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US 5,470,954 AGranted
Ultrapurification process for factor VIII
AssigneeBaxter International Inc.
InventorsGerard G. Neslund, Shu-Len Liu, Michael J. Griffith
Life of this patent
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Abstract
A process for purifying Factor VIII:C comprising contacting an immobilized antibody specifically binding a Factor VIII:C with Factor VIII: C, desorbing Factor VIII:C from the antibody which had adsorbed it, eluting Factor VIII:C from the presence of the antibody, passing the eluted Factor VIII:C through an affinity region capable of binding the Factor VIII:C, binding the Factor VIII:C in the affinity region and passing contaminants through said region, and eluting the purified Factor VIII:C.
Claims39
A method of purifying Factor VIII:C from a mixture of polypeptides and contaminants comprising:
- (a) adsorbing an antibody to a matrix to form an immunoaffinity matrix, which antibody binds by hydrophobic attraction to the Factor VIII:C to be purified to form hydrophobic bonds, adding the mixture to said antibody before or after said antibody is bound to the matrix, thereby adsorbing said Factor VIII:C in the immunoaffinity matrix;
- (b) eluting the Factor VIII:C from the immobilized antibody by treating the Factor VIII:C:immunoaffinity matrix with a desorbing substance which breaks the hydrophobic bonds between the Factor VIII:C and the immobilized antibody to desorb the Factor VIII:C from said matrix wherein the desorbing substance is a non-polar agent in a buffered salt solution;
- (c) passing the Factor VIII:C to be purified through an affinity region which is an ion-exchange region capable of binding to the Factor VIII:C, thereby binding the Factor VIII:C to the affinity material while allowing the contaminants to pass through the affinity region;
- and
- (d) eluting the purified Factor VIII:C from the affinity region.
2 more elements
A process for purifying Factor VIII:C from a complex aqueous mixture contaminated with lipid-enveloped pathogenic viruses, which comprises:
- (a) mixing the complex aqueous mixture with a virus-inactivating agent containing an organic lipid-dissolving or disrupting solvent and a detergent at any step in the process;
- (b) passing the complex aqueous mixture through an immunoaffinity matrix containing active immobilized monoclonal antibody specific to Factor VIII:C, thereby causing the Factor VIII:C to adsorb to the immunoaffinity matrix by hydrophobic attraction to said Factor VIII:C to form hydrophobic bonds;
- (c) washing the immunoaffinity matrix with an aqueous buffer to remove at least a portion of contaminants from the complex aqueous mixture, while leaving the Factor VIII:C adsorbed to the immunoaffinity matrix;
- (d) eluting the Factor VIII:C from the immunoaffinity matrix with a desorbing substance which disrupts the hydrophobic bonds formed in step (b), thereby forming a Factor VIII:C:
- desorbing substance mixture wherein the desorbing substance is a non-polar agent in a buffered salt solution;
- (e) passing the Factor VIII:C to be purified through an affinity region, thereby causing Factor VIII:C to bind thereto by hydrophilic attraction while allowing any leached monoclonal antibodies, detergents and residual contaminants to pass through the affinity region;
- (f) washing the affinity region with an aqueous buffer to remove residual contaminants from the Factor VIII:C:desorbing substance mixture while leaving Factor VIII:C adsorbed to the affinity region;
- (g) eluting the Factor VIII:C from the affinity region with an eluting solution which disrupts hydrophilic bonds between the affinity region and the Factor VIII:C to elute the Factor VIII:C substantially free of contaminants;
- and
- (h) wherein the Factor VIII:C to be purified is passed from the immunoaffinity matrix of step (d) to the affinity region of step (e) without further modification or alteration of the Factor VIII:C:
- desorbing substance mixture
8 more elements
A method of purifying Factor VIII:C from a mixture of polypeptides and contaminants comprising:
- (a) immobilizing an antibody, in a first purification region in an immunoaffinity matrix, which antibody binds by hydrophobic attraction to the Factor VIII:C to be purified to form hydrophobic bonds, adding the mixture to said antibody before or after said antibody is bound to the matrix, thereby adsorbing said Factor VIII:C in the immunoaffinity matrix;
- (b) eluting the Factor VIII:C from the immobilized antibody by treating the Factor VIII:C:immunoaffinity matrix with a desorbing substance which is non-polar or of low polarity in a buffered salt solution having a low ionic strength, thereby forming a Factor VIII:C:desorbing substance mixture;
- (c) passing the Factor VIII:C to be purified through a second purification region which is an affinity region, said affinity region being an ion-exchange region, thereby binding the Factor VIII:C to the affinity region while allowing contaminants to pass through the affinity region;
- (d) eluting the purified Factor VIII:C from the affinity region with a desorbing substance having an ionic strength sufficient to desorb the Factor VIII:C from said affinity region;
- wherein the Factor VIII:C to be purified is passed from the purification region of step (b) to the purification region of step (c) without further modification or alteration of the Factor VIII:C:desorbing substance mixture.
2 more elements
Dependencies are inferred from the claim text.