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 using an anion exchange material comprising a primary amine ligand, such as a polyamine ligand (e.g., in flow-through or weak partition chromatography mode).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 490,079, filed March 14, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure provides methods for purifying a recombinant protein from a composition comprising a recombinant protein and at least one impurity, the methods comprising using an anion exchange material comprising a primary amine ligand (e.g., a polyamine ligand). Anion exchange chromatography is performed using NH2-750F (e.g., in flow-through or weak partition chromatography mode). 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). Additionally, in some embodiments, the anion exchange chromatography unit is operated to robustly remove high molecular weight substances with high protein yields (e.g., at least about 85%) under high loading conditions (e.g., greater than about 100 g / L of anion exchange material). Background Technology

[0004] Downstream purification processes for the manufacture of active pharmaceutical ingredients (APIs) used in protein therapeutics (e.g., monoclonal antibodies (mAbs) and antibody constructs) typically include an affinity chromatography step, followed by one or more refining chromatography steps to remove product-related and process-related impurities. For products containing Fc domains, the following is commonly used to capture the protein: a protein A affinity chromatography step, in which cell culture harvest is flowed onto a protein A resin to bind the target recombinant protein, followed by elution with a low-pH buffer to desorb the protein from the protein A resin. Because the protein A pool typically has a low pH (≤ pH 5), subsequent unit operations are typically low-pH virus inactivation (VI) steps, in which the protein A pool is titrated with acid to a known low pH for inactivating enveloped viruses, held for a sufficient time to ensure VI, and then titrated with base to a higher pH suitable for product stability and / or loading to subsequent unit operations. Many downstream purification processes employ a cation exchange (CEX) chromatography step after the VI unit operation because CEX typically requires a relatively low pH to bind positively charged products and process-related impurities to negatively charged CEX resins. Additional purification chromatographic steps (such as anion exchange (AEX) chromatography, hydrophobic interaction chromatography (HIC), and mixed-mode chromatography (MMC)) are typically performed after CEX to further reduce impurities. These purification chromatographic steps can be performed in various chromatographic modes (including binding and elution modes, flow-through modes, and front-loading (i.e., "front-side") modes) depending on process requirements.

[0005] AEX chromatography enables linked processing with downstream steps such as viral filtration (VF) and ultrafiltration / distillation (UF / DF), where VF further reduces the risk of viral contamination and UF / DF buffer exchange and / or concentration of the target product to the desired conditions for pharmaceutical formulation. Furthermore, flow-through and weak partition AEX chromatography typically allows for higher column loading compared to chromatographic operations operating in binding and elution modes, which in turn reduces the column size and buffer consumption required in downstream processes. However, while flow-through AEX chromatography has proven robust in removing process-related impurities such as nucleic acids, host cell proteins, leached protein A ligands, endotoxins, and viruses, achieving removal of high molecular weight (HMW) substances and aggregates can be challenging, especially when using high protein loading to maximize process productivity (Yigzaw et al., Current Pharmaceutical Biotechnology, 2009). Achieving robust HMW removal requires optimization of AEX resin, loading pH, counterion type, and loading dilution, variables constrained by factors such as product stability and facility suitability.

[0006] Therefore, there is a need in the art for new and improved purification methods using AEX chromatography that can robustly remove HMW substances with high protein yields under high loading conditions. Summary of the Invention

[0007] One aspect of this disclosure provides a method for purifying the recombinant protein from a composition comprising the recombinant protein and at least one impurity, the method comprising:

[0008] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density greater than about 100 g / L of anion exchange material, wherein:

[0009] The composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and

[0010] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0011] Collect the purified composition containing the recombinant protein.

[0012] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0013] The composition was loaded onto an anion exchange material containing resin particles at a loading density of about 250 g / L to about 600 g / L, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, wherein:

[0014] 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

[0015] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0016] Collect the purified composition containing the recombinant protein.

[0017] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0018] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0019] 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

[0020] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0021] Collect the purified composition containing the recombinant protein, wherein:

[0022] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0023] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0024] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0025] The composition was loaded onto an anion exchange material containing polyamine ligands at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0026] 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

[0027] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0028] Collect the purified composition containing the recombinant protein, wherein:

[0029] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0030] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0031] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0032] Formic acid was used as the acid titrant for low-pH virus inactivation unit operations.

[0033] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0034] 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).

[0035] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0036] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0037] Collect the purified composition containing the recombinant protein.

[0038] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (selected from high molecular weight substances of recombinant proteins), the method comprising:

[0039] The low-pH virus inactivation unit operation was carried out using approximately 1M to approximately 2M formic acid as the acid titrant.

[0040] The composition was loaded onto an anion exchange material containing polyamine ligands at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0041] 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).

[0042] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0043] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0044] Collect the purified composition containing the recombinant protein.

[0045] Another aspect of this disclosure provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0046] Formic acid was used as the acid titrant for low-pH virus inactivation unit operations.

[0047] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0048] 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; the binding of the at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0049] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0050] Collect the purified composition containing the recombinant protein, wherein:

[0051] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0052] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading. Attached Figure Description

[0053] Figure 1AThe percentages of high molecular weight (HMW) mAb1 in the compositions loaded onto AEX columns in seven pilot-scale batches, as evaluated by SEHPLC, and in the cells recovered from the AEX columns are shown.

[0054] Figure 1B It shows Figure 1A The step yields of the AEX step in seven pilot-scale batches of mAb1 summarized in the paper demonstrate the ability of the AEX step to achieve high yields of more than 85% while providing significant impurity reduction.

[0055] Figure 2A The percentages of high molecular weight (HMW) mAb2 in the compositions loaded onto the AEX column in two pilot-scale batches, as evaluated by SE-UHPLC, and in the cells recovered from the AEX column are shown.

[0056] Figure 2B It shows Figure 2A The step yields of the AEX step in two pilot-scale batches of mAb2 summarized in the paper demonstrate the ability of the AEX step to achieve high yields of more than 85% while providing significant impurity reduction.

[0057] Figure 3 The percentage of high molecular weight molecular weight (HMW) mAb1 in two pilot-scale batches (one using 10% acetic acid as the VI titrant (PSL1) and the other using 1M formic acid as the VI titrant (PSL2)) is shown. Under similar conditions, using 1M formic acid as the VI titrant (PSL2) resulted in a lower percentage of HMW in the AEX cell compared to using 10% acetic acid as the VI titrant (PSL1). Detailed Implementation

[0058] This document discloses a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising anion exchange chromatography (e.g., in flow-through or weak partition chromatography mode) using an anion exchange material comprising a primary amine ligand (e.g., a polyamine ligand) and optionally a polymer matrix containing methacrylate.

[0059] definition:

[0060] The following definitions are provided to aid in understanding the scope of this disclosure. Unless otherwise defined, 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 pertains.

[0061] In some embodiments, when used in conjunction with a measurable numerical variable, “about” refers to the indicated value of the variable and all values ​​of the variable within the experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or within ±10% of the indicated value (whichever is greater). In some embodiments, the numerical range includes numbers (i.e., endpoints) that define the range.

[0062] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless otherwise explicitly stated in the context, to one-tenth of the lower limit unit), and any other stated or intermediate values ​​within the range, are included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges also covered in this disclosure, subject to any restrictions specifically excluded from the range. Where the range includes one or two restrictions, the range excluding any one or both of those included restrictions is also included in this disclosure.

[0063] As used herein, unless otherwise expressly indicated, the term "a (kind)" means "one or more (a and an)". Additionally, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context requires otherwise, singular terms include plural terms, and plural terms include singular terms.

[0064] As used in this article, the term "acid precipitation" refers to a harvesting operation that lowers the pH of a cell culture to induce precipitation of one or more cell culture impurities.

[0065] As used herein, the term "affinity chromatography" (also known as "capture chromatography") refers to a chromatographic operation that separates a biomolecule (e.g., a recombinant protein) from a mixture based on the selective interaction between the biomolecule and another substance (i.e., a ligand). Affinity chromatography is commonly used in biomanufacturing processes to separate and concentrate desired recombinant proteins from harvested cell culture media. In a typical affinity chromatography operation, biomolecules in the mobile phase selectively bind to or otherwise interact with the stationary phase, while the remainder of the mobile phase passes through the chromatographic material. The biomolecule is then eluted from the stationary phase by altering conditions to reduce the affinity between the ligand and the biomolecule. Non-limiting examples of affinity chromatographic materials 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 an affinity for metal ions or are engineered to have an affinity for metal ions.

[0066] In some embodiments, protein A affinity chromatography can be used to capture the target recombinant protein. Protein A ligands exhibit high selectivity for a variety of proteins containing the antibody Fc region and provide 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 and Protein ASepharoseFAST FLOW. TM MABSELECT TM PrismA (Cytiva, Marburg, Massachusetts), PROSEP-A TM (Merck Millipore, UK) HC-650F protein A (TosoHass Co., Philadelphia, PA) and AP Plus (Purolite, King Prussia, PA).

[0067] As used herein, the term "antigen-binding protein" refers to a protein or polypeptide that includes an antigen-binding region or antigen-binding moiety that has an affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include, but are not limited to, modified antigen-binding moieties of antibodies, fusion proteins, VH, VHH, VL, (s)dAb, Fv, light chains (VL-CL), Fd(VH-CH1), heavy chains, Fab, Fab', F(ab')2, or "r IgG" (a "half-antibody" composed of a heavy chain and a light chain) or full-length antibodies, such as triple-chain antibody-like molecules, antibodies with only heavy chains, single-chain variable fragments (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, biantibodies, single-chain biantibodies, tandem biantibodies (Tandab), tandem di-scFv, tandem tri-scFv, and "microantibodies" illustrated by the following structures. ": (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibody antibodies such as triabody or tetrabody, and single-domain antibodies (such as nanobodies or single variable domain antibodies containing only one variable region, which can be VHH, VH or VL, and which binds specifically to the antigen or target independently of other variable regions or domains).

[0068] As used in this article, the term "antibody" generally refers to a tetrameric immunoglobulin containing two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa).

[0069] As used herein, the term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). The immunoglobulin light chain constant domain (CL) may be a human kappa (κ) or human lambda (λ) constant domain.

[0070] As used herein, the term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising 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) from the N-terminus (N-terminus) to the C-terminus (C-terminus). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, which define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies in the IgG and IgA classes are further subdivided into several subclasses: 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). These constant domains of the immunoglobulin heavy chain can originate from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via interpeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of these two antibody heavy chains.

[0071] The variable regions of immunoglobulin chains typically exhibit the same overall structure, comprising relatively conserved framework regions (FRs) linked by three hypervariable regions (more commonly referred to as "complementarity-determining regions" or CDRs). The CDRs from each heavy and light chain pair are usually aligned via the framework regions to form a structure that specifically binds to a particular epitope on the target protein. From the N-terminus to the C-terminus, the naturally occurring light and heavy chain variable regions typically follow this order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been designed to assign numbers to the amino acids occupying positions in each of these domains. This numbering system is defined in the following literature: Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health (NIH), Bethesda, Maryland); or Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDR and FR of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include... (International ImMunoGeneTics Information System; Lefranc et al., Dev. Comp. Immunol. [Developmental and Comparative Immunology] 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. [Journal of Molecular Biology] 309(3):657-670; 2001).

[0072] Antibody digestion with papain produces two identical antigen-binding proteins (called "Fab" fragments, each with a single antigen-binding site) and a residual "Fc" fragment containing all domains except the first domain of the immunoglobulin heavy chain constant region. The Fab fragment contains variable domains from both 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. Therefore, the "Fab fragment" consists of an immunoglobulin light chain (the light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of the immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH and CH1 domains from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0073] As used herein, an “Fc fragment” or “Fc region” of an immunoglobulin typically comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally includes a CH4 domain. The Fc region may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulins. 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 functions.

[0074] As used herein, the term “F(ab')2 fragment” refers to a divalent fragment comprising two Fab' fragments connected by a disulfide bridge between the heavy chains in the hinge region.

[0075] As used herein, the term "Fv" fragment refers to the smallest fragment containing a complete antigen recognition and binding site from an antibody. This fragment consists of a dimer of an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) in tight, non-covalently associated form. It is in this conformation that the three CDRs of each variable region interact to confine the antigen binding site to the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of an Fv fragment containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, although its affinity is lower than that of the entire binding site containing both VH and VL.

[0076] As used herein, the term “single-chain variable fragment” or “scFv fragment” includes the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain, and optionally includes a peptide linker between the VH and VL regions that enables the Fv to form the desired structure for antigen binding (see, for example, Bird et al., Science, Vol. 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85: 5879-5883, 1988).

[0077] As used herein, “nanobody” refers to the variable region of the heavy chain in a heavy chain antibody. This 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 the light chain are naturally found in certain animal species, such as nurse sharks, wobbegong sharks, and camelids, including camels, dromedary camels, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, the VHH domain. These antibodies form the antigen-binding region using only the variable region of the heavy chain; that is, these functional antibodies are heavy chain homodimers possessing only the H2L2 structure (referred to as “heavy chain antibodies” or “HCAbs”). Camel-derived VHHs have been reported to recombine with constant regions of IgG2 and IgG3, which contain hinge, CH2, and CH3 domains but lack the CH1 domain. Camelized VHH domains have been found to bind to antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276: 26285-90, 2001) and exhibit 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 Publications 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be fabricated from human variable domains that more closely match those of shark V-NAR scaffolds and can provide a framework for long penetrating loop structures.

[0078] As used herein, the term "heavy-chain-only antibody" refers to an immunoglobulin protein composed of two heavy-chain polypeptides (e.g., heavy-chain polypeptides, each approximately 50-70 kDa). "Heavy-chain-only antibodies" lack the two light-chain polypeptides found in conventional antibodies. Heavy-chain antibodies account for approximately one-quarter of IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C. et al., Nature. [Nature] 363, 446-448 (1993)). These molecules are formed by two heavy chains but lack light chains. Therefore, the variable antigen-binding moiety is called the VHH domain, and it represents the smallest naturally occurring, complete antigen-binding site, only about 120 amino acids in length (Desmyter, A. et al., J. Biol. Chem. [Journal of Biochemistry] 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity for a variety of antigens can be generated through immunization (van der Linden, RH et al. Biochim. Biophys. Acta. [Chinese Journal of Biochemistry and Biophysics] 1431, 3746 (1999)), and the VHH moiety can be easily cloned and expressed in yeast (Frenken, LGJ et al. J. Biotechnol. [Journal of Biotechnology] 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of the classic F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. [Committee of the Federation of European Biochemical Societies Letters] 414, 521-526 (1997)). Sharks have also been shown to possess 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)).

[0079] In some embodiments, a "heavy-chain-only antibody" is a dimeric antibody comprising a VH antigen-binding domain and constant CH2 and CH3 domains but lacking a CH1 domain. In some embodiments, a heavy-chain-only antibody comprises a variable region antigen-binding domain comprising frames 1, CDR1, 2, CDR2, 3, CDR3, and 4. In some embodiments, a heavy-chain-only antibody comprises 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 comprises an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. This document also includes heavy-chain-only antibodies with truncated CH2 and / or CH3 domains. The heavy-chain-only antibodies described herein may belong to the IgG subclass, but heavy-chain-only antibodies belonging to other subclasses such as IgM, IgA, IgD, and IgE are also included herein. In some embodiments, the heavy-chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtypes, such as IgG1 or IgG4 subtypes. In some embodiments, the heavy-chain-only antibody is an IgG1 or IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is an IgG4 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is an IgG1 subtype, wherein one or more CH domains are modified to alter the effector function of the antibody. Modifications of the CH domains that 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.

[0080] As used herein, the term "triple-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising or consisting of three polypeptide subunits, wherein two polypeptide subunits comprise a heavy chain and a light chain of a monoclonal antibody, or an antigen-binding fragment of such antibody chain comprising an antigen-binding region and at least one CH domain, comprising or consisting of such subunits. This heavy chain / light chain pair is specific for binding to a first antigen. The third polypeptide subunit comprises or consists of an antibody consisting only of the heavy chain and one or more antigen-binding domains (e.g., two antigen-binding domains) binding to epitopes of a second antigen or different epitopes of a first antigen, comprising or consisting of such subunits. The heavy-chain-only antibody comprises an Fc portion containing CH2 and / or CH3 and / or CH4 domains but lacking a CH1 domain, wherein such binding domains are derived from or have sequence identity with a variable region of the antibody heavy or light chain. A portion of such a variable region may be derived from a V Hand / or V L Gene fragments, D and J H Gene fragments or J L Gene segments encode. Variable regions can be composed of rearranged V... H DJ H V L DJ H V H J L or V L J L Gene segment encoding.

[0081] As used herein, the term "bioreactor" means any container useful for the growth of cell cultures (e.g., mammalian cell cultures or bacterial cell cultures). "Bioreactor" herein encompasses the term "fermenter" (i.e., a container useful for the growth of bacterial cell cultures that typically includes a more robust agitator and increased gas flow rate compared to containers used for the growth of mammalian cell cultures). Non-limiting examples of bioreactors include stirred tanks, airlifts, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or sputtered bed bioreactors. In some embodiments, the example bioreactor may perform one or more (e.g., one, two, three, all) of the following steps: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium (e.g., perfusion of fresh cell culture medium and removal of used cell culture medium), separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Unless the context otherwise indicates, the bioreactor may be suitable for batch, semi-feed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable bioreactor diameter may be used. Unless the context otherwise indicates, in some embodiments, the bioreactor may have a volume between 100 mL and 50,000 L. Unless otherwise indicated, the bioreactor may be of any size, as long as it is useful for cell culture; typically, the size of the bioreactor is suitable for the volume of cell cultures grown within it. In non-limiting embodiments, unless the context otherwise indicates, the bioreactor may be at least 1 liter (L), or may be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 L, 20,000 L, or larger, or any volume in between. Internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen concentration, and temperature, can be controlled during culture. Those skilled in the art will recognize and be able to select a suitable bioreactor for the manufacturing methods disclosed herein based on relevant considerations.

[0082] As used herein, the term "cell culture" or "culture" refers to the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian and bacterial cells are known in the art. (See, for example, *Animal Cell Culture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992).) Mammalian cells can be cultured in suspension or attached to a solid substrate. In some embodiments, fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, shake flasks, and / or stirred tank bioreactors (with or without microcarriers) can be used for cell culture. In some embodiments, bioreactors of 500 L to 2000 L are used for cell culture (e.g., as part of a seed culture). In some embodiments, bioreactors of 1000 L to 2000 L are used for cell culture (e.g., as part of a seed culture).

[0083] As used herein, the term "cell culture medium" (also known as "media / 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 typically provide one or more of the following components: energy (e.g., in the form of carbohydrates, such as glucose); one or more essential amino acids (e.g., all essential amino acids; twenty basic amino acids plus cysteine); vitamins and / or other organic compounds typically required in low concentrations; lipids or free fatty acids; and trace elements typically required in extremely low concentrations (e.g., in the micromolar range), such as inorganic compounds or naturally occurring elements. As used herein, cell culture medium encompasses nutrient solutions typically used and / or known to be used with any cell culture process, including but not limited to batch, extended batch, fed batch, intensified and / or perfusion or continuous cell culture.

[0084] 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 standard viability assays (e.g., trypan blue exclusion assay). As used herein, the term “cell heap volume” (PCV), also known as “percentage of cell heap volume” (%PCV), is 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. [Biotechnology and Bioengineering] Dec 20:95(6):1228-33). Cell heap volume is a function of cell density and cell diameter; an increase in cell heap volume can be caused by an increase in cell density or cell diameter or both. Cell heap volume is a measure of the solids content in a cell culture. Because host cells vary in size and cell cultures also contain dead and dying cells as well as other cell debris, cell heap volume provides a more accurate description of the solids content in a cell culture.

[0085] As used herein, the term “connected” in relation to unit operations refers to a direct connection or mechanism that allows continuous flow between one or more unit operations in a single operation cycle.

[0086] As used herein, the term "continuous" in relation to unit operations refers to a direct connection or mechanism that allows for continuous flow between one or more unit operations across multiple operating cycles.

[0087] As used herein, the term “dynamic binding capacity” for chromatographic materials refers to the amount of product (e.g., peptide) that the material will bind under actual flow conditions before a significant breakthrough occurs in the binding of the product.

[0088] As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing the desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a specific host cell (e.g., a mammalian host cell). Vectors may include viral vectors, non-attachment mammalian vectors, plasmids, and other non-viral vectors. Expression vectors may include sequences that affect or control transcription, translation, and, if introns, affect the splicing of coding regions to which they are operably linked. "Operably linked" means that the components to which the term applies are in a relationship that allows them to perform their inherent functions. For example, the arrangement of control sequences (e.g., promoters) in a vector that is "operably linked" to a protein-coding sequence such that normal activity of the control sequence leads to transcription of the protein-coding sequence, resulting in recombinant expression of the encoded protein.

[0089] As used herein, “feed-batch culture” refers to a form of suspension culture, and more particularly, a method of culturing cells, in which additional components are provided to the culture at one or more times after the start of the culture process. The provided components typically include nutrient supplements that have been depleted for the cells during the culture process. Alternatively or additionally, the additional components may include supplemental components (e.g., cell cycle inhibitory compounds). In some embodiments, the fed-batch cell culture medium formulation may be richer or more concentrated than the basal cell culture medium formulation, which contains the components necessary for cell survival and growth and is typically used to initiate cell culture. The fed-batch culture may be stopped at a certain point in time, and the cells and / or components in the culture medium may be harvested and optionally purified.

[0090] As used herein, a "fusion protein" is a protein containing at least one polypeptide fused to or linked to a heterologous polypeptide. Typically, fusion proteins are expressed from a fusion gene, wherein the nucleotide sequence encoding a polypeptide sequence from one protein is appended to a read frame along with the nucleotide sequence encoding the polypeptide sequence, and optionally separated from the nucleotide sequence encoding a polypeptide sequence from a different protein by a linker. The fusion gene can then be expressed in a recombinant host cell to produce the fusion protein. Fusion proteins may contain fragments from immunoglobulins, such as Fc regions, fused to or linked to ligand polypeptides, receptor polypeptides, hormones, cytokines, growth factors, enzymes, or other polypeptides that are not components of immunoglobulins.

[0091] As used in this article, the “growth phase” of a cell culture refers to the period during which cells grow exponentially (i.e., the logarithmic phase), during which cells typically divide rapidly.

[0092] As used herein, the term "harvested cell culture medium" refers to a solution that has been treated with one or more methods to separate cells, cell debris, or other large particles from recombinant proteins. Such methods, as described herein, include, but are not limited to, cooling, flocculation, acidification, centrifugation, neutralization, sonic separation, and various forms of filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration). Harvested cell culture medium includes cell culture lysates and cell culture supernatants. The harvested cell culture medium may be further clarified by filtration with a membrane having a pore size of about 0.1 μm to about 0.5 μm, such as a membrane with a pore size of about 0.22 μm, to remove fine particulate matter and soluble aggregates.

[0093] As used herein, “host cell” refers to a cell that has been or is capable of being transformed with nucleic acids and thereby expresses the target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, provided the target gene is present. A host cell containing nucleic acids encoding a recombinant protein, for example, operatively linked to at least one expression control sequence (e.g., a promoter or enhancer). When cultured under appropriate conditions, the host cell can 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 host cell that produced it (if it is not secreted).

[0094] As used in this article, the term "high molecular weight" or "HMW" for the target recombinant protein refers to the dimer, oligomer, and aggregate of the recombinant protein, whose molecular weight is greater than that of the complete, fully assembled form of the recombinant protein.

[0095] As used herein, the term "impurity" refers to components other than the target recombinant protein and its associated buffer components. Impurities include, but are not limited to, process- and product-related impurities, such as host cell proteins, leached resin materials (e.g., leached protein A), nucleic acids, HMW substances of the recombinant protein, LMW substances of the recombinant protein, endotoxins, viral contaminants, cell culture medium components, etc.

[0096] As used herein, the term “load density” refers to the amount of composition in contact with a given volume of chromatographic material.

[0097] As used herein, the term "low molecular weight" or "LMW" species of a target recombinant protein refers to a fragment, truncated form, or other incomplete variant of the recombinant protein whose molecular weight is smaller than that of the complete, fully assembled form of the recombinant protein. LMW substances may include, but are not limited to, proteolytic fragments, truncated forms resulting from cellular expression of mRNA splicing variants, and single-component peptides in the case of multi-chain proteins (e.g., light-chain or heavy-chain substances only when the recombinant protein is an antibody).

[0098] 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 sufficiently intact to support cell culture during the process. In some embodiments, the perfusion cell culture medium formulation may be richer or more concentrated than the basal cell culture medium formulation to accommodate methods used to remove used culture medium. In some embodiments, the perfusion cell culture medium may be used during both the growth and production phases.

[0099] As used herein, the term "purification chromatography" refers to a chromatographic operation performed after capture or affinity chromatography to remove residual impurities and obtain compositions and / or recombinant proteins of higher purity. Common impurities removed in purification steps include, but are not limited to, product-related impurities (e.g., HMW and LMW substances), host cell proteins, DNA, leached protein A, viral contaminants, and endotoxins. Additionally, typical chromatographic techniques used for purification include, but are not limited to, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimode (or mixed-mode) chromatography (MMC).

[0100] As used herein, “anion exchange chromatography” (AEX) refers to a form of ion exchange chromatography performed on a positively charged solid-phase medium (e.g., a resin or membrane) capable of exchanging free anions with anions passing through or across an aqueous solution of the solid phase. For example, AEX chromatography is used for virus removal and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15Q, Capto...). TM Q, Q-SEPHAROSE FASTFLOW TM (Stopfan Company), FRACTOGEL EMD TMAE TM FRACTOGEL EMD DEAE TM (EMD Merck) Super and NH2-750F (Tosoh Bioscience), POROS HQ TM and POROS XQ TM (ThermoFisher).

[0101] As used herein, “cation exchange chromatography” (CEX) refers to a form of ion exchange chromatography performed on a negatively charged solid-phase medium (e.g., a resin or membrane) capable of exchanging free cations with cations passing through or across the solid phase in an aqueous solution. The charge can be provided by attaching one or more charged ligands to the solid phase (e.g., via covalent bonding). Alternatively or additionally, the charge can be an inherent property of the solid phase (e.g., silica, which has a generally 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, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., SPSEPHAROSEFAST FLOW). TM SP-SEPHAROSE FAST FLOW XL TMOr SP-SEPHAROSE HIGH PERFORMANCE TM ,CAPTOS TM CAPTO SPImpRes TM CAPTO S ImpAct TM (Situofan Company), FRACTOGEL-SO3 TM FRACTOGEL-SEHICAP TM and FRACTOPREP TM (EMD Merck, Darmstadt, Germany) XS, HS (Tosoh Biotechnology, Inc., Kingsville, Prussia, Pennsylvania), UNOsphere TM (BioRad, Hercules, California), S Ceramic Hyper TM DF (Pall Corporation, Port Washington, New York), POROS TM (Thermo Fisher Scientific, Waltham, Massachusetts) CSP and CP-FT (Millipore Sigma, Darmstadt, Germany).

[0102] As used herein, “hydrophobic interaction chromatography (HIC)” refers to chromatography performed on a solid-phase medium that utilizes the interaction 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. TM (Stopfan Corporation), Tosohhexyl (Tosoh Biotechnology Corporation) and Capto TM Phenyl (Stopfan Company).

[0103] As used herein, “mixed-mode or multi-mode chromatography” (MMC) refers to chromatography that utilizes more than one type of interaction between the stationary phase and the analyte to achieve separation. MMC differs from single-mode chromatography in that two or more interaction types (e.g., electrostatic, hydrogen bonding, and / or hydrophobic interactions) significantly contribute to the retention of the solute. Commercially available multi-mode chromatographic media include, but are not limited to, Capto. TM Adhere, Capto TMMMCimpress, Capto MMC (Stopfan), PPAHypercel, MEP Hypercell, HEAHypercell (Pall Corporation, Port Washington, New York), Eshmuno HCX (Merck Millipore) and

[0104] MX-Trp-650M (Tosoh Biotechnology Co., Ltd.)

[0105] Purification chromatography unit operations utilize materials (e.g., resins and / or membranes) containing reagents that can operate in multiple modes, including binding-elution and flow-through modes. In binding-elution chromatography, the target biomolecule is typically loaded onto the chromatographic material to maximize the dynamic binding capacity, and then washing and elution conditions are used to maximize the purity of the product in the eluent. In contrast, in flow-through chromatography, the loading conditions allow impurities to bind to the chromatographic material while allowing the target biomolecule to pass through. Flow-through chromatography allows for higher loading densities of many biomolecules compared to binding-elution chromatography.

[0106] Besides the two most common modes, weak partition chromatography, ultraloading chromatography, and frontier chromatography can also be used in purification processes. In weak partition chromatography (isocratic separation method), in addition to the binding of one or more impurities, it also identifies solution conditions (K0) that promote weak binding of biomolecules to the resin. p It is approximately 0.1 to approximately 100, compared to K in flow chromatography. pThe flow pattern can be altered by loading the target biomolecule onto the chromatographic material beyond its dynamic binding capacity (approximately 0.1). In super-loading chromatography, the target biomolecule is loaded onto the chromatographic material beyond its dynamic binding capacity. Additionally, front-line chromatography allows for continuous, high-density feeds (containing the target protein and at least one impurity) into the chromatographic medium. In front-line chromatography, the separation of the target protein from impurities and contaminants is driven by the binding affinity of the components in the feed to the chromatographic medium. The amount of target protein that can be loaded and bound to the chromatographic medium in front-line mode typically depends on the amount of higher-charged impurities / contaminants (e.g., product-related impurities) in the feed. Initially, all components in the feed will bind to the chromatographic medium. The separation of the target product from impurities / contaminants is driven by affinity for the chromatographic medium. When the chromatographic medium reaches saturation binding, those components in the feed with greater affinity for the chromatographic medium (typically product-related impurities, such as HMW substances) will displace proteins with weaker affinity (e.g., the target product), resulting in the separation of the weaker-affinity protein from the chromatographic medium. These proteins exit the column as bands in the load flow through. As loading proceeds, the bound proteins are successively replaced in order of increasing affinity for the chromatographic medium until the column reaches or approaches saturation, where these proteins have a greater affinity than the target protein.

[0107] As used herein, the term “polypeptide” refers to a polymer containing at least 50 amino acids, such as an amino acid polymer containing at least 100 amino acids.

[0108] As used herein, the term "partition factor" or "product partition factor" (K) p () refers to the molar concentration of the product (e.g., recombinant protein) bound to the stationary phase during the chromatographic step divided by the molar concentration of the product in the mobile phase.

[0109] As used herein, “production” cell culture medium refers to a cell culture medium typically used in cell culture during the transition (i.e., the “transition” and / or “product” phase) at the end of exponential growth and the takeover of protein production, and is sufficiently intact to maintain the desired cell density, viability, and / or product titer during this phase. Production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of cell culture.

[0110] As used in this article, the “production phase” of cell culture refers to the period when logarithmic cell growth has ended and recombinant protein production has become dominant.

[0111] 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 cultured in a cell culture.

[0112] As used herein, when used in connection with a composition, the term "purified" refers to a composition in which at least one impurity is present at a lower concentration relative to the composition prior to one or more unit operations. Additionally, "purified" recombinant proteins (e.g., purified antibodies) refer to recombinant proteins whose purity has been increased, such that they exist in a purer form than when they were synthesized and / or amplified in their natural environment and / or initially under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0113] As used herein, the term "titrant" refers to a solution of known concentration added to another solution during titration. "Acid titrant" refers to a titrant with a pH less than approximately 7.

[0114] As used herein, the term "unit operation" refers to a functional step performed as part of a process for purifying a recombinant protein for a specific purpose. Unit operations can be designed to achieve a single or multiple objectives, such as capture, acid precipitation, centrifugation, or chromatographic steps. Unit operations may also include storage or preservation steps between processing steps.

[0115] Non-restrictive instance features

[0116] Without limitation, some example embodiments / features disclosed herein include E1-E47:

[0117] E1. A method for purifying a recombinant protein from a composition comprising a recombinant protein and at least one impurity, the method comprising:

[0118] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density greater than about 100 g / L of anion exchange material, wherein:

[0119] The composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and

[0120] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0121] Collect the purified composition containing the recombinant protein.

[0122] E2. The method according to E1, wherein the anion exchange material comprises resin particles.

[0123] E3. The method according to E1 or E2, wherein the anion exchange material comprises resin particles, wherein at least about 80% of these resin particles have a particle size of about 30 μm to about 60 μm.

[0124] E4. The method according to any one of E1-E3, wherein the anion exchange material comprises resin particles with an average particle size of about 45 μm.

[0125] E5. The method according to any one of E1-E4, wherein the anion exchange material comprises a polyamine ligand.

[0126] E6. The method according to any one of E1-E5, wherein the anion exchange material comprises a polymer matrix containing methacrylate.

[0127] E7. The method according to any one of E1-E6, wherein the anion exchange material is NH2-750F.

[0128] E8. The method according to any one of E1-E7, wherein the loading density is less than about 600 g / L of anion exchange material.

[0129] E9. The method according to any one of E1-E7, wherein the loading density is about 200 g / L to about 600 g / L of anion exchange material.

[0130] E10. The method according to any one of E1-E9, wherein the composition has a conductivity of about 3 mS / cm to about 6 mS / cm.

[0131] E11. The method according to any one of E1-E10, wherein for the recombinant protein, the partition coefficient of the anion exchange material is about 0.1 to about 100.

[0132] E12. The method according to any one of E1-E10, wherein for the recombinant protein, the partition coefficient of the anion exchange material is about 20 to about 40.

[0133] E13. The method according to any one of E1-E12, wherein the method comprises using an equilibration buffer and / or recovery buffer on the anion exchange material, wherein:

[0134] The pH of the equilibration buffer and / or the recovery buffer is approximately 7.0 to approximately 8.0; and / or

[0135] The conductivity of the equilibration buffer and / or the recovery buffer is less than about 10 mS / cm.

[0136] E14. The method according to E13, wherein the conductivity of the equilibration buffer and / or the recovery buffer is about 2 mS / cm to about 4 mS / cm.

[0137] E15. The method according to any one of E1-E14, further comprising performing a low-pH virus inactivation unit operation prior to sample loading (e.g., one or more unit operations prior to sample loading).

[0138] E16. The method according to E15, wherein the low pH virus inactivation unit operates at a pH of about 3.5 to about 3.7.

[0139] E17. The method according to E15 or E16, wherein the low pH virus inactivation unit operation employs an acid titrator.

[0140] E18. The method according to E17, wherein the acid titrant comprises formic acid.

[0141] E19. The method according to E17 or E18, wherein the acid titrant is about 1M to about 2M formic acid (e.g., about 1M formic acid; about 2M formic acid).

[0142] E20. The method according to any one of E15-E19, wherein the low pH virus inactivation unit operation is performed for at least about 60 minutes.

[0143] E21. The method according to any one of E15-E20, wherein the low pH virus inactivation unit operates for about 60 minutes to about 12 hours.

[0144] E22. The method according to any one of E1-E21, further comprising performing one or more additional chromatographic unit operations.

[0145] E23. The method according to E22, wherein the one or more additional chromatographic unit operations include an affinity chromatographic unit operation prior to sample loading.

[0146] E24. The method according to E23, wherein the affinity chromatography unit operation is selected from protein A chromatography, protein G chromatography, protein L chromatography, and CH1 domain chromatography.

[0147] E25. The method according to E23 or E24, wherein the affinity chromatography unit operation is protein A chromatography.

[0148] E26. The method according to any one of E22-E25, wherein the one or more additional chromatographic unit operations include additional purification chromatographic unit operations performed prior to sample loading.

[0149] E27. The method according to any one of E22-E25, wherein the one or more additional chromatographic unit operations include additional purification chromatographic unit operations performed after sample loading.

[0150] E28. The method according to E26 or E27, wherein the additional refining chromatographic unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography.

[0151] E29. The method according to any one of E26-E28, wherein the additional purification chromatographic unit operation and the sample loading are continuous or connected.

[0152] E30. The method according to any one of E1-E29, further comprising performing a virus filtering unit operation and / or a UF / DF unit operation after sample loading.

[0153] E31. The method according to E30, wherein the loading and the virus filtering unit operation and / or the UF / DF unit operation are sequential or connected.

[0154] E32. The method according to any one of E1-E31, wherein less than about 5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein.

[0155] E33. The method according to any one of E1-E32, wherein less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein.

[0156] E34. The method according to any one of E1-E33, wherein less than about 1% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein.

[0157] E35. The method according to any one of E1-E34, wherein the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0158] E36. The method according to any one of E1-E35, wherein the purified composition contains at least about 90% w / w of the recombinant protein in the composition prior to loading.

[0159] E37. The method according to any one of E1-E36, wherein:

[0160] The purified composition contains less than about 1% w / w of the recombinant protein, which is a high molecular weight component of the recombinant protein; and

[0161] The purified composition contains at least about 90% w / w of the recombinant protein in the composition prior to loading.

[0162] E38. The method according to any one of E1-E37, wherein:

[0163] The loading density is approximately 100 g / L to approximately 600 g / L of anion exchange material;

[0164] The purified composition contains less than about 1% w / w of the recombinant protein, which is a high molecular weight component of the recombinant protein; and

[0165] The purified composition contains at least about 90% w / w of the recombinant protein in the composition prior to loading.

[0166] E39. The method according to any one of E1-E38, wherein the recombinant protein is an antigen-binding protein.

[0167] E40. The method according to any one of E1-E39, wherein the recombinant protein is an antibody.

[0168] E41. The method according to any one of E1-E40, wherein the at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight substances of the recombinant protein, fragments of the recombinant protein, cell culture medium components, and viral contaminants.

[0169] E42. The method according to any one of E1-E41, wherein the at least one impurity is selected from high molecular weight substances of the recombinant protein.

[0170] E43. The method according to any one of E1-E42, wherein the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with primary amine ligands.

[0171] E44. The method according to any one of E1-E42, wherein the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand.

[0172] E45. The method according to E43 or E44, wherein at least about 80% of these resin particles have a particle size of about 30 μm to about 60 μm.

[0173] E46. The method according to any one of E43, E44 or E45, wherein the resin particles have an average particle size of about 40 μm to about 50 μm.

[0174] E47. The method according to E46, wherein the resin particles have an average particle size of about 45 μm.

[0175] Anion exchange chromatography purification method

[0176] This document provides a method for purifying a recombinant protein from a composition comprising a recombinant protein and at least one impurity, the method comprising:

[0177] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density greater than about 100 g / L of anion exchange material, wherein:

[0178] The composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and

[0179] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0180] Collect the purified composition containing the recombinant protein.

[0181] In some embodiments, the anion exchange material comprises resin particles.

[0182] In some embodiments, the anion exchange material comprises resin particles, wherein at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm.

[0183] In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 30 μm to about 60 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 40 μm to about 50 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 30 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 35 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 40 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 45 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 50 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 55 μm. In some embodiments, the anion exchange material comprises resin particles with an average particle size of about 60 μm.

[0184] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0185] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate.

[0186] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate and a polyamine ligand.

[0187] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein 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, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0188] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein 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, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0189] In some embodiments, the anion exchange material is NH2-750F.

[0190] 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. In some embodiments, the loading density is less than about 150 g / L of anion exchange material.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 0.1. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 10. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 20. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 30. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 40. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 50. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 60. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 70. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 80. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 90. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is less than about 100.

[0197] In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 0.1 to about 100. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 10 to about 100. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 100. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 90. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 80. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 70. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 60. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 50. In some embodiments, the partition coefficient of the anion exchange material for recombinant proteins is from about 20 to about 40.

[0198] In some embodiments, the method includes using an equilibration buffer and / or recovery buffer on the anion exchange material, wherein:

[0199] The pH of the equilibration buffer and / or the recovery buffer is approximately 7.0 to approximately 8.0; and / or

[0200] The conductivity of the equilibration buffer and / or the recovery buffer is less than about 10 mS / cm.

[0201] In some embodiments, the equilibration buffer and / or recovery buffer have a pH of about 7.1 to about 7.9. In some embodiments, the equilibration buffer and / or recovery buffer have a pH of about 7.2 to about 7.8. In some embodiments, the equilibration buffer and / or recovery buffer have a pH of about 7.3 to about 7.7. In some embodiments, the equilibration buffer and / or recovery buffer have a pH of about 7.4 to about 7.6. In some embodiments, the equilibration buffer and / or recovery buffer have a pH of about 7.5.

[0202] In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 9 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 8 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 7 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 3 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of less than about 2 mS / cm.

[0203] In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 10 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 9.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 9 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 8.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 8 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 7.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 7 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 6.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 5.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 4.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 3.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 3 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1.5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1 mS / cm.

[0204] In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1 mS / cm to about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1 mS / cm to about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1 mS / cm to about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 1 mS / cm to about 3 mS / cm.

[0205] In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2 mS / cm to about 6 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2 mS / cm to about 5 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2 mS / cm to about 4 mS / cm. In some embodiments, the equilibration buffer and / or recovery buffer have a conductivity of about 2 mS / cm to about 3 mS / cm.

[0206] In some embodiments, the method further includes performing a low-pH virus inactivation unit operation prior to sample loading (e.g., one or more unit operations prior to sample loading). In some embodiments, the low-pH virus inactivation unit operation is performed at a pH of about 3.5 to about 3.7. In some embodiments, the low-pH virus inactivation unit operation is performed at a pH of about 3.5. In some embodiments, the low-pH virus inactivation unit operation is performed at a pH of about 3.6. In some embodiments, the low-pH virus inactivation unit operation is performed at a pH of about 3.7.

[0207] In some embodiments, the low-pH virus inactivation unit operation employs an acid titrant. In some embodiments, the acid titrant is formic acid. In some embodiments, the acid titrant is about 1M to about 2M formic acid. In some embodiments, the acid titrant is about 1M formic acid. In some embodiments, the acid titrant is about 2M formic acid.

[0208] In some embodiments, the low pH virus inactivation unit operation is performed for at least about 60 minutes. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 2 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 3 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 4 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 5 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 6 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 7 hours. In some embodiments, the low pH virus inactivation unit operation is performed for at least about 8 hours. In some embodiments, the low pH virus inactivation unit operation is performed for about 60 minutes to about 12 hours. In some embodiments, the low pH virus inactivation unit operation is performed for about 60 minutes to about 8 hours.

[0209] In some embodiments, the method further includes performing one or more additional chromatographic unit operations.

[0210] In some embodiments, one or more additional chromatographic unit operations include an affinity chromatography unit operation prior to sample 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.

[0211] In some embodiments, one or more additional chromatographic unit operations include additional purification chromatographic unit operations performed prior to sample loading. In some embodiments, one or more additional chromatographic unit operations include additional purification chromatographic unit operations performed after sample loading. In some embodiments, the additional purification chromatographic unit operations and sample loading are sequential. In some embodiments, the additional purification chromatographic unit operations and sample loading are not sequential. In some embodiments, the additional purification chromatographic unit operations and sample loading are sequential.

[0212] In some embodiments, the additional purification chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. In some embodiments, the additional purification chromatography unit operation is cation exchange chromatography. In some embodiments, the additional purification chromatography unit operation is hydrophobic interaction chromatography. In some embodiments, the additional purification chromatography unit operation is mixed-mode chromatography.

[0213] In some embodiments, the method further includes performing virus filtering unit operation and / or UF / DF unit operation after sample loading.

[0214] In some embodiments, the recombinant protein in the purified composition is a high molecular weight substance of recombinant protein at 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 2% w / w, less than about 1.5% w / w, less than about 1% w / w).

[0215] In some embodiments, the purified composition contains at least about 85% w / w (e.g., at least about 90% w / w, at least about 95% w / w) of the recombinant protein in the composition prior to loading.

[0216] In some embodiments, less than about 1% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein; and the purified composition contains 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 of anion exchange material (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); less than about 1% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein; and the purified composition contains at least about 90% w / w of the recombinant protein in the composition prior to loading.

[0217] 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.

[0218] 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.

[0219] In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody.

[0220] In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody.

[0221] In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0222] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant proteins, fragments of recombinant proteins, cell culture medium components, and viral contaminants. In some embodiments, at least one impurity is selected from high molecular weight recombinant proteins.

[0223] This document also 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 form of the recombinant protein), the method comprising:

[0224] The composition was loaded onto an anion exchange material containing resin particles at a loading density of about 250 g / L to about 600 g / L, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, wherein:

[0225] 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

[0226] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0227] Collect the purified composition containing the recombinant protein.

[0228] In some embodiments, at least about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. In some embodiments, the resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0229] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein 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, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0230] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0231] This document also 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 form of the recombinant protein), the method comprising:

[0232] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0233] 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

[0234] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0235] Collect the purified composition containing the recombinant protein, wherein:

[0236] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0237] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0238] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight substances of recombinant proteins, fragments of recombinant proteins, cell culture medium components, and viral contaminants.

[0239] In some embodiments, at least one impurity is selected from high molecular weight substances of recombinant proteins.

[0240] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0241] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate.

[0242] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate and a polyamine ligand.

[0243] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein 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, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0244] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein 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, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0245] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0246] This document further provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (e.g., a high molecular weight form of the recombinant protein), the method comprising:

[0247] The composition was loaded onto an anion exchange material containing polyamine ligands at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0248] 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

[0249] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0250] Collect the purified composition containing the recombinant protein, wherein:

[0251] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0252] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0253] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight substances of recombinant proteins, fragments of recombinant proteins, cell culture medium components, and viral contaminants.

[0254] In some embodiments, at least one impurity is selected from high molecular weight substances of recombinant proteins.

[0255] In some embodiments, the anion exchange material further comprises a polymer matrix containing methacrylate.

[0256] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0257] This document also 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 form of the recombinant protein), the method comprising:

[0258] Formic acid was used as the acid titrant for low-pH virus inactivation unit operations.

[0259] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0260] 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).

[0261] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0262] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0263] Collect the purified composition containing the recombinant protein.

[0264] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight substances of recombinant proteins, fragments of recombinant proteins, cell culture medium components, and viral contaminants.

[0265] In some embodiments, at least one impurity is selected from high molecular weight substances of recombinant proteins.

[0266] In some embodiments, the acid titrant is about 1M to about 2M formic acid. In some embodiments, the acid titrant is about 1M formic acid. In some embodiments, the acid titrant is about 2M formic acid.

[0267] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0268] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate.

[0269] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate and a polyamine ligand.

[0270] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein 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, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0271] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein 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, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0272] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0273] This document further provides a method for purifying a recombinant protein (e.g., an antigen-binding protein, such as an antibody) from a composition comprising a recombinant protein and at least one impurity (selected from high molecular weight substances of recombinant proteins), the method comprising:

[0274] The low-pH virus inactivation unit operation was carried out using approximately 1M to approximately 2M formic acid as the acid titrant.

[0275] The composition was loaded onto an anion exchange material containing polyamine ligands at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0276] 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).

[0277] The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0278] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0279] Collect the purified composition containing the recombinant protein.

[0280] In some embodiments, the acid titrant is about 1M formic acid. In some embodiments, the acid titrant is about 2M formic acid.

[0281] In some embodiments, the anion exchange material further comprises a polymer matrix containing methacrylate.

[0282] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0283] This document also 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 form of the recombinant protein), the method comprising:

[0284] Formic acid was used as the acid titrant for low-pH virus inactivation unit operations.

[0285] The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density of about 250 g / L to about 600 g / L of anion exchange material, wherein:

[0286] 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; the binding of the at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and

[0287] One or more unit operations were performed prior to sample loading to conduct a low-pH virus inactivation unit operation; and

[0288] Collect the purified composition containing the recombinant protein, wherein:

[0289] The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or

[0290] The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0291] In some embodiments, the anion exchange material comprises a polyamine ligand.

[0292] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate.

[0293] In some embodiments, the anion exchange material comprises a polymer matrix containing methacrylate and a polyamine ligand.

[0294] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein 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, wherein these resin particles comprise polymethacrylate and are functionalized with primary amine ligands, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0295] In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein 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, wherein these resin particles comprise a hydroxylated methacrylic acid polymer and are functionalized with a primary amine ligand, and further wherein these resin particles have an average particle size of about 40 μm to about 50 μm (e.g., about 45 μm).

[0296] In some embodiments, less than about 2.5% w / w of the recombinant protein in the purified composition is a high molecular weight form of the recombinant protein. In some embodiments, the purified composition contains 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 form of the recombinant protein, and the purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

[0297] host cells

[0298] 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 value. Cells may be suitable for adherent, monolayer and / or suspension culture, transfection, and expression of recombinant proteins, such as antibodies. Cells can be used for, for example, batch, fed-batch and perfusion, or continuous culture methods. Such cells are typically cell lines obtained from or derived from mammals and are capable of growth and survival when placed in monolayer or suspension cultures containing appropriate nutrients and / or other factors (such as those described herein). Host cells that can express and secrete proteins are typically selected, or host cells that can be molecularly engineered to express and secrete large quantities of a specific protein (more particularly, a target glycoprotein) into the culture medium. The selection of an appropriate host cell for expressing the recombinant protein will depend on a variety of factors, such as the desired expression level, the desired or required peptide modifications (such as glycosylation or phosphorylation) for activity, and ease of folding into a biologically active molecule. In some embodiments, the host cell that produces the recombinant protein to be purified by the methods provided herein is a mammalian host cell.

[0299] Cell lines typically originate from lineages derived from primary cultures and can be maintained indefinitely in culture. These cells may contain expression vectors (constructs), such as plasmids, introduced via transformation, transfection, infection, or injection, containing coding sequences or portions thereof encoding proteins for expression and production during culture. Such expression vectors contain the necessary elements for transcription and translation of the inserted coding sequences. Methods well known and practiced by those skilled in the art can be used to construct expression vectors containing sequences encoding desired proteins and peptides, as well as appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Such techniques are described in J. Sambrook et al., 2012. Molecular Cloning, A Laboratory Molecular Cloning Laboratory Manual 4th edition, Cold Spring Harbor Press, Plainview, NY or any previous edition; FMAusubel et al., 2013. Current Protocols in Molecular Biology (Modern Molecular Biology) method] John Wiley & Sons, New York City, New York State or any previous edition; Kaufman, RJ. Large-scale Mammalian Cell Culture All of these documents are incorporated into this paper for any purpose in 1990.

[0300] Suitable host cells include, but are not limited to, those that can be commercially available from culture collections such as DSMZ (German Microbial Culture Collection of Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0301] Example host cells include, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (e.g., Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella), as well as Bacillus (e.g., Bacillus subtilis and Bacillus licheniformis), Pseudomonas, and Streptomyces. In some embodiments, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable cloning or expression hosts for recombinant peptides. Saccharomyces cerevisiae or common baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly used and can be used in this article, such as *Pichia*, including *Pichia pastoris* and *Schizosaccharomyces pombe*; *Kluyveromyces*, *Yarrowia*; *Candida*; *Trichoderma reesia*; *Neurospora crassa*; *Schwanniomyces*, including *Schwanniomyces occidentalis*; and filamentous fungi, such as hosts of *Neurospora*, *Penicillium*, *Tolypocladium*, and *Aspergillus*, including *A. nidulans* and *A. niger*.

[0302] Vertebrate host cells are also suitable hosts for the expression of recombinant proteins. Suitable mammalian cell lines 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), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 77:4216, 1980); monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or subclones for 293 cells grown in suspension culture) (Graham et al., J. Gen Virol. [Journal of General Virology] 36:59, 1977); and young hamster kidney cells (BHK, ATCC CCL). 10); Mouse Setolly cells (TM4, Mather, Biol. Reprod. [Reproductive Biology] 23:243-251, 1980); Monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); Human lung cells (W138, ATCC CCL 75); Human liver cancer cells (Hep G2, HB 8065); Mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5-cell or FS4 cells; mammalian myeloma cells, and many other cell lines. In some embodiments, these host cells are selected from CHO cells.

[0303] 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 strain. Examples of such cells, cell lines, or cell strains include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, FIT 1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (juvenile hamster kidney cells), VERO, SP2 / 0, ΥB2 / 0, ΥO, 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 cells are 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, CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. Additionally, CHO FUT8 knockout cells are, for example,...

[0304] CHOK1 SV (Lonza, Inc.). In some embodiments, the eukaryotic cells may also be avian cells, cell lines, or cell strains, such as... Cells, EB14, EB24, EB26, EB66, or EBν13.

[0305] CHO cells (including CHOK1 cells (ATCC CCL61)) are widely used to produce complex recombinant proteins. In some embodiments, dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because highly efficient DHFR-selective and amplifiable gene expression systems allow for high levels of recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol [Enzyme Methodology] 185:537-566). Also included are glutamine synthase (GS) knockout CHOK1SV cell lines selected using methionine sulfoxide imine (MSX) based on glutamine synthase (GS). Other suitable CHO host cells include, but are not limited to, the following (ECACC accession numbers are 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).

[0306] Large-scale production of proteins for commercial applications can be carried out in suspension culture. Therefore, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted for growth in suspension culture. Various host cells known to be suitable for growth in suspension culture include mouse myeloma NS0 cells and CLIO cells derived from the CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines include, but are not limited to, mouse myeloma SP2 / 0 cells, juvenile hamster kidney BF1K-21 cells, and human... Cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or engineered from any of the cell lines disclosed herein.

[0307] In some embodiments, the eukaryotic cells are selected from lower eukaryotic cells, such as yeast cells (e.g., Pichia methanolica, Pichia kuhlii, and Pichia angusta)), Komagataella genus (e.g., Komagataella pastoris, Komagata ellapseudopastoris, or Komagataella phaffii)), Saccharomyces genus (e.g., Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum)), and Kluyveromyces genus (e.g., Kluyveromyces lactis, Kluyveromyces martensii). Cells of *Candida marxianus*, *Candida genus* (e.g., *Candida utilis*, *Candida cacaoi*, *Candida boidinii*), *Geotrichum genus* (e.g., *Geotrichum fermentans*), *Hansenula polymorpha*, *Yarrowia lipolytica*, or *Schizosaccharomyces pombe*. In some embodiments, eukaryotic cells are selected from *Pichia pastoris* strains. Non-limiting examples of *Pichia pastoris* strains include X33, GS115, KM71, KM71H, and CBS7435.

[0308] In some embodiments, the eukaryotic cells are selected from fungal cells (e.g., cells of the following: Aspergillus (e.g., Aspergillus niger, Aspergillus fumigatus, Aspergillus orzyae, Aspergillus nidus), Acremonium (e.g., Acremonium thermophilum), Chaetomium (e.g., C. thermophilum), Chrysosporium (e.g., C. thermophilum), and Chrysosporium (e.g., C. thermophilum). *C. thermophile*, *Cordyceps* (e.g., *C. militaris*), *Corynascus*, *Ctenomyces*, *Fusarium* (e.g., *F. oxysporum*), *Glomerella* (e.g., *G. graminicola*), *Hypocrea* (e.g., *C. rubrum*) *H. jecorina*, *Magnaporthe* (e.g., *M. orzyae*), *Myceliophthora* (e.g., *M. thermophile*), *Nectria* (e.g., *N. heamatococca*), *Neurospora* (e.g., *N. crassa*), *Penicillium* The genera *Sporotrichum* (e.g., *S. thermophile*), *Thielavia* (e.g., *T. terrestris*, *T. heterothallica*), *Trichoderma* (e.g., *T. reesei*), or *Verticillium* (e.g., *V. dahlia*).

[0309] In some embodiments, the eukaryotic cells are selected from insect cells (e.g., Sf9, Mimic). TM SF9, SF21, HighFive TM(BT1-TN-5BT1-4) or BT1-Ea88 cells), algal cells (e.g., like Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas) and plant cells (e.g., cells from monocotyledons (e.g., corn, rice, wheat, or foxtail grass) or cells from dicotyledons (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, sedge, or Arabidopsis thaliana)).

[0310] To generate a host cell line (e.g., a mammalian cell line) engineered to express a target recombinant protein, one or more nucleic acids encoding the recombinant protein (or a component thereof in the case of a multi-chain protein) are first inserted into one or more expression vectors. Nucleic acid control sequences in expression vectors that can be used for expression in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. A secretion signal peptide sequence may also optionally be encoded by the expression vector and operatively linked to the target coding sequence, such that the expressed protein can be secreted by the recombinant host cell, if desired, to facilitate easier isolation of the recombinant protein from the cell. The vector may also include one or more selective marker genes to facilitate the selection of host cells into which the vector is introduced. In some embodiments, the vector used employs protein fragment complementation assays using a protein reporter sequence such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964). Suitable mammalian expression vectors are known in the art and are commercially available.

[0311] Typically, vectors used in any host cell will contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcription and translation control sequences, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a natural or heterologous signal peptide sequence (lead sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a multinucleotide encoding the polypeptide to be expressed, and optional marker elements. Vectors can be constructed from starter vectors (such as commercially available vectors), and other elements can be obtained separately and ligated into the vector.

[0312] Cultivation methods

[0313] Various culture methods can be used to produce the desired recombinant protein, including but not limited to batch culture, fed-batch culture, and perfusion culture.

[0314] 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 complete harvesting. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, after which the viable cell density decreases as culture medium components are consumed and metabolic byproducts (such as lactate and ammonia) accumulate. Harvesting typically occurs when the maximum cell density is achieved (e.g., 5 x 10⁻⁶). 6 Cells / mL or higher, depending on the culture medium formulation, cell line, etc. Batch culture is the simplest culture method; however, feasible cell densities are limited by nutrient availability, and once cells reach maximum density, the culture declines and yields decrease. There is no ability to extend the production phase in batch culture because the accumulation of waste and depletion of nutrients rapidly lead to a decline in culture, typically around 3 to 7 days.

[0315] Fed-batch culture improves upon batch processes by providing feed in clumps or continuous medium to replenish those medium components that have already been consumed. Because fed-batch cultures receive additional nutrients throughout the run, they are able to achieve higher cell densities (>10 to 30 x 10⁻⁶) compared to batch methods. 6 The number of cells / mL (depending on the culture medium formulation, cell line, etc.) and the potential to increase product titers. Unlike batch processes, fed-batch cultures can be generated and maintained by manipulating feed strategies and culture medium formulations to differentiate between the cell proliferation phase (growth phase) and the suspension or slow cell growth phase (production phase) to achieve the desired cell density. Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. Typically, batch methods are used in the growth phase and fed-batch methods in the production phase, but a fed-batch feed strategy can be used throughout the process. However, unlike batch processes, bioreactor volume is a limiting factor for the amount of feed. Furthermore, as with batch methods, the accumulation of metabolic byproducts will lead to a decline in culture volume, which limits the duration of the production phase, often to around 10 to 21 days. Fed-batch culture is discontinuous, and harvest typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. Fed-batch culture can produce larger quantities of recombinant protein compared to batch culture (where no feeding occurs). (See, for example, U.S. Patent No. 5,672,502.)

[0316] Perfusion methods offer potential improvements over batch and fed-batch methods by adding fresh culture medium during culture while simultaneously removing used medium. A typical perfusion culture begins with a one- or two-day batch culture start-up, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture, while simultaneously removing used medium and retaining cells and additional high-molecular-weight compounds such as proteins (based on the filtration molecular weight cutoff). Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used 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 culture medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. [Genetic Engineering News] 22(7):62-63.

[0317] Perfusion can be continuous, stepwise, intermittent, or any combination of these. The perfusion rate can range from less than one working volume per day to many working volumes per day. Cells are retained in the culture, and the removed used culture medium is substantially cell-free or contains significantly fewer cells than in the culture. Recombinant proteins expressed in the cell culture may also be retained in the culture.

[0318] Typical large-scale commercial cell culture strategies aim to achieve high cell densities, 40–90(+) x 10⁶ cells / year. 6 Cells / mL, for example, approximately 40 x 10⁻⁶ cells / mL. 6 1 cell / mL or approximately 50 x 10⁻⁶ cells / mL 6 Cells / mL, with biomass comprising almost one-third to more than half of the reactor volume. >1 x 10⁻⁶ cells / mL has been achieved through perfusion culture. 8 Extreme cell densities of 100 cells / mL. A potential advantage of perfusion processes is that production cultures can be maintained for longer periods compared to batch or fractional culture methods. However, increased preparation, use, storage, and disposal of the culture medium are necessary to support long-term perfusion culture, especially for cultures with high cell densities that require even more nutrients. Additionally, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and increased aeration, including supplying more oxygen and removing more carbon dioxide, which may lead to greater foaming and require changes to defoaming strategies; and problems during harvest and downstream processing, where the effort required to remove excess cell material can result in product loss, negating the benefits of increased titers due to increased cell mass.

[0319] Suitable culture conditions for mammalian cells, including temperature, dissolved oxygen content, agitation rate, etc., are known in the art and can vary with the stage or period of cell culture. In some embodiments, the methods disclosed herein further include sampling during cell culture and evaluating the samples to quantitatively and / or qualitatively monitor the recombinant protein and / or the characteristics of the cell culture process. In some embodiments, process analysis techniques are used to quantitatively and / or qualitatively monitor the samples. For example, dissolved oxygen levels can be monitored during cell culture using methods known in the art, such as basic chemical analysis (titering), electrochemical analysis (diaphragm electrode method), and photochemical analysis (fluorescence method).

[0320] During recombinant protein production, a controlled system is desired in which cells grow for a desired time or to a desired density, and then the physiological state of the cells is switched to a high-productivity state of growth restriction or arrest, in which the cells use energy and substrates to produce recombinant proteins in a way that favors increased cell density. For commercial-scale cell culture and the manufacture of biotherapeutic agents, the ability to restrict or inhibit cell growth, and the ability to maintain cells in a growth-restricted or inhibited state during the production phase, is highly desirable. Such methods include, for example, temperature variations, the use of chemical inducers of protein production, nutrient restriction or starvation, and cell cycle inhibitors, used alone or in combination. Illustratively, typical cell culture involves a growth phase, which is an exponential growth period of increasing cell density. During the growth phase, cells are cultured in a cell culture medium containing necessary nutrients and additives under conditions that allow a particular cell line to achieve optimal growth (typically at a temperature of about 25°–40°C in a humid, controlled atmosphere). Cells are typically maintained in the growth phase for a period of one to eight days (e.g., three to seven days, e.g., seven days). The length of the growth phase for a particular cell line can be determined by those skilled in the art and is typically a period sufficient to allow the cells to proliferate to a viable cell density (if the culture is maintained under growth conditions) ranging from approximately 20% to 80% of the maximum possible viable cell density. Following the growth phase is a transition phase where exponential cell growth slows and protein production begins to increase. This marks the beginning of the stationary phase (production phase), in which cell density typically plateaus and product titers increase. During the production phase, culture medium is typically replenished to support continued recombinant protein production.

[0321] In some embodiments, the culture conditions used to produce recombinant proteins can be adjusted to facilitate the transition from the growth phase of cell culture to the production phase. For example, the growth phase of the cell culture can occur at a higher temperature than the production phase. In some embodiments, the growth phase can occur at a first temperature of about 35°C to about 38°C, while 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 variation from about 35°C to about 37°C to about 31°C to about 33°C can be used to facilitate the transition from the growth phase to the production phase of the culture. Chemical inducers for protein production, such as caffeine, butyrate, and hexamethylene diacetamide (HMBA), can be added before and / or after the temperature variation, or concurrently with the temperature variation. If the inducer is added after the temperature variation, it can be added from one hour to five days after the temperature variation, optionally one to two days after the temperature variation.

[0322] Additionally, any cell culture medium capable of supporting the growth of appropriate host cells in culture can be used. Typically, cell culture media contain buffers, salts, energy, amino acids, vitamins, and trace essential elements. Further supplementation of cell culture media with other components can be made to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells. These cell culture media are commercially available and include RPMI-1640, RPMI-1641, Dürbeco modified Eagle medium (DMEM), Eagle minimum essential medium, F-12K, HAM F12, Iskoff modified Dürbeco medium, McCoy's 5A medium, Leibovitz L-15 medium, and serum-free media such as EX-CELL. TM The 300 series and other media are available from the American Type Culture Collection, SAFC Biosciences, and other suppliers. The cell culture medium can be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture media can also be enriched by adding nutrients or other supplements and can be used at concentrations higher than the usual, recommended levels. In some embodiments, the culture medium used to produce recombinant proteins to be purified by the methods provided herein is a chemically defined medium, which refers to a cell culture medium in which all components have known chemical structures and concentrations. Chemically defined media are typically serum-free and free of hydrolysates or animal-derived components.

[0323] Various culture medium formulations can be used during culture, for example, to facilitate the transition from one phase (e.g., growth phase) to another (e.g., production phase) and / or to optimize conditions during cell culture (e.g., concentrated medium provided during perfusion culture). Growth medium formulations can be used to promote cell growth and minimize protein expression. Production medium formulations can be used to promote the production of the desired recombinant protein and maintain cells while minimizing the growth of new cells. Fed media are typically cell media containing more concentrated components such as nutrients and amino acids, which are consumed during the production phase of the cell culture. Fed media can be used to replenish and maintain viable cultures, particularly those operated in fed-batch, semi-perfusion, or perfusion modes. Such concentrated feed culture media can contain most cell culture media components, for example, about 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800 or even about 1000 times their normal amount.

[0324] In some embodiments, mammalian cells used to produce recombinant proteins are cultured for a defined period of time during which the mammalian cells express and secrete the recombinant proteins. 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 some embodiments, the duration of the production phase of the cell culture is about 7 days to about 28 days, about 10 days to about 30 days, about 7 days to about 14 days, about 10 days to about 18 days, about 3 days to about 15 days, about 5 days to about 8 days, about 12 days to about 15 days, about 12 days to about 18 days, or about 15 days to about 21 days. In some embodiments, the duration of the production phase of the cell culture is 7 days, 8 days, 9 days, 12 days, 15 days, 18 days, or 21 days.

[0325] In some embodiments, the biomanufacturing process for producing the recombinant protein includes a live cell density of at least 100 x 10⁻⁶ cells / mL. 5 cells / mL, for example, approximately 100 x 10⁻⁶ cells / mL 5 10 cells / mL to approximately 10 x 10⁻⁶ cells / mL 7 cells / mL, approximately 250 x 10⁻⁶ 5 1 cell / mL to approximately 900 x 10⁻⁶ cells / mL 5 cells / mL, approximately 300 x 10⁻⁶ 5 cells / mL to 800 x 10⁻⁶ 5 cells / mL, or approximately 450 x 10⁻⁶ cells / mL 5 cells / mL to 650 x 10⁻⁶ 5The production stage is cells / mL. Cell density can be measured using a hematology counter, Coulter counter, or automated cell analyzer (e.g., Cedex automated cell counter). Viable cell density can be determined by staining culture samples with trypan blue (which is absorbed only by dead cells). The viable cell density is then determined by counting the total number of cells, dividing the number of stained cells by the total number of cells, and taking the reciprocal.

[0326] In some embodiments, the upstream biomanufacturing process for producing recombinant proteins to be purified by the methods provided herein includes a production stage having a cell compaction of less than or equal to 35%. In some embodiments, the cell compaction is less than or equal to 30%.

[0327] Key properties and performance metrics of the target recombinant protein can be measured to better guide decisions regarding performance at each step during manufacturing. These key properties and performance metrics can be monitored in real-time, near real-time, and / or offline. Key parameters that can be measured during cell culture may include the levels of consumed cell culture medium components (e.g., glucose), accumulated metabolic byproducts (e.g., lactate and ammonia), and those related to cell maintenance and survival (e.g., dissolved oxygen levels). Additionally, key properties such as specific productivity, viable cell density, cell hematocrit, pH, osmolality, aggregation, yield percentage, and titer can be monitored at appropriate stages of the manufacturing process. Monitoring and measurement can be performed using known technologies and commercially available equipment.

[0328] bioreactor

[0329] In some embodiments, the growth and / or production phases of the upstream process for producing recombinant proteins are carried out within a bioreactor. Conditions supporting cell culture are provided within the bioreactor. As described above, suitable culture conditions for mammalian cells are known in the art. Bioreactor “operation” typically includes the following steps: inoculating a prepared bioreactor with a seed culture, subjecting cells to one or more growth and / or production phases until one or more predetermined parameters (e.g., time, viable cell density, cell hematocrit) are met, and then harvesting the contents of the bioreactor.

[0330] In some embodiments, one or more bioreactors for producing recombinant proteins are stainless steel bioreactors, such as built-in large stainless steel bioreactors capable of operating in volumes of about 2,000 liters to about 50,000 liters (e.g., about 2,000 liters to about 20,000 liters) or more.

[0331] In some embodiments, the bioreactors used to produce one or more recombinant proteins are single-use bioreactors. Single-use technology minimizes the infrastructure requirements associated with traditional cell culture, such as commercial-scale steel / glass containers and associated machinery. Single-use bioreactors offer manufacturing process flexibility, and on-site assembly, reconfiguration, sterilization, and validation of single-use bioreactors can be faster, easier, and less costly than traditional built-in stainless steel cell culture equipment. A single-use bioreactor comprises a single-use sterile plastic bag supported by a non-disposable support structure. The culture is agitated by a stirrer within the bag or by shaking, and air and oxygen sprayers and sensors are provided to measure and regulate various parameters of the culture, such as pH, temperature, oxygen, cell density, etc. Single-use bioreactors are commercially available, for example, from Bio. Sartorius, Göttingen, Germany; Millipore, Burlington, Massachusetts; Stofan Corporation, Marlborough, Massachusetts.

[0332] The volume of a bioreactor is divided into a working volume space and a top space. The working volume of a bioreactor refers to the volume within the bioreactor in which cell cultures are 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.

[0333] Additional harvesting and purification processes

[0334] The expressed recombinant proteins can be secreted into a culture medium, from which they can be recovered and / or collected. Some biomanufacturing processes incorporating the anion exchange chromatography operation disclosed herein may also include a harvesting operation. The harvesting operation completely or partially clarifies and / or purifies the target protein to remove at least one impurity found in the cell culture medium, such as residual cell culture medium, cells, cell debris, or culture medium components, and / or product-related impurities and / or process-related impurities.

[0335] Methods for harvesting recombinant proteins from suspension cell cultures are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation (e.g., flocculation), separation using gravity, centrifugation, acoustic separation, and filtration (including membrane filtration, ultrafiltration, microfiltration, tangential flow, alternating tangential flow, depth filters, and impeller filters).

[0336] Harvested cell culture medium (HCCF) can be stored in buffer tanks, containers, bags, or other containers suitable for feeding into a column skid and appropriate for infrastructure and / or process requirements.

[0337] Harvesting operations can be combined with additional harvesting strategies, including centrifugation, such as disc centrifugation or continuous solid discharge centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration and depth filtration; precipitation / sedimentation methods, such as flocculation; and separation based on chromatographic media.

[0338] In addition to anion exchange chromatography operations using anion exchange chromatographic materials containing primary amine ligands, this disclosure covers methods involving all known purification techniques, such as protein A purification of immunoglobulins and immunoglobulin-like biological products, as well as chromatographic separation and polishing steps, including column and alternative mode chromatographic separations via ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed-mode or multi-mode chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, size exclusion chromatography (SEC), gel filtration, or any other known form of chromatographic separation of biological and / or biochemical substances.

[0339] In some embodiments, recombinant proteins recovered from host cells or cell culture media may be further purified or partially purified to remove cell culture medium components, host cell proteins or nucleic acids, or other process or product-related impurities through one or more unit operations. Those skilled in the art can select one or more appropriate unit operations for further purification of the recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of the host cells expressing the recombinant protein, and the composition of the culture medium in which the host cells are grown. Illustratively, in some embodiments, the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, deep filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0340] Capture unit operations may include capture chromatography using resins and / or membranes containing reagents to bind to the target recombinant protein, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), solid-phase metal affinity chromatography (IMAC), etc. Such chromatographic materials are known in the art and are commercially available. For example, if the recombinant protein is an antibody or contains components derived from an antibody (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 chromatographic unit operation to further purify the recombinant protein. In other embodiments, the target recombinant protein may contain a polyhistidine tag at its amino or carboxyl terminus, followed by purification using IMAC. The recombinant protein may be engineered to include other purification tags, such as The tag or c-myc epitope is then purified by affinity chromatography using a specific antibody against such a tag or epitope.

[0341] Unit operations designed to inactivate, reduce, and / or eliminate viral contaminants may include processes to reduce viral risk by manipulating the environment and / or by using filtration. Viral mitigation measures are crucial for ensuring the safety of protein therapeutics and may be performed once or multiple times throughout the downstream purification process. Viral contaminants can originate from a variety of sources, including the use of animal-derived reagents, foreign viral contaminants in host cell lines, or system failures at GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped viruses. For enveloped viruses, the envelope allows the virus to recognize, bind, enter, and infect target host cells. Therefore, enveloped viruses are susceptible to inactivation methods. Various methods can be used to inactivate viruses, including heat inactivation / pasteurization, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, the addition of chemical inactivating agents, surfactants, and solvent / detergent treatments. Surfactants (e.g., detergents) can dissolve the membrane and may be very effective in specifically inactivating enveloped viruses. Additional unit operations for inactivating, reducing, and / or eliminating viral contaminants may include filtration processes and / or conditioning of solution conditions. One method for achieving virus inactivation is incubation at a low pH (e.g., pH < 4). Following the low-pH virus inactivation operation, a neutralization unit can be performed, which readjusts the pH of the virus-inactivated solution to better suit the requirements of subsequent unit operations. Filtration, such as depth filtration, can also be performed after the low-pH virus inactivation operation to remove any resulting turbidity or precipitation. Adjusting the temperature or chemical composition (e.g., using a cleaning agent) can also be used to achieve virus inactivation. Virus filtration can be performed using microfiltration or nanofiltration, such as those available from Asahi Kasei Corporation. and EMD Millipore Those that were obtained.

[0342] Non-enveloped viruses are not easily affected by inactivation methods that maintain product stability. Therefore, non-enveloped viruses are typically removed by filtration methods. Example processes are described in WO 2020 / 159838. Virus filtration can utilize microfilters or nanofilters (e.g., from...). (Asahi Kasei Corporation, Chicago, Illinois) (Sartorius, Göttingen, Germany) Pro (Millibosigma, Burlington, Massachusetts), Pegasus TM Prime (Pall Biotech, Port Washington, New York) and CUNO ZetaPlus VR (3M, St. Paul, Minnesota) are among those available.

[0343] Virus filtration can occur in one or more steps of a downstream operation in a biomanufacturing process. Typically, virus inactivation is performed after an affinity chromatography unit operation, and virus filtration is performed before or after an ultrafiltration / distillation (UF / DF) operation, but can also be performed after UF / DF.

[0344] In all chromatographic processes, multiple filters can be used to achieve the desired production capacity required to achieve the desired production objectives of the ultrafiltration / distillation (UF / DF) system, whether through reservoirs, skids, or physical setups.

[0345] Purification unit operations can utilize various chromatographic methods to purify target proteins and remove contaminants and impurities. The resins and / or membranes that can be used in purification chromatography unit operations contain reagents that can be used in either "flow-through mode" (where the target protein is contained in the eluent and contaminants and impurities are bound to the chromatographic medium) or "bind-elute mode" (where the target protein is bound to the chromatographic medium and elutes after contaminants and impurities flow through or are washed away from the chromatographic medium). Examples of such purification chromatographic methods include, but are not limited to, ion exchange chromatography (IEX), such as cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multi-mode chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography; and size exclusion chromatography (e.g., gel filtration).

[0346] Recombinant proteins can be formulated (i.e., buffer exchange, sterilization, batch packaging, and / or packaging for end-user use) and purified. Illustratedly, product concentration and buffer exchange of the desired recombinant protein can be accomplished by ultrafiltration and / or percolation to form a desired formulation buffer for bulk storage of the active pharmaceutical ingredient or drug product. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, Pennsylvania.

[0347] UF / DF operations can be performed at one or more stages of downstream processes. Typically, UF / DF operations are performed before bulk storage of the active pharmaceutical ingredient (API). In addition to storage, unit operations related to drug filling / completion can also be performed immediately after the UF / DF operation. Optionally, one or more stability-enhancing excipients can be added directly to a UF / DF retentate feed tank containing formulated, purified proteins to obtain the formulated API, or added to a UF / DF eluent tank. An example UF / DF process is described in WO 2020 / 159838. Filters used for UF / DF operations are well known in the art and are commercially available from a variety of sources. Many types of materials are available, such as regenerated cellulose Pellicon (Millibo Sigma, Danvers, MA), stabilized cellulose, etc.

[0348] Slice ECO Sartorius, Göttingen, Germany, and polyethersulfone (PES) membranes, Omega (Pall, Port Washington, New York).

[0349] Recombinant protein

[0350] Any type of recombinant protein (including proteins containing a single polypeptide chain or multiple polypeptide chains) can be purified according to the methods disclosed herein. Such recombinant proteins include, but are not limited to, secretory proteins, non-secretory proteins, intracellular proteins, or membrane-bound proteins. Illustratively, recombinant proteins may include, but are not limited to, cytokines, growth factors, hormones, mutant proteins, fusion proteins, antibodies, antibody fragments, peptide antibodies, T-cell conjugating molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0351] In other embodiments, the recombinant protein to be purified according to the methods disclosed herein is an antigen-binding protein. Antigen-binding proteins include, but are not limited to, antibodies, peptides, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgG scFv (see, for example, Orcutt et al., 2010, Protein Engineering Design and Selection 23:221-228), heterologous IgG (see, for example, Liu et al., 2015, J Biol Chem 290:7535-7562), mutant proteins, and... (Xencor, Inc., Monova, California). Additional antigen-binding proteins include, but are not limited to, bispecific T-cell conjugates. Bispecific T-cell conjugates with extended durations (e.g., extended half-life) (e.g., HLE BiTE molecules, HeteroIg BITE molecules, etc.), chimeric antigen receptors (CAR, CAR T), and T-cell receptors (TCRs).

[0352] In some embodiments, the antigen-binding protein binds alone or in any combination to one or more of the following: CD proteins (including, but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174); HER receptor family proteins (including, for example, HER2, HER3, HER4, and the EGF receptor EGFRvIII); and cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin). Growth factors (including, but not limited to, vascular endothelial growth factor (“VEGF”), VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müller's duct inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factors (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, T... GF-β4 or TGF-β5), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone-inducing factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein)); coagulation proteins and coagulation-related proteins (especially such as factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and Thrombopoietin receptors; colony-stimulating factors (CSFs) (especially including M-CSF, GM-CSF, and G-CSF); other blood and serum proteins (including but not limited to albumin, IgE, and blood group antigens); receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors, and T-cell receptors); neurotrophic factors (including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6)); relaxin A chain, relaxin B chain, and prorelaxin.Interferons (including, for example, interferon α, interferon β, and interferon γ); interleukins (ILs) (e.g., IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, pulmonary surfactant, and tumor necrosis factor. -α and tumor necrosis factor-β, enkephalin, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (activated and regulated normal T cell expression and secretion factors), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerator factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, growth hormone, 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), myosin, Dickkopf-1 (DKK-1), Ang2 NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFNγ, interferon-gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin Type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any of the aforementioned bioactive fragments or variants.

[0353] In other embodiments, the recombinant protein to be purified according to the methods disclosed herein is an antibody. In some embodiments, the antibody is a human antibody.

[0354] In some embodiments, the antibody is selected from abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, and ontamalimab. The antibodies are selected from panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, tafolecimab, tanezumab, tezepelumab, tremelimumab, utomilumab, and volagidemab. In some embodiments, the antibody is selected from dinosumab, errenumab, ivosumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is dinosumab. In some embodiments, the antibody is errenumab. In some embodiments, the antibody is ivosumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is terzrolumab.

[0355] 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.

[0356] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0357] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0358] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody.

[0359] Example

[0360] To provide a fuller understanding of this disclosure, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting this disclosure in any way.

[0361] Example 1: Using AEX chromatographic steps of NH2-750F

[0362] Following affinity chromatography, low-pH virus inactivation, and CEX chromatography, a mixture of... The AEX resin composed of NH2-750F (Tosoh Biotechnology Co., Ltd.) is further purified to form a composition containing one of two recombinant monoclonal antibodies, mAb1 or mAb2. NH2-750F consists of polymethacrylate beads functionalized with a proprietary primary amine (NH2) strong anion exchange group and is commercially available in 45 μm particle size (F grade). The AEX procedure operates at a pH between 7.0 and 8.0 and has an operating conductivity of less than 10 mS / cm. The AEX column is loaded with samples at densities between 250 g / L and 600 g / L.

[0363] Figure 1A The AEX step demonstrated its ability to remove total HMW from mAb1 by comparing HMW levels in seven pilot-scale batches (as assessed by SE-HPLC). The at least 0.5% HMW reduction observed in all three batches testifies to the robustness of the AEX step, even with high loading rates exceeding 500 g / L resin. Significant reductions in process-related impurities, including host cell proteins, DNA, and model viruses, were also observed in both pilot-scale operations and laboratory-scale activation studies.

[0364] Figure 1B It shows Figure 1A The step yield of the AEX step in a pilot-scale batch demonstrates the step's ability to achieve high yields while providing significant impurity reductions.

[0365] Similar high yields and significant impurity reductions were also observed for mAb2. Figure 2A The study demonstrates the high molecular weight scavenging of mAb2 in two pilot-scale batches, and Figure 2B It shows Figure 2A The step yield of the AEX step in the pilot-scale batch.

[0366] Example 2: Low pH virus inactivation using formic acid

[0367] In certain manufacturing settings with equipment limitations, it is necessary to minimize the volume of the protein A elution pool and subsequent pool titration to ensure volume compliance with container limitations and robust safety margins. As shown in Table 1, for the evaluation of VI unit operations of mAb1 with low pH incubation times of 60–90 minutes at 15–25°C, using 1M formic acid as the acid titrant compared to 10% acetic acid resulted in a lower acid volume required to reach the pH for virus inactivation, a lower alkali volume required to neutralize the VI pool to pH 5.0, and a lower net volume expansion during the virus inactivation step. This is particularly advantageous when the ability to reduce the protein A elution pool volume is limited, such as when operating under high column loading conditions.

[0368] Table 1. Effects of Titrant VI on the Volume and Conductivity of mAb1 Cell

[0369]

[0370]

[0371] Figure 3 The benefits of lower loading conductivity (resulting from the choice of VI acidifying titrant) were demonstrated, as observed for mAb1 AEX unit operations that are substantially similar to those described in Example 1. High molecular weight (HMW, detected by SE-HPLC determination of mAb1) is often a key quality attribute of mAbs (e.g., mAb1), and HMW impurity levels are typically reduced through purification chromatographic steps (e.g., AEX). Figure 3 The results show that, under similar conditions, the use of 1M formic acid VI titrant (PSL2) resulted in improved AEX step performance, leading to a lower percentage of HMW in the AEX cell, exhibiting minimal reduction in HMW material in mAb1, compared to the use of 10% acetate VI titrant (PSL1). Although the lower loading conductivity reduced the step yield (93% for PSL2 compared to 98% for PSL1) due to greater product retention on the resin, the AEX step yield remained acceptable when using 1M formic acid as the VI titrant.

[0372] All documents or portions thereof referenced in this application, including but not limited to patents, patent applications, articles, books, and monographs, are hereby expressly incorporated by reference. Unless the context clearly indicates otherwise, the content described in the embodiments of this disclosure may be combined with one or more other embodiments of this disclosure.

[0373] The subject matter disclosed is not intended to be limited to the specific embodiments described herein, which are rather intended as non-limiting illustrations of various aspects of this disclosure. Functionally equivalent methods and components are within the scope of this disclosure. In fact, various modifications to the disclosed subject matter will be apparent to those skilled in the art, in addition to those shown and described herein, based on the foregoing description and drawings. Such modifications are intended to fall within the scope of the disclosed subject matter.

[0374] The description of various embodiments and / or examples of the disclosed subject matter is presented for illustrative purposes and is 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 is chosen to best explain the principles of the embodiments, their practical application, or technical improvements to existing technologies in the market, and / or to enable others skilled in the art to understand the disclosed subject matter.

Claims

1. A method for purifying a recombinant protein from a composition comprising a recombinant protein and at least one impurity, the method comprising: The composition was loaded onto an anion exchange material containing a primary amine ligand at a loading density greater than about 100 g / L of anion exchange material, wherein: The composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and The binding of at least one impurity to the anion exchange material is stronger than the binding of the recombinant protein to the anion exchange material; and Collect the purified composition containing the recombinant protein.

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

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

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

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

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

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

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

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

10. The method of claim 9, wherein the low pH virus inactivation unit operates using an acid titrant containing formic acid.

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

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

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. The method according to any one of claims 11-13, wherein the one or more additional chromatographic unit operations include additional purification chromatographic unit operations.

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

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

17. The method according to any one of claims 1-16, wherein: The purified composition contains less than about 2.5% w / w of the recombinant protein, which is a high molecular weight form of the recombinant protein; and / or The purified composition contains at least about 85% w / w of the recombinant protein in the composition prior to loading.

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

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

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

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