目的:研究建立重组人促卵泡激素(rhFSH)氧化亚基分析方法,制备稳定的氧化亚基分析用系统适用性溶液。方法:通过加入过氧化氢氧化破坏重组人促卵泡激素,后加入甲硫氨酸终止该氧化作用,制备系统适用性溶液。采用RP-HPLC法分离分析重组人促卵泡激素氧化亚基。使用C4色谱柱(250 mm×4.6 mm,5 μm);流动相A为0.2 mol·L-1磷酸二氢钾缓冲液(pH 2.5),流动相B为80%乙腈;流速1 mL·min-1;柱温30 ℃;检测波长210 nm;梯度洗脱(0~10 min,82%A→74%A;10~25 min,74%A→60%A;25~26 min,60%A→20%A;26~56 min,20%A;56~56.1 min,20%A→82%A;56.1~75 min,82%A)。结果:加入甲硫氨酸可有效终止氧化作用,制备得到稳定的系统适用性溶液。优化建立的氧化亚基分析方法专属性良好,重复性(RSD=0.22%)、色谱柱耐用性(RSD=3.21%)、36 h内溶液稳定性(RSD=0.65%)结果良好。9批样品中氧化亚基含量范围为0.687%~1.001%。结论:优化建立的氧化亚基分析方法可对重组人促卵泡激素中的氧化亚基进行准确定量,能够用作重组人促卵泡激素的质量研究和放行检验;研究制备的系统适用性溶液可准确地对氧化亚基进行峰定位。
杨慧敏, 张伟, 孙悦, 吕萍, 王绿音, 李懿, 张慧, 李晶, 梁成罡
. 重组人促卵泡激素氧化亚基分析方法研究*[J]. 药物分析杂志, 2022
, 42(1)
: 33
-40
.
DOI: 10.16155/j.0254-1793.2022.01.04
Objective: To study and establish a method for analyzing the oxidized subunits of recombinant human follicle stimulating hormone(rhFSH), and prepare a stable system suitability solution for the analysis of oxidized subunits. Methods: Hydrogen peroxide was added to destroy rhFSH, and then methionine was added to stop oxidating for preparing the system suitability solution. The RP-HPLC method was used to separate and analyze the oxidized subunits of rhFSH. The C4 chromatographic column(250 mm×4.6 mm, 5 μm) was used, mobile phase A was 0.2 mol·L-1 potassium dihydrogen phosphate buffer (pH 2.5), mobile phase B was 80% acetonitrile. Flow rate was 1 mL·min-1, column temperature was 30 ℃, detection wavelength was 210 nm, gradient elution (0-10 min, 82%A→74%A; 10-25 min, 74%A→60%A; 25-26 min, 60%A→20%A; 26-56 min, 20%A; 56-56.1 min, 20%A→82%A; 56.1-75 min, 82%A). Results: The addition of methionine could effectively terminate the oxidation and a stable system suitability solution was prepared. The optimized analysis method of oxidized subunits had good specificity, repeatability(RSD=0.22%), column robustness(RSD=3.21%), and solution stability within 36 h(RSD=0.65%). The contents of oxidized subunits in 9 batches of samples ranged from 0.687% to 1.001%. Conclusion: The optimized method of oxidized subunits analysis could accurately quantify oxidized subunits in rhFSH, and could be used for quality research and release inspection of rhFSH. The system suitability solution could accurately locate the peaks of oxidized subunits.
[1] 曹敏, 王俊玲, 吴彦卓, 等. 重组人卵泡刺激素的研究进展[J].药物生物技术, 2014, 21(1): 86
CAO M, WANG JL, WU YZ, et al. Advances in recombinant human follicle stimulating hormone[J].Pharm Biotechol, 2014, 21(1): 86
[2] 何太平, 唐菁燕, 徐珑, 等. 重组人促卵泡激素在CHO细胞中的表达[J].中国生物制品学杂志, 2011, 24(12): 1409
HE TP, TANG JY, XU L, et al. Expression of recombinant human follicle-stimulating hormone in CHO cells[J].Chin J Biol, 2011, 24(12): 1409
[3] EP 10.0[S].2020: 2691
[4] 刘金钏, 彭贵子, 卢智俊, 等. 重组人促卵泡激素研究进展[J].中国药科大学学报, 2013, 44(3): 283
LIU JC, PENG GZ, LU ZJ, et al. Advances of recombinant human follicle stimulating hormone[J].J Chin Pharm Univ, 2013, 44(3): 283
[5] SAIRAM MR, SEIDAH NG, CHRÉTIEN M. Amino Acid Sequence of the Alpha and Beta Subunits of Ovine Pituitary Follitropin (FSH) [M]//ELZINGA M(eds). Methods in Protein Sequence Analysis. Clifton: Humana Press, 1982: 579
[6] CAHOREAU C, KLETT D, COMBARNOUS Y. Structure-function relationships of glycoprotein hormones and their subunits’ ancestors[J].Front Endocrinol, 2015, 6: 26
[7] KIM G, WEISS SJ, LEVINE RL. Methionine oxidation and reduction in proteins[J].Biochim Biophys Acta, 2014, 1840(2): 901
[8] BUTT WR, CROOKE AC, CUNNINGHAM FJ, et al. Chemical reactions which affect the biological activity of human gonadotrophins[J].Biochem J, 1961, 79: 64
[9] IRVING I, GESCHWIND, CHOH HL. The reaction of several protein hormones with perioidate[J].Endocrinology, 1958, 63(4): 449
[10] YBS00172015 国家药品标准[S].2015
YBS00172015 National Drug Standard[S].2015
[11] BASSETT RM, DRIEBERGEN R. Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH[J].Reprod Biomed Online, 2005, 10(2): 169
[12] BASSETT R, CECCARELLI D, CRISCI C, et al. Comparative analytical characterization of two commercial human follicle stimulating hormones extracted from human urine[J].Curr Med Res Opin, 2005, 21(6): 899
[13] 陈宁. 重组人促卵泡激素的质量研究[D].长春: 吉林大学, 2007
CHEN N. Quality of the Recombinant Human Follicle Stimulating Hormone[D].Changchun: Jilin University, 2007
[14] SETTI P. The importance of consistent FSH delivery in infertility treatment[J].Reprod Biomed Online, 2006, 12(4): 493
[15] JS20180100 进口药品注册标准[S].2018
JS20180100 Registration Standards for Imported Drugs[S].2018
[16] 中华人民共和国药典2020年版. 四部[S].2020: 480, 64
ChP 2020. Vol Ⅳ[S].2020: 480, 64
[17] 国家食品药品监督管理总局. 生物类似药研发与评价技术指导原则(试行) [EB/OL].(2015-02-28) [2018-08-05].https://www.nmpa.gov.cn/directory/web/nmpa/xxgk/ggtg/qtggtg/20150228155701114.html
China Food and Drug Administration. Technical Guidelines on Development and Evaluation of Biosimilar(Trial) [EB/OL].(2015-02-28) [2018-08-05].https://www.nmpa.gov.cn/directory/web/nmpa/xxgk/ggtg/qtggtg/20150228155701114.html
