Protein purification and virus inactivation with alkyl glycosides

Abstract

A process for purifying a recombinant protein comprising the steps of: i) providing a solution comprising the recombinant protein; ii) adding an alkyl glycoside to the solution; and iii) purifying the recombinant protein. The addition of the alkyl glycoside provides improved clearance of process-related impurities. The purified recombinant protein of the invention has low levels of host cell DNA, host cell protein and viral contamination.

Claims

1. A method for purifying a recombinant polypeptide comprising the steps of: i) providing a solution comprising the recombinant polypeptide; ii) adding an alkyl glycoside to the solution; and iii) purifying the recombinant polypeptide by carrying out a step of chromatography on the solution.

2. The method of claim 1, wherein the alkyl glycoside is additionally included in the wash buffer of the chromatography step.

3. The method of claim 1, wherein step (iii) results in separation of the recombinant polypeptide from host cell DNA and/or host cell protein in the solution and/or from other protein impurities in the solution.

4. The method of claim 3, wherein step (iii) results in improved separation of the recombinant polypeptide from host cell DNA and/or host cell protein in the solution compared to the same process without adding the alkyl glycoside to the solution.

5. The method of claim 1, wherein the chromatography is immunoaffinity chromatography, affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, size exclusion chromatography, or metal chelate chromatography.

6. The method of claim 5, wherein the chromatography is immunoaffinity chromatography.

7. The method of claim 1, wherein an ion exchange chromatography step is carried out on the solution before the step of adding the alkyl glycoside to the solution.

8. The method of claim 1, wherein a hydrophobic interaction chromatography step is carried out on the solution after step (iii), and/or wherein a multimodal chromatography step is carried out on the solution after step (iii).

9. The method of claim 8, wherein an ion exchange chromatography step is carried out on the solution after the hydrophobic interaction or multimodal chromatography step.

10. The method of claim 1, wherein the method is for separating the recombinant polypeptide from host cell DNA and/or host cell protein and comprises the steps of: a) providing the solution comprising the recombinant polypeptide; b) purifying the recombinant polypeptide by carrying out a step of ion exchange chromatography on the solution; c) adding an alkyl glycoside to the solution; d) purifying the recombinant polypeptide by carrying out a step of immunoaffinity chromatography on the solution; e) purifying the recombinant polypeptide by carrying out a step of hydrophobic interaction or multimodal chromatography on the solution; and f) purifying the recombinant polypeptide by carrying out a further step of ion exchange chromatography on the solution.

11. The method of claim 5, wherein the ion exchange chromatography is anion exchange chromatography.

12. The method of claim 1, wherein the method provides a solution comprising a level of host cell DNA contamination that is less than 5000 pg/ml; and/or wherein the method provides a solution of the recombinant polypeptide comprising a level of host cell DNA contamination that is reduced by a factor of at least 1.5, when compared to a reference method in which no alkyl glycoside is used or a conventional S/D treatment is used.

13. The method of claim 1, wherein the method provides a solution comprising a level of host cell protein contamination that is less than 5000 ng/ml; and/or wherein the method provides a solution of the recombinant polypeptide comprising a level of host cell protein (HCP) contamination that is reduced by a factor of at least 1.5, when compared to a reference method in which no alkyl glycoside is used or a conventional S/D treatment is used.

14. The method of claim 1, wherein step ii) further comprises incubating the solution.

15. A method for inactivating one or more viruses in a solution comprising a step of adding an alkyl glycoside to the solution and incubating the solution.

16. The method of claim 15, wherein the solution is i) a solution comprising a recombinant polypeptide, or ii) plasma-derived material.

17. The method of claim 15, wherein the incubation is carried out for between 20 minutes and 5 hours.

18. The method of claim 15, wherein the incubation is carried out at room temperature or between 4° C. and 10° C.

19. The method of claim 15, wherein the final concentration of the alkyl glycoside before the incubation is above the critical micelle concentration (CMC) of the alkyl glycoside.

20. The method of claim 15, wherein the incubation is carried without agitation.

21. The method of claim 1, wherein the recombinant polypeptide is from a cell line recombinantly producing the polypeptide.

22. The method of claim 1, wherein the recombinant polypeptide is a blood coagulation protein, albumin, an immunoglobulin, or a fusion protein.

23. The method of claim 1, wherein the solution in step (i) has between 0.1 pg/ml and 50 pg/ml of host cell DNA and/or between 50 μg/ml and 1000 μg/ml of host cell protein.

24. The method of claim 1, wherein the alkyl glycoside is n-octyl-beta-D-glucopyranoside, or is selected from the group consisting of n-decyl-beta-D-glucopyranoside, n-octyl-beta-D-maltoside, n-dodecyl-beta-D-maltoside, n-dodecyl-beta-D-glucopyranoside, and n-decyl-beta-D-maltoside.

25. The method of claim 1, wherein the solution is further treated after step (iii) and any additional purification steps by a step of viral filtration.

26. The method of claim 1, wherein the solution is further treated after step (iii) and any additional purification steps by one or more steps of ultrafiltration and/or diafiltration.

27. The method of claim 1, wherein further comprising mixing the purified recombinant polypeptide is mixed with a pharmaceutically-acceptable carrier to make a pharmaceutical composition.

28. The method of claim 1, wherein the alkyl glycoside is added without any organic solvent and/or the alkyl glycoside is added without any prior mixing with an organic solvent.

29. A solution comprising a recombinant polypeptide and an alkyl glycoside, wherein the solution is obtained from step ii) of the method of claim 1.

30. A solution comprising a purified recombinant polypeptide, wherein the solution is obtained by the method of claim 1.

31. A method for purifying a recombinant polypeptide comprising the steps of: i) providing a solution comprising the recombinant polypeptide, and ii) purifying the recombinant polypeptide by carrying out a step of chromatography on the solution, wherein an alkyl glycoside is included in the wash buffer of the chromatography step.

32. The method of claim 31, wherein step ii) results in separation of the recombinant polypeptide from host cell DNA, host cell protein, and/or other protein impurities in the solution.

33. The method of claim 32, wherein step ii) results in improved separation of the recombinant polypeptide from host cell DNA, host cell protein, and/or other protein impurities in the solution, as compared to a reference method in which the alkyl glycoside is not included in the wash buffer.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows MuLV inactivation with OG/TNBP and with OG only: open triangle show MuLV inactivation in 4.4 mg/ml D′D3-FP with 200 mM OG+0.3% TNBP at 21° C. under agitation, open square show MuLV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. without agitation.

(2) FIG. 2 shows BVDV inactivation with OG/TNBP and with OG only: open triangle show BVDV inactivation in 4.4 mg/ml D′D3-FP with 200 mM OG+0.3% TNBP 21° C. under agitation, open square show BVDV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. without agitation.

(3) FIG. 3 shows PRV inactivation with OG/TNBP and with OG only: open triangle show PRV inactivation in 4.4 mg/ml D′D3-FP with 200 mM OG+0.3% TNBP at 21° C., with agitation, open square show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. without agitation.

(4) FIG. 4 shows PRV inactivation with OG at 6° C., either with agitation or without agitation: open triangle show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. under agitation, open square show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. without agitation, open rhomb show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 6° C. without agitation.

(5) FIG. 5 shows PRV inactivation with different concentrations of OG at 5-6° C. with agitation: open square show PRV inactivation in 8.4 mg/ml D′D3-FP with 15 mM OG at 5-6° C. under agitation, open rhomb show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 5-6° C. under agitation, open triangle show PRV inactivation in 8.4 mg/ml D′D3-FP with 30 mM OG at 5-6° C. under agitation.

(6) FIG. 6 shows PRV inactivation with various concentrations of OG at 18-19° C.: open triangle show PRV inactivation in 4.4 mg/ml D′D3-FP with 10 mM OG at 21.5° C. under agitation, open square show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG at 18° C. under agitation, open rhomb show PRV inactivation in 8.4 mg/ml D′D3-FP with 30 mM OG at 18° C. under agitation, open circle show PRV inactivation in 8.4 mg/ml D′D3-FP with 40 mM OG at 18° C. under agitation, closed rhomb show PRV inactivation in 13.5 mg/ml D′D3-FP with 60 mM OG at 19° C. under agitation, closed triangle show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG stored 7d dark at 18° C. under agitation, closed square show PRV inactivation in 8.4 mg/ml D′D3-FP with 20 mM OG stored 7d light at 18° C. under agitation, closed circle show PRV inactivation in 8.4 mg/ml D′D3-FP with 10 mM OG stored 7d dark at 18° C. under agitation.

(7) FIG. 7 shows PRV inactivation with equal mM concentrations of OG at 18 or 5-6° C.: open triangle show PRV inactivation in 8.4 mg/ml D′D3-FP with 30 mM OG at 18° C. under agitation, open square show PRV inactivation in 8.4 mg/ml D′D3-FP with 30 mM OG at 5-6° C. under agitation.

(8) FIG. 8 shows the PRV inactivation capacity of a variety of alkyl glycosides: open square show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM OG, open triangle show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM n-Decyl-ß-D-glucopyranoside, open rhomb show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM n-Octyl-ß-D-maltoside, closed square show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM n-Dodecyl-ß-D-maltoside, closed triangle show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM n-Dodecyl-ß-D-glucopyranoside, closed rhomb show PRV inactivation in 11.9 mg/ml D′D3-FP at 60 mM n-Decyl-ß-D-maltoside.

(9) FIG. 9 shows the PRV inactivation capacity of n-decyl-ß-D-glucopyranoside, n-decyl-ß-D-maltoside and n-octyl-ß-D-maltoside at lower concentrations (about twice as high as their CMC values): open triangle show PRV inactivation in 11.9 mg/ml D′D3-FP at 5 mM n-Decyl-R-D-glucopyranoside, open square show PRV inactivation in 11.9 mg/ml D′D3-FP at 40 mM n-Octyl-ß-D-maltoside, open rhomb show PRV inactivation in 11.9 mg/ml D′D3-FP at 5 mM n-Decyl-ß-D-maltoside.

(10) FIG. 10 compares: a) the residual host cell protein concentration in samples of an albumin fusion protein (rD′D3-FP) obtained by immunoaffinity chromatography following treatment with n-octyl-β-D-glucopyranoside (OG), n-decyl-β-D-glucopyranoside (DG), polysorbate 80 and tri-n-butylphosphate (PS80/TNBP) or buffer control; and b) the relative improvement of host cell protein clearance (fold improvement of HCP clearance) when OG or DG are used in step ii) compared to PS80/TNBP (clear bars) or buffer control (hatched bars) treatment.

(11) FIG. 11 compares: a) the residual host cell DNA concentration in samples of an albumin fusion protein (rD′D3-FP) obtained by immunoaffinity chromatography following treatment with n-octyl-β-D-glucopyranoside (OG), polysorbate 80 and tri-n-butylphosphate (PS80/TNBP) or buffer control; and b) the relative improvement of host cell DNA clearance (fold improvement of host cell DNA clearance) when OG is used compared to PS80/TNBP (clear bar) or buffer control (striped bar) treatment.

(12) FIG. 12 shows VSV inactivation by SD treatment compared to OG: closed triangle show VSV inactivation by 40 mM OG in 10.4 mg/ml D′D3-FP at 24.5° C. and open square show VSV inactivation by 1% PS80+0.3% TNBP in 10.4 mg/ml D′D3-FP at 26.5° C.

(13) FIG. 13 shows Vaccinia virus inactivation by SD treatment compared to OG: closed triangle show Vaccinia virus inactivation by 40 mM OG in 10.4 mg/ml D′D3-FP at 24.5° C. and open square show Vaccinia virus inactivation by 1% PS80+0.3% TNBP in 10.4 mg/ml D′D3-FP at 26.5° C.

MODES FOR CARRYING OUT THE INVENTION

Example 1

(14) Comparison of n-octyl-beta-D-glucopyranoside with a Solvent/Detergent Mixture and Buffer as a Control

SUMMARY

(15) Treatment of recombinant D′D3-FP (ref. 7) with a typical solvent/detergent mixture of polysorbate 80 and tri-n-butylphosphate (PS80/TNBP) was compared with treatment with the alkyl glycoside, n-octyl-β-D-glucopyranoside (OG). Initial experiments showed that the rD′D3-FP remained stable upon treatment with the alkyl glycoside. Importantly, rapid inactivation of three model viruses was observed with the alkyl glycoside even in the absence of the tri-n-butylphoshate (TNBP). Virus inactivation was complete within one hour at temperatures as low as 5° C., with or without agitation. The efficient procedure tolerated the use of an unexpectedly wide range of process parameters resulting in efficient virus inactivation (the tested range for OG was 4-25° C., for less than or equal to 30 minutes (typically 120 min were followed up) at concentrations of more than or equal to 20 mM). Unexpectedly, the presence of the alkyl glycoside in the feedstream of a subsequent chromatography purification step resulted in significantly lower host cell DNA and host cell protein levels at the eluate stage in comparison to the use of the PS80/TNBP or control buffer solutions.

(16) Methods and Results

(17) rD′D3-FP contains the FVIII binding site of the von-Willebrand-Factor (vWF) protein generated by recombinant DNA technology. The rD′D3-FP cDNA sequence was transfected into Chinese Hamster Ovary (CHO) cells and the polypeptide was expressed to perform the investigations described. Laboratory studies were conducted to assess the virus inactivation capacity of the alkyl glycoside step with a range of representative virus models. The use of the retrovirus MuLV (murine leukemia virus) as a relevant virus is particularly relevant for CHO cell derived products as these are known to contain endogenous retrovirus-like particles. The flavivirus BVDV (bovine viral diarrhea virus) and the herpesvirus PRV (pseudorabies virus) which is in general more stable to S/D treatment were used in virus evaluation studies to demonstrate the broad virus inactivation capacity of the alkyl glycoside treatment.

(18) The virus titres of all samples studied were determined using end point dilution assays immediately after generating the sample and were calculated according to the Spearman-Kärber method as given in Ref 13.

(19) For these OG studies two process control parameters were chosen to test challenge conditions concerning virus inactivation: 1) an OG detergent concentration of 20 mM; and 2) a temperature of 6° C.±1° C. Samples of a rD. D3-FP intermediate (protein concentration of about 14 mg/ml) were diluted appropriately or used undiluted and spiked with virus to be studied resulting in the desired detergent concentration.

(20) Samples were taken before the addition of detergent (untreated) and at different time-points after the addition of detergent. The samples were assayed immediately by diluting 1:100 in cell culture medium to stop the reaction and to render the samples non-toxic in the virus assays.

(21) The following reduction factors were obtained (which for all viruses were limited only by the test system, in particular the detection limit as well as the amount of virus used for spiking):

(22) TABLE-US-00001 TABLE 1 After Kinetic Data Virus LRV Condition incubation given given Studied [log.sub.10] studied for in in MuLV ≥4.2 20 mM OG 15 minutes FIG. 1 Table 2 BVDV ≥5.3 at 6° C. 15 minutes FIG. 2 Table 3 PRV ≥6.3 60 minutes FIG. 3 Table 4

(23) The virus hold control results in FIGS. 1-3 (Tables 2-4, respectively) indicated that there was no significant reduction in the virus stability sample over the incubation period which confirms that the reduction demonstrated was due to the action of the alkyl glycoside detergent treatment. Additionally, the figures demonstrate that the virus inactivation was very rapid and in all cases greater than 4 logs of virus inactivation was observed. MuLV and BVDV were completely inactivated at a temperature of 6° C.±1° C. by an OG detergent concentration of 20 mM by the first studied 15 minutes time point while complete PRV inactivation took 60 minutes.

(24) TABLE-US-00002 TABLE 2 MuLV Inactivation by e.g. OG MuLV, 4.4 mg/ml rD′D3-FP, 200 mM MuLV, 8.4 mg/ml OG + 0.3% TNBP rD′D3-FP, 20 mM time in at 21° C., with held OG at 6° C. with- held minutes agitation control out agitation control prior OG   6.2 6.2 5.4 5.4 addition 15 n.d. n.d. ≤1.2 n.d. 30 ≤2.7 n.d. ≤1.2 n.d. 60 ≤1.7 n.d. ≤1.2 n.d. 120 ≤1.7 4.8 ≤1.2 5.2

(25) TABLE-US-00003 TABLE 3 BVDV Inactivation by e.g. OG BVDV, 4.4 mg/ml rD′D3-FP, 200 mM BVDV, 8.4 mg/ml OG + 0.3% TNBP rD′D3-FP, 20 mM time in at 21° C., with held OG at 6° C. with- held minutes agitation control out agitation control prior OG   6.5 6.5 6.5 6.5 add 15 n.d. n.d. ≤1.2 n.d. 30 ≤2.7 n.d. ≤1.2 n.d. 60 ≤1.7 n.d. ≤1.2 6.4 120 ≤1.7 6.4 n.d. n.d.

(26) TABLE-US-00004 TABLE 4 PRV Inactivation at e.g. 20 mM OG at 6° C. PRV, 4.4 mg/ml PRV, 8.4 mg/ml D′D3-FP, 200 mM held D′D3-FP, 20 mM held time in OG + 0.3% TNBP control OG at 6° C. with- control minutes at 21° C., shaken 16070521 out agitation 16111422 prior OG   8.4 8.4 7.8 7.8 add 15 n.d. n.d. 1.5 n.d. 30 ≤2.7 n.d. 1.3 n.d. 60 ≤1.7 n.d. ≤1.2 7.6 120 ≤1.7 8.0 n.d. n.d.

(27) In additional robustness studies with PRV, a more resistant enveloped virus, the following parameters were studied: Variation of protein concentration had no influence of the PRV inactivation (FIG. 3, Table 4). Agitation during the 20 mM OG incubation at 6° C. resulted in slightly faster PRV inactivation (FIG. 4, Table 5). Nevertheless, PRV was completely and reliably inactivated by 20 mM OG treatment without any agitation during OG incubation. Lowering the OG concentration to 15 mM (below CMC) at 6° C. resulted in no significant PRV inactivation (FIG. 5, Table 6). A similar observation was made at 10 mM OG at 21° C. (FIG. 6, Table 7). Increasing the OG concentration to 30 mM or higher resulted in fast PRV inactivation. Furthermore, storage of the OG stock solution for 7 days (in the dark) had no influence on the inactivation capacity. Increasing the incubation temperature from 6° C. to 18° C. at 30 mM OG resulted in no faster PRV inactivation (FIG. 7, Table 8). 60 mM of several other alkyl glycosides completely inactivates PRV very fast (FIG. 8, Table 9). Especially, low amounts of alkyl glycosides (about twice as high as the CMC) completely inactivates PRV very fast (FIG. 9, Table 10a).

(28) TABLE-US-00005 TABLE 5 PRV Inactivation at 20 mM OG by with agitation or not at 6° C. PRV, 8.4 PRV, 8.4 PRV, 8.4 mg/ml mg/ml mg/ml rD′D3-FP, rD′D3-FP, rD′D3-FP, 20 mM 20 mM OG 20 mM OG OG at at 6° C. at 6° C. time in 6° C., with held without held without held minutes agitation control agitation control agitation control prior 7.6 7.6 7.8 7.8 7.6 7.6 OG add 15 1.3 n.d. 1.5 n.d. n.d. n.d. 30 ≤1.0 n.d. 1.3 n.d. 1.3 n.d. 60 ≤1.0 7.3 ≤1.0 7.6 ≤1.2 7.7

(29) TABLE-US-00006 TABLE 6 PRV Inactivation at various mM OG at 6° C. PRV, rD′D3-FP PRV, rD′D3-FP PRV, rD′D3-FP 8.4 mg/ml, 8.4 mg/ml, 20 8.4 mg/ml, 15 mM OG mM OG at 30 mM OG time in at 5-6° C., held 5-6° C., held at 5-6° C., held minutes with agitation control with agitation control with agitation control prior 7.7 7.7 7.5 7.5 7.4 7.4 OG add 30 6.6 n.d. n.d. n.d. ≤0.9 n.d. 60 6.6 7.3 1.2 7.3 ≤0.9 7.4

(30) TABLE-US-00007 TABLE 7 PRV Load in log10 at various mM OG at 18-22° C. PRV, PRV, PRV, PRV, rD′D3-FP rD′D3-FP rD′D3-FP rD′D3-FP 4.4 mg/ml, 8.4 mg/ml, 8.4 mg/ml, 8.4 mg/ml, 10 mM OG 20 mM OG 30 mM OG 40 mM OG time at 21.5° C., at 18° C., at 18° C., at 18° C., in with held with held with held with held min agitation control agitation control agitation control agitation control prior 7.4 7.4 7.3 7.3 7.1 7.1 7.3 7.3 OG add 15 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 30 6.8 n.d. ≤0.9 n.d. ≤0.9 n.d. ≤0.9 n.d. 60 7.0 n.d. ≤0.9 7.3 ≤0.9 7.2 ≤0.9 7.2 120 6.7 7.5 n.d. n.d. n.d. n.d. n.d. n.d. PRV, PRV, PRV, PRV, 13.5 rD′D3-FP rD′D3-FP rD′D3-FP mg/ml 8.4 mg/ml, 8.4 mg/ml, 8.4 mg/ml, rD′D3-FP, 20 mM OG 20 mM OG 10 mM OG 60 mM OG stored 7 d stored 7 d stored 7 d time at 19° C. dark, at light at dark, at in without held 18° C., with held 18° C., with held 18° C., with held min agitation control agitation control agitation control agitation control prior 7.8 7.8 7.3 7.3 7.5 7.5 7.6 7.6 OG add 15 ≤1.2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. 30 ≤1.2 n.d. ≤0.9 n.d. 1.2 n.d. 7.2 n.d. 60 ≤1.2 7.5 ≤0.9 7.4 ≤0.9 7.4 7.0 7.4 120 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

(31) TABLE-US-00008 TABLE 8 PRV Inactivation at 30 mM OG at 6° C. or 18° C. PRV, rD′D3-FP PRV, rD′D3-FP 8.4 mg/ml, 30 mM 8.4 mg/ml, 30 mM prior OG at 18° C., held OG at 5-6° C., held OG add with agitation control with agitation control 0 7.1 7.1 7.4 7.4 30 ≤0.9 n.d. ≤0.9 n.d. 60 ≤0.9 7.2 ≤0.9 7.4

(32) TABLE-US-00009 TABLE 9 PRV inactivation capacity of a variety of alkyl glycosides PRV, PRV, held 60 mM n- held 60 mM n- held time in PRV, 60 con- Decyl-β-D- con- Octyl-β-D- con- minutes mM OG trol glucopyranoside trol maltoside trol prior add 7.8 7.8 7.5 7.5 7.6 7.6 30 ≤2.2 n.d. ≤2.2 n.d. 2.5 n.d. 60 ≤2.2 n.d. ≤2.2 n.d. ≤2.2 n.d. 120 ≤2.2 7.6 ≤2.2 7.5 ≤2.2 7.6 PRV, PRV, 60 PRV, 60 mM mM n- 60 mM n- n-Dodecyl- held Dodecyl-β-D- held Decyl-β-D- held time in β-D- con- gluco con- malto con- minutes D-maltoside trol pyranoside trol pyranoside trol prior add 7.8 7.8 7.7 7.7 7.6 7.6 30 ≤4.2 n.d. ≤2.2 n.d. ≤2.2 n.d. 60 ≤4.2 n.d. ≤2.2 n.d. ≤2.2 n.d. 120 ≤4.2 7.5 ≤2.2 7.5 ≤2.2 7.5

(33) TABLE-US-00010 TABLE 10a PRV inactivation capacity of n-decyl-β-D-glucopyranoside, n-decyl- β-D-maltoside and n-octyl-β-D-maltoside at lower concentrations (about twice as high as their CMC values) PRV, PRV, PRV, 40 mM 5 mM n- 5 mM n- held n-Octyl- Decyl- held time in Decyl-β-D- con- β-D- held β-D- con- minutes glucopyranoside trol maltoside control maltoside trol prior add 7.5 7.5 7.3 7.3 7.5 7.5 30 ≤2.2 n.d. 2.5 n.d. ≤2.2 n.d. 60 ≤2.2 n.d. ≤2.2 n.d. ≤2.2 n.d. 120 ≤2.2 7.5 ≤2.2 7.3 ≤2.2 7.2

(34) Under all robustness conditions evaluated the kinetics of virus inactivation were similar to the standard conditions. Overall, the OG treatment step was shown to be effective and robust and to have a high capacity to inactivate enveloped viruses, including the particularly relevant retrovirus MuLV. Also for other alkyl glycosides their capacity to effectively inactivate relevant viruses could be shown.

(35) Furthermore, robustness studies with Vaccinia virus (VACV), the most resistant enveloped virus against SD inactivation [14] and Vesicular Stomatitis Virus (VSV) were studied. The results given in FIG. 12 (Table 10b) for VSV and in FIG. 13 (Table 10c) for VACV demonstrates a much faster virus inactivation by OG compared to SD (1% PS80+0.3% TnBP). Especially only OG completely inactivates VACV very fast where SD failed to inactivate VACV effectively over a period of 4 hours (FIG. 13).

(36) TABLE-US-00011 TABLE 10b VSV (Vesicular Stomatitis Virus) inactivation by SD compared to OG treatment time in VSV, 40 held VSV, 1% PS80 + held minutes mM OG control 0.3% Tn BP control prior add 7.1 7.1 6.9 6.9 30 ≤1.5 n.d. 4.9 n.d. 60 ≤1.5 n.d. 4.7 n.d. 120 ≤1.5 n.d. 2.8 n.d. 180 ≤1.5 n.d. ≤1.5 n.d. 240 ≤1.5 6.6 ≤1.5 6.9

(37) TABLE-US-00012 TABLE 10c VACV (Vaccinia) inactivation by SD compared to OG treatment time in VACV, 40 held VACV, 1% PS80 + held minutes mM OG control 0.3% Tn BP control prior add 6.2 6.2 6.3 6.3 30 ≤1.5 n.d. 4.6 n.d. 60 ≤1.5 n.d. 4.0 n.d. 120 ≤1.5 n.d. 3.5 n.d. 180 ≤1.5 n.d. 3.3 n.d. 240 ≤1.5 6.2 3.3 6.1

CONCLUSION

(38) The rapid inactivation kinetics observed indicate that OG and other alkyl-glycosides effectively and reliably inactivate enveloped viruses.

(39) Immunoaffinity Chromatography (IAC) Experiments rD′D3-FP was treated with OG or PS80/TNBP before loading onto an IAC resin. For this purpose, the rD′D3-FP sample was diluted with OG stock solution (600 mM) to yield a virus inactivation solution including 60 mM OG. In a control experiment, PS80/TNBP stock solution (3% PS80/0.9% TNBP) was added to yield a final concentration of 1% PS80, 0.3% TNBP. Solutions were then filtered and incubated at room temperature for 2 hours. Subsequently, IAC experiments were carried out according to Table 11. For chromatography, the immunoaffinity resin CaptureSelect Human Albumin (ThermoFisher) was used. The chromatography unit used was an ÄKTA Avant system (GE Healthcare).

(40) TABLE-US-00013 TABLE 11 Experimental conditions for immunoaffinity chromatography. Step Description 1 equilibration of column with 20 mM Tris, 10 mM EDTA pH 7.4 2 Loading of 11 mg virus inactivated rD′D3-FP/mL resin, conditioned with EDTA stock solution to a final concentration of 9 mM EDTA 3 post-load wash with 20 mM Tris, 10 mM EDTA pH 7.4 (10 column volumes (CV)) 4 wash step 2 with 400 mM NaCl, 20 mM sodium phosphate pH 6.3 (5 CV) 5 pre-elution with 375 mM MgCl.sub.2, 100 mM MES pH 6.0 (3 CV) 6 elution with 1M MgCl.sub.2, 100 mM MES pH 6.0 (5 CV). Collection of two CV. 7 Column cleaning

(41) The PS80/TNBP treatment resulted in a significant loss of 60% rD′D3-FP in the pre-elution (step 5) fraction and only 40% rD′D3-FP was found in the eluate fraction. In contrast, the OG treatment gave much higher yields, and 83% of rD′D3-FP was present in the eluate fraction. Host cell DNA clearance in the eluate fraction (180 pg/mL) was 1700-fold across the purification step with OG. In other examples when different feedstock rD′D3-FP lots with higher concentrations of host cell DNA were incubated with OG, the host cell DNA content in the eluate of the immunoaffinity chromatography step were comparable resulting in purification factors of about 25,000. Compared to PS80/TNBP treatment, OG treatment yielded 7.4-fold better host cell DNA clearance. Normalized host cell protein clearance across this step was 1270-fold for OG-treated sample, resulting in 117 ppm in the eluate (1.9-fold better compared to PS80/TNBP treatment). To allow better comparability between the eluate fractions at the same rD′D3-FP concentrations after detergent or solvent/detergent treatment, step 5 (Table 11) was omitted from the protocol in a second set of experiments to avoid splitting of the rD′D3-FP in the case of PS 80/TNBP-treated feed material into two fractions.

(42) Immunoaffinity Chromatography (IAC) Experiments (without Pre-Elution Step 5)

(43) rD′D3-FP was treated with 60 mM OG or other alkyl glycosides as specified in Table 12, 1% PS 80, 0.3% TNBP or a buffer control (500 mM NaCl, 20 mM Tris pH 7.4). Solutions were then filtered and incubated at room temperature for 2 hours.

(44) TABLE-US-00014 TABLE 12 Overview alkyl glycosides used for incubation pre immunoaffinity purification. Stock concentration Final (in 500 mM NaCl, concentration Alkyl glycoside 20 mM Tris pH 7.4) after mixing n-octyl-beta-D-glucopyranoside (OG) 600 mM 60 mM n-decyl-beta-D-glucopyranoside (DG) 600 mM 60 mM 50 mM 5 mM n-dodecyl-beta-D-glucopyranoside 5 mM 0.5 mM (DDG) n-octyl-beta-D-maltoside (OM) 600 mM 60 mM n-decyl-beta-D-maltoside (DM) 600 mM 60 mM 50 mM 5 mM n-dodecyl-beta-D-maltoside (DDM) 600 mM 60 mM 5 mM 0.5 mM

(45) After 2 hours incubation, the virus inactivated samples were loaded onto a chromatography column packed with CaptureSelect Human Albumin (ThermoFisher) resin. The chromatography unit used was an ÄKTA Avant system (GE Healthcare). The chromatography protocol is described in Table 11, step 5 was omitted in this set of experiments in order to avoid yield loss in the PS80/TNBP treated fractions.

(46) All treatment options resulted in comparable yields and protein concentrations of rD′D3-FP in the eluate fractions which were analysed for host cell DNA (HC DNA) and protein (HCP) (Table 13, FIGS. 10a and 11a).

(47) TABLE-US-00015 TABLE 13 Overview analytical data obtained with different alky glycosides and control. HC HC HC Normalize HCP Eluate DNA step HCP dHCP step fractions DNA/pg/mL DNA/ppm reduction ng/mL (ppm) reduction Buffer control 18419 2091 16 5792 657 226 OG 60 mM 146 17 1978 1649 187 794 OM 60 mM 18672 2425 15 2914 379 392 DG 60 mM 50030 7829 6 288 45 3300 DG 5 mM 28401 3235 10 6528 744 200 DM 60 mM 28417 3593 10 4283 541 274 DM 5 mM 29649 4001 10 4478 535 278 DDG 0.5 mM 20149 2577 14 12119 1551 96 DDM 60 mM 37381 5065 8 5560 753 197 DDM 0.5 mM 24566 2863 12 9154 1068 139 PS80/TNBP 31293 3246 9 2381 247 601

(48) Results for host cell DNA were comparable or slightly worse compared to buffer control for all samples except when 60 mM OG were used. This led to an eluate sample with particularly low host cell DNA content which was 126-fold lower than in buffer control and 214-fold lower than in the PS80/TNBP control (FIG. 11b). Host cell DNA was cleared by a factor of about 2000 in this particular example (DNA levels normalized to protein content).

(49) Similarly, in comparison to the buffer control treatment, the OG treatment led to a 3.5-fold better reduction in levels of host cell protein. Normalized to rD′D3-FP content, the HCP reduction across the purification step was 725-fold. If PS80/TNBP was used the HCP clearance was 32% diminished compared to OG (Table 13, FIG. 10b). Of the other alkyl glycosides tested, only DG and OM at 60 mM improved HCP clearance compared to buffer control. For OM the effect was small (1.7-fold), whereas for DG a 14.6-fold improvement was found. This effect was strongly concentration dependent, as at 5 mM DG HCP content in the eluate was comparable to buffer control.

(50) In summary, the incubation of rD′D3-FP with OG prior to the following chromatography step yields an eluate sample which is significantly purer compared to buffer or PS80/TNBP control with respect to host cell DNA and host cell protein. OG treatment also allowed to use an additional wash step (Table 1, step 5) which in case of PS80/TNBP treatment yielded significant losses.

Example 2

(51) Viral Inactivation Capacity of a Variety of Alkyl Glycosides

SUMMARY

(52) Further laboratory studies were conducted similar to those described in Example 1 to assess the virus inactivation capacity of alkyl glycosides in addition to OG. n-octyl-beta-D-glucopyranoside, n-decyl-beta-D-glucopyranoside, n-octyl-beta-D-maltoside, n-dodecyl-beta-D-maltoside, n-dodecyl-beta-D-glucopyranoside and n-decyl-beta-D-maltoside were all found to be effective (FIG. 8) as well as at lower concentrations (about twice as high as their CMC values, FIG. 9).

(53) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Example 3

(54) Reducing Process-Related Impurities by Using a Wash Step Containing OG

SUMMARY

(55) Additional studies were performed to evaluate using alkyl glycosides as wash agents in a chromatography setup and study the effect on process-related impurity, in particular protein impurity clearance. In this example cell-free harvest material of rD′D3-FP from a bioreactor process was loaded onto an anion exchange column (Poros XQ, Thermo Scientific). rD′D3-FP is expressed together with the von Willebrand factor (VWF) propeptide which is cleaved off in the cells but secreted along with rD′D3-FP into the cell supernatant and is, therefore, present at levels comparable to the product. Hence, reducing the levels of this protein impurity is important. The chromatography protocol was modified and OG added to one of the wash buffers.

(56) Method and Results

(57) The purification details of the anion exchange chromatography can be found in Table 14. Two experiments were carried out. In option 1, step 5 (wash step 2) was performed without OG, in option 2 60 mM OG was additionally included in the wash buffer.

(58) TABLE-US-00016 TABLE 14 Experimental conditions for immunoaffinity chromatography. Step Description 1 Equilibration of column with 20 mM Tris, 50 mM Nal pH 7.5 2 Loading of approx. 20 mg rD′D3-FP/mL resin 3 Post-load wash with 20 mM Tris, 50 mM NaCl pH 7.5 (5 column volumes (CV)) 4 Wash step 1 with 50 mM MES, 10 mM sodium citrate, 50 mM NaCl, pH 6.0 (15 CV) 5 Wash step 2 with Option 1: 20 mM Tris, 50 mM NaCl, 10 mM EDTA, pH 7.5 (15 CV) Option 2: 20 mM Tris, 50 mM NaCl, 10 mM EDTA, 60 mM OG, pH 7.5 (15 CV) 6 Linear elution from 50 to 500 mM NaCl in 60 mM Tris, 10 mM EDTA, pH 7.5 (5 CV). Collection of three CV. 7 Column cleaning

(59) The addition of OG in the wash step reduced the content of the VWF propeptide, a major protein impurity, significantly (see Table 15).

(60) TABLE-US-00017 TABLE 15 Analytical results for main elution fractions of experiments with and without OG in the wash buffer. Amount VWF propeptide in Amount VWF Reduction factor Wash step 2 harvest (relative propeptide in eluate (relative to buffer to rD′D3-FP) (relative to rD′D3-FP) harvest material) No OG 892,985 ppm 1,041,885 ppm    None 60 mM OG 919,508 ppm 6,228 ppm 148

(61) Without the addition of OG, the VWF propeptide was not separated at all from rD′D3-FP. Upon addition of 60 mM OG to wash step 2, the concentration of VWF propeptide in the eluate was reduced almost 150-fold. As the OG presence in wash step 2 was the only variable in these experiments, the improved protein impurity clearance can be attributed to it.

REFERENCES

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