Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters.
The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.
te Booy
M P
MP
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Faber
A
A
de Jonge
E
E
Wolterink
E P
EP
Riethorst
W
W
Beugeling
T
T
Bantjes
A
A
Over
J
J
König
B W
BW
D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D005164:Factor IX / Q000032:analysis; D005169:Factor VIII / Q000302:isolation & purification*; D005170:Factor X / Q000032:analysis; D006801:Humans; D008024:Ligands; D010949:Plasma / Q000032:analysis*; D011516:Prothrombin / Q000032:analysis
D002021:Buffers; D008024:Ligands; D011516:Prothrombin; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X