Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis

Abstract

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.

Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography

“…Although HIC has been broadly implemented for the monitoring of various quality attributes for mAbs, bsAbs, and antibody-drug conjugates, [19][20][21][22][23][24][25][26][27] the use of this technique to separate N-terminal pE from main species and other variants has not been reported. In our study, pE was enriched exclusively in HIC post-peak 1 by ninefold, indicating that the retention of pE in HIC is not affected by other modifications.…”

Targeting cancer with antibody-drug conjugates: Promises and challenges

“…171 Similar analytical approaches can also be applied to other site-specific ADCs. 172,173 Lysine-linked ADCs are inherently associated with a high heterogeneity, posing an analytical challenge. For those ADCs, DAR is usually determined by measuring both the drug-specific absorbance and the mAb absorbance at 280 nm.…”

The Use of Antibody-Antibiotic Conjugates to Fight Bacterial Infections

“…Cysteine coupling is an alternative; this methodology requires a reaction between cysteine amino acid residues of the antibody and a thiol-reactive group present on the antibiotic to form a disulfide bond ( Cao et al, 2019 ; You et al, 2021 ). Nevertheless, there are no free thiols on antibodies, as virtually all cysteine amino acid residues form disulfide bonds due to their high reactivity.…”