Upstream bioprocessing has traditionally relied on a fed batch mode of operation to produce therapeutic proteins of interest. While this approach has served the industry well, the need for higher reactor yields has led to the pursuit of continuous modes of manufacturing. In this work, we describe a third mode of operation which straddles fed batch and perfusion processes. The FRESH (Fed batch reset with media exchange at short intervals) and iFRESH (FRESH process for intensified fed batch) bioprocessing strategies utilize minimal intermittent perfusion to exchange the spent media in a fed batch reactor while retaining the product and cells. This results in a 40-80% increase in volumetric productivity and improved culture health. The timing of media exchange highlights key inflection points in traditional fed batch processes where inhibitory factors might contribute to changes in cellular metabolism, cell cycle dynamics, and productivity. To better understand the nature of these inflection points and the limitations of the iFRESH approach, the timing of media exchanges and the total volume of media exchanged was optimized using a high throughput bioreactor system. This optimization demonstrated the robustness of the iFRESH approach allowing for operational flexibility to account for equipment and facility limitations. To gain further process understanding, flow cytometry was utilized to monitor the iFRESH process. Markers of early/late apoptosis and cell cycle dynamics were probed to gain further understanding of cell culture dynamics during media refresh. The insights gained from this study will guide future iFRESH development efforts in our group and provide vital information for future efforts to apply the technique to commercial scale manufacturing.
