which EU numbering 435 is His (H) and position 436 is Tyr (Y) can be used as such a first polypeptide chain
- EU polypeptide can be used as a second polypeptide chain
- a polypeptide chain in which numbering 435 is Arg (R) and 436 is Phe (F) can be used. It should be noted that positions other than EU numbering 435 or 436 may be the same as or different from natural IgG.
- the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the resin in protein A column chromatography is obtained by changing the Fc region of the Fc region-containing polypeptide that binds to the resin to the first of the Fc regions.
- the polypeptide chain of the Fc region and the second polypeptide chain of the Fc region can also be achieved by modifying the polypeptide chain of the Fc region and the second polypeptide chain of the Fc region so that the binding force to the resin is different from each other so that they become Fc regions.
- the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the resin in protein A column chromatography is the same as the Fc region of the Fc region-containing polypeptide that binds to the resin.
- the first polypeptide chain of the region binds to the resin, but the second polypeptide chain of the Fc region does not bind to the resin or has a weaker bond than the first polypeptide chain,
- modifications include, for example, specific amino acid residues at specific positions as exemplified above so that the first and second polypeptide chains of the Fc region include CH3 regions as exemplified above.
- the modification to make is, but not limited to.
- the region other than the Fc region in the Fc region-containing polypeptide used in the present invention may be homozygous or heterozygous.
- a homozygote is a homozygote having one or more single or the same antigen-binding activity (that is, when the Fc region-containing polypeptide is an antigen-binding molecule, one or more single or the same antigen An antigen-binding molecule having binding activity, such as an IgG type antibody having two identical antigen-binding sites).
- the heterozygous is preferably a heterozygote having different antigen-binding activity (that is, the Fc region-containing polypeptide is a bispecific antigen-binding molecule such as a bispecific antibody).
- the H chain is heterozygous but the L chain may be common, and the common L chain is preferably on both H chains.
- the bispecific antibody is composed of two common L chains identical to two hetero heavy chains.
- the binding capacity includes a static binding capacity (Static Binding Capacity, SBC) and a dynamic binding capacity (Dynamic Binding Capacity, DBC).
- SBC Static Binding Capacity
- DBC Dynamic Binding Capacity
- the static binding capacity represents the upper limit amount of the resin that can adsorb the polypeptide
- the dynamic binding capacity represents the extent to which the polypeptide can be recovered in a state where a solution containing the polypeptide is flowing through the column.
- the dynamic coupling capacity can be determined by the following method, for example. First, a column packed with resin is loaded into a chromatographic apparatus, and a sample solution containing a polypeptide is passed through the column at a predetermined linear flow rate. Thereafter, the absorbance of the eluate is measured, and the mass of the added polypeptide at the time when the breakthrough (BT) at a predetermined ratio (for example, 5%) of the absorbance of the added sample solution is measured is determined. Can be determined.
- -LC device AKTA AVANT25 manufactured by GE Healthcare ⁇ Software: Unicorn version6.1 manufactured by GE Healthcare ⁇ Protein A resin: GE Healthcare Mab Select SuRe (Cat No.17-5438-05 ) Or Hitrap Mab Select SuRe (Cat No. 11-0034-93) ⁇ Use buffer solution; Equilibration / pre-washing ... 20 mmol / L Na-phosphate, pH 7.5 Elution ... 50 mmol / L Acetic acid Regeneration ... 0.1 mol / L NaOH
- the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at least 5 g / L resin based on 5% BT. Preferably, they are 10 g / L resin or more, 15 g / L resin or more, 20 g / L resin or more, 25 g / L resin or more. In a specific embodiment of the present invention, the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at a contact time of 3.4 minutes based on 5% BT. , At least 5 g / L resin, preferably 10 g / L resin or more, 15 g / L resin or more, 20 g / L resin or more, 25 g / L resin or more.
- the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at least 5% BT. It is 45 g / L resin or more, preferably 50 g / L resin or more, 55 g / L resin or more, 60 g / L resin or more. In a specific embodiment of the present invention, according to the method of the present invention, the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at least 5% BT.
- a contact time of 3.4 minutes it is 50 g / L resin or more, preferably 51 g / L resin or more, 52 g / L resin or more, 53 g / L resin or more, 54 g / L resin or more, 55 g / L More than L resin.
- the Fc region-containing polypeptide may be one in which the Fc region is bound to another protein, bioactive peptide, or the like.
- proteins and physiologically active peptides include, but are not limited to, receptors, adhesion molecules, ligands (cytokines, chemokines, etc.) and enzymes.
- it may be a blood coagulation factor, and examples thereof include FIX, FIXa, and FX.
- the Fc region-containing polypeptide may be an immunoadhesin.
- the Fc region-containing polypeptide may be an antibody.
- the antibody in the present invention is not particularly limited as long as it binds to a desired antigen, and may be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced.
- the monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, monkeys, but also chimeric antibodies, humanized antibodies (reshaped humans). Artificially modified recombinant antibodies such as antibodies) and bispecific antibodies are also included.
- antibody properties may be modified to modify antibody molecule physical properties (specifically, isoelectric point (pI) modification, Fc receptor affinity modification, etc.) for the purpose of improving blood retention and pharmacokinetics.
- antibody molecule physical properties specifically, isoelectric point (pI) modification, Fc receptor affinity modification, etc.
- pI isoelectric point
- Fc receptor affinity modification etc.
- genetically engineered antibodies in which the constant region and the like are artificially modified.
- the immunoglobulin class of the antibody used in the present invention is not particularly limited, and may be any class such as IgG1, IgA, IgD, IgE, IgM such as IgG1, IgG2, IgG3, IgG4, etc. preferable.
- the antibodies used in the present invention include not only whole antibodies, but also antibody fragments such as Fv, Fab, F (ab) 2, etc., and monovalent or linked antibody variable regions with a linker such as a peptide linker. Also included are low-molecular-weight antibodies such as single-chain Fvs (dibodies such as scFv, sc (Fv) 2 and scFv dimers).
- a hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method.
- the hybridoma can be produced, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol.
- the amino acid residue can be modified by modifying one or a plurality of bases in a DNA encoding a polypeptide and expressing the DNA in a host cell as described later.
- a person skilled in the art can easily determine the number, position, and type of bases to be modified according to the type of amino acid residue after modification.
- alteration means any one of substitution, deletion, addition, insertion, or a combination thereof.
- the antibody used in the present invention may further contain additional modifications in addition to the above-described amino acid sequence modifications. Additional alterations can be selected from, for example, any of amino acid substitutions, deletions, and modifications, or combinations thereof. Specifically, any polypeptide containing the following modifications in its amino acid sequence is included in the present invention.
- human lymphocytes are sensitized with a desired antigen or cells expressing the desired antigen in vitro, and the sensitized lymphocytes are fused with human myeloma cells to obtain a desired human antibody having binding activity to the antigen.
- a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with an antigen.
- a technique for obtaining a human antibody by panning using a human antibody library is also known.
- a human antibody can be obtained by expressing a variable region of a human antibody as a single chain antibody (scFv) on the surface of the phage by the phage display method and selecting a phage that binds to the antigen.
- scFv single chain antibody
- Such human antibodies are also included in the antibodies used in the present invention.
- an antibody gene is once isolated and introduced into a suitable host to produce an antibody, a combination of a suitable host and an expression vector can be used.
- eukaryotic cells are used as hosts, animal cells, plant cells, and fungal cells can be used. Examples of animal cells include mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, and Vero. Etc. can be used.
- An antibody can be obtained by introducing a desired antibody gene into these cells by transformation and culturing the transformed cells in vitro.
- the antigen of the antibody used in the present invention is not particularly limited, and any antigen may be used.
- an antigen for example, a ligand (cytokine, chemokine, etc.), a receptor, a cancer antigen, an MHC antigen, a differentiation antigen, an immunoglobulin and an immune complex partially containing an immunoglobulin are preferably exemplified.
- a ligand cytokine, chemokine, etc.
- a receptor a cancer antigen
- an MHC antigen an MHC antigen
- a differentiation antigen an immunoglobulin and an immune complex partially containing an immunoglobulin
- an immunoglobulin and an immune complex partially containing an immunoglobulin are preferably exemplified.
- it may be a blood coagulation factor, and examples thereof include FIX, FIXa, and FX.
- the expression product is recovered when the polypeptide is secreted into the medium. If the polypeptide is produced intracellularly, the cell is first lysed and then the polypeptide is recovered.
- ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography
- Known methods including graphy and lectin chromatography can be used.
- protein A affinity chromatography is preferred.
- a purification method using a column, a separation method using a column, and chromatography may be used synonymously.
- Examples of the column using protein A resin include, but are not limited to, POROS A (manufactured by Applied Biosystems), rProtein A Sepharose F. F. (manufactured by GE), ProSep vA (manufactured by Millipore) and the like.
- a resin (resin) obtained by binding a ligand whose amino acid sequence or the like is modified from intact protein A can be used for protein A affinity chromatography. Even when such a modified protein A resin is used, the amino acid modification of the present invention causes a difference in the binding strength, and the target polypeptide multimer can be separated and purified.
- Examples of the resin to which the modified protein A is bound include mabSelect SuRE (manufactured by GE Healthcare) Hitrap MabSelect Sure (manufactured by GE Healthcare), but are not limited thereto.
- a column filled with protein A resin, a column using protein A resin, a protein A resin column, and a protein A column are synonymous.
- a purification method using a protein A resin and a purification method using a protein A column may be used synonymously.
- One embodiment of the present invention is a method for purifying an Fc region-containing polypeptide using a method for increasing the dynamic binding capacity of an Fc region-containing polypeptide to a protein A resin in protein A column chromatography. More specifically, one embodiment is a method for purifying an antibody, and another embodiment is a method for purifying a bispecific antibody.
- Another embodiment of the present invention is an Fc region-containing polypeptide purified by the purification method. More specifically, one embodiment is an antibody purified by the purification method, and another embodiment is a bispecific antibody purified by the purification method.
- another embodiment of the present invention is a protein A resin to which an Fc region-containing polypeptide is bound, which is 45 g / L resin or more with respect to 5% BT, and a column containing the resin.
- the dynamic binding capacity of the Fc region-containing polypeptide to the resin and the column containing the same is preferably 50 g / L or more, 55 g / L or more, 60 g / L or more, preferably 5% BT. 65 g / L or more.
- a specific embodiment of the present invention has a dynamic binding capacity of 50 g of the Fc region-containing polypeptide for protein A resin in protein A column chromatography at a contact time of 3.4 minutes when viewed on the basis of 5% BT.
- the dynamic binding capacity of the Fc region-containing polypeptide to the resin and the column containing the same is preferably 51 g / L resin or more and 52 g / L resin at a contact time of 3.4 minutes, based on 5% BT. More than 53 g / L resin, more than 54 g / L resin, more than 55 g / L resin.
- One embodiment of the present invention is an Fc region-containing polypeptide having an increased dynamic binding capacity for protein A resin in protein A column chromatography. More specifically, in one embodiment, the Fc region-containing polypeptide is an antibody, and in another embodiment, the Fc region-containing polypeptide is a bispecific antibody. In one embodiment of the present invention, the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at least 5 g / L resin based on 5% BT. Preferably, they are 10 g / L resin or more, 15 g / L resin or more, 20 g / L resin or more, 25 g / L resin or more.
- the increase in the dynamic binding capacity of the Fc region-containing polypeptide to the protein A resin in protein A column chromatography is at a contact time of 3.4 minutes based on 5% BT. , At least 5 g / L resin, preferably 10 g / L resin or more, 15 g / L resin or more, 20 g / L resin or more, 25 g / L resin or more.
- the Fc region-containing polypeptide having an increased dynamic binding capacity to the protein A resin has the first polypeptide chain and the second polypeptide chain contained in the Fc region bound to the protein A resin.
- the second polypeptide chain is a protein that is less protein than the first polypeptide chain.
- Polypeptide chains that bind weakly or not to the A-resin can be used.
- a polypeptide chain containing CH3 of IgG1, IgG2, or IgG4 can be used as such a first polypeptide chain, and a polypeptide chain containing CH3 of IgG3 can be used as a second polypeptide chain .
- IgG1, IG2, IgG3, and IgG4 may be natural types or may contain mutations within a range that can achieve the object of the present invention.
- a polypeptide chain in which EU numbering 435 is His (H) can be used as the first polypeptide chain, and EU numbering 435 is Arg (R) as the second polypeptide chain.
- a polypeptide chain can be used.
- a polypeptide chain in which EU numbering 435 is His (H) and position 436 is Tyr (Y) can be used as such a first polypeptide chain, and EU polypeptide can be used as a second polypeptide chain.
- a polypeptide chain in which numbering 435 is Arg (R) and 436 is Phe (F) can be used. It should be noted that positions other than EU numbering 435 or 436 may be the same as or different from natural IgG.
- One embodiment of the present invention is a method for producing an Fc region-containing polypeptide using a protein A resin, comprising the following steps: (A) a step of preparing a first polypeptide chain and a second polypeptide chain of the Fc region having different binding forces to the resin; (B) The dynamic binding capacity of the Fc region-containing polypeptide in step (a) to protein A resin in protein A column chromatography is the same as the binding force to the resin in protein A column chromatography.
- a first polypeptide chain that binds to the resin is prepared as the first polypeptide chain of the Fc region, and the second polypeptide chain of the Fc region does not bind to the resin.
- it may be a step of preparing a second polypeptide chain having a weak bond (to the resin as compared to the first polypeptide chain).
- the Fc region of the Fc region-containing polypeptide to be purified is bound to the first polypeptide chain of the Fc region, and the second polypeptide chain of the Fc region is bound to the resin.
- the modification is not particularly limited as long as it is a modification for obtaining an Fc region having the above-mentioned characteristics.
- the first polypeptide chain is a polypeptide chain containing CH3 of IgG1, IgG2, or IgG4.
- Such modifications include, for example, that EU numbering 435 of the first polypeptide chain is His (H) and EU numbering 435 of the second polypeptide chain is Arg (R). Modifications can be mentioned.
- EU numbering 435 of the first polypeptide chain is His (H)
- Tyr (Y) is 436
- EU numbering 435 of the second polypeptide chain is Arg (R )
- a modification that causes position 436 to be Phe (F) may be the same as or different from natural IgG.
- the two polypeptide chains may be any polypeptide chain as long as the binding force to the resin is substantially the same, and the degree of homology between the two polypeptide chains is high. May be low.
- an Fc region-containing polypeptide comprising two polypeptide chains that have substantially the same binding force to the resin is an Fc region-containing polypeptide comprising two of the first polypeptide chains, or two An Fc region-containing polypeptide comprising the second polypeptide chain of the book.
- two polypeptide chains having substantially the same binding force to protein A resin are, for example, a polypeptide in which each of the two polypeptide chains contains any of CH1, IgG1, IgG2 or IgG4.
- Peptide chain two polypeptide chains are both polypeptide chains containing IgG3 CH3, EU numbering at position 435 of both polypeptide chains is His (H), or both are Arg (R) Both polypeptide chain and two polypeptide chains are EU numbering 435 position His (H), position 436 is Tyr (Y) polypeptide chain and both polypeptide chains position EU numbering 435 Is a polypeptide chain in which Arg (R) and position 436 are Phe (F). “Substantially the same” does not necessarily have to be exactly the same as long as the object of the present invention is achieved, and includes the “same” case.
- “compare” in the step (b) means that the dynamic binding capacity of the Fc region-containing polypeptide of the step (a) to the protein A resin in protein A column chromatography is In protein A column chromatography, confirm that the Fc region-containing polypeptide containing two polypeptide chains that have substantially the same binding force to the resin is higher than the dynamic binding capacity to the protein A resin. It may be a process. In addition, by comparing or confirming the dynamic binding capacity, it is possible to know the maximum amount of antibody that can be added to the protein A resin column during antibody production, and efficient antibody production becomes possible.
- the sample described in the step (c) includes both the H chain of the polypeptide containing the first polypeptide chain of the Fc region and the H chain of the polypeptide containing the second polypeptide chain of the Fc region.
- a common L chain polypeptide capable of conferring binding ability to may be included, or two different L chain polypeptides may be included.
- the Fc region-containing polypeptide in the method for purifying the Fc region-containing polypeptide, is an antibody, and in another embodiment, the Fc region-containing polypeptide is a bispecific antibody. It is.
- the steps (a) to (d) are not necessarily in this order, and each step may be included a plurality of times.
- One embodiment of the present invention is a method for purifying an Fc region-containing polypeptide using a protein A resin, comprising the steps (a) to (d).
- Example 1 Preparation of antibody gene expression vector and expression of each antibody
- anti-FIXa / FX bispecific antibody H1 chain / H2 chain / L chain having FVIII function alternative activity described in WO2012 / 067176 : SEQ ID NO: 1/2/3)
- BiAb anti-FIXa / FX bispecific antibody having FVIII function alternative activity described in WO2012 / 067176 : SEQ ID NO: 1/2/3)
- the BiAb is composed of three types of four chains, each of two types of H1 and H2 chains and one type of two common L chains.
- the antibody was obtained by the method described in WO2012 / 067176.
- the bispecific antibody was expressed by integrating the antibody gene into an animal cell expression vector and transfecting it into CHO cells.
- Q homo consisting of the two L chains and two H1 chains and J homo consisting of the two L chains and two H2 chains were obtained by the above method.
- This antibody is of the IgG4 type, and His at the 435th EU numbering in the Fc region of the H1 chain is substituted with Arg. This substitution reduces or loses the binding force of the Fc region to protein A resin.
- Example 2 Evaluation Method of Dynamic Binding Capacity (DBC) In general, for DBC verification, the breakthrough curve shows how continuously loaded protein leaks from the column by UV monitoring using a purification device connected with a UV detector. (Hereinafter referred to as BTC). As an example, FIG. 1 shows a BTC chromatogram when BiAb is used. This time, DBC was evaluated by comparing the load of 5% breakthrough point (BT point) for each antibody molecule and its mixture. The following devices were used for DBC calculation.
- BTC Dynamic Binding Capacity
- Example 3 DBC of each antibody molecule alone
- MSS Hitrap MabSelect Sure (hereinafter referred to as MSS) (GE Healthcare)
- MSS Hitrap MabSelect Sure
- Load material IgG concentration of actual load CM using each antibody purification standard, Simulating pH and conductivity. IgG concentration approx. 2 g / L, pH 7.5, Conductivity 1.2 S / m J homo approx. 80%, Q homo approx. 85%, BiAb approx. 95% purity
- Contact time 3.4min (43.75 cm / h) The results are shown in Table 1.
- Example 4 (Verification of pH and contact time of BiAb against DBC) Next, the BiAb alone was used to confirm the DBC when the pH of the loading solution and the contact time with the resin were changed, and the effects of both parameters were verified. The conditions are shown below. ⁇ Column: MabSelect Sure (GE Healthcare), 1.0 x 20 cm ⁇ Load material: Diluted BiAb purified sample and imitating CM (BiAb 95%) 2g / L, pH6.5-8.0 (verification), Conductivity 1.2 S / m ⁇ Contact time: 3-8 min (verification) The results are shown in Table 2.
- Example 5 (DBC in BiAb and Homo mixture)
- HCCF culture supernatant
- both homo are regarded as BiAb competitors. Therefore, in view of actual production, it is meaningful to verify the DBA of BiAb in a situation where both homo are present to some extent in the HCCF. In order to verify this, the test was conducted under the following conditions.
- HPLC apparatus Alliance 2695/2487 made by Waters ⁇ Software: Empower3 made by Waters ⁇ CIEC column: Thermo Scientific ProPac WCX-10, Product No.054993 Column temperature: 30 degC ⁇ Injection amount: 30 ⁇ g / shot ⁇ Use buffer solution; Mobile phase A ... 9.6 mmol / L Tris, 6.0 mmol / L piperazine, 11.0 mmol / L imidazole, pH 6.0 Mobile phase B ...
- J homo has two sequences that bind strongly to the MSS ligand, so it binds to the MSS resin at two locations. In other words, the MSS ligand existing within the range occupied by J homo is not usable.
- BiAb since BiAb has only one sequence that strongly binds to MSS, it has a higher degree of spatial freedom than J homo, and it is considered that high DBC could be realized by utilizing more MSS ligands.
- the low DBC of Q homo is thought to be due to the low binding force of the molecule as a whole. Q homo and J homo are thought to competitively inhibit the binding of BiAb to MSS.
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Abstract
Description
〔1〕プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量を増加させる方法。
〔2〕前記Fc領域の第一のポリペプチド鎖及び第二のポリペプチドとして前記レジンへの結合力が互いに異なる第一のポリペプチド鎖及び第二のポリペプチド鎖を用意する工程を含む、〔1〕に記載の方法。
〔3〕前記Fc領域の第一のポリペプチド鎖として前記レジンに結合する第一のポリペプチド鎖を用意し、前記Fc領域の第二のポリペプチド鎖として前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い第二のポリペプチド鎖を用意する工程を含む、〔1〕または〔2〕に記載の方法。
〔4〕前記Fc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い、Fc領域となるよう改変する工程を含む、〔1〕~〔3〕のいずれかに記載の方法。
〔5〕前記Fc領域の第一のポリペプチド鎖はIgG1、IgG2又はIgG4のCH3を含み、前記Fc領域の第二のポリペプチド鎖はIgG3のCH3を含む、〔1〕~〔4〕のいずれかに記載の方法。
〔6〕前記Fc領域の第一のポリペプチド鎖のEUナンバリング435番目のアミノ酸はHisであり、第二のポリペプチド鎖のEUナンバリング435番目アミノ酸はArgである、〔1〕~〔5〕のいずれかに記載の方法。
〔7〕前記結合容量の増加が5 g/Lレジン以上である、〔1〕~〔6〕のいずれかに記載の方法。
〔8〕前記増加後の動的結合容量が45 g/Lレジン以上である、〔1〕~〔7〕のいずれかに記載の方法。
〔9〕前記Fc領域含有ポリペプチドが抗体である、〔1〕~〔8〕のいずれかに記載の方法。
〔10〕前記抗体が二重特異性抗体である、〔9〕に記載の方法。
〔11〕〔1〕~〔10〕に記載の方法を用いたFc領域含有ポリペプチドの精製方法。
〔12〕〔11〕に記載の方法で精製されたFc領域含有ポリペプチド。
〔13〕プロテインAカラムクロマトグラフィーにおけるFc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量が45 g/Lレジン以上である、Fc領域含有ポリペプチドが結合したプロテインAレジン。
〔14〕プロテインAカラムクロマトグラフィーにおける、プロテインAレジンに対する動的結合容量が増加したFc領域含有ポリペプチド。
〔15〕以下の工程を含む、プロテインAレジンを用いてFc領域含有ポリペプチドを製造する方法:
(a)該レジンへの結合力が互いに異なるFc領域の第一のポリペプチド鎖と第二のポリペプチド鎖とを用意する工程、
(b)プロテインAカラムクロマトグラフィーにおける、工程(a)の該Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量を、プロテインAカラムクロマトグラフィーにおける、該レジンへの結合力が実質的に同じである2本のポリペプチド鎖を含む Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量と比較する工程、
(c)Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドを含む試料を該レジンに接触させる工程;ならびに
(d)該レジンに結合した、Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドのヘテロ体を含有するFc領域含有ポリペプチドを回収する工程。
〔16〕前記工程(a)が、前記Fc領域の第一のポリペプチド鎖として前記レジンに結合する第一のポリペプチド鎖を用意し、前記Fc領域の第二のポリペプチド鎖として前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い第二のポリペプチド鎖を用意する工程である、〔15〕に記載の方法
〔17〕前記工程(a)が、精製対象である前記Fc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い、Fc領域となるよう改変する工程である、〔15〕または〔16〕に記載の方法。
〔18〕前記工程(c)に記載の試料が、前記Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよび前記Fc領域の第二のポリペプチド鎖を含有するポリペプチドの両方に結合能を与え得る共通のL鎖ポリペプチドを含む、〔15〕~〔17〕のいずれかに記載の方法。
〔19〕前記Fc領域含有ポリペプチドが抗体である、〔11〕に記載の精製方法。
〔20〕前記抗体が二重特異性抗体である、〔19〕に記載の精製方法。
〔21〕〔19〕に記載の方法で精製された抗体。
〔22〕〔20〕に記載の方法で精製された二重特異性抗体。
〔23〕〔13〕に記載のレジンを含むカラム。
本発明で使用されるFc領域含有ポリペプチドは抗体のFc領域を含んでいればよく、Fc領域と他のポリペプチドが融合したポリペプチド、例えば抗体などが含まれる。
Fc領域は、IgG1、IgG2、IgG3、Fc領域含有モノクローナル抗体等をペプシン等の蛋白質分解酵素にて部分消化した後に、プロテインAレジンに吸着された画分を再溶出することによって好適に取得され得る。かかる蛋白質分解酵素としてはpH等の酵素の反応条件を適切に設定することにより制限的にFabやF(ab')2を生じるように全長抗体を消化し得るものであれば特段の限定はされず、例えば、ペプシンやパパイン等が例示できる。
Fc領域としてはヒトIgGタイプのFcを挙げることができ、例えばIgG1、IgG2、IgG3、IgG4のアイソタイプのいずれであってもよい。
本発明のFc領域は、第一の前記ポリペプチド鎖及び第二の前記ポリペプチド鎖を含む。
本実施形態において、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドの前記レジンに対する動的結合容量の増加は、前記レジンに結合するFc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖と該Fc領域の第二のポリペプチド鎖の前記レジンへの結合力が互いに異なる、Fc領域となるように改変することによっても達成することができる。
本発明の別の実施形態において、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドの前記レジンに対する動的結合容量の増加は、前記レジンに結合するFc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは前記第一のポリペプチド鎖に比べ結合が弱い、Fc領域となるように改変することによっても達成することができる。
当該改変としては、Fc領域の第一および第二のポリペプチド鎖が上に例示したようなCH3領域を含むように、例えば上に例示したような特定の位置における特定のアミノ酸残基を含むようにする改変が挙げられるが、それらに限定されない。
ホモ体としては、一又は二以上の単一又は同じ抗原結合活性を有するホモ体である(すなわち、当該Fc領域含有ポリペプチドが抗原結合分子である場合、一又は二以上の単一又は同じ抗原結合活性を有する抗原結合分子、例えば同一の二つの抗原結合部位を有するIgG型抗体である)。
ヘテロ体としては、好ましくは、異なる抗原結合活性を有するヘテロ体である(すなわち、当該Fc領域含有ポリペプチドが二重特異性抗原結合分子、例えば二重特異性抗体である)。本発明で使用されるFc領域含有ポリペプチドが二重特異性抗体である場合、H鎖はヘテロ体であるがL鎖は共通であってもよく、当該共通L鎖は好ましくは両H鎖に結合能を与え得る共通L鎖である。尚、二重特異性抗体はIgG型の場合、ヘテロなH鎖二本と同一の共通L鎖二本から構成される。
・LC装置;GEヘルスケア社製AKTA AVANT25
・ソフトウェア;GEヘルスケア社製Unicorn version6.1
・Protein A樹脂;GEヘルスケア社製Mab Select SuRe (Cat No.17-5438-05
)またはHitrap Mab Select SuRe (Cat No. 11-0034-93)
・使用緩衝液;
平衡化/前洗浄...20 mmol/L Na-phosphate, pH 7.5
溶出...50 mmol/L Acetic acid
再生...0.1 mol/L NaOH
上記使用機器、ソフトウェア、及び樹脂を用いて、以下の手順でクロマトグラフィー操作によりDBCを算出する。以下に5%BTを指標とする場合の計算方法を示す。
(1) 負荷画分(IgG濃度P g/Lとする)をいったんカラムを通さずにLC装置に流し、100%漏出(=100%BT)時のOD280 nmの値を確認した。この値をaとする。
(2) aに0.05を掛けた値を、5%BT時のOD280 nm定義する。この値をb5%とする。
(3) 負荷画分を平衡化された一定量の樹脂(r Lとする)に流し続け、OD280 nmの値がb5%となった時の負荷画分の容量をクロマトグラムから読み取る。この値をc5% Lとする。
(4) 計算式(P x c5%)/rで得られる値を、5%BT時の動的結合容量、DBC5%として算出する。
DBC5% = (P x c5%)/r (単位;g/L resin)
DBC10%を求める際は、同じ要領でc10%を求め、算出することができる。
本発明の特定の実施形態において、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量の増加分は、5%BTを基準にみると接触時間3.4分において、少なくとも5 g/Lレジンであり、好ましくは10 g/Lレジン以上、15 g/Lレジン以上、20 g/Lレジン以上、25 g/Lレジン以上である。
本発明の特定の実施形態において、本発明の方法によれば、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量は少なくとも、5%BTを基準に見ると、接触時間3.4分において、50 g/Lレジン以上であり、好ましくは51 g/Lレジン以上、52 g/Lレジン以上、53 g/Lレジン以上、54 g/Lレジン以上、55 g/Lレジン以上である。
本発明の一つの実施形態において、Fc領域含有ポリペプチドはイムノアドヘシンであってもよい。
本発明で使用されるモノクローナル抗体としては、ヒト、マウス、ラット、ハムスター、ウサギ、ヒツジ、ラクダ、サル等の動物由来のモノクローナル抗体だけでなく、キメラ抗体、ヒト化抗体(再構成(reshaped)ヒト抗体ともいう)、二重特異性抗体(bispecific抗体)など人為的に改変した遺伝子組み換え型抗体も含まれる。さらに、血中滞留性や体内動態の改善を目的とした抗体分子の物性の改変(具体的には、等電点(pI)改変、Fc受容体の親和性改変等)を行うために抗体の定常領域等を人為的に改変した遺伝子組み換え型抗体も含まれる。
モノクローナル抗体を産生するハイブリドーマは、基本的には公知技術を使用し、以下のようにして作製できる。すなわち、所望の抗原や所望の抗原を発現する細胞を感作抗原として使用して、これを通常の免疫方法にしたがって免疫し、得られる免疫細胞を通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により、モノクローナルな抗体産生細胞(ハイブリドーマ)をスクリーニングすることによって作製できる。ハイブリドーマの作製は、たとえば、ミルステインらの方法(Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46 )等に準じて行うことができる。
アミノ酸残基の改変は、後述のようにポリペプチドをコードするDNAにおいて1又は複数の塩基を改変し、当該DNAを宿主細胞で発現させることによって行うことが出来る。当業者であれば、改変後のアミノ酸残基の種類に応じて、改変すべき塩基の数や位置、種類を容易に決定することが出来る。
本発明において改変とは、置換、欠損、付加、挿入のいずれか、又はそれらの組み合わせを意味する。
・二重特異性抗体の2種類のH鎖のヘテロ会合率を高めるためのアミノ酸の改変
・第1の抗原結合活性を有するポリペプチドと第2の抗原結合活性を有する又は抗原結合活性を有しないポリペプチド間のジスルフィド結合を安定化させるためのアミノ酸改変
・抗体の血漿中滞留性を改善するためのアミノ酸改変
・酸性条件下における安定性向上のための改変
・ヘテロジェニティー低減のための改変
・脱アミド化反応抑制のための改変
・2種類のポリペプチド間に等電点の差異を導入するための改変
・Fcγレセプターへの結合性を変化させるための改変
抗体遺伝子を一旦単離し、適当な宿主に導入して抗体を作製する場合には、適当な宿主と発現ベクターの組み合わせを使用することができる。真核細胞を宿主として使用する場合、動物細胞、植物細胞、真菌細胞を用いることができ、動物細胞としては、哺乳類細胞、例えば、CHO, COS,ミエローマ、BHK (baby hamster kidney),HeLa,Veroなどを用いることができる。これらの細胞に、目的とする抗体遺伝子を形質転換により導入し、形質転換された細胞をin vitroで培養することにより抗体が得られる。
組換え細胞培養物からポリペプチドを回収し精製するには、硫酸アンモニウム又はエタノール沈殿、酸抽出、アニオン又はカチオン交換クロマトグラフィー、ホスホセルロースクロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティクロマトグラフィー、ヒドロキシルアパタイトクロマトグラフィー及びレクチンクロマトグラフィーを含めた公知の方法を用いることができる。本発明においてはプロテインAアフィニティクロマトグラフィーが好ましい。尚、本明細書において、カラムを用いた精製方法、カラムを用いた分離方法及びクロマトグラフィーは同義に用いられる場合がある。
プロテインAレジンを用いたカラムとしては、POROS A(Applied Biosystems製), rProtein A Sepharose F. F. (GE製)、ProSep vA(Millipore製)等が挙げられるがこれらに限定されない。また、プロテインAアフィニティクロマトグラフィーには、インタクトなプロテイン Aからアミノ酸配列等を改変したリガンドを結合させたレジン(樹脂)を用いることもできる。このような改変プロテイン A樹脂を用いた場合も、本発明のアミノ酸改変により、その結合力に差が生じ、目的のポリペプチド多量体を分離・精製することが可能である。改変プロテイン Aを結合させたレジンとしては、例えば、mabSelect SuRE(GE Healthcare製) Hitrap MabSelect Sure(GE Healthcare製)が挙げられるがこれに限定されない。尚、本明細書において、プロテインAレジンが充填されたカラム、プロテインAレジンを用いたカラム、プロテインAレジンカラム及びプロテインAカラムは同義である。また、プロテインAレジンを用いた精製方法、プロテインAカラムを用いた精製方法も同義に用いられる場合がある。
また、本発明の特定の実施形態は、5%BTを基準に見ると接触時間3.4分において、プロテインAカラムクロマトグラフィーにおけるFc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量が50 g/Lレジン以上である、Fc領域含有ポリペプチドが結合したプロテインAレジン及び当該レジンを含むカラムである。Fc領域含有ポリペプチドの当該レジンおよびそれを含むカラムに対する動的結合容量は5%BTを基準に見ると、接触時間3.4分において、好ましくは51 g/Lレジン以上、52 g/Lレジン以上、53 g/Lレジン以上、54 g/Lレジン以上、55 g/Lレジン以上である。
より具体的には一つの実施形態においてFc領域含有ポリペプチドは抗体であり、また別の一つの実施形態においてFc領域含有ポリペプチドは二重特異性抗体である。
本発明における一つの実施形態において、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量の増加分は、5%BTを基準に見ると、少なくとも5 g/Lレジンであり、好ましくは10 g/Lレジン以上、15 g/Lレジン以上、20 g/Lレジン以上、25 g/Lレジン以上である。
本発明の特定の実施形態において、プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量の増加分は、5%BTを基準にみると接触時間3.4分において、少なくとも5 g/Lレジンであり、好ましくは10 g/Lレジン以上、15 g/Lレジン以上、20 g/Lレジン以上、25 g/Lレジン以上である。
プロテインAカラムクロマトグラフィーにおける、プロテインAレジンに対する動的結合容量が増加したFc領域含有ポリペプチドは、当該Fc領域に含まれる第一のポリペプチド鎖と第二のポリペプチド鎖はプロテインAレジンへの結合力が異なっていることが好ましく、例えば、第一のポリペプチド鎖としてプロテインAレジンに結合するポリペプチド鎖を用いる場合、第二のポリペプチド鎖としては第一のポリペプチド鎖に比べ、プロテインAレジンに弱く結合するか、若しくは結合しないポリペプチド鎖を用いることが出来る。そのような第一のポリペプチド鎖としてはIgG1、IgG2又はIgG4のCH3を含むポリペプチド鎖を用いることができ、第二のポリペプチド鎖としてはIgG3のCH3を含むポリペプチド鎖を用いることができる。この際、IgG1、IG2、IgG3及びIgG4は天然型でもよく、本発明の目的を達成できる範囲で変異を含んでいてもよい。また、そのような第一のポリペプチド鎖としてはEUナンバリング435位がHis(H)であるポリペプチド鎖を用いることができ、第二のポリペプチド鎖としてはEUナンバリング435位がArg(R)であるポリペプチド鎖を用いることができる。また、そのような第一のポリペプチド鎖としてはEUナンバリング435位がHis(H)、436位がTyr(Y)であるポリペプチド鎖を用いることができ、第二のポリペプチド鎖としてはEUナンバリング435位がArg(R)、436位がPhe(F)であるポリペプチド鎖を用いることができる。尚、EUナンバリング435位又は436位以外のポジションは天然型IgGと同じであってもよく、また天然型IgGとは異なっていてもよい。
(a)該レジンへの結合力が互いに異なるFc領域の第一のポリペプチド鎖と第二のポリペプチド鎖とを用意する工程;
(b)プロテインAカラムクロマトグラフィーにおける、工程(a)のFc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量を、プロテインAカラムクロマトグラフィーにおける、該レジンへの結合力が実質的に同じである2本のポリペプチド鎖を含む Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量と比較する工程、
(c)Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドを含む試料を該レジンに接触させる工程;ならびに
(d)該レジンに結合した、Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドのヘテロ体を含有するFc領域含有ポリペプチドを回収する工程。
上記工程(a)は、前記Fc領域の第一のポリペプチド鎖として前記レジンに結合する第一のポリペプチド鎖を用意し、前記Fc領域の第二のポリペプチド鎖として前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い第二のポリペプチド鎖を用意する工程であってよい。また上記工程(a)は精製対象である前記Fc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは(前記第一のポリペプチド鎖に比べ前記レジンへの)結合が弱い、Fc領域となるよう改変する工程であってもよい。当該改変は、上記のような特徴を有するFc領域を得るための改変であれば特に限定されないが、例えば、第一のポリペプチド鎖がIgG1、IgG2又はIgG4のCH3を含むポリペプチド鎖になるようにする改変、および、第二のポリペプチド鎖がIgG3のCH3を含むポリペプチド鎖になるようにする改変が挙げられる。そのような改変としては、例えば、第一のポリペプチド鎖のEUナンバリング435位がHis(H)であり、かつ第二のポリペプチド鎖のEUナンバリング435位がArg(R)であるようにする改変が挙げられる。また、別の改変として、第一のポリペプチド鎖のEUナンバリング435位がHis(H)、436位がTyr(Y)であり、かつ第二のポリペプチド鎖のEUナンバリング435位がArg(R)、436位がPhe(F)であるようにする改変が挙げられる。尚、EUナンバリング435位又は436位以外のポジションは天然型IgGと同じであってもよく、また天然型IgGとは異なっていてもよい。
前記工程(b)において、2本のポリペプチド鎖は該レジンへの結合力が実質的に同じであればいかなるポリペプチド鎖でもよく、2本のポリペプチド鎖同士の相同性の程度は高くても低くてもよい。例えば「該レジンへの結合力が実質的に同じである2本のポリペプチド鎖を含むFc領域含有ポリペプチド」は2本の前記第一のポリペプチド鎖を含む Fc領域含有ポリペプチド、又は二本の前記第二のポリペプチド鎖を含む Fc領域含有ポリペプチドである。また、プロテインAレジンへの結合力が互いに実質的に同じである2本のポリペプチド鎖とは、例えば、2本のポリペプチド鎖がいずれもIgG1、IgG2又はIgG4のCH3のいずれかを含むポリペプチド鎖、2本のポリペプチド鎖がいずれもIgG3のCH3を含むポリペプチド鎖、2本のポリペプチド鎖のEUナンバリング435位がいずれもHis(H)である、またはいずれもArg(R)であるポリペプチド鎖、2本のポリペプチド鎖のいずれもEUナンバリング435位がHis(H)、436位がTyr(Y)であるポリペプチド鎖、2本のポリペプチド鎖のいずれもEUナンバリング435位がArg(R)、436位がPhe(F)であるポリペプチド鎖を挙げることができる。
「実質的に同じ」とは、本発明の目的を達成する範囲内であれば、必ずしも全く同一である必要はなく、また「同じ」場合を含む意である。
本発明の一つの実施形態において、前記工程(b)において「比較する」とは、プロテインAカラムクロマトグラフィーにおける、工程(a)のFc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量が、プロテインAカラムクロマトグラフィーにおける、該レジンへの結合力が実質的に同じである二本のポリペプチド鎖を含む Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量よりも「高いことを確認する」工程であってもよい。
尚、動的結合容量を比較又は確認することで、抗体製造時にプロテインAレジンカラムに添加することができる最大抗体量を知ることができ、効率的な抗体製造が可能となる。
前記工程(c)に記載の試料は、前記Fc領域の第一のポリペプチド鎖を含有するポリペプチドのH鎖および前記Fc領域の第二のポリペプチド鎖を含有するポリペプチドのH鎖の両方に結合能を与え得る共通のL鎖ポリペプチドを含んでもよいし、二つの異なるL鎖ポリペプチドを含んでもよい。
本発明の一つの実施形態において、前記Fc領域含有ポリペプチドを精製する方法において、Fc領域含有ポリペプチドは抗体であり、また別の一つの実施形態においてFc領域含有ポリペプチドは二重特異性抗体である。
尚、前記工程(a)~(d)は必ずしもこの順番である必要はなく、また各工程が複数回含まれていてもよい。
本発明の一つの実施形態は、前記(a)~(d)の工程を含む、プロテインAレジンを用いてFc領域含有ポリペプチドを精製する方法である。
以下、実施例を用いて本発明をさらに具体的に説明する。ただし、本発明の技術的範囲はこれらの実施例に限定されるものではない。
本実施例では、WO2012/067176に記載されたFVIII機能代替活性を有する抗FIXa/FX二重特異性抗体(H1鎖/H2鎖/L鎖:配列番号1/2/3)を用いた(以下、「BiAb」といい、いわゆるヘテロ体である)。なお、当該BiAbは3種類の4本の鎖からなり、二種類のH1鎖及びH2鎖をそれぞれ一本、一種類の共通L鎖二本からなる。当該抗体はWO2012/067176に記載された方法で取得した。抗体遺伝子を、動物細胞発現ベクターに組み込み、それをCHO細胞へトランスフェクションすることで、二重特異性抗体を発現させた。また、前記L鎖二本とH1鎖二本からなるQ homo、前記L鎖二本とH2鎖二本からなるJ homoを上記の方法で取得した。
本抗体はIgG4型であり、H1鎖のFc領域におけるEUナンバリング435番目のHisはArgに置換されている。尚、この置換はFc領域のプロテインAレジンへの結合力を減弱若しくは失わせる。
一般にDBCの検証には、UV検出器を連結した精製装置を用いたUVモニタリングにより、継続負荷されたタンパク質がカラムから漏出する様子をブレークスルーカーブ(以下BTC)としてクロマトグラムに描くことにより行われる。一例として図1にBiAbを用いたときのBTCクロマトグラムを示した。
今回は、それぞれの抗体分子及びその混合液に対して、5%ブレークスルーポイント(BT point)の負荷量を比較することでDBCを評価した。
DBCの計算には以下の装置等を用いた。
・LC装置;GEヘルスケア社製AKTA AVANT25
・ソフトウェア;GEヘルスケア社製Unicorn version6.1
・Protein A樹脂;GEヘルスケア社製Mab Select SuRe (Cat No.17-5438-05
)またはHitrap Mab Select SuRe (Cat No.11-0034-93)
・使用緩衝液;
平衡化/前洗浄...20 mmol/L Na-phosphate, pH 7.5
溶出...50 mmol/L Acetic acid
再生...0.1 mol/L NaOH
DBCの計算方法は以下の通りに行った。
上記使用機器、ソフトウェア、及び樹脂を用いて、以下の手順でクロマトグラフィー操作によりDBCを算出した。
(1) 負荷画分(IgG濃度P g/Lとする)をいったんカラムを通さずにLC装置に流し、100%漏出(=100%BT)時のOD280 nmの値を確認した。この値をaとした。
(2) aに0.05を掛けた値を、5%BT時のOD280 nm定義した。この値をb5%とした。
(3) 負荷画分を平衡化された一定量の樹脂(r Lとする)に流し続け、OD280 nmの値がb5%となった時の負荷画分の容量をクロマトグラムから読み取った。この値をc5% Lとした。
(4) 計算式(P x c5%)/rで得られる値を、5%BT時の動的結合容量、DBC5%として算出した。
DBC5% = (P x c5%)/r (単位;g/L resin)
DBC10%を求める際は、同じ要領でc10%を求め、算出した。
Q homo, J homo及びBiAbそれぞれ単独のDBCを以下の条件で検証した。
・Column:Hitrap MabSelect Sure(以下、MSSという)(GEヘルスケア)、
0.7 x 2.5 cm
・Load material:各抗体精製標準品を用いて、実際の負荷CMのIgG濃度、
pH、導電率を模したもの。
IgG濃度約2 g/L、pH7.5、Conductivity 1.2 S/m
J homo 約80%、Q homo約85%、BiAb約95%純度のもの
を使用
・Contact time:3.4min(43.75 cm/h)
その結果を表1に示した。
次に、BiAb単独で、負荷溶液のpH、及び樹脂に対する接触時間を変化させた時のDBCを確認し、両者のパラメータの影響を検証した。
条件を以下に示した。
・Column:MabSelect Sure(GEヘルスケア)、1.0 x 20 cm
・Load material:BiAb精製標品を希釈調製しCMを模したもの(BiAb 95%)
2g/L、pH6.5-8.0(検証)、Conductivity 1.2 S/m
・Contact time:3-8 min(検証)
その結果を表2に示した。
実際のプロテインA樹脂に負荷する培養上清(以下HCCF)は、BiAbと両homoが混在している。即ち、目的物質であるBiAbを回収するという視点では、両homoはBiAbの競合物質と捉えられる。したがって、実製造を見据えた場合、両homoがHCCF中にある程度存在する状況でのBiAbのDBCを検証することは有意義である。このことを検証するため、以下の条件で試験を行った。
・Column:Hitrap MabSelect Sure(MSS)(GEヘルスケア)、0.7 x 2.5cm
・Load Material:BiAb/Homo精製標品を混合し、CMを模したもの
2 g/L、pH 7.5、Cond 1.2 S/m
Control BiAb(95%) J homo:BiAb:Q homo= 5:95:0
Mimic A J homo:BiAb:Q homo= 10:83:7
Mimic B J homo:BiAb:Q homo= 10:68:22
・Contact time:3.4 min(43.75 cm/h)
・BTCのLoadを分画し、Analytical CIECで各BT PointsのBiAb/Homo比を確認
Analytical CIECの条件は以下の通りに行った。
・HPLC装置;Waters社製Alliance 2695/2487
・ソフトウェア;Waters社製Empower3
・CIECカラム;Thermo scientific社製ProPac WCX-10、Product No.054993
・カラム温度;30 degC
・注入量;30 μg/shot
・使用緩衝液;
移動相A...9.6 mmol/L Tris, 6.0 mmol/L piperazine, 11.0 mmol/L
imidazole, pH 6.0
移動相B...9.6 mmol/L Tris, 6.0 mmol/L piperazine, 11.0 mmol/L
imidazole, 150 mmol/L NaCl, pH 10.1
・グラジエント条件;
時間(min) 流速(mL/min) %A %B
0.0 1.0 100 0
1.0 1.0 100 0
20.0 1.0 0 100
35.0 1.0 0 100
その結果を表3に示した。
・DBC;J homo ≒ Q homo < BiAb
・MSSへの親和力;Q homo < BiAb < J homo
・BiAb DBCにおけるパラメータの影響;
pH...6.5-8.0の範囲において、低いほどDBCは高い傾向があるものの、インパクトは小さい。
接触時間...3-8分の範囲において、長いほどDBCは高くなる傾向があるものの、3分ですらQ homo及びJ homoのDBCを下回ることはない。
MSSへの親和力に関しては、本発明の構成を反映した結果であり、そのことは実施例5におけるリークの順序にも表れている。
一方、DBCについては、MSS樹脂への親和力の違いとリガンドの有効利用性が働いて、このような結果になったと推察している。即ち、J homoはMSSリガンドに強く結合する配列を2か所持っているため、2か所でMSS樹脂に結合している。つまり、J homoが空間的に占める範囲内に存在するMSSリガンドは、利用できなくなる。一方、BiAbはMSSに強く結合する配列を一か所しか持たないため、空間的な自由度がJ homoより高く、より多くのMSSリガンドを有効活用して、高いDBCを実現できたと考えられる。Q homoのDBCが低いのは、単に分子全体としての結合力が低いためと考えられる。また、Q homo及びJ homoはBiAbのMSSへの結合を競合阻害していると考えられる。
Claims (17)
- プロテインAカラムクロマトグラフィーにおける、Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量を増加させる方法。
- 前記Fc領域の第一のポリペプチド鎖及び第二のポリペプチドとして前記レジンへの結合力が互いに異なる第一のポリペプチド鎖及び第二のポリペプチド鎖を用意する工程を含む、請求項1に記載の方法。
- 前記Fc領域の第一のポリペプチド鎖として前記レジンに結合する第一のポリペプチド鎖を用意し、前記Fc領域の第二のポリペプチド鎖として前記レジンに結合しないか若しくは前記第一のポリペプチド鎖に比べ前記レジンへの結合が弱い第二のポリペプチド鎖を用意する工程を含む、請求項1または2に記載の方法。
- 前記Fc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは前記第一のポリペプチド鎖に比べ前記レジンへの結合が弱い、Fc領域となるよう改変する工程を含む、請求項1~3のいずれかに記載の方法。
- 前記Fc領域の第一のポリペプチド鎖はIgG1、IgG2又はIgG4のCH3を含み、前記Fc領域の第二のポリペプチド鎖はIgG3のCH3を含む、請求項1~4のいずれかに記載の方法。
- 前記Fc領域の第一のポリペプチド鎖のEUナンバリング435番目のアミノ酸はHisであり、第二のポリペプチド鎖のEUナンバリング435番目アミノ酸はArgである、請求項1~5のいずれかに記載の方法。
- 前記結合容量の増加が5 g/Lレジン以上である、請求項1~6のいずれかに記載の方法。
- 前記増加後の動的結合容量が45 g/Lレジン以上である、請求項1~7のいずれかに記載の方法。
- 前記Fc領域含有ポリペプチドが抗体である、請求項1~8のいずれかに記載の方法。
- 前記抗体が二重特異性抗体である、請求項9に記載の方法。
- 請求項1~10に記載の方法を用いたFc領域含有ポリペプチドの精製方法。
- 請求項11に記載の方法で精製されたFc領域含有ポリペプチド。
- プロテインAカラムクロマトグラフィーにおけるFc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量が45 g/Lレジン以上である、Fc領域含有ポリペプチドが結合したプロテインAレジン。
- プロテインAカラムクロマトグラフィーにおける、プロテインAレジンに対する動的結合容量が増加したFc領域含有ポリペプチド。
- 以下の工程を含む、プロテインAレジンを用いてFc領域含有ポリペプチドを製造する方法:
(a)該レジンへの結合力が互いに異なるFc領域の第一のポリペプチド鎖と第二のポリペプチド鎖とを用意する工程、
(b)プロテインAカラムクロマトグラフィーにおける、工程(a)の該Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量を、プロテインAカラムクロマトグラフィーにおける、該レジンへの結合力が実質的に同じである2本のポリペプチド鎖を含む Fc領域含有ポリペプチドのプロテインAレジンに対する動的結合容量と比較する工程、
(c)Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドを含む試料を該レジンに接触させる工程;ならびに
(d)該レジンに結合した、Fc領域の第一のポリペプチド鎖を含有するポリペプチドおよびFc領域の第二のポリペプチド鎖を含有するポリペプチドのヘテロ体を含有するFc領域含有ポリペプチドを回収する工程。 - 前記工程(a)が、前記Fc領域の第一のポリペプチド鎖として前記レジンに結合する第一のポリペプチド鎖を用意し、前記Fc領域の第二のポリペプチド鎖として前記レジンに結合しないか若しくは前記第一のポリペプチド鎖に比べ前記レジンへの結合が弱い第二のポリペプチド鎖を用意する工程である、請求項15に記載の方法
- 前記工程(a)が、精製対象である前記Fc領域含有ポリペプチドのFc領域を、該Fc領域の第一のポリペプチド鎖は前記レジンに結合するが、該Fc領域の第二のポリペプチド鎖は前記レジンに結合しないか若しくは前記第一のポリペプチド鎖に比べ前記レジンへの結合が弱い、Fc領域となるよう改変する工程である、請求項15または16に記載の方法。
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CN108368166A (zh) | 2018-08-03 |
AU2016381992A1 (en) | 2018-05-17 |
EP3398965A1 (en) | 2018-11-07 |
KR20180091918A (ko) | 2018-08-16 |
JPWO2017115773A1 (ja) | 2018-10-18 |
IL259247A (en) | 2018-07-31 |
BR112018009312A8 (pt) | 2019-02-26 |
JP2021193136A (ja) | 2021-12-23 |
US20190330268A1 (en) | 2019-10-31 |
IL259247B (en) | 2022-10-01 |
EP3398965A4 (en) | 2019-09-18 |
BR112018009312A2 (ja) | 2018-11-06 |
AU2016381992B2 (en) | 2024-01-04 |
HK1251237A1 (zh) | 2019-01-25 |
CA3004288A1 (en) | 2017-07-06 |
JP7219005B2 (ja) | 2023-02-07 |
SG11201803989WA (en) | 2018-06-28 |
US11649262B2 (en) | 2023-05-16 |
MX2018007781A (es) | 2018-09-05 |
IL259247B2 (en) | 2023-02-01 |
CN108368166B (zh) | 2023-03-28 |
JP7308892B2 (ja) | 2023-07-14 |
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