(618g) 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.