Structural Characterization of the Aggregates of Gemtuzumab Ozogamicin

Abstract

The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in the conjugation process are elucidated. The aggregates are predominantly composed of a mixture of dimers and multimers with high drug loaded species. The level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. Studies performed on the biological activity revealed differences in binding affinity and cytotoxicity between aggregates and monomeric ADC species.