Steve Gorfien

Increasing antibody yield and modulating final product quality using the FreedomTM CHO-STM production platform

Background Cell line development (CLD) is a critical step in the generation of biotherapeutics, but it is still hindered by several pain points, including the lengthy and laborintensive workflow needed to isolate desirable clones, lack of reproducibility, as well as potential protein quality issues. Over the last decade, antibody titers in mammalian cell culture systems in excess of 3 g/L have been achieved through the use of novel media and feeds. However, it is still a challenge to consistently and rapidly create a stable cell line and a cell culture process capable of supporting both high antibody yield and acceptable post-translational modifications while managing the effort required for execution of the workflow. The goal of the study was to develop a robust and reproducible stable cell line workflow to generate scalable high-producing clones in less than 6 months, with industry-standard titers and desirable product quality using minimal effort. Using CHO-STM as the host cell line, we first evaluated if a single medium could be used for the entire CLD workflow, therefore avoiding the issues and complications of changing media during this process. We investigated if a formulation previously shown to increase titer as a production medium could in fact be used for all CLD steps, from transfection to stable pool isolation all the way through to clone productivity, without compromising titers or performance. The same rich production medium was used in limiting dilution cloning and compared to a lean cloning medium prototype. Furthermore, robustness of the workflow was verified by testing multiple molecules. We also explored reducing effort by streamlining all the steps of the workflow. Finally, we assessed top clone scalability and expressed product quality. We tested whether clones chosen only by titers responded well to scale-up and process development in a model bioreactor setting. In addition, product glycosylation from these clones was compared to the same molecule produced in CHO DG44 cells, a well-characterized production platform.

Rapid Fed-Batch Process Development in SimCell™

The SimCell™ overcomes many limitations seen in traditional systems for fed-batch process development. In this study, we demonstrated that SimCell is a robust automated tool for rapid platform process development. A platform feed and fed-batch process parameters were identified for three recombinant CHO cell lines using three consecutive SimCell experiments: Feasibility, Feed Screening and Feed Strategy Optimization. The microfluidic miniature bioreactor system was first calibrated and baseline performance was set up for each cell line. Seven chemically-defined (CD) feeds were designed based on spent media analysis and were screened in the SimCell using 378 microbioreactors. The optimal feed improved growth substantially and increased recombinant protein titers by 2 fold compared to batch cultures for all three cell lines. SimCell fed-batch process parameters associated with the feed, such as temperature shift, pH, feed schedule and volume, were optimized using 420 microbioreactors in a Design-of-Experiment (DoE) layout. Similarities and differences in fed-batch parameters were seen across the three cell lines. Recombinant protein titers were improved by 3–4 fold compared with batch cultures. For one cell line, recombinant protein titers were doubled compared to the original control process. Statistical analysis was performed to identify the optimal production parameters for each cell line.