Biological activity determination of recombinant human interleukin-2 products using reporter gene assay
Objective:To establish a cell-based reporter gene assay(RGA)for bioactivity determination of recombinant human interleukin-2(rhIL-2).Methods:A C8166 cell line that stably expressed signal transducer and activator of transcription 5(STAT5)was transfected with a lentivirus expressing luciferase.The bioassay was further optimized and validated according to the guideline of ICH Q2 and Pharmacopoeia of the People's Republic of China(ChP,2020).The assay performance characteristics of the established RGA were validated including specificity,linearity,accuracy,precision,and robustness.Results:The parameters of bioassay based on RGA were validated by optimized conditions:the incubation time was 24 h,the cells were seeded in 96-well plate with 1×105 cells per well,the initial concentration of rhIL-2 was10000 IU·mL-1 by dilution ratio of 1∶3 for 10 gradients.After incubation,the luminescence(Luc)signals were recorded.The relative bioactivity of the sample was calculated according to 4-parameter mode,which was fitted by Luc intensity to the dose-response curve.Various types of recombinant human cytokines were selected to verify the specificity of bioassays that comply with quality control requirements.The linearity and accuracy verification was performed by 5 potency concentrations,which were 50%,75%,100%,125%and 150%,respectively.The measured potency to expected potency was evaluated by linear regressing model analysis(R2 =0.9931).The recovery rate range of relative bioactivities of 5 potency concentrations was 80%～120%.The recovery range of the mixture of the reference and tested samples in 5 different proportions was 84%～111%.The repeatability of intra-day,inter-day and different people was accessed in a batch of rhIL-2 by calculating the relative bioactivity.Accessing precision was expressed by RSD%,all of which were less than 10%.The luciferase was stably expressed by S/N(Signal Noise ratio),which had no statistical difference in C8166-Luc cells between 5,26 and 78 passages.This result evidenced that different passages of C8166-Luc cells can meet the quality control requirements.Conclusion:The developed bioassay of rhIL-2 based on RGA has trong applicability and can be used in quality control release test.
recombinant human interleukin-2bioactivityreporter geneoptimized and validated
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NSTL
万方数据
维普
