Development of a fast photochemical oxidation of protein (FPOP) platform for protein therapeutics

摘要

Standard controls and FPOP experiments were performed on an improved FPOP platform (Fig. 2). Background oxidative modifications were evaluated in the control experiment (H_2O_2 only or laser only). This result can serve as baseline for all FPOP experiments performed here. The modification level significantly increases when using typical (former) FPOP conditions. The typical FPOP-induced modification pattern with multiple +16 Da shifts was observed. The result from controls and a test FPOP experiment are a standard profile for the improved platform; the improved test with model protein can be a check protocol for FPOP performance. The analyses (in quadruplicate) for each experiment can be used to determine experimental variation. Results from improved platform including mixing and from the typical setup were compared. The experimental variation was reduced from 3.1% to 0.8% by introducing mixing steps (Fig. 3 vs. Fig. 2C). With the improved FPOP platform, precision has been improved, an important requirement for for studies of protein therapeutics.