Abstract
ABSTRACTMany high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
Highlights
Biopharmaceuticals are protein-based drugs, such as monoclonal antibodies and enzymes, often with sugars attached that affect their efficacy and mechanism of action
We designed and constructed two whole-cell lactate biosensors (Fig. 2) and compared their performance in samples of M9 minimal medium with glycerol as the carbon source spiked with different concentrations of L-lactate
Negative values for fluorescence and green fluorescent protein (GFP) synthesis rates result from the data analysis procedure where values are normalized to the biosensor cultures with no added lactate
Summary
Biopharmaceuticals are protein-based drugs, such as monoclonal antibodies and enzymes, often with sugars attached that affect their efficacy and mechanism of action. They include blockbuster drugs such as Humira, Avastin, and Herceptin, with the top five selling drugs generating nearly 70 billion USD in revenue in 2013 alone. The measurement of key metabolites during biopharmaceutical production is essential to inform the design of medium and feeds and to optimize cell culture conditions for increased productivity. High concentrations of lactate can negatively affect cell growth and productivity (Li et al, 2010; Zhou et al, 2011) and can, via acidification of the medium, cause changes in glycosylation patterns (Yoon et al, 2005)
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