In situ FRET biosensors for the in vivo measurement of important metabolites during cell culture

• Main Author: Behjousiar, Alireza
• Other Authors: Kontoravdi, Cleo; Polizzi, Karen
• Published: Imperial College London 2014
• Subjects: 660
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.656554

It has long been a goal of the bioprocessing field to be able to produce proteins in a cost-effective and efficient manner. The current method of choosing high-producing mammalian cells is labour-intensive and time-consuming, representing an opportunity to employ new analytical methodologies in an effort to expedite progress. It would be advantageous to measure the intracellular concentration of key metabolites as the cells are growing. This would allow the user to have more information regarding the wellbeing and growth potential of cells. In this thesis the construction, optimisation and use of FRET biosensors for the in vivo measurement of glucose and glutamine in Chinese Hamster Ovary cells will be discussed. Experiments have been conducted in batch and fed-batch cultures as well as small-scale investigations using the BioLector™. The work presented here suggests the use of genetically encoded FRET biosensors allows for quantification of intracellular metabolite concentrations via FRET ratios during cell growth. These FRET biosensors also show that it may be possible to predict intracellular metabolite concentrations based solely on the FRET ratio of these cells. It has also been suggested that in a smaller volume micro fermentation system (BioLector™) these biosensor transfected cell lines can be used to detect changes in intracellular metabolite levels. These biosensor cell lines were then used for cell line selection, results indicate that the FRET ratio may also be used as a tool to discard various cell lines based on time of progression. Using data in the final study, it may be concluded that the early progressed cell lines may not necessarily be the highest protein producers and with, respect to scFv production, no product specific differences exist. The results indicate the usefulness of these FRET biosensors as tools in aiding the process of information gathering at an early stage during cell line selection.