Establishment and validation of a reductive peptide mapping identification method for adalimumab
Objective To establish a peptide characterization method based on high-resolution mass spectrometry and a peptide mapping identification method for quality control and release.Methods The adalimumab was denatured,reduced,alkylated and finally digested with trypsin or Lys-C to analyze the sequence coverage and confirm CDR peptides by high-resolution mass spectrometry.The liquid chromatography method for peptide mapping identification of adalimumab was established,validated and evaluated.Results All CDR peptides of adalimumab were confirmed by high-resolution mass spectrometry after trypsin or Lys-C digestion.The method had high specificity assessed by bevacizumab and rituximab.With the conserved sequence of Fe segment as reference peaks,the method's varia-bility was evaluated with relative retention time and relative peak area of CDR peptides as the indexes.For CDR peptides digested by trypsin,the RSD of RRT was 0.008％-1.097％and that of RPA was 1.212％-7.951％after five consecutive injections.Interme-diate precision test showed that the RSDs of RRT and RPA were 0.010％-0.985％and 2.306％-10.939％,respectively.The RSDs of RRT and RPA met the robustness requirements when the trypsin digestion ratio was 1∶35-1∶45 and the digestion time was 3.5-4.5 h.For Lys-C digestion,the method validation results also showed that precision and robustness were good.Different columns had different selectivity,retention,separation and intensity for peptides.Conclusion Based on CDR related peptides,the established HPLC method can be used for quality control and batch release test of adalimumab and may also provide a reference for manufacturers.
adalimumabpeptide mass fingerprintcomplementarity determining regionHPLCLC-MSmethod validation
万方数据
维普
