Methods and compositions comprising purified recombinant polypeptides

Patent metadata

Field	Value
Publication number	US-10822404-B2
Application number	US-201815902145-A
Country	US
Kind code	B2
Filing date	Feb 22, 2018
Priority date	Sep 13, 2013
Publication date	Nov 3, 2020
Grant date	Nov 3, 2020

Abstract

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.

First claim

What is claimed is:

1. A method of purifying a recombinant antibody produced in Chinese hamster ovary host cells and having phospholipase B-like 2 (PLBL2) levels greater than 100 ng/mg, wherein the method comprises performing a hydrophobic interaction chromatography (HIC) step and provides a purified preparation comprising the recombinant antibody and a residual amount of hamster PLBL2, and wherein the residual amount of hamster PLBL2 is less than 20 ng/mg.

2. The method of claim 1 , wherein the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin.

3. The method of claim 2 , wherein the HIC step comprises operating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containing column in flow-through mode.

4. The method of claim 1 , wherein the recombinant antibody is an antigen-binding antibody fragment.

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 1 , wherein the HIC step 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.

9. The method of claim 8 , 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.

10. The method of claim 8 , wherein the flow-through is collected for a maximum of 8 column volumes.

11. The method of claim 8 , further comprising an affinity chromatography step.

12. The method of claim 11 , wherein the affinity chromatography is protein A chromatography.

13. The method of claim 8 , further comprising an ion exchange chromatography step.

14. The method of claim 13 , wherein the ion exchange chromatography is anion exchange chromatography.

15. The method of claim 8 comprising a first Protein A affinity chromatography step and a second anion exchange chromatography step prior to the hydrophobic interaction chromatography (HIC) step.

16. The method of claim 15 , wherein the affinity chromatography step comprises MABSELECT SURE resin, the anion exchange chromatography step comprises Q SEPHAROSE™ Fast Flow, and the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow (high sub).

17. The method of claim 16 , 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.

18. The method of claim 1 wherein the HIC step 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 150 mM sodium acetate pH 5.0.

19. The method of claim 18 , wherein the flow-through is monitored by absorbance at 280 nanometers and the flow-through is collected beginning at 0.5 OD and for 10 column volumes.

20. The method of claim 18 , further comprising an affinity chromatography step.

21. The method of claim 20 , wherein the affinity chromatography is protein A chromatography.

22. The method of claim 18 , further comprising a mixed mode chromatography step.

23. The method of claim 18 comprising a first Protein A affinity chromatography step and a second mixed mode chromatography step prior to the hydrophobic interaction chromatography (HIC) step.

24. The method of claim 23 , wherein the affinity chromatography step comprises MABSELECT SURE resin, the mixed mode chromatography step comprises CAPTO™ Adhere, and the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow (high sub).

25. The method of claim 24 , wherein:
the affinity chromatography step comprises operating a MABSELECT SURE™ resin-containing column in bind-elute mode;
the mixed mode chromatography step comprises operating a CAPTO™ Adhere resin-containing column in flow-through mode, and
the HIC step comprises operating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containing column in flow-through mode.

26. The method of claim 1 , wherein the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay.

27. The method of claim 26 , wherein the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA.

28. The method of claim 26 , wherein the mass spectrometry assay is LC-MS/MS.

Assignees

• Genentech Inc

Inventors

• Yu X Christopher
• Fisher Susan C
• Naim Atia
• Sanchez Ailen M
• Teske Christopher A
• Vanderlaan Martin
• Amurao Annamarie
• Franklin Jayme
• Victa Corazon

Classifications

• C07K1/18 Primary

  Ion-exchange chromatography · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K16/089

  Cytomegalovirus · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title