摘要
目的:采用高效液相色谱(HPLC)、超高效液相色谱-四极杆串联飞行时间质谱(UPLC-Q-TOF MS)技术对人干扰素α2b中甲硫氨酸的氧化情况进行研究。方法:HPLC法采用Jupiter 5U C_(18)色谱柱(4.6mm×250 mm,5μm,300?),流动相A为乙腈-水-三氟乙酸(300∶700∶2),流动相B为乙腈-水-三氟乙酸(800∶200∶2),梯度洗脱,流速1.0 mL·min^(-1),检测波长210 nm。UPLC-Q-TOF MS法采用BEH300C4色谱柱(2.1 mm×50 mm,1.7μm)和BEH300 C_(18)色谱柱(2.1 mm×150 mm,1.7μm),流动相A为0.1%甲酸水溶液,流动相B为0.1%甲酸乙腈溶液,梯度洗脱,流速0.2 mL·min^(-1);采用Q-TOF MS的电喷雾正离子模式,相对分子质量测定时采用MS模式进行扫描,氨基酸序列测定时采用MS^(E)模式进行扫描。结果:对2家企业6批人干扰素原液进行了测定,2家企业产品中甲硫氨酸氧化比例差别较大,其中一家企业氧化比例较高,第16位的甲硫氨酸氧化比例达到了99%以上。结论:在人干扰素α2b相关制品中增加氧化杂质的控制是有必要的。本研究也为企业进行相关研究提供了思路。
Objective:To evaluate the oxidation of methionine in human interferonα2b using high performance liquid chromatography(HPLC)and ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-fight mass spectrometry(UPLC-Q-TOF MS).Methods:The HPLC analysis was performed on a Jupiter 5U C_(18)(4.6 mm×250 mm,5μm,300Å)column with the mobile phase A consisting of acetonitrile-water-trifluoroacetic acid(300∶700∶2)and mobile phase B consisting of acetonitrile-water-trifluoroacetic acid(800∶200∶2),with gradient elution at a flow rate of 1.0 mL·min^(-1),the detection wavelength was 210 nm.The UPLC-Q-TOF MS analysis was performed on a BEH300 C_(18)(2.1 mm×50 mm,1.7μm)column with 0.1%formic acid solution as mobile phase A and 0.1%formic acid-acetonitrile solution as mobile phase B,with gradient elution at a flow rate of 0.2 mL·min^(-1).For relative molecular mass detection,positive and MS scan mode was applied.For peptide mapping,positive and MS^(E) scan mode was applied.Results:6 batches of human interferonα2b samples from two manufactures were analyzed,and the oxidation ratio was different between two manufacturers.The oxidation ratio was much higher in one manufacturer,and more than 99%was oxidized at methionine 16 site.Conclusion:It is necessary to control oxidized interferonα2b in human interferonα2b products.In addition,the study also provides ideas for enterprises to conduct relevant research.
作者
尹红锐
张颖
徐明明
陈钢
邵泓
YIN Hong-rui;ZHANG Ying;XU Ming-ming;CHEN Gang;SHAO Hong(NMPA Key Laboratory of Quality Control of Therapeutic Monoclonal Antibodies,Shanghai Institute for Food and Drug Control,Shanghai 201203,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2021年第7期1203-1208,共6页
Chinese Journal of Pharmaceutical Analysis
关键词
人干扰素α2b
细胞因子
甲硫氨酸
氧化
高效液相色谱
超高效液相色谱-四极杆串联飞行时间质谱法
human interferonα2b
cytokine
methionine
oxidation
high performance liquid chromatography
ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-fight mass spectrometry
