Alternative surfactants for virus inactivation

Abstract

To provide an alternative surfactant for virus inactivation.SOLUTION: A surfactant for use to inactivate enveloped viruses comprising a surfactant that can be used in biomanufacturing operations and is considered environmentally friendly. Provided herein is a method of inactivating an enveloped virus in a liquid known to contain or suspected of containing at least one enveloped virus, comprising obtaining a liquid known to contain or suspected of containing at least one enveloped virus; and exposing the liquid to a surfactant of Table 1 at a concentration and for a time sufficient to cause viral inactivation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 820,330, filed March 19, 2019, which is incorporated herein by reference.

[0002] Detergents for use in inactivating enveloped viruses, including potentially environmentally friendly detergents that can be used as an alternative to Triton® X100 in biomanufacturing operations. [Background technology]

[0003] Viral inactivation process steps are important to ensure the safety of protein therapeutics (Aranha, BioProcess International 2005;17-20; Remington, Bioprocess International 2015,13(5),10-17). Viral contaminants can arise from a variety of sources, including the culture medium, the manufacturing site, or adventitious viral contaminants in Chinese hamster ovary (CHO) cell lines (Aranha, Bioprocess International 2012, 10(3), 12-17). To ensure patient safety, biomanufacturers follow guidelines specified by ICH-Q5A, which include orthogonal viral clearance steps, including inactivation and filtration processes (Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. Harmonization, ICO, Ed. 1999; Vol. Q5A). Triton X-100 (polyoxyethylene octylphenol ether) has long been used as an inactivating surfactant to inactivate enveloped viruses (Durno, and Tounekti, PDA Journal of Pharmaceutical Science and Technology, 2015, 69(1), 163-172; Standard Practice for Process Step to Inactivate Rodent Retrovirus with Triton X-100 Treatment, ASTM International. In E3042-16, ASTM, Ed. West Conshohocken, PA, 2016). Detergents solubilize the membrane lipids that make up the outer envelope layer (Liumbruno and Franchini, Journal of Thrombosis and Thrombolysis 2015, 39(1), 118-128). Therefore, detergents inactivate only enveloped viruses, not non-enveloped ones (Hellstern and Solheim, Transfusion medicine and hemotherapy, 2011, 38(1), 65-70). Although Triton X-100 potently inactivates viruses, detergents are also environmental toxins. One effect is their detrimental effect on the endocrine system of fish. Triton X-100 breaks down into octylphenol, which mimics the hormone estradiol, causing changes in the hormonal system (Laws et al., Toxicological Sciences, 2000, 54(1), 154-167).As outlined in Article 57 of REACH and Annex XIV of Commission Regulation (EU) No. 1907 / 2006 of the European Parliament and the Council on the Registration, Evaluation, Authorization, and Restriction of Chemicals ("REACH") (Union, E., Ed. 2017), the European Commission will begin banning the use of Triton X-100 by biopharmaceutical companies by 2020. Therefore, it is pertinent to identify environmentally friendly, potent, virus-inactivating alternative surfactants. Recent research by Conley et al. identified lauryldimethylamine N-oxide (LDAO), an environmentally friendly zwitterionic surfactant capable of inactivating enveloped viruses. (Conley et al., Biotechnology and Bioengineering 2017, 114(4), 813-820) and U.S. Patent Application Publication No. 20150306223. US Patent Application Publication No. 20160333046 also identifies a number of surfactants that exhibit strong viral clearance as well as ecological safety. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 20150306223 [Patent Document 2] US Patent Application Publication No. 20160333046 [Non-patent literature]

[0005] [Non-Patent Document 1] Aranha,BioProcess International 2005;17-20 [Non-patent document 2] Remington,Bioprocess International 2015,13(5),10-17 Summary of the Invention [Problem to be solved by the invention]

[0006] As such, there is a need for surfactants that are effective in inactivating enveloped viruses, particularly surfactants that are potentially environmentally friendly for use within viral clearance process steps in biomanufacturing. The invention described herein fills this need by identifying effective virus-inactivating surfactants that can be incorporated into biomanufacturing processes for multiple therapeutic modalities. [Means for solving the problem]

[0007] The present invention provides a method for inactivating an enveloped virus in a liquid known to contain or suspected to contain at least one enveloped virus, the method comprising obtaining a liquid known to contain or suspected to contain at least one enveloped virus; and exposing the liquid to a surfactant of Table 1 at a concentration and for a time sufficient to cause viral inactivation. In one embodiment, the surfactant has a CAS Registry Number of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS70504-28-8, CAS59080-45-4, CAS69984-73-2, CAS148616-91-5, CAS148565-55-3, CAS69227-93-6, CAS82494-09-5, CAS253678-67-0, CAS106402-05-5, or CAS93911-12-7. In one embodiment, the surfactant is selected from CAS 3055-99-0, CAS 3055-98-9, CAS 9043-30-5, or CAS 85618-20-8. In one embodiment, the liquid is exposed to the surfactant for at least 30 seconds to at least 60 minutes or more. In one embodiment, the liquid is exposed to the surfactant for at least 10 minutes. In one embodiment, the liquid is exposed to the surfactant for at least 30 minutes. In one embodiment, the liquid is exposed to the surfactant for at least 60 minutes or more. In one embodiment, the concentration of the surfactant is at least 2.5 times to at least 10 times or more its critical micelle concentration (CMC). In one embodiment, the concentration of the surfactant is at least 5 times its CMC. In a related embodiment, the concentration of the surfactant is at least 7.5 times its CMC. In another related embodiment, the concentration of the surfactant is at least 10 times its CMC. In one embodiment, the exposure of the liquid to the surfactant is at a temperature of at least 5° C. to 22° C. In a related embodiment, the exposure of the liquid to the surfactant is at a temperature of at least 5°C.In a related embodiment, the exposure to the liquid detergent is at a temperature of at least 15° C. In a related embodiment, the exposure to the liquid detergent is at a temperature of at least 20° C. In another embodiment, the inactivation is by TCID. 50 The concentration of the recombinant protein of interest is measured using an assay. In one embodiment, the fluid comprises the recombinant protein of interest. In one embodiment, the fluid is a harvested host cell culture fluid. In one embodiment, the fluid is from an effluent stream, eluate, pool, pool, or retentate from a unit operation comprising a harvesting, filtration, or chromatography step. In a related embodiment, the fluid is an eluate recovered from depth filtration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. In a related embodiment, the fluid is a pool containing harvested cell culture fluid, an eluate from depth filtration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multimodal chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography. In another related embodiment, the affinity chromatography is Protein A, Protein G, Protein A / G, or Protein L chromatography. In one embodiment, the concentration of the detergent is 5 times its CMC and the time is at least 10 minutes. The present invention provides a method for inactivating a recombinant protein of interest comprising obtaining a liquid containing a recombinant protein of interest that is known or suspected to contain at least one virus; exposing the liquid to at least one detergent at a concentration and for a time sufficient to cause inactivation of enveloped viruses in the liquid, wherein the detergent is selected from the group consisting of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49- and exposing the virus inactivation liquid to at least one unit operation comprising at least a filtration step or a chromatography step. In one embodiment, the surfactant is selected from CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, and CAS85618-20-8. In one embodiment, the liquid is exposed to the surfactant for at least 30 seconds to at least 60 minutes or more. In a related embodiment, the liquid is exposed to the surfactant for at least 10 minutes. In a related embodiment, the liquid is exposed to the surfactant for at least 30 minutes. In another related embodiment, the liquid is exposed to the surfactant for at least 60 minutes or more. In another embodiment, the concentration of the surfactant is at least 2.5 times to at least 10 times or more its critical micelle concentration (CMC). In a related embodiment, the concentration of the surfactant is at least 5 times its CMC. In a related embodiment, the concentration of the surfactant is at least 7.5 times its CMC. In another related embodiment, the concentration of the surfactant is at least 10 times its CMC. In a related embodiment, the exposure of the liquid to the surfactant is at a temperature of at least 5°C to 22°C.In a related embodiment, exposure to the liquid detergent occurs at a temperature of at least 5° C. In a related embodiment, exposure to the liquid detergent occurs at a temperature of at least 15° C. In another embodiment, exposure to the liquid detergent occurs at a temperature of at least 20° C. In another embodiment, inactivation occurs by TCID. 50The amount of the recombinant protein of interest is measured using an assay. In another embodiment, the liquid comprises the recombinant protein of interest. In another embodiment, the liquid is a harvested host cell culture fluid. In another embodiment, the liquid is from an effluent stream, eluate, pool, reservoir, or retentate from a unit operation comprising a harvesting, filtration, or chromatography step. In another embodiment, the liquid is an eluate recovered from depth filtration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. In another embodiment, the liquid is a pool containing harvested cell culture fluid, an eluate from depth filtration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multimodal chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography. In a related embodiment, the affinity chromatography is Protein A, Protein G, Protein A / G, or Protein L chromatography. In another embodiment, the chromatography is selected from affinity chromatography, Protein A chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography; hydrophobic interaction chromatography; mixed modal or multimodal chromatography, or hydroxyapatite chromatography. In another embodiment, the liquid is a harvested host cell culture fluid and the unit operation comprises depth filtration. In another embodiment, the liquid is a harvested host cell culture fluid and the unit operation comprises Protein A affinity chromatography. In another embodiment, the liquid is a Protein A eluate and the unit operation comprises depth filtration. In another embodiment, the unit operation comprises depth filtration. In one embodiment, the unit operation comprises microfiltration. In one embodiment, the concentration of the detergent is 5 times its CMC and the time is at least 10 minutes.

[0008] The present invention also provides a method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express the recombinant protein and culturing the cells to express the recombinant protein of interest; recovering a cell culture solution containing the recombinant protein of interest; and processing a liquid containing the recombinant protein of interest through at least two unit operations, wherein during at least one unit operation, the liquid containing the recombinant protein of interest is treated with one of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57- CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7, at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; and obtaining an isolated and purified recombinant protein of interest. In one embodiment, the surfactant is selected from CAS 3055-99-0, CAS 3055-98-9, CAS 9043-30-5, and CAS 85618-20-8. In one embodiment, the liquid is exposed to the surfactant for at least 30 seconds to at least 60 minutes or more. In a related embodiment, the liquid is exposed to the surfactant for at least 10 minutes. In a related embodiment, the liquid is exposed to the surfactant for at least 30 minutes. In another related embodiment, the liquid is exposed to the surfactant for at least 60 minutes or more. In another embodiment, the concentration of the surfactant is at least 2.5 times to at least 10 times or more its critical micelle concentration (CMC). In a related embodiment, the concentration of the surfactant is at least 5 times its CMC. In a related embodiment, the concentration of the surfactant is at least 7.5 times its CMC.In another embodiment, the concentration of the surfactant is at least 10 times its CMC. In a related embodiment, the exposure to the liquid surfactant is at a temperature of at least 5° C. to 22° C. In a related embodiment, the exposure to the liquid surfactant is at a temperature of at least 5° C. In a related embodiment, the exposure to the liquid surfactant is at a temperature of at least 15° C. In another embodiment, the exposure to the liquid surfactant is at a temperature of at least 20° C. In another embodiment, the inactivation is at least TCID. 50 The viral inactivation step is measured using an assay. In another embodiment, the at least one unit operation includes a capture chromatography step selected from affinity chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. In another embodiment, the at least one unit operation includes a polish chromatography step selected from ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. In another embodiment, the at least one unit operation includes a step selected from viral filtration, depth filtration, and UF / DF. In another embodiment, a unit operation including a viral inactivation step occurs before a unit operation including affinity chromatography. In another embodiment, a unit operation including affinity chromatography occurs before a unit operation including a viral inactivation step. In one embodiment, a unit operation including a viral inactivation step occurs before a unit operation including depth filtration. In one embodiment, a recombinant protein of interest isolated and purified according to the above method is provided. In one embodiment, a pharmaceutical composition comprising the protein of interest isolated according to the above method is provided. In one embodiment, the concentration of the surfactant is 5 times its CMC and the time is at least 10 minutes. The present invention also provides a method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express the recombinant protein and culturing the cells to express the recombinant protein of interest; harvesting a cell culture fluid containing the recombinant protein of interest; and treating the harvested fluid containing the recombinant protein of interest with a solution of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS and exposing the virus-inactivated liquid containing the recombinant protein of interest to a detergent having a CAS Registry Number of CAS 70504-28-8, CAS 59080-45-4, CAS 69984-73-2, CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7 at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; treating the virus-inactivated liquid containing the recombinant protein of interest through at least two additional unit operations; and obtaining an isolated and purified recombinant protein of interest. In one embodiment, the detergent is selected from CAS 3055-99-0, CAS 3055-98-9, CAS 9043-30-5, and CAS 85618-20-8. In one embodiment, the liquid is exposed to the surfactant for at least 30 seconds to at least 60 minutes or more. In a related embodiment, the liquid is exposed to the surfactant for at least 10 minutes. In a related embodiment, the liquid is exposed to the surfactant for at least 30 minutes. In another related embodiment, the liquid is exposed to the surfactant for at least 60 minutes or more. In another embodiment, the concentration of the surfactant is at least 2.5 times to at least 10 times or more its critical micelle concentration (CMC). In a related embodiment, the concentration of the surfactant is at least 5 times its CMC. In a related embodiment, the concentration of the surfactant is at least 7.5 times its CMC.In another related embodiment, the concentration of the surfactant is at least 10 times its CMC. In a related embodiment, the exposure to the liquid surfactant occurs at a temperature of at least 5° C. to 22° C. In a related embodiment, the exposure to the liquid surfactant occurs at a temperature of at least 5° C. In a related embodiment, the exposure to the liquid surfactant occurs at a temperature of at least 15° C. In another embodiment, the exposure to the liquid surfactant occurs at a temperature of at least 20° C. In another embodiment, the inactivation is at least TCID. 50 The viral inactivation step is measured using an assay. In another embodiment, the at least one unit operation includes a capture chromatography step selected from affinity chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. In another embodiment, the at least one unit operation includes a polish chromatography step selected from ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. In another embodiment, the at least one unit operation includes a step selected from viral filtration, depth filtration, and UF / DF. In another embodiment, a unit operation including a viral inactivation step occurs before a unit operation including affinity chromatography. In another embodiment, a unit operation including affinity chromatography occurs before a unit operation including a viral inactivation step. In one embodiment, a unit operation including a viral inactivation step occurs before a unit operation including depth filtration. In one embodiment, a recombinant protein of interest isolated and purified according to the above method is provided. In one embodiment, a pharmaceutical composition comprising the protein of interest isolated according to the above method is provided. In one embodiment, the concentration of the surfactant is 5 times its CMC and the time is at least 10 minutes.

[0009] The present invention also provides a recombinant protein of interest isolated and purified according to the methods described herein. The present invention also provides a pharmaceutical composition comprising a protein of interest isolated according to the methods described herein. [Brief explanation of the drawings]

[0010] [Figure 1] A structurally distinct class of surfactants identified to have virus-inactivating properties. [Figure 2-1] Mass spectrometry elution peaks are as follows: A: mAb elution after ProA purification. B: A blank run was performed after surfactant injection to check clearance of the surfactant from the detector. C: C12E9 surfactant (0.03%) was spiked in water. [Figure 2-2] Same as above. [Figure 3-1] Turbidity data for mixtures of surfactants and HCCF at room temperature (25°C) and 5°C. Triton X, triangles; Anapoe C12E8 (APO128), diamonds; Anapoe C12E8 (APO129), squares; control without surfactant, circles. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] %HMW and %LMW of surfactant-spiked samples before and after affinity chromatography. Triton X, triangles; Anapoe C12E8 (APO128), diamonds; Anapoe C12E8 (APO129), squares; no-surfactant control, circles; TDA-9 dashed line and stars; no-second-surfactant control, dashed line and open circles. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] %HMW and %LMW of surfactant-spiked BiTE® #1 sample before and after affinity chromatography. [Figure 6-1]%HMW and %LMW of surfactant-spiked HCCF samples after 7 days of incubation at 2–8°C. Triton X, triangles; Anapoe C12E8 (APO128), diamonds; Anapoe C12E8 (APO129), squares; no-surfactant control, circles; TDA-9, dashed line and star; no-second-surfactant control, dashed line and open circle. [Figure 6-2] Same as above. [Figure 6-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0011] The use of cell culture processes to manufacture therapeutic biological agents carries an inherent risk of transmitting viral contaminants. Such contaminants can arise from many sources, including starting materials, the use of animal-derived reagents during manufacturing, and contamination of the manufacturing system due to failures of GMP processes. As such, regulatory agencies recommend that biomanufacturing processes include dedicated viral inactivation and virus removal steps and require manufacturers to verify viral removal and inactivation from all biological products.

[0012] Provided herein is a method for inactivating an enveloped virus in a liquid known to contain or suspected to contain at least one enveloped virus, the method comprising obtaining a liquid known to contain or suspected to contain at least one enveloped virus; and exposing the liquid to a surfactant from Table 1 at a concentration and for a time sufficient to cause viral inactivation.

[0013] The present invention relates to a method for inactivating a recombinant protein of interest comprising obtaining a liquid containing a recombinant protein of interest that is known to contain or suspected to contain at least one virus; exposing the liquid to at least one detergent at a concentration and for a time sufficient to cause inactivation of enveloped viruses in the liquid, wherein the detergent is selected from the group consisting of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49- and exposing the virus inactivated liquid to at least one unit operation comprising at least a filtration step or a chromatography step.

[0014] Viruses are classified as enveloped and non-enveloped viruses. Enveloped viruses have a capsid surrounded by a lipoprotein membrane or "envelope." This envelope is composed of host cell proteins and phospholipids, as well as viral glycoproteins that coat the virus when it buds from the host cell. This envelope allows the virus to identify, bind, invade, and infect target host cells. However, because of this membrane, enveloped viruses are susceptible to inactivation methods, while non-enveloped viruses are more difficult to inactivate without risking the production of proteins, although they can be removed by filtration methods.

[0015] Examples of enveloped viruses include virus families such as Herpesviridae, Poxviridae, Hepadnaviridae, Flaviviridae, Togaviridae, Coronaviridae, Orthomyxoviridae, Deltavirus, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Filoviridae, and Retroviridae; as well as viruses such as human immunodeficiency virus, Sindbis virus, herpes simplex virus, pseudorabies virus, Sendai virus, vesicular stomatitis virus, West Nile virus, bovine viral diarrhea virus, coronavirus, equine arteritis virus, severe acute respiratory syndrome virus, Moloney murine leukemia virus, and vaccinia virus.

[0016] To ensure patient safety, viral inactivation is a necessary component of the purification process for protein therapeutics. Various methods can be employed to inactivate viruses, including heat inactivation / pasteurization, pH inactivation, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, chemical inactivation, the addition of surfactants, and solvent / detergent treatments. Surfactants, such as detergents, can be highly effective at specifically inactivating enveloped viruses because they solubilize their membranes.

[0017] Detergents solubilize the outer membrane of enveloped viruses by disrupting the protein layer with detergent micelles (Kragh-Hansen et al., Biophysical Journal 1998, 75(6), 2932-2946). The outer membrane of enveloped viruses, including xenotropic murine leukemia virus (xMuLV) and pseudorabies herpesvirus (PRV), is composed of a lipid membrane that can be disrupted by certain detergents above their critical micelle concentration (CMC). Above the CMC, detergents form micelles and disrupt the envelope layer by solubilizing the membrane lipids (Edwards and Almgren, Journal of Colloid and Interface Science 1991, 147(1), 1-21).

[0018] Detergents can be categorized into three groups based on their charge: ionic, nonionic, and zwitterionic. Both nonionic and zwitterionic detergents are mild and typically do not denature the therapeutic proteins being manufactured, while more aggressive ionic detergents can degrade them. Historically, nonionic detergents, such as the commonly used Triton X-100, have been used to inactivate viruses because they do not affect the therapeutic proteins of interest. However, ecological concerns regarding the use of certain detergents, such as Triton X-100, have driven a search to identify more "environmentally friendly" detergents that can be used to inactivate enveloped viruses in biomanufacturing processes.

[0019] As used herein, "virus inactivation," "viral inactivation," "inactivated virus," or similar such phrases refer to a process in which an enveloped virus is modified so that it is no longer able to infect, replicate, and / or propagate in cells. Surface-active substances, such as detergents, are highly effective at inactivating enveloped viruses in fluids known to or suspected of containing one or more viruses. The fluid can be obtained from an effluent stream, eluate, pool, retention, or storage vessel. In one embodiment, the fluid is obtained from a pool. In one embodiment, the pool is obtained from a recovery unit operation comprising microfiltration. In one embodiment, the fluid is obtained from an effluent stream.

[0020] The surfactant may be added to the liquid at a concentration (w / v) of 0.01% to 10%, or 0.01 to 10 times the critical micelle concentration (CMC) value of the surfactant. In certain embodiments, the concentration of the surfactant is at least 2.5 times, at least 5 times, at least 7.5 times, or at least 10 times the CMC value of the surfactant.

[0021] The liquid may be exposed to the surfactant at a temperature of at least 2°C to 22°C or higher. In certain embodiments, the liquid is exposed to the surfactant at a temperature of at least 2°C or higher, at least 5°C or higher, at least 7°C or higher, at least 10°C or higher, at least 15°C or higher, at least 20°C or higher, or at least 22°C or higher. In certain embodiments, the temperature is 2°C to 22°C, 2°C to 20°C, 2°C to 15°C, 2°C to 10°C, 2°C to 7°C, or 2°C to 5°C. In other embodiments, the temperature is 5°C to 22°C, 5°C to 20°C, 5°C to 15°C, 5°C to 10°C, or 5°C to 7°C. In other embodiments, the temperature is 7°C to 22°C, 7°C to 20°C, 7°C to 15°C, or 7°C to 10°C. In other embodiments, the temperature is 10°C to 22°C, 10°C to 20°C, or 10°C to 15°C. In other embodiments, the temperature is 10°C to 22°C, 10°C to 20°C, or 10°C to 15°C. In other embodiments, the temperature is 15°C to 22°C, or 15°C to 20°C. In one embodiment, the temperature is 20°C to 22°C. In certain embodiments, the temperature is 2°C, 5°C, 7°C, 10°C, 15°C, 20°C, or 22°C. The liquid can be exposed to the surfactant for a minimum of 30 seconds to 48 hours or more. In certain embodiments, the liquid is exposed to the surfactant for at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 90 minutes. In certain embodiments, the liquid is exposed to the surfactant for 2, 3, 4, 5, 6, 9, 10, 12, 16, 20, 24, 30, 36, 42, or 48 hours. In certain embodiments, the liquid is exposed to the surfactant for at least 30 seconds, at least 10 minutes, at least 30 minutes, at least 60 minutes, or at least 90 minutes. In certain embodiments, the concentration is 2.5 times its CMC and the time is at least 10 minutes. In certain embodiments, the concentration is 2.5 times its CMC and the time is at least 30 minutes. In certain embodiments, the concentration is 5 times its CMC and the time is at least 10 minutes. In certain embodiments, the concentration is 5 times its CMC and the time is at least 30 minutes.

[0022] Any degree of viral inactivation using the methods disclosed herein is desirable, however, it is preferred to achieve the degree of viral inactivation necessary to meet biopharmaceutical safety guidelines or regulations established by relevant regulatory agencies.

[0023] To determine the extent or effectiveness of agents, such as the detergents described herein, to inactivate viruses, one method is to monitor their effect on cytopathic effect (CPE). Cytopathic effect includes structural changes in host cells due to viral infection, such as lysis of the host cells, or cell death without lysis due to changes in the virus that affect host cell division. If no such effects are detectable in host cells after exposure to the agent, the virus is considered inactive. This is known as the TCID, which is used to determine the infectious titer of a virus that can cause cytopathic effects in cell culture for a reasonable period of time, e.g., 5-20 days, while the cells in culture remain viable. 50 This may be measured using an assay. If active virus is present, the cytopathogenic effect on the cells can be observed using a microscope. Infected cells are deformed and appear different from uninfected cells. The results are analyzed with the Spearman-Karber equation, which provides the virus titer for each sample, which can then be used to calculate the total log reduction of virus. Inactivation is considered complete when no virus is detected at the limits of the detection method being used (usually around 4 log10).

[0024] As described herein, surfactants with a wide range of structural variability, hydrophobicity, and charge were evaluated for their effectiveness in inactivating enveloped viruses. Surfactants with potent virus inactivation capabilities were identified. These include ANAPOE-C12E9; ANAPOE-C12E8; Alfonic TDA-6 ethoxylate (TDA-6); Alfonic TDA-9 ethoxylate (TDA-9); N-heptyl-β-D-thioglucopyranoside; n-octyl-β-D-thiomaltopyranoside; Sucrose monolaurate; n-decanoylsucrose; n-octyl-β-D-thioglucoside; CYMAL®-3; n-tridecyl-β-D-maltoside; CYMAL®-6; Hexaethylene glycol monooctyl ether; C-HEGA®-10; n-undecyl-β-D-maltoside; n-nonyl-β-D-thiomaltoside; HEGA®-9; ANAPOE-X-405; CYMAL®-5; Nonionic surfactants include C-HEGA®-11; Thesit; n-nonyl-bD-maltoside; MEGA-8; n-nonyl-bD-glucoside; HECAMEG®; HEGA®-10; n-octyl-bD-glucoside; HEGA®-8; MEGA-10; n-dodecyl-bD-maltoside; C8E5; MEGA-9; n-hexyl-bD-glucopyranoside; CYMAL®-7; CYMAL®-4; 2,6-dimethyl-4-heptyl-bD-malto-pyranoside; n-decyl-bD-maltoside; and C8E4. Zwitterionic detergents include NDSB-195; FOS-MEA®-10; NDSB-201; CHAPS; NDSB-211; CHAPSO; NDSB-221; Fos-Choline®-10; NDSB-256; ZWITTERGENT® 3-08; Tripao, and DDMAB.Synthetic lipid surfactants include LysoFos™ Choline 12, LysoFos™ Choline 14, Alfonic TDA-6 Ethoxylate (Novel-TDA6, IsoC13E6), and Alfonic TDA-9 Ethoxylate (Novel-TDA9, Iso-C13E9).

[0025] Among the detergents that tested positive for virus inactivation against several enveloped viruses, several are currently considered non-hazardous according to European Union Directive 67 / 548 / EC on Chemical Safety: ANAPOE-C12E9 CAS No.: 3055-99-0; ANAPOE-C12E8 CAS No.: 3055-98-9; Alfonic TDA-6 ethoxylate (TDA-6) CAS No.: 9043-30-5; Alfonic TDA-9 ethoxylate (TDA-9) CAS No.: 9043-30-5; N-heptyl-β-D-thioglucopyranoside CAS No.: 85618-20-8; CYMAL®-3 CAS No.: 181135-58-0; CYMAL®-4 CAS No.: 181135-57-9; CYMAL®-5 CAS No.: 250692-65-0; CYMAL®-6 CAS No.: 228579-27-9; CYMAL®-7 CAS No.: 349477-49-2; Fos Choline®-10 CAS No.: 70504-28-8; n-Hexyl-bD-glucopyranoside CAS No.: 59080-45-4; n-Nonyl-bD-glucoside CAS No.: 69984-73-2; n-Octyl-β-D-thiomaltopyranoside CAS No.: 148616-91-5; n-Nonyl-bD-thiomaltoside CAS No.: 148565-55-3; n-Dodecyl-bD-maltoside CAS No.: 69227-93-6; n-decyl-bD-maltoside CAS No.: 82494-09-5; n-undecyl-bD-maltoside CAS No.: 253678-67-0; n-nonyl-bD-thiomaltoside CAS No.: 106402-05-5; n-tridecyl-β-D-maltoside CAS No.: 93911-12-7; and n-octyl-bD-glucoside CAS No.: 29836-26-8.

[0026] These four groups of "eco-friendly" surfactants were selected based on similar molecular structures and labeled "CYMAL," "Fos-Choline," "Anapoe," and "Thioglucosides" (Figure 1). The surfactants in each of these groups were further analyzed for virus inactivation based on parameters such as temperature, time, concentration, product modality, virus type, and toxicity and potential clearance of the surfactant.

[0027] Based on a large set of surfactant virus inactivation data, trends affecting inactivation, including alkyl and ethoxylate chain length, were identified. Surprisingly, alkyl chain length affected virus inactivation activity. CYMAL® with alkyl chains of one or two carbons had no effect on virus inactivation, while CYMAL® with alkyl chains of three to seven carbons inactivated viruses. Similar observations were made with Fos-Cholines®. Fos-Cholines® with eight or nine carbon linker chains were found to have no effect on virus inactivation, while Fos-Cholines® with a ten carbon linker chain inactivated viruses. Anapoe surfactants are composed of alkyl and ethoxylate chains, and the number of alkyl carbons (C) and ethoxylate groups (E) is indicated in their names; for example, Anapoe C12E9 has 12 alkyl carbons and 9 ethoxylate groups. Anapoe C12E9 and Anapoe C12E8, Alfonic TDA-6 ethoxylate (TDA-6), and Alfonic TDA-9 ethoxylate (TDA-9) showed potent inactivation of the virus, but Anapoe C10E9, Anapoe C10E6, Anapoe C12E10, and C13E8 did not.

[0028] Anapoe C12E8, Anapoe C12E9, Alfonic TDA-6 ethoxylate (TDA-6), and Alfonic TDA-9 ethoxylate (TDA-9), as well as N-heptyl-β-D-thioglucopyranoside, were particularly potent at inactivating viruses in the presence of several important therapeutic modalities: monoclonal antibodies, bispecific T cell engagers (BiTEs®), and fusion proteins. These detergents demonstrated complete virus inactivation within 30 seconds to 30 minutes at temperatures as low as 5°C to 15°C.

[0029] Detergent viral inactivation can occur at one or more steps in the downstream biomanufacturing process: after harvest clarification and before the affinity chromatography step; after the affinity chromatography step and before the depth filtration and / or polish chromatography step; during the polish chromatography step; before the viral filtration and / or UF / DF step.

[0030] The terms "polynucleotide" or "nucleic acid molecule" are used interchangeably throughout and include both single-stranded and double-stranded nucleic acids, including genomic DNA, RNA, mRNA, cDNA, or any combination thereof of synthetic origin, or any combination thereof not normally associated with a sequence found in nature. The term "isolated polynucleotide" or "isolated nucleic acid molecule" specifically refers to a sequence of synthetic origin or a sequence not normally found in nature. An isolated nucleic acid molecule comprising a particular sequence may, in addition to a sequence expressing a protein of interest, also include sequences encoding up to 10 or even up to 20 other proteins or portions thereof, or may include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences. The nucleotides comprising a nucleic acid molecule may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0031] As used herein, the term "isolated" means (i) free from at least some other proteins or polynucleotides with which it is normally found; (ii) substantially free from other proteins or polynucleotides from the same source, e.g., the same species; (iii) separated from at least about 50 percent of the polypeptides, polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (iv) operably associated (by covalent or non-covalent interactions) with polypeptides or polynucleotides not naturally associated with it; or (v) not occurring in nature.

[0032] The terms "polypeptide" and "protein" are used interchangeably throughout and refer to molecules comprising two or more amino acid residues linked together by peptide bonds. Polypeptides and proteins also include polymers having one or more deletions, insertions, and / or substitutions of amino acid residues from the native sequence, i.e., polypeptides or proteins produced by naturally occurring non-recombinant cells, or molecules produced by genetically engineered or recombinant cells and having one or more deletions, insertions, and / or substitutions of amino acid residues from the amino acid sequence of the native protein. Polypeptides and proteins also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. Polypeptides and proteins also include modifications, including, but not limited to, glycosylation, lipid conjugation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The terms "isolated protein," "isolated recombinant protein," or "purified recombinant protein" may be used interchangeably and may refer to a polypeptide or protein of interest that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. In particular, drug substances and pharmaceutical products made from recombinant proteins of interest processed using the invention described herein may be referred to as "recombinant protein pharmaceuticals," "recombinant biological therapeutics."

[0033] Polypeptides and proteins may be of scientific or commercial interest, including protein therapeutics. Proteins of interest include, among others, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Proteins of interest can be produced in cell lines using the methods described herein and may be referred to interchangeably as "recombinant proteins," "recombinant proteins of interest," or "recombinant protein therapeutics." The expressed protein may be produced intracellularly or secreted into the culture medium from which it can be recovered and / or collected. Proteins of interest may include, for example, proteins that exert a therapeutic effect by binding to targets, particularly those listed below, including targets derived from, related to, and modifications thereof.

[0034] The protein of interest may comprise an "antigen-binding protein." An antigen-binding protein refers to a protein or polypeptide that contains an antigen-binding region or portion that has a strong affinity for another molecule (the antigen) to which it binds. Antigen-binding proteins include, but are not limited to, antibodies, peptibodies, antibody fragments, antibody derivatives, antibody mimetics, fusion proteins (including single-chain variable fragments (scFvs) and double-chain (bivalent) scFvs), variants, xMAbs, bispecific T-cell engagers (BiTEs®), as well as chimeric antigen receptors (CARs or CAR-Ts) and T-cell receptors (TCRs).

[0035] An scFv is a single-chain antibody fragment that contains the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Eshhar, et al., Cancer Immunol. Immunotherapy (1997) 45:131-136. An scFv retains the ability of the parent antibody to specifically interact with a target antigen.

[0036] The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or antigen-binding regions thereof that compete with the intact antibody for specific binding. Unless otherwise specified, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, hetero-IgG, bispecific, and oligomers or antigen-binding fragments thereof. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Also included are Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single-chain antibody molecules, single-domain V H Also included are proteins having antigen-binding fragments or regions, such as H, complementarity-determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide.

[0037] Also included are other antigen binding proteins, such as human, humanized, and human and humanized antibodies, that do not produce a significant adverse immune response when administered to humans.

[0038] Also included are modified proteins, such as proteins that are chemically modified non-covalently, covalently, or both covalently and non-covalently, and also proteins that further include one or more post-translational modifications, which may be made by cell engineering systems, or modifications introduced ex vivo by enzymatic and / or chemical methods, or otherwise introduced.

[0039] Proteins of interest may also include recombinant fusion proteins containing multimerization domains such as, for example, leucine zippers, coiled coils, Fc portions of immunoglobulins, etc. Also included are proteins containing all or part of the amino acid sequence of differentiation antigens (called CD proteins) or their ligands, or proteins substantially similar to any of these.

[0040] In some embodiments, the protein of interest may comprise a colony-stimulating factor, such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), and Binocrit® (epoetin Also included are erythropoiesis stimulating agents (ESAs) such as epoetin alfa, epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and molecules or variants thereof or analogs thereof and biosimilars of any of the foregoing.

[0041] In some embodiments, the protein of interest may include proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factors (CSFs), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0042] In some embodiments, the protein of interest may bind to one or more of the following, alone or in any combination: CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, CD174; HER receptor family proteins, including HER2, HER3, HER4; EGF receptor EGFRvIII; cell adhesion molecules, such as LFA-1; Mol , p150, 95, VLA-4, ICAM-1, VCAM, αv / β3 integrin, growth factors including, but not limited to, vascular endothelial growth factor ("VEGF"), VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors including, for example, aFGF and bFGF, epidermal growth factor receptors (EGFRs), and the like. Transforming growth factors (TGFs) including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, among others; insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors; insulin and insulin-related proteins, including but not limited to insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins, among others. other blood and serum proteins, including, but not limited to, coagulation and coagulation-related proteins such as Factor VIII, tissue factor, von Willebrand factor, plasminogen activators such as protein C, alpha-1-antitrypsin, urokinase, and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, thrombopoietin receptors, colony-stimulating factors (CSFs), including M-CSF, GM-CSF, and G-CSF, among others, albumin, IgE, and blood group antigens, e.g., flk2 / flt3 receptor;(x) receptors and receptor-associated proteins, including obesity (OB) receptor, growth hormone receptor, and T-cell receptor; (x) neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xi) relaxin A chain, relaxin B chain, and prorelaxin, interferons, including, for example, interferon-α, -β, and -γ, interleukins (IL), for example, IL-1 to IL-10, IL-12, IL-15, IL-17, IL- (xiv) viral antigens including, but not limited to, AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated eosinophilic peptide), and the like. on activation (normal T-cell expressed and secreted), mouse gonadotropin-related peptide, Dnase, FR-α, inhibin, and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay-accelerating factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1,Programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38 conjugate], Legionella pneumophila (Ly), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein Iib / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0043] In another embodiment, the protein of interest is selected from the group consisting of abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosuzumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab ipilimumab, ixekizumab, lerdelimumab, rumiliximab, mapatuzumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembrolizumab, penicillin This includes rituximab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargramostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and biosimilars of any of the foregoing.

[0044] Proteins of interest according to the present invention encompass all of the foregoing, and further include antibodies comprising one, two, three, four, five, or six of the complementarity-determining regions (CDRs) of any of the above antibodies. Also included are variants comprising regions that are 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, particularly 97% or more, more particularly 98% or more, and even more particularly 99% or more amino acid sequence identical to the reference amino acid sequence of the protein of interest. Identity in this regard can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those that implement the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of sequence searching and alignment. Other algorithms may also be employed, particularly where speed is an important consideration. Commonly used programs for alignment and homology matching of DNA, RNA, and polypeptides that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter of which is an implementation of the Smith-Waterman algorithm for running on massively parallel processors produced by MasPar.

[0045] Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes, or expression cassettes containing at least one of the above-described nucleic acid molecules, as well as host cells containing such expression systems or constructs. As used herein, "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, complexed DNA, etc.) suitable for use in transferring and / or transporting protein-coding information into a host cell and / or to a specific location and / or compartment within a host cell. Vectors can include viral and non-viral vectors, as well as non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors and cloning vectors. A vector can be introduced into a host cell to enable replication, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors can contain sequence components that generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be selected as needed by those skilled in the art.

[0046] A "cell" or "cells" includes any prokaryotic or eukaryotic cell. Cells may exist in isolation or as part of a higher-order structure such as a tissue or organ, either ex vivo, in vitro, or in vivo. Cells include "host cells," also referred to as "cell lines," which have been genetically engineered to express a polypeptide of commercial or scientific interest. Host cells are generally derived from lines arising from a primary culture that can be maintained in culture indefinitely. Genetic engineering of host cells involves transfecting, transforming, or transducting the cells with a recombinant polynucleotide molecule and / or otherwise modifying them (e.g., by homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) so that the host cell expresses a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, various techniques are described in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1990, and updated quarterly), Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989), and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69.

[0047] Host cells can be any prokaryotic (e.g., E. coli) or eukaryotic cell, such as yeast cells, insect cells, or animal cells (e.g., CHO cells). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

[0048] In one embodiment, the cell is a host cell. When cultured under appropriate conditions, the host cell expresses the protein of interest, which can then be recovered from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications desired or necessary for activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

[0049] "Culturing" or "culturing" refers to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. Cell culture medium and tissue culture medium are used interchangeably and refer to a medium suitable for the growth of host cells in in vitro cell culture. Typically, cell culture media contain buffers, salts, energy sources, amino acids, vitamins, and minor components. Any medium capable of supporting the growth of suitable host cells in culture can be used. Cell culture media, which can be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells, are commercially available and include RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as the EX-CELL™ 300 series, available from, among others, American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell culture media can be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell cultures can also be enriched by the addition of nutrients, sometimes at higher than normally recommended concentrations.

[0050] Various media formulations can be used during the course of the culture, for example, to facilitate the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase) and / or to optimize conditions during cell culture (e.g., concentrated media provided during perfusion culture). Growth media formulations can be used to promote cell growth and minimize protein expression. Production media formulations can be used to promote production of the protein of interest and cell maintenance and minimize the growth of new cells. A feed medium, which is a medium containing more concentrated components, typically nutrients and amino acids, that are consumed during the production phase of the cell culture, can be used to replenish and maintain active cultures, particularly cultures operated in fed-batch, semi-perfusion, or perfusion modes. Such concentrated feed medium may contain most of the components of the cell culture medium, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or even about 1000x their normal amounts.

[0051] The growth step can occur at a higher temperature than the production step. For example, the growth step can occur at a first temperature of about 35°C to about 38°C, and the production step can occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. Additionally, chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, and / or after the temperature shift. If the inducer is added after the temperature shift, it can be added between 1 hour and 5 days after the temperature shift, optionally between 1 and 2 days after the temperature shift.

[0052] Host cells can be cultured in suspension or in attached form, attached to a solid substrate. Cell cultures can be established in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers.

[0053] Cell cultures can be operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes. Mammalian cells, such as CHO cells, can be cultured on a small scale in bioreactors, from less than 100 ml to less than 1000 ml. Alternatively, large-scale bioreactors containing from 1000 ml to over 20,000 liters of medium can be used. Large-scale cell cultures, such as those for clinical and / or commercial-scale biomanufacturing of protein therapeutics, can be maintained for weeks and even months while the cells produce the desired protein.

[0054] Provided herein is a method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express the recombinant protein and culturing the cells to express the recombinant protein of interest; harvesting a cell culture fluid containing the recombinant protein of interest; and treating the harvested fluid containing the recombinant protein of interest with any of the following: CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS7 and exposing the virus to a detergent having a CAS Registry Number of CAS 0504-28-8, CAS 59080-45-4, CAS 69984-73-2, CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7 at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; treating the virus-inactivated liquid containing the recombinant protein of interest through at least two additional unit operations; and obtaining an isolated and purified recombinant protein of interest.

[0055] Provided herein is a method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express the recombinant protein and culturing the cells to express the recombinant protein of interest; recovering a cell culture solution containing the recombinant protein of interest; and processing a liquid containing the recombinant protein of interest through at least two unit operations, wherein during at least one unit operation, the liquid containing the recombinant protein of interest is treated with one of CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9. , CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS70504-28-8, CAS59080-45-4, CAS69984-73-2, CAS148616-91-5, CAS148565-55-3, CAS69227-93-6, CAS82494-09-5, CAS253678-67-0, CAS106402-05-5, or CAS93911-12-7, at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; and obtaining an isolated and purified recombinant protein of interest.

[0056] The cell culture solution containing the expressed recombinant protein can then be harvested from the cell culture in the bioreactor.Methods for harvesting proteins expressed from suspension cells are known in the art, including, but not limited to, accelerated sedimentation such as acid precipitation, flocculation, gravity separation, centrifugation, sonication, and filtration, including membrane filtration using ultrafiltration membranes, microfiltration membranes, tangential flow filtration membranes, depth filtration membranes, and sand filtration membranes (alluvial filtration filters).Recombinant proteins expressed by prokaryotes can also be recovered from inclusion bodies in the cytoplasm by redox folding processes known in the art.

[0057] One or more unit operations can then be used to purify or partially purify the recombinant protein of interest in the clarified, recovered cell culture fluid by removing any impurities, such as residual cell culture medium, cell extracts, undesirable components, host cell proteins, improperly expressed proteins, contaminants, microorganisms such as bacteria and viruses, aggregates, etc. The term "unit operation" refers to a functional step performed in a process for purifying a recombinant protein from, for example, a liquid culture medium. For example, a unit of operation can involve filtration (e.g., removal of contaminating bacteria, yeast, viruses, mycobacteria, impurities, and / or particulate matter from the liquid containing the recombinant protein), capture, epitope tag removal, purification, collection, retention, storage, polishing, recovery, viral inactivation, including detergent viral inactivation, viral filtration, adjustment of the ionic concentration and / or pH of the liquid containing the recombinant protein, and removal of unwanted salts. The invention described herein can be used in one or more steps of downstream processes, from drug substance recovery to formulation.

[0058] For example, unit operations can include, but are not limited to, steps such as recovery, capture, purification, polishing, viral inactivation, viral filtration, concentration of a recombinant protein of interest, and / or adjusting concentrations and formulations containing the recombinant protein of interest. Unit operations can also include steps of pooling, holding, and / or storing liquids, such as a capture pool after recovery, chromatography, or filtration, as well as liquids in holding or storage vessels, such as after recovery. A single unit operation can be designed to accomplish multiple objectives in the same operation, such as recovery and viral inactivation, or capture and viral inactivation.

[0059] Capture unit operations include capture chromatography, such as affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like, which utilize resins and / or membranes containing agents that bind to the recombinant protein of interest. Such materials are known in the art and commercially available. For example, affinity chromatography can include, for example, substrate-binding capture mechanisms, antibody or antibody fragment-binding capture mechanisms, aptamer-binding capture mechanisms, and cofactor-binding capture mechanisms. Exemplary affinity chromatography methods include Protein A, Protein G, Protein A / G, or Protein L. The recombinant protein of interest can be tagged with a polyhistidine tag and then purified from IMAC using imidazole or an epitope (such as FLAG®), followed by purification using a specific antibody directed against such epitope.

[0060] The one or more capture unit operations may include viral inactivation and viral filtration. In addition to the viral inactivation methods of the invention provided herein, other methods for viral inactivation may also be used, such as heat inactivation / pasteurization, pH inactivation, UV and gamma irradiation, use of high intensity broad spectrum white light, addition of chemical inactivators such as β-propiolactone, and the like.

[0061] Inactivation of viruses known or suspected to be contained in a liquid can be performed at any time. During the manufacture of a biological drug substance, viral inactivation in a liquid containing a recombinant protein of interest can be performed in one or more separate viral inactivation unit operations; as part of a recovery unit operation; before, as part of, or after one or more capture chromatography unit operations; before, as part of, or after one or more affinity chromatography unit operations; before, as part of, or after one or more polish chromatography unit operations; before, as part of, or after one or more ion exchange chromatography, hydrophobic interaction chromatography, mixed modal or multimodal chromatography, and / or hydroxyapatite chromatography unit operations; before, as part of, or after one or more viral filtration unit operations; and / or before or after an ultrafiltration / diafiltration unit operation. In one embodiment, viral inactivation occurs after recovery. In one embodiment, viral inactivation occurs following a recovery unit operation including microfiltration. In one embodiment, viral inactivation occurs before a capture chromatography unit operation. In one embodiment, viral inactivation occurs before a Protein A affinity chromatography step. Following viral inactivation, filtration (such as depth filtration) or chromatography (such as Protein A chromatography) is performed to remove inactivated viruses, inactivating agents such as detergents and surfactants, turbidity, and / or precipitates.

[0062] Virus filtration can be performed using commercially available microfiltration or nanofiltration membranes such as those from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0063] The term "polishing" is used herein to refer to one or more chromatographic processing steps performed to remove remaining contaminants and impurities, such as DNA, host cell proteins, product-specific impurities, variant products and aggregates, and virus adsorbates, from a liquid containing recombinant protein close to the final desired purity. For example, polishing can be performed in a bind-and-elute mode by passing the liquid containing the recombinant protein through a chromatography column or membrane absorber that selectively binds either the target recombinant protein or contaminants or impurities present in the liquid containing the recombinant protein. In such an example, the eluate / filtrate from the chromatography column or membrane absorber contains the recombinant protein.

[0064] Polish chromatography unit operations use chromatography resins and / or membranes containing agents that can be used in either flow-through mode (where the protein of interest is contained in the eluent that passes through the chromatography medium, while contaminants and impurities are bound to the chromatography medium) or "bind and elute mode" (where the protein of interest binds to the chromatography medium and elutes from the chromatography medium after the contaminants and impurities have passed through or been washed away from the chromatography medium). Examples of such chromatography methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and gel filtration.

[0065] While the terminology used in this application is standard in the art, definitions of certain terms are provided herein to ensure clarity and clarity of the meaning of the claims. Units, prefixes, and symbols may be denoted in the form accepted by the SI. Numerical ranges set forth herein are inclusive of the numbers defining the range and include and support each integer within the defined range. Unless otherwise specified, the methods and techniques described herein are generally carried out according to conventional methods well known in the art, and such methods and techniques are described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and journal articles, are expressly incorporated herein by reference. Anything described in one embodiment of the invention can be combined with other embodiments of the invention.

[0066] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. [Example]

[0067] Example 1 Virus stock Viral stocks of xMuLV, PRV, and MMV were produced as previously described in Romanowski et al., Bioprocess Int 2008, 6(2), 44-52. To determine cytopathic effect (CPE), virus-compatible indicator cell lines were used. For xMuLV, the cell line PG4 (a feline astrocyte cell line) was used. CPE was measured 7 days after inoculation. The cell line 324K was used to measure CPE for MMV 10 days after inoculation, and for PRV, the Vero cell line was used in a 3-day assay.

[0068] Surfactant Screening An initial screen of 96 detergents was performed using xMuLV, a murine retrovirus commonly used in viral clearance studies. Screening was performed in 96-deep-well plates from Research (HR2-406), Aliso Viejo, CA. The detergents tested had a variety of amphoteric properties, including nonionic, zwitterionic, and ionic properties. All detergents evaluated in the screening were at 10x their critical micelle concentration (CMC) values ​​or 10% w / v as indicated in the Hampton kit. Duplicate assays of each detergent were evaluated after a 5% virus spike for 30 minutes at 15°C. A control with a 5% v / v water spike was also run simultaneously to demonstrate the effectiveness of dilution in terminating detergent inactivation. Each replicate and control run was diluted 1:300 in medium to reduce cytotoxicity and interference and incubated with 25% confluent adherent PG4 cells. After the incubation period, wells were analyzed for any CPE of the inoculated PG4 cells.

[0069] The TCID50 assay is performed at 10 0 -10 -7Serial dilutions of 1:10 were performed in eight replicates from each sample. A retention control solution of 10% xMuLV spiked into the protein was retained during the assay without detergent, as was a negative control of seed medium. All samples were titrated on 25% confluent PG4 cells and incubated at 37°C for 7 days. Plates were read for CPE on day 7 and evaluated for total virus titer and log reduction value (LRV) using the Spearman-Karber equation as previously described (Romanowski et al., supra).

[0070] Expanded Virus Research Nineteen of the detergents that inactivated xMuLV are now considered non-hazardous according to European Union Directive 67 / 548 / EC on the safety of chemicals. These detergents were further tested against two more viruses, another enveloped virus, PRV, and the non-enveloped virus, MMV, at five times their CMC values. Both viruses were tested at 5% v / v, otherwise using the same conditions and assays as described in the initial detergent screen above.

[0071] Concentration studies Anapoe C12E8 (CAS No. 3055-98-9), Anapoe C12E9 (CAS No. 3055-99-90), CYMAL®-5 (CAS No. 250692-65-0), Fos-Choline®-10 (CAS No. 70504-28-8) (supplied by Anatrace, Maumee, OH), and N-heptyl-β-D-thioglucopyranoside (CAS No. 85618-20-8) (Sigma-Aldrich, St. Louis, MO) were evaluated for the lowest concentration that still inactivated the virus. Concentrations of 2.5, 5, and 7.5 times the critical micelle concentration were evaluated against xMuLV at 15°C, and the detergent and virus were incubated for 30 minutes as described above.

[0072] Temperature dependence studies Anapoe C12E8, Anapoe C12E9, CYMAL®-5, Fos-Choline®-10, and N-heptyl-β-D-thioglucopyranoside were evaluated against xMuLV and in a substrate (host cell culture medium) containing three different protein modalities: a monoclonal antibody (mAb #1), a bispecific T cell engager (BiTE® #1), and an XmAb fusion protein (fusion protein #1) at temperatures ranging from 5°C to 22°C. Time points were taken at 30 seconds, 10 minutes, and 30 minutes, and diluted 1:300. This was the dilution at which the buffer matrix caused no toxicity or interference using the Spearman-Carber method, as previously described (Romanowski et al., supra). Triton X100 control (Millipore Sigma, Temacula, CA) was run in duplicate at 18°C.

[0073] Kinetic studies of surfactants Anapoe C12E8, Anapoe C12E9, CYMAL®-5, Fos-Choline®-10, and N-heptyl-β-D-thioglucopyranoside were evaluated against xMuLV at three time points: 30 seconds, 10 minutes, and 30 minutes, and in a substrate (host cell culture medium) containing three different protein modalities: a monoclonal antibody (mAb #1), a bispecific T cell engager (BiTE® #1), and an XmAb fusion protein (fusion protein #1). The 30 seconds, 10 minutes, and 30 minutes time points were taken and diluted 1:300, a dilution that did not cause toxicity or interference from the buffer matrix using the Spearman-Carver method.

[0074] Surfactant clearance The clearance of the detergent, Anapoe C12E9, was evaluated during Protein A chromatography of a sample containing a monoclonal antibody. The protein sample (host cell culture fluid containing the monoclonal antibody) was spiked with 0.03% detergent and passed through a Protein A column. The elution peak and clearance of the detergent-spiked buffer were evaluated on a Waters BioResolve™ Polyphenol (Milford, MA) 450 Å, 2.1 x 50 mm column. The flow rate was 0.5 mL / min at 65°C. Samples were analyzed using a 5-minute gradient of 0.1% TFA / 0.1% formic acid in water as stationary phase A and 90% n-propanol as mobile phase B.

[0075] Extended range research The scope of the study was expanded to evaluate two additional alkyl ethoxylate derivatives. The alkyl ethoxylate surfactants IsoC13E6 (CMC: 29.91 mg / L) and Iso-C13E9 (CMC: 57.97 mg / L) (also available as Alfonic TDA-6 ethoxylate, Novel-TDA6, and Alfonic TDA-9 ethoxylate, Novel-TDA9, (Avantor, Radnor, PA; Sasol, Johannesburg, South Africa), CAS 9043-30-5)) were tested at three concentrations: 2.5x, 5x, and 7.5x their CMC values. Samples were incubated with host cell culture media containing either mAb #1 or bispecific T cell engager (BiTE® #1) spiked with 5% XMuLV at 5°C. Samples were diluted 1:300 to reduce cytotoxicity and interference at three time points: 30 seconds, 10 minutes, and 30 minutes.

[0076] The TCID50 assay is performed at 10 0 -10 -7Serial dilutions of 1:10 were performed in eight replicates from each sample. A retention control solution of 5% XMuLV spiked into the protein was retained during the assay without detergent, as was a negative control of seed medium. All samples were titrated on 25% confluent PG4 cells and incubated at 37°C for 7 days. Plates were read for CPE on day 7 and evaluated for total virus titer and log reduction value (LRV) using the Spearman-Karber equation as previously described (Romanowski et al., supra).

[0077] Results and Discussion Surfactant Screening An initial screening was performed against the enveloped model virus xMuLV using a selection of 96 commercially available detergents with a variety of ionic, hydrophobic, and structural properties. As shown in Table 1, 51 of these detergents did not result in CPE in either the replication or detergent controls. This group included nonionic, zwitterionic, and synthetic lipid detergents, but none of the ionic detergents tested demonstrated virus inactivation.

[0078] [Table 1-1] [Table 1-2]

[0079] Of the 51 surfactants found to be positive for virus inactivation, several were structurally similar, differing only in the length of the alkyl chain linker. By evaluating the results based on surfactant structure, trends within structural classes were observed. Nineteen of the surfactants identified are currently considered non-hazardous according to European Union Directive 67 / 548 / EC on the safety of chemicals (Table 2). These "eco-friendly" surfactants were divided into four classes based on their molecular structure and labeled "CYMAL," "Fos-Choline," "Anapoe," and "Thioglucosides" (Figure 1).

[0080] [Table 2-1] [Table 2-2]

[0081] CYMAL® surfactants contain maltoside sugars linked to cyclohexane by alkyl chains of various lengths. CYMAL® 1-7 were among 96 surfactants in the initial screening. Surprisingly, CYMAL® surfactants with alkyl chains of one or two carbons had no effect on the inactivation of xMuLV virus, while CYMAL® surfactants with alkyl chains of three to seven carbons inactivated the virus. Similar surprising results related to alkyl chain length appeared to affect Fos-Cholines and Anapoe derivatives. Fos-Cholines with eight or nine carbon linker chains were found to have no effect on virus inactivation, while Fos-Cholines with a ten carbon linker chain inactivated the virus. Anapoe surfactants are composed of alkyl and ethoxylate chains, and their names are denoted by the number of alkyl carbons (C) and ethoxylate groups (E). For example, Anapoe C12E9 has 12 alkyl carbons and 9 ethoxylate groups. Anapoe C12E9 and Anapoe C12E8 were found to potently inactivate viruses, whereas Anapoe C10E9, Anapoe C10E6, Anapoe C12E10, and Anapoe C13E8 did not (Table 3). The alkyl chain length contributes to the overall hydrophobicity of the molecule, which may ultimately affect the partitioning of the viral envelope. A specific range of lipophilicity may be required to induce disruption of the lipid envelope layer.

[0082] [Table 3]

[0083] Expanded viral research scope for virus inactivation Nineteen "environmentally friendly" detergents were further tested on a different enveloped virus, PRV, and a non-enveloped virus, minute virus of mice (MMV). All 19 detergents inactivated PRV as well as xMuLV, but had no effect on MMV. This indicates that detergents affect enveloped but not non-enveloped viruses. Triton X-100 also only affected enveloped viruses, indicating that these detergents may disrupt lipid envelopes in a similar manner.

[0084] concentration study To determine the minimum concentrations required for inactivation of xMuLV, CYMAL®-5, Anapoe C12E8, Anapoe C12E9, FosCholine®-10, and N-heptyl-β-D-thioglucopyranoside were tested in duplicate against xMuLV, PRV, and MMV at 2.5x, 5x, and 7.5x their CMC values. For all viruses tested, a CMC of 2.5x demonstrated cytopathic effect (CPE); therefore, subsequent studies were evaluated at 5x the detergent CMC values.

[0085] Temperature dependence studies Anapoe C12E8, Anapoe C12E9, N-heptyl-β-D-thioglucopyranoside, CYMAL®-5, and Fos-Choline®-10 were analyzed at various temperatures in the presence of three protein therapeutic modalities: a monoclonal antibody (mAb), a bispecific T-cell engager (BiTE®), and a fusion protein (XmAb) (Table 2). Both Anapoe detergent and N-heptyl-β-D-thioglucopyranoside showed complete inactivation in 30 minutes at both 5°C and 15°C, which represent the "worst-case scenario" temperatures of a manufacturing environment. These three detergents have LRV values ​​comparable to Triton X-100 for both BiTE® and mAb modalities and are even superior for fusion protein clearance.

[0086] The FosCholine-10 and CYMAL®-5 detergents did not produce significant inactivation of xMuLV at any temperature tested. Because viral inactivation by these detergents was observed during initial detergent screening and other experiments, there may be a negative interaction with the sample matrix in the absence of product. At a process temperature of 15°C, FosCholine®-10 and CYMAL®-5 had average LRVs of 3.8 and 0.39, respectively. To analyze the effect of temperature on the detergents, CYMAL®-5 was tested at a higher temperature (22°C), which resulted in an approximately 1-log increase in LRV. Even with this temperature increase, the LRV was still low.

[0087] [Table 4]

[0088] Kinetic studies of surfactants To assess the rate at which viral inactivation occurred, three time points were tested: 30 seconds, 10 minutes, and 30 minutes. Complete inactivation of xMuLV was observed within 30 seconds for Anapoe C12E8 and N-heptyl-β-D-thioglucopyranoside for all three treatment modalities tested. Anapoe C12E9 demonstrated complete inactivation of mAb #1 within 30 seconds and complete inactivation of fusion protein #1 and BiTE® #1 within 10 minutes (see Table 5). Anapoe C12E9 demonstrated complete clearance within 10 minutes for all three treatment modalities tested. The CMC value of Anapoe C12E9 was half that of Anapoe C12E8, meaning significantly less surfactant was required to achieve the same LRV. This characteristic is highly beneficial for commercial scale-up.

[0089] Neither Fos-Choline®-10 nor CYMAL®-5 produced significant clearance within 30 minutes.

[0090] [Table 5]

[0091] Anapoe C12E8 Clearance To ensure complete removal of the detergent after the virus inactivation process, the host cell culture medium containing the mAb was spiked with 0.03% Anapoe C12E8 detergent and affinity purified using Protein A chromatography. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the amount of detergent remaining after the chromatography run. The results show that the detergent was removed during the Protein A chromatography step (Figure 2).

[0092] Expanded Range of Alkyl Ethoxylate Surfactants To further understand the surfactant specificity required for potent viral clearance, two additional alkyl ethoxylates incorporating branched isopropyl groups were evaluated. Both surfactants also potently cleared xMuLV at concentrations five times or greater than the CMC value. High LRVs were observed with two therapeutic modalities, mAb#1 and BiTE®#1 (Table 6). These results demonstrate the potent viral inactivation capabilities of a broad range of Anapoe derivatives.

[0093] [Table 6]

[0094] Example 2 Turbidity Three surfactants were further tested to determine whether they adversely affected the turbidity, solubility, and stability of four different protein modalities as measured by high molecular weight species (HMW). Additionally, product quality was measured by size-exclusion chromatography (SEC-HPLC) for HMW and reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) for low molecular weight species (LMW).

[0095] Anapoe C12E8 (APO128), Anapoe C12E9 (APO129), and Triton X-100 were tested using one monoclonal antibody (mAb#2), two fusion proteins (Fusion#2 and Fusion#3), one bispecific (Bispecific#1), and two bispecific T cell engagers (BiTE®#1 and BiTE®#2).

[0096] Turbidity measurements were used to determine the stability of detergent-spiked samples: an increase in sample turbidity reflects the precipitation of protein molecules.

[0097] One hundred milliliters of recovered cell culture fluid (HCCF) collected from each molecule culture was exposed to four conditions: HCCF without surfactant (control), HCCF containing APO128, HCCF containing APO129, and HCCF containing Triton X-100. The surfactants were spiked at five times the critical micelle concentration (CMC) value of the surfactant: Triton-X: 1.1 mM, APO128: 0.55 mM, APO129: 0.25 mM, TDA-9: 0.48 mM.

[0098] Two sample sets were generated for each molecule; one set was kept at room temperature for 5 days, and the other set was kept at 2-8°C for 7 days. The turbidity of each sample was measured on days 0, 1, 2, 5, and 7 using a turbidimeter (Hach Turbidimeter 2100P 46500-00, Loveland, CO).

[0099] Figure 3 shows the turbidity data for the surfactant and HCCF mixtures at room temperature and 2–8 °C at various time points. The room temperature data point on day 5 shows the greatest variation, which may be due to bacterial contamination of the samples. The data points for the APO128- and APO129-spiked samples tended to be very close to those for the Triton X-100-spiked samples or were less turbid than the controls. This indicates that none of the tested surfactants caused precipitation of the different protein modalities.

[0100] Experiment 4 Product Quality Percent high molecular weight (%HMW) and percent low molecular weight (%LMW) were used to measure the stability of detergent-spiked samples. %HMW measured protein aggregation in the sample, and %LMW measured protein clipping. These product quality attributes were measured before and after affinity column purification to determine whether the presence of detergent affected the affinity purification process or the product quality of the affinity-purified material.

[0101] Cell culture media and post-affinity chromatography samples collected from one monoclonal antibody (mAb#2), two fusion proteins (Fusion#2 and Fusion#3), one bispecific (Bispecific#1), and two bispecific T cell engagers (BiTE®#1 and BiTE®#2) were tested.

[0102] 100 mL samples of the load material before HCCF affinity chromatography and the pooled material after affinity chromatography were spiked to 5x the critical micelle concentration (CMC) with Anapoe C12E8 (APO128) (0.11 mM), Anapoe C12E9 (APO129) (0.05 mM), Alfonic TDA-9 ethoxylate (TDA-9) (0.1 mM), and Triton X-100 (0.22 mM). A sample without surfactant was used as a control. The samples were stirred with the surfactant at room temperature for approximately 1 hour. They were then either held at 2-8 °C for 7 days before submission for product quality analysis or purified on an affinity column prior to submission for product quality analysis.

[0103] Post-affinity column samples were generated by subjecting the harvested cell culture fluid to Protein A affinity chromatography using GE CaptoChelating resin (Pittsburgh, PA) for BiTE#2 and GE MabSelect Sure resin (Pittsburgh, PA) for all other protein modalities.

[0104] An additional 100 mL sample of pre-HCCF affinity chromatography load material from each protein modality was spiked with surfactant and kept at 2-8 °C for 7 days.

[0105] Product quality was assessed by determining %HMW using SEC-HPLC and %LMW using rCE-SDS.

[0106] Due to the timing of material availability, two sets of experiments were performed. The first experiment consisted of four conditions: a detergent-free control, Triton X-100, APO128, and APO129. The second experiment consisted of two conditions: a detergent-free control and TDA-9.

[0107] Figure 4 shows the stability of molecules throughout the affinity capture process, as measured by %HMW and %LMW. Data were normalized by subtracting the values ​​of a surfactant-free control. For most molecules, %HMW and %LMW remained constant before and after the affinity capture step. BiTE#2 was known to be unstable during the pooling step after affinity chromatography, resulting in greater variability in %HMW.

[0108] Figure 5 shows product quality data for BiTE#1 after affinity. BiTE#1HCCF (load material) was not submitted for product quality prior to affinity chromatography.

[0109] Figure 6 shows that the stability of the molecule, as measured by %HMW and %LMW, remains relatively constant after 7 days of incubation at 2-8°C. Data were normalized by subtracting the values ​​of a control without surfactant.

[0110] The surfactant did not significantly affect product quality during affinity chromatography. The present invention provides, for example, the following items. (Item 1) 1. A method for inactivating enveloped viruses in a fluid known to contain or suspected to contain at least one enveloped virus, comprising: Obtaining a fluid known to contain or suspected to contain at least one enveloped virus; exposing the liquid to a surfactant from Table 1 at a concentration and for a time sufficient to cause viral inactivation; A method comprising: (Item 2) The surfactant is CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS70504-28-8, C 2. The method of claim 1, wherein the compound has a CAS Registry Number of CAS 59080-45-4, CAS 69984-73-2, CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7. (Item 3) 3. The method according to item 2, wherein the surfactant is selected from CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, or CAS85618-20-8. (Item 4) 2. The method of claim 1, wherein the liquid is exposed to the surfactant for at least 30 seconds to at least 60 minutes or more. (Item 5) 5. The method of claim 4, wherein the liquid is exposed to the surfactant for at least 10 minutes. (Item 6) 5. The method of claim 4, wherein the liquid is exposed to the surfactant for at least 30 minutes. (Item 7) 5. The method of claim 4, wherein the liquid is exposed to the surfactant for at least 60 minutes or more. (Item 8) 2. The method according to item 1, wherein the concentration of the surfactant is at least 2.5 times to at least 10 times or more its critical micelle concentration (CMC). (Item 9) 9. The method of claim 8, wherein the concentration of the surfactant is at least 5 times its CMC. (Item 10) 9. The method of claim 8, wherein the concentration of the surfactant is at least 7.5 times its CMC. (Item 11) 9. The method of claim 8, wherein the concentration of the surfactant is at least 10 times its CMC. (Item 12) The exposure of the liquid to the surfactant is carried out at a temperature of at least 5°C to 22°C. The method described in item 1. (Item 13) 13. The method of claim 12, wherein the exposing of the liquid to the surfactant is at a temperature of at least 5°C. (Item 14) 13. The method of claim 12, wherein exposing the liquid to the surfactant is at a temperature of at least 15°C. (Item 15) 13. The method of claim 12, wherein the exposing of the liquid to the surfactant is at a temperature of at least 20°C. (Item 16) 2. The method of claim 1, wherein the concentration is 5 times its CMC and the time period is at least 10 minutes. (Item 17) Inactivation is 50 The method according to item 1, wherein the antibody is measured using an assay. (Item 18) 2. The method of claim 1, wherein the liquid comprises a recombinant protein of interest. (Item 19) 2. The method of claim 1, wherein the liquid is a harvested host cell culture medium. (Item 20) 2. The method of claim 1, wherein the liquid is from an effluent stream, eluate, pool, storage, or retentate from a unit operation comprising a recovery, filtration, or chromatography step. (Item 21) 21. The method of claim 20, wherein the liquid is an eluate recovered from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. (Item 22) 21. The method according to item 20, wherein the fluid is a pool containing harvested cell culture fluid, an eluate from depth filtration, an eluate from microfiltration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multimodal chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography. (Item 23) 23. The method of claim 22, wherein the affinity chromatography is Protein A, Protein G, Protein A / G, or Protein L chromatography. (Item 24) 1. A method for inactivating enveloped viruses during the purification of a recombinant protein of interest, comprising: obtaining a liquid containing said recombinant protein of interest, said liquid being known to contain or suspected to contain at least one virus; exposing the liquid to at least one surfactant at a concentration and for a time sufficient to cause inactivation of enveloped viruses in the liquid, the surfactant being selected from the group consisting of CAS 3055-99-0, CAS 3055-98-9, CAS 9043-30-5, CAS 85618-20-8, CAS 181135-58-0, CAS 181135-57-9, CAS 250692-65-0 ...0, CAS 181135-57-0, CAS 181135-57-0, CAS 181135-57-0, CAS 181135-57-0, CAS 181135-57-0 CAS registration numbers S228579-27-9, CAS349477-49-2, CAS70504-28-8, CAS59080-45-4, CAS69984-73-2, CAS148616-91-5, CAS148565-55-3, CAS69227-93-6, CAS82494-09-5, CAS253678-67-0, CAS106402-05-5, or CAS93911-12-7 and having a number; exposing the viral inactivation liquid to at least one unit operation comprising at least a filtration step or a chromatography step. (Item 25) 25. The method according to item 24, wherein the surfactant is selected from CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, and CAS85618-20-8. (Item 26) 25. The method of claim 24, wherein the concentration is 5 times its CMC and the time period is at least 10 minutes. (Item 27) 25. The method of claim 24, wherein the liquid comprises a recombinant protein of interest. (Item 28) 25. The method of claim 24, wherein the liquid is a harvested host cell culture medium. (Item 29) 25. The method of claim 24, wherein the liquid is from an effluent stream, eluate, pool, storage, or retentate from a unit operation comprising a recovery, filtration, or chromatography step. (Item 30) 25. The method of claim 24, wherein the liquid is an eluate recovered from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. (Item 31) 25. The method according to item 24, wherein the fluid is a pool containing harvested cell culture fluid, an eluate from depth filtration, an eluate from microfiltration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multimodal chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography. (Item 32) 32. The method of claim 31, wherein the affinity chromatography is Protein A, Protein G, Protein A / G, or Protein L chromatography. (Item 33) 25. The method of claim 24, wherein the chromatography is selected from affinity chromatography, Protein A chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography; hydrophobic interaction chromatography; mixed modal or multimodal chromatography, or hydroxyapatite chromatography. (Item 34) 25. The method of claim 24, wherein the fluid is a harvested host cell culture fluid and the unit operation comprises depth filtration. (Item 35) 25. The method of claim 24, wherein the liquid is a harvested host cell culture fluid and the unit operation comprises microfiltration. (Item 36) 25. The method of claim 24, wherein the fluid is a harvested host cell culture fluid and the unit operation comprises Protein A affinity chromatography. (Item 37) 25. The method of claim 24, wherein the liquid is a Protein A eluate and the unit operation comprises depth filtration. (Item 38) 25. The method of claim 24, wherein the unit operation comprises depth filtration. (Item 39) 25. The method of claim 24, wherein the unit operation comprises microfiltration. (Item 40) 1. A method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express a recombinant protein and culturing the cells to express the recombinant protein of interest; recovering the cell culture medium containing the recombinant protein of interest; The recovered liquid containing the recombinant protein of interest was purified by HPLC using CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2, CAS70504-28-8, CAS59080-45- 4, exposure to a detergent having a CAS Registry Number of CAS 69984-73-2, CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7 at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; treating the virus-inactivated liquid containing the recombinant protein of interest through at least two additional unit operations; Obtaining an isolated and purified recombinant protein of interest; A method comprising: (Item 41) Item 41. The method according to item 40, wherein the surfactant is selected from CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, and CAS85618-20-8. (Item 42) 41. The method of claim 40, wherein the concentration is 5 times its CMC and the time period is at least 10 minutes. (Item 43) 41. An isolated and purified recombinant protein of interest according to item 40. (Item 44) 41. A pharmaceutical composition comprising the isolated protein of interest according to item 40. (Item 45) 1. A method for producing an isolated and purified recombinant protein of interest, comprising: establishing a cell culture in a bioreactor using host cells that express a recombinant protein and culturing the cells to express the recombinant protein of interest; recovering the cell culture medium containing the recombinant protein of interest; treating the liquid containing the recombinant protein of interest through at least two unit operations, wherein during at least one unit operation, the liquid containing the recombinant protein of interest is treated with CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, CAS85618-20-8, CAS181135-58-0, CAS181135-57-9, CAS250692-65-0, CAS228579-27-9, CAS349477-49-2 ... with a detergent having a CAS Registry Number of CAS 70504-28-8, CAS 59080-45-4, CAS 69984-73-2, CAS 148616-91-5, CAS 148565-55-3, CAS 69227-93-6, CAS 82494-09-5, CAS 253678-67-0, CAS 106402-05-5, or CAS 93911-12-7 at a detergent concentration and for a time sufficient to cause inactivation of the enveloped virus; Obtaining an isolated and purified recombinant protein of interest; A method comprising: (Item 46) Item 46. The method according to item 45, wherein the surfactant is selected from CAS3055-99-0, CAS3055-98-9, CAS9043-30-5, and CAS85618-20-8. (Item 47) 46. ​​The method of claim 45, wherein the concentration is 5 times its CMC and the time period is at least 10 minutes. (Item 48) 46. ​​The method of claim 45, wherein at least one unit operation comprises a capture chromatography step selected from affinity chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. (Item 49) 46. ​​The method of claim 45, wherein at least one unit operation comprises a polish chromatography step selected from ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. (Item 50) 46. ​​The method of claim 45, wherein at least one unit operation comprises a step selected from viral filtration, depth filtration, and UF / DF. (Item 51) 46. ​​The method of claim 45, wherein the unit operation comprising the viral inactivation step occurs before a unit operation comprising affinity chromatography. (Item 52) 46. ​​The method of claim 45, wherein a unit operation comprising affinity chromatography is performed before the unit operation comprising the viral inactivation step. (Item 53) 46. ​​The method of claim 45, wherein the unit operation comprising the viral inactivation step occurs before a unit operation comprising depth filtration. (Item 54) 46. ​​An isolated and purified recombinant protein of interest according to item 45. (Item 55) 46. ​​A pharmaceutical composition comprising the isolated protein of interest according to item 45.

Claims

[Claim 1] The invention described in the specification.

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