Troubleshooting Media Development for Bioprocessing

We recently finished our Ask the Expert discussion on troubleshooting media development for bioprocessing. This week we had many interesting questions and valuable answers and suggestions. CHO, MDCK and BHK cell lines were discussed and overall topics included media formulation matters like powder vs. liquid, stability, solubility, serum-free, chemically defined and non-defined components, and component sources. Troubleshooting tips were on protein glycosylation, fatty acid and cholesterol supplementation, using metabolism characteristics to improve production, reducing apoptosis , increasing cell viability, and handling waste issues.

This Ask the Expert Session was Sponsored by GE Healthcare Life Sciences and hosted by Peggy Lio, Director of Process Science, GE Healthcare Life Sciences. Ms. Lio is responsible for leading the technical applications strategy for the US cell culture business. She joined GE from Life Technologies where she was responsible for the development of PD-Direct Bioprocess custom cell line and cell culture media optimization service programs globally. She also led an applications team charged with scientific support of new cell line and media products as well as process development. Prior to Life Technologies, Peggy held roles of increasing responsibility with Schering-Plough Corp., where she ultimately became Section Leader with responsibility for mammalian and microbial fermentation process development for a variety of biotechnology based therapeutics. Peggy has over 30 years of extensive technical and commercial experience in bioprocessing along with multiple honors and awards from Schering and Life Technologies.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert –troubleshooting media development for bioprocessing.

Can you explain how cell metabolism characteristics can be used to optimize media in CHO cells?

The Answer:
There are several ways of utilizing cell metabolism characteristics for medium optimization. Firstly, the metabolism rate of key medium components such as amino acids and vitamins can easily be determined by spent media analysis. The metabolic profiles can then be used for design of stoichiometrically balanced medium formulations. Since metabolic characteristics are dynamic, utilization of metabolism rates measured from both the cell growth phase and stationary production phase are best for stoichiometric balancing of basal and feed media. Secondly, cellular metabolism is an interrelated network and metabolism characteristics can guide the focus of medium optimization. One example is within the glycolysis pathway and TCA cycle. For a cell line that tends towards lactate production, medium optimization can address this issue by switching the metabolic flux towards the TCA cycle. Finally, cell metabolism characteristics can also impact the balance between biomass formation and protein production. For a cell line with high growth rate but low productivity, limiting the metabolism rates through medium optimization can force the cells work on protein production thereby improving product yield.

What media components can reduce apoptosis and increase cell viability beyond critical density?

The Answer:
Apoptosis is typically caused by nutrient depletion and/or metabolite accumulation. Nutrient depletion can be prevented by proper feeding strategies. Metabolite accumulation is less of concern in a perfusion process but is often challenging to tackle in a fed-batch process. Media optimization approaches to control the accumulation of metabolites may include: reducing glutamine to control ammonia, reducing bicarbonate to modulate carbon dioxide, and use of alternate carbon sources to control lactate levels. In addition, there are reports on targeting the apoptosis pathway by the incorporation of chemicals such as caspase inhibitors or the use of silencing RNA. However, these approaches have not been incorporated into industrial processes yet.

Is there known correlation between media composition and glycosylation of proteins?

The Answer:
There are medium components known for impacting protein glycosylation through different mechanisms. For example, manganese serves as a cofactor in the glycosylation pathway and can improve glycosylation; galactose impacts glycan precursors and may improve glycosylation; high glutamine in medium formulation can cause less glycosylation due to ammonia generation.

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