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 - 46 . (canceled)
47 . A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that has been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with at least one reducing agent in a system 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:antibody in the reducing mixture is between about 46:1-about 118:1; and
b) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer coefficient (k L a*) in the system of ≤about 0.37 h −1 , said k L a* being calculated by adapting a saturation curve to an experimentally-derived dissolved oxygen curve;
wherein prior to step a) the initial percent oxygen saturation in the preparation is at least about 60%, as measured using an oxygen probe calibrated at 25° C., and wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable domain (VH) comprising the three complementary determining regions (CDRs) of the VH set forth as SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the three CDRs of the VL set forth as SEQ ID NO:10.
48 . A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that has been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with cysteine in a system to form a reducing mixture, wherein the molar ratio of cysteine:antibody in the reducing mixture is between about 46:1-about 118:1; and
b) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer coefficient (k L a*) in the system of ≤about 0.37 h −1 , said k L a* being calculated by
adapting a saturation curve to an experimentally-derived dissolved oxygen curve, wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable domain (VH) comprising the three complementary determining regions (CDRs) of the VH set forth as SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the three CDRs of the VL set forth as SEQ ID NO:10.
49 . The method according to claim 48 , wherein prior to step a) the initial percent oxygen saturation in the preparation is at least about 60%, as measured using an oxygen probe calibrated at 25° C.
50 . The method according to claim 49 , wherein the k L a* in the system during step b) is:
a. ≤about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 antibodies 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 about 240 minutes;
b. ≤about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 antibodies 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 about 300 minutes.
c. <about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is between about 46:1-about 118:1;
d. <about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is between about 54:1-about 82:1; or
e. ≤about 0.27 h −1 .
51 . The method according to claim 50 , wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is about 66:1.
52 . The method according to claim 49 , wherein the concentration of cysteine in the reducing mixture is about 4.0 mM-about 8.0 mM.
53 . The method according to any claim 48 , 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* in the system during heating is ≤about 0.69 h −1 , said k L a* being calculated by adapting a saturation curve to an experimentally-derived dissolved oxygen curve.
54 . The method according to claim 48 , wherein the reducing mixture is incubated according to step b) at a temperature between about 20° C.-about 42° C. for about 210-about 420 minutes.
55 . The method according to claim 48 , wherein an oxidized form of the reducing reagent is not added to the reducing mixture.
56 . The method according to claim 48 , wherein a denaturant is not added to the reducing mixture.
57 . A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that has been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with about 4.0 mM-about 8.0 mM cysteine in a system to form a reducing mixture;
b) heating the reducing mixture to a temperature between about 32° C.-about 42° C. for about 45-about 90 minutes, wherein the volumetric oxygen mass-transfer coefficient (k L a*) in the system during heating is <about 0.69 h −1 ; and
c) incubating the reducing mixture at a temperature between about 20° C.-about 42° C. while maintaining the k L a* in the system:
i. ≤about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 in the reducing mixture is between about 56:1-about 118:1, and wherein the reducing mixture is incubated for up to about 240 minutes;
ii. ≤about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 in the reducing mixture is between about 77:1-about 118:1, and wherein the reducing mixture is incubated for up to about 300 minutes;
iii. <about 0.37 h 1 , and wherein the molar ratio of cysteine:IL-17 in the reducing mixture is between about 46:1-about 118:1;
iv. <about 0.37 h −1 , and wherein the molar ratio of cysteine:IL-17 in the reducing mixture is between about 54:1-about 82:1; or
v. ≤about 0.27 h −1 ,
said k L a* being calculated by adapting a saturation curve to an experimentally-derived dissolved oxygen curve, wherein prior to step a) the initial percent oxygen saturation in the preparation is at least about 60%, as measured using an oxygen probe calibrated at 25° C.
58 . A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that has been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with a set of oxidation/reduction reagents selected from cysteine/cystine and cysteine/cystamine to form a reducing mixture, wherein the molar ratio of oxidation/reduction reagents in the reducing mixture is between about 4:1-about 80:1, and wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is between about 21:1-about 296:1; and
b) incubating the reducing mixture at a temperature of:
i. about 37° C. under anaerobic conditions for at least about 4 hours, or
ii. about 18° C.-about 24° C. for about 16-about 24 hours.
59 . The method according to claim 58 , wherein the set of oxidation/reduction reagents is cysteine/cystine.
60 . The method according to claim 48 , wherein the level of activity of the IL-17 antibodies in the preparation increases by at least about 10 percentage points within about 60 minutes following step b), as measured by cystamine-CEX.
61 . A method for selectively reducing CysL97 in a preparation of secukinumab antibodies that has been recombinantly produced by mammalian cells, comprising:
a) adjusting the concentration of IL-17 antibodies in the preparation to between about 4 mg/ml-about 19.4 mg/ml;
b) adjusting the percent oxygen saturation in the preparation to at least about 60%;
c) adjusting the pH of the preparation to about 7.4-about 8.5;
d) contacting the preparation with cysteine in a vessel to form a reducing mixture, wherein the concentration of cysteine in the reducing mixture is about 4.0 mM-about 8.0 mM;
e) heating the reducing mixture to a temperature between about 32° C.-about 42° C.;
f) incubating the reducing mixture from step e) at a temperature between about 20° C.-about 42° C., said incubating occurring for about 210-about 420 minutes, while maintaining a volumetric oxygen mass-transfer coefficient (k L a*) in the vessel of ≤0.37 h −1 , said k L a* being calculated by adapting a saturation curve to an experimentally-derived dissolved oxygen curve,
g) cooling the mixture resultant from step f) to a temperature between about 16° C.-about 28° C., said cooling occurring for about 45-about 90 minutes; and
h) adjusting the pH of the mixture resultant from step g) to between about 5.1-about 5.3.
62 . The method of claim 47 , wherein the IL-17 antibodies are secukinumab antibodies.
63 . The method of claim 48 , wherein the IL-17 antibodies are secukinumab antibodies.
64 . The method of claim 57 , wherein the IL-17 antibodies are secukinumab antibodies.
65 . The method of claim 58 , wherein the IL-17 antibodies are secukinumab antibodies.
66 . A purified preparation of secukinumab antibodies produced by the method of claim 63 .
67 . A purified preparation of secukinumab, wherein the level of intact secukinumab in the preparation is at least about 90%, as measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS), and wherein the level of activity of secukinumab in the preparation is at least about 90%, as measured by cystamine-CEX.