EA046732B1 - METHODS OF VIRAL INACTIVATION WITH ECOLOGICALLY COMPATIBLE DETERGENTS - Google Patents

Description

The invention relates to the field of production of recombinant protein. In particular, the invention provides a method for inactivating viruses when supplied with nutrients during the production of proteins intended for administration to a patient, such as therapeutic or diagnostic proteins. The present invention further provides a method in which an environmentally friendly detergent effectively provides antiviral activity and maintains the quality of a therapeutic or diagnostic protein. The methods provide important advantages in the production of biological products in the real world, for example, compared to methods using Triton X100 and/or methods that do not use detergent.

Detergents are key reagents during the production of therapeutic or diagnostic proteins to ensure the safety of the final biological product. Viral contamination of the final biological product can have serious clinical consequences, arising, for example, from contamination of the original cell lines or adventitious introduction of viruses at various stages of production. Thus, detergents are used to remove viral contaminants present in the feed stream during the production of a therapeutic or diagnostic protein. Once the desired protein is purified, the growth media and buffers used during the production and purification of the product are inactivated and discharged into wastewater. However, such waste containing detergents that are used in the production and cleaning process may cause environmental problems, according to the Environmental Risk Assessment (ERA) system for detergents.

Modern methods of inactivating viruses during the production of bioproducts, such as therapeutic or diagnostic proteins, largely depend on the use of the detergent Triton X-100. Triton X-100 is a polyoxyethylene ether that typically provides robust inactivation of enveloped virus greater than 4 log under a variety of experimental conditions. However, 4-(1,1,3,3-tetramethylbutyl)phenol (also known as 4-tert-octylphenol), a degradation product of Triton X-100, has been identified as a potential environmental toxin to wildlife, including possible estrogenic effects. Thus, the removal of 4-tert-octylphenol from waste streams is desirable, but will require complex, time-consuming and costly activities. For this reason, due to toxicity concerns, the EU-REACH committee voted to add Triton X-100 to Annex XIV, the EU's list of banned substances, prohibiting the use of Triton X-100 in the production of recombinant proteins after 2021. Alternative agents to replace Triton X-100 in biologics production are not well developed. As a result, there is an urgent, operational need for methods for efficiently inactivating viruses during the production of biological products, such as therapeutic or diagnostic proteins, that are less toxic to the environment than methods using Triton X-100.

Environmentally compatible alternatives to Triton X-100 for inactivating viruses, especially enveloped viruses, during protein production were investigated in WO 2019/121846 as well as WO 2015/073633. However, the effects of using different detergents to inactivate viruses may vary. Some detergents are not suitable for use in the manufacturing process because they are insoluble or difficult to remove from the feed stream. In addition, some detergents increase the turbidity and/or foaming of the nutrient stream, requiring additional measures to reduce the turbidity and/or foaming of the feed stream. Other detergents are not as effective over a wide range of temperatures and pH values and require higher concentrations to inactivate viruses. Combining these options can result in additional costs and time in the protein production process. Also, the effectiveness of many detergents in producing a feed stream of proteins of various types and sizes is insufficient.

Thus, there remains a need for methods to effectively inactivate viruses in the production of therapeutic or diagnostic proteins of various types and sizes using an environmentally friendly detergent that effectively inactivates a variety of enveloped viruses at both low and high temperatures and pH ranges, within a reasonable time frame and at reasonable concentrations of such detergent(s), and/or is aqueous, soluble and/or readily soluble, and/or does not affect the turbidity of the feed stream, making it suitable for industrial production. In addition, it is preferable that the detergent be sufficiently removed from the product feed stream (to meet regulatory requirements) during the purification step without adding complex, time-consuming and/or costly measures, and that the quality of the therapeutic or diagnostic protein product is maintained.

Accordingly, the present invention is directed to solving one or more of the above problems by providing methods for inactivating viruses during the production of therapeutic or diagnostic proteins. Surprisingly, the methods of this invention provide environmentally friendly detergents that are highly effective in inactivating viruses over a wide range of temperatures and/or pH values while producing various types of proteins, in reasonable times and in rational concentration ranges without toxic effects on the environment. Surprisingly, the methods of the current invention further provide environmentally friendly

- 1,046,732 safe detergents that readily achieve complete virus inactivation at least comparable to Triton-X 100, and in which the environmentally safe detergents have no known environmental toxicity at the concentrations used. In addition, the methods of the invention provide complete viral inactivation with a logarithmic load reduction factor (LRF) of from about greater than 3 to about greater than 6 than over a temperature range of about 4°C to at least 30°C and over a wide pH range, in the production of therapeutic or diagnostic proteins of various types and sizes in a reasonable time and at an appropriate concentration range without impact on the environment. Surprisingly, using the methods of the current invention also achieved viral inactivation in all enveloped viruses tested over a temperature range ranging from about 4°C to at least 30°C, ranging from more than 1 to about 180 minutes, over a wide pH range , in the production of therapeutic or diagnostic proteins of various types and sizes within an appropriate concentration range.

Accordingly, the present invention provides a method for inactivating viruses in a nutrient stream, comprising adding detergents to the feed stream that are environmentally compatible. The detergents that have been used in the methods of this invention are aqueous solutions that have little effect on the turbidity of the feed stream and are thus suitable for large-scale production of therapeutic or diagnostic proteins. Further, the detergents used in the current invention are removed sufficiently and effectively (in accordance with regulatory requirements) in the purification step with or without a preliminary filtration step. In addition, no complex, time-consuming and/or expensive steps are required to remove detergents from the feed stream. In addition, the detergents used in the current methods of the invention do not adversely affect the final quality of the therapeutic or diagnostic protein. The detergents used in the methods of the present invention effectively inactivate enveloped viruses over a wide range of temperatures and pH values without causing environmental toxicity.

In accordance with one aspect of the invention, a method is provided for inactivating viruses with an alkyl glucoside detergent in a feed stream during the production of therapeutic or diagnostic proteins. In accordance with another aspect of the invention, the detergent used in the methods of the invention contains more than about 40% undecyl alkyl glucoside. In another aspect of the invention, the detergent used contains more than about 40% undecyl alkyl glucoside and less than about 20% other alkyl glucosides. In yet another formulation of the invention, the detergent contains more than about 40% undecyl alkyl glucoside and less than about 10% decyl alkyl glucoside. In yet another embodiment of the invention, the detergent contains from about 40% to about 60% undecyl alkyl glucoside. Also in a further embodiment of the invention, the detergent used in the methods contains from about 53% to about 57% undecyl alkyl glucoside. In another aspect of the invention, the detergent of this invention contains more than about 50% undecyl alkyl glucoside. In another modification, the detergent used in the methods of the invention contains from about 40% to about 60% undecyl alkyl glucoside and less than about 20% other alkyl glucosides. In yet another embodiment, the detergent of the invention contains from about 40% to about 60% undecyl alkyl glucoside and less than about 10% decyl alkyl glucoside. In another embodiment, the method of this invention uses a detergent containing from about 53% to about 57% undecyl alkyl glucoside and less than about 20% other alkyl glucosides. In yet another embodiment, the detergent used in the methods of the present invention contains from about 53% to about 57% undecyl alkyl glucoside and less than about 10% decyl alkyl glucoside. In yet another embodiment of the methods of this invention, the detergent contains zero or less of 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 glucosides. In some embodiments, other alkyl glucosides include a mixture of nonyl alkyl glucoside, decyl alkyl glucoside, and/or lauryl alkyl glucoside. In another embodiment, the methods of the invention used a detergent containing zero or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 %, 10% decyl alkyl glucoside. In one embodiment of the invention, the detergent used contains undecyl alkyl glucoside.

In one embodiment of the invention, the detergent used has CAS registration number 98283-67-1. Other options involved the use of a detergent, which is SIMULSOL™ SL 11W. In another embodiment, SIMULSOL™ SL 11W used in the methods of the present invention contains more than about 40% undecyl alkyl glucoside. In yet another embodiment, SIMULSOL™ SL 11W, among the methods of this invention, contains from about 40% to about 60% undecyl alkyl glucoside. In yet another embodiment, SIMULSOL™ SL 11W used in the methods contains from about 53% to about 57% undecyl alkyl glucoside. In a further application of this invention, SIMULSOL™ SL 11W contains from about 40% to about 60% undecyl alkyl glucoside and less than about 10% decyl alkyl glucoside. Another option used in the methods of this invention involved the use of SIMULSOL™ SL 11W, which contains from about

- 2 046732

53% to about 57% undecyl alkyl glucoside and less than about 10% decyl alkyl glucoside. In other applications, SIMULSOL™ SL 11W contained zero 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 glucosides. In embodiments of the invention, the other alkyl glucosides contained in SIMULSOL™ SL 11W contained a mixture of nonyl alkyl glucoside, decyl alkyl glucoside and/or lauryl alkyl glucoside. In additional embodiments, the methods of this invention used SIMULSOL™ SL 11W containing zero or less than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% , 8%, 9%, 10% decyl alkyl glucoside. In one embodiment of the methods of this invention, SIMULSOL™ SL 11W is an undecyl alkyl glucoside.

Another method was carried out using SIMULSOL™ SL 82. In another embodiment, SIMULSOL™ SL 82 was used containing from about 40% to about 60% undecyl alkyl glucoside.

In another embodiment, the invention provides methods for inactivating viruses in a nutrient stream, including adding an environmentally friendly detergent to the stream at a final concentration of about 0.05% w/w. to about 1% w/w, wherein the environmentally friendly cleansing component comprises greater than about 40% decyl alkyl glucoside. In some embodiments, the detergent used in the methods of the current invention is added to the feed stream to a final concentration level of about 0.1% w/w. to about 1% w/w In some embodiments, the used detergent is added to the nutrient stream to a final concentration of 0.1% w/w. up to 1% w/w In some methods of this invention, detergent is added to the nutrient stream to achieve a final concentration of about 0.3% w/w. to about 1% w/w In some embodiments, the detergent used in the methods of the present invention is added to the feed stream to achieve a final concentration of 0.3% w/w. up to 1% w/w In some embodiments, used in the methods of the present invention, the detergent is added to the feed stream to a final concentration of about 0.1% w/w. to about 0.3% w/w In some embodiments of the current invention, detergent is added to the feed stream to a final concentration level of 0.1% w/w. up to 0.3% w/w In other methods of implementing the current invention, detergent is added to the feed stream to a final concentration of about 0.3% w/w. In other embodiments, the detergent used in the methods is added to the feed stream to a final concentration of 0.3%. In yet another embodiment, the detergent used in the methods of this invention is added to the feed stream to a final concentration by weight 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%.

In some embodiments of the invention, the temperature of the feed stream is from about 4°C to about 30°C. In other embodiments of the invention, the temperature of the feed stream is from 4°C to 30°C. In yet other embodiments, the temperature of the nutrient flow is from about 15°C to about 30°C. In another embodiment, the feed stream has a temperature of from about 15°C to about 20°C. In still other embodiments of the invention, the temperature of the feed stream is from about 4°C, about 15°C, about 18°C, about 20°C, about 25°C, or about 30°C.

In some embodiments, the pH of the feed stream is from about 5.5 to about 8.0. In other embodiments of the invention, the pH value of the feed stream was from 5.5 to 8.0. In still other embodiments of the invention, the feed stream has a pH of about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

In some embodiments, the feed stream is incubated with detergent for a time ranging from less than about 1 minute to about 180 minutes. In another embodiment, the feed stream is incubated with detergent for about 6 to about 180 minutes. In other methods, the feed stream is incubated with detergent for about 10 to about 180 minutes. Also, in other embodiments, the nutrient stream is incubated with detergent for about 120 to about 180 minutes. In other embodiments, the nutrient stream is incubated with detergent for about 180 minutes. In other embodiments, the feed stream is incubated with detergent for approximately 120 minutes. In other embodiments, the feed stream is incubated with detergent for about 60 minutes. In other embodiments, the feed stream is incubated with 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.

In embodiments of the invention, the detergent in the nutrient stream has a viral LRF of greater than about 1. In some embodiments, the detergent in the nutrient stream has a viral LRF of greater than about 4. In some embodiments, the detergent in the nutrient stream has a viral LRF more than about 5. In some embodiments of the invention, the detergent in the feed stream has a virus LRF of more than about 6. In some embodiments of the current invention, the detergent in the nutrient stream has a virus LRF of more than about 7. In some embodiments of the invention, the detergent in the stream

- 3 046732 raw materials ensure complete inactivation of viruses. In yet other embodiments, the detergent in the feed stream has a viral LRF greater than about 1, about 2, about 3, about 4, about 5, about 6, or about 7.

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

In some embodiments, a method of inactivating a virus in a feed stream includes the step of incubating the feed stream and an environmentally friendly detergent for about 15 minutes to about 180 minutes at a temperature of about 4°C to about 30°C. The pH was from about 5.5 to about 8.0, with detergent added to the feed stream at a final concentration of about 0.1% w/w. to about 1.0% w/w, and wherein the detergent in the feed stream has a viral LRF greater than about 2.

In some embodiments, a method of inactivating a virus in a feed stream includes the step of incubating the feed stream and an environmentally friendly detergent for about 6 minutes to about 180 minutes at a temperature of about 4°C to about 30°C. The pH was from about 5.5 to about 8.0, with detergent added to the feed stream at a final concentration of about 0.3% w/w. to about 1.0% w/w, and wherein the detergent in the feed stream has a viral LRF greater than about 4.

In some embodiments, the invention provides a method of inactivating a virus in a feed stream comprising the step of incubating the feed stream and an environmentally friendly detergent for about 180 minutes at a temperature of from about 4°C to about 30°C, and at a pH value of about 5.5 to about 8.0, wherein the detergent is added to the feed stream at a final concentration of about 0.1% w/w. to about 1.0% w/w, and wherein the detergent in the feed stream has a viral LRF greater than about 5.

In some embodiments, the method of inactivating a virus in a feed stream includes the step of incubating the feed stream and an environmentally friendly detergent for about 60 minutes to about 180 minutes at a temperature of about 4°C to about 30°C, at a pH of about 5 .5 to about 8.0, wherein the detergent is added to the feed stream at a final concentration of about 0.3% w/w and the detergent in the feed stream has a viral LRF greater than about 5.

In some embodiments, the method of inactivating a virus in a feed stream includes the step of incubating the feed stream and an environmentally friendly detergent for about 120 minutes to about 180 minutes at a temperature of about 4°C to about 30°C, at a pH value of about 5 .5 to about 8.0, wherein the detergent is added to the feed stream at a final concentration of about 0.3% w/w and the detergent in the feed stream has a viral LRF greater than about 6.

In some embodiments, the invention provides for virus inactivation in a nutrient stream, wherein the nutrient stream includes a collected cell culture fluid, a capture pool, or an extracted product pool. In some embodiments, the invention provides methods for inactivating a virus in a nutrient stream, wherein the stream includes a collected cell culture fluid, a capture pool, or a recovery pool, and where the capture pool or recovery pool is an affinity chromatography pool. In yet another embodiment, the capture pool or recovery pool is a protein A pool, protein G pool, or protein L pool. In other embodiments, the capture pool or recovery pool is a mixed mode chromatography pool.

In some embodiments, the invention provides methods where the detergent-incubated feed stream is not subjected to a filtration step. In some embodiments, the invention provides methods in which a nutrient stream incubated with a detergent is subjected to at least one filtration step. In other embodiments, the invention provides methods in which the feed stream is incubated with detergents and subjected to at least one, or at least two, or at least three stages of filtration. In some embodiments, the feed stream incubated with the detergent is subjected to a first 0.22 μm filtration step followed by a second and third 0.45 μm PVDF filtration step. According to a specific embodiment, the method of the invention uses a filter membrane in the first and/or second and/or third stages of filtration, including, but not limited to, polyvinylidene fluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone or other filter media suitable for use in a cGMP manufacturing environment. According to a preferred embodiment, the invention provides methods in which the filter membrane in both the second and third filtration stages contains PVDF. According to a specific embodiment, the invention provides a method in which the filter membrane has a pore size of about 0.45 microns in both the second and third stages of filtration. According to a preferred embodiment, the filter membrane

- 4 046732 in the second and third stages of filtration is a PVDF membrane with a pore size of 0.45 microns. In a specific application, the invention uses a method in which the filter membrane has a pore size of about 0.22 microns in the first stage of filtration.

In some embodiments, the invention provides methods in which a nutrient stream incubated with a detergent is subjected to affinity chromatography. In some embodiments, the affinity chromatography is a Protein A affinity column.

In some embodiments, the invention provides methods in which a detergent-containing feed stream is exposed to a chromatography column. In some embodiments, the chromatography column is one or more affinity columns, ion exchange columns, hydrophobic interaction columns, hydroxyapatite columns, or mixed columns. 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 invention provides methods in which the detergent is sufficiently removed from the final product. In some methods of carrying out the invention, complete removal of the detergent from the final product is ensured.

In some embodiments, the invention provides methods for inactivating a virus in a feed stream, the method comprising the step of adding detergent to said feed stream and incubating the detergent as well as the feed stream, wherein the therapeutic or diagnostic protein is an antibody, an Fc fusion protein, an immunoadhesin, enzyme, growth factor, receptor, hormone, regulatory factor, cytokine, antigen or binding agent. In additional 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 mammalian cells. In some embodiments, the mammalian cell is Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells, murine hybridoma cells, or murine myeloma cells. In some embodiments, the therapeutic or diagnostic protein is produced in bacterial cells. In other embodiments, the therapeutic or diagnostic protein is produced in yeast cells.

In several embodiments of the invention, the feed stream contains the detergent used in the methods of this invention and has low turbidity. In some embodiments, adding detergent to the feed stream does not significantly increase or change the turbidity of the nutrient stream. In another embodiment, the cleansing agent used in the methods of the invention does not affect the quality of the therapeutic or diagnostic protein product.

In some embodiments, the detergent used in the methods of the invention contains a preservative and/or stabilizer. For some embodiments, the stabilizer is monopropylene glycol. In some embodiments, the stabilizer in the detergent is from about 1% to about 5%. In a further embodiment of the invention, the stabilizing agent used in the methods of the invention does not affect the virus inactivating properties of the detergent and does not affect the quality of the therapeutic or diagnostic protein product. In a further embodiment of the invention, monopropylene glycol does not affect the virus inactivating properties of the detergent and does not affect the quality of the therapeutic or diagnostic protein product.

In embodiments of the invention, the detergent used in the methods of the invention is environmentally friendly. In one embodiment of the invention, a detergent from the methods of the invention is used that does not contain or produce 4-(1,1,3,3-tetramethylbutyl)phenol (aka 4-tert-octylphenol). In other embodiments, the detergent used in the methods of the invention does not contain or form peroxide. In other embodiments, the detergent used in the methods of the invention is biodegradable.

In one aspect of the invention, a method of inactivating a virus in a feed stream is provided, comprising the following steps:

adding to the feed stream undecyl alkyl glucoside at a concentration of about 0.1% wt./wt. up to about 0.3% w/w;

incubating the undecyl alkyl glucoside in the feed stream for about 180 minutes such that the logarithmic load reduction factor (LRF) of the undecyl alkyl glucoside in the feed stream is greater than about 5;

filtering said feed stream containing undecyl alkyl glucoside; and treating said filtered feed stream on a Protein A affinity chromatography column;

wherein during said addition and incubation step, the feed stream has a temperature of about 4°C to about 30°C and a pH of about 5.5 to about 8.0.

In one aspect of the invention, a method of inactivating a virus in a feed stream is provided, comprising the following steps:

- 5 046732 addition to the feed stream of SIMULSOL™ SL 11W in a concentration of about 0.1% w/w. up to about 0.3% w/w;

incubation of SIMULSOL™ SL 11W in the nutrient flow for about 180 minutes, while the logarithmic viral load reduction factor (LRF) of SIMULSOL™ SL 11W in the flow exceeds about 5;

filtering said stream containing SIMULSOL™ SL 11W; processing said filtered stream on a protein A affinity chromatography column;

wherein during the addition and incubation stage, the nutrient stream has a temperature of from about 4°C to about 30°C and a pH value of from about 5.5 to about 8.0. In another aspect of the invention, the filtration step in the methods of the invention includes a first filtration step using a 0.22 micron filter. In another aspect, the filtration step of the methods of the invention comprises a second filtration step with a 0.45 μm PVDF filter. In yet another aspect, the filtration step includes a third filtration step with a 0.45 μm PVDF filter. According to a certain embodiment of the method of the invention, the filter membrane used in the first and/or second and/or third stages of filtration contains, but is not limited to, polyvinylidene fluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone or other filter media suitable for use in a cGMP manufacturing environment. According to a preferred embodiment, the filter membrane contains PVDF. According to a specific embodiment, the filter membrane in both the second and third filtration stages is a membrane with a pore size of about 0.45 μm. According to a preferred embodiment, the filter membrane is a PVDF membrane with a pore size of 0.45 μm.

As used herein, the term environmentally compatible detergent or compound or substance or agent referred to herein causes a minimal and/or acceptable level of harmful effects on the environment. For example, the Organization for Economic Co-operation and Development (OECD) provides guidance on chemical safety testing. Green agent options according to current reports may also be biodegradable. Methods for determining whether an agent, such as a detergent, is compatible with the environment, for example, under protein production conditions, are known in the art. Environmental compatibility can also be assessed through environmental risk assessment, which is the process of assessing how likely the environment is to be affected by one or more environmental stressors such as chemicals, land change, disease, invasive species, and change. climate. These recommendations can be found, for example, on the US Environmental Protection Agency website. In addition, guidance on the registration, evaluation, authorization and restriction of chemicals in the EU can be found on the European Commission's REACH website.

The predicted environmental concentration is the predicted concentration of a substance in a waste discharged to a receiving water body in the environment. For example, the predicted environmental concentration of a detergent used to inactivate viruses in the preparation of a therapeutic or diagnostic protein is the concentration of the detergent in the waste stream that is released into the environment.

The term detergent as used herein refers to an agent that may contain salts of long chain aliphatic bases or acids, or hydrophilic moieties such as sugars, and which has both hydrophilic and hydrophobic properties. The detergents used here may have the ability to break down viral envelopes and inactivate viruses. The mechanism of virus inactivation by detergents is explained by the interaction between the detergent and the lipid components of the outer membrane of the virus, which leads to membrane destruction and ultimately loss of virulence. In some examples, the detergent may be a composition comprising a surfactant or surfactant and one or more other agents, such as chelating agents, preservatives or stabilizers. Detergents or surfactants can be classified according to their charge. Surfactants can be non-ionic, cationic or anionic. The surfactant used here has the ability to break down viral envelopes and inactivate viruses. Embodiments of the detergents of the present invention contain an alkyl glucoside, wherein the alkyl glucoside may be an alkyl glucoside.

An alkyl glucoside, as used herein, refers to any sugar linked by a bond to any hydrophobic alkyl. The alkyl glucoside used here consists of an alkyl group linked to a sugar, where the sugar is glucose. The alkyl glucoside may be, for example, an undecyl alkyl glucoside. An undecyl alkyl glucoside may contain an undecyl chain with an O-glycosidic bond to a mono-N-glucopyranose (e.g. n-undecyl-β-D-glucopyranose)

- 6 046732 or D-glucopyranose oligo- or polysaccharides or mixtures thereof. Chemical synthesis of alkyl glucosides, such as those used in the methods of this invention, can result in a heterogeneous mixture of compounds rather than a completely homogeneous preparation. Thus, unless otherwise indicated, references herein to a specific alkyl glucoside form mean that at least the majority of the components of any heterogeneous mixture are an alkyl glucoside form, such as undecyl alkyl glucoside. Examples of such alkyl glucosides that contain undecyl alkyl glucosides include, but are not limited to, SIMULSOL™ SL 11W and SIMULSOL™ SL 82.

A nutrient product stream or nutrient stream is a material or solution intended for a purification method that contains a therapeutic or diagnostic protein of interest and which may also contain various impurities. Non-limiting examples may include, for example, a pooled cell culture fluid, pooled cell culture material, clarified cell culture fluid, clarified cell culture material, a capture pool, a recovered pool, and/or a pooled pool containing a therapeutic or diagnostic protein of interest after one or more steps centrifugation and/or filtration steps, a capture pool, a recovered protein pool, and/or a collected pool containing a therapeutic or diagnostic protein of interest after one or more purification steps.

The term impurities refers to materials that differ from the desired protein product. Impurity includes, but is not limited to: host cell materials such as CHOP; leached protein A; nucleic acid; a variant, size variant, fragment, aggregate or derivative of the desired protein; other protein; endotoxin; viral contaminant; component of cell culture medium, etc.

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. These terms also cover an amino acid polymer that has been modified naturally or by artificial intervention; for example, due to disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling moiety. Also included in this definition are, for example, proteins containing one or more amino acid analogues (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Examples of proteins include, but are not limited to, antibodies, peptides, enzymes, receptors, hormones, regulatory factors, antigens, binding agents, cytokines, Fc fusion proteins, immunoadhesin molecules, etc.

The term antibody or antibodies is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonistic, antagonistic and neutralizing antibodies), chimeric antibodies, humanized antibodies, human antibodies, polyepitope specific antibody compositions, polyclonal antibodies, single chain antibodies, multispecific antibodies (eg, bispecific antibodies), immunoadhesins and antibody fragments as long as they exhibit the desired biological or immunological activity. The term immunoglobulin (Ig) is used here interchangeably with antibody.

The term ultrafiltration or filtration is a form of membrane filtration in which hydrostatic pressure forces liquid against a semi-permeable membrane. Suspended solids and high molecular weight solutes are retained, while water and low molecular weight solutes pass through the membrane. In some examples, ultrafiltration membranes have pore sizes ranging from 1 μm to 100 μm. The terms ultrafiltration membrane, ultrafiltration filter, filtration membrane and filtration filter can be used interchangeably. Examples of filtration membranes include, but are not limited to, polyvinylidene fluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE), polyvinyl chloride, polyethersulfone, fiberglass, or other filtration materials suitable for cGMP use in a manufacturing environment.

The numeric range used here includes the numbers that define the range.

The term enveloped virus or lipid enveloped virus refers to any virus containing a membrane or envelope including a lipid, such as, for example, an enveloped virus. The capsid of enveloped viruses is enclosed in a lipoprotein membrane or envelope. This envelope is derived from the host cell when the virus buds from its surface and consists primarily of lipids not encoded by the viral genome. Although it carries molecular determinants for attachment and entry into target cells and is essential for the infectivity of enveloped viruses, it is not susceptible to drug resistance or antigenic shift. The size of enveloped viruses varies from about 45-55 nm to about 120-200 nm. Non-limiting examples of lipid enveloped viruses that can infect mammalian cells include DNA

- 7 046732 viruses, such as herpes virus, poxvirus virus or hepadnavirus virus; RNA viruses such as flavivirus virus, togavirus virus, coronavirus virus, deltavirus virus, orthomyxovirus virus, paramyxovirus virus, rhabdovirus virus, bunyavirus virus or filovirus virus; and reverse transcription viruses such as retroviral virus or hepadnavirus virus. In one embodiment, the invention provides methods for inactivating a subviral agent in a product feed stream, comprising treating the feed stream with an environmentally friendly detergent. In some aspects, the subviral agent is a viroid or satellite. In another embodiment, the invention provides methods for inactivating a virus-like agent in a product feed stream comprising treating the feed stream with an environmentally friendly detergent.

Methods for measuring viral activity or viral infectivity are known in the art. Examples include, but are not limited to, the TCID 50 assay (ie, determination of the average tissue culture infectious dose that produces pathological changes in 50% of inoculated cell cultures) and plaque assays. Other known methods may include, for example, a transformation assay, which can be used to determine the biological activity titers of non-plaque-forming viruses that have the ability to cause cell growth transformation; or a fluorescence focus assay, which relies on the use of antibody staining techniques to detect viral antigens in infected cells in a monolayer, or a terminal dilution assay, which can be used to determine titers of many viruses, including viruses that do not infect a cell monolayer (alternatively, plaque assays ); or viral enzyme assays, which measure virus-encoded enzymes such as reverse transcriptases or viral proteases. In addition, detection of a specific virus can be done using polymerase chain reaction (PCR) using primers and probes designed to detect a specific virus.

Virus inactivation in the product feed stream can be measured using methods known in the art. In some embodiments, viral inactivation is expressed as a log reduction factor (LRF) or a log reduction value (LRV). LRF is calculated as:

VT tf = log ₁₀ ^ ¹⁰ yt , , where R is the logarithmic reduction factor (LRF); Vi - input volume, ml; Ti is the input titer of the virus; Vo is the output volume and To is the output virus titer.

Examples

Example 1. Inactivation of viruses by nonionic glycosidic detergents

Detergents and reagents

The stock solution for the example detergents listed in table. 1 (Seppic Inc. Fairfield, NJ), and 1,2-propanediol (Sigma-Aldrich, St. Louis, MO) were prepared in water at a percentage weight/volume (w/w) ratio.

Table 1 Types of detergents

Detergent name Alkyl glucoside content Physical properties SIMULSOL™ SL 11W D-glucopyranose, oligomer, undecyl glucoside (4060%), propane-1,2-diol (1-5%) Clear liquid SIMULSOL™ AS 48 2-ethylhexyl mono-O-glucopyranoside, 2-ethylhexyl di-O-glucopyranoside (40-60%) White Semi-liquid SIMULSOL™ SL4 D-glucopyranose, oligomer, butyl glucoside (40-60%) Clear liquid SIMULSOL™ SL 10 D-glucopyranose, oligomer, C10-16 (even) alkyl glucosides (40-60%) White Semi-liquid SIMULSOL™ SL 26 C D-glucopyranose, oligomer, C10-16 (even) alkyl glucosides (40-60%) White Semi-liquid SIMULSOL™ SL 82 D-glucopyranose, oligomer, undecyl glucoside (4060%), D-glucopyranose, oligomer, C10-16 (even) alkyl glucosides (10-20%), propane-1,2-diol (1-5%) Clear liquid SIMULSOL™ SL8 D-glucopyranose, oligomer, decyl octyl glucosides (40-60%) Clear liquid SIMULSOL™ SL 826 D-glucopyranose, oligomer, decyl octyl glucosides (20-40%), D-glucopyranose, oligomer, C10-16 (even)-alkyl glucosides (20-40%), (2methoxymethylethoxy)propanol (0.1-1%) Clear liquid

Viruses and indicator cells

Xenotropic murine leukemia (XMuLV), porcine parvovirus (PPV), NADL-2 strain, and pseudorabies virus (PRV) for use in these studies can be purchased from ATCC, Manassas,

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V.A. In table 2 shows the properties of these viruses.

Table 2 Virus characteristics

Virus Genome Genus Size(nm) Physicochemical resistance Shell (yes or no) Xenotropic murine leukemia DNA Retroviruses 80-100 Low Yes Pseudorabies virus RNA Herpesviruses 120-140 Low Yes Porcine parvovirus DNA Parvoviruses 18-22 High No

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

Cell-based TCID50 virus titration assay

The Tissue Culture Infectious Dose 50 (TCID50) assay is used to titrate XMuLV with PG-4 indicator cells, PPV with PK-13 indicator cells, and PRV with Vero indicator cells. 96-well tissue culture plates are seeded with 2.5 x 10 ⁴ cells/ml indicator cells at 200 µl/well and incubated at 37°C overnight or about 1624 hours. The plates are then inoculated with virus in a series of 10-fold dilutions of 50 μl/well with a minimum of n=8 per dilution. Inoculated plates are incubated for 7-9 days (XMuLV and PRV) and 10-13 days (PPV) at 37°C. The plates are then examined and scored positive or negative on the wells under a microscope for cytopathic effects (CPE), and virus titers are calculated using standard methods.

Viral cytotoxicity and interference in purified bioreactor material

The TCID50 assay exemplified above is used to determine the cytotoxic effects of viruses in clarified bioreactor harvested monoclonal antibody (mAb), bispecific antibody, and Fc fusion protein material. Bioreactor collection materials are purified by centrifugation followed by polishing filtration. Impurities such as host cell proteins, lipids and host cell DNA are also automatically concentrated during the centrifugation process. Purified collected bioreactor material for various proteins containing from 0.05% w/w. up to 1% w/w detergents, diluted in the inoculation medium 10, 50 and 100 times. Each dilution is added at n=8 to 96-well plates seeded with indicator cells and incubated according to the TCID50 assay. At the end of the incubation period, each well is examined under a microscope for the presence of CPE and the appropriate dilution required for each detergent is determined. The results showed that a 50-fold dilution was sufficient to overcome the cytotoxicity of the detergent.

Purified bioreactor collection materials for various types of proteins are assessed for any intrinsic effects of virus replication of the collected cell culture material. Each virus is diluted 10-fold in purified bioreactor harvest material and inoculated into plates seeded with indicator cells and experimented according to the TCID50 assay. A positive control (virus added to tissue culture material) was included. Virus titers are compared with positive control virus. Titers that are within ±0.5 log ₁₀ of the positive control titers do not cause viral interference. All viral test titers were within ±0.5 _log10 of the positive control titers, and thus no viral interference was observed for any of the bioreactor collection materials, regardless of protein type.

General procedure for viral inactivation

Viral inactivation of various enveloped viruses by each detergent was assessed over a range of different concentrations, temperatures and pH in purified bioreactor materials containing mAbs, bispecific and Fc fusion proteins. XMuLV, PPV or PRV viruses are added to the collected material at a concentration of no more than 5% w/w, and a detergent solution is added to the material to achieve the desired final detergent concentration. The samples are then incubated at specific temperature ranges and pH values. Inactivation of the virus begins as soon as the detergent is added to the material containing the virus. The kinetics of virus inactivation is monitored by aliquoting at different time points from the collected samples, and the virus titer is assessed using the TCID50 assay. Control matrices are included in studies to demonstrate that virus inactivation is a result of detergent treatment and not the matrix itself.

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Inactivation of XMuLV at 30°C by nonionic detergents

Viral inactivation of XMuLV by detergents listed in table. 1 are tested under optimal conditions determined for XMuLV inactivation. In accordance with the viral inactivation procedure exemplified above, each detergent was added in an amount of 0.6% w/w. to the collected purified mAb bioreactor material with the addition of XMuLV at 30°C. Virus inactivation is monitored at various time points, with a maximum inactivation time of 180 minutes after virus inoculation.

The results presented in table. 3 show that for XMuLV inactivation under optimal conditions, XMuLV was rapidly and completely inactivated for all detergents tested after 180 minutes, with the exception of 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 minutes. However, no inactivation of XMuLV was observed at elevated concentrations of SIMULSOL™ SL 4 (data not shown). Surprisingly, these results suggest that not all glucosides are capable of reliable virus inactivation or that extremely high concentrations are required to achieve reliable inactivation, as was observed for SIMULSOL™ AS 48. However, SIMULSOL™ SL 11W demonstrated complete viral inactivation greater than an LRF of 4 within 0-5 minutes of adding it to the purified harvested mAb bioreactor material. Additionally, although other glucosides were effective in inactivating XMuLV, other factors limit the applicability of some of these detergents in a large-scale production process. Such detergent characteristics included solubility, influence on the turbidity of collected material, activity at different temperatures and pH ranges, unknown or limited environmental toxicity data, and ease of handling.

Table 3

Inactivation of XMuLV at 30°C by nonionic detergents in purified collected material of the biore____________________ actor mAb________________________

Detergent (0.6% w/w) LRF achieved at 30°C 0-5 min 90 min 180 min SIMULSOL™ SL 11W >4.55 >4.55 >4.55 SIMULSOL™ AS 48 0.50 ±0.56 2.13 ±0.56 3.20 ± 0.44 SIMULSOL™ SL 4 0.88 ± 0.40 0.38 ±0.50 0.88 ± 0.40 SIMULSOL™ SL 10 >3.10 >4.62 >4.62 SIMULSOL™ SL 26 WITH >3.60 >5.12 >5.12 SIMULSOL™ SL 82 >3.60 >5.12 >5.12 SIMULSOL™ SL 8 >3.10 >4.62 >4.62 SIMULSOL™ SL 826 >3.10 >4.62 >4.62

SIMULSOL™ SL 11W turbidity in purified collected mAb bioreactor material.

Turbidity was assessed at SIMULSOL™ SL 11W concentrations of 0%, 0.05%, 0.1%, 0.3%, 0.5% and 1% w/w. in XMuLV added to purified harvested mAb bioreactor material at 18°C. Viral inactivation is monitored over 180 minutes and turbidity is measured in nephelometric turbidity units (NTU) using a 2100P portable turbidometer (Hach®).

The results shown in table. 4 show that SIMULSOL™ SL 11W at concentrations ranging from 0% to 1% w/w. was stable over time and no major precipitation was observed in the collected material for 180 minutes.

Table 4

Turbidity of SIMULSOL™ SL 11W in purified harvested mAb bioreactor material incubated _____________ at 18°C___________________

% w/w SIMULSOL™ SL 11W Turbidity (NTU) Time 0 (2 measurements) Time 180 (2 measurements) 0 7.92 7.92 7.6 7.59 0.05 11.61 I,6 15.7 16.2 ο,ι 51.5 52.1 49.4 49.1 o, s 75.8 75 91.6 91.5 0.5 355 357 377 375 1 834 836 754 760

Characterization and optimization of viral inactivation using SIMULSOL™ SL 11W

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The use of SIMULSOL™ SL 11W as a suitable environmentally friendly detergent for use in protein production processes is further evaluated on purified collected mAb material in a bioreactor according to the viral inactivation procedure described above. Briefly, the activity of SIMULSOL™ SL 11W was assessed at concentrations of 0.05%, 0.1%, 0.3%, 0.5% and 1% w/w. in purified mAb bioreactor material with the addition of XMuLV at 4 different temperatures 4°C, 18°C, 25°C and 30°C. Viral inactivation was monitored at 0, 10, 30, 60, 120 and 180 minutes after inoculation of XMuLV into SIMULSOL™ SL 11W-containing purified mAb bioreactor collection material. The results are as shown in Table. 5-9 show that SIMULSOL™ SL 11W concentration, incubation time and temperature are factors affecting XMuLV inactivation. SIMULSOL™ SL 11W achieved complete inactivation of the XMuLV virus at concentrations > 0.1% w/w. (> 3 mM) after 120 minutes and at concentrations > 0.3% w/w. after 10 minutes at all 4 temperatures assessed.

Table 5

Inactivation of XMuLV 1.5 mM (0.05%) SIMULSOL™ SL 11W in purified mAb bioreactor collection material

Point in time (min) LRF achieved 4°C 18°С 25°C 30°С 0 -0.38 0.13 0.13 0.25 10 -0.13 0.00 0.13 0.75 thirty 0.50 1.38 1.88 1.37 60 0.62 2.25 2.00 1.37 120 1.25 2.25 2.59 1.75 180 2.12 3.94 3.33 2.12

Table 6

Inactivation of XMuLV 3.0 mM (0.1%) SIMULSOL™ SL 11W in purified mAb bioreactor collection material

Point in time (min) LRF achieved 4°C 18°С 25°C 30°С 0 0.62 3.25 2.12 3.25 10 2.50 3.41 2.55 3.50 thirty 4.58 3.42 2.71 4.08 60 5.43 4.99 3.64 5.00 120 >5.42 >5.92 >4.92 >6.05 180 >5.88 >6.38 >5.38 >6.51

Table 7

Inactivation of XMuLV 9.0 mM (0.3%) SIMULSOL™ SL 11W into clarified mAb bioreactor harvest material

Point in time (min) LRF achieved 4°C 18°С 25°C 30°С 0 1.50 3.21 2.25 >5.17 10 >5.13 >5.05 >5.05 >5.17 thirty >5.30 >5.05 >5.05 >5.17 60 >5.30 >5.05 >5.05 >5.17 120 >5.30 >5.05 >5.05 >5.17 180 >5.76 >5.51 >5.51 >5.63

Table 8

Inactivation of XMuLV 15 mM (0.5%) SIMULSOL™ SL 11W in purified mAb bioreactor collection material

Point in time (min) LRF achieved 4°C 18°С 25 °C 30°С 0 2.61 5.36 >5.05 >4.55 10 >4.67 >5.05 >5.05 >4.55 thirty >4.67 >5.05 >5.05 >4.55 60 >4.67 >5.04 >5.05 >4.55 120 >4.67 >5.05 >5.05 >4.55 180 >5.13 >5.51 >5.51 >5.01

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Table 9

Inactivation of XMuLV 30 mM (1.0%) SIMULSOL™ SL 11W in purified mAb bioreactor collection material

Point in time (min) LRF achieved 4°C 18°С 25°C 30°С 0 2.91 5.61 >4.67 >5.42 10 >5.42 >5.30 >4.67 >5.42 thirty >5.42 >5.30 >4.67 >5.42 60 >5.42 >5.30 >4.67 >5.42 120 >5.42 >5.30 >4.67 >5.42 180 >5.88 >5.76 >5.13 >5.88

Inactivation of XMuLV using SIMULSOL™ SL 11W in various parent matrices

The ability of SIMULSOL™ SL 11W to inactivate virus at a concentration of 9 mM (0.3% w/w) in various starting matrices such as water, cell culture medium and Dulbecco's phosphate buffered saline (DPBS) at 4°C and 30°C. Virus inactivation was monitored at 0, 60, 120, and 180 min after XMuLV inoculation.

The results shown in table. 10 show robust inactivation of XMuLV at both 4°C and 30°C after 120 minutes in all three starting matrices. DPBS at 4°C after 180 minutes detected few live virus particles in the TCID50 assay, but an LRF of 5.63 was still achieved. Overall, these results indicate that SIMULSOL™ SL 11W is able to effectively inactivate the virus regardless of the starting matrix. This suggests the potential use of SIMULSOL™ SL 11W in biopharmaceutical production platforms and the flexibility of using SIMULSOL™ SL 11W at different stages of the protein production process.

Table 10

Inactivation of XMuLV using SIMULSOL™ SL 11W in various parent matrices

Time (min.) LRF (0.3% w/w SIMULSOL™ SL 11W) 4°С 30°С Water Cell culture medium Dulbecco's sodium phosphate buffer Water Cell culture medium Dulbecco's sodium phosphate buffer 0 4.12 2 1.5 >4.40 >4.40 4.41 60 >4.52 >4.52 4.53 >4.40 >4.40 >4.40 120 >4.52 >4.52 >4.52 >4.40 >4.40 >4.40 180 >5.31 >5.31 5.63 >5.05 >5.05 >5.05

Kinetics of XMuLV inactivation at 0.3% SIMULSOL™ SL 11W at 4°C and 30°C

The ability of SIMULSOL™ SL 11W to inactivate the virus in shorter periods of time is assessed. SIMULSOL™ SL 11W is added at a concentration of 9 mM (0.3% w/w) to the purified material collected in the bioreactor at 4°C and 30°C. Virus inactivation was monitored at 0, 2, 4, 6, 8, 10, 20, and 180 min after XMuLV inoculation.

The results shown in table. 11 show that complete inactivation of XMuLV occurs after >6 minutes at 4°C and after >2 minutes at 30°C with 9 mM (0.3% w/w) SIMULSOL™ SL 11W in purified monoclonal antibody pool material in a bioreactor.

Table 11

Inactivation of XMuLV with 9 mM (0.3%) SIMULSOL™ SL 11W at 4°C and 30°C in purified mAb___________bioreactor collection material___________

Point in time (minutes) LRF achieved 4°С 30°С 0 1.75 3.62 2 3.32 >5.05 4 4.27 >5.05 6 >4.92 >5.05 8 >4.92 >5.05 10 >4.92 >5.05 20 >4.92 >5.05 180 >5.38 >5.51

Effect of the stabilizing agent monopropylene glycol on virus inactivation

The effect of monopropylene glycol on virus inactivation at concentrations of 0.1% and 0.5% in purified mAb bioreactor collection materials at 30°C was assessed. Virus inactivation is monitored after 0 and

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180 minutes after XMuLV inoculation. SIMULSOL™ SL 11W contains from 1% to 5% monopropylene glycol. The maximum monopropylene glycol concentration given in the inactivation procedures here is 0.05%. The results shown in table. 12 show that monopropylene glycol at concentrations of 0.1% and 0.5% does not inactivate XMuLV in purified mAb bioreactor harvest material at 30°C. In addition, the concentrations of monopropylene glycol presented in Table. 11 are at least 10 times the maximum concentration that can be detected in purified mAb bioreactor collection material treated with SIMULSOL™ SL 11W. This indicates that SIMULSOL™ SL 11W is responsible for the observed virus inactivation.

Table 12

Inactivation of XMuLV by monopropylene glycol in purified mAb bioreactor collection material

Point in time (minutes) LRF at 30°C 0.1% monopropylene glycol 0.5% monopropylene glycol 0 0.25 0.00 180 0.25 0.13

Inactivation of enveloped and non-enveloped viruses with SIMULSOL™ SL 11W

The ability of SIMULSOL™ SL 11W to inactivate PRV, an enveloped virus, and PPV, a non-enveloped virus, is evaluated. SIMULSOL™ SL 11W is added at a concentration of 9 mM (0.3% w/w) to purified material collected in a bioreactor at 4°C, 18°C and 30°C. Virus inactivation is monitored at 0, 10, 30, 60, 120 and 180 minutes after inoculation.

The results shown in table. 13 show robust inactivation of PRV enveloped virus occurring after 10 minutes at all three temperatures, with robust inactivation of PRV enveloped virus occurring even at lower time points ranging from 0 to 5 minutes at 30°C. PPV inactivation was not observed for SIMULSOL™ SL 11W. These results collectively suggest that SIMULSOL™ SL 11W not only reliably inactivates a variety of enveloped viruses in clarified materials collected in the bioreactor, but also that viral inactivation by SIMULSOL™ SL 11W is specific to enveloped viruses.

Table 13

PRV inactivation 9 mM (0.3% w/w) SIMULSOL™ SL 11W in purified mAb bioreactor harvest material

Point in time (min) LRF achieved 4°C 18°С 30°С 0-5 2.93 1.30 >3.30 10 >2.92 >2.92 >3.30 thirty >2.92 >3.23 >3.30 60 >2.92 >2.92 >3.30 120 >2.92 >2.92 >3.30 180 >3.38 >3.38 >3.76

Effect of pH on XMuLV inactivation using SIMULSOL™ SL 11W

The ability of SIMULSOL™ SL 11W to inactivate XMuLV at pH 5.5 and pH 8.0 was assessed. SIMULSOL™ SL 11W is added at a concentration of 9 mM (0.3% w/w) to the purified mAb bioreactor harvest material at 18°C. Viral inactivation was monitored at 0, 10, 30, 60, 120 and 180 minutes after XMuLV inoculation using SIMULSOL™ SL 11W contained in mAb bioreactor harvest material.

The results shown in table. 14 show rapid and complete inactivation of XMuLV by SIMULSOL™ SL 11W at both pH 5.5 and pH 8.0 in 10 minutes, indicating the potential applicability of SIMULSOL™ SL 11W over a wide pH range in protein production processes.

Table 14

Inactivation of XMuLV 9 mM (0.3%) SIMULSOL™ SL 11W at 18°C, pH 5.5 and pH 8.0 in purified mAb bioreactor harvest material

Point in time (min) LRF achieved pH 5.5 pH 8.0 0 3.48 3.47 10 >5.42 >5.42 thirty >5.42 >5.42 60 >5.42 >5.42 120 >5.42 >5.42 180 >5.88 >5.88

Influence of source of harvested bioreactor material on XMuLV inactivation using SIMULSOL™ SL 11W

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The ability of SIMULSOL™ SL 11W to inactivate XMuLV in purified bioreactor materials containing various recombinant proteins such as mAbs, bispecific antibodies and Fc fusion proteins is evaluated. SIMULSOL™ SL 11W is added at a concentration of 9 mM (0.3% w/w) to purified bioreactor collection materials of various proteins at 4°C and 18°C. Virus inactivation was monitored at incubation time points of 0, 10, 30, 60, 120, and 180 minutes. The results shown in table. 15 show complete inactivation of XMuLV by SIMULSOL™ SL 11W in 120 minutes at both 4°C and 30°C in all three purified harvested bioreactor materials. In addition, complete inactivation of the Fc-fusion material and the protein mAb containing the collected material was observed after 10 minutes at 4°C. The inactivation kinetics of the bispecific antibody was slightly slower at 4°C than that of the other two purified materials collected in the bioreactor at 4°C. However, at 18°C, complete inactivation of SIMULSOL™ SL 11W was observed for all 3 proteins within 10 minutes.

Table 15

Inactivation of XMuLV 9 mM (0.3%) SIMULSOL™ SL 11W in purified recombinant protein bioreactor material________________________

Point in time (min) 4°C 18°C Fcfusion protein Bispecific antibody mAb Fcfusion protein Bispecific antibody mAb 0 2.96 1.87 1.50 3.55 2.25 >5.17 10 >5.17 2.55 >5.13 >5.17 >4.92 >5.17 thirty >5.17 3.27 >5.30 >5.17 >4.92 >5.17 60 >5.17 4.32 >5.30 >5.17 >4.92 >5.17 120 >5.17 >4.92 >5.30 >5.17 >4.92 >5.17 180 >5.63 >5.38 >5.76 >5.63 >5.38 >5.63

Effect of impurity levels on viral inactivation in purified bioreactor harvested material of various proteins

The impact of impurity levels in bioreactor harvest materials on three different recombinant proteins is assessed: an Fc fusion protein, a bispecific antibody, and a mAb.

The collected materials are purified by centrifugation followed by polishing filtration. Impurities such as host cell proteins, lipids, and host cell DNA are also automatically concentrated during the centrifugation process. A sample of purified mAb collection material is further concentrated approximately 4-fold using a tangential flow filtration (TFF) process with a molecular weight cutoff of 5 kDa. Recombinant protein, DNA and other HCP contaminants in three purified materials and concentrated TFF material are measured. The results shown in Table 16 show the recombinant protein, host cell DNA and HCP content profiles of the three purified recombinant protein materials and the concentrated purified mAb material. There was a greater than 3-fold increase in DNA and HCP contaminants in collected concentrated purified mAb material compared to unconcentrated purified collected mAb material.

Table 16

Amount of components in purified material collected in recombinant protein bioreactor

Purified protein Components of collected bioreactor material Recombinant protein (mg/ml) DNA (pg/ml) NSR (ng/ml) Fc fusion protein 1.65 72180600 94364 Bispecific mAbs 3.40 202617600 915413 mAb 3.14 142781780 969014 Concentrated mAbs 10.93 431469600 3314549

Effect of concentrated bioreactor harvested material on XMuLV inactivation using SIMULSOL™ SL 11W

The effect of SIMULSOL™ SL 11W on the inactivation of viruses in concentrated purified mAb material collected in a bioreactor is tested. The purified mAb material collected in the bioreactor, containing the mAb and other impurities, is concentrated approximately fourfold using a tangential flow filtration (TFF) process with a molecular weight cutoff of 5 kDa. The concentrated material containing the mAb and impurities is then immediately subjected to sterile filtration. SIMULSOL™ SL 11W is then added to the purified concentrated filter material at a final concentration of 0.1% w/w. (3 mM). The material containing SIMULSOL™ 11 W is then incubated

- 14 046732 at 3 different temperatures: 4°C, 18°C and 30°C. Viral inactivation at various temperatures is measured at time points ranging from 0 to 180 minutes. The results shown in table. 17 show complete inactivation of XMuLV after 180 minutes at all 3 temperatures tested, even though the inactivation kinetics are reduced at lower temperatures compared to the 30°C sample. There was no effect on the inactivation kinetics of concentrated bioreactor material compared to unconcentrated collected bioreactor material (data not shown). This suggests that increased levels of impurities do not affect virus inactivation by SIMULSOL™ SL 11W.

Table 17

Virus inactivation with 3 mM (0.1%) SIMULSOL™ SL 11W in bioreactor material collected at 4-fold concentration of purified mAb

Point in time (min) LRF achieved 4°C 18°С 30°С 0 2.25 2.50 2.25 10 2.00 5.13 5.36 thirty 3.28 5.73 >5.05 60 5.06 >5.42 >5.05 120 >5.05 >5.42 >5.05 180 >5.51 >5.88 >5.51

Example 2 Determination of removal of SIMULSOL™ SL 11W during subsequent purification of recombinant proteins

Quantitative determination of undecyl glucoside using liquid chromatography-mass spectrometry (LC-MS) - Method A

LC-MS quantitation is performed on purified mAb samples collected in a bioreactor containing SIMULSOL™ SL 11W at concentrations ranging from 0.03 mM to 3 mM.

One microliter of incubated sample is injected into a Waters Acquity® HPLC coupled to a Waters SYNAPT® G2-Si mass spectrometer. For quantitation, separation is carried out on a Varian PLRP-S reverse phase column (1 x 50 mm, 300 A, 5 µm) at 80°C using 0.05% trifluoroacetic acid (TFA) using H ₂ O 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 minute and ramped from 15% to 30% over 3 minutes, then maintained at 30% phase B for 2 minutes and then held at 100% for 1 minute. After the column is held at 100% for 1 minute, it is re-equilibrated with 15% mobile phase B. SIMULSOL™ SL 11W is separated at a rate of 0.3 ml/min, from 1 to 6 minutes and analyzed using an electrospray ionization source (ESI). The mass spectrometer operates with a positive sensitivity model, scanning range from 100 to 1000 amu, capillary voltage 3.2 kV, desolvation temperature 450°C, desolvation gas flow 900 l/h, source temperature 150°C and the sample cone voltage is 40 V. For characterization, separation is carried out on a Waters Acquity® ^UPLC OUT 300 A C4 column (2.1 x 100 mm, particle size 1.7 µm) with the same mobile phases. Equilibrate the column at 20% mobile phase B for 1 minute, increasing linearly from 20% to 25% over 14 minutes and then to 90% over 1 minute. After holding at 100% for 1.0 minutes, re-equilibrate the column with 20% mobile phase B.

The isolated peak region of saturated undecyl glucoside is measured. Under LCMS analysis conditions, a linear relationship was observed between the extracted ion peak region and SIMULSOL™ SL 11W concentrations from 0.03 to 3 mM. When the concentration of SIMULSOL™ SL 11W exceeded 3 mM, the MS detector saturated.

Quantitative determination of undecyl glucoside using liquid chromatography with tandem mass spectrometry (LC-MS/MS) - method B

Standard solutions containing SIMULSOL™ SL 11W in the concentration range from 0.0008 mM to 0.012 mM are prepared in bulk water or protein A. After addition of SIMULSOL™ SL 11W, these samples were subjected to LC-MS/MS (MRM) analysis. The incubated sample (100 µl) is injected into the LC and 1/20 of the post-column is separated onto the MS. An Agilent 1260 Infinity II HPLC coupled to a Sciex Triple Quad 5500 mass spectrometer was used for analysis. For quantitative determination, separation was performed on an Agilent Zorbax reverse phase column, 300SB-C8 (4.6 x 50 mm, 300 A, 3.5 µm) at 80°C using 0.5 mM sodium acetate in water as mobile phase A and 0.5 mM sodium acetate in a 95:5 dilution of acetonitrile:water as mobile phase B. The column is equilibrated at 20% mobile phase B for 2 minutes, linearly increase from 20% to 90% over 10 minutes, hold at 90% phase B for 2 minutes, column re-equilibrate at 20% mobile phase B. SIMULSOL™ SL 11 W separates at a rate of 1.0 ml/ min for 4.5 to 5.5 minutes and analyzed using an electrospray ionization (ESI) source. Mass spectrometer working

- 15 046732 melts in positive MRM mode with transition ion scanning (357.0 to 185.0), capillary voltage 5.5 kV, desolvation temperature 450°C and desolvation gas flow 55 l/h.

Selected Reaction Monitoring (SRM/MRM) LCMS/MS method is used for the selective detection of salt-saturated SIMULSOL™ SL 11W. SRM scan for a single precursor ion (mass of sodium SIMULSOL™ SL 11W, 357.0 m/z) and after fragmentation the product ion is detected (most predominant fragment of sodium SIMULSOL™ SL 11W, 185.0 m/z) and the peak zone corresponds to to this isolated transition ion.

Under LC-MS/MS analysis conditions, the plots are linear between concentrations of SIMULSOL™ SL 11W from 0.0008 mM to 0.012 mM in water and the bulk protein A flow. These results indicate that LCMS/MS is capable of detecting SIMULSOL™ SL 11W over a concentration range from 0.0008 to 0.012 mM, and the linearity of the graphs does not depend on the matrix. Therefore, this LC-MS/MS (SRM/MRM) method is sensitive and specific for the detection of SIMULSOL™ SL 11W and is independent of the detergent matrix. The limit of detection (LOD) is 0.001 mM, calculated from linearity.

Protein A chromatography and detection SIMULSOL™ SL 11W

Purified solutions of collected mAb bioreactor material containing 0% (control), 0.3% and 1.0% w/w. SIMULSOL™ SL 11W is subjected to MabSelect™ Protein A chromatography. Protein A chromatography is performed on unfiltered purified mAb harvest material containing SIMULSOL™ SL 11W. Once the column is loaded, six Tris/NaCl column volumes are used to wash the column before eluting the mAb with acetic/citric acid buffer. The eluted mAb bulk stream is collected and analyzed by LC-MS for the presence of SIMULSOL™ SL 11W.

Results from LC-MS analysis (Method A) show <0.03 mM levels of SIMULSOL™ SL 11W in the bulk Protein A stream from bioreactor harvest mAb solutions containing 0.3% and 1.0% w/w. SIMULSOL™ SL 11W. Using LC-MS/MS (Method B), the amount of SIMULSOL™ SL 11W was found to be 0.0005 mM in the main protein A stream from mAb bioreactor harvest solutions containing 0.3% w/w. SIMULSOL™ SL 11W, which is below the limit of detection (LOD) of 0.001 mM. These results demonstrate that Protein A chromatography is capable of removing SIMULSOL™ SL 11W from the nutrient stream to trace levels, even without filtration steps prior to column loading.

Example 3. Effect of SIMULSOL™ SL 11W on the quality of recombinant protein

The effect of SIMULSOL™ SL 11W on the quality of recombinant proteins is tested by comparing purified mAb samples collected in a bioreactor with and without 0.3% SIMULSOL™ SL 11W at 20°C for 3 hours, followed by incubation at 4°C for 16 hours. Samples are first filtered through 0.2 µm glass fiber filters, then through 0.45 µm PVDF filters, and then purified using Protein A. Samples are analyzed for analytical properties as shown in Table 1. 18, using methods known in the art, such as capillary electrophoresis (CE), size exclusion chromatography (SEC), isoelectric focusing (IEF) measurements on the iCE™ instrument. The results shown in table. 18 show that SIMULSOL™ SL 11W treatment did not have a significant effect on any of the measured qualities compared to no SIMULSOL™ SL 11W treatment.

Table 18

Analytical mAb product quality data with and without SIMULSOL™ SL 11W

Analyticity Detergent-free 0.3% SIMULSOL™ SL 11W CE reduced heavy chain (%) 65.9 65.9 CE reduced light chain (%) 31.7 31.6 CE main peak without reduction (%) 98.2 98.2 SEC monomer (%) 98.5 98.5 SEC aggregate (%) 1.5 1.5 Main Peak IEF (%) 75.5 74.8 Total acid variants IEF (%) 23.0 23.5 Total basic IEF options (%) 1.5 1.6 Chinese hamster ovary (CHO) host cell protein (HCP) (PPt) 336 283 Residual protein A (ppm) 13.3 7.3 DNA (ppb) 21527 21473

Claims (43)

1. A method for inactivating an enveloped virus in a raw material stream, comprising adding to the raw material stream an environmentally friendly detergent in a final concentration of about 0.05% wt./wt. to about 1% w/w, wherein the environmentally friendly detergent is a solution containing more than about 40% undecyl glucoside and less than about 20% other alkyl glucosides. 2. The method according to claim 1, characterized in that the detergent contains from about 40% to about 60% undecyl glucoside. 3. The method according to any one of claims 1, 2, characterized in that the detergent contains from about 53% to about 57% undecyl glucoside. 4. The method according to claim 1, characterized in that the detergent contains more than about 50% undecyl glucoside. 5. A method according to any one of claims 1 to 4, characterized in that the detergent contains less than about 15% of other alkyl glucosides. 6. A method according to any one of claims 1 to 5, characterized in that the detergent contains less than about 10% of other alkyl glucosides. 7. Method according to any one of claims 1 to 6, characterized in that the detergent is SIMULSOL™ SL 11W. 8. The method according to any one of claims 1 to 7, characterized in that the detergent is added to the feed stream in a final concentration of about 0.1% wt./wt. up to about 1% w/w 9. The method according to any one of claims 1 to 7, characterized in that the detergent is added to the feed stream in a final concentration of about 0.3% wt./wt. up to about 1% w/w 10. The method according to any one of claims 1 to 7, characterized in that the detergent is added to the feed stream in a final concentration of about 0.1% wt./wt. to about 0.3% w/w 11. The method according to any one of claims 1 to 7, characterized in that the detergent is added to the feed stream in a final concentration of about 0.3% wt./wt. 12. The method according to any one of claims 1 to 11, characterized in that during said detergent addition step the feed stream has a temperature of from about 4°C to about 30°C. 13. The method according to any one of claims 1 to 11, characterized in that during said detergent addition step the temperature of the feed stream is from about 15°C to about 30°C. 14. The method according to any one of claims 1 to 11, characterized in that during said detergent addition step the temperature of the feed stream is from about 15°C to about 20°C. 15. A method according to any one of claims 1 to 14, characterized in that during said detergent addition step, the feed stream has a pH value of from about 5.5 to about 8.0. 16. The method of any one of claims 1 to 15, further comprising the step of incubating the detergent in the feed stream for from about 1 to about 180 minutes. 17. The method of claim 16, wherein after said incubation step the detergent has a log viral load reduction factor (LRF) in the feed stream of more than about one or more than about two. 18. The method according to claim 1, characterized in that the enveloped virus is a retrovirus, herpesvirus, flavivirus, poxvirus, hepadnavirus, hepatitis virus, orthomyxovirus, paramyxovirus, rhabdovirus or togavirus. 19. The method of claim 1, further comprising the step of incubating the feed stream and detergent for about 15 minutes to about 180 minutes at a temperature of about 4°C to about 30°C, at a pH value of about 5.5 to about 8 ,0, where detergent is added to the feed stream at a final concentration of about 0.1% w/w. to about 1.0% w/w, wherein the detergent in the feed stream has a log viral load reduction factor (LRF) of greater than about 2. 20. The method of claim 1, further comprising the step of incubating the feed stream and detergent for about 6 minutes to about 180 minutes at a temperature of about 4°C to about 30°C and at a pH of about 5.5 to about 8, 0, where the detergent is added to the feed stream at a final concentration of about 0.3% w/w. to about 1.0% w/w, wherein the detergent in the feed stream has a log viral load reduction factor (LRF) of greater than about 4. 21. The method of claim 1, further comprising the step of incubating the feed stream and the detergent for about 180 minutes at a temperature of about 4°C to about 30°C and at a pH of about 5.5 to about 8.0, wherein the detergent added to the feed stream at a final concentration of about 0.1% w/w. to about 1.0% w/w, and wherein the detergent in the feed stream has a log viral load reduction factor (LRF) of greater than about 5. 22. The method of claim 1, further comprising the step of incubating the feed stream and detergent for about 60 to about 180 minutes at a temperature of about 4°C to about 30°C and a pH of about 5.5 to about 8.0 wherein the detergent is added to the feed stream at a final concentration of about 0.3% w/w, wherein the detergent in the feed stream has a logarithmic virus reduction factor - 17 046732 load (LRF) more than about 5. 23. The method according to any one of claims 1 to 22, characterized in that the feed stream is a collected cell culture fluid, a capture pool or a pool of extracted products. 24. The method according to claim 23, characterized in that the capture pool or the pool of recovered products is an affinity chromatography pool. 25. The method according to claim 24, characterized in that the affinity chromatography pool is a protein A pool, a protein G pool or a protein L pool. 26. The method of claim 23, wherein the capture pool or recovered product pool is a mixed mode chromatography pool. 27. The method according to any one of claims 1 to 26, further comprising the step of filtering a feed stream containing a detergent, wherein the feed stream is subjected to at least one filtration step. 28. The method according to claim 27, characterized in that the feed stream is subjected to at least a second filtration step. 29. The method according to claim 28, characterized in that the feed stream is subjected to at least a third stage of filtration. 30. The method according to any one of claims 27-29, further comprising the step of treating the filtered feed stream on an affinity chromatography column. 31. The method according to claim 30, characterized in that the affinity chromatography column is a protein A column. 32. Method according to any one of claims 1 to 29, characterized in that the feed stream containing the detergent is subjected to affinity chromatography. 33. The method according to any one of claims 1 to 32, characterized in that the raw material stream contains a protein, wherein 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, peptide or binding agent. 34. The method of claim 33, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment. 35. Method according to any one of claims 33-34, characterized in that the protein is produced in a mammalian cell. 36. The method according to any one of claims 1 to 35, characterized in that the step of adding detergent to the feed stream does not significantly change the turbidity of the feed stream. 37. The method according to any one of claims 1 to 35, characterized in that the step of adding detergent to the feed stream does not change the quality of the final product in the purified protein stream. 38. Method according to any one of claims 1 to 37, characterized in that the detergent contains a stabilizing agent. 39. The method according to claim 38, characterized in that the stabilizing agent is monopropylene glycol. 40. A method for inactivating an enveloped virus in a feed stream, including the following stages: adding SIMULSOL™ SL 11W to the stream at a concentration of about 0.1% w/w. up to about 0.3% w/w; incubating SIMULSOL™ SL 11W in the feed stream for about 180 minutes such that the logarithmic load reduction factor (LRF) of SIMULSOL™ SL 11W in the feed stream is greater than about 5; filtering said feed stream containing SIMULSOL™ SL 11W; and treating said filtered feed stream on a Protein A affinity chromatography column; wherein said step of adding and incubating the feed stream at a temperature of from about 4°C to about 30°C and at a pH value of from about 5.5 to about 8.0. 41. The method according to claim 40, wherein said filtration step includes a first filtration step using a 0.22 µm filter. 42. The method according to claim 41, wherein said filtration step includes a second filtration step using a PVDF filter with a pore size of 0.45 microns. 43. The method according to claim 42, wherein said filtration step includes a third filtration step using a PVDF filter with a pore size of 0.45 microns.