Abstract
The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.
Highlights
Degradation of proteins by asparagine (Asn) deamidation and aspartate (Asp) isomerization has been extensively reviewed [1,2,3,4,5,6]
The developed test system for the simultaneous identification and quantification of Asn deamidation and Asp isomerization in recombinant antibodies employs proteolytic peptide mapping at mildly acid conditions combined with quantitative UPLC-MS
For the identification of antibody critical quality attributes (CQAs) derived from Asn deamidation and Asp isomerization events, a test system was applied employing incubation at elevated temperatures combined with proteolytic peptide mapping and quantitative UPLC-MS
Summary
Degradation of proteins by asparagine (Asn) deamidation and aspartate (Asp) isomerization has been extensively reviewed [1,2,3,4,5,6]. Previous studies have identified that degradation of Asn and Asp residues in proteins can impact in vivo biological functions and in vitro stability [7,8,9,10,11]. Two independent studies reported the heavy chain Asn-55 (CDR 2) to be susceptible to deamidation in vivo [13] and to exist in a stable succinimide form at mildly acidic pH [14]. In another investigation, the light chain Asp-30 of an IgG2 antibody was found to form succinimide and iso-Asp. no significant impact on the biological function was reported [15]. Chelius et al applied accelerated degradation conditions to identify four potential deamidation sites in the conserved regions of recombinant IgG1 monoclonal antibodies [16]
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