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University of Queensland researchers have developed a fluorescent in vitro cell-based bioassay capable of continuously detecting the potency of hGH using high content imaging
About
Background Human growth hormone (hGH) is used as a prescription biologic pharmaceutical to treat children's growth disorders and adult growth hormone deficiency. Bioassays used in the manufacture and characterisation of growth hormone traditionally are expensive, laborious and require the sacrificing of rodents. Cell-based assays have the potential to improve analytical release testing for hGH drug products and be used as research tools. Cell-based assays are usually measured via colourimetric endpoints (eg. using MTT or MTS) that can be tedious to carry out, requiring additional labour and time. In addition, endpoint assays do not allow for quantification over multiple time points. University of Queensland (UQ) researchers have developed a fluorescent in vitro receptor-based bioassay capable of continuously detecting the potency of hGH using high content imaging. The technology The assay consists of a murine pro-B cell line (BaF/B03) that has been modified to express green fluorescent protein (GFP) and the hGH receptor on the cell surface. The cell line is designated Ba/F3-hGHR-GFP, and proliferation occurs in a dose dependent manner according to the concentration of hGH. The UQ team has proof of concept data that demonstrates the bioassay can detect hGH at concentrations as low as 0.05 ng/mL when using the Ba/F3-hGHR-GFP cell line. Proliferation can be determined: a) By continuous monitoring of GFP using high content imaging, where cells can be placed back in culture after analysis to allow quantification over multiple time points. b) Using an MTT cell viability assay, where absorbance (595 nm) is directly proportional to viable cell number at a single specified time point.
Key Benefits
• Highly sensitive detection of hGH (0.05 ng/mL) • Compatible with high throughput screening (384-well plates) • Cell culture can be continued after analysis to allow for time course assays • Assay adaptable to receptors for other hormones or cytokines.
Applications
The bioassay may be used as an in-process control during the manufacture of hGH or as a research tool to test novel analogues or variants for potency. It can also help identify molecules that activate or inhibit specific receptor signalling pathways. While proof of concept data has been demonstrated using hGH receptors, the cell line can be modified to express receptors for other therapeutically relevant biomolecules such as hormones and cytokines.