METHODS FOR VIRAL INACTIVATION BY ENVIRONMENTALLY COMPATIBLE DETERGENTS

Abstract

The invention provides methods of using environmentally compatible detergents in the field of recombinant protein manufacturing for inactivating viruses in a product feedstream in the manufacturing process of proteins intended for administration to a patient, such as therapeutic or diagnostic proteins. The invention further provides methods wherein the environmentally compatible detergent used in the present invention maintains product quality of the therapeutic or diagnostic protein whilst effectively inactivating viruses.

Claims

1. A method of inactivating a virus in a feedstream comprising, adding to the feedstream an environmentally compatible detergent at a final concentration of about 0.05% w/w to about 1% w/w, wherein the environmentally compatible detergent comprises greater than about 40% undecyl alkyl glycoside.

2. The method of claim 1, wherein the detergent comprises about 40% to about 60% undecyl alkyl glycoside.

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

4. The method of claim 1, wherein the detergent comprises greater than about 50% undecyl alkyl glycoside.

5. The method of claim 4, wherein the detergent comprises less than about 10% decyl alkyl glycoside.

6. The method of claim 5, wherein the detergent is an undecyl alkyl glycoside.

7. The method of claim 5, wherein the detergent is SIMULSOL™ SL 11W.

8. The method of claim 7, wherein the detergent is added to the feedstream at a concentration of about 0.1% w/w to about 1% w/w.

9. The method of claim 8, wherein the detergent is added to the feedstream at a final concentration of about 0.3% w/w.

10. The method of claim 9, wherein during said step of adding the detergent, the feedstream is at a temperature of about 15° C. to about 30° C.

11. The method of claim 10, wherein during said step of adding the detergent, the feedstream is at a temperature of about 15° C. to about 20° C.

12. The method of claim 11, wherein during said step of adding the detergent, the feedstream is at a pH of about 5.5 to about 8.0.

13. The method of claim 12, further comprising the step of incubating the detergent in the feedstream for about 1 minute to about 180 minutes.

14. The method of claim 13, wherein following said step of incubating, the detergent has a viral log reduction factor (LRF) in the feedstream of greater than about one, or greater than about two.

15. The method of claim 6, wherein the virus is a retrovirus, a herpesvirus, a flavivirus, a poxvirus, a hepadnavirus, a hepatitis virus, an orthomyxovirus, a paramyxovirus, a rhabdovirus, or a togavirus.

16. The method of claim 14, wherein the virus is a retrovirus, a herpesvirus, a flavivirus, a poxvirus, a hepadnavirus, a hepatitis virus, an orthomyxovirus, a paramyxovirus, a rhabdovirus, or a togavirus.

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

18. The method of claim 17, wherein the feedstream is a harvested cell culture fluid, a capture pool or a recovered product pool.

19. The method of claim 18, wherein the capture poor or the recovered product pool is a protein A pool, a protein G pool or a protein L pool affinity chromatography pool.

20. The method of claim 18, wherein the capture pool or recovered product pool is a mixed mode chromatography pool.

21. The method of claim 19, further comprising the step of filtering the feedstream containing the detergent, wherein the feedstream is subjected to at least one filtration step.

22. The method of claim 21, wherein the feedstream is subjected to at least a second filtration step.

23. The method of claim 21, further comprising the step of subjecting the filtered feedstream to an affinity chromatography column.

24. The method of claim 23, wherein the affinity chromatography column is a Protein A column.

25. The method of claim 24, wherein the feedstream containing the detergent is subjected to affinity chromatography.

26. The method of claim 25, wherein the feedstream comprises a protein, wherein the protein is an antibody, an Fc fusion protein, an immunoadhesin, an enzyme, a growth factor, a receptor, a hormone, a regulatory factor, a cytokine, an antigen, a peptide, or a binding agent.

27. The method of claim 26, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment produced in a mammalian cell.

28. The method of 27, wherein the step of adding the detergent to the feedstream does not significantly alter turbidity of the feedstream-and the step of adding the detergent to the feedstream does not alter final product quality of the purified feedstream protein.

29. The method of claim 28, wherein the detergent contains a stabilizing agent.

30. The method of claim 29, wherein the stabilizing agent is monopropylene glycol.

31. A method of inactivating a virus in a feedstream comprising the steps of: adding to the feedstream SIMULSOL™ SL 11W at a concentration of about 0.1% w/w to about 0.3% w/w; incubating the SIMULSOL™ SL 11W in the feedstream for about 180 minutes, wherein the viral log reduction factor (LRF) of SIMULSOL™ SL 11W in the feedstream is greater than about 5; filtering said feedstream containing the SIMULSOL™ SL 11W; and subjecting said filtered feedstream to a Protein A affinity chromatography column; wherein, said step of adding and incubating the feedstream at a temperature of about 4° C. to about 30° C. and at a pH of about 5.5 to about 8.0.

Description

EXAMPLES

Example 1. Viral Inactivation with Non-Ionic Glycoside Detergents

Detergents and Reagents

[0053] Stock solution for the exemplified detergents listed in Table 1 (Seppic Inc. Fairfield, N.J.), and 1,2-propanediol (Sigma-Aldrich, St. Louis, Mo.) are prepared in water on a weight/volume (w/w) percentage.

TABLE-US-00001 TABLE 1 Detergents Types Physical Detergent Name Alkyl glycoside content Properties SIMULSOL ™ D-Glucopyranose, oligomeric, undecyl glycoside (40-60%), Clear SL 11W propane-1,2-diol (1-5%) Liquid SIMULSOL ™ 2 ethylhexyl mono-D-glycopyranoside, 2 ethylhexyl di-D- White Soft AS 48 glycopyranoside (40-60%) Solid SIMULSOL ™ D-Glucopyranose, oligomeric, butyl glycoside (40-60%) Clear SL 4 Liquid SIMULSOL ™ D-Glucopyranose, oligomeric, C10-16 (even numbered)-alkyl White Soft SL 10 glycosides (40-60%) Solid SIMULSOL ™ D-Glucopyranose, oligomeric, C10-16 (even numbered)-alkyl White Soft SL 26 C glycosides (40-60%) Solid SIMULSOL ™ D-Glucopyranose, oligomeric, undecyl glycoside (40-60%), Clear SL 82 D-Glucopyranose, oligomeric, C10-16 (even numbered)-alkyl Liquid glycosides (10-20%), propane-1,2-diol (1-5%) SIMULSOL ™ D-Glucopyranose, oligomers, decyl octyl glycosides (40- Clear SL 8 60%) Liquid SIMULSOL ™ D-Glucopyranose, oligomers, decyl octyl glycosides (20- Clear SL 826 40%), D-glucopyranose, oligomeric, C10-16 (even Liquid numbered)-alkyl glycosides (20-40%), (2- methoxymethylethoxy)propanol (0.1-1%)

Viruses and Indicator Cells

[0054] Xenotropic Murine Leukemiavirus (XMuLV), Porcine parvovirus (PPV) NADL-2 strain and pseudorabies virus (PRV) for use in these studies may be purchased from ATCC, Manassas, Va. Table 2 shows the properties of these viruses.

TABLE-US-00002 TABLE 2 Virus characteristics Physio- Size chemical Envelope Virus Genome Genus (nm) Resistance (Yes or No) XMuLV DNA Retroviridae  80-100 Low Yes PRV RNA Herpesviradae 120-140 Low Yes PPV DNA Parvoviradae 18-22 High No

[0055] PK13 (pig epithelial cells), PG-4 (feline brain cells) and Vero (African Green Monkey cells) for use in these studies may be purchased from ATCC, Manassas, Va. PK-13 cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand Island, N.Y.). PG-4 cells are cultured in McCoy's 5a Media (ATCC, Manassas, Va.) and Vero cells are cultured in Earl's modified Eagle's Medium (EMEM, Gibco, Grand Island, N.Y.). All cells are supplemented with 10% FBS (HyClone, Logan, Utah).

Cell based TCID.sub.50 Virus Titration Assay

[0056] A cell-based tissue culture infectious dose 50 (TCID.sub.50) assay is used for viral titration for XMuLV with PG-4 indicator cells, PPV with PK-13 indicator cells and PRV with Vero indicator cells. 96-well tissue culture plates are seeded with 2.5×10.sup.4 cells/mL indicator cells at 200 μL/well and incubated at 37° C. overnight or for about 16-24 hours. The plates are then inoculated with the virus in a series of 10-fold dilutions at 50 μL/well at a minimum of n=8 per dilution. The inoculated plates are incubated for 7-9 days (XMuLV and PRV) and 10-13 days (PPV) at 37° C. The plates are then examined and scored as positive or negative well-by-well under a microscope for cytopathic effects (CPE), and viral titers are calculated by standard methods.

Viral Cytotoxicity and Interference in Clarified Bioreactor Harvest Material

[0057] The TCID.sub.50 assay exemplified above is used to determine the cytotoxic effects of viruses in clarified bioreactor harvest material of monoclonal antibody (mAb), bispecific antibody and Fc-fusion proteins. The bioreactor harvest materials are clarified by centrifugation followed by polishing filtration. Impurities such as HCPs, lipids and host cell DNA are also automatically concentrated as part of the centrifugation process. The clarified bioreactor harvest material for the different proteins containing from 0.05% w/w to 1% w/w detergents are diluted in inoculation media at 10, 50 and 100-fold. Each dilution is added at n=8 to 96 well indicator cell seeded plates and incubated as per the TCID.sub.50 assay. At the end of the incubation period, each well is observed under a microscope for CPE and the appropriate dilution needed for each detergent is determined. The results demonstrated that a 50-fold dilution factor was sufficient to overcome cytotoxicity by the detergent.

[0058] The clarified bioreactor harvest materials for the various protein types are evaluated for any intrinsic effects of the cell culture harvest material on virus replication. Each virus is diluted 10-fold in the clarified bioreactor harvest material and inoculated onto indicator cell seeded plates and the experiment is conducted as per the TCID.sub.50 assay. A positive control (virus spiked into tissue culture material) is included. The viral titers are compared to the positive control virus. Titers that are within ±0.5 log.sub.10 of the positive control titers are considered to have no viral interference occurring. All viral test titers fell within ±0.5 log.sub.10 of the positive control titers and thus no viral interference was observed for any of the bioreactor harvest materials, regardless of protein type.

Viral Inactivation General Procedure

[0059] Virus inactivation of different enveloped viruses by each detergent is evaluated at a range of different concentrations, temperatures, and pH, in clarified bioreactor harvest materials containing mAb, bispecific and Fc-fusion proteins. XMuLV, PPV or PRV viruses are spiked into the harvest material at no more than 5% w/w, and detergent stock solution is added to the spiked material to achieve a specified final detergent concentration. The samples are then incubated at specific temperature and pH ranges. The viral inactivation is initiated as soon as the detergent is added to the virus containing material. The kinetics of virus inactivation are monitored by removing aliquots at various time points from the harvest samples and viral titer are assessed by the TCID.sub.50 assay. Matrix controls are included in the studies to demonstrate that virus inactivation is a result of detergent treatment and not the matrix itself.

XMuLV Inactivation at 30° C. by Non-Ionic Detergents

[0060] Viral inactivation of XMuLV by the detergents listed in Table 1 is tested at the optimal conditions determined for XMuLV inactivation. As per the Viral Inactivation Procedure exemplified above, each detergent is added at 0.6% w/w to the XMuLV spiked clarified mAb bioreactor harvest material at 30° C. Viral inactivation is monitored at various timepoints with a maximum time of inactivation of 180 minutes post virus inoculation.

[0061] The results compiled in Table 3, show that at the optimal conditions for XMuLV inactivation, the XMuLV is rapidly and completely inactivated for all the detergents tested at 180 minutes except for SIMULSOL™ SL 4 and SIMULSOL™ AS 48. Since there was no virus inactivation observed with SIMULSOL™ SL 4, the concentration of SIMULSOL™ SL 4 is increased to 30 mM (1%) and XMuLV inactivation is monitored at 30° C. for 180 minutes. However, no XMuLV inactivation was observed at the increased concentration of SIMULSOL™ SL 4 (data not shown). Surprisingly, these results suggest that not all glycosides are capable of robust viral inactivation, or they require extremely high concentrations to achieve robust inactivation as was observed for SIMULSOL™ AS 48. However, SIMULSOL™ SL 11W showed complete viral inactivation of greater than an LRF of 4, within 0 to 5 minutes of adding it to the clarified mAb bioreactor harvest material. Furthermore, even though the other glycosides effectively inactivated XMuLV, other factors limit applicability of some of these detergents in a large-scale manufacturing process. Such detergent characteristics included solubility, effect on turbidity in the harvested material, activity at different temperature and pH ranges, unknown or limited data on environmental toxicity and ease of handling.

TABLE-US-00003 TABLE 3 XMuLV Inactivation at 30° C. by Non-Ionic Detergents in clarified mAb bioreactor harvest material Detergent LRF Achieved at 30° C. (0.6% w/w) 0-5 min 90 min 180 min SIMULSOL ™ SL 11W ≥4.55 ≥4.55 ≥4.55 SIMULSOL ™ AS 48 0.50 ± 0.56 2.13 ± 0.56 3.20 ± 0.44 SIMULSOL ™ SL 4 0.88 ± 0.40 0.38 ± 0.50 0.88 ± 0.40 SIMULSOL ™ SL 10 ≥3.10 ≥4.62 ≥4.62 SIMULSOL ™ SL 26 C ≥3.60 ≥5.12 ≥5.12 SIMULSOL ™ SL 82 ≥3.60 ≥5.12 ≥5.12 SIMULSOL ™ SL 8 ≥3.10 ≥4.62 ≥4.62 SIMULSOL ™ SL 826 ≥3.10 ≥4.62 ≥4.62

Turbidity of SIMULSOL™ SL 11W in Clarified mAb Bioreactor Harvest Material.

[0062] Turbidity is evaluated at SIMULSOL™ SL 11W concentrations of 0%, 0.05%, 0.1%, 0.3%, 0.5% and 1% w/w in XMuLV spiked into clarified mAb bioreactor harvest material at 18° C. Viral inactivation is monitored for 180 mins and the turbidity is measured in nephelometric turbidity units (NTU) using a portable turbidimeter 2100P (Hach®).

[0063] The results as demonstrated in Table 4, show that SIMULSOL™ SL 11W at concentrations ranging from 0% to 1% w/w, was stable over time and no gross precipitation was observed in the harvest material up to 180 minutes.

TABLE-US-00004 TABLE 4 Turbidity of SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material incubated at 18° C. % w/w Turbidity (NTU) SIMULSOL ™ Time 0 (2 Time 180 (2 SL 11W measurements) measurements) 0 7.92 7.92 7.6 7.59 0.05 11.61 11.6 15.7 16.2 0.1 51.5 52.1 49.4 49.1 0.3 75.8 75 91.6 91.5 0.5 355 357 377 375 1 834 836 754 760

Characterization and Optimization of Viral Inactivation by SIMULSOL™ SL 11W

[0064] The use of SIMULSOL™ SL 11W as a viable environmentally compatible detergent for use in the manufacturing processes of proteins is further evaluated in clarified mAb bioreactor harvest material as per the Viral Inactivation Procedure exemplified above. Briefly, SIMULSOL™ SL 11W activity is evaluated at concentrations of 0.05%, 0.1%, 0.3%, 0.5% and 1% w/w in XMuLV spiked clarified mAb bioreactor harvest material at 4 different temperatures of 4° C., 18° C., 25° C., and 30° C. Viral inactivation is monitored at 0, 10, 30, 60, 120- and 180-min post inoculation with XMuLV to the SIMULSOL™ SL 11W containing clarified mAb bioreactor harvest material. The results as demonstrated in Tables 5-9, show that SIMULSOL™ SL 11W concentration, incubation time and temperature are all factors that influence the inactivation of XMuLV. SIMULSOL™ SL 11W achieved complete XMuLV viral inactivation at concentrations ≥0.1% w/w (≥3 mM) by 120 min and at concentrations ≥0.3% w/w by 10 minutes at all 4 temperatures evaluated.

TABLE-US-00005 TABLE 5 XMuLV Inactivation by 1.5 mM (0.05%) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) 4° C. 18° C. 25° C. 30° C. 0 −0.38 0.13 0.13 0.25 10 −0.13 0.00 0.13 0.75 30 0.50 1.38 1.88 1.37 60 0.62 2.25 2.00 1.37 120 1.25 2.25 2.59 1.75 180 2.12 3.94 3.33 2.12

TABLE-US-00006 TABLE 6 XMuLV Inactivation by 3.0 mM (0.1%) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) 4° C. 18° C. 25° C. 30° C. 0 0.62 3.25 2.12 3.25 10 2.50 3.41 2.55 3.50 30 4.58 3.42 2.71 4.08 60 5.43 4.99 3.64 5.00 120 ≥5.42 ≥5.92 ≥4.92 ≥6.05 180 ≥5.88 ≥6.38 ≥5.38 ≥6.51

TABLE-US-00007 TABLE 7 XMuLV Inactivation by 9.0 mM (0.3%) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) 4° C. 18° C. 25° C. 30° C. 0 1.50 3.21 2.25 ≥5.17 10 ≥5.13 ≥5.05 ≥5.05 ≥5.17 30 ≥5.30 ≥5.05 ≥5.05 ≥5.17 60 ≥5.30 ≥5.05 ≥5.05 ≥5.17 120 ≥5.30 ≥5.05 ≥5.05 ≥5.17 180 ≥5.76 ≥5.51 ≥5.51 ≥5.63

TABLE-US-00008 TABLE 8 XMuLV Inactivation by 15 mM (0.5%) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) 4° C. 18° C. 25° C. 30° C. 0 2.61 5.36 ≥5.05 ≥4.55 10 ≥4.67 ≥5.05 ≥5.05 ≥4.55 30 ≥4.67 ≥5.05 ≥5.05 ≥4.55 60 ≥4.67 ≥5.04 ≥5.05 ≥4.55 120 ≥4.67 ≥5.05 ≥5.05 ≥4.55 180 ≥5.13 ≥5.51 ≥5.51 ≥5.01

TABLE-US-00009 TABLE 9 XMuLV Inactivation by 30 mM (1.0%) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) 4° C. 18° C. 25° C. 30° C. 0 2.91 5.61 ≥4.67 ≥5.42 10 ≥5.42 ≥5.30 ≥4.67 ≥5.42 30 ≥5.42 ≥5.30 ≥4.67 ≥5.42 60 ≥5.42 ≥5.30 ≥4.67 ≥5.42 120 ≥5.42 ≥5.30 ≥4.67 ≥5.42 180 ≥5.88 ≥5.76 ≥5.13 ≥5.88

XMuLV Inactivation by SIMULSOL™ SL 11W in Different Starting Matrices

[0065] The ability of SIMULSOL™ SL 11W to inactivate virus at 9 mM (0.3% w/w) in different starting matrices such as water, cell culture media and Dulbecco's Phosphate Buffered Saline (DPBS) at 4° C. and 30° C. is evaluated. Viral inactivation is monitored at 0, 60-, 120- and 180-minutes post inoculation with XMuLV.

[0066] The results as demonstrated in Table 10, show robust XMuLV inactivation at both 4° C. and 30° C. after 120 minutes in all three starting matrices. The DPBS at 4° C. after 180 minutes detected a few live viral particles in the TCID.sub.50 assay but an LRF of 5.63 was still achieved. Overall, these results suggest that SIMULSOL™ SL 11W is capable of effectively inactivating virus independent of starting matrix. This suggests the potential use of SIMULSOL™ SL 11W across biopharmaceutical manufacturing platforms and the flexibility of the use of SIMULSOL™ SL 11W at different stages of the manufacturing process of a protein.

TABLE-US-00010 TABLE 10 XMuLV inactivation by SIMULSOL ™ SL 11W in different starting matrices LRF (0.3% w/w SIMULSOL ™ SL 11W) 4° C. 30° C. Time Cell culture Cell culture (min) Water media DPBS Water media DPBS 0 4.12 2 1.5 ≥4.40 ≥4.40 4.41 60 ≥4.52 ≥4.52 4.53 ≥4.40 ≥4.40 ≥4.40 120 ≥4.52 ≥4.52 ≥4.52 ≥4.40 ≥4.40 ≥4.40 180 ≥5.31 ≥5.31 5.63 ≥5.05 ≥5.05 ≥5.05

XMuLV Inactivation Kinetics at 0.3% SIMULSOL™ SL 11W at 4° C. and 30° C.

[0067] The ability of SIMULSOL™ SL 11W to inactivate virus at lower timepoints is evaluated. SIMULSOL™ SL 11W is added at 9 mM (0.3% w/w) to clarified mAb bioreactor harvest material at 4° C. and 30° C. Virus inactivation is monitored at 0, 2, 4, 6, 8, 10 and 20- and 180-minutes post inoculation with XMuLV.

[0068] The results as demonstrated in Table 11, show complete XMuLV inactivation occurring at ≥6 mins at 4° C., and at ≥2 mins at 30° C. with 9 mM (0.3% w/w) SIMULSOL™ SL 11W in clarified mAb bioreactor harvest material.

TABLE-US-00011 TABLE 11 XMuLV inactivation by 9 mM (0.3%) SIMULSOL ™ SL 11W at 4° C. and 30° C. in clarified mAb bioreactor harvest material Time Point LRF Achieved (minutes) 4° C. 30° C. 0 1.75 3.62 2 3.32 ≥5.05 4 4.27 ≥5.05 6 ≥4.92 ≥5.05 8 ≥4.92 ≥5.05 10 ≥4.92 ≥5.05 20 ≥4.92 ≥5.05 180 ≥5.38 ≥5.51

Effects of Stabilizing Agent Monopropylene Glycol on Virus Inactivation

[0069] The effect of monopropylene glycol on viral inactivation at concentration of 0.1% and 0.5% in clarified mAb bioreactor harvest materials at 30° C. is evaluated. Viral inactivation is monitored at 0 and 180 mins post inoculation with XMuLV. SIMULSOL™ SL 11W contains 1% to 5% monopropylene glycol. The maximum concentration of monopropylene glycol found in the inactivation procedures reported herein is 0.05%. The results as demonstrated in Table 12, show that monopropylene glycol at concentrations of 0.1% and 0.5% id not inactivate XMuLV in clarified mAb bioreactor harvest material at 30° C. Furthermore, the concentrations of monopropylene glycol demonstrated in Table 11 are at least 10-fold above the maximum concentration that would be found in clarified mAb bioreactor harvest material treated with SIMULSOL™ SL 11W. This indicates that the SIMULSOL™ SL 11W is responsible for the observed virus inactivation

TABLE-US-00012 TABLE 12 XMuLV inactivation by monopropylene glycol in clarified mAb bioreactor harvest material LRF at 30° C. 0.1% 0.5% Time Point monopropylene monopropylene (minutes) glycol glycol 0 0.25 0.00 180 0.25 0.13

Enveloped and Non-Enveloped Viral Inactivation by SIMULSOL™ SL 11W

[0070] The ability of SIMULSOL™ SL 11W to inactivate PRV, an enveloped virus and PPV a non-enveloped virus is evaluated. SIMULSOL™ SL 11W is added at 9 mM (0.3% w/w) to clarified mAb bioreactor harvest at 4° C., 18° C. and 30° C. Viral inactivation is monitored at 0, 10, 30, 60, 120- and 180-minutes post inoculation

[0071] The results as demonstrated in Table 13, shows robust inactivation of PRV enveloped virus occurring by 10 minutes at all three temperatures, with robust inactivation of PRV enveloped virus occurring at even lower time points within 0 to 5 mins at 30° C. No inactivation of PPV was observed by SIMULSOL™ SL 11W. These results taken together suggest that SIMULSOL™ SL 11W not only robustly inactivates different enveloped viruses in clarified mAb bioreactor harvest materials, but that viral inactivation by SIMULSOL™ SL 11W is specific to enveloped viruses.

TABLE-US-00013 TABLE 13 PRV Inactivation by 9 mM (0.3% w/w) SIMULSOL ™ SL 11W in clarified mAb bioreactor harvest material Time point LRF Achieved (min) 4° C. 18° C. 30° C. 0-5 2.93 1.30 ≥3.30 10 ≥2.92 ≥2.92 ≥3.30 30 ≥2.92 ≥3.23 ≥3.30 60 ≥2.92 ≥2.92 ≥3.30 120 ≥2.92 ≥2.92 ≥3.30 180 ≥3.38 ≥3.38 ≥3.76
Effect of pH on XMuLV Inactivation with SIMULSOL™ SL 11W

[0072] The ability of SIMULSOL™ SL 11W to inactivate XMuLV at a pH of 5.5 and a pH of 8.0 is evaluated. SIMULSOL™ SL 11W is added at 9 mM (0.3% w/w) to clarified mAb bioreactor harvest material at 18° C. Viral inactivation is monitored at 0, 10, 30, 60, 120- and 180-minutes post inoculation with XMuLV to the SIMULSOL™ SL 11W containing clarified mAb bioreactor harvest material.

[0073] The results as demonstrated in Table 14, show rapid and complete inactivation of XMuLV by SIMULSOL™ SL 11W at both pH 5.5 and pH 8.0 by 10 mins, thus indicating potential applicability of SIMULSOL™ SL 11W over a broad pH range in the manufacturing process of proteins.

TABLE-US-00014 TABLE 14 XMuLV inactivation by 9 mM (0.3%) SIMULSOL ™ SL 11W at 18° C., pH 5.5 and pH 8.0 in clarified mAb bioreactor harvest material Time Point LRF Achieved (min) pH 5.5 pH 8.0 0 3.48 3.47 10 ≥5.42 ≥5.42 30 ≥5.42 ≥5.42 60 ≥5.42 ≥5.42 120 ≥5.42 ≥5.42 180 ≥5.88 ≥5.88
Effect of Bioreactor Harvest Material Source on Inactivation of XMuLV with SIMULSOL™ SL 11W

[0074] The ability of SIMULSOL™ SL 11W to inactivate XMuLV in clarified bioreactor harvest materials containing different recombinant proteins such as mAb, bispecific antibody, and Fc fusion protein is evaluated. SIMULSOL™ SL 11W is added at 9 mM (0.3% w/w) to the clarified bioreactor harvest materials of the different proteins at 4° C. and 18° C. Viral inactivation is monitored at incubation time points of 0, 10, 30, 60, 120 and 180 minutes. The results as demonstrated in Table 15, show complete XMuLV inactivation by SIMULSOL™ SL 11W by 120 mins at both 4° C. and 30° C. in all three clarified bioreactor harvest materials. Further, complete inactivation was observed in the Fc-fusion and mAb protein containing harvest material by 10 mins at 4° C. The inactivation kinetics for the bispecific antibody was slightly slower at 4° C. than the other two clarified bioreactor harvest materials at 4° C. However, at 18° C., complete inactivation by SIMULSOL™ SL 11W was observed for all 3 proteins within 10 mins.

TABLE-US-00015 TABLE 15 Virus inactivation by 9 mM (0.3%) SIMULSOL ™ SL 11W in clarified recombinant protein bioreactor harvest materials Time 4° C. 18° C. point Fc Fusion Bispecific Fc Fusion Bispecific (min) protein antibody mAb protein antibody mAb 0 2.96 1.87 1.50 3.55 2.25 ≥5.17 10 ≥5.17 2.55 ≥5.13 ≥5.17 ≥4.92 ≥5.17 30 ≥5.17 3.27 ≥5.30 ≥5.17 ≥4.92 ≥5.17 60 ≥5.17 4.32 ≥5.30 ≥5.17 ≥4.92 ≥5.17 120 ≥5.17 ≥4.92 ≥5.30 ≥5.17 ≥4.92 ≥5.17 180 ≥5.63 ≥5.38 ≥5.76 ≥5.63 ≥5.38 ≥5.63

Effect of Impurity Levels on Viral Inactivation in Clarified Bioreactor Harvest Material of Different Proteins

[0075] The effect of impurity levels in the bioreactor harvest materials for three different recombinant proteins: an Fc fusion protein, a bispecific antibody, and a mAb are evaluated.

[0076] The harvest materials are clarified by centrifugation followed by polishing filtration. Impurities such as HCPs, lipids and host cell DNA are also automatically concentrated as part of the centrifugation process. A sample of the clarified mAb harvest material is further concentrated about 4-fold through a 5 KD molecular weight cutoff tangential flow filtration (TFF) process. The recombinant protein, DNA and other HCP impurities in the three clarified materials and the TFF concentrated material are measured. The results as demonstrated in Table 16, show the recombinant protein, host cell DNA and HCP content profiles in the three recombinant protein clarified materials and the concentrated clarified mAb material. A greater than 3-fold increase in DNA and HCP impurities was observed in the concentrated clarified mAb harvest material when compared to the unconcentrated clarified mAb harvest material.

TABLE-US-00016 TABLE 16 Measure of components in clarified recombinant protein bioreactor harvest material Bioreactor harvest material components Recombinant Clarified protein DNA HCP Protein (mg/mL) (pg/mL) (ng/mL) Fc Fusion Protein 1.65 72180600 94364 Bispecific mAb 3.40 202617600 915413 mAb 3.14 142781780 969014 Concentrated mAb 10.93 431469600 3314549
Effect of Concentrated Bioreactor Harvest Material on Inactivation of XMuLV with SIMULSOL™ SL 11W

[0077] The effect of SIMULSOL™ SL 11W on viral inactivation in concentrated clarified mAb harvest material is tested. Clarified mAb bioreactor harvest material containing the mAb and other impurities is concentrated about four-fold, through a 5 KDa molecular weight cutoff tangential flow filtration (TFF) process. The concentrated material containing the mAb and impurities is then immediately subjected to sterile filtration. The SIMULSOL™ SL 11W is then added to the clarified concentrated filtered material at a final concentration of 0.1% w/w (3 mM). The SIMULSOL™ 11 W containing material is then incubated at 3 different temperatures of 4° C., 18° C. and 30° C. Viral inactivation at the different temperatures is measured at time points ranging from 0 minutes to 180 minutes. The results as demonstrated in Table 17, show complete inactivation of XMuLV at 180 minutes at all 3 temperatures tested, even though the inactivation kinetics decreased at the lower temperatures when compared with the 30° C. sample. No effects on inactivation kinetics of the concentrated bioreactor material when compared to the unconcentrated bioreactor harvest material was observed (data not shown). This suggests that increased impurity levels do not affect viral inactivation by SIMULSOL™ SL 11W.

TABLE-US-00017 TABLE 17 Virus inactivation by 3 mM (0.1%) SIMULSOL ™ SL 11W in 4- fold concentrated clarified mAb bioreactor harvest material Time point LRF Achieved (min) 4° C. 18° C. 30° C. 0 2.25 2.50 2.25 10 2.00 5.13 5.36 30 3.28 5.73 ≥5.05 60 5.06 ≥5.42 ≥5.05 120 ≥5.05 ≥5.42 ≥5.05 180 ≥5.51 ≥5.88 ≥5.51

Example 2. Determination of Removal of SIMULSOL™ SL 11W in Downstream

Recombinant Protein Purification

Undecyl Glycoside Quantification by Liquid Chromatography Mass Spectrometry (LC-MS)—Method A

[0078] LC-MS quantification is conducted on clarified mAb bioreactor harvest samples containing SIMULSOL™ SL 11W at concentrations ranging from 0.03 mM to 3 mM.

[0079] One microliter of the incubated sample is injected into a Waters Acquity® UPLC coupled to Waters SYNAPT® G2-Si mass spectrometer. For the quantitation, separations are performed on a Varian PLRP-S reversed-phase column (1×50 mm, 300 Å, 5 μm) at 80° C. using 0.05% trifluoroacetic acid (TFA) in H.sub.2O as mobile phase A and 0.04% TFA in acetonitrile (ACN) as mobile phase B. The column is equilibrated with 15% mobile phase B for 1 minute and is linearly increased from 15% to 30% over a 3 minute timepoint, then is held at 30% B for 2 minutes and then held at 100% over 1 minute. After the column is held at 100% for 1 minute, it is re-equilibrated at 15% mobile phase B. SIMULSOL™ SL 11W is separated at 0.3 mL/min. between 1 to 6 min and is analyzed using an electrospray ionization (ESI) source. The mass spectrometer is operated at positive, sensitivity model, scan range of 100 to 1000 amu, capillary voltage of 3.2 kV, desolvation temperature of 450° C., desolvation gas flow of 900 L/hr, source temperature of 150° C. and a sample cone of 40 V. For characterization, the separation is conducted on Waters Acquity® UPLC BHE 300 Å C4 column (2.1×100 mm, 1.7 μm particle size) with the same mobile phases. Equilibrate the column at 20% mobile phase B for 1 minute, linearly increased from 20% to 25% over 14 minutes and then to 90% over 1 minute. After holding at 100% for 1.0 minute, re-equilibrate the column at 20% mobile phase B.

[0080] The extracted peak area of sodiated undecyl glycoside is measured. Under the LC-MS analysis conditions, a linear relationship was observed between extracted ion peak area and SIMULSOL™ SL 11W concentrations of 0.03 to 3 mM. When SIMULSOL™ SL 11W concentration was greater than 3 mM, the MS detector was saturated.

Undecyl Glycoside Quantification by Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS)—Method B

[0081] Standard solutions containing SIMULSOL™ SL 11W at concentrations ranging from 0.0008 mM to 0.012 mM are prepared in water or Protein A mainstream. After addition of SIMULSOL™ SL 11W, these samples were subjected to LC-MS/MS (MRM) analysis. Incubated sample (100 μL) is injected into the LC and split 1/20 post-column to MS. An Agilent 1260 Infinity II HPLC coupled to Sciex Triple Quad 5500 mass spectrometer is utilized for the analysis. For the quantitation, separations are performed on an Agilent Zorbax, 300SB-C8 reversed-phase column (4.6×50 mm, 300 Å, 3.5 μm) at 80° C. using 0.5 mM sodium acetate in water as mobile phase A and 0.5 mM sodium acetate in 95:5 acetonitrile:water as mobile phase B. The column is equilibrated at 20% mobile phase B for 2 minute, linearly increased from 20% to 90% over 10 minutes, held at 90% B for 2 minutes, the column is re-equilibrated at 20% mobile phase B. SIMULSOL™ SL 11 W is separated at 1.0 mL/min between 4.5 to 5.5 minutes and was analyzed using an electrospray ionization (ESI) source. Mass spectrometer is operated at positive, MRM mode with scanning transition ions (357.0 to 185.0), capillary voltage of 5.5 kV, desolvation temperature of 450° C., and desolvation gas flow of 55 L/hr.

[0082] Selected reactions monitoring (SRM/MRM (multiple reaction monitoring)), an LC-MS/MS method, is used to selectively detect the sodiated SIMULSOL™ SL 11W. SRM scans for a single precursor ion (mass of sodiated SIMULSOL™ SL 11W, 357.0 m/z) and after fragmentation, product ion (the most predominant fragment of sodiated SIMULSOL™ SL 11W, 185.0 m/z) is detected, and the peak area corresponding to this extracted transition ion is obtained.

[0083] Under the LC-MS/MS analysis conditions, the plots are linear between SIMULSOL™ SL 11W concentrations of 0.0008 mM to 0.012 mM in water and Protein A mainstream. These results suggest that LC-MS/MS is able to detect SIMULSOL™ SL 11W between 0.0008 mM to 0.012 mM and linearity of plots is independent of matrix. Therefore, this LC-MS/MS (SRM/MRM) method is sensitive with specificity for detection of SIMULSOL™ SL 11W and independent of the matrix the detergent is found in. The limit of detection (LOD) is 0.001 mM calculated from linearity.

Protein a Chromatography and Detection of SIMULSOL™ SL 11W

[0084] Clarified mAb bioreactor harvest material solutions containing 0% (control), 0.3% and 1.0% w/w SIMULSOL™ SL 11W is subjected to MabSelect™ protein A chromatography. The Protein A chromatography is conducted on unfiltered clarified mAb harvest material containing SIMULSOL™ SL 11W. After the column is loaded, six column volumes of Tris/NaCl are used to wash the column prior to elution of the mAb with an acetic/citric acid buffer. The eluted mAb mainstream is collected and analyzed by LC-MS for the presence of SIMULSOL™ SL 11W.

[0085] The results of the analysis using LCMS (Method A) shows <0.03 mM of SIMULSOL™ SL 11W levels in the Protein A mainstream from the mAb bioreactor harvest material solutions containing 0.3% and 1.0% w/w SIMULSOL™ SL 11W. Using LC-MS/MS (Method B), the amount of SIMULSOL™ SL 11W is found to be 0.0005 mM in the Protein A mainstream from the mAb bioreactor harvest material solutions containing 0.3% w/w SIMULSOL™ SL 11W, which is lower than the LOD of 0.001 mM. These results indicate that Protein A chromatography is able to remove SIMULSOL™ SL 11W Simulsol SL 11W from the feedstream to a trace amount level, even without filtration steps prior to column loading.

Example 3. Effect of SIMULSOL™ SL 11W on Recombinant Protein Quality

[0086] The effect of SIMULSOL™ SL 11W on the quality of recombinant proteins, is tested by comparing clarified mAb bioreactor harvest samples with and without 0.3% SIMULSOL™ SL 11W at 20° C. for 3 h, and then incubate at 4° C. for 16 h. The samples are first filtered through 0.2 μm glass fiber filters followed by 0.45 μm PVDF filters, and then subjected to Protein A purification. The samples are analyzed for the analytical properties as shown in Table 18 using techniques known in the art, such as capillary electrophoresis (CE), Size exclusion chromatography (SEC), isoelectric focusing (IEF) measure on an iCE™ instrument. The results as shown in Table 18, indicate that treatment with SIMULSOL™ SL 11W had no significant effect on any of the measured qualities as compared to no treatment with SIMULSOL™ SL 11W.

TABLE-US-00018 TABLE 18 mAb product quality analytical data with and without SIMULSOL ™ SL 11W No 0.3% SIMULSOL ™ Analytical Property Detergent SL 11W CE reduced Heavy Chain (%) 65.9 65.9 CE reduced Light Chain (%) 31.7 31.6 CE non-reduced Main Peak (%) 98.2 98.2 SEC monomer (%) 98.5 98.5 SEC Aggregate (%) 1.5 1.5 IEF Main Peak (%) 75.5 74.8 IEF Total Acidic Variants (%) 23.0 23.5 IEF Total Basic Variants (%) 1.5 1.6 Chinese Hamster Ovary (CHO) 336 283 host cell protein (HCP) (ppm) Residual Protein A (ppm) 13.3 7.3 DNA (ppb) 21527 21473