Anion exchange chromatography process using primary amine ligands

Abstract

Disclosed herein are methods for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, the methods comprising performing anion exchange chromatography (e.g., in flow-through mode or weak partitioning chromatography mode) using an anion exchange material comprising a primary amine ligand, such as a polyamine ligand.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 490,079, filed March 14, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure provides methods for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, the method comprising performing anion exchange chromatography (e.g., in flow-through or weak partitioning chromatography mode) using an anion exchange material (e.g., TOYOPERL® NH2-750F) comprising a primary amine ligand, such as a polyamine ligand. In some embodiments, the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm (e.g., about 3 mS / cm to about 7 mS / cm). Furthermore, in some embodiments, the anion exchange chromatography unit operation enables robust high molecular weight species removal with high protein yield (e.g., at least about 85%) at high loadings (e.g., greater than about 100 g / L of anion exchange material). [Background technology]

[0003] Downstream purification processes for drug substance manufacturing of protein therapeutics, such as monoclonal antibodies (mAbs) and antibody constructs, typically involve an affinity chromatography step followed by one or more polishing chromatography steps to remove product- and process-related impurities. For products containing Fc domains, a Protein A affinity chromatography step is often used to capture the protein, in which cell culture harvest fluid is loaded onto a Protein A resin to bind the recombinant protein of interest, followed by elution with a low pH buffer that desorbs the protein from the Protein A resin. Because Protein A pools typically have a low pH (≦pH 5), the subsequent unit operation is often a low-pH virus inactivation (VI) step, in which the Protein A pool is titrated with acid to a low pH known to inactivate enveloped viruses, held for a period sufficient to ensure VI, and then titrated with base to a higher pH appropriate for product stability and / or loading into subsequent unit operations. Many downstream purification processes utilize a cation exchange (CEX) chromatography step after the VI unit operation because CEX generally requires a relatively low pH to bind positively charged product and process-related impurities to the negatively charged CEX resin. Additional polishing chromatography steps, such as anion exchange (AEX) chromatography, hydrophobic interaction chromatography (HIC), and mixed-mode chromatography (MMC), are often used after CEX to further reduce impurities. These polishing chromatography steps can be operated in a variety of chromatographic modes depending on process requirements, including bind-and-elute mode, flow-through mode, and frontal loading (i.e., "frontal") mode.

[0004] AEX chromatography can enable coupled processing with downstream steps such as virus filtration (VF), which further reduces the risk of viral contamination, and ultrafiltration / diafiltration (UF / DF), which buffer exchanges and / or concentrates the desired product to the desired condition for pharmaceutical formulation. Furthermore, flow-through and weakly partitioning AEX chromatography generally allow for higher column loads compared to chromatography operations performed in bind-and-elute mode, which in turn reduces the column size and buffer consumption required for downstream processes. However, while flow-through AEX chromatography has demonstrated robustness for the removal of process-related impurities such as nucleic acids, host cell proteins, leached Protein A ligands, endotoxins, and viruses, achieving removal of high molecular weight (HMW) species and aggregates can be difficult, especially when using high protein loads to maximize process productivity (Yigzaw et al., Current Pharmaceutical Biotechnology, 2009). Achieving robust HMW clearance requires optimization of the AEX resin, load pH, counterion type, and load dilution, and these variables are subject to constraints such as product stability and equipment compatibility. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yigzaw et al,Current Pharmaceutical Biotechnology,2009 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need in the art for new and improved purification methods utilizing AEX chromatography that allow for robust removal of HMW species with high protein yield at high loads. [Means for solving the problem]

[0007] One aspect of the present disclosure provides a method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, the method comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of greater than about 100 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering the purified composition comprising the recombinant protein.

[0008] Another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising resin particles at a loading density of about 250 g / L to about 600 g / L of anion exchange material, the resin particles comprising polymethacrylate and functionalized with primary amine ligands; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering the purified composition comprising the recombinant protein.

[0009] Yet another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0010] Yet another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising a polyamine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0011] Another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: conducting a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; and recovering the purified composition comprising the recombinant protein.

[0012] Yet another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity selected from high molecular weight species of the recombinant protein, the method comprising: conducting a low pH viral inactivation unit operation using about 1 M to about 2 M formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a polyamine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; and recovering the purified composition comprising the recombinant protein.

[0013] Yet another aspect of the present disclosure provides a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: conducting a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 shows the percentage of high molecular weight (HMW) species of mAb1, as assessed by SEHPLC, in the composition loaded onto the AEX column and in the pool recovered from the AEX column across seven pilot-scale lots. [Figure 1B] Figure 1A shows the process yields for the AEX process of seven mAb1 pilot-scale lots summarized, demonstrating the ability of the AEX process to significantly reduce impurities while achieving high yields of over 85%. [Figure 2A] Figure 1 shows the percentage of high molecular weight (HMW) species of mAb2, assessed by SE-UHPLC, in the composition loaded onto the AEX column and in the pool recovered from the AEX column across two pilot-scale lots. [Figure 2B] The process yields for the AEX process of two mAb2 pilot-scale lots summarized in Figure 2A are shown and demonstrate the ability of the AEX process to result in significant reduction of impurities while achieving high yields of over 85%. [Figure 3] Figure 1 shows the percentage of high molecular weight (HMW) species of mAb1 in two pilot-scale lots, one using 10% acetic acid as the VI titrant (PSL1) and one using 1 M formic acid as the VI titrant (PSL2). Use of 1 M formic acid VI titrant (PSL2) resulted in a lower percentage of HMW species in the AEX pool compared to use of 10% acetic acid VI titrant (PSL1) under similar conditions. DETAILED DESCRIPTION OF THE INVENTION

[0015] Disclosed herein are methods for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising performing anion exchange chromatography (e.g., in flow-through or weak partitioning chromatography mode) using an anion exchange material comprising a primary amine ligand, such as a polyamine ligand, and optionally a methacrylate-containing polymer-based matrix.

[0016] definition The following definitions are provided to facilitate understanding of the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017] In some embodiments, "about," when used in connection with a measurable, numerical variable, refers to the indicated value of the variable and all values ​​of the variable that are within experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or ±10% of the indicated value, whichever is greater. In some embodiments, numerical ranges are inclusive of the numbers (i.e., endpoints) that define the range.

[0018] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] As used herein, the terms "a" and "an" mean "one or more" unless specifically indicated otherwise. Furthermore, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context otherwise requires, singular terms include plurals and plural terms include the singular.

[0020] As used herein, the term "acid precipitation" refers to a recovery procedure in which the pH of a cell culture is lowered to induce precipitation of one or more cell culture impurities.

[0021] As used herein, the term "affinity chromatography" (also referred to as "capture chromatography") refers to a chromatographic procedure that separates a biomolecule (e.g., a recombinant protein) from a mixture based on a selective interaction between the biomolecule and another substance (i.e., a ligand). Affinity chromatography is commonly used in biomanufacturing processes to isolate and concentrate a desired recombinant protein from a harvested cell culture medium. In a typical affinity chromatography procedure, a biomolecule in the mobile phase selectively binds to or otherwise interacts with a stationary phase, and the remainder of the mobile phase passes through the chromatographic material. The biomolecule is then eluted from the stationary phase by changing conditions to reduce the affinity between the ligand and the biomolecule. Non-limiting examples of affinity chromatography include Protein A, Protein G, Protein A / G, and Protein L materials. Additionally, immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have, or have been engineered to have, affinity for metal ions.

[0022] In some embodiments, Protein A affinity chromatography can be used to capture the recombinant protein of interest. Protein A ligands are highly selective for a wide range of proteins containing antibody Fc regions, resulting in robust removal of process-related impurities and high target protein yields. Commercially available Protein A materials include, but are not limited to, MABSELECT™ SURE Protein A, Protein A Sepharose FAST FLOW™, MABSELECT™ PrismA (Cytiva, Marborough, MA), PROSEP-A™ (Merck Millipore, UK), TOYOPEARL™ HC-650F Protein A (TosoHass Co., Philadelphia, PA), and AP Plus (Purolite, King of Prussia, PA).

[0023] As used herein, the term "antigen-binding protein" refers to a protein or polypeptide that comprises an antigen-binding region or portion that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include antibodies, fusion proteins, VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "r and modified fragments of full-length antibodies, such as (IgG) ("half antibodies" consisting of heavy and light chains) or full-length antibodies, such as three-chain antibody-like molecules, heavy chain-only antibodies, single-chain variable fragments (scFv), di-scFv or bi(s)scFv, scFv-Fc, scFv-zippers, single-chain Fab (scFab), Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, (VH-VL-CH3), (scFv-CH3), (scFv)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies, which comprise only one variable region which may be a VHH, VH, or VL that specifically binds to an antigen or target independent of other variable regions or domains.

[0024] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa).

[0025] As used herein, the term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from the amino-terminus (N-terminus) to the carboxyl-terminus (C-terminus), a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain.

[0026] As used herein, the term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG-class and IgA-class antibodies are further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0027] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy-light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the CDRs and FRs of a given antibody. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0028] Digestion of an antibody with papain produces two identical antigen-binding proteins called "Fab" fragments (each of which has a single antigen-binding site) and the remaining "Fc" fragment (which contains all but the first domain of the immunoglobulin heavy chain constant region). The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fd fragment" contains the VH domain and CH1 domain from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0029] As used herein, an "Fc fragment" or "Fc region" of an immunoglobulin generally comprises two constant domains, namely, a CH2 domain and a CH3 domain, and optionally a CH4 domain. The Fc region may be derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0030] As used herein, the term "F(ab')2 fragment" refers to a bivalent fragment containing two Fab' fragments linked by an inter-heavy chain disulfide bridge at the hinge region.

[0031] As used herein, the term "Fv" fragment refers to the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy-chain variable region (VH) and one immunoglobulin light-chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light-chain or heavy-chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site containing both the VH and VL.

[0032] As used herein, the term "single-chain variable fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0033] As used herein, "nanobody" refers to the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks and nurse sharks, and Camelidae, including camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region; i.e., these functional antibodies are heavy chain homodimers with only the structure H2L2 (also referred to as "heavy chain antibodies" or "HCAbs"). Camelized VHHs contain hinge, CH2, and CH3 domains and have been reported to recombine with IgG2 and IgG3 constant regions lacking the CH1 domain. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold, providing a long, transmembrane loop structure in the framework.

[0034] As used herein, the term "heavy chain-only antibody" refers to an immunoglobulin protein consisting of two heavy chain polypeptides (e.g., each heavy chain polypeptide of approximately 50-70 kDa). A "heavy chain-only antibody" lacks the two light chain polypeptides found in conventional antibodies. Heavy chain antibodies constitute approximately one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These molecules are formed by two heavy chains but lack light chains. The resulting variable antigen-binding portion, referred to as a VHH domain, represents the smallest naturally occurring intact antigen-binding site and is only approximately 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy-chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 3746 (1999)), and VHH moieties can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, termed VNAR. (Nuttall et al.Eur.J.Biochem.270,3543-3554(2003);Nuttall et al.Function and Bioinformatics 55,187-197(2004);Dooley et al.,Molecular Immunology 40,25-33(2003)).

[0035] In some embodiments, a "heavy chain-only antibody" is a dimeric antibody comprising a VH antigen-binding domain and CH2 and CH3 constant domains, with no CH1 domain present. In some embodiments, a heavy chain-only antibody is composed of a variable region antigen-binding domain comprised of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. The heavy chain-only antibodies described herein may belong to the IgG subclass, although heavy chain-only antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included herein. In some embodiments, the heavy chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, for example, the IgG1 or IgG4 subtype. In some embodiments, the heavy chain-only antibody is of the IgG1 or IgG4 subtype, and one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG4 subtype, and one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG1 subtype, and one or more CH domains are modified to alter the effector function of the antibody. Modifications of CH domains to alter effector function are further described herein. Non-limiting examples of heavy chain-only antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0036] As used herein, the term "tri-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise one heavy chain and one light chain of a monoclonal antibody, or an antigen-binding fragment of such antibody chains, each containing an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion, absent a CH1 domain, and including CH2, and / or CH3, and / or CH4 domains, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, such binding domains comprising, essentially consisting of, or consisting of a heavy chain-only antibody derived from or sharing sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions include V H and / or V L Gene segments, D and J H Gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0037] As used herein, the term "bioreactor" refers to any vessel useful for growing cell cultures (e.g., mammalian or bacterial cell cultures). "Bioreactor" as used herein encompasses the term "fermentor" (i.e., a vessel useful for growing bacterial cell cultures that typically includes a more powerful agitator and increased gas flow compared to vessels used for growing mammalian cell cultures). Non-limiting examples of bioreactors include stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or entrained bed bioreactors. In some embodiments, an exemplary bioreactor may perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients and / or a carbon source, injecting a suitable gas (e.g., oxygen, etc.), inflow and outflow of fermentation or cell culture medium (e.g., supplying fresh cell culture medium via perfusion and removing spent cell culture medium), separation of gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Unless the context dictates otherwise, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable bioreactor diameter may be used. Unless the context dictates otherwise, in some embodiments, the bioreactor may have a volume between 100 mL and 50,000 L. Unless otherwise indicated, a bioreactor can be of any size useful for culturing cells; typically, a bioreactor is sized appropriately for the volume of cell culture to be grown therein. In a non-limiting embodiment, and unless otherwise indicated by context, a bioreactor can be at least 1 liter (L), or can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000 liters, 20,000 L or more, or any volume therebetween. Internal conditions of the bioreactor, including, but not limited to, pH, dissolved oxygen concentration, and temperature, can be controlled during the culturing period.Those skilled in the art will be able to recognize and select a suitable bioreactor for use in the manufacturing methods disclosed herein based on relevant considerations.

[0038] As used herein, the term "cell culture" or "culturing" refers to the growth and proliferation of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian and bacterial cells are known in the art. (See, e.g., Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992)). Mammalian cells may be cultured in suspension or attached to a solid culture medium. In some embodiments, fluidized-bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, and / or stirred-tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0039] As used herein, the term "cell culture medium" (also referred to as "culture medium," "culture medium," "cell culture medium," "tissue culture medium," etc.) refers to any nutrient solution used to grow cells, e.g., bacterial cells or mammalian cells. Cell culture media generally provide one or more of the following components: an energy source (e.g., in the form of carbohydrates, e.g., glucose); one or more essential amino acids (e.g., all essential amino acids, the 20 basic amino acids plus cysteine); vitamins and / or other organic compounds that are typically required in low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that are typically required in very low concentrations, e.g., concentrations in the micromolar range. As used herein, cell culture medium encompasses nutrient solutions typically used and / or known to be used in any cell culture process, including, but not limited to, batch, extended batch, fed-batch, intensified, and / or perfusion or continuous culture of cells.

[0040] As used herein, the term "cell density" refers to the number of cells in a given volume of culture medium. "Viable cell density" refers to the number of viable cells in a given volume of culture medium, as determined by a standard viability assay (e.g., trypan blue exclusion). As used herein, "packed cell volume" (PCV), also referred to as "percent packed cell volume" (%PCV), refers to the ratio of the volume occupied by cells to the total volume of the cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter, and an increase in packed cell volume can occur due to an increase in cell density, cell diameter, or both. Packed cell volume is a measure of the solid content in a cell culture. Because host cells vary in size and cell cultures also contain dead or dying cells and other cellular debris, packed cell volume can more accurately describe the solids content within a cell culture.

[0041] As used herein, the term "coupled" with respect to unit operations refers to a direct connection or mechanism that allows for continuous flow between one or more unit operations in a single operating cycle.

[0042] As used herein, the term "continuous" with respect to unit operations refers to a direct connection or mechanism that allows for continuous flow between one or more unit operations over multiple operating cycles.

[0043] As used herein, the term "dynamic binding capacity" with respect to a chromatographic material refers to the amount of product, e.g., polypeptide, that the material will bind under realistic flow conditions before significant ingress of unbound product occurs.

[0044] As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell, e.g., a mammalian host cell. Vectors can include viral vectors, non-episomal mammalian vectors, plasmids, and other non-viral vectors. Expression vectors may contain sequences that affect or control transcription, translation, and, if present, RNA splicing of the operably linked coding region. "Operably linked" means that the components to which this term is applied are in a relationship allowing them to perform their inherent functions. For example, a control sequence in a vector "operably linked" to a protein-coding sequence, e.g., a promoter, is positioned so that normal activity of the control sequence results in transcription of the protein-coding sequence and recombinant expression of the encoded protein.

[0045] As used herein, "fed-batch culture" refers to a form of suspension culture, specifically a method of culturing cells in which additional components are provided to the culture medium at some point or points after the initiation of the culture process. The provided components typically include nutritional supplements for the cells that are depleted during the culture process. Additionally or alternatively, the additional components may include supplemental components (e.g., cell cycle inhibitor compounds, etc.). In some embodiments, a fed-batch cell culture medium formulation contains components essential for cell survival and growth and may be richer or more concentrated than a basal cell culture medium formulation typically used to initiate the cell culture. The fed-batch culture may be stopped at some point, and the cells and / or components in the medium may be harvested and optionally purified.

[0046] As used herein, a "fusion protein" is a protein containing at least one polypeptide fused or linked to a heterologous polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is added in frame with a nucleotide sequence encoding a polypeptide sequence from a different protein, optionally separated from that sequence by a linker. The fusion gene can then be expressed by a recombinant host cell to produce the fusion protein. A fusion protein can include a fragment from an immunoglobulin protein, such as an Fc region fused or linked to a ligand polypeptide, receptor polypeptide, hormone, cytokine, growth factor, enzyme, or other polypeptide that is not a component of an immunoglobulin.

[0047] As used herein, the "growth phase" of a cell culture refers to the period of exponential cell growth (i.e., log phase) when cells are generally dividing rapidly.

[0048] As used herein, the term "harvested cell culture fluid" refers to a solution that has been treated by one or more operations to separate cells, cell debris, or other large particulates from recombinant protein. Such operations include, but are not limited to, chilling, flocculation, acidification, centrifugation, neutralization, sonication, and various forms of filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration), as described herein. Harvested cell culture fluid includes cell culture lysate and cell culture supernatant. Harvested cell culture fluid may be further clarified to remove small particulate matter and soluble aggregates by filtration through a membrane having a pore size of about 0.1 μm to about 0.5 μm, such as a membrane having a pore size of about 0.22 μm.

[0049] As used herein, a "host cell" refers to a cell that has been transformed, or can be transformed, with a nucleic acid and thereby expresses a gene of interest. The term includes the progeny of a parent cell, regardless of whether the morphology or genetic make-up of the progeny is identical to that of the original parent cell, so long as the gene of interest is present. For example, a host cell containing a nucleic acid encoding a recombinant protein operably linked to at least one expression control sequence (e.g., a promoter or enhancer) is a "recombinant host cell." When cultured under appropriate conditions, the host cell will synthesize the recombinant protein, which can then be collected from the culture medium (if the host cell secretes it into the medium) or directly from the producing host cell (if the host cell does not secrete it).

[0050] As used herein, "high molecular weight" or "HMW" species of a recombinant protein of interest refers to dimers, oligomers, and aggregates of the recombinant protein that have a molecular weight greater than the molecular weight of the intact, fully assembled form of the recombinant protein.

[0051] As used herein, the term "impurities" refers to components other than the recombinant protein of interest along with their associated buffer components, including, but not limited to, process and product-related impurities such as host cell proteins, leached resin materials (e.g., leached Protein A), nucleic acids, HMW species of recombinant proteins, LMW species of recombinant proteins, endotoxins, viral contaminants, cell culture media components, and the like.

[0052] As used herein, the term "loading density" refers to the amount of composition contacted with a volume of chromatographic material.

[0053] As used herein, a "low molecular weight" or "LMW" species of a recombinant protein of interest refers to a fragment, truncated, or incomplete variant of the recombinant protein that has a molecular weight less than the molecular weight of the intact, fully assembled form of the recombinant protein. LMW species can include, but are not limited to, proteolytic fragments, truncated forms resulting from cellular expression of mRNA splice variants, and single component polypeptides in the case of multi-chain polypeptide proteins (e.g., species of only the light or heavy chain when the recombinant protein is an antibody).

[0054] As used herein, "perfusion" cell culture medium refers to a cell culture medium typically used in cell cultures maintained by perfusion or continuous culture methods and that is sufficiently complete to support the cell culture during this process. In some embodiments, the perfusion cell culture medium composition may be more concentrated or more concentrated than the basal cell culture medium composition to accommodate the method used to remove spent medium. In some embodiments, the perfusion cell culture medium may be used in both the growth and production phases.

[0055] As used herein, the term "polishing chromatography" refers to a chromatographic operation performed after a capture or affinity chromatography operation to remove residual impurities and obtain a more highly purified composition and / or recombinant protein. Common impurities removed during the polishing step include, but are not limited to, product-related impurities (e.g., HMW and LMW species), host cell proteins, DNA, leached protein A, viral contaminants, and endotoxins. In addition, typical chromatographic techniques used for polishing include, but are not limited to, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimodal (or mixed-mode) chromatography (MMC).

[0056] As used herein, "anion exchange chromatography" (AEX) refers to a form of ion exchange chromatography performed on a solid-phase medium (e.g., a resin or membrane) that is positively charged and has the capacity to exchange free anions with anions in an aqueous solution passing over or through the solid phase. AEX chromatography is used, for example, in viral clearance and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL® Super Q® and TOYOPEARL® NH2-750F (Tosoh Bioscience), POROS HQ™, and POROS XQ™ (ThermoFisher).

[0057] As used herein, "cation exchange chromatography" (CEX) refers to a form of ion exchange chromatography performed on a solid phase medium (e.g., a resin or membrane) that is negatively charged and has the capacity to exchange free cations with cations in an aqueous solution passing over or through the solid phase. The charge may be imparted by attaching, e.g., covalently, one or more charged ligands to the solid phase. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g., silica, which has an overall negative charge). CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process-related impurities, and / or viral contaminants. Commercially available cation exchange media include sulfopropyl (SP) immobilized on agarose (e.g., SPSEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL™ XS, TOYOPEARL™ HS (Tosoh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), S Ceramic Hyper™ DF (Pall, Portland, FL). Examples of suitable fluoropolymers include, but are not limited to, ESHMUNO® CSP and ESHMUNO® CP-FT (Millipore Sigma, Darmstadt, Germany), POROS™ (ThermoFisher, Waltham, MA), and ESHMUNO® CP-FT (Millipore Sigma, Darmstadt, Germany).

[0058] As used herein, "hydrophobic interaction chromatography" (HIC) refers to chromatography performed on a solid phase medium that utilizes interactions between hydrophobic ligands and hydrophobic residues on the surface of a desired solute (e.g., a desired protein). Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sephrose™ (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto™ phenyl (Cytiva).

[0059] As used herein, "mixed-mode or multimodal chromatography" (MMC) refers to chromatography that utilizes two or more types of interactions between the stationary phase and the analytes to achieve separation. MMC differs from single-mode chromatography in that two or more types of interactions, such as electrostatic interactions, hydrogen bonding interactions, and / or hydrophobic interactions, contribute significantly to solute retention. Commercially available multimodal chromatography media include, but are not limited to, Capto™ Adhere, Capto™ MMC Impress, Capto MMC (Cytiva), PPA Hypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno HCX (Merk Millipore), and TOYOPEARL® MX-Trp-650M (Tosoh Bioscience).

[0060] Polishing chromatography unit operations utilize agent-containing materials (e.g., resins and / or membranes) that can be operated in a variety of ways, including bind-and-elute and flow-through modes. In bind-and-elute chromatography, the biomolecule of interest is typically loaded onto the chromatographic material to maximize dynamic binding capacity, and then specified recovery and elution conditions are used to maximize product purity in the eluate. In contrast, flow-through chromatography uses loading conditions that allow impurities to bind to the chromatographic material while the biomolecule of interest passes through. Compared to bind-and-elute chromatography, flow-through chromatography allows for higher loading densities for many biomolecules.

[0061] In addition to the two most common modes, weak partitioning, overloading, and frontal chromatography modes can also be used in the purification process. In weak partitioning, an isocratic separation method, the flow-through mode is modified by specifying solution conditions that promote weak binding of the biomolecule to the resin in addition to binding of one or more impurities (K less than about 0.1 for flow-through chromatography). p Compared to K p(where ρ is approximately 0.1 to approximately 100). In overload chromatography, the biomolecule of interest is loaded onto the chromatographic material beyond the dynamic binding capacity of the material. Furthermore, frontal chromatography mode allows for continuous high-density feed (containing the protein of interest and at least one impurity) onto the chromatographic medium. In frontal chromatography, separation of the protein of interest from impurities and contaminants is driven by the binding affinity of the components in the load feed to the chromatographic medium. The amount of protein of interest that can be loaded onto and bound to the chromatographic medium in frontal mode typically depends on the amount of more highly charged impurities / contaminants, such as product-related impurities, in the load feed. Initially, all components in the load feed bind to the chromatographic medium. Separation of the product of interest from impurities / contaminants is driven by their affinity for the chromatographic medium. When the chromatographic medium reaches saturation binding, components in the load feed with higher affinity for the chromatographic medium (typically product-related impurities such as HMW species) displace proteins with weaker affinity (e.g., the product of interest), resulting in the protein with weaker affinity being separated from the chromatographic medium. These proteins exit the column as bands in the load flow-through. As loading progresses, bound proteins are successively displaced in order of decreasing affinity for the chromatography medium until the column is saturated or near saturation with proteins that have a higher affinity than the protein of interest.

[0062] As used herein, the term "polypeptide" refers to a polymer of amino acids containing at least 50 amino acids, such as, for example, at least 100 amino acids.

[0063] As used herein, "partition coefficient" or "product partition coefficient" (K pThe term ) refers to the molar concentration of product, e.g., recombinant protein, bound to the stationary phase divided by the molar concentration of product in the mobile phase during a chromatographic step.

[0064] As used herein, "production" cell culture medium refers to a cell culture medium typically used in a cell culture during the transition when exponential growth ends and protein production becomes dominant (i.e., the "transition" and / or "production" phase), and that is sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase. The production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of the cell culture.

[0065] As used herein, the "production phase" of a cell culture refers to the period after logarithmic cell growth has ended and recombinant protein production predominates.

[0066] As used herein, the term "recombinant protein" refers to a heterologous protein produced by a host cell transfected with a nucleic acid encoding the protein when the host cell is grown in cell culture.

[0067] As used herein, the term "purified," when used in reference to a composition, refers to a composition in which at least one impurity is present in a lower concentration in the purified composition compared to the composition as it was prior to one or more unit operations. Furthermore, a "purified" recombinant protein (e.g., a purified antibody) refers to a recombinant protein that has increased purity, thereby existing in a form that is purer than it is in its natural environment and / or when originally synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0068] As used herein, the term "titrant" refers to a solution of known concentration that is added to another solution during titration. An "acid titrant" refers to a titrant that has a pH of less than about 7.

[0069] As used herein, the term "unit operation" refers to a functional step performed as part of a process to purify a recombinant protein of interest. A unit operation can be designed to accomplish a single purpose or multiple purposes, such as a capture step, an acid precipitation step, a centrifugation step, or a chromatography step. A unit operation can also include a holding or storage step between processing steps.

[0070] Non-limiting exemplary features Without limitation, some exemplary embodiments / features of the present disclosure include E1-E47 below. E1. A method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of greater than about 100 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering the purified composition comprising the recombinant protein. E2. The method of E1, wherein the anion exchange material comprises resin particles. E3. The method of E1 or E2, wherein the anion exchange material comprises resin particles, and at least 80% of the resin particles have a particle size of about 30 μm to about 60 μm. E4. The method of any one of E1-E3, wherein the anion exchange material comprises resin particles having an average particle size of about 45 μm. E5. The method of any one of E1-E4, wherein the anion exchange material comprises a polyamine ligand. E6. The method of any one of E1-E5, wherein the anion exchange material comprises a methacrylate-containing polymer-based matrix. E7. The method of any one of E1-E6, wherein the anion exchange material is TOYOPEARL® NH2-750F. E8. The method of any one of E1-E7, wherein the loading density is less than about 600 g / L of anion exchange material. E9. The method of any one of E1-E7, wherein the loading density is from about 200 g / L to about 600 g / L of anion exchange material. E10. The method of any one of E1-E9, wherein the composition has a conductivity of about 3 mS / cm to about 6 mS / cm. E11. The method according to any one of E1 to E10, wherein the partition coefficient of the anion exchange material for the recombinant protein is from about 0.1 to about 100. E12. The method according to any one of E1 to E10, wherein the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 40. E13. The method includes using an equilibration buffer and / or recovery buffer with an anion exchange material; the pH of the equilibration buffer and / or recovery buffer is about 7.0 to about 8.0; and / or The method of any one of E1-E12, wherein the conductivity of the equilibration buffer and / or recovery buffer is less than about 10 mS / cm. E14. The method according to E13, wherein the conductivity of the equilibration buffer and / or recovery buffer is about 2 mS / cm to about 4 mS / cm. E15. The method of any one of E1-E14, further comprising performing a low pH viral inactivation unit operation (e.g., one or more unit operations prior to loading) prior to loading. E16. The method of E15, wherein the low pH viral inactivation unit operation is carried out at a pH of about 3.5 to about 3.7. E17. The method of E15 or E16, wherein the low pH viral inactivation unit operation uses an acid titrant. E18. The method of E17, wherein the acid titrant comprises formic acid. E19. The method of E17 or E18, wherein the acid titrant is about 1 M to about 2 M formic acid (eg, about 1 M formic acid; about 2 M formic acid). E20. The method of any one of E15 to E19, wherein the low pH viral inactivation unit operation is carried out for at least about 60 minutes. E21. The method of any one of E15 to E20, wherein the low pH viral inactivation unit operation is carried out for about 60 minutes to about 12 hours. E22. The method of any one of E1-E21, further comprising performing one or more additional chromatographic unit operations. E23. The method of E22, wherein one or more additional chromatography unit operations comprise an affinity chromatography unit operation performed prior to loading. E24. The method of E23, wherein the affinity chromatography unit operation is selected from Protein A chromatography, Protein G chromatography, Protein L chromatography, and CH1 domain chromatography. E25. The method of E23 or E24, wherein the affinity chromatography unit operation is Protein A chromatography. E26. The method of any one of E22 to E25, wherein one or more additional chromatography unit operations comprises an additional polishing chromatography unit operation performed prior to loading. E27. The method of any one of E22 to E25, wherein the one or more additional chromatography unit operations comprises an additional polishing chromatography unit operation performed after loading. E28. The method of E26 or E27, wherein the additional polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. E29. The method of any one of E26 to E28, wherein the additional polishing chromatography unit operation and the load are sequential or linked. E30. The method of any one of E1-E29, further comprising performing a viral filtration and / or UF / DF unit operation after loading. E31. The method of E30, wherein the loading and viral filtration and / or UF / DF unit operations are sequential or linked. E32. The method of any one of E1-E31, wherein less than about 5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein. E33. The method of any one of E1-E32, wherein less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein. E34. The method of any one of E1-E33, wherein less than about 1% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein. E35. The method of any one of E1-E34, wherein the purified composition comprises at least about 85% (w / w) recombinant protein in the composition prior to loading. E36. The method of any one of E1-E35, wherein the purified composition comprises at least about 90% (w / w) recombinant protein in the composition prior to loading. E37. Approximately 1% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and The method of any one of E1-E36, wherein the purified composition comprises at least about 90% (w / w) recombinant protein in the composition prior to loading. E38. The loading density is about 100 g / L to about 600 g / L of anion exchange material; about 1% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and The method of any one of E1-E37, wherein the purified composition comprises at least about 90% (w / w) recombinant protein in the composition prior to loading. E39. The method of any one of E1-E38, wherein the recombinant protein is an antigen-binding protein. E40. The method of any one of E1 to E39, wherein the recombinant protein is an antibody. E41. The method of any one of E1-E40, wherein the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of recombinant proteins, fragments of recombinant proteins, cell culture media components, and viral contaminants. E42. The method of any one of E1-E41, wherein the at least one impurity is selected from high molecular weight species of recombinant proteins. E43. The method of any one of E1-E42, wherein the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands. E44. The method of any one of E1-E42, wherein the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with primary amine ligands. E45. The method of E43 or E44, wherein at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. E46. The method of any one of E43, E44, or E45, wherein the resin particles have an average particle size of about 40 μm to about 50 μm. E47. The method according to E46, wherein the resin particles have an average particle size of about 45 μm.

[0071] Anion exchange chromatography purification method 1. A method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of greater than about 100 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering the purified composition comprising the recombinant protein.

[0072] In some embodiments, the anion exchange material comprises resin particles.

[0073] In some embodiments, the anion exchange material comprises resin particles, wherein at least about 80% of the resin particles have a particle size between about 30 μm and about 60 μm.

[0074] In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 40 μm to about 50 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 30 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 35 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 40 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 45 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 50 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 55 μm. In some embodiments, the anion exchange material comprises resin particles having an average particle size of about 60 μm.

[0075] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0076] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix.

[0077] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix and a polyamine ligand.

[0078] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0079] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0080] In some embodiments, the anion exchange material is TOYOPEARL® NH2-750F.

[0081] In some embodiments, the loading density is less than about 750 g / L of anion exchange material. In some embodiments, the loading density is less than about 700 g / L of anion exchange material. In some embodiments, the loading density is less than about 650 g / L of anion exchange material. In some embodiments, the loading density is less than about 600 g / L of anion exchange material. In some embodiments, the loading density is less than about 550 g / L of anion exchange material. In some embodiments, the loading density is less than about 500 g / L of anion exchange material. In some embodiments, the loading density is less than about 450 g / L of anion exchange material. In some embodiments, the loading density is less than about 400 g / L of anion exchange material. In some embodiments, the loading density is less than about 350 g / L of anion exchange material. In some embodiments, the loading density is less than about 300 g / L of anion exchange material. In some embodiments, the loading density is less than about 250 g / L of anion exchange material. In some embodiments, the loading density is less than about 200 g / L of anion exchange material, hi some embodiments, the loading density is less than about 150 g / L of anion exchange material.

[0082] In some embodiments, the loading density is from about 100 g / L to about 600 g / L of anion exchange material. In some embodiments, the loading density is from about 150 g / L to about 600 g / L of anion exchange material. In some embodiments, the loading density is from about 200 g / L to about 600 g / L of anion exchange material. In some embodiments, the loading density is from about 250 g / L to about 600 g / L of anion exchange material.

[0083] In some embodiments, the composition has a pH of about 7.1 to about 7.9. In some embodiments, the composition has a pH of about 7.2 to about 7.8. In some embodiments, the composition has a pH of about 7.3 to about 7.7. In some embodiments, the composition has a pH of about 7.4 to about 7.6. In some embodiments, the composition has a pH of about 7.5.

[0084] In some embodiments, the composition has a conductivity of less than about 9 mS / cm. In some embodiments, the composition has a conductivity of less than about 8 mS / cm. In some embodiments, the composition has a conductivity of less than about 7 mS / cm. In some embodiments, the composition has a conductivity of less than about 6 mS / cm. In some embodiments, the composition has a conductivity of less than about 5 mS / cm. In some embodiments, the composition has a conductivity of less than about 4 mS / cm.

[0085] In some embodiments, the composition has a conductivity of about 10 mS / cm. In some embodiments, the composition has a conductivity of about 9.5 mS / cm. In some embodiments, the composition has a conductivity of about 9 mS / cm. In some embodiments, the composition has a conductivity of about 8.5 mS / cm. In some embodiments, the composition has a conductivity of about 8 mS / cm. In some embodiments, the composition has a conductivity of about 7.5 mS / cm. In some embodiments, the composition has a conductivity of about 7 mS / cm. In some embodiments, the composition has a conductivity of about 6.5 mS / cm. In some embodiments, the composition has a conductivity of about 6 mS / cm. In some embodiments, the composition has a conductivity of about 5.5 mS / cm. In some embodiments, the composition has a conductivity of about 5 mS / cm. In some embodiments, the composition has a conductivity of about 4.5 mS / cm. In some embodiments, the composition has a conductivity of about 4 mS / cm. In some embodiments, the composition has a conductivity of about 3.5 mS / cm. In some embodiments, the composition has a conductivity of about 3 mS / cm.

[0086] In some embodiments, the composition has a conductivity of about 3 mS / cm to about 7 mS / cm. In some embodiments, the composition has a conductivity of about 3 mS / cm to about 6 mS / cm. In some embodiments, the composition has a conductivity of about 3 mS / cm to about 5 mS / cm.

[0087] In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 0.1. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 10. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 20. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 30. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 40. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 50. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 60. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 70. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 80. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 90. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 100.

[0088] In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 0.1 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 10 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 90. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 80. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 70. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 60. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 50. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 40.

[0089] In some embodiments, the method comprises using an equilibration buffer and / or a recovery buffer with an anion exchange resin; the pH of the equilibration buffer and / or recovery buffer is about 7.0 to about 8.0; and / or The conductivity of the equilibration buffer and / or recovery buffer is less than about 10 mS / cm.

[0090] In some embodiments, the equilibration buffer and / or recovery buffer has a pH of about 7.1 to about 7.9. In some embodiments, the equilibration buffer and / or recovery buffer has a pH of about 7.2 to about 7.8. In some embodiments, the equilibration buffer and / or recovery buffer has a pH of about 7.3 to about 7.7. In some embodiments, the equilibration buffer and / or recovery buffer has a pH of about 7.4 to about 7.6. In some embodiments, the equilibration buffer and / or recovery buffer has a pH of about 7.5.

[0091] In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 9 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 8 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 7 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 3 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of less than about 2 mS / cm.

[0092] In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 10 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 9.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 9 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 8.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 8 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 7.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 7 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 6.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 5.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 4.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 3.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 3 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1 mS / cm.

[0093] In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 3 mS / cm.

[0094] In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 3 mS / cm.

[0095] In some embodiments, the method further comprises performing a low pH viral inactivation unit operation (e.g., one or more unit operations prior to loading) prior to loading. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.5 to about 3.7. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.5. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.6. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.7.

[0096] In some embodiments, the low pH viral inactivation unit operation uses an acid titrant. In some embodiments, the acid titrant is formic acid. In some embodiments, the acid titrant is about 1 M to about 2 M formic acid. In some embodiments, the acid titrant is about 1 M formic acid. In some embodiments, the acid titrant is about 2 M formic acid.

[0097] In some embodiments, the low pH viral inactivation unit operation is performed for at least about 60 minutes. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 2 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 3 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 4 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 5 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 6 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 7 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 8 hours. In some embodiments, the low pH viral inactivation unit operation is performed for between about 60 minutes and about 12 hours. In some embodiments, the low pH viral inactivation unit operation is performed for between about 60 minutes and about 8 hours.

[0098] In some embodiments, the method further comprises performing one or more additional chromatography unit operations.

[0099] In some embodiments, the one or more additional chromatography unit operations include an affinity chromatography unit operation performed prior to loading. In some embodiments, the affinity chromatography unit operation is selected from Protein A chromatography, Protein G chromatography, Protein L chromatography, and CH1 domain chromatography. In some embodiments, the affinity chromatography unit operation is Protein A chromatography. In some embodiments, the affinity chromatography unit operation is Protein G chromatography. In some embodiments, the affinity chromatography unit operation is Protein L chromatography. In some embodiments, the affinity chromatography unit operation is CH1 domain chromatography.

[0100] In some embodiments, the one or more additional chromatography unit operations include an additional polishing chromatography unit operation that is performed before the loading. In some embodiments, the one or more additional chromatography unit operations include an additional polishing chromatography unit operation that is performed after the loading. In some embodiments, the additional polishing chromatography unit operation and the load are coupled. In some embodiments, the additional polishing chromatography unit operation and the load are not coupled. In some embodiments, the additional polishing chromatography unit operation and the load are sequential. In some embodiments, the additional polishing chromatography unit operation and the load are not sequential.

[0101] In some embodiments, the additional polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. In some embodiments, the additional polishing chromatography unit operation is cation exchange chromatography. In some embodiments, the additional polishing chromatography unit operation is hydrophobic interaction chromatography. In some embodiments, the additional polishing chromatography unit operation is mixed-mode chromatography.

[0102] In some embodiments, the method further comprises performing a virus filtration unit operation and / or a UF / DF unit operation after loading.

[0103] In some embodiments, less than about 5% (w / w) (e.g., less than about 4.5% (w / w), less than about 4% (w / w), less than about 3.5% (w / w), less than about 3% (w / w), less than about 2.5% (w / w), less than about 2% (w / w), less than about 1.5% (w / w), less than about 1% (w / w)) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein.

[0104] In some embodiments, the purified composition comprises at least about 85% (w / w) (e.g., at least about 90% (w / w), at least about 95% (w / w)) recombinant protein in the composition prior to loading.

[0105] In some embodiments, less than about 1% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein; the purified composition comprises at least about 90% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, the loading density is about 100 g / L to about 600 g / L (e.g., about 150 g / L to about 600 g / L; about 200 g / L to about 600 g / L; about 250 g / L to about 600 g / L) of anion exchange material; less than about 1% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein; and the purified composition comprises at least about 90% (w / w) of the recombinant protein in the composition prior to loading.

[0106] In some embodiments, the recombinant protein is an antigen binding protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is a human antibody.

[0107] In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody.

[0108] In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody.

[0109] In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody.

[0110] In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0111] In some embodiments, the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of recombinant proteins, fragments of recombinant proteins, cell culture media components, and viral contaminants. In some embodiments, the at least one impurity is selected from high molecular weight species of recombinant proteins.

[0112] Also provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising resin particles at a loading density of about 250 g / L to about 600 g / L of anion exchange material, the resin particles comprising polymethacrylate and functionalized with primary amine ligands; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering the purified composition comprising the recombinant protein.

[0113] In some embodiments, at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm, and in some embodiments, the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0114] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0115] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0116] Also provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0117] In some embodiments, the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of recombinant proteins, fragments of recombinant proteins, cell culture media components, and viral contaminants.

[0118] In some embodiments, the at least one impurity is selected from high molecular weight species of recombinant proteins.

[0119] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0120] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix.

[0121] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix and a polyamine ligand.

[0122] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0123] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0124] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0125] Further provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: loading the composition onto an anion exchange material comprising a polyamine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; and loading at least one impurity that binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0126] In some embodiments, the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of recombinant proteins, fragments of recombinant proteins, cell culture media components, and viral contaminants.

[0127] In some embodiments, the at least one impurity is selected from high molecular weight species of recombinant proteins.

[0128] In some embodiments, the anion exchange material further comprises a methacrylate-containing polymer-based matrix.

[0129] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0130] Also provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: conducting a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; and recovering the purified composition comprising the recombinant protein.

[0131] In some embodiments, the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of recombinant proteins, fragments of recombinant proteins, cell culture media components, and viral contaminants.

[0132] In some embodiments, the at least one impurity is selected from high molecular weight species of recombinant proteins.

[0133] In some embodiments, the acid titrant is about 1 M to about 2 M formic acid. In some embodiments, the acid titrant is about 1 M formic acid. In some embodiments, the acid titrant is about 2 M formic acid.

[0134] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0135] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix.

[0136] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix and a polyamine ligand.

[0137] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0138] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0139] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0140] Further provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity selected from high molecular weight species of the recombinant protein, the method comprising: conducting a low pH viral inactivation unit operation using about 1 M to about 2 M formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a polyamine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; and recovering the purified composition comprising the recombinant protein.

[0141] In some embodiments, the acid titrant is about 1 M formic acid. In some embodiments, the acid titrant is about 2 M formic acid.

[0142] In some embodiments, the anion exchange material further comprises a methacrylate-containing polymer-based matrix.

[0143] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0144] Also provided herein is a method for purifying a recombinant protein (e.g., an antigen binding protein, such as an antibody) from a composition comprising the recombinant protein and at least one impurity (e.g., a high molecular weight species of the recombinant protein), the method comprising: conducting a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition onto an anion exchange material comprising a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and loading, wherein a low pH viral inactivation unit operation is performed on one or more unit operations prior to loading; recovering a purified composition comprising the recombinant protein, less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or and recovering the purified composition, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0145] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0146] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix.

[0147] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer-based matrix and a polyamine ligand.

[0148] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising polymethacrylate and functionalized with primary amine ligands, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0149] In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles, the resin particles comprising a hydroxylated methacrylic polymer and functionalized with a primary amine ligand, and the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0150] In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein. In some embodiments, the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading. In some embodiments, less than about 2.5% (w / w) of the recombinant protein in the purified composition is a high molecular weight species of recombinant protein, and the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to loading.

[0151] host cell The cell lines (also referred to as "cells" or "host cells") used in this disclosure are genetically engineered to express recombinant proteins of commercial or scientific interest. The cells may be suitable for adherent, monolayer, and / or suspension culture, transfection, and expression of recombinant proteins, such as antibodies. The cells may be used, for example, with batch, fed-batch, and perfusion or continuous culture methods. Such cells are typically cell lines obtained or derived from mammals and are capable of growing and surviving when placed in either monolayer or suspension culture in media containing appropriate nutrients and / or other factors, such as those described herein. Typically, host cells are selected that are capable of expressing and secreting proteins or that can be molecularly engineered to express and secrete large amounts of a particular protein, more specifically a glycoprotein of interest, into the culture medium. The selection of an appropriate host cell for expressing a recombinant protein will depend on various factors, such as the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) desired or required for activity, and the ease of folding into a biologically active molecule. In some embodiments, the host cells producing the recombinant proteins purified by the methods provided herein are mammalian host cells.

[0152] Cell lines are typically derived from lineages arising from primary cultures that can be maintained in culture for an indefinite period of time. Cells may contain, for example, cells introduced via an expression vector (construct), such as a plasmid carrying a coding sequence encoding a protein, or a portion thereof, for expression and production in a culture process, for example, by transformation, transfection, infection, or injection. Such expression vectors contain elements necessary for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding desired proteins and polypeptides and appropriate transcription and translation control elements can be constructed using methods well known and practiced by those skilled in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in detail in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4 th edition, Cold Spring Harbor Press, Plainview, NY, or any earlier edition; F.M.A.usubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or any earlier edition; Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for all purposes.

[0153] Suitable host cells include, but are not limited to, those commercially available from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0154] Examples of host cells include, but are not limited to, prokaryotes, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens. Examples of suitable host organisms include Bacillus species, such as B. marcescans and Shigella, and Bacillus species, such as B. subtilis and B. licheniformis, Pseudomonas species, and Streptomyces species. In some embodiments, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used.However, Pichia, e.g., P. pastoris, Schizosaccharomyces pombe; Kluyveromyces; Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; occidentalis); and several other genera, species, and strains of filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, are generally available and useful herein.

[0155] Vertebrate host cells are also suitable hosts for the expression of recombinant proteins. Mammalian cell lines suitable as hosts for recombinant protein expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC mammalian myeloma cells, and several other cell lines. In some embodiments, the host cell is selected from CHO cells.

[0156] In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. The mammalian cell can be, for example, a human, rodent, or bovine cell line or cell line. Examples of such cells, cell lines, or cell lines include, but are not limited to, mouse myeloma (NS0) cell lines, Chinese hamster ovary (CHO) cell lines, FIT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, YO, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK-293, VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic, or hybridoma cell lines. In some embodiments, the mammalian cell is a CHO cell line. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the mammalian cell is selected from CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN cells, and CHO-derived cells. In some embodiments, the CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. Further, the CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza, Inc.). In some embodiments, the eukaryotic cell can be, for example, an avian cell, cell line, or cell lineage, such as, for example, EBx® cells, EB14, EB24, EB26, EB66, or EBv13.

[0157] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, the dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems enable high-level recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Also included is the glutamine synthase (GS) knockout CHOK1SV cell line, which utilizes glutamine synthase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells include, but are not limited to, the following (ECACC accession numbers in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhFr- (94060607), CHO / dhFr-AC-free (05011002), and RR-CHOKI (92052129).

[0158] Large-scale production of proteins for commercial use can be carried out in suspension culture. Thus, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted to growth in suspension culture. A variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells and CLIO cells derived from the CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER.C6® cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or engineered from any of the cell lines disclosed herein.

[0159] In some embodiments, the eukaryotic cell is, for example, a yeast cell (e.g., a cell of the genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), a cell of the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), a cell of the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum), or a cell of the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum). uvarum), Kluyveromyces cells (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida cells (e.g., Candida utilis, Candida cacaoi, Candida boidinii), Geotrichum cells (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe). In some embodiments, the eukaryotic cell is selected from Pichia pastoris strains, non-limiting examples of which include X33, GS115, KM71, KM71H, and CBS7435.

[0160] In some embodiments, the eukaryotic cell is a fungal cell (e.g., a fungal cell, such as an Aspergillus (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula, etc.), an Acremonium (e.g., A. thermophilum, etc.), a Chaetomium (e.g., C. thermophilum, etc.), a Chrysosporium (e.g., C. thermophilum, etc.), a Bacillus subtilis (e.g., Bacillus subtilis ... Chrysosporium (e.g., C. thermophile, etc.), Cordyceps (e.g., C. militaris, etc.), Corynascus, Ctenomyces, Fusarium (e.g., F. oxysporum, etc.), Glomerella (e.g., G. graminicola, etc.), Hypocrea (Hypocrea pocrea (e.g., H. jecorina, etc.), Magnaporthe (e.g., M. orzyae, etc.), Myceliophthora (e.g., M. thermophile, etc.), Nectria (e.g., N. heamatococca, etc.), Neurospora (e.g., N. crassa, etc.), Penicillium llium), Sporotrichum (e.g., S. thermophile), Thielavia (e.g., T. terrestris, T. heterothallica), Trichoderma (e.g., T. reesei), or Verticillium (e.g., V. dahlia).

[0161] In some embodiments, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cells), algal cells (such as Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas), and plant cells (such as cells from monocotyledonous plants (such as maize, rice, wheat, or Setaria) or dicotyledonous plants (such as cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens). The cells are selected from the group consisting of cells from Saccharomyces cerevisiae (Saccharomyces patens) or Arabidopsis thaliana (Saccharomyces cerevisiae).

[0162] To generate a host cell line (e.g., a mammalian cell line) engineered to express a recombinant protein of interest, one or more nucleic acids encoding the recombinant protein (or its components, in the case of a multi-chain protein) are first inserted into one or more expression vectors. Nucleic acid control sequences useful in expression vectors for expression in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can optionally be encoded by the expression vector and operably linked to the coding sequence of interest, enabling the recombinant host cell to secrete the expressed protein so that it can be more easily isolated from the cells, if desired. A vector can also contain one or more selectable marker genes to facilitate selection of host cells into which the vector has been introduced. In some embodiments, vectors employing a protein fragment complementation assay using a protein reporter, such as dihydrofolate reductase, are used (see, e.g., U.S. Pat. No. 6,270,964). Suitable mammalian expression vectors are known in the art and are commercially available.

[0163] Typically, vectors used in any of the host cells contain sequences for maintaining the plasmid and for cloning and expressing exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a native or heterologous signal peptide sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. Vectors can be constructed from a starting vector, such as a commercially available vector, or additional elements can be obtained separately and ligated into the vector.

[0164] Culture method A variety of culture methods can be used to produce the recombinant protein of interest, including, but not limited to, batch culture, fed-batch culture, and perfusion culture.

[0165] Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time, followed by a total harvest. Cultures grown using batch methods undergo an increase in cell density until a maximum cell density is reached, after which the viable cell density declines as medium components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Harvesting typically occurs at maximum cell densities (e.g., 5×10 depending on medium composition, cell line, etc.). 6 The batch process is performed when the viable cell density reaches a concentration of 20 ...

[0166] Fed-batch culture improves on the batch process by using a bolus or continuous medium feed to replenish consumed medium components. Because fed-batch culture receives additional nutrients throughout the run, it can achieve higher cell densities (>10–30 × 10 depending on medium composition, cell line, etc.) compared to batch methods. 6Fed-batch culture has the potential to achieve high cell counts (cells / mL) and increased product titers. Unlike batch processes, feeding strategies and medium composition can be manipulated to create and maintain a two-phase culture, distinguishing between a cell growth phase (growth phase) to achieve a desired cell density and a period during which cell growth is halted or slow (production phase). Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. Typically, a batch method is used during the growth phase and a fed-batch method is used during the production phase, but a fed-batch feeding strategy can be used throughout the process. However, unlike batch processes, the volume of the bioreactor is a limiting factor, restricting the feed rate. Also, similar to batch processes, accumulation of metabolic by-products can lead to culture decline, which often limits the duration of the production phase to approximately 10–21 days. Fed-batch cultures are discontinuous, and harvesting is typically performed when metabolic by-product levels or culture viability reach a predetermined level. Compared to batch cultures without feeding, fed-batch cultures can produce larger amounts of recombinant protein. (See, e.g., U.S. Pat. No. 5,672,502.)

[0167] Perfusion methods offer a potential improvement over batch and fed-batch methods by adding fresh medium and simultaneously removing spent medium during cultivation. A typical perfusion culture begins with a batch culture start-up lasting 1-2 days, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture with simultaneous removal of spent medium, retaining cells and additional high-molecular-weight compounds, such as proteins (based on the filter's molecular weight cutoff), throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent medium while maintaining cell density. Non-limiting examples of filtration methods include tangential flow filtration (TFF), such as recirculating flow filtration, and alternating tangential flow (ATF) filtration. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, e.g., U.S. Pat. No. 6,544,424; Furey, 2002, Gen. Eng. News. 22(7):62-63.

[0168] Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate can be less than the working volume per day to many working volumes. The cells are maintained in culture, and the removed spent medium is substantially cell-free or has significantly fewer cells compared to the culture. Recombinant proteins expressed by the cell culture can also be maintained in the culture.

[0169] In a typical large-scale commercial cell culture strategy, approximately one-third to over one-half of the reactor volume is biomass, 40–90(+) × 10 6 cells / mL, e.g., approximately 40 x 10 6 cells / mL or approximately 50 x 10 6 We aim to achieve high cell densities of >1 × 10 cells / mL. 8Very high cell densities, up to 1000 cells / mL, have been achieved. A potential advantage of perfusion processes is the ability to maintain production cultures for longer periods than batch or fed-batch cultures. However, maintaining long-term perfusion cultures requires increased medium preparation, use, storage, and disposal, especially for cultures at high cell densities that also require more nutrients. Furthermore, high cell densities can cause issues during production, such as maintaining dissolved oxygen levels and problems with increased aeration, including more oxygen supply and more carbon dioxide removal, which can result in more foaming and the need for changes in anti-foaming strategies, as well as problems in harvesting and downstream processes, where the work required to remove excess cellular material can result in product loss, potentially negating the benefit of increased titer from increased cell mass.

[0170] Suitable culture conditions for mammalian cells, including temperature, dissolved oxygen content, agitation rate, etc., are known in the art and may vary depending on the phase or stage of the cell culture. In some embodiments, the methods disclosed herein further include withdrawing samples during the cell culture process and evaluating the samples to quantitatively and / or qualitatively monitor characteristics of the recombinant protein and / or the cell culture process. In some embodiments, the samples are monitored quantitatively and / or qualitatively using process analytical techniques. For example, dissolved oxygen levels may be monitored during the cell culture process using methods known in the art, such as, for example, elementary chemical analysis (titration), electrochemical analysis (diaphragm electrode), and photochemical analysis (fluorescence).

[0171] During recombinant protein production, it is desirable to have a controlled system that allows cells to grow for a desired time or to a desired density, and then switches the physiological state of cells to a growth-limited or arrested, high-production state that uses energy and substrates to produce the recombinant protein so that the cell density can be increased. For commercial-scale cell culture and biotherapeutic production, the ability to limit or arrest cell growth and maintain cells in a growth-limited or arrested state during the production phase is highly desirable. Such methods include, for example, temperature shift, use of chemical inducers of protein production, nutrient limitation or starvation, and cell cycle inhibitors, either alone or in combination. Illustratively, a typical cell culture undergoes a growth phase, a period of exponential growth during which cell density increases. During the growth phase, cells are cultured in cell culture medium containing necessary nutrients and additives under conditions that achieve optimal growth for the particular cell line (usually at a temperature of about 25-40°C in a humidified, controlled atmosphere). Cells are typically maintained in the growth phase for 1-8 days, e.g., 3-7 days, e.g., 7 days. The length of the growth phase for a particular cell line can be determined by one of skill in the art and is generally sufficient for the particular cells to propagate to a viable cell density within a range of approximately 20% to 80% of the maximum viable cell density possible when the culture is maintained under growth conditions. The growth phase is followed by a transition phase, during which exponential cell growth slows and protein production begins to increase. This marks the onset of a stationary phase, during which cell density typically plateaus and product titer increases. During the production phase, the medium is typically replenished to support continued production of the recombinant protein.

[0172] In certain embodiments, the culture conditions used to produce a recombinant protein can be adjusted to facilitate the transition from the growth phase to the production phase of a cell culture. For example, the growth phase of a cell culture can occur at a higher temperature than the production phase of the cell culture. In some embodiments, the growth phase can occur at a first temperature of about 35°C to about 38°C, and the production phase can occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In one embodiment, a temperature shift from about 35°C to about 37°C to a temperature of about 31°C to about 33°C can be used to drive the transition from the growth phase to the production phase of a culture. For example, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, and / or after, or instead of the temperature shift. If inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift.

[0173] Furthermore, any cell culture medium capable of supporting the growth of suitable host cells in culture can be used. Typically, cell culture media contain buffers, salts, an energy source, amino acids, vitamins, and essential trace elements. Cell culture media that can be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells are commercially available and include, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media, such as the EX-CELL™ 300 series, which can be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell culture media can be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture media can also be enriched by the addition of nutrients or other supplements, which can be used at concentrations higher than those normally recommended. In certain embodiments, the culture medium used in the production of recombinant proteins purified by the methods provided herein is a chemically defined medium, which refers to a cell culture medium in which all of the components have known chemical structures and concentrations. Chemically defined media are typically serum-free and do not contain hydrolysates or animal-derived components.

[0174] Various media formulations can be used during the culture period, for example, to drive the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase) and / or to optimize conditions during cell culture (e.g., concentrated media provided during perfusion culture). Growth media formulations can be used to promote cell growth and minimize protein expression. Production media formulations can be used to promote production of the recombinant protein of interest and cell maintenance while minimizing the growth of new cells. A feed medium is a cell culture medium that typically contains more concentrated components, such as nutrients and amino acids, that are consumed during the production phase of the cell culture. A feed medium can be used to replenish and maintain an active culture, particularly a culture operated in fed-batch, semi-perfusion, or perfusion mode. Such concentrated feed media may contain most of the components of the cell culture media, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x their normal amounts.

[0175] In some embodiments, the mammalian cells are cultured for a period during which the recombinant protein is expressed and secreted by the mammalian cells. This period (i.e., the duration of the production phase of the cell culture) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the production phase of the cell culture is about 7 to about 28 days, about 10 to about 30 days, about 7 to about 14 days, about 10 to about 18 days, about 3 to about 15 days, about 5 to about 8 days, about 12 to about 15 days, about 12 to about 18 days, or about 15 to about 21 days. In some embodiments, the duration of the production phase of the cell culture is 7, 8, 9, 12, 15, 18, or 21 days.

[0176] In some embodiments, the biomanufacturing process comprises at least 100×10 5 cells / mL, e.g., approximately 100 x 10 5 cells / mL ~ approx. 10×10 7 cells / mL, approximately 250×10 5cells / mL ~ approx. 900×10 5 cells / mL, approximately 300×10 5 cells / mL~800×10 5 cells / mL, or approximately 450 x 10 5 cells / mL~650×10 5 The production phase includes a viable cell density of cells / mL. Cell density can be measured using a hemocytometer, a Coulter counter, or an automated cell analyzer (e.g., a Cedex automated cell counter). Viable cell density can be determined by staining a culture sample with trypan blue, which is taken up only by dead cells. The total number of cells is then counted, and the viable cell density is determined by dividing the number of stained cells by the total number of cells and taking the reciprocal.

[0177] In some embodiments, the upstream biomanufacturing process producing the recombinant protein purified by the methods provided herein comprises a production phase having a packed cell volume of 35% or less, hi some embodiments, the packed cell volume is 30% or less.

[0178] Key attributes and performance indicators of a recombinant protein of interest can be measured to better inform decisions regarding the performance of each step during production. These key attributes and performance indicators can be monitored in real time, near real time, and / or offline. Key parameters that can be measured during cell culture can include levels of consumed cell culture medium components (e.g., glucose), accumulating metabolic by-products (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content. Additionally, key attributes such as specific productivity, viable cell density, packed cell volume, pH, osmolality, flocculation, percent yield, and titer can be monitored during appropriate stages in the manufacturing process. Monitoring and measurements can be performed using known techniques and commercially available equipment.

[0179] bioreactor In some embodiments, the growth and / or production phases of the upstream process used to produce the recombinant protein are carried out in a bioreactor. Conditions within the bioreactor to support cell culture. Culture conditions suitable for mammalian cells are known in the art, as described above. A bioreactor "run" typically involves inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth and / or production phases until one or more predetermined parameters (e.g., time, viable cell density, packed cell volume) are met, and then harvesting the contents of the bioreactor.

[0180] In some embodiments, one or more bioreactors used to produce recombinant proteins are stainless steel bioreactors, such as integrated, large-scale stainless steel bioreactors capable of operating at volumes of about 2,000 liters to about 50,000 liters (e.g., about 2,000 to about 20,000 liters) or greater.

[0181] In some embodiments, one or more bioreactors for recombinant protein production are disposable bioreactors. The use of disposable technology minimizes infrastructure requirements associated with traditional cell culture, such as steel / glass commercial-scale vessels and related equipment. Disposable bioreactors provide flexibility to the manufacturing process; site assembly, reconstitution, sterilization, and validation of disposable bioreactors can be faster, easier, and less costly than traditional, self-contained stainless steel cell culture plants. Disposable bioreactors include a disposable plastic sterile bag supported by a non-disposable support structure. The culture is agitated within the bag by agitation or rocking, and air and oxygen spargers and sensors are also provided to measure and adjust various parameters of the culture (e.g., pH, temperature, oxygen, cell density, etc.). Disposable bioreactors are commercially available, for example, BioSTR®, Sartorius, Goettingen Germany; MOBIUS®, Millipore, Burlington, MA; XCELLEREX®, Cytiva, Marlborough, MA.

[0182] The bioreactor volume is divided into a working volume space and a headspace. The working volume of a bioreactor refers to the volume within the bioreactor in which the cell culture is operated and is typically expressed as a percentage of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 70% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 70% to about 100% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 75% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 80% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 85% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 90% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 91% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 92% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 93% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 94% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 95% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 96% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 97% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 98% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 99% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is about 100% of the bioreactor volume.

[0183] Additional recovery and purification processes The expressed recombinant protein may be secreted into the culture medium from which it can be recovered and / or collected. Some biomanufacturing processes incorporating the disclosed anion exchange chromatography operations may also include a recovery operation. The recovery operation completely or partially clarifies and / or purifies the target protein from at least one impurity found therewith in the cell culture medium, such as residual cell culture medium, cells, cell debris, or medium components, and / or other product-related and / or process-related impurities.

[0184] Methods for recovering recombinant proteins from suspension cell cultures are known in the art and include, but are not limited to, accelerated sedimentation methods such as acid precipitation, flocculation, gravity separation, centrifugation, sonication, filtration (including membrane filtration, ultrafiltration, microfiltration, tangential flow, alternating tangential flow, depth filters, and alluvial filtration filters).

[0185] The harvested cell culture fluid (HCCF) may be adapted to provide feed to the chromatography column skid and stored in a surge tank, holding tank, bag, or other container appropriate to infrastructure and / or process requirements.

[0186] Recovery operations may be combined with additional recovery strategies, including centrifugation, such as disc stack centrifugation or continuous solids discharge centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration, and depth filtration; sedimentation / sedimentation methods, such as flocculation; and chromatographic media-based separations.

[0187] Besides anion exchange chromatography procedures using anion exchange chromatography materials comprising primary amine ligands, the present disclosure encompasses methods involving all known purification techniques, such as, for example, Protein A purification of immunoglobulins and immunoglobulin-like biologics, and chromatography-based separation and polishing steps including columns and alternative modes of chromatographic separation by ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, size exclusion chromatography (SEC), gel filtration, or other known forms of chromatographic separation of biological and / or biochemical substances.

[0188] In some embodiments, recombinant protein recovered from host cells or cell culture medium may be further purified or partially purified by one or more unit operations to remove cell culture medium components, host cell proteins, or nucleic acids, or other process- or product-related impurities. One skilled in the art can select appropriate unit operation(s) for further purifying the recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of the host cells in which the recombinant protein is expressed, and the composition of the culture medium in which the host cells are grown. Illustratively, in some embodiments, recombinant protein is purified from the harvest permeate by one or more of flocculation, sedimentation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0189] Capture unit operations can include capture chromatography, such as affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like, which utilize resins and / or membranes containing agents that bind to the recombinant protein of interest. Such chromatography materials are known in the art and commercially available. For example, if the recombinant protein is an antibody or contains an antibody-derived component (e.g., an Fc domain), affinity chromatography using ligands such as Protein A, Protein G, Protein A / G, or Protein L can be used as a capture chromatography unit operation to further purify the recombinant protein. In other embodiments, the recombinant protein of interest can contain a polyhistidine tag at its amino or carboxyl terminus and can subsequently be purified using IMAC. Recombinant proteins can be engineered to contain other purification tags, such as a FLAG® tag or a c-myc epitope, and then purified by affinity chromatography using specific antibodies against such tags or epitopes.

[0190] Unit operations aimed at inactivating, reducing, and / or eliminating viral contaminants may include steps to mitigate viral risks by manipulating the environment and / or using filtration. Viral mitigation measures are important to ensure the safety of protein therapeutics and may be implemented one or more times throughout downstream purification. Viral contaminants can arise from a variety of sources, including the use of animal-derived reagents, adventitious viral contaminants in host cell lines, or system failures in GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped viruses. With enveloped viruses, the envelope allows the virus to identify, bind, invade, and infect target host cells. Therefore, enveloped viruses are more susceptible to inactivation methods. Various methods can be employed to inactivate viruses, including heat inactivation / pasteurization, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, chemical inactivators, the addition of surfactants, and solvent / detergent treatments. Surfactants, such as detergents, can be very effective at solubilizing membranes and specifically inactivating enveloped viruses. Additional unit operations to inactivate, reduce, and / or eliminate viral contaminants may include filtration processes and / or adjustments to solution conditions. One method for achieving viral inactivation is incubation at low pH (e.g., pH <4). A low pH viral inactivation operation may be followed by a neutralization unit operation to readjust the virally inactivated solution to a pH more compatible with the requirements of subsequent unit operations. A low pH viral inactivation operation may be followed by further filtration, such as depth filtration, to remove any turbidity or precipitate that may result. Adjustments to temperature or chemical composition (e.g., the use of detergents) may also be used to achieve viral inactivation. Virus filtration may be performed using microfilters or nanofilters, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0191] Non-enveloped viruses are less susceptible to inactivation methods that maintain product stability. Therefore, non-enveloped viruses are typically removed by filtration. Exemplary processes are described in International Publication No. WO 2020 / 159838. Virus filtration can be performed using microfilters or nanofilters, such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NY), and CUNO Zeta Plus VR (3M, St. Paul, MN).

[0192] Viral filtration can occur at one or more steps in downstream operations of a biomanufacturing process. Typically, viral inactivation follows an affinity chromatography unit operation, and viral filtration precedes or follows an ultrafiltration / diafiltration (UF / DF) operation, but can also occur after UF / DF.

[0193] In all chromatographic processes, multiple filters can be used up to the capacity required to achieve the desired goal of the production process, such as a holder, skid, or the physical configuration of the ultrafiltration / diafiltration (UF / DF) system will allow.

[0194] A variety of chromatographic methods can be utilized in polishing unit operations to purify the protein of interest and remove contaminants and impurities. Polishing chromatography unit operations can utilize agent-containing resins and / or membranes that can be used in either flow-through mode (the protein of interest is contained in the eluent and contaminants and impurities are bound to the chromatographic medium) or bind-and-elute mode (the protein of interest is bound to the chromatographic medium and contaminants and impurities pass through or are washed off the chromatographic medium before being eluted). Examples of such polishing chromatography methods include, but are not limited to, ion exchange chromatography (IEX), such as cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and size-exclusion chromatography (e.g., gel filtration).

[0195] The purified recombinant protein may be formulated for the end user, i.e., buffer exchanged, sterilized, bulk packaged, and / or packaged. Illustratively, product concentration and buffer exchange of the recombinant protein of interest into a desired formulation buffer for bulk storage of a drug substance or drug product may be accomplished by ultrafiltration and / or diafiltration. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.

[0196] The UF / DF operation can be performed at one or more stages in a downstream process. Typically, the UF / DF operation is performed before bulk storage of the drug substance. Instead of storage, unit operations related to fill / finish of the drug product can also immediately follow the UF / DF operation. One or more stability-enhancing excipients can optionally be added directly to the UF / DF retentate feed tank containing the formulated purified protein, which results in the formulated drug substance, or to the UF / DF eluate pool. An exemplary UF / DF process is described in WO 2020 / 159838. Filters for use in UF / DF operations are well known in the art and are commercially available from a number of sources. There are many types of materials available, such as regenerated cellulose, Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), and polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY).

[0197] Recombinant proteins Any type of recombinant protein, including proteins containing a single polypeptide chain or multiple polypeptide chains, can be purified according to the methods of the present disclosure. Such recombinant proteins include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Exemplary recombinant proteins can include, but are not limited to, cytokines, growth factors, hormones, mutant proteins, fusion proteins, antibodies, antibody fragments, peptibodies, T-cell engaging molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0198] In other embodiments, the recombinant protein purified according to the methods of the present disclosure is an antigen-binding protein, including, but not limited to, antibodies, peptibodies, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFvs), two-chain (bivalent) scFvs, and IgGscFvs (see, e.g., Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), hetero-IgGs (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Additional antigen binding proteins include, but are not limited to, bispecific T cell engagers (BiTEs®), e.g., bispecific T cell engagers with extended, e.g., extended half-life, such as HLE BiTE molecules, HeteroIg BITE molecules, and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).

[0199] In some embodiments, the antigen binding protein binds to one or more of the following, alone or in any combination: CD proteins, such as 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, such as HER2, HER3, HER4, and EGF receptor, such as EGFRvIII; cell adhesion molecules, such as LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin; growth factors, such as but not limited to, vascular endothelial growth factor ("VEGF"); VEGFR2; growth hormone, thyroid stimulating hormone, follicle stimulating hormone, corpus luteum morphogenic hormones, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), especially TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5), insulin-like growth factors I and II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors, insulin and insulin-related proteins, such as, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding protein;(such as coagulation and coagulation-related proteins, e.g., factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, and thrombopoietin receptors, among others); colony-stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF, among others; other blood and serum proteins, such as, but not limited to, albumin, IgE, and blood group antigens; receptors and receptor-associated proteins, e.g., flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor neurotrophic factors, such as, 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, such as interferon-alpha, -beta, and -gamma, interleukins (IL), such as 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 its receptor, IL-13RA2, or IL-17 receptor, IL-1RAP;Viral antigens, such as, but not limited to, AIDS envelope viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (expressed and secreted primarily by T cells upon activation), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin, and activin, integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBPs 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, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligands, i.e., PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelial growth factor (MEGF), and IL-1. Phospho-dsFv[PE38] conjugate, Legionella pneumophila (lly), IFN-gamma, interferon-gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet-specific glycoprotein IIb / IIIb (PAC-1)), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), secretor stimulator, and biologically active fragments or variants of any of the foregoing;

[0200] In other embodiments, the recombinant protein purified according to the methods of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody.

[0201] In some embodiments, the antibody is selected from abrilumab, brazicumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplasmab, nemolizumab, ontamalimab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, taforecimab, tanezumab, tezepelumab, tremelimumab, utomilumab, and boragidemab. In some embodiments, the antibody is selected from denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody is erenumab. In some embodiments, the antibody is evolocumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is tezepelumab.

[0202] In some embodiments, the antibody is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the antibody is a human IgG1, IgG2, or IgG4 antibody.

[0203] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0204] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0205] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody. [Example]

[0206] In order that this disclosure may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the disclosure in any manner.

[0207] Example 1: AEX Chromatography Step Using TOYOPEARL® NH2-750F A composition containing one of two recombinant monoclonal antibodies, mAb1 or mAb2, was further polished using an AEX resin consisting of TOYOPEARL® NH2-750F (Tosoh Bioscience), followed by affinity chromatography, low-pH viral inactivation, and CEX chromatography. TOYOPEARL® NH2-750F is composed of polymethacrylate beads functionalized with proprietary primary amine (NH2) strong anion exchange groups and is commercially available with a 45 μm particle size (F grade). The operating pH for the AEX step was 7.0-8.0, and the operating conductivity was less than 10 mS / cm. The AEX column was loaded at a loading density of 250 g / L to 600 g / L of resin.

[0208] Figure 1A shows the removal capacity of this AEX step for total HMW species of mAb1 by comparing HMW levels (assessed by SE-HPLC) in the load and pool across seven pilot-scale lots. The observed reduction of at least 0.5% HMW across all three lots demonstrates the robustness of the AEX step, even at high loads exceeding 500 g / L of resin. Significant reductions in process-related impurities, including host cell proteins, DNA, and model viruses, have been observed in pilot-scale operations as well as bench-scale challenge studies.

[0209] FIG. 1B shows the process yield for the AEX process for the pilot scale lot of FIG. 1A, demonstrating the process's ability to achieve high yields while significantly reducing impurities.

[0210] Similar high yields and significant impurity reductions were observed for mAb2. Figure 2A shows the high molecular weight clearance for mAb2 in two pilot-scale lots, and Figure 2B shows the process yield for the AEX step for the pilot-scale lot in Figure 2A.

[0211] Example 2: Low pH virus inactivation using formic acid In certain equipment-constrained manufacturing sites, it is desirable to minimize the volume of the Protein A pool and subsequent pool titration to ensure that the volume fits within the vessel limits with a robust safety margin. As shown in Table 1, for evaluation of the VI unit operation for mAb1 using a low-pH incubation time of 60–90 minutes at 15–25°C, the use of 1 M formic acid as an acid titrant resulted in a lower acid volume to achieve the pH required for viral inactivation, a lower base volume required to neutralize the VI pool to pH 5.0, and a lower net volume expansion for the viral inactivation step compared to the use of 10% acetic acid. This is particularly advantageous when the ability to reduce the volume of the Protein A elution pool is limited, for example, when operating at high column loads.

[0212] [Table 1]

[0213] Figure 3 demonstrates the benefit of lower loading conductivity (due to the selection of a VI acidifying titrant) as observed for a mAb1 AEX unit operation substantially similar to that described in Example 1. High molecular weight (HMW, as detected by SE-HPLC assay for mAb1) is often an important quality attribute for mAbs such as mAb1, and HMW impurity levels are generally reduced by polishing chromatography steps such as AEX. Figure 3 shows improved AEX step performance resulting from the use of 1 M formic acid VI titrant (PSL2), which resulted in a lower percentage of HMW in the AEX pool, compared with the use of 10% acetic acid VI titrant (PSL1) under similar conditions, which showed minimal reduction in HMW species for mAb1. Although the lower loading conductivity reduced the process yield as a result of product retention on the resin (93% for PSL2 vs. 98% for PSL1), the AEX process yield was still acceptable when 1 M formic acid was used as the VI titrant.

[0214] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and journal articles, are hereby expressly incorporated by reference. Anything described in an embodiment of the present disclosure can be combined with one or more other embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0215] The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, but instead as non-limiting exemplifications of particular aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. Indeed, various modifications of the disclosed subject matter in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and accompanying figures. Such modifications are intended to be within the scope of the disclosed subject matter.

[0216] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for purposes of illustration and are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has been selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the marketplace, and / or to enable those skilled in the art to understand the disclosed subject matter.

Claims

1. 1. A method for purifying a recombinant protein from a composition comprising said recombinant protein and at least one impurity, said method comprising: loading the composition onto an anion exchange material comprising primary amine ligands at a loading density of greater than about 100 g / L of anion exchange material; the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and loading, wherein the at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and recovering a purified composition comprising said recombinant protein.

2. 10. The method of claim 1, wherein the anion exchange material comprises resin particles, and at least about 80% of the resin particles have a particle size of from about 30 μm to about 60 μm.

3. 3. The method of claim 1 or 2, wherein the anion exchange material comprises a polyamine ligand.

4. The method of any one of claims 1 to 3, wherein the loading density is less than about 600 g / L of anion exchange material.

5. 4. The method of any one of claims 1 to 3, wherein the loading density is from about 250 g / L of resin to about 600 g / L of resin.

6. The method of any one of claims 1 to 5, wherein the composition has a conductivity of from about 3 mS / cm to about 6 mS / cm.

7. the method comprising using an equilibration buffer and / or a recovery buffer with the anion exchange material; the pH of the equilibration buffer and / or the recovery buffer is from about 7.0 to about 8.0; and / or The method according to any one of claims 1 to 6, wherein the conductivity of the equilibration buffer and / or the recovery buffer is less than about 10 mS / cm.

8. 8. The method of claim 7, wherein the conductivity of the equilibration buffer and / or the recovery buffer is from about 2 mS / cm to about 4 mS / cm.

9. 9. The method of any one of claims 1 to 8, further comprising performing a low pH viral inactivation unit operation in one or more unit operations prior to said loading.

10. 10. The method of claim 9, wherein the low pH viral inactivation unit operation uses an acid titrant comprising formic acid.

11. 11. The method of any one of claims 1 to 10, further comprising performing one or more additional chromatographic unit operations.

12. 12. The method of claim 11 , wherein the one or more additional chromatography unit operations comprises an affinity chromatography unit operation performed prior to the loading.

13. 13. The method of claim 12, wherein the affinity chromatography unit operation is selected from Protein A chromatography, Protein G chromatography, Protein L chromatography, and CH1 domain chromatography.

14. 14. The method of any one of claims 11 to 13, wherein the one or more additional chromatography unit operations comprises an additional polishing chromatography unit operation.

15. 15. The method of claim 14, wherein the additional polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography.

16. 16. The method of any one of claims 1 to 15, further comprising performing a virus filtration unit operation and / or an ultrafiltration / diafiltration (UF / DF) unit operation after said loading.

17. less than about 2.5% (w / w) of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and / or 17. The method of any one of claims 1 to 16, wherein the purified composition comprises at least about 85% (w / w) of the recombinant protein in the composition prior to the loading.

18. The method of any one of claims 1 to 17, wherein the recombinant protein is an antigen-binding protein.

19. The method of any one of claims 1 to 18, wherein the recombinant protein is an antibody.

20. 20. The method of any one of claims 1 to 19, wherein the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight species of the recombinant protein, fragments of the recombinant protein, cell culture medium components, and viral contaminants.

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