Process Optimization For CTLA4 McAb Purification And Drug Effect Evaluation

Monoclonal antibody(McAb)drugs have become more significant in the field of tumor treatment as a novel method of treating tumor illnesses.Certain quality standards must be met for an antibody medication to be highly effective.Depth filtration,affinity chromatography,and ion chromatography are efficient ways to get rid of contaminants in protein samples.In order to effectively produce high-purity antibody drugs,this study carried out optimization experiments on the antibody purification process and successfully carried out large-scale production.1.Process optimization for CTLA4 McAb purification and scale-up of productionThe depth filtering of proteins,affinity chromatography,cation exchange chromatography,and anion exchange chromatography were among the process stages examined in this study to determine the most suitable process parameters.Finally,it was discovered that depth filtering could be accomplished using the D0SP and X0SP membrane package from Merck company.Eshmuno A was confirmed as the affinity filler and the load range was less than or equal to 40 g/L,the pH of elution buffer was 3.6±0.1.And the low pH process was inactivated at pH 3.5～3.7 for 1～1.5 h.As a cation exchange filler,Cytiva’s Capto MMC Impres was chosen,and 9%B phase was used for leaching and 29%B phase for elution.Leaching volume was 5～10 CV and loading capacity ranged from 45～55 g/L.Eshmuno Q from Merck was selected as anion exchange filler,the load range is 60～100 g/L,the sample conductance range is 5～10 mS/cm and the pH condition is 7.8±0.1.And besides,Pall’s prefiltered membrane package NP6LPRTP1(0.7 m2)and filter membrane package NP5LUPRMP1S(0.5 m2)were selected for virus removal.In the ultrafiltration process,two 1.14 m2 membrane packages were finally selected for concentration and fluid exchange.According to the analysis of the data from the pilot test 200 L scale-up production experiments and the detection datas,the process has scale-up performance,and the quality of the final product at the end of scale-up production is consistent with the quality of the final product at the process development stage.2.Drug effect evaluations of purified CTLA4 McAbThe results of pharmacokinetic test showed that there was no significant difference in the main pharmacokinetic parameters between male and female rhesus monkeys after intravenous injection of 0.3,1 and 3 mg/kg CTLA4 antibody injection(P>0.05),and as the dose was increased,so was the exposure.As the dose is increased,C0,AUClast and AUCINF_obs also increase.The results of the preclinical in vivo efficacy test revealed that all of the tested animals were active and well-fed during the administration,and their body weight increased to some extent,indicating that the animals were well tolerated by the tested products.The antitumor experiment used hCTLA4 mice transplanted with MC3 8 colon cancer model.At the end point of the experiment,the mean tumor volume was 1116±106 mm3 in G1 Anti-KLH hIgG1(1 mg/kg)group,255±88 mm3 in G2 Ipi(1 mg/kg)group,with 86.3%TGITV.The G3 CTLA4 antibody(0.1 mg/kg)group had a mean tumor volume of 736±203 mm3 and a TGITV of 38.1%,whereas the G4 CTLA4 antibody(0.3 mg/kg)and G5 CTLA4 antibody(1 mg/kg)groups had mean tumor volumes of 47±12 mm3 and 33±15 mm3,respectively.TGITV scored 107.1%and 108.5%.The results showed that CTLA4 antibody could significantly inhibit tumor growth at the dose of 0.3 mg/kg and 1 mg/kg(P<0.001),and its anticancer impact was significantly superior to that of 1 mg/kg Ipi(P<0.05).Tumor weight inhibition rate and tumor volume inhibition rate findings were mostly in agreement.All of the examined samples were safe and had no obvious toxic and side effects on animals,which gave direction for the comparative evaluation of efficacy in phase Ⅰ clinical trials.