title: Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters.
abstract: 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.
authors.affiliation: Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
authors.forename: M P
authors.initials: MP
authors.lastname: te Booy
authors.forename: A
authors.initials: A
authors.lastname: Faber
authors.forename: E
authors.initials: E
authors.lastname: de Jonge
authors.forename: E P
authors.initials: EP
authors.lastname: Wolterink
authors.forename: W
authors.initials: W
authors.lastname: Riethorst
authors.forename: T
authors.initials: T
authors.lastname: Beugeling
authors.forename: A
authors.initials: A
authors.lastname: Bantjes
authors.forename: J
authors.initials: J
authors.lastname: Over
authors.forename: B W
authors.initials: BW
authors.lastname: König
mesh_terms: 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
chemical_list: D002021:Buffers; D008024:Ligands; D011516:Prothrombin; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X