全柱成像毛细管等电聚焦电泳法评价IL-15融和蛋白的电荷异质性

Charge heterogeneity evaluation of IL-15 fusion protein by capillary isolectric focusing-whole column imaging detection

目的:建立全柱成像毛细管等电聚焦电泳(capillary isoelectric focusing-whole column imaging detection,c IEF-WCID)方法评价白细胞介素-15(IL-15)融和蛋白的电荷异质性。方法:通过比较5种不同的两性电解质,以及不同尿素浓度(0,3,6和8 mol·L-1)对实验结果的影响,优化建立了IL-15融和蛋白的全柱成像毛细管等电聚焦电泳方法。具体方法为采用3 mol·L-1尿素、0.35%甲基纤维素、4%两性电解质混合溶液作为样品缓冲液,聚焦电压为3 k V,预聚焦时间1 min,聚焦时间7 min。结果:利用新建方法测定了IL-15融和蛋白的等电点范围为6.00～6.71,分离检测到12个异构体,重复性实验中各异构体的峰面积百分比RSD为0.7%～7.4%,等电点RSD为0.0%～0.1%;稳定性实验中各异构体峰面积百分比的RSD为1.5%～10.5%,等电点RSD为0.0%～0.3%;3个不同批次IL-15融和蛋白的各异构体峰面积百分比和等电点基本一致。去N-糖基化及去唾液酸化实验结果表明,IL-15融和蛋白的电荷异构体主要是由N-糖基化不同产生,而酸性端异构体是由于唾液酸化程度不同产生的。结论:研究建立的全柱成像毛细管等电聚焦电泳方法可以有效分离IL-15融和蛋白的12种异构体,糖基化尤其是唾液酸化是引起电荷异质性的主要原因,新建方法对IL-15融和蛋白的质量控制有重要意义。

Objective： To develop a capillary isoelectric focusing-whole column imaging detection（ c IEF-WCID） to evaluate the charge heterogeneity of IL-15 fusion protein. Methods： The c IEF-WCID method for IL-15 fusion protein was established and optimized by comparing 5 different ampholytes and different working concentrations of urea（ 0,3,6 and 8 mol·L-1）. The optimized experiment parameters were using 3 mol·L-1 urea,0. 35% methylcellulose and 4% pharmalyte mixture as sample buffer,3 k V as focusing voltage,1 min as prefocusing time,and 7 min as focusing time. Results： The isoelectric point（ p I） range of IL-15 fusion protein was determined to be 6. 00 ～ 6. 71 and 12 charge isomers were separated. In repeatability test,the peak area percentage RSD values of individual isomer were 0. 7% ～ 7. 4% and p I RSD values were 0. 0% ～ 0. 1%. In stability test,the peak area percentage RSD values of individual isomer were 1. 5% ～ 10. 5% and p I RSD values were 0. 0% ～ 0. 3%.The peak area percentages and p I values of individual isomer were consistent among three batches of IL-15 fusion protein. The results of N-deglycosylation and asialo-test suggested that the charge isomers of IL-15 fusion protein were mainly caused by N-glycosylation heterogeneity and acidic isomers were mainly caused by sialic acid. Conclusion：The developed method is viable to effectively separate 12 charge isomers of IL-15 fusion protein. Glycosylation,especially sialic acid is a major cause of charge heterogeneity. This method would be of great significance to qualitycontrol of IL-15 fusion protein.