Comparison between the Theoretical and Experimental Extinction Coefficient of a Monoclonal Antibody Product
WANG Lan , WU Gang, YU Chuan-fei, ZHU Lei, GAO Kai
National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050, China
Abstract��OBJECTIVE To validate the experimental extinction coefficient of a monoclonal antibody with three METHODS and compare them with the theoretical value. METHODS The theoretical molar extinction coefficient was determined at 280 nm based on the primary sequence of the monoclonal antibody, and the theoretical extinction coefficient was calculated in combination with the molecular weight of the monoclonal antibody measured by mass spectrometry. The protein concentration of the monoclonal antibody was measured with three METHODS, and the experimental extinction coefficient was calculated according to Lambert-Beer's Law, ie, c=A/EL. Method one was calculating the extinction coefficient of the protein in its folded state (native state) based on the experimental extinction coefficient of the urea-denatured protein in its unfolded state (denatured state); method two was HPLC quantitation in combination with AccQ Fluor kit after acid hydrolysis of protein; method three was amino acid sequence analyzer quantitation after acid hydrolysis of the protein. RESULTS The theoretical extinction coefficient was 1.453 mL��mg-1��cm-1, and the experimental ones by the three METHODS were (1.442��0.009), (1.529��0.032), and (1.458��0.019) mL��mg-1��cm-1, respectively, with the average of (1.477��0.044) mL��mg-1��cm-1 and RSD of 2.979% (n=9), which demonstrated the small differences between the theoretical and experimental extinction coefficients (0.069%-7.708%). CONCLUSION The established determination and verification METHODS can be used to quantify the protein contents of monoclonal antibody products.
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WANG Lan , WU Gang, YU Chuan-fei, ZHU Lei, GAO Kai. Comparison between the Theoretical and Experimental Extinction Coefficient of a Monoclonal Antibody Product. Chinese Pharmaceutical Journal, 2016, 51(17): 1508-1512.
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