The Development Of Conjugation Process For Anti-Her2 Antibody Drug Conjugate

With the approval of kadcyla by the FDA for breast cancer in 2013,the antibody-drug conjugate(ADC)developed very fast.There are already four ADC drugs have been available,and have broad application prospects.However,enough stability of ADC in plasma must be maintained,and must be released quickly and accurately after entering the tumor cells,which made great demands on the quality of ADC drugs.The conjugation process of ADC drugs is one of the most important steps in the quality standards of ADC drugs.The lysine residue of anti-her2 antibody is linked to the small molecule toxin DM1 via SMCC by a method of chemical conjugation,researched the important element of affecting the final critical quality attributes drug-to-antibody ratio(DAR)and SEC-HPLC monomer purity.The test plans are confirmed by design of Experiments(DOE)and the correspondence analytical model is simulated.The reliability of the model is verified by the analysis of the variance,error statistics and the residuals.The weights of each key factor are analyzed by contour map and 3D surface map.Finally,the optimal conditions are simulated and verified by the model.The first step of the process is linked the lysine residue of the antibody to SMCC.Reaction time,p H,and the molar ratio of antibody to SMCC are three factors to influence the DAR value.The experimental design was carried out by the response surface.According to the results,the molar ratio of the SMCC to antibody has significant effect on the results,secondly the reaction time,finally the reaction p H.The initial conditions were as follows: p H= 6.0,reaction time 100 min,molar radio of the SMCC to antibody =8.The second step of the process is linked the product of the first step to small molecule toxic DM1.Reaction p H,time,and the molar ratio of antibody to DM1 are three factors to influence the DAR value and SEC-HPLC monomer purity.The experimental design was carried out by the response surface.According to the DAR value,the reaction p H has significant effect on the results,secondly the reaction time.The molar ratio of antibody to DM1 has little effect on the results in a limited range.According to SEC-HPLC monomer purity,the reaction pH has significant effect on the results,the reaction time and the molar ratio of antibody to DM1 have little effect on the results.Based on two aspects of the results,the selected conditions of the second step were as follows: p H= 5.2,reaction time300 min,molar radio of the DM1 to antibody =7.3.Three parallel verification tests were carried out in combined with the conditions of the first step and the second step.The final UV-DAR value was 3.52,and the SEC-HPLC monomer purity was 98.8%,which match the set quality requirements.