Objective: To establish a method to detect the biological activity of recombinant human follicle stimulating hormone Fc fusion protein(rhFSH-Fc) by transgenic cell method. Methods: Using CHO-K1-FSHR-CRE-Luc transgenic cells to seed the plate at a certain cell density. After the strain was cultured for 16-20 h, the medium was abandoned, and rhFSH-Fc was diluted in a multiple and added to the cell plate. After the drug acted for a period of time, the lucifase detection reagent was added, and the biological activity of rhFSH-Fc was detected through the lucifase detection system, and the parameters of each test condition were optimized and the methodology was verified. Results: rhFSH-Fc has a dose-effect relationship in this method and conforms to the four-parameter curve equation, R2 was greater than 0.99; after the method was optimized, the cell seed plate density was determined to be 5×105 cells·mL-1, and the initial dilution concentration of rhFSH-Fc was 750 ng·mL-1, at 1:5 times dilution factor, induction time was 4 h, sample dilution was DMEM/F12K+10% FBS. The method has good specificity. The RSD of the precision test within the day was less than 5% in 9 independent days. The recovery samples of 5 different dilution groups were measured 6 times, and the relative titers were (51±2.00)%, (78±2.05)%, (94±2.23)%, (124±3.46)% and (141±4.45)%. The corresponding average recoveries were(101±3.94)%, (104±3.00)%, (94±2.24)%, (99±2.68)%and (94±3.03)%, RSD were all less than 4%. Conclusion: The biological activity assay of rhFSH-Fc was successfully established by transgenic cells.The method is simple, reproducible and accurate. It could be used for routine detection of rhFSH-Fc products.
SUN Shuang, WANG Lü-yin, LI Jing, LÜ Ping, XU Ke-zheng, LIANG Yu-ling, ZHANG Hui, LI Zhan-jun, LIANG Cheng-gang
. Study on biological activity assay of fusion protein of rhFSH-Fc based on the reporter gene*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(1)
: 60
-67
.
DOI: 10.16155/j.0254-1793.2022.01.07
[1] WANG L, LIANG C, YANG H, et al. Development of a robust reporter gene-based assay for the bioactivity determination of recombinant human follicle stimulating hormone(rhFSH) pharmaceutical products[J].J Pharm Biomed Anal, 2020, 177: 112855
[2] SCHOLTES MC, SCHNITTERT B, HOOGSTRATEN DV, et al. A comparison of 3-day and daily follicle-stimulating hormone injections on stimulation days 1–6 in women undergoing controlled ovarian hyperstimulation[J].Fertil Steril, 2004, 81(4):996
[3] 梁成罡, 王军志. 长效蛋白生物药研究进展[J].中国新药杂志, 2014,23(20):6
LIANG CG, WANG JZ. Research progress of long-acting protein therapeutics[J].Chin J New Drugs, 2014, 23(20):6
[4] PERLMAN S, VAN DEN HAZEL B, CHRISTIANSEN J, et al. Glycosylation of an N-terminal extension prolongs the half-life and increases the in vivo activity of follicle stimulating hormone[J].J Clin Endocrinol Metab, 2003, 88(7):3227
[5] SUZUKI T, ISHII-WATABE A, TADA M, et al. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR[J].J Immunol, 2010, 184(4):1968
[6] 李湛军, 李心欣, 梁成罡. 促卵泡激素体内生物活性测定及其各国药典方法比较[J].药物分析杂志, 2014, 34(4):727
LI ZJ, LI XX, LIANG CG. Determination of the biological activity of follicle stimulating hormone in vivo and comparison of pharmacopoeia methods in various countries[J].Chin J Pharm Anal, 2014, 34(4):727
[7] 程速远, 王俊玲, 李心欣, 等. 重组人促卵泡激素体内生物活性和对幼龄大鼠超排卵作用[J].中国新药杂志, 2015, 24(17):5
CHENG SY, WANG JL, LI XX, et al. In vivo biological activity of recombinant human follicle stimulating hormone and its effect on superovulation in young rats[J].Chin J New Drugs, 2015,24(17):5
[8] 中华人民共和国药典2020年版. 四部[S].2020: 196
ChP 2020. Vol Ⅳ[S].2020: 196
[9] EP 7.0. Vol Ⅱ[S].2011: 3177
[10] WUNDER F, KALTHOF B, M#xDC;LLER T, et al. Functional cell-based assays in microliter volumes for ultra-high throughput screening[J].Comb Chem High Throughp Screen, 2008, 11(7):495
[11] ANTHONY R. MIRE-SLUIS. Progress in the use of biological assays during the development of biotechnology products[J].Pharm Res, 2001, 18(9):1239
[12] 吴彦霖, 张铭露, 张媛, 等. 基于人卵巢颗粒细胞KGN建立的卵泡刺激素体外生物活性效价测定法[J].中国新药杂志, 2021, 30(14):1316
WU YL, ZHANG ML, ZHANG Y, et al. An in vitro biological activity titer assay of follicle stimulating hormone based on human ovarian granulosa cells KGN[J].Chin J New Drugs, 2021, 30(14):1316
[13] 裴德宁, 于雷, 郭莹, 等. 报告基因法测定聚乙二醇化Ⅰ型干扰素生物学活性的研究[J].中国医药导报, 2018, 15(1):22
PEI DN, YU L, GUO Y, et al. Study on the biological activity of pegylated type Ⅰ interferon by reporter gene method[J].China Med Her, 2018, 15(1):22
[14] 牛安娜, 苏昕, 张晓鹏. 报告基因法在生物技术药物活性检测中的应用[J].生物技术通讯, 2019, 30(2):296
NIU AN, SU X, ZHANG XP. Application of reporter gene method in biotechnology drug activity detection[J].Lett Biotechnol, 2019, 30(2):296
[15] 于雷, 范文红, 王兰, 等. 报告基因法检测促胰岛素分泌肽融合蛋白生物学活性[J].药物分析杂志, 2016, 36(3):426
YU L, FAN WH, WANG L, et al. Reporter gene method to detect the biological activity of insulin secretagogue peptide fusion protein[J].Chin J Pharm Anal, 2016, 36(3):426
[16] 王威. 重组人白介素-12生物学活性检测方法的建立[J].中国生物制品学杂志, 2006, 19(5):2
WANG W. Establishment of detection method for the biological activity of recombinant human interleukin-12[J].Chin J Biol, 2006, 19(5):2
[17] WALKER WH, FUCCI L, HABENER JF. Expression of the gene encoding transcription factor cyclic adenosine 3’,5’-monophosphate (cAMP) response element-binding protein(CREB):regulation by follicle-stimulating hormone-induced cAMP signaling in primary rat Sertoli cells[J].Endocrinology, 1995, 136(8):3534
