目的: 建立人干扰素α2b(hIFN α2b)理化对照品的质控方法与质量标准。方法: 使用2020年版《中华人民共和国药典》报告基因法测定hIFN α2b理化对照品的生物学活性、福林酚法测定蛋白质含量、电泳法和高效液相色谱法测定纯度、等电聚焦电泳法测定等电点、Edman降解法测定N端氨基酸序列;液质联用法测定相对分子质量,使用C4(50 mm×2.1 mm,3.5 μm)色谱柱和QTof质谱仪;液质联用法测定质量肽图和二硫键,使用C18(100 mm×2.1 mm,1.7 μm)色谱柱和QTof质谱仪;建立hIFN α2b理化对照品的质量标准;对7批由不同企业提供的理化对照品进行检测。结果: 建立了hIFN α2b理化对照品的质量标准,7批hIFN α2b理化对照品的比活性为1.2×108~2.8×108 IU·mg-1,电泳纯度均＞96.0%,高效液相色谱纯度均＞95.0%,等电点主区带为5.2~6.6,N端氨基酸序列为(M)CDLPQTHSLGSRRT,相对分子质量、质量肽图和二硫键测定结果均与理论值相符。结论: 建立的质控方法与质量标准可以比较全面地评价hIFN α2b理化对照品的质量,各企业的理化对照品均符合质量标准的要求。
Objective: To establish the quality control method and quality standard of human interferon α2b(hIFN α2b) physical and chemical reference substance. Methods: The biological activity of hIFN α2b physical and chemical reference substance was determined by reporter gene method of the Chinese Pharmacopoeia (2020); the protein content was determined by Folin phenol method; the purity was determined by electrophoresis and high performance liquid chromatography; the isoelectric point was determined by isoelectric focusing electrophoresis; and the N-terminal amino acid sequence was determined by Edman degradation method. The relative molecular mass was determined byliquid chromatography-mass spectrometry using C4(50 mm×2.1 mm, 3.5 μm) column and QTof mass spectrometer. The peptide mass mapping and disulfide bond were determined by liquid chromatography-mass spectrometry using C18(100 mm×2.1 mm, 1.7 μm) column and QTof mass spectrometer. The quality standard of hIFN α2b physical and chemical reference substance was established. Seven batches of physical and chemical reference substance samples provided by different enterprise were tested. Results: The quality standard of hIFN α2b physical and chemical reference substance was established. The specific activity of seven batches of hIFN α2b physical and chemical reference substance was around 1.2×108-2.8×108 IU·mg-1, the electrophoresis purity was higher than 96.0%, the HPLC purities were all higher than 95.0%, the main isoelectric point band was 5.2-6.6, and the N-terminal amino acid sequence was (M)CDLPQTHSLGSRRT. The relative molecular mass, peptide mass mapping and disulfide bond determination showed the results to be consistent with the theoretical values. Conclusion: The established quality control method and quality standard can comprehensively evaluate the quality of hIFN α2b physical and chemical reference substance. The physical and chemical reference substance samples from each enterprises meets the requirements of quality standard.
[1] HOUGLUM JE.Interferon:mechanisms of action and clinical value[J].Clin Pharm, 1983, 2(1):20
[2] STANTON GJ, WEIGENT DA, FLEISCHMANN WR JR, et al. Interferon review[J].Invest Radiol, 1987, 22(3):259
[3] 中华人民共和国药典2020年版. 三部[S].2020: 294
ChP 2020. Vol Ⅲ[S].2020: 294
[4] WEISSMANN C, NAGATA S, BOLL W, et al. Structure and expression of human IFN-alpha genes[J].Philos Trans R Soc Lond B Biol Sci, 1982, 299(1094):7
[5] 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
[6] 王军志. 生物技术药物研究开发和质量控制[M].第3版.北京: 科学出版社, 2018: 468
WANG JZ. Research, Development and Quality Control of Biopharmaceuticals[M].3rd Ed. Beijing: Science Press, 2018: 468
[7] WETZEL R. Assignment of the disulphide bonds of leukocyteinterferon[J].Nature, 1981, 289(5798):606
[8] KONTSEK P. Human type I interferons: structure and function[J].ActaVirol, 1994, 38(6):345
[9] AUDAIN E, RAMOS Y, HERMJAKOB H, et al. Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences[J].Bioinformatics, 2016, 32(6):821
[10] 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 referencestandards using UPLC-MS[J].Acta Pharm Sin, 2015, 50(1):75
[11] 陶磊, 丁有学, 郭莹, 等. 液质联用分析rhTNK-tPA的一级结构[J].药学学报, 2013, 48(6):896
TAO L, DING YX, GUO Y, et al. Characterization of theprimary structure of TNK-tissue plasminogen activator using LC-MS[J].Acta Pharm Sin, 2013, 48(6):896
[12] DEUTZMANN R.Structural characterization of proteins and peptides[J].Methods Mol Med, 2004, 94:269
[13] 饶春明,陶磊,史新昌,等. 重组人干扰素α1b质量肽图分析及二硫键定位[J].药物分析杂志, 2007, 27(10):1505
RAO CM, TAO L, SHI XC, et al. Peptide mass mapping analysis of hIFN-αlb and localization of disulfide bonds[J].Chin J Pharm Anal, 2007, 27(10):1505
[14] AHSAN F, GARDNER QA, RASHID N, et al. Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli[J].Bioorg Chem, 2018, 76:294
[15] LIU YH, WYLIE D, ZHAOJ, et al. Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b[J].Anal Biochem, 2011, 408(1):105
[16] BAGHBAN R, FARAJNIA S, GHASEMI Y, et al. New developments in pichia pastoris expression system, review and update[J].Curr Pharm Biotechnol, 2018, 19(6):451
[17] 张伶俐,刘冰,杨惠洁,等. 重组人干扰素α-2b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 α-2b[J].China Pharm, 2020, 29(11):52
[18] RIGHETTI PG.Determination of the isoelectric point of proteins by capillary isoelectric focusing[J].J Chromatogr A, 2004, 1037(1-2):491
