Fluorescent bioassay to quantify the potency of human growth hormone and other hormones and cytokines
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
Key features
- 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.
Transgenic plants for recombinant manufacture of cyclotides and other cyclic peptides
Increased rigidity and stability are some of the numerous benefits of cyclic peptides. Cyclotides are a class of cyclic peptides that contain three disulfide bonds arranged in a cystine knot motif, which confers exceptional stability. Cyclotides have exhibited a range of bioactivities, from agricultural pests to human pathogens and diseases. The stable cyclotide framework can also be used as a pharmaceutical scaffold for the grafting of peptide sequences conferring bioactivity. Despite their attractive properties, efficient manufacture of cyclotides is a significant obstacle to their commercial development.
Using Agrobacterium-mediated transformation, University of Queensland (UQ) researchers have developed transgenic plants capable of producing cyclic peptides. The UQ team has demonstrated that the cyclotide precursor (Oak1) can be effectively cyclised using OaAEP1b in three plant species.
Key features
- Production of difficult to manufacture cyclic peptides in a plant-based expression system
- Improved yields over synthetic or in vitro production
- Compatible with existing molecular pharming methods (including Agrobacterium-mediated transformation)
- Peptide products with exceptional stability for therapeutic and/or agricultural applications
- Ongoing development of optimised enzyme variants and expression systems.