WO2022072291A1

WO2022072291A1 - Cation exchange chromatography process

Info

Publication number: WO2022072291A1
Application number: PCT/US2021/052268
Authority: WO
Inventors: Michael Shearer
Original assignee: Amgen Inc.
Priority date: 2020-09-30
Filing date: 2021-09-28
Publication date: 2022-04-07

Abstract

The invention relates to high molarity buffer elution from cation exchange chromatography for removal of product-related impurities during manufacture of recombinant multispecific proteins.

Description

TITLE

CATION EXCHANGE CHROMATOGRAPHY PROCESS

This application claims the benefit of U.S. Provisional Application No. 63/085,246, filed on September 30, 2020, which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein

FIELD OF DISCLOSURE

The present invention relates to the field of biopharmaceutical manufacturing. In particular, the invention relates to methods for removal of product-related impurities of bispecific antibody proteins during cation exchange purification operations.

BACKGROUND

Monoclonal antibody therapeutics are the largest sector of the biopharmaceuticals market and could easily reach hundreds of billions in sales over the next decade. The commercial development of therapeutic antibodies began in the 1980s with the approval of the first therapeutic monoclonal antibody and has continued to evolve and expand ever since. While monoclonal antibodies bind to a target with high affinity and specificity, and have been very successful for treating some indications, they also have limitations as therapeutics. Monoclonal antibodies can only bind to a single target; however, many diseases are multifactorial. In cancer immunotherapy, a treatment aimed at a single target may not be sufficient to completely destroy or immobilize cancer cells. In addition, some patients receiving monoclonal antibody therapies may fail to respond to treatment or even develop drug resistance after a time.

New antibody-like modalities such as bispecific and other multispecific antibodies have been developed to meet these challenges. They offer improvements over traditional monoclonal antibody therapeutics, such as multi-target affinity, and are proving to be effective next-generation of biotherapeutics with an enormous variety of formats that can be developed to meet even more challenging therapeutic indications.

Bispecific antibodies are the most diverse group of these antibody-like structures with an ever- increasing variety of frameworks to meet the challenges an even broader scope of therapeutic indications. These structures combine the binding properties of antibodies with additional molecular properties engineered into the frameworks to suit needs of the targeted disease indications. Development of these bispecific and multispecific antibodies brings new manufacturing challenges, particularly with regard to product instability and low expression yields. In particular, purification of bispecific and multispecific antibodies is complicated by the formation of product-related variants, such as homodimers, halfantibodies, LC mispaired species, aggregates, high and low molecular weight species and the like. These product-related impurities share similar structural and physical properties, such as charge and size, with the bispecific and multispecific antibodies of interest, making them difficult to separate during purification. These product-related impurities lower the yield, purity, and the activity of the bispecific antibody drug product.

Traditional methods for purification of monoclonal antibodies are often not ideal for purification of multispecific proteins such as bispecific antibodies which can have issues with co-elution of product- related impurities. As bispecific antibodies increase in popularity as therapeutic modalities there is a need to improve the purification processes for these proteins. The invention described herein provides such an improvement.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 2-(/V- morpholino)ethanesulfonic acid (MES); loading the composition on to the cation exchange medium in a load buffer comprises MES; washing the column with at least one wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium with a buffer gradient comprising MES. In one embodiment the equilibration buffer comprises MES pH 6.0. In one embodiment the equilibration buffer comprises 100 mM MES. In one embodiment the load buffer comprises MES pH 6.0. In one embodiment the load buffer comprises 100 mM MES. In one embodiment the wash buffer comprises MES pH 6.0-6.3. In one embodiment the wash buffer comprises MES pH 6.0. In one embodiment the wash buffer comprises 400 mM MES. In one embodiment the buffer gradient is from 100 to 400 mM MES. In one embodiment one elution buffer comprises 100 mM MES. In one embodiment one elution buffer comprises 400 mM MES. In one embodiment at least one elution buffer comprises MES pH 6.0-6.3. In one embodiment at least one elution buffer comprises MES pH 6.0. In one embodiment the elution gradient is linear. In one embodiment the multispecific protein is a bispecific protein. In one embodiment the multispecific protein is a bispecific antibody. In one embodiment the cation exchange chromatography medium is a resin. In one embodiment a purified, multispecific protein prepared by a method as described above.

The invention provides a method of reducing low pl impurities in the eluate from cation exchange chromatography, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 100 mM MES; loading the composition on to the cation exchange medium in a load buffer comprises 100 mM MES; washing the column with at least one wash buffer comprising 400 mM MES; and eluting the multispecific protein from the cation exchange chromatography medium using a buffer gradient from 100 mM MES to 400 mM MES, pH 6.0-6.23, wherein the none of the buffers comprise sodium chloride. In one embodiment the low pl impurity is a product-related impurity. In one embodiment at least one product-related impurity is a 2X, 3X, or 4X light chain-mis-assembly. The invention provides a method of performing cation exchange chromatography under high molarity elution conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition on to the cation exchange medium in a load buffer; washing the column with a wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the elution gradient is from 100 mM MES pH 6.0 to 400 mM MES pH 6.0-6.23.

The invention provides a method of producing an isolated, purified, recombinant multispecific protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer consists of MES; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer that consists of MES; washing the cation exchange medium with a wash buffer that consists of MES; eluting the multispecific protein from the cation exchange chromatography medium in a high molarity buffer gradient that comprises 100-400 mM MES; loading the cation exchange chromatography eluate comprising the recombinant multispecific protein onto a second chromatography resin in flow through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer. In one embodiment the second chromatography resin is selected from an anion exchange chromatography resin, cation exchange chromatography resin, multi-modal chromatography resin, hydrophobic interaction chromatography resin, and hydroxyapatite chromatography resin. In one embodiment an isolated, purified, recombinant multispecific protein prepared by a method according to the method above. In one embodiment a pharmaceutical composition comprising the isolated, purified, recombinant multispecific protein prepared by a method according to the method above.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the results for the HeteroIgG bispecific antibody using a salt gradient.

Fig. 2 shows the results for the IgG Fab bispecific antibody using a salt gradient. (A) shows the results of the CEX chromatography process. (B) shows the impurity profiles.

Fig. 3 shows the results of the IgG Fab bispecific antibody using a MES buffer gradient, pH 6.23.

Fig. 4 shows the results of the IgG Fab bispecific antibody using a MES step wash and buffer gradient, pH 6.23.

Fig. 5 shows the results of the IgG Fab bispecific antibody using a MES step wash and buffer gradient, pH Fig. 6 shows the results of the IgG Fab bispecific antibody using a MES step wash and buffer gradient, pH 6.0.

DETAILED DESCRIPTION OF THE INVENTION

Development of therapeutic antibody -like modalities such as multispecific proteins, particularly bispecific antibodies, is increasing and so are the challenges related to their production and manufacture. Since there is not much information related to downstream processing of bispecific antibodies, platforms developed for monoclonal antibodies are often applied (Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody -Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, pps 559-594, John Wiley & Sons, 2017).

Traditional monoclonal antibody purification processes typically make use of an ion exchange chromatography unit operation to further purify the antibody by binding the antibody to the chromatographic support in a low ionic strength buffer and eluting with a higher ionic strength buffer or by adjusting the buffer pH. Sodium chloride is typically the salt of choice used to increase the ionic strength of the buffer solution. Such conditions do not always prove ideal for purification of multispecific proteins such as bispecific antibodies. As described herein an alternate purification strategy has been developed that increases the molarity of the buffering component which allows for a slower increase in ionic strength than is possible when using a salt, such as sodium chloride. This purification strategy enables removal of product-related impurities that would otherwise co-elute with the desired product, thereby increasing the yield, purity, and desired activity. It is beneficial to separate the product-related impurities from the main product peak during elution. The invention described herein meets this need by providing conditions for removal of these product-related impurities during cation exchange chromatography unit operations.

It was found that use of 2-(JV-morpholino)ethanesulfonic acid (MES) gradient at 400 mM resulted in better yield and purity of the main product and improved the manufacturing process. Slowing and extending the elution gradient, by combining with a high molarity MES step wash, focused the product- related impurities into a more defined pre-peak such that they could be easily removed prior to the eluting the main product.

The invention provides a method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 2-(A⁷~ morpholinojethanesulfonic acid (MES); loading the composition on to the cation exchange medium in a load buffer comprises MES; washing the column with at least one wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium with a buffer gradient comprising MES. In one embodiment the invention provides the equilibration buffer comprises MES pH 6.0. In one embodiment the equilibration buffer comprises 100 mM MES. In one embodiment, the load buffer comprises MES pH 6.0. In one embodiment the load buffer comprises 100 mM MES. In one embodiment the wash buffer comprises MES pH 6.0-6.3. In a related embodiment the wash buffer comprises MES pH 6.0. In one embodiment the wash buffer comprises 400 mM MES. In one embodiment the buffer gradient is from 100 to 400 mM MES. In one embodiment one elution buffer comprises 100 mM MES. In one embodiment one elution buffer comprises 400 mM MES. In one embodiment one elution buffer comprises MES pH 6.0-6.3. In a related embodiment at least one elution buffer comprises MES pH 6.0.

In one embodiment the elution gradient is linear. In one embodiment the multispecific protein is a bispecific protein. In one embodiment the multispecific protein is a bispecific antibody. In one embodiment the cation exchange chromatography medium is a resin. In one embodiment a purified, multispecific protein prepared by a method herein.

The invention provides a method of performing cation exchange chromatography under high molarity elution conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition on to the cation exchange medium in a load buffer; washing the column with a wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the elution gradient is from 100 mM MES pH 6.0 to 400 mM MES pH 6.0-6.23.

Multipecific proteins, such as bispecific antibodies, are highly engineered and subjecting such proteins to chromatography unit operations under conditions typical for monoclonal antibodies may not be sufficient to stabilize them and/or manage the formation of product-related impurities that elute with the main product. These product-related impurities are similar in size and charge to the main product and can elute with the main product. Such an elution profile does not support the development of a robust, sustainable, commercial scale manufacturing process. As described herein a purification strategy has been developed that increases the molarity of the buffering component of the elution solution, which allows for a slower increase in ionic strength during an elution gradient than is possible when using a salt such as sodium chloride. This purification strategy enables separation and/or removal of product-related impurities, before the main peak, that would otherwise co-elute with the desired product, thereby increasing the yield, purity, and desired activity of the eluted product.

“Bispecific antibody" and “bispecific protein” are used herein to refer to proteins that are recombinantly engineered to simultaneously bind and neutralize at two different antigens or at least two different epitopes on the same antigen. For example, bispecific antibodies may be engineered to target immune effectors tn combination with targeting cytotoxic agents to tumors or infectious agents. These bispecific antibodies have been found useful for a variety of applications, such as in cancer im unotherapy, by redirecting immune effector cells to tumor cells, modifying cell signaling by blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, and as pre-targeted delivery vehicles for drugs, such as delivery of chemotherapeutic agents, radiolabels (to improve detection sensitivity) and nanopardcles (directed to specific cells/tissues, such as cancer cells).

Bispecific proteins can be grouped in two broad categories: immunoglobulin G (IgGj-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions, such as anti body -dependent cell mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC). and antibody -dependent cellular phagocytosis (ADCP), the Fc region helps improve solubility and stability and facilitate some purification operations. Non-IgG-like molecules are smaller, enhancing tissue penetration (see Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015; Williams et al., Chapter 41 Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al., eds., 2018, pages 837-855.

The formats for bispecific proteins, which include bispecific antibodies, are constantly evolving and include, but are not limited to, quadromas, knobs-in-holes, cross-Mabs, dual variable domains IgG (DVD-IgG), IgG-Fab, IgG-single chain Fv (scFv), scFv-CH3 KIH, dual action Fab (DAF), half-molecule exchange, K/.-bodics. tandem scFv, scFv-Fc, diabodies, single chain diabodies (scDiabodies), scDiabodies- CH3, triple body, miniantibody, minibody, TriBi minibody, tandem diabodies, scDiabody-HAS, Tandem scFv-toxin, dual-affinity retargeting molecules (DARTs), nanobody, nanobody-HSA, dock and lock (DNL), strand exchange engineered domain SEEDbody, Triomab, leucine zipper (LUZ-Y), XmAb®; Fabarm exchange, DutaMab, DT-IgG, charged pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(LlHl)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V V(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-scFv2, scFv- KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG- IgG, Cov-X-Body, scFvl-PEG-scFv2, bi-specific T cell engagers (BiTEs®) and half-life extended bispecific T cell engagers (HLE BiTEs) (Fan supra, Spiess supra, Sedykh supra, Seimetz et al., Cancer Treat Rev 36(6) 458-67, 2010; Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, p559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011).

The bispecific antibodies can be of scientific or commercial interest. Bispecific antibodies can be produced in various ways, most commonly by recombinant animal cell lines using cell culture methods. The bispecific antibodies may be produced intracellularly or secreted into the culture medium from which they can be recovered and/or collected and may be referred to “recombinant bispecific protein”, “recombinant bispecific antibody”. The terms “isolated bispecific protein” and “isolated bispecific antibody” refer to bispecific antibodies, that that have been purified away from other proteins, polypeptides, DNA, and/or contaminants or impurities such as product-related impurities that would interfere with the therapeutic, diagnostic, prophylactic, research, or other use of the bispecific antibody. Bispecific antibodies of interest include bispecific antibodies that exert a therapeutic effect by binding two targets, particularly targets among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.

In some embodiments, bispecific antibodies of interest bind, neutralize and/or interact specifically to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins blood group antigens; receptors, receptor-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 bispecific antibodies of interest bind, neutralize and/or interact with one or more of the following, alone or in any combination: CD proteins including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174, HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor, EGFRvIII, cell adhesion molecules, for example, LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VC AM, and alpha v/beta 3 integrin, growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-l-alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), including, among others, TGF-a and TGF-P, including TGF-pi, TGF-P2, TGF-P3, TGF-P4, or TGF-P5, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors, insulins and insulin-related proteins, including but not limited to insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor, colony stimulating factors (CSFs), including the following, among others, 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 (OB) receptor, 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 (ILs), e.g., IL-1 to 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 to the receptor, IL-13RA2, or IL-17 receptor, IL-1RAP,; viral antigens, including but not limited to, an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse 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 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, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor/hCGP, TNF, TL1A, hepatitis-C virus, mesothelin dsFv[PE38 conjugate, 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, a4p7, platelet specific (platelet glycoprotein lib/IIIb (PAC-1), transforming growth factor beta (TFGP), Zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the foregoing.

In some embodiments, bispecific antibodies 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, bispecific antibodies of interest may include bispecific antibodies that specifically bind to combinations including ILla + ILip.

The invention provides a method of reducing low pl impurities in the eluate from cation exchange chromatography, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 100 mM MES; loading the composition on to the cation exchange medium in a load buffer comprises 100 mM MES; washing the column with at least one wash buffer comprising 400 mM MES; and eluting the multispecific protein from the cation exchange chromatography medium using a buffer gradient from 100 mM MES to 400 mM MES, pH 6.0-6.23, wherein the none of the buffers comprise sodium chloride. In one embodiment, the low pl impurity is a product-related impurity. In one embodiment at least one product-related impurity is a 2X, 3X, or 4X light chain-mis-assembly.

By “purifying” is meant increasing the degree of purity of the bispecific protein in the composition by removing (partially or completely) at least one product-related impurity from the composition. Recovery and purification of bispecific antibodies is accomplished by the downstream unit operations, in particular, those operations involving cation exchange chromatography, resulting in a more “homogeneous” bispecific antibody compositions that meets yield and product quality targets (such as reduced product-related impurities and increased product quality). “Product-related impurity” refers to product-related variants of the bispecific antibody of interest. In some instances, these impurities have a charge or pl that is lower than the main product in an elution peak. Product-related impurities include, for example, light chain mis-assemblies, such as 2XLC, 3XLC, or 4XLC, high molecular weight (HMW) species, low molecular weight (LMW) species, half antibodies, aggregates, homodimers, antibody fragments and various combinations of antibody fragments. “Half antibodies” refer to a product-related impurity that can form, for example, due to incomplete assembly or disruption of the interaction between the two heavy chain polypeptides. Half antibodies comprise a single light chain polypeptide and a single heavy chain polypeptide. “Homodimers” refer to a product-related impurity, that can, for example, form when heavy and light chains having specificity for the same target recombine with each other instead of pairing to form a desired bispecific heterodimer. This typically occurs during expression in the host cell. For bispecific antibody constructs that require multiple chains (such as light chains, LCs) to pair correctly via engineered residues (such as charged paired mutations, knob-hole, etc), it is possible to still have impurities where there is a mismatch between LC and HC, wherein LC1 instead of pairing with HC1, incorrectly pairs with HC2 (2xLCl), and vice versa (2x LC2). If the multi specific protein is bivalent, having two sites for binding to each antigen of interest, it is possible to have 3X LC1, 4X LC1, and other combinations of mispaired species.

The invention provides a method of producing an isolated, purified, recombinant multispecific protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer consists of MES; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer that consists of MES; washing the cation exchange medium with a wash buffer that consists of MES; eluting the multispecific protein from the cation exchange chromatography medium in a high molarity buffer gradient that comprises 100-400 mM MES; loading the cation exchange chromatography eluate comprising the recombinant multispecific protein onto a second chromatography resin in flow through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer. In one embodiment the second chromatography resin is selected from an anion exchange chromatography resin, cation exchange chromatography resin, multi-modal chromatography resin, hydrophobic interaction chromatography resin, and hydroxyapatite chromatography resin. In one embodiment is provided an isolated, purified, recombinant multispecific protein prepared by a method described herein. In one embodiment is provided a pharmaceutical composition comprising the isolated, purified, recombinant multispecific protein prepared by a method as described herein.

Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that comprise one or more polynucleotides encoding a bispecific antibody of interest as provided herein, as well host cells comprising such expression systems or constructs. As used herein, “vector” means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport bispecific antibody encoding information into a host cell and/or to a specific location and/or compartment within a host cell. Vectors can include viral and non-viral vectors, non- episomal mammalian vectors. Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors. The vector may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components that generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.

“Cell” or “Cells” include any prokaryotic or eukaryotic cell. Cells can be either ex vivo, in vitro or in vivo, either separate or as part of a higher structure such as a tissue or organ. Cells include “host cells”, also referred to as “cell lines”, which are genetically engineered to express a bispecific antibody of commercial or scientific interest. Host cells are typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time. Genetically engineering the host cell involves transfecting, transforming or transducing the cells with a recombinant polynucleotide molecule, and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) to cause the host cell to express a desired recombinant bispecific antibody. Methods and vectors for genetically engineering cells and/or cell lines to express a bispecific antibody of interest are well known to those of skill in the art.

A host cell can be any prokaryotic cell (for example, E. coll) or eukaryotic cell (for example, yeast, insect, or animal cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

Host cells, when cultured under appropriate conditions, express the bispecific antibody of interest that can be subsequently 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 upon various factors, such as desired expression levels, protein modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

By “culture” or “culturing” is meant the growth and propagation 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 interchangeably used to refer to media suitable for growth of a host cell during in vitro cell culture. Typically, cell culture media contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. Any media capable of supporting growth of the appropriate host cell in culture can be used. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability, and/or recombinant protein production in a particular cultured host cell, are commercially available and include RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum -free media such as EX-CELL™ 300 Series, among others, which can be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell culture media can be serum-free, protein-free, growth factor-free, and/or peptone-free media. Cell culture may also be enriched by the addition of nutrients and used at greater than its usual, recommended concentrations.

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

A growth phase may occur at a higher temperature than a production phase. For example, a growth phase may occur at a first temperature from about 35° C. to about 38° C., and a production phase may occur at a second temperature 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 inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time as, before, and/or after a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.

Host cells may be cultured in suspension or in an adherent 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 a batch, fed batch, continuous, semi-continuous, or perfusion mode. Mammalian cells, such as CHO cells, may be cultured in bioreactors at a smaller scale of less than 100 ml to less than 1000 mis. Alternatively, larger scale bioreactors that contain 1000 mis to over 20,000 liters of media can be used. Large scale cell cultures, such as for clinical and/or commercial scale biomanufacturing of protein therapeutics, may be maintained for weeks and even months, while the cells produce the desired protein(s). Since product-related impurities, such as homodimers, half antibodies, and the like can resemble the desired bispecific antibody, strategies and techniques such as knob and hole, CrossMab, DVD IgG, and others have been developed to increase the selectivity for the desired bispecific antibody during cell culture. However, there will still be a portion of product-related impurities that are produced which must be removed during downstream processing.

The resulting expressed recombinant bispecific antibody can then be harvested from the cell culture media. Methods for harvesting proteins from suspension cells are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, separation using gravity, centrifugation, acoustic wave separation, filtration, including membrane filtration, using ultrafilters, microfilters, tangential flow filters, alternative tangential flow, depth, and alluvial filtration fdters. Recombinant proteins expressed by prokaryotes are retrieved from inclusion bodies in the cytoplasm by redox folding processes known in the art.

The harvested bispecific antibody can then be purified, or partially purified, away from any impurities, such as remaining cell culture media, cell extracts, undesired components, host cell proteins, improperly expressed proteins, product-related impurities, and the like, through one or more downstream unit operations.

Purification of the bispecific antibody from the harvested cell culture fluid can begin with capture chromatography. Capture chromatography makes use of mediums, such as resins, membranes, gels and the like, that will bind to the recombinant bispecific antibody of interest, for example affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like. Such materials are known in the art and are commercially available. Affinity chromatography options may comprise a substrate-binding capture mechanism, an aptamer-binding capture mechanism, or a cofactor-binding capture mechanism, for example. For bispecific antibodies containing an Fc component, an antibody- or antibody fragment-binding capture mechanism such as Protein A, Protein G, Protein A/G, and Protein L can be used. The recombinant protein of interest can be tagged with a polyhistidine tag or an epitope, such a FLAG® and subsequently purified by using a specific antibody directed to such epitope.

Additional chromatography unit operations can also be part of a downstream process. Such chromatographic steps are performed to remove remaining contaminants and impurities such as DNA, host cell proteins, product-specific impurities, variant products and aggregates, and virus adsorption from a fluid composition comprising a recombinant bispecific antibody that is close to a final desired purity. These chromatography operations can be called “intermediate” and/or “polish” chromatography and make use of chromatography mediums, such as resins and/or membranes, that contain agents that can be used in either a flow-through mode, where the bispecific antibody flows through the resin/membrane and is contained in the flow-through eluent while the contaminants and impurities are bound to the chromatography medium; frontal or overloaded chromatography mode where a solution containing the protein of interest is loaded onto a column until adsorption sites on are occupied and the species with the least affinity for the stationary phase (the protein of interest) starts to elute; or bind and elute mode where the protein of interest is bound to the chromatography medium and is eluted after the contaminants and impurities have flowed through or been washed off the chromatography medium.

It was found that by slowing and extending the elution gradient by using a high molarity buffer gradient on a cation exchange chromatography column operated in bind and elute mode, focused the product-related impurities associated with multispecific proteins into a more defined pre-peak such that they could be easily removed prior to the eluting the main product.

“Cation exchange chromatography” refers to chromatography performed on a solid phase medium that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. The charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge). Commercially available cation exchange mediums are available and include but are not limited to sulphopropyl (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™, and FRACTOPREP™ (EMD Merck).

For the inventive method, cation exchange chromatography is performed in bind and elute mode. The multispecific protein in an eluate or storage pool from a previous downstream step is loaded onto the cation exchange medium such that the multispecific protein of interest is bound to the cation exchange medium. By “binding” the multispecific protein to the cation exchange medium is meant exposing the multispecific protein to the cation exchange medium under appropriate conditions (pH/conductivity) such that the multispecific protein is reversibly immobilized in or on the cation exchange medium by virtue of ionic interactions between the multispecific protein of interest and a charged group or charged groups of the cation exchange medium. The eluate or pool may have originated from a previous unit operation, such as affinity chromatography, neutralized low pH viral inactivation, depth filtration, a harvest and/or an intermediate/polish chromatography operation. Additional buffer may be added to the eluate or pool such that the final load of the multispecific protein is at a desired concentration and/or in a desired buffer formulation at a desired molarity, conductivity and/or pH.

The loaded cation exchange chromatography medium is then subjected to at least one wash step. A wash step washes the cation exchange medium following loading and prior io eluting the bispecific antibody of interest Washing the cation exchange medium means passing an appropriate wash buffer through or over the cation exchange medium. A function of the wash buffer is to remove one or more contaminants from the cation exchange medium, without substantial elution of the multispecific protein of interest. The bound bispecific antibody is then eluted from the solid phase of the cation exchange chromatography medium. The bispecific antibody may be eluted by a gradient. As described herein, the gradient is a high molarity buffer gradient.

By "buffer” is meant a solution that resists changes in pH by the action of its acid-base conjugate components. As described herein, it was found that use a high molarity buffer gradient, such as an 2-(N- morpholinojethanesulfonic acid (MES) gradient at 400 mM resulted in better yield and purity of the main product and improved the manufacturing process. Slowing and extending the elution gradient, by combining with a high molarity step wash, focused the product-related impurities into a more defined prepeak such that they could be easily removed prior to the eluting the main product.

The eluate from the cation chromatography unit operations can be subjected to further downstream polish chromatography purification unit operations. Such operations include affinity chromatography, ion exchange chromatography (cation exchange and/or anion exchange), mixed modal chromatography, hydrophobic interaction chromatography and/or hydroxyapatite chromatography.

At any point in the downstream process virus inactivation and/or virus filtration can be performed to remove viral matter from the composition comprising the bispecific antibody of interest. One method for achieving virus inactivation is incubation at low pH or other suitable solution conditions for achieving the inactivation of viruses. Low pH virus inactivation can be followed with a neutralization operation that readjusts the viral inactivated solution to a pH more compatible with the requirements of the following unit operations. Viral inactivated or neutralized viral inactivated pools may also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitation. Viral filtration can be performed using micro- or nano-filters, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

The purified multispecific protein may be concentrated, and buffer exchanged into a desired formulation buffer, such as by an ultrafiltration/diafiltration (UFDF) unit operation for bulk storage or fill and finish of the final pharmaceutical drug product.

Critical attributes and performance parameters of the purified bispecific antibody can be measured to better inform decisions regarding performance of each step during manufacture. These critical attributes and parameters can be monitored real-time, near real-time, and/or after the fact. Key critical parameters such as media components that are consumed (such as glucose), levels of metabolic by-products (such as lactate and ammonia) that accumulate, as well as those related to cell maintenance and survival, such as dissolved oxygen content can be measured during cell culture. Critical attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, percent yield and titer may be monitored during appropriated stages in the manufacturing process. Monitoring and measurements can be done using known techniques and commercially available equipment. The pharmaceutical compositions (solutions, suspensions or 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 (e.g., aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

While the terminology used in this application is standard within the art, definitions of certain terms are provided herein to assure clarity and definiteness to the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., 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 but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference.

The present invention is not to be limited in scope by the specific embodiments described herein that are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. What is described in an embodiment of the invention can be combined with other embodiments of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

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

Example 1 Cation exchange chromatography of a hetero IgG and an IgG Fab bispecific antibody using a salt gradient.

Two bispecific antibodies to the same target, a heteroIgG and an IgG Fab were tested. Neutralized Protein A eluate pools were loaded onto two CAPTOSP IMPRES ® cation exchange chromatography resins (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 1. For loading, the Protein A pools were adjusted to pH 5.0, conductivity < 10 mS/cm.

Table 1 Conditions for cation exchange chromatography

Fig. 1 shows the results for the HeteroIgG bispecific antibody. Elution fractions were collected and assayed by E-HPLC/r-CE and nr-CE. Fractions which exceeded 2% HMW, LMW, and half Mab (Fractions 2-6) were discarded. Fractions 7-11 containing the main product were pooled. Three peaks were seen in the elution profde indicating that multiple impurities remained on the column and were eluted with the main product. The contaminates co-eluted with the main peak and could only be identified by analytical assay. These impurities included half-Mab, mispaired LC, and high molecular weight species (HMW). While not ideal, the impurity peaks were sufficiently separated from the main peak for harvesting.

Figures 2 A and B show results for the IgG Fab bispecific antibody. Elution fractions were collected and assayed by SE-HPLC/ r-CE and nr-CE. Fractions which exceeded 2% HMW, LMW, and half Mab were discarded. Main product pooling was from 90% peak max to approximately 30% peak max on the back side. There was no resolution between the product-related impurities and the main product during elution, Figure 2A. The impurity contaminates co-eluted with the main peak and could only be identified by analytical assay, Figure 2B. The results do not support a robust commercial process.

The IgG Fab bispecific antibody was more difficult to purify using a salt gradient typical for monoclonal antibody elution from cation exchange chromatography. Mispaired species were the predominant product-related impurities that were encountered, half Mab and HMW species were not significant. For the mispaired species, there was not sufficient resolution to separate these impurities from the main product, compared to the results from the heterolg bispecific antibody. Because the elution gradient conditions did not resolve the impurities from the IgG Fab main product, this gradient would not be considered sufficient to support an acceptable robust, sustainable, manufacturing process. This cation exchange chromatography elution process produced out of spec results for product quality and did not provide an acceptable yield.

Example 2 Cation Exchange Chromatography of the IgG Fab bispecific antibody with an MES buffer gradient

Three neutralized virus inactivated pools containing the IgG Fab bispecific antibody were each combined with a load buffer (100 mM MES (2-(AAnorpholino)ethanesulfonic acid) pH 6.0) at a ratio of 1:2 (1+1), giving a final conditioned load of 7 mg/ml. The conditioned samples were loaded onto three CAPTOSP IMPRES® cation exchange chromatography resins (GE Healthcare Bio-Science, Marlborough, MA), washed and eluted using MES buffers under different linear gradients described in Table 4.

Table 2 Conditions for MES linear gradient elution.

Figure 3 shows the results for Condition 1 using an 80 CV gradient. Mispaired species were the predominant product-related impurities that were encountered, half Mab and HMW species were not significant. Mispairs were confirmed using a Caliper (r-CE and nrCE) assay. Figure 4 shows the results for Condition 2 which included a step wash with 132 mM MES pH 6.23. A step wash is a wash before the elution step, used here for the purpose of removing impurities while decreasing the length of the gradient. The step wash was 33% of full gradient, 400 mM MES. Again, half Mab and HMW species were not significant, mispairs were the predominant impurity encountered. The majority of the mis-pairs were removed, down to < 2%, during the step wash. The remaining mismatched pairs may have been due to non-specific binding to the full antibody.

Figure 5 shows a repeat of Condition 2, using fresh reagents. This time there is no pre-peak, the mispairs remained bound to the column and were not removed by the wash. This result may be due to use of a buffer with inconsistent conductivity.

Figure 6 shows the elution for Condition 3, where the pH of elution buffer B was reduced to 6.0 and the step wash was performed using 120 mM MES pH 6.0. Again, half Mab and HMW species were not significant, mispairs were the predominant impurity encountered. The pH was moved farther from the isoelectric point of the bispecific antibody to promote tighter binding to the resin and provide a cleaner separation between the mispairs and the intact bispecific antibody during gradient elution.

The results show lower conductivity with the MES buffer compounds than the sodium chloride containing buffer formulation. The MES buffer gradients worked better for eluting the IgG Fab bispecific antibody than the gradient containing sodium chloride. The MES buffer allowed for manipulation of the slope and length of the gradient, making a more robust and manufacturing friendly process. Combining a MES buffer step wash allowed the elution gradient to be reduced and resulted in increased impurity removal.

Claims

What is claimed is:

1. A method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 2-(rV-morpholino)ethanesulfonic acid (MES); loading the composition on to the cation exchange medium in a load buffer comprises MES; washing the column with at least one wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium with a buffer gradient comprising MES.

2. The method according to claim 1, wherein the equilibration buffer comprises MES pH 6.0.

3. The method according to claim 1, wherein the equilibration buffer comprises 100 mM MES.

4. The method according to claim 1, wherein the load buffer comprises MES pH 6.0.

5. The method according to claim 1, wherein the load buffer comprises 100 mM MES.

6. The method according to claim 1, wherein the wash buffer comprises MES pH 6.0-6.3.

7. The method according to claim 6, wherein the wash buffer comprises MES pH 6.0.

8. The method according to claim 1, wherein the wash buffer comprises 400 mM MES.

9. The method according to claim 1, wherein the buffer gradient is from 100 to 400 mM MES.

10. The method according to claim 1, wherein one elution buffer comprises 100 mM MES.

11. The method according to claim 1, wherein one elution buffer comprises 400 mM MES.

12. The method according to claim 1, wherein at least one elution buffer comprises MES pH 6.0-6.3.

13. The method according to claim 12, wherein at least one elution buffer comprises MES pH 6.0.

14. The method according to claim 1, wherein the elution gradient is linear.

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

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

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

18. A purified, multispecific protein prepared by a method according to claim 1.

19. A method of reducing low pl impurities in the eluate from cation exchange chromatography, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 100 mM MES; loading the composition on to the cation exchange medium in a load buffer comprises 100 mM MES; washing the column with at least one wash buffer comprising 400 mM MES; and eluting the multispecific protein from the cation exchange chromatography medium using a buffer gradient from 100 mM MES to 400 mM MES, pH 6.0-6.23, wherein the none of the buffers comprise sodium chloride.

20. The method according to claim 19, wherein the low pl impurity is a product-related impurity.

21. The method according to claim 20, wherein at least one product-related impurity is a 2X, 3X, or 4X light chain-mis-assembly.

22. A method of performing cation exchange chromatography under high molarity elution conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition on to the cation exchange medium in a load buffer; washing the column with a wash buffer comprising MES; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the elution gradient is from 100 mM MES pH 6.0 to 400 mM MES pH 6.0-6.23.

23. A method of producing an isolated, purified, recombinant multispecific protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer consists of

MES; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer that consists of MES; washing the cation exchange medium with a wash buffer that consists of MES; eluting the multispecific protein from the cation exchange chromatography medium in a high molarity buffer gradient that comprises 100-400 mM MES; loading the cation exchange chromatography eluate comprising the recombinant multispecific protein onto a second chromatography resin in flow through mode; and concentrating the purified recombinant multispecific protein in a formulation buffer.

24. The method according to claim 23, wherein the second chromatography resin is selected from an anion exchange chromatography resin, cation exchange chromatography resin, multi-modal chromatography resin, hydrophobic interaction chromatography resin, and hydroxyapatite chromatography resin.

25. An isolated, purified, recombinant multispecific protein prepared by a method according to claim 23.

26. A pharmaceutical composition comprising the isolated, purified, recombinant multispecific protein prepared by a method according to claim 23.