Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wildtype CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

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Objectives To develop a recombinant FIX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8°C.Results NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation, for a wide range of formulations tested. Particular devised formulations were able to maintain rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix®). The stability study showed that rFIX remained active after 3 months when stored at 4°C, though this was not the case with samples stored at 40°C.Interestingly, particular formulations were found to show an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix® formulation in terms of particle size distribution and cake appearance.Conclusions Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at a 0.234% (w/v) concentration appear suitable for use with a CHO cell derived recombinant FIX.