Structural characterization of a monomethylauristatin-E based ADC that contains 8 drugs conjugated at interchain cysteine residues

•Antibody-drug conjugates (ADCs) are mAbs that are chemically linked to a cytotoxic payload.•8-load ADCs were formed by reduction and conjugation of IgG interchain cysteines.•The mAb and corresponding ADC were compared using a variety of biophysical techniques.•Overall, the physical stability of the ADC was similar to the unconjugated mAb.•Small changes in solvent accessibility were observed in CH2 and CH2/CH3 domain interface. Antibody-drug conjugates (ADCs) with a drug-to-antibody ratio (DAR) of 8 are attractive as therapeutic anti-cancer agents due to the higher levels of cytotoxic payload delivered to tumors. Biophysical characterization of a DAR 8 ADC fully conjugated at all interchain cysteine residues was carried out to determine if IgG1 interchain disulfide reduction and conjugation led to structural perturbations that impacted product stability. Comparisons between the DAR 8 ADC and the unconjugated parent antibody identified minor tertiary and quaternary structural changes localized to the CL, CH1, and CH2 domains and CH2-CH3 domain interface. Stability studies of the DAR 8 ADC indicated that the structural changes had minimal impacts to product stability as demonstrated by low levels of fragmentation and aggregation under nominal storage and temperature stress stability conditions. Additionally, no detectable higher order structural changes were observed by CD or DSC in the DAR 8 ADC after 3 months at (25 °C) stability conditions. The structural and stability results support the developability of DAR 8 ADCs fully conjugated to interchain cysteines residues with an optimized and clinically relevant second generation monomethylauristatin-E (MMAE) drug-linker.