Electrospray – Differential Mobility Analysis (ES-DMA) for Characterization of Heat Induced Antibody Aggregates

Abstract

Aggregation of therapeutic proteins is common and can occur during bioprocessing, shipping, storage or even during delivery to the patient. Aggregation is a major concern because it is generally believed that aggregates are immunogenic and can cause adverse responses in patients. In this study we employ electrospray- differential mobility analysis (ES-DMA) to characterize small aggregates, and microflow imaging to characterize sub-visible and visible macroscopic particles. Results are presented for four monoclonal antibodies of the IgG class: a polyclonal antibody (IgG-A), Rituxan (Rmab), and a monoclonal antibody that is glycosylated (IgG-B) and deglycosylated (IgG–C) in the Fc region. These four antibodies were systematically stressed at 70o C for up to 180 minutes, and the aggregate formation tracked. We find IgG–A to be the least stable with monomer concentration decreasing exponentially with time accompanied by the appearance of visible insoluble aggregates of a few millimeters in size. For Rmab we find a similar, but less rapid, decrease in monomer concentration and increase in aggregates. Aggregates are of the order of tens of nanometers (intermediate aggregates) while particulates are in the order of tens of micrometers. IgG–B is the most stable, however, unlike Rmab, IgG–B and C do not show any evidence of intermediate aggregates. In these two cases the aggregates are in the subvisible domain and are of the order of a few to tens of micrometers.