Development And Validation Of A Peptide Mapping Method For The Characterization Of Adalimumab With QDa Detector

With the rapid development of biotherapeutics, until now there are 61 therapeutic antibodies on the market. However, a higher drugs cost cause economic burden for national health insurance. The first generation of blockbuster antibody drug patents will be expired, cost-effective biosimilar are expected. Moreover, new technology-based therapeutic antibodies developed rapidly, including bispecific antibody, antibody-drug conjugates, fragment of antibody, fusion protein and so on, which promote the development of m Abs significantly. It is necessary to continuously monitor antibody to ensure security during research and development of therapeutic antibody. However, the platform of peptide mapping, such as LC-MS-Q-Tof and LC-TQ, there are problems of platform-expensive, operation-complicated and high requirements for sample preparation.QDa is an operation-simple, cost-effecitve and stable single quadrupole detector. It can be used as routine analysis method. Taking advantages of UPLC system, the optic-character of TUV and the MS-character of QDa, this study developed and validated a new antibody peptide mapping method. In addition, this study also investigate the application potential in the cross cotimination as a routine method of laboratory management.First, confirming the peptides of CDR of adalimumab with the LC-Q-Tof and the 8 peptides derived from CDRs of adalimumab were used as surrogates of adalimumab. The data of peptides were optimized, which the high response m/z value were collected. The digestion conditions of adalimumab were also optimized, including protein-enzyme ratio and the most excellent enzyme digestion time; the final digestion conditions of adalimumab is 37℃, 1:10 and 3h. Other 10 antibody drugs, including trastuzumab, cetuximab, denosumab, ofatumumab, golimumab, alemtuzumab, ipilimumab, nivolumab and pembrolizumab, were used to assesse the method specificity and the results demonstated that non-specificity were not found. The results showed that this method has high specificity due to the high UV-MS selectivity; thelimit of detection was 20 μg/m L; carry-over was about 1.69 %. Additionally, other validation parameters, like stability, were also evaluated. This novel LC-UV-QDa method can be a simple, cost-effective and robust peptide-mapping method for other recombinant monoclonal antibodies during routine quality control analysis.Finally, we adapted this method to detect the simulative cross contamination sample, including adalimumab, cetuximab and trastuzumab. This method successfully detected target protein, adalimumab, and track the contaminant, cetuximab and trastuzumab.