Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor

Abstract

Peptide mapping is a key method for studying the primary structure of proteins. With its sensitivity to the slightest changes in the covalent structure of a protein, this method is applicable both to medicinal product identification at the control stage and to production process stability monitoring.The aim of the study was to develop and validate a peptide-mapping procedure for the identification of a novel highly glycosylated recombinant C1 esterase inhibitor.Materials and methods. The authors studied recombinant human C1 esterase inhibitor products and trypsin. The study involved peptide mapping using reverse-phase high-performance liquid chromatography and high-resolution mass spectrometry. The following statistics were calculated to evaluate the results: mean, standard deviation, and coefficient of variation. The validation parameters included specificity, precision, and robustness.Results. The authors tested several variants of sample preparation for tryptic digests, including additional N-glycanase treatment and complete deglycosylation, and established the optimal conditions for sample preparation and chromatographic separation of C1 esterase inhibitor peptides to obtain consistent chromatographic profiles (peptide maps). The authors identified characteristic peaks and the corresponding relative retention time and area ranges. The absolute retention time of the second (characteristic) peak was approximately 16.5–16.9 minutes. The relative retention times were 2.14–2.21 for peak 9, 2.55–2.64 for peak 12, 2.97–3.14 for peak 14, 3.11–3.29 for peak 15, and 6.20–6.63 for peak 28.Conclusions. The authors developed a peptide-mapping procedure for C1 esterase inhibitors and optimised the conditions to achieve an over 18-hour reduction in sample preparation time. This procedure met the established acceptance criteria for specificity, precision, and robustness.