目的: 用人干扰素α2b相关蛋白检测方法联合质谱对人干扰素α2b相关蛋白进行一级结构分析,确认不同保留时间的相关蛋白。方法: 液相色谱条件为Agilent ZORBAX C18色谱柱(250 mm×4.6 mm,300,5 μm);流动相A为30%乙腈水溶液(含0.2%三氟乙酸),流动相B为80%乙腈水溶液(含0.2%三氟乙酸),流速为0.5 mL·min-1,柱温为室温。质谱条件为电喷雾正离子模式,采用MS模式进行扫描,毛细管电压为2 500 V,Cone电压为40 V,去溶剂气体温度为350 ℃,源温为120 ℃,去溶剂气体流速为800 L·h-1,扫描范围为m/z 400~4 000,碰撞电压为40 V,MS采集时间为20~60 min。结果: 相关蛋白的相对分子质量均与理论相对分子质量一致,3号峰为主峰,相对分子质量为19 264.81和19 395.93,为人干扰素α2b和N-末端甲硫氨酸化人干扰素α2b;1号峰相对分子质量为19 280.72,为氧化人干扰素α2b,2号峰相对分子质量为19 412.25,为(氧化+N-末端甲硫氨酸化)人干扰素α2b;4号峰相对分子质量为19 265.13,为人干扰素α2b;5号峰相对分子质量为19 322.81,为(氧化+N-末端乙酰化)人干扰素α2b;6号峰相对分子质量为19 306.79,为N-末端乙酰化人干扰素α2b。结论: 确认主峰为人干扰素α2b和N-末端甲硫氨酸化的人干扰素α2b,确认保留时间在主峰前面的相关蛋白分别为氧化和(氧化+N-末端甲硫氨酸化)修饰的人干扰素α2b,保留时间在主峰后面的相关蛋白分别为(氧化+N-末端乙酰化)和N-末端乙酰化修饰的人干扰素α2b。
朱秋媚, 刘玉林, 俞露, 王江林, 梁冠桥, 孙瑞欣, 刘琳琳, 刘景会
. 基于2020年版《中华人民共和国药典》相关蛋白检测方法并联合质谱分析人干扰素α2b相关蛋白的一级结构[J]. 药物分析杂志, 2022
, 42(6)
: 1096
-1100
.
DOI: 10.16155/j.0254-1793.2022.06.23
Objective: To analyze the primary structure of human interferon α2b related proteins by using human interferon α2b related proteins detection method combined with mass spectrometry, and to identify related proteins with different retention times. Methods: Agilent ZORBAX C18(250 mm×4.6 mm, 300, 5 μm) column was used and mobile phase A was 30% acetonitrile(0.2% TFA), mobile phase B was 80% acetonitrile(0.2% TFA). The flow rate was 0.5 mL·min-1, column temperature was room temperature. Electrospray positive ion mode with MS mode scaning was used. Capillary voltage was 2 500 V, cone voltage was 40 V, desolvation temperature was 350 ℃, source temperature was 120 ℃, desolvation gas flow was 800 L·h-1, scan scope was 400-4 000 m/z, collision voltage was 40 V and MS acquisition time was 20-60 min. Results: The molecular weight of the related proteins was consistent with the theoretical relative molecular mass. Peak 3 was the main peak, which relative molecular mass were 19 264.81 and 19 395.93. This peak was human interferon α2b and N-terminal methionine human interferon α2b. The peak 1 with relative molecular mass of 19 280.72, was oxidized human interferon α2b; and the peak 2 with relative molecular mass of 19 412.25 was (oxidation+N-terminal methioninylation) human interferon α2b. Peak 4 was human interferon α2b with relative molecular mass at 19 265.13. Peak 5 was (oxidation+N-terminal acetylation) human interferon α2b with the relative molecular mass of 19 322.81. Peak 6 was N-terminal acetylation of human interferon α2b with the relative molecular mass of 19 306.79. Conclusions: The main peak are human interferon α2b and N-terminal methionine human interferon α2b and the related proteins with retention time in front of the main peak were identified as oxidized and (oxidized+N-terminal methioninylation) modified human interferon α2b. The related proteins with retention time behind the main peak were (oxidation+N-terminal acetylation) and N-acetylated modified human interferon α2b.
[1] WALSH G. Post-translational modifications of protein biopharmaceuticals[J].Drug Discov Today, 2010, 15(17-18): 773
[2] JENKINS N, MURPHY L, TYTHER R. Post-translational modifications of recombinant proteins: significance for biopharmaceuticals[J].Mol Biotechnol, 2008, 39(2): 113
[3] TAGGART C, CERVANTES-LAUREAN D, KIM G, et al. Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity[J].J Biol Chem, 2000, 275(35): 27258
[4] HSU YR, CHANG WC, MENDIAZ EA, et al. Selective deamidation of recombinant human stem cell factor during in vitro aging: isolation and characterization of the aspartyl and isoaspartyl homodimers and hererodimers[J].Biochemistry, 1998, 37(8): 2251
[5] CORREIA IR. Stability of IgG isotypes in serum[J].MAbs, 2010, 2(3): 221
[6] HERMELING S, CROMMELIN DJ, SCHELLEKENS H, et al. Structure immunogenicity relationships of therapeutic proteins[J].Pharm Res, 2004, 21(6): 897
[7] BEERS MMCV BARDOR M. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins[J].Biotechnol J, 2012, 7(12): 1473
[8] 中华人民共和国药典2020年版. 三部[S].2020: 300
ChP 2020. Vol Ⅲ[S].2020: 300
[9] 许培, 宋礼华, 王荣海, 等. 高纯度重组人干扰素α2b的制备方法:中国,CN104356223A[P].2015-02-18
XU P, SONG LH, WANG RH, et al. Preparation method of high purity recombinant human interferon α2b: China, CN104356223A[P].2015-02-18
[10] AHRER K, JUNGBAUER A. Chromatographic and electrophoretic characteriz-ation of protein variants[J].J Chromatogr B Analyt Technol Biomed Life Sci, 2006, 841 (1-2): 110
[11] ALAHMAD Y, TRAN NT, POTIER IL, et al. A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuti-cals[J].Electrophoresis, 2011, 32(2): 292
[12] BERKOWITZ SA, ENGEN JR, MAZZEO JR, et al. Analytical tools for characterizing biopharmaceutical and the implications for biosimilars[J].Nat Rev Drug Discov, 2012, 11(7): 527
[13] EON-DUVAL A, BROLY H, GLEIXNER R. Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach[J].Biotechnol Prog, 2012, 28(3): 608
[14] STAUB A, GUILLARME D, SCHAPPLER J, et al. Intact protein analysis in the biopharmaceutical field[J].J Pharm Biomed Anal, 2011, 55(4): 810
[15] 张伶俐, 刘冰, 杨惠洁, 等. 重组人干扰素α2b相关蛋白检测和质量分析[J].中国药业, 2020, 29(11): 52
ZHANG LL, LIU B, YANG HJ, et al. Test and quality assessment of related proteins of recombinant human interferon α2b[J].China Pharm, 2020, 29(11): 52
[16] 李永红, 韩春梅, 裴德宁, 等. 利用欧洲药典方法评价国产重组人干扰素α-2原液的制品相关蛋白含量[J].药物分析杂志, 2018, 38(11): 1865
LI YH, HAN CM, PEI DN, et al. Analysis of product related protein in the bulk of recombinant human interferon α-2 produced in China using the method in European pharmacopoeia[J].Chin J Pharm Anal, 2018, 38(11): 1865
[17] 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
[18] 陶磊, 饶春明. 重组蛋白药物制品相关蛋白的分析与评价[J].药物分析杂志, 2018, 38(11): 1854
TAO L, RAO CM. Analysis and evaluation of product-related proteins in recom-binant protein therapeutics[J].Chin J Pharm Anal, 2018, 38(11): 1854
[19] AHSAN F, ARIF A, MAHMOOD N, et al. Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli[J].J Biotechnol, 2014, 184: 11
[20] 尹红锐, 张颖, 徐明明, 等. 高效液相色谱和液质联用技术用于人干扰素α2b中甲硫氨酸氧化的研究[J].药物分析杂志, 2021, 41(7): 1203
YIN HR, ZHANG Y, XU MM, et al. Study of methionine oxidization in human interferon α2b using HPLC and UPLC-Q-TOF-MS[J].Chin J Pharm Anal, 2021, 41(7): 1203
[21] GRUNE T. Oxidized protein aggregates: formation and biological effects[J].Free Radic Biol Med, 2020, 150: 120
