Methods for removing undesired components during multistage chromatographic processes

Abstract

To provide a method for purifying heterodimeric proteins.SOLUTION: High resolution affinity chromatography combining affinity resolving and affinity capture processes using a single chromatography matrix results in improved resolution between closely related molecular species and significantly enhances overall product yield for large scale commercial production of heterodimeric proteins such as bispecific antibodies. Moreover, tankage and equipment requirements are reduced via the ability to reduce salt concentration, while increasing product purity and concentration, without the need for dilution, ultrafiltration or diafiltration.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for removing unwanted components from a process stream of chromatography for purifying protein products, such as purifying a heterodimeric protein from a complex mixture of proteins by affinity chromatography. Specifically, the method involves isolating a heterodimer (such as a bispecific antibody) from a complex mixture of monomers and homodimers via affinity chromatography (such as protein A chromatography), wherein the purified heterodimer is recovered in an eluate with a low salt concentration and conductivity, facilitating further downstream processing of the heterodimeric protein.

Background Art

[0002] In the purification of protein products, various chromatography steps are often required to remove impurities such as host cell proteins, DNA, and unwanted species of the protein product. In many cases, the components of the chromatography column or buffer form part of the eluate released from each chromatography step, but these components may be undesirable in downstream process steps. For example, although using a high concentration of salt can facilitate separating a certain protein product from unwanted species of impurities or molecules, the salt may not be compatible with downstream process steps such as further chromatography steps or virus inactivation.

[0003] Various bispecific antibody formats have been proposed and are currently under development. Such a format is based on a standard fully human IgG antibody with improved pharmacokinetic profiles and minimal immunogenicity (see U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety). A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process. A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. A single common light chain and two different heavy chains are combined to form a bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*") with reduced or eliminated binding to Protein A. For example, one such Fc* sequence contains an H435R / Y436F substitution in the CH3 domain (EU numbering system; H95R / Y96F according to the IMGT exon numbering system). Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. Co-expression of the two heavy chains and the common light chain yields three products, two of which are heavy chain homodimers and one of which is the desired heterodimeric bispecific product. The Fc* sequence enables the selective purification of the FcFc* bispecific product by having an intermediate binding affinity for Protein A compared to high affinity FcFc heavy chain homodimers or weak binding Fc*Fc* homodimers in a commercially available affinity column. To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process.

[0004] To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process. To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process. To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process. To enable the purification of bispecific heterodimers on a commercial scale, good separation between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is required, along with the requirements for the amount of material to be processed, cost, and the space requirements of the equipment and materials used in the purification process. SUMMARY OF THE INVENTION

[0005] In one or more aspects or embodiments, the present invention comprises a chromatographic eluate. A method for removing a component, the method comprising: (a) performing a first chromatography step where the component is present in a first buffer applied to a chromatography column; (b) recovering an intermediate eluate from the first chromatography step, where the intermediate eluate contains a protein product and the component; (c) re-applying the intermediate eluate to a chromatography column and eluting the protein product with a second buffer containing the component at a concentration lower than the concentration in the intermediate eluate; (d) recovering a chromatography eluate from step (c), where the component is present in the chromatography eluate at a concentration lower than the concentration in the intermediate eluate; and (e) applying the chromatography eluate to a subsequent process step. In some embodiments, the component is not present in the second buffer. wherein the component is present in a first buffer applied to a chromatography column; performing said step; and (b) recovering an intermediate eluate from the first chromatography step, wherein the intermediate eluate contains a protein product and the component; re-applying the intermediate eluate to a chromatography column and eluting the protein product with a second buffer containing the component at a concentration lower than the concentration in the intermediate eluate; and (d) recovering a chromatography eluate from step (c), wherein the component is present in the chromatography eluate at a concentration lower than the concentration in the intermediate eluate; and (e) applying the chromatography eluate to a subsequent process step. In some embodiments, the component is not present in the second buffer. wherein the component is present in the chromatography eluate at a concentration lower than the concentration in the intermediate eluate; and (e) applying the chromatography eluate to a subsequent process step. In some embodiments, the component is not present in the second buffer.

[0006] In some embodiments, the component is a salt. In some cases, the salt concentration in the intermediate eluate is greater than 50 mM. In some cases, the salt concentration in the intermediate eluate is ≧100 mM, ≧250 mM, ≧500 mM, ≧600 mM, ≧700 mM, ≧800 mM, ≧900 mM, or ≧1000 mM. In some cases, the salt concentration in the intermediate eluate is 500 mM ± 50 mM. wherein the salt concentration in the intermediate eluate is greater than 50 mM. In some cases, the salt concentration in the intermediate eluate is ≧100 mM, ≧250 mM, ≧500 mM, ≧600 mM, ≧700 mM, ≧800 mM, ≧900 mM, or ≧1000 mM. In some cases, the salt concentration in the intermediate eluate is 500 mM ± 50 mM. In some cases, the salt concentration in the intermediate eluate is 500 mM ± 50 mM.

[0007] In some embodiments, the first chromatography step is selected from affinity chromatography or ion exchange chromatography. In some embodiments, the subsequent process step is a second chromatography step. In some embodiments, the first chromatography step is selected from affinity chromatography or ion exchange chromatography. In some embodiments, the subsequent process step is a second chromatography step. In that case, the subsequent process steps are selected from affinity chromatography, ion exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography , or virus inactivation.

[0008] In some embodiments, the protein product is an antibody (e.g., a bispecific antibody). .

[0009] In one or more aspects and embodiments of the present invention, a complex of proteins including homodimers and heterodimers is purified from a mixture by adopting an affinity capture and elution process, for example, a heterodimeric protein such as a bispecific antibody. The present invention relates to a method for purifying a heterodimeric protein from a complex mixture of proteins including homodimers and heterodimers, such as a bispecific antibody.

[0010] In one aspect, the present invention provides a method for purifying a heterodimeric protein, the method comprising: (a) introducing a mixture of a heterodimeric protein and impurities into an affinity matrix comprising a protein-binding ligand, wherein the heterodimeric protein comprises a first and a second polypeptide having different affinities for the protein-binding ligand, at least one impurity binds to the protein-binding ligand, and at least one impurity does not bind to the protein-binding ligand; said introduction; (b) washing the affinity matrix with a first washing buffer comprising a salt concentration of more than 200 mM and a first pH of 5 to 9, wherein the impurities are removed; said washing; (c) eluting and recovering the heterodimeric protein from the affinity matrix into a first elution buffer comprising a salt concentration of more than 200 mM and a second pH of 4 to 5, wherein the purified heterodimeric protein is in the first eluate. and (d) eluting the heterodimeric protein from the affinity matrix into a first elution buffer comprising a salt concentration of more than 200 mM and a second pH of 4 to 5, wherein the purified heterodimeric protein is in the first eluate. The washing and (c) eluting and recovering the heterodimeric protein from the affinity matrix into a first elution buffer comprising a salt concentration of more than 200 mM and a second pH of 4 to 5, wherein the purified heterodimeric protein is in the first eluate. and recovering the purified heterodimeric protein from the first eluate. The elution and recovery to obtain a heterodimeric protein, and (d) a second washing buffer containing a third pH of less than 4 Washing of the affinity matrix with a second washing buffer containing impurities removed The washing, (e) equilibration of the affinity matrix to a fourth pH of 5-9, and (f) neutralization of the first eluate to a pH of 5-9, followed by reapplication of the first eluate to the affinity matrix, and (g) washing of the affinity matrix with a third washing buffer containing less than 100 mM salt, and (h) Elution and recovery of the purified heterodimeric protein into a second eluate, wherein the second eluate contains less than 100 mM salt. The various embodiments of the method include In some embodiments, the purified heterodimeric protein is eluted and recovered into the second eluate via a third washing buffer. In some embodiments of the method, the third washing buffer contains less than 50 mM salt. In some embodiments, the impurities include homodimeric species of the first and second polypeptides. In some embodiments, the protein binding ligand is Protein A, and the affinity matrix contains a Protein A ligand immobilized on a substrate. In some cases, the Protein A ligand is a modified Protein A containing a Z domain tetramer, a modified Protein A containing a Y domain tetramer, or a modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. In some embodiments, the purified heterodimeric protein is eluted and recovered into the second eluate via a third washing buffer. In some embodiments of the method, the third washing buffer contains less than 50 mM salt. In some embodiments, the purified heterodimeric protein is eluted and recovered into the second eluate via a third washing buffer. In some embodiments of the method, the third washing buffer contains less than 50 mM salt. In some embodiments of the method, the third washing buffer contains less than 50 mM salt. In some embodiments, the third washing buffer contains less than 50 mM salt.

[0011] In some embodiments, the impurities include homodimeric species of the first and second polypeptides. Including.

[0012] In some embodiments, the protein binding ligand is Protein A, and the affinity matrix contains a Protein A ligand immobilized on a substrate. In some cases, the Protein A ligand is a modified Protein A containing a Z domain tetramer, a modified Protein A containing a Y domain tetramer, or a modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. Including. In some cases, the Protein A ligand is a modified Protein A containing a Z domain tetramer, a modified Protein A containing a Y domain tetramer, or a modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. Including the modified Protein A containing the Z domain tetramer, the modified Protein A containing the Y domain tetramer, or the modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. Including the modified Protein A containing the Z domain tetramer, the modified Protein A containing the Y domain tetramer, or the modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. Including the modified Protein A containing the Z domain tetramer, the modified Protein A containing the Y domain tetramer, or the modified Protein A lacking the D and E domains. In one embodiment, the Protein A ligand contains a Z domain tetramer. Including.

[0013] In some embodiments, the protein binding ligand is Protein G, and the affinity matrix comprises a Protein G ligand immobilized on a substrate.

[0014] In various embodiments of the method, the substrate is a particle, and the affinity matrix comprises a large number of particles with an average diameter of 2 5 μm to 100 μm. In some embodiments, the particles comprise an average diameter of 40 μm to 60 μm. In some embodiments, the particles comprise an average diameter of 45 μm to 5 5 μm. In some embodiments, the particles comprise an average diameter of about 50 μm.

[0015] In various embodiments, the substrate comprises any one or more of agarose, poly(styrene divinylbenzene), poly methacrylate, cellulose, controlled pore glass, ss, and spherical silica.

[0016] In some embodiments of the method, the first elution buffer comprises a salt at a concentration greater than 250 mM. In some cases, the salt concentration is greater than 300 mM or greater than 400 mM. In some embodiments, the salt concentration is about 500 mM.

[0017] In various embodiments of the method, the salt is (i) Cl - , Br - , I - , NO3 - , N(C H3)4 + , NH 4+ , Cs + , Rb + , K + , Na + , H + , Ca 2+ , Mg 2+ , A l 3+ selected from salts or (ii) CaCl2, MgCl2 or NaCl .

[0018] In various embodiments of the method, the second eluate contains less than 50 mM salt. In some embodiments, the second eluate contains less than 10 mM salt.

[0019] In various embodiments of the method, the first polypeptide comprises a CH3 domain capable of binding to a protein binding ligand, and the second polypeptide comprises a CH3 domain incapable of binding to a protein binding ligand. In some embodiments, the first polypeptide comprises a CH3 domain capable of binding to Protein A, and the second polypeptide comprises a CH3 domain incapable of binding to Protein A. In some embodiments, the first polypeptide comprises a CH3 domain capable of binding to Protein G, and the second polypeptide comprises a CH3 domain incapable of binding to Protein G. In various embodiments, the second polypeptide comprises a substitution from HY to RF in its CH3 domain. to a protein binding ligand, and the second polypeptide comprises a CH3 domain incapable of binding tein A. In some embodiments, the first polypeptide comprises a CH3 domain capable of binding to Protein A, and the second polypeptide comprises a CH3 domain incapable of binding to Protein A. In some embodiments, the first polypeptide comprises a CH3 domain capable of binding to Protein G, and the second polypeptide comprises a CH3 domain incapable of binding to Protein G. In various embodiments, the second polypeptide comprises a substitution from HY to RF in its CH3 domain. in its CH3 domain.

[0020] In various embodiments of the method, the first pH is 6 - 8. In some embodiments, the second pH is 4.0 - 4.25. In some cases, the second pH is 4.10 ± 0 .05. In some embodiments, the third pH is 2.8 - 3.5. In some embodiments, the fourth pH is 6 - 8.

[0021] In some embodiments, the method comprises eluting and recovering the purified heterodimeric protein into a low salt (e.g., less than 100 mM, less than 50 mM or also less than 25 mM) eluate, followed by It further includes any chromatographic step or virus inactivation step. In some cases, the conductivity of the purified composition decreases to less than 5.0 mS / cm. In some cases, the conductivity of the purified composition decreases to less than 2.0 mS / cm. In some embodiments, the further chromatographic step or virus inactivation step is carried out under conditions containing less than 100 mM salt. In some cases, the further chrom atographic step includes ion exchange chromatography. In some embodiments the ion exchange chromatography is anion exchange chromatography and is carried out under conditions containing less than 50 mM salt.

[0022] In various embodiments of the method, the heterodimeric protein is a bispecific antigen-binding protein. In some embodiments, the bispecific antigen-binding protein is a bispecific antibody.

[0023] In various embodiments, any of the features or components of the above or the embodiments discussed herein can be combined, and such combinations are included within the scope of the present disclosure. Any specific value mentioned above or herein can be combined with another related value mentioned above or herein, and ranges can be enumerated where those values represent the upper and lower limits of the range, and such ranges are included within the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1

[0025] Before the present invention is described, it is to be understood that the specific methods and experimental conditions described may vary and that the invention is not limited to such methods and conditions. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, since the scope of the invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in relation to a particular recited numerical value means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.). Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described hereinafter. All patents, applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety. In general The present invention significantly increases the overall product yield and the presence of high molecular weight species by reapplying a neutralized eluate containing a purified heterodimeric protein (e.g., a bispecific antibody) to the same affinity matrix used to purify the protein.

[0026]

[0027]

[0028] ​​​​​​​​​​​​​​It can be minimized, while on the other hand, the cost and footprint of a purification system for large-scale industrial production is at least partially based on the discovery that lint can be reduced. Considerations regarding material cost and space for large-scale manufacture and purification of therapeutic bispecific antibodies are important concerns. By reusing chromatography columns in multiple processes, both the cost of column materials (e.g., chromatography media and resins) and the space occupied by the equipment necessary to obtain the desired product are minimized. The purification of bispecific antibodies via affinity chromatography has been reported in the literature before, but these methods generally utilize two separate affinity columns (e.g., MabSelect SuR (trademark) and MabCapture A(trademark)) and also rely on ultrafiltration / diafiltration or salt-tolerant multimodal resins to remove salt from the affinity chromatography process step. The inventors have found that by using a single column for both affinity chromatography processing steps, the overall product yield can be significantly improved (compared to an average of about 77% when using two separate columns, an average of about 92%), and the salt used to reliably separate heterodimers from homodimer impurities can be removed without the need for ultrafiltration or diafiltration, thereby reducing cost and space considerations and providing a product stream for further chromatography or other sophisticated steps without the need for expensive salt-tolerant materials. The purification of bispecific antibodies via affinity chromatography has been reported in the literature before, but these methods generally utilize two separate affinity columns (e.g., MabSelect SuR (trademark) and MabCapture A(trademark)) and also rely on ultrafiltration / diafiltration or salt-tolerant multimodal resins to remove salt from the affinity chromatography process step. The inventors have found that by using a single column for both affinity chromatography processing steps, the overall product yield can be significantly improved (compared to an average of about 77% when using two separate columns, an average of about 92%), and the salt used to reliably separate heterodimers from homodimer impurities can be removed without the need for ultrafiltration or diafiltration, thereby reducing cost and space considerations and providing a product stream for further chromatography or other sophisticated steps without the need for expensive salt-tolerant materials. The inventors have found that by using a single column for both affinity chromatography processing steps, the overall product yield can be significantly improved (compared to an average of about 77% when using two separate columns, an average of about 92%), and the salt used to reliably separate heterodimers from homodimer impurities can be removed without the need for ultrafiltration or diafiltration, thereby reducing cost and space considerations and providing a product stream for further chromatography or other sophisticated steps without the need for expensive salt-tolerant materials. yield can be significantly improved (compared to an average of about 77% when using two separate columns, an average of about 92%), and the salt used to reliably separate heterodimers from homodimer impurities can be removed without the need for ultrafiltration or diafiltration, thereby reducing cost and space considerations and providing a product stream for further chromatography or other sophisticated steps without the need for expensive salt-tolerant materials. chromatography or other sophisticated steps without the need for expensive salt-tolerant materials. chromatography or other sophisticated steps without the need for expensive salt-tolerant materials.

[0029] Definition The term "antibody" refers to four polypeptides interconnected by disulfide bonds The lock, that is, it encloses an immunoglobulin molecule consisting of two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH in this specification) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as LCVR or VL in this specification) and a light chain constant region. The light chain constant region contains one domain (CL1). The VL H region and the VL region can be further divided into hypervariable regions called complementarity determining regions (CDRs) and intervening conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus, namely FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "high affinity" antibody has, for example, at least 10 M, at least 10 M, at least 10 -9 M, or at least -1 10 -11 M, or at least 10 -12 M of binding affinity to its target, as measured by surface plasmon resonance,

[0030] The phrase "bispecific antibody" encompasses antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally contain two different heavy chains, and each heavy chain specifically binds to different molecules (e.g., multiple antigens) or different epitopes on the same molecule (e.g., the same antigen). When a bispecific antibody binds to two different epitopes (a first epitope and a second epitope), it specifically binds to different epitopes on the same molecule (e.g., the same antigen). When a bispecific antibody binds to two different epitopes (a first epitope and If it can selectively bind to the second epitope), the affinity of the first heavy chain for the first epitope is generally one to two digits, or three or four digits lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by bispecific antibodies can be on the same or different targets (e.g., the same or different proteins). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, a nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs followed by (from the N-terminus to the C-terminus) a CH1 domain, a hinge, a CH2 domain and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can bind to one or more epitopes that can associate with each heavy chain and are bound by the heavy chain antigen-binding region, or can enable the binding of one or both heavy chains to one or both epitopes. In various embodiments of the methods discussed herein, heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. can be of isotype IgG. In some cases, heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG1, IgG2, IgG3 or IgG4. In some cases A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. Typical bispecific antibodies have two heavy chains, each having three heavy chain CDRs followed by (from the N-terminus to the C-terminus) a CH1 domain, a hinge, a CH2 domain and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can bind to one or more epitopes that can associate with each heavy chain and are bound by the heavy chain antigen-binding region, or can enable the binding of one or both heavy chains to one or both epitopes. A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. A nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding different heavy chain constant regions, and such a sequence can be expressed in cells that express immunoglobulin light chains. Have.

[0031] In various embodiments of the methods discussed herein, heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. can be of isotype IgG. In some cases, heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG. In some cases, heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG1, IgG2, IgG3 or IgG4. In some cases Heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of the isotype IgG1. In some cases, heterodimeric proteins, bispecific anti- bodies, Fc-containing proteins, etc. are of the isotype IgG4. In various embodiments heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are fully human form.

[0032] The phrase "heavy chain" or "immunoglobulin heavy chain" encompasses immunoglobulin heavy chain constant region sequences from any organism, and, unless otherwise specified, also includes the heavy chain variable domain. The heavy chain variable domain, unless otherwise specified, includes three heavy chain CDRs and four FR regions . Fragments of the heavy chain include the CDRs, the CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain, (from the N-terminus to the C-terminus) a CH1 domain, a hinge, a CH2 domain and a CH3 domain. Functional fragments of the heavy chain are capable of specifically recognizing an antigen (e.g., recognizing an antigen with a KD in the micromolar, nanomolar or picomolar range) and

[0033] being expressed and secreted from cells, and include fragments that contain at least one CDR. The phrase "light chain" encompasses immunoglobulin light chain constant region sequences from any organism, and, unless otherwise specified, also includes human kappa and lambda light chains. The light chain variable (VL Towards the VL comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a light chain constant domain. Examples of light chains that can be used in the present invention include those that do not selectively bind to either the first or second antigen selectively bound by the antigen-binding protein . Suitable light chains can be screened from the most commonly used light chains in existing antibody libraries ( wet libraries or in silico), and the light chains can be identified by screening, and the light chains do not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein . Suitable light chains include those that can bind to one or both epitopes bound by the antigen-binding region of the antigen-binding protein .

[0034] The phrase "variable domain" (unless otherwise specified) refers to the amino acid regions in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus of an immunoglobulin light or heavy chain (optionally modified), and the "variable domain" includes an amino acid sequence that can fold into a canonical domain (VH or VL) having a double β-sheet structure, and the β-sheet is linked by a disulfide bond between the residues of the first β sheet and the residues of the second β-sheet .

[0035] The phrase "complementary determining region" or the term "CDR" usually (i.e., in wild type animals) appears between two framework regions within the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor), and is the immunoglobulin gene of an organism ​​It includes the amino acid sequence encoded by the nucleic acid sequence of the child. CDRs can be encoded, for example, by the sequences of the germline sequences or the sequences before or after rearrangement, and, for example, by naive or mature B or T cells. In some situations (e.g., for CDR3), the CDRs may not be adjacent (e.g., in the nucleic acid sequence before rearrangement), but in the B cell nucleic acid sequence, they may be adjacent as a result of, for example, splicing or ligation of sequences (e.g., V-D-J recombination to form the heavy chain CDR3), and can be encoded by two or more sequences (e.g., germline sequences).

[0036] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, heterodimeric proteins and immunoadhesins, and other binding proteins that contain at least the functional portions of the immunoglobulin CH2 and CH3 regions. The "functional portion" refers to the CH2 and CH3 regions that can bind to Fc receptors (e.g., FcγR, or FcRn, i.e., the neonatal Fc receptor), and / or can be involved in the activation of complement. Deletions, substitutions, insertions or other modifications that prevent binding to Fc receptors and activation of complement are included in the CH 2 and CH3 regions, and such CH2 and CH3 regions do not have functionality.

[0037] Fc-containing proteins can include modifications (e.g., modifications that affect FcγR binding, FcRn binding and the resulting half-life, and / or CDC activity) in one or more effector functions of the binding protein in the immunoglobulin domain. Such modifications include, but are not limited to, those in the immunoglobulin constant region in the EU numbering 238, 239, 248, 249, 250, 252, 254, 255, 256, 258 , 265, 267, 268, 269, 270, 272, 276, 278, 280, 283 , 285, 286, 289, 290, 292, 293, 294, 295, 296, 297 , 298, 301, 303, 305, 307, 308, 309, 311, 312, 315 , 318, 320, 322, 324, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 337, 338, 339, 340, 342, 344 , 356, 358, 359, 360, 361, 362, 373, 375, 376, 378 , 380, 382, 383, 384, 386, 388, 389, 398, 414, 416 , 419, 428, 430, 433, 434, 435, 437, 438 and 439, and modifications thereof, are included.

[0038] Non-limiting examples of such Fc modifications include, for example, at position 250 (e.g., E or Q) , at positions 250 and 428 (e.g., L or F), at position 252 (e.g., L / Y / F / W or T), at position 254 (e.g., S or T) and at position 256 (e.g., S / R / Q / E / D or T) modifications, or at positions 428 and / or 433 (e.g., H / L / R / SI / P / Q or K) and / or at position 434 (e.g., H / F or Y) modifications, or at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F) and at position 434. In one embodiment, the modification is 428L (e.g., M428L) and 434S (e.g., N43 Modifications of 4S), 428L, 259I (e.g., V259I) and 308F (e.g., V30 8F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254 and 256 (e.g., 252Y, 254T and 256E), 25 0Q and 428L (e.g., T250Q and M428L), 307 and / or modifications of 308 (e.g., 308F or 308P) are included.

[0039] The terms "star substitution", "Fc*", and "HC*" refer to any molecule, immunoglobulin heavy chain, Fc fragment, Fc-containing molecule, heterodimeric protein, etc. that contain an array that inactivates protein A binding within the CH3 domain. Specific modifications such as H95R and Y96F that can attenuate or inactivate protein A binding in the CH3 domain are described in US8,586,713. This dipeptide mutation is called "star substitution".

[0040] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Such cells include prokaryotic and eukaryotic (single cell or multiple cells), bacterial cells (e.g., strains such as E. coli, Bacillus spp., Streptomyces sp p.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevi siae, S. pombe, P. pastoris, P. methanolica, etc.) , plant cells, insect cells (SF-9, SF-21, insect cells infected with baculovirus, Trichoplusia ni, etc.), non-human animal cells, human cells, or fusion cells such as hybridomas or quadromas. In some embodiments, the cell ​ The cell is a cell of a human, monkey, ape, hamster, rat or mouse. In some embodiments, the cell is eukaryotic and is the following cells: CHO (e.g., CHOK1 , DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells , Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, M DCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo20 5, HB8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A4 31 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS -0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells , myeloma cells, tumor cells, and cell lines derived from the above cells, and is selected from. In some embodiments, the cell contains one or more viral genes, for example, retinal cells (e.g., PER.C6™ cells) that express viral genes.

[0041] The phrase "mobile phase modifier" includes moieties that reduce or disrupt the effects of non-specific (i.e., non-affinity) ionic and other non-covalent interactions between proteins. Examples of "mobile phase modifiers" include, for example, salts, ionic combinations of Group I and Group II metals with acetate, bicarbonate, carbonate, halogen (e.g., chloride or fluoride), nitrate, phosphate or sulfate. A non-limiting exemplary list of "mobile phase modifiers" includes beryllium, lithium, sodium and potassium salts of acetic acid, sodium bicarbonate and potassium and, for example, combinations with acetate, bicarbonate, carbonate, halogen (e.g., chloride or fluoride), nitrate, phosphate or sulfate. Ums, lithium, sodium, potassium and cesium salts of carbonic acid. Lithium, sodium Lithium, potassium, cesium and magnesium salts, sodium fluoride and potassium fluoride Sodium, potassium and calcium salts of nitric acid, sodium phosphate and phosphate Potassium, as well as calcium sulfate and magnesium sulfate.

[0042] "Mobile phase modifier" also includes chaotropic agents that weaken or otherwise interfere with non-covalent bonds and increase the entropy within the biomolecule system. Non-limiting examples of chaotropic agents include butanol, calcium chloride, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, dodecyl sodium sulfate, thiourea and urea. Chaotropic agents include salts that affect the solubility of proteins. More chaotropic anions include, for example, chloride, nitrate, bromide, chlorate, iodide, perchlorate and thiocyanate. More chaotropic cations include, for example, lithium, magnesium, calcium and guanidinium.

[0043] "Mobile phase modifier" also affects ionic or other non-covalent interactions and provides an extension of the pH unit distance between elutions of homodimeric IgG and heterodimeric IgG (e.g., wild-type human IgG and the same IgG but having one or more modifications of the CH 3 domains described herein) when added to a pH gradient or pH step, or during equilibration of the protein A support in the "mobile phase modifier" and application of the pH step or pH gradient. ​​​​​A portion is included. The appropriate concentration of the "mobile phase modifier" is the same column, pH step, or while using the same pH gradient, the maximum pH distance is obtained at a given pH step or pH gradient It can be determined by increasing the concentration of the "mobile phase modifier" until it is obtained. The "mobile phase Modifier" may also include non-polar modifiers such as propylene glycol and ethylene glycol .

[0044] As used herein, "affinity chromatography" refers to the specific reversible interaction between biomolecules rather than the general properties of biomolecules such as isoelectric point, hydrophobicity, or size. It is a chromatographic method that performs chromatographic separation by utilizing the specific reversible interaction between biomolecules. "Protein A Affinity chromatography" or "Protein A chromatography" refers to A specific affinity chromatography method that utilizes the affinity of the IgG binding domain of Protein A for the Fc portion of the immunoglobulin molecule. This Fc portion contains the human or animal immunoglobulin constant domains CH2 and CH3, or immunoglobulin domains that are substantially similar thereto. Protein A includes natural proteins derived from the cell wall of Staphyloc occus aureus, Protein A produced by recombinant or synthetic methods, and mutants that retain the ability to bind to the Fc region are included . In practice, Protein A chromatography involves the use of Protein A immobilized on a solid phase support. Gagnon, "Protein A Affinity Chr omotography, Purification Tools for Monoc lonal Antibodies", pp. 155 - 198, Validated B In fact, Protein A chromatography involves the use of Protein A immobilized on a solid phase support. in A. Gagnon, "Protein A Affinity Chr omotography, Purification Tools for Monoc lonal Antibodies", pp. 155 - 198, Validated B ​See iosystems, 1996. Protein G and Protein L can also be used in affinity chromatography. The solid phase carrier is a non-aqueous matrix to which Protein A adheres. Such carriers include agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose, and other suitable materials. Such materials are well known in the art. The second protein can be immobilized on the solid phase carrier using any suitable method. Methods for immobilizing proteins on suitable solid phase carriers are well known in the art. For example, see Ostrove, “in Guide to Protein Purification”, Methods in Enzymology, 182: 357-371, 1990. Such solid phase carriers can be obtained easily as commercial products from many vendors such as Vector Laboratories (Burlingame, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences (a division of GE Healthcare, Uppsala, Sweden), Pall (Port Washington, NY), and EMD-Millipore (Billerica, Mass.). Protein A immobilized on a porous glass matrix is commercially available as PROSEP®-A (Millipore). The solid phase can also be an agarose-based matrix. Protein A immobilized on an agarose matrix is MABSELECT™ (A that can be used in affinity chromatography. The solid phase carrier is a non-aqueous matrix to which Protein A adheres such as agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose, and other suitable materials materials are well known in the art. The second protein can be immobilized on the solid phase carrier using any suitable method Methods for immobilizing proteins on suitable solid phase carriers are well known in the art. For example, see Ostrove, “in Guide to Protein Purification”, Methods in Enzymology, 182: 357-371, 1990 such solid phase carriers can be obtained easily as commercial products from many vendors such as Vector Laboratories (Burlingame, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences (a division of GE Healthcare, Uppsala, Sweden), Pall (Port Washington, NY), and EMD-Millipore (Billerica, Mass.) Protein A immobilized on a porous glass matrix is commercially available as PROSEP®-A (Millipore) The solid phase can also be an agarose-based matrix Protein A immobilized on an agarose matrix is MABSELECT™ (A that can be used in affinity chromatography. The solid phase carrier is a non-aqueous matrix to which Protein A adheres such as agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose, and other suitable materials materials are well known in the art. The second protein can be immobilized on the solid phase carrier using any suitable method Methods for immobilizing proteins on suitable solid phase carriers are well known in the art. For example, see Ostrove, “in Guide to Protein Purification”, Methods in Enzymology, 182: 357-371, 1990 such solid phase carriers can be obtained easily as commercial products from many vendors such as Vector Laboratories (Burlingame, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences (a division of GE Healthcare, Uppsala, Sweden), Pall (Port Washington, NY), and EMD-Millipore (Billerica, Mass.) Protein A immobilized on a porous glass matrix is commercially available as PROSEP®-A (Millipore) The solid phase can also be an agarose-based matrix Protein A immobilized on an agarose matrix is MABSELECT™ (A that can be used in affinity chromatography. The solid phase carrier is a non-aqueous matrix to which Protein A adheres agarose matrix is MABSELECT™ (A is commercially available as mersham Biosciences).

[0045] Affinity chromatography also selectively binds to antibodies, antibody fragments, or chimeric fusion proteins containing immunoglobulin domains and / or sequences, and thus includes media that can be used to purify them. Antibodies include IgG, IgA, IgM, IgY, IgD, and IgE types. Antibodies also include single-chain antibodies such as camel antibodies, modified camel antibodies, single-chain antibodies, single-domain antibodies, nanobodies, etc. Antibody fragments include VH, VL, CL, and CH sequences. Examples of fusion proteins containing antibody fragments and antibody sequences include, for example, F(ab’)3, F(ab’)2, Fab, Fc, Fv, dsFv, (scFv)2, scFv, scAb, minibodies (m inibody), diabodies, triabodies (triabody ), tetrabody, Fc fusion proteins, trap molecules, etc. (see Ayyar et al., Methods 56(2012):116-1 29). Such affinity chromatography media can include ligands that selectively bind to antibodies, their fragments, and fusion proteins containing those fragments. Such ligands include antibody-binding proteins, bacterial-derived receptors, antigens, lectins, or anti-antibodies to target molecules. Antibodies that require purification. For example, any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC -lambda, IgG3 / 4, IgA, IgM, etc. against camel-derived For example, camel-derived against any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. Such ligands can include antibody-binding proteins, bacterial-derived receptors, antigens, lectins, or anti-antibodies to target molecules. Antibodies that require purification, for example, any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. against camel-derived can include ligands that selectively bind to antibodies, their fragments, and fusion proteins containing those fragments. Such ligands include antibody-binding proteins, bacterial-derived receptors, antigens, lectins, or anti-antibodies to target molecules. Antibodies that require purification, for example, any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. against camel-derived antigens, lectins, or anti-antibodies to target molecules. Antibodies that require purification, for example, any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. against camel-derived For example, against any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. camel-derived For example, any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. against camel-derived An affinity ligand that can be used as such an affinity ligand (CAPTUR commercially available as an ESELECT chromatography resin, Life Technologie s, Inc., Carlsbad, Calif.).

[0046] A method for removing unwanted components from a purification stream Embodiments of the method of the present invention include a method for removing unwanted components from a process stream for the purification of a protein product. This method aims to remove one or more components (e.g., chemical components or salts) that may be inconvenient for downstream process steps from the eluate generated from a chromatography step. In some embodiments, the method comprises: (a) performing a first chromatography step, wherein the component is present in a first buffer applied to the chromatography column; (b) recovering an intermediate eluate from the first chromatography step, wherein the intermediate eluate contains the protein product and the component; (c) re-applying the intermediate eluate to the chromatography column and eluting the protein product with a second buffer containing the component at a concentration lower than the concentration in the intermediate eluate; (d) recovering the chromatography eluate from step (c), wherein the component is present in the chromatography eluate at a concentration lower than the concentration in the intermediate eluate; and (e) applying the chromatography eluate to a subsequent process step. In some embodiments, the component is not present in the second buffer. This will be discussed in more detail below in connection with a method for purifying a heterodimeric protein.

[0047] Thus, in some embodiments, the component is a salt. In some cases, the salt concentration in the intermediate eluate is greater than 50 mM. In some cases, the salt concentration in the intermediate eluate is ≧ 10 0 mM, ≧ 150 mM, ≧ 200 mM, ≧ 250 mM, ≧ 300 mM, ≧ 350 mM, ≧ 400 mM, ≧ 450 mM, ≧ 500 mM, ≧ 600 mM, ≧ 700 mM, ≧ 800 mM , ≧ 900 mM, or ≧ 1000 mM. In some cases, the salt concentration in the intermediate eluate is 500 mM ± 50 mM. In some embodiments, the salt concentration in the intermediate eluate is , 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM or 600 mM, each value including a variation range of ± 10%.

[0048] In some embodiments, the first chromatography step is selected from affinity chromatography or ion exchange chromatography. In some embodiments , the subsequent process step is a second chromatography step. In some cases, the subsequent process step is selected from affinity chromatography, ion exchange chromatography, mixed-mode chromatography, or virus inactivation. In some cases, the methods discussed herein can be utilized between two ion exchange steps, where the first ion exchange step increases the conductivity of the pool beyond the upper limit at which the subsequent ion exchange step can be properly performed. In some cases, the methods discussed herein can be utilized between two chromatography steps, where in the first step, removal is performed for the proper implementation of the subsequent chromatography step to increase the conductivity of the pool beyond the upper limit at which the subsequent ion exchange step can be properly performed. In some cases, the methods discussed herein can be utilized between two chromatography steps, where in the first step, removal is performed for the proper implementation of the subsequent chromatography step so that the subsequent chromatography step can be properly performed. Chemically necessary components are introduced. In some cases, the methods discussed herein can be utilized prior to virus filtration to reduce conductivity, thereby improving filtration performance and shortening the implementation time. In some cases, the methods discussed herein can be utilized prior to mixed-mode chromatography (MMC) to remove chemically necessary components that interfere with or degrade the MMC medium. For example, it is desirable to remove citrate from the stream that is incompatible with ceramic hydroxyapatite. In some cases, the methods discussed herein can be utilized prior to virus filtration to reduce conductivity, thereby improving filtration performance and shortening the implementation time. In some cases, the methods discussed herein can be utilized prior to virus filtration to reduce conductivity, thereby improving filtration performance and shortening the implementation time. In some cases, the methods discussed herein can be utilized prior to mixed-mode chromatography (MMC) to remove chemically necessary components that interfere with or degrade the MMC medium. In some cases, the methods discussed herein can be utilized prior to mixed-mode chromatography (MMC) to remove chemically necessary components that interfere with or degrade the MMC medium. For example, it is desirable to remove citrate from the stream that is incompatible with ceramic hydroxyapatite. In some cases, the methods discussed herein can be utilized prior to mixed-mode chromatography (MMC) to remove chemically necessary components that interfere with or degrade the MMC medium. For example, it is desirable to remove citrate from the stream that is incompatible with ceramic hydroxyapatite.

[0049] In some embodiments, the protein product is an antibody (e.g., a bispecific antibody).

[0050] Method for purifying a heterodimeric protein Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, (e) equilibrating the affinity matrix to a pH of 5 to 9, (f) neutralizing the eluate containing the heterodimeric protein to a pH of 5 to 9 and re-applying the neutralized eluate to the affinity matrix, and (g) washing the affinity matrix with a third wash buffer containing less than 100 mM salt. Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, (e) equilibrating the affinity matrix to a pH of 5 to 9, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, (e) equilibrating the affinity matrix to a pH of 5 to 9, (f) neutralizing the eluate containing the heterodimeric protein to a pH of 5 to 9 and re-applying the neutralized eluate to the affinity matrix, Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, (e) equilibrating the affinity matrix to a pH of 5 to 9, (f) neutralizing the eluate containing the heterodimeric protein to a pH of 5 to 9 and re-applying the neutralized eluate to the affinity matrix, and (g) washing the affinity matrix with a third wash buffer containing less than 100 mM salt. Embodiments of the method of the present invention include the steps shown in FIG. 1. For example, a method for purifying a heterodimeric protein includes (a) loading a mixture of the heterodimeric protein and impurities onto an affinity matrix, (b) washing the affinity matrix with a first wash buffer having a pH of 5 to 8 and a salt concentration of more than 200 mM, (c) eluting and recovering the heterodimeric protein with a first elution buffer having a pH of 4 to 5 and a salt concentration of more than 200 mM, (d) washing the affinity matrix with a second wash buffer having a pH of less than 4, (e) equilibrating the affinity matrix to a pH of 5 to 9, (f) neutralizing the eluate containing the heterodimeric protein to a pH of 5 to 9 and re-applying the neutralized eluate to the affinity matrix, and (g) washing the affinity matrix with a third wash buffer containing less than 100 mM salt. (h) washing the heterodimeric protein with an eluate containing less than 100 mM salt. and eluting and recovering the affinity material. The method further includes an initial equilibration step in which the eluate is equilibrated to a pH between 5 and 9. In an embodiment, the method further comprises the steps of: washing with a first wash buffer followed by elution of the heterodimeric protein; Prior to elution and recovery, wash the affinity matrix with a wash buffer containing less than 100 mM salt. The method further includes washing the ricks.

[0051] In various embodiments, the affinity of a mixture of heterodimeric proteins and impurities is Loading onto the matrix involves the use of nucleotide sequences encoding heterodimeric proteins. Clarified cell culture fluid from one or more bioreactors containing cells expressing the sequence is then added to the column. For example, the cells can be tagged with a bispecific antibody (e.g., CD3xC D20 bispecific antibody, MET×M, in which two arms bind distinct epitopes on MET ET bispecific antibody, CD3 x BCMA bispecific antibody, CD22 x CD28 bispecific antibody Antibody, PSMA×CD28 bispecific antibody, CD3×PSMA bispecific antibody, CD3× heavy chains that form the MUS16 bispecific antibody, CD3×STEAP2 bispecific antibody, etc. In some cases, a duplex may be expressed, which may contain nucleotides encoding a light chain and a light chain, respectively. Each of the antigen-binding arms of the specific antibody comprises a common light chain. contains heterodimeric species along with other impurities such as homodimeric species, host cell proteins, and DNA. In some cases, heterodimeric proteins are included, such as bispecific antibodies. The protein is produced in eukaryotic cells, for example Chinese hamster ovary (CHO) cells. is possible.

[0052] In some embodiments, the mixture loaded onto the affinity matrix comprises: (i) a first homodimer comprising two copies of a first polypeptide, (ii) a heterodimer comprising the first polypeptide and a second polypeptide, and (iii) a second homodimer comprising two copies of the second polypeptide. The first and second polypeptides have different affinities for the affinity matrix, such that the first homodimer, heterodimer, and second homodimer can be separated based on differences in their binding to the affinity matrix. The differences in binding to the affinity matrix can be manipulated, inter alia, by changing the pH and / or ionic strength of the solution passing through the affinity matrix.

[0053] In various embodiments, the salts discussed herein in the context of buffers or eluents (or others) are salts comprising Cl - , Br - , I - , NO3 - , N(CH3)4 + , NH 4+ , Cs + , Rb + , K + , Na + , H + , Ca 2+ , Mg 2+ or Al 3+ . In some embodiments, the salt comprises Na , H + , Ca + , Mg 2+ or Al 2+ . 3+ ​​ . In some embodiments, the salt is Cl - , Br - , I - , NO3 - or ClO4 - . . In some embodiments, the salt contains Na + , H + , Ca 2+ , Mg 2+ or Al 3 + and a combination with Cl - , Br - , I - , NO3 - or ClO4 - . . In some embodiments, the salt is selected from CaCl2, MgCl2 or NaCl. . In some embodiments, the salt is NaCl. In some embodiments, the salt is CaCl2 . In some embodiments, the salt is MgCl2.

[0054] . After loading the clarified cell culture, the affinity matrix is washed with a wash buffer containing more than 200 mM salt and a pH of 5 - 9 (the first wash buffer in Figure 1). . In some cases, the pH of the wash buffer is 6 - 8. In some cases , the pH of the wash buffer is about 7 - about 7.5. In various embodiments, the pH of the wash buffer is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5. 8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6. 8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7. 8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8. 8, 8.9 or 9.0, or approximately any of them. In some cases In combination, the pH of the wash buffer is 7.2 or about 7.2. In various embodiments, the buffer can be any buffer that can maintain the pH within a desired numerical point or a desired numerical range. In various embodiments, the concentration of the buffer can be from about 5 mM to about 100 mM. In some cases, the concentration of the buffer is from about 5 mM to about 15 mM. In some cases, the concentration of the buffer is from about 5 mM to about 50 mM. In some cases, the buffer concentration is from about 10 mM to about 25 mM. In some cases, the buffer concentration is from about 20 mM to about 40 mM. In some cases, the buffer concentration is from about 30 mM to about 50 mM. In various embodiments, the concentration of the buffer is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or 50 mM, or approximately any of them. In some embodiments, the concentration of the wash buffer is about 10 mM, or about 10 mM. In some embodiments, the concentration of the wash buffer is about 40 mM, or about 40 mM. In some embodiments, the wash buffer is sodium phosphate.

[0055] In some cases, the wash buffer contains a salt at a concentration of about 200 mM to about 800 mM​ Well. In some cases, the wash buffer contains salt at a concentration of from about 250 mM to about 750 mM In some cases, the wash buffer contains salt at a concentration of from about 300 mM to about 700 mM In some cases, the wash buffer contains salt at a concentration of from about 350 mM to about 650 mM In some cases, the wash buffer contains salt at a concentration of from about 400 mM to about 600 mM In some cases, the wash buffer contains salt at a concentration of from about 450 mM to about 550 mM In some cases, the wash buffer contains salt at a concentration of about 200 mM, 210 mM, 220 mM, 225 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM 275 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 325 mM 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 425 mM, 430 mM 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 490 mM 500 mM, 510 mM, 520 mM, 525 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 575 mM, 580 mM, 590 mM, 600 mM 610 mM, 620 mM, 625 mM, 630 mM, 640 mM, 650 mM, 660 mM 670 mM, 675 mM, 680 mM, 690 mM, 700 mM, 710 mM, 720 mM, 725 mM, 730 mM, 740 mM, 750 mM, 760 mM, 770 mM 780 mM, 790 mM or 800 mM, or approximately any concentration thereof In some embodiments, the salt concentration of the wash buffer is about 500 mM or or about 500 mM. In some embodiments, the wash buffer is about 500 m M NaCl. In some cases, washing of this affinity matrix removes host cell proteins, DNA, homodimeric species, etc. that have little or no affinity for the affinity matrix material (such as Protein A). unbound impurities.

[0056] In some embodiments, the method optionally includes a second wash with a wash buffer having little (<25 mM) or no salt at a pH of 5- 9, prior to elution of the heterodimeric protein. In some embodiments, this wash buffer contains about 10 mM- about 50 mM Tris (tris(hydroxymethyl)aminomethane), sodium phosphate , or acetate, or combinations thereof. In various embodiments, this wash buffer has a pH equal to the pH of the first wash buffer discussed above. Following one or more of the above washes, the heterodimeric protein is eluted into an elution buffer (the first elution buffer of FIG. 1) from the affinity matrix

[0057] and recovered in the eluate. The pH of the elution buffer is from about 4 to about 5 and contains a salt at a concentration greater than 200 mM. In some embodiments, the pH of the elution buffer is from about 4.0 to about 4.2. In some embodiments, the pH of the elution buffer is from about 4.4 to about 4.6. In various embodiments, the pH of the elution buffer is 4.0, 4.05, 4.1, 4.15, 4.2, 4.25, 4 .3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4 .8. In various embodiments, .3, 4.35, 4.4, 4.45, 4.5, 4.55, 4.6, 4.65, 4.7, 4 It is 0.75, 4.8, 4.85, 4.9, 4.95 or 5.0, or approximately any of them. In some embodiments, the pH of the elution buffer is 4.1. In some embodiments, the pH of the elution buffer is 4.55. In various embodiments , the buffer can be any buffer that can maintain the pH within a desired numerical point or a desired numerical range. In various embodiments, the concentration of the buffer can be from about 5 mM to about 100 mM. In some cases, the concentration of the buffer is from about 25 mM to about 55 mM. In some cases, the concentration of the buffer is from about 30 mM to about 50 mM. In various embodiments , the concentration of the buffer is about 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or 50 mM or approximately any of them. In some embodiments, the concentration of the elution buffer is about 40 mM or about 40 mM. In some embodiments, the elution buffer is acetic acid. In some embodiments, the elution buffer is acetate.

[0058] In some cases, the elution buffer contains a salt at a concentration of from about 200 mM to about 800 mM. In some cases, the elution buffer contains a salt at a concentration of from about 250 mM to about 750 mM. In some cases, the elution buffer contains a salt at a concentration of from about 300 mM to about 700 mM. In some cases, the elution buffer contains a salt at a concentration of from about 350 mM to about 650 mM. In some cases, the elution buffer contains a salt at a concentration of from about 400 mM to about 600 mM. contains salts. In some cases, the elution buffer contains salts at a concentration of about 450 mM to about 550 mM. In some cases, the elution buffer contains salts at a concentration of 200 mM, 210 mM, 2 20 mM, 225 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM , 275 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 3 80 mM, 390 mM, 400 mM, 410 mM, 420 mM, 425 mM, 430 mM , 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 525 mM, 530 mM, 540 mM, 5 50 mM, 560 mM, 570 mM, 575 mM, 580 mM, 590 mM, 600 mM , 610 mM, 620 mM, 625 mM, 630 mM, 640 mM, 650 mM, 660 mM, 670 mM, 675 mM, 680 mM, 690 mM, 700 mM, 710 mM, 7 20 mM, 725 mM, 730 mM, 740 mM, 750 mM, 760 mM, 770 mM , 780 mM, 790 mM or 800 mM, or contains salts at approximately any of those concentrations. In some embodiments, the salt concentration of the elution buffer is about 500 mM or is about 500 mM. In some embodiments, the elution buffer contains about 5 00 mM of NaCl. In some embodiments, the elution buffer contains about 500 mM of CaCl2. In some embodiments, the elution buffer contains about 500 mM of Mg Cl2.

[0059] After eluting and recovering the heterodimeric protein from the affinity matrix, a Wash the affinity matrix with a wash buffer having a pH of less than about 4 (the second wash buffer of FIG. 1). In some embodiments, the pH of the wash buffer is from about 2.5 to about 3 .5. In some embodiments, the pH of the wash buffer is 3.0 ± 0.2 . In various embodiments, the pH of the wash buffer is 2.0, 2.1, 2.2, 2.3, 2 .4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3 .4, 3.5, 3.6, 3.7, 3.8 or 3.9, or approximately any of them is off. The wash buffer can include any suitable material for providing the above pH or pH range. In some embodiments, the wash buffer is about 20 mM to about 60 mM acetic acid. In some embodiments, the wash buffer is about 30 mM to about 50 mM acetic acid. In some cases, the wash buffer contains about 40 m M acetic acid. In some cases, washing of this affinity matrix removes impurities previously bound to the affinity matrix material (such as protein A) with a higher affinity than the heterodimeric protein, such as the homodimeric species. In some cases, the method of the present invention also includes a buffer having a lower pH ( e.g., 2.45 ± 0.2) and a higher buffer concentration (e.g., 500 mM acetic acid) than the wash buffer described above. The affinity matrix may be further washed with the buffer. After removing further impurities with one or more of the above wash solutions, re-equilibrate the affinity matrix to a pH of 5 - 9. In various embodiments, the affinity matrix

[0060] ​​is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5. 9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6. 9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7. 9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8. 9 or 9.0, or equilibrated to approximately any of those pH values. In some embodiments the affinity matrix is equilibrated to a pH of about 7.2. Equilibration can be carried out using an equilibration buffer having the desired pH. In various embodiments the buffer can be any buffer capable of maintaining the pH within a desired numerical point or range. In various embodiments the concentration of the buffer can be from about 5 mM to about 100 mM In some cases, the concentration of the buffer is from about 10 mM to about 30 mM. In some cases, the concentration of the buffer is from about 30 mM to about 50 mM. In some cases the concentration of the buffer is from about 40 mM to about 60 mM. In various embodiments the concentration of the buffer is 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM 57 mM, 58 mM, 59 mM or 60 mM, or approximately any of them It is. In some embodiments, the concentration of the buffer is about 20 mM or about 20 mM. In some embodiments, the concentration of the buffer is about 40 mM or about 40 mM. In some embodiments, the concentration of the buffer is about 50 mM or about 50 mM. In some embodiments, the buffer is sodium phosphate . In some embodiments, this buffer contains about 10 mM to about 50 mM Tri s, sodium phosphate or acetate, or a combination thereof.

[0061] Following equilibration of the affinity matrix, the neutralized eluate containing the heterodimeric protein (purified from homodimer contaminants and other impurities) is at a pH of 5 - 9 and is reapplied to the same affinity matrix as used in the above purification process steps. In various embodiments, the neutralized eluate is reapplied to the affinity matrix at a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0, or approximately at any of those pH values, and reapplied to the affinity matrix. In some embodiments, the pH is about 7.2 or about 7.2. After reapplying the neutralized eluate to the affinity matrix, the matrix is washed with a wash buffer at neutral pH and a salt concentration of less than 100 mM (the third wash buffer in Figure 1)

[0062] After reapplying the neutralized eluate to the affinity matrix, the matrix is washed with a wash buffer at neutral pH and a salt concentration of less than 100 mM (the third wash buffer in Figure 1) ) Wash with it. Generally, the pH of this wash buffer closely corresponds to the pH of the neutralized eluate reapplied to the affinity matrix as described above. In various embodiments, the salt concentration of this wash buffer is considered to be from about 0 mM to about 100 mM, from about 0 mM to about 75 mM, from about 0 mM to about 50 mM, from about 0 mM to about 25 mM, or from about 0 mM to about 10 mM. In various embodiments, the salt concentration of this wash buffer is 99 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or approximately any concentration less than those, or 0 mM. In various embodiments, the buffer can be any buffer that can maintain the pH within a desired numerical point or a desired numerical range. In various embodiments, the concentration of the buffer can be from about 5 mM to about 100 mM. In some cases, the concentration of the buffer is from about 10 mM to about 30 mM. In some cases, the concentration of the buffer is from about 30 mM to about 50 mM. In some cases, the concentration of the buffer is from about 40 mM to about 60 mM. In various embodiments, the concentration of the buffer is 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. It closely corresponds to the pH of the neutralized eluate reapplied to the affinity matrix. In various embodiments, the salt concentration of this wash buffer is from about 0 mM to about 100 mM, from about 0 mM to about 75 mM, from about 0 mM to about 50 mM, from about 0 mM to about 25 mM, or from about 0 mM to about 10 mM. is considered to be. In various embodiments, the salt concentration of this wash buffer is 99 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or approximately any concentration less than those, or 0 mM. M, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or approximately any concentration less than those, or 0 mM. M, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or approximately any concentration less than those, or 0 mM. M, 5 mM or less, or approximately any concentration less than those, or 0 mM. In various embodiments, the buffer can be any buffer that can maintain the pH within a desired numerical point or a desired numerical range. In various embodiments, the buffer can be any buffer that can maintain the pH within a desired numerical point or a desired numerical range. In various embodiments, the concentration of the buffer can be from about 5 mM to about 100 mM. In some cases, the concentration of the buffer is from about 10 mM to about 30 mM. In some cases, the concentration of the buffer is from about 30 mM to about 50 mM. In some cases, the concentration of the buffer is from about 40 mM to about 60 mM. M, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. M, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. M, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. M, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. M, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. M, 56 mM, 57 mM, 58 mM, 59 mM or 60 mM, or approximately any of them is. In some embodiments, the concentration of the washing buffer is about 20 mM or about 20 mM. In some embodiments, the concentration of the washing buffer is about 40 mM or about 40 mM. In some embodiments, the concentration of the washing buffer is about 50 mM or about 50 mM. In some embodiments, the washing buffer is sodium phosphate. In some embodiments, this washing buffer is about 10 mM to about 50 mM Tris, sodium phosphate or acetate, or a combination thereof is included.

[0063] Following the above washing, from the affinity matrix, the purified heterodimeric protein is eluted and recovered in an eluate containing less than about 100 mM salt. In various embodiments the salt concentration of the eluate is about 99 mM, 95 mM, 90 mM, 85 mM, 80 mM, 7 5 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 3 5 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or approximately any of their concentrations or less, or 0 mM. Generally, the elution of the heterodimeric protein is performed using a buffer with a pH of less than about 4. In some embodiments, the pH of the elution buffer is about 2.5 to about 3.5. In some embodiments, the pH of the elution buffer is 3.0 ± 0.2. In various embodiments, the pH of the elution buffer is about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9, or approximately any of them It is. The elution buffer can contain any suitable material for providing the above pH or pH range. In some embodiments, the elution buffer contains acetic acid at a concentration of about 20 mM to about 6 0 mM. In some embodiments, the elution buffer contains acetic acid at a concentration of about 30 mM to about 50 mM. In some cases, the elution buffer contains about 40 mM acetic acid.

[0064] In various embodiments, loading of the affinity matrix from clarified cell culture or from a neutralized eluate containing a heterodimeric protein can involve addition of the material at up to about 75 g / L of the affinity matrix resin. In various embodiments, the affinity matrix is loaded with the material at 65 g / L, 60 g / L, 55 g / L, or 50 g / L or less.

[0065] In various embodiments of the methods discussed herein, neither diafiltration nor ultrafiltration is used to remove salts from the purified heterodimeric protein eluate. Nor is it necessary to dilute the product of interest to lower the salt concentration for subsequent processing. This reduction in salt concentration without performing this dilution, ultrafiltration or diafiltration reduces the footprint for installation of the equipment and tanks required for purification of these bispecific antibodies.

[0066] In some embodiments, the affinity matrix contains a ligand (such as Protein A) immobilized on a substrate. In some cases, the substrate is beads or particles, and as a result, the affinity matrix is a plurality of particles with the ligand attached. Various In some embodiments, the ligand is Protein A or Protein G. When the ligand is Protein A, Protein A can be a naturally occurring or modified Protein A derived from Staphylococcus, or a modified Protein A. The modified Protein A can be, for example, a Z domain tetramer, a Y domain tetramer, or a modified Protein A lacking the D and E domains. These exemplified modified Protein As cannot bind (or can bind only with very low affinity) to the VH3 domain of immunoglobulins, but can bind to the CH3 domains of IgG1, IgG2, and IgG4. When it is Protein A, Protein A can be a naturally occurring or modified Protein A derived from the genus Staphylococcus, or a modified Protein A. When it is Protein A, Protein A can be a naturally occurring or modified Protein A derived from Staphylococcus, or a modified Protein A. The modified Protein A can be, for example, a Z domain tetramer, a Y domain tetramer, or a modified Protein A lacking the D and E domains. The modified Protein A can be, for example, a Z domain tetramer, a Y domain tetramer, or a modified Protein A lacking the D and E domains. These exemplified modified Protein As cannot bind (or can bind only with very low affinity) to the VH3 domain of immunoglobulins, but can bind to the CH3 domains of IgG1, IgG2 and IgG4. IgG2 and IgG4.

[0067] In some cases, the affinity matrix substrate includes or is made of agarose, poly(styrene divinylbenzene), polymethacrylate, controlled pore glass, spherical silica, cellulose, etc. In embodiments where the substrate is formed as beads or particles, the average diameter of the particles is 25 μm ~100 μm. In some embodiments, the average diameter of the particles is about 40 μm to about 60 μm. In some embodiments, the average diameter of the particles is about 45 μm to about 55 μm. μm. In some embodiments, the average diameter of the particles is about 45 μm to about 55 μm. μm. In some embodiments, the average diameter of the particles is about 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 μm. μm. In some cases, the average diameter of the particles is about 45 μm. In some cases, the average diameter of the particles is about 50 μm. In some embodiments, the particles are 35 μm, 45 μm . is about 50 μm. In some embodiments, the particles are 35 μm, 45 μm , having an average diameter of 60 μm, 75 μm or 85 μm. In some embodiments, the particles contain pores having an average diameter of about 1000 Å, 1050 Å, 1100 Å, 1150 Å or 1200 Å . In some embodiments, the particles contain pores having an average diameter of about 1100 Å .

[0068] In some embodiments of the method, the heterodimeric protein comprises a first polypeptide comprising a CH3 domain ("Fc") capable of binding to protein A, and a second polypeptide comprising a CH3 domain ("Fc*") incapable of binding to protein A, and is a bispecific antibody . In some cases, the second polypeptide contains an H435R / Y436F (H95R / Y96F in the EU numbering system, IMGT exon numbering system) substitution (also known as "Fc*" or "star substitution") in its CH3 domain. Thus, in some embodiments , the first homodimer is a monospecific antibody having two unsubstituted CH3 domains (i.e., FcFc), the second homodimer is a monospecific antibody having two H435R / Y436F-substituted CH3 domains (i.e., Fc*Fc*), and the heterodimeric protein is a bispecific antibody having one unsubstituted CH3 domain and one H435R / Y436F-substituted CH3 domain (i.e., Fc*Fc).

[0069]

Examples

[0069] Example 1: Purification of CD3xCD20 bispecific antibody (BsAb1) The purification of BsAb1 was carried out as a two-step process involving an affinity separation chromatography step and an affinity capture chromatography step. The a In the affinity separation step, the bispecific antibody was captured from the clarified conditioned medium, thereby reducing the volume of the product, increasing the protein concentration, and enhancing the purity of the bispecificity through the removal of Fc*Fc* and FcFc homodimer species. Following the elution of the bispecific antibody from the affinity separation process, the eluate was adjusted to a higher pH (~7.2) in preparation for reloading onto the same affinity column for buffer exchange and virus inactivation purposes. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In any of the chromatography steps, the same matrix containing MabSelect SuRe™ pcc resin (GE Healthcare Life Sciences), which contains the tetramer of the Z domain, was employed. Thereby reducing the volume of the product, increasing the protein concentration, and enhancing the purity of the bispecificity through the removal of Fc*Fc* and FcFc homodimer species. Following the elution of the bispecific antibody from the affinity separation process, the eluate was adjusted to a higher pH (~7.2) in preparation for reloading onto the same affinity column for buffer exchange and virus inactivation purposes. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. ~7.2) for buffer exchange and virus inactivation purposes. Thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In the affinity capture step, the bispecific antibody was captured from the neutral affinity separation protocol, thereby reducing the volume of the product, increasing the protein concentration, and removing salts from the eluate used in the affinity separation step, but without the need for ultrafiltration, diafiltration, or dilution, which would require more equipment and tank requirements. In any of the chromatography steps, the same matrix containing MabSelect SuRe™ pcc resin (GE Healthcare Life Sciences), which contains the tetramer of the Z domain, was employed. In any of the chromatography steps, the same matrix containing MabSelect SuRe™ pcc resin (GE Healthcare Life Sciences), which contains the tetramer of the Z domain, was employed. In any of the chromatography steps, the same matrix containing MabSelect SuRe™ pcc resin (GE Healthcare Life Sciences), which contains the tetramer of the Z domain, was employed.

[0070] Following the affinity capture process, virus inactivation was performed, including holding the bispecific antibody pool at a pH of 3.50 - 3.65 (with 0.25 M glycine HCl) for 30 - 50 minutes. Following the affinity capture process, virus inactivation was performed, including holding the bispecific antibody pool at a pH of 3.50 - 3.65 (with 0.25 M glycine HCl) for 30 - 50 minutes. Following virus inactivation at neutral pH, filtration of the bispecific antibody pool was performed. Following virus inactivation at neutral pH, filtration of the bispecific antibody pool was performed.

[0071] The purification process included the affinity separation and affinity capture steps shown in Tables 1 and 2, respectively. The purification process included the affinity separation and affinity capture steps shown in Tables 1 and 2, respectively.

Table 1

Table 2

[0072] Results: By performing multiple purifications, an average bispecific purity of 97.1% was obtained, and a bispecific yield of 92.8% of BsAb1 was achieved by filtration following virus inactivation. A decrease in conductivity from approximately 1.79 mS / cm to <2.0 mS / cm was made possible. Even when 500 mM CaCl2 at pH 4.45 was used instead of sodium chloride described in Table 1 in the elution step of the affinity separation process, equivalent results were obtained for BsAb1. Results: By performing multiple purifications, an average bispecific purity of 97.1% was obtained, and a bispecific yield of 92.8% of BsAb1 was achieved by filtration following virus inactivation. A decrease in conductivity from approximately 1.79 mS / cm to <2.0 mS / cm was made possible. Even when 500 mM CaCl2 at pH 4.45 was used instead of sodium chloride described in Table 1 in the elution step of the affinity separation process, equivalent results were obtained for BsAb1. 1.79 mS / cm to <2.0 mS / cm was made possible. In the elution step of the affinity separation process, even when 500 mM CaCl2 at pH 4.45 was used instead of sodium chloride described in Table 1, equivalent results were obtained for BsAb1. In the elution step of the affinity separation process, even when 500 mM CaCl2 at pH 4.45 was used instead of sodium chloride described in Table 1, equivalent results were obtained for BsAb1. Even when 500 mM CaCl2 at pH 4.45 was used instead of sodium chloride described in Table 1 in the elution step of the affinity separation process, equivalent results were obtained for BsAb1. equivalent results were obtained for BsAb1.

[0073] Example 2: Purification of MET×MET (different epitope) bispecific antibody (BsAb2) The purification of BsAb2 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 3 and 4, respectively. The purification of BsAb2 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 3 and 4, respectively.

Table 3

Table 4

[0074] Results: By performing multiple purifications, an average bispecific purity of 96.0% ± 0.7% was obtained, and a bispecific yield of 90.9% of BsAb2 was achieved by filtration following virus inactivation. A decrease in conductivity from approximately 70.75 mS / cm to <2.0 mS / cm was made possible. Results: By performing multiple purifications, an average bispecific purity of 96.0% ± 0.7% was obtained, and a bispecific yield of 90.9% of BsAb2 was achieved by filtration following virus inactivation. A decrease in conductivity from approximately 70.75 mS / cm to <2.0 mS / cm was made possible. Results: By performing multiple purifications, an average bispecific purity of 96.0% ± 0.7% was obtained, and a bispecific yield of 90.9% of BsAb2 was achieved by filtration following virus inactivation. A decrease in conductivity from approximately 70.75 mS / cm to <2.0 mS / cm was made possible. .

[0075] Example 3: Purification of BCMA×CD3 bispecific antibody (BsAb3) The purification of BsAb3 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 5 and 6, respectively. [Table 5] [Table 6]

[0076] Result: By performing multiple purifications, an average bispecific purity of 97.4% ± 0.5% was obtained and, by filtration following virus inactivation, the bispecific yield of BsAb3 was 93.4% The conductivity was reduced from approximately 72.80 mS / cm to <2.0 mS / cm .

[0077] Example 4: Purification of BCMA×CD3 bispecific antibody (BsAb4) The purification of BsAb4 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 7 and 8, respectively. [Table 7] [Table 8]

[0078] Result: By performing multiple purifications, an average bispecific purity of 94.6% ± 1.0% was obtained and, by filtration following virus inactivation, the bispecific yield of BsAb4 was 86.0% The conductivity was reduced from approximately 70.78 mS / cm to <2.0 mS / cm

[0079] Example 5: Purification of PSMA×CD28 bispecific antibody (BsAb5) The purification of BsAb5 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 9 and 10, respectively. [Table 9] [Table 10]

[0080] Results: By performing multiple purifications, an average bispecific purity of 96.1% ± 0.9% was obtained and, by filtration following virus inactivation, the bispecific yield of BsAb5 was 92.4% The conductivity was reduced from approximately 75.09 mS / cm to <2.0 mS / cm .

[0081] Example 6: Purification of CD22×CD28 bispecific antibody (BsAb6) The purification of BsAb6 was carried out according to the process described in Example 1, including the affinity separation and affinity capture steps shown in Tables 1 1 and 12, respectively. . [Table 11] [Table 12]

[0082] Results: By performing multiple purifications, an average bispecific purity of 96.5% ± 0.7% was obtained and, by filtration following virus inactivation, the bispecific yield of BsAb6 was 92.8% The conductivity was reduced from approximately 71.78 mS / cm to <2.0 mS / cm .

[0083] Example 7: Quality improvement of the product obtained by affinity desalting compared to UFDF Measurement of high molecular weight (HMW) species present in the composition of BsAb1 (CD3×CD20) was not by using ultrafiltration / diafiltration (UFDF), but by taking advantage of the re - application of the purified heterodimer product to an existing affinity - column, showing further advantages. As shown in Table 13 below, when the bispecific antibody purified on an existing affinity column was re - applied, the HMW species decreased by 0.88%, while when using UFDF for the same purified bispecific antibody, the HMW increased by 0.12%.

Table 13

[0084] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various changes to the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such changes are intended to be included within the scope of the appended claims. ​​​​

Claims

1. A method for removing components from a chromatographic eluate, comprising: (a) performing a first chromatographic step, wherein the component is present in a first buffer applied to a chromatographic column; (b) recovering an intermediate eluate from the first chromatographic step, wherein the intermediate eluate contains a protein product and the component;

2. The method according to claim 1, wherein the component is not present in the second buffer.

3. The method according to claim 1 or 2, wherein the component is a salt.

4. The method according to claim 3, wherein the concentration of the salt in the intermediate eluate is greater than 50 mM.

5. The method according to claim 4, wherein the concentration of the salt in the intermediate eluate is ≧ 100 mM, ≧ 250 mM, or ≧ 500 mM.

6. The method according to any one of claims 1 to 5, wherein the first chromatographic step is selected from affinity chromatography or ion exchange chromatography.

7. The method according to any one of claims 1 to 6, wherein the subsequent process step is a second chromatographic step.

8. The method according to any one of claims 1 to 7, wherein the subsequent process step is selected from affinity chromatography, ion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, or virus inactivation.

9. The method according to any one of claims 1 to 8, wherein the protein product is an antibody.

10. A method for purifying a heterodimeric protein, comprising: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ A first and a second polypeptide having different affinities for a protein-binding ligand comprising, at least one impurity binds to the protein-binding ligand and at least one impurity does not bind to the protein-binding ligand, introduction, and (b) washing of the affinity matrix with a first wash buffer comprising a salt concentration of more than 200 mM and a first pH of 5 to 9, wherein impurities are removed, washing, and (c) elution and recovery of the heterodimeric protein from the affinity matrix into a first elution buffer comprising a salt concentration of more than 200 mM and a second pH of 4 to 5, wherein a purified heterodimeric protein is obtained in the first eluate, elution and recovery, and recovery, and (d) washing of the affinity matrix with a second wash buffer comprising a third pH of less than 4, wherein impurities are removed, washing, and (e) equilibration of the affinity matrix to a fourth pH of 5 to 9, and and (f) neutralization of the first eluate to a pH of 5 to 9 and subsequent reapplication of the first eluate to the affinity matrix, and and and and and and and (g) washing of the affinity matrix with a third wash buffer comprising less than 100 mM salt, and 。 (h) elution and recovery of the purified heterodimeric protein into a second eluate, wherein the second eluate comprises less than 100 mM salt, elution and recovery, comprising a method The method according to claim 10, wherein the third wash buffer comprises less than 50 mM salt.

12. The method according to claim 10 or 11, wherein the impurities comprise homodimeric species of the first and second polypeptides. or 11.

13. The method according to any one of claims 10 to 12, wherein the protein-binding ligand is Protein A and the affinity matrix comprises the Protein A ligand attached to a substrate. item.

14. The method according to claim 13, wherein the Protein A ligand is a modified Protein A comprising a Z domain tetramer, a modified Protein A comprising a Y domain tetramer, or a modified Protein A lacking domains D and E. modified Protein A.

15. The method according to any one of claims 10 to 12, wherein the protein-binding ligand is Protein G and the affinity matrix comprises the Protein G ligand attached to a substrate. The method according to the item.

16. The substrate is a particle, and the affinity matrix comprises a number of the particles having an average diameter of 25 μm to 100 μm. The method according to any one of claims 13 to 15.

17. The method according to claim 16, wherein the particles have an average diameter of 40 μm to 60 μm.

18. The method according to claim 17, wherein the particles have an average diameter of 45 μm to 55 μm.

19. The method according to claim 18, wherein the particles have an average diameter of about 50 μm.

20. The substrate comprises one or more of agarose, poly(styrene divinylbenzene), polymethacrylate, cellulose, controlled pore glass, and spherical silica. The method according to any one of claims 13 to 19.

21. The method according to any one of claims 13 to 20, wherein the particles comprise pores having an average diameter of about 1100 Å.

22. The method according to any one of claims 10 to 21, wherein the first elution buffer contains a salt at a concentration greater than 250 mM. The method according to any one of claims 10 to 21.

23. The method according to claim 22, wherein the salt concentration is greater than 300 mM or greater than 400 mM.

24. The method according to claim 23, wherein the salt concentration is about 500 mM.

25. wherein the salt is (i) Cl － , Br － , I － , NO ３ － , N(CH ３ ) ４ ＋ , NH ４＋ , C s ＋ , Rb ＋ , K ＋ , Na ＋ , H ＋ , Ca ２＋ , Mg ２＋ , Al ３＋ and a salt containing (ii) Na ＋ 、H ＋ 、Ca ２＋ 、Mg ２＋ or Al ３＋ and, Cl － 、Br － 、I － 、NO ３ － or ClO ４ － in combination with, or (iii) CaCl ２ , MgCl ２ if Or NaCl, and is selected from the group consisting of any one of claims 1 to 15.

26. The method according to any one of claims 10 to 25, wherein the second eluate contains a salt at a concentration less than 50 mM. The method according to any one of claims 10 to 25.

27. The method according to claim 26, wherein the second eluate contains a salt at a concentration less than 10 mM.

28. The method according to any one of claims 10 to 27, wherein the purified heterodimeric protein is eluted into and recovered from the second eluate via the third washing buffer. The method according to any one of claims 10 to 27.

29. The first polypeptide comprises a CH3 domain capable of binding to the protein-binding ligand, and the second polypeptide comprises a CH3 domain incapable of binding to the protein-binding ligand. The method according to any one of claims 10 to 28.

30. The first polypeptide comprises a CH3 domain capable of binding to Protein A, and the second polypeptide comprises a CH3 domain incapable of binding to Protein A. The method according to any one of claims 10 to 29.

31. The first polypeptide includes a CH3 domain capable of binding to protein G, and the second polypeptide includes a CH3 domain incapable of binding to protein G, according to any one of claims 10 to 29. **Claim 32** The method according to claim 30, wherein the second polypeptide includes a substitution from HY to RF in its CH3 domain. **Claim 33** The method according to any one of claims 10 to 32, wherein the first pH is 6 to 8. **Claim 34** The method according to any one of claims 10 to 33, wherein the second pH is 4.0 to 4.

25. **Claim 35** The method according to claim 34, wherein the second pH is 4.10 ± 0.

05. **Claim 36** The method according to any one of claims 10 to 35, wherein the third pH is 2.8 to 3.

5. **Claim 37** The method according to any one of claims 10 to 36, wherein the fourth pH is 6 to 8. **Claim 38** The method according to any one of claims 10 to 37, further comprising a further chromatography step or a virus inactivation step following step (h). **Claim 39** The method according to claim 38, wherein the further chromatography step or the virus inactivation step is carried out under conditions of a salt concentration of less than 100 mM. **Claim 40** The method according to claim 39, wherein the further chromatography step comprises ion exchange chromatography. **Claim 41** The method according to claim 40, wherein the ion exchange chromatography is anion exchange chromatography and is carried out under conditions of a salt concentration of less than 50 mM. **Claim 42** The method according to any one of claims 10 to 41, wherein the heterodimeric protein is a bispecific antigen-binding protein. **Claim 43** The method according to claim 42, wherein the bispecific antigen-binding protein is a bispecific antibody. ​ ​ ​ ​ ​ ​ ​ ​ ​

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