Abstract
Purpose
The purpose of this paper was to identify the location of a succinimide and determine the rate of its formation and hydrolysis in a recombinant human monoclonal IgG2 antibody aged in mildly acidic buffers at elevated temperatures.
Materials and Methods
Cation exchange (CEX) HPLC separated multiple Main Peaks and high levels (up to 50%) of basic variants, the identification of which was an analytical challenge and required several complementary techniques. The relative abundance of the CEX basic variants was used to quantify the percentage of succinimide and to study the rates of its formation and hydrolysis.
Results
Mass decrease by approximately 18 Da for intact antibodies from the CEX basic fractions suggested succinimide formation from aspartic acid as the major modification. Reversed-phase HPLC/MS of the reduced and trypsin-digested samples detected an isoaspartate 30 (isoD30) in the light chain peptide A25-R37. Direct evidence that isoD30 was from succinimide was obtained by performing succinimide hydrolysis in \( {\text{H}}_{{\text{2}}} {}^{{{\text{18}}}}{\text{O}}\) followed by tryptic digestion in \( {\text{H}}_{{\text{2}}} {}^{{{\text{16}}}}{\text{O}}\).
Conclusions
Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.
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Acknowledgements
We would like to thank Thomas Dillon for help with the RP HPLC of the intact IgG2 antibody; Alexis Lueras for her assistance with preparation of some of the aged protein samples; Barbara Norwood for her assistance in bioactivity measurements; Tiansheng Li for his support and David Brems for his comments and suggestions.
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Chu, G.C., Chelius, D., Xiao, G. et al. Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures. Pharm Res 24, 1145–1156 (2007). https://doi.org/10.1007/s11095-007-9241-4
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DOI: https://doi.org/10.1007/s11095-007-9241-4