Recombinant Anti-CD20 Monoclonal Antibody ELISA Methodology Establishment And Application

Rituximab,a monoclonal anti-CD20 on B lymphocytes capable of specific binding molecules,resulting in apoptosis of B cells,made recombinant anti-CD20 monoclonal antibody rituximab monoclonal antibody experiments comparing observed both in food cynomolgus monkey in vivo pharmacokinetics.According to China’s relevant provisions of the new development of the drug for domestic recombinant anti-CD20 monoclonal antibody drug before clinical pharmacokinetic study of relevant information.The experimental study of the methods of recombinant anti-CD20 monoclonal antibody of the school establishment and in cynomolgus monkeys in vivo pharmacokinetics.1、the establishment of a double-antibody sandwich ELISA for recombinant anti-CD20 monoclonal antibody quantitative determination method(for detecting food cynomolgus monkeys in vivo pharmacokinetics of the drug and toxicokinetics).A goat anti-monkey serum adsorption using anti-human IgG coated ELISA plate,two anti-biotin labeled RAT ANTI RITUXIMAB,using TMB one-component color liquid,in microplate 450 nm/560 nm Reading.The extent and depth of color in the sample is proportional to the concentration of the drug.After validation methodology,the test requirements are met by ELISA recombinant anti-CD20 monoclonal antibody in cynomolgus monkeys pharmacokinetic study requirements.2、cynomolgus monkeys were intravenous infusion of low,after the medium and high dose(10,30,100 mg · kg-1)made of recombinant anti-CD20 monoclonal antibody,which ends with a half-life t1/2 was 128.4 ± 26.5,155.3 ± 40.7 and 210.2 ± 39.2h;AUC(0-480h)was 13,957.4 ± 2,972.6,38,564.7 ± 4,492.2,and 132,195.4 ±35,213.1μg · h · mL-1,comparison shows that there are significant differences(P among data<0.05);clearance CLS each dose group were 0.7 ± 0.2,0.7 ± 0.1 and 0.7 ±0.2mL · h-1 · kg-1,meaning comparison shows no significant difference(P>0.05);each dose group The highest concentration time Tmax occurs after needle injection point or needle injection 5min,the highest concentration Cmax was 194.4 ±37.7,450.2 ± 84.2 and 1,622.4 ± 250.7μg/ml.Dose ratio of 1:3:10 growth AUC was 1:2.8:9.5,increase Cmax was 1:2.3:8.3 comparison shows that each dose group CLS systemic clearance rate was not significant,and the end phase half-life t1/2 is similar to the performance characteristics of linear pharmacokinetics.Cynomolgus monkeys single intravenous infusion of the reference monoclonal antibody rituximab 30mg · kg-1,the average value of the same dose of drug concentrations of recombinant anti-CD20 monoclonal antibody and the reference average drug concentration value during the same time point the difference was not statistically significant(P>0.05).It is found in cynomolgus monkeys intravenous reference monoclonal antibody rituximab(30mg · kg-1)after the end of the half-life t1/2 was 130.7 ± 6.0 h;AUC(0-480h)was 39,716.3 ± 7,818.4μg· h · ml-1;CLS systemic clearance was 0.7 ± 0.1mL · kg-1 · h-1;the highest concentration Tmax time after needle injection point or needle injection at 5min,the peak concentration was 402.3 ± 94.2p,g · mL-1.The reference monoclonal antibody rituximab and in both dose groups was not statistically significant(P>0.05)in CL,Vss,Cmax,the end of the phase half-life t1/2 was not statistically significance difference(P>0.05),the average resident The residence time was no significant difference(P>0.05).Therefore,pharmacokinetic behavior of the production of recombinant anti-CD20 monoclonal antibody with Roche’s monoclonal antibody rituximab is very similar.Cynomolgus monkeys in a row four times,once weekly intravenous infusion of 30 mg · kg-1TSLXK.First and last dose peak concentrations were 378.0 ± 187.7 and 525.4 ± 177.7 g · mL-1,no significant difference(P>0.05)between the two.For the first time and after the last administration AUC(0-168h)was 20,754.9 ± 8,079.3 and 27,022.0 ± 15,814.0 g · h · mL-1,accumulation factors(AUC 4 weeks/AUC the first week)was 1.7 ± 1.1.Showed that continuous administration of 4 times,no significant drug accumulation.There above,cynomolgus monkey intravenous infusion 10～100 mg/kg of recombinant anti-CD20 monoclonal performance within the scope of the anti-linear pharmacokinetic profile with rituximab pharmacokinetics of Roche’s very similar for consecutive no significant accumulation of recombinant anti-CD20 mAb show and Roche’s Rituxan has a similar pharmacokinetic behavior.