TNF-α单抗分子大小变异体与电荷变异体色谱行为的比较研究

Comparative study of size and charge heterogeneities of anti-TNF-αantibodies by high performance liquid chromatography

目的：分析对比原研TNF-α全人源单克隆抗体（单抗）和4个生物类似药的分子大小和电荷异质性差异。方法采用体积排阻色谱（SEC-HPLC）分析抗体大小异质性，采用弱阳离子交换色谱（WCX-HPLC）分析单抗电荷异质性。结合羧肽酶B（CpB）处理单抗，分析单抗主要电荷异质性来源。结果经体积排阻色谱检测发现，4个生物类似药与原研单抗在单体比例上基本一致，其中两个生物类似药（SBP-3，SBP-4）聚合物比例较原研单抗略高。离子交换色谱检测结果显示，4个生物类似药与原研单抗的电荷分布在碱区以及主峰比例上有明显差异。羧肽酶B处理样品后，样品碱区峰比例明显减少，提示4个生物类似药在碱区上的差异主要由C末端赖氨酸引起。结论4个生物类似药虽然在单抗分子大小异质性上与原研单抗无明显差别，然而在电荷变异体组成上却存在较大差异。在对单抗质量控制中应重视电荷异质性检测的重要性。

Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products （ SBP ） .Methods The size exclusion chromatography （ SEC-HPLC ） and weak cation exchange chromatography （ WCX-HPLC ） were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B （CpB） treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .