抗EGFR/c-Met双特异性抗体药物的质量控制OA

Objective To investigate and establish quality control methods for the critical quality attributes(CQAs)of anti-EGFR/c-Met bispecific antibody(BsAb).Methods The peptide mapping was used to identify the anti-EGFR/c-Met BsAb by reversed-phase ultra-high performance liquid chromatography(RP-UPLC)technology.Purity analysis was conducted using size exclusion chromatogra-phy(SEC),sodium dodecyl sulfate capillary electrophoresis(CE-SDS)and imaging capillary isoelectric focusing electrophoresis(icIEF).The contents of homodimeric impurities were analyzed by hydrophobic liquid chromatography(HIC).The contents of isomeri-zation and oxidation modifications were analyzed by multi-attribute method(MAM).Binding activity to the c-Met antigen was analyzed using time-resolved fluorescence resonance energy transfer assay(TR-FRET).Biological activity was determined by luciferase reporter gene assay(LRGA).Results The peptide mapping successfully identified all the characteristic peptides of the anti-EGFR/c-Met BsAb.The purity of anti-EGFR/c-Met BsAb was(99.24±0.00)%and(98.44±0.10)%by SEC and CE-SDS,respectively.The area per-centages of main peak,acidic peaks and basic peaks were(74.44±0.55)%,(22.91±0.28)%and(2.64±0.31)%by icIEF,respec-tively.The contents of homodimers were all below the limits of quantification(LOQ)by HIC.The contents of isomerization and oxida-tion modifications were(0.62±0.01)%and(2.35±0.03)%by MAM,respectively.Compared to the reference sample,the binding activity and the biological activity of anti-EGFR/c-Met BsAb were(99.00±10.78)%and(103.50±10.09)%,respectively.Conclusion Based on the physicochemical and biological characteristics of the anti-EGFR/c-Met BsAb,the quality control methods for its CQAs are successfully developed and validated,which ensures the clinical safety and efficacy of anti-EGFR/c-Met BsAb products from the perspective of quality controllability.