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Recombinant factor VIII SQ--inactivation kinetics in aqueous solution and the influence of disaccharides and sugar
A Fatouros1, T Osterberg, M Mikaelsson
1Department of Pharmaceutical Technology, Pharmacia & Upjohn AB, Stockholm, Sweden. angelica.fatouros@eu.pnu.com
Purpose:
To investigate the influence of various nonreducing disaccharides and sugar alcohols on the inactivation kinetics of recombinant factor VIII SQ (r-VIII SQ) in aqueous solution not containing albumin as a stabiliser.
Methods:
The stability of r-VIII SQ was followed using measurement of activity (VIII:C) and HPLC gel filtration at different temperatures. The thermal stability was investigated using differential scanning calorimetry (DSC).
Results:
The decline in VIII:C followed pseudo-first order kinetics. However, the Arrhenius plot was not linear for formulations without carbohydrate, demonstrating a distinct, reproducible curvature. The reaction rate at 5 degrees C was faster than expected from the Arrhenius kinetics. The energy of activation (Ea) for formulations without added carbohydrates, derived from the linear part of the Arrhenius plot, varied between 77 and 86 kJ/mole in the temperature range 20-37 degrees C. The addition of 600 mg/ml sucrose increased the Ea to 104 kJ/mole. DSC measurements showed that Tm' was 64.2 +/- 0.2 degrees C for r-VIII SQ without stabiliser. This value increased linearly with increasing concentrations of carbohydrate. This stabilising effect is most probably explained by the theory of preferential hydration.
Conclusions:
The inactivation kinetics of r-VIII SQ in aqueous solution without addition of carbohydrates followed pseudo-first order kinetics but the Arrhenius plot was nonlinear. Sucrose and sorbitol both had highly stabilising effects on r-VIII SQ at concentrations above 300 mg/ml. The preparation containing 600 mg/ml sucrose was stable for at least 12 months at 5 degrees C and 6 months at 25 degrees C.