Bleed recycling is a novel method to increase the yield of steady-state perfusion processes by concentrating process bleed to selectively remove biomass and recycle the liquid fraction. This results in significant product saving which otherwise would go to waste. As long as cells can be concentrated and separated, existing cell separation devices can be used for such an application. However, limited information comparing operation modes and efficiency for bleed recycling applications is available. For the first time, inclined gravity settling has been used as bleed recycling technology and was compared to acoustic separation. Except for lower debris removal, inclined gravity settling showed similar bleed recycling efficiency and no negative impact on cell viabilities, nutrient and metabolite levels and product quality. Additionally considering reduced system complexity and facilitated scale-up, inclined gravity settling was the preferred technology for further evaluation during a 42-day lab-scale perfusion process. Up to a 3.5-fold bleed reduction and an average harvest rate increase of 19% was achieved. Scalability was subsequently tested with a large-scale inclined gravity settler suitable for a 2000 L perfusion process confirming performance of lab-scale experiments. Bleed recycling characterization data from screening experiments combined with scalability demonstration facilitates decision making when considering bleed recycling for novel perfusion process settings to reduce perfusion waste, increase process sustainability and boost overall process yield.
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