Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

Abstract

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies

“…The Asn384/389 deamidation rate ranged from 70 ~ 144 x 10 −3 day −1 , which was the highest among all Asn sites. 1 The R square values for all three mAbs were >0.99.…”

“…Deamidation of several Asn residues in the complementary-determining regions (CDRs) of IgG mAbs have been reported to affect antigen binding. 1–4 Recent studies have also shown that changes in the charge distribution on the protein surface could alter mAb pharmacokinetics (PK) by affecting FcRn-IgG dissociation. 5,6 Thus, the potential risk of deamidation at each site needs to be evaluated to ensure product stability.…”

Cysteinylation of a monoclonal antibody leads to its inactivation

“…Carboxyl-terminal lysine processing, deamidation, oxidation, isomerization, mutation, and changes in glycosylation are examples of modifications that can be extremely small in size (Dalton range), but can have a substantial effect on how the antibody functions. 3 To fully characterize the nature and effects of post-translational modifications, a comprehensive analytical approach combining bioanalytical and structural studies should be considered. Analytical chromatography methods such as ion exchange and hydrophobic interaction chromatography (HIC) are effective analytical tools for uncovering changes in antibody charge variance or changes in hydrophobicity that can accompany post-translational modifications.…”

In vivo characterization of therapeutic monoclonal antibodies

“…After administration, the accumulated succinimide hydrolyzes to Asp and/or isoAsp under the physiologic pH. In mAbs, deamidation has been reported in the Fc regions [4,5] and the CDRs [6][7][8][9][10][11][12]. Deamidation in the CDR regions could affect drug efficacy [10,12].…”

“…In mAbs, deamidation has been reported in the Fc regions [4,5] and the CDRs [6][7][8][9][10][11][12]. Deamidation in the CDR regions could affect drug efficacy [10,12]. Deamidation can accumulate more rapidly in in vitro and in vivo serum samples than that in PBS or formulation buffers [7,13,14], which raises concerns about loss of potency and the potential immunogenicity of the therapeutics after administration.…”