Defoaming agent compositions and methods of using the same

Abstract

The present disclosure provides compositions and methods for inactivating viruses in a product feed stream during the manufacture of a therapeutic agent using an antifoam agent. 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 relate to methods for inactivating viruses in a eukaryotic cell culture of a product feed stream, the methods comprising contacting the cell culture with an antifoam agent. In some aspects, the antifoam agent comprises an emulsifier, such as a non-ionic emulsifier, and optionally a silicone polymer.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 562,205, filed March 6, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure provides information on the use of defoamers to inactivate viruses in cell cultures (i.e., product feed streams) during the manufacture of therapeutic proteins, viral particles, nucleic acid molecules, or any combination thereof. Background Technology

[0003] Viral contamination can arise from the cell line itself when the virus is endogenously produced by cells or when there is latent or persistent infection. Alternatively, the virus may be introduced during recombinant production due to the use of contaminated reagents or viral vectors. To ensure patient safety, downstream purification processes require robust virus removal strategies to ensure the removal of potential viral contamination. For enveloped viruses, typical virus inactivation processes use low pH conditions or chemical inactivators as key components of the removal strategy. For biopharmaceuticals that are unstable under low pH conditions, chemical inactivators provide the necessary virus inactivation without stability issues. Historically, the nonionic surfactant Triton X-100 has provided excellent virus inactivation. However, given recent safety concerns regarding the degradation products of Triton X-100, novel virus removal methods are needed in the art.

[0004] Defoamers are widely used in the biopharmaceutical industry to prevent unwanted foam formation in bioreactors and fermenters. Some defoamers also have lubricating properties, reducing shear forces on cells caused by aeration and agitation conditions. Typically, defoaming chemicals used in cell culture contain emulsions of polydimethylsiloxane (PDMS) or nonionic detergents. Variations in defoamers are caused by the emulsifiers or detergents used. Because defoaming chemicals are already used in biopharmaceutical manufacturing, they have established supply chains, regulatory familiarity, and known removal and detection strategies. Summary of the Invention

[0005] Some aspects of this disclosure relate to a method for inactivating a virus in a product feed stream during a downstream manufacturing process of a therapeutic agent, the method comprising contacting the product feed stream with an antifoaming agent. In some aspects, the therapeutic agent comprises a therapeutic protein, viral particles, nucleic acid molecules, or any combination thereof.

[0006] Some aspects of this disclosure relate to a method for inactivating viruses in eukaryotic cell cultures via an inactivation product feed stream, the method comprising contacting the cell culture with an antifoaming agent.

[0007] In some respects, eukaryotic cell cultures contain mammalian or insect cells. In other respects, mammalian cells are selected from CHO cells and HEK cells.

[0008] In some respects, defoamers contain emulsifiers. In some respects, the emulsifier is a nonionic emulsifier. In some respects, the emulsifier contains one or more hydrogenated glycerol esters or PEG esters.

[0009] In some aspects, the defoamer further comprises a silicone polymer. In some aspects, the silicone polymer comprises polydimethylsiloxane (PDMS). In some aspects, the defoamer 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% of a silicone polymer. In some aspects, the defoamer comprises about 10% of a silicone polymer.

[0010] In some aspects, the defoamer comprises ANTIFOAM B™. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01%. In some aspects, the product feed stream 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%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01% to about 10%. In some aspects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, and approximately 3.0%. Approximately 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% of ANTIFOAM B™ contact.

[0011] In some aspects, the defoamer comprises defoamer SE-15. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with defoamer 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%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of at least about 0.01% to about 10%. In some aspects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately 3%. 2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5% or approximately 10.0% of defoamer SE-15 in contact with defoamer SE-15.

[0012] In some aspects, the defoamer comprises defoamer L-30. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with defoamer 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%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.01% to about 10%. In some respects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately 3 0.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5% or approximately 10.0% of defoamer L-30 in contact with defoamer L-30.

[0013] In some respects, defoamers contain one or more organic non-silicone polypropylene-based polyethers.

[0014] In some aspects, the defoamer comprises defoamer 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with defoamer 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%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of at least about 0.01% to about 10%. In some respects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately... 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% of defoamer 204 in contact with defoamer 204.

[0015] In some respects, defoamers comprise one or more bifunctional block copolymer surfactants. In some respects, bifunctional block copolymer surfactants comprise terminal secondary hydroxyl groups.

[0016] In some aspects, the bifunctional block copolymer surfactant comprises PLURONIC®. In some aspects, the bifunctional block copolymer surfactant comprises PLURONIC® 31R1. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01%. In some aspects, the product feed stream 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%, or at least about 0.01% to about 10%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01% to about 10%. In other aspects, the product feed stream is contacted with concentrations 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%, and about 3.0%. Approximately 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% of PLURONIC® 31R1 contacts.

[0017] In some respects, the product feed stream does not come into contact with the Triton X-100.

[0018] In some respects, the product feed stream comprises harvested material from the bioreactor, chromatographic loading solution, chromatographic eluent, filtrate, or any combination thereof. In some respects, the chromatographic eluent is protein A chromatographic eluent.

[0019] In some respects, viruses include lipid-enveloped viruses. In some respects, lipid-enveloped viruses are retroviruses. In some respects, retroviruses are A-MuLV. In some respects, lipid-enveloped viruses are herpesviruses. In some respects, herpesviruses are HSV-1.

[0020] In some respects, the inactivated virus includes a logarithmic decrease 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 follows:

[0021] .

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

[0023] In some respects, the contact lasted 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 respects, therapeutic proteins include antibodies, antibody fragments, fusion proteins, naturally occurring proteins, chimeric proteins, or any combination thereof. In some respects, therapeutic proteins are fusion proteins. In some respects, fusion proteins include an Fc moiety.

[0025] In some respects, the therapeutic agent contains non-enveloped viral particles. In some respects, the non-enveloped viral particles contain AAV.

[0026] Some aspects of this disclosure relate to a method for treating a disease or condition, the method comprising administering a therapeutic protein to a subject, the therapeutic protein being manufactured through a process including a virus inactivation step according to the method disclosed herein.

[0027] Some aspects of this disclosure relate to pharmaceutical compositions manufactured by a process including a virus inactivation step according to the methods disclosed herein.

[0028] Some aspects of this disclosure relate to a method for manufacturing therapeutic proteins, which includes a virus inactivation step according to the method disclosed herein. Attached Figure Description

[0029] Figure 1A-1B This is a scatter plot illustrating the effect of A-MulV ( ) when treated with a set of different agents. Figure 1A ) or X-MulV ( Figure 1B Comparison of the viral log decrease value (LRV) of retrovirus-like particles (RVLP) and retrovirus-like particles (RVLP).

[0030] Figure 2This is a schematic diagram of an experimental procedure for quantifying virus inactivation using retrovirus-like particles (RVLPs).

[0031] Figures 3A-3B This is a bar chart showing data related to the defoamer test results: the defoamer test panel compared the results with known detergents ( Figure 3A ), and the concentration dependence of virus inactivation on the active ingredient of the defoamer ( Figure 3B ). Detailed Implementation

[0032] This disclosure relates to methods for inactivating viruses in cell cultures, including contacting the cell cultures with an antifoaming agent. Some aspects of this disclosure relate to methods for inactivating viruses in a product feed stream, including contacting the product feed stream with an antifoaming agent. In some aspects, the product feed stream is in a downstream manufacturing process of a therapeutic agent, including contacting the product feed stream with an antifoaming agent. Therefore, some aspects of this disclosure relate to methods for inactivating viruses in a product feed stream during a downstream manufacturing process of a therapeutic agent, including contacting the product feed stream with an antifoaming agent.

[0033] Some aspects of this disclosure relate to methods for inactivating viruses in eukaryotic cell cultures via a product feed stream, methods including contacting the cell cultures with an antifoaming agent. In some aspects, the eukaryotic cell cultures comprise mammalian or insect cells. In some aspects, the mammalian cells comprise CHO cells or HEK cells. I. Terminology

[0034] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning stated below. Further definitions are set forth throughout the application.

[0035] It should be understood that any aspect described in the text is “comprising”, and other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0036] As used herein, the term "approximately," when applied to one or more values ​​of interest, means a value similar to the specified reference value. In some respects, unless otherwise specified or otherwise apparent from the context (except where such a number would exceed 100% of the possible value), the term "approximately" means a range of values ​​falling within (greater than or less than) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the stated reference value.

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

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide general dictionaries for those skilled in the art regarding many of the terms used in this disclosure.

[0039] Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form. Numerical ranges include the values ​​that define the range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino to carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to this specification as a whole. Accordingly, the terms defined below are defined more fully by reference to the entire contents of this specification.

[0040] As used herein, the terms “antiviral,” “inactivated virus,” or “virus inactivation” refer to the process by which a virus ceases to infect cells, replicate, and multiply, and is itself removed. Therefore, the term “virus inactivation” generally refers to the process of rendering the fluids disclosed herein completely free of infectious viral contaminants. It is desirable to use the methods disclosed herein to achieve any degree of virus inactivation. However, it is expected to achieve the level of virus inactivation required to meet stringent drug safety guidelines. These guidelines are issued by the WHO and are well known to those skilled in the art.

[0041] As used herein, the term "defoamer" refers to a class of agents traditionally used to prevent foam formation, for example, in cell cultures. Defoamers typically comprise (i) a carrier fluid, such as silicone, mineral oil, ester, and / or polyol; and / or (ii) a hydrophobic solid, such as wax, fatty alcohol, fatty acid, and / or silica. In some aspects, the defoamers disclosed herein comprise emulsifiers. As used herein, "emulsifier" refers to an agent that promotes the mixing 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 glycerol esters or PEG esters. In some aspects, the defoamer further comprises a silicone polymer. As used herein, the term "silicone polymer" or "polysiloxane" refers to a substance whose molecules consist of polymer chains of a polymeric siloxane having alternating silicon and oxygen atoms (e.g., -O-Si-O-Si-...). In some respects, silicone polymers contain polydimethylsiloxane (PDMS).

[0042] The methods disclosed herein can be used, for example, to generate biological substances such as antibodies or fusion proteins. As used herein, an "antibody" (Ab) should include, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding portion. Each H chain contains a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), in which more conserved regions called framework regions (FRs) are scattered. Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0043] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may contain modified amino acids. These terms also cover polymers of amino acids that have been naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. The definition also includes, for example, one or more analogues containing amino acids (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine) and other modified polypeptides known in the art.

[0044] A “product feed stream” or alternatively a “feed stream” is a material or solution provided for a process purification method that contains the target therapeutic agent and may also contain various impurities. Non-limiting examples may include, for example, harvested cell culture medium (HCCF), or a pool containing the target therapeutic protein collected after one or more purification process steps. It should be understood that the uses of the compositions and methods disclosed herein are not limited to the manufacture of biological feed streams and can be used to inactivate enveloped viruses in any solution or medium in which viral inactivation is desired. In some aspects, viral inactivation can be carried out in solution. In some aspects, viral inactivation can be carried out on a solid surface. In some aspects, viral inactivation can be carried out on the body surface of a mammal. In some aspects, the product feed stream includes harvested material (e.g., harvested cell culture medium), loading solution (e.g., chromatographic or filtered loading solution, i.e., chromatographic loading solution or filtered loading solution), eluent (e.g., chromatographic eluent), filtrate (e.g., a solution collected after filtration of a solution), or any combination thereof. Generally, the methods disclosed herein can be used to inactivate viruses in any solution containing or suspected of containing viruses.

[0045] "Impurities" refer to materials that differ from the desired polypeptide product. Impurities include, but are not limited to: host cell material, such as host cell proteins (HCP); leached protein A; nucleic acids; variants, size variants, fragments, aggregates, or derivatives of the desired polypeptide; another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc.

[0046] The term “chromatography” refers to any technique that separates a target protein from other molecules (e.g., contaminants) present in a mixture, wherein the target protein is separated from other molecules (e.g., contaminants) due to the different rates at which individual molecules in the mixture migrate through the stationary medium under the influence of the mobile phase or during binding and elution.

[0047] The term "aggregation" refers to the tendency of a polypeptide (such as an antibody or fusion protein) to form a complex with other molecules (such as other molecules of the same polypeptide), thereby forming a high molecular weight (HMW) aggregate. Exemplary methods for measuring aggregate formation include analytical size exclusion chromatography as described in the examples herein. The relative amount of aggregates can be determined relative to a reference compound, for example, to identify polypeptides with reduced aggregation. The relative amount of aggregates can also be determined relative to a reference formulation.

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

[0049] The term "detergent" refers to a reagent or combination thereof that may contain a salt or hydrophilic portion (such as a sugar) of a long-chain aliphatic base or acid and possess both hydrophilic and hydrophobic properties. Detergents, by virtue of their hydrophilic and hydrophobic properties, can perform specific functions. As used herein, detergents have the ability to disrupt viral envelopes and inactivate viruses.

[0050] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and guinea pigs). In a preferred aspect, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0051] As used herein, the term "treat (treating and treatment)" refers to any type of intervention or process performed on or administered to a subject with the goal of reversing, reducing, improving, inhibiting, slowing, or preventing the progression, development, severity, or recurrence of disease-related symptoms, complications, conditions, or biochemical indicators, or enhancing overall survival. Treatment may be directed at subjects with the disease or subjects without the disease (e.g., for prevention).

[0052] The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination of alternatives. As used herein, the indefinite article "a or an" should be understood to mean "one or more" of any enumerated or listed components.

[0053] The terms “about” or “substantially constitute” refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled 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 “substantially constitute” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” or “substantially constitute” may mean a range up to 10%. Furthermore, particularly for biological systems or processes, these terms may mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of “about” or “substantially constitute” should be assumed to be within an acceptable margin of error for that particular value or composition.

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

[0055] The various aspects of this disclosure are described in more detail in the following subsections. II. Methodology of this Disclosure

[0056] Some aspects of this disclosure relate to methods for inactivating viruses in a product feed stream, including contacting the product feed stream with an antifoaming agent. In some aspects, the product feed stream is present in a downstream manufacturing process of a therapeutic agent, including contacting the product feed stream with an antifoaming agent. Therefore, some aspects of this disclosure relate to methods for inactivating viruses in a product feed stream during a downstream manufacturing process of a therapeutic agent, including contacting the product feed stream with an antifoaming agent. In some aspects, the therapeutic agent comprises a therapeutic protein, viral particles, nucleic acid molecules, or any combination thereof.

[0057] Some aspects of this disclosure relate to methods for inactivating viruses in eukaryotic cell cultures via a product feed stream, methods including contacting the cell culture with an antifoaming agent. In some aspects, the eukaryotic cell culture comprises mammalian or insect cells. In some aspects, the eukaryotic cells are mammalian cells. In some aspects, the eukaryotic cells are human cells. In some aspects, the eukaryotic cells are insect cells. In some aspects, the mammalian cells are selected from CHO cells (e.g., CHO-K1 CHKDG44 or CHO DXB11), HEK cells, Namalwa cells, HeLa cells, WI-28 cells, MRC-5 cells, HepG2 cells, MEF cells (e.g., 3T3), L-929 cells, myeloma cells, BHK-21 cells, COS cells (e.g., COS-7), and Vero cells. In some aspects, the eukaryotic cells comprise CHO cells. In some aspects, the eukaryotic cells comprise HEK cells. In some aspects, the eukaryotic cells comprise HEK293 cells.

[0058] In some respects, the product feed stream does not come into contact with the Triton X-100.

[0059] In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate lipid-enveloped viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate herpesviridae viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate poxviridae viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate hepatoviridae viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate RNA viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate flaviviridae viruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate viruses of the Wombaviridae family. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate viruses of the Coronaviridae family. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate deltaviruses. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate viruses of the Orthomyxoviridae family. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate viruses of the Paramyxoviridae family. In some aspects, the virus inactivation methods disclosed herein include contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate viruses of the Rhabdoviridae family. In some aspects, the virus inactivation methods disclosed herein involve contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate Bunyaviridae viruses. In some aspects, the virus inactivation methods disclosed herein involve contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate Filoviridae viruses. In some aspects, the virus inactivation methods disclosed herein involve contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate retroviruses. In some aspects, the virus inactivation methods disclosed herein involve contacting the feed stream or cell culture with the defoaming agent disclosed herein to inactivate retroviridae viruses. II.A. Defoamer

[0060] Some aspects of this disclosure relate to methods for inactivating viruses in cell cultures by contacting cell cultures with an antifoaming agent. In some aspects, the antifoaming agent comprises an emulsifier. Any emulsifier may be used in the methods disclosed herein. In some aspects, the emulsifier is a nonionic emulsifier. Non-limiting examples of nonionic emulsifiers that may 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, and polysorbate esters (e.g., polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85). In some respects, the emulsifier contains one or more hydrogenated glycerol esters. In other respects, the emulsifier contains one or more PEG esters.

[0061] In some aspects, the defoamer contains 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 defoamer contains at least about 1% emulsifier. In some aspects, the defoamer contains at least about 2% emulsifier. In some aspects, the defoamer contains at least about 3% emulsifier. In some aspects, the defoamer contains at least about 4% emulsifier. In some aspects, the defoamer contains at least about 5% emulsifier. In some aspects, the defoamer contains at least about 10% emulsifier. In some aspects, the defoamer contains at least about 15% emulsifier. In some aspects, the defoamer contains at least about 20% emulsifier. In some aspects, the defoamer contains at least about 25% emulsifier. In some aspects, the defoamer contains at least about 30% emulsifier. In some aspects, the defoamer contains at least about 25% emulsifier. In some aspects, the defoamer contains at least about 40% emulsifier. In some aspects, the defoamer contains at least about 45% emulsifier. In some aspects, the defoamer contains at least about 50% emulsifier. In some aspects, the defoamer contains at least about 55% emulsifier. In some aspects, the defoamer contains at least about 60% emulsifier. In some aspects, the defoamer contains at least about 65% emulsifier. In some aspects, the defoamer contains at least about 70% emulsifier. In some aspects, the defoamer contains at least about 75% emulsifier. In some respects, the defoamer contains at least about 80% emulsifier. In some respects, the defoamer contains at least about 85% emulsifier. In some respects, the defoamer contains at least about 90% emulsifier. In some respects, the defoamer contains at least about 95% emulsifier.

[0062] In some aspects, the defoamer contains 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 glycerol ester. In some aspects, the defoamer contains at least about 1% hydrogenated glycerol ester. In some aspects, the defoamer contains at least about 2% hydrogenated glycerol ester. In some aspects, the defoamer contains at least about 3% hydrogenated glycerol ester. In some aspects, the defoamer contains at least about 4% hydrogenated glycerol ester. In some aspects, the defoamer contains at least about 5% hydrogenated glycerol ester. In some aspects, the defoamer contains at least about 10% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 15% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 20% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 25% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 30% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 35% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 40% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 45% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 50% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 55% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 60% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 65% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 70% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 75% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 80% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 85% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 90% hydrogenated glycerol esters. In some aspects, the defoamer contains at least about 95% hydrogenated glycerol esters.

[0063] In some aspects, the defoamer contains 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 defoamer contains at least about 1% PEG ester. In some aspects, the defoamer contains at least about 2% PEG ester. In some aspects, the defoamer contains at least about 3% PEG ester. In some aspects, the defoamer contains at least about 4% PEG ester. In some aspects, the defoamer contains at least about 5% PEG ester. In some aspects, the defoamer contains at least about 10% PEG ester. In some aspects, the defoamer contains at least about 15% PEG ester. In some aspects, the defoamer contains at least about 20% PEG ester. In some aspects, the defoamer contains at least about 25% PEG ester. In some aspects, the defoamer contains at least about 30% PEG ester. In some aspects, the defoamer contains at least about 35% PEG ester. In some aspects, the defoamer contains at least about 40% PEG ester. In some aspects, the defoamer contains at least about 45% PEG ester. In some aspects, the defoamer contains at least about 50% PEG ester. In some aspects, the defoamer contains at least about 55% PEG ester. In some aspects, the defoamer contains at least about 60% PEG ester. In some aspects, the defoamer contains at least about 65% PEG ester. In some aspects, the defoamer contains at least about 70% PEG ester. In some aspects, the defoamer contains at least about 75% PEG ester. In some aspects, the defoamer contains at least about 80% PEG ester. In some aspects, the defoamer contains at least about 85% PEG ester. In some cases, defoamers contain at least about 90% PEG esters. In others, defoamers contain at least about 95% PEG esters.

[0064] In some aspects, the defoamer further comprises a silicone polymer. Any silicone polymer may 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 simethicone (PDMS plus silica).

[0065] In some aspects, the defoamer 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%, and about 0.1%. The defoamer comprises about 0.01% 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% of a silicone polymer, such as PDMS. In some aspects, the defoamer comprises about 0.01% to about 50% of a silicone polymer, such as PDMS. In some aspects, the defoamer comprises about 0.1% to about 50% of a silicone polymer, such as PDMS. In some aspects, the defoamer comprises about 1% to about 50% of a silicone polymer, such as PDMS.

[0066] In some aspects, the defoamer contains about 0.01% to about 40% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 40% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 40% of a silicone polymer, such as PDMS.

[0067] In some aspects, the defoamer contains about 0.01% to about 30% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 30% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 30% of a silicone polymer, such as PDMS.

[0068] In some aspects, the defoamer contains about 0.01% to about 25% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 25% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 25% of a silicone polymer, such as PDMS.

[0069] In some aspects, the defoamer contains about 0.01% to about 20% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 20% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 20% of a silicone polymer, such as PDMS.

[0070] In some aspects, the defoamer contains about 0.01% to about 15% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 15% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 15% of a silicone polymer, such as PDMS.

[0071] In some aspects, the defoamer contains about 0.01% to about 10% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 10% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 10% of a silicone polymer, such as PDMS.

[0072] In some aspects, the defoamer contains about 0.01% to about 5% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 0.1% to about 5% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains about 1% to about 5% of a silicone polymer, such as PDMS.

[0073] In some aspects, the defoamer contains about 0.01% to about 1% of a silicone polymer, such as PDMS.

[0074] In some aspects, the defoamer 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 defoamer comprises at least about 1% silicone polymer. In some aspects, the defoamer comprises at least about 2% silicone polymer. In some aspects, the defoamer comprises at least about 3% silicone polymer. In some aspects, the defoamer comprises at least about 4% silicone polymer. In some aspects, the defoamer comprises at least about 5% silicone polymer. In some aspects, the defoamer comprises at least about 6% silicone polymer. In some aspects, the defoamer comprises at least about 7% silicone polymer. In some aspects, the defoamer contains at least about 8% silicone polymer. In some aspects, the defoamer contains at least about 9% silicone polymer. In some aspects, the defoamer contains at least about 10% silicone polymer. In some aspects, the defoamer contains at least about 11% silicone polymer. In some aspects, the defoamer contains at least about 12% silicone polymer. In some aspects, the defoamer contains at least about 13% silicone polymer. In some aspects, the defoamer contains at least about 14% silicone polymer. In some aspects, the defoamer contains at least about 15% silicone polymer. In some aspects, the defoamer contains at least about 16% silicone polymer. In some aspects, the defoamer contains at least about 17% silicone polymer. In some aspects, the defoamer contains at least about 18% silicone polymer. In some aspects, the defoamer contains at least about 19% silicone polymer. In some aspects, the defoamer contains at least about 20% silicone polymer. In some aspects, the defoamer contains at least about 25% silicone polymer. In some aspects, the defoamer contains at least about 30% silicone polymer. In some aspects, the defoamer contains at least about 35% silicone polymer. In some aspects, the defoamer contains at least about 40% silicone polymer. In some aspects, the defoamer contains at least about 45% silicone polymer. In some aspects, the defoamer contains at least about 50% silicone polymer. In some aspects, the defoamer contains at least about 55% silicone polymer. In some aspects, the defoamer contains at least about 60% silicone polymer. In some aspects, the defoamer contains at least about 65% silicone polymer. In some aspects, the defoamer contains at least about 70% silicone polymer. In some aspects, the defoamer contains at least about 75% silicone polymer. In some aspects, the defoamer contains at least about 80% silicone polymer. In some aspects, the defoamer contains at least about 85% silicone polymer. In some aspects, the defoamer contains at least about 90% silicone polymer. In some aspects, the defoamer contains at least about 95% silicone polymer.

[0075] In some aspects, the defoamer contains 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% PDMS. In some aspects, the defoamer contains at least about 1% PDMS. In some aspects, the defoamer contains at least about 2% PDMS. In some aspects, the defoamer contains at least about 3% PDMS. In some aspects, the defoamer contains at least about 4% PDMS. In some aspects, the defoamer contains at least about 5% PDMS. In some aspects, the defoamer contains at least about 6% PDMS. In some aspects, the defoamer contains at least about 7% PDMS. In some aspects, the defoamer contains at least about 8% PDMS. In some aspects, the defoamer contains at least about 9% PDMS. In some aspects, the defoamer contains at least about 10% PDMS. In some aspects, the defoamer contains at least about 11% PDMS. In some aspects, the defoamer contains at least about 12% PDMS. In some aspects, the defoamer contains at least about 13% PDMS. In some aspects, the defoamer contains at least about 14% PDMS. In some aspects, the defoamer contains at least about 15% PDMS. In some aspects, the defoamer contains at least about 16% PDMS. In some aspects, the defoamer contains at least about 17% PDMS. In some aspects, the defoamer contains at least about 18% PDMS. In some aspects, the defoamer contains at least about 19% PDMS. In some aspects, the defoamer contains at least about 20% PDMS. In some aspects, the defoamer contains at least about 25% PDMS. In some aspects, the defoamer contains at least about 30% PDMS. In some aspects, the defoamer contains at least about 35% PDMS. In some aspects, the defoamer contains at least about 40% PDMS. In some aspects, the defoamer contains at least about 45% PDMS. In some aspects, the defoamer contains at least about 50% PDMS. In some aspects, the defoamer contains at least about 55% PDMS. In some aspects, the defoamer contains at least about 60% PDMS. In some aspects, the defoamer contains at least about 65% PDMS. In some aspects, the defoamer contains at least about 70% PDMS. In some aspects, the defoamer contains at least about 75% PDMS. In some aspects, the defoamer contains at least about 80% PDMS. In some aspects, the defoamer contains at least about 85% PDMS. In some aspects, the defoamer contains at least about 90% PDMS. In some aspects, the defoamer contains at least about 95% PDMS.

[0076] In some aspects, the defoamer contains at least 0.1% and less than 100% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 30% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 29% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 28% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 27% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 26% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 25% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 24% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 23% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 22% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 21% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 20% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 19% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 18% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 17% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 16% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 15% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 14% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 13% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 12% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 11% of a silicone polymer, such as PDMS. In some aspects, the defoamer contains at least 0.1% and less than about 10% of a silicone polymer, such as PDMS.

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

[0078] In some respects, the defoamer comprises one or more organic non-silicone polypropylene-based polyethers. In some respects, the defoamer comprises defoamer 204.

[0079] In some aspects, the defoamer comprises one or more bifunctional block copolymer surfactants. In some aspects, the bifunctional block copolymer surfactant comprises a terminal secondary hydroxyl group. In some aspects, the bifunctional block copolymer surfactant comprises PLURONIC. In some aspects, the bifunctional block copolymer surfactant comprises PLURONIC® 31R1. II.A.1.ANTIFOAM B™

[0080] In some aspects, the defoamer contains ANTIFOAM B™ (SIGNMA-ALDRICH). ANTIFOAM B™ is a silicone-based defoamer, marketed as a 10% aqueous emulsion of PDMS. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at concentrations 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%.

[0081] In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of at least about 0.1%. In some aspects, the product feed stream 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%.

[0082] In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of at least about 1%. In some aspects, the product feed stream 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%.

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

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

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

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

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

[0088] In some aspects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, and approximately 3.0%. Approximately 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% ANTIFOAM B™ are contacted. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 0.01%. In some aspects, the product feed stream is contacted with ANTIFOAMB™ at a concentration of approximately 0.1%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 1%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 2%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 3%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 4%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 5%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 6%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of about 7%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 8%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 9%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 10%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 11%.In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 12%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 13%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 14%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 15%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 16%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 17%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 18%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 19%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 20%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 25%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 30%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 35%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 40%. In some aspects, the product feed stream is contacted with ANTIFOAM B™ at a concentration of approximately 45%. In some respects, the product feed stream is brought into contact with ANTIFOAM B™ at a concentration of approximately 50% ANTIFOAMB™. II.A.2 Defoamer SE-15

[0089] In some aspects, the defoamer includes defoamer SE-15 (Sigma-Aldrich). Defoamer SE-15 is a silicone-based defoamer, marketed as an aqueous emulsion containing 10% active silicone and a nonionic emulsifier. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with defoamer SE-15 at concentrations 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%.

[0090] In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of at least about 0.1%. In some aspects, the product feed stream is contacted with defoamer 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%.

[0091] In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of at least about 1%. In some aspects, the product feed stream is contacted with defoamer 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%.

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

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

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

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

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

[0097] In some aspects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately 3%. The product feed stream is contacted with defoamer SE-15 at a concentration of about 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%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 0.01%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 0.1%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 1%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 2%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 3%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 4%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 5%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 6%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 7%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 8%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 9%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 10%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 11%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 12%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 13%.In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 14%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 15%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 16%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 17%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 18%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 19%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 20%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 25%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 30%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 35%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 40%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 45%. In some aspects, the product feed stream is contacted with defoamer SE-15 at a concentration of about 50%. II.A.3. Defoamer L-30

[0098] In some aspects, the defoamer includes defoamer L-30 (Sigma-Aldrich). Defoamer L-30 is a silicone-based defoamer and is sold as a 30% aqueous emulsion. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.01%. In some aspects, the product feed stream is contacted with defoamer L-30 at concentrations 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%.

[0099] In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.1%. In some aspects, the product feed stream is contacted with defoamer 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%.

[0100] In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 1%. In some aspects, the product feed stream is contacted with defoamer L-30 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%.

[0101] In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.01% to about 50%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 0.1% to about 150%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of at least about 1% to about 50%.

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

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

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

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

[0106] In some respects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately 3 The product feed stream is contacted with defoamer L-30 at a concentration of about 0.01% defoamer L-30. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 0.1% defoamer L-30. In other aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 0.1% defoamer L-30. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 1%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 2%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 3%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 4%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 5%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 6%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 7%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 8%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 9%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 10%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 11%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 12%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 13%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 14%.In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 15%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 16%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 17%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 18%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 19%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 20%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 25%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 30%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 35%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 40%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 45%. In some aspects, the product feed stream is contacted with defoamer L-30 at a concentration of about 50%. II.A.4. Defoamer 204

[0107] In some respects, the defoamer comprises defoamer 204 (Sigma-Aldrich). Defoamer 204 is sold as a mixture of organic non-silicone polypropylene-based polyether dispersions, which is free of mineral oil and other surfactants, and is synthetic and not derived from animal or plant sources. In some respects, the product feed stream is contacted with defoamer 204 at a concentration of at least about 0.01%. In some aspects, the product feed stream is brought into contact with defoamer 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%.

[0108] In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of at least about 0.1%. In some aspects, the product feed stream is contacted with defoamer 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%.

[0109] In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of at least about 1%. In some aspects, the product feed stream is contacted with defoamer 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%.

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

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

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

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

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

[0115] In some respects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3.0%, approximately 3.1%, and approximately... The defoamer 204 is contacted at concentrations of approximately 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0%. In some aspects, the product feed stream is contacted with the defoamer 204 at a concentration of approximately 0.01%. In some aspects, the product feed stream is contacted with the defoamer 204 at a concentration of approximately 0.1%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% 16% 17% 18% 19% 10 ... In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 9% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 10% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 11% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 12% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 13% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 14% 204. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 15% 204.In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 16%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 17%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 18%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 19%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 20%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 25%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 30%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 35%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 40%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 45%. In some aspects, the product feed stream is contacted with defoamer 204 at a concentration of about 50%. II.A.5.PLURONIC® 31R1

[0116] In some respects, the defoamer contains PLURONIC® 31R1 (BASF). PLURONIC® 31R1 is marketed as a reverse block copolymer of ethylene oxide and propylene oxide. In some respects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.01%. In some aspects, the product feed stream 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%.

[0117] In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of at least about 0.1%. In some aspects, the product feed stream 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%.

[0118] In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of at least about 1%. In some aspects, the product feed stream 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%.

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

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

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

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

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

[0124] In some aspects, the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, and approximately 3.0%. Approximately 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% of PLURONIC® 31R1. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of approximately 0.01%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 0.1%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 1%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 2%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 3%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 4%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 5%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 6%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 7%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 8%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 9%.In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 10%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 11%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 12%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 13%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 14%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 15%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 16%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 17%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 18%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 19%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 20%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 25%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 30%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 35%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 40%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 45%. In some aspects, the product feed stream is contacted with PLURONIC® 31R1 at a concentration of about 50%. II.B. Cell Culture and Product Feed Flow

[0125] In some aspects, the product feed stream comprises harvested material (e.g., from a bioreactor), loading solution (e.g., loading solution from a chromatographic column or filter column or other filtration device), eluent (e.g., chromatographic eluent), filtrate, or a combination thereof. Generally, the methods and compositions disclosed herein can be used to inactivate viruses in any solution containing, suspected of containing, or readily containing viruses. In some aspects, the feed stream is harvested cell culture medium. In some aspects, the feed stream comprises a capture cell or a recovered product cell. In some aspects, the capture cell or recovered product cell is a chromatographic cell. In some aspects, the capture cell or recovered product cell is an affinity chromatographic cell. In some aspects, the capture cell or recovered product cell is a protein A cell, a protein G cell, or a protein L cell. In some aspects, the product feed stream is protein A column eluent. In some aspects, the product feed stream is, for example, filtrate from a filtration step in a downstream purification process. In some aspects, the product feed stream is a loading solution, such as a loading solution from a chromatographic column or filtration system.

[0126] In some aspects, this disclosure provides a method for inactivating viruses in a product feed stream during the manufacture of therapeutic proteins using the defoamer disclosed herein, wherein the feed stream (e.g., harvest, loading solution, eluent, or filtrate) is subjected to chromatography after the addition of the defoamer. In some aspects, the chromatography is one or more of affinity chromatography, ion exchange chromatography (e.g., cation exchange and / or anion exchange), hydrophobic interaction chromatography, hydroxyapatite chromatography, or mixed-mode chromatography.

[0127] Examples of affinity chromatographic materials include, but are not limited to, chromatographic materials derived from protein A or protein G. Examples of affinity chromatographic materials include, but are not limited to, Prosep-VA, Prosep-VA Ultra Plus, Protein A Agarose Rapid Flow, Tyopearl Protein A, MAbSelect, MAbSelect SuRe, and MAbSelect SuRe LX. In some aspects, affinity chromatographic materials are affinity columns. In some aspects, affinity chromatographic materials are affinity membranes. Examples of anion exchange chromatographic materials include, but are not limited to, Poros HQ 50, Poros PI 50, Poros D, Mustang Q, Q Agarose FF, and DEAE Agarose. Examples of cation exchange materials include, but are not limited to, Mustang S, Sartobind S, SO3 Monolith, S Ceramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Agarose XL (SPXL), CM Agarose Rapid Flow, Capto S, Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. Examples of HIC chromatographic materials include, but are not limited to, Toyopearl hexyl 650, Toyopearl butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source, Resource, Hi-Trap agarose, octyl agarose, and phenyl agarose. Examples of hydroxyapatite chromatographic materials include, but are not limited to, HA Ultrogel and CHT hydroxyapatite. Examples of mixed-mode chromatographic materials include, but are not limited to, Capto Adhere, QMA, MEP Hypercel, HEA Hypercel, PPA Hypercel, and Capto MMC.

[0128] Lipid-enveloped viruses that can infect mammalian cells include DNA viruses, such as those from the Herpesviridae, Poxviridae, or Hepatoviridae families; RNA viruses, such as those from the Flaviviridae, Wombaviridae, Coronaviridae, Deltaviruses, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, or Filoviridae families; and retroviruses, such as those from the Retroviridae or Hepatoviridae families. Non-limiting examples of lipid-enveloped viruses include human immunodeficiency virus (HIV), Sindbis virus, herpes simplex virus, pseudorabies virus, Sendai virus, vesicular stomatitis virus, West Nile virus, bovine viral diarrhea virus, coronaviruses, equine arthritis virus, severe acute respiratory syndrome virus, Moloney murine leukemia virus, or vaccine viruses. In some respects, lipid-enveloped viruses are selected from the group consisting of: retroviruses, flaviviruses, orthomyxoviruses, herpesviruses, paramyxoviruses, arenaviruses, poxviruses, hepatoviral viruses, hepatitis viruses, rhabdoviruses, and wombaviridae viruses. In some respects, viruses include lipid-enveloped viruses, such as retroviruses like A-MuLV and herpesviruses like HSV-1.

[0129] Virus inactivation can be quantified using the logarithmic decrease value (LRV). Therefore, in some aspects, the methods disclosed herein for inactivating viruses in a feed stream of inactivated products (e.g., harvest, loading solution, eluent, or filtrate) include determining the logarithmic decrease value (LRV) of the number of viruses in the feed stream or the solution containing the virus. 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, the LRV is calculated according to the following formula:

[0130] The term "pfu" or "plaque-forming unit" is a virology measure used to describe the number of viral particles per unit volume capable of forming plaques. In some respects, LRV is at least about 4. In some respects, 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] Detection of live lipid-coated viruses can be accomplished using any technique that can qualitatively or quantitatively measure the presence or activity of live lipid-coated viruses. Typically, cell culture-based assays are used to determine viral titers, but in vivo infectivity assays can also be employed. Detection of viral amplification can be performed, for example, by microscopic examination (in the presence of clearly visible cytopathic effects), PCR-based assays, or antibody-based assays. Therefore, in some respects, LRV is calculated based on infectivity assays.

[0132] One non-limiting example is an in vitro infectiousness assay called the tissue culture infectious dose 50 (TCID50) assay. In this assay, a fluid sample and its serial dilutions are dispensed into 96-well plates inoculated with cells that can serve as hosts for the lipid-coated virus being measured. After inoculation, the plates are incubated at a time and temperature sufficient to allow viral replication in the host cells. After incubation, the cells are examined under a microscope for signs of infection, such as lysed cells, cells exhibiting cytopathic effects, or any other criteria indicating viral infection. Viral titers are calculated from a pattern of positive (virally infected) and negative (no viral infection) wells. The absence of any well showing positive signs of infection indicates that the fluid is substantially free of lipid-coated virus. Therefore, in some respects, the infectiousness assay used to calculate LRV is the TCID50 assay.

[0133] Another cell culture-based assay is the plaque assay, in which the virus-inducing effect in the cell culture layer is visible as plaques or macroscopically visible after treatment. No plaque-indicating fluid is substantially free of lipid-coated virus. In some respects, plaque assays are used to calculate LRV infectivity.

[0134] In some respects, the defoamer disclosed herein comes into contact with the product feed stream (e.g., harvest, loading solution, eluent, or filtrate) or any solution containing or suspected of containing a virus 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 respects, the defoamer disclosed herein comes into contact with the product feed stream or the virus-containing solution for about 10 to about 20 minutes, about 20 to about 30 minutes, about 30 to about 40 minutes, about 40 to about 50 minutes, about 50 to about 60 minutes, about 60 to about 70 minutes, about 70 to about 80 minutes, about 80 to about 90 minutes, about 90 to about 100 minutes, about 100 to about 110 minutes, about 110 to about 120 minutes, about 15 to about 30 minutes, about 30 to about 45 minutes, about 45 to about 60 minutes, about 60 to about 75 minutes, about 75 to about 90 minutes, about 90 to about 105 minutes, about 105 to about 120 minutes, about 30 to about 60 minutes, about 60 to about 90 minutes, about 90 to about 120 minutes, or about 60 to about 120 minutes. In some respects, the feed stream may be subjected to the defoamer disclosed herein for more than 2 hours, such as 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 feed stream (e.g., harvest, loading solution, eluent, or filtrate) is contacted with the defoamer disclosed herein at about 4°C to about 30°C. In some aspects, the feed stream is contacted with the defoamer disclosed herein at about 10°C to about 25°C. In some aspects, the feed stream is contacted with the defoamer disclosed herein at about 15°C to about 20°C. In some aspects, the feed stream is contacted with the defoamer disclosed herein at about 20°C. In some aspects, the feed stream is contacted with the defoamer disclosed herein at about ambient temperature. In some aspects, the feed stream is contacted with the defoamer disclosed herein at about 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, or 30°C.

[0136] In some respects, in the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamers disclosed herein to inactivate the virus in the feed stream does not result in an increase in the amount of protein aggregates (high molecular weight substances) exceeding the acceptable protein aggregation parameters of the total protein in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100.

[0137] In some aspects, during the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in changes in glycosylation levels exceeding acceptable glycosylation parameters of the total protein in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100. In some aspects, during the manufacture of therapeutic proteins, treating the feed stream with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in changes in glycosylation patterns; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100.

[0138] In some respects, during the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamers disclosed herein to inactivate viruses in the feed stream does not result in an increase in the deamidation of the therapeutic protein product exceeding acceptable protein deamidation parameters for the total protein in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or use Triton X-100. The deamidated products include proteins in which one or more glutamine and / or asparagine residues are deamidated. In some respects, the deamidation of the therapeutic protein product leads to a change in peptide charge. Methods for analyzing deamidated variants of therapeutic proteins are known in the art. For example, by pH-mediated ion-exchange chromatography or isoelectric focusing.

[0139] In some respects, during the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in an increase in the oxidation of the therapeutic protein product exceeding acceptable protein oxidation parameters of the total protein in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100. Oxidation products include proteins in which one or more oxygen-reactive amino acid residues (such as methionine, cysteine, and tyrosine) are oxidized. In some respects, oxidation of the product peptide leads to a change in peptide charge. Methods for analyzing oxidized variants of peptides are known in the art. For example, the oxidation level in a given peptide can be determined by LC-mass spectrometry.

[0140] In some respects, in the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in an increase in process impurities exceeding acceptable protein impurity parameters for the total protein in the product feed stream; for example, compared to a manufacturing process that does not use a defoamer or detergent or uses Triton X-100. Therefore, in some respects, treating the feed stream with the defoamer disclosed herein to inactivate the virus in the feed stream during the manufacture of therapeutic proteins does not alter the removal of process impurities during the manufacturing process.

[0141] For example, compared to the manufacturing process of therapeutic proteins using Triton X-100 to inactivate viruses or without the use of defoamers or detergents, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamers disclosed herein does not alter the removal of impurities during manufacturing. In some aspects, the use of the defoamers disclosed herein does not alter the removal of process impurities at specific steps in the manufacturing process (e.g., chromatographic steps, filtration steps, concentration steps, etc.). In some aspects, the use of the defoamers disclosed herein does not alter the removal of process impurities throughout the overall manufacturing process of the therapeutic protein. Process impurities include host cell proteins (HCPs), nucleic acids, leached protein A, peptides other than the desired peptide, endotoxins, viral contaminants, cell culture medium components, and variants, fragments, aggregates, or derivatives of the desired therapeutic protein.

[0142] In some respects, in the manufacture of therapeutic proteins, treating the feed stream (e.g., harvest, loading solution, eluent, or filtrate) with the defoamers disclosed herein to inactivate the virus in the feed stream does not result in an increase in HCP exceeding the acceptable protein HCP level in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100.

[0143] In some respects, in the manufacture of therapeutic proteins, treating the feed stream with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in an increase in residual DNA levels exceeding acceptable HCP levels in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100.

[0144] In some respects, in the manufacture of therapeutic proteins, treating the feed stream with the defoamer disclosed herein to inactivate the virus in the feed stream does not result in an increase in residual protein A levels exceeding acceptable levels of protein A in the product feed stream; for example, compared to manufacturing processes that do not use defoamers or detergents or that use Triton X-100. II.C. Therapeutic Protein

[0145] As disclosed above, in some respects, therapeutic proteins prepared according to the virus inactivation methods disclosed herein include, for example, antibodies, antibody fragments, the Fc portion of antibodies and their fusions, the antigen-binding portion 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 respects, the therapeutic proteins are produced in mammalian cells. In some respects, the mammalian cell lines are Chinese hamster ovary (CHO) cells or young hamster kidney (BHK) cells, mouse hybridoma cells, or mouse myeloma cells. Compared to corresponding processes using, for example, Triton X-100, the process of manufacturing therapeutic proteins using the defoamers disclosed herein does not adversely affect the product quality of the therapeutic proteins.

[0146] Any therapeutic protein that can be expressed in a host cell can be generated according to this disclosure and can be present in the provided composition. The therapeutic protein can be expressed from an endogenous gene in the host cell or from a gene introduced into the host cell through genetic engineering.

[0147] The methods and compositions provided can employ any cells suitable for growth and / or production of therapeutic proteins in a culture medium, including animal, yeast, or insect cells. In one aspect, the cells are any mammalian cells or cell types suitable for cell culture and expression of polypeptides. Therefore, the methods (e.g., methods for inactivating viruses) and compositions provided herein can employ any suitable cell type, including animal cells. In one aspect, these methods and compositions employ mammalian cells. These methods and compositions can also employ hybridoma cells. In one aspect, the mammalian cells are non-hybridoma mammalian cells that have been transformed with exogenously isolated nucleic acids encoding the desired therapeutic protein. In one respect, these methods and compositions employ mammalian cells selected from the group consisting of: human retinal cells (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 lines transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones for growth in suspension medium, Graham et al., J. Gen Virol. [Journal of General Virology], 36:59 (1977)); juvenile hamster kidney cells (BHK. ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 77:4216 (1980)); mouse supporting cells (TM4, Mather. Biol. Reprod. [Reproductive Biology], 23:243-251). (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences], 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In some respects, these methods and compositions employ CHO cells. In some aspects, the culture of CHO cell lines and the expression of therapeutic proteins derived from CHO cell lines are employed.Therapeutic proteins can be secreted into a culture medium from which they can be isolated and / or purified, or they can be released into the culture medium by lysing cells containing isolated nucleic acids encoding the therapeutic protein. II.D. Manufacturing methods and treatment methods

[0148] Some aspects of this disclosure relate to a method for treating a disease or condition, the method comprising administering a therapeutic protein to a subject, the therapeutic protein being manufactured by a process including a virus inactivation step according to a virus inactivation method disclosed herein (e.g., a virus inactivation method including the use of an antifoaming agent disclosed herein). A pharmaceutical composition is also provided, the pharmaceutical composition being manufactured by a process including a virus inactivation step according to a virus inactivation method disclosed herein (e.g., a virus inactivation method including the use of an antifoaming agent disclosed herein). This disclosure also provides a method for manufacturing a therapeutic protein, the method comprising a virus inactivation step according to a virus inactivation method disclosed herein (e.g., a virus inactivation method including the use of an antifoaming agent disclosed herein). II.E. Reagent Kits and Manufacturing Products

[0149] Some aspects of this disclosure relate to reagent kits or manufactured products that contain the defoaming agent disclosed herein and, optionally, instructions for inactivating viruses according to the methods disclosed herein, in one or more containers. Those skilled in the art will readily recognize that the defoaming agent disclosed herein can be readily incorporated into one of the established reagent kit formats well known in the art. In some aspects, the reagent kit or manufactured product includes instructions for virus inactivation according to the methods disclosed herein.

[0150] All references cited above, as well as all references cited in this paper, are incorporated herein by reference in their entirety.

[0151] The following examples are provided in an illustrative rather than restrictive manner. Example Example 1

[0152] Exogenous viruses remain a critical issue in the production of biologics. To ensure patient safety, downstream purification processes require robust virus removal strategies to ensure the removal of potential viral contamination. For enveloped viruses, low pH conditions or chemical inactivators are key components of the removal strategy in virus inactivation processes. For biologics with poor stability under low pH conditions, chemical inactivators provide the necessary virus inactivation without stability issues. Historically, the nonionic surfactant Triton X-100 has provided excellent virus inactivation. However, due to safety concerns regarding Triton X-100 degradation products, the European Chemicals Agency (ECHA) has mandated its replacement. To aid in the evaluation of new virus inactivation agents, a novel approach using retrovirus-like particles (RVLPs) has been developed to determine the log-viral decrease value (LRV) of the inactivation agent. RVLPs are endogenously generated, non-infectious, membrane-bound viral particles from the CHO. They possess properties comparable to those of infectious membrane-bound viral particles traditionally used in infectiousity studies to validate virus inactivation conditions. This method was validated against two industry standards for these viruses (A-MulV and X-MulV), which showed high comparability. Figure 1A-1B Based on the physiological characteristics of RVLP compared to other related membrane-bound viruses and its comparability with model viruses, RVLP is expected to serve as a good model for membrane-bound viruses as a whole.

[0153] After validation, this method was used as a platform to study new candidates for virus inactivation agents. Defoaming chemicals were identified as a potentially advantageous class of molecules for virus inactivation. Defoamers are currently widely used in the biopharmaceutical industry to prevent unwanted foam generation in bioreactors and fermenters. Some defoamers also possess lubricating properties, reducing shear forces on cells caused by aeration and agitation conditions. Typically, defoaming chemicals used in cell culture contain emulsions of polydimethylsiloxane (PDMS) or nonionic detergents. Variations in defoamers are caused by the emulsifiers or detergents used. Because defoaming chemicals are already used in biopharmaceutical manufacturing, they have established supply chains, regulatory familiarity, and known removal and detection strategies. Interestingly, they also offer the possibility of using the same products in cell culture and virus inactivation. The use of defoamer A, defoamer B emulsion, defoamer C emulsion, Pluronic F-68 nonionic surfactant, Pluronic 31R1, defoamer SE-15, defoamer L-30 emulsion, and defoamer 204 was investigated (Table 1). To the best of our knowledge, this study is the first to investigate the use of defoamers for virus inactivation.

[0154] Materials and methods

[0155] RVLP-based inactivation test method

[0156] An internal method for characterizing enveloped viruses using retrovirus-like particles (RVLPs) has been developed. Figure 2 This method was developed by adapting our previous work to Phi6 phage (Feroz, H. et al., Surrogate model to screen for inactivation-based clearance of enveloped viruses during biotherapeutics process development. Biotechnology Journal, 2021. 16(12): p. 2100176.). This method allows for the differentiation between damaged and intact viral particles.

[0157] Incorporate a 1% RVLP viral particle solution into a clear cell culture harvest of monoclonal antibody (mAb) or fusion protein. Add the target inactivation reagent or detergent, proteinase K to a final concentration of 10.8 mAU / mL, and PMAxx to a final concentration of 0.24 mM to each sample. For control samples, omit the target inactivation reagent or detergent and use water instead. Gently mix the solutions and incubate at room temperature under LED illumination at 465–475 nm for the required incubation period. Proteinase K degrades the membrane-damaged viral particles, and PMAxx intercalates into free RNA and covalently binds to free RNA under light exposure, preventing it from participating in RT-qPCR. After the incubation period, extract viral RNA using the MockV RNA Extraction Kit. RNA extraction is performed using the provided kit materials and methods after the RNA precipitation reaction (MockV RVLP Kit Guide. 2023, Cygnus Technologies.). In addition, the kit provides an RNA standard, which is serially diluted eight times and extracted along with the target sample, allowing for the determination of a quantifiable range. After RNA extraction, RT-qPCR was performed using the MockV RVLP qPCR kit with a FAM reporter gene, following the instructions provided in the kit (MockV RVLP Kit Guide. 2023, Cygnus Technologies.). A standard curve was used to determine the RNA concentrations of the inactivated reagent-treated or untreated control.

[0158] Then use the following equation (Equation 1) to calculate the logarithmic decrease value (LRV). Equation 1.

[0159] Defoamer test

[0160] The virus inactivation capabilities of a group of defoaming chemicals were tested. The defoamers tested and their manufacturers are listed (Table 1). Table 1. Virus inactivation ability of the tested defoamers.

[0161] result

[0162] The virus inactivation ability of defoamers was successfully screened. A wide range of defoamers were tested (Table 1), and the number of LRVs was compared with known virus inactivation chemicals. Figure 3A Defoamer B and defoamer 204 showed performance comparable to known detergents, with LRVs of 4.71 and 3.98, respectively, while defoamers SE-15, L-30, and Pluronic 31R1 showed clearance rates close to two logarithms. Defoamers A, C, and Pluronic F-68 did not show significant virus inactivation. The concentration dependence of the virus inactivation capabilities of defoamers A, B, C, and 204 was also tested. Figure 3B The defoamer conditions and concentrations tested, along with the raw RT-qPCR results and calculated LRV, were recorded (Table 2). Based on the results, the virus inactivation ability of defoamer B may not solely originate from PDMS. Defoamer A, composed entirely of PDMS but without emulsifiers, performed poorly. Similarly, the poor performance of defoamer C may be due to the different emulsifiers used for suspending PDMS. SE-15 and L-30 may have similar mechanisms to defoamer B. Defoamer 204 is an organic, non-silicone polypropylene-based polyether dispersion. The surfactant properties of defoamer 204 may be the mechanism by which it acts as a virus inactivator. Pluronic 31R1 is also a non-silicone ethylene oxide and propylene oxide reverse block copolymer. Table 2. Experimental results of the defoamer experiment.

[0163] It should be understood that the Detailed Description section, rather than the Summary and Abstract section, is intended to be used to interpret the claims. The Summary and Abstract section may set forth one or more, but not all, exemplary aspects of this disclosure as contemplated by the inventors, and is therefore not intended to limit this disclosure and the appended claims in any way.

[0164] This disclosure has been described above using functional building blocks that illustrate the implementation of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries can be defined as long as the specified functions and their relationships are properly executed.

[0165] The foregoing description of the specific aspects so fully reveals the general nature of this disclosure that others can readily modify and / or adapt such specific aspects for various applications by applying knowledge within the scope of the art, without excessive experimentation and without departing from the general concepts of this disclosure. Therefore, based on the teachings and guidance presented herein, such modifications and adaptations are intended to be within the meaning and scope of equivalent solutions to the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive rather than limiting purposes, and therefore the terminology or terminology in this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0166] The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the following claims and their equivalents.

[0167] All references cited in this application (including bibliographic references, U.S. or foreign patent or patent application information, and websites) are hereby expressly incorporated by reference as if they were written in their entirety herein for any purpose. In the event of any inconsistency, the material disclosed herein shall prevail.

Claims

1. A method for inactivating a virus in a product feed stream during a downstream manufacturing process of a therapeutic agent, the method comprising contacting the product feed stream with an antifoaming agent.

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 for inactivating viruses in a eukaryotic cell culture via an inactivation product feed stream, the method comprising contacting the cell culture with an antifoaming agent.

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

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

6. The method of any one of claims 1 to 5, wherein the defoamer 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 glycerol esters or PEG esters.

9. The method of any one of claims 6 to 8, wherein the defoamer 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 defoamer 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% of a silicone polymer.

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

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

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

15. The method of claim 13 or 14, wherein the product feed stream 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%.

16. The method of any one of claims 13 to 15, wherein the product feed stream 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 feed stream and concentration are made to be 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%, approximately 3.0%, approximately 3.1%, approximately 3.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5%, or approximately 10.0% ANTIFOAM B™ contact.

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

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

20. The method of claim 18 or 19, wherein the product feed stream is contacted with defoamer 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%.

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

22. The method of any one of claims 18 to 21, wherein the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, or approximately 3.0%. Approximately 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% of defoamer SE-15 in contact with defoamer SE-15.

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

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

25. The method of claim 23 or 24, wherein the product feed stream is contacted with defoamer 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%.

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

27. The method of any one of claims 23 to 26, wherein the product feed stream and concentration are made to be 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%, or about 3.0%. %, approximately 3.1%, approximately 3.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5% or approximately 10.0% of defoamer L-30 in contact with defoamer L-30.

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

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

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

31. The method of claim 29 or 30, wherein the product feed stream is contacted with defoamer 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%.

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

33. The method of any one of claims 29 to 32, wherein the product feed stream and concentration are approximately 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.25%, approximately 0.5%, approximately 0.75%, approximately 1.0%, approximately 1.1%, approximately 1.2%, approximately 1.3%, approximately 1.4%, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 2.2%, approximately 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, or approximately 3%. 0%, approximately 3.1%, approximately 3.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5% or approximately 10.0% of defoamer 204 in contact with defoamer 204.

34. The method of any one of claims 1 to 33, wherein the defoamer comprises one or more bifunctional block copolymer surfactants.

35. The method of claim 34, wherein the bifunctional block copolymer surfactant comprises a terminal secondary hydroxyl group.

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

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

38. The method of claim 37, wherein the product feed stream 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 feed stream 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%, or at least about 0.01% to about 10%.

40. The method of any one of claims 37 to 39, wherein the product feed stream 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 feed stream and concentration are made to be 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%, approximately 3.0%, approximately 3.1%, approximately 3.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, approximately 7.0%, approximately 7.5%, approximately 8.0%, approximately 8.5%, approximately 9.0%, approximately 9.5%, or approximately 10.0% of PLURONIC® 31R1 contacts.

42. The method of any one of claims 1 to 41, wherein the product feed stream does not come into contact with the Triton X-100.

43. The method of any one of claims 1 to 42, wherein the product feed stream comprises harvested material from a bioreactor, chromatographic loading solution, chromatographic eluent, filtration loading solution, filtrate, or any combination thereof.

44. The method of claim 43, wherein the chromatographic eluent is protein A chromatographic eluent.

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 logarithmic decrease 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 follows: 。 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 contact 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, an 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 moiety.

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

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

59. A method of treating a disease or ailment, the method comprising administering a therapeutic protein to a subject, the therapeutic protein being manufactured by a process including a virus inactivation step according to any one of claims 1 to 58.

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

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

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