Methods for virus inactivation with environmentally compatible detergents

Abstract

The present invention provides methods for using environmentally friendly detergents in the field of recombinant protein production to inactivate product viruses in the feedstream in processes for producing proteins intended for administration to patients, such as therapeutic or diagnostic proteins. The present invention further provides methods where the environmentally friendly detergents used in the present invention effectively inactivate viruses while maintaining the product quality of the therapeutic or diagnostic protein.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant protein production. More specifically, the present invention provides a method for inactivating viruses in the feedstream in a process for producing a protein intended for administration to a patient, such as a therapeutic or diagnostic protein. The present invention further provides a method in which the eco-friendly detergent of the present invention effectively provides antiviral activity and maintains the product quality of the therapeutic or diagnostic protein. This method provides important real-world biological product manufacturing advantages, for example, compared to methods using Triton X-100 and / or methods that do not use detergents. [Background technology]

[0002] Detergents are important reagents in therapeutic or diagnostic protein manufacturing processes to ensure the safety of the final biological product. Viral contamination of the final biological product can have serious clinical consequences, for example resulting from contamination of the source cell line itself or from accidental viral contamination at various manufacturing stages. Thus, detergents are used to remove viral contaminants present in the feedstream during therapeutic or diagnostic protein manufacturing processes. After purifying the protein of interest, the growth media and buffers used in the product manufacturing and purification process are inactivated and discharged as wastewater. However, such discharges containing detergents used in the manufacturing and purification process may raise environmental concerns, as estimated by the Environmental Risk Assessment (ERA) for detergents.

[0003] Current methods for inactivating viruses in manufacturing processes for biological products such as therapeutic or diagnostic proteins rely heavily on the use of the detergent Triton X-100. Triton X-100 is a polyoxyethylene ether that typically achieves potent enveloped virus inactivation of 4 logs or more under a variety of experimental conditions. However, the degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl)phenol (also known as 4-tert-octylphenol), has been identified as a potential environmental toxin for wildlife, including possible estrogenic effects. Removal of 4-tert-octylphenol from waste streams is therefore desirable, but would require complex, lengthy and costly measures. Therefore, due to toxicity concerns, the EU-REACH Committee decided to add Triton X-100 to Annex XIV, the list of substances banned in the EU, and to prohibit its use in recombinant protein manufacturing from 2021 onwards. Alternatives to Triton X-100 in biologics manufacturing are not well established, and therefore there is an urgent and time-critical need for methods to effectively inactivate viruses during manufacturing processes for biologics, such as therapeutic or diagnostic proteins, that are less environmentally toxic than methods incorporating Triton X-100.

[0004] Environmentally compatible alternatives to Triton X-100 for inactivating viruses, particularly enveloped viruses, in protein production methods have been explored, such as in WO2019 / 121846 and WO2015 / 073633. However, the impact of using different detergents for virus inactivation is not consistent. Some detergents are not suitable for use in production methods because of their insolubility or because they are difficult to remove from the feed stream. In addition, some detergents increase the turbidity and / or foaming of the feed stream and therefore require additional measures to reduce the turbidity and / or foaming of the feed stream. Other detergents are ineffective over a wide temperature and pH range and require higher concentrations for virus inactivation. The combination of these variations can result in additional costs and time for protein production methods. Furthermore, the effectiveness of many detergents in production feedstreams of various types and sizes of proteins is not sufficient.

[0005] Thus, there remains a need for methods to effectively inactivate viruses during the production of therapeutic or diagnostic proteins of various types and sizes using environmentally compatible detergents that effectively inactivate a variety of enveloped viruses at both low and high temperatures and pH ranges, and / or that are aqueous, soluble, and / or easily solubilized, and / or that do not affect the turbidity of the feedstream, and thus are suitable for large-scale production, for a reasonable amount of time and at a reasonable concentration of such detergent. Furthermore, the detergent is sufficiently removed from the product feedstream (to meet regulatory requirements) during purification steps without the addition of complex, time-consuming, and / or costly measures to maintain the product quality of the therapeutic or diagnostic protein. Summary of the Invention

[0006] Thus, the present invention addresses one or more of the above problems by providing a method for inactivating viruses in the production of therapeutic or diagnostic proteins. Surprisingly, the method of the present invention provides an environmentally compatible detergent that is highly effective in inactivating viruses in a wide temperature and / or pH range, in a reasonable time, at a reasonable concentration, without toxic effects on the environment, in the production of different types of proteins. Surprisingly, the method of the present invention provides the use of an environmentally compatible detergent, at least comparable to Triton X-100, that easily achieves complete virus inactivation, which has no known environmental toxicity at the concentrations used. Furthermore, the method of the present invention achieves complete virus inactivation during the production of therapeutic or diagnostic proteins of various types and sizes, in a reasonable time, at a reasonable concentration, without toxic effects on the environment, at a temperature range of about 4° C. to at least about 30° C. and a wide pH range, with a virus log reduction factor ("LRF") of about 3 or more to about 6 or more. Surprisingly, the methods of the present invention also achieved viral inactivation within more than 1 minute to about 180 minutes for all enveloped viruses tested, at reasonable concentration ranges, over a wide pH range, and at temperatures ranging from about 4°C to at least about 30°C during the production of therapeutic or diagnostic proteins of various types and sizes.

[0007] Thus, the present invention provides a method for inactivating viruses in a feedstream, comprising adding an environmentally compatible detergent to the feedstream. The exemplary detergent used in the method of the present invention is an aqueous solution that does not significantly affect the turbidity of the feedstream, and is therefore suitable for large-scale production of therapeutic or diagnostic proteins. In addition, the detergent used in the present invention is sufficiently and effectively removed (to meet regulatory requirements) in the purification step, with or without a prior filtration step. Furthermore, no complex, time-consuming, and / or expensive means are required to remove the detergent from the feedstream. In addition, the exemplary detergent used in the method of the present invention does not adversely affect the final quality of the therapeutic or diagnostic protein. The detergent used in the method of the present invention effectively inactivates enveloped viruses over a wide temperature and pH range without toxic effects on the environment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] According to one aspect of the present invention, a method is provided for inactivating viruses with alkyl glycoside detergents in a feedstream in a method for producing a therapeutic or diagnostic protein. According to another aspect of the present invention, the detergent used in the method of the present invention comprises about 40% or more undecyl alkyl glycosides. In another aspect of the present invention, the detergent used in the method of the present invention comprises about 40% or more undecyl alkyl glycosides and less than about 20% other alkyl glycosides. In another aspect of the present invention, the detergent used in the method of the present invention comprises about 40% or more undecyl alkyl glycosides and less than about 10% decyl alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 40% to about 60% undecyl alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 53% to about 57% undecyl alkyl glycosides. In another aspect of the present invention, the detergent used in the method of the present invention comprises about 50% or more undecyl alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 40% to about 60% undecyl alkyl glycosides and less than about 20% other alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 40% to about 60% undecyl alkyl glycosides and less than about 10% decyl alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 53% to about 57% undecyl alkyl glycosides and less than about 20% other alkyl glycosides. In yet another embodiment, the detergent used in the method of the present invention comprises about 53% to about 57% undecyl alkyl glycosides and less than about 10% decyl alkyl glycosides. In still other embodiments, the detergent used in the methods of the invention comprises 0 or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% other alkyl glycosides. In some embodiments of the invention, the other alkyl glycosides comprise a mixture of nonyl alkyl glycosides, decyl alkyl glycosides, and / or lauryl alkyl glycosides.In yet other embodiments, the detergent used in the methods of the invention comprises 0 or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% decyl alkyl glycoside.In one embodiment of the invention, the detergent used in the methods of the invention comprises undecyl alkyl glycoside.

[0009] In one embodiment of the present invention, the detergent used in the method of the present invention has a CAS Registry Number of CAS 98283-67-1. In yet another embodiment, the detergent used in the method of the present invention is SIMULSOL® SL11W. In a further embodiment, the SIMULSOL® SL11W used in the method of the present invention comprises about 40% or more undecyl alkyl glycosides. In yet another embodiment, the SIMULSOL® SL11W used in the method of the present invention comprises about 40% to about 60% undecyl alkyl glycosides. In yet another embodiment, the SIMULSOL® SL11W used in the method of the present invention comprises about 53% to about 57% undecyl alkyl glycosides. In yet another embodiment, the SIMULSOL® SL11W used in the method of the present invention comprises about 40% to about 60% undecyl alkyl glycosides and less than about 10% decyl alkyl glycosides. In yet another embodiment, SIMULSOL® SL11W used in the methods of the invention contains about 53% to about 57% undecyl alkyl glycosides and less than about 10% decyl alkyl glycosides. In yet another embodiment, SIMULSOL® SL11W used in the methods of the invention contains 0 or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% other alkyl glycosides. In an embodiment of the invention, the other alkyl glycosides in SIMULSOL® SL11W used in the methods of the invention include a mixture of nonyl alkyl glycosides, decyl alkyl glycosides, and / or lauryl alkyl glycosides. In yet other embodiments, the SIMULSOL® SL11W used in the methods of the invention contains 0 or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% decyl alkyl glycosides. In one embodiment of the invention, the SIMULSOL® SL11W used in the methods of the invention is an undecyl alkyl glycoside.

[0010] In yet another embodiment, the detergent used in the methods of the invention is SIMULSOL® SL 82. In yet another embodiment, the SIMULSOL® SL 82 used in the methods of the invention contains about 40% to about 60% undecyl alkyl glycoside.

[0011] In further embodiments, the present invention provides a method for inactivating viruses in a feedstream comprising adding an environmentally compatible detergent to the feedstream at a final concentration of about 0.05% to about 1% w / w, the environmentally compatible detergent comprising about 40% or more decyl alkyl glycoside. In some embodiments, the detergent used in the methods of the present invention is added to the feedstream to a final concentration of about 0.1% to about 1% w / w. In some embodiments, the detergent used in the methods of the present invention is added to the feedstream to a final concentration of 0.1% to 1% w / w. In some embodiments, the detergent used in the methods of the present invention is added to the feedstream to a final concentration of about 0.3% to about 1% w / w. In some embodiments, the detergent used in the methods of the present invention is added to the feedstream to a final concentration of 0.3% to 1% w / w. In some embodiments, the detergent used in the methods of the present invention is added to the feedstream to a final concentration of about 0.1% to about 0.3% w / w. In some embodiments, the detergent used in the methods of the invention is added to the feed stream to a final concentration of 0.1% to 0.3% w / w. In yet other embodiments, the detergent used in the methods of the invention is added to the feed stream to a final concentration of about 0.3% w / w. In yet other embodiments, the detergent used in the methods of the invention is added to the feed stream to a final concentration of 0.3%. In yet other embodiments, the detergent used in the methods of the invention is added to the feed stream to a final w / w concentration of about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.5%, about 0.9%, or about 1%.

[0012] In some embodiments of the invention, the feed stream is at about 4° C. to about 30° C. In other embodiments of the invention, the feed stream is at about 4° C. to about 30° C. In yet other embodiments, the feed stream is at about 15° C. to about 30° C. In another embodiment, the feed stream is at about 15° C. to about 20° C. In yet other embodiments of the invention, the feed stream is at about 4° C., about 15° C., about 18° C., about 20° C., about 25° C., or about 30° C.

[0013] In some embodiments of the invention, the feed stream is at a pH of about 5.5 to about 8.0. In other embodiments of the invention, the feed stream is at a pH of about 5.5 to about 8.0. In yet other embodiments of the invention, the feed stream is at a pH of about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0014] In some embodiments, the feedstream is incubated with the detergent for less than about 1 minute to about 180 minutes. In other embodiments, the feedstream is incubated with the detergent for about 6 minutes to about 180 minutes. In other embodiments, the feedstream is incubated with the detergent for about 10 minutes to about 180 minutes. In still other embodiments, the feedstream is incubated with the detergent for about 120 minutes to about 180 minutes. In still other embodiments, the feedstream is incubated with the detergent for about 180 minutes. In still other embodiments, the feedstream is incubated with the detergent for about 120 minutes. In still other embodiments, the feedstream is incubated with the detergent for about 60 minutes. In still other embodiments, the feedstream is incubated with the detergent for about 1, about 5, about 6, about 10, about 15, about 30, about 45, about 60, about 75, about 90, about 105, about 120, about 135, about 150, about 165, or about 180 minutes.

[0015] In embodiments of the invention, the detergent in the feedstream has a viral LRF of about 1 or more. In some embodiments of the invention, the detergent in the feedstream has a viral LRF of about 4 or more. In some embodiments of the invention, the detergent in the feedstream has a viral LRF of about 5 or more. In some embodiments of the invention, the detergent in the feedstream has a viral LRF of about 6 or more. In some embodiments of the invention, the detergent in the feedstream has a viral LRF of about 7 or more. In some embodiments of the invention, the detergent in the feedstream achieves complete viral inactivation. In still other embodiments of the invention, the detergent in the feedstream has a viral LRF of about 1, about 2, about 3, about 4, about 5, about 6 or about 7 or more.

[0016] In some embodiments of the invention, the virus is an enveloped virus. In some embodiments, the virus is a retrovirus, a herpesvirus, a flavivirus, a poxvirus, a hepadnavirus, a hepatitis virus, an orthomyxovirus, a paramyxovirus, a rhabdovirus, or a togavirus.

[0017] In some embodiments, the invention provides a method for inactivating viruses in a feedstream, the method comprising incubating the feedstream with an environmentally compatible detergent at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0 for about 15 minutes to about 180 minutes, wherein the detergent is added to the feedstream at a final concentration of about 0.1% w / w to about 1.0% w / w, and wherein the detergent in the feedstream has a viral LRF of about 2 or greater.

[0018] In some embodiments, the invention provides a method for inactivating viruses in a feedstream, the method comprising incubating the feedstream with an environmentally compatible detergent at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0, for about 6 minutes to about 180 minutes, wherein the detergent is added to the feedstream at a final concentration of about 0.3% w / w to about 1.0% w / w, and wherein the detergent in the feedstream has a viral LRF of about 4 or greater.

[0019] In some embodiments, the invention provides a method for inactivating viruses in a feedstream, the method comprising incubating the feedstream with an environmentally compatible detergent at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0 for about 180 minutes, where the detergent is added to the feedstream at a final concentration of about 0.1% w / w to about 1.0% w / w, and where the detergent in the feedstream has a viral LRF of about 5 or greater.

[0020] In some embodiments, the invention provides a method for inactivating viruses in a feedstream, the method comprising incubating the feedstream with an environmentally compatible detergent at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0 for about 60 minutes to about 180 minutes, where the detergent is added to the feedstream at a final concentration of about 0.3 w / w%, and where the detergent in the feedstream has a viral LRF of about 5 or greater.

[0021] In some embodiments, the present invention provides a method for inactivating viruses in a feedstream, the method comprising incubating the feedstream with an environmentally compatible detergent at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0 for about 120 minutes to about 180 minutes, where the detergent is added to the feedstream at a final concentration of about 0.3 w / w%, and where the detergent in the feedstream has a viral LRF of about 6 or greater.

[0022] In some embodiments, the present invention provides a method for inactivating viruses in a feedstream, the feedstream comprising a harvested cell culture fluid, a capture pool, or a harvested product pool. The present invention provides a method for inactivating viruses in a feedstream, the feedstream comprising a harvested cell culture fluid, a capture pool, or a harvested product pool, the capture pool or the harvested product pool being an affinity chromatography pool. In yet another embodiment, the capture pool or the harvested product pool is a protein A pool, a protein G pool, or a protein L pool. In other embodiments, the capture pool or the harvested product pool is a mixed-mode chromatography pool.

[0023] In some embodiments, the present invention provides a method in which the feedstream incubated with detergent is not subjected to a filtration step. In some embodiments, the present invention provides a method in which the feedstream incubated with detergent is subjected to at least one filtration step. In other embodiments, the present invention provides a method in which the feedstream incubated with detergent is subjected to at least one, or at least two, or at least three filtration steps. In some embodiments, the present invention provides a method in which the feedstream incubated with detergent is subjected to a first filtration step using a 0.22 μm filter, followed by a second and a third filtration step using a 0.45 μm PVDF filter. In certain embodiments, the present invention provides a method in which the filter membrane in the first and / or second and / or third filtration steps includes, but is not limited to, vinylidene difluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone, glass fiber filters, or other filter media suitable for use in a cGMP manufacturing environment. In a preferred embodiment, the present invention provides a method in which the filter membrane in both the second and third filtration steps comprises PVDF. In a particular embodiment, the present invention provides a method in which the filter membrane in both the second and third filtration steps comprises a membrane with a pore size of about 0.45 μm. In a particular embodiment, the present invention provides a method in which the filter membrane in both the second and third filtration steps comprises a PVDF membrane with a pore size of 0.45 μm. In a particular embodiment, the present invention provides a method in which the filter membrane in the first filtration step comprises a membrane with a pore size of about 0.22 μm.

[0024] In some embodiments, the invention provides methods in which the feedstream incubated with the detergent is subjected to affinity chromatography, hi some embodiments, the affinity chromatography is a Protein A affinity column.

[0025] In some embodiments, the present invention provides a method in which a feed stream containing a detergent is subjected to a chromatography column. In some embodiments, the chromatography column is one or more of an affinity column, an ion exchange column, a hydrophobic interaction column, a hydroxyapatite column, or a mixed mode column. In some embodiments, the affinity chromatography column is a protein A column, a protein G column, or a protein L column. In other embodiments, the ion exchange chromatography column is an anion exchange column or a cation exchange column. In some embodiments, the present invention provides a method in which the detergent is substantially removed from the final product. In some embodiments, the present invention provides a method in which the detergent is completely removed from the final product.

[0026] In some embodiments, the present invention provides a method for inactivating viruses in a feedstream, the method comprising adding a detergent to the feedstream and incubating the detergent with the feedstream, wherein the therapeutic or diagnostic protein is an antibody, an Fc fusion protein, an immunoadhesin, an enzyme, a growth factor, a receptor, a hormone, a regulator, a cytokine, an antigen, or a binding agent. In further embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment. In some embodiments, the therapeutic or diagnostic protein is produced in a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell, or a baby hamster kidney (BHK) cell, a mouse hybridoma cell, or a mouse myeloma cell. In some embodiments, the therapeutic or diagnostic protein is produced in a bacterial cell. In other embodiments, the therapeutic or diagnostic protein is produced in a yeast cell.

[0027] In some embodiments of the invention, the detergent-containing feed stream used in the methods of the invention is of low turbidity. In some embodiments, the addition of the detergent to the feed stream does not significantly increase or change the turbidity of the feed stream. In further embodiments, the detergent used in the methods of the invention does not affect the product quality of the therapeutic or diagnostic protein.

[0028] In some embodiments, the detergent used in the methods of the invention includes a preservative and / or stabilizer. In some embodiments, the stabilizer is monopropylene glycol. In some embodiments, the stabilizer in the detergent comprises about 1% to about 5%. In further embodiments, the stabilizer does not affect the virus inactivation properties of the detergent used in the methods of the invention and does not affect the product quality of the therapeutic or diagnostic protein. In further embodiments, monopropylene glycol does not affect the virus inactivation properties of the detergent used in the methods of the invention and does not affect the product quality of the therapeutic or diagnostic protein.

[0029] In an embodiment of the present invention, the detergent used in the method of the present invention is environmentally compatible. In an embodiment of the present invention, the detergent used in the method of the present invention does not contain or form 4-(1,1,3,3-tetramethylbutyl)phenol (also known as 4-tert-octylphenol). In another embodiment, the detergent used in the method of the present invention does not contain or form peroxides. In another embodiment of the present invention, the detergent used in the method of the present invention is biodegradable.

[0030] In one aspect, the present invention provides a method for inactivating viruses in a feedstream, comprising the steps of: adding to the feed stream an undecyl alkyl glycoside at a concentration of about 0.1 w / w% to about 0.3 w / w%; incubating the undecyl alkyl glycoside in the feed stream for about 180 minutes, wherein the undecyl alkyl glycoside in the feed stream has a viral log reduction factor (LRF) of about 5 or greater; filtering the feedstream containing undecyl alkyl glycosides, and then subjecting the filtered feedstream to a Protein A affinity chromatography column, comprising: The adding and incubating steps are carried out in the feed stream at a temperature of about 4° C. to about 30° C. and a pH of about 5.5 to about 8.0.

[0031] In one aspect, the present invention provides a method for inactivating viruses in a feedstream, comprising the steps of: adding SIMULSOL® SL11W to the feed stream at a concentration of about 0.1 w / w% to about 0.3 w / w%; incubating SIMULSOL® SL11W in the feedstream for about 180 minutes, wherein the viral log reduction factor (LRF) of SIMULSOL® SL11W in the feedstream is about 5 or greater; filtering the feedstream containing SIMULSOL® SL11W and then subjecting the filtered feedstream to a Protein A affinity chromatography column, The adding and incubating steps are carried out in the feed stream at a temperature of about 4°C to about 30°C and a pH of about 5.5 to about 8.0. In yet another aspect of the invention, the filtration step in the method of the invention comprises a first filtration step using a 0.22 μm filter. In yet another aspect of the invention, the filtration step in the method of the invention comprises a second filtration step using a 0.45 μm PVDF filter. In yet another aspect, the filtration step in the method of the invention comprises a third filtration step using a 0.45 μm PVDF filter. In a further embodiment, the filter membrane in the first and / or second and / or third filtration steps in the method of the invention comprises, but is not limited to, vinylidene difluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone, glass fiber filters, or other filter media suitable for use in a cGMP manufacturing environment. In a preferred embodiment, the filter membrane comprises PVDF. In a particular embodiment, the filter membrane in both the second and third filtration steps is a membrane having a pore size of about 0.45 μm. In a preferred embodiment, the filter membrane is a PVDF membrane having a pore size of 0.45 μm.

[0032] As used herein, an "environmentally compatible" detergent or compound or substance or agent referred to herein causes minimal and / or acceptable levels of harmful effects to the environment. For example, the Organization for Economic Cooperation and Development ("OECD") provides guidelines for testing the safety of chemicals. An embodiment of an environmentally compatible agent according to the present disclosure may also be biodegradable. Methods for determining whether an agent, such as a detergent, is environmentally compatible under conditions of, for example, protein production, are known in the art. Environmental compatibility may also be evaluated by ecological risk assessment, which is a process that evaluates the likelihood that the environment will be affected as a result of exposure to one or more environmental stressors, such as chemicals, land changes, diseases, invasive species, and climate change. These guidelines can be found, for example, on the U.S. Environmental Protection Agency website. Additionally, guidelines for the registration, evaluation, authorization, and restriction of chemicals in the EU can be found on the European Commission's REACH website.

[0033] "Predicted Environmental Concentration" or "PEC" is the predicted concentration of a substance in a waste stream discharged into a receiving body of water in the environment. For example, the predicted environmental concentration of a detergent used to inactivate viruses in the preparation of therapeutic or diagnostic proteins is the concentration of the detergent in the waste stream discharged into the environment.

[0034] The term "detergent" as used herein refers to an agent that may contain a hydrophilic moiety, such as a salt of a long-chain aliphatic base or acid, or a sugar, and has both hydrophilic and hydrophobic properties. As used herein, a detergent may have the ability to disrupt the viral envelope and inactivate the virus. The mechanism of virus inactivation by detergents is due to the interaction between the detergent and lipid components of the viral outer membrane, resulting in the disruption of the membrane and ultimately loss of toxicity. In some examples, a detergent may be a composition that includes a "surfactant" or "surface acting agent" and one or more other agents, such as a chelating agent, a preservative, or a stabilizer. Detergents or surfactants may be classified based on charge. Surfactants may be nonionic, cationic, or anionic. As used herein, a detergent has the ability to disrupt the viral envelope and inactivate the virus. An embodiment of a detergent according to the present disclosure includes an alkyl glycoside, which may be an alkyl glucoside.

[0035] An "alkyl glycoside" according to the present disclosure refers to any sugar attached by a bond to any hydrophobic alkyl. As used herein, an "alkyl glucoside" consists of an alkyl group attached to a sugar, the sugar being glucose. The alkyl glycoside may be, for example, an undecyl alkyl glycoside. The undecyl alkyl glycoside may include an undecyl chain with an O-glycosidic linkage to a mono-D-glucopyranose (e.g., n-undecyl-β-D-glucopyranose), or a D-glucopyranose oligosaccharide or polysaccharide, or a mixture thereof. Chemical synthesis of alkyl glycosides as used in the methods of the present invention may result in a heterogeneous mixture of compounds rather than a completely homogeneous preparation. Thus, unless otherwise specified, a reference to a particular form of alkyl glycoside as used herein means that at least the majority component of any heterogeneous mixture is that form of alkyl glycoside, such as an undecyl alkyl glycoside. Examples of such alkyl glycosides, including undecyl alkyl glycosides, include, but are not limited to, SIMULSOL® SL11W and SIMULSOL® SL82.

[0036] A "product feedstream" or "feedstream" is a material or solution provided to a process purification method that contains the therapeutic or diagnostic protein of interest and may also contain various impurities. Non-limiting examples can include, for example, harvested cell culture fluid (HCCF), harvested cell culture material, clarified cell culture fluid, clarified cell culture material, capture pools, recovery pools, and / or recovery pools containing the therapeutic or diagnostic protein of interest after one or more centrifugation and / or filtration steps, where the capture pool, recovered protein pool, and / or recovery pool contains the therapeutic or diagnostic protein of interest after one or more purification steps.

[0037] The term "impurities" refers to materials other than the desired protein product. Impurities include, but are not limited to, host cell materials such as CHOP, leached protein A, nucleic acids, mutants, size variants, fragments, aggregates or derivatives of the desired protein, other proteins, endotoxins, viral contaminants, cell culture media components, etc.

[0038] The terms "protein" and "polypeptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, proteins that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), and other modifications known in the art. Examples of proteins include, but are not limited to, antibodies, peptides, enzymes, receptors, hormones, regulators, antigens, binding agents, cytokines, Fc fusion proteins, immunoadhesin molecules, and the like.

[0039] The term "antibody" or "antibodies" is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), chimeric antibodies, humanized antibodies, human antibodies, antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), immunoadhesins, and fragments of antibodies so long as they exhibit the desired biological and immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0040] The term "ultrafiltration" or "filtration" is a form of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. High molecular weight suspended solids and solutes are retained while water and low molecular weight solutes pass through the membrane. In some instances, ultrafiltration membranes have pore sizes ranging from 1 μm to 100 μm. The terms "ultrafiltration membrane", "ultrafiltration filter", "filtration membrane" and "filtration filter" may be used interchangeably. Examples of filtration membranes include, but are not limited to, polyvinylidene fluoride (PVDF) membranes, cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone, glass fiber, or other filtration materials suitable for use in a cGMP manufacturing environment.

[0041] As used herein, numerical ranges are inclusive of the numbers defining the range.

[0042] The term "enveloped virus" or "lipid coat-containing virus" refers to any virus that contains a lipid-containing membrane or envelope, such as, for example, enveloped viruses. Enveloped viruses have a capsid, or envelope, surrounded by a lipoprotein membrane. This envelope originates from the host cell as the virus "buds" from its surface and is composed of lipids that are mostly not encoded by the viral genome. It carries the molecular determinants for binding and entry into target cells and is essential for the infectivity of enveloped viruses, but is not subject to drug resistance or antigenic shifts. Enveloped viruses range in size from about 45-55 nm to about 120-200 nm. Non-limiting examples of lipid-coat-containing viruses that may infect mammalian cells include DNA viruses such as Herpesviridae, Poxviridae, or Hepadnaviridae; RNA viruses such as Flaviviridae, Togaviridae, Coronaviridae, Deltaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, or Filoviridae; and reverse transcribing viruses such as Retroviridae or Hepadnaviridae. In a variation, the present invention provides a method for inactivating subviral agents in a product feedstream, comprising subjecting the feedstream to an environmentally compatible detergent. In some embodiments, the subviral agent is a viroid or a satellite. In another variation, the present invention provides a method for inactivating virus-like agents in a product feedstream, comprising subjecting the feedstream to an environmentally compatible detergent.

[0043] Methods for measuring viral activity or viral infectivity are known in the art. Examples include TCID 50These include, but are not limited to, the 50% tissue culture infectious dose assay (i.e., the determination of the 50% tissue culture infectious dose that produces pathological changes in 50% of the inoculated cell cultures) and the plaque assay. Other known methods include, for example, the transformation assay, which can be used to determine the titer of the biological activity of non-plaque-forming viruses that have the ability to cause cell growth transformation, or the fluorescent focus assay, which uses antibody staining to detect viral antigens in monolayers of infected cells, or the end-point dilution assay, which can be used to determine the titer of many viruses, including viruses that do not infect monolayer cells (as an alternative to the plaque assay), or the viral enzyme assay, which measures virally encoded enzymes such as reverse transcriptase or viral protease. Additionally, detection of a specific virus can be achieved by polymerase chain reaction (PCR) using primers and probes designed to detect the specific virus.

[0044] Viral inactivation in product feedstreams can be measured using methods known in the art. In some embodiments of the invention, viral inactivation is expressed as a log reduction factor ("LRF") or log reduction value ("LRV"). LRF is calculated as follows:

number

[0045] Example 1. Virus inactivation by non-ionic glycoside detergents Detergents and Reagents Stock solutions of the exemplary detergents listed in Table 1 (Seppic Inc. Fairfield, NJ), and 1,2-propanediol (Sigma-Aldrich, St. Louis, MO) are prepared in water at weight / volume (w / w) percentages. [Table 1]

[0046] Viruses and indicator cells Xenotropic murine leukemia virus (XMuLV), porcine parvovirus (PPV) strain NADL-2, and pseudorabies virus (PRV) used in these studies can be purchased from ATCC, Manassas, Va. Table 2 shows the characteristics of these viruses. [Table 2]

[0047] PK13 (porcine epithelial cells), PG-4 (feline brain cells), and Vero (African green monkey cells) used in these studies can be purchased from ATCC, Manassas, VA. PK-13 cells are cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY). PG-4 cells are cultured in McCoy's 5a medium (ATCC, Manassas, VA), and Vero cells are cultured in Earle's modified Eagle's medium (EMEM, Gibco, Grand Island, NY). All cells are supplemented with 10% FBS (HyClone, Logan, UT).

[0048] Cell-based TCID 50 Virus titration assay Cell-based 50% Tissue Culture Infective Dose (TCID 50 The ) assay is used for virus titration for XMuLV using PG-4 indicator cells, PPV using PK-13 indicator cells, and PRV using Vero indicator cells. 2.5 × 10 4Indicator cells at 10 cells / mL are seeded at 200 µL / well and incubated at 37 °C overnight or for approximately 16-24 hours. Plates are then inoculated with virus in a series of 10-fold dilutions at 50 µL / well with a minimum of n=8 per dilution. Inoculated plates are incubated at 37 °C for 7-9 days (XMuLV and PRV) and 10-13 days (PPV). Plates are then inspected and scored under a microscope for cytopathic effect (CPE) as positive or negative per well, and virus titers are calculated using standard methods.

[0049] Viral cytotoxicity and interference in depuration bioreactor harvest material The TCID shown above 50 The assay is used to determine the cytotoxic effect of viruses in clarified bioreactor harvest material of monoclonal antibodies (mAbs), bispecific antibodies, and Fc fusion proteins. Bioreactor harvest material is clarified by centrifugation followed by abrasive filtration. Impurities such as HCPs, lipids, and host cell DNA are also automatically concentrated as part of the centrifugation process. Clarified bioreactor harvest material of various proteins containing 0.05 w / w% to 1 w / w% detergent is diluted 10, 50, and 100-fold in inoculation medium. Each dilution is added to a 96-well indicator cell-seeded plate with n=8 and TCID 50 Incubate according to the assay. At the end of the incubation period, observe each well under a microscope for CPE and determine the appropriate dilution required for each detergent. Results showed that a 50-fold dilution factor was sufficient to overcome cytotoxicity by the detergent.

[0050] The clarified bioreactor harvest material for the various protein types is evaluated for the inherent effects of the cell culture harvest material on viral replication. Each virus is diluted 10-fold in the clarified bioreactor harvest material and inoculated into indicator cell seeded plates, with the TCID 50Run the experiment according to the assay. Include a positive control (tissue culture material spiked with virus). Compare the virus titer to the positive control virus. ±0.5 log of the positive control titer. 10 Titers within ±0.5 log of the positive control titer are considered to be free of viral interference. All virus test titers are within ±0.5 log of the positive control titer. 10 within 100% of the total protein content and therefore no viral interference was observed for any of the bioreactor harvest materials, regardless of protein type.

[0051] General procedure for virus inactivation Viral inactivation of various enveloped viruses by each detergent is evaluated at various concentrations, temperatures, and pH ranges in clarified bioreactor harvest material containing mAb, bispecific, and Fc fusion proteins. XMuLV, PPV, or PRV viruses are spiked into the harvest material at 5% w / w or less, and detergent stock solutions are added to the spiked material to achieve the specified final detergent concentration. The samples are then incubated at specific temperatures and pH ranges. Viral inactivation begins as soon as the detergent is added to the virus-containing material. The kinetics of viral inactivation are monitored by removing aliquots from the harvest sample at various time points, and viral titers are measured in TCID 50 A matrix control is included in the study to demonstrate that viral inactivation is a result of the detergent treatment and not the matrix itself.

[0052] Inactivation of XMuLV by non-ionic detergents at 30°C Viral inactivation of XMuLV by the detergents listed in Table 1 is tested at the optimal conditions determined for XMuLV inactivation. Following the viral inactivation procedure exemplified above, each detergent is added at 0.6 w / w% to the XMuLV-spiked clarified mAb bioreactor harvest material at 30° C. Viral inactivation is monitored at various time points with a maximum inactivation time of 180 min after viral inoculation.

[0053] The results summarized in Table 3 show that at the optimal conditions for XMuLV inactivation, XMuLV is rapidly and completely inactivated by all detergents tested at 180 min, except for SIMULSOL® SL 4 and SIMULSOL® AS 48. Since no virus inactivation was observed with SIMULSOL® SL 4, the concentration of SIMULSOL® SL 4 is increased to 30 mM (1%) and the inactivation of XMuLV is monitored at 30° C. for 180 min. However, no inactivation of XMuLV was observed with increasing concentrations of SIMULSOL® SL 4 (data not shown). Surprisingly, these results suggest that not all glycosides allow potent virus inactivation or require very high concentrations to achieve potent inactivation as observed with SIMULSOL® AS 48. However, SIMULSOL® SL11W demonstrated complete viral inactivation with an LRF of 4 or greater within 0-5 minutes of addition to purified mAb bioreactor harvest material. Furthermore, even if other glycosides effectively inactivated XMuLV, other factors limit the applicability of some of these detergents in large-scale production methods. Such detergent properties include solubility, effect on turbidity in harvest material, activity at different temperatures and pH ranges, unknown or limited data on environmental toxicity, and ease of handling. [Table 3]

[0054] Turbidity of SIMULSOL® SL11W in clarified mAb bioreactor harvest material. Turbidity is assessed in XMuLV spiked into clarified mAb bioreactor harvest material at 18° C. at concentrations of 0%, 0.05%, 0.1%, 0.3%, 0.5%, and 1% w / w of SIMULSOL® SL11W. Viral inactivation is monitored for 180 minutes and turbidity is measured in nephelometric turbidity units (NTU) using a handheld turbidimeter 2100P (Hach®).

[0055] The results, shown in Table 4, indicate that SIMULSOL® SL11W at concentrations ranging from 0 w / w% to 1 w / w% was stable over time, with no total precipitation observed in the harvest up to 180 minutes. [Table 4]

[0056] Characterization and Optimization of Viral Inactivation by SIMULSOL® SL11W The use of SIMULSOL® SL11W as a viable environmentally compatible detergent for use in protein production processes is further evaluated in clarified mAb bioreactor harvest material following the virus inactivation procedure described above. Briefly, SIMULSOL® SL11W activity is evaluated in XMuLV-spiked clarified mAb bioreactor harvest material at concentrations of 0.05%, 0.1%, 0.3%, 0.5%, and 1 w / w% at four different temperatures: 4°C, 18°C, 25°C, and 30°C. Viral inactivation is monitored 0, 10, 30, 60, 120, and 180 minutes after inoculating XMuLV into SIMULSOL® SL11W containing clarified mAb bioreactor harvest material. The results shown in Tables 5-9 demonstrate that SIMULSOL® SL11W concentration, incubation time, and temperature are all factors affecting the inactivation of XMuLV. SIMULSOL® SL11W achieved complete XMuLV viral inactivation by 120 minutes at concentrations ≧0.1 w / w% (≧3 mM) and by 10 minutes at concentrations ≧0.3 w / w% at all four temperatures evaluated. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0057] Inactivation of XMuLV by SIMULSOL® SL11W in different starting matrices The ability of SIMULSOL® SL11W to inactivate virus at 9 mM (0.3 w / w%) in different starting matrices, including water, cell culture medium, and Dulbecco's phosphate buffered saline (DPBS), is evaluated at 4° C. and 30° C. Viral inactivation is monitored at 0, 60, 120, and 180 minutes after inoculation with XMuLV.

[0058] The results, shown in Table 10, demonstrate robust XMuLV inactivation at both 4°C and 30°C after 120 min in all three starting matrices. DPBS at 4°C after 180 min inactivated 10% of the TCID 50 Although some live virus particles were detected in the assay, an LRF of 5.63 was still achieved. Overall, these results suggest that SIMULSOL® SL11W can effectively inactivate viruses regardless of the starting matrix. This suggests the potential use of SIMULSOL® SL11W across biopharmaceutical manufacturing platforms, and the flexibility of using SIMULSOL® SL11W at different stages of protein manufacturing processes. [Table 10]

[0059] Inactivation kinetics of XMuLV with 0.3% SIMULSOL® SL11W at 4°C and 30°C The ability of SIMULSOL® SL11W to inactivate virus at lower time points is evaluated. SIMULSOL® SL11W is added at 9 mM (0.3 w / w%) to clarified mAb bioreactor harvest material at 4° C. and 30° C. Viral inactivation is monitored at 0, 2, 4, 6, 8, 10, 20 and 180 minutes after inoculation with XMuLV.

[0060] The results, shown in Table 11, indicate that complete XMuLV inactivation occurs in ≧6 minutes at 4° C. and ≧2 minutes at 30° C. with 9 mM (0.3 w / w%) SIMULSOL® SL11W in the clarified mAb bioreactor harvest material. [Table 11]

[0061] Effect of the stabilizer monopropylene glycol on virus inactivation The effect of monopropylene glycol at concentrations of 0.1% and 0.5% on viral inactivation in clarified mAb bioreactor harvest material at 30°C is evaluated. Viral inactivation is monitored at 0 and 180 min after inoculation with XMuLV. SIMULSOL® SL11W contains 1% to 5% monopropylene glycol. The maximum concentration of monopropylene glycol seen in the inactivation procedure reported here is 0.05%. The results shown in Table 12 indicate that monopropylene glycol at concentrations of 0.1% and 0.5% does not inactivate XMuLV in clarified mAb bioreactor harvest material at 30°C. Furthermore, the concentrations of monopropylene glycol shown in Table 11 are at least 10-fold greater than the maximum concentration seen in clarified mAb bioreactor harvest treated with SIMULSOL® SL11W. This indicates that SIMULSOL® SL11W is responsible for the observed viral inactivation. [Table 12]

[0062] Inactivation of enveloped and non-enveloped viruses by SIMULSOL® SL11W The ability of SIMULSOL® SL11W to inactivate the enveloped virus PRV and the non-enveloped virus PPV is evaluated. SIMULSOL® SL11W is added at 9 mM (0.3 w / w%) to the clarified mAb bioreactor harvest material at 4° C., 18° C., and 30° C. Viral inactivation is monitored at 0, 10, 30, 60, 120, and 180 minutes post-inoculation.

[0063] The results shown in Table 13 show strong inactivation of PRV enveloped virus occurring by 10 minutes at all three temperatures, with even lower inactivation of PRV enveloped virus occurring within 0-5 minutes at 30°C. No inactivation of PPV was observed with SIMULSOL® SL11W. Taken together, these results suggest that SIMULSOL® SL11W not only strongly inactivates different enveloped viruses in the clarified mAb bioreactor harvest material, but that viral inactivation by SIMULSOL® SL11W is specific to enveloped viruses. [Table 13]

[0064] Effect of pH on inactivation of XMuLV by SIMULSOL® SL11W The ability of SIMULSOL® SL11W to inactivate XMuLV at pH 5.5 and pH 8.0 is evaluated. SIMULSOL® SL11W is added at 9 mM (0.3 w / w%) to the clarified mAb bioreactor harvest material at 18° C. Viral inactivation is monitored at 0, 10, 30, 60, 120, and 180 minutes after inoculation of XMuLV into SIMULSOL® SL11W containing the clarified mAb bioreactor harvest material.

[0065] The results shown in Table 14 demonstrate rapid and complete inactivation of XMuLV by SIMULSOL® SL11W at both pH 5.5 and pH 8.0 by 10 minutes, thus demonstrating the potential application of SIMULSOL® SL11W over a broad pH range in protein production methods. [Table 14]

[0066] Effect of bioreactor harvest material source on inactivation of XMuLV by SIMULSOL® SL11W The ability of SIMULSOL® SL11W to inactivate XMuLV in clarified bioreactor harvest materials containing various recombinant proteins such as mAb, bispecific antibody, and Fc fusion protein is evaluated. SIMULSOL® SL11W is added at 9 mM (0.3 w / w%) to clarified bioreactor harvest materials of different proteins at 4°C and 18°C. Viral inactivation is monitored at 0, 10, 30, 60, 120, and 180 minutes of incubation. The results shown in Table 15 show complete inactivation of XMuLV by SIMULSOL® SL11W in all three clarified bioreactor harvest materials at both 4°C and 30°C by 120 minutes. Furthermore, complete inactivation was observed by 10 minutes at 4°C in harvests containing Fc fusion protein and mAb protein. The inactivation kinetics of the bispecific antibody was slightly slower at 4° C. than the other two clarified bioreactor harvest materials at 4° C. However, at 18° C., complete inactivation by SIMULSOL® SL11W was observed for all three proteins within 10 minutes. [Table 15]

[0067] Effect of impurity levels on virus inactivation in clarified bioreactor harvest material of different proteins The impact of impurity levels in the bioreactor harvest material of three different recombinant proteins is evaluated: an Fc fusion protein, a bispecific antibody, and a mAb.

[0068] The harvest material is clarified by centrifugation followed by polishing filtration. Impurities such as HCPs, lipids, and host cell DNA are also automatically concentrated as part of the centrifugation process. A sample of the clarified mAb harvest material is further concentrated approximately 4-fold by a 5 KDa molecular weight cutoff tangential flow filtration (TFF) process. The recombinant protein, DNA and other HCP impurities in the three clarified materials and the TFF concentrated material are measured. The results shown in Table 16 show the recombinant protein, host cell DNA, and HCP content profiles in the three recombinant protein purified materials and the concentrated purified mAb material. A greater than 3-fold increase in DNA and HCP impurities was observed in the concentrated clarified mAb harvest material when compared to the non-concentrated clarified mAb harvest material. [Table 16]

[0069] Effect of concentrated bioreactor harvest material on inactivation of XMuLV by SIMULSOL® SL11W The effect of SIMULSOL® SL11W on virus inactivation in concentrated clarified mAb harvest material is tested. The clarified mAb bioreactor harvest material containing mAb and other impurities is concentrated approximately 4-fold by a 5KDa molecular weight cutoff tangential flow filtration (TFF) process. The concentrated material containing mAb and impurities is then immediately subjected to sterile filtration. SIMULSOL® SL11W is then added to the clarified concentrated filter medium at a final concentration of 0.1 w / w% (3mM). The material containing SIMULSOL® 11W is then incubated at three different temperatures: 4°C, 18°C, and 30°C. The virus inactivation at various temperatures is measured at time points ranging from 0 minutes to 180 minutes. The results, shown in Table 17, indicate that XMuLV was completely inactivated at 180 minutes at all three temperatures tested, although the inactivation rate was reduced at lower temperatures when compared to the 30° C. samples. No effect on inactivation kinetics was observed for concentrated bioreactor material when compared to unconcentrated bioreactor harvest material (data not shown), suggesting that increasing impurity levels do not affect viral inactivation by SIMULSOL® SL11W. [Table 17]

[0070] Example 2. Determining the removal of SIMULSOL® SL11W in downstream recombinant protein purification Quantitation of undecyl glycosides by liquid chromatography-mass spectrometry (LC-MS) - Method A LC-MS quantification is performed on clarified mAb bioreactor harvest samples containing SIMULSOL® SL11W at concentrations ranging from 0.03 mM to 3 mM.

[0071] One microliter of the incubated sample is injected into a Waters Acquity® UPLC coupled to a Waters SYNAPT® G2-Si mass spectrometer. For quantification, separation is performed on a Varian PLRP-S reversed-phase column (1×50 mm, 300 Å, 5 μm) at 80° C. using 0.05% trifluoroacetic acid (TFA) in H2O as mobile phase A and 0.04% TFA in acetonitrile (ACN) as mobile phase B. The column is equilibrated with 15% mobile phase B for 1 min, linearly increased from 15% to 30% over 3 min, then held at 30% B for 2 min, then held at 100% for 1 min. The column is held at 100% for 1 min, then re-equilibrated with 15% mobile phase B. Separation is performed on a SIMULSOL® SL11W at 0.3 mL / min for 1-6 min and analyzed using an electrospray ionization (ESI) source. The mass spectrometer is operated in positive sensitivity mode, with a scan range of 100-1000 amu, a capillary voltage of 3.2 kV, a desolvation temperature of 450 °C, a desolvation gas flow rate of 900 L / h, a source temperature of 150 °C, and a sample cone of 40 V. For characterization, separations are performed on a Waters Acquity® UPLC BHE 300 Å C4 column (2.1 × 100 mm, 1.7 μm particle size) using the same mobile phase. The column is equilibrated with 20% mobile phase B for 1 min, linearly increased from 20% to 25% in 14 min, then to 90% in 1 min. After a 1.0 min hold at 100%, the column is re-equilibrated with 20% mobile phase B.

[0072] The extracted peak area of ​​sodiated undecyl glycoside was measured. Under LC-MS analysis conditions, a linear relationship was observed between the extracted ion peak area and SIMULSOL® SL11W concentrations from 0.03 to 3 mM. When the SIMULSOL® SL11W concentration exceeded 3 mM, the MS detector became saturated.

[0073] Quantitation of undecyl glycosides by liquid chromatography with tandem mass spectrometry (LC-MS / MS) - Method B Standard solutions containing SIMULSOL® SL11W at concentrations ranging from 0.0008 mM to 0.012 mM are prepared in a mainstream of water or protein A. After the addition of SIMULSOL® SL11W, these samples were subjected to LC-MS / MS (MRM) analysis. The incubated samples (100 μL) are injected into the LC with a 1 / 20 post-column split to the MS. For the analysis, an Agilent 1260 Infinity II HPLC coupled to a Sciex Triple Quad 5500 mass spectrometer is used. Quantification is performed on an Agilent Zorbax, 300SB-C8 reversed-phase column (4.6 × 50 mm, 300 Å, 3.5 μm) at 80 °C using 0.5 mM sodium acetate in water as mobile phase A and 95:5 acetonitrile:water as mobile phase B. The column is equilibrated with 20% mobile phase B for 2 min, linearly increased from 20% to 90% over 10 min, held at 90% B for 2 min, and equilibrated with 20% mobile phase B. SIMULSOL® SL11W was separated at 1.0 mL / min between 4.5 and 5.5 min and analyzed using an electrospray ionization (ESI) source. The mass spectrometer is operated in positive MRM mode scanning transition ions (357.0-185.0), a capillary voltage of 5.5 kV, a desolvation temperature of 450 °C, and a desolvation gas flow rate of 55 L / h.

[0074] Selected reaction monitoring (SRM / MRM), an LC-MS / MS method, is used to selectively detect sodiated SIMULSOL® SL11W. SRM scans a single precursor ion (mass of sodiated SIMULSOL® SL11W, 357.0 m / z) and detects the product ion after fragmentation (the most predominant fragment of sodiated SIMULSOL® SL11W, 185.0 m / z) and obtains the peak area corresponding to this extracted transition ion.

[0075] Under LC-MS / MS analytical conditions, the plot is linear between SIMULSOL® SL11W concentrations of 0.0008 mM to 0.012 mM in water and the predominant amount of protein A. These results show that LC-MS / MS can detect SIMULSOL® SL11W at concentrations of 0.0008 mM to 0.012 mM and that the linearity of the plot is matrix independent. Thus, this LC-MS / MS (SRM / MRM) method is specific for the detection of SIMULSOL® SL11W and is independent of the matrix in which the detergent is present. The limit of detection (LOD) is 0.001 mM calculated from the linearity.

[0076] Protein A Chromatography and Detection with SIMULSOL® SL11W The clarified mAb bioreactor harvest material solution containing 0% (control), 0.3%, 1.0 w / w% SIMULSOL® SL11W is subjected to MabSelect® Protein A chromatography. Protein A chromatography is performed on the unfiltered clarified mAb harvest material containing SIMULSOL® SL11W. After loading the column, it is washed using 6 column volumes of Tris / NaCl before eluting the mAb with acetate / citrate buffer. The main stream of eluted mAb is collected and analyzed by LC-MS for the presence of SIMULSOL® SL11W.

[0077] Results of the analysis using LCMS (Method A) show SIMULSOL® SL11W levels of <0.03 mM in the protein A mainstream from mAb bioreactor harvest material solutions containing 0.3% and 1.0 w / w% SIMULSOL® SL11W. Using LC-MS / MS (Method B), the amount of SIMULSOL® SL11W was found to be 0.0005 mM in the protein A mainstream from mAb bioreactor harvest material solutions containing 0.3 w / w% SIMULSOL® SL11W, which is lower than the LOD of 0.001 mM. These results indicate that Protein A chromatography can remove SIMULSOL® SL11W Simulsol SL11W from the feed stream to trace levels without a filtration step prior to column loading.

[0078] Example 3. Effect of SIMULSOL® SL11W on recombinant protein quality The impact of SIMULSOL® SL11W on recombinant protein quality is tested by comparing clarified mAb bioreactor harvest samples with and without 0.3% SIMULSOL® SL11W, incubated at 20° C. for 3 hours, then at 4° C. for 16 hours. Samples are first filtered through a 0.2 μm glass fiber filter, followed by a 0.45 μm PVDF filter, and then subjected to Protein A purification. Samples are analyzed for analytical properties shown in Table 18 using techniques known in the art, such as capillary electrophoresis (CE), size exclusion chromatography (SEC), and isoelectric focusing (IEF) measurements on an iCE® instrument. The results shown in Table 18 indicate that treatment with SIMULSOL® SL11W did not significantly affect any of the measured qualities compared to no treatment with SIMULSOL® SL11W. [Table 18]

Claims

1. 1. A method for inactivating viruses in a feedstream, comprising adding an environmentally compatible detergent to the feedstream at a final concentration of 0.05% to 1% w / w, wherein the environmentally compatible detergent comprises 50% or more undecyl alkyl glycoside, and wherein the feedstream is a harvested cell culture fluid, a protein A pool, a protein G pool, a protein L pool, an affinity chromatography pool, or a mixed mode chromatography pool.

2. The method of claim 1, wherein the detergent comprises 53% to 57% undecyl alkyl glycoside.

3. 3. The method of claim 1 or 2, wherein the detergent comprises less than 10% decyl alkyl glycosides.

4. The method according to any one of claims 1 to 3, wherein the detergent is SIMULSOL® SL11W.

5. The method according to any one of claims 1 to 4, wherein the detergent is an undecyl alkyl glycoside.

6. A method according to any one of claims 1 to 5, wherein the detergent is added to the feed stream at a concentration of from 0.1% to 1% w / w.

7. The method of any one of claims 1 to 5, wherein the detergent is added to the feed stream at a final concentration of 0.3% w / w.

8. A method according to any one of claims 1 to 7, wherein the feed stream is at a temperature of from 15°C to 30°C during addition of the detergent.

9. A method according to any one of claims 1 to 8, wherein the feed stream is at a temperature of from 15°C to 20°C during addition of the detergent.

10. 10. The method of any one of claims 1 to 9, wherein the feed stream is at a pH of from 5.5 to 8.0 during addition of the detergent.

11. The method of any one of claims 1 to 10, further comprising incubating the detergent in the feed stream for between 1 minute and 180 minutes.

12. 12. The method of claim 11, wherein after the incubating step, the detergent has a virus log reduction factor (LRF) in the feedstream of 1 or more, or 2 or more.

13. The method of any one of claims 1 to 12, wherein the virus is a retrovirus, a herpesvirus, a flavivirus, a poxvirus, a hepadnavirus, a hepatitis virus, an orthomyxovirus, a paramyxovirus, a rhabdovirus, or a togavirus.

14. 10. The method of claim 1, further comprising incubating the feedstream with the detergent at a temperature between 4° C. and 30° C. and a pH between 5.5 and 8.0 for 60 minutes to 180 minutes, wherein the detergent is added to the feedstream at a final concentration of 0.3% w / w, and wherein the detergent has a viral log reduction factor (LRF) of 5 or greater in the feedstream.

15. 15. The method of any one of claims 1 to 14, further comprising the step of filtering the feed stream containing the detergent, wherein the feed stream is subjected to at least one filtering step.

16. 16. The method of claim 15, wherein the feed stream is subjected to at least a second filtration step.

17. 17. The method of claim 15 or 16, further comprising subjecting the filtered feed stream to an affinity chromatography column.

18. 18. The method of claim 17, wherein the affinity chromatography column is a Protein A column.

19. The method of any one of claims 1 to 16, wherein the feed stream containing the detergent is subjected to affinity chromatography.

20. 20. The method of any one of claims 1 to 19, wherein the feedstream comprises a protein, and the protein is an antibody, an Fc fusion protein, an immunoadhesin, an enzyme, a growth factor, a receptor, a hormone, a regulatory factor, a cytokine, an antigen, a peptide, or a binding agent.

21. 21. The method of claim 20, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment produced in a mammalian cell.

22. 22. The method of any one of claims 1-21, wherein adding said detergent to said feed stream does not significantly change the turbidity of said feed stream, and wherein adding said detergent to said feed stream does not change the final product quality of purified feed stream protein.

23. The method of any one of claims 1 to 22, wherein the detergent contains a stabiliser.

24. 24. The method of claim 23, wherein the stabilizer is monopropylene glycol.

25. 1. A method for inactivating viruses in a feedstream, comprising adding an environmentally compatible detergent to the feedstream at a final concentration of 0.05% to 1% w / w, wherein the environmentally compatible detergent is an aqueous solution comprising 40% or more undecyl alkyl glycoside and 1-5% propane-1,2-diol, and wherein the feedstream is a harvested cell culture fluid, a protein A pool, a protein G pool, a protein L pool, an affinity chromatography pool, or a mixed mode chromatography pool.

26. 26. The method of claim 25, wherein the detergent comprises 40% to 60% undecyl alkyl glycoside.

27. 27. The method of claim 25 or 26, wherein the detergent comprises 53% to 57% undecyl alkyl glycoside.

28. 26. The method of claim 25, wherein the detergent comprises 50% or more of undecyl alkyl glycoside.

29. A method according to any one of claims 25 to 28, wherein the detergent comprises less than 10% decyl alkyl glycosides.

30. 30. The method of any one of claims 25 to 29, wherein the detergent is SIMULSOL® SL11W.

31. The method according to any one of claims 25 to 30, wherein the detergent is an undecyl alkyl glycoside.

32. A method according to any one of claims 25 to 31, wherein the detergent is added to the feed stream at a concentration of from 0.1% to 1% w / w.

33. 33. The method of any one of claims 25 to 32, wherein the detergent is added to the feed stream at a final concentration of 0.3% w / w.

34. A method according to any one of claims 25 to 33, wherein the feed stream is at a temperature of from 15°C to 30°C during addition of the detergent.

35. A method according to any one of claims 25 to 34, wherein the feed stream is at a temperature of from 15°C to 20°C during addition of the detergent.

36. A method according to any one of claims 25 to 35, wherein the feed stream is at a pH of from 5.5 to 8.0 during addition of the detergent.

37. 37. The method of any one of claims 25 to 36, further comprising incubating the detergent in the feed stream for between 1 minute and 180 minutes.

38. 38. The method of claim 37, wherein after the incubating step, the detergent has a virus log reduction factor (LRF) in the feedstream of 1 or more, or 2 or more.

39. 39. The method of any one of claims 25 to 38, wherein the virus is a retrovirus, a herpesvirus, a flavivirus, a poxvirus, a hepadnavirus, a hepatitis virus, an orthomyxovirus, a paramyxovirus, a rhabdovirus, or a togavirus.

40. 26. The method of claim 25, further comprising incubating the feedstream with the detergent at a temperature between 4° C. and 30° C. and a pH between 5.5 and 8.0 for 60 minutes to 180 minutes, wherein the detergent is added to the feedstream at a final concentration of 0.3% w / w, and wherein the detergent has a viral log reduction factor (LRF) of 5 or greater in the feedstream.

41. 41. The method of any one of claims 25 to 40, further comprising the step of filtering the feed stream containing the detergent, wherein the feed stream is subjected to at least one filtering step.

42. 42. The method of claim 41, wherein the feed stream is subjected to at least a second filtration step.

43. 43. The method of claim 41 or 42, further comprising subjecting the filtered feed stream to an affinity chromatography column.

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

45. The method of any one of claims 25 to 42, wherein the feed stream containing the detergent is subjected to affinity chromatography.

46. 46. ​​The method of any one of claims 25-45, wherein the feedstream comprises a protein, and the protein is an antibody, an Fc fusion protein, an immunoadhesin, an enzyme, a growth factor, a receptor, a hormone, a regulatory factor, a cytokine, an antigen, a peptide, or a binding agent.

47. 47. The method of claim 46, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment produced in a mammalian cell.

48. 48. The method of any one of claims 25-47, wherein adding said detergent to said feed stream does not significantly change the turbidity of said feed stream, and wherein adding said detergent to said feed stream does not change the final product quality of purified feed stream protein.

49. The method of any one of claims 25 to 48, wherein the detergent contains a stabiliser.

50. 50. The method of claim 49, wherein the stabilizer is monopropylene glycol.

51. 1. A method for inactivating viruses in a feedstream, comprising: adding SIMULSOL® SL11W to said feed stream at a concentration of 0.1% to 0.3% w / w; incubating the SIMULSOL® SL11W in the feedstream for 180 minutes, wherein the viral Log Reduction Factor (LRF) of SIMULSOL® SL11W in the feedstream is 5 or greater; filtering the feed stream containing the SIMULSOL® SL11W; and subjecting the filtered feed stream to a Protein A affinity chromatography column. wherein said adding and incubating steps are performed at a temperature of said feed stream between 4° C. and 30° C. and a pH of between 5.5 and 8.0, and said feed stream is a harvested cell culture fluid, a Protein A pool, a Protein G pool, a Protein L pool, an affinity chromatography pool, or a mixed mode chromatography pool.

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