Abstract
A humanized anti-cocaine mAb (h2E2) under development for the treatment of cocaine use disorders was deglycosylated enzymatically, and analyzed by differential scanning fluorimetry (DSF) using two distinct extrinsic dyes, and by FITC fluorescent labeling, to assess domain stability changes induced by deglycosylation of the Fc domain. The deglycosylated Fc fragment of the mAb was generated by proteolysis, and purified by ion exchange. Deglycosylation caused thermal destabilization of the glycosylated domain of the mAb, but there was no change in antigen (cocaine) binding or affinity. FITC fluorescent labeling of the deglycosylated mAb was suitable for qualitative assessment of cocaine binding. The thermal destabilization of the deglycosylated Fc fragment could be assessed by FITC labeling-based DSF, unlike the FITC-labeled control Fc fragment. This difference is due to the exposure of a secondary limited proteolysis site on the deglycosylated Fc, which increased accessibility and fluorescence labeling of Fc fragment lysine residues. Many of the methods and results in this study may be generalizable to numerous therapeutic mAbs, since the IgG1 anti-cocaine mAb shares the same glycosylation site and fully human heavy chain Fc sequence with other therapeutic human and humanized mAbs.