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
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