Selective reduction of cysteine residues in IL-17 antibodies
The present disclosure relates to methods for selectively reducing CysL97 in a preparation of IL-17 antibodies or antigen binding fragments thereof (e.g., a preparation of secukinumab antibodies) that have been recombinantly produced by mammalian cells. Also provided are purified preparations of IL-17 antibodies or antigen binding fragments thereof produced by such methods, e.g, purified preparations of secukinumab, wherein the level of intact IL-17 antibodies or antigen binding fragments thereof (e.g., secukinumab) in the preparation is high, e.g., at least about 90%, as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS), and wherein the level of activity of IL-17 antibodies or antigen binding fragments thereof (e.g., secukinumab) in the preparation is high, e.g., at least about 92%, as measured by cation exchange chromatograph (CEX).
1. A method for selectively reducing CysL97 in a preparation of secukinumab antibody that have been recombinantly produced by mammalian cells comprising:
a) contacting the preparation with at least one reducing agent to form a reducing mixture, wherein the at least one reducing agent is a thiol-containing reducing agent having a standard oxidation-reduction potential, E°, of about −0.20 V-about −0.23 V at pH 7.0, as measured by thiol-disulfide exchange, and wherein the molar ratio of reducing agent to antibody in the reducing mixture is between about 46:1-about 118:1; and
b) incubating the reducing mixture at a temperature of between about 32° C. to about 42° C. while maintaining a volumetric oxygen mass-transfer coefficient (k L a*)<0.37 h −1 ;
wherein prior to step a) the initial percent oxygen saturation in the preparation is at least 60%, as measured using an oxygen probe calibrated at 25° C.
2. The method according to claim 1 , wherein the at least one reducing agent is cysteine, and wherein the molar ratio of cysteine:antibody in the reducing mixture is between about 46:1-about 118:1.
3. The method according to claim 2 , wherein the k L a* is:
a. <0.37 h −1 , and wherein the molar ratio of cysteine to secukinumab antibody in the reducing mixture is between about 56:1-about 118:1, and wherein the reducing mixture is incubated according to step b) for up to 240 minutes;
b. <0.37 h −1 , and wherein the molar ratio of cysteine to secukinumab antibody in the reducing mixture is between about 77:1-about 118:1, and wherein the reducing mixture is incubated according to step b) for up to 300 minutes;
c. <0.37 h −1 , and wherein the molar ratio of cysteine to secukinumab antibody in the reducing mixture is between about 46:1-about 118:1;
d. <0.37 h −1 , and wherein the molar ratio of cysteine to secukinumab antibody in the reducing mixture is between about 54:1-about 82:1; or
e. <0.27 h −1 .
4. The method according to claim 3 , wherein the molar ratio of cysteine to secukinumab antibody in the reducing mixture is about 66:1.
5. The method according to claim 3 , wherein the concentration of cysteine in the reducing mixture is at about 4.0 mM-about 8.0 mM.
6. The method according to claim 2 , wherein prior to step a), the concentration of secukinumab antibody in the preparation is about 4 mg/ml to about 19.4 mg/ml.
7. The method according to claim 6 , wherein prior to step a) the pH of the preparation is about 7.3 to about 8.5.
8. The method according to claim 7 , wherein prior to step a) the initial percent oxygen saturation in the preparation is at least 80%, as measured using an oxygen probe calibrated at 25° C.
9. The method according to claim 2 , wherein between step a) and step b), the reducing mixture is heated to a temperature that is between about 32° C. to 42° C.
10. The method according to claim 9 , wherein the reducing mixture is heated for about 45 to about 90 minutes.
11. The method according to claim 9 , wherein the k L a* during heating is ≤0.69 h −1 .
12. The method according to claim 2 , wherein between step a) and step b), the reducing mixture is heated to a temperature between about 32° C.-about 42° C. for about 45-about 90 minutes, wherein the k L a* during heating is ≤0.69 h −1 .
13. The method according to claim 2 , wherein during step b) the reducing mixture is stirred for ≤15 minutes per hour.
14. The method according to claim 2 , wherein the activity of secukinumab antibody in the preparation increases by at least 10% within about 60 minutes following step b), as measured by cystamine-cation exchange chromatography (cystamine-CEX).
15. The method according to claim 2 , further comprising:
c) after step b), cooling the reducing mixture to room temperature.
16. The method according to claim 15 , wherein the mixture is cooled according to step c) for about 45 to about 90 minutes.
17. The method according to claim 15 , wherein the k L a* during step c) is ≤0.69 h −1 .
18. The method according to claim 2 , further comprising:
c) adjusting the pH of the mixture resultant from step b) to between about 5.1 to about 5.3.
19. The method according to claim 18 , wherein adjusting step c) comprises adding o-phosphoric acid to the mixture resultant from step b).
20. The method according to claim 18 , wherein at least 90% of the secukinumab antibody in the mixture resultant from step c) is intact, as measured by CE-SDS.
21. The method according to claim 18 , wherein at least 90% of secukinumab antibody in the mixture resultant from step c) is active, as measured by cystamine-CEX.
22. The method according to claim 2 , wherein the reducing mixture further comprises about 0.8 mM to about 1.3 mM EDTA.
23. The method according to claim 2 , wherein an oxidized form of the reducing reagent is not added to the reducing mixture.
24. The method according to claim 2 , wherein a denaturant is not added to the reducing mixture.
25. The method according to claim 1 , wherein the reducing mixture is incubated according to step b) for about 210 to about 420 minutes.