Modulation of charge variants in a monoclonal antibody composition

• US 10,696,735 B2
• Filed: 01/21/2016
• Issued: 06/30/2020
• Est. Priority Date: 01/21/2015
• Status: Active Grant

• Current Assignee: Outlook Therapeutics, Inc.
• Original Assignee: Outlook Therapeutics, Inc.
• Inventors: Yonan, Chris, Caroselli, Christine, Dendamrongvit, Wiphusanee, Gangloff, Scott
• Primary Examiner(s): Kolker, Daniel E
• Assistant Examiner(s): Rogers, James L
• Application Number:
  US15/545,271
• Publication Number:
  US 20180009876A1
• Time in Patent Office: 1,622 Days
• Field of Search: None
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/362 Cation-exchange
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01D 15/3847 Multimodal interactions
• CPC Class Codes: C07K 1/165 mixed-mode chromatography
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Abstract

Combinations of different chromatography modalities with particularly refined conditions significantly reduce acid charge variants in a preparation of monoclonal antibodies. The process for reducing acid charge variants utilizes a combination of anion exchange and hydrophobic interaction chromatography, followed by cation exchange chromatography polishing, whereby the levels of acidic or basic charge species of the monoclonal antibodies may be modulated to a desired level.

Claims

1. A process for removing acid charge variants from a monoclonal antibody, comprising

(a) loading a mammalian cell-expressed monoclonal antibody preparation onto a Protein A support, and eluting the monoclonal antibody from the Protein A support, thereby producing a first eluate comprising the monoclonal antibody;

(b) loading the first eluate from step (a) onto an anion exchange and hydrophobic interaction (AEX/HIC) chromatography support, and allowing the first eluate to flow through the support, thereby producing a flow-through pool comprising the monoclonal antibody;

(c) loading the flow-through pool comprising the monoclonal antibody onto a cation exchange (CEX) chromatography support having an antibody binding capacity of from about 25 g/L to about 65 g/L and performing wash steps comprising;

(i) performing a first wash step comprising a wash buffer having a pH from about 5.8 to about 6.6 and a conductivity target from about 1.0 mS/cm to about 3.6 mS/cm;

(ii) performing a second wash step comprising a wash buffer having a pH from about 5.8 to about 6.6, a conductivity target from about 6.6 mS/cm to about 7.6 mS/cm, and a sodium chloride concentration from about 30 mM to about 60 mM;

wherein the second wash step comprises determining when the absorbance units measured at UV A280 decrease from about 7% to about 14% from the peak absorbance units measured at UV A280; and

(iii) performing a third wash step comprising a wash buffer having a pH from about 5.8 to about 6.6 and a conductivity target from about 1.0 mS/cm to about 3.6 mS/cm; and

(d) eluting the monoclonal antibody from the CEX chromatography support in step with an elution buffer having a pH of from about 6.0 to about 6.4 and a conductivity target of from about 10 mS/cm to about 14 mS/cm, thereby producing a second eluate comprising the monoclonal antibody and from about 10% to about 20% by weight of acid charge variants of the monoclonal antibody.

2. The method of claim 1, wherein the monoclonal antibody specifically binds to tumor necrosis factor (TNF) alpha.

3. The method of claim 1, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:

1 and a light chain comprising the amino acid sequence of SEQ ID NO;

2.

4. The method of claim 1, wherein the the wash buffer of steps (c)(i), (c)(ii) and (c)(iii) comprises a pH of from about 5.9 to about 6.5.

5. The method of claim 1, wherein the the wash buffer of steps (c)(i), (c)(ii) and (c)(iii) comprises a pH of from about 6 to about 6.4.

6. The method of claim 1, wherein the the wash buffer of steps (c)(i), (c)(ii) and (c)(iii) comprises a pH of from about 6.1 to about 6.3.

7. The method of claim 1, wherein the the wash buffer of steps (c)(i), (c)(ii) and (c)(iii) comprises a pH of about 6.2.

8. The method of claim 1, wherein the wash buffer of wash step (c)(ii) comprises a conductivity target of from about 6.8 mS/cm to about 7.4 mS/cm.

9. The method of claim 1, wherein the wash buffer of wash step (c)(ii) comprises a conductivity target of from about 6.9 mS/cm to about 7.3 mS/cm.

10. The method of claim 1, wherein the wash buffer of wash step (c)(ii) comprises a conductivity target of from about 7 mS/cm to about 7.2 mS/cm.

11. The method of claim 1, wherein the wash buffer of wash step (c)(ii) comprises a conductivity target of about 7.2 mS/cm.

12. The method of claim 1, wherein the elution buffer comprises a pH of from about 6.1 to about 6.3.

13. The method of claim 1, wherein the elution buffer comprises a pH of about 6.2.

14. The method of claim 1, wherein the elution step comprises a conductivity target of from about 11 mS/cm to about 14 mS/cm.

15. The method of claim 1, wherein the elution step comprises a conductivity target of from about 12 mS/cm to about 14 mS/cm.

16. The method of claim 1, wherein the elution step comprises a conductivity target of from about 12 mS/cm to about 13 mS/cm.

17. The method of claim 1, wherein the elution step comprises a conductivity target of from about 12.3 mS/cm to about 13.3 mS/cm.

18. The method of claim 1, wherein the elution step comprises a conductivity target of from about 12.6 mS/cm to about 13 mS/cm.

19. The method of claim 1, wherein the elution step comprises a conductivity target of about 12.8 mS/cm.

20. The method of claim 1, wherein the second eluate comprises the monoclonal antibody and from about 9% to about 15% by weight of acid charge variants of the monoclonal antibody.

21. The method of claim 1, wherein the second eluate comprising the monoclonal antibody and from about 10% to about 14% by weight of acid charge variants of the monoclonal antibody.

22. The method of claim 1, wherein the antibody preparation loaded onto the Protein A support comprises from about 20% to about 30% by weight of acid charge variants of the monoclonal antibody.

23. The of claim 1, wherein the antibody preparation loaded onto the Protein A support comprises from about 22% to about 28% by weight of acid charge variants of the monoclonal antibody.

24. The method of claim 1, wherein the antibody preparation loaded onto the Protein A support comprises from about 24% to about 26% by weight of acid charge variants of the monoclonal antibody.

25. The method of claim 1, wherein the antibody preparation loaded onto the Protein A support comprises about 25% by weight of acid charge variants of the monoclonal antibody.

26. The method of claim 1, further comprising filtering the second eluate with a filter having a pore size of from about 15 nm to about 20 nm.

27. The method of claim 1, further comprising concentrating and diafiltrating the second eluate.

28. The method of claim 1, wherein the elution buffer comprises sodium chloride and step (d) comprises increasing the sodium chloride concentration of the elution buffer according to a linear gradient.

29. The method of claim 1, wherein the elution buffer comprises sodium chloride and step (d) comprises increasing the sodium chloride concentration of the elution buffer according to a step-wise gradient.

30. The method of claim 28, wherein the sodium chloride concentration is increased to about 100 mM.