Effect of Linker-Drug Properties and Conjugation Site on the Physical Stability of ADCs

Abstract

The physical stability of antibody drug conjugates is dictated by the properties of the antibody, linker-drug, and conjugation site. Two linker-drugs were chosen that are different in terms of hydrophobicity and polar surface area to evaluate the effect of linker-drug properties on antibody-drug conjugate (ADC) behavior. Site-specific and non–site-specific conjugation was used to investigate the role of conjugation site in conformational and colloidal stability. Finally, 2 antibodies were selected to determine if the observed results were antibody-specific. The conformational stability is affected, with the highest degree of destabilization observed when conjugation results in the removal of interchain disulfide bonds. Although conformational destabilization occurred in the domain in which conjugation occurred and domains distinct from the conjugation site, no correlation could be drawn between linker-drug properties and conformational stability. Evaluation of aggregation by size exclusion HPLC confirmed a relationship between linker-drug hydrophobicity and aggregation propensity under thermal stress in all ADCs tested. The extent of aggregation was far greater in the conjugates generated with a more hydrophobic antibody, illustrating that the properties of both the antibody and linker-drug contribute to aggregation. These studies emphasize that the distinct properties of the molecule as a whole warrant a case-by-case evaluation of each ADC.