人源化抗VEGF单克隆抗体制品的大小异质性分析

Size heterogeneity analysis of monoclonal antibody products

目的：比较十二烷基磺酸钠聚丙烯酰胺凝胶电泳（sodiumdodecylsulfatepolyacrylam-idegelelectrophoresis，CE-SDS）和分子排阻高效液相（sizeexclusionhighperformanceliquidchromatogra-phy，SE-HPLC）在单克隆抗体（单抗）大小异质性分析中的区别。方法应用非还原和还原CE-SDS以及常规、变性和变性还原SE-HPLC评价人源化抗血管内皮细胞生长因子（VEGF）单抗的大小异质性。结果对于多聚体的分析，虽然非还原CE-SDS结果显示其含量为0．82％±0．01％，显著低于常规SE-HPLC的分析结果（5．08％±0．10％），但与变性SE-HPLC的分析结果（1．05％±0．02％）则基本一致；在非还原状态下对单抗的片段分析，非还原CE-SDS结果显示其含量为7．12％±0．04％，显著高于常规SE-HPLC的分析结果（0．02％±0．01％）以及变性SE-HPLC的分析结果（0．62％±0．01％）；而在还原状态下对单抗的片段分析，还原CE-SDS结果显示其含量为（3．19％±0．50％），其含量同样高于变性还原SE-HPLC的分析结果（0．07％±0．01％）。结论在分析多聚体含量时，由于常规SE-HPLC对共价和非共价连接的多聚体形式均可显示，相比CE-SDS相比更加客观；而在分析片段含量时，非还原CE-SDS可显示非共价键连接的片段，还原CE-SDS可显示降解片段，所以CE-SDS相比常规SE-HPLC更具优势。两种不同原理分析方法的组合有效保障了单抗制品大小异质性的质量控制。

Objective To compare the capability of capillary electrophoresis-sodium dodecyl sul-fate （ CE-SDS） and size exclusion-high performance liquid chromatography （ SE-HPLC） for analysis of size heterogeneity of monoclonal antibody products .Methods The size heterogeneity of one humanized anti-VEGF monoclonal antibody was analyzed by using non-reduced and reduced CE-SDS, and conventional , de-natured and denatured reduced SE-HPLC.Results The percentage of aggregates detected by non-reduced CE-SDS （0.82%±0.01%） was equal to that by using denatured SE-HPLC （1.05%±0.02%）, but it was significantly lower than that by using conventional SE-HPLC analysis （5.08%±0.10%）.With regard to fragments analyzed with non-reduced antibodies, its percentage was （7.12±0.04）% measured by non-re-duced CE-SDS analysis that was significantly higher than that by conventional SE -HPLC analysis （0.02%± 0.01%） and denatured SE-HPLC analysis （0.62%±0.01%）.Using reduced antibodies , the percentage of fragments was （3.19±0.50）%tested by reduced CE-SDS analysis that was significantly higher than that by using denatured reduced SE-HPLC analysis （0.07%±0.01%）.Conclusion Conventional SE-HPLC was more objective than CE-SDS for content analysis of aggregates , as both the covalent and non-covalent forms of aggregates could be detected .Non-reduced CE-SDS could demonstrate the content of clips , while reduced CE-SDS showed the degraded fragments .Therefore, CE-SDS had an advantage over conventional SE-HPLC for content analysis of fragments .The use of the two analytical methods in combination provided solid techni-cal supports for the quality control of size heterogeneity of monoclonal antibodies .