重组人促红素理化对照品的质量评价

目的：建立重组人促红细胞生成素的理化对照品的质量标准，对8家企业的促红素理化对照品进行质量评价。方法：用正常小鼠网织红细胞计数法测定体内生物学活性，使用LC-MS绘制质量肽图谱。色谱条件：采用BEH300 C₄色谱柱（2.1 mm×50 mm，3.5 μm），流动相为水（A）-乙腈（B）-1%甲酸水溶液（C），梯度洗脱（0~60 min，1% B→60% B，10% C；60~61 min，60% B→90% B，10% C；61~75 min，90% B→1% B，10% C），流速0.2 mL·min^-1，柱温为40℃，样品保存温度为10℃，上样量5 μL；质谱条件：MS^E模式采集数据，毛细管电压3 000 V，Cone电压40 V，去溶剂气体温度350℃，源温120℃，去溶剂气体流速800 L·h^-1，扫描范围m/z 50~2 000。确证一级结构，二硫键链接方式及糖基化位点；绘制N糖图谱，分析寡糖结构；用毛细管区带电泳分析异构体。采用压力进样3.45 kPa；进样时间10 s，分离电压15.4 kV，分离时间80 min，检测波长214 nm。其余检测项目按中国药典2015年版三部方法进行。结果：建立的方法适合促红素理化对照品的检测，各样品的氨基酸序列均与理论一致；二硫键连接方式均为Cys7-Cys161、Cys29-Cys33；O-糖基化位点均为Ser126，N-糖基化位点均为Asn24、Asn38、Asn83；各样品的糖基修饰方式存在差异，不同糖型的相对比例不一致。等电聚焦结果为5~8条电荷异构体条带，不同样品的电荷异构体的组成比例有所差异，蛋白质含量、体内比活性、电泳纯度、液相纯度、等电聚焦、唾液酸含量、N端氨基酸序列、肽图结果均符合拟定的理化对照品质量标准。结论：建立了理化对照品的质量标准，不同企业的理化对照品质量总体稳定一致，但糖基化水平存在一定差异，分析比较了不同企业的促红素理化对照品在生物比活性，糖基化，异构体分布、电荷异质性等方面的差异。

Objective: To establish the quality standard of physicochemical reference for recombinant human erythropoietin(rhEPO), and to evaluate the quality of rhEPO physicochemical references from eight pharmaceutical companies.Methods: The biological activity in vivo was measured by normal mouse reticulocyte count method, and the peptide map was drawn by LC-MS.Chromatographic conditions:BEH300 C4 column (2.1 mm×50 mm, 3.5 μm)was adopted, the mobile phase was water(A)-acetonitrile(B)-1% formic acid (C)with gradient elution(0-60 min, 1%B → 60%B, 10%C; 60-61 min, 60%B → 90%B, 10%C; 61-75 min, 90%B → 1%B, 10%C)at the flow rate of 0.2 mL·min^-1, the column temperature was 40℃, the sample storage temperature was 10℃, the sample volume was 5 μL; Mass spectrometry conditions:The data were collected by MSE model, the capillary voltage was 3000 V, the cone voltage was 40 V, the solvent gas temperature was 350℃, the source temperature was 120℃, the solvent gas flow rate was 800 L·h^-1, the scan range was m/z 500-2 000.The primary structure, the disulfide bond linkage and glycosylation sites were confirmed.N-linked glycosylation maps were drawn and the oligosaccharide structures were analyzed by LC-MS, and the isomers were analyzed by capillary zone electrophoresis.The sample inject pressure was 3.45 kPa, the inject time was 10 s, the separate voltage was 15.4 kV, the separate time was 80 min and the detection wavelength was 214 nm.The rest of the test items were in accordance with the 2015(3)edition of Chinese Pharmacopoeia.Results: The established methods were compared and applied to the tests of rhEPO physicochemical references, and the results met the proposed quality standard for rhEPO physicochemical references.All the samples had consistent amino acid sequence, disulfide bonds(Cys7-Cys161, Cys29-Cys33), O-glycosylation site(Ser126)and N-glycosylation sites(Asn24, Asn38, Asn83), glycosylation patterns of the samples were different, and the relative proportions of the different glycoforms were not consistent.Isoelectric focusing results showed 5-8 charge isomers bands, composition charge isomers of different samples were different.The content of protein, specific activityin vivo, electrophoresis purity, liquid phase purity, isoelectric focusing, sialic acid content, N terminal amino acid sequence and peptide mapping results were in line with the physicochemical quality control standard.Conclusion: The quality standard for rhEPO physicochemical reference is established, and the qualities of physicochemical references from different companies are generally stable and consistent, but there are some differences in the glycosylation level. Analysis and comparison of differences between rhEPO physicochemical reference from different companies in biological activity.

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