Characterization analysis of capsid protein of recombinant adeno-associated virus 6 using reversed phase liquid chromatography-mass spectrometry / 中国生物制品学杂志

ABSTRACT

@#Objective To characterize the capsid proteins of recombinant adeno-associated virus 6(rAAV6)vectors by reversed phase liquid chromatography-mass spectrometry(RPLC-MS),including primary structure and post-translational modification(PTM).Methods The mobile phase A consisted of 0. 1% aqueous solution of difluoroacetic acid(DFA),while the mobile phase B was 0. 1% DFA acetonitrile solution. The column temperature was maintained at 80 ℃,and the gradient elution lasted for 10 min(0→10 min,mobile phase B 15%→45%). The ESI-Q-TOF mass spectrometry detection operated in positive ion mode with the scanning range of 400-4 000 m/z,the scanning frequency of 2 Hz,the cone voltage at 80 V,the capillary voltage at 3. 0 kV,and the ion source temperature at 120 ℃.Results The measured relative molecular mass of the AAV capsid proteins VP1,VP2,and VP3 was 81 255. 9,66 062. 9,and 59 488. 6,respectively. The deviations from the theoretical values were 8. 1 ppm for VP1,3. 8 ppm for VP2,and 36 ppm for VP3. Mass peptide profile analysis of the enzymatically digested rAAV6 sample indicated a sequence coverage of about 89% with detected PTMs mainly including deamidation,N-terminal acetylation,ubiquitination,and phosphorylation;no glycosylation modification sites were found. Tandem mass spectrometry confirmed the N-terminal and C-terminal sequences of the rAAV6 capsid protein as well as the N-terminal PTM.Conclusion The complete relative molecular mass of rAAV6 capsid protein was analyzed by RPLCMS technique,and the PTM of rAAV6 capsid protein was analyzed by tandem mass spectrometry at the peptide level,which has a certain significance for the quality control of AAV gene therapy products and the improvement of production process.