Establishment And Application Of Detection Method For CHO Cell Host Protein Residues In Monoclonal Antibody Drugs

At present,monoclonal antibody drugs,with the advantages of strong targeting,clear curative effect and weak side effects,are rapidly expanding into new therapeutic fields such as autoimmune diseases,infectious diseases,cardiovascular diseases,diabetes,and so on,and have become the fastest growing field in biopharmaceuticals The gold standard of the monoclonal antibody drug expression system is the Chinese Hamster Ovary cell system.The CHO cell expression system constructed by recombinant DNA technology will secret endogenous protein(host cell protein,HCP)with production of the monoclonal antibody drug.These HCP residues may cause serious immune response when it enters into the human body with drug use.In addition,it may affect the stability of the products.To ensure the quality of the drug,the residue of the HCP in the antibody drug must be controlled and monitored,so a stable and sensitive detection method is necessary for detecting the HCP residues in the antibody drugHCP antigens were prepared by CHO null cells fermentation and then New Zealand White Rabbits were immunized several times under the skin.The final anti-serum titer can reach 640000 after the last immunization and anti-serum was collected through carotid artery.The antibody was purified by protein A chromatography and HCP antibody was obtain with a concentration of 15.2 and a purity of 96.9%.The coverage of self-made CHO HCP antibodies to CHO HCP was 54.6%by two-dimensional electrophoresis(two-dimensional electrophoresis,2-DE);while the coverage of commercial antibodies to CHO HCP antibodies was only 28.8%,indicating that self-made antibodies were more suitable for HCP residue detection in our laboratory than commercial antibodiesThe HCP standard was calibrated by BCA method,the calibration result was 1.159 mg/ml.The HCP accompanying control was established by diluting the HCP standard by 20000 times with 1%BSA solution.The concentration range of the accompanying control should be within 50.573?65.755 mg/ml which determined by mean±3 SD value of 20 samples,as the basis for the stability of the of the CHO HCP standard.The optimal working concentration of coated antibody and the secondary antibodies were determined by chessboard titration to be 1:5000 times dilution and 1:500 times dilution,respectively,and the double antibody sandwich ELISA method was established.This method showed good specificitywithout blank interference,while the monoclonal antibody drug products and non CHO cell host protein also had no interference.The RSD values(n=6)of 6 repetitive solutions detection by this method under low,medium and high concentration were 2.65%,0.70%,1.59%,respectively,while the RSD values(n=6)of 6 samples of intermediate precision solution were 3.02%,0.97%,1.15%,respectively.And the RSD values(n=12)of 12 samples between repeatability and intermediate precision were 3.25%,0.81%,1.89%,respectively,indicating the good precision of the method.The linear range of this method is 6.25?200 ng/ml,the four-parameter fitting equation is Y=3.45-3.129/(1[(X/77.5)]^1.36)(R2=0.999),while the quantitative limit is 6.25 ng/ml.The recovery range of this method at 25 ng/ml,50ng/ml and 75ng/ml was 110.4%?112.1%,100.8%?101.6%,98.0%?103.0%,respectively,and the RSD values of the recovery were 0.74%,0.39%,2.45%,respectively.The incubation time of the second antibody had no influence on this method within 1?2 h as the color development time within 3?10 minThe determination results of the CHO HCP standard by the self-built method was basically consistent with commercial kit.The self-built double-antibody sandwich ELISA method and the market-based kit were used to detect 4 batches of monoclonal antibody drug substance,the test results showed that the commercial kit detection results were much lower than the self-built method,indicating that the self-built method was stricter to control the HCP residues,which was more suitable for the CHO HCP detection in our platform.