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Updated: May 22, 2025
Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model
Published on: October 6, 2023
Development and Validation of Two Cell-Based Reporter-Gene Assays for Determining the Bioactivity of Recombinant
Lyuyin Wang1, Jing Gao2, Kaixin Xu2
1National Institutes for Food and Drug Control, No. 31, Huatuo Road, DaXing District, Beijing 102629, China.
Abstract:
To develop a cell-based in vitro thyroid-stimulating hormone (TSH) biological activity assay that can simulate in vivo pharmacodynamic mechanisms, we constructed two HEK293-TSHR cell lines based on two main cell signaling pathways (Gαs-cAMP-PKA and Gαq/11-PLC-Ca2+) that TSH depends on for its in vivo physiological function. These cell lines stably expressed the luciferase reporter driven by the cAMP response element (CRE) and nuclear factor of activated T cells (NFAT) response element, and two reporter-gene assays (RGAs) were correspondingly established and validated. The two transgenic genes could measure signals produced from the simulation of the in vivo effects of TSH from the Gαs-cAMP and Gαq/11-PLC pathways after TSH activation. TSH showed a good dose-response relationship in these two cell lines and conformed to the four-parameter model. We optimized the critical experimental parameters of these two methods and performed comprehensive methodological validation according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, the Chinese Pharmacopoeia, and the United States Pharmacopoeia. The two methods showed good specificity, accuracy, precision, and linearity and can be used to aid in assessments of the biological activity of TSH drugs, product characterization, final product release, stability studies, and comparability studies for biosimilar applications.
Insights
Developed novel cell-based assays to measure thyroid-stimulating hormone (TSH) biological activity, simulating in vivo effects for drug development and characterization. These validated reporter-gene assays offer reliable assessment of TSH drug potency and quality.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Thyroid-stimulating hormone (TSH) is crucial for thyroid function.
- Existing assays may not fully capture in vivo pharmacodynamic mechanisms.
- Need for robust in vitro methods to assess TSH biological activity.
Purpose of the Study:
- To develop and validate cell-based in vitro assays for TSH biological activity.
- To simulate in vivo pharmacodynamic mechanisms of TSH action.
- To support TSH drug development, characterization, and quality control.
Main Methods:
- Constructed two HEK293-TSHR cell lines targeting Gαs-cAMP-PKA and Gαq/11-PLC-Ca2+ pathways.
- Established and validated two reporter-gene assays (RGAs) using luciferase reporters.
- Optimized experimental parameters and performed validation per ICH, Chinese Pharmacopoeia, and USP guidelines.
Main Results:
- TSH demonstrated a good dose-response relationship in both cell lines, conforming to a four-parameter model.
- The developed RGAs showed good specificity, accuracy, precision, and linearity.
- Validated assays effectively measure TSH-mediated signaling through key in vivo pathways.
Conclusions:
- The developed cell-based assays accurately simulate TSH's in vivo signaling pathways.
- These validated RGAs are suitable for assessing TSH drug biological activity, product characterization, release, and stability.
- The assays support biosimilar comparability studies and enhance TSH therapeutic product quality assessment.
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