Establishment And Application Of Two Kinds Of Activity Detection Methods For PCSK9 Monoclonal Antibody

Background:A large number of studies have shown that elevated circulating LDL-C levels are important risk factors for atherosclerosis and related cardiovascular diseases.Statins,which are widely used clinically for lowering blood lipids,couldn’t reduce the level of blood LDL-C to ideal levels of prevention and treatment of cardiovascular disease in all populations.Based on this background,a research and development boom has been launched at home and abroad for PCSK9 targeted monoclonal antibody for lipid-lowering.At present,there has been no published publication of methods of antigen binding activity and biological activity that can be directly applied to the development and production of PCSK9 monoclonal antibodies(mAbs).Objective:To establish respectively an antigen binding activity and biological activity determination methods for PCSK9 mAbs.They are intended to be used for quality control of active in the research and development of PCSK9 mAbs and to provide reference for the development of activity assay methods for similar monoclonal antibody drugs.Methods:1 On the basis of previous research in this laboratory,the antibody protein stock solution was obtained through fermentation and purification.The isoelectric point,purity,content,impurities(protein A and endotoxin)and pH value of the protein stock solution were measured by CIEF,CE-SDS,UV spectrophotometry,gel limit test,acidity test to control the quality.2.According to the experimental procedure of indirect ELISA,three key experimental factors were studied:1)three kinds of secondary antibodies(HRP-goat anti-human IgG Fc,HRP-goat anti-human IgG(H+L)and HRP-goat anti-human Kappa Light Chain Fab);2)two coating strategies(direct coating and coating captured by the streptavidin-biotin system captures);3)dilution ratios of the secondary antibody and concentration sequence of PCSK9 mAb.3.HepG2 hepatoma cells were cultured,and the key experimental factors were examined one by one according to a set procedure:1)FBS content of plating medium,cell culture time and time of Dil-LDL incubation;2)density of plating cells;3)PCSK9 protein concentration;4)Dil-LDL concentration;5)Concentration gradient of the test sample.Results:1.After purification,1.26L of PCSK9 antibody stock solution(123.5g/L)was obtained.The isoelectric point of the solution was 7.059;the purity in CE-SDS(NR)was 94.0%,CE-SDS(R)was 95.9%,SE-HPLC was 98.9%;PH was5.28;endotoxin was less 10 eu/ml;protein A was 0.61ppm.All above met the PCSK9mAbs quality standard.2.Considering the OD value,the fitting of the four-parameter curve and the cost and efficiency of test,the key experimental parameters were selected:1)PCSK9 coated bythe streptavidin-biotin system;2)Antibody concentration gradient:1350 to 0.023ng/ml(3 fold gradient);3)Secondary antibody:HRP-goat anti-human IgG(H+L)(1:10000).3.Similarly to the above considerations,the following key experimental parameters of biological activity were determined:1)10%FBS plating medium,cell culture for 24 hours,Dil-LDL incubation for 20 hours;2)Cell plating density 5×10~5cells/ml;3)PCSK9 protein concentration 20μg/ml;4)Dil-LDL concentration 16μg/ml;5)PCSK9mAb gradient:200,52,20,12,8,4,0.8μg/ml.4.After being verified separately,the specificity,accuracy,precision,and durability of the two established methods met the verification criteria,and within a range of 50%to 150%of the potency,all exhibited a good linear relationship.5.The antigen binding activity of the three batches of antibody proteins determined by the established method were 74.7%,88.9%,and 76.2%(RSD 9.8%);the biological activity was 80.4%,76.3%,and 75.5%(RSD 3.4%),All met the quality standards.Conclusions:The methods were successfully established for the determination of antigen binding activity and biological activity of PCSK9 mAbs,which could be used for the quality control of activity for PCSK9 mAbs.