Process for preparing agents containing virus-inactivated vitamin k- dependent plasma components as well as protein c and protein s by membrane chromatography

Abstract

Disclosed is a process for preparing agents containing virus-inactivated vitamin K-dependent plasma components as well as protein C, protein S, factors II, VII, IX and/or X as well as combinations thereof, such as, for example, PPSB preparations, wherein a source containing these components is subjected to appropriate separation procedures, especially by using membrane-chromatographic methods.

1 . A process for preparing agents containing virus-inactivatd vitamin k-dependent plasma components as well as protein c, protein s, factors II, VII, IX and/or X as well as combinations thereof, such as, for example, PPSB preparations, wherein a source containing these components is subjected to the following process steps: 
 a) Solid phase extraction of the sources containing the components to be separated on anion exchanger materials in loose bulk, on membranes and/or on compact disks under conditions of relatively low ionic strength, removal of the resulting effluent and, if desired, supplying the effluent to further work-up procedures for the recovery of other materials;    b) elution of the material adsorbed on the solid phase;    c) optionally, adjustment of the ionic strength and/or of the pH value of the fraction containing the eluate to the condition of the following purification step;    d) virus-inactivation by means of ionic and/or non-ionic detergents in the presence of di- or trialkyl phosphate compounds such as tri-n-butyl phosphate and optionally a heat treatment;    e) a ion exchange membrane chromatography, affinity membrane chromatography with immobilized high or low molecular weight substances possessing a high affinity to vitamin K-dependent plasma components as well as protein C and protein S or hydrophobic interaction chromatography;    f) fractionation of individual components by stepwise elution by changing the ionic strength, the polarity and/or the pH value;    g) optionally membrane affinity chromatography;    h) optionally collecting the eluate that contains the products which have not already been removed, and optionally the further fractionation thereof by repeating step f), wherein appropriate affinity materials are used;    i) elution of the substances bound to the affinity material under conditions reversing the affinity bond, followed by concentration of the eluate, optionally with reducing the amount of the agent used for the elution from the affinity material;    or the virus inactivation tades place after step f) followed by a membrane chromatography step and optionally a heat treatment and/or a filtration for the removal of viruses takes place at a suitable stage.    
     
     
         2 . The process according to  claim 1 , wherein the source containing vitamin K-dependent plasma components such as protein C and protein S is blood plasma in fresh or thawed condition.  
     
     
         3 . The process according to claims  1  and/or  2 , wherein the solid phase extraction is effected by means of a cross-linked polysaccharide modified with basic groups, such as Sephadex® or by means of anion exchanger membranes appropriately modified.  
     
     
         4 . The process according to at least one of  claims 1  to  3 , wherein the membrane chrmatography is carried out by using membranes which have been modified with DEAE or quaternary amines, or by using membranes which have been modified by low or high molecular weight affinity ligands capable of specifically binding to the desired plasma components (membrane affinity chromatography).  
     
     
         5 . The process according to at least one of  claims 1  to  4 , wherein the affinity chromatography is a heparin membrane affinity chromatography, a membrane immunoaffinity chromatography with immobilized antibodies against the substance to be isolated or a membrane affinity chromatography using membranes with hydrophobic ligands.  
     
     
         6 . The process according to  claim 5 , wherein the hydrophobic ligands for modifying the chromatographic carrier are 2-hydroxyaminoalkyl groups such as 2-hydroxyaminopropyl and/or hydrophobic ligands such as propyl, butyl, phenyl groups and other similar ligands exhibiting some gradual change in hydrophobicity.  
     
     
         7 . The process according to at least one of  claims 1  to  6 , wherein the adjustment of the ionic strength after the chromatographic steps is effected by dilution or desalting procedures such as dia- or ultrafiltration or by the addition of agents increasing the ionic strength.  
     
     
         8 . The process according to at least one of  claims 1  to  7 , wherein the virus-inactivation is effected by heating the fraction at from 55° C. to 70° C. for a period of from 5 to 30 hours in the presence of stabilizers such as sugar, amino acids, bivalent cations and/or heparin.  
     
     
         9 . The process according to  claim 8 , wherein the stabilizers are removed by desalting procedures such as diafiltration or ultrafiltration, by heparin affinity chromatography or anion exchanger chromatography or on carriers modified with DEAE or quaternary ammonium compounds or carriers modified with hydrophobic ligands.  
     
     
         10 . The process according to at least one of  claims 1  to  9 , wherein the chromatography material is a particulate material arranged as loose bulk, a material embedded in membranes and/or compact disks made of the respective materials.  
     
     
         11 . The process according to at least one of  claims 1  to  10 , wherein the fractions obtained are concentrated by lyophilization or spray drying.