PROBING BIOPHYSICAL PROPERTIES OF THERAPEUTIC PROTEIN AGGREGATES WITH INTERFEROMETRIC SCATTERING MICROSCOPY

Abstract

A growing biopharmaceutical market increases the importance of therapeutic proteins, of which monoclonal antibodies (mAb) are the largest category. Protein aggregation in biopharmaceutical production has important consequences in mAb immunogenicity. Submicron (100-1000 nm) protein aggregates in particular are a key challenge due to their higher immunogenicity and the relative lack of analytical methods capable of characterizing them. We propose the use of interferometric scattering (IFS) microscopy as a simple and potentially high-throughput orthogonal characterization method of submicron aggregates. We demonstrate its utility by testing two variants of IFS microscopy: (1) Correlative IFS and fluorescence microscopy (2) hyperspectral interferometric scattering (h-IFS) microscopy. Using correlative IFS and fluorescence microscopy, we characterize the size and surface structure of a stirred protein aggregate sample. We find that smaller protein aggregates (~100 nm) have higher surface concentrations of Fc domains and hydrophobic regions. Then, we demonstrate the usage of h-IFS microscopy to differentiate and quantify protein aggregates and contaminants in biologic drugs.