High salt load conditioning during cation exchange chromatography to remove product related impurities

Abstract

To provide a method for high salt load conditioning during cation exchange chromatography to remove low isoelectric point product-related impurities during the production of recombinant multispecific proteins.SOLUTION: A step of equilibrating a cation exchange chromatography media with an equilibration buffer comprising 94 to 105mM sodium chloride, a step of loading the composition onto the cation exchange media in a loading buffer comprising 94 to 105mM sodium chloride, a step of washing the column with at least one wash buffer comprising 94 to 105mM sodium chloride, and a step of eluting the multispecific proteins from the cation exchange chromatography media.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 931,863, filed November 7, 2019, which is incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceutical manufacturing. In particular, the present invention relates to a method for removing low isoelectric point product-related impurities during cation exchange purification operations. [Background technology]

[0003] Antibody products represent the largest sector of the biopharmaceutical market and will easily reach hundreds of billions of dollars in sales within the next decade. Commercial development of therapeutic monoclonal antibodies began in the 1980s with the approval of the first therapeutic monoclonal antibody and has continued to evolve and expand ever since. While monoclonal antibodies bind to their targets with high affinity and specificity and have been extremely successful therapeutic treatments for some indications, they also have limitations. Monoclonal antibodies bind to a single target; however, many diseases are multifactorial. In cancer immunotherapy, single-target therapy may not be sufficient to destroy or immobilize cancer cells. Furthermore, some patients receiving monoclonal antibody therapy may fail to respond to treatment or may develop drug resistance.

[0004] Novel antibody-like structures, such as antibody Fab fragments, Fc fusion proteins, antibody-drug conjugates, glycol-engineered antibodies, and most particularly bispecific and other multispecific antibody-like structures, have been developed to address these challenges. These antibody-like structures, particularly bispecific antibodies, offer improvements over traditional monoclonal antibody therapeutics and are proving to be effective next-generation biological therapeutics with a vast array of formats that can be developed to address even more challenging therapeutic indications.

[0005] Bispecific antibodies are the most diverse group of these antibody-like structures, with an ever-increasing variety of frameworks to meet the needs of therapeutic indications. These structures combine the binding properties of antibodies with additional molecular properties tailored to the desired disease indication. Bispecific antibodies are being developed for a variety of applications and uses, such as for immune responses against cancer, redirecting immune effector cells to tumor cells, blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, crossing the blood-brain barrier, diagnostic assays, and as therapeutic and delivery agents for pathogens. (Sedykh et al.,Drug Design,Development and Therapy 18(12),195-208,2018;Walsh,Nature Biotechnology,32(10),992-1000,2014;Ecker et al.,mAbs 7(1),9-14,2015;Spiess et al.,Mol Immunol 67,95-106,2015;Fan et al.,J Hematol&Oncology 8:130-143,2015;Williams et al.,Process Design for Bispecific Antibodies,Biopharmaceutical Processing,Development,Design and Implementation of Processes,Jagschies et al.,editors,Elsevier Ltd,pages 837-855,2018).

[0006] The development of these multispecific proteins poses new biopharmaceutical manufacturing challenges, particularly with regard to product instability and low expression yields. Specifically, the purification of multispecific proteins is complicated by the formation of product-associated variants, such as homodimers, half-antibodies, aggregates, and high and low molecular weight species. These variants share similar structural and physical properties, such as charge, with the multispecific protein of interest, making them difficult to separate during purification. These product-associated impurities reduce the yield and activity of multispecific formulations and affect the robustness of the manufacturing process. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sedykh et al.,Drug Design, Development and Therapy 18(12),195-208,2018 [Non-patent document 2] Walsh,Nature Biotechnology,32(10),992-1000,2014 [Non-patent document 3] Ecker et al.,mAbs 7(1),9-14,2015 [Non-patent document 4] Spiess et al.,Mol Immunol 67,95-106,2015 [Non-Patent Document 5] Fan et al.,J Hematol & Oncology 8:130-143,2015 [Non-patent document 6] Williams et al.,Process Design for Bispecific Antibodies,Biopharmaceutical Processing,Development,Design and Implementation of Processes,Jagschies et al.,editors,Elsevier Ltd,pages 837-855,2018 Summary of the Invention [Problem to be solved by the invention]

[0008] Product-related impurities with a similar charge (isoelectric point) to the multispecific protein of interest can co-elute with the multispecific protein during cation exchange chromatography operation, complicating purification and reducing yield. It would be beneficial to separate low pI product-related impurities prior to elution. The invention described herein fulfills this need by providing high salt loading conditioning during cation exchange chromatography to remove these low pI impurities. [Means for solving the problem]

[0009] The present invention provides a method for purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading the composition onto the cation exchange medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium. In one embodiment, the loading buffer comprises 94-96 mM sodium chloride. In a related embodiment, the loading buffer comprises 96-105 mM sodium chloride. In a related embodiment, the loading buffer comprises 94 mM sodium chloride. In a related embodiment, the loading buffer comprises 96 mM sodium chloride. In a related embodiment, the loading buffer comprises 105 mM sodium chloride. In one embodiment, the loading buffer comprises acetate. In a related embodiment, the loading buffer comprises acetate at a pH of 4.9-5.1. In a related embodiment, the loading buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, the loading buffer comprises 100 mM acetate. In one embodiment, the loading buffer comprises acetate and 94 mM to 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises 94 to 96 mM sodium chloride. In one related embodiment, at least one wash buffer comprises 96 to 105 mM sodium chloride. In one related embodiment, at least one wash buffer comprises 94 mM sodium chloride. In one related embodiment, at least one wash buffer comprises 96 mM sodium chloride. In one related embodiment, at least one wash buffer comprises 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.0 to 5.1. In a related embodiment, the at least one wash buffer comprises acetate at a pH of 5.0±0.05 to 5.0 to 0.1. In a related embodiment, the at least one wash buffer comprises 100 mM acetate.In one embodiment, at least one wash buffer comprises acetate and 94 mM to 105 mM sodium chloride. In one embodiment, at least one additional wash buffer comprises 0 mM to 26 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate and 94 mM to 96 mM sodium chloride, followed by at least one additional wash buffer comprising acetate and 25 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate and 105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate. In one embodiment, at least one equilibration buffer comprises 94 mM to 96 mM sodium chloride. In a related embodiment, at least one equilibration buffer comprises 96 mM to 105 mM sodium chloride. In a related embodiment, at least one equilibration buffer comprises 94 mM sodium chloride. In a related embodiment, at least one equilibration buffer comprises 96 mM sodium chloride. In a related embodiment, at least one equilibration buffer comprises 105 mM sodium chloride. In one embodiment, the equilibration buffer comprises acetate. In a related embodiment, the equilibration buffer comprises acetate at a pH of 4.9 to 5.1. In a related embodiment, the equilibration buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, the equilibration buffer comprises 100 mM acetate. In one embodiment, the equilibration buffer comprises acetate and 94 mM to 105 mM sodium chloride. In one embodiment, the composition is loaded at 10 to 27 g / L. In one embodiment, the composition is loaded at 15 to 27 g / L. In one embodiment, the multispecific protein is eluted from the cation exchange resin by a gradient. In a related embodiment, the gradient is linear. In one embodiment, the gradient is a salt gradient. In one embodiment, the multispecific protein is a bispecific protein. In one embodiment, the multispecific protein is a bispecific antibody. In one embodiment, a purified multispecific protein prepared by the above method is provided. In one embodiment, the cation exchange chromatography medium is a resin.

[0010] The present invention provides a method for reducing low pI impurities in an eluate from cation exchange chromatography, comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer containing 94-105 mM sodium chloride; loading a composition onto the cation exchange chromatography medium in a loading buffer containing 94-105 mM sodium chloride; washing the column with at least one wash buffer containing 94-105 mM sodium chloride; and eluting a multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pI impurities compared to a cation exchange chromatography eluate collected in a corresponding method in which sodium chloride is not used in the equilibration, loading, and washing steps. In one embodiment, the low pI impurities are product-related impurities. In one embodiment, the at least one product-related impurity is a half antibody or 2X, 3X, or 4X light chain misassembly.

[0011] The present invention provides a method of performing cation exchange chromatography under high salt load conditions to reduce product-related impurities, comprising the steps of: equilibrating a cation exchange chromatography medium with an equilibration buffer; loading a composition onto the cation exchange chromatography medium in a loading buffer; washing the column with first and second wash buffers; and eluting the multispecific protein from the cation exchange chromatography medium, wherein the equilibration, loading, and wash buffers comprise 94-105 mM sodium chloride. In one embodiment, the second wash buffer comprises 0-26 mM sodium chloride.

[0012] The present invention provides a method for producing isolated and purified recombinant multispecific proteins, comprising the steps of: establishing a cell culture in a bioreactor using host cells that express the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating viruses at a low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; equilibrating a load buffer comprising 94-105 mM sodium chloride. The present invention provides a method comprising the steps of loading a neutralized, affinity-purified recombinant multispecific protein onto a cation exchange medium equilibrated in a buffer; washing the cation exchange medium with a wash buffer containing 94-105 mM sodium chloride, followed by a second wash buffer containing 0-26 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography medium; loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto a second chromatography resin in flow-through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer. In one embodiment, the second chromatography resin is selected from an anion exchange chromatography resin, a cation exchange chromatography resin, a multimodal chromatography resin, a hydrophobic interaction chromatography resin, and a hydroxyapatite chromatography resin. In a related embodiment, an isolated and purified recombinant multispecific protein prepared by the above method is provided. In a related embodiment, a pharmaceutical composition comprising the isolated and purified recombinant multispecific protein prepared by the above method is provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows that impurities (half antibody and 2× LC) co-elute with the major product, Bispecific #1. [Figure 2]Figure 1 shows that after high salt loading conditioning, low pI impurities passed through the column between the loading step and the first wash step. The second wash returned the UV baseline to zero before elution. During elution of bispecific #1, the elution peaks decreased from four peaks to one peak. [Figure 3] Figure 1 shows a single elution peak resulting from high loading density, no salt loading conditions with steep elution gradient for bispecific #2. Low pI product-related impurities do not resolve from the main product under high loading density and are mostly found in fractions 1-3. [Figure 4] FIG. 1 shows that a lower loading density (10 vs. 25 g / L) and a shallower gradient (8 vs. 16 mM / CV) allowed separation of the major low pI product impurity into a distinct peak formed by fractions 1-4 for bispecific #2. [Figure 5] FIG. 1 shows that under high salt loading conditions, there was a reduction in the number of impurity peaks in the elution profile from two peaks to a single peak for bispecific #2, with a small shoulder (fractions 1-3) still containing some mismatched species (LC1 / LC2=2-3). DETAILED DESCRIPTION OF THE INVENTION

[0014] Because there is little information in the literature regarding the downstream processing of multispecific proteins, platforms developed for monoclonal antibodies are often applied (Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, pp. 559-594, John Wiley & Sons, 2017). When multispecific proteins were subjected to cation exchange chromatography (CEX) in bind-and-elute mode under conditions typical for antibodies and antibody-like proteins, numerous impurity peaks were observed in the elution profile. These impurities had both lower and higher isoelectric points than the main product. Impurities with low pIs eluted ahead of the main product as pre-peaks. This elution profile would not support the development of a robust, sustainable, commercial-scale manufacturing method.

[0015] The properties of multispecific proteins can make them susceptible to the formation of product-related impurities, and cell culture conditions can significantly affect the amount of such impurities. These impurities can complicate purification and reduce the yield and activity of the desired multispecific protein. A high-salt loading strategy was found to improve the yield of the main product in the CEX eluate pool by removing low-pI impurities prior to the elution step. By targeting a final sodium chloride concentration of 94-105 mM for the equilibration buffer, final conditioning loading buffer, and first wash buffer, low-pI impurities pass through the column, reducing the number of peaks in the elution profile and the amount of product-related impurities in the CEX eluate. A second wash step was further added to ensure full binding conditions for the desired multispecific protein and to reestablish a UV baseline to zero before the start of elution, thereby fixing the elution profile and resulting in much more efficient collection and better quality of the main product. For example, high salt loading conditioning unexpectedly reduced the elution length for the bispecific protein from 44 CV (column volumes) to 20.3 CV, a reduction in the CEX eluate pool volume that is highly desirable for time and resource savings and process effectiveness and robustness in midstream downstream equipment operations. For both bispecific species, high salt loading conditions enabled efficient removal of impurities prior to elution, reduced the amount of product-related impurities in the CEX eluate, and simplified elution collection criteria, thus providing a robust manufacturing process.

[0016] The present invention provides a method for purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading the composition onto the cation exchange chromatography medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.

[0017] The present invention also provides a method for reducing low pI impurities in an eluate from cation exchange chromatography, comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading a composition onto the cation exchange chromatography medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pI impurities compared to a cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not used in the equilibration, loading, and washing steps.

[0018] The present invention also provides a method of performing cation exchange chromatography under high salt load conditions to reduce product-related impurities, comprising the steps of: equilibrating a cation exchange chromatography medium with an equilibration buffer; loading a composition onto the cation exchange chromatography medium in a loading buffer; washing the column with first and second wash buffers; and eluting the multispecific protein from the cation exchange chromatography medium, wherein the equilibration, loading, and first wash buffers comprise 94-105 mM sodium chloride.

[0019] The present invention provides a method for producing an isolated and purified recombinant multispecific protein of interest, comprising the steps of: establishing a cell culture in a bioreactor using host cells expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating viruses at a low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer containing 94-105 mM sodium chloride; loading the neutralized affinity-purified recombinant multispecific protein onto an equilibrated cation exchange medium in a loading buffer containing 94-105 mM sodium chloride; washing the cation exchange medium with a wash buffer containing 94-105 mM sodium chloride followed by a second wash buffer containing 0-26 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography medium; loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto a second chromatography resin in flow-through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer.

[0020] In one embodiment, the loading buffer comprises 94-105 mM sodium chloride. In one embodiment, the loading buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment, the loading buffer comprises 94 mM sodium chloride. In one embodiment, the loading buffer comprises 96 mM sodium chloride. In one embodiment, the loading buffer comprises 98 mM sodium chloride. In one embodiment, the loading buffer comprises 105 mM sodium chloride.

[0021] In one embodiment of the present invention, the loading buffer comprises acetate. In one embodiment, the loading buffer comprises acetate at a pH of 4.9 to 5.1. In one embodiment, the loading buffer comprises acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the loading buffer comprises acetate at a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, the loading buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, the loading buffer comprises acetate at a pH of 5.0. In one embodiment, the loading buffer comprises 100 mM acetate. In one embodiment, the loading buffer comprises 100 mM acetate at a pH of 4.9 to 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at pH 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at pH 5.0±0.05% to pH 5.0±0.1%.

[0022] In one embodiment, the loading buffer comprises acetate and 94 mM to 105 mM sodium chloride. In one embodiment, the loading buffer comprises acetate and 94 mM to 96 mM sodium chloride. In one embodiment, the loading buffer comprises acetate and 96 mM to 105 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 94 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 96 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 98 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 105 mM sodium chloride. In a related embodiment, the concentration of acetate is 100 mM.

[0023] In a related embodiment, the loading buffer comprises acetate, 94-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the loading buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the loading buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment, the loading buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 5.0. In a related embodiment, the concentration of acetate is 100 mM.

[0024] In one embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9 to 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9, 5.0, or 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0.

[0025] In one embodiment, at least one wash buffer comprises 94-96 mM sodium chloride. In one embodiment, at least one wash buffer comprises 96-105 mM sodium chloride. In one embodiment, at least one wash buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises 94 mM sodium chloride. In one embodiment, at least one wash buffer comprises 96 mM sodium chloride. In one embodiment, at least one wash buffer comprises 98 mM sodium chloride. In one embodiment, at least one wash buffer comprises 105 mM sodium chloride.

[0026] In one embodiment of the present invention, at least one wash buffer comprises acetate. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.9-5.1. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, at least one wash buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, at least one wash buffer comprises 100 mM acetate. In one embodiment, at least one wash buffer comprises 100 mM acetate at a pH of 4.9-5.1. In one embodiment, at least one wash buffer comprises 100 mM acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at pH 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, at least one wash buffer comprises 100 mM acetate at pH 5.0±0.05% to pH 5.0±0.1%.

[0027] In one embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94 mM to 96 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 96 mM to 105 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 94 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 96 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 98 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 105 mM sodium chloride. In a related embodiment, the concentration of acetate is 100 mM.

[0028] In one embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0±0.05 to 5.0±0.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0. In a related embodiment, the concentration of acetate is 100 mM.

[0029] In one embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9, 5.0, or 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 5.0.

[0030] In one embodiment, there is at least one additional wash step using a different wash buffer. In one embodiment, the at least one additional wash is a second wash. In one embodiment, the at least one additional wash buffer comprises 0-26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 0, 23, 24, 25, or 26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 0 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 23 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 24 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 25 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 26 mM sodium chloride.

[0031] In one embodiment of the present invention, at least one additional wash buffer comprises acetate. In one embodiment, at least one additional wash buffer comprises acetate at a pH of 4.9-5.1. In one embodiment, at least one additional wash buffer comprises acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, at least one additional wash buffer comprises acetate at a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, at least one additional wash buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, at least one additional wash buffer comprises 100 mM acetate. In one embodiment, at least one additional wash buffer comprises 100 mM acetate at a pH of 4.9-5.1. In one embodiment, at least one additional wash buffer comprises 100 mM acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, the at least one additional wash buffer comprises 100 mM acetate at pH 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, the at least one wash buffer comprises 100 mM acetate at pH 5.0±0.05% to pH 5.0±0.1%.

[0032] In one embodiment, at least one wash buffer comprises acetate and sodium chloride, followed by at least one additional wash. In one embodiment, at least one wash buffer comprises acetate and 94-105 mM sodium chloride, followed by at least one additional wash. In one embodiment, at least one wash buffer comprises acetate and 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 23-26 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 0 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 23 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 24 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 25 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises 26 mM sodium chloride.

[0033] In one embodiment, at least one wash buffer comprises acetate and sodium chloride, followed by at least one additional wash. In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash comprising acetate. In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 23-26 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 0 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 23 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 24 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 25 mM sodium chloride. In a related embodiment, at least one additional wash buffer comprises acetate, 26 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94 mM sodium chloride, followed by at least one additional wash buffer comprising 23-24 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 96 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 25 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 98 mM sodium chloride, followed by an additional wash buffer comprising acetate, 26 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 0 mM sodium chloride. In one embodiment, the concentration of acetate is 100 mM.

[0034] In one embodiment, the additional wash is a second wash. In a related embodiment, the second wash buffer comprises 0-26 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by a second wash buffer comprising acetate, 0-26 mM sodium chloride. In one embodiment, the concentration of acetate is 100 mM.

[0035] In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM sodium chloride, where the pH of the buffers is the same or different. In one embodiment, the pH of one or more wash buffers is between pH 4.9 and 5.1. In one embodiment, the pH of one or more wash buffers is between pH 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, the pH of one or more wash buffers is between pH 4.9, 5.0, or 5.1. In one embodiment, the pH of one or more wash buffers is pH 5.0. In one embodiment, the pH of one or more wash buffers is between pH 5.0±0.05% and pH 5.0±0.1%. In one embodiment, the acetate concentration of one or more wash buffers is 100 mM.

[0036] In one embodiment, the equilibration buffer contains 94 to 96 mM sodium chloride. In one embodiment, the equilibration buffer contains 96 to 105 mM sodium chloride. In one embodiment, the equilibration buffer contains 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment, the equilibration buffer contains 94 mM sodium chloride. In one embodiment, the equilibration buffer contains 96 mM sodium chloride. In one embodiment, the equilibration buffer contains 105 mM sodium chloride.

[0037] In one embodiment of the present invention, the equilibration buffer comprises acetate. In one embodiment, the equilibration buffer comprises acetate at a pH of 4.9 to 5.1. In one embodiment, the equilibration buffer comprises acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the equilibration buffer comprises acetate at a pH of 5.0. In one embodiment, the equilibration buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, the equilibration buffer comprises 100 mM acetate. In one embodiment, the equilibration buffer comprises 100 mM acetate at a pH of 4.9 to 5.1. In one embodiment, the equilibration buffer comprises 100 mM acetate at a pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment, the equilibration buffer comprises 100 mM acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the equilibration buffer comprises 100 mM acetate at pH 5.0±0.05% to pH 5.0±0.1%.

[0038] In one embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride. In one embodiment, the equilibration buffer comprises acetate, 94 mM to 96 mM sodium chloride. In one embodiment, the equilibration buffer comprises acetate, 96 mM to 105 mM sodium chloride. In a related embodiment, the equilibration buffer comprises acetate, 94 mM sodium chloride. In a related embodiment, the equilibration buffer comprises acetate, 96 mM sodium chloride. In a related embodiment, the equilibration buffer comprises acetate, 105 mM sodium chloride. In one embodiment, the concentration of acetate is 100 mM.

[0039] In one embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0±0.05 to 5.0±0.1. In a related embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0. In one embodiment, the concentration of acetate is 100 mM.

[0040] In one embodiment, the equilibration buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, the equilibration buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9 to 5.1. In a related embodiment, the equilibration buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9, 4.95, 5.0, 5.01, or 5.1. In a related embodiment, the equilibration buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 4.9, 5.0, or 5.1. In a related embodiment, the equilibration buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, and a pH of 5.0.

[0041] In one embodiment, the composition is loaded at 10-27 g / L. In a related embodiment, the composition is loaded at 10-25 g / L. In a related embodiment, the composition is loaded at 10-23 g / L. In a related embodiment, the composition is loaded at 10-15 g / L. In a related embodiment, the composition is loaded at 15-27 g / L. In a related embodiment, the composition is loaded at 15-25 g / L. In a related embodiment, the composition is loaded at 15-23 g / L. In a related embodiment, the composition is loaded at 23-27 g / L. In a related embodiment, the composition is loaded at 23-25 ​​g / L. In a related embodiment, the composition is loaded at 25-27 g / L. In one embodiment, the composition is loaded at 10, 15, 23, 25, or 27 g / L. In one embodiment, the composition is loaded at 10 g / L. In one embodiment, the composition is loaded at 15 g / L. In one embodiment, the composition is loaded at 23 g / L, in one embodiment, the composition is loaded at 25 g / L, in one embodiment, the composition is loaded at 27 g / L.

[0042] In one embodiment, the multispecific protein is eluted from the cation exchange resin by a gradient. In a related embodiment, the gradient is linear. In a related embodiment, the gradient is a salt gradient.

[0043] In a related embodiment, the low pI impurities are product-related impurities. In a related embodiment, at least one product-related impurity is a half antibody or a 2X, 3X, or 4X light chain misassembly.

[0044] In one embodiment, the multispecific protein is a bispecific protein. In one embodiment, the multispecific protein is a bispecific antibody.

[0045] In one embodiment, the cation exchange chromatography medium is a resin. In one embodiment, the second chromatography medium is a resin. In a related embodiment, the second chromatography resin is selected from an anion exchange chromatography resin, a cation exchange chromatography resin, a multimodal chromatography resin, a hydrophobic interaction chromatography resin, and a hydroxyapatite chromatography resin.

[0046] The present invention provides purified multispecific proteins produced according to the methods described herein.The present invention provides isolated and purified recombinant multispecific proteins made according to the methods described herein.

[0047] The present invention provides pharmaceutical compositions comprising a recombinant multispecific protein of interest isolated and purified according to the methods described herein.

[0048] The terms "multispecific," "multispecific protein," and "multispecific antibody" are used interchangeably herein to refer to proteins engineered to simultaneously bind to and neutralize at least two different antigens or at least two different epitopes on the same antigen. For example, multispecific proteins can be engineered to target immune effectors and cytotoxic agents to tumors or infectious agents. These multispecific proteins have been found to be useful in a variety of applications, such as redirecting immune effector cells to tumor cells, blocking signaling pathways to alter cell signaling, targeting tumor angiogenesis, and cancer immunotherapy by blocking cytokines, as well as for the delivery of chemotherapeutic agents, radiolabeling (to improve detection sensitivity), and as pretargeting delivery vehicles for drugs such as nanoparticles (targeted to specific cells / tissues, such as cancer cells).

[0049] The most common and most diverse multispecific proteins are those that bind two antigens and are referred to herein as "bispecifics," "bispecific proteins," and "bispecific antibodies." Bispecific proteins can be classified into two broad categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), and the Fc region improves solubility and stability and helps facilitate some purification procedures. Non-IgG-like molecules are smaller, enhancing tissue penetration. (See Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015; Williams et al., Chapter 41 Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al., eds., 2018, pages 837-855.) Bispecific proteins may have binding specificities for different antigens or several epitopes and be used as a framework for additional components that increase the binding specificity of the molecule.

[0050] Formats for bispecific proteins, including bispecific antibodies, are constantly evolving and include quadromas, knob-in-holes, cross-mAbs, dual variable domain IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CH3 KIH, dual acting Fab (DAF), half molecule exchange, κλ-bodies, tandem scFv, scFv-Fc, diabodies, single chain diabodies (sc diabodies), sc diabody-CH3, triple bodies, miniantibodies, minibodies, TriBi minibodies, tandem diabodies, sc diabody-HAS, tandem scFv-toxins, dual affinity retargeting molecules (DART), nanobodies, nanobody-HSA, and docks. -and-lock (DNL), strand exchange engineered domain SEED body, Triomab, Leucine Zipper (LUZ-Y), XmAb®; Fab-arm exchange, DutaMab, DT-IgG, charge pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-VV(L)-IgG, KIH IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (four functions), Fab-scFv, scFv-CH-CL-scFV, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, single chain bispecific antibody construct, single chain bispecific T cell engager (BITE), bispecific T cell engager and half-life extended bispecific T cell engager (HLE BiTE) (Fan supra; Spiess supra; Sedykh supra; Seimetz et al., Cancer Treat Rev 36(6)458-67, 2010; Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011).

[0051] In some embodiments, the bispecific protein is selected from the group consisting of blinatumomab, catumaxomab, ertumaxomab, solitomab, targomiR, rutikizumab (ABT981), vanucizumab (RG7221), memtolumab (ABT122), ozoralizumab (ATN103), flotetuzumab (MGD006), pasotuximab (AMG112, MT112), lymphomunin (FBTA0 5), (ATN-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, Indium-labeled IMP-2 05, xm734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013(ACE910), RG7597(MEDH7945A ), RG7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352 and molecules or variants or analogs thereof and biosimilars of any of the above.

[0052] Multispecific proteins also include triabodies, tetravalent bispecific antibodies, multispecific proteins without antibody components such as diabodies, triabodies or tetrabodies, minibodies, and single-chain proteins that can bind to multiple targets (Coloma, MJ, et al., Nature Biotech. 15 (1997) 159-163).

[0053] In some embodiments, the multispecific protein of interest specifically binds to, neutralizes, and / or interacts with one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factors (CSFs), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxin, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0054] In some embodiments, the multispecific protein of interest is any of the following, alone or in any combination: CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including HER2, HER3, HER4; EGF receptor EGFRvIII; cell adhesion molecules, such as LFA-1, Mol, p150; ,95, VLA-4, ICAM-1, VCAM and αv / β3 integrin, growth factors including, but not limited to, vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors including, for example, aFGF and bFGF, epidermal growth factor ( EGF), crypto, transforming growth factors (TGFs) including TGF-α and TGF-β including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, among others, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone morphogenetic factors, insulin and insulin-related proteins including, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins, among others, Factor VIII. clotting and coagulation-related proteins such as tissue factor, von Willebrand factor, plasminogen activators such as protein C, alpha-1-antitrypsin, urokinase, and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, thrombopoietin receptors, colony-stimulating factors (CSFs) including M-CSF, GM-CSF, and G-CSF, among others, albumin, other blood and serum proteins including, but not limited to, IgE and blood group antigens, e.g., flk2 / flt3 receptor, obesity (OB) receptor,receptors and receptor-associated proteins, including growth hormone receptors, and T-cell receptors; neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin; interferons, including, for example, interferon-α, -β, and -γ; interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL- IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13RA2, which is a receptor for IL-13, or IL-1RAP, which is an IL-17 receptor, viral antigens including, but not limited to, AIDS envelope virus antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation (normal T-cell expressed and secreted), mouse gonadotropin-related peptide, Dnase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay-accelerating factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1,Binds to, neutralizes, and / or interacts with one or more of: mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38 conjugate, Legionella pneumophila (ll), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein Iib / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0055] In some embodiments, a multispecific protein of interest may comprise a bispecific antibody that specifically binds to a combination comprising CD3 and CD19, EpCAM, CEA, PSA, CD33, BCMA, Her2, CD20, P-cadherin, CD123, gpA33, or B7H3, hi some embodiments, a bispecific antibody of interest may comprise a bispecific antibody that specifically binds to a combination comprising IL1α+IL1β.

[0056] Multispecific proteins can be proteins of scientific or commercial interest, particularly bispecific-based therapeutics. Multispecific proteins can be produced by a variety of methods, most commonly by recombinant animal cell lines using cell culture techniques. Multispecific proteins can be produced intracellularly or secreted into the culture medium from which they can be recovered and / or collected, and can be interchangeably referred to as "recombinant multispecific proteins" and "recombinant multispecific antibodies." The terms "isolated multispecific proteins" and "isolated recombinant multispecific antibodies" refer to multispecific proteins that have been purified from proteins, polypeptides, DNA, and / or other contaminants or impurities that would interfere with their therapeutic, diagnostic, prophylactic, research, or other uses. Also included are "recombinant bispecific proteins," "recombinant bispecific antibodies," "isolated recombinant bispecific isolates," and "isolated recombinant bispecific antibodies." Multispecific proteins of interest particularly include multispecific antibodies that exert a therapeutic effect by binding to two or more targets, including those listed below and derived from, related to, and modified versions of, them.

[0057] The present invention provides a method for purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading the composition onto the cation exchange chromatography medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.

[0058] "Purifying" means increasing the purity of the multispecific protein in a composition by removing (partially or completely) at least one impurity from the product. Multispecific protein recovery and purification is carried out by downstream equipment operations, particularly those involving ion exchange chromatography, that result in a more "homogeneous" multispecific protein composition that meets yield and product quality targets (e.g., reduced product-related impurities, increased product quality, etc.).

[0059] "Product-related impurities" refer to product-related variants of the multispecific protein of interest. In some instances, these impurities have a lower pI than the main product in the elution peak. Product-related impurities include, for example, homodimers, half antibodies, aggregates, antibody fragments, and various combinations of antibody fragments, as well as light chain misassemblies, such as 2XLC, 3XLC, or 4XLC, high molecular weight (HMW) species, and low molecular weight (LMW) species. "Half antibodies" refer to product-related impurities that can form, for example, due to incomplete assembly or disruption of the interaction between two heavy chain polypeptides. Half antibodies generally contain one light chain polypeptide and one heavy chain polypeptide. "Homodimers" refer to product-related impurities that can form, for example, when heavy and light chains with specificity for the same target recombine with each other instead of pairing to form the desired bispecific heterodimer, which typically occurs during expression in host cells. For multispecific constructs that require multiple chains (light chains, LCs, etc.) to pair correctly via engineered residues (charge-pairing mutations, knob-holes, etc.), it is still possible to have impurities where there is a mismatch between the LC and HC, but where LC1, instead of pairing with HC1, incorrectly pairs with HC2 (2 x LC1), and vice versa (2 x LC2). If the multispecific protein is bivalent, with two sites for binding each antigen of interest, it is conceivable to have 3 XLC1s, 4 XLC1s, and other combinations of mismatched species.

[0060] The present invention provides a method for reducing low pI impurities in an eluate from cation exchange chromatography, comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading a composition onto the cation exchange chromatography medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pI impurities compared to a cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not used in the equilibration, loading, and washing steps.

[0061] As disclosed herein, the pI of product-related impurities can be similar to the pI of the desired multispecific protein. These product-related impurities are found in the eluate peak comprising the main product. Because they have a slightly lower pI, they are eluted as a pre-peak, just before the main product. The "isoelectric point" or "pI" of a protein refers to the pH at which a positive charge equilibrates with the negative charge of the protein. pI can be calculated / determined using known methods, such as from the net charge of the amino acid residues of the protein or by isoelectric focusing. Product-related impurities with a lower pI than the main product are much more acidic than the main product.

[0062] The present invention provides a method for producing an isolated and purified recombinant multispecific protein of interest, comprising the steps of: establishing a cell culture in a bioreactor using host cells that express the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating viruses at a low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94 to 105 mM sodium chloride; The method includes the steps of loading a neutralized, affinity-purified recombinant multispecific protein onto a cation exchange medium equilibrated in a loading buffer; washing the cation exchange medium with a wash buffer containing 94-105 mM sodium chloride followed by a second wash buffer containing 0-26 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography medium; loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto a second chromatography resin in flow-through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer.

[0063] Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes, or expression cassettes containing at least one nucleic acid encoding a multispecific protein, as well as host cells containing such expression systems or constructs. As used herein, "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, complexed DNA, etc.) suitable for use in transferring and / or transporting multispecific protein-encoding information to a host cell and / or to a specific location and / or compartment within a host cell. Vectors can include viral and non-viral vectors, as well as non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors and cloning vectors. A vector can be introduced into a host cell to allow the vector to replicate itself, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors can contain sequence components that generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These factors can be selected as needed by those skilled in the art.

[0064] A "cell" includes any prokaryotic or eukaryotic cell. Cells may exist ex vivo, in vitro, or in vivo, either in isolation or as part of a higher-order structure such as a tissue or organ. Cells include "host cells," also referred to as "cell lines," that have been engineered to express a multispecific protein of commercial or chemical interest. Host cells are typically derived from lines arising from a primary culture that can be maintained in culture indefinitely. Genetic modification of host cells involves transfecting, transforming, or transducing the cells with a recombinant polynucleotide molecule and / or otherwise modifying (e.g., by homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) the host cell to express the desired recombinant multispecific protein. Methods and vectors for genetically modifying cells and / or cell lines to express a multispecific protein of interest are well known to those skilled in the art.

[0065] Host cells can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., yeast cells, insect cells, or mammalian cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

[0066] In one embodiment, the cell is a host cell. When cultured under appropriate conditions, the host cell expresses the multispecific protein of interest, which can then be recovered from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, protein modifications (such as glycosylation or phosphorylation) that are desirable or essential for activity, and the ease of folding into a biologically active molecule.

[0067] "Culture" or "culturing" refers to the growth and proliferation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. Cell culture medium and tissue culture medium are used interchangeably and refer to a medium suitable for the growth of host cells in in vitro cell culture. Typically, cell culture medium contains buffers, salts, energy sources, amino acids, vitamins, and essential trace elements. Any medium capable of supporting the growth of suitable host cells in culture may be used. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells, are commercially available and include, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as the EX-CELL™ 300 series, which can be obtained from the American Type Culture Collection or SAFC Biosciences and other vendors. Cell culture media may be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell cultures may also be enriched by the addition of nutrients, and may be used at concentrations higher than their normally recommended concentrations.

[0068] Various media formulations may be used during the culture period, for example, to facilitate the transition from one stage (e.g., growth stage or phase) to another (e.g., production stage or phase) and / or to optimize conditions in the cell culture (e.g., concentrated media provided during perfusion culture). Growth media preparations may be used to promote cell growth and minimize protein expression. Production media preparations may be used to promote production of the protein of interest and maintenance of the cells with minimal new cell growth. Feed media consumed during the production phase of the cell culture, typically media containing more concentrated components such as nutrients and amino acids, may be used to supplement and maintain active cultures, particularly cultures operated in fed-batch, semi-perfusion, or perfusion modes. Such concentrated feed media may contain most of the components of cell culture media, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or about 1000x their normal amounts.

[0069] The growth phase can occur at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to about 38°C, and the production phase can occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. Additionally, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, and / or after the temperature shift. If inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift. The pH can also be varied during the culture, either independently or in combination with other methods.

[0070] Host cells can be cultured in suspension or in an attached form attached to a solid culture medium. Cell cultures can be established in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors with or without microcarriers.

[0071] Cell cultures may be operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes. Mammalian cells, such as CHO cells, may be cultured in bioreactors on a small scale, less than 100 ml to less than 1000 ml. Alternatively, large-scale bioreactors containing 1000 ml to over 20,000 liters of medium may be used. Large-scale cell cultures, such as those for clinical and / or commercial-scale biomanufacturing of protein therapeutics, can be maintained for weeks and months while the cells produce the desired protein.

[0072] Because product-related impurities such as homodimers, half antibodies, 2X LC-misalignments, etc. can mimic the desired multispecific proteins, strategies and techniques such as knobs and holes, CrossMab, DVD IgG, and others have been developed to increase selectivity for the desired multispecific proteins in cell culture. However, there will still be some amount of generated product-related impurities that must be removed during downstream processing.

[0073] The resulting expressed recombinant multispecific protein can then be obtained from the cell culture medium. Methods for harvesting proteins from suspension cells are known in the art and include accelerated sedimentation such as acid precipitation, flocculation, gravity separation, centrifugation, sonication, membrane filtration, including but not limited to filtration using ultrafiltration membranes, microfilters, tangential flow filters, alternative tangential flow, depth, and sedimentation filters. Recombinant proteins expressed by prokaryotes are recovered from inclusion bodies in the cytoplasm by a redox folding process known in the art.

[0074] The obtained multispecific protein can then be purified or partially purified from any impurities such as residual cell culture medium, cell extracts, unwanted components, host cell proteins, improperly expressed proteins, product-related impurities, etc. using one or more downstream purification steps.

[0075] Purification of multispecific proteins from harvested cell culture fluid can begin with capture chromatography, e.g., affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), etc., which utilize resins and / or membranes containing agents that will bind to the recombinant protein of interest. Such materials are known in the art and commercially available. Affinity chromatography options can include substrate-binding capture mechanisms, aptamer-binding capture mechanisms, and cofactor-binding capture mechanisms. For multispecific proteins containing an Fc component, antibody- or antibody fragment-binding capture mechanisms such as Protein A, Protein G, and Protein A / G, Protein L, etc. can be used.

[0076] At any point in the downstream process, viral inactivation and / or viral filtration can be performed to remove viral material from the purified multispecific protein solution. One method for achieving viral inactivation is incubation at low pH or other solution conditions to achieve viral inactivation. Low pH viral inactivation can be followed by a neutralization operation to readjust the virally inactivated solution to a pH more compatible with the requirements of the next device operation. Typically, neutralization occurs at a pH of 5-7. The viral inactivated or neutralized viral inactivation pool can then be further subjected to filtration, such as depth filtration, to remove any resulting turbidity or precipitate. Sterile filtration is typically performed in conjunction with depth filtration. Viral filtration can be performed using microfiltration or nanofiltration membranes, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0077] The term "polishing" is used herein to refer to one or more chromatographic steps performed to remove remaining contaminants and impurities such as DNA, host cell proteins, product-specific impurities, mutant products and aggregates, as well as viral adsorption, from a liquid finally containing a recombinant multispecific protein close to the desired purity. Polish chromatography utilizes resins and / or membranes containing agents that can be used in flow-through mode (the protein of interest passes through the resin / membrane, contaminants and impurities bind to the chromatography medium, and the protein of interest is contained in the eluate), frontal or overload chromatography mode (a solution containing the protein of interest is loaded onto the column until the adsorption sites are occupied, and species with the least affinity for the stationary phase (protein of interest) begin to elute), or bind and elute mode (the protein of interest is bound to the chromatography medium and eluted after contaminants and impurities have passed through or been washed off the chromatography medium). Examples of such chromatographic methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and gel filtration. In one embodiment, the chromatographic method is cation exchange chromatography. In one embodiment, the cation exchange medium is a resin.

[0078] The present invention provides a method for performing cation exchange chromatography under high salt load conditions to reduce product-related impurities, comprising the steps of: equilibrating a cation exchange chromatography medium with an equilibration buffer; loading a composition onto the cation exchange chromatography medium in a loading buffer; washing the column with first and second wash buffers; and eluting the multispecific protein from the cation exchange chromatography medium, wherein the equilibration, loading, and wash buffers comprise 94-105 mM sodium chloride.

[0079] "Cation exchange chromatography" refers to chromatography performed on a solid phase material that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase, e.g., by covalent bonding. Alternatively, or in addition, the charge can be an inherent property of the solid phase (e.g., as is the case for silica, which has an overall negative charge). Commercially available cation exchange materials can be used, including resin and membrane absorber media, weak cation exchangers, strong cation exchangers, sulfopropyl (SP) immobilized on agar (e.g., SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, among others, from GE Healthcare), CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (GE Healthcare), FRACTOGEL-SO™, FRACTOGEL-SE HICAP™, FRACTOPREP™ (EMD Merck), Fractogel® EMD SO™ (M), Fractogel® EMD SE Hicap™ (M), Eshmuno® CPX, Eshmuno® S resin, Fractogel® EMD COO™ (M), Mustang S These include, but are not limited to, Acrodisc with Mustang S, AcroPrep with Mustang S, CM Ceramic HyperD® F, and AcroSep with CM Ceramic HyperD® F.

[0080] For the methods of the present invention, cation exchange chromatography is performed in bind and elute mode. The eluate or pool containing the multispecific protein of interest is loaded onto an equilibrated cation exchange medium such that the multispecific protein of interest binds to the cation exchange medium. "Binding" the multispecific protein to the cation exchange material means exposing the multispecific protein to the cation exchange material under appropriate conditions (pH / conductivity) such that the multispecific protein is reversibly immobilized in or on the cation exchange material by ionic interactions between the multispecific protein of interest and one or more charged groups of the cation exchange material. The multispecific protein may be present in an eluate or pool originating from a previous device operation, such as affinity chromatography, neutralized low-pH viral inactivation, depth filtration, or polish chromatography operation.

[0081] The performance of cation exchange chromatography in bind and elute mode for the methods of the invention consists of several steps. In preparation for loading a multispecific protein onto a cation exchange medium, the medium is equilibrated prior to loading with a buffer composition identical to the multispecific protein composition. An "equilibration buffer" is a buffer used to equilibrate the chromatographic material prior to loading a composition containing the multispecific protein of interest.

[0082] After equilibration, the eluate or pool containing the multispecific protein of interest from the previous device run is titrated with a high salt load formulation so that the final conditioned load buffer of the composition contains sodium chloride at the desired concentration. "Loading buffer" and "final conditioned load buffer" are used interchangeably herein. The loading buffer has a suitable formulation so that the multispecific protein of interest binds to the cation exchange material.

[0083] The loaded and bound cation exchange chromatography material is then subjected to multiple washes. Washing the cation exchange material refers to passing an appropriate wash buffer through or over the cation exchange material. The wash buffer removes one or more contaminants, including product-related impurities, without substantial elution of the multispecific protein of interest from the cation exchange material. According to one embodiment of the present invention, there are two wash steps. In one embodiment, there is a "first wash buffer" and a "second wash buffer." The wash buffer is used to wash or re-equilibrate the cation exchange material before eluting the multispecific protein of interest. The one or more wash buffer preparations may be the same as the equilibration and / or final conditioning load buffer preparation. The terms "first wash" and "second wash" should not be construed as referring to one or more additional washes or other buffers between the load and the first and / or second wash steps. Preferably, by the end of the second wash step, the UV baseline has returned to or is at approximately zero prior to the start of elution.

[0084] The present invention provides that the equilibration buffer, the final conditioning loading buffer, and at least one wash buffer have a high salt load formulation, and in one embodiment, all have the same high salt load formulation buffer. In one embodiment, the equilibration buffer, the final conditioning loading buffer, and at least one wash buffer provide 94-105 mM sodium chloride.

[0085] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. In one embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is a 100 mM acetate buffer. In one embodiment, the pH of the buffer is in the range of 5±0.05 to 5.0±0.1. In one embodiment, the range is pH 4.9 to 5.1. In one embodiment, the range is pH 4.95 to 5.05.

[0086] The equilibration buffer, the final conditioning loading buffer, and the at least one wash buffer each further comprise a salt. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is sodium chloride in an amount of about 94 mM to about 105 mM.

[0087] In one embodiment, the loading buffer comprises 96-105 mM sodium chloride. In one embodiment, the loading buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment, the loading buffer comprises 94 mM sodium chloride. In one embodiment, the loading buffer comprises 96 mM sodium chloride. In one embodiment, the loading buffer comprises 105 mM sodium chloride.

[0088] In one embodiment of the present invention, the loading buffer comprises acetate. In one embodiment, the loading buffer comprises acetate at a pH of 4.9 to 5.1. In one embodiment, the loading buffer comprises acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the loading buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, the loading buffer comprises 100 mM acetate. In one embodiment, the loading buffer comprises 100 mM acetate at a pH of 4.9 to 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at a pH of 4, 9, 5.0, or 5.1. In one embodiment, the loading buffer comprises 100 mM acetate at a pH of 5.0±0.05% to 5.0±0.1%.

[0089] In one embodiment, the loading buffer comprises acetate and 94 mM to 105 mM sodium chloride. In one embodiment, the loading buffer comprises acetate and 94 mM to 96 mM sodium chloride. In one embodiment, the loading buffer comprises acetate and 96 mM to 105 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 94 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 96 mM sodium chloride. In a related embodiment, the loading buffer comprises acetate and 105 mM sodium chloride. In one embodiment, the equilibration buffer comprises acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, the loading buffer comprises acetate, 94 mM to 96 mM sodium chloride at a pH of 5.0±0.05. In a related embodiment, the loading buffer comprises acetate, 96 mM to 105 mM sodium chloride at a pH of 5.0±0.05. In a related embodiment, the loading buffer comprises acetate, 96 mM sodium chloride, pH 5.0±0.05. In a related embodiment, the loading buffer comprises acetate, 105 mM sodium chloride, pH 5.0±0.1. In a related embodiment, the loading buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, the loading buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the loading buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0. In a related embodiment, the loading buffer comprises acetate, 94 mM to 96 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, the loading buffer comprises acetate, 96 mM sodium chloride, pH 5.0±0.05. In a related embodiment, the loading buffer comprises acetate, 105 mM sodium chloride, pH 5.0±0.1.

[0090] In one embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, pH 5.0 ± 0.05 to 5.0 ± 0.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 96 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, the loading buffer comprises 100 mM acetate, 96 mM to 105 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, the loading buffer comprises 100 mM acetate, 96 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, the loading buffer comprises 100 mM acetate, 105 mM sodium chloride, pH 5.0 ± 0.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9, 5.0, or 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0. In a related embodiment, the loading buffer comprises 100 mM acetate, 94 mM to 96 mM sodium chloride at a pH of 4.9 to 5.1. In a related embodiment, the loading buffer comprises 100 mM acetate, 96 mM sodium chloride at a pH of 5.0±0.05. In a related embodiment, the loading buffer comprises 100 mM acetate, 105 mM sodium chloride at a pH of 5.0±0.1.

[0091] In one embodiment, at least one wash buffer comprises 94-96 mM sodium chloride. In one embodiment, at least one wash buffer comprises 96-105 mM sodium chloride. In one embodiment, at least one wash buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises 94 mM sodium chloride. In one embodiment, at least one wash buffer comprises 96 mM sodium chloride. In one embodiment, at least one wash buffer comprises 105 mM sodium chloride.

[0092] In one embodiment of the present invention, at least one wash buffer comprises acetate. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.9 to 5.1. In one embodiment, at least one wash buffer comprises acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, at least one wash buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.1. In one embodiment, at least one wash buffer comprises 100 mM acetate. In one embodiment, at least one wash buffer comprises 100 mM acetate at a pH of 4.9 to 5.1. In one embodiment, at least one wash buffer comprises 100 mM acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, at least one wash buffer comprises 100 mM acetate at a pH of 5.0±0.05% to 5.0±0.1%.

[0093] In one embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94 mM to 96 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 96 mM to 105 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 94 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 96 mM sodium chloride. In a related embodiment, at least one wash buffer comprises acetate, 105 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0±0.05 to 5.0±0.1. In a related embodiment, at least one wash buffer comprises acetate, 94 to 96 mM sodium chloride at a pH of 5.0±0.05. In a related embodiment, at least one wash buffer comprises acetate, 96 mM to 105 mM sodium chloride, pH 5.0±0.05. In a related embodiment, at least one wash buffer comprises acetate, 96 mM sodium chloride, pH 5.0±0.05. In a related embodiment, at least one wash buffer comprises acetate, 105 mM sodium chloride, pH 5.0±0.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride, pH 5.0. In a related embodiment, at least one wash buffer comprises acetate, 94 mM to 96 mM sodium chloride, pH 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises acetate, 96 mM sodium chloride, pH 5.0±0.05. In a related embodiment, at least one wash buffer comprises acetate, 105 mM sodium chloride, pH 5.0±0.1.

[0094] In one embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride, pH 5.0 ± 0.05 to 5.0 ± 0.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 96 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 96 mM to 105 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 96 mM sodium chloride, pH 5.0 ± 0.05. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 105 mM sodium chloride, pH 5.0 ± 0.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 4.9, 5.0, or 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 105 mM sodium chloride at a pH of 5.0. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 94 mM to 96 mM sodium chloride at a pH of 4.9 to 5.1. In a related embodiment, at least one wash buffer comprises 100 mM acetate, 96 mM sodium chloride at a pH of 5.0±0.05. In a related embodiment, the at least one wash buffer comprises 100 mM acetate, 105 mM sodium chloride at pH 5.0±0.1.

[0095] In one embodiment, there is at least one additional wash step using a different wash buffer. In one embodiment, the at least one additional wash is a second wash. In one embodiment, the at least one additional wash buffer comprises 0-26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 23-26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 23, 24, 25, or 26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 23 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 24 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 25 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 26 mM sodium chloride. In one embodiment, the at least one additional wash buffer comprises 25 mM sodium chloride at a pH of 5.0±0.05. In one embodiment, the at least one wash buffer comprises acetate and sodium chloride, followed by at least one additional wash. In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash. In one embodiment, at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM sodium chloride. In one embodiment, the additional wash is a second wash. In a related embodiment, the second wash buffer comprises 0-26 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate, 94-96 mM sodium chloride, followed by at least one additional wash buffer comprising 25 mM sodium chloride. In one embodiment, at least one wash buffer comprises acetate and 105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate.

[0096] In one embodiment, at least one wash buffer comprises 100 mM acetate, 94-105 mM sodium chloride at a pH of 5.0±0.05, followed by at least one additional wash. In a related embodiment, the additional wash is a second wash. In a related embodiment, the second wash buffer comprises 0-26 mM sodium chloride. In one embodiment, at least one wash buffer comprises 100 mM acetate, 94-96 mM sodium chloride at a pH of 5.0±0.05, followed by at least one additional wash buffer comprising 100 mM hydrochloride, 25 mM sodium chloride at a pH of 5.0±0.05. In one embodiment, at least one wash buffer comprises 100 mM acetate, 105 mM sodium chloride at a pH of 5.0±0.1, followed by at least one additional wash comprising 100 mM hydrochloride at a pH of 5.0±0.1.

[0097] The bound multispecific protein is then eluted from the cation exchange chromatography material. The multispecific protein may be eluted by a gradient. The multispecific protein may be eluted from the cation exchange material by a linear or step gradient. Preferably, the gradient is a salt gradient. The gradient is created using at least two elution buffers, where the combination of these buffers has a substantially increased conductivity such that the multispecific protein of interest is eluted from the cation exchange material. Preferably, the conductivity of the gradient is greater than the equilibrium conductivity and the equilibrium conductivity of each of the preceding buffers.

[0098] The cation exchange chromatography eluate can be subjected to further polish chromatography purification steps, preferably at least one additional polish chromatography step. Preferably, the multispecific protein of interest is applied to the chromatographic material in flow-through mode.

[0099] Buffer exchange into the desired formulation buffer for concentration of the purified multispecific protein and bulk storage of the drug substance can be achieved by ultrafiltration and diafiltration operations. Viral filtration can also be performed at any point during the downstream process.

[0100] Key attributes and performance parameters of purified multispecific proteins can be measured to better inform decisions regarding the performance of each step during production. These key attributes and parameters can be monitored in real time, near real time, and / or after the fact. Key parameters such as consumed medium components (e.g., glucose), metabolic levels of accumulating by-products (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content, can be measured during cell culture. Key attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, yield, titer, concentration, viability, activity, etc. can be monitored during appropriate stages in the manufacturing process. Monitoring and measurement can be performed using known techniques and commercially available equipment.

[0101] Pharmaceutical compositions (solutions, suspensions, etc.) may include, but are not limited to, one or more of the following: buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, dextran, mannitol, etc.; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0102] While the terminology used herein is standard within the art, definitions of certain terms are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI-accepted form. Numerical ranges recited herein are inclusive of the numbers defining the range and include and support each integer within the defined range. Unless otherwise specified, the methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and journal articles, are expressly incorporated herein by reference. Anything described in an embodiment of the invention may be combined with other embodiments of the invention.

[0103] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. [Example]

[0104] Example 1 Salt-Free Bispecific #1 The neutralized Protein A pool containing fully human bispecific, engineered immunoglobulin (bispecific #1) in acetate buffer was loaded onto Eshmuno CP-FT® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 1.

[0105] [Table 1]

[0106] Figure 1 shows that several impurities remained on the column and eluted with the main product. These impurities were similar in charge (isoelectric point) to the main product and included half antibodies, 2X light chain misassemblies, and high molecular weight (HMW) impurities.

[0107] Example 2 High Salt Conditioning Bispecific #1 The neutralized virus inactivated pool containing bispecific #1 (100 mM acetate, pH 5.0) was combined with loading buffer (100 mM acetate, 350 mM sodium chloride, pH 5.00) in a ratio of 1:0.378 to yield a final conditioned load of 100 mM acetate, 96 mM sodium chloride, pH 5.00. The conditioned load was loaded onto Capto-SP ImpRes® cation exchange chromatography resin under the conditions described in Table 2.

[0108] [Table 2]

[0109] When high-salt loading conditions were used, it was found that low-pI impurities were removed from the resin before elution. Figure 2 shows that high-salt loading conditions resulted in a reduction in the number of impurity peaks in the elution profile from four to one (the center point is shown). The majority of impurities were removed between loading and the second wash step. Half the mAb flowed through the resin during loading or only slightly bound to the column. 2X LC was removed from the column or only slightly bound to the column after the first wash step. The second wash step provided full binding conditions for remaining protein species, reestablished the UV baseline to zero before the start of elution, and stabilized the elution profile, resulting in much more efficient collection and better quality of the main product.

[0110] The equilibration buffer, final conditioning load, and first wash buffer were tested at sodium chloride concentrations ranging from 94 to 98 mM with similar results. The pH of the equilibration buffer, final conditioning load, and two wash buffer preparations were tested at various concentrations ranging from 4.95 to 5.05 with similar results. Load concentrations ranging from 5 to 27 mg / ml were tested with similar results.

[0111] Overall, the high-salt load had a lower yield (approximately 60%) compared to the no-salt load condition. This is because the no-salt load run allows for fractionation of the elution, allowing for greater precision and accuracy in controlling the quality of the final product. However, the no-salt load had a steep gradient of 8 mM sodium chloride / CV, which is not optimal for a robust manufacturing process, while the high-salt load had a salt gradient of 16 mM sodium chloride / CV. The high-salt load conditioning allowed for a reduction in the elution length from 44 to 20 column volumes. This reduction in column volume saves time and resources, as the elution gradient is shorter, and also results in a more robust manufacturing process.

[0112] Example 3: High Load Density, Salt-Free Load Conditioning, Bispecific #2 The neutralized Protein A eluate pool containing the fully human, engineered IgG / Fab fusion protein (bispecific #2) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 3.

[0113] [Table 3]

[0114] Figure 3 shows one elution peak resulting from high loading density, salt-free loading conditions with a steep elution gradient. The low pI product-related impurity did not resolve from the main product under high loading density, as evidenced by the CE-SDS LC1:LC2 ratio (mismatched LC1 species) decreasing from 4 to 7 and 2–4% LMW species, mostly in fractions 1, 2, and 3.

[0115] Example 4 Low Load Density, No Salt Load Conditioning, Bispecific #2 The neutralized Protein A eluate pool containing the fully human, engineered IgG / Fab fusion protein (bispecific #2) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 4.

[0116] [Table 4]

[0117] The high loading density, steep elution conditions of Example 3 did not provide sufficient resolution of the main product from the low-pI product-related impurities, so the loading density and gradient conditions were reduced. Figure 4 shows that a lower loading density (10 vs. 25 g / L) and a gentler gradient (8 vs. 16 mM / CV) allowed separation of the major low-pI product impurity into a distinct peak formed by fractions 1–4. This fraction exhibited an LC1:LC2 ratio of 3:10, indicating a mismatched LC1 species. In contrast, the main peak exhibited an accumulated LC1:LC2 ratio of 1.2. While resolution was better, increasing yield from 44% to 73%, it still required automated pooling based on OD, and therefore it would still be necessary to collect the eluate by starting above the highest OD for the pre-peak, reducing yield and making this process unsuitable for use in manufacturing operations.

[0118] Example 5 High Salt Conditioning, Bispecific #2 The neutralized virus inactivated pool containing bispecific #2 was combined with a high salt load buffer (100 mM acetate, 500 mM sodium chloride, pH 5.00) to yield a final conditioned load buffer of 100 mM acetate, 105 mM sodium chloride, pH 5.00. The conditioned load was loaded onto Capto-SP ImpRes® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 4.

[0119] [Table 5]

[0120] When high-salt loading conditions were used, it was found that low-pI impurities were removed from the CEX column prior to elution. These impurities likely corresponded to mismatched LC1 species, given that the LC1 to LC2 ratio for the collected first and second washes was 5.0, compared to the expected ratio of 1 if LC1 and LC2 were exactly similarly paired. Figure 5 shows that under high-salt loading conditions, there was a reduction in the number of impurity peaks in the elution profile from two peaks to a single peak, with a small shoulder (fractions 1–3) still containing the mismatched species (LC1 / LC2 = 2–3). The second wash step reestablished the UV baseline to zero before the start of elution, fixing the elution profile and resulting in much more efficient collection and better quality of the main product. The optimized procedure, with a low loading level and gentle gradient coupled with a high salt conditioning load, allowed for an increased CEX purification yield from 44% to 58%, while allowing for an elution profile to collect purified pools using low levels of mismatched LC1 species (evidenced by an LC1 to LC2 ratio close to 1), HMW and LMW, and absorbance-based pooling criteria.

Claims

1. 1. A method for purifying a multispecific protein from a composition comprising said multispecific protein and at least one product-related impurity, comprising: equilibrating the cation exchange chromatography medium with an equilibration buffer containing 94-105 mM sodium chloride; loading the composition onto the cation exchange medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.

2. 10. The method of claim 1, wherein the loading buffer comprises 94-96 mM sodium chloride.

3. 3. The method of claim 2, wherein the loading buffer comprises 94-105 mM sodium chloride.

4. 3. The method of claim 2, wherein the loading buffer comprises 94 mM sodium chloride.

5. 3. The method of claim 2, wherein the loading buffer comprises 96 mM sodium chloride.

6. 3. The method of claim 2, wherein the loading buffer comprises 98 mM sodium chloride.

7. 3. The method of claim 2, wherein the loading buffer comprises 105 mM sodium chloride.

8. The method of claim 1 , wherein the loading buffer comprises acetate.

9. 9. The method of claim 8, wherein the loading buffer comprises acetate at a pH of 4.9 to 5.

1.

10. 9. The method of claim 8, wherein the loading buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.

1.

11. 9. The method of claim 8, wherein the loading buffer comprises 100 mM acetate.

12. 2. The method of claim 1, wherein the loading buffer comprises acetate, 94-105 mM sodium chloride.

13. 10. The method of claim 1, wherein the at least one wash buffer comprises 94 to 105 mM sodium chloride.

14. The method of claim 13, wherein the at least one wash buffer comprises 94 to 96 mM sodium chloride.

15. The method of claim 13, wherein the at least one wash buffer comprises 96 to 105 mM sodium chloride.

16. 14. The method of claim 13, wherein the at least one wash buffer comprises 94 mM sodium chloride.

17. 14. The method of claim 13, wherein at least one wash buffer comprises 96 mM sodium chloride.

18. 14. The method of claim 13, wherein at least one wash buffer comprises 98 mM sodium chloride.

19. 14. The method of claim 13, wherein at least one wash buffer comprises 105 mM sodium chloride.

20. 10. The method of claim 1, wherein at least one wash buffer comprises acetate.

21. 21. The method of claim 20, wherein the at least one wash buffer comprises acetate at a pH of 4.9 to 5.

1.

22. 21. The method of claim 20, wherein the at least one wash buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.

1.

23. 21. The method of claim 20, wherein at least one wash buffer comprises 100 mM acetate.

24. 10. The method of claim 1, wherein the at least one wash buffer comprises acetate, 94 mM to 105 mM sodium chloride.

25. The method of claim 1 , wherein the method comprises at least one additional wash buffer.

26. 26. The method of claim 25, wherein the at least one additional wash buffer is a second wash buffer.

27. 26. The method of claim 25, wherein the at least one additional wash buffer comprises 0 to 26 mM sodium chloride.

28. 26. The method of claim 25, wherein at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 0-25 mM sodium chloride.

29. 10. The method of claim 1, wherein the at least one equilibration buffer comprises 94 to 105 mM sodium chloride.

30. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 94 to 96 mM sodium chloride.

31. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 96 to 105 mM sodium chloride.

32. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 94 mM sodium chloride.

33. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 96 mM sodium chloride.

34. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 98 mM sodium chloride.

35. 30. The method of claim 29, wherein the at least one equilibration buffer comprises 105 mM sodium chloride.

36. The method of claim 1 , wherein the equilibration buffer comprises acetate.

37. 37. The method of claim 36, wherein the equilibration buffer comprises acetate at a pH of 4.9 to 5.

1.

38. 37. The method of claim 36, wherein the equilibration buffer comprises acetate at a pH of 5.0±0.05 to 5.0±0.

1.

39. 37. The method of claim 36, wherein the equilibration buffer comprises 100 mM acetate.

40. 10. The method of claim 1, wherein the composition is loaded at 10 to 27 g / L.

41. 41. The method of claim 40, wherein the composition is loaded at 15 to 27 g / L.

42. 10. The method of claim 1, wherein the multispecific protein is eluted from the cation exchange resin by a gradient.

43. 43. The method of claim 42, wherein the gradient is linear.

44. 43. The method of claim 42, wherein the gradient is a salt gradient.

45. 10. The method of claim 1, wherein the multispecific protein is a bispecific protein.

46. The method of claim 1 , wherein the multispecific protein is a bispecific antibody.

47. A purified multispecific protein prepared by the method of claim 1.

48. 10. The method of claim 1, wherein the cation exchange chromatography medium is a resin.

49. 1. A method for reducing low pI impurities in an eluate from cation exchange chromatography, comprising: equilibrating the cation exchange chromatography medium with an equilibration buffer containing 94-105 mM sodium chloride; loading the composition onto the cation exchange medium in a loading buffer comprising 94-105 mM sodium chloride; washing the column with at least one wash buffer containing 94-105 mM sodium chloride; and eluting said multispecific protein from said cation exchange chromatography medium; wherein said cation exchange chromatography eluate has reduced low pI impurities compared to said cation exchange chromatography eluate collected in a corresponding method in which sodium chloride is not used in the equilibration, loading and washing steps.

50. 50. The method of claim 49, wherein the low pI impurity is a product-related impurity.

51. 51. The method of claim 50, wherein the at least one product-related impurity is a half antibody or a 2X, 3X, or 4X light chain misassembly.

52. 1. A method for performing cation exchange chromatography under high salt loading conditions to reduce product-related impurities, comprising: equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition onto the cation exchange medium in a loading buffer; washing the column with first and second wash buffers; and eluting said multispecific protein from said cation exchange chromatography medium, wherein said equilibration, loading and first wash buffer comprises 94-105 mM sodium chloride.

53. 53. The method of claim 52, wherein the second wash buffer comprises 0 to 26 mM sodium chloride.

54. 1. A method for producing an isolated and purified recombinant multispecific protein, comprising: establishing a cell culture in a bioreactor with host cells that express the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating viruses in the eluate pool from the affinity purification at a low pH to neutralize the pool; equilibrating the cation exchange chromatography medium with an equilibration buffer containing 94-105 mM sodium chloride; loading the neutralized affinity-purified recombinant multispecific protein onto the equilibrated cation exchange medium in a loading buffer containing 94-105 mM sodium chloride; washing the cation exchange medium with a wash buffer containing 94-105 mM sodium chloride followed by a second wash buffer containing 0-26 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography medium; loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto a second chromatography resin in flow-through mode; and concentrating said purified recombinant multispecific protein in a formulation buffer.

55. 48. The method of claim 47, wherein the second chromatography resin is selected from an anion exchange chromatography resin, a cation exchange chromatography resin, a multimodal chromatography resin, a hydrophobic interaction chromatography resin, and a hydroxyapatite chromatography resin.

56. 56. An isolated and purified recombinant multispecific protein prepared by the method of claim 55.

57. 56. A pharmaceutical composition comprising an isolated and purified recombinant multispecific protein prepared by the method of claim 55.

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