High-salt washing in cation exchange chromatography to remove product-related impurities

Abstract

This invention provides a method for removing low-isoelectric point product-related impurities during cation exchange purification operations. [Solution] A method for purifying a multispecific protein is provided, comprising the steps of: loading a sample containing the multispecific protein onto a cation exchange chromatography medium; washing the cation exchange medium with at least one washing buffer containing 100 to 147 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography resin.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 931,874, filed on November 7, 2019, which is incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceutical manufacturing. Specifically, the present invention relates to a method for removing product - related impurities of multispecific proteins during cation - exchange chromatography purification operations.

Background Art

[0003] Antibody products are the largest sector of the biopharmaceutical market and will easily reach hundreds of billions of dollars in sales within the next decade. The commercial development of therapeutic antibodies began in the 1980s with the approval of the first therapeutic monoclonal antibody and has since continued to evolve and expand. Monoclonal antibodies bind to targets with high affinity and specificity and were a therapeutic treatment with very good results for some applications, but they also have limitations. Monoclonal antibodies can only bind to a single target; however, many diseases are multifactorial. In cancer immunotherapy, a treatment targeting a single target may not be sufficient to destroy or immobilize cancer cells. Furthermore, some patients receiving monoclonal antibody therapy may not respond to the treatment or may eventually develop drug resistance.

[0004] To address these issues, novel antibody - like structures such as antibody Fab fragments, Fc - fusion proteins, antibody - drug conjugates, glycol - modified antibodies, and most particularly bispecific and other multispecific antibody - like structures have been developed. These antibody - like structures, particularly bispecific antibodies, offer improvements over traditional monoclonal antibody therapeutics such as multispecific target affinity and are being proven to be effective next - generation biological therapeutics with a vast number of formats that can be developed to address even more difficult therapeutic applications.

[0005] Bispecific antibodies represent the most diverse group of antibody-like structures, featuring an ever-growing array of frameworks to address the challenges of a broader range of therapeutic applications. These structures combine the binding properties of an antibody with additional molecular properties modified within the framework to suit the needs of the target disease application. Bispecific antibodies are being developed for a wide range of applications and uses, such as for immune responses against cancer, for example, 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 presents challenges in the production of novel biopharmaceuticals, particularly in terms of product instability and low expression yields. Specifically, the purification of multispecific proteins is complicated by the formation of product-related variants such as homodimers, semi-antibodies, aggregates, high-molecular-weight species, and low-molecular-weight species. These product-related variants share similar structural and physical properties, such as charge, with the multispecific protein of interest, making their separation during purification difficult. These product-related impurities reduce the yield and activity of multispecific drug formulations. [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 [Overview of the project] [Problems that the invention aims to solve]

[0008] Product-related impurities having a similar charge (isoelectric point, pI) to the multispecific protein of interest may co-elute with the multispecific protein during cation exchange chromatography operations, complicating purification and reducing yield. It would be beneficial to separate low-pI product-related impurities before elution. The present invention, as described herein, satisfies this need by providing a high-salt washing conditioning for removing these low-pI impurities during cation exchange chromatography. [Means for solving the problem]

[0009] The present invention provides a method for purifying a multispecific protein, comprising the steps of: loading a sample containing the multispecific protein onto a cation exchange chromatography medium; washing the cation exchange medium with at least one washing buffer containing 100 to 147 mM sodium chloride; and eluting the multispecific protein from a cation exchange chromatography resin. In one embodiment, at least one washing buffer contains 100 to 125 mM sodium chloride. In one embodiment, at least one washing buffer contains 100 to 105 mM sodium chloride. In one embodiment, at least one washing buffer contains 105 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains 105 to 125 mM sodium chloride. In one embodiment, at least one washing buffer contains 125 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate. In a related embodiment, at least one washing buffer contains acetate at pH 5.0 ± 0.05% to pH 5.0 ± 0.1%. In one related embodiment, at least one wash buffer contains an acetate with a pH of 4.91 to 5.1. In another related embodiment, at least one wash buffer contains an acetate with a pH of 4.9, 5.0, or 5.1. In yet another related embodiment, the wash buffer contains 100 mM acetate. In one embodiment, at least one wash buffer contains an acetate and 100 to 125 mM sodium chloride. In one embodiment, at least one wash buffer contains an acetate and 100 to 105 mM sodium chloride. In one embodiment, at least one wash buffer contains an acetate and 105 to 147 mM sodium chloride. In one embodiment, at least one wash buffer contains an acetate and 105 to 125 mM sodium chloride. In one embodiment, at least one wash buffer contains an acetate and 125 to 147 mM sodium chloride.

[0010] In one embodiment, the cation exchange medium is washed using at least two types of washing buffers. In another embodiment, the cation exchange medium is washed using at least three types of washing buffers.

[0011] In one embodiment, the cation exchange medium is washed with at least two wash buffers, at least one of which contains 0 to 147 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with at least two wash buffers, at least one of which contains 0 to 70 mM sodium chloride. In one embodiment, the cation exchange medium is washed with at least two wash buffers, at least one of which contains acetate and 0 mM sodium chloride, and the next wash buffer contains acetate and 100 to 147 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with a wash buffer containing acetate and 0 mM sodium chloride, and the next wash buffer is selected from the group consisting of a wash buffer containing acetate and 100 mM sodium chloride, a wash buffer containing acetate and 105 mM sodium chloride, or a wash buffer containing acetate and 125 mM sodium chloride. In one embodiment, the cation exchange medium is washed with a washing buffer containing acetate and 100-147 mM sodium chloride, and then washed with a washing buffer containing acetate and 0-70 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with a washing buffer containing acetate and 100-147 mM sodium chloride, and then washed with a washing buffer containing acetate and 0 mM sodium chloride. In another related embodiment, the cation exchange medium is washed with a washing buffer containing acetate and 100-147 mM sodium chloride, and then washed with a washing buffer containing 70 mM sodium chloride.

[0012] In one embodiment, the cation exchange medium is washed with at least three wash buffers, the first wash buffer containing acetate and 0 mM sodium chloride, followed by a second wash buffer containing acetate and 100-147 mM sodium chloride, followed by a third wash buffer containing acetate and 0 mM sodium chloride, or a wash buffer containing acetate and 70 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with a first wash buffer containing acetate and 0 mM sodium chloride, followed by a second wash buffer selected from the group consisting of a wash buffer containing acetate and 100 mM sodium chloride, a wash buffer containing acetate and 105 mM sodium chloride, a wash buffer containing acetate, or a wash buffer containing acetate and 125 mM sodium chloride, followed by a third wash buffer containing acetate and 0 mM sodium chloride. In one related embodiment, the cation exchange medium is washed with a first washing buffer containing acetate and 0 mM sodium chloride, followed by a second washing buffer containing acetate and 147 mM sodium chloride, then a washing buffer containing acetate, and then a third washing buffer containing acetate and 70 mM sodium chloride.

[0013] In one embodiment, the cation exchange medium is washed with a 2.5 mM / CV washing buffer containing 147 mM sodium chloride.

[0014] In one embodiment, the multispecific protein is eluted from the cation exchange resin by a gradient. In one related embodiment, the gradient is linear or stepwise. In one related embodiment, the gradient is a salt gradient. In one related embodiment, the buffer used to form the elution gradient contains 0 to 1 M sodium chloride. In another related embodiment, at least one buffer used to form the elution gradient contains 70 to 500 mM sodium chloride. In yet another related embodiment, at least one buffer used to form the elution gradient contains 125 mM sodium chloride. In one related embodiment, at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

[0015] In one embodiment, the cation exchange medium is loaded with at least 10 g / L of multispecific protein. In another embodiment, the cation exchange medium is loaded with 10 g / L to 40 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 15 g / L to 30 g / L. In a related embodiment, the cation exchange medium is loaded with 25 g / L to 40 g / L. In one embodiment, the cation exchange medium is loaded with 10 g / L of multispecific protein, washed with a wash buffer containing 105 mM sodium chloride, and eluted with a salt gradient of 8 mM / CV. In another embodiment, the cation exchange medium is loaded with 15 g / L to 30 g / L of multispecific protein and washed with a wash buffer containing 147 mM sodium chloride. In another embodiment, the cation exchange medium is loaded with 25 g / L to 40 g / L of multispecific protein, where at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

[0016] In one embodiment, at least one product-related impurity is a homodimer, a high molecular weight species, a semi-antibody, an aggregate, a low molecular weight species, an antibody fragment, or a light chain misassembly. In one embodiment, a purification process including instrumentation involving cation exchange chromatography was carried out according to the method described above.

[0017] In one embodiment, the above method further includes one or more instrumental operations for purifying multispecific proteins, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography columns, and / or mixed-mode chromatography columns, before and / or after the cation exchange chromatography step.

[0018] In one embodiment, the multispecific protein is a bispecific protein. In one embodiment, the multispecific protein is a bispecific antibody. In one embodiment, the purified multispecific protein is produced according to the method described above. In one embodiment, the cation exchange chromatography medium is a resin.

[0019] The present invention provides a method for reducing low pI product-related impurities in an eluate from cation exchange chromatography, comprising the steps of: loading a composition containing a multispecific protein and at least one product-related impurity having a lower pI than the multispecific protein onto a cation exchange chromatography medium; washing the cation exchange medium with a first washing buffer; washing the cation exchange medium with a second washing buffer containing 100 to 147 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography resin; wherein the cation exchange chromatography eluate has reduced pI product-related impurities compared to a cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not included in the washing buffer preparation. In one embodiment, the cation exchange medium is washed with three washing buffers.

[0020] The present invention provides a method for performing cation exchange chromatography under high-salt washing conditions to reduce product-related impurities, comprising the steps of: loading a composition containing a multispecific protein and at least one product-related impurity onto an equilibrated cation exchange column; washing the cation exchange medium with at least two washing buffers, one of which contains 100 to 147 mM sodium chloride; and eluting the bound multispecific protein from the cation exchange chromatography resin. In one embodiment, before loading the composition, the cation exchange medium is equilibrated with a buffer that does not contain sodium chloride.

[0021] The present invention provides a method for producing an isolated and purified recombinant multispecific protein, comprising the steps of: establishing cell culture in a bioreactor using a host cell expressing the multispecific protein; culturing the host cell to express the multispecific protein; harvesting the recombinant multispecific protein from the cell culture; affinity purifying the recombinant multispecific protein; loading the affinity-purified recombinant multispecific protein onto a cation exchange chromatography resin; washing the cation exchange resin with at least one washing buffer containing 100 to 147 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography resin; and loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto an additional chromatography medium in flow-through mode. In one embodiment, the additional chromatography medium is selected from a cation exchange chromatography medium, a multimodal chromatography medium, a hydrophobic interaction chromatography medium, and a hydroxyapatite chromatography medium. In one embodiment, the affinity-purified multispecific protein is in an eluate pool, subjected to low pH virus inactivation, and then followed by neutralization prior to the step of loading onto the cation exchange medium. In one embodiment, the flow-through from the third chromatography medium is subjected to ultrafiltration and dialysis unit operation. In one embodiment, the isolated and purified recombinant multispecific protein is produced according to the method described above. In one embodiment, a pharmaceutical composition containing the isolated and purified recombinant multispecific protein is produced using the above method. Brief Description of the Drawings

[0022] [Figure 1] It is a diagram showing product-related impurities (homodimer, NCG) eluted together with the bispecific #1 as the main product. [Figure 2]Impurities related to the total product, which had a lower pI than the main product, were washed away from the column between the second and third washing steps after high-salt washing conditions. The baseline recovered to zero before elution. The elution peak decreased to a single peak. [Figure 3] This figure shows that multiple impurities remained on the column and were eluted along with the main product. These impurities included half-antibodies (fractions 1-4, containing approximately 50% half-antibodies), 2X light chain misassemblies (fractions 5 and 6), and high molecular weight molecules (fractions 12-21). [Figure 4] The figure shows that high-salinity washing resulted in a reduction in the number of peaks in the elution profile from four peaks to a single peak with a small shoulder still containing 2X LC1 / LC2 mispairing species (LC1 / LC2 <0.11 compared to the predicted ratio of 1). [Figure 5] This figure shows a single elution peak resulting from a high loading density with a steep elution gradient. Product-related impurities with low pI do not dissipate from the main product under high loading densities and are mostly located within fractions 1-3, as evidenced by the reduced CE-SDS. [Figure 6] The process of reducing the load density using a gentler gradient resulted in the separation of a distinct peak containing mispaired LC1 of the main product impurities with low pI. [Figure 7] This figure shows the results of high-salinity washing. The number of impurity peaks in the elution profile decreased, indicating that most of the impurities were removed during the second and third washing steps. The third washing step re-established 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. [Figure 8] After high-salinity washing conditions, all product-related impurities with low pI (homodimeric species with pI of 6.8 and any agglutinating species with low pI) were either flushed through the column into the waste or collected separately in a “wash pool” for testing their content. The baseline recovered to zero before elution. The elution peak decreased to a single peak. [Modes for carrying out the invention]

[0023] Due to limited literature on 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). Multispecific proteins are highly modified, and the process of subjecting such proteins to cation exchange chromatography (CEX) in binding and elution modes under typical antibody conditions may not be sufficient to stabilize the multispecific proteins and / or control product-related impurities eluted with the main product. These product-related impurities have both lower and higher isoelectric points than the main product. Those product-related impurities with lower pI than the main product have been found to elute as pre-peaks along with the main product. Such elution profiles do not support the development of robust, sustainable, commercial-scale manufacturing methods.

[0024] The use of a high-salt washing strategy was found to improve the manufacturing process by resulting in better yield and purity of the main product. The addition of high-salt washing reduced lower pI product-related impurities in the eluate pool by removing them before the elution step. The addition of a washing step using sodium chloride in the range of 105–147 mM resulted in product-related impurities with lower pI than the main product being washed away before the elution step or eluted from the cation exchange medium, reducing the number of peaks in the elution profile. Automated pooling of eluates based on OD becomes difficult when pre-peaks are present. Incorporating a high-salt washing step into the cation exchange chromatography protocol was found to reduce pre-peaks associated with low pI product-related impurities before elution, which would likely result in lower yields and a less robust manufacturing process, thus reducing the need to use more conservative strategies during the collection of the main product during elution.

[0025] The present invention provides a method for purifying multispecific proteins, comprising the steps of: loading a sample containing multispecific proteins onto a cation exchange chromatography medium; washing the cation exchange medium with at least one washing buffer containing 100 to 147 mM sodium chloride; and eluting the multispecific proteins from a cation exchange chromatography resin.

[0026] The present invention provides a method for reducing low pI product-related impurities in an eluate from cation exchange chromatography, comprising the steps of: loading a composition containing a multispecific protein and at least one product-related impurity having a lower pI than the multispecific protein onto a cation exchange chromatography medium; washing the cation exchange medium with a first washing buffer; washing the cation exchange medium with a second washing buffer containing 100 to 147 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography resin; wherein the cation exchange chromatography eluate has reduced levels of low pI product-related impurities compared to a cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not present in the washing buffer preparation.

[0027] The present invention provides a method for performing cation exchange chromatography under high-salt washing conditions to reduce product-related impurities, comprising the steps of: loading a composition containing a multispecific protein and at least one product-related impurity onto an equilibrated cation exchange column; washing the cation exchange medium with at least two washing buffers, wherein one washing buffer contains 100 to 147 mM sodium chloride; and eluting the bound multispecific protein from the cation exchange chromatography resin.

[0028] 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 expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein from the cell culture; affinity purification of the recombinant multispecific protein; loading the affinity purified recombinant multispecific protein onto a cation exchange chromatography resin; washing the cation exchange chromatography resin with at least one washing buffer containing 100 to 147 mM sodium chloride; eluting the multispecific protein from the cation exchange chromatography resin; and loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto a third chromatography medium in flow-through mode.

[0029] The present invention provides a purification process comprising instrumental operations including cation exchange chromatography carried out according to the method described above.

[0030] The present invention provides purified multispecific proteins prepared according to the methods disclosed herein.

[0031] The present invention provides a pharmaceutical composition comprising an isolated and purified recombinant multispecific protein prepared according to any of the methods described herein.

[0032] In one embodiment, at least one washing buffer contains 0 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains 70 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains 100 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains 100 to 125 mM sodium chloride. In one embodiment, at least one washing buffer contains 100 to 105 mM sodium chloride. In one embodiment, at least one washing buffer contains 105 to 147 mM sodium chloride. In one embodiment, at least one washing buffer contains 105 to 125 mM sodium chloride. In one embodiment, at least one washing buffer contains 125 to 147 mM sodium chloride. In one embodiment, at least one wash buffer contains 0 mM sodium chloride. In one embodiment, at least one washing buffer contains 70 mM sodium chloride. In one embodiment, at least one washing buffer contains 100 mM sodium chloride. In one embodiment, at least one washing buffer contains 105 mM sodium chloride. In one embodiment, at least one washing buffer contains 125 mM sodium chloride. In one embodiment, at least one washing buffer contains 147 mM sodium chloride.

[0033] In one embodiment, at least one washing buffer contains acetate. In one embodiment, at least one washing buffer contains acetate at pH 5.0±0.05 to 5.0±0.1. In one embodiment, at least one washing buffer contains acetate at pH 4.9 to 5.1. In one embodiment, at least one washing buffer contains acetate at pH 4.9, 5.0, or 5.1. In one embodiment, at least one washing buffer contains 100 mM acetate. In one embodiment, at least one washing buffer contains acetate at pH 5.0±0.05% to pH 5.0±0.1%. In one embodiment, at least one washing buffer contains acetate at pH 4.9 to 5.1. In one embodiment, at least one washing buffer contains acetate at pH 4.9, 5.0, or 5.1. In one embodiment, at least one washing buffer contains acetate at pH 5.0. The washing buffer may have a pH range of 0.05 to 0.1 higher or lower, accompanied by variations in conductivity, in order to robustly remove product-related impurities.

[0034] In one embodiment, at least one washing buffer contains acetate and 0-147 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 70-147 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 100-147 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 100-125 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 100-105 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 105-147 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 105-125 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 125-147 mM sodium chloride. In one related embodiment, the concentration of acetate is 100 mM.

[0035] In one embodiment, at least one washing buffer contains acetate and 0 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 70 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 100 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 105 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 125 mM sodium chloride. In one embodiment, at least one washing buffer contains acetate and 147 mM sodium chloride. In a related embodiment, the concentration of acetate is 100 mM.

[0036] In one embodiment, at least one washing buffer contains acetate and 0 to 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one embodiment, at least one washing buffer contains acetate and 70 to 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one embodiment, at least one washing buffer contains acetate and 100 to 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one embodiment, at least one washing buffer contains acetate and 100 to 125 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In a related embodiment, at least one washing buffer contains acetate and 100 to 105 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one related embodiment, at least one washing buffer contains acetate and 105 to 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one related embodiment, at least one washing buffer contains acetate and 105 to 125 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one related embodiment, at least one washing buffer contains acetate and 125 to 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one related embodiment, the concentration of acetate is 100 mM.

[0037] In one related embodiment, at least one washing buffer contains acetate and 0 mM sodium chloride at a pH of 5.0±0.05% to 5.0±0.1%. In one related embodiment, at least one washing buffer contains acetate and 70 mM sodium chloride at a pH of 5.0±0.05% to 5.0±0.1%. In one related embodiment, at least one washing buffer contains acetate and 100 mM sodium chloride at a pH of 5.0±0.05% to 5.0±0.1%. In one related embodiment, at least one washing buffer contains acetate and 105 mM sodium chloride at a pH of 5.0±0.05% to 5.0±0.1%. In one related embodiment, at least one washing buffer contains acetate and 125 mM sodium chloride at a pH of 5.0±0.05% to 5.0±0.1%. In one related embodiment, at least one washing buffer contains an acetate and 147 mM sodium chloride at a pH of 5.0 ± 0.05% to 5.0 ± 0.1%. In one related embodiment, the concentration of the acetate is 100 mM.

[0038] In one related embodiment, at least one washing buffer contains acetate and 0 to 147 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 70 to 147 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 to 147 mM sodium chloride at pH 4.9 to 5.1. In one embodiment, at least one washing buffer contains acetate and 100 to 125 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 to 105 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 105 to 147 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 105-125 mM sodium chloride at pH 4.9-5.1. In another related embodiment, at least one washing buffer contains acetate and 125-147 mM sodium chloride at pH 4.9-5.1. In yet another related embodiment, the concentration of acetate is 100 mM.

[0039] In one related embodiment, at least one washing buffer contains acetate and 0 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 70 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 105 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 125 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, at least one washing buffer contains acetate and 147 mM sodium chloride at pH 4.9 to 5.1. In one related embodiment, the concentration of acetate is 100 mM.

[0040] In one related embodiment, at least one washing buffer contains acetate and 0 to 147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 70 to 147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 to 147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one embodiment, at least one washing buffer contains acetate and 100 to 125 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 to 105 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 105 to 147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 105-125 mM sodium chloride at pH 4.9, 5.0, or 5.1. In another related embodiment, at least one washing buffer contains acetate and 125-147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In yet another related embodiment, the concentration of acetate is 100 mM.

[0041] In one related embodiment, at least one washing buffer contains acetate and 0 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 70 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 100 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 105 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 125 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, at least one washing buffer contains acetate and 147 mM sodium chloride at pH 4.9, 5.0, or 5.1. In one related embodiment, the concentration of acetate is 100 mM.

[0042] In one embodiment, at least one washing buffer contains 100 mM acetate and 100 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment, at least one washing buffer contains 100 mM acetate and 105 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment, at least one washing buffer contains 100 mM acetate and 125 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment, at least one washing buffer contains 100 mM acetate and 147 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment, at least one washing buffer contains 100 mM acetate and 70 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 100 mM sodium chloride at a pH of 5.0 ± 0.5% to 5.0 ± 0.1.

[0043] In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 0 mM to 147 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 70 mM to 147 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 100 mM to 125 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 100 mM to 105 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 105 mM to 147 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 105 mM to 125 mM sodium chloride at pH 5.0. In one embodiment of the present invention, at least one washing buffer contains 100 mM acetate and 125 mM to 147 mM sodium chloride at pH 5.0. The washing buffer may have a pH unit higher or lower by 0.05 to 0.1, with variations in conductivity, in order to robustly remove product-related impurities.

[0044] In one embodiment, the cation exchange medium is washed with at least two types of washing buffers. In one embodiment, the washing buffer contains 0 to 147 mM sodium chloride. In one embodiment, at least one type of washing buffer contains 0, 70, 100, 105, 125, or 147 mM sodium chloride. In one embodiment, the washing buffer contains acetate. In one embodiment, the washing buffer contains 100 mM acetate. In one embodiment, the pH of at least one type of washing buffer is 5.0 ± 0.05% to pH 5.0 ± 0.1%. In one embodiment, the pH of at least one type of washing buffer is 4.9 to 5.1. In one embodiment, the pH of at least one type of washing buffer is 4.9, 5.0, or 5.1. In one embodiment, the cation exchange medium is washed with at least two types of washing buffers, at least one type of washing buffer contains acetate and 0 to 70 mM sodium chloride, and at least one type of washing buffer contains acetate and 100 to 147 mM sodium chloride. In one embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, at least one of which contains acetate and 0 mM sodium chloride, followed by a wash buffer containing acetate and 100–147 mM sodium chloride. In another embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, one of which contains acetate and 100–147 mM sodium chloride, followed by a wash buffer containing acetate and 0–70 mM sodium chloride. In yet another embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, the first wash buffer contains acetate and 0 mM sodium chloride, and the second wash buffer contains acetate and 100–147 mM sodium chloride. In yet another embodiment, the sodium chloride concentration of the second wash buffer is selected from 100, 105, 125, and 147 mM sodium chloride. In one embodiment, the cation exchange chromatography medium is washed with at least two washing buffers, the first washing buffer containing acetate and 100-147 mM sodium chloride, and the second washing buffer containing acetate and 0-70 mM sodium chloride.In one embodiment, the sodium chloride concentration of the first wash buffer is selected from 100, 105, and 147 mM sodium chloride. In another embodiment, the concentration of the first wash buffer is 100 mM acetate and 0 mM sodium chloride, and the concentration of the second wash buffer is 100 mM acetate and 125 mM sodium chloride.

[0045] In one embodiment, the cation exchange medium is washed with at least three types of washing buffers. In one embodiment, the washing buffer contains 0 to 147 mM sodium chloride. In one embodiment, at least one type of washing buffer contains 0, 70, 100, 105, 125, or 147 mM sodium chloride. In one embodiment, the washing buffer contains acetate. In one embodiment, at least one type of washing buffer contains 100 mM acetate. In one embodiment, the pH of at least one type of washing buffer is 5.0 ± 0.05% to pH 5.0 ± 0.1%. In one embodiment, the pH of at least one type of washing buffer is 4.9 to 5.1. In one embodiment, the pH of at least one type of washing buffer is 4.9, 5.0, or 5.1.

[0046] In one embodiment, the cation exchange medium is washed with at least three wash buffers, at least two of which contain acetate and 0-70 mM sodium chloride, and at least one of which contains acetate and 100-147 mM sodium chloride. In one embodiment, the cation exchange chromatography medium is washed with at least three wash buffers, one of which contains acetate and 0 mM sodium chloride, followed by a wash buffer containing acetate and 100-147 mM sodium chloride; followed by a wash buffer containing acetate and 0-70 mM sodium chloride. In one embodiment, the first wash buffer contains acetate and 0 mM NaCl; the second wash buffer contains acetate and 100-147 mM sodium chloride; and the third wash buffer contains acetate and 0-70 mM sodium chloride. In one embodiment, the sodium chloride concentration of the first wash buffer is 0 mM sodium chloride. In one embodiment, the sodium chloride concentration of the second wash buffer is selected from 100, 105, and 147 mM sodium chloride. In one embodiment, the sodium chloride concentration of the third wash buffer is selected from 0 and 70 mM sodium chloride. In one embodiment, the first wash buffer is 100 mM acetate and 0 mM sodium chloride; the second wash buffer is selected from 100 mM acetate, 100 mM sodium chloride and 100 mM acetate and 105 mM sodium chloride; and the third wash buffer is 100 mM acetate and 0 mM sodium chloride. In one embodiment, the first wash buffer is 100 mM acetate and 0 mM sodium chloride; the second wash buffer is 100 mM acetate and 147 mM sodium chloride; and the third wash buffer is 100 mM acetate and 70 mM sodium chloride.

[0047] In one embodiment, the cation exchange resin is washed with a 2.5 mM / CV washing buffer containing 147 mM sodium chloride.

[0048] In one embodiment, the multispecific protein is eluted from the cation exchange resin by a gradient. In one related embodiment, the gradient is linear or stepwise. In one related embodiment, the gradient is a salt gradient. In one related embodiment, at least one buffer used to form the elution gradient contains 0 to 1 M sodium chloride. In one related embodiment, at least one buffer used to form the elution gradient contains 70 to 500 mM sodium chloride.

[0049] In one related embodiment, at least one buffer solution used to form the elution gradient contains 125 mM sodium chloride. In another related embodiment, at least one wash buffer solution and one elution buffer solution contain 125 mM sodium chloride.

[0050] In one embodiment, the cation exchange medium is equilibrated with a buffer that does not contain sodium chloride before loading the composition.

[0051] In one embodiment, the cation exchange medium is loaded with 10 g / L of multispecific protein, washed with a washing buffer containing 105 mM sodium chloride, and eluted with a salt gradient of 8 mM / CV.

[0052] In one embodiment, at least one product-related impurity is a homodimer, a high molecular weight species, a semi-antibody, an aggregate, a low molecular weight species, an antibody fragment, or a light chain misassembly.

[0053] In one embodiment, affinity-purified multispecific proteins are in an eluate pool, subjected to low-pH virus inactivation, and then neutralized before being loaded onto a cation exchange medium.

[0054] In one embodiment, the cation exchange chromatography medium is a resin.

[0055] In one embodiment, the third chromatographic medium is selected from cation exchange chromatographic medium, multimodal chromatographic medium, hydrophobic interaction chromatographic medium, and hydroxyapatite chromatographic medium. In one embodiment, the flow-through from the third chromatographic medium undergoes ultrafiltration and dialysis.

[0056] In one embodiment, one or more instrumental operations for purifying multispecific proteins are provided, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography columns, and / or mixed-mode chromatography columns, before and / or after a cation exchange chromatography step.

[0057] In one embodiment, the multispecific protein is a bispecific protein. In another embodiment, the multispecific protein is a bispecific antibody.

[0058] "Multispecificity," "multispecificity protein," and "multispecificity antibody" are used herein to refer to proteins that have been recombinantly modified to simultaneously bind to and neutralize at least two different antigens or at least two different epitopes on the same antigen. For example, multispecificity proteins may be modified in combination with targeted cytotoxic agents to target immune effectors to tumors or infectious pathogens. These multispecificity proteins have been found useful in a variety of applications, including in cancer immunotherapy by redirecting immune effector cells to tumor cells, modifying cell signaling by blocking signaling pathways, targeting tumor angiogenesis, and blocking cytokines, as well as as pre-targeting delivery vehicles for drugs such as chemotherapeutic agents, radiolabeled (to improve detection sensitivity), and nanoparticles (to be directed to specific cells / tissues such as cancer cells).

[0059] The most common and diverse group of multispecific proteins are those that bind to two antigens, and are referred to herein as “bispecificity,” “bispecificity proteins,” and “bispecificity antibodies.” Bispecificity 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 cell phagocytosis (ADCP), and the Fc region enhances solubility and stability and facilitates some purification operations. Non-IgG-like molecules are smaller and enhance tissue permeability (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 have binding specificity to different antigens or several epitopes and may be used as a framework for additional components to increase the binding specificity of a molecule.

[0060] The forms of bispecific proteins, including bispecific antibodies, are constantly evolving, including quadromas, knob-in-holes, cross-Mab, bivariable domain IgG (DVD-IgG), IgG-single-strand Fv (scFv), scFv-CH3 KIH, dual-acting Fab (DAF), half-body exchange, κλ-body, tandem scFv, scFv-Fc, diabody, single-strand diabody (scdiabody), scdiabody-CH3, triplebody, mini-antibody, minibody, TriBi minibody, tandem diabody, scdiabody-HAS, tandem scFv-toxin, biaffinity retargeting molecule (DART), nanobody, nanobody-HSA, and dock. • AND ROCK (DNL), Strand Exchange Modified Domain SEED Body, Triomab, Leucine Zipper (LUZ-Y), XmAb (Registered Trademark); Fab-Arm Exchange, DutaMab, DT-IgG, Charged 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, sc-diabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, bispecific T cell engager (BITE®) and bispecific T cell engager with extended half-life (HLE BiTE®) (Fan above; Spiess above; Sedykh above; Seimetz et al.)This includes, but is not limited to, the following publications: 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 Gottswachalk editor, pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011).

[0061] In some embodiments, the bispecific protein is blinatumomab, catumakisomab, erzumakisomab, solitomab, targomiR, lutikizumab (ABT981), vanucizumab (RG7221), memtolumab (ABT122), ozoralizumab (ATN103), fluotetuzumab (MGD006), pasotuxizumab (AMG112, MT112), lymphomun (FBTA05), (A TN-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-205, x m734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013(ACE910), RG7597(MEDH7945A), R G7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM This may include 141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141;BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, and their molecules or variants or analogs, and any of the above biosimilars.

[0062] Multispecific proteins also include triplicate antibodies capable of binding to multiple targets, tetravalent bispecific antibodies, multispecific proteins without antibody components such as diabodies, triabodies, tetrabodies, or minibodies, and single-chain proteins. (Coloma, MJ, et. al., Nature Biotech. 15(1997) 159-163).

[0063] In some embodiments, the 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 factor, fibroblast growth factor, transform growth factor (TGF), insulin-like growth factor, bone-inducing factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptor; neurotrophic factors, neurotrophins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, adresins, regulatory proteins, and immunoadhesins.

[0064] In some embodiments, the multispecific proteins of interest, either alone or in any combination, include but are not limited to the following CD proteins: 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, and HER4; the EGF receptor EGFRvIII; and cell adhesion molecules such as LFA-1, Mol, p150, and 9. 5. Growth factors including, but not limited to, VLA-4, ICAM-1, VCAM, αv / β3 integrins, e.g., vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müller inhibitor, 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 aFGF and bFGF, epidermal growth factor (EGF). Among the many, transform growth factors (TGF) including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factors-I and-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone formation factors, insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding proteins, insulin and insulin-related proteins, among the many, factor VIII, tissue Coagulation and coagulation-related proteins such as factors, von Willebrand factor, protein C, α-1-antitrypsin, urokinase and tissue plasminogen activators such as "t-PA", bombazine, thrombin, thrombopoietin and thrombopoietin receptors, colony-stimulating factors (CSFs) including M-CSF, GM-CSF and G-CSF, albumin, IgE and other blood and serum proteins including but not limited to blood group antigens, such as flk2 / flt3 receptors and obesity (OB) receptors.Receptors and receptor-related 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 interferon-α, -β, and -γ; interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, and IL-12 / IL-2. 3, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor IL-13RA2, or IL-17 receptor IL-1RAP; AIDS enveloped virus antigen, lipoprotein, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, including but not limited to these viral antigens, enkephalinase, BCMA, STEAP1, IgKappa, ROR-1, ERBB2, mesoserine, 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, disintegration promoter (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, Adresin, 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, Granglioside 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,It binds to, neutralizes, and / or interacts with one or more of the following bioactive fragments or variants: mannose receptor / hCGβ, TNF, TL1A, hepatitis C virus, mesoserine dsFv[PE38 conjugate, Legionella pneumophila (lly), IFNγ, interferon-γ-inducing protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphocyte neoplastic factor (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 any one or more of the aforementioned bioactive fragments or variants.

[0065] In some embodiments, the multispecific protein of interest may include a bispecific antibody that specifically binds to a combination of CD3 and CD19, EpCAM, CEA, PSA, CD33, BCMA, Her2, CD20, P-cadherin, CD123, gpA33, or B7H3. In some embodiments, the bispecific antibody of interest may include a bispecific antibody that specifically binds to a combination of IL1α + IL1β.

[0066] Multispecific proteins are proteins of scientific or commercial interest, particularly bispecificity-based therapeutics. Multispecific proteins can be produced by recombinant animal cell lines using various methods, most commonly cell culture methods. Multispecific proteins can be produced intracellularly, or secreted into culture media from which they can be recovered and / or collected, and can be interchangeably referred to as “recombinant multispecific proteins,” “recombinant multispecific antibodies,” “recombinant bispecific proteins,” and “recombinant bispecific antibodies.” The terms “isolated multispecific proteins,” “isolated recombinant multispecific antibodies,” “isolated bispecific proteins,” and “isolated bispecific antibodies” refer to bispecific proteins, including bispecific proteins purified from proteins, polypeptides, DNA, and / or other contaminants or impurities, such as product-related impurities that would interfere with their therapeutic, diagnostic, prophylactic, research, or other use, particularly product-related impurities with low pI. Multispecific proteins of interest include multispecific antibodies that exert therapeutic effects by binding to two or more targets, including those listed below, derived from them, associated targets, and modifications thereof.

[0067] The "purification process" means increasing the purity of the multispecific protein in the composition by removing (partially or completely) at least one type of impurity from the product. The recovery and purification of the multispecific protein is carried out by downstream instrumentation operations, particularly those including ion exchange chromatography, which produce a more "homogeneous" multispecific protein composition that satisfies yield and product quality targets (e.g., reduced product-related impurities and increased product quality).

[0068] "Product-related impurities" refer to product-related variants of the multispecific protein of interest. In some cases, these impurities have a lower pI than the main product in the elution peak. Examples of product-related impurities include homodimers, high molecular weight (HMW) species, semi-antibodies, aggregates, low molecular weight (LMW) species, antibody fragments and various combinations of antibody fragments, as well as light chain misassemblies such as 2XLC, 3XLC, or 4XLC. "Semi-antibodies" refer to product-related impurities that may form, for example, due to incomplete assembly or the breakdown of the interaction between two heavy chain polypeptides. Semi-antibodies generally contain one light chain polypeptide and one heavy chain polypeptide. "Homodimers" refer to product-related impurities that may form, for example, when molecules recombine with each other instead of pairing to form a desired bispecific heterodimer, even if they have specificity for the same target. This typically occurs during expression in host cells. For multispecific constructs that require multiple chains (light chains, LCs, etc.) to correctly pair via modified residues (charge pairing mutations, knobs, holes, etc.), there is still a possibility of impurities containing mismatches between LCs and HCs. Here, LC1 may incorrectly pair with HC2 (2×LC1) instead of HC1, and vice versa (2×LC2). If a multispecific protein is bivalent, having two sites for binding to each antigen of interest, it is possible to have 3XLC1, 4XLC1, and other combinations of mispair species.

[0069] As disclosed herein, the pI of product-related impurities may be lower than the pI of the desired multispecific protein and may elute together with the main product. Product-related impurities with low pI elute as pre-peaks, preceding the main product. The "isoelectric point" or "pI" of a protein refers to the pH at which the positive charge equilibrium with the negative charge of the protein. The pI can be calculated / determined from the net charge of the amino acid residues of the protein, or by known methods such as isoelectric focusing electrophoresis. In one embodiment, the difference between the pI of product-related impurities and the main product is at least 0.5 pI units. In another embodiment, the difference is 0.5 to 3 pI units. In another embodiment, the difference is 0.5 to 2 pI units. In another embodiment, the difference is 0.5 to 1 pI unit. In another embodiment, the difference is 1 to 3 pI units. In another embodiment, the difference is 2 to 3 pI units. In another embodiment, the difference is 0.5, 1, 2, 3 or more pI units.

[0070] This specification provides expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes, or expression cassettes containing one or more polynucleotides encoding a multispecific protein of interest, as well as host cells containing such expression systems or constructs. As used herein, “vector” means any molecule or entity suitable for use in translocating and / or transporting a multispecific protein encoding information into a host cell and / or a specific location and / or compartment within the host cell (e.g., nucleic acids, plasmids, bacteriophages, transposons, cosmids, chromosomes, viruses, viral capsids, virions, naked DNA, complexed DNA, etc.). Vectors may include viral and nonviral vectors, and non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors and cloning vectors. A vector may be introduced into a host cell to allow replication of the vector itself, thereby amplifying copies of the polynucleotides it contains. Cloning vectors may contain sequence components, including but not limited to replication origins, promoter sequences, transcription start sequences, enhancer sequences, and selectable markers. These elements may be selected as needed by those skilled in the art.

[0071] "Cells" include any prokaryotic or eukaryotic cells. Cells may exist ex vivo, in vitro, or either separately or as part of a higher-order structure such as a tissue or organ. Cells include "host cells," also called "cell lines," that have been modified to express a multispecific protein of commercial or chemical interest. Host cells typically originate from a line arising from a primary culture that can be maintained in culture without time constraints. Genetic engineering of host cells includes transfecting, transforming, or transducing the host cells with recombinant polynucleotide molecules and / or modifying them in other ways (e.g., by homologous recombination and gene activation, or by fusion of recombinant and non-recombinant cells) to express a desired recombinant multispecific protein. Methods and vectors for genetically engineering cells and / or cell lines to express a multispecific protein of interest are well known to those skilled in the art.

[0072] The host cell can be any prokaryotic cell (e.g., Escherichia coli) or eukaryotic cell (e.g., yeast cell, insect cell, or animal cell (e.g., CHO cell)). The vector DNA can be introduced into the prokaryotic or eukaryotic cell by conventional transformation or gene transfer techniques.

[0073] When cultured under appropriate conditions, host cells express the 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 producing it (if it does not secrete it). The selection of a suitable host cell depends on various factors, including the desired expression level, protein modifications desirable or essential for activity (such as glycosylation or phosphorylation), and the ease of folding into a bioactive molecule.

[0074] "Culture" or "culturing" means 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 media and tissue culture media are used interchangeably to refer to media suitable for the growth of host cells in in vitro cell culture. Typically, cell culture media contain buffers, salts, energy sources, amino acids, vitamins, and trace amounts of essential elements. Any medium capable of promoting the growth of suitable host cells in culture may be used. Cell culture media that may be further supplemented with other components to maximize cell proliferation, cell viability, and / or recombinant protein production in specific cultured host cells are commercially available and include, among others, serum-free media such as RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham F12 medium, Iskov Modified Dulbecco's medium, McCoy's 5A medium, Leibovitz L-15 medium, and EX-CELL® 300 series, which can be obtained from 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 eutrophicated by the addition of nutrients and may be used at concentrations higher than their usual recommended concentrations.

[0075] Various culture medium formulations may be used during the culture period to, for example, facilitate the transition from one stage (e.g., growth stage or phase) to another (e.g., production stage or phase) and / or optimize conditions in the cell culture (e.g., concentrated medium provided during perfusion culture). Growth medium formulations may be used to promote cell proliferation and minimize protein expression. Production medium formulations can be used to promote the production of the protein of interest and the maintenance of cells while minimizing the proliferation of new cells. Supply media consumed during the production phase of cell culture, typically 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 manner. Such concentrated supply media may contain most of the components of cell culture media in amounts of, for example, about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or about 1000× of their normal amounts.

[0076] The growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of approximately 35°C to 38°C, and the production phase may occur at a second temperature of approximately 29°C to 37°C, or optionally, approximately 30°C to 36°C or approximately 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with, before, and / or after, the temperature change. If the inducers are added after the temperature change, they may be added 1 hour to 5 days after the temperature change, or optionally, 1 to 2 days after the temperature change.

[0077] Host cells can be cultured in suspension or in an adherent form bound to a solid culture medium. Cell cultures can be established in fluid-bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors, with or without microcarriers.

[0078] Cell cultures may be operated in batch, fed-batch, continuous, semi-continuous, or perfusion manner. Mammalian cells such as CHO cells may be cultured in bioreactors in small quantities of less than 100 ml to less than 1000 ml. Alternatively, large bioreactors containing 1000 ml to more than 20,000 liters of culture medium may be used. Large-scale cell cultures, such as those for the bioproduction of clinical and / or commercial-scale protein therapeutics, may be maintained for several weeks and months while the cells produce the desired protein.

[0079] Product-related impurities such as homodimers and half-antibodies can resemble the desired multispecific protein; therefore, strategies and techniques such as knobs and holes, CrossMab, DVD IgG, and others have been developed to increase selectivity for the desired multispecific protein during cell culture. However, there will still be some product-related impurities that must be removed during downstream processes.

[0080] The resulting expressed recombinant multispecific proteins can then be harvested from the cell culture medium. Methods for collecting proteins from suspended cells are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation such as cottony sedimentation, separation using gravity, centrifugation, ultrasonic separation, and membrane filtration, and include filtration using ultrafiltration membranes, microfilters, tangential flow filters, and alternative tangential flow, depth, and sediment filtration filters. Recombinant proteins expressed by prokaryotes are recovered in cytoplasmic inclusion bodies by redox folding processes known in the art.

[0081] Next, the acquired multispecific proteins can be purified, or partially purified, using one or more downstream instrumental operations to remove any impurities such as residual cell culture medium, cell extracts, unwanted components, host cell proteins, improperly expressed proteins, and product-related impurities.

[0082] The purification of multispecific proteins from harvested cell cultures can be initiated using capture chromatography. Capture chromatography methods such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), and immobilized metal affinity chromatography (IMAC) utilize media such as resins, membranes, and gels that will bind to the recombinant multispecific protein of interest. Such materials are known and commercially available in the art. Affinity chromatography options may include, for example, substrate-binding capture mechanisms, aptamer-binding capture mechanisms, and cofactor-binding capture mechanisms. For multispecific proteins containing an Fc component, antibody-binding or antibody-fragment-binding capture mechanisms such as those for Protein A, Protein G, Protein A / G, and Protein L can be used. The recombinant protein of interest can be labeled using an epitope such as a polyhistidine tag or FLAG®, and subsequently purified using a specific antibody directed to such an epitope.

[0083] At any point in the downstream process, viral inactivation and / or viral filtration can be performed to remove viral material from a composition containing the multispecific protein of interest. One method for achieving viral inactivation is incubation at a low pH or other solution conditions for achieving viral inactivation. Following low-pH viral inactivation, a neutralization operation may follow to readjust the pH of the virus-inactivated solution to a level more suitable for the requirements of the next apparatus operation. Typically, neutralization occurs at pH 5–7. The viral-inactivated or neutralized viral-inactivated pool may then be subjected to filtration, such as depth filtration, to remove any resulting turbidity or precipitate. Viral filtration can be performed using microfiltration or nanofiltration membranes, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0084] The term "polishing" is used herein to refer to one or more chromatographic steps performed to remove residual contaminants and impurities, such as DNA, host cell proteins, product-specific impurities, mutant compositions, and aggregates, as well as viral adsorption, from a liquid composition containing recombinant multispecific protein of a near-desired purity. For example, polishing may be performed by passing a liquid containing recombinant multispecific protein through a chromatographic column or membrane absorber that selectively binds to either the target recombinant multispecific protein or contaminants or impurities present in the liquid composition, in binding and eluate modes. In such an example, the eluate / filtrate from the chromatographic column or membrane absorber contains the recombinant multispecific protein.

[0085] Polish chromatography utilizes a medium such as a resin and / or membrane containing a substance that can be used in flow-through mode (in which case multispecific proteins pass through the resin / membrane and are contained in the flow-through eluate, while contaminants and impurities bind to the chromatography medium), frontal or overload chromatography mode (in which case the solution containing the protein of interest is loaded onto the column until the adsorption sites are occupied, and the species with the least affinity to the stationary phase (the protein of interest) begins to elute), or binding and elution mode (in which case the protein of interest binds to the chromatography medium, and elutes after contaminants and impurities have passed through the chromatography medium or have been washed away from the chromatography medium). Examples of such chromatographic methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multi-mode chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, size exclusion chromatography (SEC), and gel filtration. In one embodiment, the chromatography method is cation exchange chromatography. In one embodiment, the cation exchange medium is a resin.

[0086] "Cation exchange chromatography" refers to chromatography performed on a solid-phase medium that is negatively charged and has free cations for exchange with cations in an aqueous solution that has passed over or through the solid phase. The charge can be provided, for example, by covalent bonding, by attaching one or more charged ligands to the solid phase. Alternatively, or in addition, the charge may be an intrinsic property of the solid phase (as in the case of silica, which has a total negative charge, for example). Commercially available cation exchange media can be used, including sulfopropyl (SP) immobilized on agar (e.g., SP-SEPHAROSE FAST FLOW®, SP-SEPHAROSE FAST FLOW XL®, or SP-SEPHAROSE HIGH PERFORMANCE® from GE Healthcare), CAPTO S®, CAPTO SP ImpRes®, CAPTO S ImpAct® (GE Healthcare), FRACTOGEL-SO3®, FRACTOGEL-SE HICAP®, FRACTOPREP® (EMD Merck), Fractogel® EMD SO3-(M), Fractogel® EMD SE Hicap(M), Eshmuno® CPX, Eshmuno® S resin, Fractogel® EMD COO-(M), Mustang S Acrodisc with Mustang S AcroPrep with Mustang S, and CM Ceramic. Examples include, but are not limited to, HyperD®F AcroSep with CM Ceramic HyperD®F.

[0087] For the method of the present invention, cation exchange chromatography is performed in binding and elution modes. Multispecific proteins in the eluate and storage pool from a previous downstream process are loaded onto the cation exchange medium so that the multispecific protein of interest binds to the cation exchange medium. "Binding" the multispecific protein to the cation exchange medium means exposing the multispecific protein to the cation exchange medium under appropriate conditions (pH / conductivity) such that the multispecific protein is reversibly immobilized in or on the cation exchange medium by ionic interactions between the multispecific protein of interest and one or more charged groups of the cation exchange medium. The eluate or pool may result from previous instrument operations such as affinity chromatography, neutralized low-pH virus inactivation, depth filtration or harvesting and / or polish chromatography operations. Additional buffers may be added to the eluate or pool so that the final loading of the multispecific protein is at the desired concentration.

[0088] The loaded cation exchange chromatography medium is then subjected to at least one washing step. Washing the cation exchange medium means passing a suitable washing buffer through or over the cation exchange medium. The function of the washing buffer is to remove one or more contaminants from the cation exchange medium without substantial elution of the multispecific protein of interest. "Buffer" means a solution that resists changes in pH due to the action of its acid-base conjugate component. In one embodiment, the buffer is an acetate. In one embodiment, 100 mM acetate is provided. In one embodiment, the pH of the washing buffer is in the range of 5.0 ± 0.05% to 5.0 ± 0.1%. In one embodiment, the pH of the buffer is in the range of 4.9 to 5.1. In one embodiment, the pH of the washing buffer is 4.9, 5.0, or 5.1. In one embodiment, at least one washing buffer contains an acetate at pH 5.0. The washing buffer may have a pH range of 0.05 to 0.1 higher or lower, accompanied by variations in conductivity, in order to robustly remove product-related impurities.

[0089] At least one type of washing buffer further contains a salt. In one embodiment, the salt is sodium chloride. In one embodiment, the concentration of sodium chloride in the washing buffer is 0 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 70 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 100 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 100 mM to 125 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 100 mM to 105 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 105 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 105 mM to 125 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 125 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 0 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 70 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 100 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 105 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 125 mM. In one embodiment, the concentration of sodium chloride in the washing buffer is 147 mM.

[0090] The washing step washes the cation exchange medium after loading and before elution of the multispecific protein of interest. The present invention provides at least one washing step comprising a high-salt washing buffer. High-salt washing buffers have been found to wash or elute product-related impurities with low pI from the cation exchange medium before elution of the main product. In addition to the high-salt washing step, there may be one or more additional washing steps using a buffer containing no salt and / or a salt at a lower concentration compared to the high-salt washing buffer. Preferably, the UV baseline is restored to very close to zero or very close to zero at the end of the final washing step before the start of elution. According to one embodiment of the present invention, there are at least two washing steps. In one embodiment, there are a "first washing buffer" and a "second washing buffer". According to one embodiment of the present invention, there are at least three washing steps. In one embodiment, there are a "first washing buffer", a "second washing buffer", and a "third washing buffer". The terms “first wash,” “second wash,” and / or “third wash” should not be interpreted as excluding one or more additional washes or the use of other buffers between one or more additional steps. Wash buffers are used to wash or re-equilibrium the cation exchange material before eluting the multispecific protein of interest. One or more wash buffer preparations may be identical to the equilibration and / or final conditioning load buffer preparations.

[0091] In one embodiment of the present invention, the first wash includes a wash buffer containing acetate at pH 5.0±0.5 to pH 5.0±0.1%. In one embodiment of the present invention, the first wash buffer contains acetate at pH 4.9 to 5.1. In one embodiment of the present invention, the first wash buffer contains acetate at pH 4.9, 5.0, or 5.1. In one embodiment of the present invention, the first wash buffer contains acetate at pH 5.0. In one embodiment of the present invention, the first wash buffer contains 100 mM acetate. In one embodiment of the present invention, the first wash buffer contains 100 mM acetate at pH 5.0. In one embodiment of the present invention, the first wash buffer contains acetate and 0 to 147 mM sodium chloride. In one embodiment of the present invention, the first wash includes a wash buffer containing 100 mM acetate and 0 mM sodium chloride at pH 5.0±0.5 to pH 5.0±0.1%.

[0092] In one embodiment of the present invention, the second wash includes a wash buffer containing acetate at a pH of 5.0 ± 0.5 to 5.0 ± 0.1%. In one embodiment of the present invention, the second wash buffer contains acetate at a pH of 4.9 to 5.1. In one embodiment of the present invention, the second wash buffer contains acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate at a pH of 5.0.

[0093] In one related embodiment of the present invention, the second washing buffer contains 0 to 147 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 70 to 147 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 100 to 147 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 100 to 125 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 100 to 105 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 105 to 147 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 105 to 125 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 125 to 147 mM sodium chloride. In one related embodiment of the present invention, the second washing buffer contains 125 mM sodium chloride.

[0094] In one embodiment of the present invention, the second washing buffer contains 100 mM acetate and 0 to 147 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In one embodiment of the present invention, the second washing buffer contains 100 mM acetate and 70 to 147 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In one embodiment of the present invention, the second washing buffer contains 100 mM acetate and 100 to 147 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In one embodiment of the present invention, the second washing buffer contains 100 mM acetate and 100 to 125 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In one embodiment, the second washing buffer contains 100 mM acetate and 100 to 105 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In another embodiment, the second washing buffer contains 100 mM acetate and 105 to 147 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In a related embodiment, the second washing buffer contains 100 mM acetate and 105 to 125 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In a related embodiment, the second washing buffer contains 100 mM acetate and 125 to 147 mM sodium chloride at a pH of 5.0 ± 0.05 to 5.0 ± 0.1. In one related embodiment, the second washing buffer contains 100 mM acetate and 125 mM sodium chloride with a pH of 5.0 ± 0.05 to 5.0 ± 0.1.

[0095] In one related embodiment, the second washing buffer contains 100 mM acetate and 0 mM sodium chloride at pH 5.0. In one related embodiment, the second washing buffer contains 100 mM acetate and 70 mM sodium chloride at pH 5.0. In one related embodiment, the second washing buffer contains 100 mM acetate and 100 mM sodium chloride at pH 5.0. In one related embodiment, the second washing buffer contains 100 mM acetate and 105 mM sodium chloride at pH 5.0. In one related embodiment, the second washing buffer contains 100 mM acetate and 125 mM sodium chloride at pH 5.0. In one related embodiment, the second washing buffer contains 100 mM acetate and 147 mM sodium chloride at pH 5.0.

[0096] In one embodiment of the present invention, the third wash includes a wash buffer containing acetate at a pH of 5.0±0.05% to 5.0±0.1%. In one embodiment of the present invention, the third wash buffer contains acetate at a pH of 4.9 to 5.1. In one embodiment of the present invention, the third wash buffer contains 100 mM acetate. In one embodiment of the present invention, the third wash buffer contains 100 mM acetate at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment of the present invention, the third wash buffer contains 0 to 147 mM sodium chloride. In one embodiment of the present invention, the third wash buffer contains 0 mM sodium chloride. In one embodiment of the present invention, the third wash buffer contains 100 mM acetate and 0 mM sodium chloride at a pH of 5.0±0.5% to 5.0±0.1. In one embodiment of the present invention, the third wash buffer contains 70 mM sodium chloride. In one embodiment of the present invention, the third washing buffer contains 100 mM acetate and 70 mM sodium chloride at a pH of 5.0 ± 0.5% to 5.0 ± 0.1.

[0097] In one embodiment, the cation exchange chromatography medium is washed with at least three wash buffers, one of which contains acetate and 0 mM sodium chloride, followed by a wash buffer containing acetate and 100–147 mM sodium chloride; and then a wash buffer containing acetate and 0–70 mM sodium chloride. In one embodiment, the first wash buffer contains acetate and 0 mM NaCl; the second wash buffer contains acetate and 100–147 mM sodium chloride; and the third wash buffer contains acetate and 0–70 mM sodium chloride. In one embodiment, the sodium chloride concentration of the first wash buffer is 0 mM sodium chloride. In one embodiment, the sodium chloride concentration of the second wash buffer is selected from 100, 105, and 147 mM sodium chloride. In one embodiment, the sodium chloride concentration of the third wash buffer is selected from 0 and 70 mM sodium chloride. In one embodiment, the first wash buffer is 100 mM acetate and 0 mM sodium chloride; the second wash buffer is selected from 100 mM acetate, 100 mM sodium chloride and 100 mM acetate and 105 mM sodium chloride; and the third wash buffer is 100 mM acetate and 0 mM sodium chloride. In another embodiment, the first wash buffer is 100 mM acetate and 0 mM sodium chloride; the second wash buffer is 100 mM acetate and 147 mM sodium chloride; and the third wash buffer is 100 mM acetate and 70 mM sodium chloride.

[0098] In one embodiment of the present invention, the cation exchange medium is loaded with at least 10 g / L of multispecific protein. In one embodiment of the present invention, the cation exchange medium is loaded with 10 g / L to 40 g / L of multispecific protein. In one related embodiment of the present invention, the cation exchange medium is loaded with 15 g / L to 40 g / L of multispecific protein. In one related embodiment of the present invention, the cation exchange medium is loaded with at least 20 g / L to 40 g / L of multispecific protein. In one related embodiment of the present invention, the cation exchange medium is loaded with 25 g / L to 40 g / L of multispecific protein. In one related embodiment of the present invention, the cation exchange medium is loaded with 35 g / L to 40 g / L of multispecific protein. In one related embodiment, the cation exchange medium is loaded with 15 g / L to 35 g / L of multispecific protein. In one related embodiment, the cation exchange medium is loaded with 15 g / L to 25 g / L of multispecific protein. In one embodiment, the cation exchange medium is loaded with 15 g / L to 20 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 15 g / L to 35 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 20 g / L to 35 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 20 g / L to 30 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 20 g / L to 25 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 25 g / L to 35 g / L of multispecific protein. In a related embodiment, the cation exchange medium is loaded with 25 g / L to 30 g / L of multispecific protein.

[0099] In one embodiment, the present invention provides a method in which a cation exchange medium is loaded with a 10 g / L multispecific protein, washed with at least one washing buffer containing 105 mM sodium chloride, and eluted with a salt gradient of 8 mM / CV.

[0100] In one embodiment, the present invention provides that a cation exchange medium is loaded with a multispecific protein at a concentration of 15 g / L to 30 g / L and washed with at least one washing buffer containing 147 mM sodium chloride.

[0101] In one embodiment, the present invention provides that a cation exchange medium is loaded with a multispecific protein at a concentration of 25 g / L to 40 g / L, wherein at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

[0102] The bound multispecific proteins are then eluted from the solid phase of the cation exchange chromatography medium. Multispecific proteins can be eluted by a gradient. The gradient can be linear or stepwise. The gradient can be a salt gradient. For salt gradients, the salt concentration (ionic strength) fluctuates over time during the gradient. An appropriate salt concentration is required to break the binding of the multispecific proteins and release them into the eluate. Examples of usable salts include sodium chloride, potassium chloride, and acetate.

[0103] The cation exchange chromatography eluate can then be subjected to subsequent downstream polish chromatography purification. In one embodiment, after cation exchange chromatography, the multispecific protein of interest is subjected to polish chromatography in flow-through mode.

[0104] Following polish chromatography, concentration of the purified multiple isomers and exchange of the buffer to the desired formulation buffer for bulk storage of the formulation raw materials can be achieved by ultrafiltration and diafiltration.

[0105] Key attributes and performance parameters of purified multispecific proteins can be measured to better communicate performance decisions at each stage of the manufacturing process. These key attributes and parameters can be monitored in real time, near real time, and / or retrospectively. Key parameters such as the metabolic levels of consumed culture medium components (e.g., glucose), accumulated 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, gravimetric osmolality, appearance, color, aggregation, yield, and titer can be monitored at appropriate stages in the manufacturing process. Monitoring and measurement can be performed using known methods and commercially available equipment.

[0106] Pharmaceutical compositions (liquids, suspensions, etc.) may contain, but are not limited to, one or more of the following: buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, non-volatile oils such as synthetic monoglycerides or diglycerides that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and active ingredients for preparing isotonicity such as sodium chloride or dextrose. Parenteral formulations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0107] The terminology used in this application is standard in the art; however, definitions of certain terms are provided herein to ensure clarity and unambiguity with respect to the meaning of the claims. Units, prefixes, and symbols may be given in their SI-recognized forms. Numerical ranges described herein include the number defining the range, each integer within the defined range, and supporting it. Unless otherwise stated, methods and techniques described herein are generally carried out in accordance with common methods well known in the art, such methods and techniques are described in the various general and specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); 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 parts of documents cited herein, including but not limited to patents, patent applications, papers, books, and academic articles, are expressly incorporated herein by reference.

[0108] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. What is described in one embodiment of the invention can be combined with other embodiments of the invention. In fact, in addition to those illustrated and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0109] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Examples]

[0110] Example 1: Single salt-free load washing, dual specificity #1 A neutralized Protein A pool containing fully human bispecific, modified immunoglobulin (bispecificity #1) in acetate buffer was loaded onto Capto-SP ImpRes® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 1.

[0111] [Table 1]

[0112] Figure 1 shows three peaks in the elution profile, indicating that multiple impurities remained on the column and were eluted along with the main product. These impurities included homodimers, NCGs (non-consensus glycosylated) and high molecular weight species (HMWs).

[0113] Example 2: High-salt washing, dual specificity #1 A neutralized virus inactivation pool containing bispecificity #1 in acetate buffer was loaded onto Capto-SP ImpRes® cation exchange chromatography resin under the conditions described in Table 2.

[0114] [Table 2]

[0115] With the addition of high-salt washing, it was found that low-pI impurities, previously removed in the eluate, were now removed from the resin during the pre-elution wash. Figure 2 shows that the addition of high-salt washing reduced the number of impurity peaks in the elution profile from three to one. The majority of impurities were ≥68% NCG and ≥80% homodimers, which were removed between the second and third washing steps. The homodimers and NCG were largely removed from the resin during the second wash, or bound to the resin only in very small amounts. The third washing step re-established 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 addition of the high-salt washing step optimized the purification, making it a robust and manufacturing-friendly process that maintained an acceptable yield while reducing deviations from the end-use results regarding product quality.

[0116] These conditions were effective using a bispecific #1 loading concentration of 15–30 g / L.

[0117] Furthermore, washing buffers with pH values ​​above 0.05 and below 5.0 (pH 4.95-5.05) were tested and found to be effective in removing product-related impurities with low pI values, consistent with the results above.

[0118] It was found that using a second wash buffer at a column volume of 2.5 was sufficient to wash / elute product-related impurities from the cation exchange medium. Smaller column volumes were less effective in removing product-related impurities, and using a column volume larger than 2.5 began to elute the main product.

[0119] Example 3: Single salt-free wash, dual specificity #2 A neutralized protein A eluate pool (100 mM acetate, pH 5.0) containing a modified fully human anti-hetero-IgG bispecific antibody (bispecificity #2) was loaded onto Capto-SP ImpRE® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 3.

[0120] [Table 3]

[0121] Figure 3 shows that multiple impurities remained on the column and eluted along with the main product. These impurities included half-antibodies (fractions 1, 2, 3, and 4 containing approximately 50% half-antibodies), 2X light chain misassemblies (fractions 5 and 6 with an LC1:LC2 ratio of <0.4, indicating that LC2 improperly assembled with HC1), and high molecular weight (HMW, fractions 12–21). The resolution of the chromatographic separation is acceptable with a separate pre-peak containing impurities, but the manufacturing process utilized OD-based automated pooling; therefore, it would be necessary to collect the eluate by starting above the highest OD for the pre-peak, reducing the yield, and making this process insufficient for use in the manufacturing operation.

[0122] Example 4: High-salt washing, dual specificity #2 A neutralized protein A eluate pool containing bispecificity #2 (100 mM acetate, pH 5.0) was loaded onto Capto-SP ImpRE® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 4.

[0123] [Table 4]

[0124] When a high-salt washing step was added (a second wash using 100 mM sodium chloride), the half-antibody impurities were removed from the CEX resin before elution (100% of the half-antibodies were detected in the collected second and third washes). Figure 4 shows that the high-salt washing resulted in a reduction in the number of peaks in the elution profile from four peaks to a single peak with a small shoulder still containing 2X LC2 mispairing species (LC1 / LC2 < 0.11, compared to when LC1 and LC2 assembled to a predicted ratio of 1 when they accurately assembled to HC1 and HC2, respectively). The third washing step further re-established 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 using higher-salt washing is suitable for use in the manufacturing field. The purification yield of CEX increased from 65% to 73%, accompanied by a suitable elution profile for collecting a purification pool with low levels of half-antibody, mispaired LC2 species (as evidenced by an LC1:LC2 ratio close to 1), HMW and LMW, and absorbance-based pooling criteria.

[0125] Example 5 Single salt-free wash, dual specificity #3 A neutralized protein A eluate pool containing fully human, modified IgG / Fab fusion protein (bispecificity #3) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 5.

[0126] [Table 5]

[0127] Figure 5 shows a single elution peak resulting from a high loading density with a steep elution gradient. Product-related impurities with low pI values ​​do not dissipate from the main product under high loading densities, as evidenced by the decreased CE-SDS LC1:LC2 ratio (mismatched LC species) from 4 to 7 and 2-4% LMW species, and are mostly contained within fractions 1, 2, and 3.

[0128] Example 6: Low load density, single salt-free wash, dual specificity #3 A neutralized protein A eluate pool containing fully human, modified IgG / Fab fusion protein (bispecificity #3) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 6.

[0129] [Table 6]

[0130] The high loading density and steep gradient elution conditions of Example 5 did not provide sufficient resolution of the main product from low pI product-related impurities, so the loading density and gradient conditions were reduced. Figure 6 shows that a lower loading density (10 vs. 25 g / L) and a gentler gradient (8 vs. 16 mM / CV) allowed the separation of the main low pI product impurities into distinct peaks formed by fractions 1-4. This fraction showed an LC1:LC2 ratio of 3:10, indicating a mismatched LC1 species. In contrast, the main peak showed an accumulated LC1:LC2 ratio of 1.2. The resolution was better and the yield increased from 44% to 73%, although it still required automated pooling based on OD. However, collecting the eluate would still be necessary, as it would start beyond the highest OD relative to the pre-peak, reducing the yield and making this process insufficient for use in manufacturing operations.

[0131] Example 7: High-salt washing, dual specificity #3 A neutralized virus inactivation pool containing bispecificity #3 was loaded onto Capto-SP ImpRE® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 7.

[0132] [Table 7]

[0133] When a high-salinity washing step was added (second wash), it was found that impurities were removed from the CEX resin before elution. These impurities likely corresponded to mispaired LC1 species, assuming an LC1 to LC2 ratio of 8.0 for the collected second and third washes compared to a predicted ratio of 1, assuming that LC1 and LC2 paired exactly similarly. Figure 7 shows that the high-salinity washing resulted in a reduction in the number of impurity peaks in the elution profile from two peaks to a single peak, with a small shoulder still containing mispaired species (LC1 / LC2 = 2-4). The majority of impurities were removed between the second and third washes. The third wash further re-established 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. This optimized procedure using high-salinity washing, combined with low loading levels and a gentle gradient, is suitable for use in the manufacturing field. The purification yield of CEX increased from 44% to 66%, accompanied by elution profiles for collecting a purification pool with low levels of mispaired LC1 species (evidence of an LC1 to LC2 ratio close to 1), HMW and LMW, and absorbance-based pooling criteria.

[0134] Example 8: High-salt washing, dual specificity #4 A neutralized, low-pH viral-inactivated protein A eluate pool containing a fully human-modified immunoglobulin bispecific antibody (bispecificity #4) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin under the conditions described in Table 7.

[0135] [Table 8]

[0136] When a washing step with a high salt concentration was added (second wash), it was found that low pI product impurities (homodimers and aggregated species) were removed from the cation exchange medium before elution. Figure 8 shows that high-salt washing resulted in a reduction of the number of impurity peaks in the elution profile to one peak. The first wash, which did not contain sodium chloride, returned the conductivity to baseline. Low-pI product impurities were removed during the high-salt loading washing step, returning the conductivity baseline to zero before elution. Conductivity was maintained using elution buffer A, which had the same high-salt preparation as the high-salt washing buffer. This allowed for the maintenance of stable conductivity before initiating elution and fixing the elution profile, and since the separation was pI-based, it resulted in a much more robust and efficient collection and better quality of the main product.

[0137] Furthermore, washing buffers with pH levels above 0.1 and below 5.0 (pH 4.9-5.1) were tested and found to be effective in removing product-related impurities with low pI values.

[0138] Furthermore, we tested loading concentrations of 25-40 g / Lr for bispecificity #4 and found that they exhibited similar clearance to the 35 g / Lr condition described above.

Claims

1. A method for purifying multispecific proteins: A step of loading a sample containing multispecific proteins onto a cation exchange chromatography medium; A step of washing the cation exchange medium with at least one washing buffer containing 100 to 147 mM sodium chloride; and A method comprising the step of eluting the multispecific protein from the cation exchange chromatography resin.

2. The method according to claim 1, wherein at least one washing buffer contains 100 to 125 mM sodium chloride.

3. The method according to claim 1, wherein at least one washing buffer contains 100 to 105 mM sodium chloride.

4. The method according to claim 1, wherein at least one washing buffer contains 105 to 147 mM sodium chloride.

5. The method according to claim 1, wherein at least one washing buffer contains 105 to 125 mM sodium chloride.

6. The method according to claim 1, wherein at least one washing buffer contains 125 to 147 mM sodium chloride.

7. The method according to claim 1, wherein at least one washing buffer comprises an acetate.

8. The method according to claim 7, wherein at least one washing buffer contains an acetate with a pH of 5.0 ± 0.05% to 5.0 ± 0.1%.

9. The method according to claim 7, wherein at least one washing buffer contains an acetate with a pH of 4.9 to 5.

1.

10. The method according to claim 7, wherein at least one washing buffer comprises an acetate with a pH of 4.9, 5.0, or 5.

1.

11. The method according to claim 7, wherein the washing buffer contains 100 mM acetate.

12. The method according to claim 1, wherein at least one washing buffer comprises an acetate and 100 to 125 mM sodium chloride.

13. The method according to claim 1, wherein at least one washing buffer comprises an acetate and 100 to 105 mM sodium chloride.

14. The method according to claim 1, wherein at least one washing buffer comprises an acetate and 105 to 147 mM sodium chloride.

15. The method according to claim 1, wherein at least one washing buffer comprises an acetate and 105 to 125 mM sodium chloride.

16. The method according to claim 1, wherein at least one washing buffer comprises an acetate and 125 to 147 mM sodium chloride.

17. The method according to claim 1, wherein the cation exchange medium is washed using at least two types of washing buffers.

18. The method according to claim 1, wherein the cation exchange medium is washed with at least three types of washing buffers.

19. The method according to claim 18, wherein the cation exchange medium is washed with at least two washing buffers, and at least one of the washing buffers contains 0 to 147 mM sodium chloride.

20. The method according to claim 19, wherein the cation exchange medium is washed with at least two washing buffers, and at least one of the washing buffers contains 0 to 70 mM sodium chloride.

21. The method according to claim 1, wherein the cation exchange medium is washed with at least two washing buffers, at least one of the washing buffers comprising an acetate and 0 mM sodium chloride, followed by a washing buffer comprising an acetate and 100 to 147 mM sodium chloride.

22. The method according to claim 21, wherein the cation exchange medium is washed with a washing buffer containing acetate and 0 mM sodium chloride, and the washing buffer is then selected from the group consisting of a washing buffer containing acetate and 100 mM sodium chloride, a washing buffer containing acetate and 105 mM sodium chloride, or a washing buffer containing acetate and 125 mM sodium chloride.

23. The method according to claim 21, wherein the cation exchange medium is washed with a washing buffer containing acetate and 100 to 147 mM sodium chloride, followed by a washing buffer containing acetate and 0 to 70 mM sodium chloride.

24. The method according to claim 23, wherein the cation exchange medium is washed with a washing buffer containing acetate and 100 to 147 mM sodium chloride, followed by a washing buffer containing acetate and 0 mM sodium chloride.

25. The method according to claim 23, wherein the cation exchange medium is washed with a washing buffer containing an acetate and 100 to 147 mM sodium chloride, followed by a washing buffer containing 70 mM sodium chloride.

26. The cation exchange medium is A first washing buffer containing acetate and 0 mM sodium chloride, then A second washing buffer containing acetate, 100–147 mM sodium chloride, then The method according to claim 18, wherein the washing is performed using at least three washing buffers, each containing acetate and a third washing buffer containing 0 mM sodium chloride, or one washing buffer containing acetate and 70 mM sodium chloride.

27. The cation exchange medium is A first washing buffer containing acetate and 0 mM sodium chloride, then A second washing buffer selected from the group consisting of a washing buffer containing acetate and 100 mM sodium chloride, a washing buffer containing acetate and 105 mM sodium chloride, or a washing buffer containing acetate and 125 mM sodium chloride, then The method according to claim 26, wherein the product is washed with a third washing buffer containing acetate and 0 mM sodium chloride.

28. The cation exchange medium is A first washing buffer containing acetate and 0 mM sodium chloride, then A second washing buffer containing acetate and 147 mM sodium chloride, then The method according to claim 26, wherein the product is washed with a third washing buffer containing acetate and 70 mM sodium chloride.

29. The method according to claim 1, wherein the cation exchange medium is washed with a 2.5 mM / CV washing buffer containing 147 mM sodium chloride.

30. The method according to claim 1, wherein the multispecific protein is eluted from the cation exchange medium by gradient.

31. The method according to claim 30, wherein the gradient is linear or stepped.

32. The method according to claim 30, wherein the gradient is a salt gradient.

33. The method according to claim 30, wherein at least one of the buffers used to form the elution gradient comprises 0 to 1 M sodium chloride.

34. The method according to claim 33, wherein at least one of the buffers used to form the elution gradient contains 70 to 500 mM sodium chloride.

35. The method according to claim 33, wherein at least one of the buffers used to form the elution gradient comprises 125 mM sodium chloride.

36. The method according to claim 1, wherein at least one washing buffer and one elution buffer contain 125 mM sodium chloride.

37. The method according to claim 1, wherein at least 10 g / L of the multispecific protein is loaded onto the cation exchange medium.

38. The method according to claim 1, wherein the cation exchange medium is loaded with the multispecific protein in a concentration of 10 g / L to 40 g / L.

39. The method according to claim 38, wherein the cation exchange medium is loaded with 15 g / L to 30 g / L of the multispecific protein.

40. The method according to claim 38, wherein the cation exchange medium is loaded with the multispecific protein in a concentration of 25 g / L to 40 g / L.

41. The method according to claim 1, wherein the cation exchange medium is loaded with 10 g / L of the multispecific protein, washed with a washing buffer containing 105 mM sodium chloride, and eluted with a salt gradient of 8 mM / CV.

42. The method according to claim 1, wherein the cation exchange medium is loaded with 15 g / L to 30 g / L of the multispecific protein and washed with a washing buffer containing 147 mM sodium chloride.

43. The method according to claim 1, wherein 25 g / L to 40 g / L of the multispecific protein is loaded onto the cation exchange medium, and at least one washing buffer and one elution buffer contain 125 mM sodium chloride.

44. The method according to claim 1, wherein at least one product-related impurity is a homodimer, a high molecular weight species, a semi-antibody, an aggregate, a low molecular weight species, an antibody fragment, or a light chain misassembly.

45. A purification process comprising instrumental operations including cation exchange chromatography carried out according to the method of claim 1.

46. The method according to claim 1, further comprising one or more instrumental operations for purifying the multispecific protein, including affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography column and / or mixed-mode chromatography column, before and / or after the cation exchange chromatography step.

47. The method according to claim 1, wherein the multispecific protein is a bispecific protein.

48. The method according to claim 1, wherein the multispecific protein is a bispecific antibody.

49. A purified multispecific protein produced according to the method of claim 1.

50. The method according to claim 1, wherein the cation exchange chromatography medium is a resin.

51. A method for reducing low pI product-related impurities in the eluate from cation exchange chromatography, comprising: A step of loading a composition containing a multispecific protein and at least one product-related impurity having a pI lower than that of the multispecific protein onto a cation exchange chromatography medium; A step of washing the cation exchange medium with a first washing buffer; A step of washing the cation exchange medium with a second washing buffer containing 100 to 147 mM sodium chloride; The process includes eluting the multispecific protein from the cation exchange chromatography resin; Herein, the cation exchange chromatography eluate is obtained in a method that reduces product-related impurities with a lower pI compared to a cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not included in the washing buffer preparation.

52. The method according to claim 51, wherein the cation exchange medium is washed with a third washing buffer.

53. A method for performing cation exchange chromatography under high-salt washing conditions to reduce product-related impurities, comprising: A step of loading a composition containing a multispecific protein and at least one product-related impurity onto an equilibrated cation exchange column; A step of washing the cation exchange medium using at least two types of washing buffers, wherein at least one of the washing buffers contains 100 to 147 mM sodium chloride; and A method comprising the step of eluting the bound multispecific protein from the cation exchange chromatography resin.

54. The method according to claim 53, wherein the cation exchange medium is equilibrated with a buffer that does not contain sodium chloride before loading the composition.

55. A method for producing isolated and purified recombinant multispecific proteins: A step of establishing cell culture in a bioreactor using host cells expressing the aforementioned multispecific protein; A step of culturing the host cells to express the multispecific protein; A step of harvesting the recombinant multispecific protein from the cell culture; A step of affinity purification of the recombinant multispecific protein; A step of loading the affinity-purified recombinant multispecific protein onto a cation exchange chromatography resin; A step of washing the cation exchange resin with at least one washing buffer containing 100 to 147 mM sodium chloride; A step of eluting the multispecific protein from the cation exchange chromatography resin; and A method comprising the step of loading the cation exchange chromatography eluate containing the recombinant multispecific protein onto an additional chromatography medium in flow-through mode.

56. The method according to claim 55, wherein the additional chromatographic medium is selected from a cation exchange chromatographic medium, a multimodal chromatographic medium, a hydrophobic interaction chromatographic medium, and a hydroxyapatite chromatographic medium.

57. The method according to claim 55, wherein the affinity-purified multispecific protein is in an eluate pool and subjected to low-pH virus inactivation, followed by neutralization before being loaded onto the cation exchange medium.

58. The method according to claim 56, wherein the flow-through from the third chromatographic medium undergoes ultrafiltration and dialysis.

59. A multispecific protein isolated and purified by the method of claim 55.

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

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