Study On Digestion Of β-HCG Monoclonal Antibodies And Purification Of Fragments

Now,monoclonal antibodies have been widely used in the treatment and diagnosis of diseases.Because of its high molecular weight,monoclonal antibodies could easy cause immunogenicity during treatment and false positive in diagnosis.Fab or F(ab’)2 fragments from monoclonal antibody have smaller molecular weight,so they have stronger penetrability and lower immunogenicity,which was safer and more effective when used.In this paper,the methods about digestion of monoclonal antibodies and purification of fragments were investigated base on β-HCG monoclonal antibodies as the model protein,which has important research significance and application value.First,HCG monoclonal antibodies was purified by ammonium sulfate precipitation,caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography from mice ascites.The results show that:the purity of HCG antibody was 67.8%and the antibody protein recovery was 9.47%when purified by ammonium sulfate precipitation;the purity of HCG antibody was 84.5%and the antibody protein recovery was 13.25%when purified by caprylic acid-ammonium sulfate precipitation;the purity of HCG antibody was 98.0%and the antibody protein recovery was 10.60%when purified by protein A affinity chromatography and its recovery was 3.76mg/mL ascites.Comprehensive consideration,it chose protein A affinity chromatography to purify HCG monoclonal antibodies from mice ascites.Second,it chose papain,pepsin and trypsin to digest HCG monoclonal antibodies.The results show that:papain is the best to digest HCG antibody.Different kinds of carriers used to immobilize papain were studied.The results show that:the NHS-PNIPAM was the best carrier for papain immobilized.The conditions of papain immobilized by NHS-PNIPAM was optimized.The best condition is that:lmg NHS-PNIPAM was used to immobilize 0.6mg papain for 2 hours at 22℃,60r/min in borate buffer solution(pH8.5).To investigate properties of the prepared immobilized papain found that the optimal pH value and temperature were 7.5 and 65 C respectively.The immobilized papain left was 84.1%of the activity after 30 thermal cycles.After stored at 4 C for 60 d,the immobilized papain activity retained 74.1%.The Michaelis constant was 3.63 mg/mL.HCG antibodies was digested by NHS-PNIPAM-immobilized papain and the digestive Process was optimized.The results show that:1mg HCG antibodies was digested by 40U immobilized papain for 3 hours at 30℃,pH 7.0,180 r/min in 1ml phosphate buffer solution(containing 10 mmol/L cysteine,2 mmol/L EDTA),the conversion of HCG antibodies could reach more than 98.0%and Fab fragments was a major product.Finally,Fab fragments were purified by gel chromatography,ion exchange chromatography,protein A affinity chromatography and protein L affinity chromatography respectively from the digestion mixture.Among them,the purity of Fab fragments was 85.3%and the recovery rate was 42.7%when purified by gel chromatography;the purity of Fab fragments was 93.2%and the recovery rate was 51.6%when purified by ion exchange chromatography;the purity of Fab fragments was 91.5%and the recovery rate was 63.1%when purified by protein A affinity chromatography;the purity of Fab fragments was 98.6%and the recovery rate was 74.5%when purified by protein L affinity chromatography.Comprehensive consideration,Protein L affinity chromatography is the best method to purify Fab fragments.