Anion Exchange Chromatography Processes Using a Primary Amine Ligand

Abstract

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

Description

1 / 112 Anion Exchange Chromatography Processes Using a Primary Amine Ligand CROSS-REFERENCE TO RELATED REQUESTS

[0001] This application claims the priority benefit of Provisional Application No. U.S. 63 / 490,079, filed March 14, 2023, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates to methods for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, wherein the methods comprise performing anion exchange chromatography (e.g., in flow or weak partition chromatography mode) using an anion exchange material comprising a primary amine ligand, such as a polyamine ligand (e.g., TOYOPEARL® NH2-750F). 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 unit operation of anion exchange chromatography allows robust removal of high molecular weight species with high protein yield (such as, for example, at least about 85%) at high loadings (such as, for example, greater than about 100 g / l of anion exchange material). BACKGROUND

[0003] Downstream purification processes for the manufacture of pharmaceutical substances from protein therapeutic agents, such as monoclonal antibodies (mAbs) and Petition 870250082204, dated 12 / 09 / 2025, page 17 / 138 2 / 112 antibody constructs generally include an affinity chromatography step followed by one or more polishing chromatography steps to remove product-related and process-related impurities. For products containing an Fc domain, a Protein A affinity chromatography step in which cell culture harvest fluid is flowed into a Protein A resin to bind to the recombinant protein of interest, followed by elution with a low pH buffer that desorbs the protein from the Protein A resin, is frequently used to capture the protein.Since Protein A clusters generally have a low pH (< pH 5), the subsequent unit operation is often a low pH viral inactivation (VI) step, in which the Protein A cluster is titrated with an acid at a low pH known to inactivate enveloped viruses, held for a sufficient period to ensure VI, and then titrated with a base at a higher pH appropriate for product stability and / or loading into subsequent unit operations. Many downstream purification processes utilize a cation exchange chromatography (CEX) step after the VI unit operation, since CEX generally requires a relatively low pH to bind to the positively charged product and product-related impurities in the negatively charged CEX resin.Additional polishing chromatography steps, such as anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and mixed-mode chromatography (MMC), are frequently employed post-CEX to further reduce impurities. These polishing chromatography steps can be operated in various modes. Petition 870250082204, dated 12 / 09 / 2025, page 18 / 138 3 / 112 chromatographic, depending on process requirements, including binding and elution mode, continuous flow mode and front loading (i.e., frontal) mode.

[0004] AEX chromatography can enable connected processing with downstream steps such as viral filtration (VF), which further reduces the risk of viral contamination, and ultrafiltration / diafiltration (UF / DF), where buffer exchanges and / or concentrates the product of interest to the desired conditions for drug product formulation. Furthermore, continuous flow and weak partitioning AEX chromatography generally allow for higher column loadings compared to chromatography operations performed in ligation-elution mode, which in turn reduces the required column sizes and buffer consumption in downstream processes.However, while flow AEX chromatography has demonstrated robustness for removing process-related impurities such as nucleic acids, host cell proteins, leached protein A ligand, endotoxins, and viruses, achieving high molecular weight (HMW) species and aggregate removal can be challenging, especially when high protein loads are used to maximize process throughput (Yigzaw et al., Current Pharmaceutical Biotechnology, 2009). Achieving robust HMW clearance requires optimization of the AEX resin, loading pH, contraion type, and loading dilution—variables subject to constraints such as product stability and plant tuning.

[0005] Consequently, there is a need in the art for new and improved purification methods using Petition 870250082204, dated 12 / 09 / 2025, page 19 / 138 4 / 112 AEX chromatography that allows robust removal of HMW species with high protein yield at high loadings. SUMMARY

[0006] One aspect of the disclosure relates to a method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, wherein the method is characterized in that it comprises: loading the composition onto an anion exchange material comprising a primary amine ligand at a charge 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein.

[0007] Another aspect of the disclosure provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: loading the composition into an anion exchange material comprising resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with Petition 870250082204, dated 12 / 09 / 2025, page 20 / 138 5 / 112 a primary amine ligand, at a charge density of about 250 g / l, approximately 600 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 (e.g., about 3 mS / cm to about 6 mS / cm); and at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein.

[0008] Yet another aspect of the disclosure provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: loading the composition into an anion exchange material comprising a primary amine ligand at a charge density of about 250 g / l to about 600 g / l of anion exchange material, wherein: 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein, wherein: Petition 870250082204, dated 12 / 09 / 2025, page 21 / 138 6 / 112 less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading.

[0009] Yet another aspect of the disclosure provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: loading the composition into an anion exchange material comprising a polyamine binder at a charge density of about 250 g / l to about 600 g / l of anion exchange material, wherein: 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein, wherein: Less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or Petition 870250082204, dated 12 / 09 / 2025, page 22 / 138 7 / 112 The purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading.

[0010] Another aspect of the disclosure provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: Perform a low-pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition into an anion exchange material comprising a primary amine ligand at a charge density of about 250 g / l to about 600 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 10 mS / cm (for example, about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and The low pH viral inactivation unit operation is performed one or more times prior to loading; and a purified composition comprising the recombinant protein is collected.

[0011] Yet another aspect of the revelation provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, a Petition 870250082204, dated 12 / 09 / 2025, page 23 / 138 8 / 112 antibody) from a composition comprising the recombinant protein and at least one impurity selected from a high molecular weight species of the recombinant protein, wherein the method comprises: To perform a low pH viral inactivation unit operation using approximately 1 M to approximately 2 M formic acid as an acid titrant; loading the composition into an anion exchange material comprising a polyamine binder at a charge density of about 250 g / l to about 600 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 10 mS / cm (for example, about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and The low pH viral inactivation unit operation is performed one or more times prior to loading; and a purified composition comprising the recombinant protein is collected.

[0012] Yet another aspect of the disclosure provides a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: Petition 870250082204, dated 12 / 09 / 2025, page 24 / 138 9 / 112 perform a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition into an anion exchange material comprising a primary amine ligand at a charge density of about 250 g / l to about 600 g / l of anion exchange material, wherein: The composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and The low-pH viral inactivation unit operation is performed one or more times prior to loading; and a purified composition comprising recombinant protein is collected, wherein: Less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A shows the percentage of high molecular weight (HMW) species of mAb1, as assessed by SEHPLC, in a composition loaded onto an AEX column and in a cluster recovered from the AEX column through seven pilot-scale batches. Petition 870250082204, dated 12 / 09 / 2025, page 25 / 138 10 / 112

[0014] Figure 1B shows the step-to-step yield for the AEX step for the seven mAbl pilot-scale batches summarized in Figure 1A, demonstrating the AEX step's ability to achieve high yields of over 85% while providing significant impurity reduction.

[0015] Figure 2A shows the percentage of high molecular weight (HMW) mAb2 species, as assessed by SEUHPLC, in a composition loaded onto an AEX column and in a cluster recovered from the AEX column via two pilot-scale batches.

[0016] Figure 2B shows the step-to-step yield for the AEX step for the two pilot-scale mAb2 batches summarized in Figure 2A, demonstrating the AEX step's ability to achieve high yields of over 85% while providing significant impurity reduction.

[0017] Figure 3 shows the percentage of high molecular weight (HMW) species of mAb1 in two pilot-scale batches, one in which 10% acetic acid was used as a VI titrant (PSL1) and one in which 1M formic acid was used as a VI titrant (PSL2). The use of a 1M formic acid VI titrant (PSL2) led to a lower percentage of HMW in the AEX cluster vs. the use of a 10% acetic acid VI titrant (PSL1) under similar conditions. DETAILED DESCRIPTION

[0018] Methods for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein) are disclosed in this document. Petition 870250082204, dated 12 / 09 / 2025, page 26 / 138 11 / 112 methods comprising performing anion exchange chromatography (e.g., in continuous flow chromatography or weak partitioning mode) using an anion exchange material comprising a primary amine ligand, such as a polyamine ligand, and optionally a methacrylate-containing polymer base matrix. DEFINITIONS:

[0019] The following definitions are provided to assist in understanding the scope of this disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains.

[0020] In some embodiments, “about,” when used in connection with a measurable numerical variable, refers to the stated value of the variable and to all values ​​of the variable that are within the experimental error of the stated value (e.g., within the 95% confidence interval for the mean) or ± 10% of the stated value, whichever is greater. In some embodiments, the numerical ranges include the numbers defining the range (i.e., the endpoints).

[0021] Where a range of values ​​is provided, it is understood that each intervening value, up to the tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range and any other value indicated or intervening in that indicated range is included within the disclosure. The upper and lower limits of those smaller ranges may independently be included in the smaller ranges also included within the disclosure, subject to any limit. Petition 870250082204, dated 12 / 09 / 2025, p. 27 / 138 12 / 112 specifically excluded in the indicated range. Where the indicated range includes one or both limits, the ranges excluding either or both of the included limits are also included in the disclosure.

[0022] As used herein, the terms “a” and “an” mean one or more unless specifically indicated otherwise. Additionally, “one or more” and “at least one” are used interchangeably herein. Furthermore, unless otherwise required by the context, singular terms include plurals and plural terms include the singular.

[0023] As used in this document, the term acid precipitation refers to a harvesting operation in which the pH of the cell culture is reduced to induce precipitation of one or more cell culture impurities.

[0024] As used in this document, the term affinity chromatography (also called capture chromatography) refers to a chromatography operation in which a biomolecule (e.g., a recombinant protein) is separated from a mixture based on a selective interaction between the biomolecule and another substance (i.e., a ligand). Affinity chromatography is commonly used in biofabrication processes to isolate and concentrate desirable recombinant proteins from collected cell culture fluid. In a typical affinity chromatography operation, a biomolecule in a mobile phase selectively binds to or otherwise interacts with a stationary phase while the rest of the mobile phase passes through the chromatography material. The biomolecule is then eluted from the stationary phase by altering the conditions in a way that reduces the affinity between the ligand and the biomolecule. Examples Petition 870250082204, dated 12 / 09 / 2025, page 28 / 138 13 / 112 Non-limiting affinity chromatography materials include Protein A, Protein G, Protein A / G, or Protein L materials. Additionally, immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have, or are designed to have, an affinity for metal ions.

[0025] In some embodiments, Protein A affinity chromatography can be employed to capture a recombinant protein of interest. Protein A ligands are highly selective for a wide range of proteins containing an antibody Fc region and provide robust removal of process-related impurities with high yields of target protein. Commercially available Protein A materials include, but are not limited to, MABSELECT™ SURE Protein A, Sepharose FAST FLOW™ Protein A, MABSELECT™ PrismA (Cytiva, Marlborough, MA), PROSEP-A™ (Merck Millipore, UK), TOYOPEARL® HC-650F Protein A (TosoHass Co., Philadelphia, PA), and AP Plus (Purolite, King of Prussia, PA).

[0026] As used in this document, the term “antigen-binding protein” refers to a protein or polypeptide comprising an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen). Antigen-binding proteins include, but are not limited to, antibodies, VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or “r IgG” fusion proteins (“half antibody consisting of a heavy chain and a light chain”) or an antigen-binding portion of a full-length antibody, such as, for example, Petition 870250082204, dated 12 / 09 / 2025, page 29 / 138 14 / 112 example, triple-chain antibody-like molecule, heavy-chain-only antibody, single-chain variable fragment (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-closure, single-chain Fab (scFab), Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, minibodies exemplified by a structure that is as follows: (VHVL-CH3)2, (scFv-CH3)2, ((scFv)2—CH3 + CH3), ((scFv)2—CH3) or (scFv-CH3-scFv)2, multibodies, such as triabodies or tetrabodies, and single-domain antibodies, such as nanobodies or single variable domain antibodies comprising merely a variable region, which It could be VHH, VH, or VL, which binds specifically to an antigen or target independently of other variable regions or domains.

[0027] As used herein, the term antibody generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (about 25 kDa each) and two heavy chain polypeptides (about 50-70 kDa each).

[0028] As used in this document, the term light chain or immunoglobulin light chain refers to a polypeptide comprising, from the amino terminus (N-terminal) to the carboxyl terminus (C-terminal), a single immunoglobulin light chain (VL) variable region and a single immunoglobulin light chain (CL) constant domain. The immunoglobulin light chain (CL) constant domain may be a human kappa (κ) or human lambda (λ) constant domain. Petition 870250082204, dated 12 / 09 / 2025, page 30 / 138 15 / 112

[0029] As used in this document, the term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising, from the amino terminus (N-terminal) to the carboxyl terminus (C-terminal), a single variable region of the immunoglobulin heavy chain (VH), a constant domain of the immunoglobulin heavy chain 1 (CH1), an immunoglobulin hinge region, a constant domain of the immunoglobulin heavy chain 2 (CH2), a constant domain of the immunoglobulin heavy chain 3 (CH3), and optionally a constant domain of the immunoglobulin heavy chain 4 (CH4). The heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively.The heavy chains in IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains in IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). The constant domains of immunoglobulin heavy chains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked together through interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0030] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved structural regions (FRs) joined together. Petition 870250082204, dated 12 / 09 / 2025, page 31 / 138 16 / 112 by three hypervariable regions, more frequently called complementarity-determining regions or CDRs. The CDRs of the two chains of each heavy chain and light chain pair are typically aligned by the structure regions to form a structure that specifically binds to a specific epitope on the target protein. From the N-terminus to the C-terminus, the variable regions of naturally occurring light and heavy chains typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system was devised to assign numbers to amino acids occupying positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883. The CDRs and FRs of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29: 185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309 (3): 657-670; 2001).

[0031] 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 but the first constant region domain of the immunoglobulin heavy chain. The Fab fragment contains the variable domains of the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1). Petition 870250082204, dated 12 / 09 / 2025, page 32 / 138 17 / 112 of the heavy chain. Thus, a “Fab fragment” comprises an immunoglobulin light chain (variable region of the light chain (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of an immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The “Fd fragment” comprises the VH and CH1 domains of an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0032] As used in this document, an “Fc fragment” or “Fc region” of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc region may be an Fc region of an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises CH2 and CH3 domains of a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector function, such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0033] As used in this document, the term “F(ab')2 fragment” refers to a bivalent fragment including two Fab' fragments linked by a disulfide bridge between the heavy chains in the hinge region.

[0034] As used in this document, the “Fv” fragment refers to the minimal fragment containing a complete antigen recognition and antibody binding site. This fragment consists of a dimer of a region Petition 870250082204, dated 12 / 09 / 2025, page 33 / 138 18 / 112 Immunoglobulin heavy chain (VH) variable region and an immunoglobulin light chain (VL) variable region in close, non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single variable region of the light or heavy chain (or half of an Fv fragment comprising only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, although at a lower affinity than the entire binding site comprising both VH and VL.

[0035] As used in this document, the term “single-chain variable fragment” or “scFv fragment” comprises the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain and optionally comprising a peptide linker between the VH and VL regions that allows 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).

[0036] As used in this document, a “nanobody” refers to a variable heavy chain region of a heavy chain antibody. Such variable domains are the fully functional smaller antigen-binding fragment of such heavy chain antibodies with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64: 2853-57, 2004. Functional heavy chain antibodies devoid of light chains occur naturally in certain animal species, such as nurse sharks, wobbegong sharks, and Camelidae, such Petition 870250082204, dated 12 / 09 / 2025, page 34 / 138 19 / 112 such as camels, dromedaries, alpacas, and llamas. The antigen-binding site is reduced to a single domain, the VHH domain, in these animals. These antibodies form antigen-binding regions using only the variable region of the heavy chain; that is, these functional antibodies are heavy-chain homodimers having only the H2L2 structure (referred to as "heavy-chain antibodies" or "HCAbs"). The camelized VHH supposedly recombines with constant regions of IgG2 and IgG3 that contain hinge domains, CH2 and CH3, and lack a CH1 domain. Camelized VHH domains have been found to bind to antigen with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and possess high stability in solution (Ewert et al., Biochemistry, Vol. 41: 3628-36, 2002). Methods for generating antibodies having camelized heavy chains are described, for example, in Patent Publications No. US 2005 / 0136049 and 2005 / 0037421.Alternative supports can be prepared from human-variable-like domains that closely resemble the shark V-NAR support and can provide a framework for a long-penetrating loop structure.

[0037] As used herein, the term “heavy chain-only antibody” refers to an immunoglobulin protein consisting of two heavy chain polypeptides (such as, for example, heavy chain polypeptides that are about 50–70 kDa each). A “heavy chain-only antibody” lacks the two light chain polypeptides found in a conventional antibody. Heavy chain antibodies constitute about a quarter of the IgG antibodies produced by camelids, for example, camels and Petition 870250082204, dated 12 / 09 / 2025, page 35 / 138 20 / 112 llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These molecules are formed by two heavy chains, but are devoid of light chains. As a consequence, the variable antigen-binding portion is called the VHH domain and represents the smallest naturally occurring intact antigen-binding site, with only about 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 3746 (1999)), and the VHH portion can be readily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of classic F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)).It has also been shown that sharks have a unique VH-type 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)).

[0038] In some embodiments, a “heavy chain-only antibody” is a dimeric antibody comprising an HV antigen-binding domain and the constant CH2 and CH3 domains, in the absence of the CH1 domain. In some embodiments, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of structure 1, CDR1, structure 2, CDR2, structure 3, CDR3, and structure 4. In some embodiments, an antibody Petition 870250082204, dated 12 / 09 / 2025, page 36 / 138 21 / 112 heavy chain-only antibodies consist of an antigen-binding domain, at least part of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody consists of an antigen-binding domain, at least part of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody consists of an antigen-binding domain, at least part of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included here. The heavy chain-only antibodies described here may belong to the IgG subclass, but heavy chain-only antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also included here. In some embodiments, a heavy chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, for example, the IgG1 or IgG4 subtype.In some embodiments, the heavy chain-only antibody is of the IgG1 or IgG4 subtype, in which one or more of the CH domains are modified to alter an effector function of the antibody. In some embodiments, a heavy chain-only antibody is of the IgG4 subtype, in which one or more of the CH domains are modified to alter an effector function of the antibody. In some embodiments, a heavy chain-only antibody is of the IgG1 subtype, in which one or more of the CH domains are modified to alter an effector function of the antibody. Effector function-altering CH domain modifications are further described herein. Non-limiting examples of heavy chain-only antibodies are described, for example. Petition 870250082204, dated 12 / 09 / 2025, p. 37 / 138 22 / 112 example, in document no. WO2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0039] As used herein, the term “three-chain antibody-like molecule” or “TCA” refers to an antibody-like molecule comprising, essentially consisting of, or consisting of three polypeptide subunits, two of which comprise, essentially consist of, or consist of a heavy chain and a light chain of a monoclonal antibody, or antigen-binding fragments of such antibody chains comprising an antigen-binding region and at least one CH domain. This heavy chain / light chain pair has binding specificity for a first antigen.The third polypeptide subunit comprises, essentially consists of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and one or more antigen-binding domains (such as, for example, two antigen-binding domains) that bind to an epitope of a second antigen or to a different epitope of the first antigen, wherein such binding domain is derived from, or has sequence identity with, the variable region of an antibody heavy or light chain. Portions of such variable region may be encoded by segments of Vh and / or Vl genes, segments of D and Jh genes, or segments of Jl genes. The variable region may be encoded by rearranged VhDJh, VlDJh, VhJl, or VlJl gene segments.

[0040] As used herein, the term “bioreactor” means any vessel useful for growing a cell culture (e.g., a mammalian cell culture or Petition 870250082204, dated 12 / 09 / 2025, page 38 / 138 23 / 112 a bacterial cell culture). Bioreactor encompasses the term fermenter (i.e., a vessel useful for growing a bacterial cell culture, typically containing a more rigorous agitator and increased gas flow compared to a vessel used for growing a mammalian cell culture) herein. Non-limiting examples of bioreactors include stirred tank bioreactors, air transport, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed and / or spouted bed.In some embodiments, an exemplary 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 gas (such as, for example, oxygen), inflow and outflow of fermentation medium or cell culture (e.g., perfusion of fresh cell culture medium into and removal of spent cell culture medium), separation of gas and liquid phases, temperature maintenance, maintenance of oxygen and CO2 levels, pH level maintenance, agitation (e.g., stirring) and / or cleaning / sterilization. Unless otherwise indicated by the context, a bioreactor may be suitable for batch, fed-batch, fed-batch, perfusion and / or continuous fermentation processes. Any suitable bioreactor diameter may be used.Unless otherwise indicated by the context, in some embodiments, the bioreactor may have a volume between about 100 ml and about 50,000 l. Unless otherwise indicated, a bioreactor may be of any size provided it is useful for cell culture; typically, a bioreactor is sized appropriately to the volume of... Petition 870250082204, dated 12 / 09 / 2025, pp. 39 / 138 24 / 112 cell culture being grown inside it. In non-limiting embodiments and unless otherwise indicated by the context, a bioreactor may have at least 1 liter (L) or may have 2, 5, 10, 50, 100, 200, 250, 500, 1000, 1500, 2000, 2500, 5000, 8000, 10000, 12000 liters, 20000 L or more or any intermediate volume. The internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen concentration, temperature, may be controlled during the culture period. Those skilled in the art will be aware of, and will be able to select, bioreactors suitable for use in the manufacturing methods disclosed herein based on relevant considerations.

[0041] As used herein, the term “cell culture” or “culture” refers to the growth and propagation of cells outside of 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, D. Rickwood, ed., Oxford University Press, New York (1992)). Mammalian cells can be cultured in suspension or while attached to a solid substrate. In some embodiments, fluidized bed bioreactors, hollow fiber bioreactors, rolling bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 l to 2000 l bioreactors are used for cell culture (e.g., as part of a seed train).In some embodiments, bioreactors of 1000 L to 2000 L are used for cell culture (e.g., as part of a seed train). Petition 870250082204, dated 12 / 09 / 2025, p. 40 / 138 25 / 112

[0042] As used herein, the term cell culture medium (also known as medium, culture medium, cell culture medium, tissue culture medium and the like) refers to any nutrient solution used to grow cells, for example, bacterial or mammalian cells. Cell culture medium generally provides one or more of the following components: an energy source (e.g., in the form of a carbohydrate, such as, for example, glucose); one or more essential amino acids (e.g., all essential amino acids; the twenty basic amino acids plus cysteine); vitamins and / or other organic compounds typically required at low concentrations; lipids or free fatty acids; and trace elements, such as, for example, inorganic compounds or naturally occurring elements that are typically required at very low concentrations, such as, for example, concentrations in the micromolar range.As used herein, cell culture medium encompasses nutrient solutions that are typically employed and / or known for use in any cell culture process, including, but not limited to, batch, extended batch, fed-batch, intensified and / or perfusion, or continuous cell culture.

[0043] 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 live cells in a given volume of culture medium, as determined by standard viability assays (such as, for example, the trypan blue dye exclusion method). As used in this document, the term cell volume Petition 870250082204, dated 12 / 09 / 2025, page 41 / 138 Packed cell volume (PCV), also referred to as “percentage of packed cell 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. Dec. 20;95(6):122833). Packed cell volume is a function of cell density and cell diameter; increases in packed cell volume can arise from increases in cell density or cell diameter or both. Packed cell volume is a measure of the solid content in the cell culture. Since host cells vary in size and cell cultures also contain dead and dying cells and other cell debris, packed cell volume can describe with a higher degree of accuracy the solid content within a cell culture.

[0044] As used in this document, the term “connected,” in reference to unit operations, refers to a direct connection or mechanism that allows continuous flow between one or more unit operations in a single operational cycle.

[0045] As used in this document, the term “connected,” in reference to unit operations, refers to a direct connection or mechanism that allows continuous flow between one or more unit operations through multiple operational cycles.

[0046] As used in this document, the term dynamic binding capacity, in reference to a chromatography material, refers to the amount of product, for example, polypeptide, the material will bind under conditions of Petition 870250082204, dated 12 / 09 / 2025, page 42 / 138 27 / 112 actual flow before a significant discovery of the unbound product occurs.

[0047] As used in this document, the term “expression vector” or “expression construct” refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the operationally linked expression of the coding sequence in a particular host cell, for example, a mammalian host cell. Vectors may include viral vectors, non-episomic mammalian vectors, plasmids, and other non-viral vectors. An expression vector may include sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of an operationally linked coding region. “Operationally linked” means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions.For example, a control sequence, such as a promoter, in a vector that is "operationally linked to a protein-coding sequence" is arranged such that the normal activity of the control sequence leads to transcription of the protein-coding sequence, resulting in recombinant expression of the encoded protein.

[0048] As used herein, a “fed-batch culture” refers to a form of suspension culture, specifically a method of cell culture in which additional components are supplied to the culture at a time or times subsequent to the start of the culture process. The components supplied typically comprise Petition 870250082204, dated 12 / 09 / 2025, page 43 / 138 28 / 112 Nutritional supplements for cells that have been depleted during the culture process. Alternatively, the additional components may include supplementary components (such as, for example, a cell cycle inhibitor compound). In some embodiments, fed-batch cell culture media formulations may be richer or more concentrated than base cell culture media formulations, which contain components essential for cell survival and growth and are typically used to initiate a cell culture. A fed-batch culture may be stopped at some point, and the cells and / or components in the medium may be collected and optionally purified.

[0049] As used in this document, a fusion protein is a protein containing at least one polypeptide fused to or linked to a heterologous polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence of one protein is frame-attached to, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence of a different protein. The fusion gene can then be expressed by a recombinant host cell to produce the fusion protein. The fusion protein may comprise a fragment of an immunoglobulin protein, such as an Fc region, fused to or linked to a linker polypeptide, a receptor polypeptide, a hormone, cytokine, growth factor, enzyme, or other polypeptide that is not a component of an immunoglobulin. Petition 870250082204, dated 12 / 09 / 2025, page 44 / 138 29 / 112

[0050] As used herein, a “growth phase of a cell culture” refers to the period of exponential cell growth (i.e., the logarithmic phase) in which cells typically divide rapidly.

[0051] As used in this document, the term harvested cell culture fluid refers to a solution that has been processed by one or more operations to separate cells, cell debris, or other large particles from recombinant protein. Such operations, as described in this document, include, but are not limited to, cooling, flocculation, acidification, centrifugation, neutralization, acoustic wave separation, and various forms of filtration (e.g., depth filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration). Harvested cell culture fluid includes cell culture lysates as well as cell culture supernatants.The collected cell culture fluid can be further clarified to remove fine particulate matter and soluble aggregates by filtration with a membrane having a pore size between about 0.1 µm and about 0.5 µm, such as, for example, a membrane having a pore size of about 0.22 µm.

[0052] As used in this document, a “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene of interest. The term includes the offspring of the parent cell, whether or not the offspring are identical in morphology or genetic makeup to the original parent cell, provided that the gene of interest Petition 870250082204, dated 12 / 09 / 2025, page 45 / 138 30 / 112 is present. A host cell comprising a nucleic acid encoding a recombinant protein, for example, operationally linked to at least one expression control sequence (e.g., promoter or enhancer) is a “recombinant host cell.” A host cell, when cultured under appropriate conditions, can synthesize a recombinant protein that can be subsequently collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted).

[0053] As used in this document, “high molecular weight” or “HMW” species of a recombinant protein of interest refer to dimers, oligomers, and aggregates of the recombinant protein that have a molecular weight greater than the molecular weight of the intact, fully assembled form of the recombinant protein.

[0054] As used in this document, the term “impurity” refers to a component other than the recombinant protein of interest, together with its associated buffer components. Impurities include, but are not limited to, process- and product-related impurities such as, for example, host cell proteins, leached resin materials (such as, for example, leached protein A), nucleic acids, HMW species of the recombinant protein, LMW species of the recombinant protein, endotoxins, viral contaminants, cell culture media components, and the like.

[0055] As used herein, the term “charge density” refers to the amount of composition brought into contact with a volume of chromatography material. Petition 870250082204, dated 12 / 09 / 2025, page 46 / 138 31 / 112

[0056] As used in this document, low molecular weight or LMW species of a recombinant protein of interest refers to fragments, truncated forms, or other incomplete variants of the recombinant protein that have a molecular weight lower than the molecular weight of the intact, fully assembled form of the recombinant protein. LMW species may include, but are not limited to, proteolytic fragments, truncated forms resulting from cellular expression of mRNA splicing variants, and single-component polypeptides in the case of multi-polypeptide chain proteins (such as, for example, light-chain only or heavy-chain only species when the recombinant protein is an antibody).

[0057] As used herein, a perfusion cell culture medium refers to a cell culture medium that is typically used in cell cultures that are maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. In some embodiments, perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate the method used to remove spent medium. In some embodiments, perfusion cell culture medium may be used during both the growth and production phases.

[0058] As used in this document, the term polishing chromatography refers to a chromatography operation performed after a capture or affinity chromatography operation to remove remaining impurities and obtain a more highly chromatographic composition. Petition 870250082204, dated 12 / 09 / 2025, page 47 / 138 32 / 112 purified and / or recombinant protein. Common impurities removed during polishing steps include, but are not limited to, product-related impurities (e.g., HMW and LMW species), host cell proteins, DNA, leached protein A, viral contaminants, and endotoxins. Furthermore, typical chromatographic techniques used for polishing include, but are not limited to, ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimodal (or mixed-mode) chromatography (MMC).

[0059] As used in this document, “anion exchange chromatography (AEX) refers to a form of ion exchange chromatography performed on a solid-phase medium (e.g., resin or membrane) that is positively charged and has the ability to exchange free anions for anions in an aqueous solution passed over or through the solid phase. AEX chromatography is used, for example, for viral clearance and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL EDM TMAE™, FRACTOGEL EDM DEAE™ (EMD)). Merck), TOYOPEARL® Super Q® and TOYOPEARL® NH2-750F (Tosoh Bioscience), POROS HQ™ and POROS XQ™ (ThermoFisher).

[0060] As used in this document, “cation exchange chromatography (CEX) refers to a form of ion exchange chromatography performed on a solid-phase material (e.g., resin or membrane) that is negatively charged and has the ability to exchange free cations for cations in an aqueous solution passed over or through the solid phase. The charge may be Petition 870250082204, dated 12 / 09 / 2025, page 48 / 138 33 / 112 provided by attaching one or more charged ligands to the solid phase, for example, through covalent bonding. Alternatively or additionally, the charge may be an inherent property of the solid phase (e.g., silica, which has an overall negative charge). CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process-related impurities, and / or viral contaminants.Commercially available cation exchange media include, but are not limited to, sulfopropyl (SP) immobilized in agarose (e.g., SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™ or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO3™, FRACTOGEL-SE HICAP™ and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL® XS, TOYOPEARL® HS (Tosoh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), S Ceramic Hyper™ DF (Pall, Port Washington, NY), POROS™ (ThermoFisher, Waltham, MA), ESHMUNO® CSP and ESHMUNO® CP-FT (Millipore Sigma, Darmstadt, Germany).

[0061] As used in this document, hydrophobic interaction chromatography (HIC) refers to chromatography performed in a solid-phase medium that makes use of 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™ (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto™ phenyl (Cytiva).

[0062] As used in this document, mixed-mode or multimodal chromatography (MMC) refers to Petition 870250082204, dated 12 / 09 / 2025, page 49 / 138 34 / 112 refers to chromatography that uses more than one form of interaction between the stationary phase and the analyte to achieve separation. MMC differs from single-mode chromatography because two or more types of interaction, such as electrostatic, hydrogen bonding, and / or hydrophobic interactions, contribute significantly to solute retention. Commercially available multimodal chromatography media include, but are not limited to, Capto™ Adhere, Capto™ MMC Impress, Capto MMC (Cytiva), PPA Hypercell, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno HCX (Merk Millipore), and TOYOPEARL® MX-Trp-650M (Tosoh Bioscience).

[0063] Polishing chromatography unit operations make use of materials (e.g., resins and / or membranes) containing agents that can be operated in a variety of modes, including binding and elution mode and continuous flow mode. In binding and elution chromatography, a biomolecule of interest is typically loaded onto the chromatography material to maximize dynamic binding capacity, and then specified recovery and elution conditions are used to maximize the purity of the product in the eluate. In contrast, in continuous flow chromatography, loading conditions are employed that allow impurities to bind to the chromatography material while the biomolecule of interest passes through. Compared to binding and elution chromatography, continuous flow chromatography allows for higher loading densities for many biomolecules.

[0064] In addition to the two most common modes, weak partition chromatography, overload chromatography and modes of Petition 870250082204, dated 12 / 09 / 2025, page 50 / 138 35 / 112 Forward chromatography can also be employed in purification processes. In weak partition chromatography, an isocratic separation method, the flow mode is altered by identifying solution conditions that promote weak binding of a biomolecule to the resin (Kp is about 0.1 to about 100, compared to a Kp less than about 0.1 for continuous flow chromatography), in addition to the binding of one or more impurities. In overload chromatography, the biomolecule of interest is loaded onto the chromatography material beyond the dynamic binding capacity of the material. Additionally, forward chromatography allows for a continuous high-density feed (containing the protein of interest and at least one impurity) into the chromatography medium. In forward chromatography, the separation of the protein of interest from impurities and contaminants is driven by the binding affinity of the components in the feed to the chromatography medium.The amount of protein of interest that can be loaded and bound to the chromatography medium in forward mode is typically dependent on the amount of highly loaded impurities / contaminants, such as product-related impurities, in the feedstock. Initially, all components in the feedstock will bind to the chromatography medium. The separation of the product of interest from the impurities / contaminants is driven by affinity for the chromatography medium. When the chromatography medium reaches saturation binding, those components in the feedstock that have a higher affinity for the chromatography medium (typically product-related impurities, such as a type of HMW) will displace. Petition 870250082204, dated 12 / 09 / 2025, page 51 / 138 36 / 112 proteins that have a weaker affinity (the product of interest), resulting in the separation of the weaker-affinity proteins from the chromatography medium. These proteins exit the column in the loading stream as a band. As loading progresses, the bound proteins are continuously displaced in order of increasing affinity into the chromatographic medium until the column is at or near saturation with proteins having a higher affinity than the protein of interest.

[0065] As used in this document, the term polypeptide refers to a polymer of amino acids comprising at least 50 amino acids, such as, for example, at least 100 amino acids.

[0066] As used in this document, the term partition coefficient or product partition coefficient (Kp) refers to the molar concentration of the product, for example, recombinant protein, bound to the stationary phase divided by the molar concentration of the product in the mobile phase during a chromatography step.

[0067] As used herein, a production cell culture medium refers to a cell culture medium that is typically used in a cell culture during the transition when exponential growth is ending and protein production takes over (i.e., transition and / or product phases) and is sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase. A production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of the cell culture. Petition 870250082204, dated 12 / 09 / 2025, page 52 / 138 37 / 112

[0068] As used in this document, a “production phase of a cell culture” refers to the period of time during which logarithmic cell growth has ended and recombinant protein production is predominant.

[0069] As used in this document, the term “recombinant protein” refers to a heterologous protein produced by a host cell transfected with a nucleic acid that encodes the protein when the host cell is grown in cell culture.

[0070] As used in this document, the term “purified,” when used in relation to a composition, refers to a composition in which at least one impurity is present at a lower concentration in the purified composition compared to the composition as it existed before one or more unit operations. Additionally, a “purified” recombinant protein (e.g., a purified antibody) refers to a recombinant purity that has been increased in purity so that it exists in a form that is purer than it exists in its natural environment and / or when initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0071] As used in this document, the term “titrant” refers to a solution of known concentration that is added to another solution during a titration. An “acidic titrant” refers to a titrant with a pH less than about 7.

[0072] As used in this document, the term “unit operation” refers to a functional step that is Petition 870250082204, dated 12 / 09 / 2025, page 53 / 138 38 / 112 performed as part of the purification process of a recombinant protein of interest. Unit operations may be designed to achieve a single objective or multiple objectives, such as capture, acid precipitation, centrifugation, or chromatography steps. Unit operations may also include retention or storage steps between processing steps. NON-LIMITING EXAMPLE CHARACTERISTICS:

[0073] Without limitation, some exemplary features / features of the present disclosure include E1-E47: E1. A method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity comprising: loading the composition onto an anion exchange material comprising a primary amine ligand at a charge 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein. E2. The method according to E1, wherein the anion exchange material comprises resin particles. E3. The method according to E1 or E2, wherein the anion exchange material comprises resin particles, in which Petition 870250082204, dated 12 / 09 / 2025, page 54 / 138 39 / 112 less about 80% of the resin particles have a particle size of about 30 μm to about 60 μm. 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. E5. The method according to E1-E4, wherein the anion exchange material comprises a polyamine ligand. E6. The method according to any one of E1-E5, wherein the anion exchange material comprises a methacrylate-containing polymer base matrix. E7. The method according to any of E1-E6, wherein the anion exchange material is TOYOPEARL® NH2-750F. E8. The method according to any of E1-E7, wherein the charge density is less than about 600 g / l of anion exchange material. E9. The method according to any of E1-E7, where the charge density is about 200 g / l to about 600 g / l of anion exchange material. E10. The method according to any of E1-E9, in which the composition has a conductivity of about 3 mS / cm to about 6 mS / cm. E11. The method according to any of E1-E10, wherein the partition coefficient of the anion exchange material for the recombinant protein is about 0.1 to about 100. E12. The method follows any of E1-E10, where the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 40. E13. The method according to any one of E1-E12, wherein the method comprises using an equilibrium buffer and / or a recovery buffer with the anion exchange material, wherein: Petition 870250082204, dated 12 / 09 / 2025, page 55 / 138 40 / 112 the pH of the equilibrium buffer and / or the recovery buffer is about 7.0 to about 8.0; and / or the conductivity of the equilibrium buffer and / or the recovery buffer is less than about 10 mS / cm. E14. The method according to E13, in which the conductivity of the equilibrium buffer and / or recovery buffer is about 2 mS / cm to about 4 mS / cm. E15. The method according to any one of E1-E14, additionally comprising performing a low pH viral inactivation unit operation prior to loading (e.g., one or more unit operations prior to loading). E16. The method according to E15, in which the low pH viral inactivation unit operation is carried out at a pH of about 3.5 to about 3.7. E17. The method according to E15 or E16, wherein the low pH viral inactivation unit operation employs an acidic titrant. E18. The method according to E17, wherein the acid titrant comprises formic acid. E19. The method according to E17 or E18, wherein the acid titrant is formic acid of about 1 M to about 2 M (e.g., formic acid of about 1 M; formic acid of about 2 M). E20. The method according to any of E15-E19, in which the low pH viral inactivation unit operation is performed for at least about 60 minutes. E21. The method follows any of E15-E20, in which the low pH viral inactivation unit operation is performed for approximately 60 minutes to approximately 12 hours. Petition 870250082204, dated 12 / 09 / 2025, p. 56 / 138 41 / 112 E22. The method according to any of E1-E21 which additionally comprises performing one or more additional chromatographic unit operations. E23. The method according to E22, wherein one or more additional chromatography unit operations comprise an affinity chromatography unit operation performed before loading. E24. The method according to E23, wherein the affinity chromatography unit operation is selected from protein chromatography A, protein chromatography G, protein chromatography L and CH1 domain chromatography. E25. The method according to E23 or E24, wherein the affinity chromatography unit operation is protein chromatography A. E26. The method according to any one of E22-E25, wherein one or more additional chromatography unit operations comprise an additional polishing chromatography unit operation performed before loading. E27. The method according to any one of E22-E25, wherein one or more additional chromatography unit operations comprise an additional polishing chromatography unit operation performed after loading. E2 8. The method according to E2 6 or E2 7, wherein the polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography and mixed-mode chromatography. Petition 870250082204, dated 12 / 09 / 2025, page 57 / 138 42 / 112 E2 9. The method according to any one of E26-E28, wherein the polishing chromatography unit operation and loading are continuous or connected. E30. The method according to any of E1-E29, further comprising performing a viral filtration unit operation and / or a UF / DF unit operation after loading. E31. Method, according to E30, in which the loading and the viral filtration unit operation and / or the UF / DF unit operation are continuous or connected. E32. The method according to any of E1-E31, wherein less than about 5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. E33. The method according to any of E1-E32, wherein less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. E34. The method according to any of E1-E33, wherein less than about 1% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. E35. The method according to any one of E1-E34, wherein the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. E36. The method according to any one of E1-E35, wherein the purified composition comprises at least about 90% by w / w of the recombinant protein in the composition before loading. Petition 870250082204, dated 12 / 09 / 2025, page 58 / 138 43 / 112 E37. The method according to any one of E1-E36, wherein: less than about 1% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and the purified composition comprises at least about 90% by w / w of the recombinant protein in the composition prior to loading. E38. The method according to any one of E1-E37, where: the charge density is approximately 100 g / l, while the anion exchange material is approximately 600 g / l; Less than about 1% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and the purified composition comprises at least about 90% by w / w of the recombinant protein in the composition prior to loading. E39. The method according to any one of E1-E38, wherein the recombinant protein is an antigen-binding protein. E40. The method according to any one of E1-E39, where the recombinant protein is an antibody. E41. The method according to any of E1-E40, wherein at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components, and viral contaminants. Petition 870250082204, dated 12 / 09 / 2025, page 59 / 138 44 / 112 E42. The method according to any one of E1-E41, wherein at least one impurity is selected from a high molecular weight species of recombinant protein. 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 a primary amine linker. 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 polymer and are functionalized with a primary amine ligand. E45. The method according to E43 or E44, wherein at least 80% of the resin particles have a particle size of about 30 µm to about 60 µm. E46. The method, according to any one of E43, E44 or E45, in which the resin particles have an average particle size of about 40 µm to about 50 µm. E47. The method according to E46, wherein the resin particles have an average particle size of about 45 µm. Anion Exchange Chromatography Purification Methods

[0074] A method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity is provided herein, wherein the method comprises: loading the composition onto an anion exchange material comprising a primary amine ligand at a charge density greater than about 100 g / L of anion exchange material, wherein: Petition 870250082204, dated 12 / 09 / 2025, page 60 / 138 45 / 112 the composition has a pH of about 7.0 to about 8.0 and a conductivity of less than about 10 mS / cm; and at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein.

[0075] In some embodiments, the anion exchange medium comprises resin particles.

[0076] In some embodiments, the anion exchange medium comprises resin particles, wherein at least 80% of the resin particles have a particle size of about 30 µm to about 60 µm.

[0077] 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 Petition 870250082204, dated 12 / 09 / 2025, page 61 / 138 46 / 112 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.

[0078] In some embodiments, the anion exchange medium comprises a polyamine ligand.

[0079] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix.

[0080] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix and a polyamine binder.

[0081] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, 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 the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, wherein the resin particles have an average particle size of Petition 870250082204, dated 12 / 09 / 2025, page 62 / 138 47 / 112 about 40 pm to about 50 pm (such as, for example, about 45 pm).

[0082] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, 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 the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, wherein the resin particles have an average particle size of about 40 µm to about 50 µm (such as, for example, about 45 µm).

[0083] In some embodiments, the anion exchange medium is TOYOPEARL® NH2-750F.

[0084] In some embodiments, the charge density is less than about 750 g / l of anion exchange material. In some embodiments, the charge density is less than about 700 g / l of anion exchange material. In some embodiments, the charge density is less than about 650 g / l of anion exchange material. In some embodiments, the charge density is less than about 600 g / l of anion exchange material. In some embodiments, the charge density is less than about 550 g / l of anion exchange material. Petition 870250082204, dated 12 / 09 / 2025, page 63 / 138 48 / 112 anionic. In some embodiments, the charge density is less than about 500 g / l of anionic exchange material. In some embodiments, the charge density is less than about 450 g / l of anionic exchange material. In some embodiments, the charge density is less than about 400 g / l of anionic exchange material. In some embodiments, the charge density is less than about 350 g / l of anionic exchange material. In some embodiments, the charge density is less than about 300 g / l of anionic exchange material. In some embodiments, the charge density is less than about 250 g / l of anionic exchange material. In some embodiments, the charge density is less than about 200 g / l of anionic exchange material. In some embodiments, the charge density is less than about 150 g / l of anionic exchange material.

[0085] In some embodiments, the charge density is about 100 g / l of anion exchange material. In some embodiments, the charge density is about 150 g / l of anion exchange material. In some embodiments, the charge density is about 200 g / l of anion exchange material. In some embodiments, the charge density is about 250 g / l of anion exchange material.

[0086] 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 Petition 870250082204, dated 12 / 09 / 2025, p. 64 / 138 49 / 112 pH of approximately 7.4 to approximately 7.6. In some forms, the composition has a pH of approximately 7.5.

[0087] In some modalities, the composition has a conductivity of less than about 9 mS / cm. In some modalities, the composition has a conductivity of less than about 8 mS / cm. In some modalities, the composition has a conductivity of less than about 7 mS / cm. In some modalities, the composition has a conductivity of less than about 6 mS / cm. In some modalities, the composition has a conductivity of less than about 5 mS / cm. In some modalities, the composition has a conductivity of less than about 4 mS / cm.

[0088] 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 forms, the composition has a conductivity of approximately 5 mS / cm. In some forms, the composition has a conductivity of approximately 4.5 mS / cm.In some forms, the composition has a conductivity of... Petition 870250082204, dated 12 / 09 / 2025, p. 65 / 138 50 / 112 approximately 4 mS / cm. In some forms, the composition has a conductivity of approximately 3.5 mS / cm. In some forms, the composition has a conductivity of approximately 3 mS / cm.

[0089] 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.

[0090] In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 0.1. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 10. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 20. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 30. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 40. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 50. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 60.In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is less than about 70. In some embodiments, the partition coefficient of the exchange material... Petition 870250082204, dated 12 / 09 / 2025, page 66 / 138 The anionic partition coefficient for recombinant protein is less than about 80. In some embodiments, the partition coefficient of the anionic exchange material for recombinant protein is less than about 90. In some embodiments, the partition coefficient of the anionic exchange material for recombinant protein is less than about 100.

[0091] In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 0.1 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 10 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 100. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 90. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 80. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 70. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 60.In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 50. In some embodiments, the partition coefficient of the anion exchange material for the recombinant protein is about 20 to about 40. Petition 870250082204, dated 12 / 09 / 2025, page 67 / 138 52 / 112

[0092] In some embodiments, the method comprises using an equilibrium buffer and / or a recovery buffer with the anion exchange material, wherein: The pH of the equilibrium buffer and / or the recovery buffer is about 7.0 to about 8.0; and / or the conductivity of the equilibrium buffer and / or the recovery buffer is less than about 10 mS / cm.

[0093] In some embodiments, the equilibrium buffer and / or recovery buffer has a pH of about 7.1 to about 7.9. In some embodiments, the equilibrium buffer and / or recovery buffer has a pH of about 7.2 to about 7.8. In some embodiments, the equilibrium buffer and / or recovery buffer has a pH of about 7.3 to about 7.7. In some embodiments, the equilibrium buffer and / or recovery buffer has a pH of about 7.4 to about 7.6. In some embodiments, the equilibrium buffer and / or recovery buffer has a pH of about 7.5.

[0094] In some embodiments, the balance buffer and / or recovery buffer has a conductivity of less than about 9 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of less than about 8 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of less than about 7 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of less than about 6 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of less than about 5 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity Petition 870250082204, dated 12 / 09 / 2025, p. 68 / 138 53 / 112 less than about 4 mS / cm. In some modalities, the equilibrium buffer and / or recovery buffer has a conductivity less than about 3 mS / cm. In some modalities, the equilibrium buffer and / or recovery buffer has a conductivity less than about 2 mS / cm.

[0095] In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 10 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 9.5 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 9 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 8.5 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 8 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 7.5 mS / cm. In some embodiments, the balance buffer and / or recovery buffer has a conductivity of about 7 mS / cm. In some modalities, the equilibrium buffer and / or recovery buffer has a conductivity of approximately 6.5 mS / cm.In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of approximately 6 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of approximately 5.5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of approximately 5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of approximately... Petition 870250082204, dated 12 / 09 / 2025, p. 69 / 138 54 / 112 of 4.5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 4 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 3.5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 3 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 2.5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 2 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1.5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1 mS / cm.

[0096] In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 6 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 5 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 4 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 1 mS / cm to about 3 mS / cm.

[0097] In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 6 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 5 mS / cm. In Petition 870250082204, dated 12 / 09 / 2025, p. 70 / 138 55 / 112 In some embodiments, the equilibrium buffer and / or the recovery buffer has a conductivity of about 2 mS / cm to about 4 mS / cm. In some embodiments, the equilibrium buffer and / or recovery buffer has a conductivity of about 2 mS / cm to about 3 mS / cm.

[0098] In some embodiments, the method further comprises performing a low pH viral inactivation unit operation prior to loading (e.g., one or more unit operations prior to loading). In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.5 to about 3.7. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.5. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.6. In some embodiments, the low pH viral inactivation unit operation is performed at a pH of about 3.7.

[0099] In some embodiments, the low pH viral inactivation unit operation employs an acidic titrant. In some embodiments, the acidic titrant is formic acid. In some embodiments, the acidic titrant is about 1 M to about 2 M formic acid. In some embodiments, the acidic titrant is about 1 M formic acid. In some embodiments, the acidic titrant is about 2 M formic acid.

[0100] In some embodiments, the low pH viral inactivation unit operation is performed for at least about 60 minutes. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 2 hours. In some embodiments, the unit operation Petition 870250082204, dated 12 / 09 / 2025, p. 71 / 138 56 / 112 low pH viral inactivation is performed for at least about 3 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 4 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 5 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 6 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 7 hours. In some embodiments, the low pH viral inactivation unit operation is performed for at least about 8 hours. In some embodiments, the low pH viral inactivation unit operation is performed for about 60 minutes to about 12 hours. In some embodiments, the low pH viral inactivation unit operation is performed for about 60 minutes to about 8 hours.

[0101] In some embodiments, the method further comprises performing one or more additional chromatography unit operations.

[0102] In some embodiments, one or more additional chromatography unit operations comprise an affinity chromatography unit operation performed prior to loading. In some embodiments, the affinity chromatography unit operation is selected from protein A chromatography, protein G chromatography, protein L chromatography, and CH1 domain chromatography. In some embodiments, the affinity chromatography unit operation is protein A chromatography. In some embodiments, the affinity chromatography unit operation is protein G chromatography. In some Petition 870250082204, dated 12 / 09 / 2025, page 72 / 138 In 57 / 112 embodiments, the unit operation of affinity chromatography is L-protein chromatography. In some embodiments, the unit operation of affinity chromatography is CH1-domain chromatography.

[0103] In some embodiments, one or more additional chromatography unit operations comprise an additional polishing chromatography unit operation performed before loading. In some embodiments, one or more additional chromatography unit operations comprise an additional polishing chromatography unit operation performed after loading. In some embodiments, the additional polishing chromatography unit operation and the load are connected. In some embodiments, the additional polishing chromatography unit operation and the load are not connected. In some embodiments, the additional polishing chromatography unit operation and the load are continuous. In some embodiments, the additional polishing chromatography unit operation and the load are not continuous.

[0104] In some embodiments, the polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. In some embodiments, the additional polishing chromatography unit operation is cation exchange chromatography. In some embodiments, the additional polishing chromatography unit operation is hydrophobic interaction chromatography. In some embodiments, the additional polishing chromatography unit operation is mixed-mode chromatography. Petition 870250082204, dated 12 / 09 / 2025, page 73 / 138 58 / 112

[0105] In some embodiments, the method additionally comprises performing a viral filtration unit operation and / or a UF / DF unit operation after loading.

[0106] In some embodiments, less than about 5% w / w (for example, 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) of the recombinant protein in the purified composition is high molecular weight recombinant protein species.

[0107] In some embodiments, the purified composition comprises at least about 85% w / w (e.g., at least about 90% w / w, at least about 95% w / w) of the recombinant protein in the composition before loading.

[0108] In some embodiments, less than about 1% w / w of the recombinant protein in the purified composition is high molecular weight species of the recombinant protein; and the purified composition comprises at least about 90% w / w of the recombinant protein in the composition before loading.In some embodiments, the loading density is about 100 g / l to about 600 g / l (e.g., about 150 g / l to about 600 g / l; about 200 g / l to about 600 g / l; about 250 g / l to about 600 g / l) of anion exchange material; less than about 1% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and the purified composition comprises at least about 90% by w / w of the recombinant protein in the composition before loading. Petition 870250082204, dated 12 / 09 / 2025, p. 74 / 138 59 / 112

[0109] 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.

[0110] 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.

[0111] In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody.

[0112] In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody.

[0113] In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0114] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components, and viral contaminants. In some embodiments, at least one impurity is selected from high molecular weight recombinant protein species.

[0115] Also provided in this document is a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, Petition 870250082204, dated 12 / 09 / 2025, page 75 / 138 60 / 112 (a type of high molecular weight recombinant protein), wherein the method comprises: loading the composition into an anion exchange material comprising resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine linker, to a loading density of about 250 g / l to about 600 g / l of anion exchange material, wherein: The composition has a pH of approximately 7.0 to approximately 8.0 and a conductivity of less than about 10 mS / cm (e.g., about 3 mS / cm to about 6 mS / cm); and at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein.

[0116] 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 (such as, for example, about 45 µm).

[0117] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with an amine ligand. Petition 870250082204, dated 12 / 09 / 2025, page 76 / 138 61 / 112 primary and, additionally, wherein at least about 80% of the resin particles have a particle size of about 30 pm to about 60 pm. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise hydroxylated methacrylic polymer and are functionalized with a primary amine ligand and, additionally, wherein the resin particles have an average particle size of about 40 pm to about 50 pm (such as, for example, about 45 pm).

[0118] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. In some embodiments, the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species and the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading.

[0119] Also provided in this document is a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: to load the composition into an anion exchange material comprising a primary amine ligand at a density Petition 870250082204, dated 12 / 09 / 2025, page 77 / 138 62 / 112 of charge of about 250 g / l to about 600 g / l of anion exchange material, wherein: 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein, wherein: Less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading.

[0120] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components, and viral contaminants.

[0121] In some embodiments, at least one impurity is selected from high molecular weight species of the recombinant protein.

[0122] In some embodiments, the anion exchange medium comprises a polyamine ligand.

[0123] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix. Petition 870250082204, dated 12 / 09 / 2025, page 78 / 138 63 / 112

[0124] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix and a polyamine binder.

[0125] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, 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 the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, the resin particles have an average particle size of about 40 µm to about 50 µm (such as, for example, about 45 µm).

[0126] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, 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 material. Petition 870250082204, dated 12 / 09 / 2025, page 79 / 138 64 / 112 anion exchange comprises resin particles, wherein the resin particles comprise hydroxylated methacrylic polymer and are functionalized with a primary amine ligand and, furthermore, wherein the resin particles have an average particle size of about 40 pm to about 50 pm (such as, for example, about 45 pm).

[0127] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. In some embodiments, the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species and the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading.

[0128] A method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein) is further provided in this document, wherein the method comprises: loading the composition into an anion exchange material comprising a polyamine binder at a charge density of about 250 g / l to about 600 g / l of anion exchange material, wherein: Petition 870250082204, dated 12 / 09 / 2025, page 80 / 138 65 / 112 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collect a purified composition comprising the recombinant protein, wherein: Less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading.

[0129] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components, and viral contaminants.

[0130] In some embodiments, at least one impurity is selected from high molecular weight species of the recombinant protein.

[0131] In some embodiments, the anion exchange material additionally comprises a methacrylate-containing polymer base matrix.

[0132] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. In some embodiments, the purified composition comprises Petition 870250082204, dated 12 / 09 / 2025, page 81 / 138 66 / 112 at least about 85% by w / w of recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of recombinant protein in the purified composition is high molecular weight recombinant protein species and the purified composition comprises at least about 85% by w / w of recombinant protein in the composition before loading.

[0133] Also provided in this document is a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: Perform a low-pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition into an anion exchange material comprising a primary amine ligand at a charge density of about 250 g / l to about 600 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 10 mS / cm (for example, about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and The low pH viral inactivation unit operation is performed one or more unit operations prior to loading; and Petition 870250082204, dated 12 / 09 / 2025, page 82 / 138 67 / 112 collect a purified composition comprising recombinant protein.

[0134] In some embodiments, at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components, and viral contaminants.

[0135] In some embodiments, at least one impurity is selected from high molecular weight species of the recombinant protein.

[0136] In some embodiments, the acid titrant is about 1 M to about 2 M formic acid. In some embodiments, the acid titrant is about 1 M formic acid. In some embodiments, the acid titrant is about 2 M formic acid.

[0137] In some embodiments, the anion exchange medium comprises a polyamine ligand.

[0138] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix.

[0139] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix and a polyamine binder.

[0140] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine linker. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles Petition 870250082204, dated 12 / 09 / 2025, page 83 / 138 68 / 112 comprise polymethacrylate and are functionalized with a primary amine ligand and, additionally, wherein at least about 80% of the resin particles have a particle size of about 30 pm to about 60 pm. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand and, additionally, the resin particles have an average particle size of about 40 pm to about 50 pm (such as, for example, about 45 pm).

[0141] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand.In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, 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 the resin particles comprise hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, the resin particles have an average particle size of about 40 µm to about 50 µm (such as, for example, about 45 µm).

[0142] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. Petition 870250082204, dated 12 / 09 / 2025, page 84 / 138 69 / 112 In some embodiments, the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species, and the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading.

[0143] A method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity selected from a high molecular weight species of the recombinant protein is further provided herein, wherein the method comprises: To perform a low pH viral inactivation unit operation using approximately 1 M to approximately 2 M formic acid as an acid titrant; loading the composition into an anion exchange material comprising a polyamine binder at a charge density of about 250 g / l to about 600 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 10 mS / cm (for example, about 3 mS / cm to about 6 mS / cm); at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and Petition 870250082204, dated 12 / 09 / 2025, page 85 / 138 70 / 112 The low pH viral inactivation unit operation is performed one or more times prior to loading; and a purified composition comprising the recombinant protein is collected.

[0144] In some embodiments, the acid titrant is approximately 1 M formic acid. In some embodiments, the acid titrant is approximately 2 M formic acid.

[0145] In some embodiments, the anion exchange material additionally comprises a methacrylate-containing polymer base matrix.

[0146] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. In some embodiments, the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species and the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading.

[0147] Also provided in this document is a method for purifying a recombinant protein (for example, an antigen-binding protein, such as, for example, an antibody) from a composition comprising the recombinant protein and at least one impurity (for example, a high molecular weight species of the recombinant protein), wherein the method comprises: Petition 870250082204, dated 12 / 09 / 2025, page 86 / 138 71 / 112 perform a low pH viral inactivation unit operation using formic acid as an acid titrant; loading the composition into an anion exchange material comprising a primary amine ligand at a charge density of about 250 g / l to about 600 g / l of anion exchange material, wherein: The composition has a pH of about 7.0 to about 8.0 and a conductivity of about 3 mS / cm to about 6 mS / cm; at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and The low-pH viral inactivation unit operation is performed one or more times prior to loading; and a purified composition comprising recombinant protein is collected, wherein: Less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition prior to loading.

[0148] In some embodiments, the anion exchange medium comprises a polyamine ligand.

[0149] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix. Petition 870250082204, dated 12 / 09 / 2025, page 87 / 138 72 / 112

[0150] In some embodiments, the anion exchange material comprises a methacrylate-containing polymer base matrix and a polyamine binder.

[0151] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, 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 the resin particles comprise polymethacrylate and are functionalized with a primary amine ligand, and additionally, the resin particles have an average particle size of about 40 µm to about 50 µm (such as, for example, about 45 µm).

[0152] In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand. In some embodiments, the anion exchange material comprises resin particles, wherein the resin particles comprise a hydroxylated methacrylic polymer and are functionalized with a primary amine ligand, and additionally, 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 material. Petition 870250082204, dated 12 / 09 / 2025, pp. 88 / 138 73 / 112 anion exchange comprises resin particles, wherein the resin particles comprise hydroxylated methacrylic polymer and are functionalized with a primary amine ligand and, furthermore, wherein the resin particles have an average particle size of about 40 pm to about 50 pm (such as, for example, about 45 pm).

[0153] In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species. In some embodiments, the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. In some embodiments, less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species and the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading. Host Cells

[0154] The cell lines (also referred to as cells or “host cells”) used in the present disclosure are genetically modified to express a recombinant protein of commercial or scientific interest. The cells may be suitable for culture, transfection and expression of adherent, monolayer and / or suspension recombinant proteins, such as, for example, antibodies. The cells may be used, for example, with batch, fed-batch and perfusion or continuous culture methods. Such cells are typically cell lines obtained or derived from mammals and are capable of growing and surviving when placed in monolayer or continuous culture. Petition 870250082204, dated 12 / 09 / 2025, page 89 / 138 74 / 112 Suspension culture in a medium containing appropriate nutrients and / or other factors, such as those described in this document. Host cells are typically selected that can express and secrete proteins, or that can be molecularly manipulated to express and secrete large amounts of a particular protein, more particularly, a glycoprotein of interest, into the culture medium. The selection of an appropriate host cell for the expression of a recombinant protein will depend on several factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule. In some embodiments, the host cell producing the recombinant protein to be purified by a method provided in this document is a mammalian host cell.

[0155] Cell lines are typically derived from a primary culture line that can be maintained in culture indefinitely. The cells may contain, introduced, for example, through transformation, transfection, infection, or injection, expression vectors (constructs), such as plasmids and the like, which harbor coding sequences, or portions thereof, that encode proteins for expression and production in the culture process. Such expression vectors contain the elements necessary for the transcription and translation of the inserted coding sequence. Methods that are well known and practiced by those skilled in the art can be used to construct expression vectors containing Petition 870250082204, dated 12 / 09 / 2025, pp. 90 / 138 75 / 112 sequences encoding the desired proteins and polypeptides, as well as the appropriate transcriptional and translational control elements. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainview, NY, or any of the earlier editions; FM Ausubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or any of the earlier editions; Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for any purpose.

[0156] Host cells include, but are not limited to, those that are commercially available, for example, from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0157] Exemplary 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, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. In some embodiments, eukaryotic microbes such as filamentous fungi or yeasts are cloning or expression hosts. Petition 870250082204, dated 12 / 09 / 2025, pp. 91 / 138 76 / 112 suitable for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful in the present document, such as Pichia, for example, P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis, and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0158] Vertebrate host cells are also suitable hosts for expressing recombinant proteins. Mammalian cell lines available as hosts for 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 cells (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216, 1980); SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 subcloned cells for growth in suspension culture, Graham et al., J. Gen Virol. 36; 59, 1977); Neonatal hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod.23: 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL. Petition 870250082204, dated 12 / 09 / 2025, page 92 / 138 77 / 112 2); canine renal cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells and various other cell lines. In some embodiments, the host cells are selected from among CHO cells.

[0159] In some embodiments, the host cells are eukaryotic cells, such as mammalian cells. Mammalian cells may be, for example, human, rodent, or bovine cell lines or strains. Examples of such cells, cell lines, or cell strains include, but are not limited to, mouse myeloma cell line (NSO), Chinese hamster ovary cell line (CHO), FIT 1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (neonatal hamster kidney cell), VERO, SP2 / 0, HB2 / 0, H0, C127, L cell, COS, for example, COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the mammalian cells are CHO cell lines. In some embodiments, the mammalian cells are CHO cells.In some embodiments, mammalian cells are selected from among CHOK1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knock-out cells, CHO FUT8 GS knock-out cells, CHOZN cells, and CHO-derived cells. In some embodiments, a CHO GS knock-out cell (such as, for example, a GSKO cell) is, for example, a cell. Petition 870250082204, dated 12 / 09 / 2025, pp. 93 / 138 78 / 112 CHO-K1 SV GS knockout. Additionally, the CHO FUT8 knockout cell is, for example, Potelligent® CHOK1 SV (Lonza, Inc.). In some embodiments, the eukaryotic cells may also be avian cells, cell lines or cell strains, such as, for example, EBx®, EB14, EB24, EB26, EB66 or EBν13 cells.

[0160] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, the dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficient amplifiable and selectable DHFR gene expression system allows high-level recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Also included are CHOK1SV cell lines with glutamine synthetase (GS) knockout, making use of methionine sulfoxide-based glutamine synthetase (GS) selection (MSX).Other suitable CHO host cells include, but are not limited to, the following (ECACC accession numbers in parentheses): CHO (85050302); CHO (PROTEIN FREE) (00102307); CHO-K1 (85051005); CHO-K1 / SF (93061607); CHO / dhFr- (94060607); CHO / dhFr-AC-free (05011002); and RR-CHOKI (92052129).

[0161] 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 do not need to, be adapted. Petition 870250082204, dated 12 / 09 / 2025, pp. 94 / 138 79 / 112 to growth in suspension culture. A variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells and CLIO cells from CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines include, but are not limited to, mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER.C6® cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or manipulated from any of the cell lines disclosed herein.

[0162] In some embodiments, eukaryotic cells are chosen from among lower eukaryotic cells, such as, for example, yeast cells (e.g., genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluiveri and Pichia angusta), of the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris or Komagataella phaffii), cells of the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum), cells of the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), cells of the genus Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), cells of the genus Geotrichum (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 Χ33, GS115, KM71, KM71H, and CBS7435.

[0163] In some embodiments, eukaryotic cells are selected from fungal cells (for example, Petition 870250082204, dated 12 / 09 / 2025, pp. 95 / 138 80 / 112 cells of Aspergillus (such as, for example, A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium (such as, for example, A. thermophilum), Chaetomium (such as, for example, C. thermophilum), Chrysosporium (such as, for example, C. thermophile), Cordyceps (such as, for example, C. militaris), Corynascus, Ctenomyces, Fusarium (e.g. F. oxysporum), Glomerella (e.g. for example, G. graminicola), Hypocrea (such as, for example, H. jecorina), Magnaporthe (such as, for example, M. orzyae), Myceliophthora (such as, for example, M. thermophile), Nectria (such as, for example, N. heamatococca), Neurospora (such as, for example, N. crassa), Penicillium, Sporotrichum (such as, for example, S. thermophile), Thielavia (such as, for example, T. terrestris, T. heterothallica), Trichoderma (such as, for example, T. reesei) or Verticillium (such as, for example, V. dahlia)).

[0164] In some embodiments, eukaryotic cells are selected from insect cells (such as, for example, Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN5B1-4) or BT1-Ea88 cells), algal cells (such as, for example, from the genera Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina or Ochromonas) and plant cells (such as, for example, cells from monocotyledonous plants (such as, for example, maize, rice, wheat or Setaria); or cells from a dicotyledonous plant (such as, for example, cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens or Arabidopsis)). Petition 870250082204, dated 12 / 09 / 2025, pp. 96 / 138 81 / 112

[0165] To generate host cell lines (e.g., mammalian cell lines) engineered to express a recombinant protein of interest, one or more nucleic acids encoding the recombinant protein (or components thereof in the case of multi-stranded proteins) are initially inserted into one or more expression vectors. Useful nucleic acid control sequences in expression vectors for mammalian cell expression include promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence may also optionally be encoded by the expression vector operationally linked to the coding sequence of interest, such that the expressed protein can be secreted by the recombinant host cell, for easier isolation of the recombinant protein from the cell, if desired.Vectors may also include one or more selectable marker genes to facilitate the selection of host cells into which the vectors have been introduced. In some embodiments, vectors employing protein fragment complementation assays using protein reporters such as dihydrofolate reductase are used (see, for example, U.S. Patent No. 6,270,964). Suitable mammalian expression vectors are known in the art and are also commercially available.

[0166] Typically, the vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences will normally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a Petition 870250082204, dated 12 / 09 / 2025, pp. 97 / 138 82 / 112 origin of replication, transcriptional and translational control sequences, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a native or heterologous signal peptide sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinkage region for inserting the polynucleotide encoding the polypeptide to be expressed, and a selectable marker element. Vectors can be constructed from a starter vector such as a commercially available vector, and additional elements can be individually sourced and ligated to the vector. Culture Methods

[0167] Various culture methods can be used to produce a recombinant protein of interest, including, but not limited to, batch culture, fed-batch culture, and perfusion culture.

[0168] Batch culture is a discontinuous method where cells are grown in a fixed volume of culture media for a short period of time followed by a complete harvest. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, followed by a decline in viable cell density as media components are consumed and levels of metabolic byproducts (such as lactate and ammonia) accumulate. Harvesting typically occurs at the point where maximum cell density is reached (e.g., 5 x 10⁶ cells / ml or higher, depending on the formulation). Petition 870250082204, dated 12 / 09 / 2025, pp. 98 / 138 83 / 112 medium, cell line, etc.). The batch process is the simplest culture method; however, the density of viable cells is limited by nutrient availability, and as soon as the cells reach maximum density, the culture declines and production decreases. There is no ability to prolong a production phase in batch culture because the accumulation of waste and nutrient depletion rapidly lead to culture decline, typically around 3 to 7 days.

[0169] Fed-batch culture improves upon the batch process by providing boluses or continuous feedings of media to replenish those media components that have been consumed. Since fed-batch cultures receive additional nutrients throughout the operation, they have the potential to achieve higher cell densities (>10 to 30x10⁶ cells / ml, depending on media formulation, cell line, etc.) and increased product titers compared to the batch method. Unlike the batch process, a biphasic culture can be created and sustained by manipulating feeding strategies and media formulations to distinguish the period of cell proliferation to achieve the desired cell density (the growth phase) from the period of suspended or slow cell growth (the production phase).As such, fed-batch crops have the potential to achieve higher yields compared to non-batch crops. Typically, a batch method is used during the growth phase and a fed-batch method is used during the production phase, but one... Petition 870250082204, dated 12 / 09 / 2025, pp. 99 / 138 84 / 112 fed-batch strategy can be used throughout the entire process. However, unlike the batch process, the bioreactor volume is a limiting factor that restricts the amount of feed. Similarly, as with the batch method, the accumulation of metabolic byproducts will lead to culture decline, which limits the duration of the production phase, often around 10 to 21 days. Fed-batch cultures are discontinuous, and harvesting typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. Compared to a batch culture, in which no feeding occurs, a fed-batch culture can produce larger quantities of recombinant protein. (See, for example, U.S. Patent No. 5,672,502).

[0170] Perfusion methods offer potential improvements over batch and fed-batch methods by adding fresh media and simultaneously removing spent media during culture. Typical perfusion cultures begin with a batch culture lasting one or two days followed by the continuous, gradual, and / or intermittent addition of fresh feed media to the culture and simultaneous removal of spent media, retaining cells and additional high molecular weight compounds such as proteins (based on the molecular weight limit of the filter) throughout the growth and production phases of the culture. Several methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent media while maintaining cell density. Exemplary, non-limiting filtration methods include Petition 870250082204, dated 12 / 09 / 2025, pp. 100 / 138 85 / 112 tangential flow filtration (TFF), as well as recirculating flow filtration and alternating tangential flow filtration (ATF). Alternating tangential flow is maintained by pumping the medium through hollow fiber filter modules. See, for example, US Patent No. 6,544,424; Furey, 2002, Gen. Eng. News.. 22 (7):62-63.

[0171] Perfusion can be continuous, gradual, intermittent, or a combination of any or all of these. Perfusion rates can be less than a working volume for many working volumes per day. Cells are retained in the culture, and the spent medium that is removed is substantially cell-free or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture may also be retained in the culture.

[0172] Typical large-scale commercial cell culture strategies strive to achieve high cell densities, 40–90 (+) x 10⁶ cells / ml, such as approximately 40 x 10⁶ cells / ml or approximately 50 x 10⁶ cells / ml, where almost one-third to more than half of the reactor volume is biomass. With perfusion culture, extreme cell densities of >1 x 10⁸ cells / ml have been achieved. A potential advantage of the perfusion process is that the production culture can be maintained for longer periods than batch or fed-batch culture methods. However, increased media preparation, use, storage, and disposal are required to support a long-term perfusion culture, particularly for a high-density cell culture, which also requires even more nutrients. Petition 870250082204, dated 12 / 09 / 2025, pp. 101 / 138 86 / 112 Additionally, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and problems with increased gasification, including supplying more oxygen and removing more carbon dioxide, which could result in more foam formation and the need for changes in antifoaming strategies; as well as during harvesting and downstream processing where the efforts required to remove excess cell material may result in product loss, negating the benefit of increased titer due to increased cell mass.

[0173] Suitable culture conditions, including temperature, dissolved oxygen content, agitation rate, and the like, for mammalian cells are known in the art and may vary by the phase or stage of cell culture. In some embodiments, the methods disclosed herein further comprise taking samples during cell culture processes, evaluating the samples to quantitatively and / or qualitatively monitor recombinant protein and / or cell culture process characteristics. In some embodiments, the samples are quantitatively and / or qualitatively monitored using process analytical techniques.For example, dissolved oxygen levels can be monitored during cell culture processes using methods known in the art, such as, for instance, a basic chemical analysis method (titration method), an electrochemical analysis method (diaphragm electrode method), and a photochemical analysis method (fluorescence method). Petition 870250082204, dated 12 / 09 / 2025, pp. 102 / 138 87 / 112

[0174] During recombinant protein production, it is desirable to have a controlled system where cells are cultured for a desired time or until a desired density is reached, and then the physiological state of the cells is altered to a state of limited or arrested growth, of high productivity, where the cells use energy and substrates to produce the recombinant protein in favor of increased cell density. For commercial-scale cell culture and the manufacture of biological therapeutic products, the ability to limit or stop cell growth and maintain cells in a growth-limited or arrested state during the production phase is highly desirable. Such methods include, for example, temperature changes, the use of chemical inducers of protein production, nutrient limitation or starvation, and cell cycle inhibitors, alone or in combination.Illustratively, a typical cell culture undergoes a growth phase, a period of exponential growth where cell density is increased. During the growth phase, cells are grown in a cell culture medium containing the necessary nutrients and additives under conditions (usually at around a temperature of 25°C-40°C, in a controlled humidified atmosphere) so that optimal growth is achieved for the particular cell line. Cells are typically maintained in the growth phase for a period between one and eight days, for example, between three and seven days, for example, seven days. The length of the growth phase for a particular cell line can be determined by a person of ordinary skill in the technique and will generally be the period of time sufficient to allow for... Petition 870250082204, dated 12 / 09 / 2025, pp. 103 / 138 88 / 112 as individual cells reproduce at a viable cell density within a range of about 20%–80% of the maximum viable cell density if the culture was maintained under growth conditions. The growth phase is followed by a transition phase when the exponential growth of the cells is slowing down and protein production begins to increase. This marks the beginning of the stationary phase, a production phase, where cell density typically stabilizes and product titer increases. During the production phase, the medium is usually supplemented to support continued recombinant protein production.

[0175] In certain embodiments, the culture conditions used to produce a recombinant protein can be adjusted to facilitate the transition from the cell culture growth phase to the production phase. For example, a cell culture growth phase can occur at a higher temperature than a cell culture production phase. In some embodiments, a growth phase can occur at a first temperature of about 35 °C to about 38 °C and a production phase can occur at a second temperature of about 29 °C to about 37 °C, optionally from about 30 °C to about 36 °C or from about 30 °C to about 34 °C. In one embodiment, a temperature change from about 35 °C to about 37 °C to a temperature of about 31 °C to about 33 °C can be employed to facilitate the transition from the cell culture growth phase to the production phase.Chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added simultaneously, before and / or after a [treatment / process]. Petition 870250082204, dated 12 / 09 / 2025, pp. 104 / 138 89 / 112 temperature change or in place of a temperature change. If inductors are added after a temperature deviation, they can be added from one hour to five days after the temperature deviation, optionally from one to two days after the temperature deviation.

[0176] Additionally, any cell culture medium capable of supporting the growth of the appropriate host cell in culture may be used. Typically, cell culture media contain a buffer, salts, energy source, amino acids, vitamins, and essential trace elements. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in a particular cultured host cell, are commercially available and include RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco Modified Eagle Medium (DMEM), Eagle Essential Minimum Medium, F-12K Medium, Ham F12 Medium, Iscove Modified Dulbecco Medium, McCoy 5A Medium, Leibovitz L-15 Medium, and serum-free media such as EX-CELL™ 300 Series, among others, which may be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other suppliers.Cell culture media can be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture media can also be enriched by the addition of nutrients or other supplements, which may be used in concentrations higher than those recommended. In certain embodiments, the culture medium used in the production of a recombinant protein to be purified by a method provided in this document is... Petition 870250082204, dated 12 / 09 / 2025, pp. 105 / 138 90 / 112 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 do not contain hydrolysates or animal-derived components.

[0177] Various media formulations can be used during the life of the culture, for example, to facilitate the transition from one stage (e.g., the growth stage or phase) to another (e.g., the production stage or phase) and / or to optimize conditions during cell culture (e.g., concentrated media supplied during a perfusion culture). A growth medium formulation can be used to promote cell growth and minimize protein expression. A production medium formulation can be used to promote the production of the recombinant protein of interest and the maintenance of cells, with minimal growth of new cells. A feed medium is typically a cell culture medium containing more concentrated components, such as nutrients and amino acids, that are consumed during the production phase of the cell culture.A feeding medium can be used to supplement and maintain an active culture, particularly a culture operated in fed-batch, semi-perfusion, or perfusion mode. Such a concentrated feeding medium may contain most of the components of the cell culture medium at, for example, approximately 5χ, 6χ, 7χ, θχ, 9χ, 10χ, 12χ, 14χ, 16χ, 20χ, 30χ, 50x, 100χ, 200χ, 400χ, 600χ, 800x, or even approximately 1000x of their normal value. Petition 870250082204, dated 12 / 09 / 2025, pp. 106 / 138 91 / 112

[0178] In some embodiments, the mammalian cell used to produce a recombinant protein is cultured for a defined period of time during which the recombinant protein is expressed and secreted by the mammalian cell. This period of time (i.e., the duration of the cell culture production phase) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the cell culture production phase 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 methods, the duration of the cell culture production phase is 7 days, 8 days, 9 days, 12 days, 15 days, 18 days, or 21 days.

[0179] In some embodiments, the biofabrication process to produce a recombinant protein comprises a production phase with a viable cell density of at least 100 x 10⁵ cells / ml, for example, between about 100 x 10⁵ cells / ml and about 10 x 10⁷ cells / ml, between about 250 x 10⁵ cells / ml and about 900 x 10⁵ cells / ml, between about 300 x 10⁵ cells / ml and 800 x 10⁵ cells / ml, or between about 450 x 10⁵ cells / ml and 650 x 10⁵ cells / ml. Cell density can be measured using a hemocytometer, a Coulter counter, or an automated cell analyzer (e.g., Cedex automated cell counter). Viable cell density can be determined by staining a culture sample with blue Petition 870250082204, dated 12 / 09 / 2025, pp. 107 / 138 92 / 112 Trypan, which is absorbed only by dead cells. 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 obtaining the reciprocal.

[0180] In some embodiments, the upstream biofabrication process that produces a recombinant protein to be purified by a method provided in this document comprises a production phase with a packed cell volume less than or equal to 35%. In some embodiments, the packed cell volume is less than or equal to 30%.

[0181] Critical attributes and performance indicators of the recombinant protein of interest can be measured to better inform decisions about the performance of each step during manufacturing. These critical attributes and performance indicators can be monitored in real-time, near real-time, and / or offline. Critical parameters that can be measured during cell culture may include components of the cell culture medium that are consumed (such as glucose), levels of metabolic byproducts (such as lactate and ammonia) that accumulate, as well as those related to cell maintenance and survival, such as dissolved oxygen content. Additionally, critical attributes such as specific productivity, viable cell density, packed cell volume, pH, osmolality, aggregation, percent yield, and titer can be monitored during appropriate stages in the manufacturing process.Monitoring and measurements can be performed using known techniques and commercially available equipment. Petition 870250082204, dated 12 / 09 / 2025, pp. 108 / 138 93 / 112 Bioreactors

[0182] In some embodiments, the growth and / or production phase of an upstream process used to produce a recombinant protein is conducted within a bioreactor. The conditions within the bioreactor are designed to support cell culture. Suitable culture conditions for mammalian cells are known in the art, as described above. A bioreactor run typically comprises the steps of inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth and / or production phases until one or more predetermined parameters are reached (e.g., time, viable cell density, packed cell volume), and then collecting the contents of the bioreactor.

[0183] In some embodiments, one or more bioreactors used to produce a recombinant protein are stainless steel bioreactors, such as, for example, a large-scale embedded stainless steel bioreactor capable of operating in volumes of about 2000 liters to about 50000 liters (e.g., about 2000 liters to about 20000 liters) or more.

[0184] In some embodiments, one or more bioreactors for producing a recombinant protein constitute a single-use bioreactor. Single-use technology minimizes the infrastructure requirements associated with traditional cell culture, such as commercial-scale steel / glass vessels and associated machinery. Single-use bioreactors provide flexibility in the manufacturing process and allow for on-site assembly, reconfiguration, sterilization, and validation. Petition 870250082204, dated 12 / 09 / 2025, pp. 109 / 138 94 / 112 for single-use bioreactors can be faster, easier, and less expensive than traditional built-in stainless steel cell culture plants. Single-use bioreactors comprise sterile, disposable plastic bags supported by a non-disposable support structure. The culture is agitated by an agitator inside the bag or by rocking; air and oxygen sprayers are also provided, as well as sensors to measure and adjust various culture parameters such as pH, temperature, oxygen, cell density, and the like. Single-use bioreactors are commercially available, for example, Bio STR®, Sartorius, Göttingen, Germany; MOBIUS®, Millipore, Burlington, MA; XCELLEREX®, Cytiva, Marlborough, MA.

[0185] The bioreactor volume is divided into working volume and free space. The bioreactor working volume refers to the volume within the bioreactor in which the cell culture is operated, typically expressed as a percentage of the bioreactor volume. In some embodiments, the bioreactor working volume is at least about 70% of the bioreactor volume. In some embodiments, the bioreactor working volume is at least about 70% to about 100% of the bioreactor volume. In some embodiments, the bioreactor working volume is at least about 75% of the bioreactor volume. In some embodiments, the bioreactor working volume is at least about 80% of the bioreactor volume. In some embodiments, the bioreactor working volume 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 Petition 870250082204, dated 12 / 09 / 2025, pp. 110 / 138 95 / 112 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 approximately 99% of the bioreactor volume.In some models, the bioreactor's workload is approximately... 100% of the bioreactor volume. Additional Collection and Purification Processes

[0186] Expressed recombinant proteins can be secreted into the culture medium from which they can be recovered and / or collected. Some biofabrication processes incorporating an anion-exchange chromatography operation of the present disclosure may also include a harvesting operation. The harvesting operation clarifies and / or purifies the protein of interest wholly or partially from at least one impurity with which it is found in the cell culture fluid, such as remaining cell culture media, cells, Petition 870250082204, dated 12 / 09 / 2025, pp. 111 / 138 96 / 112 cell remnants or media components and / or other impurities related to the product and / or process.

[0187] Methods for collecting recombinant proteins from cell cultures in suspension are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, separation using gravity, centrifugation, separation by acoustic waves, filtration, including membrane filtration, ultrafilters, microfilters, tangential flow, alternative tangential flow, depth filters and alluvial filtration filters.

[0188] The collected cell culture fluid (HCCF) can be stored in buffer tanks, holding tanks, bags, or other containers that are adapted to provide feed to a chromatography column rack and are appropriate for the infrastructure and / or process requirements.

[0189] Harvesting operations can be combined with additional harvesting strategies, including centrifugation, such as disc stack centrifugation or continuous solid discharge centrifugation; filtration, including tangential flow filtration, microfiltration, ultrafiltration and depth filtration; precipitation / sedimentation methods, such as flocculation; and chromatography-based separations.

[0190] In addition to an anion exchange chromatography operation using an anion exchange chromatography material comprising a primary amine ligand, the present disclosure covers methods involving all known purification technologies, such as, by Petition 870250082204, dated 12 / 09 / 2025, pp. 112 / 138 97 / 112 example, purification of protein A from immunoglobulins and immunoglobulin-like biologics, as well as chromatography-based separations and polishing steps that include column modes and alternative chromatographic separations by ion-exchange chromatography (IEX), including anion-exchange chromatography (AEX) and / or cation-exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), multimodal or mixed-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.

[0191] In some embodiments, recombinant protein recovered from host cells or cell culture medium may be further purified or partially purified to remove components of cell culture media, host cell proteins or nucleic acids, or other process- or product-related impurities by one or more unit operations. An individual of ordinary skill in the art may select the appropriate unit operation(s) for further purification of a recombinant protein based on the characteristics of the recombinant protein to be purified, the characteristics of the host cell from which the recombinant protein is expressed, and the composition of the culture medium in which the host cells were grown. Illustratively, in some embodiments, the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, Petition 870250082204, dated 12 / 09 / 2025, pp. 113 / 138 98 / 112 depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0192] A capture unit operation may include capture chromatography that makes use of resins and / or membranes containing agents that will bind to the recombinant protein of interest, for example, affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like. These 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 may be employed as a capture chromatography unit operation to further purify the recombinant protein.In other embodiments, the recombinant protein of interest may comprise a polyhistidine tag at its amino or carboxyl terminus and subsequently purified using IMAC. Recombinant proteins may be engineered to include other purification tags, such as a FLAG® tag or c-myc epitope, and subsequently purified by affinity chromatography using a specific antibody targeting such a tag or epitope. Petition 870250082204, dated 12 / 09 / 2025, pp. 114 / 138 99 / 112

[0193] Unit operations aimed at inactivating, reducing, and / or eliminating viral contaminants may include processes that mitigate viral risk by manipulating the environment and / or using filtration. Viral mitigation measures are critical to ensuring the safety of protein therapeutics and may be performed one or more times during downstream purification. Viral contaminants can arise from a variety of sources, including the use of reagents of animal origin, adventitious viral contaminants in host cell lines, or system failures in GMP manufacturing facilities. Viruses are classified as enveloped and non-enveloped viruses. With enveloped viruses, the envelope allows the virus to identify, bind to, enter, and infect target host cells. As such, enveloped viruses are susceptible to inactivation methods.Several methods can be employed for virus inactivation, including heat inactivation / pasteurization, UV and gamma irradiation, use of high-intensity broad-spectrum white light, addition of chemical inactivating agents, surfactants, and solvent / detergent treatments. Surfactants, such as detergents, solubilize membranes and can be very effective in the specific inactivation of enveloped viruses. Additional unit operations to inactivate, reduce, and / or eliminate viral contaminants may include filtration processes and / or adjustment of solution conditions. One method to achieve viral inactivation is incubation at low pH (e.g., pH < 4). Low pH virus inactivation can be followed by a neutralization unit operation that readjusts the inactivated viral solution to a pH more compatible with the requirements of subsequent operations. Petition 870250082204, dated 12 / 09 / 2025, pp. 115 / 138 100 / 112 unit. A low pH viral inactivation operation can also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitation. Adjusting the temperature or chemical composition (e.g., using detergents) can also be used to achieve viral inactivation. Viral filtration can be performed using micro or nanofilters, such as those available from Asahi Kasei (Plavona®) and EMD Millipore (VPro®).

[0194] Non-enveloped viruses are less susceptible to inactivation methods that preserve product stability. As such, non-enveloped viruses are typically removed by filtration methods. An exemplary process is described in document no. WO2020 / 159838. Viral filtration can be performed using micro or nanofilters, such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NI), and CUNO Zeta Plus VR (3M, St. Paul, Mn).

[0195] Viral filtration can occur in one or more steps in the downstream operations of a biofabrication process. Typically, viral inactivation follows an affinity chromatography unit operation and viral filtration precedes or follows an ultrafiltration / diafiltration (UF / DF) operation, but it can also occur after UF / DF.

[0196] In all chromatography processes, multiple filters can be used up to the capacity that the supports, racks or physical configuration of the ultrafiltration / diafiltration (UF / DF) system will allow or are Petition 870250082204, dated 12 / 09 / 2025, pp. 116 / 138 101 / 112 needed to achieve the desired objectives of a production process.

[0197] A polishing unit operation can make use of various chromatographic methods for the purification of the protein of interest and the removal of contaminants and impurities. The polishing chromatography unit operation can make use of resins and / or membranes containing agents that can be used in a “continuous flow mode, in which the protein of interest is contained in the eluent and the contaminants and impurities are bound to the chromatographic medium, or a “binding and elution mode, in which the protein of interest is bound to the chromatographic medium and eluted after the contaminants and impurities have flowed or been washed from the chromatographic medium.Examples of such polishing chromatography methods include, but are not limited to, ion exchange chromatography (IEX), such as cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed or multimodal chromatography (MM); hydroxyapatite (HA) chromatography; reversed-phase chromatography; and size exclusion chromatography (e.g., gel filtration).

[0198] Purified recombinant proteins can be formulated, i.e., subjected to buffer exchange, sterilized, bulk packaged, and / or packaged for an end user. Illustratively, product concentration and buffer exchange of the recombinant protein of interest in a desired formulation buffer for bulk storage of the active ingredient or medicinal product can be performed by ultrafiltration and / or diafiltration. Suitable formulations for pharmaceutical compositions include those Petition 870250082204, dated 12 / 09 / 2025, pp. 117 / 138 102 / 112 descritas em Remington's Pharmaceutical Sciences, 18aed. 1995, Mack Publishing Company, Easton, PA.

[0199] A UF / DF operation can occur in one or more steps in a downstream process. Typically, a UF / DF operation is performed before bulk storage of the pharmaceutical substance. Instead of storage, unit operations related to filling / finishing the pharmaceutical product may also immediately follow a UF / DF operation. One or more stability-enhancing excipients may optionally be added directly to the UF / DF retentate feed tank containing the purified formulated protein resulting in the formulated pharmaceutical substance or added to the UF / DF eluate pool. An exemplary UF / DF process is described in WO 2020 / 159838. Filters for use in a UF / DF operation are well known in the art and are commercially available from many sources.There are many types of materials available, such as Pellicon regenerated cellulose (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), and polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY). Recombinant Proteins

[0200] Any type of recombinant protein, including proteins containing single or multiple polypeptide chains, can be purified according to the methods of the present disclosure. Such recombinant proteins include, but are not limited to, secreted proteins, non-secreted proteins, proteins Petition 870250082204, dated 12 / 09 / 2025, pages 118 / 138 103 / 112 intracellular or membrane-bound proteins. Illustratively, recombinant proteins may include, but are not limited to, cytokines, growth factors, hormones, muteins, fusion proteins, antibodies, antibody fragments, pepticobodies, T-cell engagement molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0201] In other embodiments, the recombinant protein to be purified according to a method of the present disclosure is an antigen-binding protein. Antigen-binding proteins include, but are not limited to, antibodies, pepticobodies, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFvs), double-chain (divalent) scFvs and IgGscFv (see, for example, Orcutt et al., 2010, Protein Eng Des Sel 23: 221-228)), hetero-IgGs (see, for example, Liu et al., 2015, J Biol Chem 290: 7535-7562), muteins and XmAb® (Xencor, Inc., Monrovia, CA).Additional antigen-binding proteins include, but are not limited to, bispecific T-cell couplers (BiTE®), bispecific T-cell couplers having extensions, such as half-life extensions, such as, for example, HLE BiTE molecules, HeteroIg BITE molecules and others, chimeric antigen receptors (CARs, CAR Ts) and T-cell receptors (TCRs).

[0202] In some embodiments, the antigen-binding protein binds to one or more of the following, alone or in any combination: CD proteins which include, but are not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25,. Petition 870250082204, dated 12 / 09 / 2025, pp. 119 / 138 104 / 112 CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171 and CD174, proteins of the HER receptor family, which include, for example, HER2, HER3, HER4 and the EGF receptor, EGFRvIII, cell adhesion molecules, for example, LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM and integrin alpha v / beta 3, growth factors, which include, but are not limited to, for example, vascular endothelial growth factor (VEGF); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, which include, for example, aFGF and bFGF, epidermal growth factor (EGF), Cryptocoryne-3, transforming growth factors (TGF), which include,among others, TGF-α and TGF-β, which include TGF-β1, TGF-32, TGF-33, TGF-34 or TGF-35, insulin-like growth factors-I and -II (IGFI and IGF-II), des(1-3)-IGF-I (brain IGF-I) and osteoinductive factors, insulins and insulin-related proteins, which include, but are not limited to, insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding proteins; (coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (t-PA), bombazine, thrombin, Petition 870250082204, dated 12 / 09 / 2025, pp. 120 / 138 105 / 112 thrombopoietin and thrombopoietin receptor, colony-stimulating factors (CSFs), including, but not limited to, M-CSF, GM-CSF, and G-CSF, other blood and serum proteins, including, but not limited to, albumin antigens, IgE and blood groups, receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity receptor (OB), growth hormone receptors and T-cell receptors; neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain and prorelaxin, interferons, including, for example, interferon-alpha, -beta and -gamma, interleukins (ILs), for example, IL-1 to IL-10, IL-12, IL-15, IL-17, IL23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 receptor or IL-17 receptor, IL-1RAP;Viral antigens, including, but not limited to, an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor alpha and beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated upon activation of normally expressed and secreted T cells), mouse gonadotropin-associated peptide, DNase, RF-alpha, inhibin and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay-accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-A, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins; Petition 870250082204, dated 12 / 09 / 2025, pages 121 / 138 106 / 112 immunoadhesins, antigen-binding proteins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGp, hepatitis C virus, dsFv[PE38] mesothelin conjugate, Legionella pneumophila (lly), IFN-gamma, protein induced by interferon gamma 10 (IP10), IFNAR, TALL-1 lymphopoietin, thymic stromal lymphopoietin (TSLP), proprotein convertase / kexin type 9 subtilisin (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1)), transforming growth factor beta (TFGp),Sperm-binding protein zona pellucida 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the aforementioned.

[0203] In other embodiments, the recombinant protein to be purified according to a method of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody.

[0204] In some embodiments, the antibody is selected from among abrilumab, brazikumab, brodalumab, crizanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplazumab, nemolizumab, ontamalimab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, Petition 870250082204, dated 12 / 09 / 2025, pages 122 / 138 107 / 112 satralizumab, tafoleciumab, tanezumab, tezepelumab, tremelimumab, utomilumab, and volagidumab. In some embodiments, the antibody is selected from denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody is erenumab. In some embodiments, the antibody is evolocumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is tezepelumab.

[0205] In some embodiments, the antibody is a human IgG1, IgG2, or IgG4 antibody.

[0206] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0207] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0208] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody. EXAMPLES

[0209] In order for this disclosure to be more fully understood, 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. Petition 870250082204, dated 12 / 09 / 2025, pp. 123 / 138 108 / 112 Example 1: AEX Chromatography Step using TOYOPEARL® NH2750F

[0210] An AEX resin consisting of TOYOPEARL® NH2-750F (TOSOH Bioscience) was used to further polish compositions comprising one of two recombinant monoclonal antibodies, mAb1 or mAb2, after affinity chromatography, low pH viral inactivation, and CEX chromatography. TOYOPEARL® NH2-750F is composed of polymethacrylate microspheres that have been functionalized with proprietary primary amine (NH2) strong anion exchange groups and is commercially available in a 45 µm particle size (grade F). The operating pH for the AEX step was between 7.0 and 8.0, and the operating conductivity was less than 10 mS / cm. The AEX column was loaded at a loading density between 250 g / l of resin and 600 g / l of resin.

[0211] Figure 1A shows the removal capabilities of this AEX step for general HMW species from mAbl by comparing HMW levels (as assessed by SE-HPLC) in the feedstock and pooling in seven pilot-scale batches. The observed HMW reduction of at least 0.5% in all three batches demonstrates the robustness of the AEX step even at high feedstocks of more than 500 g / L resin. Significant reductions in process-related impurities, including host cell protein, DNA, and model viruses, were also observed in pilot-scale operations as well as bench-scale challenge studies.

[0212] Figure 1B shows the step yield for the AEX step for the pilot-scale batches of Figure 1A, demonstrating the step's ability to achieve high yields. Petition 870250082204, dated 12 / 09 / 2025, pages 124 / 138 109 / 112 yields while providing significant impurity reduction.

[0213] Similar high yields and significant impurity reduction were observed for mAb2. Figure 2A shows a high molecular weight clearance for mAb2 in two pilot-scale batches, and Figure 2B shows the step yield for the AEX step for the pilot-scale batches of Figure 2A. Example 2: Low pH viral inactivation using formic acid

[0214] In certain manufacturing sites with equipment constraints, it is desirable to minimize the volume of Protein A clustering and subsequent cluster titrations in order to ensure that volumes fit within vessel limitations with a robust safety margin. As shown in Table 1, for an evaluation of unit operation VI for mAb1 with a low pH incubation time of 60–90 minutes at 15–25 °C, the use of 1M formic acid vs. 10% acetic acid as the acid titrant resulted in a lower acid volume required to reach the pH for viral inactivation, a lower base volume required to neutralize cluster VI to pH 5.0, and a lower liquid volume expansion over the viral inactivation step.This is particularly advantageous when there is limited capacity to reduce the elution pool volume of Protein A, for example, when operating at high column loads. Petition 870250082204, dated 12 / 09 / 2025, pages 125 / 138 110 / 112 TABLE 1. Impact of Titrant VI on Volume and Conductivity of mAb1 Cluster Test 1 (PSL1) Test 2 (PSL2, Batch 2 of Example 1) % Reduction (Formic Acid vs. Acetic Acid) Starting Material mAb1 Protein A Cluster mAb1 Protein A Cluster Initial pH 4.4 4.4 Acid Titrant Acetic Acid 10% Formic Acid 1M Volume of acid added (ml / l) 107.3 36.1 66% Acidified pH 3.6 ~3.6 Base Titrant 2M Tris 2M Tris Volume of base added (ml / l) 67.6 24.6 64% Final pH 5.0 5.0 Final Conductivity (mS / cm) 6.4 4.5 30% Net volume expansion upon viral inactivation 18% 6% 11%

[0215] FIG. 3 demonstrates the benefits of lower charge conductivity (resulting from the choice of acidification titrant VI), as observed for a mAb1 AEX unit operation substantially similar to that described. Petition 870250082204, dated 12 / 09 / 2025, pp. 126 / 138 111 / 112 in Example 1. High molecular weight (HMW, detected by SE-HPLC assay for mAb1) is often a critical quality attribute for mAbs, such as mAb1, and HMW impurity levels are generally reduced by polishing chromatography steps, such as AEX. Figure 3 shows improved AEX step performance resulting from the use of a 1M formic acid titrant VI (PSL2), leading to a lower percentage of HMW in the AEX cluster vs. the use of a 10% acetic acid titrant VI (PSL1) under similar conditions, which exhibited minimal reduction in HMW species from mAb1. While the lower charge conductivity reduced the step yield (93% for PSL2 versus 98% for PSL1) as a result of greater product retention in the resin, the AEX step yield when 1M formic acid was used as a VI titrant was still acceptable.

[0216] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books and treatises, are hereby expressly incorporated by reference. What is described in one disclosure embodiment may be combined with one or more other disclosure embodiments unless the context clearly indicates otherwise.

[0217] The disclosed matter is not intended to be limited in scope by the specific modalities described herein, which are in fact intended as non-limiting illustrations of individual aspects of the disclosure. Functionally equivalent methods and components are within the scope of the disclosure. In fact, various modifications of the disclosed matter, in addition to those shown and described herein, will be evident to those skilled in the art from the description. Petition 870250082204, dated 12 / 09 / 2025, pp. 127 / 138 112 / 112 previous and attached drawing(s). Such modifications are intended to remain within the scope of the disclosed matter.

[0218] The descriptions of the various embodiments and / or examples of the disclosed matter have been presented for illustrative purposes only, but are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the embodiments described. The terminology used in this document has been chosen to better explain the principles of the embodiments, the practical application or technical improvement in relation to technologies found on the market, and / or to enable other persons of ordinary skill in the art to understand the disclosed matter. Petition 870250082204, dated 12 / 09 / 2025, pp. 128 / 138

Claims

1 / 4 Claims 1. A method for purifying a recombinant protein from a composition comprising the recombinant protein and at least one impurity, wherein the method is characterized in that it comprises: loading the composition onto an anion exchange material comprising a primary amine ligand at a charge 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 at least one impurity binds to the anion exchange material more strongly than the recombinant protein binds to the anion exchange material; and collecting a purified composition comprising the recombinant protein.

2. A method according to claim 1, characterized in that the anion exchange medium comprises resin particles, wherein at least 80% of the resin particles have a particle size of about 30 µm to about 60 µm.

3. A method according to claim 1 or 2, characterized in that the anion exchange material comprises a polyamine ligand.

4. Method, according to any one of claims 1, 2 or 3, characterized in that the charge density is less than about 600 g / l of anion exchange material. Petition 870250082204, dated 12 / 09 / 2025, pp. 129 / 138 2 / 4 5. Method, according to any one of claims 1, 2 or 3, characterized in that the filler density is about 250 g / l to about 600 g / l of resin.

6. A method according to any one of claims 1, 2, 3, 4 or 5, characterized in that the composition has a conductivity of about 3 mS / cm to about 6 mS / cm.

7. A method according to any one of claims 1, 2, 3, 4, 5 or 6, characterized in that the method comprises an equilibrium buffer and / or recovery buffer with an anion exchange material, wherein: the pH of the equilibrium buffer and / or recovery buffer is about 7.0 to about 8.0; and / or the conductivity of the equilibrium buffer and / or recovery buffer is less than about 10 mS / cm.

8. Method according to claim 7, characterized in that the conductivity of the equilibrium buffer and / or the recovery buffer is about 2 mS / cm to about 4 mS / cm.

9. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that it further comprises performing one or more unit operations prior to loading, performing a low pH viral inactivation unit operation.

10. Method according to claim 9, characterized in that the low pH viral inactivation unit operation employs an acidic titrant comprising formic acid.

11. Method, according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized by the fact that Petition 870250082204, dated 12 / 09 / 2025, pp. 130 / 138 3 / 4, further comprises performing one or more additional chromatography unit operations.

12. Method according to claim 11, characterized in that one or more additional chromatography unit operations comprise an affinity chromatography unit operation performed prior to loading.

13. Method according to claim 12, characterized in that the affinity chromatography unit operation is selected from protein chromatography A, protein chromatography G, protein chromatography L and CH1 domain chromatography.

14. A method according to any one of claims 11, 12 or 13, characterized in that one or more additional chromatography unit operations comprise an additional polishing chromatography unit operation.

15. Method according to claim 14, characterized in that the polishing chromatography unit operation is selected from cation exchange chromatography, hydrophobic interaction chromatography and mixed-mode chromatography.

16. Method, according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, characterized in that it further comprises performing a viral filtration unit operation and / or an ultrafiltration / diafiltration (UF / DF) unit operation after loading. Petition 870250082204, dated 12 / 09 / 2025, pp. 131 / 138 4 / 4 17. Method, according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, characterized in that: less than about 2.5% by w / w of the recombinant protein in the purified composition is high molecular weight recombinant protein species; and / or the purified composition comprises at least about 85% by w / w of the recombinant protein in the composition before loading.

18. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, characterized in that the recombinant protein is an antigen-binding protein.

19. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, characterized in that the recombinant protein is an antibody.

20. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, characterized in that at least one impurity is selected from host cell proteins, nucleic acids, high molecular weight recombinant protein species, recombinant protein fragments, cell culture media components and viral contaminants. Petition 870250082204, dated 12 / 09 / 2025, pp. 132 / 138

Images