Development and validation of a mechanism-based Ba/F3-hGHR bioassay for the potency assessment of recombinant human...

The biological potency of recombinant human growth hormone (rhGH) is a critical quality attribute (CQA) for ensuring clinical efficacy. However, current pharmacopoeial methods, such as the USP in vitro Nb2-11 cell proliferation assay, lack specificity as they measure lactogenic activity via prolactin receptors rather than the growth hormone receptor (GHR). In this study, we developed a highly specific and robust potency assay for rhGH by standardizing and validating a murine pro-B lymphocyte (Ba/F3) cell platform stably expressing human GHR (hGHR). While GHR-transfected cell models have been previously described for basic research, this work bridges the gap between research-grade tools and pharmaceutical quality control (QC). We established a strictly characterized monoclonal cell line to ensure the long-term genetic and functional stability essential for routine commercial testing. The engineered Ba/F3-hGHR cells exhibited a dose-dependent proliferative response exclusively triggered by hGHR signaling. Through systematic optimization of assay parameters-including cell seeding density, serum concentrations, and incubation kinetics-we established a high-performance analytical protocol. The method was rigorously validated according to ICH Q2(R2) guidelines, demonstrating superior intermediate precision (GCV 8.4-18.3%), high accuracy, and the sensitivity required to detect subtle potency shifts in degraded samples. Our findings provide a mechanism-based, validated alternative to current regulatory methods, offering a reliable approach for the accurate quantification of rhGH biological activity in biopharmaceutical manufacturing.