An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics

Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H₂O₂-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP spectrum as a template to report the unmodified level. We also tested different buffer systems for this modified FPOP platform with cytochrome c. The outcome is a standard oxidation profile that can be compared between different laboratories and regulatory agencies that wish to adopt FPOP for quality control purposes. [Figure not available: see fulltext.]