抗HER2单抗注射液(皮下注射)的质量控制

Quality control of anti-HER2 monoclonal antibody for subcutaneous injection

目的 研究并建立针对抗HER2单抗注射液(皮下注射)的关键质量属性质控方法。方法 分别采用胰蛋白酶或Lys-C酶切结合反相高效液相色谱法(reversed-phase high performance liquid chromatography, RP-HPLC)的肽图分析法进行抗HER2单抗的特异性鉴别。采用还原/非还原十二烷基硫酸钠毛细管电泳(capillary electrophoresis-sodium dodecyl sulfonate, CE-SDS)和分子排阻色谱(size-exclusion chromatography, SEC)法进行纯度控制。采用阳离子交换色谱(ion-exchange chromatography, IEC)法进行电荷异质性分析。采用亲水相互作用液相色谱(hydrophilic interaction liquid chromatography, HILIC)法进行寡糖图谱分析。采用酶活力测定法分析透明质酸酶含量。采用BT474细胞增殖抑制法测定生物学活性。采用紫外分光光度法测定蛋白含量。结果 两种肽图检测法均获得特征图谱,对抗HER2单抗发挥很好的特异性鉴别作用。还原CE-SDS的重链和轻链峰面积之和百分比为(98.63±0.14)%,非还原CE-SDS主峰面积百分比为(98.20±0.11)%,前峰面积百分比总和为(1.47±0.04)%。SEC测得的单体峰面积百分比为(99.82±0.05)%。IEC测定的主要活性成分(峰3*+峰3)以及产品相关杂质(峰4)的峰面积百分比分别为(75.01±0.04)%和(6.72±0.03)%。寡糖图谱分析aFuc、G2和Man5的面积百分比分别为(9.20±0.01)%,(6.33±0.02)%和(1.92±0.01)%。透明质酸酶含量为(2 021.02±238.41)U/ml。供试品相对于参比品的生物学活性为(95.87±6.10)%,蛋白含量为(115.60±0.45)mg/ml。结论 针对抗HER2单抗注射液(皮下注射)关键质量属性的质控方法的建立可以有效确保该类产品的安全性和有效性。

Objective To study and establish a quality control method for critical quality attributes of recombinant anti-HER2 monoclonal antibody for subcutaneous injection.Methods The anti-HER2 mAb was specifically identified by peptide mapping method based on trypsin or Lys-C digestion combined with reversed-phase high performance liquid chromatography(RP-HPLC).The purity control was performed by reducing/non-reducing capillary electrophoresis sodium dodecyl sulfate(CE-SDS)and size-exclusion chromatography(SEC).The charge heterogeneity was analyzed by ion-exchange chromatography(IEC).The oligosaccharide profiling was assayed by hydrophilic interaction liquid chromatography(HILIC)-UPLC.The hyaluronidase content was also detected by enzyme activity assay.Biological activity was determined by BT474 cell proliferation inhibition assay,and the protein concentration was measured by UV spectrophotometry.Results Both trypsin and Lys-C digestion followed by RP-HPLC provided specific chromatogram for anti-HER2 antibody.The percentage of the peak area of heavy chain and light chain of reduced CE-SDS was(98.63±0.14)%,while the percentages of the main peak area and the front peak area of non-reduced CE-SDS were(98.20±0.11)%and(1.47±0.04)%,respectively.The percentage of main peak area in SEC was(99.82±0.05)%.The area percentages of major bioactive components(peak 3+peak 3)and product-related impurity(peak 4)determined by IEC were(75.01±0.04)%and(6.72±0.03)%,respectively.The percentages of aFuc,G2 and Man5 areas were(9.20±0.01)%,(6.33±0.02)%and(1.92±0.01)%by oligosaccharide mapping analysis,respectively.The hyaluronidase concentration was(2021.02±238.41)U/ml.The biological activity of the test product relative to the reference product was(95.87±6.10)%,and the protein concentration was up to(115.60±0.45)mg/ml.Conclusion The establishment of quality control methods for key quality attributes of HER2 monoclonal antibody(subcutaneous injection)can effectively ensure the safety and effectiveness of such products.