Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof

1. A method of making a pharmaceutical formulation comprising combining:

(i) a composition comprising:

(a) a main species HER2 antibody comprising light chain and heavy chain amino acid sequences set forth in SEQ ID Nos. 15 and 16, respectively; and

(b) acidic variants of the main species antibody, comprising a disulfide reduced variant, with:

(ii) a pharmaceutically acceptable carrier.

2. The method of claim 1 wherein the percentage of acidic variants as compared to the main species antibody in the pharmaceutical formulation is less than 25%.

3. The method of claim 1 wherein the disulfide reduced variant is detectable by non-reduced capillary electrophoresis with sodium dodecyl sulfate (CE-SDS).

4. The method of claim 3 wherein the disulfide reduced variant comprises 1% to 6% of the pharmaceutical formulation, when measured by non-reduced CE-SDS.

5. The method of claim 1 wherein the disulfide reduced variant is detectable by hydrophobic interaction chromatography.

6. The method of claim 1 wherein the acidic variants further comprise a glycated variant.

7. The method of claim 1 wherein the acidic variants further comprise a non-reducible variant.

8. The method of claim 1 wherein the acidic variants further comprise a sialylated variant.

9. The method of claim 1 wherein the acidic variants further comprise a deamidated variant.

10. The method of claim 1 wherein the acidic variants further comprise a glycated variant, a deamidated variant, a sialylated variant, and a non-reducible variant.

11. The method of claim 10 wherein the disulfide reduced variant, glycated variant, deamidated variant, sialylated variant, and non-reducible variant constitute at least 75 to 80% of the acidic variants in the pharmaceutical formulation.

12. The method of claim 1 wherein the composition further comprises a variant selected from the group consisting of: an amino acid-terminal leader extension of the main species antibody, wherein the amino acid-terminal leader extension comprises or consists of VHS-; an antibody comprising a C-terminal lysine residue on one or both heavy chains thereof; and an antibody with one or more oxidized methionine residues.

13. The method of claim 1 wherein the light and heavy chains are expressed by Chinese Hamster Ovary (CHO) cells and the composition is recovered from the CHO cells.

14. The method of claim 13 comprising incubating the composition with cell culture media for 12 days, wherein the cell culture media comprises glucose or peptone.

15. The method of claim 1 further comprising confirming the pharmaceutical formulation is effective for treating HER2 positive cancer in a patient comprising analyzing potency, monomer content, or pharmacokinetics of the pharmaceutical formulation.

16. The method of claim 1 further comprising confirming the pharmaceutical formulation is effective for treating HER2 positive cancer in a patient comprising analyzing potency, monomer content, and pharmacokinetics of the pharmaceutical formulation.

17. A method of making a pharmaceutical composition comprising preparing a composition comprising a main species HER2 antibody comprising light chain and heavy chain amino acid sequences set forth in SEQ ID Nos. 15 and 16, respectively, and acidic variants of the main species antibody, comprising a disulfide reduced variant, wherein the disulfide reduced variant comprises 1% to 6% of the composition, when measured by non-reduced CE-SDS.

18. The method of claim 17 wherein the percentage of acidic variants as compared to the main species antibody in the composition is less than 25%.

19. The method of claim 17 further comprising combining the composition with a pharmaceutically acceptable carrier.

20. The method of claim 17 wherein the acidic variants further comprise a glycated variant.

21. The method of claim 17 wherein the acidic variants further comprise a non-reducible variant.

22. The method of claim 17 wherein the acidic variants further comprise a sialylated variant.

23. The method of claim 17 wherein the acidic variants further comprise a deamidated variant.

24. The method of claim 17 wherein the acidic variants further comprise a glycated variant, a deamidated variant, a sialylated variant, and a non-reducible variant.

25. A method of making a pharmaceutical formulation comprising combining:

(i) a composition comprising:

(a) a main species HER2 antibody comprising light chain and heavy chain amino acid sequences set forth in SEQ ID Nos. 15 and 16, respectively; and

(b) variants that elute earlier than the main species antibody upon separation by cation exchange chromatography, the variants comprising a disulfide reduced variant, with:

(ii) a pharmaceutically acceptable carrier.

26. The method of claim 25 wherein the percentage of acidic variants as compared to the main species antibody in the pharmaceutical formulation is less than 25%.

27. The method of claim 25 wherein the disulfide reduced variant comprises 1% to 6% of the pharmaceutical formulation, when measured by non-reduced CE-SDS.

28. A method of making a pharmaceutical composition comprising preparing a composition comprising a main species HER2 antibody comprising light chain and heavy chain amino acid sequences set forth in SEQ ID Nos. 15 and 16, respectively, and variants that elute earlier than the main species antibody upon separation by cation exchange chromatography, the variants comprising disulfide reduced variant, glycated variant, deamidated variant, sialylated variant, and non-reducible variant constituting at least 75 to 80% of the acidic variants in the composition.

29. The method of claim 28 wherein the percentage of acidic variants as compared to the main species antibody in the composition is less than 25%.

30. The method of claim 28 wherein the disulfide reduced variant comprises 1% to 6% of the composition, when measured by non-reduced CE-SDS.