Hikmat Bushnaq-Josting, technical manager at SAFC Biosciences, described how product titers can be increased through media and feed optimization. Titer is related to viable cell density, time in the fermentor, and the productivity of the individual cell. Factors related to the cell itself include the vector used, transfection method, and clone selection, while those related to the process include oxygen, agitation, and pH.
SAFC Biosciences has a CHO cGMP media library reflecting the diversity in clones and clonal nutritional needs. Screening is accomplished either by looking at pure or mixed media or using a more rational design approach based on nutritional depletion, which analyzes spent media. The method chosen depends upon the time available to find an appropriate media for a client project, explained Bushnaq-Josting.
Since every clone is different, this process needs to be done fresh each time. “Each new project for a customer feeds back into the library—we may dispense with some media, but we also add new ones from our genomics and proteomics experiments,” he said.
For instance, in one high-throughput screen of the library, nine CHO cell lines and 30 different media were investigated in 50 mL culture tubes; this showed that for each cell line, use of different media can lead to widely differing performance in cell culture.
Bushnaq-Josting described a case history involving a CHO cell line that produced an IgG for which the customer wanted a 20–30% increase in titer via media development. Four media from the SAFC library gave a higher titer than the customer media, so these were used as the basis for a design-of-experiment approach that involved a pyramid of mixtures. This revealed three mixtures that gave a titer increase of 100–150%—at least at the small scale. The customer found the media selection was scalable to the bioreactor and led to a satisfactory increase in yield to 3,300 mg/L.
One issue that came up in this study was the dilemma of process time. Titer can be improved by increasing culture time in the fermentor. But in a business where every additional day in a large-scale fermentor adds costs, this is not always desirable. There are also questions about the quality of product that spent a long time in the fermentor.
When it comes to feed development, SAFC finds that it is better to look at groups of components and try to assess which ones the culture actually needs most. It is a more time-intensive approach than just adding concentrated media (with all components) but generally gives a better yield of product.
In a second case study, five feed formulations—two complete, three with groups of components—were tested on two clones with two different media using 30 mL bioreactor tubes. The results showed that there is no general rule; the feeds were ranked differently according to performance, depending on the clone and media involved. The best combination gave a yield improved by 67%.
Finally, Bushnaq-Josting described how SAFC, having sequenced the CHO cell line, is now using transcriptome-, proteome-, and metabolome-based tools to optimize cell culture. In one study, SAFC looked at what soy and yeast hydrolysates can add to the performance of a CHO culture. Some CHO genes are expressed in response to both hydrolysates, others uniquely to one or the other. These findings have led to the development of a chemically defined hydrolysate that will be launched soon.
It is hoped that this new product will overcome the problems associated with the fact that although most companies prefer to have a chemically defined (hydrolysate-free) media, it has been observed that many cell lines have a higher titer when being fed with plant-based hydrolysates. With the new CD hydrolysate, the best of both worlds can be combined, explained Bushnaq-Josting.