Establishment, verification and application of biological activity assay method of recombinant human TNF receptorⅡFc fusion protein based on reporter gene
10.13200/j.cnki.cjb.004398
- VernacularTitle:基于报告基因的重组人Ⅱ型肿瘤坏死因子受体抗体融合蛋白生物学活性测定方法的建立、验证及应用
- Author:
FAN Wenhong
- Publication Type:Journal Article
- Keywords:
Recombinant human tumor necrosis factor receptorⅡFc fusion protein(rhTNFRⅡ-Fc);
Nuclear factor-κB(NF-κB);
Reporter gene;
Biological activity;
Luciferase assay system;
International standard
- From:
Chinese Journal of Biologicals
2025;38(1):73-79
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a simple and rapid method for the determination of biological activity of recombinant human tumor necrosis factor receptorⅡ(rhTNFRⅡ) Fc fusion protein(rhTNFRⅡ-Fc) products, and to verify and apply the method, in order to lay a foundation for the process stability evaluation of this kind of products.Methods Using HEK293TNF-κB-Luc transgenic cells, different concentrations of rhTNFRⅡ-Fc fusion protein can inhibit the transcriptional activation of nuclear factor(NF)-kB induced by TNF-α in transgenic cells to different degrees. The biological activity of rhTNFRⅡ-Fc was detected by Bright-Glo~(TM)-Luciferase Assay System, the experimental parameters were optimized, and the specificity,accuracy, linearity and precision of the method were validated. Using the international standard for biological activity assay of rhTNFRⅡ-Fc fusion protein products as the reference, this method was used to determine the specific activity of rhTNFRⅡ-Fc fusion protein products from three different domestic enterprises(A, B and C) and the original drug Etanercept.Results rhTNFRⅡ-Fc fusion protein had a dose-effect relationship in this method and conformed to the four-parameter curve equation with R~2 greater than 0. 99. After optimization, the cell seed plate density was determined to be 4 × 10~5/mL,the action concentration of TNF-α was 10 ng/mL, the initial dilution concentration of rhTNFRⅡ-Fc was 200 ng/mL, 1∶1. 5times the dilution, the action time was 4-5 h, and the sample diluting solution was DMEM + 1% FBS. Only Etanercept inhibited the activation of NF-κB transcription in HEK293T-NF-κB-Luc cells induced by TNF-α, while other fusion proteins had no inhibition on the induction of TNF-α. The recovery samples of five different dilution groups were measured three times, and the average recoveries were(101. 54 ± 4. 63)%,(99. 67 ± 6. 41)%,(101. 20 ± 5. 58)%,(101. 44 ± 6. 80)% and(100. 72 ±6. 15)%, respectively, with the relative standard deviations(RSDs) lower than 7% and the linear fitting R~2 of 0. 999 8. The RSDs of verification for inter-plate and inter-day precision were less than 8. 0%. Using this method for determination, the specific activity of the original drug was 2. 01 × 10~6IU/mg, 2. 59 × 10~6IU/mg for enterprise A, 1. 56 × 10~6IU/mg for enterprise B, and 2. 04 × 10~6IU/mg for enterprise C.Conclusion Using HEK293T-NF-κB-Luc transgenic cells, a method for the determination of biological activity of rhTNFRⅡ-Fc fusion protein was successfully established, which is simple and rapid with strong specificity, high accuracy and good precision, and can be used for the routine detection of biological activity of this kind of products in different enterprises.
