Methods of manufacturing a high molecular weight heparin compound

Abstract

A method of manufacturing a high molecular weight heparin (HMWH) compound is disclosed. The method comprises dissolving heparin to form a heparin solution and fractionating the heparin solution via tangential flow filtration (TFF) using a membrane with a molecular weight cut off (MWCO) between about 8 kDa and about 12 kDa. The TFF yields a retentate comprising fractionated heparin with a weight average molecular weight of about 20 kDa or greater, i.e., a high molecular weight heparin compound. A substantial proportion of heparin chains in the fractionated heparin may have a high molecular weight, e.g., 50% of the heparin chains or greater may have a molecular weight of 20 kDa or greater.

1 .- 33 . (canceled) 
     
     
         34 . A method of manufacturing fractionated heparin, the method comprising:
 dissolving heparin in a solvent to form a heparin solution;   filtering the heparin solution through a submicron membrane to sterilize the heparin solution; and   fractionating the heparin solution by tangential flow filtration using a fractionation membrane with a molecular weight cut off between about 8 kDa and about 12 kDa, thereby yielding fractionated heparin with a weight average molecular weight of 20 kDa or greater,   wherein at least 50% of heparin chains in the fractionated heparin have a molecular weight of 18 kDa or greater.   
     
     
         35 . The method of  claim 34 , wherein the heparin is USP heparin. 
     
     
         36 . The method of  claim 34 , wherein the solvent is a sodium chloride (NaCl) solution. 
     
     
         37 . The method of  claim 36 , where the NaCl solution has a concentration of about 100 mM. 
     
     
         38 . The method of  claim 34 , wherein the molecular weight cut off of the fractionation membrane is about 10 kDa. 
     
     
         39 . The method of  claim 34 , wherein fractionating the heparin solution by tangential flow filtration comprises permeating at least some of the heparin solution through the fractionation membrane under an applied pressure to yield a retentate comprising the fractionated heparin. 
     
     
         40 . The method of  claim 39 , wherein the applied pressure is about 29 psi to about 30 psi. 
     
     
         41 . The method of  claim 39 , wherein fractionating the heparin solution by tangential flow filtration further comprises adding an additional quantity of the solvent to maintain a volume of the retentate. 
     
     
         42 . The method of  claim 34 , further comprising desalting the fractionated heparin. 
     
     
         43 . The method of  claim 42 , wherein desalting the fractionated heparin comprises performing tangential flow filtration using a desalting membrane with a molecular weight cut off between about 1 kDa and about 5 kDa. 
     
     
         44 . The method of  claim 34 , further comprising drying the fractionated heparin. 
     
     
         45 . The method of  claim 44 , wherein drying the fractionated heparin comprising lyophilizing the fractionated heparin. 
     
     
         46 . A method of manufacturing a high molecular weight (HMW) heparin, the method comprising:
 dissolving a heparin salt in a salt solution to form a heparin solution;   sterilizing the heparin solution by filtering through a sterilization membrane having a pore size of about 0.2 μm, thereby yielding a sterilized heparin solution;   fractionating the sterilized heparin solution by tangential flow filtration using a fractionation membrane with a pore size of about 5 nm, thereby yielding fractionated heparin with a weight average molecular weight of 20 kDa or greater, wherein at least 50% of heparin chains in the fractionated heparin have a molecular weight of 18 kDa or greater;   desalting the fractionated heparin by tangential flow filtration using a desalting membrane with a pore size of about 3 nm, thereby yielding desalted heparin; and   drying the desalted heparin by lyophilization to yield the HMW heparin.   
     
     
         47 . The method of  claim 46 , wherein the heparin salt is selected from the group consisting of heparin sodium and heparin calcium. 
     
     
         48 . The method of  claim 46 , wherein the heparin salt is a USP heparin salt. 
     
     
         49 . The method of  claim 46 , wherein the salt solution is a sodium chloride (NaCl) solution having a concentration of about 100 mM. 
     
     
         50 . The method of  claim 49 , wherein fractionating the sterilized heparin solution by tangential flow filtration comprises permeating at least some of the sterilized heparin solution through the fractionation membrane under an applied pressure of about 29 psi to about 30 psi to yield a retentate comprising the fractionated heparin. 
     
     
         51 . The method of  claim 46 , wherein fractionating the sterilized heparin solution by tangential flow filtration further comprises adding an additional quantity of the salt solution to maintain a volume of the retentate. 
     
     
         52 . The method of  claim 46 , wherein the molecular weight cut off of the desalting membrane is about 3 kDa. 
     
     
         53 . The method of  claim 46 , wherein fractionating the sterilized heparin solution by tangential flow filtration comprises permeating at least some of the sterilized heparin solution through the fractionation membrane under a transmembrane pressure of about 30 psi such that (1) heparin chains in the sterilized heparin having a molecular weight of less than 18 kDa permeate through the filtration membrane as a filtrate, and (2) heparin chains in the sterilized heparin having a molecular weight of 18 kDa or greater do not permeate the filtration membrane, thereby yielding the retentate comprising the fractionated heparin,
 wherein the heparin salt comprises heparin sodium and the salt solution comprises sodium chloride (NaCl) solution in a concentration of about 100 mM.   
     
     
         54 . A method of manufacturing fractionated heparin, the method comprising:
 dissolving heparin in a solvent to form a heparin solution; and   fractionating the heparin solution by tangential flow filtration using a fractionation membrane with a molecular weight cut off between about 8 kDa and about 12 kDa, thereby yielding fractionated heparin with a weight average molecular weight of about 20 kDa or greater,   wherein at least 50% of heparin chains in the fractionated heparin have a molecular weight of 18 kDa or greater.