Protein purification methods to reduce acidic species

Patent metadata

Field	Value
Publication number	US-9957318-B2
Application number	US-201615149898-A
Country	US
Kind code	B2
Filing date	May 9, 2016
Priority date	Apr 20, 2012
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification.

First claim

The invention claimed is:

1. A method of making a pharmaceutical composition, comprising mixing
(a) a composition comprising adalimumab, wherein the composition comprises less than 10% total acidic species of adalimumab, wherein the acidic species of adalimumab correspond to the peaks that elute earlier than the main peak in a WCX-10 HPLC chromatogram of adalimumab, wherein the WCX-10 HPLC chromatogram is generated using a first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein the WCX-10 HPLC chromatogram is generated using detection at 280 nm; and
(b) a pharmaceutically acceptable carrier, thereby making a pharmaceutical composition.

2. The method of claim 1 , wherein the acidic species of adalimumab comprise a first acidic region (AR1) and a second acidic region (AR2).

3. The method of claim 1 , wherein the adalimumab is produced in a mammalian host cell grown in cell culture.

4. The method of claim 3 , wherein the mammalian host cell is selected from the group consisting of a CHO cell, an NSO cell, a COS cell, and an SP2 cell.

5. The method of claim 1 , wherein the low acidic species composition comprises less than 3.8% total acidic species of adalimumab.

6. The method of claim 2 , wherein the low acidic species composition comprises 0.8% AR1 and 3.0% AR2.

7. The method of claim 1 , wherein the low acidic species composition comprises less than 2.4% total acidic species of adalimumab.

8. The method of claim 2 , wherein the low acidic species composition comprises 0.2% AR1 and 2.2% AR2.

9. The method of claim 1 , wherein the low acidic species composition comprises 4.7%-8.3% total acidic species of adalimumab.

10. The method of claim 1 , wherein adalimumab is present in the pharmaceutical composition at a concentration of 0.1-250 mg/mL.

11. The method of claim 1 , further comprising filling a syringe with the pharmaceutical composition.

12. The method of claim 1 , wherein the pharmaceutically acceptable carrier comprises one or more excipient selected from the group consisting of a buffer, a surfactant and a polyalcohol, or a combination thereof.

13. The method of claim 12 , wherein the buffer is an amino acid.

14. The method of claim 13 , wherein the amino acid is histidine.

15. The method of claim 12 , wherein the buffer is sodium citrate.

16. The method of claim 12 , wherein the surfactant is polysorbate 80.

17. The method of claim 12 , wherein the polyalcohol is mannitol.

18. The method of claim 1 , wherein the pharmaceutically acceptable carrier comprises sodium chloride.

19. A method of making a pharmaceutical composition, comprising mixing
(a) a composition comprising adalimumab, wherein the composition comprises less than 10% total acidic species of adalimumab, wherein the acidic species of adalimumab are quantified based on the relative area percent of peaks that elute earlier than the main peak in a WCX-10 HPLC chromatogram of adalimumab wherein the WCX-10 HPLC chromatogram is generated using a first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5) and wherein the WCX-10 HPLC chromatogram is generated using detection at 280 nm; and
(b) a pharmaceutically acceptable carrier comprising a surfactant and a polyalcohol, thereby making a pharmaceutical composition; and
filling a syringe with the pharmaceutical composition.

20. The method of claim 19 , wherein the acidic species of adalimumab comprise a first acidic region (AR1) and a second acidic region (AR2).

21. The method of claim 19 , wherein the adalimumab is produced in a mammalian host cell grown in cell culture.

22. The method of claim 21 , wherein the mammalian host cell is selected from the group consisting of a CHO cell, an NSO cell, a COS cell, and an SP2 cell.

23. The method of claim 19 , wherein the low acidic species composition comprises less than 3.8% total acidic species of adalimumab.

24. The method of claim 20 , wherein the low acidic species composition comprises 0.8% AR1 and 3.0% AR2.

25. The method of claim 19 , wherein the low acidic species composition comprises less than 2.4% total acidic species of adalimumab.

26. The method of claim 20 , wherein the low acidic species composition comprises 0.2% AR1 and 2.2% AR2.

27. The method of claim 19 , wherein the low acidic species composition comprises 4.7%-8.3% total acidic species of adalimumab.

28. The method of claim 19 , wherein the surfactant is polysorbate 80.

29. The method of claim 19 , wherein the polyalcohol is mannitol.

30. The method of claim 19 , wherein the pharmaceutically acceptable further comprises sodium chloride.

Assignees

• Abbvie Inc

Inventors

• Ramasubramanyan Natarajan
• Yang Lihua
• Herigstad Matthew Omon
• Yang Hong
• Chumsae Christopher

Classifications

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• C07K2317/14

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

• C07K2317/21

  from primates, e.g. man · CPC title

• A61K39/39591

  Stabilisation, fragmentation · CPC title