(126c) Elucidating Therapeutic Monoclonal Antibody Aggregation

A strong interest in protein aggregation has emerged in the pharmacology industry for therapeutic monoclonal antibodies (MAB). We report combined chromatographic and multi-spectroscopic studies of such MAB aggregation. Results from different spectroscopic methods agree well with each other, and they are used in a combinatorial way to get a complete picture of protein aggregation. SEC-HPLC was used to separate monomer from aggregates. Spectroscopic techniques consisting of intrinsic tryptophan fluorescence and extrinsic fluorophore binding were also applied, as well as static-light scattering. Both steady-state and time-resolved fluorescence spectroscopy, including fluorescence lifetime, anisotropy, quenching and FRET were used. Based on monomer peak loss in SEC-HPLC at elevated temperatures, aggregation mechanisms were elucidated, and a kinetic protein aggregation model, based on nucleation and growth polymerization, is proposed to predict in vitro therapeutic monoclonal antibody aggregation at low storage temperatures. According to our model, MAB first partially unfolds with at least one unfolding intermediate and forms an aggregation prone monomeric structure. Having said that, aggregation model is not limited to the formation of such structure, and in fact, aggregation could occur from any type of monomer. In addition, we found that aggregation of studied MABs show temperature dependent aggregation regimes. Based on accelerated studies, a kinetic model used for the prediction of MAB aggregation at low storage temperatures is proposed.