Antifoam compositions and methods of use thereof

Abstract

The present disclosure provides compositions and methods of inactivating a virus in a product feedstream in a manufacturing process of a therapeutic agent using an antifoam reagent. In some aspects, the therapeutic agent comprises a therapeutic protein, a viral particle, a nucleic acid molecule, or any combination thereof. Other aspects of the present disclosure are directed to methods of inactivating a virus in a eukaryotic cell culture of a product feedstream, comprising contacting the cell culture with an antifoam reagent. In some aspects, the antifoam reagent comprises an emulsifier, e.g., a nonionic emulsifier, and optionally a silicone polymer.

Description

ANTIFOAM COMPOSITIONSAND METHODS OF USE THEREOFCROSS-REFERENCE TO RELA TED AP PLICA TIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 562,205, filed March 6, 2024, which is incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure provides of inactivating viruses in a cell culture, i.e., a product feed stream in a manufacturing process of a therapeutic protein, a viral particle, a nucleic acid molecule, or any combination thereof, using an antifoam agent.BACKGROUND OF THE DISCLOSURE

[0003] Viral contamination may arise from the cell line itself when cells produce viruses endogenously or have latent or persistent infection. Alternatively, virus may be introduced during the recombinant production process due to the use of contaminated reagents or viral vectors. To ensure patient safety, the downstream purification process requires a robust viral clearance strategy to ensure the process can remove potential viral contamination. For enveloped viruses, typical viral inactivation processes use either low pH conditions or chemical inactivation agents as a key component of the removal strategy. For biologic drugs with poor stability at low pH conditions, chemical inactivation agents provide the necessary viral inactivation without stability concerns. Historically, the non-ionic surfactant, Triton X-100, provided excellent viral inactivation. However, in view of recent safety concerns with the degradation products of Triton X-100, there is a need in the art for novel means of viral clearance.

[0004] Antifoam reagents currently have broad use in the biopharmaceutical industry to prevent unwanted foam production in bioreactors and fermenters. Some antifoams also have lubricating properties that reduce sheer forces on cells introduced from aeration and agitation conditions. Often, antifoam chemicals used in cell culture contain either an emulsion of polydimethylsiloxane (PDMS) or nonionic detergents. The variation in the antifoam arises from the emulsifier or detergent used. As antifoam chemicals are already used in biologies manufacturing, they have established supply chains, regulatory familiarity, and known clearance and detection strategies.SUMMARY OF THE DISCLOSURE

[0005] Some aspects of the present disclosure are directed to a method of inactivating a virus in a product feedstream in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent. In some aspects, the therapeutic agent comprises a therapeutic protein, a viral particle, a nucleic acid molecule, or any combination thereof.

[0006] Some aspects of the present disclosure are directed to a method of inactivating a virus in a eukaryotic cell culture of a product feedstream, comprising contacting the cell culture with an antifoam reagent.

[0007] In some aspects, the eukaryotic cell culture comprises a mammalian cell or an insect cell. In some aspects, the mammalian cell is selected from a CHO cell and a HEK cell.

[0008] In some aspects, the antifoam reagent comprises an emulsifier. In some aspects, the emulsifier is a nonionic emulsifier. In some aspects, the emulsifier comprises one or more hydrogenated glyceride or PEG ester.

[0009] In some aspects, the antifoam reagent further comprises a silicone polymer. In some aspects, the silicone polymer comprises polydimethylsiloxane (PDMS). In some aspects, the antifoam reagent comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% silicone polymer. In some aspects, the antifoam reagent comprises about 10% silicone polymer.

[0010] In some aspects, the antifoam reagent comprises ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 10% ANTIFOAM B™. In some aspects, the product feedstream is contactedwith ANTIFOAM B™ at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% ANTIFOAM B™.

[0011] In some aspects, the antifoam reagent comprises antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 10% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam SE-15.

[0012] In some aspects, the antifoam reagent comprises antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% toabout 15%, or at least about 0.01% to about 10% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 10% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam L-30.

[0013] In some aspects, the antifoam reagent comprises one or more organic non-silicone polypropylene-based polyether.

[0014] In some aspects, the antifoam reagent comprises antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 10% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam 204.

[0015] In some aspects, the antifoam reagent comprises one or more difunctional block copolymer surfactant. In some aspects, the difunctional block copolymer surfactant comprises terminal secondary hydroxyl groups.

[0016] In some aspects, the difunctional block copolymer surfactant comprises a PLURONIC®. In some aspects, the difunctional block copolymer surfactant comprises PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 3 IRlat a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 10% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% PLURONIC® 31R1.

[0017] In some aspects, the product feedstream is not contacted with Triton X-100.[00 IS] In some aspects, the product feedstream comprises a harvest from a bioreactor, a chromatography load, a chromatography eluate, a filtration load, a filtrate, or any combination thereof. In some aspects, the chromatography eluate is a Protein A chromatography eluate.

[0019] In some aspects, the virus comprises a lipid-enveloped virus. In some aspects, the lipid-enveloped virus is a retrovirus. In some aspects, the retrovirus is A-MuLV. In some aspects, the lipid-enveloped virus is a herpesvirus. In some aspects, the herpesvirus is HSV-1.

[0020] In some aspects, inactivating the virus comprises a log reduction value (LRV) of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, and wherein the LRV is calculated as:[Virus in pfu]Product pooi

[0021] LRV = —logw[Virus in pfu]Load / Feed '

[0022] In some aspects, the LRV is at least about 2. In some aspects, the LRV is at least about 4.

[0023] In some aspects, the contacting occurs for at least about 15 minutes, at least about 30 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, or at least about 120 minutes.

[0024] In some aspects, the therapeutic protein comprises an antibody, antibody fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof. In some aspects, the therapeutic protein is a fusion protein. In some aspects, the fusion protein comprises an Fc portion.

[0025] In some aspects, the therapeutic agent comprises a non-enveloped virus particle. In some aspects, the non-enveloped virus particle comprises an AAV.

[0026] Some aspects of the present disclosure are directed to a method to treat a disease or condition comprising administering to a subject a therapeutic protein manufactured by a process comprising a viral inactivation step according to a method disclosed herein.

[0027] Some aspects of the present disclosure are directed to a pharmaceutical composition manufactured by a process comprising a viral inactivation step according to a method disclosed herein.

[0028] Some aspects of the present disclosure are directed to a method of manufacturing a therapeutic protein comprising a viral inactivation step according to a method disclosed herein.BRIEF DESCRIPTION OF THE DRA WINGS

[0029] FIGs. 1 A-1B are scatter plots, illustrating the comparison of the viral log reduction values (LRVs) of A-MulV (FIG. 1 A) or X-MulV (FIG. IB) and Retrovirus-Like Particles (RVLP) when treated with a panel of different agents.

[0030] FIG. 2 is a schematic of the experimental procedure for quantifying viral inactivation using Retrovirus-Like Particles (RVLP).

[0031] FIGs. 3 A-3B are bar graphs representing data related to the antifoam testing results: the antifoam testing panel compared against known detergents (FIG. 3 A) and the antifoam active ingredient concentration dependence for viral inactivation (FIG. 3B).DETAILED DESCRIPTION OF THE DISCLOSURE

[0032] The present disclosure is directed to methods of inactivating a virus in a cell culture comprising contacting the cell culture with an antifoam agent. Some aspects of the present disclosure are directed to methods of inactivating a virus in a product feedstream, comprising contacting the product feedstream with an antifoam reagent. In some aspects, the product feedstream is in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent. As such, some aspects of the present disclosure are directed to methods of inactivating a virus in a product feedstream in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent.

[0033] Some aspects of the present disclosure are directed to methods of inactivating a virus in a eukaryotic cell culture of a product feedstream, comprising contacting the cell culture with an antifoam reagent. In some aspects, the eukaryotic cell culture comprises a mammalian cell or an insect cell. In some aspects, the mammalian cell comprises a CHO cell or a HEK cell.I. Terms

[0034] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0035] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and / or "consisting essentially of' are also provided.

[0036] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0037] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0039] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0040] As used herein, the terms "antiviral," "inactivating a virus" or "virus inactivation" refer to a process where a virus can no longer infect cells, replicate, and propagate, and per se virus removal. As such, the term "virus inactivation" refers generally to the process of making a fluid disclosed herein completely free of infective viral contaminants. Any degree of viral inactivation using the methods disclosed herein is desirable. However, it is desirable to achieve the degree of viral inactivation necessary to meet strict safety guidelines for pharmaceuticals. These guidelines are set forth by the WHO and well known to those of skill in the art.

[0041] As used herein, the term "antifoam" refers to a class of reagents that are traditionally used to prevent the formation of foam, such as in a cell culture. Antifoam reagents typically comprise (i) a carrier fluid such, as silicone, mineral oil, an ester, and / or polyol; and / or (ii) ahydrophobic solid, such as a wax, a fatty alcohol, a fatty acid, and / or a silica. In some aspects, an antifoam reagent disclosed herein comprises an emulsifier. An "emulsifier," as used herein, refers to agent that facilitates the mixture of two liquids that typically separate when mixed in solution. In some aspects, the emulsifier is a nonionic emulsifier. In some aspects, the emulsifier is an ionic emulsifier. In some aspects, the emulsifier comprises one or more hydrogenated glyceride or PEG ester. In some aspects, the antifoam reagent further comprises a silicone polymer. As used herein, the term "silicon polymer" or "polysiloxane" refers to a substance whose molecules consist of polymeric chains of polymerized siloxane, having alternate silicon and oxygen atoms (e.g., -O-Si- O-Si- . . .). In some aspects, the silicone polymer comprises poly dimethyl siloxane (PDMS):

[0042] The methods disclosed herein can be used, e.g., for the production of a biological such as an antibody or a fusion protein. As use herein, the term "antibody" (Ab) shall include, without limitation, a glycoprotein immunoglobulin that binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0043] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0044] A "product feedstream" or alternatively, "feedstream" is the material or solution provided for a process purification method which contains a therapeutic agent of interest and which may also contain various impurities. Non-limiting examples may include, for example, harvested cell culture fluid (HCCF), or the collected pool containing the therapeutic protein of interest after one or more purification process steps. It is understood that the uses of the compositions and methods of the present disclosure are not limited to manufacture feedstreams of biological, and can be used to inactivate enveloped virus in any solution or medium in which viral inactivation is desired. In some aspects, viral inactivation can be conduct in a solution. In some aspects, viral inactivation can be conducted on a solid surface. In some aspects, viral inactivation can be conducted on the surface of the body of a mammal. In some aspects, the product feedstream comprises a harvest (e.g., a harvested cell culture fluid), a load (e.g., a loading solution of a chromatography or filtration, i.e., a chromatography load or a filtration load), an eluate (e g., a chromatography eluate), a filtrate (e.g., a solution collected after a solution has been filtered), or any combination thereof. In general, the methods disclosed herein can be used for viral inactivation in any solution comprising a virus or suspected to contain a virus.

[0045] "Impurities" refer to materials that are different from the desired polypeptide product The impurity includes, without limitation: host cell materials, such as host cell protein (HCP); leached Protein A; nucleic acid; a variant, size variant, fragment, aggregate or derivative of the desired polypeptide; another polypeptide; endotoxin; viral contaminant; cell culture media component, etc.

[0046] The term "chromatography" refers to any kind of technique which separates a protein of interest from other molecules (e.g., contaminants) present in a mixture, in which the protein of interest is separated from other molecules (e.g., contaminants) as a result of differencesin rates at which the individual molecules of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.

[0047] The term "aggregation" refers to the tendency of a polypeptide, e g., an antibody or a fusion protein, to form complexes with other molecules (such as other molecules of the same polypeptide) thereby forming high molecular weight (HMW) aggregates. Exemplary methods of measuring the formation of aggregates include analytical size exclusion chromatography as described in the Examples herein. Relative amounts of aggregation may be determined with respect to a reference compound, e.g., to identify a polypeptide having reduced aggregation. Relative amounts of aggregation can also be determined with respect to a reference formulation.

[0048] As used herein the terms "HMW" refers to any one or more unwanted proteins present in a mixture with a molecular weight generally higher than that of the desired protein of interest. High molecular weight proteins can include dimers, trimers, tetramers, or other multimers. These proteins can be either covalently or non-covalently linked, and can also, for example, consist of misfolded monomers in which hydrophobic amino acid residues are exposed to a polar solvent, and can cause aggregation.

[0049] The term "detergent" refers to an agent or combination thereof that may comprise salts of long-chain aliphatic bases or acids, or hydrophilic moi eties such as sugars, and that possess both hydrophilic and hydrophobic properties. Having both hydrophilic and hydrophobic properties, the detergent can exert particular effects. As used herein, detergents have the ability to disrupt viral envelopes and inactivate viruses.

[0050] A "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. In preferred aspects, the subject is a human. The terms, "subject" and "patient" are used interchangeably herein.

[0051] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis).

[0052] The use of the alternative (e.g. , "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0053] The terms "about" or "comprising essentially of refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of should be assumed to be within an acceptable error range for that particular value or composition.

[0054] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0055] Various aspects of the disclosure are described in further detail in the following subsections.II. Methods of the Disclosure

[0056] Some aspects of the present disclosure are directed to methods of inactivating a virus in a product feedstream, comprising contacting the product feedstream with an antifoam reagent. In some aspects, the product feedstream is in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent. As such, some aspects of the present disclosure are directed to methods of inactivating a virus in a product feedstream in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent. In some aspects, the therapeutic agent comprises a therapeutic protein, a viral particle, a nucleic acid molecule, or any combination thereof.

[0057] Some aspects of the present disclosure are directed to methods of inactivating a virus in a eukaryotic cell culture of a product feedstream, comprising contacting the cell culturewith an antifoam reagent. In some aspects, the eukaryotic cell culture comprises a mammalian cell or an insect cell. In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the eukaryotic cell is a human cell. In some aspects, the eukaryotic cell is an insect cell. In some aspects, the mammalian cell is selected from a CHO cell (e.g., CH0-K1 CHK DG44, or CHO DXB11), HEK cell, Namalwa cell, HeLa, cell, WI-28 cell, MRC-5 cell, HepG2 cell, MEF cell (e.g., 3T3), L-929 cell, myeloma cell, BHK-21 cell, COS cell (e.g., COS-7), and vero cell. In some aspects, the eukaryotic cell comprises a CHO cell. In some aspects, the eukaryotic cell comprises a HEK cell. In some aspects, the eukaryotic cell comprises a HEK293 cell.

[0058] In some aspects, the product feedstream is not contacted with Triton X-100.

[0059] In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a lipid-enveloped viruses. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a herpesviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a poxviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a hepadnaviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a RNA virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a flaviviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a togaviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a coronaviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a deltavirus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a orthomyxoviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a paramyxoviridae virus. In some aspects, the methods of viral inactivation disclosedherein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a rhabdoviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a bunyaviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a filoviridae virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a reverse transcribing virus. In some aspects, the methods of viral inactivation disclosed herein comprise contacting a feedstream or cell culture with an antifoam agent, as disclosed herein, to inactivate a retroviridae virus.ILA. Antifoam Reagents

[0060] Some aspects of the present disclosure are related to methods of inactivating a virus in a cell culture by contacting the cell culture with an antifoam reagent. In some aspects, the antifoam agent comprises an emulsifier. Any emulsifier can be used in the methods disclosed herein. In some aspects, the emulsifier is a nonionic emulsifier. Non-limiting examples of nonionic emulsifiers that can be used in the compositions and methods disclosed herein include hydrogenated glycerides, sorbitan esters (e.g., sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate), PEG esters, polysorbates (e.g., polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85). In some aspects, the emulsifier comprises one or more hydrogenated glyceride. In some aspects, the emulsifier comprises one or more PEG ester.

[0061] In some aspects, the antifoam reagent comprises at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% emulsifier. In some aspects, the antifoam reagent comprises at least about 1% emulsifier. In some aspects, the antifoam reagent comprises at least about 2% emulsifier. In some aspects, the antifoam reagent comprises at least about 3% emulsifier. In some aspects, the antifoam reagent comprises at least about 4% emulsifier. In some aspects, theantifoam reagent comprises at least about 5% emulsifier. In some aspects, the antifoam reagent comprises at least about 10% emulsifier. In some aspects, the antifoam reagent comprises at least about 15% emulsifier. In some aspects, the antifoam reagent comprises at least about 20% emulsifier. In some aspects, the antifoam reagent comprises at least about 25% emulsifier. In some aspects, the antifoam reagent comprises at least about 30% emulsifier. In some aspects, the antifoam reagent comprises at least about 25% emulsifier. In some aspects, the antifoam reagent comprises at least about 40% emulsifier. In some aspects, the antifoam reagent comprises at least about 45% emulsifier. In some aspects, the antifoam reagent comprises at least about 50% emulsifier. In some aspects, the antifoam reagent comprises at least about 55% emulsifier. In some aspects, the antifoam reagent comprises at least about 60% emulsifier. In some aspects, the antifoam reagent comprises at least about 65% emulsifier. In some aspects, the antifoam reagent comprises at least about 70% emulsifier. In some aspects, the antifoam reagent comprises at least about 75% emulsifier. In some aspects, the antifoam reagent comprises at least about 80% emulsifier. In some aspects, the antifoam reagent comprises at least about 85% emulsifier. In some aspects, the antifoam reagent comprises at least about 90% emulsifier. In some aspects, the antifoam reagent comprises at least about 95% emulsifier.

[0062] In some aspects, the antifoam reagent comprises at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 1% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 2% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 3% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 4% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 5% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 10% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 15% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 20% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 25% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 30% hydrogenated glyceride. In some aspects, the antifoam reagentcomprises at least about 35% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 40% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 45% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 50% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 55% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 60% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 65% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 70% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 75% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 80% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 85% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 90% hydrogenated glyceride. In some aspects, the antifoam reagent comprises at least about 95% hydrogenated glyceride.

[0063] In some aspects, the antifoam reagent comprises at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% PEG ester. In some aspects, the antifoam reagent comprises at least about 1% PEG ester. In some aspects, the antifoam reagent comprises at least about 2% PEG ester. In some aspects, the antifoam reagent comprises at least about 3% PEG ester. In some aspects, the antifoam reagent comprises at least about 4% PEG ester. In some aspects, the antifoam reagent comprises at least about 5% PEG ester. In some aspects, the antifoam reagent comprises at least about 10% PEG ester. In some aspects, the antifoam reagent comprises at least about 15% PEG ester. In some aspects, the antifoam reagent comprises at least about 20% PEG ester. In some aspects, the antifoam reagent comprises at least about 25% PEG ester. In some aspects, the antifoam reagent comprises at least about 30% PEG ester. In some aspects, the antifoam reagent comprises at least about 35% PEG ester. In some aspects, the antifoam reagent comprises at least about 40% PEG ester. In some aspects, the antifoam reagent comprises at least about 45% PEG ester. In some aspects, the antifoam reagent comprises at least about 50% PEG ester. In some aspects, the antifoam reagent comprises at least about 55% PEG ester. In some aspects, the antifoam reagent comprises at least about 60% PEG ester. In some aspects, theantifoam reagent comprises at least about 65% PEG ester. In some aspects, the antifoam reagent comprises at least about 70% PEG ester. In some aspects, the antifoam reagent comprises at least about 75% PEG ester. In some aspects, the antifoam reagent comprises at least about 80% PEG ester. In some aspects, the antifoam reagent comprises at least about 85% PEG ester. In some aspects, the antifoam reagent comprises at least about 90% PEG ester. In some aspects, the antifoam reagent comprises at least about 95% PEG ester.

[0064] In some aspects, the antifoam reagent further comprises a silicone polymer. Any silicone polymer can be used in the compositions and methods disclosed herein. In some aspects, the silicone polymer comprises a siloxane. In some aspects, the silicone polymer comprises polydimethylsiloxane (PDMS). In some aspects, the silicone polymer comprises simeticone (PDMS plus silicon dioxide.

[0065] In some aspects, the antifoam reagent comprises about 0.01% to about 50%, about 0.01% to about 45%, about 0.01% to about 40%, about 0.01% to about 35%, about 0.01% to about 30%, about 0.01% to about 25%, about 0.01% to about 20%, about 0.01% to about 15%, about 0.01% to about 10%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, or about 1% to about 10% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.01% to about 50% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 50% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 50% silicone polymer, e.g., PDMS.

[0066] In some aspects, the antifoam reagent comprises about 0.01% to about 40% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 40% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 40% silicone polymer, e.g., PDMS.

[0067] In some aspects, the antifoam reagent comprises about 0.01% to about 30% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 30% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 30% silicone polymer, e.g., PDMS.

[0068] In some aspects, the antifoam reagent comprises about 0.01% to about 25% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 25% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 25% silicone polymer, e.g., PDMS.

[0069] In some aspects, the antifoam reagent comprises about 0.01% to about 20% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 20% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 20% silicone polymer, e.g., PDMS.

[0070] In some aspects, the antifoam reagent comprises about 0.01% to about 15% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 15% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 15% silicone polymer, e.g., PDMS.

[0071] In some aspects, the antifoam reagent comprises about 0 01% to about 10% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 10% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 10% silicone polymer, e.g., PDMS.

[0072] In some aspects, the antifoam reagent comprises about 0.01% to about 5% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 5% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 1% to about 5% silicone polymer, e.g., PDMS.

[0073] In some aspects, the antifoam reagent comprises about 0.01% to about 1% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises about 0.1% to about 1% silicone polymer, e.g., PDMS.

[0074] In some aspects, the antifoam reagent comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% silicone polymer. In some aspects, the antifoam reagent comprises at least about 1% silicone polymer. In some aspects, the antifoam reagent comprises at least about 2% silicone polymer. In some aspects, the antifoam reagent comprises at least about 3% siliconepolymer. In some aspects, the antifoam reagent comprises at least about 4% silicone polymer. In some aspects, the antifoam reagent comprises at least about 5% silicone polymer. In some aspects, the antifoam reagent comprises at least about 6% silicone polymer. In some aspects, the antifoam reagent comprises at least about 7% silicone polymer. In some aspects, the antifoam reagent comprises at least about 8% silicone polymer. In some aspects, the antifoam reagent comprises at least about 9% silicone polymer. In some aspects, the antifoam reagent comprises at least about 10% silicone polymer. In some aspects, the antifoam reagent comprises at least about 11% silicone polymer. In some aspects, the antifoam reagent comprises at least about 12% silicone polymer. In some aspects, the antifoam reagent comprises at least about 13% silicone polymer. In some aspects, the antifoam reagent comprises at least about 14% silicone polymer. In some aspects, the antifoam reagent comprises at least about 15% silicone polymer. In some aspects, the antifoam reagent comprises at least about 16% silicone polymer. In some aspects, the antifoam reagent comprises at least about 17% silicone polymer. In some aspects, the antifoam reagent comprises at least about 18% silicone polymer. In some aspects, the antifoam reagent comprises at least about 19% silicone polymer. In some aspects, the antifoam reagent comprises at least about 20% silicone polymer. In some aspects, the antifoam reagent comprises at least about 25% silicone polymer. In some aspects, the antifoam reagent comprises at least about 30% silicone polymer. In some aspects, the antifoam reagent comprises at least about 35% silicone polymer. In some aspects, the antifoam reagent comprises at least about 40% silicone polymer. In some aspects, the antifoam reagent comprises at least about 45% silicone polymer. In some aspects, the antifoam reagent comprises at least about 50% silicone polymer. In some aspects, the antifoam reagent comprises at least about 55% silicone polymer. In some aspects, the antifoam reagent comprises at least about 60% silicone polymer. In some aspects, the antifoam reagent comprises at least about 65% silicone polymer. In some aspects, the antifoam reagent comprises at least about 70% silicone polymer. In some aspects, the antifoam reagent comprises at least about 75% silicone polymer. In some aspects, the antifoam reagent comprises at least about 80% silicone polymer. In some aspects, the antifoam reagent comprises at least about 85% silicone polymer. In some aspects, the antifoam reagent comprises at least about 90% silicone polymer. In some aspects, the antifoam reagent comprises at least about 95% silicone polymer.

[0075] In some aspects, the antifoam reagent comprises at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% PDMS. In some aspects, the antifoam reagent comprises at least about 1% PDMS. In some aspects, the antifoam reagent comprises at least about 2% PDMS. In some aspects, the antifoam reagent comprises at least about 3% PDMS. In some aspects, the antifoam reagent comprises at least about 4% PDMS. In some aspects, the antifoam reagent comprises at least about 5% PDMS. In some aspects, the antifoam reagent comprises at least about 6% PDMS. In some aspects, the antifoam reagent comprises at least about 7% PDMS. In some aspects, the antifoam reagent comprises at least about 8% PDMS. In some aspects, the antifoam reagent comprises at least about 9% PDMS. In some aspects, the antifoam reagent comprises at least about 10% PDMS. In some aspects, the antifoam reagent comprises at least about 11% PDMS. In some aspects, the antifoam reagent comprises at least about 12% PDMS. In some aspects, the antifoam reagent comprises at least about 13% PDMS. In some aspects, the antifoam reagent comprises at least about 14% PDMS. In some aspects, the antifoam reagent comprises at least about 15% PDMS. In some aspects, the antifoam reagent comprises at least about 16% PDMS. In some aspects, the antifoam reagent comprises at least about 17% PDMS. In some aspects, the antifoam reagent comprises at least about 18% PDMS. In some aspects, the antifoam reagent comprises at least about 19% PDMS. In some aspects, the antifoam reagent comprises at least about 20% PDMS. In some aspects, the antifoam reagent comprises at least about 25% PDMS. In some aspects, the antifoam reagent comprises at least about 30% PDMS. In some aspects, the antifoam reagent comprises at least about 35% PDMS. In some aspects, the antifoam reagent comprises at least about 40% PDMS. In some aspects, the antifoam reagent comprises at least about 45% PDMS. In some aspects, the antifoam reagent comprises at least about 50% PDMS. In some aspects, the antifoam reagent comprises at least about 55% PDMS. In some aspects, the antifoam reagent comprises at least about 60% PDMS. In some aspects, the antifoam reagent comprises at least about 65% PDMS. In some aspects, the antifoam reagent comprises at least about 70% PDMS. In some aspects, the antifoam reagent comprises at least about 75% PDMS. In some aspects, the antifoam reagent comprises at least about 80% PDMS. In some aspects, the antifoam reagent comprises at least about 85% PDMS. In some aspects, the antifoam reagent comprises at least about 90% PDMS. In some aspects, the antifoam reagent comprises at least about 95% PDMS.

[0076] In some aspects, the antifoam reagent comprises at least 0.1% and less than 100% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 30% silicone polymer, e. ., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 29% silicone polymer, e. ., PDMS. In some aspects, the antifoamreagent comprises at least 0.1% and less than about 28% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 27% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 26% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 25% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 24% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 23% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 22% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 21% silicone polymer, e. ., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 20% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 19% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 18% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 17% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 16% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 15% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 14% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 13% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 12% silicone polymer, e.g, PDMS. In some aspects, the antifoam reagent comprises at least 0.1% and less than about 11% silicone polymer, e.g., PDMS. In some aspects, the antifoam reagent comprises at least 0. 1% and less than about 10% silicone polymer, e.g., PDMS.

[0077] In some aspects, the antifoam reagent is selected from ANTIFOAM B™, antifoam SE-15, or antifoam L-30.

[0078] In some aspects, the antifoam reagent comprises one or more organic non-silicone polypropylene-based polyether. In some aspects, the antifoam agent comprises antifoam 204.

[0079] In some aspects, the antifoam reagent comprises one or more difunctional block copolymer surfactant. In some aspects, the difunctional block copolymer surfactant comprises terminal secondary hydroxyl groups. In some specs, the difunctional block copolymer surfactant comprises a PLURONIC. In some aspects, the difunctional block copolymer surfactant comprises PLURONIC® 31R1.

[0080] In some aspects, the antifoam reagent comprises ANTIFOAM B™ (SIGNMA- ALDRICH). ANTIFOAM B™ is silicone-based antifoam, marketed as a 10% aqueous emulsion of PDMS. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, at least about 0.01% to about 10%, at least about 0.01% to about 5%, or at least about 0.01% to about 1% ANTIFOAM B™.

[0081] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0. 1% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 50%, at least about 0.1% to about 45%, at least about 0.1% to about 40%, at least about 0.1% to about 35%, at least about 0.1% to about 30%, at least about 0. 1% to about 25%, at least about 0.1% to about 20%, at least about 0.1% to about 15%, at least about 0.1% to about 10%, at least about 0.1% to about 5%, or at least about 0. 1% to about 1% ANTIFOAM B™.

[0082] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 50%, at least about 1% to about 45%, at least about 1% to about 40%, at least about 1% to about 35%, at least about 1% to about 30%, at least about 1% to about 25%, at least about 1% to about 20%, at least about 1% to about 15%, at least about 1% to about 10%, or at least about 1% to about 5% ANTIFOAM B™.

[0083] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 50% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 150% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 50% ANTIFOAM B™.

[0084] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 40% ANTIFOAM B™. In some aspects, the productfeedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 40% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 40% ANTIFOAM B™.

[0085] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 30% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 30% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 30% ANTIFOAM B™.

[0086] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 20% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 20% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 20% ANTIFOAM B™.

[0087] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 10% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1% to about 10% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 1% to about 10% ANTIFOAM B™.

[0088] In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 0.01% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 0.1% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 1% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration ofabout 2% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 3% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 4% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 5% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 6% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 7% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 8% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 9% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 10% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 11% ANTIFOAM B™ In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 12% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 13% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 14% ANTIFOAM B™ In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 15% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 16% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 17% ANTIFOAM B™ In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 18% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 19% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 20% ANTIFOAM B™ In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 25% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 30% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 35% ANTIFOAM B™ In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 40% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 45% ANTIFOAM B™. In some aspects, the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 50% ANTIFOAM B™.ILA.2. Antifoam SE-15

[0089] In some aspects, the antifoam reagent comprises antifoam SE-15 (SIGMA- ALDRICH). Antifoam SE-15 is silicone-based antifoam, marketed as an aqueous emulsion containing 10% active silicon and non-ionic emulsifiers. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, at least about 0.01% to about 10%, at least about 0.01% to about 5%, or at least about 0.01% to about 1% antifoam SE-15.

[0090] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 50%, at least about 0.1% to about 45%, at least about 0.1% to about 40%, at least about 0.1% to about 35%, at least about 0.1% to about 30%, at least about 0. 1% to about 25%, at least about 0.1% to about 20%, at least about 0.1% to about 15%, at least about 0.1% to about 10%, at least about 0.1% to about 5%, or at least about 0.1% to about 1% antifoam SE-15.

[0091] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 50%, at least about 1% to about 45%, at least about 1% to about 40%, at least about 1% to about 35%, at least about 1% to about 30%, at least about 1% to about 25%, at least about 1% to about 20%, at least about 1% to about 15%, at least about 1% to about 10%, or at least about 1% to about 5% antifoam SE- 15.

[0092] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 50% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 150% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 50% antifoam SE-15.

[0093] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 40% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 40% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 40% antifoam SE-15.

[0094] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 30% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 30% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 30% antifoam SE-15.

[0095] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 20% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 20% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 20% antifoam SE-15.

[0096] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 10% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.1% to about 10% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 1% to about 10% antifoam SE-15.

[0097] In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 0.01% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 0.1% antifoam SE-15. In some aspects, the product feedstream iscontacted with antifoam SE-15 at a concentration of about 1% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 2% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 3% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 4% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 5% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 6% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 7% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 8% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 9% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 10% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 11% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 12% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 13% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 14% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 15% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 16% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 17% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 18% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 19% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 20% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 25% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 30% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 35% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 40% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 45% antifoam SE-15. In some aspects, the product feedstream is contacted with antifoam SE-15 at a concentration of about 50% antifoam SE-15.ILA.3. Antifoam L-30

[0098] In some aspects, the antifoam reagent comprises antifoam L-30 (SIGMA- ALDRICH). Antifoam L-30 is silicone-based antifoam, marketed as a 30% aqueous emulsion. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, at least about 0.01% to about 10%, at least about 0 01% to about 5%, or at least about 0.01% to about 1% antifoam L-30.

[0099] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.1% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0. 1% to about 50%, at least about 0.1% to about 45%, at least about 0.1% to about 40%, at least about 0.1% to about 35%, at least about 0.1% to about 30%, at least about 0.1% to about 25%, at least about 0.1% to about 20%, at least about 0.1% to about 15%, at least about 0. 1% to about 10%, at least about 0. 1% to about 5%, or at least about 0.1% to about 1% antifoam L-30.

[0100] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 50%, at least about1% to about 45%, at least about 1% to about 40%, at least about 1% to about 35%, at least about1% to about 30%, at least about 1% to about 25%, at least about 1% to about 20%, at least about1% to about 15%, at least about 1% to about 10%, or at least about 1% to about 5% antifoam L-30.

[0101] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 50% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0. 1% to about 150% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 50% antifoam L-30.

[0102] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 40% antifoam L-30. In some aspects, the productfeedstream is contacted with antifoam L-30 at a concentration of at least about 0.1% to about 40% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 40% antifoam L-30.

[0103] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 30% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.1% to about 30% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 30% antifoam L-30.

[0104] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 20% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.1% to about 20% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 20% antifoam L-30.

[0105] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 10% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.1% to about 10% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of at least about 1% to about 10% antifoam L-30.

[0106] In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 0.01% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 0.1% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 1% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 2% antifoam L-30. In someaspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 3% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 4% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 5% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 6% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 7% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 8% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 9% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 10% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 11% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 12% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 13% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 14% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 15% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 16% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 17% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 18% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 19% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 20% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 25% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 30% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 35% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 40% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 45% antifoam L-30. In some aspects, the product feedstream is contacted with antifoam L-30 at a concentration of about 50% antifoam L-30.ILA.4. Antifoam 204

[0107] In some aspects, the antifoam reagent comprises antifoam 204 (SIGMA- ALDRICH). Antifoam 204 is marketed as a mixture of organic non-silicone polypropylene-based polyether dispersions, which does not contain mineral oil, which does not contain other surfactants, and which is synthetic and not derived from animal or plant sources. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, at least about 0.01% to about 10%, at least about 0.01% to about 5%, or at least about 0.01% to about 1% antifoam 204.

[0108] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 50%, at least about 0.1% to about 45%, at least about 0.1% to about 40%, at least about 0.1% to about 35%, at least about 0.1% to about 30%, at least about 0.1% to about 25%, at least about 0.1% to about 20%, at least about 0.1% to about 15%, at least about 0.1% to about 10%, at least about 0.1% to about 5%, or at least about 0.1% to about 1% antifoam 204.

[0109] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 50%, at least about 1% to about 45%, at least about 1% to about 40%, at least about 1% to about 35%, at least about 1% to about 30%, at least about 1% to about 25%, at least about 1% to about 20%, at least about 1% to about 15%, at least about 1% to about 10%, or at least about 1% to about 5% antifoam 204.

[0110] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 50% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 150% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 50% antifoam 204.

[0111] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 40% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 40% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 40% antifoam 204.

[0112] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 30% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 30% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 30% antifoam 204.

[0113] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 20% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 20% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 20% antifoam 204.

[0114] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 10% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.1% to about 10% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of at least about 1% to about 10% antifoam 204.

[0115] In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 0.01% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 0.1% antifoam 204. In some aspects, the product feedstream is contactedwith antifoam 204 at a concentration of about 1% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 2% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 3% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 4% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 5% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 6% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 7% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 8% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 9% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 10% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 11% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 12% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 13% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 14% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 15% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 16% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 17% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 18% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 19% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 20% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 25% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 30% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 35% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 40% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 45% antifoam 204. In some aspects, the product feedstream is contacted with antifoam 204 at a concentration of about 50% antifoam 204.ILA.5. PLURONIC® 31R1

[0116] In some aspects, the antifoam reagent comprises PLURONIC® 31R1 (BASF). PLURONIC® 31R1 is marketed as an ethylene oxide and propylene oxide reverse block copolymer. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of atleast about 0.01% PLURONIC® 31 Rl . In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, at least about 0.01% to about 10%, at least about 0.01% to about 5%, or at least about 0.01% to about 1% PLURONIC® 31R1.

[0117] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0. 1% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 50%, at least about 0.1% to about 45%, at least about 0. 1% to about 40%, at least about 0.1% to about 35%, at least about 0.1% to about 30%, at least about 0.1% to about 25%, at least about 0.1% to about 20%, at least about 0.1% to about 15%, at least about 0.1% to about 10%, at least about 0.1% to about 5%, or at least about 0.1% to about 1% PLURONIC® 31R1.

[0118] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 50%, at least about 1% to about 45%, at least about 1% to about 40%, at least about 1% to about 35%, at least about 1% to about 30%, at least about 1% to about 25%, at least about 1% to about 20%, at least about 1% to about 15%, at least about 1% to about 10%, or at least about 1% to about 5% PLURONIC® 31R1.

[0119] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 50% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 150% PLURONIC® 31RL In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 50% PLURONIC® 31R1 .

[0120] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 40% PLURONIC® 31R1. In some aspects, theproduct feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 40% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 40% PLURONIC® 31R1.

[0121] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 30% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 30% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 30% PLURONIC® 31R1.

[0122] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 20% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 20% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 20% PLURONIC® 31R1.

[0123] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 10% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1% to about 10% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 1% to about 10% PLURONIC® 31R1.

[0124] In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 0.01% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 0.1% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 1% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC®31R1 at a concentration of about 2% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 3% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 4% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 5% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 6% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 7% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 8% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 9% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 10% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 11% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 12% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 13% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 14% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 15% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 16% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 17% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 18% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 19% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 20% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 25% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 30% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 35% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 40% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about45% PLURONIC® 31R1. In some aspects, the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 50% PLURONIC® 31R1. n.B. Cell Culture and Product Feedstream

[0125] In some aspects, the product feedstream comprises a harvest (e.g., from a bioreactor), a load (e.g., the load of a chromatography or filtration column or other filtration device), an eluate (e.g., a chromatography eluate), a filtrate, or a combination thereof. In general, the methods and compositions disclosed herein can be used to inactivate viruses in any solution containing viruses, suspected of containing viruses, or susceptible of containing viruses. In some aspects, the feedstream is a harvested cell culture fluid. In some aspects, the feedstream comprises a capture pool or a recovered product pool. In some aspects, the capture pool or recovered product pool is a chromatography pool. In some aspects, the capture pool or recovered product pool is an affinity chromatography pool. In some aspects, the capture pool or recovered product pool is a protein A pool, a protein G pool or a protein L pool. In some aspects, the product feedstream is a Protein A chromatography column eluate. In some aspects, the product feedstream is a filtrate, e g., from a filtering step in a downstream purification process. In some aspects, the product feedstream is a load, e g., the load of a chromatography column or a filtration system.

[0126] In some aspects, the present disclosure provides a method of inactivating virus in a product feedstream in a manufacturing process of a therapeutic protein using an antifoam reagent disclosed herein, wherein the feedstream (e g., a harvest, load, eluate, or filtrate) is subject to chromatography after addition of the antifoam reagent. In some aspects, the chromatography is one or more of one or more of an affinity chromatography, an ion exchange chromatography (e.g., cation exchange and / or anion exchange), a hydrophobic interaction chromatography, a hydroxyapatite chromatography, or a mixed mode chromatography.

[0127] Examples of affinity chromatography materials include, but are not limited to chromatography materials derivatized with protein A or protein G. Examples of affinity chromatography material include, but are not limited to, Prosep-VA, Prosep-VA Ultra Plus, Protein A sepharose fast flow, Tyopearl Protein A. MAbSelect, MAbSelect SuRe and MAbSelect SuRe LX. In some aspects, the affinity chromatography material is an affinity chromatography column. In some aspects, the affinity chromatography material is an affinity chromatography membrane. Examples of anion exchange chromatography materials include, but are not limited to Poros HQ 50, Poros PI 50, Poros D, Mustang Q, Q Sepharose FF, and DEAE Sepharose. Examples of cation exchange materials include, but are not limited to Mustang S, Sartobind S, SO3 Monolith, SCeramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Sepharose XL (SPXL), CM Sepharose Fast Flow, Capto S, Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. Examples of HIC chromatography materials include, but are not limited to, Toyopearl hexyl 650, Toyopear butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source, Resource, Sepharose Hi-Trap, Octyl sepharose, phenyl sepharose. Examples of hydroxyapatite chromatography material include but are limited to HA Ultrogel, and CHT hydroxyapatite. Examples of mixed mode chromatography materials include, but are not limited to Capto Adhere, QMA, MEP Hypercel, HEA Hypercel, PPA Hypercel, Capto MMC.

[0128] Lipid-enveloped viruses that can infect mammalian cells include DNA viruses like a herpesviridae virus, a poxviridae virus, or a hepadnaviridae virus; RNA viruses like a flaviviridae virus, a togaviridae virus, a coronaviridae virus, a deltavirus virus, an orthomyxoviridae virus, a paramyxoviridae virus, a rhabdoviridae virus, a bunyaviridae virus, or a filoviridae virus; and reverse transcribing viruses like a retroviridae virus or a hepadnaviridae virus. Non-limiting examples of lipid-enveloped viruses include a human immunodeficiency virus, a sindbis virus, a herpes simplex virus, a pseudorabies virus, a sendai virus, a vesicular stomatitis virus, a West Nile virus, a bovine viral diarrhea virus, a corona virus, an equine arthritis virus, a severe acute respiratory syndrome virus, Moloney murine leukemia virus, or a vaccinia virus. In some aspects, the lipid-enveloped virus is selected from the group consisting of retroviruses, flaviviruses, orthomyxoviruses, herpes viruses, paramyxoviruses, arena viruses, poxviruses, hepadnaviruses, hepatitis viruses, rhabdoviruses, and togavirus. In some aspect, the virus comprises a lipid-enveloped virus, e.g., a retrovirus such as A-MuLV, a herpesvirus such asHSV- 1.

[0129] Virus inactivation can be quantitated using log reduction value (LRV). Thus, in some aspects, the methods of inactivating a virus in a product feedstream (e.g., a harvest, load, eluate, or filtrate) disclosed herein comprise determining a log reduction value (LRV) of the number of virus in the feedstream or virus-containing solution. In some aspects, the LRV value is at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10. In some aspects, LRV is calculated according to the following formula:

[0130] The term “pfu” or “plaque-forming unit” is a measure used in virology to describe the number of virus particles capable of forming plaques per unit volume. In some aspects, the LRV is at least about 4. In some aspects, the LRV is between about 3 and about 4, between about 4 and about 5, between about 5 and about 6, between about 6 and about 7, between about 7 and about 8, between about 8 and about 9, between about 9 and about 10, between about 3 and about 5, between about 4 and about 6, between about 5 and about 7, between about 6 and about 8, between about 7 and about 9, between about 8 and about 10, between about 3 and about 6, between about 4 and about 7, between about 5 and about 8, between about 6 and about 9, between about 7 and about 10, between about 3 and about 7, between about 4 and about 8, between 5 and about 9, between 6 and about 10, between about 3 and about 8, between 4 and about 9, or between 5 and about 10.

[0131] Detecting a viable lipid-coat containing virus can be accomplished by any technique that can qualitatively or quantitatively measure the presence or activity of a viable lipid-coat containing virus. Typically, a cell-culture based assay is used to determine titer levels of a virus, but in vivo infectivity assays can also be employed. Detection of virus amplification may be done, e.g., by microscopic examination (in case of a clearly visible cytopathogenic effect), a PCR-based detection assay, or an antibody -based detection assay. Thus, in some aspects, the LRV is calculated based on an infectivity assay.

[0132] One non-limiting example is an in vitro infectivity assay called the Tissue Culture Infectious Dose 50 (TCID50) assay. In this assay, fluid samples and serial dilutions thereof are dispensed into 96-well plates seeded with cells that can serve as hosts for the lipid-coat containing virus being assayed. After inoculation, the plates are incubated at a time and temperature sufficient to allow the virus to replicate in the host cells. After incubation, the cells are examined by microscope for signs of infection, such as, e g., lysed cells, cells exhibiting a cytopathogenic effect, or any other criteria indicative of viral infection. From the pattern of positive (viral infection) and negative (no viral infection) wells the virus titer is calculated. The absence of any wells showing positive signs of infection is indicative of a fluid that is essentially free of a lipid-coat containing virus. Accordingly, in some aspects, the infectivity assay used to calculate LRV is a TCID50 assay.

[0133] Another cell-culture based assay is a plaque assay, where virus-induced effects in the cell culture layer are visible or made visible macroscopically as plaques. The absence of any plaques is indicative of a fluid that is essentially free of a lipid-coat containing virus. In some aspects, the infectivity assay used to calculate LRV is a plaque assay.

[0134] In some aspects, the contacting of the antifoam reagent disclosed herein with the product feedstream (e.g., a harvest, load, eluate, or filtrate) or any virus-containing solution or suspected to contain a virus occurs for at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, or at least about 120 minutes. In some aspects, the contacting of the antifoam reagent disclosed herein with the product feedstream or virus- containing solution or suspected to contain a virus occurs for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes. In some aspects, the contacting of the antifoam reagent disclosed herein with the product feedstream or virus-containing solution occurs for between about 10 minutes and about 20 minutes, between about 20 minutes and about 30 minutes, between about 30 minutes and about 40 minutes, between about 40 minutes and about 50 minutes, between about 50 minutes and about 60 minutes, between about 60 minutes and about 70 minutes, between about 70 minutes and about 80 minutes, between about 80 minutes and about 90 minutes, between about 90 minutes and about 100 minutes, between about 100 minutes and about 110 minutes, between about 110 minutes about 120 minutes, between about 15 minutes and about 30 minutes, between about 30 minutes and about 45 minutes, between about 45 minutes and about 60 minutes, between about 60 minutes and about 75 minutes, between about 75 minutes and about 90 minutes, between about 90 minutes and about 105 minutes, between about 105 minutes and about 120 minutes, between about 30 minutes about 60 minutes, between about 60 minutes and about 90 minutes, between about 90 and about 120 minutes, or between about 60 minutes and about 120 minutes. In some aspects, the feedstream can be subjected to a antifoam reagent disclosed herein for more than 2 hours, e.g., 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 16 hours, 20 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

[0135] In some aspects, the feedstream (e.g., a harvest, load, eluate, or filtrate) is contacted with an antifoam reagent of the present disclosure at about 4° C. to about 30° C. In some aspects, the feedstream is contacted with the antifoam reagent of the present disclosure at about 10° C. to about 25° C. In some aspects, the feedstream is contacted with the antifoam reagent of the present disclosure at about 15° C. to about 20° C. In some aspects, the feedstream is contacted with the antifoam reagent of the present disclosure at about 20° C. In some aspects, the feedstream is contacted with the antifoam reagent of the present disclosure at about ambient temperature. Insome aspects, the feedstream is contacted with the antifoam reagent of the present disclosure at about 4° C , 5° C., 10° C., 15° C., 20° C., 25° C., or 30° C.

[0136] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase in the amount of protein aggregates (high molecular weight species) beyond the acceptable protein aggregation parameters for total protein in the product feedstream; for example, compared to the manufacturing process without an antifoam agent or a detergent, or with Triton X- 100.

[0137] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in a change in glycosylation amount beyond the acceptable glycosylation parameters for total protein in the product feedstream; for example, compared to the manufacturing process without an antifoam agent or a detergent, or with Triton X-100. In some aspects, treatment of the feedstream in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in a change in glycosylation pattern; for example, compared to the manufacturing process without an antifoam agent or a detergent, or with Triton X-100.

[0138] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase in the deamidation of the therapeutic protein product beyond the acceptable protein deamidation parameters for total protein in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or a detergent, or with Triton X-100. Deamidation products include proteins where one or more glutamine and / or asparagine residues have been deamidated. In some aspects, deamidation of a product therapeutic protein results in a change in the charge of the polypeptide. Methods to analyze therapeutic proteins for deamidated variants are known in the art. For example, by pH- mediated ion exchange chromatography or isoelectric focusing.

[0139] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase in the oxidation of the therapeutic protein product beyond the acceptable protein oxidation parameters for total protein in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or adetergent, or with Triton X-100. Oxidation products include proteins where one or more oxygenreactive amino acid residues, such as methionine, cysteine and tyrosine, have been oxidized. In some aspects, oxidation of a product polypeptide results in a change in the charge of the polypeptide. Methods to analyze polypeptides for oxidized variants are known in the art. For example, levels of oxidation in a given polypeptide may be determined by LC-mass spectroscopy.

[0140] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase in process impurities beyond the acceptable protein impurity parameters for total protein in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or a detergent, or with Triton X-100. Thus, in some aspects, treatment of the feedstream in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not alter the clearance of process impurities during the manufacturing process.

[0141] For example, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) with an antifoam reagent disclosed herein does not alter clearance of impurities in the manufacturing process compared to a manufacturing process of the therapeutic protein using Triton X-100 to inactivate virus or a manufacturing process of the therapeutic protein that does not use an antifoam reagent or a detergent,. In some aspects, the use of the antifoam reagents disclosed herein does not alter the clearance of process impurities in a particular step in the manufacturing process, such as a chromatography step, a filtration step, a concentration step and the like In some aspects, the use of the antifoam reagents of the present disclosure does not alter the clearance of process impurities in the overall in the manufacturing process of the therapeutic protein. Process impurities include host cell proteins (HCP), nucleic acids, leached protein A, polypeptides other than the desired polypeptide, endotoxin, viral contaminant, cell culture media component, and variants, fragments, aggregates or derivatives of the desired therapeutic protein.

[0142] In some aspects, treatment of the feedstream (e.g., a harvest, load, eluate, or filtrate) in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase in HCP beyond the acceptable protein HCP levels in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or a detergent, or with Triton X-100.

[0143] In some aspects, treatment of the feedstream in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstreamdoes not result in an increase of residual DNA level beyond the acceptable HCP level in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or a detergent, or with Triton X-100.

[0144] In some aspects, treatment of the feedstream in a manufacturing process of a therapeutic protein with an antifoam reagent disclosed herein to inactivate virus in the feedstream does not result in an increase of residual Protein A level beyond the acceptable Protein A level in the product feedstream; for example, compared to the manufacturing process without an antifoam reagent or a detergent, or with Triton X-100. n.C. Therapeutic Proteins

[0145] As disclosed above, in some aspects, the therapeutic proteins that can be prepared according to the viral inactivation methods disclosed herein comprise, for example, antibodies, antibody fragments, Fc portions of antibodies and fusions thereof, antigen binding portions of antibodies, fusion proteins, naturally occurring proteins, recombinant proteins, chimeric proteins, immunoadhesins, enzymes, growth factors, receptors, hormones, regulatory factors, cytokines, or any combination thereof. In some aspects, the therapeutic protein is produced in mammalian cells. In some aspects, the mammalian cell line is a Chinese Hamster Ovary (CHO) cells, or baby hamster kidney (BHK) cells, murine hybridoma cells, or murine myeloma cells. Manufacturing processes of therapeutic proteins using an antifoam reagent disclosed herein do not adversely affect product quality of the therapeutic protein compared to corresponding processes using, e.g., TritonX-100.

[0146] Any therapeutic protein that is expressible in a host cell can be produced in accordance with the present disclosure and may be present in the compositions provided. The therapeutic protein can be expressed from a gene that is endogenous to the host cell, or from a gene that is introduced into the host cell through genetic engineering.

[0147] The methods and compositions provided can employ any cell that is suitable for growth and / or production of a therapeutic protein in a culture medium, including animal, yeast or insect cells. In one aspect, the cell is any mammalian cell or cell type suitable to cell culture and to expression of polypeptides. The methods provided herein (e.g., methods of inactivating virus) and compositions can therefore employ any suitable type of cell, including an animal cell. In one aspect, the methods and compositions employ a mammalian cell. The methods and compositions can also employ hybridoma cells. In one aspect, the mammalian cell is a non-hybridoma mammalian cell, which has been transformed with exogenous isolated nucleic acid encoding a desired therapeuticprotein. In one aspect, the methods and compositions employ mammalian cells selected from the group consisting of human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 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); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); 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-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some aspects, the methods and compositions employ CHO cells. In some aspects, the culturing of CHO cell lines and expression of therapeutic proteins from CHO cell lines is employed. The therapeutic protein can be secreted into the culture medium from which the therapeutic protein can be isolated and / or purified or the therapeutic protein can be released into the culture medium by lysis of a cell comprising an isolated nucleic acid encoding the therapeutic protein. n.D. Methods of Manufacture and Methods of Treatment

[0148] Some aspects of the present disclosure are directed to a method to treat a disease or condition comprising administering to a subject a therapeutic protein manufactured by a process comprising a viral inactivation step according to the viral inactivation methods disclosed herein, e.g., a viral inactivation method comprising the use of an antifoam reagent of the present disclosure. Also provided is a pharmaceutical composition manufactured by a process comprising a viral inactivation step according to the viral inactivation methods disclosed herein, e.g., a viral inactivation method comprising the use of an antifoam reagent of the present disclosure. The present disclosure also provides a method of manufacture a therapeutic protein comprising a viral inactivation step according to the viral inactivation methods disclosed herein, e.g., a viral inactivation method comprising the use of an antifoam reagent of the present disclosure. n.E. Kits and Products of Manufacture

[0149] Some aspects of the present disclosure are directed to a kit or product manufacture comprising an antifoam reagent disclosed herein, in one or multiple containers and optionally instructions for inactivating a virus according to the methods disclosed herein. A person of ordinaryskill in the art would readily recognize that an antifoam reagent of the present disclosure can be readily incorporated into one of the established kit formats which are well known in the art. In some aspects, the kit or product manufacture comprising instructions for viral inactivation according to the methods of the present disclosure.

[0150] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0151] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1

[0152] Adventitious viruses remain a key concern in the production of biologies drugs. To ensure patient safety, the downstream purification process requires a robust viral clearance strategy to ensure the process can remove potential viral contamination. For enveloped viruses, viral inactivation processes use either low pH conditions or chemical inactivation agents as a key component of the removal strategy. For biologic drugs with poor stability at low pH conditions, chemical inactivation agents provide the necessary viral inactivation without stability concerns. Historically, the non-ionic surfactant, Triton X-100, provided excellent viral inactivation. However, due to safety concerns with the degradation products of Triton X-100, the European Chemicals Agency (ECHA) has mandated the replacement of Triton X-100. To aid in the evaluation of new viral inactivation reagents, a new method using Retrovirus-like Particles (RVLP) was developed to determine viral log reduction values (LRVs) for inactivation reagents. RVLPs are CHO endogenously produced, non-infectious, and membrane-bound viral particles. They have comparable properties to infectious, membrane-bound viral particles traditionally used in infectivity studies to validate viral inactivation conditions. The present method was validated against two of the industry standards of these viruses (A-MulV and X-MulV), which showed high comparability (FIGs. 1A-1B). Based on the physiological properties of RVLP compared to other relevant membrane bound viruses and the comparability with model viruses, RVLP was expected to act as a good model for membrane bound viruses as a whole.

[0153] Upon validation, the present method was used as a platform to investigate new candidates for viral inactivation reagents. The antifoam chemicals were identified as a potentially advantageous class of molecules for use in viral inactivation. Antifoam reagents currently havebroad use in the biopharmaceutical industry to prevent unwanted foam production in bioreactors and fermenters. Some antifoams also have lubricating properties that reduce sheer forces on cells introduced from aeration and agitation conditions. Often, antifoam chemicals used in cell culture contain either an emulsion of polydimethylsiloxane (PDMS) or nonionic detergents. The variation in the antifoam arises from the emulsifier or detergent used. As antifoam chemicals are already used in biologies manufacturing, they have established supply chains, regulatory familiarity, and known clearance and detection strategies. Interestingly, they also offer the possibility to use the same product in both cell culture and viral inactivation. The use of Antifoam A, Antifoam B emulsion, Antifoam C Emulsion, Pluronic F-68 Non-ionic Surfactant, Pluronic 31R1, Antifoam SE-15, Antifoam L-30 Emulsion, and Antifoam 204 were investigated (Table 1). Based on our knowledge, the present study is the first to investigate the use of antifoam reagents for viral inactivation.

[0154] Materials and Methods

[0155] RVLP Based Inactivation Testing Method

[0156] An internal method was developed for characterizing viral inactivation of enveloped viruses using the Retrovirus-like Particle (RVLP) (FIG. 2). This method was developed by adapting our previous work with the Phi6 bacteriophage (Feroz, H., et al., Surrogate model to screen for inactivation - based clearance of enveloped viruses during biotherapeutics process development. Biotechnology loumal, 2021. 16(12): p. 2100176.). This method allows differentiation between compromised and intact virus particles.

[0157] A 1% solution of RVLP viral particles is spiked into clarified cell culture harvest of either monoclonal antibody (mAb) or fusion protein. The inactivation reagent or detergent of interest, Proteinase K at a final concentration of 10.8 mAU / mL, and PMAxx at a final concentration of 0.24 mM are added to each sample. For the control sample, the inactivation reagent or detergent of interest is omitted and replaced instead with water. The solution is gently mixed and incubated at room temperature under LED light illumination at 465-475 nm for the required incubation period. Proteinase K degrades viral particles with compromised membranes and PMAxx intercalates with free RNA and with light exposure covalently binds to the free RNA, rendering it unable to participate in RT-qPCR. After the incubation period, the MockV RNA Extraction Kit is used to extract the viral RNA. The RNA extraction is performed following an RNA precipitation reaction using the provided kit materials and method (MockV RVLP Kit Guide. 2023, CygnusTechnologies.). Additionally, the kit provides an RNA standard that is serially diluted eight logs and is extracted alongside the samples of interest allowing for the quantifiable range to be determined. Following RNA extraction, RT-qPCR is performed using the MockV RVLP qPCR kit following the provided method from the kit using a FAM reporter MockV RVLP Kit Guide. 2023, Cygnus Technologies ). Using the standard curve, the concentration of RNA is determined for the inactivation reagent treated or untreated control.

[0158] The log reduction value (LRV) is then calculated using the following equation(Equation 1).Equation 1.

[0159] Antifoam Testing

[0160] A set of antifoam chemicals were tested for their viral inactivation capacity. The antifoams tested and their manufacturer are listed (Table 1)Table 1. Antifoams tested for their viral inactivation capability.

[0161] Results

[0162] Antifoams were successfully screened for viral inactivation capability. A broad panel of antifoams were tested (Table 1) and were compared against known viral inactivating chemicals for number of LRVs (FIG. 3A). Antifoam B and Antifoam 204 show comparable performance to known detergents with 4.71 and 3.98 LRVs respectively, while Antifoam SE-15, Antifoam L-30, and Pluronic 31R1 demonstrated nearly 2 logs of clearance. Antifoam A, Antifoam C, and Pluronic F-68 showed no appreciable viral inactivation. Antifoam A, B, C, and 204 weretested as well for concentration dependence on viral inactivation capability (FIG. 3B). Tested antifoam conditions and concentrations along with both raw RT-qPCR results and LRVs calculated are recorded (Table 2). Based on the results, Antifoam B’s viral inactivation capability is likely not from PDMS alone. Antifoam A, which is composed entirely of PDMS but contains no emulsifiers, did not perform well. Likewise, the poor performance of Antifoam C likely results from the difference in emulsifiers used to suspend the PDMS. SE-15 and L-30 possibly have a similar mechanism to Antifoam B. Antifoam 204 is an organic, non-silicone polypropylene-based polyether dispersion. The surfactant qualities of Antifoam 204 are likely the mechanism by which the antifoam acts a viral inactivation agent. Pluronic 31R1 likewise is a non-silicone ethylene oxide and propylene oxide reverse block copolymer.Table 2. Experimental results from antifoam experiments.* *

[0163] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0164] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0165] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0166] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0167] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

Claims

WHAT IS CLAIMED IS:

1. A method of inactivating a virus in a product feedstream in a downstream manufacturing process of a therapeutic agent, comprising contacting the product feedstream with an antifoam reagent.

2. The method of claim 1, wherein the therapeutic agent comprises a therapeutic protein, a viral particle, a nucleic acid molecule, or any combination thereof.

3. A method of inactivating a virus in a eukaryotic cell culture of a product feedstream, comprising contacting the cell culture with an antifoam reagent.

4. The method of claim 3, wherein the eukaryotic cell culture comprises a mammalian cell or an insect cell.

5. The method of claim 4, wherein the mammalian cell is selected from a CHO cell and a HEK cell.

6. The method of any one of claims 1 to 5, wherein the antifoam reagent comprises an emulsifier.

7. The method of claim 6, wherein the emulsifier is a nonionic emulsifier.

8. The method of claim 6 or 7, wherein the emulsifier comprises one or more hydrogenated glyceride or PEG ester.

9. The method of any one of claims 6 to 8, wherein the antifoam reagent further comprises a silicone polymer.

10. The method of claim 9, wherein the silicone polymer comprises polydimethylsiloxane (PDMS).

11. The method of claim 9 or 10, wherein the antifoam reagent comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% silicone polymer.

12. The method of any one of claims 9 to 11, wherein the antifoam reagent comprises about 10% silicone polymer.

13. The method of any one of claims 1 to 12, wherein the antifoam reagent comprises ANTIFOAM B™.

14. The method of claim 13, wherein the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% ANTIFOAM B™.

15. The method of claim 13 or 14, wherein the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% ANTIFOAM B™.

16. The method of any one of claims 13 to 15, wherein the product feedstream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 10% ANTIFOAM B™.

17. The method of any one of claims 13 to 16, wherein the product feedstream is contacted with ANTIFOAM B™ at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% ANTIFOAM B™.

18. The method of any one of claims 1 to 12, wherein the antifoam reagent comprises antifoam SE-15.

19. The method of claim 18, wherein the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% antifoam SE-15.

20. The method of claim 18 or 19, wherein the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% antifoam SE-15.

21. The method of any one of claims 18 to 20, wherein the product feedstream is contacted with antifoam SE-15 at a concentration of at least about 0.01% to about 10% antifoam SE- 15.

22. The method of any one of claims 18 to 21, wherein the product feedstream is contacted with antifoam SE-15 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam SE-15.

23. The method of any one of claims 1 to 12, wherein the antifoam reagent comprises antifoam L-30.

24. The method of claim 23, wherein the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% antifoam L-30.

25. The method of claim 23 or 24, wherein the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% antifoam L-30.

26. The method of any one of claims 23 to 25, wherein the product feedstream is contacted with antifoam L-30 at a concentration of at least about 0.01% to about 10% antifoam L-30.

27. The method of any one of claims 23 to 26, wherein the product feedstream is contacted with antifoam L-30 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam L-30.

28. The method of any one of claims 1 to 5, wherein the antifoam reagent comprises one or more organic non-silicone polypropylene-based polyether.

29. The method of claim 28, wherein the antifoam reagent comprises antifoam 204.

30. The method of claim 29, wherein the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% antifoam 204.

31. The method of claim 29 or 30, wherein the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% antifoam 204.

32. The method of any one of claims 29 to 31, wherein the product feedstream is contacted with antifoam 204 at a concentration of at least about 0.01% to about 10% antifoam 204.

33. The method of any one of claims 29 to 32, wherein the product feedstream is contacted with antifoam 204 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% antifoam 204.

34. The method of any one of claims 1 to 33, wherein the antifoam reagent comprises one or more difunctional block copolymer surfactant.

35. The method of claim 34, wherein the difunctional block copolymer surfactant comprises terminal secondary hydroxyl groups.

36. The method of claim 34 or 35, wherein the difunctional block copolymer surfactant comprises a PLURONIC®.

37. The method of any one of claims 34 to 36, wherein the difunctional block copolymer surfactant comprises PLURONIC® 31R1.

38. The method of claim 37, wherein the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% PLURONIC® 31R1.

39. The method of claim 37 or 38, wherein the product feedstream is contacted with PLURONIC® 31Rlat a concentration of at least about 0.01% to about 50%, at least about 0.01% to about 45%, at least about 0.01% to about 40%, at least about 0.01% to about 35%, at least about 0.01% to about 30%, at least about 0.01% to about 25%, at least about 0.01% to about 20%, at least about 0.01% to about 15%, or at least about 0.01% to about 10% PLURONIC® 31R1.

40. The method of any one of claims 37 to 39, wherein the product feedstream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 10% PLURONIC® 31R1.

41. The method of any one of claims 37 to 40, wherein the product feedstream is contacted with PLURONIC® 31R1 at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%,about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, or about 10.0% PLURONIC® 31R1.

42. The method of any one of claims 1 to 41, wherein the product feedstream is not contacted with Triton X-100.

43. The method of any one of claims 1 to 42, wherein the product feedstream comprises a harvest from a bioreactor, a chromatography load, a chromatography eluate, a filtration load, a filtrate, or any combination thereof.

44. The method of claim 43, wherein the chromatography eluate is a Protein A chromatography eluate.

45. The method of any one of claims 1 to 44, wherein the virus comprises a lipid-enveloped virus.

46. The method of claim 45, wherein the lipid-enveloped virus is a retrovirus.

47. The method of claim 46, wherein the retrovirus is A-MuLV.

48. The method of claim 45, wherein the lipid-enveloped virus is a herpesvirus.

49. The method of claim 48, wherein the herpesvirus is HSV-1.

50. The method of any one of claims 1 to 49, wherein inactivating the virus comprises a log reduction value (LRV) of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10, and wherein the LRV is calculated as:[Virus in pfu]producpooiLRV = —logw[Virus in pfu}Load / Feed'51. The method of claim 50, wherein the LRV is at least about 2.

52. The method of claim 50, wherein the LRV is at least about 4.

53. The method of any one of claims 1 to 52, wherein the contacting occurs for at least about 15 minutes, at least about 30 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, or at least about 120 minutes.

54. The method of any one of claims 2 and 6 to 53, wherein the therapeutic protein comprises an antibody, antibody fragment, a fusion protein, a naturally occurring protein, a chimeric protein, or any combination thereof.

55. The method of any one of claims 2 and 6 to 54, wherein the therapeutic protein is a fusion protein.

56. The method of claim 55, wherein the fusion protein comprises an Fc portion.

57. The method of any one of claims 1, 2, and 6 to 53, wherein the therapeutic agent comprises a non-enveloped virus particle.

58. The method of claim 57, wherein the non-enveloped virus particle comprises an AAV.

59. A method to treat a disease or condition comprising administering to a subj ect a therapeutic protein manufactured by a process comprising a viral inactivation step according to the method of any one of claims 1 to 58.

60. A pharmaceutical composition manufactured by a process comprising a viral inactivation step according to the method of any one of claims 1 to 58.

61. A method of manufacturing a therapeutic protein comprising a viral inactivation step according to the method of any one of claims 1 to 56.