EA046702B1 - METHODS FOR PURIFYING BISPECIFIC ANTIBODIES USING CATION EXCHANGE CHROMATOGRAPHY USING A HIGH SALT WASH - Google Patents

Description

This application claims priority to U.S. Provisional Application No. 62/931874, filed November 7, 2019, which is hereby incorporated by reference.

Field of technology to which the invention relates

The present invention relates to the field of production of biopharmaceuticals. In particular, the present invention relates to methods for removing product-related impurities from cation exchange purification operations of polyspecific proteins.

State of the art

Antibody products represent the largest sector of the biopharmaceutical market and could easily reach hundreds of billions of dollars in sales over the next decade. Commercial development of therapeutic antibodies began in the 1980s with the approval of the first therapeutic monoclonal antibody and has continued to develop and expand since then. Although monoclonal antibodies are capable of binding to their target with high affinity and specificity and have been very successful for the treatment of some indications, they also have limitations as therapeutic agents. Monoclonal antibodies can bind to only one target; however, many diseases are multifactorial. In cancer immunotherapy treatments, treatments directed at a single target may not be sufficient to completely kill or immobilize cancer cells. In addition, some patients receiving monoclonal antibody therapies may not respond to treatment or may even develop resistance to the drug over time.

To address these challenges, new antibody-like structures have been developed, such as Fab antibody fragments, Fc fusion proteins, antibody-drug conjugates, engineered glycosylation antibodies, and, in particular, bispecific and other multispecific antibody-like structures. Such antibody-like structures, in particular bispecific antibodies, offer improvements over traditional monoclonal antibody therapeutics, such as affinity for multiple targets, and have proven to be effective next-generation biotherapeutics with a huge variety of formats that can be developed to meet even more difficult to treat therapeutic indications.

Bispecific antibodies represent the most diverse group of such antibody-like structures, with a steadily increasing variety of scaffolds to address the challenges of an ever-wider range of therapeutic indications. Such structures combine the binding properties of antibodies with the properties of additional molecules engineered into scaffolds to meet the needs of the target disease indication. Bispecific antibodies are being developed for a variety of indications and applications, such as redirecting immune effector cells to tumor cells for an immune response against cancer, blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, crossing the blood-brain barrier, diagnostic assays, treating diseases caused by pathogens, and as delivery vehicles. (Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Walsh, Nature Biotechnology, 32(10), 9921000, 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).

The development of such multispecific proteins introduces new challenges in biomanufacturing, particularly with regard to product instability and low expression yields. In particular, the purification of multispecific proteins is complicated by the formation of product-associated variants such as homodimers, semi-antibodies, aggregates, high and low molecular weight species, and the like. Such product-associated variants have structural and physical properties, such as charge, similar to those of the polyspecific protein of interest, making them difficult to separate during purification. Such product-associated impurities reduce the yield and activity of the multispecific drug product.

Product-bound impurities having a charge (isoelectric point, pI) similar to that of the polyspecific protein of interest may co-elute with the polyspecific protein during separate cation exchange chromatography steps, complicating purification and reducing yield. It would be useful to separate low pI product-bound impurities before elution. The present invention described herein satisfies this need by providing high salt wash conditions to remove such low pI product related impurities during cation exchange chromatography.

The essence of the invention

The present invention provides a method for purifying a multispecific protein, and more specifically, a bispecific antibody, comprising loading a sample containing the bispecific antibody into a cation exchange chromatography medium; washing the cation exchange medium with at least one wash buffer with a pH of 4.9 to 5.1 containing 100-147 mM chloride per

- 1 046702 tria; and eluting the bispecific antibody from the cation exchange chromatography resin. In one embodiment, the at least one wash buffer contains 100-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains 100-105 mM sodium chloride. In one embodiment, the at least one wash buffer contains 105-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 105-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains 125-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate. In a related embodiment, the at least one wash buffer contains acetate, pH 5.0±0.05% to pH 5.0±0.1%. In a related embodiment, the at least one wash buffer contains acetate, pH 4.91-5.1. In a related embodiment, the at least one wash buffer contains acetate, pH 4.9, 5.0, or 5.1. In another related embodiment, the wash buffer contains 100 mM acetate. In one embodiment, the at least one wash buffer contains acetate, 100-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 100-105 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 105-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 105-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 125-147 mM sodium chloride.

In one embodiment, the cation exchange medium is washed with at least two wash buffers. In one embodiment, the cation exchange medium is washed with at least three wash buffers.

In one embodiment, the cation exchange medium is washed with at least two wash buffers, at least one of the wash buffers containing 0-147 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with at least two wash buffers, at least one of the wash buffers containing 070 mM sodium chloride. In one embodiment, the cation exchange medium is washed with at least two wash buffers, at least one wash buffer containing acetate, 0 mM sodium chloride, and then a wash buffer containing acetate, 100-147 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with a wash buffer containing acetate, 0 mM sodium chloride, and then a wash buffer selected from the group consisting of a wash buffer containing acetate, 100 mM sodium chloride, a wash buffer containing acetate, 105 mM sodium chloride or wash buffer containing acetate, 125 mM sodium chloride. In one embodiment, the cation exchange medium is washed with a wash buffer containing acetate, 100-147 mM sodium chloride, followed by a wash buffer containing acetate, 0-70 mM sodium chloride. In a related embodiment, the cation exchange medium is washed with a wash buffer containing acetate, 100-147 mM sodium chloride, followed by a wash buffer containing acetate, 0 mM sodium chloride. In one related embodiment, the cation exchange medium is washed with a wash buffer containing acetate, 100-147 mM sodium chloride, and then a wash buffer containing 70 mM sodium chloride.

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

In one embodiment, the cation exchange medium is washed with 2.5 mM/CV wash buffer containing 147 mM sodium chloride.

In one embodiment, the bispecific antibody is eluted from the cation exchange medium via a gradient. In a related embodiment, the gradient is a linear or step gradient. In a related embodiment, the gradient is a salt gradient. In a related embodiment, the buffers used to create the elution gradient contain 0-1 M sodium chloride. In another related embodiment, at least

- 2 046702 one of the buffers used to create the elution gradient contains 70-500 mM sodium chloride. In another related embodiment, at least one of the buffers used to create the elution gradient contains 125 mM sodium chloride. In a related embodiment, at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

In one embodiment, at least 10 g/L of bispecific antibody is loaded into the cation exchange medium. In one embodiment, 10 g/L to 40 g/L of bispecific antibody is loaded into the cation exchange medium. In a related embodiment, 15 g/L to 30 g/L is charged into the cation exchange medium. In a related embodiment, 25 g/L - 40 g/L is charged to the cation exchange medium. In one embodiment, the cation exchange medium is loaded with 10 g/L of bispecific antibody, washed with wash buffer containing 105 mM sodium chloride, and eluted on a salt gradient at 8 mM/CV. In one embodiment, the cation exchange medium is loaded with 15 g/L to 30 g/L of bispecific antibody, washed with wash buffer containing 147 mM sodium chloride. In one embodiment, the cation exchange medium is loaded with 25 g/L to 40 g/L of bispecific antibody, wherein at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

In one embodiment, the at least one product-associated impurity is a homodimer, high molecular weight species, semi-antibody, aggregate, low molecular weight species, antibody fragment, or light chain misassembly. In one embodiment, the purification method, which includes a separate step involving cation exchange chromatography, is performed in accordance with the method described above.

In one embodiment, the above method further includes, before and/or after the cation exchange chromatography step, one or more separate steps for purifying the bispecific antibody, comprising affinity chromatography, ion exchange chromatography, hydrophobic interaction column chromatography, and/or mixed mode column chromatography.

In the present invention, the multispecific protein is a bispecific protein, and more specifically, is a bispecific antibody. In one embodiment, the purified bispecific antibody is prepared in accordance with the method described above. In one embodiment, the cation exchange chromatography medium is a resin.

The present invention provides a method for reducing low pI product related impurities in a cation exchange chromatography eluate, the method comprising feeding a composition comprising a bispecific antibody and at least one product related impurity having a lower pI than the bispecific antibody to medium for cation exchange chromatography; washing the cation exchange medium with a first wash buffer, washing the cation exchange medium with a second wash buffer with a pH of 4.9 to 5.1 containing 100-147 mM sodium chloride; eluting the bispecific antibody from the cation exchange chromatography resin; wherein the cation exchange chromatography eluate has a reduced content of low pI product-related impurities compared to the cation exchange chromatography eluate recovered in a corresponding process in which sodium chloride is not included in the wash buffer. In one embodiment, the cation exchange medium is further washed with a third wash buffer.

The present invention provides a method of performing cation exchange chromatography under high salt wash conditions to reduce product-related impurities, the method comprising loading a composition comprising a bispecific antibody and at least one product-related impurity onto an equilibrated cation exchange column; washing the cation exchange medium with at least two wash buffers, where one wash buffer has a pH of 4.9 to 5.1 and contains 100-147 mM sodium chloride; and eluting the bound bispecific antibody from the cation exchange chromatography resin. In one embodiment, the cation exchange medium is equilibrated with a sodium chloride-free buffer before loading the composition.

The present invention provides a method for producing an isolated, purified recombinant bispecific antibody, the method comprising creating a cell culture in a bioreactor with a host cell expressing the bispecific antibody; culturing host cells to express the bispecific antibody; collecting the recombinant bispecific antibody from the cell culture; affinity purification of the recombinant bispecific antibody; loading the affinity purified recombinant bispecific antibody onto a cation exchange chromatography resin; washing the cation exchange resin with at least one wash containing 100-147 mM sodium chloride; eluting the bispecific antibody from the cation exchange chromatography resin and loading the cation exchange chromatography eluate containing the recombinant bispecific antibody into additional chromatography media in a flow mode. In one embodiment, the additional chromatography medium is selected from

- 3 046702 for cation exchange chromatography, multimodal chromatography media, hydrophobic interaction chromatography media and hydroxyapatite chromatography media. In one embodiment, the affinity purified bispecific antibody is in the eluate pool and is virally inactivated at low pH and then neutralized before loading into the cation exchange medium. In one embodiment, the filtrate from the third chromatographic medium is subjected to a separate ultrafiltration and diafiltration step. In one embodiment, the isolated, purified recombinant bispecific antibody is prepared in accordance with the method described above.

Brief description of graphic materials

In fig. 1 shows product-related impurities (homodimer, NCG) eluting with the main product, bispecific molecule #1.

In fig. Figure 2 shows that under high salt wash conditions, all product-associated impurities whose pI is lower than that of the main product are washed out of the column between the second and third wash steps. Before elution, the initial level was ensured to return to zero. The elution peak was reduced to a single peak.

In fig. Figure 3 shows that numerous impurities remained in the column and eluted with the main product. These impurities included semi-antibodies (fractions 1-4 with ~50% semi-antibodies), 2X light chain misassemblies (fractions 5 and 6), and high molecular weight species (fractions 12-21).

In fig. Figure 4 shows that the high salt wash resulted in a reduction in the number of peaks in the elution profile from four peaks to one peak with a small shoulder that still contained 2X LC2 mismatched species (LC1/LC2 <0.11 compared to the expected ratio 1).

In fig. Figure 5 shows a single elution peak resulting from a high loading density with a steep elution gradient. Product-associated low pI impurities were not separated from the main product at high loading densities and were mainly found in fractions 1-3 as evidenced by the decrease in CE-SDS.

In fig. Figure 6 shows that decreasing loading density with a flatter gradient resulted in the separation of major low pI product-related impurities into a distinct peak containing LC1 mismatched species.

In fig. Figure 7 shows the results of the high salt wash. A decrease in the number of impurity peaks was observed in the elution profile, with most of the impurities being removed during the second and third washing steps. The third washing step ensured that the original UV spectrum was restored to zero before elution began, tightening the elution profile, resulting in much more efficient collection and better quality of the main product.

In fig. Figure 8 shows that under high salt wash conditions, all impurities related to the low pI products (homodimer species with a pI of 6.8 and any aggregated low pI species) passed through the column to waste, or were collected separately in the wash pool to test their composition. Before elution, the initial level was ensured to return to zero. The elution peak was reduced to a single peak.

Detailed Description of the Invention

Since there is not much information in the literature regarding the downstream processing of multispecific proteins, platforms developed for monoclonal antibodies are often used (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, p559-594, John Wiley & Sons, 2017). Multispecific proteins are highly technical, and subjecting such proteins to cation exchange chromatography (CEX) in a binding and eluting mode under typical antibody conditions may not be sufficient to stabilize the multispecific proteins and/or handle product-related impurities that elute with the main product. Such product-associated impurities are characterized by isoelectric points that are both lower and higher than those of the main product. Product-bound impurities with a pI lower than that of the main product were found to elute with the main product as prepeaks. This elution profile does not support the development of a reliable, stable production process on an industrial scale.

It was found that the use of a high salt washing strategy resulted in better yield and purity of the main product and an improvement in the production process. The addition of a high-salt wash reduced the content of lower pI product-related impurities in the eluate pool by removing them before the elution step. The addition of a wash step containing sodium chloride in the range of 105-147 mM caused product-associated impurities with a pI that was lower than that of the main product to be washed out or eluted from the cation exchange medium before the elution step, reducing the number of peaks in the elution profile . Automatic OD-based eluate pooling becomes difficult in the presence of prepeaks. It was found that the inclusion of a washing step with cation exchange chromatography before elution

- 4 046702 high salt content reduced the number of prepeaks associated with low pI product-related impurities, reducing the need for a more conservative strategy to be applied during the collection of the main product during elution, which would likely result in lower yields and less stable production process.

The present invention provides a method for purifying a polyspecific protein, comprising loading a sample containing the polyspecific protein into a cation exchange chromatography medium; washing the cation exchange medium with at least one wash buffer containing 100-147 mM sodium chloride; and eluting the polyspecific protein from the cation exchange chromatography resin.

The present invention provides a method for reducing the content of product-related low pI impurities in an eluate from cation exchange chromatography, the method comprising loading a composition containing a polyspecific protein (hereinafter in the description of the present invention the term polyspecific protein refers mainly to bispecific proteins, and more specifically, a bispecific antibody) and at least one product-related impurity having a pI lower than that of the multispecific protein into the cation exchange chromatography medium; washing the cation exchange medium with a first wash buffer, washing the cation exchange medium with a second wash buffer containing 100-147 mM sodium chloride; eluting the polyspecific protein from the cation exchange chromatography resin; wherein the cation exchange chromatography eluate has a reduced content of low pI product-related impurities compared to the cation exchange chromatography eluate recovered in a corresponding process in which sodium chloride is not included in the wash buffer.

The present invention provides a method of performing cation exchange chromatography under high salt wash conditions to reduce product-associated impurities, the method comprising loading a composition comprising a polyspecific protein and at least one product-associated impurity onto an equilibrated cation exchange column; washing the cation exchange medium with at least two wash buffers, where one buffer contains 100-147 mM sodium chloride; and eluting the bound polyspecific protein from the cation exchange chromatography resin.

The present invention provides a method for producing an isolated, purified recombinant polyspecific protein, the method comprising creating a cell culture in a bioreactor with a host cell expressing the polyspecific protein; culturing host cells to express a polyspecific protein; collection of recombinant polyspecific protein from cell culture; affinity purification of recombinant polyspecific protein; loading the affinity purified recombinant polyspecific protein onto a cation exchange chromatography resin; washing the cation exchange resin with at least one wash containing 100-147 mM sodium chloride; eluting the polyspecific protein from the cation exchange chromatography resin; and loading the cation exchange chromatography eluate containing the recombinant polyspecific protein into a third chromatography medium in flow mode.

The present invention provides a purification method that includes a separate step involving cation exchange chromatography performed in accordance with the methods disclosed herein.

The present invention provides a purified multispecific protein obtained in accordance with the methods disclosed herein.

The present invention provides a pharmaceutical composition containing an isolated, purified recombinant polyspecific protein obtained in accordance with any of the methods described herein.

In one embodiment, the at least one wash buffer contains 0-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 70-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 100-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 100-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains 100-105 mM sodium chloride. In one embodiment, the at least one wash buffer contains 105-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 105-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains 125-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 0, 70, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 1, 142, 142, 144, 145, 146 or 147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 0 mM sodium chloride. In one embodiment, the at least one wash buffer contains 70 mM sodium chloride. In one embodiment, the at least one wash buffer contains 100 mM chloride per

- 5 046702 tria. In one embodiment, the at least one wash buffer contains 105 mM sodium chloride. In one embodiment, the at least one wash buffer contains 125 mM sodium chloride. In one embodiment, the at least one wash buffer contains 147 mM sodium chloride.

In one embodiment of the present invention, at least one wash buffer contains acetate. In one embodiment, the at least one wash buffer contains acetate, pH from 5.0±0.05 to 5.0±0.1. In one embodiment, the at least one wash buffer contains acetate, pH 4.9-5.1. In one embodiment, the at least one wash buffer contains acetate at a pH of 4.9, 5.0, or 5.1. In one embodiment, the at least one wash buffer contains 100 mM acetate. In one embodiment, the at least one wash buffer contains acetate, pH 5.0±0.05% to pH 5.0±0.1%. In one embodiment, the at least one wash buffer contains acetate, pH 4.9-5.1. In one embodiment, the at least one wash buffer contains acetate at a pH of 4.9, 5.0 or 5.1. In one embodiment, the at least one wash buffer contains acetate at pH 5.0. To consistently remove product-bound impurities, wash buffers can be pH units 0.05-0.1 units higher and lower with varying conductivities.

In one embodiment, the at least one wash buffer contains acetate, 0147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 70-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 100-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 100-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 100-105 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 105-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 105-125 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 125-147 mM sodium chloride. In a related embodiment, the acetate concentration is 100 mM.

In one embodiment, the at least one wash buffer contains acetate, 0 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 70 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 100 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 105 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 125 mM sodium chloride. In one embodiment, the at least one wash buffer contains acetate, 147 mM sodium chloride. In a related embodiment, the acetate concentration is 100 mM.

In one embodiment, the at least one wash buffer contains acetate, 0147 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In one embodiment, the at least one wash buffer contains acetate, 70-147 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In one embodiment, the at least one wash buffer contains acetate, 100-147 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In one embodiment, the at least one wash buffer contains acetate, 100-125 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In a related embodiment, the at least one wash buffer contains acetate, 100-105 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In a related embodiment, the at least one wash buffer contains acetate, 105-147 mM sodium chloride, pH 5.0±0.05% to pH 5.0±0.1%. In a related embodiment, the at least one wash buffer contains acetate, 105-125 mM sodium chloride, pH 5.00±0.05% to pH 5.0±0.1%. In a related embodiment, the at least one wash buffer contains acetate, 125,147 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the acetate concentration is 100 mM.

In a related embodiment, the at least one wash buffer contains acetate, 0 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the at least one wash buffer contains acetate, 70 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the at least one wash buffer contains acetate, 100 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the at least one wash buffer contains acetate, 105 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the at least one wash buffer contains acetate, 125 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the at least one wash buffer contains acetate, 147 mM sodium chloride, pH 5.00 ± 0.05% to pH 5.0 ± 0.1%. In a related embodiment, the acetate concentration is 100 mM.

- 6 046702

In a related embodiment, the at least one wash buffer contains acetate, 0-147 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 70-147 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 100-147 mM sodium chloride, pH 4.9-5.1. In one embodiment, the at least one wash buffer contains acetate, 100-125 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 100-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 105-147 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 105-125 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 125-147 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the acetate concentration is 100 mM.

In a related embodiment, the at least one wash buffer contains acetate, 0 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 70 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 100 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 105 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 125 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the at least one wash buffer contains acetate, 147 mM sodium chloride, pH 4.9-5.1. In a related embodiment, the acetate concentration is 100 mM.

In a related embodiment, the at least one wash buffer contains acetate, 0-147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 70-147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 100-147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In one embodiment, at least one wash buffer contains acetate, 100-125 mM sodium chloride, pH 4.9, 5.0 or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 100-105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 105-147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 105-125 mM sodium chloride, pH 4.9, 5.0 or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 125,147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the acetate concentration is 100 mM.

In a related embodiment, the at least one wash buffer contains acetate, 0 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 70 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 100 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 125 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the at least one wash buffer contains acetate, 147 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment, the acetate concentration is 100 mM.

In one embodiment, the at least one wash buffer contains 100 mM acetate, 100 mM sodium chloride, pH 5.0±0.5% to pH 5.0±0.1. In one embodiment, the at least one wash buffer contains 100 mM acetate, 105 mM sodium chloride, pH 5.0±0.5% to pH 5.0±0.1. In one embodiment, the at least one wash buffer contains 100 mM acetate, 125 mM sodium chloride, pH 5.0 ± 0.5% to ± 0.1. In one embodiment, the at least one wash buffer contains 100 mM acetate, 147 mM sodium chloride, pH 5.0 ± 0.5% to pH 5.0 ± 0.1. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 70 mM sodium chloride, pH 5.0±0.5% to pH 5.0±0.1. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 0 mM sodium chloride, pH 5.0±0.5% to pH 5.0±0.1.

In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 0-147 mM sodium chloride, pH 5.0. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 70-147 mM sodium chloride, pH 5.0. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 100-125 mM sodium chloride, pH 5.0. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 100-105 mM sodium chloride, pH 5.0. In one embodiment of the present invention

- 7 046702 at least one wash buffer contains 100 mM acetate, 105-147 mM sodium chloride, pH 5.0. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 105-125 mM sodium chloride, pH 5.0. In one embodiment of the present invention, at least one wash buffer contains 100 mM acetate, 125,147 mM sodium chloride, pH 5.0. To consistently remove product-bound impurities, wash buffers can be pH units 0.05-0.1 units higher and lower with varying conductivities.

In one embodiment, the cation exchange medium is washed with at least two wash buffers. In one embodiment, the wash buffers contain 0-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 0, 70, 100, 105, 125, or 147 mM sodium chloride. In one embodiment, the wash buffers contain acetate. In one embodiment, the wash buffer contains 100 mM acetate. In one embodiment, the pH of the at least one wash buffer is between 5.0±0.05% and pH 5.0±0.1%. In one embodiment, the pH of the at least one wash buffer is 4.9-5.1. In one embodiment, the pH of the at least one wash buffer is 4.9, 5.0, or 5.1. In one embodiment, the cation exchange medium is washed with at least two wash buffers, wherein at least one wash buffer contains acetate, 0-70 mM sodium chloride, and at least one wash buffer contains acetate, 100-147 mM sodium. In one embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, one wash buffer containing acetate, 0 mM sodium chloride, and then a wash buffer containing acetate, 100-147 mM sodium chloride. In one embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, one wash buffer containing acetate, 100-147 mM sodium chloride, and then a wash buffer containing acetate, 0-70 mM sodium chloride. In one embodiment, the cation exchange chromatography medium is washed with at least two wash buffers, the first wash buffer containing acetate, 0 mM NaCl, and the second wash buffer containing acetate, 100-147 mM sodium chloride. In one embodiment, the concentration of sodium chloride in 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 wash buffers, the first wash buffer containing acetate, 100-147 mM NaCl, and the second wash buffer containing acetate, 0-70 mM sodium chloride. In one embodiment, the concentration of sodium chloride in the first wash buffer is selected from 100, 105 and 147 mM sodium chloride. In one embodiment, the concentration of the first buffer is 100 mM acetate, 0 mM sodium chloride, and the concentration of the second wash buffer is 100 mM acetate, 125 mM sodium chloride.

In one embodiment, the cation exchange medium is washed with at least three wash buffers. In one embodiment, the wash buffers contain 0-147 mM sodium chloride. In one embodiment, the at least one wash buffer contains 0, 70, 100, 105, 125, or 147 mM sodium chloride. In one embodiment, the wash buffer contains acetate. In one embodiment, the at least one wash buffer contains 100 mM acetate. In one embodiment, the pH of the at least one wash buffer is between 5.0±0.05% and pH 5.0±0.1%. In one embodiment, the pH of the at least one wash buffer is 4.9-5.1. In one embodiment, the pH of the at least one wash buffer is 4.9, 5.0, or 5.1.

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

- 8 046702 The sodium wash buffer is 100 mM acetate, 70 mM sodium chloride.

In one embodiment, the cation exchange resin is washed with 2.5 mM/CV wash buffer containing 147 mM sodium chloride.

In one embodiment, the polyspecific protein is eluted from the cation exchange medium via a gradient. In a related embodiment, the gradient is a linear or step gradient. In a related embodiment, the gradient is a salt gradient. In a related embodiment, at least one of the buffers used to create the elution gradient contains 0-1 M sodium chloride. In a related embodiment, at least one of the buffers used to create the elution gradient contains 70,500 mM sodium chloride.

In one embodiment, at least one of the buffers used to create the elution gradient contains 125 mM sodium chloride. In a related embodiment, at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

In one embodiment, the cation exchange medium is equilibrated with a sodium chloride-free buffer before loading the composition.

In one embodiment, the cation exchange medium is loaded with 10 g/L of the polyspecific protein, washed with wash buffer containing 105 mM sodium chloride, and eluted on a salt gradient at 8 mM/CV.

In one embodiment, the at least one product-associated impurity is a homodimer, high molecular weight species, semi-antibody, aggregate, low molecular weight species, antibody fragment, or light chain misassembly.

In one embodiment, the affinity purified polyspecific protein is in the eluate pool and is virally inactivated at low pH and then neutralized before loading into the cation exchange medium.

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

In one embodiment, the third chromatography medium is selected from cation exchange chromatography medium, multimodal chromatography medium, hydrophobic interaction chromatography medium, and hydroxyapatite chromatography medium. In one embodiment, the filtrate from the third chromatographic medium is subjected to a separate ultrafiltration and diafiltration step.

In one embodiment, before and/or after the cation exchange chromatography step, one or more separate steps are provided for purifying the polyspecific protein, comprising affinity chromatography, ion exchange chromatography, hydrophobic interaction column chromatography, and/or mixed-mode column chromatography.

In one embodiment, the polyspecific protein is a bispecific protein. In one embodiment, the polyspecific protein is a bispecific antibody.

Polyspecific, polyspecific protein and polyspecific antibody are used herein to refer to proteins that are recombinantly engineered to simultaneously bind and neutralize at least two different antigens or at least two different epitopes of the same antigen. For example, multispecific proteins can be engineered to target immune effectors in combination with targeting cytotoxic agents against tumors or infectious agents. Such multispecific proteins have been found to be useful in a variety of applications, such as cancer immunotherapy by redirecting immune effector cells to tumor cells, modifying cell signaling by blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, and as pretargeting agents. delivery vehicles for drugs, such as delivery of chemotherapeutic agents, radioactive tags (to improve detection sensitivity) and nanoparticles (targeted to specific cells/tissues, such as cancer cells).

The most common and most diverse group of multispecific proteins are those proteins that bind two antigens, referred to herein as bispecifics, bispecific proteins and bispecific antibodies. Bispecific proteins can be grouped into two broad categories: immunoglobulin G (IgG)-like molecules and non-immunoglobulin G (IgG)-like molecules. While IgG-like molecules retain Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), the Fc region helps improve solubility and stability and simplify some purification operations. Molecules other than IgG-like molecules are smaller in size, which improves their penetration into tissues (see Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018;

- 9 046702

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 are sometimes used as a scaffold for additional components characterized by binding specificity to different antigens or a large number of epitopes, which increases the binding specificity of the molecule.

Formats for bispecific proteins, which include bispecific antibodies, are constantly evolving and include, but are not limited to, quadromas, peak-to-valve structures, CrossMab, double variable domain IgG (DVD-IgG), IgG single chain Fv (scFv), scFv-SN3 KIH, Dual Action Fab (DAF), Half Exchange Structure, κλ Bodies, Tandem scFv, scFv-Fc, Diabodies, Single Chain Diabodies (sc-Dibodies), sc-Dibodies-CH3, Tribody, Minibody, Minibody, TriBi Minibody , tandem diabodies, sc-diabody-HAS, scFv-toxin tandem structure, dual affinity retargeting molecules (DART), nanobody, nanobody-HSA, docking and anchoring (DNL) structure, SEEDbody based on engineered strand exchange domains, Triomab, Leucine Zipper (LUZ-Y), XmAb®; structure with exchange of Fab arms, DutaMab, DT-IgG, structure with charged pairs, 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 in one), Fab-scFv, scFv-CH-CL-scFV, F(ab') ₂ -scFv ₂ , 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-attracting activators (BITE®), and bispecific T-cell-attracting activators with extended half-life (ULE BITE ®) (Fan above; Spiess above; Sedykh above; Seimetz et al., Cancer Treat Rev 36(6) 458-67, 2010; Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, p559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011).

In some embodiments, the bispecific proteins may include blinatumomab, catumaxomab, ertumaxomab, solitomab, targomiR, luticizumab (ABT981), vanucizumab (RG7221), remtolumab (ABT122), ozoralixumab (ATN103), floteuzmab (MGD006), pasotuxizumab (AMG112, MT 112), lymphomune (FBTA05), (ATN-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, indium-labeled IMP-205, xm734, LY3164530, OMP305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013 (ACE910), RG7597 (MEDH7945A), RG7802, RG7813 (RO6895882 ), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM111, MM141, MOR209/ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, as well as molecules or variants thereof or analogs and biosimilars of any of the above.

Multispecific proteins also include trispecific antibodies, tetravalent bispecific antibodies, multispecific proteins without antibody components, such as dia-, tria- or tetrabodies, minibodies and single-chain proteins capable of binding multiple targets. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163.

In some embodiments, the polyspecific proteins of interest bind to, neutralize, and/or specifically interact with one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation proteins and coagulation-related proteins, colony-stimulating factors (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins and immunoadhesins.

In some embodiments, the polyspecific proteins of interest bind to, neutralize, and/or interact with one or more of the following, individually or in any combination: CD proteins, including, but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171 and CD174, proteins of the HER receptor family, including, for example, HER2, HER3, HER4 and the EGF receptor, EGFRvIII, cell adhesion molecules, e.g., LfA-1, Mol, p150.95, VLA-4, ICaM-1, VCAM and integrin alpha-y/beta 3, growth factors, including but not limited to, e.g., endothelial growth factor blood vessels (VEGF); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parati

- 10 046702 rheoid hormone, Müllerian inhibitory factor, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factor such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factor, including including, for example, aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), including, but not limited to, TGF-α and TGF-β, including TGF-pI, TGF-e2, TGF-e3, TGF^4 or TGF-e5, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and osteoinductive factors , insulins and insulin-related proteins, including restrictions on insulin, insulin A-chain, insulin B-chain, proinsulin and insulin-like growth factor binding proteins; coagulation proteins and coagulation-related proteins such as, but not limited to, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1 antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator (t-PA), bombasin , thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factors (CSF), including but not limited to M-CSF, GM-CSF and G-CSF, other blood and serum proteins, including but not limited to albumin, IgE and blood group antigens, receptors and receptor-associated proteins, including, for example, flk2/flt3 receptor, obesity receptor (OB), growth hormone receptors and T-cell receptors; neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6); relaxin A-chain, relaxin B-chain and prorelaxin, interferons, including, for example, interferon-alpha, -beta and -gamma, interleukins (IL), for example, IL-1-IL-10, IL-12, IL -15, IL-17, IL-23, IL-12/IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and/or IL-13 receptors, IL13RA2 or IL-17 receptor , IL-1RAP; viral antigens, including but not limited to AIDS virus envelope antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factors alpha and beta, enkephalinase, BCMA, STEAP1, kappa chain Ig, ROR-1, ERBB2, mesothelin, RANTES (activation-regulated protein expressed and secreted by normal T cells), murine gonadotropin-associated peptide, DNase, FR-alpha, inhibin and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP ), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS virus envelope, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins , antigen-binding proteins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, glanglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, cKit, B7RP-1, PSMA, NKG2D- 1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor/hCGe, TNF, TL1A, hepatitis C virus, mesothelin conjugate dsFv[PE38, Legionella pneumophila (lly), IFN-gamma, interferon-gamma-induced protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin/kexin type 9 (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, protein , platelet-specific (platelet glycoprotein Iib/IIIb (PAC-1)), transforming growth factor beta (TFGe), sperm-binding protein 3 zona pellucida (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRa), sclerostin and biologically active fragments or variants of any of the above.

In some embodiments, the polyspecific proteins of interest may include bispecific antibodies that specifically bind to combinations including CD3 and CD19, EpCAM, CEA, PSA, CD33, BCMA, Her2, CD20, P-cadherin, CD123, gpA33, or B7H3. In some embodiments, the bispecific antibodies of interest may include bispecific antibodies that specifically bind to combinations comprising IL1a+IL1e.

Polyspecific proteins may be of scientific or commercial interest, in particular, they may be bispecific therapeutic agents. Multispecific proteins can be produced in a variety of ways, most commonly using recombinant animal cell lines using cell culture techniques. Multispecific proteins can be produced intracellularly or secreted into a culture medium from which they can be recovered and/or collected, and may be referred to as recombinant multispecific protein, recombinant multispecific antibody, recombinant bispecific protein, and recombinant bispecific antibody. The terms isolated polyspecific protein, isolated recombinant polyspecific antibody, isolated bispecific protein and isolated bispecific antibody refer to a multispecific protein, including bispecific proteins, that have been purified from proteins, polypeptides, DNA and/or other contaminants or impurities such as those associated with the product impurities, in particular product-associated low pI impurities that could interfere with its therapeutic, diagnostic, prophylactic, research or other use

- 11 046702 neniya. Multispecific proteins of interest include multispecific antibodies that exert a therapeutic effect by binding to two or more targets, particularly targets listed below, including targets derived from them, targets related to them, and modifications thereof.

By purification we mean increasing the purity of a polyspecific protein in a composition by removing (partially or completely) at least one product-related impurity from the composition. Extraction and purification of polyspecific proteins is accomplished by subsequent separate steps, particularly those involving ion exchange chromatography, resulting in a more homogeneous composition of the polyspecific proteins that meets yield and product quality targets (such as reducing product-related impurities and improving product quality).

A product-associated impurity refers to product-associated variants of a multispecific protein of interest. In some cases, these impurities have a pI lower than that of the main product at the elution peak. Product-associated impurities include, for example, homodimers, high molecular weight species (HMW), semi-antibodies, aggregates, low molecular weight species (LMW), antibody fragments and various combinations of antibody fragments, as well as light chain misassemblies such as 2XLC, 3XLC or 4XLC. Half-antibodies refer to a product-associated impurity that may be formed, for example, due to incomplete assembly or disruption of the interaction between two heavy chain polypeptides. Hemiantibodies contain one light chain polypeptide and one heavy chain polypeptide. Homodimers refer to a product-associated impurity that, for example, can be formed when heavy and light chains having specificity for the same target recombine with each other instead of pairing to form the desired bispecific heterodimer. Typically, this occurs during expression in the host cell. In the case of polyspecific constructs that require multiple chains (such as light chains, LCs) to be paired correctly using designed residues (such as charged pairwise mutations, peak-to-valve, etc.), it is still possible that impurities if there is an erroneous pairing between LC and HC, with LC1, instead of forming a pair with HC1, forming an incorrect pair with HC2 (2xLC1) and vice versa (2xLC2). If the polyspecific protein is bivalent, having two binding sites for each antigen of interest, 3X LC1, 4X LC1, and other combinations of mismatched species may be present.

As disclosed herein, the pI of product-associated impurities may be lower than the pI of the desired polyspecific protein and elute with the main product. Product-bound impurities, characterized by a lower pI, elute before the main product as prepeaks. The isoelectric point or pI of a protein refers to the pH at which the positive charge balances the negative charge of the protein, the pI can be calculated/determined using known methods such as based on the net charge of the amino acid residues of the protein or by isoelectric focusing. In one embodiment, the difference between the pI of the product-associated impurity and the main product is at least 0.5 pI units. In another embodiment, the difference is from 0.5 to 3 pI units. In another embodiment, the difference is between 0.5 and 2 pI units. In another embodiment, the difference is between 0.5 and 1 pI units. In another embodiment, the difference is between 1 and 3 pI units. In another embodiment, the difference is 2 to 3 pI units. In another embodiment, the difference is at least 0.5, 1, 2, 3 or more pI units.

Presented herein are expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that contain one or more polynucleotides encoding a polyspecific protein of interest, as well as host cells containing such expression systems or constructs. As used herein, vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, deproteinized DNA, complexed DNA, etc.) that is suitable for use in transferring and/or transporting information encoding a polyspecific protein into a host cell and/or to a specific location and/or compartment within the host cell. Vectors may include viral and non-viral vectors, nonepisomal vectors for mammalian cells. Vectors are often referred to as expression vectors, such as recombinant expression vectors and cloning vectors. The vector can be introduced into a host cell to cause the vector itself to replicate and thereby amplify copies of the polynucleotide contained therein. Cloning vectors may contain sequence components, which typically include, but are not limited to, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. If necessary, such elements can be selected by a person skilled in the art.

Cell or cells include any prokaryotic or eukaryotic cell. Cells

- 12 046702 can be found either ex vivo, in vitro or in vivo, and be either separate or part of a higher level of organization such as a tissue or organ. Cells include host cells, also called cell lines, that are genetically engineered to express a polyspecific protein of commercial or scientific interest. Host cells are typically derived from a line derived from a primary culture that can be maintained in culture indefinitely. Genetic engineering of a host cell involves transfecting, transforming or transducing cells with a recombinant polynucleotide molecule and/or otherwise altering (for example, by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) to cause the host cell to express the desired recombinant polyspecific protein. Methods and vectors for genetically engineering cells and/or cell lines to provide expression of polyspecific proteins of interest are well known to those skilled in the art.

The host cell can be any prokaryotic cell (eg, E. coli) or eukaryotic cell (eg, yeast, insect or animal cells (eg, CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques.

When cultured under appropriate conditions, the host cells express the multispecific protein of interest, which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the levels of expression required, modifications of the protein that are required or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

Culture or culturing refers to the growth and reproduction 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 growing host cells during in vitro cell culture. Typically, cell culture media contain a buffer, salts, an energy source, amino acids, vitamins and essential microelements. Any media capable of supporting the growth of the appropriate host cell in culture can be used. Cell culture media, which may also be supplemented with other components to maximize cell growth, cell viability and/or recombinant protein production in a particular cultured host cell, are commercially available and include, but are not limited to, RPMI-1640 media, RPMI-1640 media, 1641, Dulbecco's modified Eagle's medium (DMEM), minimally enriched Eagle's medium, F-12K medium, Ham's F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibowitz's L-15 medium, and serum-free media such as the series EX-CELL™ 300, which are available from the American Type Culture Collection or SAFC Biosciences, as well as other suppliers. Cell culture media may be serum-free media, protein-free media, growth factor-free media, and/or peptone-free media. Cell culture can also be enriched by adding nutrients, and these can be used in concentrations higher than the usual recommended concentrations.

During the life cycle of a culture, different media compositions can be used, for example, to facilitate the transition from one stage (e.g., growth stage or phase) to another (e.g., production stage or phase) and/or to optimize conditions during cell culture (e.g. During perfusion culture, a concentrated medium is provided). The composition of the growth medium can be used to promote cell growth and minimize protein expression. The production medium composition can be used to promote production of the protein of interest and maintain cells while minimizing new cell growth. A culture medium, typically a medium containing more concentrated components such as nutrients and amino acids that are consumed during the production phase of cell culture, can be used to supplement and maintain an active culture, in particular a culture operated in a fed-batch, semi-perfusion or perfusion mode mode. Such a concentrated culture medium may contain most of the components of the cell culture medium in an amount of, for example, about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x or even approximately 1000x of their normal amount.

The growth phase can occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of from about 35°C to about 38°C, and the production phase may occur at a second temperature of from about 29°C to about 37°C, optionally from about 30°C to about 36°C or from about 30°C to about 34°C. In addition chemical

- 13 046702 protein production inducers, such as, for example, caffeine, butyrate and hexamethylene bisacetamide (HMBA), can be added simultaneously, before and/or after the temperature change. If inductors are added after a temperature change, they can be added at a time interval of one hour to five days after the temperature change, optionally one to two days after the temperature change.

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

Cell cultures can be operated in batch, fed, continuous, semi-continuous or perfusion mode. Mammalian cells, such as CHO cells, can be cultured in bioreactors at smaller scales ranging from less than 100 ml to less than 1000 ml. Alternatively, larger scale bioreactors that contain from 1000 ml to over 20,000 liters of media can be used. Large-scale cell cultures, such as for the bioproduction of protein therapeutics on a clinical and/or commercial scale, can be maintained for weeks and even months while the cells produce the required protein(s).

Since product-associated impurities, such as homodimers, semi-antibodies, etc., may be similar to the desired multispecific protein, strategies and techniques such as knob-to-valley, CrossMab, DVD IgG and others have been developed to improve selectivity in regarding the required polyspecific protein during cell culture. However, there will still be a portion of product-related impurities that are produced and that must be removed during subsequent processing.

The resulting expressed recombinant polyspecific protein can then be collected from the cell culture medium. Methods for collecting proteins from suspension cells are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, gravity separation, centrifugation, acoustic wave separation, filtration, including membrane filtration using ultrafilters, microfilters, tangential flow filters, variable tangential flow filters, depth filters and precoat filters. Recombinant proteins expressed by prokaryotes are recovered from inclusion bodies in the cytoplasm by redox folding processes known in the art.

The collected polyspecific protein can then be purified or partially purified from any contaminants, such as remaining cell culture medium, cell extracts, unwanted components, host cell proteins, misexpressed proteins, product-related contaminants, and the like, using one or more subsequent individual operations.

Purification of the polyspecific protein from the cell culture fluid collected can be initiated using capture chromatography. Capture-based chromatography uses media such as resins, membranes, gels, etc., which will bind to the recombinant polyspecific protein of interest, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal ion affinity chromatography (IMAC), etc. Such materials are known in the art and are commercially available. Affinity chromatography variations may include, for example, a substrate-binding-based capture mechanism, an aptamer-binding-based capture mechanism, or a cofactor-binding-based capture mechanism. For multispecific proteins containing an Fc component, a capture mechanism can be used based on binding to an antibody or antibody fragment, such as protein A, protein G, protein A/G and protein L. The recombinant protein of interest can be tagged with a polyhistidine tag or epitope such as FLAG®, and subsequently purified using a specific antibody targeting such an epitope.

At any point in subsequent processing, viral inactivation and/or filtration may be performed to remove viral material from the composition containing the polyspecific protein of interest. One method to achieve viral inactivation is to incubate at low pH or other suitable solution conditions to achieve viral inactivation. Viral inactivation at low pH may be followed by a neutralization step in which the inactivated virus solution is readjusted to a pH more compatible with the requirements of the following individual steps. As a rule, neutralization occurs at pH 5-7. Pools that have undergone viral inactivation or viral inactivation followed by neutralization may also be subjected to filtration, such as depth filtration, to remove any turbidity or sediment that has formed. Viral filtration can be accomplished using micro- or nanofilters such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

- 14 046702

The term fine purification is used herein to refer to one or more chromatographic steps performed to remove remaining contaminants and impurities such as DNA, host cell proteins; product-specific impurities, product variants and aggregates, and adsorption of viruses from a liquid composition containing a recombinant polyspecific protein that is close to the required final purity. For example, fine purification can be performed in the binding and elution mode by passing a liquid containing the recombinant polyspecific protein through chromatographic column(s) or membrane adsorber(s) that selectively binds either the target recombinant polyspecific protein, or with contaminants or impurities present in the liquid composition. In such an example, the eluate/filtrate from the chromatography column(s) or membrane adsorber(s) contains the recombinant polyspecific protein.

Fine purification chromatography uses media such as resins and/or membranes containing agents that can be applied either in a flow mode (where the polyspecific protein flows through the resin/membrane and is contained in the flow eluent, and contaminants and impurities are bound to the chromatographic medium), frontal or overload chromatography mode (where a solution containing the protein of interest is loaded onto the column until adsorption sites are occupied and the species with the lowest affinity for the stationary phase (the protein of interest) begins to elute), or bind-and-elute mode (if the protein of interest binds to the chromatography medium and elutes after contaminants and impurities have flowed through or been washed out of the chromatography medium). Examples of such chromatography operations include ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and/or cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed mode chromatography or multimodal chromatography (MM), chromatography with hydroxyapatite (HA); reverse 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.

Cation exchange chromatography refers to chromatography performed on a solid phase medium that is negatively charged and has free cations to exchange with cations in an aqueous solution passed over or through the solid phase. Charge can be provided by attaching one or more charged ligands to the solid phase, for example by forming a covalent bond. Alternatively or in addition, the charge may be an intrinsic property of the solid phase (eg, as in the case of silica, which is characterized by an overall negative charge). Cation exchange media are commercially available and include, but are not limited to, sulfopropyl (SP) immobilized on agarose (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 resins, Fractogel® EMD COO-(M), Mustang S Acrodisc with Mustang S AcroPrep with Mustang S, CM Ceramic HyperD® F AcroSep with CM Ceramic HyperD® F.

In the method of the present invention, cation exchange chromatography is performed in the binding and elution mode. The polyspecific protein in the eluate or storage pool from a previous downstream step is loaded into a cation exchange medium such that the polyspecific protein of interest binds to the cation exchange medium. By binding a polyspecific protein to a cation exchange medium is meant exposing the polyspecific protein to the cation exchange medium under appropriate conditions (pH/conductivity) such that the polyspecific protein is reversibly immobilized in or on the cation exchange medium due to ionic interactions between the polyspecific protein of interest and a charged group or charged groups. cation exchange medium. The eluate or pool may come from a previous separate operation, such as affinity chromatography, low pH viral inactivation followed by neutralization, depth filtration, or a collection and/or fine chromatography operation. Additional buffer can be added to the eluate or pool to ensure that the final polyspecific protein load matches the desired concentration.

The loaded cation exchange chromatography medium is then subjected to at least one washing step. Washing the cation exchange medium means passing an appropriate wash buffer through or over the cation exchange medium. The function of the wash buffer is to remove one or more contaminants from the cation exchange medium without significantly eluting the polyspecific protein of interest. A buffer is a solution that resists changes in pH through the action of its components, which are acid-base conjugates. In one embodiment, the buffer is acetate. In one embodiment, 100 mM acetate is provided. In one embodiment, the pH buffer for

- 15 046702 flushing is in the range from 5.0±0.05% to 5.0±0.1%. In one embodiment, the pH of the wash buffer is in the range of 4.9 to 5.1. In one embodiment, the pH of the wash buffer is 4.9, 5.0, or 5.1. In one embodiment of the present invention, the at least one wash buffer contains acetate, pH 5.0. To consistently remove product-bound impurities, wash buffers can be pH units 0.05 to 0.1 units higher and lower with varying conductivities.

At least one wash buffer also contains salt. In one embodiment, the salt is sodium chloride. In one embodiment, the concentration of sodium chloride in the wash buffer is from 0 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is from 70 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is from 100 mM to 147 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is from 100 mM to 125 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is from 100 mM to 105 mM. In one embodiment, the sodium chloride concentration in the wash buffer is between 105 mM and 147 mM. In one embodiment, the sodium chloride concentration in the wash buffer is between 105 mM and 125 mM. In one embodiment, the sodium chloride concentration in the wash buffer is between 125 mM and 147 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is 0 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is 70 mM. In one embodiment, the concentration of sodium chloride in the wash buffer is 100 mM. In one embodiment, the sodium chloride concentration in the wash buffer is 105 mM. In one embodiment, the sodium chloride concentration in the wash buffer is 125 mM. In one embodiment, the sodium chloride concentration in the wash buffer is 147 mM.

In the washing step, the cation exchange medium is washed after loading and before elution of the polyspecific protein of interest. The present invention provides at least one wash step comprising a wash buffer with a high salt concentration. The high salt wash buffer was found to wash out or elute low pI product related impurities from the cation exchange medium before eluting the main product. In addition to the high salt wash step, there may be one or more additional wash steps that use buffers that do not contain salt and/or buffers that contain salt at a lower concentration than the high salt wash buffer. Preferably, at the end of the last washing step before the start of elution, the original level of the UV spectrum returned to zero or was very close to it. In accordance with one embodiment of the present invention, there are at least two washing stages. In one embodiment, there is a first wash buffer and a second wash buffer. In accordance with one embodiment of the present invention, there are at least three washing stages. In one embodiment, there is a first wash buffer, a second wash buffer, and a third wash buffer. The terms first wash, second wash and/or third wash should not be interpreted as excluding the use of one or more additional washes or other buffers between one or more washing steps. A wash buffer is used to wash or re-equilibrate the cation exchange material before eluting the polyspecific protein of interest. One or more of the wash buffer compositions may be the same as the batch equilibration and/or final conditioning buffer compositions.

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

In one embodiment of the present invention, the second wash contains a wash buffer containing acetate, from pH 5.0±0.5 to pH 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

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100 mM acetate. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, pH 5.0.

In a related embodiment of the present invention, the second wash buffer contains 0-147 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 70-147 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 100-147 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 100-125 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 100-105 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 105-147 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 105-125 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 125,147 mM sodium chloride. In a related embodiment of the present invention, the second wash buffer contains 125 mM sodium chloride.

In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 0-147 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 70-147 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 100-147 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 100-125 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 100-105 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In one embodiment of the present invention, the second wash buffer contains 100 mM acetate, 105-147 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In a related embodiment, the second wash buffer contains 100 mM acetate, 105-125 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In a related embodiment, the second wash buffer contains 100 mM acetate, 125-147 mM sodium chloride, pH 5.0±0.05 to pH 5.0±0.1. In a related embodiment, the second wash buffer contains 100 mM acetate, 125 mM sodium chloride, pH 5.0 ± 0.05 to pH 5.0 ± 0.1.

In a related embodiment, the second wash buffer contains 100 mM acetate, 0 mM sodium chloride, pH 5.0. In a related embodiment, the second wash buffer contains 100 mM acetate, 70 mM sodium chloride, pH 5.0. In a related embodiment, the second wash buffer contains 100 mM acetate, 100 mM sodium chloride, pH 5.0. In a related embodiment, the second wash buffer contains 100 mM acetate, 105 mM sodium chloride, pH 5.0. In a related embodiment, the second wash buffer contains 100 mM acetate, 125 mM sodium chloride, pH 5.0. In a related embodiment, the second wash buffer contains 100 mM acetate, 147 mM sodium chloride, pH 5.0.

In one embodiment of the present invention, the third wash contains a wash buffer containing acetate, pH 5.0±0.05 to pH 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, pH 5.0 ± 0.5% to pH 5.0 ± 0.1. In one embodiment of the present invention, the third wash buffer contains 0-147 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, 0 mM sodium chloride, pH 5.0 ± 0.5% to pH 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 wash buffer contains 100 mM acetate, 70 mM sodium chloride, pH 5.0 ± 0.5% to pH 5.0 ± 0.1.

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

- 17 046702 sodium chloride; the second wash buffer is selected from 100 mM acetate, 100 mM sodium chloride and 100 mM acetate, 105 mM sodium chloride; and the third wash buffer is 100 mM acetate, 0 mM sodium chloride. In one embodiment, the first wash buffer is 100 mM acetate, 0 mM sodium chloride; the second wash buffer is 100 mM acetate, 147 mM sodium chloride, and the third wash buffer is 100 mM acetate, 70 mM sodium chloride.

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

In one embodiment of the present invention, the cation exchange medium is charged with 10 g/L of the polyspecific protein, washed with at least one wash buffer containing 105 mM sodium chloride, and eluted with a salt gradient at 8 mM/CV.

In one embodiment of the present invention, the cation exchange medium is charged with 15 g/L to 30 g/L of the polyspecific protein and washed with at least one wash buffer containing 147 mM sodium chloride.

In one embodiment of the present invention, the cation exchange medium is loaded with 25 g/L to 40 g/L of the polyspecific protein, wherein at least one wash buffer and one elution buffer contain 125 mM sodium chloride.

The bound polyspecific protein is then eluted from the solid phase of the cation exchange chromatography medium. The polyspecific protein can be eluted via a gradient. The gradient can be a linear or step gradient. The gradient may be a salt gradient. The gradient may be a linear salt gradient or a stepped salt gradient. In the case of a salt gradient, the salt concentration (ionic strength) changes with time along the gradient. Proper salt concentration is required to disrupt the binding of the polyspecific protein and release it into the eluate. Examples of salts that can be used include sodium chloride, potassium chloride and acetate.

The cation exchange chromatography eluate can then be subjected to subsequent separate purification steps with fine purification chromatography. In one embodiment, after cation exchange chromatography, the polyspecific protein of interest is applied to a fine purification chromatography medium in a flow-through mode.

After fine purification chromatography operations, concentration of the purified polyspecific protein and replacement of the buffer with the required formulation buffer designed to store the drug substance in bulk can be accomplished using an ultrafiltration and diafiltration operation.

To help make more informed decisions regarding the performance of each step during production, critical metrics and performance parameters of the purified multispecific protein can be measured. These critical metrics and parameters can be monitored in real time, near real time and/or after the fact. During cell culture, key critical indicators can be measured, such as components of the medium that are consumed (for example, glucose), levels of accumulated metabolic by-products (such as lactate and ammonium), and indicators associated with cell maintenance and survival, such as dissolved oxygen. Critical parameters such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, percentage yield and titer can be monitored during relevant stages of the production process. Tracking and Measuring

- 18 046702 rhenium can be carried out using known techniques and commercially available equipment.

Pharmaceutical compositions (solutions, suspensions, and the like) may include one or more of the following: buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide) and preservatives; sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride solution, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose. The drug for parenteral administration can be contained in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Although the terminology used in this invention is standard within the art, certain terms are defined herein to provide clarity and certainty to the meaning of the claims. Units, prefixes and symbols may be expressed in their SI form. Numeric ranges referred to herein include numbers that define the boundaries of the range and include and support each integer within the specified range. The methods and techniques described herein are generally performed in accordance with conventional methods well known in the art and described in various general and more specialized literature references which are cited and discussed throughout this application unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). All documents or portions of documents cited in this application, including, without limitation, patents, patent applications, articles, books and treatises, are hereby expressly incorporated by reference.

The present invention is not to be limited in scope to the specific embodiments described herein, which are intended as separate illustrations of individual aspects of the present invention, and functionally equivalent methods and components are within the scope of the present invention. The embodiment described in the present invention may be combined with other embodiments of the present invention. Of course, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. It is intended that such modifications fall within the scope of the appended claims.

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

Examples

Example 1: Single wash without salt, bispecific molecule #1.

The protein A purified and neutralized pool containing fully human bispecific engineered immunoglobulin (bispecific molecule #1) in acetate buffer was loaded onto Capto-SP ImpRes® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA, USA) under the conditions noted in table. 1.

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Table 1

Conditions for Cation Exchange Chromatography Using Low Salt Wash

Bispecific Molecule No. 1 Column size and speed 20±2 cm, 180 cm/h ± 10% Concentration, g/l 20 Preliminary balancing 500 mM acetate, 1.75 M sodium chloride, pH 4.9 U balancing 100 mM acetate, pH 5.0 Flushing 1 100 mM acetate, pH 5.0, CV 2.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100 Elution gradient duration, CV 31.25 Salt elution gradient, mM/CV 16.0 CV Column Volumes 17.5 Exit 70% Impurities in the recovered eluate The entire elution step was collected into fractions. The best factions had 0.9% HMW, 0% NCG.

In fig. Figure 1 shows three peaks in the elution profile, indicating that numerous impurities remained in the column and were eluting with the main product. These impurities included homodimer, NCG (mismatched glycosylation) and high molecular weight (HMW) species.

Example 2: High Salt Washes, Bispecific Molecule #1.

The pool, after undergoing viral inactivation and neutralization, containing bispecific molecule No. 1 in acetate buffer, was loaded onto Capto-SP ImpRes® cation exchange chromatography resin under the conditions described in table. 2.

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table 2

Conditions for cation exchange chromatography using high salt washes

Bispecific Molecule No. 1 Column size and speed 20±2 cm, linear speed 180 cm/h Concentration 30 g/l Pre-equilibration 500 mM acetate, 1.75 M sodium chloride, pH 4.9 Balancing 100 mM acetate, pH 5.0 Flushing 1 100 mM acetate, pH 5.0 Flushing 2 100 mM acetate, 147 mM sodium chloride, pH 5.0, CV 2.5 Flushing 3 100 mM acetate, 70 mM sodium chloride, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.00 Elution Buffer B 100 mM acetate, 350 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 20 Elution gradient at the end, %V 100 Elution gradient duration, CV 17.5 Salt elution gradient, mM/CV 16.0 CV Column Volumes 17.5 Exit 70% Impurities in the recovered eluate 1.6% HMW 0.5% NCG

It was found that by adding a high salt wash, lower pI impurities that were previously removed from the eluate during elution were now removed from the resin during the pre-elution wash. In fig. Figure 2 shows that the addition of a high salt wash reduced the number of impurity peaks in the elution profile from three peaks to one peak. Most of the impurities were removed between the second and third washing steps, with >68% NCG and >80% homodimer removed. Homodimers and NCG were predominantly removed from the resin during the second wash or minimal amounts remained bound to the resin. The third washing step ensured that the original UV spectrum was restored to zero before elution began, tightening the elution profile, resulting in much more efficient collection and better quality of the main product. The addition of a high-salt wash step optimized purification into a more stable and process-friendly process, reducing out-of-spec results in terms of product quality and maintaining acceptable yield.

These conditions were effective at loading concentrations of 15-30 g/L, bispecific molecule no.

In addition, wash buffers were tested at 0.05 pH units above and below pH 5.0 (pH 4.95

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5.05) and found them to be effective in removing low pI product-related impurities, which is consistent with the above results.

It was found that using 2.5 column volumes of second wash buffer was sufficient to wash/elute product-bound impurities from the cation exchange medium. Smaller column volumes were not as effective in removing product-bound impurities, and using more than 2.5 column volumes resulted in major product being eluted.

Example 3: Single wash without salt, bispecific molecule #2.

The pool of eluates, after undergoing Protein A purification and neutralization (100 mM acetate, pH 5.0), containing an engineered fully human bispecific hetero-IgG antibody (bispecific molecule #2), was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, Massachusetts, USA) under the conditions specified in table. 3.

Table 3

Conditions . for cation exchange chromatography under low salt wash conditions

Bispecific Molecule No. 2 Column size and speed 4.66 ml, linear speed 140% (cm/h) Concentration Loading level (g/l) 25 Preliminary balancing 500 mM acetate, 1.75 M sodium chloride, pH 4.9 Balancing 100 mM acetate, pH 5.0 Flushing 100 mM acetate, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100 Elution gradient duration, CV 31 Salt elution gradient, mM/CV 16 Pool column volumes 2.5 Yield (based on fractions 7-11 combined) 65% Impurities in extracted eluate (based on combined fractions 7-11) SE-HPLC HMW (%)=1.5% SE-HPLC LMW (%)=0.9% No antibodies=0% LC1/LC2 ratio under reduced CE- SDS=l.40

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In fig. Figure 3 shows that numerous impurities remained in the column and eluted with the main product. These impurities included hemi-antibodies (fractions 1, 2, 3, 4 with ~50% hemi-antibodies), 2x light chain misassemblies (fractions 5 and 6 with LC1 to LC2 ratio <0.4, indicating that LC2 is misassembled with HC1), and high molecular weight species (HMW, fractions with 12=21). Although the resolution of chromatographic separation was acceptable with distinct prepeaks containing impurities, the production process used OD-based automated pooling; so it was necessary to collect the eluate starting from the highest OD for the prepeaks, which reduces the yield and makes the process insufficient for use in a production operation.

Example 4: High Salt Wash, Bispecific Molecule #2.

The pool of eluates, after undergoing Protein A purification and neutralization (100 mM acetate, pH 5.0), containing bispecific molecule No. 2, was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA, USA ) under the conditions specified in table. 4.

Table 4

Conditions for cation exchange chromatography under high salt wash conditions

Bispecific Molecule No. 2 Column size and speed 4.66 ml, linear speed 140% (cm/h) Concentration Loading level (g/l) 20 Preliminary balancing 500 mM acetate, 1.75 M sodium chloride, pH 4.9 Balancing 100 mM acetate, pH 5.0 Flushing 1 100 mM acetate, pH 5.0 Flushing 2 100 mM acetate, 100 mM sodium chloride, pH 5.0 Flushing 3 100 mM acetate, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100

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Elution gradient duration, CV 31 Salt elution gradient, mM/CV 16.0 Pool column volumes 3 Yield (based on combined fractions 5-10) 73% Impurities in the extracted eluate (corresponding to fractions 5-10) SE-HPLC HMW (%)=1.4 SE-HPLC LMW (%)=0.6 Semi-antibodies=0.4% LC1/LC2 ratio under reduced CD-SDS conditions=0.8

It was found that by adding a high salt wash step (Wash 2 with 100 mM sodium chloride), hemiantibody impurities were removed from the CEX resin prior to elution (100% of the semiantibodies were detected in the collected washes 2 and 3). In fig. Figure 4 shows that the high salt wash resulted in a reduction in the number of peaks in the elution profile from four peaks to one peak with a small shoulder that still contained 2x LC2 mismatched species (LC1/LC2<0.11 compared to the expected ratio 1 when LC1 and LC2 are correctly assembled with HC1 and HC2, respectively). The third washing step also ensured that the original UV spectrum was restored to zero before elution began, tightening the elution profile, resulting in much more efficient collection and better quality of the base product. This optimized procedure with a higher salt rinse is suitable for use on the production floor. CEX purification yield increased from 65% to 73% with the proper elution profile to collect the purified pool with low levels of semiantibodies, LC2 mismatched species (as evidenced by an LC1 to LC2 ratio close to 1), HMW and LMW, and pooling criteria on basis of the takeover.

Example 5: Single wash without salt, bispecific molecule #3.

The Protein A purification and neutralization pool of eluates containing the fully human engineered IgG/Fab fusion protein (bispecific molecule #3) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA, USA ) under the conditions specified in table. 5.

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Table 5

Conditions for high density cation exchange chromatography without conditioning with ______________________________salt loading______________________________

Bispecific Molecule No. 3 Column size and speed 4.66 ml Linear speed 140 cm/h. Concentration Loading level (mg/ml) 25 Preliminary balancing 500 mM acetate, 1.75 M sodium chloride, pH 4.9 Balancing 100 mM acetate, pH 5.0 Flushing 100 mM acetate, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100 Elution gradient duration, CV 31 Salt gradient elution, mM/CV 16 Pool column volumes 1.5 Exit 44% Impurities in the extracted eluate (correspond to fractions 3, 5 and 6) LC1/LC2 ratio under reduced conditions CR/SDS=0.9 SE-HPLC HMW (%)=1.7 SE-HPLC LMW (%)=0.9 Monomer SE-HPLC=97.4%

)ig. Figure 5 shows one elution peak resulting from high loading density

On c with a steep elution gradient. Product-associated low pI impurities were not separated from the main product at high loading densities and were primarily found in fractions 1, 2 and 3, as seen by the decrease in LC1 to LC2 ratios at CE-SDS from 4 to 7 (LC mismatched varieties ) and LMW varieties, ranging from 2 to 4%.

Example 6: Lower Load Density, Single Wash Without Salt, Bispecific Molecule #3.

The protein A purified and neutralized pool containing the fully human engineered IgG/Fab fusion protein (bispecific molecule #3) was loaded onto Capto-SP ImpREs® cation exchange resin (GE Healthcare Bio-Science, Marlborough, MA, USA) under the conditions specified in table. 6.

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Table 6

Conditions for cation exchange chromatography with lower loading densities in the absence of ___________________________salt loading________________________

Bispecific Molecule No. 3 Column size and speed 4.66 ml Linear speed 140 cm/h. Concentration Loading level (mg/ml) 10 Preliminary balancing 500 mM acetate, 1.75 M sodium chloride, pH 4.9 Balancing 100 mM acetate, pH 5.0 Flushing 100 mM acetate, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100 Elution gradient duration, CV 62 Salt elution gradient, mM/CV 8 Pool column volumes h,o Exit 73% Impurities in the recovered eluate (corresponding to fractions 5, 6, 7, 8, 9 and 10) LC1/LC2 ratio under reduced CESDS conditions = 1.2 SE-HLPC HMW (%)=1.4 SE-HPLC LMW (%)=0.1 Monomer SE-HPLC=98.4%

Because the high loading density, steep elution gradient conditions in Example 5 did not provide sufficient separation of the main product and product-associated low pI impurities, the loading density and gradient conditions were reduced. In fig. Figure 6 shows that the lower loading density (10 vs. 25 g/L) and flatter gradient (8 vs. 16 mM/CV) provided separation of the main impurities of the product with a low pI in the form of a distinct peak formed by fractions 1-4 . This fraction showed LC1 to LC2 ratios of 3 to 10, indicating LC1 mismatched varieties. In contrast, the main peak showed a combined LC1 to LC2 ratio of 1.2. Although the resolution was better and increased the yield from 44% to 73%, since automatic pooling based on OD was still required, it was still necessary to collect the eluate starting from the highest OD for the prepeak, which reduces the yield and makes this process insufficient for application in a manufacturing operation.

Example 7: High Salt Wash, Bispecific Molecule #3.

The protein A purified and neutralized pool containing bispecific molecule No. 3 was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA, USA) under the conditions shown in Table 1. 7.

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Table 7

Conditions for cation exchange chromatography under high salt loading conditions

Bispecific Molecule No. 3 Column size and speed 4.66 ml Linear speed 140 cm/h. Concentration, g/l 10 Pre-equilibration 500 mM acetate, 1.75 M sodium chloride, pH 4.9 U balancing 100 mM acetate, pH 5.0 Flushing 1 100 mM acetate, pH 5.0 Flushing 2 100 mM acetate, 105 mM sodium chloride, pH 5.0 Flushing 3 100 mM acetate, pH 5.0 Elution Buffer A 100 mM acetate, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100 Elution gradient duration, CV 62 Salt elution gradient, mM/CV 8.1 Column volumes pool h,o Exit 66 Impurities in the extracted eluate (corresponding to fractions 7, 8, 9, 10, I and 12) The ratio of LC1 and LC2 under conditions of restored CESDS=1.1 SE-HPLC HMW=1.4% SE HPLC LMW=0.0 Monomer SE-HPLC=98.6%

It was found that by adding a high salt wash step (Wash 2), impurities were removed from the CEX resin prior to elution. These contaminants likely corresponded to LC1 mispairing varieties, given that the ratio of LC1 to LC2 in collected Wash 2 and Wash 3 was 8.0 compared to the expected ratio of 1 when LC1 and LC2 are correctly assembled and paired. In fig. Figure 7 shows that the high salt wash resulted in a reduction in the number of impurity peaks in the elution profile from two peaks to one peak with

- 27 046702 by a small shoulder that still contained incorrectly paired species (LC1/LC2=ot 2 to 4). Most of the impurities were removed during the second and third washing stages. The third washing step also ensured that the original UV spectrum was restored to zero before elution began, tightening the elution profile, resulting in much more efficient collection and better quality of the base product. This optimized procedure using a higher salt rinse combined with a lower loading level and flatter gradient is suitable for use on the production floor. CEX purification yield increased from 44% to 66% with an elution profile to collect the purified pool with low levels of LC1 mismatched species (as evidenced by an LC1 to LC2 ratio close to 1), HMW and LMW, and absorbance-based pooling criteria.

Example 8: High Salt Wash, Bispecific Molecule #4.

The eluate pool, after undergoing Protein A purification, low pH viral inactivation, and neutralization, containing a fully human engineered immunoglobulin bispecific antibody (bispecific molecule #4) was loaded onto Capto-SP ImpREs® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, Massachusetts) under the conditions specified in table. 7.

Table 7

Conditions for cation exchange chromatography under high salt wash conditions

Bispecific Molecule No. 4 Column speed 20 cm, linear speed 180 cm/h Concentration, g/l 35 Pre-equilibration 200 mM acetate, 1.0 M sodium chloride, pH 5.0 Balancing 100 mM acetate, pH 5.0 Flushing 1 100 mM acetate, pH 5.0 Flushing 2 100 mM acetate, 125 mM sodium chloride, pH 5.0 Elution Buffer A 100 mM acetate, 125 mM sodium chloride, pH 5.0 Elution Buffer B 100 mM acetate, 500 mM sodium chloride, pH 5.0 Elution gradient at the beginning, %V 0 Elution gradient at the end, %V 100

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Claims (52)

Elution gradient duration, CV 10 Salt elution gradient, mM/CV 37.5 Average pool column volumes (CV) 2.5 2-3 (second cycle) Exit 66% 77.7% (second cycle) Impurities in the recovered eluate SE-HPLC HMW=1.3% (second cycle 1.2) SE HPLC LMW=0.6 (second cycle 0.5) % main peak=98.1% (second cycle 98.3) % post peaks=0, 0 for both cycles % prepeaks=0.0 for both cycles It was found that by adding a high salt wash step (Wash 2), low pI product associated impurities (homodimers and aggregated species) were removed from the cation exchange medium prior to elution. In fig. Figure 8 shows that the high salt wash reduced the number of impurity peaks in the elution profile to a single peak. The first flush without sodium chloride brought the conductivity back to its original level. Product-associated low pI impurities were removed in a subsequent high-salt wash step and the original conductivity level was returned to zero before elution. Conductivity was maintained using elution buffer A, which had the same high-salt composition as the high-salt wash buffer. This ensured that the conductivity was maintained before elution began and the elution profile was narrowed, resulting in much more stable and efficient collection and better quality of the main product since the separation was based on pI. Additionally, wash buffers were tested at 0.1 pH units above and below pH 5.0 (pH 4.9-5.1) and found to be equally effective in removing low pI product-related impurities. Additionally, loading concentrations of Bispecific Molecule No. 4 from 25 to 40 g/L were tested and found to exhibit impurity removal similar to the 35 g/L condition described above. CLAIM 1. A method for purifying a bispecific antibody, comprising: loading the sample containing the bispecific antibody into a cation exchange chromatography medium; washing the cation exchange medium with at least one wash buffer with a pH of 4.9 to 5.1 containing 100-147 mM sodium chloride; and eluting the bispecific antibody from the cation exchange chromatography resin. 2. The method of claim 1, further comprising, before and/or after the cation exchange chromatography step, one or more separate steps for purifying the bispecific antibody, comprising affinity chromatography, ion exchange chromatography, hydrophobic interaction column chromatography, and/or mixed-mode column chromatography. 3. The method according to claim 1, wherein the cation exchange chromatography medium is a resin. 4. The method according to claim 1, wherein at least one wash buffer contains 100-125 mM sodium chloride. 5. The method according to claim 1, wherein at least one wash buffer contains 100-105 mM sodium chloride. 6. The method according to claim 1, wherein at least one wash buffer contains 105-147 mM sodium chloride. 7. The method according to claim 1, wherein at least one wash buffer contains 105-125 mM chloride per - 29 046702 triya. 8. The method according to claim 1, wherein at least one wash buffer contains 125-147 mM sodium chloride. 9. The method of claim 1, wherein the at least one wash buffer contains acetate. 10. The method of claim 9, wherein the at least one wash buffer contains acetate, pH 5.0±0.05% to pH 5.0±0.1%. 11. The method according to claim 9, wherein at least one wash buffer contains acetate, pH 4.91-5.1. 12. The method of claim 9, wherein the at least one wash buffer contains acetate, pH 4.9, 5.0 or 5.1. 13. The method according to claim 7, where the wash buffer contains 100 mM acetate. 14. The method according to claim 1, wherein at least one wash buffer contains acetate, 100-125 mM sodium chloride. 15. The method according to claim 1, wherein at least one wash buffer contains acetate, 100-105 mM sodium chloride. 16. The method according to claim 1, wherein at least one wash buffer contains acetate, 105-147 mm sodium chloride. 17. The method according to claim 1, wherein at least one wash buffer contains acetate, 105-125 mM sodium chloride. 18. The method according to claim 1, wherein at least one wash buffer contains acetate, 125-147 mm sodium chloride. 19. The method of claim 1, wherein the cation exchange medium is washed with at least two wash buffers. 20. The method of claim 1, wherein the cation exchange medium is washed with at least three wash buffers. 21. The method of claim 19, wherein the cation exchange medium is washed with at least two wash buffers, at least one of the wash buffers containing 0-147 mM sodium chloride. 22. The method of claim 21, wherein the cation exchange medium is washed with at least two wash buffers, at least one of the wash buffers containing 0-70 mM sodium chloride. 23. The method according to claim 1, where the cation exchange medium is washed with at least two wash buffers, namely, at least one wash buffer containing acetate, 0 mm sodium chloride, and then a wash buffer containing acetate, 100- 147 mM sodium chloride. 24. The method of claim 23, wherein the cation exchange medium is washed with a wash buffer containing acetate, 0 mM sodium chloride, and then a wash buffer selected from the group consisting of a wash buffer containing acetate, 100 mM sodium chloride, wash containing acetate, 105 mM sodium chloride, or wash buffer containing acetate, 125 mM sodium chloride. 25. The method according to claim 23, where a third washing buffer containing acetate, 0-70 mM sodium chloride is additionally used. 26. The method according to claim 25, where the cation exchange medium is washed with a wash buffer containing acetate, 100-147 mM sodium chloride, and then with a wash buffer containing acetate, 0 mM sodium chloride. 27. The method according to claim 25, where the cation exchange medium is washed with a wash buffer containing acetate, 100-147 mM sodium chloride, and then a wash buffer containing 70 mM sodium chloride. 28. The method according to claim 20, where the cation exchange medium is washed with at least three wash buffers, namely: a first wash buffer containing acetate, 0 mM sodium chloride, and then a second wash buffer containing acetate, 100-147 mM sodium chloride, and then a third wash buffer containing acetate, 0 mM sodium chloride, or wash buffer, containing acetate, 70 mM sodium chloride. 29. The method of claim 28, wherein the cation exchange medium is washed with a first wash buffer containing acetate, 0 mM sodium chloride, and then a second wash buffer selected from the group consisting of a wash buffer containing acetate, 100 mM sodium chloride, a wash buffer containing acetate, 105 mM sodium chloride, or a wash buffer containing acetate, 125 mM sodium chloride, followed by a third wash buffer containing acetate, 0 mM sodium chloride. 30. The method of claim 28, wherein the cation exchange medium is washed with a first wash buffer containing acetate, 0 mM sodium chloride, and then a second wash buffer containing acetate, 147 mM sodium chloride, and then a third wash buffer containing acetate, 70 mM sodium chloride. 31. The method of claim 1, wherein the cation exchange medium is washed with 2.5 mM/CV wash buffer containing 147 mM sodium chloride. 32. The method of claim 1, wherein the bispecific antibody is eluted from the cation exchange medium via a gradient. - 30 046702 33. The method according to claim 32, where the gradient is a linear or step gradient. 34. The method of claim 32, wherein the gradient is a salt gradient. 35. The method according to claim 32, where at least one of the buffers used to create the elution gradient contains 0-1 M sodium chloride. 36. The method of claim 35, wherein at least one of the buffers used to create the elution gradient contains 70-500 mM sodium chloride. 37. The method of claim 35, wherein at least one of the buffers used to create the elution gradient contains 125 mM sodium chloride. 38. The method of claim 1, wherein at least one wash buffer and one elution buffer contain 125 mM sodium chloride. 39. The method according to claim 1, where at least 10 g/l of bispecific antibody is loaded into the cation exchange medium. 40. The method according to claim 1, where 10 to 40 g/l of bispecific antibody is loaded into the cation exchange medium. 41. The method according to claim 38, where from 15 to 30 g/l is loaded into the cation exchange medium. 42. The method according to claim 38, where 25-40 g/l is loaded into the cation exchange medium. 43. The method according to claim 1, where 10 g/L of bispecific antibody is loaded into the cation exchange medium, washed with a washing buffer containing 105 mM sodium chloride, and eluted in a salt gradient at 8 mM/CV. 44. The method according to claim 1, where 15 to 30 g/l of bispecific antibody is loaded into the cation exchange medium and washed with a washing buffer containing 147 mM sodium chloride. 45. The method according to claim 1, where 25 to 40 g/L of bispecific antibody is loaded into the cation exchange medium, where at least one washing buffer and one elution buffer contain 125 mM sodium chloride. 46. The method of claim 1, wherein the bispecific antibody is purified from at least one bispecific antibody-associated impurity selected from a homodimer, high molecular weight species, half-antibody, aggregate, low molecular weight species, antibody fragment, or light chain misassembly. 47. A method for purifying a bispecific antibody, which includes a separate step, including cation exchange chromatography, performed in accordance with the method according to claim 1. 48. A method for reducing the content of low pI impurities associated with a bispecific antibody in an eluate from cation exchange chromatography, the method comprising: loading a composition containing a bispecific antibody and at least one bispecific antibody-associated impurity having a pI lower than that of the bispecific antibody into a cation exchange chromatography medium; washing the cation exchange medium with a first wash buffer, washing the cation exchange medium with a second wash buffer having a pH of 4.9 to 5.1 containing 100-147 mM sodium chloride; eluting the bispecific antibody from the cation exchange chromatography resin; wherein the cation exchange chromatography eluate has a reduced content of bispecific antibody-associated low pI impurities compared to the cation exchange chromatography eluate recovered in a corresponding method in which sodium chloride is not included in the wash buffer. 49. The method of claim 48, wherein the cation exchange medium is further washed with a third wash buffer. 50. A method of performing cation exchange chromatography under high salt wash conditions to reduce the content of bispecific antibody-associated impurities, comprising: loading a composition containing a bispecific antibody and at least one bispecific antibody-associated impurity into a balanced cation exchange column; washing the cation exchange medium with at least two wash buffers, where one wash buffer has a pH of 4.9 to 5.1 and contains 100-147 mM sodium chloride; and eluting the bound bispecific antibody from the cation exchange chromatography resin. 51. The method according to claim 50, where before loading the composition, the cation exchange medium is equilibrated with a buffer not containing sodium chloride. 52. A method for producing isolated purified recombinant bispecific antibodies, including: creating a cell culture in a bioreactor with a host cell expressing a bispecific antibody; culturing host cells to express the bispecific antibody; collecting the recombinant bispecific antibody from the cell culture; affinity purification of the recombinant bispecific antibody; -