Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody

Abstract

The last decade has witnessed a rapid growth in the development of protein pharmaceuticals for diagnostic and therapeutic purposes. The biopharmaceutical industry increasingly demands thorough characterization of protein conformation and conformational dynamics to ensure product quality and consistency. Here we present a chromatography-based method that is able to characterize protein conformation and conformational dynamics at peptide level resolution in a high-throughput manner. The surface lysine residues of the protein were labeled with a fluorescent dye prior to trypsin and Glu-C digestion. The resulting peptide maps were monitored by fluorescence detection and the peak areas of the respective peptides were normalized to protein concentration. The normalized fluorescence peak area for a specific peptide represents the individual lysine solvent accessibility. A higher normalized fluorescence peak area indicates higher solvent accessibility at a specific site. The identity of the peak of interested was determined by LC-MS/MS analysis. We first demonstrated this method is suitable for probing protein surface/conformation by studying the effect of deglycosylation on a recombinant monoclonal antibody (mAb), IgG 1. The results from this method were consistent with previous results obtained by H/D-exchange. We then applied our method to study the interaction of the mAb with a common excipient, polysorbate-20 (PS-20). The interaction between PS-20 and the mAb was generally weak. The presence of PS-20 increased the fluorescent labeling of several lysine residues on the mAb. These lysine residues localized near the protein domains of relatively high hydrophobicity. This result provides a first insight into PS-20-mAb interaction at peptide level resolution.