Conference Dates

October 6-10, 2019


Hydrocyclones (HC) are very compact devices that promote solid-liquid separation under the action of a centrifugal field. Despite the small size, HCs have a large processing capacity and do not suffer from clogging. Therefore, several publications explored HCs as a potential cell retention device in perfusion applications in the last 20 years, but limited to non-disposable lab-scale bioreactors and to relatively low cell densities (up to ~10 million cells/mL).

Even though the absence of moving parts may streamline the HC manufacturing, the performance of solid-liquid separation is highly dependent on the HC internal geometry. Said that, hydrocyclones can be produced by 3D printing, making them a promising alternative for the integration of cell retention devices in single-use bioreactor bags. The performance of hydrocyclones also depends on the attachment configuration to the bioreactor and cell concentration of the feed suspension. In this work, at first rapid batch tests were carried out to evaluate the impact of: (i) cell concentration; (ii) diameter of connector installed in the recirculation loop; and (iii) controlled harvest flow rate enabled by a peristaltic pump (520U model, Watson Marlow). The main response considered was their effect on HC separation efficiency. The stainless-steel HC2015 designed for mammalian cell separation (Pinto et al., 2008) was selected for the preliminary batch tests, and also used as a benchmark for plastic prototypes produced by 3D-printing techniques. Afterwards, the same HC2015 was installed in a 50-L single-use bag (XDR50 Xcellerex, GE Healthcare) specially customized for a perfusion cultivation with a mAb producer CHO cell line.

The stainless-steel HC2015 when operating at 2.3 bar provided a total separation efficiency (Et) up to 96%, and a centrifugal separation efficiency (E´) of 82% for a CHO cell suspension at 24E6 viable cells per mL Concentrated cells recovered by the underflow port did not show decrease in viability compared to the feed suspension. The reduction of a TC connector size from 19.7 to 12.7 mm resulted in the total filling of the recirculation loop with liquid, disrupting the formation of the desirable umbrella-pattern discharge of the underflow and reducing cell retention. The use of a peristaltic pump to control the overflow flow rate equivalent to perfusion rates of 1 and 2 RV (reactor volume) per day in 40-L bioreactor working volume resulted in a reduction of the E´ values and a consequent increase of cell concentration in the harvest stream. The reduction in the separation efficiency was probably due to a disturbance of the liquid flow pattern inside the HC, since it was observed that the typical gas core coming out from the overflow was absent. These features were taken into account in the HC operation in the single-use 50-L perfusion bioreactor, and a cell concentration of 50E6 cells per mL was successfully achieved with a cell-specific perfusion rate (CSPR) as low as 20 pL per cell per day. The harvest stream consisted of a natural cell bleeding leaving the overflow outlet. Moreover, the lower cell viability and average diameter in the overflow evidenced the preferential retention of viable cells returning into the bioreactor, thus providing a healthier culture environment. A 3D-printed hydrocyclone with equivalent geometry to the stainless-steel HC2015 was made and presented slightly lower separation efficiencies. Further studies proposing materials with a smoother surface and investigating further 3D-printing techniques are currently ongoing.

Pinto, R. C.V., Medronho, R. A., Castilho, L. R. (2008). Separation of CHO cells using hydrocyclones. Cytotechnology, 56(1), 57–67. doi:10.1007/s10616-007-9108-x

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