Factor VIII separation

Abstract

The claims describe methods for isolating functionally active Factor VIII using a membrane-based separation system containing a separation membrane to create a first and second interstitial volume between at least two restriction membranes. One or more stabilizing agents are added to the sample and/or an interstitial volume. A solvent in the first interstitial volume maintains FVIII in a desired charge state. Applying a potential between the first and second interstitial volumes separates FVIII on one side of the separation membrane from unwanted molecules on the other side of the separation membrane. These methods may also be used as a substitute for one or more steps in a conventional purification scheme for the separation of native or recombinant FVIII.

1 . A method for obtaining Factor VIII (FVIII) from a sample comprising: 
 (a) placing the sample containing FVIII in an interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off different from the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to one or more of the sample, the first interstitial volume, or the second interstitial volume.    (c) selecting a solvent having a defined pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes wherein an end product containing FVIII is located on one side of the separation membrane while unwanted molecules are substantially located on the other side of the separation membrane; and    (e) maintaining step (d) until the desired amount of FVIII is located on one side of the separation membrane.    
   
   
       2 . A method for obtaining Factor VIII (FVIII) from a sample comprising: 
 (a) placing the sample containing FVIII in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off greater than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a defined pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume forming an end product containing FVIII while unwanted molecules are substantially prevented from entering the second interstitial volume; and    (e) maintaining step (d) until the desired amount of FVIII is moved to the second interstitial volume.    
   
   
       3 . A method for obtaining Factor VIII (FVIII) from a sample comprising: 
 (a) placing the sample containing FVIII in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off less than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both the sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of components in the sample other than FVIII in the first interstitial volume through the separation membrane into the second interstitial volume while FVIII is substantially prevented from entering the second interstitial volume and retained in the first interstitial volume as an end product; and    (e) maintaining step (d) until the desired amount components in the sample are removed from the first interstitial volume and the FVIII is retained in the first interstitial volume.    
   
   
       4 . The method according to  claim 1 ,  2  or  3  wherein the sample is plasma or fraction thereof, cryoprecipitate or a fraction thereof, a source of recombinant FVIII, or combinations or mixtures thereof.  
   
   
       5 . The method according to  claim 1 ,  2  or  3  wherein the stabilizing agent is one or more buffering constituents, molarity altering components, proteins, amino acids, or sugars.  
   
   
       6 . The method according to  claim 1 ,  2  or  3  wherein the stabilizing agent is selected from the group consisting of sorbitol, salt, glycerol, sugars such as sucrose, lactose, and dextran/dextrose, glycine, gelatin, potassium acetate, azide, and protease inhibitors.  
   
   
       7 . The method according to  claim 5  wherein the stabilizing agent is albumin, a mixture of amino acids, sucrose, or a mixture thereof.  
   
   
       8 . The method according to  claim 7  wherein albumin is used at a concentration of at least about 2 mg/mL (2 g/L), mixture of amino acids is used at a concentration of at least about 0.01 g/mL (10 g/L), and sucrose is used at a concentration of at least about 1% (10 g/L).  
   
   
       9 . The method according to  claim 7  wherein albumin is used at a concentration of about 10 mg/mL (10 g/L), mixture of amino acids is used at a concentration of about 0.05 g/mL (50 g/L), and sucrose is used at a concentration of about 5% (50 g/L).  
   
   
       10 . The method according to  claim 1 ,  2  or  3  wherein step (c) utilizes a buffer such that FVIII has a net negative charge.  
   
   
       11 . The method according to  claim 1 ,  2  or  3  wherein step (c) utilizes a buffer having a pH between 6.5-7.0.  
   
   
       12 . The method according to  claim 10  wherein the buffer is MES/Histidine pH 6.5, the separation membrane having a molecular mass cut off of about 1500 kDa and the restriction membranes having a molecular mass cut off of about 5 kDa.  
   
   
       13 . The method according to  claim 1 ,  2  or  3  wherein the end product FVIII retains at least about 40% specific activity.  
   
   
       14 . The method according to  claim 13  wherein the specific activity of the end product FVIII is at least about 60%.  
   
   
       15 . The method according to  claim 13  wherein the specific activity of the end product FVIII is greater than about 75%.  
   
   
       16 . The method according to  claim 1 ,  2 , or  3  wherein the sample is a cell supernatant or cell lysate containing a recombinant (rFVIII).  
   
   
       17 . A method for separating toxin, pathogen or infectious agent contamination from a sample containing FVIII comprising: 
 (a) placing the sample containing FVIII and toxin, pathogen or infectious agent contamination in a first interstitial volume of an apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off greater than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both the sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume forming an end product while unwanted molecules and toxin, pathogen or infectious agent contaminants are prevented from entering the second interstitial volume; and    (e) maintaining step (d) until the desired amount of FVIII is moved to the second interstitial volume.    
   
   
       18 . A method for separating toxin, pathogen or infectious agent contamination from a sample containing FVIII and toxin, pathogen or infectious agent contamination, comprising: 
 (a) placing the sample containing FVIII and toxin, pathogen or infectious agent contamination in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off greater than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a defined pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume forming an end product containing FVIII while unwanted molecules are prevented from entering the second interstitial volume;    (e) maintaining step (d) until the desired amount of FVIII is moved to the second interstitial volume;    (f) placing the sample obtained from step (e) in a first interstitial volume of an apparatus comprising a separation membrane having a defined pore size having a molecular mass cut off greater than that of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (g) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (h) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume while the toxin, pathogen or infectious agent contaminants are prevented from entering the second interstitial volume; and    (i) maintaining step (h) until the desired amount of FVIII is moved to the second interstitial volume.    
   
   
       19 . A method for separating toxin, pathogen or infectious agent contamination from a sample containing FVIII, comprising: 
 (a) placing the sample containing FVIII and toxin, pathogen or infectious agent contamination in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off less than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both the sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of components in the sample other than FVIII in the first interstitial volume through the separation membrane into the second interstitial volume while FVIII is prevented from entering the second interstitial volume and retained in the first interstitial volume as an end product;    (e) maintaining step (d) until the desired amount components in the sample are removed from the first interstitial volume and the FVIII is retained in the first interstitial volume;    (f) placing the sample obtained from (e) in a first interstitial volume of an apparatus comprising a separation membrane having a defined pore size having a molecular mass cut off greater than that of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (g) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (h) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume while the toxin, pathogen or infectious agent contaminants are prevented from entering the second interstitial volume; and    (i) maintaining step (h) until the desired amount of FVIII is moved to the second interstitial volume.    
   
   
       20 . A method for separating toxin, pathogen or infectious agent contamination from a sample containing FVIII, comprising: 
 (a) placing the sample containing FVIII and toxin, pathogen or infectious agent contaminationin in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off greater than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a defined pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of FVIII in the first interstitial volume through the separation membrane into the second interstitial volume forming an end product containing FVIII while unwanted molecules are substantially prevented from entering the second interstitial volume;    (e) maintaining step (d) until the desired amount of FVIII is moved to the second interstitial volume;    (f) placing the sample obtained from (e) in a first interstitial volume of an apparatus comprising a separation membrane having a defined pore size having a molecular mass cut off greater than the toxin, pathogen or infectious agent, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (g) selecting a solvent for the first interstitial volume having a pH such that the toxin, pathogen, infectious agent contaminants has a desired charge state;    (h) applying an electric potential between the first and second interstitial volumes causing movement of toxin, pathogen or infectious agent contaminants in the first interstitial volume through the separation membrane into the second interstitial volume while FVIII is prevented from entering the second interstitial volume; and    (i) maintaining step (h) until the desired amount of FVIII is in the first interstitial volume.    
   
   
       21 . A method for obtaining FVIII substantially free from toxin, pathogen or infectious agent contamination, the method comprising: 
 (a) placing the sample containing FVIII and toxin, pathogen or infectious agent contamination in a first interstitial volume of an electrophoresis apparatus comprising a separation membrane having a defined pore size with a molecular mass cut off less than the molecular mass of FVIII, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (b) providing one or more stabilizing agents to either or both the sample and the second interstitial volume;    (c) selecting a solvent for the first interstitial volume having a pH such that the FVIII has a desired charge state;    (d) applying an electric potential between the first and second interstitial volumes causing movement of components in the sample other than FVIII in the first interstitial volume through the separation membrane into the second interstitial volume while FVIII is substantially prevented from entering the second interstitial volume and retained in the first interstitial volume as an end product;    (e) maintaining step (d) until the desired amount components in the sample are removed from the first interstitial volume and the FVIII is retained in the first interstitial volume;    (f) placing the FVIII with toxin, pathogen or infectious agent contaminant in a first interstitial volume of an apparatus comprising a separation membrane having a defined pore size having a molecular mass cut off greater than the toxin, pathogen or infectious agent, a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween, and a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween;    (g) selecting a solvent for the first interstitial volume having a pH such that the toxin, pathogen, infectious agent contaminants has a desired charge state;    (h) applying an electric potential between the first and second interstitial volumes causing movement of toxin, pathogen or infectious agent contaminants in the first interstitial volume through the separation membrane into the second interstitial volume while FVIII is prevented from entering the second interstitial volume; and    (i) maintaining step (h) until the desired amount of FVIII is in the first interstitial volume.    
   
   
       22 . The method according to claims  17 ,  18 ,  19 ,  20 , or  21  wherein the toxin, pathogen or infectious agent is selected from the group consisting of endotoxin, prion, viral, bacterial, fungal, yeast, and protozoan.  
   
   
       23 . The method according to claims  17 ,  18 ,  19 ,  20 , or  21  wherein the pathogen or infectious agent is a virus.