# Development and Validation of Two Cell-Based Reporter-Gene Assays for Determining the Bioactivity of Recombinant Human Thyroid-Stimulating Hormone Pharmaceutical Products

## TL;DR

Researchers developed two cell-based assays to measure the biological activity of thyroid-stimulating hormone drugs by mimicking their effects in the body.

## Contribution

The novel contribution is the development and validation of two reporter-gene assays based on distinct TSH signaling pathways for drug activity assessment.

## Key findings

- The two reporter-gene assays showed a good dose–response relationship with TSH and conformed to a four-parameter model.
- The assays demonstrated specificity, accuracy, precision, and linearity suitable for drug activity evaluation and biosimilar studies.

## 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.