重组人干扰素β1b中ox-IFN β1b含量测定
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摘要
目的:建立重组人干扰素β1b(IFN β1b)中氧化型干扰素(ox-IFN β1b)的测定方法。方法:RP-HPLC色谱条件:RP-C8色谱柱(Grace Vydac,4.6 mm×250 mm,5μm),柱温40℃,0.1%TFA-10%乙腈-水溶液为流动相A,0.1%TFA-乙腈溶液为流动相B,梯度洗脱,流速为1.0 mL·min~(-1),检测波长214 nm。结果:ox-IFN β1b峰与峰3的分离度约为4.0,理论塔板数大于20 000;6次进样的RSD小于5.0%;2个厂家共6批IFN β1b原液中均检出ox-IFN β1b,用面积归一化法计算百分含量为0.47%～0.96%。结论:新建方法可有效分离和测定不同厂家IFN β1b原液中的ox-IFN β1b,对IFN β1b质量标准的提高具有重要意义。
Objective:To develop a method for determination of ox-IFN β1 b in recombinant human interferon β1 b(IFN β1 b). Methods:The RP-HPLC was carried out on an RP-C8 column(Grace Vydac,4.6 mm×250 mm,5μm),at the temperature of 40 ℃. The mobile phase consisting of solution A(0.1% TFA-10% ACN-ddH2 O) and solution B(0.1% TFA-ACN as solution B) was applied with a gradient elution at a flow rate of 1.0 mL·min~(-1). The detection wavelength was 214 nm. Results:The resolution of the ox-IFNβ1 b peak relative to peak 3 was about 4.0 and theoretical plate number was more than 20 000. RSD of 6 injections was below 5.0%. ox-IFNβ1 b was detected in 6 batches of IFN β1 b bulks from two different manufacturers,and percentage contents were between 0.47%and 0.96%,which were determined by area normalization. Conclusion:The developed method could effectively separate and determine ox-IFNβ1 b in IFN β1 b bulks from different manufacturers,which was of great significance to the improvement of quality control of IFNβ1 b.
Objective:To develop a method for determination of ox-IFN β1 b in recombinant human interferon β1 b(IFN β1 b). Methods:The RP-HPLC was carried out on an RP-C8 column(Grace Vydac,4.6 mm×250 mm,5μm),at the temperature of 40 ℃. The mobile phase consisting of solution A(0.1% TFA-10% ACN-ddH2 O) and solution B(0.1% TFA-ACN as solution B) was applied with a gradient elution at a flow rate of 1.0 mL·min~(-1). The detection wavelength was 214 nm. Results:The resolution of the ox-IFNβ1 b peak relative to peak 3 was about 4.0 and theoretical plate number was more than 20 000. RSD of 6 injections was below 5.0%. ox-IFNβ1 b was detected in 6 batches of IFN β1 b bulks from two different manufacturers,and percentage contents were between 0.47%and 0.96%,which were determined by area normalization. Conclusion:The developed method could effectively separate and determine ox-IFNβ1 b in IFN β1 b bulks from different manufacturers,which was of great significance to the improvement of quality control of IFNβ1 b.
引文
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[17]FORSTENLEHNER IC,HOLZMANN J,TOLL H,et al.Sitespecific characterization and absolute quantification of pegfilgrastim oxidation by top-down high-performance liquid chromatographymass spectrometry[J].Anal Chem,2015,87(18):9336
[18]HAUSBERGER A,LAMANNA WC,HARTINGER M,et al.Identification of low-level product-related variants in filgrastim products presently available in highly regulated markets[J].BioDrugs,2016,30(3):233
[2]GARTNER J,BRUCK W,WEDDIGE A,et al.Interferon beta-1b in treatment-na?ve paediatric patients with relapsing-remitting multiple sclerosis:two-year resu lts from the BETAPAEDIC study[J].Mult Scler J Exp Transl Clin,2017,3(4):1842799705
[3]DOSHI A,CHATAWAY J.Multiple sclerosis,a treatable disease[J].Clin Med(Lond),2017,17(6):530
[4]杨云凯,马昱,刘明,等.注射用重组人干扰素β1b规模化分离纯化工艺的建立及其产品质量的检定[J].中国生物制品学杂志,2017,30(12):1321YANG YK,MA Y,LIU M,et al.Establishment of process for large-scale purification of recombinant human interferonβ1b for injection and quality control of products[J].Chin J Biol,2017,30(12):1321
[5]张书艳,柳文科,陈忠义.不同剂量的干扰素-β对多发性硬化(MS)的疗效探讨[J].中外医疗,2015(23):94ZHANG SY,LIU WK,CHEN ZY.Observe the curative effect of different doses of interferon-βon multiple sclerosis[J].Chin Foreign Med Treat,2015(23):94
[6]ZETTL U,BAUERSTEINHU SEN U,GLASER T,et al.Adherence to long-term interferon beta-1b injection therapy in patients with multiple sclerosis using an electronic diary[J].Adv Ther,2016,33(5):834
[7]陶磊,丁有学,刘兰,等.应用串联质谱技术分析几种重组蛋白药物的翻译后修饰[J].中国药学杂志,2015,50(19):1726TAO L,DING YX,LIU L,et al.Identification of several kinds ofpost-translationalmodificationsinrecombinantprotein pharmaceuticals using MS/MS[J].Chin Pharm J,2015,50(19):1726
[8]MAHJOUBI N,FAZELI MR,DINARVAND R,et al.Preventing aggregation of recombinant interferon beta-1b in solution by additives:approach to an albumin-free formulation[J].Adv Pharm Bull,2015,5(4):497
[9]DEEHAN M,GARCES S,KRAMER D,et al.Managi ng unwanted immunogenicity of biologicals[J].Autoimmun Rev,2015,14(7):569
[10]LAROCQUE L,BLIU A,XU R,et al.Bioactivity determination of native and variant forms of therapeutic interferons[J].J Biomed Biotechnol,2011,2011:174615
[11]SKRLIN A,KOSOR K,GOSAK D,et al.Correlation of liquid chromatographic and biological assay for potency assessment of filgrastim and related impurities[J].J Pharm Biomed Anal,2010,53(3):262
[12]Van BEERS MMC,SAUERBORN M,GILLI F,et al.Oxidized a nd aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice[J].Pharm Res,2011,28(10):2393
[13]ABDOLVAHAB MH,FAZELI A,HALIM A,et al.Immunogenicity of recom binant human interferon beta-1b in immune-tolerant transgenic mice corresponds with the biophysical characteristics of aggregat es[J].J Interferon Cytokine Res,2016,36(4):247
[14]杨云凯,马昱,孙玉杰,等.反相高效液相色谱法测定注射用重组人干扰素β1b中干扰素β1b的含量[J].中国生物制品学杂志,2015,28(10):1053.YANG YK,MA Y,SUN YJ,et al.Determination of interferonβ1b content in recombinant human interferonβ1b for injection by reversed phase high performance liquid chromatography[J].Chin J Biol,2015,28(10):1053
[15]TOROSA NTUCCIR,SHAROVVS,VanBEERSM,etal.Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon beta-1a:potential implications for protein aggregation and i mmunogenicity[J].Mol Pharm,2013,10(6):2311
[16]LIU H,NEILL A,PONNIAH G,et al.Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis[J].Anal Chem,2013,85(24):11705
[17]FORSTENLEHNER IC,HOLZMANN J,TOLL H,et al.Sitespecific characterization and absolute quantification of pegfilgrastim oxidation by top-down high-performance liquid chromatographymass spectrometry[J].Anal Chem,2015,87(18):9336
[18]HAUSBERGER A,LAMANNA WC,HARTINGER M,et al.Identification of low-level product-related variants in filgrastim products presently available in highly regulated markets[J].BioDrugs,2016,30(3):233