Method Development For The Analysis Of Therapeutic Monoclonal Antibody And Its Glycosylation By Mass Spectrometry

PURPOSEThe peptide mapping and characterization of glycosylation of bevacizumab was analyzed based on proteomic approach. A method for the quantitation analysis of bevacizumab and its glycosylation was established. The bevacizumab concentrations in mice plasma with different intravenous injection doses of bevacizumab were detected and the concentration-time curves were obtained. The glycosylation of bevacizumab was simultaneously quantified to support the study of pharmacokinetics of bevacizumab and its glycosylation, the drug development and the safe clinical drug application. METHODThe molecular weight of bevacizumab was analyzed by MALDI-TOF MS. The bevacizumab and its glycosylation were characterized at the level of intact protein and heavy chain, light chain by SDS-PAGE. Based on shot-gun strategy, the trypsin digest of bevacizumab was analyzed and the peptide mapping was obtained. Combined high resolution mass spectrometry with database searching, the glycosylation was characterized after trypsin digest and deglycosylation using PNGase F.A unique peptide of bevacizumab was selected and a parallel reaction monitoring(PRM) method was established for the quantification of bevacizumab by detecting the unique peptide. The sample preparation includes trypsin digestion and solid phase extraction(SPE). With synthetic stable isotope peptide as internal standard, the calibration curve of bevacizumab in mice plasma was established. Then the bevacizumab concentrations in mice plasma with different intravenous injection doses of bevacizumab were detected and the concentration-time curves were obtained.A PRM method was established for the quantification of glycosylation of bevacizumab. The sample preparation includes trypsin digestion and SPE. Then the bevacizumab glycopeptides in mice plasma with different intravenous injection doses of bevacizumab were detected and their relative amounts were obtained. A HILIC method for the enrichment of glycopeptides was optimized and the glycopeptides which were not directly detected were detected after HILIC enrichment of glycopeptides. RESULTSA method for the qualitative analysis of bevacizumab and its glycosylation was established. It was demonstrated that the molecular weight of bevacizumab, light chains, heavy chains and glycans are about 150 k Da, 25 k Da, 50 k Da and 2-4 k Da, respectively. The N-glycosylation site of bevacizumab is Asn303 of heavy chains. The peptide sequence of trypsin digest of the site is EEQYNSTYR. Seventeen glycans were characterized including H3N3, H4N4, H3N4, H4N3F1, H4N4F1, H3N3F1, H3N4F1, H3N5F1, H5N2, H6N2, H5N4F1, H8N2, H5N5, H4N3F1S1, H5N3F1, H6N3F1 and H4N5.The sequence of unique peptide is FTFSLDTSK and the stable isotope labeling peptide was synthesized. A PRM method for the quantitation of bevacizumab in complex matrix was established. The precursor ions of the unique peptide and internal standard by PRM mode were at m/z 523.26 and 526.88, respectively. The extracted ions were at m/z 797.39 and 805.41, respectively. The dynamic range was from 0.005 to 1.2 μg/μL, the created calibration curve was y = 0.0684449x- 0.000239987(r2 = 0.9980). The detection results of high and low doses of the drug in the mice plasma samples showed that the drug concentration-time curve trend was consistent.A method for the simultaneous quantitation of seventeen kinds of glycoforms was established. The results demonstrated that the metabolism of different glycoforms was different. A HILIC enrichment method was also established to provide a method for better analysis of low abundance glycoforms.Through the above study, qualitative and quantitative analysis methods of therapeutic monoclonal antibodies and their glycosylation were established. And a novel platform was provided for the pharmacokinetic research.