靶向组织因子的抗体偶联药物的质量控制OA

Objective To investigate and establish quality control methods for the critical quality attributes(CQAs)of anti-tissue fac-tor antibody-drug conjugate(anti-TF-ADC).Methods The quality control of the anti-TF-ADC was performed from peptide mapping,purity and impurities,charge heterogeneity,and activities.Reversed-phase high-performance liquid chromatography(RP-HPLC)was used for peptide mapping identification.Purity analysis was conducted using size-exclusion chromatography(SEC)and sodium dodecyl sulfate capillary electrophoresis(CE-SDS).Charge heterogeneity was assessed by imaged capillary isoelectric focusing(icIEF).The drug-to-antibody ratio(DAR)was determined using hydrophobic interaction chromatography(HIC).Free cytotoxin residues were quan-tified by RP-HPLC.Binding activity and biological activity were evaluated using enzyme-linked immunosorbent assay(ELISA),ampli-fied luminescent proximity homogeneous assay(AlphaLISA),and cell proliferation inhibition assay.Results For the anti-TF-ADC drug,peptide mapping successfully identified key peptides,enabling specific identification.The purities of anti-TF-ADC drug were 98.89%and 97.99%by SEC and CE-SDS,respectively.The area percentages of main peak,acidic peak,and basic peak were 61.73%,29.24%,and 9.03%by icIEF,respectively.The DAR value determined by HIC was 3.92,with DAR4 as the dominant form(accounting for 49.2%).No residual free cytotoxin was detected by RP-HPLC.The data obtained from the determination of binding activity and biological activity of anti-TF-ADC drug all conformed to the four-parameter equation.The EC₅₀ value for the binding of Fab region to the target was 13.13 ng/mL.The EC₅₀ values for the binding of Fc region to Fc receptors CD16a,CD32a,and CD64 were 2 695.83 ng/mL,14 517.67 ng/mL,and 191.73 ng/mL,respectively.The EC₅₀ value for its cellular biological activity was 18.95 ng/mL.Conclusion Based on the physicochemical and biological characteristics of the anti-TF-ADC,quality control methods for its CQAs are successfully developed and validated,which ensures the clinical safety and efficacy of anti-TF ADC products from a quality control perspective.