RECOMBINANT FACTOR-VIII SQ - INACTIVATION KINETICS IN AQUEOUS-SOLUTION AND THE INFLUENCE OF DISACCHARIDES AND SUGAR ALCOHOLS

Purpose, To investigate the influence of various nonreducing disacchar ides and sugar alcohols on the inactivation kinetics of recombinant fa ctor 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 differenti al scanning calorimetry (DSC). Results. The decline in VIII:C followed pseudo-first order kinetics. However, the Arrhenius plot was not line ar for formulations without carbohydrate, demonstrating a distinct, re producible curvature. The reaction rate at 5 degrees C was faster than expected from the Arrhenius kinetics. The energy of activation (Ea) f or formulations without added carbohydrates, derived from the linear p art of the Arrhenius plot, varied between 77 and 86 kJ/mole in the tem perature range 20-37 degrees C. The addition of 600 mg/ml sucrose incr eased 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 increas ed linearly with increasing concentrations of carbohydrate. This stabi lising effect is most probably explained by the theory of preferential hydration. Conclusions. The inactivation kinetics of r-VIII SQ in aqu eous solution without addition of carbohydrates followed pseudo-first order kinetics but the Arrhenius plot was nonlinear. Sucrose and sorbi tol both had highly stabilising effects on r-VIII SQ at concentrations above 300 mg/ml. The preparation containing 600 mg/ml sucrose was sta ble for at least 12 months at 5 degrees C and 6 months at 25 degrees C .