Washing agents and methods for purifying biological products

Abstract

Provided herein is an environmentally friendly detergent for use in the purification of recombinantly produced biological products. This environmentally friendly detergent contains laureth-9 and can be used for virus clearance, cell lysis, and removal of impurities such as host cell proteins and endotoxins. Advantageously, laureth-9 does not adversely affect product quality.

Description

[Technical Field]

[0001] Provided herein are environmentally friendly cleaning agents for use in the purification of biological therapeutics. These environmentally friendly cleaning agents are suitable for virus elimination, cell lysis, and removal of impurities such as proteins and endotoxins from host cells, and do not adversely affect product quality. [Background technology]

[0002] Biologics (e.g., macromolecular therapies such as polypeptides, proteins, antibodies, polynucleotides, and viral vectors) are rapidly growing as a therapeutic option available to physicians for the treatment of a wide range of diseases and disorders. One reason for the success of biologics is their high target specificity and superior safety profile compared to many small molecule therapies.

[0003] Biological products are typically manufactured using biological materials such as cell lines, cell culture media, and tissue fluid or body fluids, and carry risks such as viral contamination from contaminated cell banks (endogenous contamination) or viral contamination from the introduction of viruses during manufacturing (exogenous contamination).

[0004] Viruses contain DNA or RNA, are single-stranded or double-stranded, and are enveloped or non-enveloped. The infectious unit of a non-enveloped virus, the byron, contains a capsid protein and nucleic acid (DNA or RNA). Enveloped viruses contain a lipid bilayer surrounding the capsid, which contains membrane-associated proteins encoded by the virus.

[0005] The manufacturing process for biological products typically includes steps to remove viruses from the product stream, as well as other impurities such as host cell proteins and endotoxins. The viral elimination process may include physical removal steps and / or viral inactivation steps. Physical removal is generally achieved by viral filtration or column chromatography. Viral inactivation refers to a process that causes irreversible destruction or denaturation of the viral structure and can be achieved by incubating the product stream with a solvent or detergent, by heating the product stream (pasteurization), or by exposure to a low (acidic) pH. Purification processes often include both physical removal and chemical inactivation steps to enhance viral elimination.

[0006] Viruses enclosed by an outer membrane (enveloped viruses) are generally susceptible to exposure to solvents / cleaning agents and low pH, while non-enveloped viruses with an external protein capsid have higher physiological and chemical resistance and generally require physical removal. Non-patent document 1.

[0007] Triton X-100 is a non-ionic detergent that has been commonly used by manufacturers in the production of commercially available biological preparations for more than 30 years due to its effectiveness in viral inactivation, and typically achieves more than 4 log of enveloped virus inactivation under various sets of experimental conditions. Non-Patent Document 1. However, recent ecological studies have suggested that Triton X-100 and octylphenol, a decomposition product of Triton X-100, may act as endocrine-disrupting chemicals in aquatic organisms, raising concerns from the perspective of environmental impact. Non-Patent Document 1. In 2012, the European Chemicals Agency (ECHA) included Triton X-100 on the candidate list of Substances of Very High Concern (SVHC) under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation. In 2019, ECHA included Triton X-100 on the authorization list (Annex XIV) and ordered the replacement of Triton X-100 in manufacturing processes by the "sunset date" of January 4, 2021.

[0008] Patent Document 1 (METHODS FOR VIRAL INACTIVATION USING ECO-FRIENDLY DETERGENTS) describes the use of Triton CG-110, an environmentally compatible detergent, as an alternative to Triton X-100 for viral inactivation.

[0009] Patent Document 2 (METHODS OF INACTIVATION OF VIRUSES USING N-METHYLGLUCAMIDE AND ITS DERIVATIVES) describes the use of N-methylglucamide as an alternative to Triton X-100 for viral inactivation.

[0010] Lauryl dimethylamine N-oxide (LDAO) is an environmentally friendly zwitterionic detergent that replaces Triton X-100 and has also been evaluated for enveloped virus inactivation. See Non-Patent Document 1.

Prior Art Literature

Patent Literature

[0011]

Patent Document 1

Patent Document 2

Non-Patent Literature

[0012]

Non-Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0013] However, there still remains a need for alternative detergents that are environmentally friendly and effective for virus inactivation during the production of commercially available biological products.

Means for Solving the Problem

[0014] Provided herein is an environmentally compatible detergent for use in the manufacture of a biological product of interest. Specifically, an environmentally compatible detergent for use in the purification of recombinantly produced biological products is provided. In one aspect, the environmentally compatible detergent is used for viral inactivation, cell lysis, removal of impurities such as host cell proteins and endotoxins, or a combination thereof. In one aspect, the environmentally compatible detergent does not adversely affect product quality. In one aspect, the environmentally compatible detergent includes laureth-9.

[0015] In one aspect, a method for inactivating an enveloped virus is provided. In one aspect, a method for inactivating an enveloped virus in a mixture comprising a recombinant biological product of interest is provided. In one aspect, the method comprises adding to the mixture a solution comprising a virus-inactivating amount of laureth-9, and incubating the mixture to inactivate the enveloped virus. In one aspect, the solution comprising laureth-9 provides about 1 log 10 , 2 log 10 , 3 log 10 , or 4 log 10 greater log reduction value (LRV) as compared to a control that does not contain a surfactant. In one aspect, the solution comprising laureth-9 provides about 1 log 10 , 2 log 10 , 3 log 10 , or 4 log 10 greater log reduction value (LRV) as compared to a control that does not contain Triton X-100. In one aspect, the solution comprising laureth-9 provides about 1 log 10 , 2 log 10 , 3 log 10 , or 4 log 10This results in a logarithmically reduced value (LRV). In one embodiment, the LRV is determined by quantitative PCR or an infectivity assay. In one embodiment, the LRV is determined by a plaque assay. In one embodiment, the solution contains about 0.1% to about 2% (w / v) laureth-9. In one embodiment, this mixture is incubated with laureth-9 for at least about 1 minute. In one embodiment, this mixture is incubated with laureth-9 for at least about 5 minutes. In one embodiment, this mixture is incubated with laureth-9 for about 1 minute to about 120 minutes. In one embodiment, this mixture is incubated with laureth-9 at room temperature.

[0016] In one embodiment, the enveloped virus is a DNA virus or an RNA virus. In another embodiment, the enveloped virus is a single-stranded virus or a double-stranded virus. In one embodiment, the enveloped virus may belong to the families Herpesviridae, Hepadnaviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, Arteriviridae, or a combination thereof.

[0017] In one embodiment, a method for lysing cells is provided. In one embodiment, a method for lysing cells expressing a recombinant biological agent of interest is provided. In one embodiment, the method comprises contacting a mixture containing the cells with a solution containing a cell-destroying amount of laureth-9, and incubating the mixture to lyse the cells. In one embodiment, after incubation of the mixture with the laureth-9 solution, at least about 75%, 80%, 85%, 90%, or 95% of the cells in the mixture are lysed. In one embodiment, the solution contains about 0.1% to about 2% (w / v) laureth-9. In one embodiment, the mixture is incubated with laureth-9 for at least about 1 minute. In one embodiment, the mixture is incubated with laureth-9 for at least about 5 minutes. In one embodiment, the mixture is incubated with laureth-9 for about 1 minute to about 120 minutes. In one embodiment, the mixture is incubated with laureth-9 at room temperature.

[0018] In one embodiment, a method is provided for removing endotoxin from a mixture containing a biological agent of interest. In one embodiment, the method comprises contacting the mixture containing the biological agent of interest with a solution containing an endotoxin-removing amount of laureth-9, and incubating the mixture. In one embodiment, the endotoxin concentration is reduced to less than about 1 EU / mg. In one embodiment, the solution contains about 0.1% to about 2% (w / v) laureth-9.

[0019] In one embodiment, the method includes filtering a mixture containing the recombinant biological product of interest. In one embodiment, the filtering is performed before incubating the enveloped virus with a solution containing laureth-9. In one embodiment, the filtering is performed after incubating the enveloped virus with a solution containing laureth-9. In one embodiment, the filtering includes ultrafiltration or deep filtration.

[0020] In one embodiment, the method comprises one or more chromatography steps. In one embodiment, one or more chromatography steps comprise loading a mixture containing the biological agent of interest onto a chromatography carrier and washing the chromatography carrier with a washing solution containing laureth-9. In one embodiment, the washing solution contains about 0.1% to about 2 (w / v) laureth-9.

[0021] In one embodiment, a method for purifying a biological product of interest is provided. In one embodiment, the method comprises loading a mixture containing the biological product of interest and one or more impurities onto a chromatographic carrier; washing the chromatographic carrier with a washing solution containing laureth-9; and eluting the biological product of interest from the chromatographic carrier to obtain a purified eluate containing the biological product of interest.

[0022] In one embodiment, the washing solution contains a host cell protein removal amount of laureth-9. In one embodiment, the purified eluate has a reduced host cell protein content compared to the host cell protein content in the eluate from a chromatography support that was not washed with a washing solution containing laureth-9. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is reduced by at least about 50%, 45%, 40%, 35%, 30%, or 25% compared to the host cell content of the mixture applied to the column. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is about 2000 ng HCP / mg protein, 1700 ng / mg, 1500 ng / mg, 1200 ng / mg, 1000 ng / mg, 500 ng / mg, or less than 100 ng / mg.

[0023] In one embodiment, the washing solution contains an amount of laureth-9 sufficient to remove endotoxins. In another embodiment, the purified eluate has an endotoxin concentration of less than approximately 1 EU / mg.

[0024] In one embodiment, the cleaning solution contains approximately 0.1% to approximately 2% (w / v) laureth-9.

[0025] In one embodiment, the recombinant biological product of interest is a therapeutic macromolecule. In one embodiment, this therapeutic macromolecule is a therapeutic polypeptide or therapeutic polynucleotide. In one embodiment, the recombinant biological product of interest is an enzyme, a soluble receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen-binding antibody fragment, an antibody-drug conjugate, a fusion polypeptide, or a non-enveloped virus. In one embodiment, the recombinant biological product of interest is an antibody or an antigen-binding antibody fragment. In one embodiment, this antibody is a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, this non-enveloped virus is an adenovirus or adeno-associated virus (AAV).

[0026] In one embodiment, the recombinant biological agent of interest is produced by cells. In one embodiment, the recombinant biological agent of interest is produced by eukaryotic cells. In one embodiment, the recombinant biological agent of interest is produced by prokaryotic cells. In one embodiment, the recombinant biological agent of interest is produced by mammalian cells. In one embodiment, these mammalian cells are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, mouse hybridoma cells, or mouse myeloma cells.

[0027] In one embodiment, the chromatography is selected from one or more of affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and mixed-mode chromatography. In one embodiment, affinity chromatography is mentioned as the chromatography. In one embodiment, affinity chromatography is performed using a protein A chromatography step, a protein G chromatography step, a protein L chromatography step, or a camel antibody ligand (V HH) Selected from affinity chromatography steps. In one embodiment, ion exchange chromatography is selected from anion exchange chromatography and cation exchange chromatography.

[0028] In one embodiment, the method includes a step of filtering the product stream. In one embodiment, the filtering includes ultrafiltration or deep filtration.

[0029] In one embodiment, the target recombinant biological product has a biological activity of approximately 25%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of that of the control recombinant biological product. In one embodiment, the control recombinant biological product has not been in contact with laureth-9.

[0030] In one embodiment, a method for producing a recombinant non-enveloped virus is provided. In one embodiment, the method comprises culturing host cells containing the genome of a non-enveloped virus; and contacting the host cells with a solution containing laureth-9. In one embodiment, the host cells are contacted with a solution containing about 0.1% to about 2% laureth-9. In one embodiment, the host cells are contacted with the solution containing laureth-9 for about 1 minute to about 120 minutes. In one embodiment, the method comprises preparing a cell paste of host cells expressing a non-enveloped virus; and contacting the cell paste with a solution containing a cell-lysate amount of laureth-9. In one embodiment, the non-enveloped virus is an adenovirus or adeno-associated virus (AAV). In one embodiment, the purified non-enveloped virus has a physical titer of about 10 log 10 The infectivity titer is greater than vg / ml. In one embodiment, the purified non-enveloped virus has an infectivity titer of approximately 8 log. 10 The TCID is greater than 50 / mL. In one embodiment, viral infectivity was not significantly reduced compared to a control that had not been in contact with a solution containing laureth-9.

[0031] In one embodiment, a method for producing a recombinant non-enveloped virus is provided. In one embodiment, the method comprises the steps of: loading a mixture containing the non-enveloped virus onto a chromatography carrier; washing the chromatography carrier with a washing solution containing laureth-9; and eluting the non-enveloped virus from the chromatography carrier to obtain a purified eluate containing the non-enveloped virus. In one embodiment, the washing solution contains about 0.1% to about 2% (w / v) laureth-9.

[0032] In one embodiment, a method for purifying a non-enveloped virus is provided. In one embodiment, the method comprises contacting a mixture containing the non-enveloped virus with a solution containing laureth-9, and filtering the mixture. In one embodiment, filtration is performed before contacting the mixture with the solution containing laureth-9. In one embodiment, filtration is performed after contacting the mixture with the solution containing laureth-9. In one embodiment, filtration includes ultrafiltration or deep filtration. In one embodiment, the solution contains about 0.1% to about 2% (w / v) laureth-9. [Brief explanation of the drawing]

[0033] [Figure 1] The results show a reduction in Log10 viral load obtained with a 1% surfactant solution of Brij L9 (Laureth-9, obtained from Croda International Plc., East Yorkshire, United Kingdom), Polidocanol 600 (Laureth-9, obtained from Schaerer & Schlaepfer AG, Rothrist, Switzerland), Myrj S25, or Brij S20. [Figure 2]This report shows the Log10 viral reduction obtained for two antibody samples (BisAb and BisFusion) using various amounts of laureth-9 from different sources. BisAb was treated with a 1% solution of Brij L9 (laureth-9, obtained from Croda International Plc., East Yorkshire, United Kingdom) or a 1% solution of Polidocanol 600 (laureth-9, obtained from Schaerer & Schlaepfer AG, Rothrist, Switzerland). BisFusion was treated with a 1% or 0.1% solution of Brij L9 (laureth-9 obtained from Croda International Plc). Inactivation of more than 4Log10 was observed in all samples at 1 minute. [Modes for carrying out the invention]

[0034] Provided herein is a method for purifying biological products using environmentally friendly cleaning agents.

[0035] definition Overall, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry, as well as hybridization, as described herein are publicly known and commonly used in the art. In this specification, amino acids may be referred to by either a commonly known three-letter code or a single-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by a commonly accepted single-letter code.

[0036] As used herein, “a” or “an” (one) may mean one or more. As used herein and in the claims, when used together with the word “comprising,” “a” or “an” may mean one or more. As used herein, “another” or “further” may mean at least two or more.

[0037] Throughout this specification, the term “approximately” is used to indicate that a value includes inherent variations in error relating to the method / apparatus used to determine that value, or variations that exist between the test subjects. Typically, the term means, depending on the context, to include variations of approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0038] The use of the term “or” in the claims is used to mean “and / or” unless expressly otherwise, or unless such substitutes are mutually exclusive; however, this disclosure supports the definitions of substitutes only and “and / or” as applicable.

[0039] Where used herein and in the claims, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude any additional unquoted elements or steps of the method. Any embodiment considered herein is intended to be performed with respect to any method, system, host cell, expression vector, and / or composition of the Disclosure. Furthermore, the methods and proteins of the Disclosure can be obtained using the compositions, systems, host cells, and / or vectors of the Disclosure.

[0040] The use of the term “for example” and its corresponding abbreviation “eg” (whether italicized or not) means that the terms cited refer to representative examples and embodiments of the Disclosure, unless otherwise explicitly stated, are not intended to be limited to the specific examples referenced or cited.

[0041] The term "cleansing agent" refers to an amphiphilic molecule that has a hydrophilic (polar) head group and a hydrophobic (nonpolar) tail group. The amphiphilic structure of a cleaning agent allows it to interact with other molecules, such as proteins or enveloped viruses, in aqueous solutions. Cleansing agents can be included in the manufacturing process of biological products for various purposes, for example, as solubilizers; as stabilizers, for example, to prevent aggregation of biomolecules; for inactivating viruses; for removing impurities (e.g., host cell proteins or endotoxins); or in combination thereof. Cleansing agents can be classified into three groups according to their charge: Anionic cleaning agents contain a negatively charged hydrophilic group; Cationic cleaning agents contain a positively charged hydrophilic group; Nonionic cleaning agents have neither a positive nor a negative charge. Advantageously, nonionic cleaning agents help preserve the structure and activity of biomolecules. Zwitterionic surfactants, also known as amphoteric surfactants, possess both positive and negative charges, which can be permanent or pH-dependent.

[0042] The critical micelle concentration (CMC) is a concentration at which a detergent forms micelles. Below the CMC, the surface tension decreases as the concentration of the surfactant increases. Above the CMC, any further detergent added to the system will form micelles.

[0043] The term "surfactant" or "surface active agent" refers to a compound that contains both hydrophobic and hydrophilic groups and is semi-soluble in both organic solvents and aqueous solvents. Surfactants can be nonionic, cationic, anionic, or zwitterionic.

[0044] As used herein, “environmentally compatible” refers to a substance that has a low burden or impact on the natural environment. In one embodiment, “environmentally compatible” refers to a substance that conforms to regulatory guidelines or standards relating to environmental protection. In one embodiment, an “environmentally compatible” substance does not behave as an endocrine-disrupting chemical.

[0045] The term “bioactive agent” may refer to any substance suitable for therapeutic, prophylactic, or diagnostic use and may be used interchangeably with the term “therapeutic agent.” Bioactive agents may include “biological” products in which the active substance is derived from a biological origin. Examples of biological products include macromolecules, such as therapeutic polynucleotides or polypeptides. Examples of biological products include, but are not limited to, proteins and hormones, monoclonal antibodies (mAbs), cytokines, growth factors, gene therapy products, viral vectors, vaccines, and gene silencing / editing therapies. Bioactive agents may include, but are not limited to, macromolecules that are naturally occurring or manufactured by recombinant processes. In one embodiment, a bioactive agent is of therapeutic, scientific, or commercial interest.

[0046] The term "cell culture" refers to the growth and proliferation of host cells outside of a multicellular organism or tissue, and includes culturing host cells in suspension and / or attached to a solid substrate. Host cells may be cultured in small-scale cultures (e.g., in a laboratory environment) or large-scale cultures (e.g., in a commercial-scale bioreactor). Cell cultures may include continuous culture systems, batch culture systems, and fed-batch culture systems. Host cells may be cultured, for example, with or without microcarriers, in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors, and may be operated in batch mode, fed-batch mode, continuous mode, semi-continuous mode, or perfusion mode.

[0047] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. These polymers may be linear or branched and may contain modified amino acids, unnatural amino acids, or be interrupted by non-amino acids. In one embodiment, the terms “polypeptide” and “protein” refer to antibodies or antigen-binding antibody fragments.

[0048] The term "polynucleotide" refers to polymers of ribonucleic acid, polymers of deoxyribonucleic acid (DNA), or DNA / RNA hybrid polymers. Polynucleotides may be single-stranded or double-stranded and may contain sense or antisense polynucleotide sequences of DNA or RNA. Examples of DNA or RNA molecules include complementary DNA (cDNA), genomic DNA, synthetic DNA, or hybrids thereof, or RNA molecules (e.g., mRNA) containing untranslated and translated regions. In one embodiment, polynucleotides may include non-standard nucleotides (e.g., inosine, thiouridine, or pseudouridine) or chemically modified nucleotides.

[0049] An "isolated" polypeptide, protein, peptide, or polynucleotide is a molecule extracted from its natural environment. In one embodiment, an "isolated polypeptide" is recovered from cells or cell cultures in which the polypeptide is expressed. It should be understood that an "isolated" polypeptide, protein, peptide, or polynucleotide may be compounded with excipients (e.g., diluents or adjuvants) and still be considered isolated.

[0050] A "purified" polypeptide, protein, peptide, or polynucleotide is a molecule that has been purified to a degree that is purer than it would be in its natural environment and / or purer than it would be if it were originally produced, synthesized, or amplified. Purity is a relative term and does not necessarily require absolute purity.

[0051] "Product flow" refers to the material or solution containing the target biopharmaceutical molecule during a manufacturing or purification process. Examples of product flow include, but are not limited to, cell culture media, recovered cell culture fluid (HCCF), or a pool containing one or more post-purification biopharmaceutical processes.

[0052] In one embodiment, the product stream contains one or more impurities. "Impurities" refer to substances present in the product stream that are not the desired biological product. In one embodiment, the product stream contains one or more process-related impurities, including, but not limited to, host cell proteins (HCPs), host cell DNA, viral contaminants, endotoxins, extracts from resins and filters, and leached protein A. Viral contaminants may include viral fragments, viral nucleic acids, and exogenous or endogenous viruses. In one embodiment, product-related impurities include, but are not limited to, size variants, e.g., aggregates or fragments; charge variants; glycosylated variants; or variants resulting from oxidation, deamidation, or denaturation.

[0053] The term “recombinant,” when used in reference to polynucleotides, peptides, polypeptides, or proteins, means a combination of substances not known to exist naturally, or arising from such a combination. Recombinant molecules may be produced by any of the known techniques available in the field of recombinant technology, including, but not limited to, polymerase chain reaction (PCR), gene splicing (e.g., gene splicing using restriction endonucleases), and solid-phase synthesis of nucleic acid molecules, peptides, or proteins. The term “recombinantly expressed” means that the protein of interest is expressed in a “recombinant host cell” that has been genetically modified or can be genetically modified by the introduction of exogenous polynucleotides, such as recombinant plasmids or vectors. It should be understood that such terms are intended to refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain mutations may occur in the progeny due to either mutation or environmental influences, but they are still included in the scope of the term “recombinant host cell” as used herein.

[0054] "Virus removal" refers to the removal or inactivation of viruses from a mixture (e.g., a product stream containing the biological agent of interest). Virus removal refers to a reduction in the number of virus particles in the sample and can be achieved by methods such as affinity chromatography or filtration. "Virus inactivation" refers to the process of rendering viruses contained in a composition nonfunctional. Methods of virus inactivation are known and include, for example, thermal activation, pH inactivation, and chemical inactivation using, for example, surfactants.

[0055] Log 10 "Reduction Value" or "LRV" refers to the calculated ratio between the viral titer in the starting material and the viral titer in the associated product fraction. LRV can be used to describe the ability of a process step to remove or inactivate the virus. LRV can be determined using known model viruses such as mouse leukemia virus (MuLV) or mouse microvirus (MVM), and can be calculated by quantitative PCR (qPCR) or by infectivity assay.

[0056] The term “bioactivity” refers to the function of a molecule and may encompass biological, biochemical, physical, and chemical functions. Examples of bioactivity include, but are not limited to, enzymatic activity; the ability to interact with or bind to another molecule; the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of another molecule; and the ability to localize to a specific location within a cell. As used herein, “bioactivity” with respect to a polynucleotide (e.g., a gene) or related polypeptide may refer to a specific function that the polynucleotide, gene, or polypeptide may have in vivo. Examples include, but are not limited to, the production of a specific protein, enzymatic activity, the conferral of resistance, and the like. Bioactivity may be measured using techniques known in the art. Bioactivity may be evaluated using in vitro, in vivo, and / or in situ assays that demonstrate activity for a particular biological agent.

[0057] Filtration is the process of separating particles from a suspension by passing the liquid through the pores of a filter. The liquid that passes through the filter is called the filtrate. The retaining liquid is the portion that is held by the filter.

[0058] overview Provided herein are environmentally friendly cleaning agents for use in the manufacture of biological products. In one embodiment, the biological product is manufactured by recombinant DNA. This environmentally friendly cleaning agent can be used at any step in the manufacturing process. In one embodiment, the environmentally friendly cleaning agent is used in a downstream purification process. In one embodiment, the environmentally friendly cleaning agent is used in the purification process of the biological product of interest. In one embodiment, environmentally friendly cleaning agents are provided for virus elimination, cell lysis, and removal of impurities (host cell proteins and endotoxins). In one embodiment, the environmentally friendly cleaning agent does not adversely affect the quality of the product.

[0059] Advantageously, the product quality of the biological product is maintained after contact with the environmentally friendly cleaning agent. In one embodiment, the biological activity of the biological product is within approximately 25%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the activity of the control biological product (e.g., a control biological product not in contact with a surfactant). In one embodiment, the biological activity of the biological product is within approximately 25%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the activity of the control biological product (e.g., a control biological product not in contact with Triton X-100). In one embodiment, the biological activity of the biological product is within approximately 25%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the biological activity of the control biological product (e.g., a control biological product not in contact with Laureth-9). Methods for determining the biological activity of biological agents are known, including, for example, in vitro, in vivo, and / or in situ assays that demonstrate the activity of a particular biological agent.

[0060] Nonionic detergents to replace Triton X-100 were evaluated based on hydrophilic-lipophilic balance (HLB), water solubility, virus inactivation kinetics, availability, safety, and environmental compatibility. Surprisingly, in contrast to the findings of Conley et al. (2017) “Evaluation of eco-friendly zwitterionic Detergents for Enveloped Virus Inactivation” Biotech. and Bioeng. 114(4):813-820, which concluded that Brij 35 (polyoxyethylene(23) lauryl ether), a surfactant structurally related to laureth-9 but with a longer polyoxyethylene chain, is not effective in virus inactivation, the environmentally friendly nonionic surfactant laureth-9 demonstrated process performance and product quality equivalent to Triton X-100 in terms of virus inactivation, cell lysis, host cell protein (HCP) elimination, and endotoxin elimination.

[0061] Laureth-9 In one embodiment, an environmentally friendly cleaning agent is provided for use in the purification of biological products. In one embodiment, the biological product is manufactured by recombinant DNA. In one embodiment, the environmentally friendly cleaning agent contains laureth-9.

[0062] Laureth-9 (CAS numbers: 3055-99-0; 9002-92-0; 68439-50-9) is a nonionic detergent (HLB value approximately 13.3) with the chemical formula C 30 H 62 O 10 (Due to its polymerizability, it exhibits an average empirical formula); its average molecular weight is 580 g / mol; and its IUPAC chemical name is 3,6,9,12,15,18,21,24,27-nonoxanonatricontan-1-ol. Formula 1 below shows a typical structure of laureth-9. [ka]

[0063] Laureth-9 can be produced by the reaction of alcohol with ethylene oxide, which typically produces a mixture of ethoxylates with different numbers of ethylene oxide units. Laureth-9 has an average alkyl chain of 12 to 14 carbon atoms (C 12~14 It has an average ethylene oxide chain (EO9) consisting of 9 ethylene oxide units.

[0064] Laureth-9 is also known as: Brij L9; polidokanol 600; polidokanol; polyoxyethylene(9) lauryl ether; polyethylene glycol 450 lauryl ether; PEG-9 lauryl ether; nonaoxyethylene monododecyl ether; nonaethylene glycol monododecyl ether; nonaethylene glycol monolauryl ether; lauromacrogol 400; macrogol 9 lauryl ether; polyoxyl 9 lauryl ether; Aethoxy-sklerol; polyoxyl 9 lauryl ether; and macrogol lauryl ether 9.

[0065] In one embodiment, a solution containing laureth-9 is provided. In one embodiment, the solution contains laureth-9, a solvent, and one or more other agents such as chelating agents or preservatives. In one embodiment, a solution containing laureth-9 and one or more additional cleaning agents is provided. In one embodiment, the solution does not contain Triton X-100. In one embodiment, a solution is provided containing at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) of laureth-9 and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) of laureth-9. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9.

[0066] Virus inactivation Virus elimination is one of the goals of downstream purification processes. Methods for virus elimination include physical removal of virus particles and virus inactivation. Methods for removing virus particles include filtration, which can remove both enveloped and non-enveloped viruses. Methods for virus inactivation include heating (pasteurization), low pH inactivation, and / or washing. In one embodiment, one or more virus inactivation methods are used to reduce the level of enveloped virus in the product stream containing the biological agent of interest.

[0067] In one embodiment, a method for inactivating a virus is provided. In one embodiment, a method for inactivating an enveloped virus is provided. In one embodiment, viral inactivation is carried out in a mixture containing the biological product of interest. In one embodiment, a method for inactivating an enveloped virus during the manufacture of the biological product of interest is provided. In one embodiment, the mixture may include, but may not include, a product stream from a purification process of the biological product of interest. In one embodiment, the product stream may include, but is not limited to, cell culture media, recovered cell culture fluid (HCCF), a capture pool, a flow-through pool, or a filtrate.

[0068] Enveloped viruses are viruses that typically contain a coat derived from a portion of the host cell membrane, which also contains several viral glycoproteins. These glycoproteins on the surface of the coat help the virus enter the host cell by binding to receptor sites on the host cell membrane. The virus infects the host cell by fusion of its coat with the host cell membrane. The lipid bilayer coat of the virus is relatively sensitive to cleaning agents. In one embodiment, an environmentally friendly cleaning agent is provided that inactivates enveloped viruses by damaging the viral coat (for example, by dissolving the viral coat).

[0069] Enveloped viruses include DNA viruses and RNA viruses, and these enveloped viruses can be single-stranded or double-stranded. Examples of enveloped viruses include, but are not limited to, the following: Herpesviridae, e.g., herpes simplex virus, varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus; Hepadnaviridae, e.g., hepatitis B virus; Togaviridae, e.g., rubella virus; Arenaviridae, e.g., lymphocytic choriomeningitis virus; Flaviviridae, e.g., dengue virus, hepatitis C virus, and yellow fever virus; Orthomyxoviridae, e.g., influenza virus A, influenza virus B, influenza virus C, etc. Saviruses and sogothoviruses; Paramyxoviridae, e.g., measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, and canine distemper virus; Bunyaviridae, e.g., California encephalitis virus and hantavirus; Filoviridae, e.g., Ebola virus and Marburg virus; Coronaviridae, e.g., coronavirus; Astroviridae, e.g., astrovirus; Bornaviridae, e.g., Borna disease virus; and Arteriviridae, e.g., Arterivirus and equine arteritis virus.

[0070] In one embodiment, an enveloped virus is inactivated by incubation of a mixture containing the biological agent of interest with an environmentally friendly cleaning agent. In one embodiment, the environmentally friendly cleaning agent contains laureth-9. In one embodiment, the environmentally friendly cleaning agent contains a virus-inactivating amount of laureth-9. With respect to the environmentally friendly cleaning agent, “virus-inactivating amount” refers to the amount (e.g., concentration, volume, or weight ratio) of the cleaning agent that is effective in solubilizing at least a portion of the membrane proteins that form the protein coat surrounding the enveloped virus, thereby causing the lysis of the virus. In one embodiment, “virus-inactivating amount” refers to the amount of cleaning agent that damages and / or dissolves the protein coat of the virus without adversely affecting the biological agent of interest. In one embodiment, a mixture containing the biological agent of interest is brought into contact with a solution containing at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, this solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, this mixture is incubated with the laureth-9 solution for about 1 minute to about 120 minutes. In one embodiment, the mixture is incubated with a solution containing laureth-9 for at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes and up to about 60, 90, or 120 minutes. In one embodiment, the mixture is incubated with a solution containing laureth-9 for up to about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, the mixture is incubated with a solution containing laureth-9 for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 60, 90, or 120 minutes. In one embodiment, the mixture is incubated with a solution containing laureth-9 for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, the mixture is incubated with laureth-9 for about 1 minute.In one embodiment, the mixture is incubated with laureth-9 for at least about 5 minutes. In one embodiment, the mixture is incubated with laureth-9 at a temperature of about 4°C to about 42°C. In one embodiment, the mixture is incubated with laureth-9 at a temperature of about 4°C to about 25°C. In one embodiment, the mixture is incubated with laureth-9 at room temperature (e.g., about 20°C to about 25°C). In one embodiment, the mixture is brought into contact with a solution containing laureth-9 at a pH of about 4.5 to about 8.5. In one embodiment, the mixture is brought into contact with a solution containing laureth-9 at a pH of about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8, or 8.5.

[0071] In one embodiment, viral inactivation is expressed as a logarithmic reduction value (LRV). In one embodiment, the logarithmic reduction value is LRV = log 10 It can be calculated as × (total virus in the product stream after treatment with Laureth-9 / total virus in the product stream before treatment with Laureth-9). In one embodiment, the logarithmic reduction value (LRV) obtained with this detergent solution is approximately 1 log 10 It is extremely high. In one embodiment, the LRV is about 1 log compared to a control that was not incubated with the surfactant. 10 , 2log 10 , 3log 10 , or 4log 10 It is extremely high. In one embodiment, LRV is about 1 log compared to a control that was not incubated with a surfactant containing Triton X-100. 10 , 2log 10 , 3log 10 , or 4log 10 It is extremely high. In one embodiment, the LRV is about 1 log compared to a control that was not incubated with a surfactant containing laureth-9. 10 , 2log 10 , 3log 10 , or 4log 10It is extremely rare. Methods for determining LRV are known, such as quantitative PCR or infectivity assays. In one embodiment, LRV is determined by a plaque assay. In one embodiment, viral inactivation can be determined by adding a test virus such as X-MuLV to a sample, incubating the sample, and determining viral inactivation using, for example, a plaque assay.

[0072] In one embodiment, the method includes a step of filtering a mixture containing the biological agent of interest. In one embodiment, the filtering is performed before incubating the enveloped virus with a solution containing laureth-9. In one embodiment, the filtering is performed after incubating the enveloped virus with a solution containing laureth-9. In one embodiment, the filtering includes ultrafiltration. In one embodiment, the filtering includes deep filtration.

[0073] Virus inactivation using a washing agent such as laureth-9 can be carried out at any step in the manufacturing process. In one embodiment, virus inactivation includes contacting a product stream from the purification process of the biological product with a solution containing laureth-9. In one embodiment, cell culture medium is contacted with a solution containing laureth-9. In one embodiment, recovered cell culture medium is contacted with a solution containing laureth-9. In one embodiment, a capture pool from a chromatography resin containing the biological product of interest is contacted with a solution containing laureth-9. In one embodiment, a flow-through pool from a chromatography resin containing the biological product of interest is contacted with a solution containing laureth-9. In one embodiment, a filtrate containing the biological product of interest is contacted with a solution containing laureth-9. In one embodiment, a retention solution containing the biological product of interest is contacted with a solution containing laureth-9.

[0074] cell culture In one embodiment, the biological product is manufactured by cell culture. In one embodiment, the biological product is expressed from a gene that is endogenous to the host cell. In one embodiment, the biological product is expressed from a gene that has been introduced into the host cell, for example, by genetic engineering. In one embodiment, the biological product may be naturally occurring. In one embodiment, the biological product may be manufactured or engineered by recombinant DNA. In one embodiment, the biological product is constructed from segments that are individually naturally occurring. In one embodiment, the biological product includes one or more segments that are not naturally occurring.

[0075] In one embodiment, the biological agent is produced in adherent cell culture. In another embodiment, the biological agent is produced in suspension cell culture. In one embodiment, the biological agent is produced in prokaryotic cells. In one embodiment, the prokaryotic cell is Escherichia coli. In one embodiment, the biological agent is produced in eukaryotic cells. In one embodiment, the biological agent is produced in animal cells. In one embodiment, the biological agent is produced in yeast cells. In one embodiment, the yeast cells are selected from Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica. In another embodiment, the biological agent is produced in insect cells. In one embodiment, the insect cells are Spodoptera frugiperda (Sf9) insect cells. In another embodiment, the biological agent is produced in mammalian cells. In one embodiment, the mammalian cells are selected from the following: human retinoblast cells (PER.C6); human embryonic kidney cells (HEK-293); baby hamster kidney cells (BHK); Chinese hamster ovary cells (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HeLa); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT); TRI cells (Mather et al., Annals NYAcad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma strain (Hep G2). In one embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells. In another embodiment, the cells are hybridoma cells.

[0076] In one embodiment, the biological agent is secreted into the cell culture medium. In another embodiment, the biological agent is expressed in the cytoplasm of a host cell. In another embodiment, the biological agent is expressed in the host cell membrane. In another embodiment, the host cell is lysed to release the biological agent. In another embodiment, the cell culture medium is clarified, for example by centrifugation, to remove cells and cell debris, thereby forming a clarified cell culture medium. In another embodiment, the cell culture medium is purified, for example, by one or more chromatography steps, one or more filtration steps, or a combination thereof.

[0077] Cell lysis In one embodiment, a method for lysing host cells is provided. In one embodiment, the host cells are present in a cell culture. In one embodiment, the host cells are present in a cell slurry. In one embodiment, the host cells are lysed by contacting the cells with a nonionic surfactant. In one embodiment, the host cells are lysed by contacting the cells with laureth-9. In one embodiment, the host cells are lysed by contacting the cells with a solution containing laureth-9. In one embodiment, the host cells are lysed by contacting a mixture containing the cells with a cell-destroying amount of laureth-9. With respect to environmentally friendly detergents, "cell-destroying amount" refers to the amount of detergent effective (e.g., concentration, volume, or weight ratio) to rupture the cell membrane and release at least some or all of the cell contents. In one embodiment, "cell-destroying amount" refers to the amount of detergent that ruptures the cell membrane and releases all or part of the cell contents without adversely affecting the biological product of interest.

[0078] In one embodiment, host cells are dissolved by contacting a mixture containing these cells with a solution containing about 0.1% to about 2% laureth-9. In another embodiment, host cells are dissolved by contacting a mixture containing these cells with a solution containing at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In yet another embodiment, host cells are dissolved by contacting a mixture containing these cells with a solution containing about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the mixture containing these cells is incubated with laureth-9 for about 1 minute to about 120 minutes. In one embodiment, the mixture is incubated with laureth-9 for at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes and up to about 60, 90, or 120 minutes. In one embodiment, the mixture is incubated with a solution containing laureth-9 for up to about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, the mixture is incubated with laureth-9 for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 60, 90, or 120 minutes. In one embodiment, the mixture is incubated with a solution containing laureth-9 for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, the mixture is incubated with laureth-9 for about 1 minute. In another embodiment, the mixture is incubated with laureth-9 for at least about 5 minutes. In another embodiment, the mixture is incubated with laureth-9 at a temperature of about 4°C to about 42°C. In another embodiment, the mixture is incubated with laureth-9 at a temperature of about 4°C to about 25°C. In another embodiment, the mixture is incubated with laureth-9 at room temperature (for example, at a temperature of about 20°C to about 25°C). In another embodiment, the mixture is brought into contact with a solution containing laureth-9 at a pH of about 4.5 to about 8.5.In one embodiment, the mixture is brought into contact with a solution containing laureth-9 at a pH of about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5. In one embodiment, after incubation of the mixture with the laureth-9 solution, at least about 75%, 80%, 85%, 90%, or 95% of the cells in the mixture are dissolved. In one embodiment, after incubation of the mixture with the laureth-9 solution, at least about 80% of the cells in the mixture are dissolved.

[0079] Endotoxin removal In one embodiment, a method is provided for removing endotoxins from a mixture containing a biological agent of interest. In one embodiment, the mixture is a product stream containing the biological agent of interest. In one embodiment, the method comprises contacting the mixture with an endotoxin-removing amount of an environmentally friendly cleaning agent. In one embodiment, the method comprises contacting the product stream with an endotoxin-removing amount of an environmentally friendly cleaning agent. In one embodiment, the method comprises contacting the product stream with an endotoxin-removing amount of a solution containing laureth-9.

[0080] Endotoxins are lipopolysaccharides (LPS) derived from the cell membranes of Gram-negative bacteria and are contaminants that may be found in recombinantly produced biological preparations. Even small amounts of endotoxin in biological preparations can trigger systemic inflammatory responses, leading to side effects such as endotoxin shock, tissue damage, and death. Endotoxin contamination has been found in connection with biological preparations produced using Gram-negative bacteria such as Escherichia coli, and may be introduced during the manufacturing process, for example, under non-sterilization process conditions. Many purification methods have been developed for endotoxin removal, including: affinity chromatography, size exclusion chromatography, membrane ultrafiltration, membrane microfiltration, anion exchange chromatography, cation exchange chromatography, hydroxyapatite, hydrophobic interaction, reversed phase, and thiophilic adsorption. Other methods for endotoxin removal include microfiltration and ultrafiltration. Endotoxins exhibit a significant ability to interact with biomolecules (e.g., biological preparations such as biotherapeutic proteins).

[0081] In one embodiment, “endotoxin removal amount” with respect to an environmentally friendly cleaning agent refers to the amount (e.g., concentration, volume, or weight ratio) of the cleaning agent effective in dissociating endotoxins from the biological product of interest in a mixture (e.g., a product stream from a purification process). In another embodiment, “endotoxin removal amount” refers to the amount of cleaning agent effective in dissociating endotoxins from the biological product of interest without adversely affecting the biological product of interest.

[0082] In one embodiment, the method involves contacting a mixture (e.g., a product stream) with a solution containing an environmentally friendly cleaning agent in an amount effective for dissociating endotoxins from the biological product of interest. In another embodiment, the method involves contacting a mixture containing the biological product of interest with a solution containing laureth-9 in an amount effective for dissociating endotoxins from the biological product of interest. In yet another embodiment, the method involves contacting a product stream with a solution containing laureth-9 in an amount effective for dissociating endotoxins from the biological product of interest. In yet another embodiment, the method involves contacting a mixture with a solution containing about 0.1% to about 2% (w / v) laureth-9. In one embodiment, the washing solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, this solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9.

[0083] In one embodiment, the method includes filtering a mixture (e.g., a product stream from a purification process) to remove endotoxins. In one embodiment, the method includes contacting the product stream with a solution containing laureth-9, and then filtering the mixture. In one embodiment, the mixture is filtered through a molecular weight cutoff filter having a pore size effective for retaining the biological preparation of interest and allowing the dissociated bacterial endotoxins to pass through.

[0084] In one embodiment, the method includes one or more chromatography steps. In one embodiment, the chromatography washing solution contains a surfactant such as laureth-9 to increase the removal of endotoxins during the chromatography process.

[0085] purification In one embodiment, the cell culture medium is collected and clarified. In another embodiment, the clarified cell culture medium is subjected to one or more purification processes to separate one or more impurities from the target biological product. In one embodiment, the biological product is held in the product stream. In another embodiment, one or more impurities are discarded into the waste stream.

[0086] As used herein, the term “mixture” may include “product stream.” The term “product stream” refers to the output obtained from cell culture containing the biological product of interest, and may include the results of one or more purification process steps, for example, product stream after centrifugation, chromatography, filtration, or other after other steps in the purification process, and may include, for example, cell culture media, recovered cell culture media, clarified cell culture media, capture pool eluates from chromatography resin, flow-through pools from chromatography resin, supernatant, filtrate, retention solution, and the like.

[0087] In one embodiment, the target biological agent in the product stream is contacted with an environmentally friendly surfactant containing laureth-9 during the purification process of the target biological agent. Advantageously, the quality of the biological agent product is maintained during this process. In one embodiment, treatment of the product stream during the purification process of the biological agent with an environmentally friendly detergent containing laureth-9 does not increase the amount of product variants in the product stream compared to, for example, a purification process without surfactants or a purification process using Triton X-100 as the surfactant. In one embodiment, these product variants may include size variants, charge variants, oxidized variants, deamidated variants, glycated variants, or variants with altered glycan profiles. In one embodiment, product variants may include, for example, size variants resulting from the degradation or aggregation of the product. Methods for detecting the presence of size variants are known, such as size exclusion chromatography and gel electrophoresis. In one embodiment, treatment of the product stream in the purification process of a biological product with an environmentally friendly detergent containing laureth-9 results in a change of less than approximately 5% in the product variants of the total product in the product stream compared to a purification process that does not use a surfactant or uses Triton X-100 as a surfactant. In one embodiment, the environmentally friendly detergent results in an increase of less than approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in the product variants of the total protein in the product stream compared to a manufacturing process that does not use an environmentally friendly detergent containing laureth-9 (e.g., a process that does not use a surfactant or uses Triton X-100 as a surfactant).

[0088] chromatography In one embodiment, the product stream is subjected to one or more chromatographic steps to separate impurities from a biological preparation, for example, based on charge, hydrophobicity, size, or affinity. In column chromatography, a liquid mobile phase is passed through a stationary phase (chromatographic resin), and molecules in this mobile phase are separated based on differences in their interactions with the stationary phase. For example, the interaction with the stationary phase may be based on molecular size (size exclusion chromatography), charge (ion exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), specific binding interactions (affinity chromatography), or a combination thereof (mixed-mode chromatography). One or more chromatographic columns may be used in the purification process, and the number and order of chromatographic columns may vary.

[0089] Examples of column chromatography resins include affinity chromatography, ion exchange column chromatography, mixed-mode column chromatography, hydroxyapatite chromatography, and hydrophobic interaction column chromatography. Examples of affinity chromatography resins include protein A, protein G, and protein L. Other examples of affinity chromatography resins include resins having ligands containing recombinant camel single-domain antibodies. In one embodiment, LambaFabSelect chromatography or KappaSelect chromatography are used as affinity chromatography. Examples of ion exchange chromatography include anion exchange chromatography and cation exchange chromatography. In one embodiment, CaptoAdhere chromatography is used as mixed-mode chromatography.

[0090] The chromatography process includes a "flow-through" process and a "binding and elution" process. In the "flow-through" chromatography process, the desired biological agent may flow through the chromatography and be collected in a "flow-through pool," while impurities are retained on the chromatography resin. In the "binding and elution" chromatography process, the desired biological agent is retained by the chromatography resin, while impurities pass through. The desired biological agent can then be eluted from the chromatography resin and collected in a "capture pool."

[0091] In one embodiment, the product stream is a flow-through pool. In another embodiment, the product stream is a capture pool. In another embodiment, the flow-through pool or capture pool is an affinity chromatography pool. In another embodiment, the flow-through pool or capture pool is a protein A pool, a protein G pool, or a protein L pool. In another embodiment, the flow-through pool or capture pool is a Capto AVB affinity chromatography pool. In another embodiment, the flow-through pool or capture pool is an anion exchange chromatography pool. In another embodiment, the flow-through pool or capture pool is a cation exchange chromatography pool. In another embodiment, the flow-through pool or capture pool is a mixed-mode chromatography pool. In another embodiment, the flow-through pool or capture pool is a CaptoAdhere pool. In another embodiment, the flow-through pool or capture pool is a hydroxyapatite pool. In another embodiment, the flow-through pool or capture pool is a hydrophobic interaction column pool.

[0092] When used in binding and elution modes, column chromatography typically involves an equilibration step in which an equilibration buffer suitable for the biological agent of interest and the chromatography resin is passed through the column resin. Generally, about 5 to about 10 column volumes (CV) of equilibration buffer are used. The sample is then loaded onto the column. In many cases, the same loading buffer as the equilibration buffer is used. Once the biological agent of interest is immobilized on the stationary phase of the column, impurities that are slightly interacting with the column resin can be removed by washing the column with 1 or more column volumes of washing solution. In one embodiment, the washing solution has the same composition as the equilibration buffer. In another embodiment, the washing solution contains components that help disrupt the weak interactions between the impurities and the column resin. In one embodiment, the column is washed until impurities are undetectable in the eluate. After washing the impurities away from the chromatography resin, molecules such as the biological agent can be eluted with elution buffer. Generally, the elution buffer has a different composition from the equilibration buffer and / or washing solution.

[0093] In one embodiment, the washing solution contains a surfactant. In another embodiment, the washing solution contains a surfactant including laureth-9. In one embodiment, the inclusion of laureth-9 in the washing solution enhances the removal of viruses from the product stream during the chromatographic purification process. In one embodiment, the inclusion of laureth-9 in the washing solution increases the removal of host cell proteins (HCPs) from the product stream. In another embodiment, the inclusion of laureth-9 in the washing solution increases the removal of endotoxins from the product stream. In one embodiment, the inclusion of an intermediate wash after washing with a surfactant but before elution of the target biological product from the chromatographic resin increases the yield of the biological product in the eluate, in addition to removing residual surfactants from the chromatographic support.

[0094] When used in flow-through mode, chromatographic conditions are selected such that the target biological agent flows through the column rather than binds to the chromatographic resin, while impurities remain bound to the column resin. The flow-through chromatography process may include a washing step to recover the weakly bound biological agent from the column resin.

[0095] In one embodiment, a method for purifying a biological agent of interest is provided. In one embodiment, the method comprises loading a mixture containing the biological agent of interest and one or more impurities onto a chromatography support. In one embodiment, the method comprises washing the chromatography support with a washing solution containing an environmentally friendly cleaning agent. In one embodiment, the environmentally friendly cleaning agent contains laureth-9. In one embodiment, the method comprises eluting the biological agent of interest from the chromatography support to obtain a purified eluate containing the biological agent of interest.

[0096] In one embodiment, the washing solution contains about 0.1% to about 2% (w / v) laureth-9. In one embodiment, the washing solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, surfactants such as laureth-9 are included in the chromatography washing solution, increasing virus elimination. In one embodiment, a surfactant such as laureth-9 is included in the chromatography washing solution, and the removal of host cells is increased. In another embodiment, a surfactant such as laureth-9 is included in the chromatography washing solution, and the removal of endotoxins is increased.

[0097] In one embodiment, a surfactant such as laureth-9 is included in the chromatography washing buffer to increase virus elimination. In one embodiment, a chromatography resin holding a desired biological agent is washed with a washing solution containing a surfactant such as laureth-9. In one embodiment, the surfactant-containing washing solution is applied to the column before elution of the desired biological agent from the chromatography resin. In one embodiment, the chromatography resin is an affinity chromatography resin. In one embodiment, the washing solution contains a virus-inactivating amount of laureth-9. In one embodiment, the washing solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9.

[0098] In one embodiment, a surfactant such as laureth-9 is included in the chromatography wash buffer, increasing the removal of host cell proteins from the product stream. In one embodiment, a chromatography resin holding the desired biological agent is washed with a washing solution containing a surfactant such as laureth-9. In one embodiment, the surfactant-containing washing solution is applied to the column before elution of the desired biological agent from the chromatography resin. In one embodiment, the chromatography resin is an affinity chromatography resin. In one embodiment, the affinity chromatography resin contains a recombinant camel single-domain antibody. In one embodiment, LambaFabSelect or KappaSelect are mentioned as the affinity chromatography resin. In one embodiment, protein A chromatography resin, protein G chromatography resin, or protein L chromatography resin are mentioned as the affinity chromatography resin.

[0099] In one embodiment, the washing solution contains a host cell protein removal amount of laureth-9. In one embodiment, the purified eluate has a reduced host cell protein content compared to the host cell protein content in the eluate from a chromatography support that was not washed with a washing solution containing laureth-9. In one embodiment, the washing solution contains about 0.1% to about 2% (w / v) laureth-9. In one embodiment, the washing solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a laureth-9 washing solution is reduced by at least approximately 50%, 45%, 40%, 35%, 30%, or 25% compared to the host cell content of the mixture applied to the column. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a laureth-9 washing solution is reduced by at least approximately 30% compared to the host cell content of the mixture applied to the column. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is approximately 75%, 70%, 65%, 60%, 55%, or less than 50% of the host cell protein content in the mixture applied to the column. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is approximately 65% ​​of the host cell protein content in the mixture applied to the column. In one embodiment, the host cell protein content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is approximately 2000 ng / mg, 1700 ng / mg, 1500 ng / mg, 1200 ng / mg, 1000 ng / mg, 500 ng / mg, or less than 100 ng / mg.

[0100] In one embodiment, the washing solution contains an endotoxin-removing amount of laureth-9. In one embodiment, the purified eluate has a reduced endotoxin content compared to the endotoxin content in the eluate from a chromatography support that was not washed with a washing solution containing laureth-9. In one embodiment, the washing solution contains about 0.1% to about 2% (w / v) laureth-9. In one embodiment, the washing solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is reduced by at least approximately 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% compared to the endotoxin content of the mixture applied to the column. In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is reduced by at least approximately 99.9% compared to the endotoxin content of the mixture applied to the column. In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is less than approximately 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% of the endotoxin content in the mixture applied to the column. In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is less than approximately 0.1% of the endotoxin content in the mixture applied to the column. In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is less than approximately 0.05% of the endotoxin content in the mixture applied to the column.In one embodiment, the endotoxin content in the purified eluate from a chromatography support washed with a washing solution containing laureth-9 is less than approximately 0.01% of the endotoxin content in the mixture applied to the column. In one embodiment, the purified eluate has an endotoxin concentration of less than approximately 1 EU / mg, 0.9 EU / mg, 0.8 EU / mg, 0.7 EU / mg, 0.6 EU / mg, 0.5 EU / mg, 0.4 EU / mg, 0.3 EU / mg, 0.2 EU / mg, or 0.1 EU / mg. In one embodiment, the purified eluate has an endotoxin concentration of less than approximately 1 EU / mg. In one embodiment, the purified eluate has an endotoxin concentration of less than approximately 0.5 EU / mg. Methods for determining the endotoxin concentration are known, such as the limulus amebocyte lysate assay (LAL assay). In one embodiment, ion exchange chromatography is used as the chromatography method. In one embodiment, cation exchange chromatography is used as the chromatography method.

[0101] filtration In one embodiment, the purification process includes a filtration step. In one embodiment, the mixture is filtered to remove virus particles. In one embodiment, the mixture is the product flow from the purification process. In one embodiment, the purification process includes a filtration step for concentrating the product formulation. In one embodiment, ultrafiltration is used as the filtration. In one embodiment, dialysfiltration is used as the filtration. In one embodiment, deep filtration is used as the filtration. In one embodiment, one or more impurities (e.g., virus particles) are removed from a mixture such as the product flow using ultrafiltration, deep filtration, or a combination thereof. In one embodiment, the product flow may be a cell culture medium, a clarified cell culture medium, a capture pool, or a flow-through pool. In one embodiment, the method includes contacting the product flow with an environmentally friendly cleaning agent and then filtering the product flow. In one embodiment, the method includes contacting the product flow with laureth-9 and then filtering the product flow. In one embodiment, the method involves contacting the product stream with a solution containing at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9, and then filtering the product stream. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the product stream is filtered at least about 15 minutes to at least about 48 hours after the addition of laureth-9. In one embodiment, the product stream is filtered at least about 1 hour to at least about 3 hours after the addition of laureth-9.

[0102] sample In one embodiment, the sample is brought into contact with a solution containing laureth-9. In one embodiment, the sample is a product stream from the purification process of a biological product. In one embodiment, the sample is a product stream from the purification process of a recombinantly produced biological product. In one embodiment, the sample is brought into contact with a solution containing at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the sample is brought into contact with a solution containing about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9.

[0103] In one embodiment, the sample is a recovered cell medium. In another embodiment, the sample is a clarified cell recovery medium. Cells can be recovered from the cell culture medium using any conventional means (e.g., centrifugation or microfiltration, but not limited to these). In one embodiment, the sample is whole cells. In another embodiment, the sample is a cell lysate. In another embodiment, the sample is a cell paste slurry formed by centrifuging and purifying a biological preparation from cell lysate and cell culture medium. In another embodiment, the cell paste slurry is formed by pelletizing cells from a cell culture and removing the supernatant to form a cell paste. In another embodiment, the sample is a capture pool from a chromatography resin. In another embodiment, the sample is a flow-through pool from a chromatography resin. In another embodiment, the sample is a filtrate. In another embodiment, the sample is a retention solution.

[0104] Activating agent The method described herein can be used in the manufacturing process of a wide variety of compositions, such as compositions having any of the following properties: pharmaceutical properties, diagnostic properties, agricultural properties, and / or various other properties useful for commercial, experimental, or other uses. In one embodiment, the method is used in the manufacturing process of a therapeutic agent. In one embodiment, the method is used in the manufacturing process of a biological product. In one embodiment, the biological product is naturally occurring. In one embodiment, the biological product is manufactured by recombinant DNA. In one embodiment, the biological product is homologous to the host cell. In one embodiment, the biological product is heterologous to the host cell, i.e., exogenous or foreign.

[0105] In one embodiment, the biological agent is a therapeutic macromolecule, such as a therapeutic polynucleotide or polypeptide. In another embodiment, the bioactive agent is a chemically modified macromolecule, such as a pegylated macromolecule or a macromolecule to which a diagnostic, targeted, or therapeutic moiety has been added. In another embodiment, the bioactive agent is of therapeutic, scientific, or commercial interest.

[0106] In one embodiment, the bioactive agent is a therapeutic protein, which includes: enzymes or enzymatically active polypeptides, soluble receptors or soluble ligands, hormones, neurotransmitters, coagulation / clotting factors, growth factors, integrins, cytokines, regulatory factors, interferons, antigens, secreted proteins, or fragments thereof. In one embodiment, the bioactive agent is an antibody, an antigen-binding antibody fragment, an Fc fusion protein, an antibody-drug conjugate (ADC), an immunoadhesin, or an antigen conjugate. In one embodiment, the biological agent is a gene therapy product; a vaccine, e.g., a virus-based vaccine; and a non-enveloped virus therapeutic agent. In one embodiment, the therapeutic protein is a fusion protein or a proteolysis-targeting chimera (protac). In one embodiment, the bioactive agent is a therapeutic peptide. As used herein, “therapeutic peptide” refers to a bioactive polypeptide containing fewer than approximately 500, 250, 100, 50, or 20 amine acids and having a molecular weight of less than approximately 100 kDa or 50 kDa.

[0107] In one embodiment, the recombinant biological product of interest is an antibody or an antigen-binding antibody fragment. In one embodiment, an antibody-like protein is used as the bioactive agent. As used herein, the terms “antibody” and “immunoglobulin” may be used interchangeably and refer to one polypeptide or group of polypeptides comprising at least one binding domain formed by the folding of a polypeptide chain having a three-dimensional binding space with an inner surface shape and charge distribution complementary to the characteristic features of the antigenic determinant of the antigen. Naturally occurring antibodies or their antigen-binding fragments typically have a tetrameric form with two pairs of polypeptide chains, each pair having one “light” chain and one “heavy” chain. The variable region of each light / heavy chain pair forms the antibody-binding site. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds differs among the heavy chains of various immunoglobulin isotypes. Each of the heavy and light chains also has regularly separated interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains (CH), and each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Light chains are classified as lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. Immunoglobulin molecules can be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, e.g., G1m(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)).

[0108] The term "antigen-binding fragment" refers to a fragment of an antibody that contains at least one antigen-binding site and retains the ability to specifically bind to an antigen. Examples of antigen-binding antibody fragments that can be produced by recombination include, but are not limited to, fragments containing variable heavy and light chain domains, e.g., single-chain Fv(scFv), single-chain antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, dimeric variable regions (bispecific antibodies), and disulfide-bonded variable regions (dsFv). Antibody fragments may also include epitope-binding fragments or derivatives of any of the antibodies listed above.

[0109] Antibodies may be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, camelized antibodies, CDR-transplanted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, fully human antibodies, anti-idiotype antibodies, and intracellular antibodies, either alone or in combination with other amino acid sequences, as well as fragments produced by recombinant means, such as fragments exhibiting desired biological activity, such as epitope-binding fragments, variants or derivatives thereof.

[0110] In one embodiment, the bioactive agent is an IgG fragment crystallizable (Fc) region or domain, which comprises a set of pairs of antibody heavy chain domains, each containing a heavy chain constant domain 2 (CH2) and a heavy chain constant domain 3 (CH3), forming a structure of approximately 50 kDa. The Fc region interacts with cell surface receptors called Fc receptors and some proteins of the complement system, enabling the antibody to activate the immune system.

[0111] In one embodiment, the biological agent is a polynucleotide. In one embodiment, the biological agent is a viral vector. In one embodiment, a non-enveloped virus is an example of the biological agent. In one embodiment, a viral vector such as an adenovirus vector or an adeno-associated virus (AAV) vector is an example of the biological agent.

[0112] Non-enveloped virus biological agents In one embodiment, the biological product is a non-enveloped virus. In one embodiment, the biological product is a single-stranded non-enveloped virus. In one embodiment, the biological product is an adenovirus. In one embodiment, the biological product is an adeno-associated virus (AAV). One method is provided for purifying a non-enveloped virus. In one embodiment, a method is provided in which enveloped viruses present in the product stream are inactivated, but non-enveloped viruses remain intact.

[0113] In one embodiment, the non-enveloped virus vector is produced in cell culture. In one embodiment, the non-enveloped virus vector is recovered from the cell culture. In one embodiment, the non-enveloped virus particles accumulate in the cytoplasm of host cells and in the cell culture medium. In one embodiment, the host cells are lysed to increase the yield. In one embodiment, the host cells are collected, for example, by centrifugation and / or filtration. The collected cells may be cryopreserved or further processed for the purification of the biological product. In one embodiment, the collected cells are resuspended in a buffer to form a cell slurry. In one embodiment, the resuspension buffer is selected from Tris-HCl, sodium acetate, potassium citrate, monobasic sodium phosphate or dibasic sodium phosphate, or a mixture thereof.

[0114] In one embodiment, cells in a slurry are brought into contact with a solution containing an environmentally friendly cleaning agent. In one embodiment, cells in a cell slurry are brought into contact with a solution containing laureth-9. In one embodiment, cells in a slurry are brought into contact with a solution containing laureth-9 to lyse these cells. In one embodiment, enveloped viruses are inactivated by bringing cells in a slurry into contact with a solution containing laureth-9. In one embodiment, the solution comprises laureth-9 and one or more additional cleaning agents. In one embodiment, the solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, these cells are incubated with the laureth-9-containing solution for about 1 minute to about 120 minutes. In one embodiment, these cells are incubated with the laureth-9-containing solution for at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 minutes and up to about 60, 90, or 120 minutes. In one embodiment, this mixture is incubated with the laureth-9-containing solution for up to about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, these cells are incubated with a solution containing laureth-9 for approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 60, 90, or 120 minutes. In one embodiment, this mixture is incubated with a solution containing laureth-9 for approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, or 48 hours. In one embodiment, these cells are incubated with laureth-9 for approximately 1 minute. In one embodiment, these cells are incubated with laureth-9 for at least approximately 5 minutes. In one embodiment, these cells are incubated with laureth-9 at a temperature of approximately 4°C to approximately 42°C. In one embodiment, these cells are incubated with laureth-9 at a temperature of approximately 4°C to approximately 25°C.In one embodiment, these cells are incubated with laureth-9 at room temperature (e.g., at a temperature of approximately 20°C to approximately 25°C). In one embodiment, this mixture is brought into contact with a solution containing laureth-9 at a pH of approximately 4.5 to approximately 8.5. In another embodiment, this mixture is brought into contact with a solution containing laureth-9 at a pH of approximately 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5.

[0115] In one embodiment, the titer is determined by the concentration of viral particles containing the viral genome (physical titer). In one embodiment, this physical titer is determined by quantitative PCR (qPCR), digital droplet PCR (ddPCR), or other DNA quantification methods. The physical titer may be expressed as viral genome per 1 ml (vg / ml). In one embodiment, the titer of purified AAV is approximately 10 log 10 The concentration is greater than vg / ml. In one embodiment, the physical titer is not reduced compared to the control that was not in contact with laureth-9. In another embodiment, the physical titer is approximately 0.05 log compared to the control that was not in contact with laureth-9. 10 vg / mL, 0.1 log 10 vg / mL, 0.2 log 10 vg / mL, or 0.3log 10 The concentration is within vg / mL. In one embodiment, this control includes cells lysed using the freeze-law method. In another embodiment, this control includes cells lysed using Triton-X.

[0116] In one embodiment, the titer is determined by the concentration of viral particles that can transduce cells (infectious titer). In another embodiment, the infectious titer is quantified by a cell transduction assay such as TCID50 (median infectious dose in tissue culture). In another embodiment, the titer of purified AAV is approximately 8 log 10 TCID is greater than 50 / mL. In one embodiment, the infectivity titer is not reduced compared to the control that has not been in contact with laureth-9. In another embodiment, the infectivity titer is approximately 0.1 log compared to the control that has not been in contact with laureth-9. 10TCID50 / mL, 0.2 log 10 TCID50 / mL, 0.3 log 10 TCID50 / mL, or 0.7 log 10 The TCID is within 50 / mL. In one embodiment, this control includes cells lysed using the freeze-law method. In another embodiment, this control includes cells lysed using Triton-X.

[0117] In one embodiment, the method comprises loading a mixture containing a non-enveloped virus and one or more impurities onto a chromatography carrier; washing the chromatography carrier with a washing solution containing laureth-9; and eluting the non-enveloped virus from the chromatography carrier to obtain a purified eluate containing the non-enveloped virus. In one embodiment, the solution comprises laureth-9 and one or more additional washing agents. In one embodiment, the solution contains at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (w / v) and up to 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9. In one embodiment, the solution contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or 10% (w / v) laureth-9.

[0118] Embodiments of the present invention Aspects of the present invention are further described in the following sections: [Section 1] A method for purifying a recombinant biological product of interest in a mixture containing the biological product of interest and one or more impurities, comprising contacting the mixture with a solution containing laureth-9, and incubating the mixture. [Section 2] The method according to item 1, wherein the mixture comprises an enveloped virus, and the method comprises contacting the mixture with a solution containing a virus-inactivating amount of laureth-9. [Section 3] The solution containing laureth-9 showed approximately 1 log improvement compared to the control without the surfactant. 10 , 2log 10 , 3log 10 , or 4log 10 The method described in item 2 above, which yields a logarithmically decreasing value (LRV). [Section 4] The solution containing Laureth-9 showed approximately 2 log improvement compared to the control without Triton X-100. 10 , 3log 10 , or 4log 10 The method described in item 2 above, which yields a logarithmically decreasing value (LRV). [Section 5] The solution containing laureth-9 showed approximately 2 log improvement compared to the control without laureth-9. 10 , 3log 10 , or 4log 10 The method described in item 2 above, which yields a logarithmically decreasing value (LRV). [Section 6] The method according to any one of items 3 to 5 above, wherein LRV is determined by quantitative PCR or an infectivity assay. [Section 7] The method according to any one of items 3 to 5 above, wherein LRV is determined by a plaque assay. [Section 8] The method according to any one of items 1 to 7 above, wherein the enveloped virus includes a DNA virus or an RNA virus. [Section 9] The method according to any one of items 1 to 8 above, wherein the enveloped virus includes a single-stranded virus or a double-stranded virus. [Section 10] The method according to any one of items 1 to 9 above, wherein the enveloped virus includes the families Herpesviridae, Hepadnaviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, Arteriviridae, or a combination thereof. [Section 11] The method according to item 1, comprising contacting a mixture containing the cells with a solution containing a cell-destroying amount of laureth-9. [Section 12] The method according to item 11, wherein, after incubation of the mixture with the laureth-9 solution, at least about 75%, 80%, 85%, 90%, or 95% of the cells in the mixture are dissolved. [Section 13] The method according to any one of items 1 to 12, wherein the mixture is incubated with laureth-9 for at least about 1 minute. [Section 14] The method according to any one of items 1 to 13, wherein the mixture is incubated with laureth-9 for at least about 5 minutes. [Section 15] The method according to any one of items 1 to 14, wherein the mixture is incubated with laureth-9 for about 1 minute to about 120 minutes. [Section 16] The method according to any one of items 1 to 15, wherein the mixture is incubated with laureth-9 at room temperature. [Section 17] The method according to item 1, comprising contacting the mixture with a solution containing an amount of laureth-9 to remove endotoxins. [Section 18] The method described in item 17 above, wherein the endotoxin concentration is reduced to less than approximately 1 EU / mg. [Section 19] The solution is the method according to any one of items 1 to 18 above, comprising about 0.1% to about 2% (w / v) laureth-9. [Section 20] The method according to any one of items 1 to 19, further comprising the step of filtering the mixture containing the recombinant biological preparation for the above purpose. [Section 21] The method according to item 20, wherein filtering is performed before incubating the mixture with the solution containing laureth-9. [Section 22] The method according to item 20, wherein filtering is performed after incubating the mixture with the solution containing laureth-9. [Section 23] Filtration is the method described in any one of items 20 to 22 above, including ultrafiltration or deep filtration. [Section 24] The method according to any one of the above items 1 to 23, further comprising one or more chromatography steps. [Section 25] The method according to item 24, wherein one or more chromatography steps include loading a mixture containing the biological agent of the objective onto a chromatography carrier, and washing the chromatography carrier with a washing solution containing laureth-9. [Section 26] A method for purifying a target biological product, (a) Loading a mixture containing the biological preparation of the objective and one or more impurities onto a chromatographic carrier; (b) Wash the chromatography support with a washing solution containing laureth-9; and (c) Elute the target biological agent from the chromatography carrier to obtain a purified eluate containing the target biological agent. A method that includes this. [Section 27] The method according to item 26, wherein the washing solution contains a host cell protein removal amount of laureth-9, and the purified eluate has a reduced host cell protein content compared to the host cell protein content in the eluate from a chromatography support that was not washed with the washing solution containing laureth-9. [Section 28] The method according to item 27, wherein the host cell protein content in the purified eluate from the chromatography support washed with a washing solution containing laureth-9 is reduced by at least about 50%, 45%, 40%, 35%, 30%, or 25% compared to the host cell content of the mixture applied to the column. [Section 29] The method according to item 27, wherein the host cell protein content in the purified eluate from the chromatography carrier washed with a washing solution containing laureth-9 is about 2000 ng / mg, 1700 ng / mg, 1500 ng / mg, 1200 ng / mg, 1000 ng / mg, 500 ng / mg, or less than 100 ng / mg. [Section 30] The washing solution is the method according to item 26, wherein the washing solution contains an amount of laureth-9 sufficient to remove endotoxins. [Section 31] The method according to item 30, wherein the purified eluate has an endotoxin concentration of less than approximately 1 EU / mg. [Section 32] The washing solution is the method according to any one of items 26 to 31 above, comprising about 0.1% to about 2% (w / v) laureth-9. [Section 33] The recombinant biological preparation for the above purpose is a therapeutic macromolecule, as described in any one of items 1 to 32 above. [Section 34] The method according to item 33, wherein the therapeutic macromolecule is a therapeutic polypeptide or a therapeutic polynucleotide. [Section 35] The method according to any one of items 1 to 32 above, wherein the recombinant biological product for the purpose is an enzyme, a soluble receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen-binding antibody fragment, an antibody-drug conjugate, a fusion polypeptide, or a non-enveloped virus. [Section 36] The method according to item 35, wherein the recombinant biological product for the purpose is an antibody or an antigen-binding antibody fragment. [Section 37] The method according to item 36, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, or a human antibody. [Section 38] The method according to item 35, wherein the non-enveloped virus is an adenovirus or adeno-associated virus (AAV). [Section 39] The recombinant biological product for the above purpose is produced by cells, as described in any one of items 1 to 38 above. [Section 40] The recombinant biological agent for the above purpose is produced by eukaryotic cells, as described in item 39 above. [Section 41] The recombinant biological preparation for the above purpose is produced by prokaryotic cells, as described in item 39 above. [Section 42] The recombinant biological preparation for the above purpose is produced by mammalian cells, as described in item 39 above. [Section 43] The method according to item 42, wherein the mammalian cells include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, mouse hybridoma cells, or mouse myeloma cells. [Section 44] The method according to any one of items 26 to 43 above, wherein the chromatography includes one or more of affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and mixed-mode chromatography. [Section 45] Chromatography is the method described in item 44 above, including affinity chromatography. [Section 46] Affinity chromatography is the method described in item 45 above, selected from protein A chromatography, protein G chromatography, or protein L chromatography. [Section 47] Ion exchange chromatography is the method described in item 44 above, selected from anion exchange chromatography and cation exchange chromatography. [Section 48] The method according to any one of items 26 to 47 above, further comprising the step of filtering the product stream. [Section 49] Filtration is the method described in item 48 above, including ultrafiltration or deep filtration. [Section 50] The method according to any one of items 1 to 49 above, wherein the recombinant biological product for the purpose has a biological activity of about 25%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of that of the control recombinant biological product. [Section 51] The method according to item 50, wherein the control recombinant biological product has not been in contact with laureth-9. [Section 52] A method for producing recombinant non-enveloped viruses, (a) Culturing host cells containing the genome of a non-enveloped virus; (b) Contacting the host cells with a solution containing laureth-9. A method that includes this. [Section 53] The method according to item 52, wherein the host cells are brought into contact with a solution containing approximately 0.1% to approximately 2% laureth-9. [Section 54] The method according to item 52 or 53, wherein the host cells are brought into contact with the solution containing laureth-9 for approximately 1 minute to approximately 120 minutes. [Section 55] The method according to item 52, comprising preparing a cell paste containing host cells expressing the non-enveloped virus, and contacting the cell paste with a solution containing a cell-lysing amount of laureth-9. [Section 56] The method according to any one of items 51 to 55 above, wherein the non-enveloped virus is an adenovirus or an adeno-associated virus (AAV). [Section 57] The purified non-enveloped virus had a physical titer of approximately 10 log. 10 The method according to item 56 above, wherein the concentration is greater than vg / ml. [Section 58] The purified non-enveloped virus described above has an infectivity titer of approximately 8 log. 10 The method according to item 56 above, wherein the TCID is greater than 50 / mL. [Section 59] The method according to any one of items 51 to 58 above, wherein viral infectivity is not significantly reduced compared to a control that has not been in contact with a solution containing laureth-9. [Section 60] A method for purifying a target non-enveloped virus, (a) Loading the mixture containing the non-enveloped virus onto a chromatography carrier; (b) Wash the chromatography support with a washing solution containing laureth-9; and (c) Elute the non-enveloped virus from the chromatography carrier to obtain a purified eluate containing the non-enveloped virus. A method that includes this. [Section 61] The washing solution is the method described in item 60 above, comprising about 0.1% to about 2% (w / v) laureth-9. [Section 62] A method for purifying a target non-enveloped virus, (a) Contacting the mixture containing the non-enveloped virus with a solution containing laureth-9; and (b) Filter the mixture containing the non-enveloped virus. A method that includes this. [Section 63] Filtration is the method described in item 62 above, including ultrafiltration or deep filtration. [Section 64] The method according to item 62 or 63, wherein filtration is performed before contacting the mixture with the solution containing laureth-9. [Section 65] The method according to item 62 or 63, wherein filtration is performed after contacting the mixture with the solution containing laureth-9. [Section 66] The solution is the method according to any one of items 62 to 65 above, comprising about 0.1% to about 2% (w / v) laureth-9. [Section 67] A method for inactivating one or more enveloped viruses in a sample containing a biological agent of interest, (a) Contacting a sample containing the biological preparation of the subject with a solution containing laureth-9; and (b) Incubate the sample with the solution containing laureth-9 for approximately 1 minute to approximately 120 minutes. A method that includes this. Embedding by reference All references cited herein (e.g., patents, patent applications, articles, textbooks, and similar materials), and references cited therein, are incorporated herein by reference in their entirety unless they have already been cited. [Examples]

[0119] Example 1. Virus inactivation using a nonionic surfactant. The viral inactivation of four washing agents was investigated using cell recovery materials derived from CHOs containing a monovalent bispecific human IgG1 monoclonal antibody. Brij L9 (Laureth-9), Myrj S25, and Brij S20 were obtained from Croda International Plc. (East Yorkshire, United Kingdom). Polidocanol 600 (Laureth-9) was obtained from Schaerer & Schlaepfer AG (Rothrist, Switzerland). After adding a stock of mouse heterotropic leukemia virus-associated virus (XMuLV) to the recovery medium, the washing agents were added.

[0120] 8.5-log XMuLV was added to conditioned cell recovery medium containing a monovalent, bispecific human IgG1 monoclonal antibody (BisAb). Samples of this virus-supplemented recovery medium were taken and used as retention controls to determine the viral inactivation rate without the use of washing agents.

[0121] A 10% (w / w) stock solution was prepared by dissolving the washing agent in 50 mM Tris pH 7.4. An appropriate amount of the 10% stock solution was added to cell recovery samples in BisAb medium supplemented with XMuLV to a washing agent concentration of 1%. Retention controls and reaction samples were incubated at room temperature. Samples were collected at 1, 10, 60, and 120 minutes and diluted 1:50 with McCoy medium to stop the virus inactivation reaction. Diluted samples and retention controls were assayed for virus concentration using a plaque assay. As shown in Table 1, the virus concentration decreased from 7.79 to less than 3.18 in less than 1 minute for samples supplemented with Brij L9 and Polidocanol 600, and decreased by 4 Log in 1 minute with the washing agents Brij L9 and Polidocanol 600. 10 We observed the inactivation of the superhydrogen (Figure 1 and Table 2).

[0122] The results shown in Tables 1 and 2 demonstrate that not all structurally similar detergents, or detergents belonging to the same polyoxyethylene alkyl ether class, are necessarily effective in inactivating viruses. Brij S20 (polyoxyethylene(20) stearyl ether), a surfactant structurally related to laureth-9, showed a significantly slower, and therefore significantly undesirable, virus inactivation kinetics compared to laureth-9, as shown in Tables 1 and 2. Only Brij L9 showed an improved and desirable virus inactivation kinetics.

[0123] [Table 1]

[0124] [Table 2]

[0125] Example 2. Virus inactivation using Laureth-9 The tests were carried out essentially as described in Example 1, and for BisAb, 1% Brij L9 (laureth-9, obtained from Croda International Plc., East Yorkshire, United Kingdom) and 1% Polidodecanol 600 (laureth-9, obtained from Schaerer & Schlaepfer AG, Rothrist, Switzerland) were used as detergents on the conditioned medium derived from CHO to examine viral inactivation. For bispecific fusion proteins (BisFusion), 1% or 0.1% Brij L9 was used as the detergent to examine viral inactivation on the conditioned medium derived from CHO. As shown in Figure 2, 4Log was observed in all samples at 1 minute. 10 Further inactivation was observed. As shown in Table 3, for all four mixtures, the virus concentration decreased from 7.00 (BisAb) or 7.21 (BisFusion) to less than 2.88 in less than 1 minute.

[0126] [Table 3]

[0127] [Table 4]

[0128] Example 3. Purification of adeno-associated virus (AAV) AAV6.2 cell paste slurry was prepared and stored at -80°C. Brij L9 (Laureth-9) was obtained from Croda International Plc. (East Yorkshire, United Kingdom). Triton X-100 was obtained from Spectrum Chemical (New Brunswick, NJ). Capto AVB resin was obtained from GE Healthcare (Piscataway, NJ). C0SP filters were obtained from EMD Millipore (Burlington, MA). Sartopore 2 XLG, 0.8 / 0.2um filters were obtained from Sartorius Stedim Biotech GmbH (Gottingen, Germany). All buffer salts were obtained from Avantor Performance Chemicals (Center Valley, Pennsylvania).

[0129] For dissolution with a washing agent, the AAV6.2 cell paste was diluted to a 10% slurry with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, and pH 7.5. A 10% (w / w) washing agent stock solution was added to the cell slurry to a final washing agent concentration of 0.5% (v / w). Benzonase was added to the cell slurry mixture to a maximum of 10 U / ml and gently mixed at RT for 1 hour.

[0130] For freeze-thaw and thawing, the cell paste slurry was subjected to two freeze-thaw cycles at -80°C and RT. The AAV lysate was diluted to a 10% slurry with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, and pH 7.5. Benzoase was added to the cell slurry mixture up to 10 U / ml and gently mixed at RT for 1 hour.

[0131] AAV purification was performed using a Capto AVB affinity column. Purification was controlled using Unicorn 7.0 software on an AKTA AVANT purchased from GE Healthcare (Piscataway, NJ). The C0SP POD filter was washed with 20 mM Tris, 500 mM NaCl, 2 mM MgCl2, pH 7.5. 10% AAV6.2 cell lysates were purified with C0SP, followed by purification with a Sartopore 2 XLG, 0.8 / 0.2 μm filter. The Capto AVB column was equilibrated at 3 column volumes (CV) with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 7.5. The purified cell lysates were loaded with a residence time of 6 minutes. The column was re-equilibriumated at 4 CV with 20 mM Tris, 200 mM NaCl, 2 mM MgCl2, pH 7.5. The product was eluted from the column using 50 mM citric acid, 500 mM NaCl, and 2 mM MgCl2 at pH 2.5. The pH of the eluate was adjusted to pH 8.0 with 1 M Tris / HCl at pH 8.0.

[0132] The AAV titer was determined by centrifuging the 10% lysate at 13,000 rpm for 10 minutes and collecting the supernatant. The AAV titer was determined by an in-house qPCR method.

[0133] As shown in Table 5, Brij L9 (Laureth-9), like Triton X-100, is effective for cell lysis for AAV purification.

[0134] [Table 5]

[0135] As shown in Table 6, Brij L9 (Laureth-9) does not have any adverse effect on AAV infectivity.

[0136] [Table 6]

[0137] Example 4. Elimination of host cell proteins (HCPs) Cell recovery materials derived from CHOs containing bispecific antibodies were used. Brij L9 (Laureth-9) was obtained from Croda International Plc. (East Yorkshire, United Kingdom). Triton X-100 was obtained from Spectrum Chemicals (New Brunswick, NJ). LambdaFabSelect resin was obtained from GE Healthcare (Piscataway, NJ). All buffer salts were obtained from Avantor Performance Chemicals (Center Valley, Pennsylvania).

[0138] A 10% (w / w) stock solution was prepared by dissolving the detergent in USP water. An appropriate amount of this stock solution was added to the cell recovery material so that the final detergent concentration was 2% (w / w).

[0139] For chromatography experiments, purification was controlled using Unicorn 7.0 software on an AKTA AVANT purchased from GE Healthcare (Piscataway, NJ). Cell recovery material with added washing agent was loaded onto a LambdaFabSelect column. For washing experiments, cell recovery material without added washing agent was loaded onto a LambdaFabSelect column. Equilibration was performed using 50 mM Tris, pH 7.4 in 3CVs during protein binding. For washing experiments, a washing solution containing 1% washing agent (w / w) in equilibration buffer was applied to the column in 3CVs. Following this, 3CV washing with equilibration buffer was performed, and then proteins were eluted from the resin using 25 mM sodium acetate, pH 3.6 buffer. The HCP content in the eluate was measured using an in-house HCP ELISA assay.

[0140] As shown in Table 7, Brij L9 (laureth-9) exhibits performance comparable to that of Triton X-100, and can be used as a washing solution in affinity chromatography media for removing HCP.

[0141]

Table 7

[0142] As shown in Table 8, Brij L9 (laureth-9) exhibits performance comparable to that of Triton X-100, and can reduce HCP content when added to a cell recovery material before loading onto an affinity chromatography medium.

[0143]

Table 8

[0144] Example 5. Endotoxin Removal A CHO-derived monoclonal antibody was used. Lysozyme and bovine serum albumin (BSA) were obtained from Sigma Aldrich (St. Louis, MO). Endotoxin extracted from Escherichia coli (E. coli) serotype O55:B5 was obtained from Sigma Aldrich (St. Louis, MO). Brij L9 (laureth-9) was obtained from Croda International Plc. (East Yorkshire, United Kingdom). Hi-Trap MabSelectSuRe, Hi-Trap Q HP, and Hi-Trap SP Sepharose Fast Flow were obtained from GE Healthcare (Piscataway, NJ). Phosphate-buffered saline (PBS) was obtained from Sigma Aldrich (St. Louis, MO); all other buffer salts were obtained from Avantor Performance Chemicals (Center Valley, Pennsylvania).

[0145] An endotoxin standard was prepared by dissolving 25 mg of O55:B5in in 5 mL of UP-grade water. This standard was then diluted 100-fold with USP-grade water to prepare the final additive standard. All protein solutions were added to obtain a final endotoxin concentration of approximately 1000 EU / ml. All initial endotoxin levels were measured and reported.

[0146] For chromatography experiments, purification was controlled using Unicorn 7.0 software on an AKTA AVANT purchased from GE Healthcare (Piscataway, NJ). A known amount of endotoxin was added to the protein solution and conjugated to various chromatography resins. Upon protein conjugation, a washing solution of equilibration buffer containing 1% laureth-9 was applied to the column in volumes exceeding 10. This was followed by 10 CV washing with equilibration buffer, after which the protein was eluted from the resin. Lysozyme-endotoxin material was conjugated to an SP Fast Flow with 1×PBS, pH 7.2 equilibration buffer, followed by washing, equilibration, and elution with 1×PBS, 500 mM sodium chloride, pH 7.2. Monoclonal antibodies containing endotoxin were conjugated to MabSelectSuRe in 1×PBS, pH 7.2, and then washed, equilibrated, and eluted with 50 mM acetate and 30 mM sodium chloride, pH 3.5.

[0147] Endotoxin concentrations were measured using an Endosafe PTS100, purchased from Charles River Laboratories (Charleston, SC), which has a sensitivity of up to 0.1 EU / mL.

[0148] Protein concentrations were measured using Nanodrop, purchased from Thermo Fisher Scientific (Rockville, MD). The protein concentration of each sample was analyzed by absorbance at 280 nm. After calibration to zero absorbance using a blank buffer solution, each protein sample was measured for absorbance at A280. The absorbance was divided by the extinction coefficient of each protein.

[0149] As shown in Figure 9, laureth-9 reduces endotoxin to less than 1 EU / mg (a typical requirement in in vivo studies) and can therefore be used as a washing solution for cation exchange chromatography media to remove endotoxin from lysozyme solutions.

[0150] [Table 9]

[0151] As shown in Table 10, Brij L9 (Laureth-9) reduces endotoxin to less than 1 EU / mg (a typical requirement in in vivo studies) and can therefore be used as a washing solution for protein A affinity chromatography media to remove endotoxin from mAb solutions.

[0152] [Table 10]

Claims

1. A method for purifying a target non-enveloped virus, wherein the non-enveloped virus is an adeno-associated virus (AAV), and the method is: (a) Contacting the mixture containing the non-enveloped virus with a solution containing laureth-9; and (b) Filter the mixture containing the non-enveloped virus. A method that includes this.

2. (a) Filtration includes ultrafiltration or deep filtration; and / or (b) Filtration is performed before or after contact between the mixture and the solution containing laureth-9; and / or (c) The solution contains about 0.1% to about 2% (w / v) of laureth-9. The method according to claim 1.

3. The method according to claim 1, wherein the preparation of the mixture containing the non-enveloped virus comprises culturing host cells containing the genome of the non-enveloped virus; and contacting the mixture with a solution containing laureth-9 comprises contacting the host cells with a solution containing laureth-9.

4. (a) Loading the mixture containing the non-enveloped virus onto a chromatography carrier; (b) Wash the chromatography support with a washing solution containing about 0.1% to about 2% (w / v) of laureth-9; and (c) Elute the non-enveloped virus from the chromatography carrier to obtain a purified eluate containing the non-enveloped virus. The method according to claim 1, including the method described in claim 1.

5. The method according to claim 4, wherein the chromatography comprises one or more of affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and mixed-mode chromatography.

6. The method according to claim 5, wherein affinity chromatography is selected from protein A chromatography, protein G chromatography, or protein L chromatography.

7. The method according to claim 5, wherein the ion exchange chromatography is selected from anion exchange chromatography and cation exchange chromatography.

8. The method according to any one of claims 4 to 7, further comprising the step of filtering the product stream.

9. The method according to claim 8, further comprising ultrafiltration or deep filtration.

10. The purified non-enveloped virus had a physical titer of approximately 10 log １０ The method according to any one of claims 1 to 9, wherein the concentration is greater than vg / ml.

11. The purified non-enveloped virus had an infectivity titer of approximately 8 log. １０ The method according to any one of claims 1 to 9, wherein the TCID is greater than 50 / mL.

12. The method according to any one of claims 1 to 11, wherein the viral infectivity is not significantly reduced compared to a control that has not been in contact with the solution containing laureth-9.

13. A method for inactivating one or more enveloped viruses in a sample containing a biological agent of interest, wherein the biological agent of interest contains a non-enveloped virus, the non-enveloped virus is an adeno-associated virus (AAV), and the method is: (a) Contacting a sample containing the biological preparation of the objective with a solution containing laureth-9; and (b) Incubate the sample with the solution containing laureth-9 for approximately 1 minute to approximately 120 minutes. A method that includes this.

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