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 smallest active form, a metal ion-linked 80 + 90 kDa heterodimer; present in therapeutic factor VIII concentrates. The stability of r-VIII SQ was investigated in aqueous solution, without albumin (human) as a stabiliser. 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 (DSC). There was a rapid loss of activity when r-VIII SQ was stored in solution in vials containing air in the headspace. The stability was markedly 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 correlated with dissociation of the light (80 kDa) and heavy (90 kDa) chain complex. The separation of these chains was partly prevented by addition of calcium or strontium ions in the concentration range 1-10 mM. The transition point for heat denaturation (T'(m)) was 64 +/- 0.2 degrees 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 and 0.2 mg/ml polysorbate 80. The stability results presented here show premise and prompt further investigations into the development of a stable solution of r-VIII SQ. (C) 1997 Elsevier Science B.V.