Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

OBJECTIVES: To develop a recombinant human factor IX (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 maintained 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). 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 had 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 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

Separation of CHO cells using hydrocyclones.

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 wild-type 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.

Effect of suramin on trypomastigote forms of Trypanosoma cruzi: changes on cell motility and on the ultrastructure of the flagellum-cell body attachment region.

Suramin has been previously reported to inhibit distinct cell enzymes and to affect the synthesis and distribution of cytoskeleton proteins. Our study indicates that prolonged incubation of Trypanosoma cruzi infected-LLC-MK2 cells in the presence of 500 microM suramin during the intracellular development of the parasite caused morphological changes on trypomastigote forms characterized by a partial or complete detachment of the flagellum from the cell body, besides an accentuated decrease on parasite motility. Immunofluorescence analysis of the region of adhesion between the cell body and the flagellum on trypomastigotes obtained from suramin-treated host cells after the completion of cell cycle did not show any difference in the localization of FAZ antigens recognized by 4D9 and L3B2 monoclonal antibodies despite the presence of a detached flagellum. On the other hand, suramin caused a significant increase on the phenotypic expression of FRA antigen, which was observed throughout the surface of trypomastigotes. Cytochemical localization of cationized ferritin in trypomastigotes obtained from suramin-treated host cells showed that anionic particles gained access to the space between the cell and flagellar membranes, as well as to the flagellar pocket, indicating an alteration on extracellular components of the region of adhesion between the cell body and the flagellum.