重组抗淀粉样β肽单克隆抗体药物的质量控制研究

Quality control study of recombinant anti-amyloid-βmonoclonal antibody

目的:研究并建立针对抗淀粉样肽(amyloid-β,Aβ)单克隆抗体的关键质量属性质量控制方法。方法:基于胰蛋白酶酶切和反相高效液相色谱法(RP-HPLC)进行肽指纹图谱分析,分子排阻色谱法(SE-HPLC)进行单体聚体纯度分析,采用还原/非还原十二烷基硫酸钠毛细管电泳法(CE-SDS)进行大小变异体纯度分析,采用阳离子色谱法(CEX-HPLC)进行电荷变异体纯度分析,采用HILIC-UPLC法进行寡糖分析,采用ELISA法测定Aβ抗原相对结合活性。其他各项指标均应符合《中华人民共和国药典》2020年版以及其他相关要求。结果:肽指纹图谱起到良好的鉴别作用;SE-HPLC法中单体相对百分含量为(99.40±0.00)%,聚体的相对百分含量为(0.55±0.00)%;还原CE-SDS法中轻重链之和的相对百分含量为(98.11±0.08)%,非还原CE-SDS法的主峰相对百分含量为(96.57±0.15)%,HHL峰相对百分含量为(1.91±0.10)%;CEX-HPLC法中主成分相对百分含量为(75.08±0.06)%,酸性变异体的相对百分含量为(16.12±0.05)%,碱性变异体的相对百分含量为(8.80±0.05)%;HILIC-UPLC寡糖图谱分析的(G0F+G1F+G2F),Man5,G0相对百分含量分别为(91.56±0.04)%,(1.21±0.03)%,(2.60±0.01)%;ELISA法中与Aβ抗原的相对结合活性为(101.40±0.53)%。结论:针对抗Aβ单抗的质量属性,研究建立质控方法并进行质量研究,确保产品安全有效和质量可控,为该类单抗产品的质量控制方法和策略提供参考依据。

Objective:To study and establish a quality control method for the critical quality attributes of anti-amyloid-βmonoclonal antibody(anti-AβmAb).Methods:Peptide fingerprints assay based on trypsin digestion and reversed-phase HPLC(RP-HPLC)was used for identification.The purities of monomers and polymers were analyzed by size-exclusion HPLC(SE-HPLC).Reducing/non-reducing capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS)was used to analyze the purities of the size variants.The purities of the charge variants were analyzed by cationic chromatography(CEX-HPLC).The oligosaccharides were analyzed by HILIC-UPLC.The relative binding activity of Aβantigen was determined by ELISA.All other indicators should comply with the current version of the Pharmacopoeia of the People's Republic of China and other relevant requirements.Results:The fingerprint of peptide achieved good identification.The relative percentage of SE-HPLC monomer was(99.40±0.00)%and the relative percentage of polymers was(0.55±0.00)%.The relative percentage of light and heavy chains in reduced CE-SDS was(98.11±0.08)%,the relative percentage of main peak was(96.57±0.15)%and the relative percentage of HHL peak was(1.91±0.10)%in non-reduced CE-SDS.The relative percentages of principal component,acid variant and basic variant were(75.08±0.06)%,(16.12±0.05)%and(8.80±0.05)%respectively in CEX-HPLC.The relative percentages of(G0F+G1F+G2F),Man5 and G0 were(91.56±0.04)%,(1.21±0.03)%and(2.60±0.01)%,respectively.The relative binding activity to Aβantigen by ELISA was(101.40±0.53)%.Conclusion:According to the quality attribute of anti-Aβmonoclonal antibody,the quality control methods were established to ensure the safety,effectiveness and quality control of the product and provide a reference for the quality control methods and strategies of this kind of monoclonal antibody products.The study is of great significance to be a representative quality control reference for anti-AβmAbs.