Sensitive Detection of Immunoglobulin G Stability Using in Real-Time Isothermal Differential Scanning Fluorimetry: Determinants of Protein Stability for Antibody-Based Therapeutics

Abstract

Protein instability is a major obstacle in the production and delivery of monoclonal antibody–based therapies for cancer. This study presents real-time isothermal differential scanning fluorimetry as an emerging method to evaluate the stability of human immunoglobulin G protein with high sensitivity. The stability of polyclonal human immunoglobulin G against urea-induced denaturation was assessed following: (1) oxidation by the free-radical generator 2,2-Azobis[2-amidinopropane]dihydrochloride and (2) in selected storage buffers. Significant differences in immunoglobulin G stability were detected by real-time isothermal differential scanning fluorimetry when the immunoglobulin G was stored in 1,4-Piperazinediethanesulfonic acid buffer compared to phosphate-buffered saline, with half-maximal rate of denaturation occurring at a higher urea concentration in 1,4-Piperazinediethanesulfonic acid than phosphate-buffered saline (Knd;PIPES = 3.56 ± 0.09 M, Knd;PBS = 2.94 ± 0.08 M; P