CE-LIF方法对重组人促红细胞生成素中N-寡糖的快速分析

目的：建立毛细管电泳-激光诱导荧光检测（CE-LIF）的方法对重组人促红细胞生成素（recombinant human erythropoietin，rhEPO）的N-寡糖进行快速分析。方法：以N-糖苷酶酶切rhEPO样品，采用磁珠辅助法收集酶切下来的N-寡糖，以APTS对酶切下来的N-寡糖进行荧光标记，利用CE-LIF实现分离检测，通过GU数据库进行糖型鉴定。CE-LIF条件：采用未涂层熔融石英毛细管（有效长度20 cm，总长度30 cm，内径50 μm），电迁移进样，电压设为-2 kV，进样时间2 s；毛细管分离温度设为25℃，毛细管分离电压为-30 kV，分离时间5.5 min；LIF检测器的激发波长及发射波长分别为488 nm和520 nm。结果：新建方法在60 min内完成样品前处理，5 min内完成分离分析检测；FA2BG2S2、FA2（3）G1S1和A2B 3个峰的迁移时间的RSD均小于0.2%，校正峰面积百分比RSD均小于2.6%（n=5）。结论：本研究建立的CE-LIF快速糖分析法高效快速，重复性好，可对糖型进行实时鉴定，适用于rhEPO制品的N-寡糖分析。

Objective: To develop a capillary electrophoresis with laser induced fluorescence (CE-LIF)method for rapid analysis of N-glycan in recombinant human erythropoietin (rhEPO). Methods: The N-glycan of rhEPO samples was released by PNGase F,collected by magnetic microparticles,labeled by fluorescence indicator APTS,and then separated by CE-LIF and identified by GU database. The experiment parameters for CE-LIF were using an uncoated fused silica capillary tube (effective length:20 cm;total length:30 cm;internal diameter:50 μm)with separation temperature being 25℃,separation voltage being -30 kV and separation time being 5.5 min. Electrophoretic injection with injection voltage was -2 kV and injection time was 2 s. The excitation wavelength and emission wavelength of LIF was 488 nm and 520 nm,respectively. Results: For the developed method,the preparation of N-glycan could be completed in 60 minutes and analysis could be finished in 5 minutes. For FA2BG2S2,FA2 (3)G1S1 and A2B peaks,the RSD of retention time was below 0.2% and the RSD of corrected peak area ratio was below 2.6% (n=5). Conclusion: The developed CE-LIF method is rapid,efficient and with good repeatability,which is capable of on-line identification of N-glycan and is viable to N-glycan analysis of rhEPO products.

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