重组抗CD38单克隆抗体的质量研究

目的：针对抗白细胞分化抗原38（cluster of differentiation 38，CD38）单抗的关键质量属性建立质控方法。方法：采用肽图法进行鉴别；采用还原/非还原十二烷基磺酸钠毛细管电泳（capillary electrophoresis-sodium dodecyl sulfonate，CE-SDS）和尺寸排阻色谱（size exclusion-high performance liquid chromatography，SEC-HPLC）进行纯度控制；采用成像毛细管等电聚焦电泳（imaging capillary isoelectric focusing electrophoresis，iCIEF）测定电荷异质性；采用酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）测定结合活性；采用补体依赖的细胞毒作用（complement dependent cytotoxicity，CDC）和抗体依赖的细胞介导的细胞毒作用（antibody-dependent cell-mediated cytotoxicity，ADCC）的方法测定生物学活性。其他各项指标均应符合2015年版《中华人民共和国药典》以及其他相关要求。结果：肽图检测具有特征图谱，起到很好的鉴别作用。还原CE-SDS的轻链与重链峰面积之和百分比为（97.87±0.72）%，非还原CE-SDS主峰峰面积百分比为（97.70±0.35）%；SEC-HPLC单体的峰面积百分比为（99.15±0.01）%，高分子量物质的峰面积百分比为（0.47±0.002）%，低分子量物质的峰面积百分比为（0.37±0.01）%。iCIEF分析的主要亚型的峰面积百分比为（63.54±0.37）%，酸性亚型的峰面积百分比为（23.07±0.50）%，碱性亚型的峰面积百分比为（13.38±0.12）%。结合活性的半最大效应浓度（concentration for 50% of maximal effect，EC₅₀）值为（7.35±0.06）ng·mL^-1。CDC活性EC₅₀值为（270.67±12.42）ng·mL^-1，ADCC活性EC₅₀值为（8.71±1.04）ng·mL^-1。结论：针对抗CD38单抗的质量属性，研究建立质控方法并进行质量研究，确保产品的安全、有效和质量可控，对抗CD38单抗的质量控制具有借鉴意义。

Objective:To establish the quality control methods for the key quality attributes of the anti-leukocyte differentiation antigen 38 (cluster of differentiation 38,CD38) monoclonal antibody(mAb). Methods:Peptide map analysis was used for identification of an anti-CD38 mAb. The purity was measured by reduced/non-reduced capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS) and size exclusion-high performance liquid chromatography(SEC-HPLC). Charge heterogeneity was measured by imaging capillary isoelectric focusing electrophoresis (iCIEF). Binding potency was measured by ELISA assay. The biological activity was determined by complement dependent cytotoxicity (CDC) method and antibody-dependent cell-mediated cytotoxicity (ADCC) method. Other quality control items complied with corresponding requirements of Chinese Pharmacopeia 2015 edition and related guidelines. Results:Peptide mapping showed the specific spectrum of the anti-CD38 mAb,which could be adopted for the identification test. The sum percentage of light chain and heavy chain peak area detected by reduced CE-SDS was (97.87±0.72)%;the area percentage of main peak detected by non-reduced CE-SDS was (97.70±0.35)%. The area percentage of monomer peak,high molecular mass peak and low molecular mass peak detected by SEC-HPLC were (99.15±0.01)%,(0.47±0.002)% and (0.37±0.01)%,respectively. The area percentage of major subtypes peak,acidic subtypes peak and alkaline subtypes peak detected by iCIEF were (63.54±0.37)%,(23.07±0.50)% and (13.38±0.12)%,respectively. The concentration for 50% of maximal effect (EC₅₀) of binding activity was (7.35±0.06)ng·mL^-1. The EC₅₀ value of CDC activity was (270.67±12.42)ng·mL^-1 and the EC₅₀ value of ADCC activity was (8.71±1.04)ng·mL^-1. Conclusion:A series of quality control methods for anti-CD38 mAb have been established,which can be adaptable in ensuring the safety,effectiveness and quality controllability for the product. It has reference significance for the quality control of anti-CD38 mAb. 

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