RECOMBINANT FACTOR-VIII SQ - INFLUENCE OF OXYGEN, METAL-IONS, PH AND IONIC-STRENGTH ON ITS STABILITY IN AQUEOUS-SOLUTION

Recombinant factor VIII SQ (r-VIII SQ) is a derivative of human factor VIII in which the B-domain has been deleted. It corresponds to the sm allest active form, a metal ion-linked 80 + 90 kDa heterodimer; presen t in therapeutic factor VIII concentrates. The stability of r-VIII SQ was investigated in aqueous solution, without albumin (human) as a sta biliser. Activity assay (VIII:C), visual inspection and gel filtration were performed after storage at different temperatures. The influence of oxygen, metal ions, pH and ionic strength was studied. The thermal stability was investigated using differential scanning calorimetry (D SC). There was a rapid loss of activity when r-VIII SQ was stored in s olution in vials containing air in the headspace. The stability was ma rkedly improved by reducing the oxygen content, pH 6.5-7.0 was optimal for stability at both low and high ionic strengths. The best results were obtained at high ionic strengths, since r-VIII SQ precipitated at sodium chloride concentrations below 5 mg/ml. The loss of VIII:C corr elated with dissociation of the light (80 kDa) and heavy (90 kDa) chai n complex. The separation of these chains was partly prevented by addi tion of calcium or strontium ions in the concentration range 1-10 mM. The transition point for heat denaturation (T'(m)) was 64 +/- 0.2 degr ees C in a formulation containing 9500 I r-VIII SQ/ml, 18 mg/ml sodium chloride, 0.5 mg/ml calcium chloride dihydrate, 3 mg/ml L-histidine a nd 0.2 mg/ml polysorbate 80. The stability results presented here show premise and prompt further investigations into the development of a s table solution of r-VIII SQ. (C) 1997 Elsevier Science B.V.