Thorough characterisation is essential for efficient and knowledge-led cell culture process development in biomanufacturing. Despite diverse applications of rocked bag bioreactors, there is currently little understanding of the fundamental determinants of fluid mixing and mass transfer, and the effects that these would have on cell culture kinetics, product quality and cell physiology. A rocked bag bioreactor has been fully evaluated at 10 to 50 L scale. Under typical operating conditions, single-use rocked bag bioreactor tm were found to vary from 7-71 s, kLa(O2) from 3.5-29 h -1 and kLa(CO2) from 0.6-2.7 h -1 , with the rocking rate found to cause gas entrainment above 20 min-1 . A GS-CHO cell line cultured under controlled fed-batch conditions at low rocking rate to produce surface aeration achieved significantly higher cell specific antibody productivities. However, these cells were significantly less robust at harvest than cells cultured in the presence of a dispersed gas phase in rocked bags or stirred tanks. A fabricated rocked bag mimic was fluid dynamically characterised using particle image velocimetry. It was found that increasing rocking rate from 25 to 42 min-1 produced an 8-fold increase in turbulence kinetic energy, giving the rocked bag similar fluid dynamic characteristics to a stirred tank. The gas entrainment noted at higher rocking rates was connected to the fluid transitioning out of phase at higher rocking rates. A detailed cell culture kinetic, physiological and transcriptomic evaluation demonstrated that cells cultured in the rocked bag operated to entrain gas matched very closely those cultured in a stirred tank. Cells cultured in a bubble free environment exhibited several indications of higher stress, despite identical cell culture kinetics to the stirred tank. In a second industrial GS-CHO cell line, the specific productivity of the cells cultured in entrained gas phase bags was again found to be lower than those cells cultured in surface aerated bags, however the product quality was not significantly impacted. Abstract 5 In summary, this work demonstrates the flexibility of rocked bags as alternative single-use bioreactor designs.
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