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1. (WO2019028382) METHOD OF CONJUGATION OF CYS-MABS
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CLAIMS

What is claimed is:

1. A method for preparing an antibody conjugate or antibody fragment conjugate, the method comprising the steps of;

a) obtaining a composition comprising an antibody or antibody fragment;

b) exposing the antibody or antibody fragment to a cysteine blocking agent, wherein the cysteine blocking agent forms a stable mixed-disulfide with at least one cysteine residue of the antibody or antibody fragment;

c) adding a reducing agent to the composition to form a reduction mix and allowing a reduction reaction to occur such that the reduction mix comprises a reduced antibody or reduced antibody fragment;

d) adding an oxidizing agent to the reduction mix to form an oxidized mix and allowing an oxidizing reaction to occur such that the oxidizing mix comprises an oxidized antibody or oxidized antibody fragment; and

e) adding an activated chemical moiety to the oxidized mix to form a conjugation mix and allowing a conjugation reaction to occur such that an antibody conjugate or antibody fragment conjugate is formed.

2. The method according to claim 1, wherein the mixed disulfide is an antibody or antibody fragment with a capped free cysteine.

3. The method according to claim 2, wherein the antibody or antibody fragment with a capped free cysteine comprises a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione.

4. The method according to claim 1, wherein following step b) and before step c) cation exchange chromatography is performed to remove excess cysteine blocking agent.

5. The method according to any previous claim, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

6. The method according to claim 5, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4: 1 (mole/mole).

7. The method according to any previous claim, wherein following step c) and before step d) a buffer exchange step is performed to remove the reducing agent.

8. The method according to claim 7, wherein the buffer exchange step is ultrafiltration/diafiltration.

9. The method according to any previous claim, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

10. The method according to claim 9, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6: 1 (mole/mole).

11. The method according to any previous claim, wherein the activated chemical moiety is a peptide comprising a halogen, wherein the halogen is selected from the group consisting of Br, I, and CI.

12. The method according to claim 11, wherein the ratio of activated chemical moiety to antibody or antibody fragment is 2 to 3: 1 (mole/mole).

13. The method according to any previous claim, wherein following step e), a purification step is performed to remove the activated chemical moiety.

14. The method according to claim 13, wherein the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration/diafiltration, or hydrophobic interaction chromatography ("HIC") followed by ultrafiltration/diafiltration.

15. A method for preparing an antibody conjugate or antibody fragment conjugate, the method comprising the steps of;

a) obtaining a composition comprising a mixed disulfide comprising an antibody or antibody fragment;

b) adding a reducing agent to the composition to form a reduction mix and allowing a reduction reaction to occur such that the reduction mix comprises a reduced antibody or reduced antibody fragment;

c) adding an oxidizing agent to the reduction mix to form an oxidized mix and allowing an oxidizing reaction to occur such that the oxidizing mix comprises an oxidized antibody or oxidized antibody fragment; and

d) adding an activated chemical moiety to the oxidized mix to form a conjugation mix and allowing a conjugation reaction to occur such that an antibody conjugate or antibody fragment conjugate is formed.

16. The method according to claim 15, wherein the mixed disulfide is an antibody or antibody fragment with a capped free cysteine.

17. The method according to claim 16, wherein the antibody or antibody fragment with a capped free cysteine comprises a cap selected from the group consisting of cysteine, cysteamine, cystamine, and glutathione.

18. The method according to claim 15, wherein following step a) and before step b) cation exchange chromatography is performed to remove excess cysteine blocking agent.

19. The method according to any one of claims 15-18, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

20. The method according to claim 19, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4: 1 (mole/mole).

21. The method according to any one of claims 15-20, wherein following step b) and before step c) a buffer exchange step is performed to remove the reducing agent.

22. The method according to claim 21, wherein the buffer exchange step is ultrafiltration/diafiltration.

23. The method according to any one of claims 15-22, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

24. The method according to claim 23, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6: 1 (mole/mole).

25. The method according to any one of claims 15-24, wherein the activated chemical moiety is a peptide comprising a halogen, wherein the halogen is selected from the group consisting of Br, I, and CI.

26. The method according to claim 25, wherein the ratio of activated chemical moiety to antibody or antibody fragment is 2 to 3: 1 (mole/mole).

27. The method according to any one of claims 15-26, wherein following step d), a purification step is performed to remove the activated chemical moiety.

28. The method according to claim 27, wherein the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration/diafiltration, or hydrophobic interaction chromatography ("HIC") followed by ultrafiltration/diafiltration.

29. A method for preparing an antibody conjugate or antibody fragment conjugate, the method comprising the steps of;

a) obtaining a composition comprising a reduction mix comprising a reduced antibody or reduced antibody fragment;

b) adding an oxidizing agent to the reduction mix to form an oxidized mix and allowing an oxidizing reaction to occur such that the oxidizing mix comprises an oxidized antibody or oxidized antibody fragment; and

c) adding an activated chemical moiety to the oxidized mix to form a conjugation mix and allowing a conjugation reaction to occur such that an antibody conjugate or antibody fragment conjugate is formed.

30. The method according to claim 29, wherein the reducing agent is selected from the group consisting of triphenylphosphine-3,3',3"-trisulfonate ("TPPTS"), tris(2-carboxyethyl)phosphine ("TCEP"), and triphenylphosphine-3,3'-disulfonate ("TPPDS").

31. The method according to claim 30, wherein the ratio of reducing agent to antibody or antibody fragment is 2 to 4: 1 (mole/mole).

32. The method according to any one of claims 29-31, wherein following step a) and before step b) a buffer exchange step is performed to remove the reducing agent.

33. The method according to claim 32, wherein the buffer exchange step is ultrafiltration/diafiltration.

34. The method according to any one of claims 29-33, wherein the oxidizing agent is dehydroascorbic acid ("DHAA").

35. The method according to claim 34, wherein the ratio of oxidizing agent to antibody or antibody fragment is 3 to 6: 1 (mole/mole).

36. The method according to any one of claims 29-35, wherein the activated chemical moiety is a peptide comprising a halogen, wherein the halogen is selected from the group consisting of Br, I, and CI.

37. The method according to claim 36, wherein the ratio of activated chemical moiety to antibody or antibody fragment is 2 to 3: 1 (mole/mole).

38. The method according to any one of claims 29-37, wherein following step c), a purification step is performed to remove the activated chemical moiety.

39. The method according to claim 38, wherein the purification step includes hydrophobic interaction chromatography ("HIC"), ultrafiltration/diafiltration, or hydrophobic interaction chromatography ("HIC") followed by ultrafiltration/diafiltration.

40. The method according to any previous claim, wherein the antibody or antibody fragment comprises a cysteine residue at a position selected from the group consisting of D70 of the antibody light chain relative to reference sequence SEQ ID NO: 7; E276 of the antibody heavy chain relative to reference sequence SEQ ID NO: 8; and T363 of the antibody heavy chain relative to reference sequence SEQ ID NO: 8.