Virus filtration operation using a large virus prefilter

Abstract

A method for removing at least one viral contaminant from a concentrated composition is disclosed herein, comprising filtering the concentrated composition through a viral prefilter and a viral filter over one or more filtration cycles, wherein the ratio of the viral prefilter area to the viral filter area in each filtration cycle is at least about 2:1, and a viral filtration skid for use in such a method is disclosed herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 489,857, filed March 13, 2023, which is incorporated in its entirety by reference herein.

[0002] This disclosure provides a method for removing at least one viral contaminant from a concentrated composition (for example, a composition containing recombinant protein such as at least about 10 g / L, for example, at least about 15 g / L), comprising filtering the concentrated composition through a viral prefilter and a viral filter over one or more filtration cycles, wherein the ratio of viral prefilter area to viral filter area in each filtration cycle is at least about 2:1, and provides a viral filtration skid for use in such a method. [Background technology]

[0003] Mammalian cells used in the manufacture of recombinant protein therapeutics are susceptible to viral infection and proliferation. To remove potential exogenous and endogenous viral contaminants, downstream purification processes for therapeutic proteins generally include dedicated viral clearance operations, such as viral inactivation and viral filtration. For example, low pH or detergent-based viral inactivation is commonly used to denature enveloped viruses. Furthermore, viral retention filtration, a complementary unit operation, is robust and removes various viruses from the recovered cell culture medium, mostly through size-based mechanisms. Specifically, viral filters with complex pore structures retain viral particles while allowing other solutes to pass through the polymer membrane.

[0004] Viral filtration is often a costly unit operation. To improve the throughput that can be achieved and thereby reduce the required filter size, associated costs, and footprint, in-line pre-filters are commonly used in conjunction with viral filters to remove certain contaminants in the product pool or eluate stream before applying the pool or eluate to the viral filter. However, despite the improvements in viral filtration economics associated with the use of pre-filters, viral filtration remains one of the most expensive unit operations in protein purification. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, there is a need in this field for novel and improved virus filtration methods that enable robust removal of virus particles at reduced costs in high-throughput processes. [Means for solving the problem]

[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 viral prefilter and a viral filter, wherein the ratio of the viral prefilter area to the viral filter area is at least about 2:1.

[0007] Another aspect of the present disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over one or more filtration cycles. 2 Loaded up to, and The present invention provides a method in which the ratio of virus prefilter area to 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 recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / L of recombinant protein. In some embodiments, the composition comprises at least about 15 g / L of recombinant protein.

[0009] In some embodiments, the virus filter is loaded up to at least about 2000 L / m over one or more filtration cycles 2 up to.

[0010] In some embodiments, the virus filter is loaded from about 1500 L / m 2 to about 3000 L / m 2 over one or more filtration cycles. In some embodiments, the virus filter is loaded from about 2000 L / m 2 to about 3000 L / m 2 over one or more filtration cycles. In some embodiments, the virus filter is loaded from about 2500 L / m 2 to about 3000 L / m 2 over one or more filtration cycles.

[0011] Yet another aspect of the present disclosure is a method of removing at least one viral contaminant from a composition, comprising filtering the composition through a virus prefilter and a virus filter, where the virus filter is loaded up to at least about 30,000 g / m over one or more filtration cycles, and 2 and the ratio of the virus prefilter area to 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 recombinant protein. In some embodiments, the composition comprises at least about 12.5 g / L of recombinant protein. In some embodiments, the composition comprises at least about 15 g / L of recombinant protein.

[0013] A further aspect of this disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over one or more filtration cycles. 2 and / or at least about 30,000 g / m² 2 Loaded up to, The virus prefilter is a depth filter, The virus filter consists of at least one flat sheet, and The present invention provides a method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:1.

[0014] In some embodiments, the composition contains at least about 10 g / L of recombinant protein. In some embodiments, the composition contains at least about 12.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 15 g / L of recombinant protein.

[0015] In some embodiments, the virus filter can filter at least about 2000 L / m³ over one or more filtration cycles. 2 It will be loaded up to this point.

[0016] In some embodiments, the virus filter can filter approximately 1500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter can handle approximately 2000 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

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

[0018] A further aspect of this disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over at least two filtration cycles. 2 and / or at least about 30,000 g / m² 2 The virus prefilter is loaded up to a certain point, and optionally replaced after one or more filtration cycles. The virus prefilter is a depth filter, The virus filter consists of at least one flat sheet, and The present invention provides a method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:1.

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

[0020] In some embodiments, the virus filter can filter at least about 2000 L / m³ over at least two filtration cycles. 2 It will be loaded up to this point.

[0021] In some embodiments, the virus filter can filter approximately 1500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2000 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

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

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

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

[0026] In some embodiments, the composition has a pH of less 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 of 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 present disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The virus prefilter is replaced after each filtration cycle. The virus filter can filter at least approximately 1500 L / m³ over at least two filtration cycles. 2 Loaded up to, and The present invention provides a method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:1.

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

[0031] In some embodiments, the virus filter can filter at least about 2000 L / m³ over at least two filtration cycles. 2 It will be loaded up to this point.

[0032] In some embodiments, the virus filter can filter approximately 1500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2000 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

[0033] A further aspect of the present disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The virus prefilter is replaced after each filtration cycle. The virus filter will filter at least approximately 30,000 g / m³ over at least two filtration cycles. 2 Loaded up to, and The present invention provides a method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:1.

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

[0035] Another aspect of the present disclosure is a method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The virus prefilter is replaced after each filtration cycle. The virus filter can filter at least approximately 1500 L / m³ over at least two filtration cycles. 2 and / or at least about 30,000 g / m² 2 Loaded up to, The virus prefilter is a depth filter, The virus filter consists of at least one flat sheet, and The present invention provides a method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:1.

[0036] In some embodiments, the composition contains at least about 10 g / L of recombinant protein. In some embodiments, the composition contains at least about 12.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 15 g / L of recombinant protein.

[0037] In some embodiments, the virus filter can filter at least about 2000 L / m³ over at least two filtration cycles. 2 It will be loaded up to this point.

[0038] In some embodiments, the virus filter can filter approximately 1500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2000 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

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

[0040] In some embodiments, the composition has a pH of less 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 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.

[0043] Further aspects of the present disclosure provide a virus filtration skid for use in the method described herein. Further aspects of the present disclosure provide a virus filtration skid comprising a virus prefilter and a virus filter, wherein the ratio of the virus prefilter area to the virus filter area is at least about 2:1. [Brief explanation of the drawing]

[0044] [Figure 1] This shows the viral filter flow rate versus load from a bench-scale proof-of-concept run using a 2.9:1 viral pre-filter to viral filter area ratio. [Figure 2] For each filtration cycle, bench-scale results regarding the virus filter inlet pressure versus load are shown for three filtration cycles using a virus pre-filter to virus filter area ratio of 2.9:1 (i.e., a net virus pre-filter to virus filter area ratio of 8.7:1) with the same virus filter running at a "constant flow rate" (250 L / m2 / hour (LMH)). [Figure 3A]For each filtration cycle, the differential pressure versus elapsed time during viral filtration at a constant flow rate (250 LMH) over two filtration cycles (Cycle 1 (Figure 3A), Cycle 2 (Figure 3B)) in a bench-scale evaluation using a viral pre-filter to viral filter area ratio of 2.9:1 (i.e., a net viral pre-filter to viral filter area ratio of 5.8:1) is shown. [Figure 3B] For each filtration cycle, the differential pressure versus elapsed time during viral filtration at a constant flow rate (250 LMH) over two filtration cycles (Cycle 1 (Figure 3A), Cycle 2 (Figure 3B)) in a bench-scale evaluation using a viral pre-filter to viral filter area ratio of 2.9:1 (i.e., a net viral pre-filter to viral filter area ratio of 5.8:1) is shown. [Figure 4] For each filtration cycle, the virus filter differential pressure measurements at the end of each filtration cycle are shown, using a virus pre-filter to virus filter area ratio of 2.6:1, in a pilot-scale run with three filtration cycles per virus filter (i.e., a net virus pre-filter to virus filter area ratio of 7.8:1). [Figure 5] For each filtration cycle, the virus filter inlet pressure measurements at the end of each filtration cycle are shown in a pilot-scale run using three filtration cycles with a virus pre-filter to virus filter area ratio of 2.6:1 (i.e., a net virus pre-filter to virus filter area ratio of 7.8:1). [Modes for carrying out the invention]

[0045] A method for removing at least one viral contaminant from a concentrated composition (for example, a composition containing at least about 10 g / L of recombinant protein), comprising filtering the concentrated composition through a viral prefilter and a viral filter over one or more filtration cycles, wherein the ratio of viral prefilter area to viral filter area in each filtration cycle is at least about 2:1, and a viral filtration skid for use in such a method is disclosed herein.

[0046] Definition: The following definitions are provided to facilitate understanding of the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0047] In some embodiments, "approximately" when used in relation to a measurable numerical variable refers to all values ​​of the variable that are greater than the indicated value of the variable and the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or ±10% of the indicated value. In some embodiments, the numerical range includes a number (i.e., endpoints) that defines the range.

[0048] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediary value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated values ​​or intermediary values ​​within that stated range, are included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges also included in this disclosure, subject to the limits specifically excluded in the stated range. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0049] As used herein, the terms “a” and “an” mean “one or more” unless otherwise indicated. Furthermore, “one or more” and “at least one” are used interchangeably herein. In addition, unless the context requires otherwise, singular terms include plural forms, and plural terms include singular forms.

[0050] As used herein, the term “acid precipitation” refers to a recovery operation that lowers the pH of a cell culture to induce the precipitation of one or more cell culture impurities.

[0051] As used herein, the term “affinity chromatography” (also known as “capture chromatography”) refers to a chromatographic operation that separates a biomolecule (e.g., recombinant protein) from a mixture based on the selective interaction between the biomolecule and another substance (i.e., ligand). Affinity chromatography is commonly used in biomanufacturing processes to isolate and concentrate a desired recombinant protein from a recovered cell culture medium. In a typical affinity chromatography operation, a biomolecule in the mobile phase selectively binds to or otherwise interacts with the stationary phase, while the rest of the mobile phase passes through the chromatographic material. The biomolecule is then eluted from the stationary phase by changing the conditions to reduce the affinity between the ligand and the biomolecule. Non-limiting examples of affinity chromatography include protein A, protein G, protein A / G, and protein L materials. Furthermore, immobilized metal affinity chromatography (IMAC) may be used to capture proteins that have an affinity for metal ions, or that have been manipulated to have an affinity for metal ions.

[0052] In some embodiments, protein A affinity chromatography can be used to capture the recombinant protein of interest. Protein A ligands are highly selective for a wide range of proteins containing antibody Fc regions, resulting in robust removal of process-related impurities and high target protein yield. Commercially available protein A materials include, but are not limited to, MABSELECT® SURE Protein A, Protein A Sepharose FAST FLOW®, MABSELECT® Prism A (Cytiva, Marborough, MA), PROSEP-A® (Merck Millipore, UK), TOYOPEARL® HC-650F Protein A (TosoHass Co., Philadelphia, PA), and AP Plus, Purolite, King of Prussia, PA.

[0053] As used herein, the term “antigen-binding protein” refers to a protein or polypeptide that contains an antigen-binding region or antigen-binding moiety that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include antibodies, fusion proteins, VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd(VH-CH1), heavy chain, Fab, Fab', F(ab')2, or “r IgG” (a “half-antibody” consisting of a heavy chain and a light chain), or modified fragments of full-length antibodies, such as tri-chain antibody-like molecules, heavy chain-only antibodies, single-chain variable fragments (scFv), di-scFv or bi(s)scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, (VH-VL-CH3)2, (scFv-CH3). 2. Examples include, but are not limited to, "mini-bodies" exemplified by structures such as ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multi-bodies, such as tria-bodies or tetra-bodies, and single-domain antibodies, such as nano-bodies or single-variable-domain antibodies containing only one variable region, which may be VHH, VH, or VL that specifically binds to an antigen or target independently of other variable regions or domains.

[0054] As used herein, the term “antibody” generally refers to a tetrameric immunoglobulin protein comprising two light-chain polypeptides (each about 25 kDa) and two heavy-chain polypeptides (each about 50–70 kDa).

[0055] As used herein, the terms “light chain” or “immunoglobulin light chain” refer to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). The immunoglobulin light chain constant domain (CL) may be a human kappa (κ) constant domain or a human lambda (λ) constant domain.

[0056] As used herein, the terms “heavy chain” or “immunoglobulin heavy chain” refer to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). The constant domains of immunoglobulin heavy chains may originate from any immunoglobulin isotype, including subtypes. The antibody chains are linked via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain) and between the hinge regions of the two antibody heavy chains.

[0057] The variable regions of immunoglobulin chains generally exhibit an identical overall structure, including a relatively conserved framework region (FR) linked by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs, derived from the two chains of each pair of heavy and light chains, are typically aligned by the framework region to form a structure that specifically binds to a particular epitope of the target protein. From the N-terminus to the C-terminus, both the naturally occurring light and heavy chain variable regions typically correspond to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids occupying positions within each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the CDR and FR of a given antibody. 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).

[0058] When an antibody is digested with papain, two identical antigen-binding proteins called "Fab" fragments (each possessing a single antigen-binding site) and the remaining "Fc" fragment (containing all but the first domain of the constant region of the immunoglobulin heavy chain) are produced. The Fab fragment contains the variable domains derived from the light chain and heavy chain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the "Fab fragment" consists of one immunoglobulin light chain (variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH domain and CH1 domain derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0059] An immunoglobulin “Fc fragment” or “Fc region” generally contains two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain. The Fc region may be derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region contains CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector functions.

[0060] The "F(ab')2 fragment" is a divalent fragment containing two Fab' fragments linked by disulfide bridges between heavy chains in the hinge region.

[0061] The "Fv" fragment is the smallest fragment containing a complete antigen recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in a tightly covalently bound state. In this configuration, the three CDRs of each variable region interact to define the antigen-binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of the Fv fragment containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, but has lower affinity than the entire binding site containing both VH and VL.

[0062] A "single-chain variable fragment" or "scFv fragment" comprises the VH and VL regions of an antibody, which are present on a single polypeptide chain and optionally include a peptide linker between the VH and VL regions, thereby enabling Fv to form a 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).

[0063] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking a light chain occur naturally in certain animal species, such as nurse sharks, pygmy sharks, and camelids, such as camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is a single-domain VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding domain; that is, these functional antibodies are homodimers of the heavy chain (also called "heavy chain antibodies" or "HCAb") that have only the structure H2L2. Camelized VHH contains a hinge domain, a CH2 domain, and a CH3 domain, and has been reported to recombine with the constant regions of IgG2 and IgG3 lacking a CH1 domain. Camelized VHH domains have been shown to bind to antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276: 26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41: 3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be constructed from human variable-like domains that are more closely compatible with shark V-NAR scaffolds, potentially providing long, penetrating loop structures to the framework.

[0064] As used herein, the term “heavy-chain-only antibody” refers to an immunoglobulin protein consisting of two heavy-chain polypeptides (e.g., heavy-chain polypeptides, each approximately 50–70 kDa). “Heavy-chain-only antibodies” lack the two light-chain polypeptides found in conventional antibodies. Heavy-chain antibodies constitute about a quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446–448 (1993)). These molecules are formed by two heavy chains but lack light chains. As a result, the variable antigen-binding region is called the VHH domain, representing the smallest naturally occurring intact antigen-binding site, with a length of only about 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276, 26285–26290 (2001)). Highly specific and affinity heavy chain antibodies can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 3746 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, 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 possess a single VH-like domain called VNAR in their antibodies. (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)).

[0065] In some embodiments, a “heavy chain-only antibody” is a dimer antibody containing a VH antigen-binding domain and CH2 and CH3 constant domains, lacking a CH1 domain. In some embodiments, a heavy chain-only antibody consists of a variable region antigen-binding domain comprising Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3, and Framework 4. In some embodiments, a heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. While heavy chain-only antibodies described herein may belong to the IgG subclass, heavy chain-only antibodies belonging to other subclasses such as the IgM, IgA, IgD, and IgE subclasses are also included herein. In some embodiments, the heavy-chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, for example, the IgG1 or IgG4 subtype. In some embodiments, the heavy-chain-only antibody is of the IgG1 or IgG4 subtype, and one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is of the IgG4 subtype, and one or more CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy-chain-only antibody is of the IgG1 subtype, and one or more CH domains are modified to alter the effector function of the antibody. Modifications of CH domains that alter effector function are further described herein. Non-limiting examples of heavy-chain-only antibodies are described, for example, in International Publication No. 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0066] As used herein, the terms “triple-chain antibody-like molecule” or “TCA” refer to an antibody-like molecule comprising, essentially consisting of, or comprising three polypeptide subunits, two of which comprise a single heavy chain and a single light chain of a monoclonal antibody, or an antigen-binding fragment of such an antibody chain, comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity to a first antigen. The third polypeptide subunit comprises an Fc portion containing CH2, and / or CH3, and / or CH4 domains in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of a first antigen, wherein such binding domains comprise, essentially consisting of, or comprising an antibody consisting only of a heavy chain having sequence identity with, or derived from or having sequence identity with, a variable region of such an antibody heavy or light chain. H and / or V L Gene segments, D and J H Gene segment or J L It can be encoded by gene segments. The variable region is a rearranged V H DJ H , V L DJ H , V H J L or V L J L It can be encoded by a gene segment.

[0067] As used herein, the term “bioreactor” means any vessel useful for growing cell cultures (e.g., mammalian cell cultures or bacterial cell cultures). “Bioreactor” as used herein also encompasses the term “fermenter” (i.e., a vessel useful for growing bacterial cell cultures, typically including a more powerful agitator and increased gas flow compared to vessels used for growing mammalian cell cultures). Non-limiting examples of bioreactors include agitated tank type, airlift type, fiber type, microfiber type, hollow fiber type, ceramic matrix type, fluidized bed type, fixed bed type, and / or jet bed type bioreactors. In some embodiments, an exemplary bioreactor may perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients (e.g., in perfusion cell culture) and / or supplying a carbon source; injecting a suitable gas (e.g., oxygen); fermentation or inflow and outflow of cell medium (e.g., supplying fresh cell medium by perfusion and removing used cell medium); separating gas and liquid phases; maintaining temperature; maintaining oxygen and CO2 levels; maintaining pH levels; stirring (e.g., agitation) and / or washing / sterilization. Unless otherwise indicated by context, the bioreactor may be suitable for batch processes, semi-fed batch processes, fed batch processes, perfusion processes and / or continuous fermentation processes. Any suitable bioreactor diameter may be used. Unless otherwise indicated by context, in some embodiments, the bioreactor may have a volume of 100 mL to 50,000 L. Unless otherwise indicated, a bioreactor may be of any size, as long as it is useful for culturing cells, and typically, a bioreactor is sized to be appropriate for the volume of cell culture to be grown inside it. In a non-limiting embodiment and unless otherwise indicated by context, a bioreactor may be at least 1 liter (L), or 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000, 20,000 liters or more, or any volume in between.Internal conditions of a bioreactor, including but not limited to pH, dissolved oxygen (DO), and temperature, can be controlled during the incubation period. Those skilled in the art can identify and select a suitable bioreactor based on the relevant considerations.

[0068] As used herein, the terms “cell culture” or “culture” refer to the proliferation and multiplication 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 attached to solid culture media. In some embodiments, fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks and / or agitated tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0069] As used herein, the term “cell culture medium” (also referred to as “culture medium,” “culture medium,” “cell culture medium,” “tissue culture medium,” etc.) refers to any nutrient solution used to grow cells, such as bacterial or mammalian cells. A cell culture medium generally provides one or more of the following components: an energy source (e.g., in the form of carbohydrates such as glucose), one or more essential amino acids (e.g., all essential amino acids, the 20 basic amino acids plus cysteine), vitamins and / or other organic compounds, lipids or free fatty acids, which are usually required at low concentrations, and trace elements such as inorganic compounds or naturally occurring elements, which are usually required at very low concentrations, such as in the micromolar range. As used herein, a cell culture medium encompasses any nutrient solution typically used and / or known to be used in any cell culture process, including but not limited to batch culture, extended batch culture, fed batch culture, intensive culture, and / or perfusion culture or continuous culture of cells.

[0070] As used herein, the term “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of viable cells in a given volume of culture medium, determined by a standard viability assay (e.g., trypan blue exclusion), and can be measured at any point in time during a particular stage of the cell culture process. As used herein, “packed cell volume” (PCV), also referred to as “percentage of packed cell volume” (%PCV), is the ratio of the volume occupied by cells to the total volume of the cell culture, and is expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter, and an increase in packed cell volume may occur due to an increase in cell density, cell diameter, or both. Packed cell volume is a measure of the solid content in the cell culture. Since host cells vary in size and cell cultures contain dead or dying cells and other cellular debris, the filled cell volume can more accurately describe the solid content within the cell culture.

[0071] As used herein, the term “continuous” in relation to a unit operation refers to a direct connection or mechanism that enables a continuous flow between one or more unit operations.

[0072] As used herein, the term “dynamic binding capacity” in relation to chromatographic materials refers to the amount of products, such as polypeptides, to which the material will bind under actual flow conditions before significant intrusion of unbound products occurs.

[0073] As used herein, the terms “expression vector” or “expression construct” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid regulatory sequences necessary for the expression of an operably ligated coding sequence in a particular host cell, e.g., a mammalian host cell. Vectors may include viral vectors, non-episomal mammalian vectors, plasmids, and other nonviral vectors. Expression vectors may contain sequences that act on or control transcription, translation, and, where introns are present, act on RNA splicing of the coding region operably ligated thereto. “Operatably ligated” means that the components to which this term applies are related in such a way that they can perform their intrinsic functions. For example, a regulatory sequence in a vector “opertably ligated” to a protein coding sequence, e.g., a promoter, is positioned such that the normal activity of the regulatory sequence leads to the transcription of the protein coding sequence and the recombinant expression of the encoded protein.

[0074] As used herein, “fed-batch culture” refers to a form of suspension culture, specifically a method of culturing cells in which additional components are provided to the culture medium at some or more points after the start of the culture process. The supplied components typically include nutrient supplements for cells depleted during the culture process. In addition or alternatively, the additional components may include supplemental components (e.g., cell cycle inhibitors). In some embodiments, the fed-batch cell culture medium formulation may contain components essential for cell survival and proliferation and may be richer or more concentrated than the basal cell culture medium formulation typically used to initiate cell culture. The fed-batch culture may be stopped at some point, and the cells and / or components in the medium may be recovered and optionally purified.

[0075] As used herein, “fusion protein” is a protein comprising 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 from one protein is added in frame together with a nucleotide sequence encoding a polypeptide sequence from a different protein, and optionally separated from that sequence by a linker. The fusion gene may then be expressed by a recombinant host cell to produce a fusion protein. A fusion protein may include an Fc region fused to or linked to a fragment derived from an immunoglobulin protein, such as a ligand polypeptide, a receptor polypeptide, a hormone, a cytokine, a growth factor, an enzyme, or another polypeptide that is not a component of immunoglobulin.

[0076] As used herein, the “proliferative phase” of a cell culture refers to the period of exponential cell growth (i.e., logarithmic phase) in which cells are generally dividing rapidly.

[0077] As used herein, the term “recovered cell culture medium” refers to a solution treated by one or more operations for separating cells, cell debris, or other large particles from recombinant proteins. Such operations include, but are not limited to, cooling, agglutination, acidification, centrifugation, neutralization, ultrasonic separation, and various forms of filtration (e.g., deep filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration), as described above. The recovered cell culture medium includes cell culture lysates and cell culture supernatants. The recovered cell culture medium may be further clarified to remove fine particulate matter and soluble aggregates by filtration through a membrane with a pore size of about 0.1 μm to about 0.5 μm, for example, a membrane with a pore size of about 0.22 μm.

[0078] As used herein, “host cell” means a cell that is transformed with a nucleic acid, or can be transformed, to express the gene of interest. The term includes offspring of a parent cell, regardless of whether the offspring’s morphology or genetic structure is identical to that of the original parent cell, as long as the gene of interest is present. For example, a host cell containing nucleic acid encoding a recombinant protein, operably ligated to at least one expression regulatory sequence (e.g., a promoter or enhancer), is a “recombinant host cell.” When cultured under appropriate conditions, the host cell synthesizes a recombinant protein, which can then be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if the host cell does not secrete it).

[0079] As used herein, the “high molecular weight” or “HMW” species of the recombinant protein of interest refers to dimers, oligomers, and aggregates of the recombinant protein having a molecular weight greater than that of the intact, fully assembled form of the recombinant protein.

[0080] As used herein, the term “impurities” refers to components other than the recombinant protein of interest. Examples of impurities include, but are not limited to, host cell proteins, leaching resin materials (e.g., leaching protein A), nucleic acids, HMW species of recombinant protein, LMW species of recombinant protein, endotoxins, viral contaminants, cell culture medium components, and similar process and product-related impurities.

[0081] As used herein, the term “load density” refers to the amount of composition in contact with a certain volume of chromatographic material.

[0082] As used herein, the “low molecular weight” or “LMW” species of the recombinant protein of interest refers to a fragment, truncated form, or incomplete variant of the recombinant protein having a molecular weight smaller than that 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 the cellular expression of mRNA splice variants, and single-component polypeptides in the case of multi-chain polypeptide proteins (e.g., light-chain or heavy-chain-only species if the recombinant protein is an antibody).

[0083] As used herein, “perfused” cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. In some embodiments, the perfused cell culture medium composition may be more concentrated or higher in concentration than the basic cell culture medium composition to accommodate the method used to remove used medium. In some embodiments, the perfused cell culture medium may be used in both the growth phase and the production phase.

[0084] As used herein, the term "polypeptide" refers to a polymer of amino acids containing at least 50 amino acids, such as at least 100 amino acids.

[0085] As used herein, “production” cell culture medium refers to a cell culture medium typically used in cell culture during the transition (i.e., the “transition” phase and / or “production” phase) when exponential growth has ended and protein production becomes dominant, and which is sufficiently complete to maintain the desired cell density, viability, and / or product titer during this phase. The production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of the cell culture.

[0086] As used herein, the “productive phase” of a cell culture refers to the period after logarithmic cell proliferation has ended and recombinant protein production becomes dominant.

[0087] As used herein, the term "polishing chromatography" refers to a chromatographic operation performed after a capture chromatography or affinity chromatography operation to remove residual impurities and obtain a more highly purified composition and / or recombinant protein. Common impurities removed during the polishing process 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).

[0088] Anion exchange chromatography (AEX) refers to a form of ion exchange chromatography performed on a positively charged solid-phase medium (e.g., a resin or membrane) capable of anion exchange with anions in an aqueous solution passing 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 15Q, Capto(trademark)Q, Q-SEPHAROSE FAST FLOW(trademark)(Cytiva), FRACTOGEL TMAE(trademark), FRACTOGEL EDM DEAE(trademark),(EMD Merck), TOYOPEARL Super Q(registered trademark), and TOYOPEARL NH2-750F(Tosoh Bioscience), POROS HQ(trademark) and POROS XQ(trademark),(ThermoFisher).

[0089] Cation exchange chromatography (CEX) refers to a form of ion exchange chromatography performed on a solid-phase medium (e.g., a resin or membrane) that is negatively charged and capable of cation exchange with cations in an aqueous solution passing over or through the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase, for example, by covalent bonds. Alternatively or in addition, the charge may be an inherent property of the solid phase (e.g., silica with a total negative charge). CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process-related impurities, and / or viral contaminants. Commercially available cation exchange media include sulfopropyl (SP) immobilized on agarose (e.g., SPSEPHAROSE FAST FLOW (trademark), SP-SEPHAROSE FAST FLOW XL (trademark), or SP-SEPHAROSE HIGH PERFORMANCE (trademark), CAPTO S (trademark), CAPTO SP ImpRes (trademark), CAPTO S ImpAct (trademark) (Cytiva), FRACTOGEL-SO3 (trademark), FRACTOGEL-SE HICAP (trademark), and FRACTOPREP (trademark) (EMD Merck, Darmstadt, Germany), TOYOPEARL (registered trademark) XS, TOYOPEARL (registered trademark) HS (Tosoh Bioscience, King of Prussia, PA), UNOsphere (trademark) (BioRad, Hercules, CA), and S Ceramic Hyper (trademark) DF (Pall, Port). Examples include, but are not limited to, Washington, NY; POROS (Trademark) (ThermoFisher, Waltham, MA); ESHMUNO (Registered Trademark) CSP; and ESHMUNO (Registered Trademark) CP-FT (Millipore Sigma, Darmstadt, Germany).

[0090] Hydrophobic interaction chromatography (HIC) refers to chromatography performed on a solid-phase medium that utilizes the interaction between a hydrophobic ligand and a hydrophobic residue on the surface of a solute. Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sephrose® (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto® phenyl (Cytiva).

[0091] Mixed-mode or multimodal chromatography (MMC) refers to chromatography that achieves separation by utilizing two or more forms of interaction between the stationary phase and the analyte. MMC differs from single-mode chromatography in that two or more interactions, such as electrostatic interactions, hydrogen bonding interactions, and / or hydrophobic interactions, significantly contribute to solute retention. Commercially available multimodal chromatography media include, but are not limited to, Capto® Adhere, Capto® MMC Impress, Capto MMC (Cytiva), PPA Hypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno® HCX (Merk Millipore), and Toyopearl® MX-Trp-650M (Tosoh Bioscience).

[0092] Polishing chromatography unit operations utilize materials containing agents (e.g., resins and / or membranes) that can be operated in various modes, two of which are binding and elution mode and flow-through mode. In binding and elution chromatography, the biomolecules of interest are typically loaded onto the chromatographic material to maximize dynamic binding capacity, and then washing and elution conditions are used to maximize the purity of the product in the eluate. In contrast, flow-through chromatography uses loading conditions that allow impurities to bind to the chromatographic material while the biomolecules of interest pass through. Compared to binding and elution chromatography, flow-through chromatography allows for higher loading densities for a wider range of biomolecules.

[0093] In addition to the two most common modes, weak partition chromatography, overload chromatography, and frontal chromatography modes can also be used in the purification process. In isocratic separation, which is weak partition chromatography, the flow-through mode is modified with a low product partition coefficient ranging from 0.1 to 20 by identifying solution conditions that promote the weak binding of biomolecules to the resin, in addition to the binding of one or more impurities. In overload chromatography, the biomolecules of interest are loaded onto the chromatography material beyond the dynamic binding capacity of the material. Furthermore, frontal chromatography mode allows for a continuous, high-density feed (containing the protein of interest and at least one impurity) onto the chromatography medium. In frontal chromatography, the separation of the protein of interest from impurities and contaminants is facilitated by the binding affinity of the components in the load feed to the chromatography medium. The amount of protein of interest that can be loaded onto the chromatography medium and bound to it in frontal mode typically depends on the amount of more highly charged impurities / contaminants, such as product-related impurities, in the load feed. Initially, all components in the load feed bind to the chromatography medium. The separation of the product of interest from impurities / contaminants is driven by its affinity to the chromatography medium. When the chromatographic medium reaches saturation binding, components in the load feed with a higher affinity for the chromatographic medium (typically product-related impurities such as HMW species) displace proteins with a weaker affinity (e.g., the product of interest), resulting in the separation of the weaker-affinity proteins from the chromatographic medium. These proteins exit the column during load flow-through. As loading progresses, bound proteins are successively replaced in order of their affinity for the chromatographic medium, from least to most, until the column is saturated with proteins that have a higher affinity than the product of interest.

[0094] As used herein, the term "partition coefficient" or "product partition coefficient" (K pThe term refers to the molar concentration of a product bound to the stationary phase, such as recombinant protein, divided by the molar concentration of the product in the mobile phase, during the chromatography process.

[0095] As used herein, 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 compared to a composition in which it remained present before one or more unit operations. Furthermore, “purified” recombinant protein (e.g., purified antibody) refers to a recombinant protein whose purity has been increased, thereby being present in a form that is purer than that which exists in its natural environment and / or when it was first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0096] As used herein, the term “recombinant protein” refers to a heterologous protein produced when a host cell transfected with a protein-coding nucleic acid is cultured in a cell culture.

[0097] As used herein, the term “unit operation” refers to a functional step performed as part of a process for purifying a recombinant protein of interest. Unit operations may be designed to achieve one or more objectives, such as a capture step and a virus inactivation step. Unit operations may also include holding or preserving steps between processing steps.

[0098] As used herein, the term “virus prefilter” refers to a filter located upstream of a virus filter and in fluid communication with the virus filter, which can bind to one or more bioprocess impurities to increase the flow rate and / or throughput of the virus filter.

[0099] As used herein, the term “filtration cycle” refers to a period of time (e.g., a continuous period, e.g., a continuous period before a scheduled interruption) during which a composition is filtered through a single virus filter. A single virus filter may be used for one or more filtration cycles (e.g., 1, 2, 3, 4 or more), with interruptions occurring between each filtration cycle, and the virus prefilter being optionally replaced in each filtration cycle.

[0100] As used herein, the phrase “viral prefilter area to viral filter area ratio” refers to the ratio of the viral prefilter area to the viral filter area in a given filtration cycle.

[0101] As used herein, “net ratio of virus prefilter area to virus filter area” means the cumulative ratio of virus prefilter area to virus filter area over one or more filtration cycles (i.e., the ratio of all virus prefilter area to virus filter area used during the lifespan 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., if the virus prefilter is not replaced during the lifespan of the virus filter) or different (e.g., if the virus prefilter is replaced during one or more filtration cycles during the lifespan of the virus filter).

[0102] Non-restrictive exemplary features Without limiting them, some exemplary embodiments / features of this disclosure include: E1. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, wherein the ratio of the viral prefilter area to the viral filter area is at least about 2:1. E2. The virus prefilter is a depth filter, as described in E1. E3. The virus prefilter is a diatomaceous earth-based depth filter, as described in E1 or E2. E4. The method according to E3, further comprising rinsing the diatomaceous earth-based depth filter with water or buffer before filtering the composition. E5. The virus filter consists of at least one flat sheet, as described in any one of E1-E4. E6. The virus filter is a method according to any one of E1-E5, containing polyethersulfone (PES). E7. The virus filter is a flat sheet PES membrane, according to any one of the methods described in E1 to E6. E8. The virus filter is a small virus filter, and is a method described in any one of E1-E7. E9. The virus filter is a large virus filter, and is a method described in one of E1-E7. E10. The ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 4:1, as described in any one of E1 to E9. E11. The ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 3:1, as described in any one of E1 to E10. E12. The ratio of virus pre-filter area to virus filter area is approximately 2.5:1 to approximately 3:1, as described in any one of E1 to E11. E13. The method according to any one of E1 to E12, further comprising adjusting the pH of the composition to less than approximately 7.2 before filtration. E14. The method according to any one of E1 to E12, further comprising adjusting the pH of the composition to about 5 to about 7 before filtration. E15. The method according to any one of E1 to E14, further comprising adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. E16. The method according to any one of E1 to E15, further comprising adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. E17. The method according to any one of E1 to E15, further comprising adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration. E18. The method according to any one of E1 to E17, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after each filtration cycle. E19. The method according to any one of E1 to E17, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after each filtration cycle. E20. The method according to any one of E1 to E17, comprising at least two filtration cycles, wherein the virus prefilter is replaced after each filtration. E21. The method according to any one of E1 to E17, comprising 3 filtration cycles, wherein the net ratio of virus pre-filter area to virus filter area is at least about 6:1. E22. The method described in E21, wherein the net ratio of virus pre-filter area to virus filter area is approximately 6:1 to approximately 9:1. E23. The composition is approximately 100 L / m³ 2 / hour ~ approx. 500L / m 2 A method according to one of E1-E22, wherein the virus is filtered through a virus filter at a flow rate of / hour. E24. The composition is approximately 200 L / m³ 2 / hour ~ approx. 300L / m 2 A method according to any one of E1-E23, in which the virus is filtered through a virus filter at a flow rate of / hour. E25. The composition is approximately 250 L / m³ 2 A method according to one of E1-E24, where the virus is filtered through a virus filter at a flow rate of / hour. E26. The composition is filtered through a virus filter at a pressure of approximately 10 psi to approximately 60 psi, according to any one of E1 to E25. E27. The method according to any one of E1 to E26, wherein the composition is filtered through a virus filter at a pressure of approximately 30 psi. E28. At least approximately 1500 L / m³ over one or more filtration cycles2 A method according to any one of E1 to E27, including loading the virus filter up to that point. E29. At least approximately 1500 L / m³ over 3 filtration cycles 2 A method according to any one of E1 to E28, including loading the virus filter up to that point. E30. At least approximately 500 L / m³ for each of the three filtration cycles. 2 The method described in E29, which includes loading the virus filter up to that point. E31. The composition yields at least about 250 L / m³ for each of the three filtration cycles. 2 The method described in E29 or E30, wherein the virus is filtered through a virus filter at a flow rate of / hour. E32. At least approximately 30,000 g / m³ over one or more filtration cycles. 2 A method according to any one of E1 to E31, including loading the virus filter up to that point. E33. At least approximately 30,000 g / m³ over 3 filtration cycles. 2 The method described in E32, which includes loading the virus filter up to that point. E34. The method according to any one of E1 to E33, wherein at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. E35. A composition comprising a recombinant protein, as described in any one of E1 to E34. E36. The method according to E35, wherein the composition contains at least about 15 g / L of recombinant protein. E37. The composition according to E35 or E36, comprising approximately 15 g / L to approximately 25 g / L of recombinant protein. E38. The method according to any one of E1 to E37, wherein 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. E39. The method according to any one of E1 to E38, wherein the 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. E40. A virus filtration skid comprising a virus prefilter and a virus filter, wherein the ratio of the virus prefilter area to the virus filter area is at least about 2:1. E41. The virus pre-filter is a depth filter, as described in E40. E42. The virus pre-filter is a diatomaceous earth-based depth filter, as described in E40 or E41. E43. The virus filter is a virus filtration skid described in any one of E40-E42, consisting of at least one flat sheet. E44. The virus filter is a virus filtration skid containing polyethersulfone (PES) as described in any one of E40-E43. E45. The virus filter is a flat sheet PES membrane, as described in any one of E40-E44. E46. The virus filter is a small virus filter, a virus filtration skid described in one of the following E40-E45. E47. The virus filter is a large virus filter, a virus filtration skid described in one of the following E40-E45. E48. The ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 4:1, as described in any one of E40 to E47. E49. The ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 3:1, as described in any one of E40 to E48. E50. The ratio of virus pre-filter area to virus filter area is approximately 2.5:1 to approximately 3:1, as described in any one of E40 to E49. E51. A virus filtration skid as described in any one of E40-E50, further comprising at least one inline monitoring system. E52. A virus filtration skid as described in E51, comprising at least one in-line monitoring system that monitors at least one characteristic selected from pressure, flow, pH, conductivity, and UV. E53. The virus pre-filter is a virus filtration skid described in any one of E40-E52, which is placed in series with the virus filter.

[0103] Furthermore, some further exemplary embodiments / features of this disclosure, though not limited to them, include: F1. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over one or more filtration cycles. 2 Loaded up to, and The ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1, according to the method. F2. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can withstand at least approximately 30,000 g / m³ over one or more filtration cycles. 2 Loaded up to, and The ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1, according to the method. F3. The method according to F1 or F2, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after one or more filtration cycles. F4. The method according to any one of F1-F3, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after each filtration cycle. F5. The method according to F1 or F2, comprising at least two filtration cycles, wherein the virus prefilter is replaced after one or more filtration cycles. F6, the method according to F1 or F2, comprising at least two filtration cycles, wherein the virus prefilter 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 viral prefilter and a viral filter over at least two filtration cycles, wherein the viral prefilter is replaced after each filtration cycle. The virus filter can filter at least approximately 1500 L / m³ over at least two filtration cycles. 2 Loaded up to, and The ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1, according to the method. F8. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, wherein the viral prefilter is replaced after each filtration cycle. The virus filter will filter at least approximately 30,000 g / m³ over at least two filtration cycles. 2 Loaded up to, and The ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1, according to the method. F9. The virus pre-filter is a depth filter, and is configured in any of the methods described in F1-F8. F10. The virus pre-filter is a diatomaceous earth-based depth filter, as described in any one of F1-F9. F11. The method according to F10, further comprising rinsing a diatomaceous earth-based depth filter with water or a buffer solution before filtering the composition. F12. The virus filter consists of at least one flat sheet, as described in any one of F1 to F11. F13. The virus filter is a method according to any one of F1-F12, containing polyethersulfone (PES). F14. The virus filter is a flat sheet PES membrane, according to one of the methods described in F1 to F13. F15. The method according to any one of F1 to F14, wherein the virus pre-filter is a depth filter and the virus filter is a flat sheet PES membrane. F16. The method according to any one of F1-F8, wherein the virus pre-filter is a diatomaceous earth-based depth filter and the virus filter is a flat sheet PES membrane. F17. The virus filter is a small virus filter, and is a method of one of F1-F8. F18. The virus filter is a large virus filter, and is a method of one of F1-F8. F19. The method according to any one of F1 to F18, wherein the ratio of virus pre-filter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 4:1. F20. The ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 3:1, as described in any one of F1 to F19. F21. The method according to any one of F1 to F20, wherein the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.5:1 to approximately 3:1. F22. The method according to any one of F1 to F21, further comprising adjusting the pH of the composition to less than approximately 7.2 before filtration. F23. The method according to any one of F1 to F22, further comprising adjusting the pH of the composition to about 5 to about 7 before filtration. F24. The method according to any one of F1 to F23, further comprising adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. F25. The method according to any one of F1 to F24, further comprising adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. F26. The method according to any one of F1 to F23, further comprising adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration. The method according to any one of F1 to F26, comprising three filtration cycles, wherein the net ratio of virus pre-filter area to virus filter area over the three filtration cycles is at least about 6:1. F28. The net ratio of virus pre-filter area to virus filter area is approximately 6:1 to approximately 9:1, as described in F27. F29. The composition is approximately 100 L / m³. 2 / hour ~ approx. 500L / m 2 A method using one of F1-F28, where the virus is filtered through a virus filter at a flow rate of / hour. F30. The composition is approximately 200 L / m³. 2 / hour ~ approx. 300L / m 2 A method using one of F1-F29, where the virus is filtered through a virus filter at a flow rate of / hour. F31. The composition is approximately 250 L / m³. 2 A method using one of F1 to F30, where the virus is filtered through a virus filter at a flow rate of / hour. F32. The composition is filtered through a virus filter at a pressure of approximately 10 psi to approximately 60 psi, according to any one of F1 to F31. F33. The method according to any one of F1 to F32, wherein the composition is filtered through a virus filter at a pressure of approximately 30 psi. F34. At least approximately 1500 L / m³ over 3 filtration cycles. 2 (For example, at least about 2000 L / m 2 , about 1500L / m 2 ~About 3000L / m 2 A method of any one of F1 to F33, which includes loading the virus filter up to ). F35. At least approximately 500 L / m³ for each of the three filtration cycles. 2 The method of F34, which includes loading the virus filter up to that point. F36. The composition yields at least approximately 250 L / m³ for each of the three filtration cycles. 2 The method described in F34 or F35, wherein the virus is filtered through a virus filter at a flow rate of / hour. F37. At least approximately 30,000 g / m³ over 3 filtration cycles. 2 A method according to any one of F1 to F36, including loading a virus filter up to that point. F38. The method according to any one of F1 to F37, wherein at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. F39. The composition is the method according to any one of F1 to F38, comprising a recombinant protein. F40. The recombinant protein is an antibody (e.g., an IgG2 antibody), as described in F39. F41. The virus pre-filter is a diatomaceous earth-based depth filter. The virus filter contains polyethersulfone (PES), and The method according to any one of F1-F16 or F19-F40, further comprising rinsing a diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. F42. The virus pre-filter is a diatomaceous earth-based depth filter. The virus filter contains polyethersulfone (PES), The method further comprises rinsing a diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition, and The method according to F41, wherein, after filtering the composition, 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, between about 5 μg / L and less than 15 μg / L, between about 5 μg / L and less than 12.5 μg / L, between about 5 μg / L and less than 10 μg / L). F43. The carbonate-containing solution is the method according to F41 or F42, comprising sodium carbonate, potassium carbonate, or a mixture thereof. F44. After filtering the composition, the composition has a β-glucan concentration of less than approximately 10 μg / L, according to any one of F41 to F43. The F45 virus pre-filter is a diatomaceous earth-based depth filter. The virus filter contains polyethersulfone (PES), and The method according to any one of F1-F16 or F19-F40, further comprising rinsing a diatomaceous earth-based depth filter with sodium carbonate before filtering the composition. F46. The method according to F45, wherein after filtering the composition, 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, between about 5 μg / L and less than 15 μg / L, between about 5 μg / L and less than 12.5 μg / L, between about 5 μg / L and less than 10 μg / L). F47. The method according to any one of F1 to F40, wherein, after filtering the composition, 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 10 μg / L). F48. The composition according to any one of F1 to F47, wherein the composition contains at least about 10 g / L of recombinant protein. F49. The method according to any one of F1 to F47, wherein the composition contains at least about 12.5 g / L of recombinant protein. F50. The composition according to any one of F1 to F47, comprising at least about 15 g / L of recombinant protein. F51. The composition is one of the methods according to F1 to F47, comprising approximately 15 g / L to approximately 25 g / L of recombinant protein. F52. The composition is the method according to any one of F1 to F47, comprising approximately 15 g / L to approximately 20 g / L of recombinant protein. F53. The method according to any one of F1 to F52, wherein 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. F54. The method according to any one of F1 to F53, wherein the filtration results in a logarithmic reduction value (LRV) of at least one viral contaminant of at least approximately 4 for each of three filtration cycles. A virus filtration skid for use in any one of the methods described in F55.F1~F54.

[0104] Virus filtration method using a large pre-filter A method for removing at least one viral contaminant from a composition is provided herein, comprising filtering the composition through a viral prefilter and a viral filter, wherein the ratio of the viral prefilter area to the viral filter area is at least about 2:1.

[0105] 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 containing cationic imine groups.

[0106] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with water or a buffer before filtering the composition.

[0107] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 μm. In some embodiments, the virus prefilter is a membrane of about 0.1 μm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0108] In some embodiments, the virus prefilter is an absorption membrane. In some embodiments, the virus prefilter is an absorption membrane having an ion exchange function.

[0109] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane whose surface has been modified by a crosslinked polymer sulfonic acid cation exchange chemical reaction. In some embodiments, the virus prefilter is a flat sheet membrane having a PES membrane whose surface has been modified by a crosslinked mixed-mode chemical reaction.

[0110] In some embodiments, the virus prefilter is a three-layer flat sheet membrane. In some embodiments, the virus prefilter is a three-layer flat sheet membrane made of polyamide.

[0111] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane containing nylon. In some embodiments, the virus prefilter is a pleated sheet membrane containing hydrophilic acrylate-modified PVDF.

[0112] 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 contains hydrophilic copper-ammonium regenerated cellulose.

[0113] In some embodiments, the virus filter consists of at least one flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet and the virus filter includes hydrophilic polyethersulfone (PES).

[0114] In some embodiments, the virus filter consists of at least one pleated sheet.

[0115] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and includes hydrophilic PES.

[0116] In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet. In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet and the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet and the virus filter includes surface-modified PES. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet and the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet and the virus filter includes hydrophilic polyvinylidene fluoride (PVDF).

[0117] In some embodiments, the virus filter includes a symmetrical two-layer pleated sheet. In some embodiments, the virus filter includes a symmetrical two-layer pleated sheet and the virus filter includes hydrophilic acrylate-modified PVDF. In some embodiments, the virus filter includes a symmetrical three-layer pleated sheet. In some embodiments, the virus filter includes a symmetrical three-layer pleated sheet and the virus filter includes hydrophilic acrylate-modified PVDF.

[0118] In some embodiments, the virus filter is composed of 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 contains hydrophilic copper-ammonium regenerated cellulose. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and contains modified PVDF. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and contains hydrophilic PES.

[0119] In some embodiments, the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes surface-modified PES.

[0120] In some embodiments, the virus filter includes modified PVDF. In some embodiments, the virus filter includes hydrophilic PVDF. In some embodiments, the virus filter includes hydrophilic acrylate-modified PVDF.

[0121] In some embodiments, the virus filter includes hydrophilic copper-ammonium regenerated cellulose.

[0122] In some embodiments, the virus filter is a miniature virus filter.

[0123] In some embodiments, the virus filter is a large virus filter.

[0124] In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 4:1. In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 3:1. In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2.5:1 to approximately 3:1.

[0125] In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.4:1. In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.6:1. In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.9:1.

[0126] In some embodiments, the method further includes pre-preparing the composition before filtration. In some embodiments, the method further includes pre-preparing the composition in-line before filtration.

[0127] In some embodiments, the method further includes adjusting the pH of the composition to less than about 7.5 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to less than about 7.2 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to less than about 7 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to about 5 to about 7 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to about 6 to about 7 before filtration.

[0128] In some embodiments, the method further includes adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. In some embodiments, the method further includes adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. In some embodiments, the method further includes adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration.

[0129] In some embodiments, the composition is filtered through a virus prefilter in normal flow filtration mode. In some embodiments, the composition is filtered through a virus filter in tangential flow filtration mode.

[0130] In some embodiments, the composition is filtered through a virus prefilter and a virus filter in a normal flow filtration mode.

[0131] In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 100 L / m 2 / hour to about 500 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 200 L / m 2 / hour to about 400 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 200 L / m 2 / hour to about 300 L / m 2 / hour.

[0132] In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 100 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 150 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 200 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 250 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 300 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 350 L / m 2 / hour. In some embodiments, the composition is filtered through a virus pre-filter at a flow rate of about 400 L / m 2 / hour.

[0133] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi to about 50 psi.

[0134] In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 10 psi / psid to approximately 60 psi / psid. In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 20 psi / psid to approximately 50 psi / psid.

[0135] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi to about 50 psi.

[0136] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psid to about 60 psid. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psid to about 50 psid.

[0137] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 60 psi.

[0138] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 60 psi.

[0139] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 30 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 40 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 50 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 60 psids.

[0140] In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 10 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 20 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 30 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 40 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 50 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 60 psi / psid.

[0141] In some embodiments, this method yields at least about 1500 L / m³ over one or more filtration cycles. 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields at least about 1500 L / m³ over a single filtration cycle. 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields at least about 1500 L / m³ over two filtration cycles. 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields at least about 1500 L / m³ over three filtration cycles. 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields at least about 1500 L / m³ over more than three filtration cycles. 2 This includes loading the virus filter.

[0142] In some embodiments, this method yields approximately 1500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 This includes loading the virus filter up to a certain level. In some embodiments, this method yields approximately 1500 L / m³ over a single filtration cycle. 2 ~About 3000L / m2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over two filtration cycles. 2 ~About 3000L / m 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over three filtration cycles. 2 ~About 3000L / m 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over more than three filtration cycles. 2 ~About 3000L / m 2 This includes loading the virus filter.

[0143] In some embodiments, this method yields approximately 1500 L / m³ over one or more filtration cycles. 2 ~About 2000L / m 2 This includes loading the virus filter up to a certain level. In some embodiments, this method yields approximately 1500 L / m³ over a single filtration cycle. 2 ~About 2000L / m 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over two filtration cycles. 2 ~About 2000L / m 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over three filtration cycles. 2 ~About 2000L / m 2 This includes loading the virus filter until it reaches a certain level. In some embodiments, this method yields approximately 1500 L / m³ over more than three filtration cycles. 2 ~About 2000L / m 2 This includes loading the virus filter.

[0144] In some embodiments, this method can achieve a maximum of approximately 3000 L / m³ over one or more filtration cycles. 2This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 3000 L / m³ over a single filtration cycle. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 3000 L / m³ over two filtration cycles. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 3000 L / m³ over three filtration cycles. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 3000 L / m³ over more than three filtration cycles. 2 This includes loading the virus filter.

[0145] In some embodiments, this method can achieve a maximum of approximately 2000 L / m³ over one or more filtration cycles. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 2000 L / m³ over a single filtration cycle. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 2000 L / m³ over two filtration cycles. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 2000 L / m³ over three filtration cycles. 2 This includes loading the virus filter up to a certain point. In some embodiments, this method can achieve a maximum of approximately 2000 L / m³ over more than three filtration cycles. 2 This includes loading the virus filter.

[0146] In some embodiments, this method yields at least about 30,000 g / m³ over one or more filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, the method loads at least about 30,000 g / m³ over a single filtration cycle. 2This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 30,000 g / m³ over two filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 30,000 g / m³ over three filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 30,000 g / m³ over more than three filtration cycles. 2 This includes loading the virus filter.

[0147] In some embodiments, this method yields at least about 50,000 g / m³ over one or more filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, the method loads at least about 50,000 g / m³ over a single filtration cycle. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 50,000 g / m³ over two filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 50,000 g / m³ over three filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method allows for at least about 50,000 g / m³ of filtration over more than three filtration cycles. 2 This includes loading the virus filter.

[0148] In some embodiments, this method yields at least about 60,000 g / m³ over one or more filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, the method loads at least about 60,000 g / m³ over a single filtration cycle. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 60,000 g / m³ over two filtration cycles. 2This includes loading the virus filter up to a certain level. In some embodiments, this method loads at least about 60,000 g / m³ over three filtration cycles. 2 This includes loading the virus filter up to a certain level. In some embodiments, this method allows for at least about 60,000 g / m³ of filtration over more than three filtration cycles. 2 This includes loading the virus filter.

[0149] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one contaminant is parvovirus. In some embodiments, at least one contaminant is retrovirus. In some embodiments, at least one contaminant is pseudorabies virus. In some embodiments, at least one contaminant is reovirus.

[0150] 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 an antibody fragment. In some embodiments, the recombinant protein is a single-chain variable fragment. In some embodiments, the recombinant protein is a fusion protein.

[0151] In some embodiments, the composition contains at least about 15 g / L of recombinant protein. In some embodiments, the composition contains at least about 17.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 20 g / L of recombinant protein. In some embodiments, the composition contains at least about 25 g / L of recombinant protein. In some embodiments, the composition contains at least about 30 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 50 g / L of recombinant protein.

[0152] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three or more filtration cycles.

[0153] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three or more filtration cycles.

[0154] In some embodiments, filtration yields 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 yields 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 yields 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 yields 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 embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three or more filtration cycles.

[0155] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of more than three filtration cycles.

[0156] A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over one or more filtration cycles. 2 and / or at least about 30,000 g / m² 2 Loaded up to, and A method is provided herein in which the ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1.

[0157] In some embodiments, the virus filter can filter at least about 1500 L / m³ over one or more filtration cycles. 2 It is loaded up to a certain extent. In some embodiments, the virus filter is loaded at least about 2000 L / m³ over one or more filtration cycles. 2 It will be loaded up to this point.

[0158] In some embodiments, the virus filter can filter approximately 1500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2is loaded up to. In some embodiments, the virus filter is about 2000 L / m over one or more filtration cycles 2 ~ about 3000 L / m 2 is loaded up to. In some embodiments, the virus filter is about 2500 L / m over one or more filtration cycles 2 ~ about 3000 L / m 2 is loaded up to.

[0159] In some embodiments, the virus filter is loaded with at least about 30,000 g / m over one or more filtration cycles 2 is loaded up to.

[0160] In some embodiments, the composition comprises a recombinant protein.

[0161] In some embodiments, the composition comprises at least about 10 g / L (e.g., 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 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) of recombinant protein.

[0162] In some embodiments, the composition comprises at least about 12.5 g / L of recombinant protein. In some embodiments, the composition comprises at least about 15 g / L of recombinant protein.

[0163] In some embodiments, the composition contains at least about 17.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 20 g / L of recombinant protein. In some embodiments, the composition contains at least about 25 g / L of recombinant protein. In some embodiments, the composition contains at least about 30 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 50 g / L of recombinant protein.

[0164] In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 20 g / L of recombinant protein.

[0165] In some embodiments, the method includes at least two filtration cycles, and the virus prefilter is optionally replaced after one or more filtration cycles.

[0166] In some embodiments, the method includes at least two filtration cycles, and the virus prefilter is optionally replaced after each filtration cycle.

[0167] 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 containing cationic imine groups.

[0168] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with water or a buffer before filtering the composition.

[0169] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of at least 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0170] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0171] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0172] In some embodiments, 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 10 μg / L). In some embodiments, after filtering the composition, the composition has a β-glucan concentration of less than about 10 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 15 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 12.5 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than about 10 μg / L.

[0173] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 μm. In some embodiments, the virus prefilter is a membrane of about 0.1 μm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0174] In some embodiments, the virus prefilter is an absorption membrane. In some embodiments, the virus prefilter is an absorption membrane having an ion exchange function.

[0175] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane whose surface has been modified by a crosslinked polymer sulfonic acid cation exchange chemical reaction. In some embodiments, the virus prefilter is a flat sheet membrane having a PES membrane whose surface has been modified by a crosslinked mixed-mode chemical reaction.

[0176] In some embodiments, the virus prefilter is a three-layer flat sheet membrane. In some embodiments, the virus prefilter is a three-layer flat sheet membrane made of polyamide.

[0177] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane containing nylon. In some embodiments, the virus prefilter is a pleated sheet membrane containing hydrophilic acrylate-modified PVDF.

[0178] 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 contains hydrophilic copper-ammonium regenerated cellulose.

[0179] In some embodiments, the virus filter consists of at least one flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet and the virus filter includes hydrophilic polyethersulfone (PES).

[0180] In some embodiments, the virus filter consists of at least one pleated sheet.

[0181] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and includes hydrophilic PES.

[0182] In some embodiments, the virus filter comprises an asymmetric two-layer pleated sheet. In some embodiments, the virus filter comprises an asymmetric two-layer pleated sheet, and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises an asymmetric two-layer pleated sheet, and the virus filter comprises surface-modified PES. In some embodiments, the virus filter comprises an asymmetric three-layer pleated sheet. In some embodiments, the virus filter comprises an asymmetric three-layer pleated sheet, and the virus filter comprises hydrophilic PES. In some embodiments, the virus filter comprises an asymmetric three-layer pleated sheet, and the virus filter comprises hydrophilic polyvinylidene fluoride (PVDF).

[0183] In some embodiments, the virus filter comprises a symmetric two-layer pleated sheet. In some embodiments, the virus filter comprises a symmetric two-layer pleated sheet, and the virus filter comprises hydrophilic acrylate-modified PVDF. In some embodiments, the virus filter comprises a symmetric three-layer pleated sheet. In some embodiments, the virus filter comprises a symmetric three-layer pleated sheet, and the virus filter comprises hydrophilic acrylate-modified PVDF.

[0184] In some embodiments, the virus filter is composed of 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.

[0185] In some embodiments, the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes surface-modified PES.

[0186] In some embodiments, the virus filter includes modified PVDF. In some embodiments, the virus filter includes hydrophilic PVDF. In some embodiments, the virus filter includes hydrophilic acrylate-modified PVDF.

[0187] In some embodiments, the virus filter includes hydrophilic copper-ammonium regenerated cellulose.

[0188] In some embodiments, the virus filter is a miniature virus filter.

[0189] In some embodiments, the virus filter is a large virus filter.

[0190] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 3:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.5:1 to approximately 3:1.

[0191] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.6:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.9:1.

[0192] In some embodiments, the method further includes pre-preparing the composition before filtration. In some embodiments, the method further includes pre-preparing the composition in-line before filtration.

[0193] In some embodiments, the method further includes adjusting the pH of the composition to less than about 7.5 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to less than about 7.2 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to less than about 7 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to about 5 to about 7 before filtration. In some embodiments, the method further includes adjusting the pH of the composition to about 6 to about 7 before filtration.

[0194] In some embodiments, the method further includes adjusting the conductivity of the composition to at least about 10 mS / cm before filtration. In some embodiments, the method further includes adjusting the conductivity of the composition to at least about 12 mS / cm before filtration. In some embodiments, the method further includes adjusting the conductivity of the composition to about 10 mS / cm to about 20 mS / cm before filtration.

[0195] 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 between about 5 and about 7. In some embodiments, the pH of the composition is between about 6 and about 7.

[0196] 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 about 10 mS / cm to about 20 mS / cm.

[0197] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm.

[0198] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm.

[0199] In some embodiments, the composition is filtered through a virus prefilter in normal flow filtration mode. In some embodiments, the composition is filtered through a virus filter in tangential flow filtration mode.

[0200] In some embodiments, the composition is filtered through a virus prefilter and a virus filter in a normal flow filtration mode.

[0201] In some embodiments, the composition is approximately 100 L / m³ 2 / hour ~ approx. 500L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 400L / m 2The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 300L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0202] In some embodiments, the composition is approximately 100 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 150 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 250 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 300 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 350 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 400 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0203] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi to about 50 psi.

[0204] In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 10 psi / psid to approximately 60 psi / psid. In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 20 psi / psid to approximately 50 psi / psid.

[0205] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi to about 50 psi.

[0206] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psid to about 60 psid. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psid to about 50 psid.

[0207] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 60 psi.

[0208] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 60 psi.

[0209] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 30 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 40 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 50 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 60 psids.

[0210] In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 10 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 20 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 30 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 40 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 50 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 60 psi / psid.

[0211] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one contaminant is parvovirus. In some embodiments, at least one contaminant is retrovirus. In some embodiments, at least one contaminant is pseudorabies virus. In some embodiments, at least one contaminant is reovirus.

[0212] 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.

[0213] In some embodiments, the recombinant protein is a bispecific antibody.

[0214] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a single-chain variable fragment.

[0215] In some embodiments, the recombinant protein is a fusion protein.

[0216] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three or more filtration cycles.

[0217] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three or more filtration cycles.

[0218] In some embodiments, filtration yields 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 yields 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 yields 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 yields 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 embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three or more filtration cycles.

[0219] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of more than three filtration cycles.

[0220] A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The virus filter can filter at least approximately 1500 L / m³ over one or more filtration cycles. 2 and / or at least about 30,000 g / m² 2 Loaded up to, The virus prefilter is a depth filter, The virus filter consists of at least one flat sheet, and A method is also provided herein in which the ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1.

[0221] In some embodiments, the virus filter can filter at least about 2000 L / m³ over one or more filtration cycles. 2 It will be loaded up to this point.

[0222] In some embodiments, the virus filter can filter approximately 1500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter can handle approximately 2000 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over one or more filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

[0223] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter contains polyethersulfone (PES). In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus filter contains polyethersulfone (PES).

[0224] In some embodiments, the composition includes recombinant protein.

[0225] In some embodiments, the composition contains at least about 10 g / L (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 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, Approximately 13g / L, approximately 13.5g / L, approximately 14g / L, approximately 14.5g / L, approximately 15g / L, approximately 15.5g / L, approximately 16g / L, approximately 16.5g / L, approximately 17g / L, approximately 17.5g / L, approximately 18g / L, approximately 18.5g / L, approximately 19g / L, approximately 19 .5g / L, about 20g / L, about 20.5g / L, about 21g / L, about 21.5g / L, about 22g / L, about 22.5g / L, about 23g / L, about 23.5g / L, about 24g / L, about 24.5g / L, about 25g / L) of recombinant protein.

[0226] In some embodiments, the composition contains at least about 12.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 15 g / L of recombinant protein.

[0227] In some embodiments, the composition contains at least about 17.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 20 g / L of recombinant protein. In some embodiments, the composition contains at least about 25 g / L of recombinant protein. In some embodiments, the composition contains at least about 30 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 50 g / L of recombinant protein.

[0228] In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 20 g / L of recombinant protein.

[0229] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of at least 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0230] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0231] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0232] In some embodiments, 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 10 μg / L). In some embodiments, after filtering the composition, the composition has a β-glucan concentration of less than about 10 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 15 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 12.5 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than about 10 μg / L.

[0233] In some embodiments, the virus filter can filter at least about 1500 L / m³ over one or more filtration cycles. 2 It will be loaded up to this point.

[0234] In some embodiments, the virus filter can withstand at least about 30,000 g / m³ over one or more filtration cycles. 2 It will be loaded up to this point.

[0235] In some embodiments, the method includes at least two filtration cycles, and the virus prefilter is optionally replaced after one or more filtration cycles.

[0236] In some embodiments, the method includes at least two filtration cycles, and the virus prefilter is optionally replaced after each filtration cycle.

[0237] 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 containing cationic imine groups.

[0238] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with water or a buffer before filtering the composition.

[0239] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane whose surface has been modified by a crosslinked polymer sulfonic acid cation exchange chemical reaction. In some embodiments, the virus prefilter is a flat sheet membrane having a PES membrane whose surface has been modified by a crosslinked mixed-mode chemical reaction.

[0240] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane surface.

[0241] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 3:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.5:1 to approximately 3:1.

[0242] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.6:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.9:1.

[0243] 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 between about 5 and about 7. In some embodiments, the pH of the composition is between about 6 and about 7.

[0244] 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 about 10 mS / cm to about 20 mS / cm.

[0245] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm.

[0246] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm.

[0247] In some embodiments, the composition is filtered through a virus prefilter in normal flow filtration mode. In some embodiments, the composition is filtered through a virus filter in tangential flow filtration mode.

[0248] In some embodiments, the composition is filtered through a virus prefilter and a virus filter in a normal flow filtration mode.

[0249] In some embodiments, the composition is approximately 100 L / m³ 2 / hour ~ approx. 500L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 400L / m 2The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 300L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0250] In some embodiments, the composition is approximately 100 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 150 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 250 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 300 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 350 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 400 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0251] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi to about 50 psi.

[0252] In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 10 psi / psid to approximately 60 psi / psid. In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 20 psi / psid to approximately 50 psi / psid.

[0253] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi to about 50 psi.

[0254] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psid to about 60 psid. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psid to about 50 psid.

[0255] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 60 psi.

[0256] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 60 psi.

[0257] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 30 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 40 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 50 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 60 psids.

[0258] In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 10 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 20 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 30 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 40 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 50 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 60 psi / psid.

[0259] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one contaminant is parvovirus. In some embodiments, at least one contaminant is retrovirus. In some embodiments, at least one contaminant is pseudorabies virus. In some embodiments, at least one contaminant is reovirus.

[0260] 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.

[0261] In some embodiments, the recombinant protein is a bispecific antibody.

[0262] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a single-chain variable fragment.

[0263] In some embodiments, the recombinant protein is a fusion protein.

[0264] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three or more filtration cycles.

[0265] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three or more filtration cycles.

[0266] In some embodiments, filtration yields 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 yields 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 yields 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 yields 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 embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three or more filtration cycles.

[0267] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of more than three filtration cycles.

[0268] A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The virus prefilter is replaced after each filtration cycle. The virus filter can filter at least approximately 1500 L / m³ over at least two filtration cycles. 2and / or at least about 30,000 g / m² 2 Loaded up to, and A method is also provided herein in which the ratio of the virus prefilter area to the virus filter area in each filtration cycle is at least about 2:1.

[0269] In some embodiments, the composition includes recombinant protein.

[0270] In some embodiments, the composition contains at least about 10 g / L (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 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, Approximately 13g / L, approximately 13.5g / L, approximately 14g / L, approximately 14.5g / L, approximately 15g / L, approximately 15.5g / L, approximately 16g / L, approximately 16.5g / L, approximately 17g / L, approximately 17.5g / L, approximately 18g / L, approximately 18.5g / L, approximately 19g / L, approximately 19 .5g / L, about 20g / L, about 20.5g / L, about 21g / L, about 21.5g / L, about 22g / L, about 22.5g / L, about 23g / L, about 23.5g / L, about 24g / L, about 24.5g / L, about 25g / L) of recombinant protein.

[0271] In some embodiments, the composition contains at least about 12.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 15 g / L of recombinant protein.

[0272] In some embodiments, the composition contains at least about 17.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 20 g / L of recombinant protein. In some embodiments, the composition contains at least about 25 g / L of recombinant protein. In some embodiments, the composition contains at least about 30 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 50 g / L of recombinant protein.

[0273] In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 20 g / L of recombinant protein.

[0274] In some embodiments, the virus filter can filter at least about 2000 L / m³ over at least two filtration cycles. 2 It will be loaded up to this point.

[0275] In some embodiments, the virus filter can filter approximately 1500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2000 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It is loaded up to a certain level. In some embodiments, the virus filter is loaded up to approximately 2500 L / m³ over at least two filtration cycles. 2 ~About 3000L / m 2 It will be loaded up to this point.

[0276] In some embodiments, the virus prefilter is a depth filter, and the virus filter consists of at least one flat sheet.

[0277] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter. In some embodiments, the virus filter contains polyethersulfone (PES). In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus filter contains polyethersulfone (PES).

[0278] In some embodiments, the virus prefilter is a depth filter comprising diatomaceous earth, cellulose fibers, and a binder containing cationic imine groups.

[0279] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with water or a buffer before filtering the composition.

[0280] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane whose surface has been modified by a crosslinked polymer sulfonic acid cation exchange chemical reaction. In some embodiments, the virus prefilter is a flat sheet membrane having a PES membrane whose surface has been modified by a crosslinked mixed-mode chemical reaction.

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

[0282] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth filter with a carbonate-containing solution before filtering the composition, and after filtering the composition, the composition has a β-glucan concentration of at least 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0283] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition. 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 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.

[0284] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the virus filter contains polyethersulfone (PES), and the method further comprises rinsing the diatomaceous earth-based depth filter with sodium carbonate before filtering the composition.

[0285] In some embodiments, 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 to less than 15 μg / L, about 5 μg / L to less than 12.5 μg / L, about 5 μg / L to less than 10 μg / L). In some embodiments, after filtering the composition, the composition has a β-glucan concentration of less than about 10 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 15 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than 12.5 μg / L. In some embodiments, after filtering the composition, the composition has a β-glucan concentration of about 5 μg / L to less than about 10 μg / L.

[0286] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2:1 to approximately 3:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.5:1 to approximately 3:1.

[0287] In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.4:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.6:1. In some embodiments, the ratio of virus prefilter area to virus filter area in each filtration cycle is approximately 2.9:1.

[0288] 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 between about 5 and about 7. In some embodiments, the pH of the composition is between about 6 and about 7.

[0289] 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 about 10 mS / cm to about 20 mS / cm.

[0290] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 10 mS / cm.

[0291] In some embodiments, the pH of the composition is less than approximately 7.5 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7.2 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is less than approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 5 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm. In some embodiments, the pH of the composition is approximately 6 to approximately 7 and the conductivity of the composition is at least approximately 12 mS / cm.

[0292] In some embodiments, the composition is filtered through a virus prefilter in normal flow filtration mode. In some embodiments, the composition is filtered through a virus filter in tangential flow filtration mode.

[0293] In some embodiments, the composition is filtered through a virus prefilter and a virus filter in a normal flow filtration mode.

[0294] In some embodiments, the composition is approximately 100 L / m³ 2 / hour ~ approx. 500L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 400L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³. 2 / hour ~ approx. 300L / m 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0295] In some embodiments, the composition is approximately 100 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 150 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 200 L / m³.2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 250 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 300 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 350 L / m³. 2 The virus is filtered through a pre-filter at a flow rate of / hour. In some embodiments, the composition is approximately 400 L / m³ 2 The virus is filtered through a pre-filter at a flow rate of / hour.

[0296] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi to about 50 psi.

[0297] In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 10 psi / psid to approximately 60 psi / psid. In some embodiments, the virus is filtered through a pre-filter at an inlet pressure and / or differential pressure of approximately 20 psi / psid to approximately 50 psi / psid.

[0298] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi to about 60 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi to about 50 psi.

[0299] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psid to about 60 psid. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psid to about 50 psid.

[0300] In some embodiments, the composition is filtered through a virus filter at a pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter at a pressure of about 60 psi.

[0301] In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 10 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 20 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 30 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 40 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 50 psi. In some embodiments, the composition is filtered through a virus filter in a constant flow mode at an inlet pressure of about 60 psi.

[0302] In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 10 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 20 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 30 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 40 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 50 psids. In some embodiments, the composition is filtered through a virus filter in constant pressure mode with a differential pressure of about 60 psids.

[0303] In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 10 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 20 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 30 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 40 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 50 psi / psid. In some embodiments, the composition is filtered through a virus filter at an inlet pressure and / or differential pressure of about 60 psi / psid.

[0304] In some embodiments, at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus, and reovirus. In some embodiments, at least one contaminant is parvovirus. In some embodiments, at least one contaminant is retrovirus. In some embodiments, at least one contaminant is pseudorabies virus. In some embodiments, at least one contaminant is reovirus.

[0305] 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.

[0306] In some embodiments, the recombinant protein is a bispecific antibody.

[0307] In some embodiments, the recombinant protein is an antibody fragment. In some embodiments, the recombinant protein is a single-chain variable fragment.

[0308] In some embodiments, the recombinant protein is a fusion protein.

[0309] In some embodiments, the composition contains at least about 17.5 g / L of recombinant protein. In some embodiments, the composition contains at least about 20 g / L of recombinant protein. In some embodiments, the composition contains at least about 25 g / L of recombinant protein. In some embodiments, the composition contains at least about 30 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 25 g / L of recombinant protein. In some embodiments, the composition contains about 15 g / L to about 50 g / L of recombinant protein.

[0310] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of at least about 4 for each of three or more filtration cycles.

[0311] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of at least about 4 for each of three or more filtration cycles.

[0312] In some embodiments, filtration yields 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 yields 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 yields 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 yields 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 embodiments, filtration yields a logarithmic reduction value (LRV) of at least one viral contaminant of about 4 to about 7 for each of three or more filtration cycles.

[0313] In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for one filtration cycle. In some embodiments, filtration yields 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 yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of three filtration cycles. In some embodiments, filtration yields a logarithmic reduction value (LRV) of at least one parvovirus of about 4 to about 7 for each of more than three filtration cycles.

[0314] Virus filtration skid A virus filtration skid for use in the method described herein is provided herein. For example, a virus filtration skid comprising a virus prefilter and a virus filter, wherein the ratio of the virus prefilter area to the virus filter area is at least about 2:1 is provided herein.

[0315] 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 containing cationic imine groups.

[0316] In some embodiments, the virus prefilter is a diatomaceous earth-based depth filter, and the method further comprises rinsing the diatomaceous earth-based depth filter with water or a buffer before filtering the composition.

[0317] In some embodiments, the virus prefilter is a microfiltration membrane. In some embodiments, the virus prefilter is a membrane of about 0.2 μm. In some embodiments, the virus prefilter is a membrane of about 0.1 μm. In some embodiments, the virus prefilter is a membrane of about 75 nm.

[0318] In some embodiments, the virus prefilter is an absorption membrane. In some embodiments, the virus prefilter is an absorption membrane having an ion exchange function.

[0319] In some embodiments, the virus prefilter is a flat sheet membrane. In some embodiments, the virus prefilter is a flat sheet membrane having a polyethersulfone (PES) membrane whose surface has been modified by a crosslinked polymer sulfonic acid cation exchange chemical reaction. In some embodiments, the virus prefilter is a flat sheet membrane having a PES membrane whose surface has been modified by a crosslinked mixed-mode chemical reaction.

[0320] In some embodiments, the virus prefilter is a three-layer flat sheet membrane. In some embodiments, the virus prefilter is a three-layer flat sheet membrane made of polyamide.

[0321] In some embodiments, the virus prefilter is a pleated sheet membrane. In some embodiments, the virus prefilter is a pleated sheet membrane containing nylon. In some embodiments, the virus prefilter is a pleated sheet membrane containing hydrophilic acrylate-modified PVDF.

[0322] 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 contains hydrophilic copper-ammonium regenerated cellulose.

[0323] In some embodiments, the virus filter consists of at least one flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet. In some embodiments, the virus filter includes an asymmetrical double-layer flat sheet and the virus filter includes hydrophilic polyethersulfone (PES).

[0324] In some embodiments, the virus filter consists of at least one pleated sheet.

[0325] In some embodiments, the virus filter is a pleated sheet. In some embodiments, the virus filter is a pleated sheet and includes hydrophilic PES.

[0326] In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet. In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet and the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes an asymmetrical double-layer pleated sheet and the virus filter includes surface-modified PES. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet and the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes an asymmetrical triple-layer pleated sheet and the virus filter includes hydrophilic polyvinylidene fluoride (PVDF).

[0327] In some embodiments, the virus filter includes a symmetrical two-layer pleated sheet. In some embodiments, the virus filter includes a symmetrical two-layer pleated sheet and the virus filter includes hydrophilic acrylate-modified PVDF. In some embodiments, the virus filter includes a symmetrical three-layer pleated sheet. In some embodiments, the virus filter includes a symmetrical three-layer pleated sheet and the virus filter includes hydrophilic acrylate-modified PVDF.

[0328] In some embodiments, the virus filter is composed of 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 contains hydrophilic copper-ammonium regenerated cellulose. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and contains modified PVDF. In some embodiments, the virus filter is an asymmetric single-layer hollow fiber membrane and contains hydrophilic PES.

[0329] In some embodiments, the virus filter includes hydrophilic PES. In some embodiments, the virus filter includes surface-modified PES.

[0330] In some embodiments, the virus filter includes modified PVDF. In some embodiments, the virus filter includes hydrophilic PVDF. In some embodiments, the virus filter includes hydrophilic acrylate-modified PVDF.

[0331] In some embodiments, the virus filter includes hydrophilic copper-ammonium regenerated cellulose.

[0332] In some embodiments, the virus filter is a miniature virus filter.

[0333] In some embodiments, the virus filter is a large virus filter.

[0334] In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 4:1. In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2:1 to approximately 3:1. In some embodiments, the ratio of virus pre-filter area to virus filter area is approximately 2.5:1 to approximately 3:1.

[0335] In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.4:1. In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.6:1. In some embodiments, the ratio of virus prefilter area to virus filter area is approximately 2.9:1.

[0336] In some embodiments, the virus filtration skid further includes at least one inline monitoring system. In some embodiments, the at least one inline monitoring system monitors at least one characteristic selected from pressure, flow, pH, conductivity, and UV.

[0337] In some embodiments, the virus prefilter and the virus filter are arranged in series.

[0338] host cell The compositions used in the viral filtration methods disclosed herein may be derived from a biomanufacturing process (e.g., a cell culture medium or product pool recovered after one or more unit operations). Cell lines used in such biomanufacturing processes (also called “cells” or “host cells”) are genetically engineered to express recombinant proteins of commercial or scientific interest. Cells may be suitable for the adhesion, monolayer and / or suspension culture, transfection, and expression of recombinant proteins, such as antibodies. Cells may be used in conjunction with batch culture, fed-batch culture, and perfusion or continuous culture methods, for example. Such cells are typically cell lines obtained from or derived from mammals and can grow and survive when placed in either monolayer culture or suspension culture in a medium containing appropriate nutrients and / or other factors, e.g., those described herein. Typically, host cells are selected that can express and secrete proteins, or that can be molecularly engineered to express and secrete large quantities of specific proteins, more specifically glycoproteins of interest, into the culture medium. The selection of appropriate host cells for expressing recombinant proteins will depend on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. In some embodiments, the host cells used to produce recombinant proteins are mammalian host cells.

[0339] Cell lines typically originate from lines resulting from primary cultures that can be maintained in culture without time constraints. Cells may contain cells introduced via expression vectors (constructs), such as plasmids containing a coding sequence or a portion thereof that encodes a protein for expression and production in a culture process, for example, through transformation, transfection, infection, or injection. Such expression vectors contain elements necessary for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding desired proteins and polypeptides, as well as appropriate transcription and translation regulatory elements, can be constructed using methods well known to and practiced by those skilled in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4. th Disclosed in either the edition of Cold Spring Harbor Press, Plainview, NY or an earlier edition; FMAusubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY or an earlier edition; and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for all purposes.

[0340] Suitable host cells include, but are not limited to, those commercially available from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0341] Examples of host cells include, but are not limited to, prokaryotes, yeasts, or higher eukaryotic cells. Examples of prokaryotic host cells include bacteria, such as Gram-negative or Gram-positive microorganisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli, such as Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, and Serratia, such as Serratia marcescens. Examples include the genera *Marcescans*, *Shigella*, and *Bacillus*, such as *B. subtilis* and *B. licheniformis*, as well as the genera *Pseudomonas* and *Streptomyces*. In some embodiments, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for recombinant polypeptides. Among lower eukaryotic host microorganisms, *Saccharomyces cerevisiae*, i.e., common baker's yeast, is the most commonly used.However, genera such as Pichia (e.g., P. pastoris), Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, and Schwanniomyces (e.g., Schwanniomyces occidentalis) are not included. Hosts of filamentous fungi (such as *Occidentalis*), including the genera *Neurospora*, *Penicillium*, *Tolypocladium*, and *Aspergillus*, such as *A. nidulans* and *A. niger*, are generally available and useful herein.

[0342] Vertebrate host cells are also suitable hosts for recombinant protein expression. Suitable mammalian cell lines as hosts for recombinant protein expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but are not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44 and Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980), monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651), human fetal kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL 10) Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocellular carcinoma cells (Hep G2, HB 8065), mouse mammary cancer cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982), MRC Examples include 5 cells or FS4 cells, mammalian myeloma cells, and several other cell lines. In some embodiments, host cells are selected from CHO cells.

[0343] In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. The mammalian cell may be, for example, a human, rodent, or bovine cell line or cell lineage. Examples of such cells, cell lines, or cell lineages include, but are not limited to, the mouse myeloma (NSO) cell line, the Chinese hamster ovary (CHO) cell line, FIT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (baby hamster kidney cell), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the mammalian cell is a CHO cell line. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, mammalian cells are selected from CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN cells, and CHO-derived cells. In some embodiments, CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. Furthermore, CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza, Inc.). In some embodiments, eukaryotic cells may also be avian cells, cell lines, or cell lines such as EBx® cells, EB14, EB24, EB26, EB66, or EBv13.

[0344] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplified gene expression systems allow for high levels of recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Glutamine synthase (GS) knockout CHOK1SV cell lines utilizing methionine sulfoximine (MSX) selection based on glutamine synthase (GS) are also included. Other CHO host cells suitable for use in bioreactors 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).

[0345] Large-scale production of proteins for commercial use can be carried out in suspension culture. Therefore, the mammalian host cells used to produce the recombinant mammalian cells described herein can, but do not need to, be adapted for growth in suspension culture. Various host cells adapted for growth in suspension culture are known, including mouse myeloma NS0 cells and CLIO cells derived from CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER.C6® cells, human fetal kidney F1EK-293 cells, and cell lines derived from or manipulated from any of the cell lines described herein.

[0346] In some embodiments, eukaryotic cells are, for example, yeast cells (e.g., Pichia species (e.g., methanol-assimilating yeast (Pichia pastoris), Pichia methanolica, Pichia kluyveri, and Pichia angusta)), Komagataella species (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii)), Saccharomyces species (e.g., budding yeast (Saccharomyces cerevisae), Saccharomyces kluyveri, Saccharomyces ubarum) The cells are selected from lower eukaryotic cells such as *Pichia 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 fission yeast (*Schizosaccharomyces pombe*). In some embodiments, the eukaryotic cells are selected from methanol-assimilating yeast strains (*Pichia pastoris*). Non-exclusive examples of methanol-assimilating yeast (Pichia pastoris) strains include X33, GS115, KM71, KM71H, and CBS7435.

[0347] In some embodiments, eukaryotic cells are fungal cells (e.g., Aspergillus (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula, etc.), Acremonium (e.g., A. thermophilum, etc.), Chaetomium (e.g., C. thermophilum, etc.), Chrysosporium (Chrysosporium) (e.g., C. thermophile), Cordyceps (e.g., C. militaris), Corynascus, Ctenomyces, Fusarium (e.g., F. oxysporum), Glomerella (e.g., G. graminicola), Hypoclea pocrea (e.g., H. jecorina), Magnaporthe (e.g., M. orzyae), Myceliophthora (e.g., M. thermophile), Nectria (e.g., N. heamatococca), Neurospora (e.g., N. crassa), Penicillium (Penic) Cells are selected from the genera (e.g., *T. illium*), *Sporotrichum* (e.g., *S. thermophile*), *Thielavia* (e.g., *T. terrestris*, *T. heterothallica*), *Trichoderma* (e.g., *T. reesei*), or *Verticillium* (e.g., *V. dahlia*).

[0348] In some embodiments, eukaryotic cells are insect cells (e.g., Sf9, Mimic® Sf9, Sf21, High Cells from Five (trademark) (BT1-TN-5B1-4) or BT1-Ea88 cells, algal cells (e.g., Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas), and plant cells (e.g., monocotyledonous plants (e.g., maize, rice, wheat, or Setaria), or dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella). Cells are selected from (Patens) or (Arabidopsis, etc.).

[0349] To construct a host cell line (e.g., a mammalian cell line) engineered to express a target recombinant protein, one or more nucleic acids encoding the recombinant protein (or, in the case of a multichain protein, its components) are first inserted into one or more expression vectors. Useful nucleic acid regulatory sequences for expression vectors in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. Optionally, secretion signal peptide sequences may also be encoded by the expression vector and operably linked to the target coding sequence, thereby causing the recombinant host cell to secrete the expressed protein, allowing for easier isolation of the recombinant protein from the cell as needed. The vector may also include one or more selection marker genes to facilitate the selection of host cells into which the vector has been introduced. In some embodiments, vectors are used with protein fragment complementation assays using protein reporters such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964). Suitable mammalian expression vectors are known in the art and are commercially available.

[0350] Typically, a vector used in any host cell contains a sequence for maintaining the plasmid and a sequence for cloning and expressing the exogenous nucleotide sequence. Such a sequence typically includes the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional regulatory sequence and a translational regulatory sequence, a transcription termination sequence, a complete intron sequence containing a donor splice site and an 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 polylinker region for inserting the polynucleotide encoding the polypeptide to be expressed, and one or more selective marker elements. The vector may be constructed from a starting vector, such as a commercially available vector, and further elements may be obtained individually and ligated into the vector.

[0351] bioreactor The compositions used in the viral filtration methods disclosed herein may originate from biomanufacturing processes carried out in one or more bioreactors. In some embodiments, the growth and / or production phases of such biomanufacturing processes are carried out in the bioreactor. Suitable culture conditions for mammalian cells are known in the art. A bioreactor "run" typically includes the steps of seeding a seed culture in a prepared bioreactor, subjecting the cells to one or more growth and / or production phases until one or more predetermined parameters (e.g., time, viable cell density, packed cell volume) are met, and then collecting the contents of the bioreactor.

[0352] In some embodiments, one or more bioreactors used in the bioproduction process are stainless steel bioreactors, such as built-in large-scale stainless steel bioreactors that can operate with capacities ranging from approximately 2,000 liters to approximately 50,000 liters or more.

[0353] In some embodiments, one or more bioreactors used in the biofabrication process are disposable bioreactors. Using disposable technology minimizes the infrastructure requirements associated with conventional cell culture, such as commercial-scale steel / glass containers and associated equipment. Disposable bioreactors offer flexibility and adaptability to the manufacturing process. On-site assembly, reconfiguration, sterilization, and validation of disposable bioreactors can be faster, easier, and less expensive than conventional built-in stainless steel cell culture plants. A disposable bioreactor includes a disposable plastic sterile bag supported by a non-disposable support structure. The culture is agitated or shaken within the bag, and air and oxygen spargers, as well as sensors for measuring and adjusting various culture parameters (e.g., pH, temperature, oxygen, cell density, etc.), are also supplied. Disposable bioreactors are commercially available, such as Bio STR®, Sartorius, Goettingen Germany, MOBIUS®, Millipore, Burlington, MA, XCELLEREX®, and Cytiva, Marlborough, MA.

[0354] The bioreactor volume is divided into working volume space and headspace. The working volume of a bioreactor refers to the volume within the bioreactor where cell cultures are operated and is typically expressed as a percentage of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 70% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 70% to about 100% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 75% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 80% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 85% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 90% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 91% of the bioreactor volume. In some embodiments, the working volume of a bioreactor is at least about 92% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 93% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 94% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 95% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 96% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 97% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 98% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is at least about 99% of the bioreactor volume. In some embodiments, the working volume of the bioreactor is about 100% of the bioreactor volume.

[0355] Virus prefilter Viral prefilters, commercially available from several suppliers (e.g., MilliporeSigma, Sartorius, Pall, Asahi Kasei), remove protein aggregates and other process impurities that could otherwise contaminate the viral filter. Common viral prefilters utilize various membrane chemical reactions, such as PES membranes surface-modified by crosslinked polymer sulfonate cation exchange chemical reactions (Viresolve® Shield, MilliporeSigma) and PES membranes surface-modified by crosslinked mixed-mode chemical reactions (Viresolve® Shield-H, MilliporeSigma). Furthermore, depth filters (Viresolve® Prefilter, MilliporeSigma), pleated sheet membranes (Pegasus® Protect, Pall, Pegasus® grade UL6, Pall), and three-layer flat sheet membranes (Virosart® Max, Sartorius), composed of diatomaceous earth, cellulose fibers, and a binder containing cationic imine groups, are commonly used as prefilters.

[0356] Virus prefilters generally operate at least partially by size exclusion, and have size exclusion cutoff values ​​of approximately 75 nm to approximately 0.2 μm (e.g., 75 nm (Planova 75N, Asahi Kasei), 0.1 μm (Virosart® Max, Sartorius, Viresolve® Prefilter, MilliporeSigma), 0.2 μm (Viresolve® Shield, MilliporeSigma, Viresolve® Shield-H, MilliporeSigma, Pegasus® Protect, Pall)).

[0357] Virus filter Viral filters are polymer membranes with complex pore structures designed to retain various membrane chemical structures and viral particles. Viral filters are commercially available from various manufacturers (e.g., MilliporeSigma, Sartorius, Pall, and Asahi Kasei) and include large and small viral filters. Large viral filters are designed to retain viruses larger than 60 nm, while small viral filters are designed to retain viruses larger than 20 nm. Viral filters can be used in either normal flow filtration (NFF) mode or tangential flow filtration (TFF) mode. In either TFF or NFF mode, filtration is performed under conditions that retain viral contaminants (e.g., viruses with a diameter of 20-100 nm) on the membrane surface while allowing recombinant proteins to pass through the filtration membrane.

[0358] Non-limiting examples of virus filters include those formed from regenerated cellulose (e.g., copper ammonium regenerated cellulose), polyethersulfone, polyacryl sulfone, polysulfone, polyimide, polyamide, and polyvinylidene fluoride (PVDF). Non-limiting examples of virus filters include VIRESOLVE® membranes and RETROPORE® membranes available from EMD Millipore, Billerica, Mass. These can be supplied either in cartridge (NFF) form, such as VIRESOLVE® NFP virus filters, or as cassettes (for TFF), such as PELLICON® cassettes available from EMD Millipore, Billerica, Mass.

[0359] A further non-limiting example is the Sartorius Virosart® CPV miniature virus filter, which contains 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. Additional non-limiting exemplary filters include Planova® BioEX filters and Viresolve® NFP filters, both of which contain polyvinylidene fluoride (PVDF) membranes, and Asahi Kasei's 15N, 20N, and 35N filters, which contain copper-ammonium regenerated cellulose membranes (e.g., hollow fiber copper-ammonium regenerated cellulose membranes). Alternatives to virus filters are known in the art and include, for example, hydrophilic acrylate-modified PVDF membranes manufactured by Pall, such as Ultipor® VF grade DV20, Ultipor® VF grade DV50, and Pegasus® grade PV4.

[0360] Viral contaminants Non-exclusive examples of viral contaminants of concern in biomanufacturing 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 type 1 (HSV-1), bovine herpesvirus type 1 (IBRV), porcine enterovirus (PEV), pseudorabies virus (PRV), reovirus type 3 (Reo-3), Semryki forest fever virus (SFV), Sindbisvirus (SINV), Simian virus 40 (SV40), Tyler's mouse encephalomyelitis virus (TMEV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and heterotropic mouse leukemia virus (xMuLV).

[0361] Culture method Various culture methods, including but not limited to batch culture, fed-batch culture, perfusion culture, and aggregate cell culture, can be used to produce the recombinant protein of the desired outcome. The compositions subjected to the viral filtration methods disclosed herein can be obtained from any of these culture methods and may be subjected to one or more unit operations before viral filtration.

[0362] Batch culture is a discontinuous method in which cells are grown for a short period in a fixed volume of culture medium, and then the entire volume is harvested. Cultures grown using the batch method increase in cell density until they reach their maximum cell density, after which the viable cell density decreases as the culture medium components are consumed and metabolic by-products (such as lactate and ammonia) accumulate. Harvesting is typically performed when the maximum cell density (e.g., 5 × 10⁶ depending on the culture medium composition, cell line, etc.) is reached. 6 This process is performed when the cell density reaches a certain level (above or below cells / mL). While batch culture is the simplest culture method, the viable cell density is limited by nutrient availability, and the culture declines and production decreases once the cell density reaches its maximum. In batch culture, the production phase cannot be extended due to the accumulation of waste and rapid decline of the culture due to nutrient depletion, and is usually about 3 to 7 days.

[0363] Fed-batch culture improves upon the batch process by providing bolus or continuous medium supply to replenish consumed media components. Because fed-batch culture receives additional nutrients throughout the operation, it results in higher cell densities (>10-30 × 10⁶ cells, depending on medium composition, cell line, etc.) compared to batch methods. 6Fed batch culture has the potential to achieve a higher cell density (cells / mL) and increased product titer. Unlike batch processes, by manipulating the feeding strategy and culture medium composition, a two-phase culture can be created and maintained, allowing for the distinction between a cell proliferation phase (growth phase) to achieve the desired cell density and a period of cell growth arrest or slowing (production phase). Therefore, fed batch culture has the potential to achieve higher product titer compared to batch culture. Typically, the batch method is used during the growth phase and the fed batch method is used during the production phase, but a fed batch feeding strategy can be used throughout the entire process. However, unlike batch processes, the volume of the bioreactor becomes a limiting factor, restricting the amount of feed. Similar to batch methods, the accumulation of metabolic byproducts can also lead to culture decline, which often limits the production phase to approximately 10-21 days. Fed batch culture is discontinuous, and harvesting is typically performed when metabolic byproduct levels or culture viability reach predetermined levels. Compared to batch culture without feeding, fed batch culture can produce larger amounts of recombinant protein. (For example, see U.S. Patent No. 5,672,502.)

[0364] Perfusion culture offers potential improvements over batch and fed-batch methods by adding fresh medium during culture and simultaneously removing used medium. A typical perfusion culture begins with a batch culture startup lasting one or two days, followed by continuous, stepwise, and / or intermittent addition of fresh feeding medium to the culture, along with simultaneous removal of used medium, to retain cells and additional high molecular weight compounds (based on the filter's molecular weight cutoff value) such as proteins throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell density. Non-limiting examples of filtration methods include alternating tangential flow filtration and recirculating tangential flow. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424, Furey, 2002, Gen.Eng.News.22(7):62-63.

[0365] Perfusion may be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate may be less than or greater than the daily working volume. Cells are retained in the culture, and the used medium removed contains substantially no cells or significantly fewer cells compared to the culture. Recombinant proteins expressed by cell culture may also be retained in the culture.

[0366] In a typical large-scale commercial cell culture strategy, biomass accounts for approximately one-third to more than one-half of the reactor volume, e.g., 30-90(+) × 10 6 The goal is to achieve a high cell density of cells / mL. In perfusion culture, >1 × 10 8A very high cell density of cells / mL is achieved. A potential advantage of the perfusion process is that it allows for the maintenance of productive cultures for longer periods than batch or fed-batch cultures. However, maintaining long-term perfusion cultures requires increased preparation, use, storage, and disposal of the culture medium, especially in the case of high-cell-density cultures which also require more nutrients. All of these factors can increase production costs compared to batch and fed-batch methods. Furthermore, high cell densities can lead to problems in recovery and downstream processes, such as issues associated with increased aeration, including more oxygen supply and more carbon dioxide removal, which may lead to problems during production, such as maintaining dissolved oxygen levels, and the need for more changes to foaming and defoaming strategies, as well as product loss due to the work required to remove excess cellular material, which may negate the benefit of increased titer due to increased cell volume.

[0367] Suitable culture conditions for mammalian cells, including temperature, dissolved oxygen content, and stirring speed, are known in the art and may vary depending on the stage or phase of cell culture. In some embodiments, the methods disclosed herein further include taking a sample during the cell culture process and evaluating the sample to quantitatively and / or qualitatively monitor the characteristics of recombinant proteins and / or the cell culture process. In some embodiments, the sample is monitored quantitatively and / or qualitatively using process analysis techniques. For example, dissolved oxygen levels may be monitored during the cell culture process using methods known in the art, such as basic chemical analysis (titration), electrochemical analysis (diaphragm electrode method), and photochemical analysis (fluorescence method).

[0368] It is desirable to have a controlled system that switches the physiological state of cells to a high-productivity state in which growth is restricted or stopped, using energy and substrates to produce recombinant proteins so that the cells can increase cell density, while growing cells for a desired period of time or to a desired density. For commercial-scale cell culture and the manufacture of biopharmaceuticals, the ability to restrict or stop cell growth and maintain cells in a growth-restricted or stopped state during the production phase is highly desirable. Such methods include, for example, temperature shifts, the use of chemical inducers of protein production, nutrient restriction or starvation, and cell cycle inhibitors, either alone or in combination. Exemplarily, a typical cell culture goes through a growth phase, which is a period of exponential growth in which cell density increases. During the growth phase, cells are cultured in a cell culture medium containing the necessary nutrients and additives under conditions (usually a temperature of about 25-40°C in a humidified controlled atmosphere) in which optimal growth of a particular cell line is achieved. Cells are typically maintained for 1-8 days, e.g., 3-7 days, e.g., 7 days, during the growth phase. The length of the growth phase of a particular cell line can be determined by those skilled in the art, and is generally sufficient for the cells to proliferate to a viable cell density in the range of approximately 20% to 80% of the maximum viable cell density possible if the culture is maintained under growth conditions. The growth phase is followed by a transitional phase in which exponential cell growth slows down and protein production begins to increase. This marks the beginning of a quiescent phase in which the cell density typically plateaus and the product titer increases. During the production phase, the culture medium is usually replenished to support the continued production of recombinant proteins.

[0369] In certain embodiments, culture conditions may be adjusted to promote the transition from the growth phase to the production phase of a cell culture. For example, the growth phase of a cell culture may occur at a higher temperature than the production phase. In some embodiments, the growth phase may occur at a first temperature of about 35°C to about 38°C, and the production phase may occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In one embodiment, a temperature shift from about 35°C to about 37°C to about 31°C to about 33°C may be used to promote the transition from the growth phase to the production phase of the culture. For example, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with the temperature shift, before and / or after, or instead of the temperature shift. If the inducers are added after the temperature shift, they may be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift.

[0370] Furthermore, any cell culture medium that can support the growth of suitable host cells in culture may be used. Typically, cell culture media contain buffers, salts, energy sources, amino acids, vitamins, and trace essential elements. Cell culture media are commercially available that may be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells, including, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham F12 medium, Iskov Modified Dulbecco's medium, McCoy's 5A medium, Leibowitz L-15 medium, and serum-free media, such as the EX-CELL® 300 series, which can be obtained from American Type Culture Collection or SAFC Biosciences and other vendors. Cell culture media may be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture media may also be eutrophicated by the addition of nutrients or other supplements, which may be used at concentrations higher than the usual recommended concentrations. In certain embodiments, the culture medium is a known-composition medium, which refers to a cell culture medium in which all components have known chemical structures and concentrations. A known-composition medium is typically serum-free and does not contain hydrolysates or animal-derived components.

[0371] Various culture medium formulations may be used during the culture period, for example, to facilitate the transition from one stage (e.g., the growth stage or growth phase) to another stage (e.g., the production stage or production phase) and / or to optimize the conditions during cell culture (e.g., concentrated medium provided during perfusion culture). Growth medium formulations may be used to promote cell growth and minimize protein expression. Production medium formulations may be used to promote the production of the target recombinant protein and cell maintenance while minimizing the growth of new cells. Supply mediums are typically cell culture media containing more concentrated components such as nutrients and amino acids that are consumed during the production phase of a cell culture. Supply mediums may be used to supplement and maintain active cultures, particularly cultures operated in fed-batch mode, semi-perfusion mode, or perfusion mode. Such concentrated supply media may contain most of the components of cell culture media in amounts approximately 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or even about 1000 times their normal amounts.

[0372] In some embodiments, mammalian cells are cultured for a predetermined period during which recombinant proteins are expressed and secreted by the mammalian cells. This period (i.e., the duration of the productive phase of the cell culture) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the productive phase of the cell culture is about 7 to 28 days, about 10 to 30 days, about 7 to 14 days, about 10 to 18 days, about 3 to 15 days, about 5 to 8 days, about 12 to 15 days, about 12 to 18 days, or about 15 to 21 days. In some embodiments, the duration of the productive phase of the cell culture is 7 days, 8 days, 9 days, 12 days, 15 days, 18 days, or 21 days.

[0373] In some embodiments, the biomanufacturing process includes at least 100 × 10 5 Cells / mL, e.g., approximately 100 × 10⁶ 5 cells / mL ~ approx. 10×10 7 cells / mL, approximately 250×10 5cells / mL ~ approx. 900×10 5 cells / mL, approximately 300×10 5 cells / mL~800×10 5 Cells / mL or approximately 450 × 10 5 cells / mL~650×10 5 This includes the production phase with a viable cell density of cells / mL. Cell density can be measured using a hemocytometer, Coulter counter, or automated cell analyzer (e.g., Cedex automated cell counter). Viable cell density can be determined by staining the culture sample with trypan blue, which is taken up only by dead cells. The viable cell density is then determined by counting the total number of cells, dividing the number of stained cells by the total number of cells, and taking the reciprocal.

[0374] In some embodiments, the biomanufacturing process includes a production phase having a packed cell volume of 35% or less. In some embodiments, the packed cell volume is 30% or less.

[0375] Key attributes and performance indicators of the target recombinant protein may be measured to better communicate performance decisions at each stage of the manufacturing process. These key attributes and parameters may be monitored in real time, near real time, and / or offline. Key parameters that may be measured during cell culture may include the levels of cell culture medium components consumed (e.g., glucose), the levels of metabolic by-products accumulated (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content. Furthermore, key attributes such as specific productivity, viable cell density, compressed cell mass, pH, molar osmotic concentration, aggregation, percentage yield, and titer may be monitored at appropriate stages in the manufacturing process. Monitoring and measurement may be performed using known techniques and commercially available instruments.

[0376] Purification process The compositions used in the viral filtration methods disclosed herein may originate from cell culture processes and may be subjected to one or more purification processes before viral filtration. Exemplarily, expressed recombinant proteins may be secreted into culture media and recovered and / or collected therefrom. Recovery operations, including acid precipitation, may be combined with additional recovery strategies, including centrifugation such as disk stack centrifugation, intermittent discharge centrifugation, or continuous solid discharge centrifugation; filtration including tangential flow filtration, microfiltration, ultrafiltration, and deep filtration; precipitation / settlement methods, such as agglutination; and chromatography-based separation.

[0377] Furthermore, this disclosure encompasses all known post-recovery techniques, such as the purification of protein A from immunoglobulins and immunoglobulin-like biological products, as well as methods involving chromatography-based separation and polishing steps, including ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and / or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, size exclusion chromatography (SEC), gel filtration, or alternative modes of column and chromatographic separation by any other known form of chromatographic separation of biological and / or biochemical substances. Such post-recovery techniques may be used before or after viral filtration.

[0378] In some embodiments, recombinant proteins recovered from host cells or cell culture media can be further purified or partially purified by one or more unit operations before or after viral filtration to remove cell culture medium components, host cell proteins or nucleic acids, or other process or product-related impurities. Those skilled in the art can select appropriate unit operations for further purification of the recombinant proteins based on the characteristics of the recombinant proteins to be purified, the characteristics of the host cells expressing the recombinant proteins, and the composition of the culture medium in which the host cells were grown. Exemplarily, in some embodiments, recombinant proteins are purified from the osmotic recovery by one or more of the following: aggregation, precipitation, centrifugation, deep filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

[0379] Capture unit operations include capture chromatography using resins and / or membranes containing agents that bind to the recombinant protein of interest, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), and immobilized metal affinity chromatography (IMAC). Such chromatography materials are known in the art and are commercially available. For example, if the recombinant protein is an antibody or contains antibody-derived components (e.g., an Fc domain), affinity chromatography using ligands such as protein A, protein G, protein A / G, or protein L may be used as a capture chromatography unit operation for further purification of the recombinant protein. In other embodiments, the recombinant protein of interest may contain a polyhistidine tag at its amino or carboxyl terminus and can subsequently be purified using IMAC. The recombinant protein can be manipulated to contain other purification tags such as a FLAG® tag or a c-myc epitope, and then purified by affinity chromatography using a specific antibody against such tag or epitope.

[0380] Additional unit operations to inactivate, reduce, and / or eliminate viral contaminants may include filtration processes and / or adjustments to solution conditions. One method for achieving viral inactivation is incubation at a low pH (e.g., pH < 4). Following the low-pH viral inactivation operation, a neutralization unit operation may follow to readjust the pH of the virus-inactivated solution to a level more suitable for the requirements of subsequent unit operations. After the low-pH viral inactivation operation, further filtration, such as deep filtration, may follow to remove any resulting turbidity or precipitate. Adjustments to temperature or chemical composition (e.g., the use of detergents) may also be used to achieve viral inactivation.

[0381] In polishing unit operations, various chromatographic methods can be used to purify the target protein and remove contaminants and impurities. Polishing chromatographic unit operations can utilize resins and / or membranes containing agents that can be used in either flow-through mode (the target protein is contained in the eluent and contaminants and impurities are bound to the chromatographic medium) or binding and elution mode (the target protein is bound to the chromatographic medium and eluted after contaminants and impurities have passed through or been washed out of the chromatographic medium). Examples of such polishing chromatographic 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-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, and size exclusion chromatography (e.g., gel filtration).

[0382] UF / DF Purified recombinant proteins (e.g., recombinant proteins subjected to one or more purification processes in addition to viral filtration) can be formulated, i.e., buffer exchange, sterilization, bulk packaging, and / or packaging for the end user. Exemplarily, concentration of the product and buffer exchange of the recombinant protein of interest to the desired formulation buffer for bulk storage of the active pharmaceutical ingredient or drug can be performed by ultrafiltration and / or diafiltration. Suitable formulations for pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.

[0383] UF / DF operations may be performed at one or more stages in the downstream process. Typically, UF / DF operations are performed before bulk storage of the active pharmaceutical ingredient (API). Instead of storage, unit operations related to filling / finishing the drug product may also follow immediately after the UF / DF operation. One or more stability-enhancing excipients may optionally be added directly to the UF / DF residue feed tank containing formulated purified protein resulting in the formulated API, or to the UF / DF eluate pool. An exemplary UF / DF process is described in International Publication No. 2020 / 159838. Filters for use in UF / DF operations are well known in the art and are commercially available from many suppliers. Many types of materials are available, including regenerated cellulose, Pellicon® (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), and polyethersulfone (PES) membranes, Omega (Pall Corporation, Port Washington, NY).

[0384] Recombinant protein Compositions comprising any type of recombinant protein, including proteins containing a single polypeptide chain or multiple polypeptide chains, can be purified according to the methods of this disclosure. Such recombinant proteins include, but are not limited to, secretory proteins, non-secretory proteins, intracellular proteins, or membrane-bound proteins. Exemplary recombinant proteins may include, but are not limited to, cytokines, growth factors, hormones, mutant proteins, fusion proteins, antibodies, antibody fragments, peptide bodies, T cell engagement molecules, and multispecific antigen-binding proteins. In some embodiments, the recombinant protein is a fusion protein.

[0385] In other embodiments, the recombinant protein in the composition purified by the method of the present disclosure is an antigen-binding protein. Antigen-binding proteins include, but are not limited to, antibodies, peptide bodies, antibody derivatives, antibody analogs, fusion proteins (e.g., single-chain variable fragments (scFv), double-chain (bivalent) scFv and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), heteroIgG molecules (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), mutant proteins, and XmAb® (Xencor, Inc., Monrovia, CA). Additional antigen-binding proteins include, but are not limited to, bispecific T cell engagers (BiTE®), such as HLE BiTE molecules and HeteroIg BITE molecules, which have extended half-lives, as well as chimeric antigen receptors (CAR, CAR T) and T cell receptors (TCR).

[0386] In some embodiments, antigen-binding proteins bind to one or more of the following, either alone or in any combination: CD proteins, but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptors, such as EGFRvIII; and cell adhesion molecules, such as LFA. -1, Mol, p150, 95, VLA-4, ICAM-1, VCAM and alpha-v / beta-3 integrin, growth factors, not limited to, but such as vascular endothelial growth factor ("VEGF"), VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian duct inhibitors, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet-derived growth factor (PDGF), for example a Fibroblast growth factors such as FGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factor (TGF), particularly TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5), insulin-like growth factors I and II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone induction factors, insulin and insulin-related proteins, such as, but not limited to, insulin, insulin A chain, insulin B chain, and proinsulin. and insulin-like growth factor-binding proteins, (coagulation and coagulation-related proteins, such as plasminogen activators such as factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, urokinase, and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, and thrombopoietin receptors), colony-stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF, and other blood and serum proteins, but not limited to these.Albumin, IgE and blood group antigens, receptors and receptor-related proteins such as the flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor and T cell receptor, neurotrophic factors, but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophins-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), relaxin A chain, relaxin B chain and prorelaxin, interferons such as interferon-alpha, -beta and -gamma, interleukins (IL) such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 to its receptor, IL-13RA2 or IL -17 receptor, IL-1RAP, viral antigens (not limited to AIDS enveloped virus antigens), lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (mainly expressed and secreted by cells when T cells are activated), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, degeneration factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, Adresin, Regulatory Protein, Immunoadhesin, Antigen-binding Protein, Somatropin, CTGF, CTLA4, Eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, Ganglioside GD2, Ganglioside GM2, BAFF, OPGL (RANKL), Myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 Receptor, Hepatocyte Growth Factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1,Programmed cell death protein 1 and ligands, namely PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (lly), IFN gamma, interferon gamma-inducing protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphocyte neoplastic factor (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet-specific glycoprotein IIb / IIIb (PAC-1), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), secretin, and any of the aforementioned biologically active fragments or variants.

[0387] In other embodiments, the recombinant protein in the composition purified by the method of the present disclosure is an antibody. In some embodiments, the antibody is a human antibody.

[0388] In some embodiments, the antibody is selected from abrilumab, brazicumab, brodalumab, chryzanlizumab, denosumab, eculizumab, erenumab, evolocumab, fremanezumab, meplasmab, nemolizumab, ontamalimab, panitumumab, prezalumab, ravulizumab, rilotumumab, romosozumab, satralizumab, taforesimab, tanezumab, tezeperumab, tremelimumab, utomirumab, and boragidemab. In some embodiments, the antibody is selected from denosumab, erenumab, evolocumab, panitumumab, romosozumab, and tezeperumab. In some embodiments, the antibody is denosumab. In some embodiments, the antibody is erenumab. In some embodiments, the antibody is evolocumab. In some embodiments, the antibody is panitumumab. In some embodiments, the antibody is romosozumab. In some embodiments, the antibody is tezeperumab.

[0389] In some embodiments, the antibody is an IgG1, IgG2, or IgG4 antibody.

[0390] In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0391] In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is a human IgG2 antibody.

[0392] In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a human IgG4 antibody. [Examples]

[0393] Examples are provided below to allow for a more detailed understanding of this disclosure. These examples are for illustrative purposes only and should not be construed as limiting the disclosure.

[0394] Example 1. Use of a large pre-filter to enable robust virus clearance with a high-concentration (>15 g / L) feed stream over multiple filtration cycles. To achieve high flow rates, throughput, and cycling capabilities for highly concentrated human IgG2 antibody ("mAb") feed streams, a prefilter of the viral filter, which has a significantly larger surface area than the viral filter, was used in viral filtration operations at bench-scale and pilot run scales. Specifically, all tested ratios of prefilter area to viral filter area exceeded 2:1 for each filtration cycle (2.4:1, 2.6:1, 2.9:1), and the prefilter was replaced after each filtration cycle (i.e., the listed prefilter area to viral filter area ratios are the ratios 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) membrane viral filter (Viresolve® Pro, EMD Millipore).

[0395] The mAb feed stream was adjusted before filtration. Before adjusting the load conditions, the mAb feed stream was characterized by low conductivity (<6 mS / cm), high concentration (>20 g / L), and high pH (7.5), and contained buffer species including Tris and acetate. In contrast, the pre-adjusted stream was characterized by a lower pH (6.7) achieved by titration with acetate and a higher conductivity (>12 mS / cm) achieved by the addition of sodium chloride.

[0396] Figure 1 shows the load from a bench-scale proof-of-concept run with viral filter flow rate versus non-viral additive load. The bench-scale evaluation was performed using pre-adjusted, non-frozen loading material (i.e., "fresh" load as shown in the legend), a pre-filter to viral filter area ratio of 2.9:1, and a constant differential pressure of 30 psid. The evaluation was performed at 1800 L / m³. 2 Volume measurement throughput exceeding 250 L / m³ 2 An average flow rate per hour was achieved, demonstrating the capability of the viral filtration scheme to enable the cycling of viral filters.

[0397] Figure 2 shows bench-scale results regarding virus filter inlet pressure versus load for a “constant flow” run using the same virus filter for three filtration cycles. This evaluation was conducted using fresh, pre-conditioned load material, a pre-filter to virus filter area ratio of 2.9:1 for each filtration cycle, and 250 L / m². 2 The procedure was performed using a constant flow rate per hour. In the first two virus filtration cycles, the flow rate was approximately 500 L / m³. 2 High throughput was achieved. The load for the third filtration cycle continued until the entire material was loaded onto the filter, and the total volumetric throughput was 3400 L / m³. 2 It exceeded expectations.

[0398] Figures 3A, 3B, and Table 1 show the viral clearance results obtained in bench-scale runs performed with a pre-filter:viral filter area ratio of 2.9:1 for each of two filtration cycles, using fresh loading material to which the model virus, mouse microvirus (MMV), was added at different volume percentage values ​​summarized in Table 1. During the viral clearance test, the flow rate was 1,000 L / m³. 2 The target cycle load was set. As shown in Figures 3A and 3B, the inlet pressure was 1,000 L / m³. 2 When run up to the target load, each of the two filtration cycles was well controlled. The pressure trends for filtration cycles 1 and 2 show periods of low pressure due to filtration pauses. 2,000 L / m 2 The net throughput of the virus filter exceeding this value is 37,000 g / m². 2 This represents a significant improvement, achieving high throughput with a single virus filter. Furthermore, a logarithmic reduction value (LRV) of more than 4 was obtained for each of the two filtration cycles, and no viral breakthrough was observed.

[0399] [Table 1]

[0400] Figures 4 and 5 show the measured viral filter differential pressure and inlet pressure, respectively, during a pilot-scale run using three filtration cycles per viral filter, where a pre-filter:viral filter area ratio of 2.6:1 was used for each filtration cycle. The target viral filter load was 500 L / m². 2 The cycle was 1 / cycle, and the pressure was well controlled against the limits, as observed during the virus clearance test. The differential pressure and inlet pressure were measured at the end of each filtration cycle.

[0401] Table 2 shows the beta-glucan levels in the final active pharmaceutical ingredient (API) from three different pilot-scale runs (PSRs). The intent of this evaluation was to verify that the large pre-filter does not result in unacceptable beta-glucan leaching, a risk associated with some cellulose-based pre-filters that could affect the safety of the final API. The beta-glucan levels are within the range previously recorded for mAb production, demonstrating the feasibility of this filtration scheme combined with a risk mitigation strategy of sodium carbonate flushing each pre-filter before viral filtration.

[0402] [Table 2]

[0403] Based on these evaluations, the filtration scheme using a large prefilter yields 1500 L / m³ by volume over three filtration cycles at a flow rate of 250 LMH (liters / m² / hour) and a feed product concentration exceeding 18 g / L. 2 (30,000 g / m³ based on the mass of the product) 2This enables the above-mentioned virus filter load. Therefore, using a large pre-filter facilitates enhanced virus filtration operations that address: (1) high load capacity to provide high throughput in a relatively small footprint, (2) high flow rate to enable connected processing, which leads to dematerialization (i.e., lower costs through efficient use of raw materials and reduce manufacturing footprint), and reduces footprint compared to individual processing, (3) the ability to circulate the virus filter, thereby resulting in dematerialization, and (4) robust performance with a high concentration (>15g / L) feed flow that helps ensure high throughput in a small footprint.

[0404] All documents or parts of documents cited herein, including but not limited to patents, patent applications, papers, books, and academic articles, are expressly incorporated herein by reference. The embodiments described herein may be combined with one or more other embodiments of the herein unless the context expressly indicates otherwise.

[0405] The subject matter disclosed is not intended to be limited in scope by the specific embodiments described herein, but rather is intended as a non-limiting illustration of the individual aspects of this disclosure. Functionally equivalent methods and components are within the scope of this disclosure. In fact, various modifications of the disclosed subject matter, in addition to those shown and described herein, will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be within the scope of the disclosed subject matter.

[0406] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for illustrative purposes only and are not intended to be exhaustive or restrictive. Many variations and modifications will be apparent to those skilled in the art without departing from the scope and spirit of the embodiments described. The terms used herein have been chosen to best describe the principles of the embodiments, their practical applications, or technical improvements to the art found in the market, and / or to enable those skilled in the art to understand the disclosed subject matter.

Claims

1. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The aforementioned virus filter can filter at least about 1500 L / m³ over one or more filtration cycles. ２ Loaded up to, and A method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:

1.

2. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter, The aforementioned virus filter has a filtration capacity of at least about 30,000 g / m³ over one or more filtration cycles. ２ Loaded up to, and A method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:

1.

3. The method according to claim 1 or 2, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after one or more filtration cycles.

4. The method according to any one of claims 1 to 3, comprising at least two filtration cycles, wherein the virus prefilter is optionally replaced after each filtration cycle.

5. The method according to claim 1 or 2, comprising at least two filtration cycles, wherein the virus prefilter is replaced after one or more filtration cycles.

6. The method according to claim 1 or 2, comprising at least two filtration cycles, wherein the virus prefilter is replaced after each filtration cycle.

7. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The aforementioned virus prefilter is replaced after each filtration cycle. The virus filter filters at least about 1500 L / m³ over the at least two filtration cycles. ２ Loaded up to, and A method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:

1.

8. A method for removing at least one viral contaminant from a composition, comprising filtering the composition through a viral prefilter and a viral filter over at least two filtration cycles, The aforementioned virus prefilter is replaced after each filtration cycle. The virus filter absorbs at least about 30,000 g / m³ over the at least two filtration cycles. ２ Loaded up to, and A method in which the ratio of virus prefilter area to virus filter area in each filtration cycle is at least about 2:

1.

9. The aforementioned virus prefilter is a depth filter and / or The method according to any one of claims 1 to 8, wherein the virus filter is composed of at least one flat sheet.

10. The aforementioned virus prefilter is a diatomaceous earth-based depth filter, and / or The method according to any one of claims 1 to 9, wherein the virus filter comprises polyethersulfone (PES).

11. The aforementioned virus prefilter is a diatomaceous earth-based depth filter, and The method according to any one of claims 1 to 10, wherein the virus filter comprises polyethersulfone (PES).

12. The method according to any one of claims 1 to 11, wherein the ratio of the virus prefilter area to the virus filter area in each filtration cycle is about 2:1 to about 3:

1.

13. Adjust the pH of the composition to less than approximately 7.2 before filtration, and / or Before filtration, adjust the conductivity of the composition to at least about 10 mS / cm. The method according to any one of claims 1 to 12, further comprising:

14. Before filtration, adjust the pH of the composition to less than approximately 7.2, and Before filtration, adjust the conductivity of the composition to at least about 10 mS / cm. The method according to any one of claims 1 to 13, further comprising:

15. The composition is approximately 100 L / m³ ２ / hour ~ approx. 500L / m ２ The method according to any one of claims 1 to 14, wherein the virus is filtered through the virus filter at a flow rate of / hours.

16. The method according to any one of claims 1 to 15, wherein the composition is filtered through the virus filter at a pressure of about 10 psi to about 60 psi.

17. The method according to any one of claims 1 to 16, comprising three filtration cycles, wherein the net ratio of the virus prefilter area to the virus filter area over the three filtration cycles is at least about 6:

1.

18. The method according to any one of claims 1 to 17, wherein the net ratio of the virus prefilter area to the virus filter area over the filtration is about 6:1 to about 9:

1.

19. The aforementioned virus prefilter is a diatomaceous earth-based depth filter. The virus filter comprises polyethersulfone (PES), and The method according to any one of claims 1 to 18, further comprising rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition.

20. The method according to any one of claims 1 to 19, wherein, after filtering the composition, the composition has a β-glucan concentration of less than about 15 μg / L.

21. The aforementioned virus prefilter is a diatomaceous earth-based depth filter. The aforementioned virus filter contains polyethersulfone (PES), The method further comprises rinsing the diatomaceous earth-based depth filter with a carbonate-containing solution before filtering the composition, and The method according to any one of claims 1 to 20, wherein, after filtering the composition, the composition has a β-glucan concentration of less than about 10 μg / L.

22. The method according to any one of claims 1 to 21, wherein the composition comprises at least about 10 g / L of recombinant protein.

23. The method according to any one of claims 1 to 22, wherein the composition comprises at least about 15 g / L of recombinant protein.

24. The method according to any one of claims 1 to 23, wherein the at least one viral contaminant is selected from parvovirus, retrovirus, pseudorabies virus and reovirus.

25. The method according to any one of claims 1 to 24, wherein 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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