Hydrocyclones as cell retention device for CHO perfusion processes in single‐use bioreactors

Abstract

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50‐L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low‐viability late fed‐batch culture, and viability was restored. The successive runs allowed optimization of the HC‐bag configuration, and cultivations with 20–25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

Hydrocyclones as cell retention devices for an N‐1 perfusion bioreactor linked to a continuous‐flow stirred tank production bioreactor

“…The lower viability in the N‐1 bioreactor is likely due to the high shear environment created by the hydrocyclone. The cell densities achieved in the N‐1 perfusion bioreactor in the experiments presented here were somewhat lower than those achieved by other researchers performing perfusion with CHO cells using hydrocyclones as cell retention devices (Bettinardi et al, 2020).…”

“…Although early work with the hydrocyclones has shown promise with separation efficiencies reaching as high as 99% (Pinto et al, 2008), these experiments were performed at low cell densities that are not representative of current perfusion processes. More recently, viable cell densities of up to 50 × 10 6 cells/ml were achieved in a sustainable perfusion process utilizing a hydrocyclone (Bettinardi et al, 2020). In experiments performed in our laboratory, compared to membrane cell retention, lower separation efficiencies and shear damage resulting from multiple passes through the hydrocyclone have generally rendered the devices not, or only marginally, suitable for N‐1 (seed bioreactor) or production bioreactors.…”

A common framework for integrated and continuous biomanufacturing

“…Recently the product sieving & fouling has been successfully mitigated to allow more than 85% transmission for 40 days by the use of wide pore filters (Pinto et al, 2020; Wang et al, 2019). Despite these improvements, it is clear that opportunities remain to improve the understanding of fouling and the potential for pursuit of non‐membrane approaches (Bettinardi et al, 2020; Kwon et al, 2017).…”

Miniature auto‐perfusion bioreactor system with spiral microfluidic cell retention device

“…However, they suffer from various practical challenges such as cell damage, separation efficiency, and reliability in long‐term continuous operations (Castilho, 2015). For example, hydrocyclone requires a high perfusate flow rate, limiting its applications in small‐scale cultures (Bettinardi et al, 2020; Pinto et al, 2008). Acoustic‐based cell aggregation technology has also been widely explored as a highly efficient cell retention method for perfusion bioreactors systems in cell manufacturing and tissue engineering (Li et al, 2014; Trampler et al, 1994).…”