10601686

pubmed:Citation

1

The main aim of this paper was to investigate the influence of temperature, pH and ionic interactions on the structural stability and surface adsorption of a recombinant factor VIII product, r-VIII SQ. The interaction of r-VIII SQ with glass and air interfaces, and possible means of increasing the stability of the formulation, were also investigated. The stability of r-VIII SQ was followed by measuring the biological activity (VIII:C), by circular dichroism (CD) studies and by the measurement of surface tension using the 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 increased temperatures. Far UV CD spectra were not influenced in the range 5-55 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 rapidly in formulations without a macromolecular additive. Nonionic surfactants such as polysorbate 80 and polysorbate 20 protected VIII:C to an equally high degree against surface adsorption. Albumin was less effective, but it is possible that this is because it is a protein itself and may have been affected 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 theory of preferential hydration. Near UV CD spectra at acidic or basic pH mainly indicated changes around 242 nm, especially at low ionic strength. Addition of 10 mM EDTA at pH 7 resulted in similar changes. This effect was completely reversed by the addition of an excess of Ca(2+), Sr(2+) or Mg(2+) ions. In conclusion, CD spectra and surface tension measurements of r-VIII SQ did not reveal any temperature-induced conformational changes in the temperature range 5-20 degrees C; changes were first noted at elevated temperatures. Surface adsorption of r-VIII SQ during agitation was prevented by the addition of a nonionic surfactant. Preferential hydration improved the storage stability of the protein but did not directly prevent its surface adsorption. The structural integrity of the molecule was preserved at pH 7, at an increased ionic strength and in the presence of some divalent metal ions (Ca(2+), Sr(2+) or Mg(2+)).

http://linkedlifedata.com/resource/pubmed/journal/7804127

http://linkedlifedata.com/resource/pubmed/chemical/Cations, Divalent, http://linkedlifedata.com/resource/pubmed/chemical/Factor VIII, http://linkedlifedata.com/resource/pubmed/chemical/Polysorbates, http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Surface-Active Agents

Jan

pubmed-author:FatourosAA, pubmed-author:SjöströmBB

20

NLM

69-79

pubmed-meshheading:10601686-Adsorption, pubmed-meshheading:10601686-Animals, pubmed-meshheading:10601686-CHO Cells, pubmed-meshheading:10601686-Cations, Divalent, pubmed-meshheading:10601686-Circular Dichroism, pubmed-meshheading:10601686-Cricetinae, pubmed-meshheading:10601686-Drug Stability, pubmed-meshheading:10601686-Factor VIII, pubmed-meshheading:10601686-Glass, pubmed-meshheading:10601686-Hydrogen-Ion Concentration, pubmed-meshheading:10601686-Polysorbates, pubmed-meshheading:10601686-Protein Conformation, pubmed-meshheading:10601686-Protein Denaturation, pubmed-meshheading:10601686-Recombinant Proteins, pubmed-meshheading:10601686-Surface Properties, pubmed-meshheading:10601686-Surface Tension, pubmed-meshheading:10601686-Surface-Active Agents, pubmed-meshheading:10601686-Temperature

Recombinant factor VIII SQ--the influence of formulation parameters on structure and surface adsorption.

Journal Article