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A multi-platform approach to accelerate spent media analysis

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The AMBR 250: Automated Parallel Bioreactor platform

Current biopharmaceutical process development requires the use of living cell lines with highly specific nutritional and environmental needs, which poses a number of complex challenges – least of all finding the optimal cell culture media formulation. But getting the media formulation right is crucial; the nutritional composition has a direct effect on cell growth – as well as the yield and quality of the biotherapeutic molecules they produce. By fully optimizing media, biopharmaceutical developers can dramatically improve productivity and cost-efficiency. 

As with all scientific processes, the key to successful optimization is understanding data – in this case, it’s about understanding how each medium component influences the cells. This data can be collected using a technique known as “spent media analysis.” This iterative, empirical approach compares levels of different components in media samples before, during, and after cell growth to provide insight into component utilization over time, which then informs  optimization of the media. Spent media analysis can identify a huge number of molecules that are taken up or secreted by the cells such as amino acids, vitamins, organic acids, nucleosides, nucleotides and polyamines to name a few.

Spent media analysis relies on an iterative approach to identify how media components are being used by cells, and then use this insight to optimize the media.

For example, consider a process where the amino acid serine is rapidly depleted despite relatively low consumption by the synthesized therapeutic protein. In this scenario, a hypothesis for where the serine is used could be developed and tested using spent media analysis. Based on the data output, our Biologics colleagues can then undertake further investigations to determine whether to add the missing component to their media, rather than more (potentially unnecessary) serine.

In undertaking these investigations, we make use of two work-horse technologies – the AMBR automated parallel bioreactor platform combined with LC-MS.  To make full use of both these technologies, there is an onus upon us to think big in terms of potential applications. For example, the use of spent media analysis is not restricted to new media optimization projects; it can also be applied to existing processes to enable efficient and reliable media troubleshooting in the event of unexplained product and process variations. By applying these techniques collaboratively at different workflow stages, our clients and partners not only benefit from a significantly improved process, but also from an expedited development timeline and, in turn, an accelerated speed to market.

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LC-MS: Our Agilent AdvanceBio 6545XT Q-TOF.

The typical workflow is as follows: Samples are automatically pulled from the AMBR and stored on-board (chilled) until they can easily be collected, centrifuged to remove cell mass and delivered to our analytical scientists for analysis. After that, samples are treated to protein precipitation by the addition of acetonitrile in a 96-well filter plate. This is a high-throughput method enabling up to 200 samples to be tested by LC-MS within a few hours. The LC-MS method uses HILIC chromatography and either high-resolution identification of intact analyte molecules, or tandem MS to identify analytes based on their unique fragmentation pattern. 

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APC’s Accelerated Approach:  Performing sample preparation on 24 Ambr bioreactors across 15 days each = 360 samples is no easy task UNLESS, we use high-throughput sample preparation technologies….from delivery of 360 samples to delivery of 360 data points in < 24 hours !

At APC, our multidisciplinary team is consistently working to push the boundaries of process and analytical development to help our clients accelerate the delivery of their medicines to patients.  In this regard, spent media analysis has become a standard client offering and represents one of the next wave of Biologics-Analytical synergies enabling a much deeper understanding of cellular metabolism dynamics.  We are currently working on other exciting breakthrough techniques including using flow cytometry to link cell-cycle analysis to protein titer/quality. So watch this space for more breakthrough innovations to be announced in the near future!