Recombinant factor VIIISQ - the influence of formulation parameters on structure and surface adsorption

The main aim of this paper was to investigate the influence of temperature, pH and ionic interactions on thr: structural stability and surface adsorpt ion of a recombinant factor VIII product, r-VIII SQ. The interaction of r-V III SQ with glass and air interfaces, and possible means of increasing the stability of the formulation, were also investigated. The stability of r-VI II SQ was followed by measuring the biological activity (VIII:C), by circul ar dichroism(CD) studies and by the measurement of surface tension using th e pendant drop method. The results show that the surface tension decreased exponentially with time: this decrease was more pronounced above 20 degrees C, indicating increased conformational flexibility of the protein with inc reased temperatures. Far UV CD spectra were not influenced in the range 5-5 5 degrees C and near UV CD measurements did not indicate structural changes below 45 degrees C. During agitation at 25 degrees C, VIII:C was lost rapi dly in formulations without a macromolecular additive. Nonionic surfactants such as polysorbate 80 and polysorbate 20 protected VIII:C to an equally h igh degree against surface adsorption. Albumin was less effective, but it i s possible that this is because it is a protein itself and may have been af fected by the agitation. The addition of 300 mg/ml of sucrose improved the long term stability of VIII:C, a finding most likely explained by the theor y of preferential hydration. Near UV CD spectra at acidic or basic pH mainl y indicated changes around 242 nm, especially at low ionic strength. Additi on of 10 mM EDTA at pH 7 resulted in similar changes. This effect was compl etely reversed by the addition of an excess of Ca2+, Sr2+ or Mg2+ ions. In conclusion, CD spectra and surface tension measurements of r-VIII SQ did no t reveal any temperature-induced conformational changes in the temperature range 5-20 degrees C; changes were first noted at elevated temperatures. Su rface adsorption of r-VIII SQ during agitation was prevented by the additio n of a nonionic surfactant. preferential hydration improved the storage sta bility of the protein but did not directly prevent its surface adsorption. The structural integrity of the molecule was preserved at DH 7, at an incre ased ionic strength and in the presence of some divalent metal ions (Ca2+, Sr2+ or Mg2+). (C) 2000 Published by Elsevier Science B.V. All rights reser ved.