Virus filtration operations using an oversized virus pre-filter.

Abstract

Disclosed herein are methods for removing at least one viral contaminant from a concentrated composition, comprising filtering the concentrated composition through a virus prefilter and a virus filter over one or more filtration cycles, wherein a ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1, as well as viral filtration skids for use in such methods.

Description

Virus filtration operations using an oversized virus pre-filter. CROSS-REFERENCE TO RELATED REQUESTS

[0001] This application claims the priority benefit of Provisional Application No. U.S. 63 / 489,857, filed March 13, 2023, which is incorporated herein by reference in its entirety. FIELD

[0002] The present invention provides methods for removing at least one viral contaminant from a concentrated composition (for example, a composition comprising at least about 10 g / l, such as, for example, at least about 15 g / l, of a recombinant protein), comprising filtering the concentrated composition through a virus pre-filter and a virus filter over one or more filtration cycles, wherein the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is at least about 2:1, as well as viral filtration skids for use in such methods. BACKGROUND

[0003] Mammalian cells used in the production of recombinant protein therapeutic agents are susceptible to viral infection and spread. To remove potential adventitious and endogenous viral contaminants, downstream purification processes for therapeutic proteins generally include dedicated viral clearance operations, such as viral inactivation and viral filtration operations. For example, low pH or detergent-based viral inactivation is commonly employed to denature enveloped viruses. Additionally, retentive virus filtration, an operation Petition 870250080997, dated 09 / 09 / 2025, page 17 / 193 2 / 164 complementary unitary, removes a strip of virus from harvested cell culture fluid through a robust, largely size-based mechanism. Specifically, virus filters with complex pore structures retain viral particles while allowing other solutes to pass through a polymeric membrane.

[0004] Virus filtration is often an expensive unit operation. To extend the achievable throughput and thus reduce the required filter size, associated cost, and area occupied, in-line pre-filters are commonly used in conjunction with virus filters to remove certain contaminants in the product cluster or eluate stream before applying the cluster or eluate to the virus filter. However, despite the improvements in virus filtration economics associated with the use of pre-filters, virus filtration remains one of the most expensive unit operations in protein purification.

[0005] Consequently, there is a need in the field for new and improved virus filtration methods that allow for the robust removal of viral particles in high-throughput processes at reduced cost. SUMMARY

[0006] One aspect of the present disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein the ratio of the virus pre-filter area to the virus filter area is at least about 2:1. Petition 870250080997, dated 09 / 09 / 2025, p. 18 / 193 3 / 164

[0007] Another aspect of the present disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: The virus filter is loaded to at least about 1500 l / m2 over one or more filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0008] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0009] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over one or more filtration cycles.

[0010] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over one or more filtration cycles.

[0011] Yet another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: Petition 870250080997, dated 09 / 09 / 2025, p. 19 / 193 4 / 164 The virus filter is loaded to at least about 30,000 g / m2 over one or more filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0012] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0013] Yet another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: The virus filter is loaded to at least about 1500 l / m2e and / or at least about 30000 g / m2 over one or more filtration cycles; The virus pre-filter is a depth filter; The virus filter comprises at least one flat sheet, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least approximately 2:1.

[0014] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein. Petition 870250080997, dated 09 / 09 / 2025, p. 20 / 193 5 / 164

[0015] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over one or more filtration cycles.

[0016] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over one or more filtration cycles.

[0017] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter comprises polyethersulfone (PES). In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the virus filter comprises polyethersulfone (PES).

[0018] Yet another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: The virus filter is loaded to at least about 1500 l / m2e and / or at least about 30000 g / m2 over at least two filtration cycles, whereby the virus pre-filter is optionally replaced after one or more filtration cycles; The virus pre-filter is a depth filter; The virus filter consists of at least one flat sheet and Petition 870250080997, dated 09 / 09 / 2025, p. 21 / 193 6 / 164 a ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0019] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0020] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over at least two filtration cycles.

[0021] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles.

[0022] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter. In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0023] In some embodiments, the virus filter comprises polyethersulfone (PES). In some embodiments, the virus pre-filter is an earth-based depth filter. Petition 870250080997, dated 09 / 09 / 2025, p. 22 / 193 7 / 164 diatomaceous earth and the virus filter comprises polyethersulfone (PES). In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with sodium carbonate prior to filtering the composition.

[0024] In some embodiments, the virus pre-filter is replaced after one or more filtration cycles.

[0025] In some embodiments, the virus pre-filter is optionally replaced after each filtration cycle.

[0026] In some embodiments, the composition has a pH lower than about 7.2. In some embodiments, the composition has a pH of about 5 to about 7.

[0027] In some embodiments, the composition has a conductivity of at least about 10 mS / cm. In some embodiments, the composition has a conductivity of at least about 12 mS / cm.

[0028] In some embodiments, the composition has a pH less than about 7.2 and a conductivity of at least about 10 mS / cm. In some embodiments, the composition has a pH of about 5 to about 7 and a conductivity of at least about 12 mS / cm.

[0029] Another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, where: The virus pre-filter is replaced after each filtration cycle; Petition 870250080997, dated 09 / 09 / 2025, p. 23 / 193 8 / 164 The virus filter is loaded to at least about 1500 l / m2 over at least two filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0030] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0031] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over at least two filtration cycles.

[0032] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles.

[0033] Yet another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein: The virus pre-filter is replaced after each filtration cycle; Petition 870250080997, dated 09 / 09 / 2025, p. 24 / 193 9 / 164 The virus filter is loaded to at least about 30,000 g / m2 over at least two filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0034] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0035] Another aspect of the disclosure provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, where: The virus pre-filter is replaced after each filtration cycle; The virus filter is loaded to at least approximately 1500 l / m2e or at least approximately 30000 g / m2 over at least two filtration cycles; The virus pre-filter is a depth filter; The virus filter comprises at least one flat sheet, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least approximately 2:1.

[0036] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein. In Petition 870250080997, dated 09 / 09 / 2025, p. 25 / 193 10 / 164 In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0037] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over at least two filtration cycles.

[0038] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles.

[0039] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter comprises polyethersulfone (PES). In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the virus filter comprises polyethersulfone (PES).

[0040] In some embodiments, the composition has a pH lower than about 7.2. In some embodiments, the composition has a pH of about 5 to about 7.

[0041] In some embodiments, the composition has a conductivity of at least about 10 mS / cm. In some embodiments, the composition has a conductivity of at least about 12 mS / cm.

[0042] In some embodiments, the composition has a pH of less than about 7.2 and a conductivity of at least about Petition 870250080997, dated 09 / 09 / 2025, p. 26 / 193 11 / 164 mS / cm. In some forms, the composition has a pH of about 5 to about 7 and a conductivity of at least about 12 mS / cm.

[0043] Yet another aspect of the disclosure provides a viral filtration skid for use in a method described in this document. Yet another aspect of the disclosure provides a viral filtration skid comprising a virus pre-filter and a virus filter, wherein the ratio of the area of ​​the virus pre-filter to the area of ​​the virus filter is at least about 2:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIGURE 1 shows the virus filter flow versus load of a bench-scale proof of concept using a virus prefilter area to virus filter ratio of 2.9:1.

[0045] FIGURE 2 shows bench-scale results for virus filter inlet pressure versus load for “constant flow (250 l / m2 / h (LMH))” runs using the same virus filter for three filtration cycles using an area ratio between the virus pre-filter and the virus filter of 2.9:1 in each filtration cycle (i.e., with a net ratio of 8.7:1 between the virus pre-filter area and the virus filter area).

[0046] FIGURES 3A and 3B show the delta pressure versus elapsed time during constant flow viral filtration (250 LMH) over two filtration cycles (cycle 1 (FIGURE 3A); cycle 2 (FIGURE 3B)) in a bench-scale analysis using an area ratio between the virus pre-filter and the virus filter of 2.9:1 for each cycle. Petition 870250080997, dated 09 / 09 / 2025, page 27 / 193 12 / 164 filtration (i.e., with a net area ratio between the virus pre-filter and the virus filter of 5.8:1).

[0047] FIGURE 4 shows differential pressure measurements of the virus filter at the end of each filtration cycle in pilot-scale runs with three filtration cycles per virus filter using an area ratio between the virus pre-filter and the virus filter of 2.6:1 for each filtration cycle (i.e., with a net area ratio of the virus pre-filter to the virus filter of 7.8:1).

[0048] FIGURE 5 shows the virus filter inlet pressure measurements at the end of each filtration cycle in pilot-scale runs with three filtration cycles per virus filter using an area ratio between the virus pre-filter and the virus filter of 2.6:1 for each filtration cycle (i.e., with a net virus pre-filter area ratio between the virus and the virus filter of 7.8:1). DETAILED DESCRIPTION

[0049] Methods for removing at least one viral contaminant from a concentrated composition (for example, a composition comprising at least about 10 g / l of a recombinant protein) are disclosed in this document, comprising filtering the concentrated composition through a virus pre-filter and a virus filter over one or more filtration cycles, wherein the ratio of virus pre-filter area to virus filter area in each filtration cycle is at least about 2:1, as well as viral filtration skids for use in such methods. DEFINITIONS:

[0050] The following definitions are provided to aid in understanding the scope of this disclosure. Unless Petition 870250080997, dated 09 / 09 / 2025, page 28 / 193 13 / 164 otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by a person of ordinary skill in the art to which this disclosure pertains.

[0051] 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, numerical ranges include the numbers defining the range (i.e., the endpoints).

[0052] When a range of values ​​is provided, it is understood that each intermediate value, up to the tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other stated or intermediate value in that stated range is covered by the disclosure. The upper and lower limits of those smaller ranges may be independently included in the smaller ranges also covered by the disclosure, subject to any limit specifically excluded in the stated range. Where the stated range includes one or both limits, the ranges excluding either or both of the included limits are also included in the disclosure.

[0053] As used in this document, the terms “a” and “an” mean “one or more” unless specifically indicated otherwise. Additionally, “one or more” and “at least one” are used interchangeably in this document. Furthermore, unless otherwise indicated. Petition 870250080997, dated 09 / 09 / 2025, p. 29 / 193 14 / 164 mode required by the context, singular terms include plurals and plural terms include singular.

[0054] 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.

[0055] As used in this document, the term “affinity chromatography” (also referred to as “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.Non-limiting examples of 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.

[0056] In some embodiments, Protein A affinity chromatography can be employed to capture a protein Petition 870250080997, dated 09 / 09 / 2025, page 30 / 193 15 / 164 recombinant 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).

[0057] 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 an 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” (“half antibody consisting of a heavy chain and a light chain”) fusion proteins, or an antigen-binding portion of a full-length antibody, such as, for 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, Faba, 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. Petition 870250080997, dated 09 / 09 / 2025, page 31 / 193 16 / 164 (scFv-CH3-scFv)2, multibody, such as triabodies or tetrabodies, and single-domain antibodies, such as nanobodies or single variable-domain antibodies comprising merely a variable region, which could be VHH, VH, or VL, that binds specifically to an antigen or target independently of other variable regions or domains.

[0058] As used in this document, 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).

[0059] 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.

[0060] 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 domain Petition 870250080997, dated 09 / 09 / 2025, page 32 / 193 17 / 164 constant heavy chain 4 (CH4) of immunoglobulin. 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, 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 via 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.

[0061] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved structural regions (FRs) joined by three hypervariable regions, most often 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 structural 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 has been devised. Petition 870250080997, dated 09 / 09 / 2025, page 33 / 193 18 / 164 to assign numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883. 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).

[0062] 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) 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. Petition 870250080997, dated 09 / 09 / 2025, page 34 / 193 19 / 164

[0063] The “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.

[0064] An “F(ab')2 fragment” is a bivalent fragment including two Fab' fragments linked by a disulfide bridge between the heavy chains in the hinge region.

[0065] The “Fv” fragment is the minimal fragment containing a complete antigen recognition and antibody binding site. This fragment consists of a dimer of an 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 light chain or heavy chain variable region (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. Petition 870250080997, dated 09 / 09 / 2025, page 35 / 193 20 / 164

[0066] “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) .

[0067] A “nanobody” is the variable region of the heavy chain of a heavy chain antibody. Such variable domains are the smallest fully functional 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 lacking light chains occur naturally in certain animal species, such as nurse sharks, wobbegong sharks, and Camelidae, 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, i.e., these functional antibodies are heavy chain homodimers having only the H2L2 structure (referred to as “heavy chain antibodies” or “HCAbs”).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). Petition 870250080997, dated 09 / 09 / 2025, page 36 / 193 21 / 164 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 US Patent publications 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be prepared from human variable-like domains that closely resemble the shark V-NAR scaffold and can provide a framework for a long penetrating loop structure.

[0068] As used in this document, the term “heavy chain-only antibody” refers to an immunoglobulin protein consisting of two heavy chain polypeptides (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 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, measuring 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 absorbed. Petition 870250080997, dated 09 / 09 / 2025, p. 37 / 193 22 / 164 cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Its expression levels, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a unique VH-like domain in their antibodies, termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0069] In some embodiments, a “heavy chain-only antibody” is a dimeric antibody comprising an HV antigen-binding domain and 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, a heavy chain-only antibody is composed 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 is composed of an antigen-binding domain, at least part of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region, and a CH3 domain.Heavy chain-only antibodies in which the CH2 and / or CH3 domain is truncated are also included in this document. The heavy chain-only antibodies described in this document may belong to the subclass. Petition 870250080997, dated 09 / 09 / 2025, page 38 / 193 23 / 164 of IgG, but heavy chain-only antibodies belonging to other subclasses, such as IgM, IgA, IgD, and IgE subclasses, are also included in this document. 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 in this document.Non-limiting examples of heavy chain-only antibodies are described, for example, in document no. WO2018 / 039180, the disclosure of which is incorporated herein in its entirety by reference.

[0070] As used in this document, the term “three-chain antibody-like molecule” or “TCA” refers to antibody-like molecules comprising, essentially consisting of, or consisting of three polypeptide subunits, two of which comprise, essentially consist of, or consist of a heavy and a light chain of a monoclonal antibody, or antigen-binding fragments of such antibody chains comprising an antigen-binding region and at least Petition 870250080997, dated 09 / 09 / 2025, page 39 / 193 24 / 164 a 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 an epitope different from the first antigen, where 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 VH and / or VL gene segments, D and JH gene segments, or JL gene segments. The variable region may be encoded by rearranged VhDJh, VlDJh, VhJl, or VlJl gene segments.

[0071] As used in this document, the term bioreactor means any vessel useful for growing a cell culture (e.g., a mammalian cell culture or a bacterial cell culture). Bioreactor encompasses the term fermenter (i.e., a vessel useful for growing a bacterial cell culture, which typically contains a more rigorous agitator and increased gas flow compared to a vessel used for growing a mammalian cell culture) in this document. Non-limiting examples of bioreactors include stirred tank, air-transport, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. In some embodiments, an exemplary bioreactor may perform a Petition 870250080997, dated 09 / 09 / 2025, page 40 / 193 25 / 164 or more (e.g., one, two, three, all) of the following steps: feeding of nutrients (such as, for example, in perfusion cell culture) 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 cell culture being grown within 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, 20000 liters or more or any intermediate volume. The internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen (DO), and temperature, can be controlled during the culture period. People with common skills in... Petition 870250080997, dated 09 / 09 / 2025, page 41 / 193 26 / 164 technicians will be aware of, and will be able to select, suitable bioreactors based on relevant considerations.

[0072] As used in this document, 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, can be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0073] As used in this document, the term “cell culture medium” (also referred to 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 (for example, in the form of a carbohydrate, such as glucose); one or more amino acids. Petition 870250080997, dated 09 / 09 / 2025, page 42 / 193 27 / 164 essential (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 are known to be used in any cell culture process, including, but not limited to, batch, extended batch, fed-batch and / or perfusion or continuous cell culture.

[0074] As used in this document, 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) and can be measured at any point during a specific phase of a cell culture process. As used in this document, the term “packed cell volume (PCV), also referred to as “percentage of packed cell volume (% PCV),” is the ratio of the volume occupied by the cells to the total volume of the cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20;95(6):1228-33). Packed cell volume is a function of cell density and cell diameter; increases in cell volume Petition 870250080997, dated 09 / 09 / 2025, page 43 / 193 Packed cells (28 / 164) can arise from increases in cell density or cell diameter, or both. Packed cell volume is a measure of the solid content in 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.

[0075] 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.

[0076] 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 actual flow conditions before significant discovery of unbound product occurs.

[0077] 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 bound 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 a region. Petition 870250080997, dated 09 / 09 / 2025, page 44 / 193 29 / 164 operationally linked coding sequence. Operationally linked means that the components to which the term is applied are in a relationship that allows them to carry out 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.

[0078] As used in this document, a fed-batch culture refers to a form of suspension culture, specifically a cell culture method in which additional components are supplied to the culture at one or more subsequent times after the start of the culture process. The supplied components typically comprise nutritional supplements for the 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 medium formulations may be richer or more concentrated than the base cell culture medium formulations, which contain components essential for cell survival and growth and are typically used to initiate a cell culture.A fed-batch culture can be stopped at some point, and the cells and / or components in the medium can be collected and optionally purified. Petition 870250080997, dated 09 / 09 / 2025, page 45 / 193 30 / 164

[0079] As used in this document, a “fusion protein” is a protein that contains 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 with, 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.

[0080] As used in this document, 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.

[0081] 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 above, include, but are not limited to, cooling, flocculation, acidification, centrifugation, neutralization, acoustic wave separation, and various forms of filtration (e.g., depth filtration, microfiltration, Petition 870250080997, dated 09 / 09 / 2025, page 46 / 193 31 / 164 ultrafiltration, tangential flow filtration and alternating tangential flow filtration). The harvested cell culture fluid includes cell culture lysates as well as cell culture supernatants. The collected cell culture fluid may 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.

[0082] 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 the gene of interest 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).

[0083] 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 Petition 870250080997, dated 09 / 09 / 2025, page 47 / 193 32 / 164 is the molecular weight of the intact, fully assembled form of the recombinant protein.

[0084] As used in this document, the term impurity refers to a component other than the recombinant protein of interest. Impurities include, but are not limited to, process- and product-related impurities such as host cell proteins, leached resin materials (such as 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.

[0085] As used in this document, the term charge density refers to the amount of composition brought into contact with a volume of chromatography material.

[0086] 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 (e.g., light-chain only or heavy-chain only species when the recombinant protein is an antibody). Petition 870250080997, dated 09 / 09 / 2025, page 48 / 193 33 / 164

[0087] As used in this document, 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.

[0088] 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.

[0089] 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.

[0090] As used herein, a production phase of a cell culture refers to the period of Petition 870250080997, dated 09 / 09 / 2025, page 49 / 193 34 / 164 time during which logarithmic cell growth has ended and recombinant protein production is predominant.

[0091] As used in this document, the term “polishing chromatography” refers to a chromatographic operation performed after a capture or affinity chromatography operation to remove remaining impurities and obtain a more highly purified composition 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. In addition, typical chromatographic techniques used for polishing include, but are not limited to, ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimodal (or mixed-mode) chromatography (MMC).

[0092] “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 TMAE™, FRACTOGEL EDM DEAE™, (EMD Merck), TOYOPEARL Super Q® and TOYOPEARL NH2-750F (Tosoh Bioscience), POROS HQ™ and POROS XQ™, (ThermoFisher). Petition 870250080997, dated 09 / 09 / 2025, page 50 / 193 35 / 164

[0093] 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 cations for cations in an aqueous solution passed over or through the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase, for example, via covalent bonding. Alternatively or additionally, the charge can 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).

[0094] 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 solute. The available hydrophobic interaction chromatography media Petition 870250080997, dated 09 / 09 / 2025, page 51 / 193 36 / 164 commercially available products include, but are not limited to, Phenyl Sephrose™ (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto™phenyl (Cytiva).

[0095] Mixed-mode or multimodal chromatography (MMC) 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).

[0096] 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, two of which are the binding and elution mode and the 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 washing 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. Regarding binding and elution chromatography, chromatography Petition 870250080997, dated 09 / 09 / 2025, page 52 / 193 37 / 164 continuous flow allows for higher charge densities for many biomolecules.

[0097] In addition to the two most common modes, weak partition chromatography, overload chromatography, and forward chromatography modes can also be employed in purification processes. In weak partition chromatography, an isocratic separation method, the continuous flow mode is altered by identifying solution conditions that promote weak binding of a biomolecule to the resin, in addition to the binding of one or more impurities, with a low product partition coefficient in the range of 0.1-20. In overload chromatography, the biomolecule of interest is loaded onto the chromatography material beyond the dynamic binding capacity of the material. Additionally, forward chromatography mode allows for continuous feeding of high-density material (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 feedstock 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 more 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 the affinity for the chromatography medium. When the chromatography medium reaches saturation binding, those... Petition 870250080997, dated 09 / 09 / 2025, page 53 / 193 38 / 164 Components in the feed that have a higher affinity for the chromatography medium (typically product-related impurities, such as a type of HMW) will displace 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 feed stream. As the feed progresses, 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.

[0098] 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.

[0099] 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 Petition 870250080997, dated 09 / 09 / 2025, p. 54 / 193 39 / 164 laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0100] 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.

[0101] As used in this document, the term unit operation refers to a functional step that is 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 virus capture and inactivation steps. Unit operations may also include retention or storage steps between processing steps.

[0102] As used in this document, the term virus prefilter refers to an upstream filter in fluid communication with a virus filter, which is capable of binding one or more bioprocess impurities to increase the flow and / or productivity of the virus filter.

[0103] As used in this document, the term filtration cycle refers to a period of time (such as, for example, a continuous period of time before a scheduled interval) during which a composition is filtered through a virus filter. A virus filter may be used for one or more (e.g., one, two, three, four or more) filtration cycles, wherein an interruption occurs between each filtration cycle and the virus pre-filter is optionally replaced for each filtration cycle. Petition 870250080997, dated 09 / 09 / 2025, page 55 / 193 40 / 164

[0104] As used in this document, the phrase the ratio between the virus prefilter area and the virus filter area refers to the ratio between the virus prefilter area and the virus filter area in a given filtration cycle.

[0105] As used in this document, the phrase net ratio of virus prefilter area to virus filter area refers to the ratio of the cumulative area of ​​the virus prefilter to the area of ​​the virus filter in one or more filtration cycles (i.e., the ratio of the area of ​​all virus prefilters used during the lifetime of a virus filter to the area of ​​the virus filter). The ratio of virus prefilter area to virus filter area and the net ratio of virus prefilter area to virus filter area may be the same (e.g., when the virus prefilter is not replaced during the lifetime of the virus filter) or different (e.g., when the virus prefilter is replaced between one or more filtration cycles during the lifetime of the virus filter). NON-LIMITING EXAMPLES:

[0106] Without limitation, some exemplary features / resources of the present disclosure include: E1. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein the ratio of the virus pre-filter area to the virus filter area is at least about 2:1. E2. The method according to E1, where the virus pre-filter is a depth filter. Petition 870250080997, dated 09 / 09 / 2025, p. 56 / 193 41 / 164 E3. The method according to E1 or E2, wherein the virus pre-filter is a diatomaceous earth-based depth filter. E4. The method according to E3, which additionally comprises washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition. E5. The method according to any of E1 to E4, wherein the virus filter comprises at least one flat sheet. E6. The method according to any of E1 to E5, wherein the virus filter comprises polyethersulfone (PES). E7. The method conforms to any of E1 to E6, where the virus filter is a flat sheet PES membrane. E8. The method is in accordance with any of E1 to E7, where the virus filter is a small virus filter. E9. The method is in accordance with any of E1 to E7, where the virus filter is a large virus filter. E10. The method follows any of E1 to E9, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is approximately 2:1 to approximately 4:1. E11. The method follows any of E1 to E10, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is approximately 2:1 to approximately 3:1. E12. The method follows any of E1 to E11, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is approximately 2.5:1 to approximately 3:1. E13. The method according to any of E1 to E12, which additionally comprises adjusting the pH of the composition to less than about 7.2 before filtration. Petition 870250080997, dated 09 / 09 / 2025, p. 57 / 193 42 / 164 E14. The method according to any of E1 to E12, which additionally comprises adjusting the pH of the composition to about 5 to about 7 before filtration. E15. The method according to any of E1 to E14, which additionally comprises adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. E16. The method according to any of E1 to E15, which additionally comprises adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. E17. The method according to any of E1 to E15, which additionally comprises adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration. E18. The method in accordance with any of E1 to E17, comprising at least two filtration cycles, in which the virus pre-filter is optionally replaced after each filtration cycle. E19. The method in accordance with any of E1 to E17, comprising at least two filtration cycles, in which the virus pre-filter is optionally replaced after each filtration cycle. E20. The method conforming to any of E1 to E17, comprising at least two filtration cycles, in which the virus pre-filter is replaced after each filtration cycle. E21. The method according to any of E1 to E17, which comprises three filtration cycles, where the net ratio Petition 870250080997, dated 09 / 09 / 2025, p. 58 / 193 The ratio between the virus pre-filter area and the virus filter area is at least approximately 6:1. E22. The method according to E21, where the net ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is about 6:1 to about 9:1. E23. The method according to any of E1 to E22, in which the composition is filtered through the virus filter at a flow of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. E24. The method according to any of E1 to E23, in which the composition is filtered through the virus filter at a flow of approximately 200 l / m2 / ha or approximately 300 l / m2 / h. E25. The method according to any of E1 to E24, in which the composition is filtered through the virus filter at a flow rate of approximately 250 l / m2 / h. E26. The method according to any of E1 to E25, in which the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). E27. The method according to any of E1 to E26, in which the composition is filtered through the virus filter at a pressure of about 0.20 MPa (30 psi). E28. The method according to any one of E1 to E27, wherein the method comprises loading the virus filter to at least about 1500 l / m2 over one or more filtration cycles. E29. The method according to any one of E1 to E28, wherein the method comprises loading the virus filter to at least about 1500 l / m2 over three filtration cycles. E30. The method according to E29, wherein the method comprises loading the virus filter to at least about 500 l / m2 for each of the three filtration cycles. Petition 870250080997, dated 09 / 09 / 2025, p. 59 / 193 44 / 164 E31. The method according to E29 or E30, in which the composition is filtered through the virus filter at a flow rate of at least about 250 l / m2 / h for each of the three filtration cycles. E32. The method according to any of E1 to E31, wherein the method comprises loading the virus filter to at least about 30000 g / m2 over one or more filtration cycles. E33. The method according to E32, wherein the method comprises loading the virus filter to at least about 30000 g / m2 over three filtration cycles. E34. The method according to any of E1 to E33, in which at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus and reovirus. E35. The method according to any of E1 to E34, wherein the composition comprises a recombinant protein. E36. The method according to E35, wherein the composition comprises at least about 15 g / l of recombinant protein. E37. The method according to E35 or E36, wherein the composition comprises approximately 15 g / l to approximately 25 g / l of recombinant protein. E38. The method according to any of E1 to E37, in which filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. E39. The method according to any of E1 to E38, wherein filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of the three filtration cycles. Petition 870250080997, dated 09 / 09 / 2025, p. 60 / 193 45 / 164 E40. A viral filtration skid comprising a virus pre-filter and a virus filter, wherein the ratio of the area of ​​the virus pre-filter to the area of ​​the virus filter is at least about 2:1. E41. The viral filtration skid according to E40, where the virus pre-filter is a depth filter. E42. The viral filtration skid according to E40 or E41, where the virus pre-filter is a diatomaceous earth-based depth filter. E43. The viral filtration skid conforms to any one of E40 to E42, wherein the virus filter is composed of at least one flat sheet. E44. The viral filtration skid conforms to any one of E40 to E43, wherein the virus filter comprises polyethersulfone (PES). E45. The viral filtration skid conforms to any of E40 to E44, where the virus filter is a flat sheet PES membrane. E46. The viral filtration skid conforms to any of E40 to E45, where the virus filter is a small virus filter. E47. The viral filtration skid conforms to any of E40 to E45, where the virus filter is a large virus filter. E48. The viral filtration skid conforms to any of E40 to E47, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is approximately 2:1 to approximately 4:1. E49. The viral filtration skid corresponds to any of E40 to E48, where the ratio between the area of ​​the virus pre-filter is... Petition 870250080997, dated 09 / 09 / 2025, page 61 / 193 46 / 164 and the virus filter area is approximately 2:1 to approximately 3:1. E50. The viral filtration skid corresponds to any of E40 to E49, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is approximately 2.5:1 to approximately 3:1. E51. The viral filtration skid conforming to any of E40 to E50, which additionally comprises at least one online monitoring system. E52. The viral filtration skid according to E51, wherein at least one online monitoring system monitors at least one property selected from pressure, flow, pH, conductivity and UV. E53. The viral filtration skid conforms to any of E40 to E52, where the virus pre-filter is positioned in series with the virus filter.

[0107] Additionally, without limitation, some other examples of features / resources of the present disclosure include: F1. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: The virus filter is loaded to at least about 1500 l / m2 over one or more filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1. F2. A method for removing at least one viral contaminant from a composition, comprising filtering the composition. Petition 870250080997, dated 09 / 09 / 2025, p. 62 / 193 47 / 164 through a virus pre-filter and a virus filter, where: The virus filter is loaded to at least about 30,000 g / m2 over one or more filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1. F3. The method according to F1 or F2, comprising at least two filtration cycles, wherein the virus pre-filter is optionally replaced after one or more filtration cycles. F4. The method according to any of F1 to F3, comprising at least two filtration cycles, in which the virus pre-filter is optionally replaced after each filtration cycle. F5. The method according to F1 or F2, which comprises at least two filtration cycles, in which the virus pre-filter is replaced after one or more filtration cycles. F6. The method according to F1 or F2, which comprises at least two filtration cycles, in which the virus pre-filter is replaced after each filtration cycle. F7. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein the virus pre-filter is replaced after each filtration cycle, in which: the virus filter is loaded to at least approximately 1500 l / m2 over at least two filtration cycles and Petition 870250080997, dated 09 / 09 / 2025, page 63 / 193 48 / 164 a ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1. F8. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein the virus pre-filter is replaced after each filtration cycle, in which: The virus filter is loaded to at least about 30,000 g / m2 over at least two filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1. F9. The method is in accordance with any of F1 to F8, where the virus pre-filter is a depth filter. F10. The method is consistent with any of F1 to F9, where the virus pre-filter is a diatomaceous earth-based depth filter. F11. The method according to F10, which additionally includes washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition. F12. The method according to any of F1 to F11, in which the virus filter is comprised of at least one flat sheet. F13. The method conforms to any of F1 to F12, wherein the virus filter comprises polyethersulfone (PES). Petition 870250080997, dated 09 / 09 / 2025, page 64 / 193 49 / 164 F14. The method conforms to any of F1 to F13, where the virus filter is a flat sheet PES membrane. F15. The method conforms to any of F1 to F14, where the virus pre-filter is a depth filter and the virus filter is a flat sheet PES membrane. F16. The method conforms to any of F1 to F8, where the virus pre-filter is a diatomaceous earth-based depth filter and the virus filter is a flat sheet PES membrane. F17. The method is consistent with any of F1 through F8, where the virus filter is a small virus filter. F18. The method is consistent with any of F1 through F8, where the virus filter is a large virus filter. F19. The method follows any of F1 to F18, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is approximately 2:1 to approximately 4:1. F20. The method follows any of F1 to F19, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is approximately 2:1 to approximately 3:1. F21. The method follows any of F1 to F20, where the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is approximately 2.5:1 to approximately 3:1. F22. The method according to any of F1 to F21, which additionally comprises adjusting the pH of the composition to less than about 7.2 before filtration. Petition 870250080997, dated 09 / 09 / 2025, p. 65 / 193 50 / 164 F23. The method according to any of F1 to F22, which additionally comprises adjusting the pH of the composition to about 5 to about 7 before filtration. F24. The method according to any of F1 to F23, which additionally comprises adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. F25. The method according to any of F1 to F24, which additionally comprises adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. F2 6. The method according to any of F1 to F23, which additionally comprises adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration. F27. The method according to any of F1 to F26, comprising three filtration cycles, where the net ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter over the three filtration cycles is at least about 6:1. F28. The method according to F27, in which the net ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter is about 6:1 to about 9:1. F29. The method is in accordance with any of F1 to F28, in which the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. F30. The method is in accordance with any of F1 to F29, in which the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha or approximately 300 l / m2 / h. Petition 870250080997, dated 09 / 09 / 2025, p. 66 / 193 51 / 164 F31. The method according to any of F1 to F30, in which the composition is filtered through the virus filter at a flow rate of approximately 250 l / m2 / h. F32. The method according to any of F1 to F31, in which the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). F33. The method according to any of F1 to F32, in which the composition is filtered through the virus filter at a pressure of about 0.20 MPa (30 psi). F34. The method according to any of F1 to F33, wherein the method comprises loading the virus filter to at least about 1500 l / m2 (for example, at least about 2000 l / m2; about 1500 l / m2 to about 3000 l / m2) over three filtration cycles. F35. The method according to F34, wherein the method comprises loading the virus filter to at least about 500 l / m2 for each of the three filtration cycles. F36. The method according to F34 or F35, wherein the composition is filtered through the virus filter at a flow rate of at least about 250 l / m2 / h for each of the three filtration cycles. F37. The method according to any of F1 to F36, wherein the method comprises loading the virus filter to at least about 30000 g / m2 over three filtration cycles. F38. The method according to any of F1 to F37, in which at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. F39. The method conforms to any of F1 to F38, wherein the composition comprises a recombinant protein. Petition 870250080997, dated 09 / 09 / 2025, page 67 / 193 52 / 164 F40. The method according to F39, wherein the recombinant protein is an antibody (e.g., an IgG2 antibody). F41. The method according to either F1-F16 or F19-F40, wherein: The virus pre-filter is a depth filter based on diatomaceous earth; The virus filter comprises polyethersulfone (PES); The method further involves washing the diatomaceous earth-based depth filter with a solution containing carbonate before filtering the composition. F42. The method according to F41, where: The virus pre-filter is a depth filter based on diatomaceous earth; The virus filter comprises polyethersulfone (PES); The method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition and after filtering the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l); F43. The method according to F41 or F42, wherein the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. F44. The method according to any of F41 to F43, in which, after filtration of the composition, the composition has a β-glucan concentration of less than about 10 pg / l. Petition 870250080997, dated 09 / 09 / 2025, p. 68 / 193 53 / 164 F45. The method according to any of F1-F16 or F19-F40, where: The virus pre-filter is a depth filter based on diatomaceous earth; The virus filter comprises polyethersulfone (PES); The method further involves washing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition. F46. The method according to F45, wherein, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; F47. The method according to any of F1 to F40, wherein, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l, less than about 15 pg / l; F48. The method conforms to any of F1 to F47, wherein the composition comprises at least about 10 g / l of a recombinant protein. F49. The method according to any of F1 to F47, wherein the composition comprises at least about 12.5 g / l of a recombinant protein. Petition 870250080997, dated 09 / 09 / 2025, p. 69 / 193 54 / 164 F50. The method conforms to any of F1 to F47, wherein the composition comprises at least about 15 g / l of a recombinant protein. F51. The method according to any of F1 to F47, wherein the composition comprises approximately 15 g / l to approximately 25 g / l of a recombinant protein. F52. The method according to any of F1 to F47, wherein the composition comprises approximately 15 g / l to approximately 20 g / l of a recombinant protein. F53. The method according to any of F1 to F52, in which filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. F54. The method according to any of F1 to F53, wherein filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of the three filtration cycles. F55. A viral filtration skid for use in a method conforming to any of F1 to F54. Viral Filtration Methods Using an Oversized Pre-filter

[0108] A method for removing at least one viral contaminant from a composition is provided herein, comprising filtering the composition through a virus pre-filter and a virus filter, wherein the ratio of the virus pre-filter area to the virus filter area is at least about 2:1.

[0109] In some embodiments, the virus pre-filter is a depth filter. In some embodiments, the virus pre-filter is a ground-based depth filter. Petition 870250080997, dated 09 / 09 / 2025, page 70 / 193 55 / 164 diatomaceous earth. In some embodiments, the virus prefilter is a depth filter comprising diatomaceous earth, cellulose fibers, and a binder comprising cationic imine groups.

[0110] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition.

[0111] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 pm. In some embodiments, the virus prefilter is a membrane of about 0.1 pm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0112] In some embodiments, the virus prefilter is an absorbent membrane. In some embodiments, the virus prefilter is an absorbent membrane with ion exchange functionality.

[0113] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface modified by crosslinked polymeric sulfonic acid cation exchange chemistry. In some embodiments, the virus prefilter is a flat sheet membrane with a crosslinked mixed-mode PES membrane surface modified by crosslinked mixed-mode chemistry.

[0114] In some embodiments, the virus prefilter is a triple-layer flat sheet membrane. In some Petition 870250080997, dated 09 / 09 / 2025, page 71 / 193 In the 56 / 164 modalities, the virus pre-filter is a three-layer polyamide flat sheet membrane.

[0115] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane comprising nylon. In some embodiments, the virus prefilter is a pleated sheet membrane comprising PVDF modified with hydrophilic acrylate.

[0116] In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane and the virus prefilter comprises hydrophilic cuprammonium regenerated cellulose.

[0117] In some embodiments, the virus filter comprises at least one flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet and the virus filter comprises hydrophilic polyethersulfone (PES).

[0118] In some embodiments, the virus filter comprises at least one pleated sheet.

[0119] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and the virus filter comprises hydrophilic PES.

[0120] In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises Petition 870250080997, dated 09 / 09 / 2025, page 72 / 193 57 / 164 Hydrophilic PES. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises surface-modified PES. In some embodiments, the virus filter comprises a triple-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a triple-layer asymmetric pleated sheet and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises a triple-layer asymmetric pleated sheet and the virus filter comprises hydrophilic polyvinylidene fluoride (PVDF).

[0121] In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate.

[0122] In some embodiments, the virus filter comprises hollow fibers. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic cuprammonium regenerated cellulose. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises modified PVDF. In some embodiments, Petition 870250080997, dated 09 / 09 / 2025, page 73 / 193 58 / 164 the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic PES.

[0123] In some embodiments, the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises surface-modified PES.

[0124] In some embodiments, the virus filter comprises modified PVDF. In some embodiments, the virus filter comprises hydrophilic PVDF. In some embodiments, the virus filter comprises PVDF modified with hydrophilic acrylate.

[0125] In some embodiments, the virus filter comprises hydrophilic cuprammonium regenerated cellulose.

[0126] In some forms, the virus filter is a small virus filter.

[0127] In some forms, the virus filter is a large virus filter.

[0128] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2:1 to about 4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2:1 to about 3:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2.5:1 to about 3:1.

[0129] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.6:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.9:1. Petition 870250080997, dated 09 / 09 / 2025, page 74 / 193 59 / 164

[0130] In some embodiments, the method further comprises preconditioning the composition before filtration. In some embodiments, the method further comprises online preconditioning of the composition before filtration.

[0131] In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7.5 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7.2 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to about 5 to about 7 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to about 6 to about 7 before filtration.

[0132] In some embodiments, the method further comprises adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. In some embodiments, the method further comprises adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. In some embodiments, the method further comprises adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration.

[0133] In some modes, the composition is filtered through the virus filter in normal flow filtering mode. In some modes, the composition is filtered Petition 870250080997, dated 09 / 09 / 2025, page 75 / 193 60 / 164 through the virus filter in tangential flow filtering mode.

[0134] In some embodiments, the composition is filtered through the virus pre-filter and the virus filter in normal flow filtration mode.

[0135] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 400 l / m2 / h. In some methods, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 300 l / m2 / h.

[0136] In some embodiments, the composition is filtered through the virus filter at a flow rate of about 100 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 150 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 200 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 250 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 300 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 350 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of about 400 l / m² / h.

[0137] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the filter of Petition 870250080997, dated 09 / 09 / 2025, p. 76 / 193 61 / 164 viruses at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0138] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid.

[0139] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0140] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid) to about 413685.6 Pa (60 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at an inlet pressure of about 137895.2 Pa (20 psid) to about 344738 Pa (50 psid).

[0141] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered Petition 870250080997, dated 09 / 09 / 2025, p. 77 / 193 62 / 164 through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi).

[0142] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of approximately 0.06 MPa (10 psi).

[0143] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the filter of Petition 870250080997, dated 09 / 09 / 2025, page 78 / 193 63 / 164 viruses in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid).

[0144] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of approximately 0.06 MPa (10 psi) / psid. Petition 870250080997, dated 09 / 09 / 2025, p. 79 / 193 64 / 164

[0145] In some embodiments, the method comprises loading the virus filter to at least about 1500 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 1500 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter to at least about 1500 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 1500 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 1500 l / m2 over more than three filtration cycles.

[0146] In some embodiments, the method comprises loading the virus filter with about 1500 l / m2 to about 3000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter with about 1500 l / m2 to about 3000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter with about 1500 l / m2 to about 3000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter with about 1500 l / m2 to about 3000 l / m2 over three filtration cycles. In some embodiments, the method involves loading the virus filter with approximately 1500 l / m2 to approximately 3000 l / m2 over more than three filtration cycles.

[0147] In some embodiments, the method comprises loading the virus filter with approximately 1500 l / m2 to approximately 2000 l / m2 over one or more filtration cycles. In some Petition 870250080997, dated 09 / 09 / 2025, p. 80 / 193 In some embodiments, the method comprises loading the virus filter with approximately 1500 l / m2 to approximately 2000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter with approximately 1500 l / m2 to approximately 2000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter with approximately 1500 l / m2 to approximately 2000 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter with approximately 1500 l / m2 to approximately 2000 l / m2 over more than three filtration cycles.

[0148] In some embodiments, the method comprises loading the virus filter with a maximum of about 3,000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of about 3,000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter with a maximum of about 3,000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of about 3,000 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of about 3,000 l / m2 over more than three filtration cycles.

[0149] In some embodiments, the method comprises loading the virus filter with a maximum of about 2,000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of about 2,000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the filter Petition 870250080997, dated 09 / 09 / 2025, p. 81 / 193 66 / 164 of viruses with a maximum of approximately 2000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of approximately 2000 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter with a maximum of approximately 2000 l / m2 over more than three filtration cycles.

[0150] In some embodiments, the method comprises loading the virus filter to at least about 30,000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 30,000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter to at least about 30,000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 30,000 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 30,000 l / m2 over more than three filtration cycles.

[0151] In some embodiments, the method comprises loading the virus filter to at least about 50,000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 50,000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter to at least about 50,000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 50,000 l / m2 over three filtration cycles. In some Petition 870250080997, dated 09 / 09 / 2025, p. 82 / 193 67 / 164 modalities, the method comprises loading the virus filter to at least approximately 50,000 l / m2 over more than three filtration cycles.

[0152] In some embodiments, the method comprises loading the virus filter to at least about 60,000 l / m2 over one or more filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 60,000 l / m2 over one filtration cycle. In some embodiments, the method comprises loading the virus filter to at least about 60,000 l / m2 over two filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 60,000 l / m2 over three filtration cycles. In some embodiments, the method comprises loading the virus filter to at least about 60,000 l / m2 over more than three filtration cycles.

[0153] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one viral contaminant is a parvovirus. In some embodiments, at least one viral contaminant is a retrovirus. In some embodiments, at least one viral contaminant is a pseudorabies virus. In some embodiments, at least one viral contaminant is a reovirus.

[0154] In some embodiments, the composition comprises a recombinant protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is a bispecific antibody. In some embodiments, the recombinant protein is a fragment Petition 870250080997, dated 09 / 09 / 2025, page 83 / 193 68 / 164 antibody. In some embodiments, the recombinant protein is a variable single-chain fragment. In some embodiments, the recombinant protein is a fusion protein.

[0155] In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 17.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 20 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 25 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 30 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 50 g / l of a recombinant protein.

[0156] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of Petition 870250080997, dated 09 / 09 / 2025, p. 84 / 193 69 / 164 at least about 4 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of more than three filtration cycles.

[0157] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three filtration cycles. In some methods, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of more than three filtration cycles.

[0158] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about Petition 870250080997, dated 09 / 09 / 2025, p. 85 / 193 70 / 164 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of the more than three filtration cycles.

[0159] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of three filtration cycles. In some methods, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the more than three filtration cycles.

[0160] This document provides a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: Petition 870250080997, dated 09 / 09 / 2025, p. 86 / 193 71 / 164 the virus filter is loaded to at least about 1500 l / m2e / or at least about 30000 g / m2 in one or more filtration cycles and a ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0161] In some embodiments, the virus filter is loaded to at least about 1500 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded to at least about 2000 l / m2 over one or more filtration cycles.

[0162] In some embodiments, the virus filter is loaded with about 1500 l / m2 to about 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with about 2000 l / m2 to about 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with about 2500 l / m2 to about 3000 l / m2 over one or more filtration cycles.

[0163] In some embodiments, the virus filter is loaded to at least about 30000 g / m2 over one or more filtration cycles.

[0164] In some embodiments, the composition comprises a recombinant protein.

[0165] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein (for example, at least about 10.5 g / l, at least about 11 g / l, at least about 11.5 g / l, at least about 12 g / l, at least about 12.5 g / l, at least about 13 g / l, at least about 13.5 g / l, at least about 14 Petition 870250080997, dated 09 / 09 / 2025, p. 87 / 193 72 / 164 g / l, at least about 14.5 g / l, at least about 15 g / l; about 10 g / l; about 10.5 g / l, about 11 g / l; about 11.5 g / l; about 12 g / l; about 12.5 g / l; about 13 g / l; about 13.5 g / l; about 14 g / l; about 14.5 g / l; about 15 g / l; about 15.5 g / l; about 16 g / l; about 16.5 g / l; about 17 g / l; about 17.5 g / l; about 18 g / l; about 18.5 g / l; about 19 g / l; about 19.5 g / l; about 20 g / l; about 20.5 g / l; about 21 g / l; about 21.5 g / l; about 22 g / l; about 22.5 g / l; about 23 g / l; about 23.5 g / l; about 24 g / l; about 24.5 g / l; about 25 g / l).

[0166] In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0167] In some embodiments, the composition comprises at least about 17.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 20 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 25 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 30 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 50 g / l of a recombinant protein.

[0168] In some embodiments, the composition comprises approximately 15 g / l to approximately 25 g / l of a recombinant protein. In some embodiments, the composition Petition 870250080997, dated 09 / 09 / 2025, p. 88 / 193 73 / 164 comprises approximately 15 g / l to approximately 20 g / l of a recombinant protein.

[0169] In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is optionally replaced after one or more filtration cycles. In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is replaced after one or more filtration cycles.

[0170] In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is optionally replaced after each filtration cycle. In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is replaced after each filtration cycle.

[0171] In some embodiments, the virus prefilter is a depth filter. In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus prefilter is a depth filter comprising diatomaceous earth, cellulose fibers, and a binder comprising cationic imine groups.

[0172] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition.

[0173] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with a solution that Petition 870250080997, dated 09 / 09 / 2025, p. 89 / 193 74 / 164 contains carbonate before filtration of the composition. In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter; the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtration of the composition, and after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l); In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0174] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, Petition 870250080997, dated 09 / 09 / 2025, pp. 90 / 193 75 / 164 less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; about 5 pg / l less than about 10 pg / l). In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0175] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0176] In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 10 pg / l. In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of about 5 pg / l less than about 15 pg / l. In some embodiments, after filtration From the composition, the composition has a β-glucan concentration of approximately 5 pg / l, less than approximately 12.5 pg / l. In some embodiments, after filtration of the composition, the composition has a Petition 870250080997, dated 09 / 09 / 2025, pp. 91 / 193 76 / 164 β-glucan concentration of approximately 5 pg / l less than approximately 10 pg / l.

[0177] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 pm. In some embodiments, the virus prefilter is a membrane of about 0.1 pm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0178] In some embodiments, the virus prefilter is an absorbent membrane. In some embodiments, the virus prefilter is an absorbent membrane with ion exchange functionality.

[0179] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface modified by crosslinked polymeric sulfonic acid cation exchange chemistry. In some embodiments, the virus prefilter is a flat sheet membrane with a crosslinked mixed-mode PES membrane surface modified by crosslinked mixed-mode chemistry.

[0180] In some embodiments, the virus prefilter is a triple-layer flat sheet membrane. In some embodiments, the virus prefilter is a three-layer polyamide flat sheet membrane.

[0181] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane comprising nylon. In some embodiments, the prefilter of Petition 870250080997, dated 09 / 09 / 2025, pp. 92 / 193 77 / 164 virus is a pleated sheet membrane comprising PVDF modified with hydrophilic acrylate.

[0182] In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane and the virus prefilter comprises hydrophilic cuprammonium regenerated cellulose.

[0183] In some embodiments, the virus filter comprises at least one flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet and the virus filter comprises hydrophilic polyethersulfone (PES).

[0184] In some embodiments, the virus filter comprises at least one pleated sheet.

[0185] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and the virus filter comprises hydrophilic PES.

[0186] In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises surface-modified PES. In some embodiments, the virus filter comprises a triple-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a pleated sheet Petition 870250080997, dated 09 / 09 / 2025, page 93 / 193 78 / 164 asymmetric triple-layer pleated sheet and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises an asymmetric triple-layer pleated sheet and the virus filter comprises hydrophilic polyvinylidene fluoride (PVDF).

[0187] In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate.

[0188] In some embodiments, the virus filter comprises hollow fibers. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic cuprammonium regenerated cellulose. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises modified PVDF. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic PES.

[0189] In some embodiments, the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises surface-modified PES. Petition 870250080997, dated 09 / 09 / 2025, page 94 / 193 79 / 164

[0190] In some embodiments, the virus filter comprises modified PVDF. In some embodiments, the virus filter comprises hydrophilic PVDF. In some embodiments, the virus filter comprises PVDF modified with hydrophilic acrylate.

[0191] In some embodiments, the virus filter comprises hydrophilic cuprammonium regenerated cellulose.

[0192] In some forms, the virus filter is a small virus filter.

[0193] In some forms, the virus filter is a large virus filter.

[0194] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 3:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2.5:1 to about 3:1.

[0195] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.6:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.9:1.

[0196] In some embodiments, the method additionally comprises the preconditioning of the composition before Petition 870250080997, dated 09 / 09 / 2025, page 95 / 193 80 / 164 filtration. In some embodiments, the method additionally includes in-line preconditioning of the composition prior to filtration.

[0197] In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7.5 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7.2 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to less than about 7 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to about 5 to about 7 before filtration. In some embodiments, the method further comprises adjusting the pH of the composition to about 6 to about 7 before filtration.

[0198] In some embodiments, the method further comprises adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. In some embodiments, the method further comprises adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. In some embodiments, the method further comprises adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration.

[0199] In some embodiments, the pH of the composition is less than about 7.5. In some embodiments, the pH of the composition is less than about 7.2. In some embodiments, the pH of the composition is less than about 7. In some embodiments, the pH of the composition is from about 5 to about 7. In some Petition 870250080997, dated 09 / 09 / 2025, pp. 96 / 193 81 / 164 modalities, the pH of the composition is about 6 to about 7.

[0200] In some embodiments, the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the conductivity of the composition is from about 10 mS / cm to about 20 mS / cm.

[0201] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 5 to about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 6 to about 7 and the conductivity of the composition is at least about 10 mS / cm.

[0202] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is from about 5 to about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the Petition 870250080997, dated 09 / 09 / 2025, p. 97 / 193 The composition of 82 / 164 is about 6 to about 7, and the conductivity of the composition is at least about 12 mS / cm.

[0203] In some embodiments, the composition is filtered through the virus filter in normal flow filtration mode. In some embodiments, the composition is filtered through the virus filter in tangential flow filtration mode.

[0204] In some embodiments, the composition is filtered through the virus pre-filter and the virus filter in normal flow filtration mode.

[0205] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 400 l / m2 / h. In some methods, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 300 l / m2 / h.

[0206] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 150 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 200 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 250 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 300 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 350 l / m² / h. In some Petition 870250080997, dated 09 / 09 / 2025, p. 98 / 193 In the 83 / 164 modes, the composition is filtered through the virus filter at a flow rate of approximately 400 l / m2 / h.

[0207] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0208] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid.

[0209] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0210] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid) to about 413685.6 Pa (60 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at an inlet pressure of about 137895.2 Pa (20 psid) to about 344738 Pa (50 psid). Petition 870250080997, dated 09 / 09 / 2025, p. 99 / 193 84 / 164

[0211] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi).

[0212] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of approximately 0.06 MPa (10 psi). Petition 870250080997, dated 09 / 09 / 2025, pp. 100 / 193 85 / 164

[0213] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid).In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid).

[0214] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition Petition 870250080997, dated 09 / 09 / 2025, pp. 101 / 193 86 / 164 is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid.

[0215] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one viral contaminant is a parvovirus. In some embodiments, at least one viral contaminant is a retrovirus. In some embodiments, at least one viral contaminant is a pseudorabies virus. In some embodiments, at least one viral contaminant is a reovirus.

[0216] In some embodiments, the composition comprises a recombinant protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody. Petition 870250080997, dated 09 / 09 / 2025, p. 102 / 193 87 / 164

[0217] In some embodiments, the recombinant protein is a bispecific antibody.

[0218] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a variable single-chain fragment.

[0219] In some embodiments, the recombinant protein is a fusion protein.

[0220] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of more than three filtration cycles.

[0221] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one cycle Petition 870250080997, dated 09 / 09 / 2025, pp. 103 / 193 88 / 164 filtration. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of more than three filtration cycles.

[0222] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of the more than three filtration cycles.

[0223] In some modes, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of one or Petition 870250080997, dated 09 / 09 / 2025, p. 104 / 193 89 / 164 plus filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the more than three filtration cycles.

[0224] Also provided in this document is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter, wherein: The virus filter is loaded to at least about 1500 l / m2e and / or at least about 30000 g / m2 over one or more filtration cycles; The virus pre-filter is a depth filter; The virus filter comprises at least one flat sheet, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least approximately 2:1.

[0225] In some embodiments, the virus filter is loaded to at least about 2000 l / m2 over one or more filtration cycles. Petition 870250080997, dated 09 / 09 / 2025, page 105 / 193 90 / 164

[0226] In some embodiments, the virus filter is loaded with about 1500 l / m2 to about 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with about 2000 l / m2 to about 3000 l / m2 over one or more filtration cycles. In some embodiments, the virus filter is loaded with about 2500 l / m2 to about 3000 l / m2 over one or more filtration cycles.

[0227] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter comprises polyethersulfone (PES). In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the virus filter comprises polyethersulfone (PES).

[0228] In some embodiments, the composition comprises a recombinant protein.

[0229] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein (for example, at least about 10.5 g / l, at least about 11 g / l, at least about 11.5 g / l, at least about 12 g / l, at least about 12.5 g / l, at least about 13 g / l, at least about 13.5 g / l, at least about 14 g / l, at least about 14.5 g / l, at least about 15 g / l; about 12.5 g / l; 14 g / l; about 14.5 g / l; about 15 g / l; about 15.5 g / l; about 16 g / l; about 16.5 g / l; about 17 g / l; about 17.5 g / l; about 18 g / l; about 18.5 g / l; about 19 g / l; about 19.5 g / l; about 20 g / l; about 20.5 g / l; Petition 870250080997, dated 09 / 09 / 2025, pp. 106 / 193 91 / 164 about 21.5 g / l; about 22 g / l; about 22.5 g / l; about 23 g / l; about 23.5 g / l; about 24 g / l; about 24.5 g / l; about 25 g / l).

[0230] In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0231] In some embodiments, the composition comprises at least about 17.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 20 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 25 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 30 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 50 g / l of a recombinant protein.

[0232] In some embodiments, the composition comprises about 15 g / l to about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 20 g / l of a recombinant protein.

[0233] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus pre-filter is a filter of Petition 870250080997, dated 09 / 09 / 2025, p. 107 / 193 92 / 164 depth based on diatomaceous earth, the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition and, after filtering the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l); In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0234] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l); Petition 870250080997, dated 09 / 09 / 2025, p. 108 / 193 93 / 164 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; about 5 pg / l less than about 10 pg / l). In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0235] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0236] In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 10 pg / l. In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of about 5 pg / l less than about 15 pg / l. In some embodiments, after filtration Based on the composition, the composition has a β-glucan concentration of approximately 5 pg / L, with less than approximately 12.5 pg / L.In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of about 5 pg / l less than about 10 pg / l. Petition 870250080997, dated 09 / 09 / 2025, p. 109 / 193 94 / 164

[0237] In some embodiments, the virus filter is loaded to at least about 1500 l / m2 over one or more filtration cycles.

[0238] In some embodiments, the virus filter is loaded to at least about 30,000 g / m2 over one or more filtration cycles.

[0239] In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is optionally replaced after one or more filtration cycles. In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is replaced after one or more filtration cycles.

[0240] In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is optionally replaced after each filtration cycle. In some embodiments, the method comprises at least two filtration cycles, in which the virus pre-filter is replaced after each filtration cycle.

[0241] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus prefilter is a depth filter comprising diatomaceous earth, cellulose fibers, and a binder comprising cationic imine groups.

[0242] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition.

[0243] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the prefilter Petition 870250080997, dated 09 / 09 / 2025, pages 110 / 193 95 / 164 virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface modified by cation exchange chemistry with crosslinked polymeric sulfonic acid. In some embodiments, the virus prefilter is a flat sheet membrane with a PES membrane surface modified by crosslinked mixed-mode chemistry.

[0244] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface.

[0245] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 3:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2.5:1 to about 3:1.

[0246] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.6:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.9:1.

[0247] In some embodiments, the pH of the composition is less than about 7.5. In some embodiments, the pH of the composition Petition 870250080997, dated 09 / 09 / 2025, p. 111 / 193 96 / 164 is less than about 7.2. In some embodiments, the pH of the composition is less than about 7. In some embodiments, the pH of the composition is from about 5 to about 7. In some embodiments, the pH of the composition is from about 6 to about 7.

[0248] In some embodiments, the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the conductivity of the composition is from about 10 mS / cm to about 20 mS / cm.

[0249] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 5 to about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 6 to about 7 and the conductivity of the composition is at least about 10 mS / cm.

[0250] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is about Petition 870250080997, dated 09 / 09 / 2025, p. 112 / 193 97 / 164 from 5 to about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is from about 6 to about 7 and the conductivity of the composition is at least about 12 mS / cm.

[0251] In some embodiments, the composition is filtered through the virus filter in normal flow filtration mode. In some embodiments, the composition is filtered through the virus filter in tangential flow filtration mode.

[0252] In some embodiments, the composition is filtered through the virus pre-filter and the virus filter in normal flow filtration mode.

[0253] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 400 l / m2 / h. In some methods, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m2 / ha approximately 300 l / m2 / h.

[0254] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 150 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 250 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 300 l / m² / h. In some embodiments, the composition is filtered through the filter of Petition 870250080997, dated 09 / 09 / 2025, pp. 113 / 193 98 / 164 viruses at a flow rate of approximately 350 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 400 l / m² / h.

[0255] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0256] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid.

[0257] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0258] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid) to about 413685.6 Pa (60 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at an inlet pressure of about 137895.2 Pa (20 psid) to about 344738 Pa (50 psid). Petition 870250080997, dated 09 / 09 / 2025, pp. 114 / 193 99 / 164

[0259] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi).

[0260] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of approximately 0.06 MPa (10 psi). Petition 870250080997, dated 09 / 09 / 2025, pp. 115 / 193 100 / 164

[0261] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid).In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid).

[0262] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition Petition 870250080997, dated 09 / 09 / 2025, pages 116 / 193 101 / 164 is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or differential pressure of about 0.06 MPa (10 psi) / psid.

[0263] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one viral contaminant is a parvovirus. In some embodiments, at least one viral contaminant is a retrovirus. In some embodiments, at least one viral contaminant is a pseudorabies virus. In some embodiments, at least one viral contaminant is a reovirus.

[0264] In some embodiments, the composition comprises a recombinant protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody. Petition 870250080997, dated 09 / 09 / 2025, p. 117 / 193 102 / 164

[0265] In some embodiments, the recombinant protein is a bispecific antibody.

[0266] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a variable single-chain fragment.

[0267] In some embodiments, the recombinant protein is a fusion protein.

[0268] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of more than three filtration cycles.

[0269] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one cycle Petition 870250080997, dated 09 / 09 / 2025, pp. 118 / 193 103 / 164 filtration. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of more than three filtration cycles.

[0270] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of the more than three filtration cycles.

[0271] In some modes, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of one or Petition 870250080997, dated 09 / 09 / 2025, p. 119 / 193 104 / 164 plus filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the more than three filtration cycles.

[0272] Also provided in this document is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein: The virus pre-filter is replaced after each filtration cycle; The virus filter is loaded to at least about 1500 l / m2e or at least about 30000 g / m2 over at least two filtration cycles, and the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is at least about 2:1.

[0273] In some embodiments, the composition comprises a recombinant protein.

[0274] In some embodiments, the composition comprises at least about 10 g / l of a recombinant protein (per Petition 870250080997, dated 09 / 09 / 2025, pages 120 / 193 105 / 164 example, hair less about 10.5 g / l, hair less about 11 g / l, hair less about 11.5 g / l, hair less about 12 g / l, hair less about 12.5 g / l, hair less about 13 g / l, hair less about 13.5 g / l, hair less about 14 g / l, hair less about 14.5 g / l, hair less about 15 g / l; about 10 g / l; about 10.5 g / l, about 11 g / l; about 11.5 g / l; about 12 g / l; about 12.5 g / l; about 13 g / l; about 13.5 g / l; about 14 g / l; about 14.5 g / l; about 15 g / l; approximately 15.5 g / l; approximately 16 g / l; approximately 16.5 g / l; approximately 17 g / l; approximately 17.5 g / l; approximately 18 g / l; approximately 18.5 g / l; approximately 19 g / l; approximately 19.5 g / l; approximately 20 g / l; approximately 20.5 g / l; approximately 21 g / l; approximately 21.5 g / l; approximately 22 g / l; approximately 22.5 g / l; approximately 23 g / l; approximately 23.5 g / l; approximately 24 g / l; approximately 24.5 g / l; approximately 25 g / l).

[0275] In some embodiments, the composition comprises at least about 12.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 15 g / l of a recombinant protein.

[0276] In some embodiments, the composition comprises at least about 17.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 20 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 25 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 30 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l or about 25 g / l of a recombinant protein. In some embodiments, the composition Petition 870250080997, dated 09 / 09 / 2025, pp. 121 / 193 106 / 164 comprises approximately 15 g / l to approximately 50 g / l of a recombinant protein.

[0277] In some embodiments, the composition comprises about 15 g / l to about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l to about 20 g / l of a recombinant protein.

[0278] In some embodiments, the virus filter is loaded to at least about 2,000 l / m2 over at least two filtration cycles.

[0279] In some embodiments, the virus filter is loaded with approximately 1500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2000 l / m2 to approximately 3000 l / m2 over at least two filtration cycles. In some embodiments, the virus filter is loaded with approximately 2500 l / m2 to approximately 3000 l / m2 over at least two filtration cycles.

[0280] In some embodiments, the virus pre-filter is a depth filter and the virus filter comprises at least one flat sheet.

[0281] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter comprises polyethersulfone (PES). In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the virus filter comprises polyethersulfone (PES).

[0282] In some embodiments, the virus pre-filter is a depth filter comprising diatomaceous earth, Petition 870250080997, dated 09 / 09 / 2025, pp. 122 / 193 107 / 164 cellulose fibers and a binder comprising cationic imine groups.

[0283] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with water or buffer before filtering the composition.

[0284] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface modified by crosslinked polymeric sulfonic acid cation exchange chemistry. In some embodiments, the virus prefilter is a flat sheet membrane with a crosslinked mixed-mode PES membrane surface modified by crosslinked mixed mode chemistry.

[0285] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter and the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface.

[0286] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition and, after filtering the composition, Petition 870250080997, dated 09 / 09 / 2025, pp. 123 / 193 108 / 164 the composition has a β-glucan concentration of less than about 15 μg / l (for example, less than about 14 μg / l, less than about 13 μg / l, less than about 12 μg / l, less than about 11 μg / l, less than about 10 μg / l; about 5 μg / l less than about 15 μg / l; about 5 μg / l less than about 12.5 μg / l; about 5 μg / l less than about 10 μg / l). In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a mixture thereof. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0287] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition.In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of less than about 15 μg / l (e.g., less than about 14 μg / l, less than about 13 μg / l, less than about 12 μg / l, less than about 11 μg / l, less than about 10 μg / l; about 5 μg / l, less than about 15 μg / l); In some embodiments, the carbonate-containing solution comprises sodium carbonate, potassium carbonate, or a... Petition 870250080997, dated 09 / 09 / 2025, pp. 124 / 193 109 / 164 mixture of them. In some embodiments, the carbonate-containing solution comprises sodium carbonate.

[0288] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter, the virus filter comprises polyethersulfone (PES), and the method further comprises washing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0289] In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l (e.g., less than about 14 pg / l, less than about 13 pg / l, less than about 12 pg / l, less than about 11 pg / l, less than about 10 pg / l; about 5 pg / l less than about 15 pg / l; about 5 pg / l less than about 12.5 pg / l; In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of less than about 10 pg / l. In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of about 5 pg / l less than about 15 pg / l. In some embodiments, after filtration Based on the composition, the composition has a β-glucan concentration of approximately 5 pg / L, with less than approximately 12.5 pg / L.In some embodiments, after filtration of the composition, the composition has a β-glucan concentration of about 5 pg / l less than about 10 pg / l.

[0290] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and Petition 870250080997, dated 09 / 09 / 2025, p. 125 / 193 In 110 / 164, the virus filter area in each filtration cycle is approximately 2:1 to approximately 3:1. In some embodiments, the ratio between the virus pre-filter area and the virus filter area in each filtration cycle is approximately 2.5:1 to approximately 3:1.

[0291] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.6:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter in each filtration cycle is about 2.9:1.

[0292] In some embodiments, the pH of the composition is less than about 7.5. In some embodiments, the pH of the composition is less than about 7.2. In some embodiments, the pH of the composition is less than about 7. In some embodiments, the pH of the composition is from about 5 to about 7. In some embodiments, the pH of the composition is from about 6 to about 7.

[0293] In some embodiments, the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the conductivity of the composition is from about 10 mS / cm to about 20 mS / cm.

[0294] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 10 mS / cm. In some Petition 870250080997, dated 09 / 09 / 2025, pp. 126 / 193 In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 5 to about 7 and the conductivity of the composition is at least about 10 mS / cm. In some embodiments, the pH of the composition is from about 6 to about 7 and the conductivity of the composition is at least about 10 mS / cm.

[0295] In some embodiments, the pH of the composition is less than about 7.5 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7.2 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is less than about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is from about 5 to about 7 and the conductivity of the composition is at least about 12 mS / cm. In some embodiments, the pH of the composition is from about 6 to about 7 and the conductivity of the composition is at least about 12 mS / cm.

[0296] In some embodiments, the composition is filtered through the virus filter in normal flow filtration mode. In some embodiments, the composition is filtered through the virus filter in tangential flow filtration mode.

[0297] In some embodiments, the composition is filtered through the virus pre-filter and the virus filter in normal flow filtration mode.

[0298] In some forms, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m2 / ha or approximately 500 l / m2 / h. In some forms, the composition Petition 870250080997, dated 09 / 09 / 2025, p. 127 / 193 112 / 164 is filtered through the virus filter at a flow rate of approximately 200 l / m² / ha or approximately 400 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 200 l / m² / ha or approximately 300 l / m² / h.

[0299] In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 100 l / m² / h. In some embodiments, the composition is filtered through the virus filter at a flow rate of approximately 150 l / m² / h. In some In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 200 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 250 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 300 l / m² / h. In some embodiments, the composition is filtered through a virus filter at a flow rate of approximately 350 l / m² / h. In some embodiments, the composition is filtered through a filter of Viruses at a flow rate of approximately 400 l / m2 / h.

[0300] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0301] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid. In some embodiments, the composition is filtered through the virus filter at a Petition 870250080997, dated 09 / 09 / 2025, pp. 128 / 193 113 / 164 inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid to about 0.41 MPa (60 psi) / psid.

[0302] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

[0303] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid) to about 413685.6 Pa (60 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at an inlet pressure of about 137895.2 Pa (20 psid) to about 344738 Pa (50 psid).

[0304] In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi). Petition 870250080997, dated 09 / 09 / 2025, p. 129 / 193 114 / 164

[0305] In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of about 0.06 MPa (10 psi). In some embodiments, the composition is filtered through the virus filter in a constant flow mode at an inlet pressure of approximately 0.06 MPa (10 psi).

[0306] In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of about 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the filter of Petition 870250080997, dated 09 / 09 / 2025, pp. 130 / 193 115 / 164 viruses in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid). In some embodiments, the composition is filtered through the virus filter in a constant pressure mode at a differential pressure of approximately 68947.6 Pa (10 psid).

[0307] In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of about 0.06 MPa (10 psi) / psid. In some embodiments, the composition is filtered through the virus filter at an inlet pressure and / or a differential pressure of approximately 0.06 MPa (10 psi) / psid.

[0308] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one viral contaminant is a parvovirus. In some embodiments, at least one viral contaminant is a retrovirus. In some embodiments, at least one viral contaminant is a pseudorabies virus. In some Petition 870250080997, dated 09 / 09 / 2025, pp. 131 / 193 In 116 / 164 modalities, at least one viral contaminant is a reovirus.

[0309] In some embodiments, the composition comprises a recombinant protein. In some embodiments, the recombinant protein is an antibody. In some embodiments, the recombinant protein is an IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is an IgG1 antibody. In some embodiments, the recombinant protein is an IgG2 antibody. In some embodiments, the recombinant protein is an IgG4 antibody. In some embodiments, the recombinant protein is a human antibody. In some embodiments, the recombinant protein is a human IgG1, IgG2, or IgG4 antibody. In some embodiments, the recombinant protein is a human IgG1 antibody. In some embodiments, the recombinant protein is a human IgG2 antibody. In some embodiments, the recombinant protein is a human IgG4 antibody.

[0310] In some embodiments, the recombinant protein is a bispecific antibody.

[0311] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a variable single-chain fragment.

[0312] In some embodiments, the recombinant protein is a fusion protein.

[0313] In some embodiments, the composition comprises at least about 17.5 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 20 g / l of a recombinant protein. In some embodiments, the composition comprises at least about 25 g / l of a recombinant protein. In some embodiments, the Petition 870250080997, dated 09 / 09 / 2025, pp. 132 / 193 117 / 164 composition comprises at least about 30 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l or about 25 g / l of a recombinant protein. In some embodiments, the composition comprises about 15 g / l or about 50 g / l of a recombinant protein.

[0314] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of more than three filtration cycles.

[0315] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration results in Petition 870250080997, dated 09 / 09 / 2025, pp. 133 / 193 118 / 164 a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of more than three filtration cycles.

[0316] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three filtration cycles. In some modes, filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of the more than three filtration cycles.

[0317] In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of one or more filtration cycles. In some embodiments, filtration Petition 870250080997, dated 09 / 09 / 2025, pp. 134 / 193 119 / 164 results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the two filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the three filtration cycles. In some embodiments, filtration results in a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of the more than three filtration cycles. Viral Filtration Skids

[0318] A viral filtration skid is provided herein for use in a method described herein. For example, a viral filtration skid is provided herein comprising a virus prefilter and a virus filter, wherein the ratio of the area of ​​the virus prefilter to the area of ​​the virus filter is at least about 2:1.

[0319] In some embodiments, the virus prefilter is a depth filter. In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus prefilter is a depth filter comprising diatomaceous earth, cellulose fibers, and a binder comprising cationic imine groups.

[0320] In some embodiments, the virus pre-filter is a diatomaceous earth-based depth filter and the method further comprises washing the filter with Petition 870250080997, dated 09 / 09 / 2025, pp. 135 / 193 120 / 164 depth based on diatomaceous earth with water or buffer before filtering the composition.

[0321] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 pm. In some embodiments, the virus prefilter is a membrane of about 0.1 pm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0322] In some embodiments, the virus prefilter is an absorbent membrane. In some embodiments, the virus prefilter is an absorbent membrane with ion exchange functionality.

[0323] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane with a polyethersulfone (PES) membrane surface modified by crosslinked polymeric sulfonic acid cation exchange chemistry. In some embodiments, the virus prefilter is a flat sheet membrane with a crosslinked mixed-mode PES membrane surface modified by crosslinked mixed mode chemistry.

[0324] In some embodiments, the virus prefilter is a triple-layer flat sheet membrane. In some embodiments, the virus prefilter is a three-layer polyamide flat sheet membrane.

[0325] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane comprising nylon. In some embodiments, the prefilter of Petition 870250080997, dated 09 / 09 / 2025, pages 136 / 193 121 / 164 virus is a pleated sheet membrane comprising PVDF modified with hydrophilic acrylate.

[0326] In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus prefilter is an asymmetric single-layer hollow fiber membrane and the virus prefilter comprises hydrophilic cuprammonium regenerated cellulose.

[0327] In some embodiments, the virus filter comprises at least one flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet. In some embodiments, the virus filter comprises a double-layer asymmetric flat sheet and the virus filter comprises hydrophilic polyethersulfone (PES).

[0328] In some embodiments, the virus filter comprises at least one pleated sheet.

[0329] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and the virus filter comprises hydrophilic PES.

[0330] In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises a double-layer asymmetric pleated sheet and the virus filter comprises surface-modified PES. In some embodiments, the virus filter comprises a triple-layer asymmetric pleated sheet. In some embodiments, the virus filter comprises a pleated sheet Petition 870250080997, dated 09 / 09 / 2025, pages 137 / 193 122 / 164 asymmetric triple-layer pleated sheet and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises an asymmetric triple-layer pleated sheet and the virus filter comprises hydrophilic polyvinylidene fluoride (PVDF).

[0331] In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical double-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet. In some embodiments, the virus filter comprises a symmetrical triple-layer pleated sheet and the virus filter comprises PVDF modified with hydrophilic acrylate.

[0332] In some embodiments, the virus filter comprises hollow fibers. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic cuprammonium regenerated cellulose. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises modified PVDF. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and the virus filter comprises hydrophilic PES.

[0333] In some embodiments, the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises surface-modified PES. Petition 870250080997, dated 09 / 09 / 2025, pp. 138 / 193 123 / 164

[0334] In some embodiments, the virus filter comprises modified PVDF. In some embodiments, the virus filter comprises hydrophilic PVDF. In some embodiments, the virus filter comprises PVDF modified with hydrophilic acrylate.

[0335] In some embodiments, the virus filter comprises hydrophilic cuprammonium regenerated cellulose.

[0336] In some forms, the virus filter is a small virus filter.

[0337] In some forms, the virus filter is a large virus filter.

[0338] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2:1 to about 4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2:1 to about 3:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is from about 2.5:1 to about 3:1.

[0339] In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.4:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.6:1. In some embodiments, the ratio between the area of ​​the virus prefilter and the area of ​​the virus filter is about 2.9:1.

[0340] In some embodiments, the viral filtration skid additionally comprises at least one online monitoring system. In some embodiments, the at least one online monitoring system monitors at least one Petition 870250080997, dated 09 / 09 / 2025, pp. 139 / 193 124 / 164 property selected from pressure, flow, pH, conductivity and UV.

[0341] In some embodiments, the virus pre-filter and the virus filter are positioned in series. Host Cells

[0342] The compositions subjected to the virus filtration methods disclosed in this document may derive from a biofabrication process (such as, for example, harvested cell culture fluid or a grouping of products after one or more unit operations). The cell lines (also called cells or “host cells”) used in such biofabrication processes 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 methods or continuous culture.These cells are typically cell lines obtained or derived from mammals and are capable of growing and surviving when placed in monolayer culture or 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 quantities of a particular protein, more particularly, a glycoprotein of interest, into the culture medium. The selection of a host cell appropriate for the expression of a protein. Petition 870250080997, dated 09 / 09 / 2025, pp. 140 / 193 125 / 164 recombinant 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 used to produce a recombinant protein is a mammalian host cell.

[0343] Cell lines are typically derived from a line originating from a primary culture that can be maintained in culture for an unlimited time. 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 the 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 skilled individuals can be used to construct expression vectors containing sequences that encode 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 later editions. Petition 870250080997, dated 09 / 09 / 2025, pages 141 / 193 126 / 164 previous; Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for any purpose.

[0344] 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).

[0345] 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 suitable cloning or expression hosts 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 this 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. Petition 870250080997, dated 09 / 09 / 2025, pages 142 / 193 127 / 164 example, Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

[0346] 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 to 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 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 a number of other cell lines. In some modalities, the host cells are selected from among CHO cells. Petition 870250080997, dated 09 / 09 / 2025, pages 143 / 193 128 / 164

[0347] In some embodiments, the host cells are eukaryotic cells, such as mammalian cells. Mammalian cells may be, for example, cell lines or strains of human, rodent or bovine cells. 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 knockout cells, CHO FUT8 GS knockout cells, CHOZN cells, and CHO-derived cells. In some embodiments, a CHO GS knockout cell (such as a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. Additionally, the CHO FUT8 knockout cell is, for example, Potelligent®CHOK1 SV (Lonza, Inc.). In some embodiments, eukaryotic cells may also be avian cells, cell lines, or cell strains, such as, for example, EBx®, EB14, EB24, EB26, EB66, or EB13 cells.

[0348] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, the Petition 870250080997, dated 09 / 09 / 2025, pages 144 / 193 129 / 164 mutant dihydrofolate reductase (DHFR) deficient cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable host CHO cell lines because the efficient amplifiable and selectable DHFR gene expression system allows for 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) (MSX) selection. Other CHO host cells suitable for use in a bioreactor 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).

[0349] Large-scale production of proteins for commercial applications can be performed 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 for growth in suspension culture. A variety of host cells adapted for growth in suspension culture are known, including mouse myeloma NS0 cells and CFIO-S, DG44, and DXB11 cell lines CHO cells. Other suitable cell lines include, but are not limited to, mouse myeloma SP2 / 0 cells, neonatal hamster kidney BF1K-21 cells, human PER.C6® cells, and F1EK-293 cells. Petition 870250080997, dated 09 / 09 / 2025, pages 145 / 193 130 / 164 of human embryonic kidney and cell lines derived from or manipulated from any of the cell lines described in this document.

[0350] 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), Yarrowia lipolytica or Schizosaccharomyces pombe. In some embodiments, eukaryotic cells are selected from Pichia pastoris strains. Non-limiting examples of Pichia pastoris strains include X33, GS115, KM71, KM71H, and CBS7435.

[0351] In some embodiments, eukaryotic cells are selected from fungal cells (e.g., Aspergillus cells (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 (such as, for example, F. oxysporum), Glomerella (such as, for example, G. graminicola), Petition 870250080997, dated 09 / 09 / 2025, pages 146 / 193 131 / 164 Hypocrea (such as H. jecorina), Magnaporthe (such as M. orzyae), Myceliophthora (such as M. thermophile), Nectria (such as N. heamatococca), Neurospora (such as N. crassa), Penicillium, Sporotrichum (such as S. thermophile), Thielavia (such as T. terrestris, T. heterothallica), Trichoderma (such as T. reesei), or Verticillium (such as V. dahlia).

[0352] In some embodiments, eukaryotic cells are selected from insect cells (such as, for example, Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5BT1-4) 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)).

[0353] 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 Petition 870250080997, dated 09 / 09 / 2025, pages 147 / 193 132 / 164 can 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.

[0354] Typically, 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, an 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 Petition 870250080997, dated 09 / 09 / 2025, pages 148 / 193 133 / 164 a starting vector, such as a commercially available vector, and additional elements can be individually obtained and linked to the vector. Bioreactors

[0355] The compositions subjected to the virus filtration methods disclosed in this document may derive from a biofabrication process carried out in one or more bioreactors. In some embodiments, the growth and / or production phase of such a biofabrication process is conducted within a bioreactor. Suitable culture conditions for mammalian cells are known in the art. 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.

[0356] In some embodiments, one or more bioreactors used in the biofabrication method are stainless steel bioreactors, such as, for example, a large-scale embedded stainless steel bioreactor on-site, capable of operating in volumes of about 2000 liters to about 50000 liters or more.

[0357] In some embodiments, one or more bioreactors used in the biofabrication method are single-use bioreactors. 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 Petition 870250080997, dated 09 / 09 / 2025, pages 149 / 193 134 / 164 Flexibility in the manufacturing process and on-site assembly, reconfiguration, sterilization, and validation 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.

[0358] 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 Petition 870250080997, dated 09 / 09 / 2025, pages 150 / 193 135 / 164 In some embodiments, the working volume of the bioreactor is at least about 90% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 91% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 92% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 93% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 94% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 95% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 96% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 97% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 98% of the bioreactor volume.In some embodiments, the working volume of the bioreactor is at least about 99% of the bioreactor volume. In some... In these modes, the working volume of the bioreactor is approximately 100% of the bioreactor's volume. Virus Pre-filters

[0359] Virus prefilters, which are commercially available from various sources (e.g., MilliporeSigma, Sartorius, Pall, Asahi Kasei), remove protein aggregates and other process impurities that can foul virus filters. Common virus prefilters use a variety of membrane chemistries, such as a chemically modified PES membrane surface. Petition 870250080997, dated 09 / 09 / 2025, pages 151 / 193 136 / 164 crosslinked polymeric sulfonic acid cation exchange (Viresolve® Shield, MilliporeSigma) and a crosslinked mixed-mode chemically modified PES membrane surface (Viresolve® Shield-H, MilliporeSigma). Additionally, depth filters composed of diatomaceous earth, cellulose fibers and a binder comprising cationic imine groups (Viresolve® Prefilter, MilliporeSigma), pleated sheet membranes (Pegasus™ Protect, Pall; Pegasus™ UL6 grade, Pall) and triple-layer flat sheet membranes (Virosart® Max, Sartorius) are commonly used as prefilters.

[0360] Virus prefilters generally act at least in part by size exclusion, for example, with size exclusion limits between about 75 nm and about 0.2 pm (e.g., 75 nm (Planova 75N, Asahi Kasei); 0.1 pm (Virosart® Max, Sartorius; Viresolve® Prefilter, MilliporeSigma); 0.2 pm (Viresolve® Shield, MilliporeSigma; Viresolve® Shield-H, MilliporeSigma; Pegasus™ Protect, Pall)). Virus Filters

[0361] Virus filters are polymeric membranes with diverse chemical compositions and complex pore structures designed to retain viral particles. Virus filters are commercially available from various manufacturers (e.g., MilliporeSigma, Sartorius, Pall, and Asahi Kasei) and include large and small virus filters. Large virus filters are designed to retain viruses larger than 60 nm, while small virus filters are designed to retain viruses larger than 20 nm. Virus filters can be used in filtration mode of Petition 870250080997, dated 09 / 09 / 2025, pages 152 / 193 137 / 164 normal flow filtration (NFF) or tangential flow filtration (TFF). In TFF or NFF mode, filtration is conducted under conditions to retain viral contaminants (e.g., a virus with a diameter of 20 to 100 nm) on the membrane surface, allowing the passage of a recombinant protein through the filter membrane.

[0362] Non-limiting examples of virus filters include those formed from regenerated cellulose (e.g., cuprammonium-regenerated cellulose), polyethersulfone, polyaryl sulfones, polysulfone, polyimide, polyamide, polyvinylidene difluoride (PVDF), and the like. For example, non-limiting examples of virus filters include VIRESOLVE® membranes and RETROPORE™ membranes available from EMD Millipore, Billerica, Massachusetts. They may be supplied in cartridge form (NFF), as VIRESOLVE® NFP virus filters, or as cassettes (for TFF), such as PELLICON® cassettes, available from EMD Millipore, Billerica, Massachusetts.

[0363] A further non-limiting example is the Sartorius Virosart® CPV small virus filter, which comprises a polyethersulfone membrane. Other PES membrane filters include, but are not limited to, Viresolve® NFR, Viresolve® Pro, Virosart® CPV, Virosart® HF, Virosart® HC, and Pegasus™ grade Prime. Further non-limiting exemplary filters include the Planova™ BioEX filter and the Viresolve® NFP filter, both composed of a polyvinylidene fluoride (PVDF) membrane, as well as the Asahi Kasei 15N, 20N, and 35N filters, which are composed of a cuprammonium-regenerated cellulose membrane (e.g., a hollow fiber cuprammonium-regenerated cellulose membrane). Examples Petition 870250080997, dated 09 / 09 / 2025, pages 153 / 193 138 / 164 alternative virus filters are known in the art and include Pall's hydrophilic acrylate-modified PVDF membranes, such as Ultipor® VF grade DV20, Ultipor® VF grade DV50 and Pegasus™ grade PV4. Viral Contaminants

[0364] Non-limiting examples of viral contaminants of concern in biofabrication processes include bovine enterovirus (BEV), bovine parvovirus (BPV), bovine viral diarrhea virus (BVDV), encephalomyocarditis virus (EMCV), feline calicivirus (FCV), hepatitis A virus (HAV), human immunodeficiency virus (HIV), human poliovirus-1 (HPV-1), human herpesvirus 1 (HSV-1), bovine herpesvirus 1 (IBRV), porcine enterovirus (PEV), pseudorabies virus (PRV), reovirus type 3 (Reo-3), Semliki forest virus (SFV), Sindbis virus (SINV), simian virus 40 (SV40), Theiler's murine encephalomyelitis virus (TMEV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and xenotropic murine leukemia virus. (xMuLV). Culture Methods

[0365] Various culture methods can be used to produce a recombinant protein of interest, including, but not limited to, batch culture, fed-batch culture, perfusion culture, and intensified cell culture. The compositions subjected to the virus filtration methods disclosed herein may derive from any culture method and may be subjected to one or more unit operations prior to virus filtration.

[0366] Batch culture is a discontinuous method where cells are grown in a fixed volume of media. Petition 870250080997, dated 09 / 09 / 2025, pages 154 / 193 139 / 164 culture 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 medium formulation, cell line, etc.). The batch process is the simplest culture method; however, viable cell density is limited by nutrient availability, and as soon as cells are at maximum density, the culture slows down and production decreases.There is no ability to prolong a production phase in discontinuous cropping because the accumulation of residues and the depletion of nutrients quickly lead to crop decline, typically within 3 to 7 days.

[0367] Fed-batch culture improves upon the batch process by providing boluses or continuous feeds of media to replenish those media components that have been consumed. Since fed-batch cultures receive additional nutrients throughout the run, 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 established and sustained by Petition 870250080997, dated 09 / 09 / 2025, pp. 155 / 193 140 / 164 manipulation of 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 cultures have the potential to achieve higher product titers compared to batch cultures. Typically, a batch method is used during the growth phase and a fed-batch method is used during the production phase, but a fed-batch feeding strategy can be used throughout the 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 to around 10 to 21 days. Fed-batch cultures are discontinuous, and harvesting typically occurs when levels of metabolic byproducts 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).

[0368] Perfusion methods offer potential improvements over batch and fed-batch methods by adding new media and simultaneously removing spent media during culture. Typical perfusion cultures begin with the start of batch culture with Petition 870250080997, dated 09 / 09 / 2025, pp. 156 / 193 141 / 164 duration of 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 with the retention of additional cells and 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. Various methods, such as sedimentation, centrifugation or filtration, can be used to remove spent media while maintaining cell density. Non-limiting examples of filtration methods include alternating tangential flow filtration and recirculating tangential flow. Alternating tangential flow is maintained by pumping the media through hollow fiber filter modules. See, for example, US Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22 (7): 62-63.

[0369] 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.

[0370] Typical large-scale commercial cell culture strategies strive to achieve high cell densities, such as 30–90(+) x 10⁶ cells / ml, where almost a third to more than half of the reactor volume is biomass. With perfusion culture, extreme cell densities of >1 x 10⁸ have been achieved. Petition 870250080997, dated 09 / 09 / 2025, pp. 157 / 193 142 / 164 cells / ml. 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 preparation, use, storage, and disposal of media are required to support a long-term perfusion culture, particularly for a high-density cell culture, which also requires even more nutrients. All of this can increase production costs compared to batch and fed-batch methods.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.

[0371] 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, analyzing the samples to monitor quantitatively and / or qualitatively recombinant protein characteristics and / or the process of Petition 870250080997, dated 09 / 09 / 2025, pages 158 / 193 143 / 164 cell culture. In some embodiments, 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 example, a basic chemical analysis method (titration method), an electrochemical analysis method (diaphragm electrode method), and a photochemical analysis method (fluorescence method).

[0372] 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.For example, 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 specific conditions (generally). Petition 870250080997, dated 09 / 09 / 2025, pages 159 / 193 144 / 164 at approximately a temperature of 25°-40°C, in a humidified controlled 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 art and will generally be the period of time sufficient to allow the particular cells to reproduce to a viable cell density within a range of about 20%-80% of the maximum viable cell density if the culture were maintained under the 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.

[0373] In certain embodiments, the culture conditions can be adjusted to facilitate the transition from the growth phase of the cell culture to the production phase. For example, a growth phase of the cell culture can occur at a higher temperature than a production phase of the cell culture. 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 Petition 870250080997, dated 09 / 09 / 2025, pp. 160 / 193 145 / 164 °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 may be employed to facilitate the transition from the growth phase of the culture to the production phase. Chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time, before and / or after a temperature change, or in place of a temperature change. If inducers are added after a temperature shift, they may be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.

[0374] 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. Petition 870250080997, dated 09 / 09 / 2025, pages 161 / 193 146 / 164 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 recommended. In certain embodiments, the culture medium is a chemically defined medium, which refers to a cell culture medium in which all components have known chemical structures and concentrations. Chemically defined media are typically serum-free and do not contain hydrolysates or animal-derived components.

[0375] 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.The 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 feeding medium... Petition 870250080997, dated 09 / 09 / 2025, pp. 162 / 193 147 / 164 concentrate may contain most of the components of the cell culture medium at, for example, about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x or even about 1000x of their normal value.

[0376] In some embodiments, the mammalian cell 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.

[0377] In some embodiments, the biofabrication process 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., cell counter). Petition 870250080997, dated 09 / 09 / 2025, pages 163 / 193 148 / 164 automated Cedex). Viable cell density can be determined by staining a culture sample with Trypan blue, 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 taking the reciprocal.

[0378] In some embodiments, the biofabrication process 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%.

[0379] 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 parameters 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 870250080997, dated 09 / 09 / 2025, pages 164 / 193 149 / 164 Purification processes

[0380] The compositions subjected to the virus filtration methods disclosed in this document may derive from a cell culture process and may be subjected to one or more purification processes prior to virus filtration. Illustratively, the expressed recombinant proteins may be secreted into the culture medium from which they may be recovered and / or collected. Harvesting operations comprising acid precipitation may be combined with additional harvesting strategies including centrifugation, such as stacked disc centrifugation, intermittent discharge 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 media separations.

[0381] Additionally, the present disclosure covers methods involving all known post-harvest recovery technologies, such as, for 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 separation. Petition 870250080997, dated 09 / 09 / 2025, pages 165 / 193 150 / 164 chromatographic analysis of biological and / or biochemical substances. Such post-harvest recovery technologies can be employed before or after virus filtration.

[0382] 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, before or after virus filtration. A person of ordinary skill in the art may select 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 the following: flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0383] A unit capture 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, Petition 870250080997, dated 09 / 09 / 2025, pages 166 / 193 151 / 164 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 can 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 can 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 that tag or epitope.

[0384] 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). A low-pH viral inactivation operation may be followed by a neutralization unit operation that readjusts the virus-inactivated solution to a pH more compatible with the requirements of subsequent unit operations. A low-pH viral inactivation operation may also be followed by filtration, such as depth filtration, to remove any turbidity or Petition 870250080997, dated 09 / 09 / 2025, pages 167 / 193 152 / 164 resulting precipitation. Adjusting the temperature or chemical composition (e.g., using detergents) can also be used to achieve viral inactivation.

[0385] 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 Polish chromatography methods include, but are not limited to, ion-exchange chromatography (IEX), such as anion-exchange chromatography (AEX) and cation-exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography and size exclusion chromatography (e.g., gel filtration). UF / DF

[0386] Purified recombinant protein (e.g., recombinant protein that has undergone one or more purification processes in addition to virus filtration) can be formulated, i.e., buffer exchanged, sterilized, bulk packaged, and / or packaged for an end user. Illustratively, the concentration of the product and the buffer exchange Petition 870250080997, dated 09 / 09 / 2025, pages 168 / 193 153 / 164 Buffering of the recombinant protein of interest into a buffer of the desired formulation for bulk storage of the active ingredient or drug product can be carried out by ultrafiltration and / or diafiltration. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.

[0387] 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 active pharmaceutical ingredient. 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 active pharmaceutical ingredient or added to the UF / DF eluate pool. An exemplary UF / DF process is described in document no. 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 regenerated cellulose, Pellicon™ (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), and polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY). Recombinant Proteins

[0388] Compositions comprising any type of recombinant protein, including proteins containing chains Petition 870250080997, dated 09 / 09 / 2025, pages 169 / 193 154 / 164 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, intracellular proteins, 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.

[0389] In other embodiments, the recombinant protein in a composition 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-IgG molecules (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 HLE BiTE molecules, HeteroIg BITE molecules and others, chimeric antigen receptors (CARs, CAR Ts) and T-cell receptors (TCRs). Petition 870250080997, dated 09 / 09 / 2025, pages 170 / 193 155 / 164

[0390] In some embodiments, the antigen-binding protein binds to one or more of the following, alone or in any combination: CD proteins including, but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171 and CD174, HER receptor family proteins including, for 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 including, but not limited to, for example, vascular endothelial growth factor (VEGF); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, 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), Crypto, transforming growth factors (TGF), which include, among others, TGF-α and TGF-β, which include TGF-01, TGF-32, TGF-33, TGF-34 or TGF-35, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (cerebral 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, Petition 870250080997, dated 09 / 09 / 2025, pp. 171 / 193 156 / 164 among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator (t-PA), bombazine, thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factors (CSFs), including but not limited to the following, MCSF, 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; Petition 870250080997, dated 09 / 09 / 2025, pages 172 / 193 157 / 164 dismutase, surface membrane proteins, decay-accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-A, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, antigen-binding proteins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, 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, cell death protein programmed receptor 1 and ligand, PD1 and PDL1, mannose receptor / hCGp, hepatitis C virus, dsFv[PE38] mesothelin conjugate, Legionella pneumophila (lly), IFN-gamma, interferon-gamma-induced protein 10 (IP10), IFNAR, TALL-1 lymphopoietin, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin Type 9 (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), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the aforementioned.

[0391] In other embodiments, the recombinant protein in a composition to be purified according to a method of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody. Petition 870250080997, dated 09 / 09 / 2025, pages 173 / 193 158 / 164

[0392] 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, satralizumab, tafoleciumab, tanezumab, tezepelumab, tremelimumab, utomilumab, and volagidumab. In some embodiments, the antibody is selected from among denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezepelumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody The antibody is erenumab. In some modalities, the antibody is evolocumab. In some modalities, the antibody is panitumumab. In some modalities, the antibody is romosozumab. In some modalities, the antibody is... tezepelumab.

[0393] In some embodiments, the antibody is a human IgG1, IgG2, or IgG4 antibody.

[0394] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0395] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0396] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody. Petition 870250080997, dated 09 / 09 / 2025, pp. 174 / 193 159 / 164 EXAMPLES

[0397] In order that the present revelation may be more fully understood, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting this revelation in any way. Example 1. Use of an Oversized Pre-Filter to Enable Robust Viral Clearance with High Concentration Feed Streams (> 15 g / l) Across Multiple Filtration Cycles

[0398] To achieve high flow rate, throughput, and cycling for a highly concentrated feed stream of a human IgG2 antibody (mAb), a virus filter prefilter with an area oversized relative to the virus filter was employed in bench-scale and pilot-scale virus filtration operations. Specifically, all tested ratios of prefilter area to virus filter area were greater than 2:1 (2.4:1, 2.6:1, 2.9:1) for each filtration cycle, where the prefilter was changed after each filtration cycle (i.e., the listed ratio of prefilter area to virus filter area is the ratio tested in each filtration cycle). In all runs, a diatomaceous earth-based depth filter (Viresolve® Prefilter (VPF), EMD Millipore) was used as a prefilter in combination with a flat sheet polyethersulfone (PES) virus membrane filter (Viresolve® Pro, EMD Millipore).

[0399] The mAb feed stream was conditioned before pre-filtration. Before load conditioning, the mAb feed streams were characterized by Petition 870250080997, dated 09 / 09 / 2025, pages 175 / 193 160 / 164 had low conductivity (<6 mS / cm), high concentration (>20 g / l) and high pH (7.5) and contained buffering species, including tris and acetate. In contrast, the preconditioned streams were characterized by a lower pH (6.7), obtained by titration with acetate, and higher conductivity (>12 mS / cm), obtained by adding sodium chloride.

[0400] FIGURE 1 shows the virus filter flow versus load of a bench-scale proof of concept performed with increased non-viral load. The bench-scale analysis was conducted with preconditioned and non-frozen load material (i.e., fresh load, as indicated in the legend), a pre-filter to virus filter area ratio of 2.9:1, and a constant differential pressure of 206842.8 Pa (30 psid). In the analysis, a volumetric throughput greater than 1800 l / m2 and an average flow of approximately 250 l / m2 / h were achieved, demonstrating the ability of the virus filtration scheme to allow virus filter cycling.

[0401] FIGURE 2 shows bench-scale results for virus filter inlet pressure versus load for constant flow runs using the same virus filter for three filtration cycles. This analysis was conducted with fresh and preconditioned feedstock, a 2.9:1 area ratio between the pre-filter and virus filter for each filtration cycle, and a constant flow rate of 250 l / m² / h. High productivities of approximately 500 l / m² were achieved for the first two virus filtration cycles. Loading for the third filtration cycle continued until all material was loaded into the filter, with an overall volumetric productivity exceeding 3400 l / m². Petition 870250080997, dated 09 / 09 / 2025, pages 176 / 193 161 / 164

[0402] FIGURE 3A, FIGURE 3B and Table 1 show the viral clearance results obtained in bench-scale runs conducted with a prefilter:virus filter area ratio of 2.9:1 for each of the two filtration cycles, with fresh feedstock enriched with mouse tiny virus (MMV), a model virus, at different volume percentage values ​​summarized in Table 1. During the viral clearance studies, a feed rate of 1000 l / m2 / cycle was targeted. As shown in FIGURE 3A and FIGURE 3B, the inlet pressure was well controlled for each of the two filtration cycles when run with a target feed rate of 1000 l / m2. The pressure trends for filtration cycles 1 and 2 show periods of low pressure due to filtration pauses. The net productivity of the virus filter, exceeding 2000 l / m2, equated to more than 37000 g / m2, a high productivity for a single virus filter.Additionally, logarithmic reduction rate (LRV) values ​​greater than 4 were obtained for each of the two filtration cycles, with no viral passage observed. Table 1. Summary of Viral Clearance Results using an Oversized Prefilter. Filtration Cycle Load (l / m2) % of Peak MMV LRV achieved 1 1000 0.01 Not measured 2 1300 0.01 4.04 ± 0.25

[0403] FIGURE 4 and FIGURE 5 show differential pressure and virus filter inlet pressure measurements, respectively, in pilot-scale runs with three virus filter filtration cycles, where a prefilter:virus filter area ratio of 2.6:1 was employed in each cycle. Petition 870250080997, dated 09 / 09 / 2025, pages 177 / 193 162 / 164 filtration. The target virus filter load was 500 l / m2 / cycle and the pressures were well controlled within the limits, similar to what was observed during the viral clearance studies. Differential and inlet pressures were measured at the end of each filtration cycle.

[0404] Table 2 shows the beta-glucan levels in the final active ingredient for three different pilot-scale runs (PSRs). The aim of this analysis was to verify whether the oversized pre-filter did not lead to unacceptable leaching of beta-glucans, a risk associated with some cellulose-based pre-filters, which could affect the safety of the final active ingredient. Beta-glucan levels were within historical ranges for mAb manufacturing, demonstrating the viability of this filtration scheme in combination with a risk mitigation strategy of a sodium carbonate flush in each pre-filter prior to viral filtration. Table 2. Beta-Glucan Levels in the Final mAb Active Ingredient for Pilot-Scale Trials Performance Level of Beta Glucan in the Active Ingredient (g / l) Historical Ranges 6.6 — 11.1 PSR1 4.1 PSR2 2.8 PSR3 9.8

[0405] Based on the results of these analyses, a filtration scheme employing an oversized pre-filter allows virus filter loads greater than or equal to 1500 l / m2 on a volumetric basis (greater than or equal to 30000 g / m2 on a product mass basis) over three cycles of Petition 870250080997, dated 09 / 09 / 2025, pp. 178 / 193 163 / 164 filtration, at a flow rate of 250 LMH (liters per square meter per hour) and with a product concentration in the feed stream greater than 18 g / l. Consequently, the use of an oversized pre-filter can facilitate intensified virus filtration operations that accommodate: (1) high loads to provide high throughput in a relatively small space; (2) high flow rate to enable connected processing, thus leading to dematerialization (i.e., efficient use of raw materials to reduce costs and reduce manufacturing space) and reduced space versus discrete processing; (3) ability to cycle the virus filter, thus leading to dematerialization; and (4) robust performance with high concentration feed streams (> 15 g / l), which helps ensure high throughput in a small space.

[0406] All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books and treatises, are expressly incorporated herein in their entirety by way of reference. What is described in one embodiment of disclosure may be combined with one or more other embodiments of disclosure unless the context clearly indicates otherwise.

[0407] 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 in Petition 870250080997, dated 09 / 09 / 2025, pp. 179 / 193 164 / 164 of this document, will be evident to persons skilled in the art from the preceding description and the attached drawing(s). Such modifications are intended to remain within the scope of the disclosed subject matter.

[0408] The descriptions of the various modalities 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 persons of ordinary skill in the art without departing from the scope and spirit of the modalities described. The terminology used in this document has been chosen to better explain the principles of the modalities, 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.

Claims

CLAIMS 1. A method for removing at least one viral contaminant from a composition, characterized in that it comprises filtering the composition through a virus pre-filter and a virus filter, wherein: the virus filter is loaded to at least about 1500 l / m2 over one or more filtration cycles and a ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is at least about 2:

1.

2. A method for removing at least one viral contaminant from a composition, characterized in that it comprises filtering the composition through a virus pre-filter and a virus filter, wherein: the virus filter is loaded to at least about 30,000 g / m2 over one or more filtration cycles and the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is at least about 2:

1.

3. A method according to claim 1 or 2, characterized in that it comprises at least two filtration cycles, wherein the virus pre-filter is optionally replaced after one or more filtration cycles.

4. A method according to any one of claims 1, 2 or 3, characterized in that it comprises at least two filtration cycles, wherein the virus pre-filter is optionally replaced after each filtration cycle.

5. Method, according to claim 1 or 2, characterized in that it comprises at least two filtration cycles, wherein the virus pre-filter is replaced after one or more filtration cycles.

6. Method according to claim 1 or 2, characterized in that it comprises at least two filtration cycles, wherein the virus pre-filter is replaced after each filtration cycle.

7. A method for removing at least one viral contaminant from a composition, characterized in that it comprises filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein: the virus pre-filter is replaced after each filtration cycle; the virus filter is loaded to at least about 1500 l / m2 over the at least two filtration cycles; and the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is at least about 2:

1.

8. A method for removing at least one viral contaminant from a composition, characterized in that it comprises filtering the composition through a virus pre-filter and a virus filter over at least two filtration cycles, wherein: the virus pre-filter is replaced after each filtration cycle; the virus filter is loaded to at least about 30,000 g / m2 over at least two filtration cycles; and a ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is at least about 2:

1.

9. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that: the virus pre-filter is a depth filter and / or the virus filter comprises at least one flat sheet.

10. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that: the virus pre-filter is a diatomaceous earth-based depth filter and / or the virus filter comprises polyethersulfone (PES).

11. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized in that: the virus pre-filter is a diatomaceous earth-based depth filter and the virus filter comprises polyethersulfone (PES).

12. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, characterized in that the ratio between the area of ​​the virus pre-filter and the area of ​​the virus filter in each filtration cycle is from about 2:1 to about 3:

1.

13. Method, according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, characterized in that it further comprises: adjusting the pH of the composition to less than about 7.2 before filtration and / or adjusting the conductivity of the composition to at least about 10 mS / cm before filtration.

14. A method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, characterized in that it further comprises: adjusting the pH of the composition to less than about 7.2 before filtration and adjusting the conductivity of the composition to at least about 10 mS / cm before filtration.

15. Method, according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, characterized in that the composition is filtered through the virus filter at a flow rate of about 100 l / m2 / ha or about 500 l / m2 / h.

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 the composition is filtered through the virus filter at a pressure of about 0.06 MPa (10 psi) to about 0.41 MPa (60 psi).

17. A 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 it comprises three filtration cycles, wherein the net ratio between the area of ​​the virus prefilter and the area of ​​the virus filter over the three filtration cycles is at least about 6:

1.

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 net ratio of the area of ​​the virus pre-filter to the area of ​​the virus filter over the filtration is from about 6:1 to about 9:

1.

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 virus pre-filter is a diatomaceous earth-based depth filter; the virus filter comprises polyethersulfone (PES); and the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition.

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, after filtration of the composition, the composition has a β-glucan concentration of less than about 15 pg / l.

21. 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, 18, 19 or 20, characterized in that: the virus pre-filter is a diatomaceous earth-based depth filter; the virus filter comprises polyethersulfone (PES); the method further comprises washing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition and after filtering the composition, the composition has a β-glucan concentration of less than about 10 pg / l.

22. 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, 19, 20 or 21, characterized in that the composition Petition 870250080997, dated 09 / 09 / 2025, page 185 / 193 6 / 6 comprises at least about 10 g / l of a recombinant protein.

23. 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, 19, 20 or 22, characterized in that the composition comprises at least about 15 g / l of a recombinant protein.

24. 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, 18, 19, 20, 21, 22 or 23, characterized in that at least one viral contaminant is selected from parvoviruses, retroviruses, pseudorabies viruses and reoviruses.

25. 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, 18, 19, 20, 21, 22, 23 or 24, characterized in that the filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of one or more filtration cycles.

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