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 C18 色谱柱（4.6 mm×250 mm,5 μm,300 Å）,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BEH300 C4 色谱柱（2.1 mm×50 mm,1.7 μm）和BEH300 C18 色谱柱（2.1 mm×150 mm,1.7 μm）,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MSE 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 interferon α2b 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 C18（4.6 mm×250 mm,5 μm,300 Å） 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 C18（2.1 mm×50 mm,1.7 μm） 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 MSE scan mode was applied. Results： 6 batches of human interferon α2b 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 interferon α2b in human interferon α2b products. In addition,the study also provides ideas for enterprises to conduct relevant research.
[1] 赵广荣. 重组人干扰素的研究进展[J]. 中国生物制品学杂志, 2010,23(12):1384
ZHAO GR.Progress in research on recombinant human interferon[J]. Chin J Biol,2010,23(12):1384
[2] 张德芳. 重组干扰素α-2b 合成研究进展[J]. 海峡药学,2017, 29(7):57
ZHANG DF.Progress in research on recombinant human interferon α-2b[J]. Strait Pharml J,2017,29(7):57
[3] RADWANSKI E,PERENTESIS G,JACOBS S,e t a l . Pharmacokinetics of interferon α-2b in healthy volunteers[J]. J Clin Pharmacol,1987,27(5):432
[4] ALKESH A,SHALIGRAM R,MANOJ D,et al.Development and validation of RP-UPLC method for quantification of interferon in pharmaceutical preparation of interferon[J]. Int J Pharm Biol Sci, 2012,2(2):135
[5] GITLIN G,TSARBOPOULOS A,PATEL S,et al.Isolation and characterization of a monomethioninesulfoxide variant of interferon α-2b[J]. Pharm Res,1996,13(5):762
[6] CHOU DK,KRISHNAMURTHY R,MANNING MC,et al.Effects of solution conditions on methionine oxidation in albinterferon α-2b and the role of oxidation in its conformation and aggregation[J]. J Pharm Sci,2013,102(2):660
[7] EP 9.0[S]. 2017:2780
[8] 陶磊,裴德宁,韩春梅,等. 液质联用进行干扰素理化对照品的一级结构鉴定及比对研究[J]. 药学学报,2015,50(1):75
TAO L,PEI DN,HAN CM,et al.Characterization and comparison of interferon reference standards using UPLC-MS[J]. Acta Pharm Sin,2015,50(1):75
[9] LI SH,SCHONEICH C,BORCHARDT RT.Chemical instability of protein pharmaceuticals – Mechanisms of oxidation and strategies for stabilization[J]. Biotechnol Bioeng,1995,48(5):490
[10] CHU JW,YIN J,WANG DIC,et al.Molecular dynamics simulations and oxidation rates of methionine residues of granulocyte colony-stimulating factor at different pH values[J]. Biochemistry,2004, 43(4):1019
[11] VOLKIN DB,MACH H,MIDDAUGH CR.Degradative covalent reactions important to protein stability[J]. Mol Biotechnol,1997,8(2):105
[12] CHU JW,BROOKS BR,TROUT BL.Oxidation of methionine residues in aqueous solutions:free methionine and methionine in granulocyte colony-stimulating factor[J]. J Am Chem Soc,2004, 126(50):16601
[13] CINDRIC M,GALIC N,VULETIC M,et al.Evaluation of recombinant human interferon α2b structure and stability by in-gel tryptic digestion,H/D exchange and mass spectrometry[J]. J Pharm Biomed Anal,2006,40(3):781
[14] RADHAKRISHNAN R,WALTER LJ,HRUZA A,et al.Zinc mediated dimer of human interferon-α(2b) revealed by X-ray crystallography[J]. Structure,1996,4(12):1453
[15] 中华人民共和国药典2020 年版. 三部[S]. 2020:300
ChP 2020. Vol Ⅲ[S]. 2020:300
