Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells
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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.
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.
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.
KeywordsExcipients Formulation Freeze-drying Lyophilization Recombinant factor IX Stability study
The authors would like to thank CNPq, Capes, FAPERJ, BNDES, and Hemobras for the financial support of this research project, and the University of Kent for their support and laboratory facilities to enable these experiments. CMS thanks the Royal Society for an Industrial Fellowship (IF130004).
Supplementary Table 1—Excipients and concentration ranges used in the Design of Experiments (DoE).
Supplementary Table 2—Two-level fractional factorial design of experiment (resolution IV) showing the 3 categorical factors and the 5 numeric factors (concentration ranges).
Supplementary Fig. 1—DSC thermal analysis of rFIX samples formulated with Benefix (A) and Rixubis (B) excipients.
Supplementary Fig. 2—Cake appearance after the lyophilisation of formulations A to D, rFIX formulated with Benefix excipients and the condition with no excipients (just rFIX).
Supplementary Fig. 3—Particle size profiles (by DLS) of formulations A to D, rFIX formulated with Benefix excipients and the condition with no excipients for all three time points analyzed.
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