Molecular biomarkers identification and applications in CHO bioprocessing.

Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy.

Combined approach of selective and accelerated cloning for microfluidic chip-based system increases clone specific productivity.

Improving current cell line development workflows can either focus on increasing the specific productivity of the cell lines or shortening timelines to reach the clinic as fast as possible. In this work, using the Beacon platform, we have combined two distinct protocols - early cloning with low-viability pools, and IgG membrane staining-, to concomitantly reach both objectives, and generate highly productive CHO clones in shorter timelines. Fast-sorting approaches using low-viability pools in combination with the Beacon platform have recently been reported to shorten CLD timelines. However, the low recovery led to a drastic reduction in the clone number obtained postcloning. Here, we report a combined approach of fast-sorting and fluorescent membrane staining. With this new protocol, the cells reach a correct recovery, allowing to fully exploit the Beacon screening capacities. In addition, by using a fluorescent staining recognizing the secreted IgG, we were able to enrich the fraction of highly secreting cells prior to cloning and we obtained significant increases in the cell's specific productivity. The combination of these two protocols has a synergistic effect, and as they help discarding the dead and nonproducing populations prior to cloning, they increase the throughput power of the Beacon platform and the detection of super productive clones.