Application of a new statistical approach to optimize the immunopurification of antihemophilia A factor.
Our aim was to optimize the immunopurification process of human factor VIII. This purification was performed using a mouse monoclonal anti-factor VIII light-chain antibody. Previous dissociation of the factor VIII-von Willebrand factor complex with CaCl2 led to a 50% increase of the factor VIII adsorption on the immunosorbent. The optimization of the elution step required the analysis of the effects of two parameters, pH and ionic strength, on four different responses: elution yield, concentration, specific activity and stability of factor VIII. For this purpose, a multifunctional method using Doehlert matrices for statistically designed experiments was applied. This methodology allowed us to obtain, with only seven experiments, a 60% increase of the elution yield and a two-fold increase of the specific activity of factor VIII.
Bihoreau
N
N
T.M. Innovation (Centre National de Transfusion Sanguine-Institut Mérieux, Les Ulis, France.
Layet
S
S
Fontaine-Aupart
M P
MP
Paolantonacci
P
P
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000302:isolation & purification; D015944:Factor VIIIa / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D008954:Models, Biological; D013223:Statistics as Topic
D000911:Antibodies, Monoclonal; D015944:Factor VIIIa; D005169:Factor VIII