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 preparation comprising anti-IL-17 antibodies in a reduced state at cysteine at light chain position 97 (CysL97), comprising a heavy chain of SEQ ID NO: 15 with or without the C-terminal lysine and a light chain of SEQ ID NO:14, in which the anti-IL-17 antibodies have been recombinantly produced by CHO cells, and the preparation of anti-IL-17 antibodies exhibits at least 90% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS and at least 90% activity.
2 . The preparation of anti-IL-17 antibodies of claim 1 , wherein the activity is measured by cystamine-CEX.
3 . The preparation of anti-IL-17 antibodies of claim 1 , wherein the activity is measured by a cell-based assay.
4 . The preparation of anti-IL-17 antibodies of claim 3 , wherein the cell-based assay comprises an assay for inhibition of IL-17-dependent release of IL-6.
5 . The preparation of anti-IL-17 antibodies of claim 1 , wherein the preparation exhibits at least 95% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS and at least 95% activity.
6 . The preparation of anti-IL-17 antibodies of claim 5 , wherein the activity is measured by cystamine-CEX.
7 . A preparation comprising anti-IL-17 antibodies in a reduced state at cysteine at light chain position 97 (CysL97) and not in a reduced state at the conserved cysteine residues, comprising a heavy chain of SEQ ID NO:15 with or without the C-terminal lysine and a light chain of SEQ ID NO:14, in which the anti-IL-17 antibodies have been recombinantly produced by mammalian cells, and the preparation of anti-IL-17 antibodies exhibits at least 90% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS and at least 90% activity.
8 . The preparation of anti-IL-17 antibodies of claim 7 , wherein the activity is measured by cystamine-CEX.
9 . The preparation of anti-IL-17 antibodies of claim 7 , wherein the activity is measured by a cell-based assay.
10 . The preparation of anti-IL-17 antibodies of claim 9 , wherein the cell-based assay comprises an assay for inhibition of IL-17-dependent release of IL-6.
11 . The preparation of anti-IL-17 antibodies of claim 7 , wherein the preparation exhibits at least 95% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS and at least 95% activity.
12 . The preparation of anti-IL-17 antibodies of claim 11 , wherein the activity is measured by cystamine-CEX.
13 . The preparation of anti-IL-17 antibodies of claim 12 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells
14 . The preparation of anti-IL-17 antibodies of claim 8 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells.
15 . A preparation comprising anti-IL-17 antibodies in a reduced state at cysteine at light chain position 97 (CysL97) and not in a reduced state at the conserved cysteine residues, comprising a heavy chain of SEQ ID NO: 15 with or without the C-terminal lysine and a light chain of SEQ ID NO:14, in which the anti-IL-17 antibodies have been recombinantly produced by mammalian cells, and the preparation of anti-IL-17 antibodies exhibits at least 90% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS, and exhibits increased activity after reduction of CysL97 as compared to before reduction.
16 . The preparation of anti-IL-17 antibodies of claim 15 , wherein the activity is measured by cystamine-CEX.
17 . The preparation of anti-IL-17 antibodies of claim 15 , wherein the activity is measured by a cell-based assay.
18 . The preparation of anti-IL-17 antibodies of claim 17 , wherein the cell-based assay comprises an assay for inhibition of IL-17-dependent release of IL-6.
19 . The preparation of anti-IL-17 antibodies of claim 16 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells.
20 . The preparation of anti-IL-17 antibodies of claim 18 , wherein the preparation exhibits at least 95% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS.
21 . The preparation of anti-IL-17 antibodies of claim 16 , wherein the preparation exhibits an increased activity of at least 15% after reduction of CysL97 as compared to before reduction.
22 . The preparation of anti-IL-17 antibodies of claim 21 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells.
23 . A preparation comprising anti-IL-17 antibodies in a reduced state at cysteine at light chain position 97 (CysL97) and not in a reduced state at the conserved cysteine residues, comprising a heavy chain of SEQ ID NO: 15 with or without the C-terminal lysine and a light chain of SEQ ID NO:14, in which the anti-IL-17 antibodies have been recombinantly produced by mammalian cells, and the preparation of anti-IL-17 antibodies exhibits at least 90% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS, and exhibits at least 90% activity, wherein the anti-IL-17 antibodies exhibit no more than 80% activity in the absence of reduction of CysL97.
24 . The preparation of anti-IL-17 antibodies of claim 23 , wherein the activity is measured by cystamine-CEX.
25 . The preparation of anti-IL-17 antibodies of claim 23 , wherein the activity is measured by a cell-based assay.
26 . The preparation of anti-IL-17 antibodies of claim 25 , wherein the cell-based assay comprises an assay for inhibition of IL-17-dependent release of IL-6.
27 . The preparation of anti-IL-17 antibodies of claim 24 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells.
28 . The preparation of anti-IL-17 antibodies of claim 23 , wherein the preparation exhibits at least 95% intact anti-IL-17 antibodies as measured by non-reducing CE-SDS and at least 95% activity.
29 . The preparation of anti-IL-17 antibodies of claim 28 , wherein the activity is measured by cystamine-CEX.
30 . The preparation of anti-IL-17 antibodies of claim 29 , wherein the anti-IL-17 antibodies have been recombinantly produced by CHO cells.