Objective To establish quality control methods for PEGylated recombinant anti-tumor necrosis factor α(TNFα)monoclonal antibody(PEG-anti-TNFα mAb).Methods Peptide mapping was used for identification of the PEG-anti-TNFαmAb.Size exclusion-high performance liquid chromatography(SEC-HPLC)methods were used to measure the molecular size heterogeneity,and cation exchange-high performance liquid chromatography(CEX-HPLC)was used to test the charge heterogeneity.The residual polyethylene glycol(PEG)and mismatched isomers were determined by reversed-phase-ultra performance liquid chromatography(RP-UPLC).The potency was measured by HEK293 cells stably transfected with reporter gene driven by downstream reaction elements of TNF α associated receptors.Results Peptide mapping detection of PEG-anti-TNFα mAb had the corresponding characteristic map,which played a good role in the identification.The peak area percentages of monomer and high molecular weight(HMW)determined by SEC-HPLC were(99.66±0.01)％and(0.31±0.01)％,respectively,and that of low molecular weight(LMW)was lower than the limit of quantification.The peak area percentages of the main subtypes,the acid subtype,and the basic subtype analyzed by CEX-HPLC were(98.31±0.10)％,(1.31±0.04)％and(0.38±0.07)％,respectively.The residual PEG was lower than the limit of quantification and the content of mismatched isomers was(0.76±0.007)％.The concentration for 50％of maximal effect(EC50)of PEG-anti-TNFα mAb in bioassay was(2.22±0.11)ng/mL.Conclusion According to the key quality attri-butes of PEG-anti-TNFα mAb,the corresponding quality control methods were established,which can ensure its safety,effectiveness and controllable quality,and provide reference for the quality control methods and strategies of PEG-anti-TNFα mAb products.
Polyethylene glycol(PEG)Tumor necrosis factor α(TNFα)Monoclonal antibody(mAb)Quality control
NETL
NSTL
万方数据
