On 29 November 2018, Matthew Smonskey (senior scientist in the Gibco Custom Media Services team at Thermo Fisher Scientific) presented an “Ask the Expert” webinar on resources available to help developers identify media limitations and options for designing custom media formulations that maximize protein yield.
Gibco’s goal is to solve its clients’ bioproduction problems: e.g., creating a chemically defined medium, increasing end-point titer, modifying protein quality attributes, and so on. Designing custom media using spent-media analysis might solve some such problems.
Some cultures might not perform up to expectation. All immortalized cell lines experience genetic and epigenetic drift throughout their periods of use. Evolutionary pressure leads to hundreds of thousands of small mutations and larger structural variances, many biologically active in the cells. A final cell line could be very different from the starting cell line it came from, with unique nutritional requirements.
Smonskey presented a case study of two experiments. In the first, a 14-day shake-flask batch culture using chemically defined media, the goal was to identify what media components were most heavily consumed (and therefore limited overall culture performance). Cell count, viability, titer, glucose, and other major metabolite levels were measured daily. Spent-media analyses monitored amino acids and select water-soluble vitamins throughout.
The second experiment was a 17-day ambr15 batch-fed culture to compare three ThermoFisher media and feeds against a competitor medium and feed a client had been using. The goal was to determine whether a base formulation and feeding strategy (nonoptimized) would be sufficient to meet a cell line’s unique nutritional requirements. The same data were collected as in the first experiment.
Results from the first experiment showed a low peak viable cell density (VCD) on Day 3 — nearly 1.5 × 106 cells/mL — suggesting that some critical medium component was exhausted at that point. Another problem was production of a high amount of ammonia. Spent-media analysis showed that cysteine was exhausted by Day 3, probably the cause of the poor growth. Asparagine also was exhausted, probably having deamidated to form ammonia. Thiamine also was depleted by Day 6.
Results of the second experiment showed that the catalog media and feeds — especially the new QpCHO product — supported higher VCD (>15 × 106 cells/mL) than did the competitor media (~10 × 106 cells/mL). Peak VCD was achieved on Days 7 and 8, with higher titers (a 50% increase) for the catalog media.
Recommendations for improving performance included assigning supplementation or a custom formulation to deliver higher concentrations of cysteine and thiamine and lowering asparagine exposure to cells through process optimization or a custom formulation.
Using a suite of ultrahigh-performance and high-performance liquid chromatography (UHPLC, HPLC) and inductively coupled plasma mass spectrometry (ICP-MS), Gibco Custom Media Analytical Services can quantitate amino acids, vitamins, and trace elements. The service can help bioproduction clients from early stage cell-line development and clone selection to media development, process optimization, and scaling up to prototype media.
Questions and Answers
What other media components are testable? We also can examine trace-metal contamination, lactate production, and other factors that affect a culture’s overall health. Beyond those, our services can analyze specific supplements, polyamines, cholesterol, select fatty acids, peptides, proteins (e.g., insulin), bovine serum albumin, transferrin, growth factors, and protein quality attributes.
On what days should you sample your cultures for spent media? Some clients have submitted terminal samples after a 14-day run. But feeding continues throughout a culture, with components changing over time. So I strongly recommend having good coverage throughout a run, starting before the first feed so that you can see how the base formulation supports growth sufficiently. Then I would like good coverage through exponential growth to prevent limiting overall biomass prematurely through amino-acid depletion. Following that would be some coverage through the plateau and into productivity phase to ensure that you are providing all the necessary protein building blocks.
How scalable are results? We do a lot of work at the small scale for custom formulations. It should hold fairly well scaling up, but you would have to confirm that at scale.
The full presentation of this webcast — with slides and additional questions and answers — can be found on the BioProcess International website at the link below.