Enhanced cell‐specific productivity through delayed supplementation of antioxidants in intensified processes

Abstract Antioxidant supplementation to serum‐free culture media is a common strategy to enhance productivity through oxidative stress alleviation. In this study, it was hypothesized that certain antioxidants can improve the specific productivity of a CHO‐GS cell line expressing a bi‐specific antibody. A fed‐batch (FB) screening study investigated several antioxidants and revealed rosmarinic acid (RoA) and retinyl acetate (RAc), to a lesser extent, improved cell productivity. Contrary to the previous literature reports, the addition of RoA and/or RAc resulted in slower cell growth and reduced peak viable cell density, counteracting the enhanced specific productivity. We hypothesized that supplementing RoA/RAc after the exponential growth phase would increase titer through enhanced specific productivity without substantially impeding cell growth. This hypothesis was tested in three different ways: (1) supplementing RoA/RAc to the feed, rather than the basal media, in the FB process; (2) implementing the intensified fed‐batch (iFB) process mode which started with high seeding VCD, bypassing the exponential cell growth phase; (3) supplementing RoA/RAc to the production phase perfusion media, rather than the growth phase perfusion media, in the perfusion‐based continuous manufacturing (CM) process. All three methods were proven effective in titer improvement, which supported the hypothesis. Additionally, RoA/RAc significantly impacted product quality, with variations depending on the process mode and components. Overall, their supplementation led to decreased N‐glycan mannose percentage and increased product fragmentation and aggregation. These changes do not fully align with the previous reports, highlighting that the supplementation strategy needs to be evaluated carefully based on cell line and expressed molecule type.