Method for improving the resolution of heterodimeric proteins from impurities using affinity chromatography

Abstract

Disclosed is a method for purifying a heterodimeric protein (e.g., a bispecific antibody) from impurities through a series of chromatography cycles. In various embodiments, the pH of the elution buffer is increased with increasing cycles in the series to keep contamination by bound impurities to a minimum and without significant loss of recovery of the heterodimeric protein.

Description

[Technical field]

[0001] The present invention relates to improving the resolution lifetime of affinity chromatography columns for the purification of protein products, such as the purification of heterodimeric proteins from complex mixtures of proteins. Specifically, the method involves performing a series of chromatography cycles utilizing increased elution pH in the increased cycles to minimize contamination by impurities while minimizing loss of recovery of the heterodimeric protein (e.g., bispecific antibody). [Background technology]

[0002] Purification of protein products often requires the use of various chromatographic steps to remove impurities such as host cell proteins, DNA, and undesired species of the protein product.

[0003] Heterodimeric proteins, including multispecific or bispecific antibodies, can be formatted for purification using affinity chromatography. One such format is based on a standard fully human IgG antibody with improved pharmacokinetic profile and minimal immunogenicity (see U.S. Patent No. 8,586,713, incorporated herein in its entirety). A single common light chain and two separate heavy chains combine to form the bispecific antibody. One of the heavy chains contains a substituted Fc sequence (hereinafter "Fc*"), which reduces or eliminates binding of Fc* to Protein A. For example, one such Fc* sequence contains H435R / Y436F (according to the EU numbering system; H95R / Y96F according to the IMGT exon numbering system) substitutions in the CH3 domain. Co-expression of the two heavy chains with the common light chain results in three products, two of which are homodimers for the heavy chain and one of which is the desired heterodimeric bispecific product. The Fc* sequence allows for the selective purification of FcFc* bispecific products on commercially available affinity columns due to their intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimers, or the weakly binding Fc*Fc* homodimers.

[0004] To achieve commercial-scale purification of heterodimeric proteins (e.g., bispecific antibodies), good resolution between FcFc homodimers, Fc*Fc heterodimers, and Fc*Fc* homodimers is necessary. However, repeated use of affinity columns over many cycles generally leads to increased contamination with bound impurities that can lead to batch failure. Although such issues can be addressed by replacement of the column's resin (protein-binding ligands affixed to a substrate), column replacement is expensive (approximately $15,000 per L of resin) and results in delays associated with column unpacking and repacking times. For purification via affinity chromatography, the cost of producing purified heterodimeric proteins is a function of the number of cycles that can be performed on the affinity resin while maintaining acceptable purity and recovery. Thus, methods that improve column function over a greater number of cycles are desirable. Summary of the Invention

[0005] In one aspect of the disclosure, the present invention provides a method for purifying a heterodimeric protein, the method comprising: (a) performing a series of chromatography cycles, wherein each cycle comprises: (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, wherein the heterodimeric protein comprises first and second polypeptides 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; (ii) washing the affinity matrix with a first wash buffer at a first pH between 5 and 9 to remove unbound impurities; (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; and (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities, wherein the second pH is a preliminary series of cycles within the series of chromatography cycles. (b) performing a preparatory pH cycle (a preparatory pH) and the second pH is increased to a subsequent pH higher than the preparatory pH during a subsequent series of chromatography cycles, the preparatory pH and the subsequent pH being within the range of 4.0 to 5.2; and (b) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0006] In some embodiments, the preliminary series of cycles consists of 20 cycles. In some embodiments, the preliminary series of cycles consists of 30 cycles. In some embodiments, the preliminary series of cycles consists of 40 cycles. In some embodiments, the preliminary series of cycles consists of 50 cycles. In some embodiments, the preliminary series of cycles consists of at least 50 cycles, at least 60 cycles, at least 70 cycles, or at least 80 cycles, or more.

[0007] In some embodiments, the subsequent series of cycles consists of at least 20 cycles. In some embodiments, the subsequent series of cycles consists of at least 50, at least 60, at least 70, or at least 80 cycles.

[0008] In some embodiments, the preliminary pH is 4.0-4.2. In some cases, the preliminary pH is 4.1±0.05. In some embodiments, the subsequent pH is 4.3-4.7. In some cases, the subsequent pH is 4.5±0.05.

[0009] In one aspect of the disclosure, the present invention provides a method for purifying a heterodimeric protein, the method comprising: (a) performing a series of chromatography cycles, where each cycle comprises: (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, where the heterodimeric protein comprises first and second polypeptides 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; (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities; and (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH. (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities; (b) measuring a level of bound impurities in the eluate containing the heterodimeric protein after any one or more cycles in the series of chromatography cycles and comparing the measured level of bound impurities with a reference level of bound impurities, where if the measured level of bound impurities exceeds the reference level of bound impurities, increasing the second pH of a subsequent cycle in the series of chromatography cycles, the second pH being within the range of 4.0 to 5.2 during each cycle or subsequent cycles in the series of chromatography cycles; and (c) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0010] In some embodiments, the reference level of bound impurities is between 2% and 10%. In some cases, the reference level of bound impurities is between 3% and 7%. In some cases, the reference level of bound impurities is 5%±0.5%.

[0011] In some embodiments, the level of bound impurities in the eluate is measured after each cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured after every fifth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured after every tenth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured following the twentieth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured following the fortieth or fiftieth cycle in the series of chromatography cycles. In some cases, the eluate is collected over a series of cycles (e.g., five or ten cycles) and the level of bound impurities is measured in the combined eluate pool.

[0012] In some embodiments, the second pH is increased from a range of 4.0-4.2 to a range of 4.3-4.7 if the measured level of the bound impurity exceeds the reference level of the bound impurity, in some cases, the second pH is increased from 4.1±0.05 to 4.5±0.05 if the measured level of the bound impurity exceeds the reference level of the bound impurity.

[0013] In one aspect of the disclosure, the present invention provides a method for purifying a heterodimeric protein, the method comprising: (a) performing a series of chromatography cycles, wherein each cycle comprises: (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, wherein the heterodimeric protein comprises first and second polypeptides 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; (ii) washing the affinity matrix with a first wash buffer at a first pH between 5 and 9 to remove unbound impurities; (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; and (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities, wherein the second pH is a primary pH during a primary series of cycles in the series of chromatography cycles. (b) performing a chromatography cycle in which the second pH is increased to a secondary pH higher than the primary pH during a secondary series of cycles following the primary series of cycles in the series of chromatography cycles, and the secondary pH is increased to a tertiary pH higher than the secondary pH during a tertiary series of cycles following the secondary series of cycles in the series of chromatography cycles, where the primary pH, secondary pH, and tertiary pH are within the range of 4.0 to 5.2; and (b) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0014] In some embodiments, the primary series of cycles includes 5 to 50 cycles. In some cases, the primary series of cycles includes up to 20 cycles. In some cases, the primary series of cycles includes up to 40 cycles.

[0015] In some embodiments, the secondary series of cycles includes 5 to 50 cycles. In some cases, the secondary series of cycles includes 10 to 25 cycles.

[0016] In some embodiments, the tertiary series of cycles includes 5 to 50 cycles. In some cases, the tertiary series of cycles includes 10 to 25 cycles.

[0017] In some embodiments, the primary pH is in the range of 4.0-4.2. In some cases, the primary pH is 4.1±0.05. In some embodiments, the secondary pH is in the range of 4.2-4.4. In some cases, the secondary pH is 4.3±0.05. In some embodiments, the tertiary pH is in the range of 4.4-4.6. In some cases, the tertiary pH is 4.5±0.05.

[0018] In some embodiments, the second pH is increased to a fourth pH higher than the tertiary pH during a fourth series of cycles following a tertiary series of cycles in the series of chromatography cycles, where the fourth pH is in the range of 4.0 to 5.2.

[0019] In some embodiments, the second pH is increased to a fifth pH higher than the fourth pH during a fifth series of cycles following a fourth series of cycles in the series of chromatography cycles, where the fifth pH is in the range of 4.0 to 5.2.

[0020] In some embodiments, the second pH is increased to a sixth pH higher than the fifth pH during a sixth series of cycles following a fifth series of cycles in the series of chromatography cycles, where the sixth pH is in the range of 4.0 to 5.2.

[0021] In some cases, the secondary pH is 0.1-0.9 higher than the primary pH, the tertiary pH is 0.1-0.9 higher than the secondary pH, the fourth pH is 0.1-0.9 higher than the tertiary pH, the fifth pH is 0.1-0.9 higher than the fourth pH, ​​and / or the sixth pH is 0.1-0.9 higher than the fifth pH, ​​and the primary pH is in the range of 4.0-4.2. In some embodiments, the primary pH is 4.1±0.05.

[0022] In some embodiments, the first series of cycles, the second series of cycles, the tertiary series of cycles, the fourth series of cycles, the fifth series of cycles, and / or the sixth series of cycles each include between 5 and 50 cycles within the series of chromatography cycles.

[0023] In various embodiments of any of the methods described above or discussed herein, the impurity comprises a homodimeric species of the first and second polypeptides.

[0024] In various embodiments of any of the methods described above or discussed herein, the protein binding ligand is Protein A and the affinity matrix comprises a Protein A ligand affixed to a substrate.

[0025] In some cases, the Protein A ligand is an engineered Protein A comprising a Z domain tetramer, an engineered Protein A comprising a Y domain tetramer, or an engineered Protein A lacking the D and E domains.

[0026] In some cases, the substrate is a particle and the affinity matrix comprises a multiplicity of particles with an average diameter between 25 μm and 100 μm. In some embodiments, the particles have an average diameter between 40 μm and 60 μm. In some embodiments, the particles have an average diameter between 45 μm and 55 μm. In some embodiments, the particles have an average diameter of 50 μm.

[0027] In some cases, the matrix comprises any one or more of agarose, poly(styrenedivinylbenzene), polymethacrylate, cellulose, controlled pore glass, and spherical silica.

[0028] In some cases, the particles comprise pores having an average diameter of about 1100 Å.

[0029] In various embodiments of any of the methods discussed above or herein, the elution buffer comprises a salt at a concentration of at least 250 mM. In some cases, the salt concentration is greater than 300 mM or greater than 400 mM. In some cases, the salt concentration is about 500 mM.

[0030] In some embodiments, the salt is: (i) Cl - , Br - , I - , NO3 - , N(CH3)4 + , NH4 + , Cs + , Rb + , K + , Na + , H + , Ca 2+ , Mg 2+ , Al 3+ , (ii) Na + , H + , Ca 2+ , Mg 2+ , or Al 3+ And, Cl - , Br - , I - , NO3 - , or ClO4 - or (iii) a salt comprising CaCl2, MgCl2, or NaCl.

[0031] In various embodiments of any of the methods described above or discussed herein, the first polypeptide comprises a CH3 domain capable of binding to a protein-binding ligand, and the second polypeptide comprises a CH3 domain that cannot bind to a protein-binding ligand.

[0032] In various embodiments of any of the methods described above or discussed herein, where the protein-binding ligand is Protein A, the first polypeptide comprises a CH3 domain that is capable of binding to Protein A and the second polypeptide comprises a CH3 domain that is not capable of binding to Protein A. In some cases, the second polypeptide comprises a H435R modification and a Y436F modification (EU numbering) in the CH3 domain.

[0033] In various embodiments of any of the methods described above or discussed herein, the first pH is between 6 and 8.

[0034] In various embodiments of any of the methods described above or discussed herein, the third pH is between 2.8 and 3.5.

[0035] In various embodiments of any of the methods discussed above or herein, the heterodimeric protein is an antibody. In various embodiments of the methods, the heterodimeric protein is a bispecific antigen-binding protein. In some embodiments, the bispecific antigen-binding protein is a bispecific antibody.

[0036] In various embodiments of any of the above or herein discussed methods, at least 85% of the heterodimeric protein is recovered in the eluate in each cycle within a series of chromatography cycles.In some cases, at least 87% of the heterodimeric protein is recovered in the eluate in each cycle within a series of chromatography cycles.In some cases, at least 89% of the heterodimeric protein is recovered in the eluate in each cycle within a series of chromatography cycles.

[0037] In various embodiments of any of the methods described above or discussed herein, the series of chromatography cycles comprises 100 or more cycles.

[0038] In various embodiments of any of the methods discussed above or herein, the affinity matrix may be contacted with a basic solution having a pH of at least 11 after each cycle. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 3 cycles. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 5 cycles. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 7 cycles. In some embodiments, the pH of the basic solution is at least 12. In some embodiments, the basic solution comprises a base at a concentration of 0.1N to 0.5N. In some cases, the base concentration is 0.1N to 0.3N. In some embodiments, the basic solution comprises NaOH.

[0039] In various embodiments of any of the methods discussed above or herein, each cycle may further comprise (v) washing the affinity matrix by contacting it with a basic solution having a pH of at least 11. In some cases, the pH of the basic solution is at least 12. In some embodiments, the basic solution comprises a base at a concentration of 0.1N to 0.5N. In some cases, the concentration is 0.1N to 0.3N. In some embodiments, the basic solution comprises NaOH. In some embodiments, at least 75% of the heterodimeric protein is recovered in the eluate at each cycle in the series of chromatography cycles, and the bound impurities do not exceed 6.5%. In some cases, at least 78% of the heterodimeric protein is recovered in the eluate at each cycle in the series of chromatography cycles. In some cases, at least 80% of the heterodimeric protein is recovered in the eluate at each cycle in the series of chromatography cycles.

[0040] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another associated value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 is an illustration of an exemplary heterodimeric protein (e.g., bispecific Fc*Fc) and associated impurities (homodimeric species) according to embodiments of the present disclosure. The heterodimeric protein comprises one polypeptide that binds to a protein-binding ligand and one polypeptide that does not bind to the protein-binding ligand (Φ). The two impurities shown are homodimers: an unbound impurity composed of two polypeptides that do not bind to the protein-binding ligand (Φ), and a bound impurity composed of two polypeptides that bind to the protein-binding ligand.

[0042] [Diagram 2] 2 is a diagram of an exemplary chromatography cycle according to an embodiment of the present disclosure. As shown, the cycle involves loading a mixture of heterodimeric protein and impurities onto an affinity matrix, washing the affinity matrix to remove unbound impurities, eluting the heterodimeric protein, and washing the affinity matrix to remove bound impurities. Binding of the bound impurities and heterodimeric protein to the protein-binding ligands in the affinity matrix is ​​shown in the first two panels.

[0043] [Diagram 3]FIG. 3 shows the relationship between elution pH and the presence of bound impurities in the eluate, and the corresponding recovery of a heterodimeric protein (eg, a bispecific antibody) in a naive chromatography column.

[0044] [Figure 4] Figures 4A and 4B show the effect of increasing elution pH during naive (7 cycles) and cycled (84 cycles) columns on the presence of bound impurities in the eluate (Figure 4A) and the corresponding recovery of a heterodimeric protein (e.g., a bispecific antibody) (Figure 4B). The "target <5%" shown in Figure 4A for bound impurity levels is exemplary and may vary depending on the heterodimeric protein being purified.

[0045] [Diagram 5] Figures 5A and 5B show design diagnostic parameters, including power analysis (Figure 5A) and percentage of design space plot (Figure 5B). Power analysis determines the probability that a proposed design can distinguish parameter effects of a particular size. As shown in Figure 5A, the power of the main effect term is >0.7. As shown in Figure 5B, the relative prediction variance is less than 0.32 across 50% of the design space.

[0046] [Figure 6-1] 6 shows a grayscale map of the correlations evaluated in Example 3. As shown, all correlations are less than 0.6, indicating a fully orthogonal design. A table of data corresponding to the maps is also included in FIG. [Figure 6-2] 6 shows a grayscale map of the correlations evaluated in Example 3. As shown, all correlations are less than 0.6, indicating a fully orthogonal design. A table of data corresponding to the maps is also included in FIG.

[0047] [Figure 7]Figures 7A and 7B show the model predicted profiler of bispecific yield % (Figure 7A) and bound impurities % (Figure 7B). As the pH of the separation elution buffer is decreased and the hydroxide contact time is increased, both the bispecific yield and bound impurity levels increase. Furthermore, as the column load is increased, the bispecific yield increases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are used to describe only specific embodiments, and are not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value can vary by 1% or less from the referenced value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0050] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0051] General Provisions Purification of bispecific antibodies via affinity chromatography has been described above. Briefly, a heterodimeric protein of interest, comprising one polypeptide that binds to a protein-binding ligand and one that does not bind to the protein-binding ligand, is introduced onto an affinity matrix (containing the protein-binding ligand) together with homodimeric impurities. As will be understood, the two homodimeric species comprise either a pair of polypeptides that bind to the protein-binding ligand of the affinity matrix, or a pair of polypeptides that do not bind to the protein-binding ligand of the affinity matrix (see, for example, FIG. 1).

[0052] Repeated use of affinity chromatography columns over many cycles results in loss of functional protein-ligand density, resulting in increased impurity levels. Without intending to be bound by any particular theory, the loss of functional protein-ligand density is believed to be due to accumulation of impurities, changes in structural ligands, and / or physical loss of ligands. In some cases, the loss of functional protein-ligand density is believed to be related, at least in part, to exposure to hydroxide ions (e.g., from NaOH) used to periodically clean the chromatography columns. Regardless of the cause, the loss of functional protein-ligand density results in reduced avidity for the affinity matrix for both bound impurities and the heterodimeric protein of interest. Lower avidity, coupled with a reduced likelihood of rebinding events, may result in early removal of the heterodimeric protein of interest with unbound impurities, or early removal of bound impurities with the heterodimeric protein of interest during elution.

[0053] The present invention is predicted, at least in part, based on the unexpected discovery that increasing the elution pH in a circulated affinity chromatography column can improve the separation of heterodimeric proteins (e.g., bispecific antibodies) from bound impurities while maintaining high recovery of the heterodimeric protein. The cost of materials for large-scale commercial production and purification of therapeutic proteins (e.g., bispecific antibodies) is a significant concern, and the cost of replacing a 100L column can easily exceed $1.5 million, slowing down the purification process. Therefore, extending the usable life of an affinity chromatography column over a greater number of cycles can achieve significant cost benefits.

[0054] As discussed in more detail below, methods for extending the resolution of affinity columns and maintaining recovery of heterodimeric proteins include: (i) performing a preliminary series of cycles at a preliminary elution pH and subsequent series of cycles at subsequent (and higher) pHs; (ii) performing a series of cycles in which impurity levels in the eluate are measured after each cycle or periodically, and increasing the elution pH in subsequent cycles to maintain minimum impurity levels throughout the series of cycles; and (iii) performing a series of cycles in which the elution pH is increased stepwise over a set of multiple cycles (e.g., the elution pH is increased by 0.1 to 1 points after every 5, 10, 15, 20, or 25 cycles). Additionally, in some embodiments, the resolution of affinity columns can also be extended by reducing the frequency of washing of the chromatography column (e.g., by contacting the column with a basic solution having a pH of at least 11) or by reducing the concentration of base in the solution used to wash the chromatography column.

[0055] definition The term "antibody", as used herein, includes immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH), 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 herein as LCVR or VL), and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3. The term "high affinity" antibodies refers to antibodies having a binding affinity for their target of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -9 M, at least 10 -1 M, at least 10 -11 M, or at least 10 -12 This refers to an antibody that is M.

[0056] The phrase "bispecific antibodies" includes antibodies that can selectively bind two or more epitopes. Bispecific antibodies typically contain two different heavy chains, each of which specifically binds to a different epitope - either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two orders of magnitude lower, or three or four orders of magnitude lower, than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by bispecific antibodies can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences 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 (N-terminus to C-terminus) by 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 bind each heavy chain, or can bind each heavy chain and bind one or more of the epitopes bound by the heavy chain antigen binding region, or can bind each heavy chain and bind one or both of the heavy chains to one or both of the epitopes.

[0057] In various embodiments of the methods discussed herein, the heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. may be of isotype IgG. In some cases, the heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG1, IgG2, IgG3, or IgG4. In some cases, the heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG1. In some cases, the heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are of isotype IgG4. In various embodiments, the heterodimeric proteins, bispecific antibodies, Fc-containing proteins, etc. are fully human.

[0058] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain constant region sequences from any organism, including heavy chain variable domains unless otherwise specified. Unless otherwise specified, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain followed (N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of heavy chains include those that can specifically recognize an antigen (e.g., recognize an antigen with a KD in the micromolar, nanomolar, or picomolar range), can be expressed and secreted from a cell, and contain at least one CDR.

[0059] The term "light chain" includes immunoglobulin light chain constant region sequences from any organism, including human kappa light chains and human lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. In general, a full-length light chain includes a VL domain including, from the amino terminus to the carboxyl terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used in the present invention include, for example, light chains that do not selectively bind to either the first antigen or the second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), which 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 capable of binding one or both of the epitopes bound by the antigen-binding region of the antigen-binding protein.

[0060] The term "variable domain" includes the amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions from N-terminus to C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" includes an amino acid sequence that can fold into a canonical domain (VH or VL) with a bipartite beta-sheet structure, the beta-sheets being linked by disulfide bonds between residues of the first and second beta-sheets.

[0061] The phrase "complementarity determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene of an organism, which is normally (i.e., in wild-type animals) found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, for example, by naive B cells or mature B cells or T cells. In some circumstances (e.g., for CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences), which are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B cell nucleic acid sequence, for example, as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form heavy chain CDR3).

[0062] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, heterodimeric proteins, immunoadhesins, and other binding proteins that contain at least a functional portion of an immunoglobulin CH2 and CH3 region. "Functional portion" refers to the CH2 and CH3 regions that can bind an Fc receptor (e.g., FcγR or FcRn, i.e., fetal Fc receptor) and / or participate in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions, or other modifications that render them incapable of binding any Fc receptor and incapable of complement activation, the CH2 and CH3 regions are not functional.

[0063] Fc-containing proteins can contain modifications in immunoglobulin domains, including those that affect one or more effector functions of the binding protein (e.g., modifications that affect FcγR binding, FcRn binding and therefore half-life and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of immunoglobulin constant regions: 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.

[0064] For example, and not intended to be limiting, the binding protein is an Fc-containing protein that exhibits improved serum half-life (compared to the same Fc-containing protein without the described modifications) and has modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In another example, the modifications can include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); 307 and / or 308 modifications (e.g., 308F or 308P).

[0065] The terms "star substitution", "Fc*" and "HC*" include any molecule, immunoglobulin heavy chain, Fc fragment, Fc-containing molecule, heterodimeric protein, etc., that contains a sequence in the CH3 domain that prevents binding to Protein A. Specific modifications such as H95R and Y96F that can reduce or prevent Protein A binding in the CH3 domain are discussed in U.S. Patent No. 8,586,713. This dipeptide mutation is designated as a "star substitution".

[0066] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., strains of E. coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Streptomyces niger, etc.), non-human animal cells, human cells, or cell fusions, such as, for example, hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), 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 aforementioned cells.

[0067] The phrase "mobile phase modifiers" includes moieties that reduce or disrupt the effects of non-specific (i.e., non-affinity) ionic and other non-covalent interactions between proteins. "Mobile phase modifiers" include, for example, salts and 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 acetate; sodium and potassium bicarbonate; lithium, sodium, potassium, and cesium carbonate; lithium, sodium, potassium, and cesium chloride; sodium and potassium fluoride; sodium, potassium, and calcium nitrate; sodium and potassium phosphate, and calcium and magnesium sulfate.

[0068] "Mobile phase modifiers" also include chaotropic agents that weaken or otherwise interfere with non-covalent binding forces and increase entropy within a biomolecular system. Non-limiting examples of chaotropic agents include butanol, calcium chloride, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, and urea. Chaotropic agents include salts that affect protein solubility. 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.

[0069] "Mobile phase modifiers" include moieties that affect ionic or other non-covalent interactions that result in an increase in pH unit distance during elution of homodimeric and heterodimeric IgG (e.g., wild-type human IgG and the same IgG but with one or more modifications of its CH3 domain as described herein) upon addition of a pH gradient or step or equilibration of the Protein A support in the "mobile phase modifier" and application of a pH step or gradient. The appropriate concentration of the "mobile phase modifier" can be determined by its concentration using the same column, pH step, or gradient, by increasing the concentration of the "mobile phase modifier" until a maximum pH distance is reached at a given pH step or pH gradient. "Mobile phase modifiers" can also include non-polar modifiers, including, for example, propylene glycol, ethylene glycol, and the like.

[0070] As used herein, "affinity chromatography" is a chromatographic method that exploits specific, reversible interactions between biomolecules to effect chromatographic separation, rather than general properties of biomolecules such as isoelectric point, hydrophobicity, or size. "Protein A affinity chromatography" or "Protein A chromatography" refers to a specific affinity chromatography method that exploits the affinity of the IgG binding domain of Protein A for the Fc portion of immunoglobulin molecules. The Fc portion includes human or animal immunoglobulin constant domains CH2 and CH3, or immunoglobulin domains substantially similar thereto. Protein A encompasses native proteins from the cell wall of Staphylococcus aureus, Protein A produced by recombinant or synthetic methods, and variants that retain the ability to bind to the Fc region. In practice, Protein A chromatography involves the use of Protein A immobilized on a solid support. See Gagnon, Protein A Affinity Chromatography, Purification Tools for Monoclonal Antibodies, pp.155-198, Validated Biosystems, 1996. Protein G and Protein L can also be used for affinity chromatography. The solid support is a non-aqueous matrix to which Protein A is attached. Such supports include agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose, and any other suitable material. Such materials are known in the art. The second protein can be attached to the solid support using any suitable method. Methods for attaching proteins to suitable solid supports are well known in the art. See, for example, Ostrove, in Guide to Protein Purification, Methods in Enzymology, 182:357-371, 1990.Such solid supports, with or without immobilized Protein A, are readily available from a number of commercial sources, such as Vector Laboratories (Burlingame, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Bio-Rad (Hercules, CA), Cytiva (Marlborough, MA), Pall (Port Washington, NY), and EMD-Millipore (Billerica, MA). Protein A immobilized on a small pore glass matrix is ​​commercially available as PROSEP®-A (Millipore). The solid phase may also be an agarose-based matrix. Protein A immobilized on an agarose matrix is ​​commercially available as MABSELECT™ (Cytiva).

[0071] Affinity chromatography also includes media that can be used to selectively bind and therefore purify antibodies, antibody fragments, or chimeric fusion proteins containing immunoglobulin domains and / or sequences. Antibodies include IgG, IgA, IgM, IgY, IgD, and IgE types. Antibodies also include single chain antibodies such as camelid antibodies, engineered camelid antibodies, single chain antibodies, single domain antibodies, nanobodies, etc. Antibody fragments include VH, VL, CL, CH sequences. Antibody fragments and fusion proteins containing antibody sequences include, for example, F(ab')3, F(ab')2, Fab, Fc, Fv, dsFv, (scFv)2, scFv, scAb, minibodies, diabodies, triabodies, tetrabodies, Fc fusion proteins, trap molecules, etc. (see Ayyar et al., Methods 56(2012):116-129). Such affinity chromatography media may contain ligands that selectively bind to antibodies, fragments thereof, and fusion proteins containing fragments thereof. Such ligands include antibody-binding proteins, bacterial receptors, antigens, lectins, or anti-antibodies directed against the target molecule (i.e., the molecule requiring purification). For example, camelid-derived affinity ligands directed against any one or more of IgG-CH1, IgG-Fc, IgG-CH3, IgG1, LC-kappa, LC-lambda, IgG3 / 4, IgA, IgM, etc. may be used as affinity ligands (commercially available as CAPTURESELECT chromatography resins, Life Technologies, Carlsbad, Calif.).

[0072] Methods for purifying heterodimeric proteins Embodiments of methods for purifying heterodimeric proteins (via extending the resolution of an affinity column and maintaining recovery of the heterodimeric protein) according to the present disclosure include: (i) performing a preliminary series of cycles at a preliminary elution pH and performing a subsequent series of cycles at a subsequent (and higher) pH; (ii) performing a series of cycles in which impurity levels in the eluate are measured after each cycle or periodically, and the elution pH is increased in subsequent cycles to maintain minimum impurity levels throughout the series of cycles; and (iii) performing a series of cycles in which the elution pH is increased stepwise over a set of multiple cycles (e.g., the elution pH is increased by 0.5, 0.75, or 1 point after every 10, 15, 20, or 25 cycles).

[0073] Each of the methods for purifying a heterodimeric protein includes: (a) performing a series of chromatography cycles, where each cycle includes (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, where the heterodimeric protein includes first and second polypeptides having different affinities for the protein-binding ligand, and where at least one impurity binds to the protein-binding ligand and at least one impurity does not bind to the protein-binding ligand, (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities, (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH, and (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities, where the second pH is between 4.0 and 5.2.

[0074] In various embodiments, loading the mixture of heterodimeric protein and impurities onto the affinity matrix includes loading clarified cell culture from one or more bioreactors containing cells expressing a nucleotide sequence encoding the heterodimeric protein. For example, the cells may express nucleotides encoding each of the heavy and light chains that form a bispecific antibody. In some cases, each of the antigen-binding arms of the bispecific antibody comprises a common light chain. The clarified cell culture contains the heterodimeric protein (e.g., bispecific antibody) along with impurities such as homodimeric species, host cell proteins, and DNA. In some cases, the heterodimeric protein may be produced in eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells.

[0075] In some embodiments, the mixture loaded onto the affinity matrix comprises a mixture of proteins including (i) a first homodimer comprising two copies of a first polypeptide, (ii) a heterodimer comprising a first polypeptide and a second polypeptide, and (iii) a second homodimer comprising two copies of a 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 differential binding to the affinity matrix. Differential binding to the affinity matrix can be manipulated, inter alia, by altering the pH and / or ionic strength of the solution passed through the affinity matrix.

[0076] After loading of the clarified cell culture, the affinity matrix is ​​washed with a wash buffer (first wash buffer) having a pH of 5 to 9. In some cases, the pH of the wash buffer is 6 to 8. In some cases, the pH of the wash buffer is about 7 to 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 is 9.0 or about 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. In some embodiments, the pH of the wash buffer is 7.2 or about 7.2. In various embodiments, the buffer may be any buffer capable of maintaining the pH at a desired point or within a desired range. In various embodiments, the buffer concentration may be about 5 mM to about 100 mM. In some cases, the buffer concentration is about 5 mM to about 15 mM. In some cases, the buffer concentration is about 5 mM to about 50 mM. In some cases, the buffer concentration is about 10 mM to about 25 mM. In some cases, the buffer concentration is about 20 mM to about 40 mM. In some cases, the buffer concentration is about 30 mM to about 50 mM.In various embodiments, the buffer concentration 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. mM or is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mM. In some embodiments, the wash buffer concentration is 10 mM or about 10 mM. In some embodiments, the wash buffer concentration is 40 mM or about 40 mM. In some embodiments, the wash buffer is sodium phosphate. The wash buffer (first wash buffer) may also contain a salt as discussed below.

[0077] In some cases, the wash buffer comprises a salt concentration of about 200 mM to about 800 mM. In some cases, the wash buffer comprises a salt concentration of about 250 mM to about 750 mM. In some cases, the wash buffer comprises a salt concentration of about 300 mM to about 700 mM. In some cases, the wash buffer comprises a salt concentration of about 350 mM to about 650 mM. In some cases, the wash buffer comprises a salt concentration of about 400 mM to about 600 mM. In some cases, the wash buffer comprises a salt concentration of about 450 mM to about 550 mM.In some cases, the wash buffer may be 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, Concentrations of 490mM, 500mM, 510mM, 520mM, 525mM, 530mM, 540mM, 550mM, 560mM, 570mM, 575mM, 580mM, 590mM, 600mM, 610mM, 620mM, 625mM, 630mM, 640mM, 650mM, 660mM, 670mM, 675mM, 680mM, 690mM, 700mM, 710mM, 720mM, 725mM, 730mM, 740mM, 750mM, 760mM, 770mM, 780mM, 790mM, or 800mM. or 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, and containing salt at a concentration of 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. In some embodiments, the salt concentration of the wash buffer is or is about 500 mM. In some embodiments, the wash buffer comprises about 500 mM NaCl.In some cases, this washing of the affinity matrix removes unbound impurities such as host cell proteins, DNA, and homodimeric species that have little or no affinity for the affinity matrix material (e.g., Protein A).

[0078] In some embodiments, the method includes an optional second wash with little (<25 mM) or no salt at a pH of 5 to 9 prior to elution of the heterodimeric protein. In some embodiments, the wash buffer includes about 10 mM to about 50 mM Tris [tris(hydroxymethyl)aminomethane]], sodium phosphate, or acetate, or a combination thereof. In various embodiments, the wash buffer has a pH equal to that of the first wash buffer described above.

[0079] After the above-mentioned washes, the heterodimeric protein is eluted from the affinity matrix in an elution buffer and collected in the eluate. The elution buffer has a pH of about 4 to about 5.2 (or 4.0 to 4.9) and contains a salt concentration of greater than 200 mM. As discussed in more detail below in connection with various methods, in some embodiments, the pH of the elution buffer is about 4.0 to about 4.2. In some embodiments, the pH of the elution buffer is about 4.4 to about 4.6. In various embodiments, the pH of the elution buffer is or is about 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, 4.75, 4.8, 4.85, 4.9, 4.95, or 5.0. In some embodiments, the pH of the elution buffer is 4.1±0.05. In some embodiments, the pH of the elution buffer is 4.5±0.05. In various embodiments, the buffer may be any buffer capable of maintaining the pH at a desired point or within a desired range. In various embodiments, the buffer concentration may be from about 5 mM to about 100 mM. In some cases, the buffer concentration is from about 25 mM to about 55 mM. In some cases, the buffer concentration is from about 30 mM to about 50 mM. In various embodiments, the buffer concentration is at or 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. In some embodiments, the elution buffer concentration is at or about 40 mM. In some embodiments, the elution buffer is acetic acid, in some embodiments, the elution buffer is acetate.

[0080] In some cases, the elution buffer comprises a salt concentration of about 200 mM to about 800 mM. In some cases, the elution buffer comprises a salt concentration of about 250 mM to about 750 mM. In some cases, the elution buffer comprises a salt concentration of about 300 mM to about 700 mM. In some cases, the elution buffer comprises a salt concentration of about 350 mM to about 650 mM. In some cases, the elution buffer comprises a salt concentration of about 400 mM to about 600 mM. In some cases, the elution buffer comprises a salt concentration of about 450 mM to about 550 mM.In some cases, the elution buffer may be 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, Concentrations of 490mM, 500mM, 510mM, 520mM, 525mM, 530mM, 540mM, 550mM, 560mM, 570mM, 575mM, 580mM, 590mM, 600mM, 610mM, 620mM, 625mM, 630mM, 640mM, 650mM, 660mM, 670mM, 675mM, 680mM, 690mM, 700mM, 710mM, 720mM, 725mM, 730mM, 740mM, 750mM, 760mM, 770mM, 780mM, 790mM, or 800mM. or 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, and containing salt at a concentration of 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. In some embodiments, the salt concentration of the elution buffer is or is about 500 mM. In some embodiments, the elution buffer comprises about 500 mM NaCl.In some embodiments, the elution buffer comprises about 500 mM CaCl. In some embodiments, the elution buffer comprises about 500 mM MgCl.

[0081] After elution and collection of the heterodimeric protein from the affinity matrix, the affinity matrix is ​​washed with a wash buffer (second wash buffer) at a pH of less than about 4. In some embodiments, the pH of the wash buffer is 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 or is about 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. The wash buffer can include any suitable material to provide the above-mentioned pH or pH range. In some embodiments, the wash buffer includes acetic acid at a concentration of about 20 mM to about 60 mM. In some embodiments, the wash buffer includes acetic acid at a concentration of about 30 mM to about 50 mM. In some cases, the wash buffer comprises about 40 mM acetic acid. In some cases, this washing of the affinity matrix removes previously bound impurities, such as homodimeric species, which have greater affinity for the affinity matrix material (e.g., Protein A) than the heterodimeric proteins. In some cases, the methods of the invention may also include further washing the affinity matrix with a buffer comprising a lower pH (e.g., 2.45±0.2) and a higher concentration of buffer material (e.g., 500 mM acetate) than the wash buffer discussed immediately above.

[0082] After the above-mentioned washes to remove additional impurities, the affinity matrix may be re-equilibrated to a pH of 5-9 before beginning the next cycle. In various embodiments, the affinity matrix may be 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. The affinity matrix is ​​equilibrated to a pH of about 7.0 or about 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. In some embodiments, the affinity matrix is ​​equilibrated to a pH of about 7.2. Equilibration can be performed using an equilibration buffer having a desired pH. In various embodiments, the buffer can be any buffer capable of maintaining the pH at the desired point or within the desired range. In various embodiments, the buffer concentration may be from about 5 mM to about 100 mM. In some cases, the buffer concentration is from about 10 mM to about 30 mM. In some cases, the buffer concentration is from about 30 mM to about 50 mM. In some cases, the buffer concentration is from about 40 mM to about 60 mM.In various embodiments, the buffer concentration 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, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM M, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, or 60 mM or about 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, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 5 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. In some embodiments, the buffer concentration is 20 mM or about 20 mM. In some embodiments, the buffer concentration is 40 mM or about 40 mM. In some embodiments, the buffer concentration is 50 mM or about 50 mM. In some embodiments, the buffer is sodium phosphate. In some embodiments, the buffer comprises about 10 mM to about 50 mM Tris, sodium phosphate, or acetate, or a combination thereof.

[0083] After equilibration of the affinity matrix, the neutralized eluate containing the heterodimeric protein (now purified from homodimeric contaminants and other impurities) is reapplied to the same affinity matrix used in the purification process steps discussed above at a pH of 5 to 9. In various embodiments, the neutralized eluate is reapplied to the same affinity matrix used in the purification process steps discussed above 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. is reapplied to the affinity matrix at a pH of about 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. In some embodiments, the pH is 7.2 or about 7.2.

[0084] In some embodiments, a method of purifying a heterodimeric protein includes (a) performing a series of chromatography cycles, where each cycle includes (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, where the heterodimeric protein comprises first and second polypeptides 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, and (ii) washing the affinity matrix with a first wash buffer at a first pH between 5 and 9 to remove unbound impurities. (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; and (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities, where the second pH is a preparatory pH during a preparatory series of cycles in the series of chromatography cycles and the second pH is increased to a subsequent pH higher than the preparatory pH during a subsequent series of cycles in the series of chromatography cycles, the preparatory pH and the subsequent pH being within the range of 4.0 to 5.2; and (b) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0085] In various embodiments, the preliminary series of cycles consists of 20 cycles. In some embodiments, the preliminary series of cycles consists of 30 cycles. In some embodiments, the preliminary series of cycles consists of 40 cycles. In some embodiments, the preliminary series of cycles consists of 50 cycles. In some embodiments, the preliminary series of cycles consists of 60 cycles. In some embodiments, the preliminary series of cycles consists of 70 cycles. In some embodiments, the preliminary series of cycles consists of 80 cycles. In some cases, the preliminary series of cycles includes or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 cycles.

[0086] In some embodiments, the subsequent series of cycles consists of at least 20 cycles. In some embodiments, the subsequent series of cycles consists of at least 50, at least 60, at least 70, or at least 80 cycles. In some cases, the subsequent series of cycles consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, or 100.

[0087] In some embodiments, the preliminary pH is 4.0-4.2. In some cases, the preliminary pH is 4.1±0.05. In some cases, the preliminary pH is 4.0, 4.025, 4.05, 4.075, 4.1, 4.125, 4.15, 4.175, or 4.2. In some embodiments, the subsequent pH is 4.3-4.7. In some cases, the subsequent pH is 4.5±0.05. In some cases, the subsequent pH is 4.4, 4.425, 4.45, 4.475, 4.5, 4.525, 4.55, 4.575, or 4.6.

[0088] In some embodiments, a method of purifying a heterodimeric protein includes (a) performing a series of chromatography cycles, where each cycle includes (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, where 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, (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities, and (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH. (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities; (b) measuring a level of bound impurities in the eluate containing the heterodimeric protein after any one or more cycles in the series of chromatography cycles and comparing the measured level of bound impurities with a reference level of bound impurities, where if the measured level of bound impurities exceeds the reference level of bound impurities, increasing the second pH of a subsequent cycle in the series of chromatography cycles, the second pH being within the range of 4.0 to 5.2 during each cycle or subsequent cycles in the series of chromatography cycles; and (c) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0089] In some embodiments, the reference level of bound impurities is between 2% and 10%. In some cases, the reference level of bound impurities is between 3% and 7%. In some cases, the reference level of bound impurities is 5%±0.5%. In various embodiments, the reference level of bound impurities is at or about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0090] In some embodiments, the level of bound impurities in the eluate is measured after each cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured after every fifth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured after every tenth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured following the twentieth cycle in the series of chromatography cycles. In some embodiments, the level of bound impurities in the eluate is measured following the fortieth or fiftieth cycle in the series of chromatography cycles. In various embodiments, the level of bound impurities in the eluate is determined in cycles 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 11 and / or after cycle 100. In various embodiments, the level of bound impurities in the eluate is measured after every 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles. In some cases, eluate is collected over a series of cycles (e.g., 5 or 10 cycles) and the level of bound impurities is measured in the combined eluate pool.In various embodiments, the combined elution pool comprises a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 cycles or more are collected.

[0091] In some embodiments, the second pH is increased from a range of 4.0-4.2 to a range of 4.2-5.2 (or 4.3-4.7) if the measured level of the bound impurity exceeds the reference level of the bound impurity. In some cases, the second pH is increased from 4.1±0.05 to 4.5±0.05 if the measured level of the bound impurity exceeds the reference level of the bound impurity. In some cases, the second pH is 4.0, 4.025, 4.05, 4.075, 4.1, 4.125, 4.15, 4.175, or 4.2 and increased to 4.4, 4.425, 4.45, 4.475, 4.5, 4.525, 4.55, 4.575, or 4.6 if the measured level of the bound impurity exceeds the reference level of the bound impurity.

[0092] In some embodiments, the second pH is between 4.0 and 4.2 and is increased by 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 points in subsequent cycles if the measured level of bound impurity exceeds the reference level of bound impurity. Thus, in some cases, the second pH may be increased stepwise in a subsequent cycle if the measured level of bound impurity exceeds the reference level of bound impurity, and then increased stepwise again (and optionally repeatedly) if the measured level of bound impurity exceeds the reference level of bound impurity in the next cycle. In this way, the elution pH may be maintained at a level that minimizes bound impurities in the eluate while maintaining maximum recovery of the heterodimeric protein over the course of a series of chromatography cycles.

[0093] In some embodiments, a method for purifying a heterodimeric protein includes (a) performing a series of chromatography cycles, where each cycle includes (i) introducing a mixture of the heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, where the heterodimeric protein comprises first and second polypeptides 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, (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities, and (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH. (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities, where the second pH is a first pH during a first series of cycles in the series of chromatography cycles, the second pH is increased to a second pH higher than the first pH during a second series of cycles following the first series of cycles in the series of chromatography cycles, and the second pH is increased to a tertiary pH higher than the second pH during a tertiary series of cycles following the second series of cycles in the series of chromatography cycles, where the first pH, the second pH, and the tertiary pH are within the range of 4.0 to 5.2; and (b) collecting the heterodimeric protein from the affinity matrix in the eluate.

[0094] In some embodiments, the primary series of cycles includes 5 to 50 cycles. In some cases, the primary series of cycles includes up to 20 cycles. In some cases, the primary series of cycles includes up to 40 cycles. In some cases, the primary series of cycles includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles. cycles or up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles.

[0095] In some embodiments, the secondary series of cycles includes 5 to 50 cycles. In some cases, the secondary series of cycles includes 10 to 25 cycles. In some cases, the secondary series of cycles includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles. cycles or up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles.

[0096] In some embodiments, the tertiary series of cycles includes 5 to 50 cycles. In some cases, the tertiary series of cycles includes 10 to 25 cycles. In some cases, the tertiary series of cycles includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles. cycles or up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles.

[0097] In some embodiments, the primary pH is in the range of 4.0-4.2. In some cases, the primary pH is 4.1±0.05. In some cases, the primary pH is 4.0, 4.025, 4.05, 4.075, 4.1, 4.125, 4.15, 4.175, or 4.2. In some embodiments, the secondary pH is in the range of 4.2-4.4. In some cases, the secondary pH is 4.3±0.05. In some cases, the secondary pH is 4.2, 4.225, 4.25, 4.275, 4.3, 4.325, 4.35, 4.375, or 4.4. In some embodiments, the tertiary pH is in the range of 4.4-4.6. In some cases, the tertiary pH is 4.5±0.05. In some cases, the tertiary pH is 4.4, 4.425, 4.45, 4.475, 4.5, 4.525, 4.55, 4.575, or 4.6.

[0098] In some embodiments, the second pH is increased to a fourth pH higher than the tertiary pH during a fourth series of cycles following a tertiary series of cycles in the series of chromatography cycles, where the fourth pH is in the range of 4.0 to 5.2.

[0099] In some embodiments, the second pH is increased to a fifth pH higher than the fourth pH during a fifth series of cycles following a fourth series of cycles in the series of chromatography cycles, where the fifth pH is in the range of 4.0 to 5.2.

[0100] In some embodiments, the second pH is increased to a sixth pH higher than the fifth pH during a sixth series of cycles following a fifth series of cycles in the series of chromatography cycles, where the sixth pH is in the range of 4.0 to 5.2.

[0101] In some cases, the secondary pH is 0.1-0.9 higher than the primary pH, the tertiary pH is 0.1-0.9 higher than the secondary pH, the fourth pH is 0.1-0.9 higher than the tertiary pH, the fifth pH is 0.1-0.9 higher than the fourth pH, ​​and / or the sixth pH is 0.1-0.9 higher than the fifth pH, ​​and the primary pH is in the range of 4.0-4.2. In some embodiments, the primary pH is 4.1±0.05.

[0102] In some embodiments, the pH of the secondary, tertiary, quaternary, quintic, or septiary (or septiary, octetary, ninth, etc.) is increased by 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or even 5 points from the previous pH in the next series of cycles (e.g., the secondary pH is increased relative to the primary pH, the tertiary pH is increased relative to the secondary pH, etc.). Thus, in some cases, the elution pH may be increased stepwise in each subsequent series of cycles. In this way, the elution pH can be maintained at a level that minimizes bound impurities in the eluate while maintaining maximum recovery of the heterodimeric protein over the course of a series of chromatography cycles.

[0103] In some embodiments, the primary series of cycles, secondary series of cycles, tertiary series of cycles, quaternary series of cycles, quinary series of cycles, and / or sextuple series of cycles (and further series of cycles, if desired) each include 5 to 50 cycles within the series of chromatography cycles. In various embodiments, each series of cycles includes or at least includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, or 75 cycles or more.

[0104] In various embodiments, loading of the affinity matrix from the clarified cell culture or from the neutralized eluate containing the heterodimeric protein can include a material loading of up to about 75 g / L of affinity matrix resin, hi various embodiments, the affinity matrix is ​​loaded with no more than 65 g / L, no more than 60 g / L, no more than 55 g / L, or no more than 50 g / L of material.

[0105] In some embodiments, the affinity matrix includes a ligand (e.g., protein A) attached to a substrate. In some cases, the substrate is a bead or particle, such that the affinity matrix is ​​a plurality of particles attached with a ligand. In various embodiments, the ligand is protein A or protein G. When the ligand is protein A, the protein A can be naturally occurring or modified staphylococcal protein A, or engineered protein A. The engineered protein A can be, for example, a Z domain tetramer, a Y domain tetramer, or engineered protein A lacking the D and E domains. These examples of engineered protein A cannot bind (or, if they can, bind with very low affinity) to the VH3 domain of immunoglobulins, but can still bind to the CH3 domains of IgG1, IgG2, and IgG4.

[0106] In some cases, the affinity matrix substrate contains or consists of agarose, poly(styrenedivinylbenzene), polymethacrylate, controlled pore glass, spherical silica, cellulose, and the like. In embodiments in which the substrate is shaped as beads or particles, the particles have an average diameter of 25 μm to 100 μm. In some embodiments, the particles have an average diameter of about 40 μm to about 60 μm. In some embodiments, the particles have an average diameter of about 45 μm to about 55 μm. In some embodiments, the particles have an average diameter of about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 μm. In some cases, the particles have an average diameter of about 45 μm. In some cases, the particles have an average diameter of about 50 μm. In some embodiments, the particles have an average diameter of 35 μm, 45 μm, 60 μm, 75 μm, or 85 μm. In some embodiments, the particles include pores having an average diameter of about 1000 Å, 1050 Å, 1100 Å, 1150 Å, or 1200 Å. In some embodiments, the particles include pores having an average diameter of about 1100 Å.

[0107] In various embodiments, the elution buffer or wash buffer may contain a salt. In some cases, the salt is Cl. - , Br - , I - , NO3 - , N(CH3)4 + , NH4 + , Cs + , Rb + , K + , Na + , H + , Ca 2+ , Mg 2+ , or Al 3+ In some embodiments, the salt comprises Na + , H + , Ca 2+ , Mg 2+ , or Al 3+ In some embodiments, the salt comprises Cl - , Br - , I - , NO3 - , or ClO4 - In some embodiments, the salt comprises Na + , H + , Ca 2+ , Mg 2+ , or Al 3+ And, 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.

[0108] In some embodiments of the method, the heterodimeric protein is a bispecific antibody comprising a first polypeptide comprising a CH3 domain capable of binding Protein A ("Fc") and a second polypeptide comprising a CH3 domain incapable of binding Protein A ("Fc*"). In some cases, the second polypeptide comprises a H435R / Y436F (according to the EU numbering system; H95R / Y96F according to the IMGT exon numbering system) substitution in its CH3 domain (also known as an "Fc*" or "star substitution"). 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).

[0109] In some embodiments of the method, the frequency at which the chromatographic column is subjected to washing (e.g., by contacting the column with a basic solution having a pH of at least 11) may be reduced to minimize effects on protein-ligand function. Similarly, in some embodiments of the method, the concentration of base in the solution used to wash the chromatographic column may be reduced to the range of 0.1 N to 0.5 N to maximize column separation capacity over a greater number of cycles.

[0110] In various embodiments, the affinity matrix may be contacted with a basic solution having a pH of at least 11 after each cycle. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 3 cycles. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 5 cycles. In some cases, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after every 7 cycles. In various embodiments, the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 only following every 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles.

[0111] In some embodiments, the pH of the basic solution is at least 12. In some embodiments, the pH of the basic solution is at least 11, at least 11.1, at least 11.2, at least 11.3, at least 11.4, at least 11.5, at least 11.6, at least 11.7, at least 11.8, at least 11.9, at least 12, at least 12.1, at least 12.2, at least 12.3, at least 12.4, at least 12.5, at least 12.6, at least 12.7, at least 12.8, at least 12.9, or at least 13.

[0112] In some embodiments, the basic solution includes a base at a concentration of 0.1N to 0.5N. In some cases, the base concentration is 0.1N to 0.3N. In some cases, the base concentration is 0.1N, 0.15N, 0.2N, 0.25N, 0.3N, 0.35N, 0.4N, 0.45N, or 0.5N. In some embodiments, the basic solution includes an alkali metal hydroxide. In some cases, the base is NaOH. In some cases, the base is KOH. EXAMPLES

[0113] Example 1: Evaluation of elution pH on the presence of bound impurities and recovery of heterodimeric proteins in affinity chromatography A 16.2 mL MabSelect SuRe™ pcc column (1.0 cm internal diameter, 20.6 cm bed height) was packed with naive resin and integrated into an AKTA Avant 25 benchtop liquid chromatography controller for this experiment. The affinity separation process was performed as outlined in Table 1 below, with the elution pH varying from 3.90 to 4.30.

[0114] [Table 1] a Bound species refers to the bispecific and bound impurity species. Binding titers were used to determine column loading. b Eluate collection began 0.5 CV into the elution block. CV, column volume; RODI, reverse osmosis deionized water

[0115] The affinity separation eluate was fractionated to allow for the preparation of mock pools representing eluate composition at elution lengths of 5, 6, and 7 CV. Eluate collection began 0.5 CV into the elution block. CVs 0.5-5 were collected in bulk, followed by CVs 5-6 and 6-7 collected individually. After fractionation, appropriate volumes were combined from each fraction to generate mock pools of 6 CV and 7 CV, and the 5, 6, and 7 CV pools were then statistically evaluated as separate runs.

[0116] The concentration of each mock pool was determined by UV (280 nm) using a Solo VPE instrument. Each mock pool was analyzed for bispecific purity measured using a mixed-mode chromatography assay. The eluate volume, eluate protein concentration, bound impurities, and unbound impurities data for each mock pool were used to calculate the affinity separation bispecific yield for each run. A model was generated using factors selected from a backward stepwise regression tool with an entry probability of 0.25, an exit probability of 0.05, and a p-value threshold stopping rule set at 95% and used to calculate bound impurity levels and heterodimeric protein recovery.

[0117] As shown in Figure 3, both the percentage of bound impurities in the eluate and the heterodimeric protein recovery decreased with increasing pH of the naive column (0 cycles prior). As shown, pH 4.1 results in minimal levels of bound impurities in the eluate (e.g., 2.0%) while maintaining significant levels of heterodimeric protein recovery (e.g., 92.5%). Notably, increasing the pH of the elution buffer to 4.2 dramatically reduces the recovery of the heterodimeric protein (e.g., to about 80%).

[0118] Example 2: Evaluation of elution pH on the presence of bound impurities and recovery of heterodimeric proteins on naive and cycled affinity chromatography columns The experiments were carried out using a MabSelect SuRe™ pcc column (1.0 cm internal diameter, 20 cm bed height) installed on an Akta Avant 25 (Cytiva) liquid chromatography system. The affinity separation process was carried out as outlined in Table 2 below, but with varying elution pH and cycle number as shown in Table 3 below.

[0119] [Table 2]

[0120] [Table 3]

[0121] As shown in Figure 4A, increasing the elution pH (from 4.1 to 4.5) in the naive column (≦6 cycles) only slightly reduces the percentage of bound impurities in the eluate (from 2.7% to 1.2%), whereas increasing the elution pH (from 4.1 to 4.5) in the cycled column (78–83 cycles) dramatically and unexpectedly reduces the percentage of bound impurities in the eluate (from 17.4% to 2.0%). Figure 4B shows that increasing the elution pH (from 4.1 to 4.5) also negatively impacts the recovery of heterodimeric proteins (e.g., bispecific antibodies), but the reduction in recovery in the cycled column is unexpectedly very small (about 10-fold compared to the naive column). As shown in Figure 4B, when the elution pH was increased from 4.1 to 4.5, the recovery of the heterodimeric protein was reduced by about 40% in the naive column, whereas the reduction in recovery in the cycled column was only about 4% for the same pH increase.

[0122] Example 3: Evaluation of input parameters versus measured output in pH dissolution tests The experiments were carried out using three MabSelect SuRe™ pcc columns (1.0 cm internal diameter, 21 cm bed height; 16.5 mL column volume) separately installed on an AKTA pure 150 (Cytiva) liquid chromatography system. The affinity separation process was carried out as outlined in Table 4 below, but with various column loadings (33-55 g of bound species (bispecific + bound impurities) per L of resin), elution pH (4.0-4.5), and hydroxide cycles or hydroxide exposure times (1-109 cycles or 0.28-30.56 hours), as shown in Table 5 below. Yields (% of bispecific + bound impurities), bound impurities (%), and aggregation (SE-UPLC high molecular weight %) were measured in relation to column loading, elution pH, and hydroxide cycles (or exposure times).

[0123] [Table 4]

[0124] [Table 5]

[0125] Experimental runs were performed in the order listed in Table 5 above. Design diagnostics are shown in Figures 5A, 5B, and 6. Experiments were performed across three columns with low (1-5), medium (50-56), or high (105-109) numbers of hydroxide cycles as described above. Hydroxide cycles were converted to hydroxide cycle times to facilitate analysis. Hydroxide contact time was 16.82 min (0.28 h) per cycle. Resolving elution buffer was prepared within a pH tolerance of ±0.05. Eluate collection began 0.5 CV into the elution block.

[0126] The concentration of each pool was determined by UV (280 nm) using a Solo VPE instrument. The eluate volume and eluate protein concentration were used to calculate the affinity separation bispecific yield for each run, assuming that the pool contained only the bispecific protein (i.e., the resulting yield could be measured at >100% due to impurities (bound impurities). Bispecific purity was measured using a hydrophobic interaction chromatography (HIC) assay. Aggregation was measured using a size-exclusion ultra-performance liquid chromatography assay (SE-UPLC).

[0127] Models were generated using factors selected from a backward stepwise regression tool starting with the full model and left as is combining rules and a p-value threshold of 0.05. Process knowledge and / or further statistical analysis were also used to add or remove model terms as necessary. Regression analysis was performed for bispecific step yield (%), bound impurities (%), and aggregation (%HMW).

[0128] The model prediction profiler is shown in Figures 7A and 7B. Models with significant terms were created for bispecific yield and bound impurities. No significant terms were found for aggregation. Lower pH and higher column load, as well as hydroxide contact time, resulted in higher bispecific yield. The main component of this higher yield was due to the presence of increased bound impurities, as shown in Figure 7B, which followed the same trend for pH and hydroxide contact time.

[0129] Three validation runs were performed to evaluate the ability of these models to predict new data. Clarified cell culture was purified on each of the three columns using an average column loading of 44 g per L of resin. The models were used to predict what pH should be used to target fixed bound impurity levels (approximately 6%) on the column at different stages of resin life. The results are shown in Table 6 below.

[0130] [Table 6]

[0131] The bispecific yield model predicted consistently low over the range evaluated, but within 7% of actual. Column loading was not a significant factor in predicting bound impurities. Bound impurity model predictions were higher than actual, but within 0.5%.

[0132] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for purifying a heterodimeric protein, comprising: (a) performing a series of chromatography cycles, each cycle comprising: (i) introducing a mixture of a heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, wherein the heterodimeric protein comprises first and second polypeptides having different affinities for the protein-binding ligand, and wherein at least one impurity binds to the protein-binding ligand and at least one impurity does not bind to the protein-binding ligand; (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities; (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities; the second pH is a preliminary pH during a preliminary series of cycles in the series of chromatography cycles, and the second pH is increased during a subsequent series of cycles in the series of chromatography cycles to a subsequent pH that is higher than the preliminary pH, and the preliminary pH and the subsequent pH are within the range of 4.0 to 5.2; and (b) collecting said heterodimeric protein from said affinity matrix in an eluate.

2. 10. The method of claim 1, wherein the preliminary series of cycles consists of 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, or 80 cycles.

3. 3. The method of claim 2, wherein the subsequent series of cycles consists of at least 20 cycles, at least 50, at least 60, at least 70, or at least 80 cycles.

4. 2. The method of claim 1, wherein the preliminary pH is 4.0 to 4.2, or 4.1±0.

05.

5. 10. The method of claim 1, wherein the subsequent pH is 4.3 to 4.7, or 4.5±0.

05.

6. 1. A method for purifying a heterodimeric protein, comprising: (a) performing a series of chromatography cycles, each cycle comprising: (i) introducing a mixture of a heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, wherein the heterodimeric protein comprises first and second polypeptides having different affinities for the protein-binding ligand, and wherein at least one impurity binds to the protein-binding ligand and at least one impurity does not bind to the protein-binding ligand; (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities; (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities; (b) measuring a level of bound impurities in an eluate containing the heterodimeric protein after any one or more of the cycles in the series of chromatography cycles, and comparing the measured level of bound impurities with a reference level of bound impurities, wherein if the measured level of bound impurities exceeds the reference level of bound impurities, increasing the second pH in subsequent cycles in the series of chromatography cycles, wherein the second pH is within the range of 4.0 to 5.2 during each cycle or subsequent cycles in the series of chromatography cycles; and (c) collecting the heterodimeric protein from the affinity matrix in the eluate.

7. 7. The method of claim 6, wherein the reference level of bound impurities is between 2% and 10%, or between 3% and 7%, or is 5%±0.5%.

8. 7. The method of claim 6, wherein the level of bound impurities in the eluate is measured after each cycle in the series of chromatography cycles, after every fifth cycle in the series of chromatography cycles, after every tenth or every twentieth cycle in the series of chromatography cycles, or following the fortieth or fiftieth cycle in the series of chromatography cycles.

9. 7. The method of claim 6, wherein the level of bound impurities in the eluate is measured in a combined eluate pool collected from a series of cycles.

10. 7. The method of claim 6, wherein the second pH is increased from a range of 4.0 to 4.2 to a range of 4.3 to 4.7, or increased from 4.1±0.05 to 4.5±0.05, if the measured level of bound impurities exceeds the reference level of bound impurities.

11. 2. The method of claim 1, wherein the impurities comprise homodimeric species of the first and second polypeptides.

12. 2. The method of claim 1, wherein the protein-binding ligand is Protein A and the affinity matrix comprises the Protein A ligand affixed to a substrate, or the Protein A ligand is an engineered Protein A comprising a Z domain tetramer, an engineered Protein A comprising a Y domain tetramer, or an engineered Protein A lacking the D and E domains. (a) the substrate is a particle, and the affinity matrix comprises a multiplicity of said particles having an average diameter of 25 μm to 100 μm, 40 μm to 60 μm, 45 μm to 55 μm, or about 50 μm; (b) the substrate comprises any one or more of agarose, poly(styrenedivinylbenzene), polymethacrylate, cellulose, controlled pore glass, and spherical silica; or (c) the substrate is a particle, and the affinity matrix comprises a multiplicity of the particles with pores having an average diameter of about 1100 Å; The method of claim 12.

14. 10. The method of claim 1, wherein the elution buffer comprises a salt concentration of at least 250 mM, greater than 300 mM, or greater than 400 mM, or about 500 mM.

15. The salt is (i) Cl － ,Br － , I － , NO ３ － , N(CH ３ ) ４ ＋ , N.H. ４ ＋ , Cs ＋ , Rb ＋ , K. ＋ , Na ＋ , Ca ２＋ , Mg ２＋ , Al ３＋ , (ii) Na ＋ , Ca ２＋ , Mg ２＋ , or Al ３＋ And Cl － ,Br － , I － , NO ３ － , or ClO ４ － or (iii) CaCl ２ , MgCl ２ 15. The method of claim 14, wherein the salt is selected from the group consisting of NaCl, NaCl, and NaCl.

16. 2. The method of claim 1, wherein the first polypeptide comprises a CH3 domain capable of binding to the protein-binding ligand, and the second polypeptide comprises a CH3 domain that cannot bind to the protein-binding ligand.

17. 13. The method of claim 12, wherein the first polypeptide comprises a CH3 domain capable of binding to Protein A and the second polypeptide comprises a CH3 domain that cannot bind to Protein A, or the second polypeptide comprises a H435R modification and a Y436F modification (EU numbering) in the CH3 domain.

18. 6. The method of claim 5, wherein the first pH is from 6 to 8, or the third pH is from 2.8 to 3.

5.

19. 2. The method of claim 1, wherein the heterodimeric protein is an antibody, a bispecific antigen-binding protein, or a bispecific antibody.

20. 2. The method of claim 1, wherein at least 85%, at least 87%, or at least 89% of the heterodimeric protein is recovered in the eluate in each cycle within the series of chromatography cycles.

21. 10. The method of claim 1, wherein the series of chromatography cycles comprises 100 or more cycles.

22. 10. The method of claim 1, wherein the affinity matrix is ​​contacted with a basic solution having a pH of at least 11 after each cycle, after every third cycle, after every fifth cycle, or after every seventh cycle.

23. 23. The method of claim 22, wherein the pH of the basic solution is at least 12, wherein the basic solution comprises a base at a concentration of 0.1 N to 0.5 N, wherein the basic solution comprises a base at a concentration of 0.1 N to 0.3 N, or wherein the basic solution comprises NaOH.

24. 10. The method of claim 1, wherein each cycle further comprises (v) washing the affinity matrix by contacting the affinity matrix with a basic solution having a pH of at least 11.

25. 25. The method of claim 24, wherein the pH of the basic solution is at least 12, wherein the basic solution comprises a base at a concentration of 0.1 N to 0.5 N, wherein the basic solution comprises a base at a concentration of 0.1 N to 0.3 N, or wherein the basic solution comprises NaOH.

26. 25. The method of claim 24, wherein at least 75%, at least 78%, or at least 80% of the heterodimeric protein is recovered in the eluate in each cycle within the series of chromatography cycles, and the bound impurities do not exceed 6.5%.

27. A method for purifying a heterodimeric protein, comprising: (a) performing a series of chromatography cycles, each cycle comprising: (i) introducing a mixture of a heterodimeric protein and impurities into an affinity matrix containing a protein-binding ligand, wherein the heterodimeric protein comprises first and second polypeptides having different affinities for the protein-binding ligand, and wherein at least one impurity binds to the protein-binding ligand and at least one impurity does not bind to the protein-binding ligand; (ii) washing the affinity matrix with a first wash buffer at a first pH of 5 to 9 to remove unbound impurities; (iii) eluting the heterodimeric protein from the affinity matrix in a first elution buffer at a second pH; (iv) washing the affinity matrix with a second wash buffer at a third pH less than 4 to remove bound impurities; the second pH is a first pH during a first series of cycles in the series of chromatography cycles, the second pH is increased to a second pH higher than the first pH during a second series of cycles following the first series of cycles in the series of chromatography cycles, and the second pH is increased to a tertiary pH higher than the second pH during a tertiary series of cycles following the second series of cycles in the series of chromatography cycles, the first pH, the second pH, and the tertiary pH being within the range of 4.0 to 5.2; and (b) collecting said heterodimeric protein from said affinity matrix in an eluate.

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