Controlling Residual Protein Impurities in Active Pharmaceutical Ingredients (APIs) by Derivative Size-Exclusion High-Performance Liquid Chromatography (SEC-HPLC).

This study aimed to develop a reliable protein labeling strategy for detecting enzymes in chemically complex active pharmaceutical ingredient (API)-rich environments where dimethyl sulfoxide (DMSO) is commonly used as the solvent. We compared two lysine targeting fluorescent dyes and identified NHS-fluorescein as superior to fluorescein isothiocyanate (FITC), yielding stronger signals and consistent linearity between protein concentration and absorbance intensity. Optimization experiments revealed that higher protein and dye concentrations improved labeling efficiency, with 500 μM NHS-fluorescein providing reproducible signals. Importantly, labeling remained robust across varying conditions: DMSO enhanced performance at 20-25% but reduced efficiency at 50%, while detergent effects varied by type, with zwitterionic and non-ionic detergents supporting efficient labeling and ionic detergent nearly abolishing it. The optimized protocol proved applicable to real industrial enzyme (Protein X) and maintained quantitative measurability even in the presence of diverse small molecules. Notably, these experiments could be transferred easily from size exclusion chromatography (SEC)-HPLC to SEC-UPLC. Together, these results establish a versatile, sensitive, and broadly applicable approach for protein labeling and detection in chemically challenging environments, with strong potential for applications in pharmaceutical research, compound screening, protein quantification, and industrial quality control.