Protein purification and virus inactivation with alkyl glycosides
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.
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.