Novel Biosensors for Bioprocess Development

Novel biosensors for bioprocess development

It has long been a goal of the bioprocessing field to be able to produce proteins in a cost effective and efficient manner. The most basic stage of protein production is to optimize the growth of the cells in question to give higher and more specific protein production. The current method of choosing high producing stable mammalian cells is labour intensive and time consuming, representing an opportunity to employ new analytical methodologies in an effort to expedite progress. The established cell line construction and selection methods rely heavily on achieving high cell growth rates, assessed in terms of confluence, in the early stages of cell line development. We suggest the use of genetically encoded FRET biosensors, which will allow us to use a low volume assay at an earlier stage in cell selection to measure key metabolites in cell culture as signs of well being of these cells.

The proposed system is able to monitor the concentrations of key intracellular metabolites including glucose and glutamine in real time, in a non-invasive way. Glucose and glutamine are key energy sources for cultured cells, and therefore vital metabolites to monitor in cell line selection. Therefore for efficient cell growth to occur glucose and glutamine must be present in a steady supply and not used up to quickly. The system consists of CHO-S cells stably transfected with biosensors for glucose/glutamine, which measure metabolites in the relevant range for healthily growing cells. The fluorescent signal of these biosensors has been calibrated against intracellular glucose and glutamine concentration measurements to produce in vivo calibration curves (Behjousiar et al., 2012).

We have further established the use of these biosensors for bioprocess development in a high throughput culture system (BioLector, m2p labs).  Using cells transfected with the glutamine biosensor we performed a medium optimisation experiment using CD-CHO and different amounts of L-glutamine versus two different types of glutamine containing dipeptides.  We monitored pH, DOT, and glutamine utilisation in 48 well plates and assessed the growth kinetics, integral viable cell concentration, and glutamine utilisation rates.

The proposed system for real-time, in situ monitoring of intracellular metabolites will allow us to make informed decisions regarding which cell lines to progress to subsequent stages of selection. Importantly, it will enable the selection of cell lines that present better characteristics in terms of manufacturability at an earlier stage than conventional methods. Our work also presents data to support the usefulness of the BioLector as a valid tool for cell maintenance and metabolic monitoring in an automatic, high throughput manner.