Risk-based strategy for evaluating and controlling residual protein impurities in enzymatically synthesized calcium gluconate: Bridging analytical detection to safety assessment.

Regulatory bodies (e.g., FDA, NMPA, EDQM) mandate control of residual protein impurities in enzymatically produced pharmaceuticals, yet a standardized, risk-informed methodology for their safety assessment is lacking. This study addresses this gap for calcium gluconate, a critical injectable API. We compared three techniques for target residual protein (GOD and CAT); ELISA uniquely achieved ppb-level detection, outperforming HPLC-MS and HPLC-FD (ppm-level). Crucially, the target enzymes constituted only ≈0.04% of the total residual protein, necessitating a multi-method approach (Kjeldahl, BCA, Bradford, AAA) to quantify the total burden at 192-269 ppm. Ultrafiltration-based profiling revealed >80% of residues were low molecular weight (<3 kDa), while the >3 kDa fraction represented the primary immunogenic risk. We therefore propose a novel, risk-based control strategy that integrates specific enzyme verification with total residual protein monitoring and advocates deducting the low-risk <3 kDa fraction. This refines the control target from >200 ppm to <50 ppm, providing a practical framework that links analytical detection to safety assessment for regulatory compliance and quality assurance of enzymatically synthesized drugs.