Methods and compositions comprising purified recombinant polypeptides
View Patent ↗Purified recombinant polypeptides isolated from Chinese hamster ovary host cells, including antibodies, such as therapeutic antibodies, and methods of making and using such polypeptides are provided.
1. A method of reducing phospholipase B-like 2 (PLBL2) levels in a preparation of recombinant polypeptide produced in Chinese hamster ovary host cells and having PLBL2 levels of greater than 100 ng/mg, comprising:
subjecting the preparation of recombinant polypeptide produced in Chinese hamster ovary host cells and having PLBL2 levels of greater than 100 ng/mg to hydrophobic interaction chromatography (HIC) with a resin-containing column, and
obtaining a purified preparation comprising the recombinant polypeptide and a residual amount of hamster PLBL2,
wherein the recombinant polypeptide is an anti-IL13 monoclonal antibody comprising three heavy chain CDRs, CDR-H1 comprising the amino acid sequence of SEQ ID NO.: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1 comprising the amino acid sequence of SEQ ID NO.: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO.: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO.: 6.
2. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 20 ng/mg.
3. The method of claim 1 , wherein the resin comprises PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin.
4. The method of claim 3 , wherein the PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containing column is operated in flow-through mode.
5. The method of claim 1 , wherein the antibody is a humanized monoclonal antibody.
6. The method of claim 5 , wherein the antibody is IgG1, or IgG2, or IgG3, or IgG4.
7. The method of claim 6 , wherein the antibody is IgG4.
8. The method of claim 7 , wherein the antibody is lebrikizumab.
9. The method of claim 1 , wherein the HIC comprises operating a resin-containing column in flow-through mode and an equilibration buffer and a wash buffer, wherein each of the equilibration buffer and the wash buffer comprise 50 mM sodium acetate pH 5.0.
10. The method of claim 9 , wherein the flow-through is monitored by absorbance at 280 nanometers and the flow-through is collected between 0.5 OD to 1.5 OD.
11. The method of claim 9 , wherein the flow-through is collected for a maximum of 8 column volumes.
12. The method of claim 9 , further comprising an affinity chromatography step.
13. The method of claim 12 , wherein the affinity chromatography is protein A chromatography.
14. The method of claim 9 , further comprising an ion exchange chromatography step.
15. The method of claim 14 , wherein the ion exchange chromatography is anion exchange chromatography.
16. The method of claim 9 comprising a first Protein A affinity chromatography step and a second anion exchange chromatography step prior to the hydrophobic interaction chromatography (HIC) step.
17. The method of claim 16 , wherein the affinity chromatography step comprises MAB SELECT SURE™ resin, the anion exchange chromatography step comprises Q SEPHAROSE™ Fast Flow, and the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow (high sub).
18. The method of claim 17 , wherein:
the affinity chromatography step comprises operating a MABSELECT SURE™ resin-containing column in bind-elute mode;
the anion exchange chromatography step comprises operating a Q SEPHAROSE™ Fast Flow resin-containing column in bind-elute mode, and
the HIC step comprises operating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containing column in flow-through mode.
19. The method of claim 2 , wherein the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay.
20. The method of claim 19 , wherein the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA.
21. The method of claim 19 , wherein the mass spectrometry assay is LC-MS/MS.
22. The method of claim 6 , wherein the antibody is IgG1.
23. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 15 ng/mg.
24. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 10 ng/mg.
25. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 8 ng/mg.
26. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 5 ng/mg.
27. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 3 ng/mg.
28. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 2 ng/mg.
29. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 1 ng/mg.
30. The method of claim 1 , wherein the residual amount of hamster PLBL2 is less than 0.5 ng/mg.
31. The method of claim 1 , wherein the anti-IL13 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO.: 7 or SEQ ID NO: 8.
32. The method of claim 1 , wherein the anti-IL13 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO.: 9.
33. The method of claim 1 , wherein the anti-IL13 antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs.: 10 to 13.
34. The method of claim 1 , wherein the anti-IL13 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO.: 14.
35. The method of claim 1 , wherein the anti-IL13 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO.: 7 or SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO.: 9.
36. The method of claim 1 , wherein the anti-IL13 antibody comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs.: 10 to 13 and a light chain comprising the amino acid sequence of SEQ ID NO.: 14.
37. The method of claim 1 , wherein the PLBL2 levels are greater than 200 ng/mg.
38. The method of claim 1 , wherein the PLBL2 levels are greater than 300 ng/mg.