US2019339236A1PendingUtilityA1

Methods of detecting glycosaminoglycans

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Assignee: BIOVERATIV THERAPEUTICS INCPriority: Apr 11, 2012Filed: Apr 16, 2019Published: Nov 7, 2019
Est. expiryApr 11, 2032(~5.7 yrs left)· nominal 20-yr term from priority
Inventors:Zoran SosicAndrew BlumBing GuanBoris Boumajny
G01N 33/6842C08B 37/0003C08B 37/0063G01N 2030/027G01N 30/60G01N 2400/40C08B 37/0021
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Claims

Abstract

In one aspect, the disclosure provides methods of distinguishing a glycosaminoglycan from one or more other components in a sample by subjecting the sample to size-exclusion chromatography using a mobile phase having a pH of 6.8 or lower. A mobile phase having a pH of 6.8 or lower is found to improve the separation of glycosaminoglycans from proteins during size exclusion chromatography. In some embodiments, improved separation is due to the low pH of the mobile phase causing elution of less dispersed fractions of the protein and/or glycosaminoglycan. In some embodiments, the overlap between protein and/or glycosaminoglycan fractions is reduced.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method of distinguishing a glycosaminoglycan from one or more other components in a sample, the method comprising:
 subjecting a sample to size-exclusion chromatography using a mobile phase having a pH of 6.8 or lower.   
     
     
         2 . The method of  claim 1 , further comprising using a detection device to determine an amount of glycosaminoglycan in the mobile phase that has been subjected to the size-exclusion chromatography, thereby determining the amount of glycosaminoglycan in the sample. 
     
     
         3 . A method of determining if a sample contains glycosaminoglycan, the method comprising:
 applying a sample to a size-exclusion column,   applying a mobile phase to run the sample through the size-exclusion column, wherein the mobile phase has a pH of 6.8 or lower,   using a detection device to determine if glycosaminoglycan is present in the mobile phase that has run through the column, thereby determining if the sample contains glycosaminoglycan.   
     
     
         4 . The method of  claim 2 , further comprising determining if the amount of glycosaminoglycan in the sample is above a threshold level. 
     
     
         5 . The method of  claim 4 , wherein the threshold level is 2 microgram/mi. 
     
     
         6 . The method of  claim 1 , wherein the sample comprises protein. 
     
     
         7 . The method of  claim 1 , wherein the sample is unpurified. 
     
     
         8 . The method of  claim 1 , wherein the sample is partially purified. 
     
     
         9 . The method of  claim 1 , wherein the sample comprises one or more blood cascade proteins. 
     
     
         10 . The method of  claim 9 , wherein the sample comprises Factor VIIIFc or Factor IXFc. 
     
     
         11 . The method of  claim 1 , wherein the glycosaminoglycan is negatively charged. 
     
     
         12 . The method of  claim 1 , wherein the glycosaminoglycan is dextran sulfate. 
     
     
         13 . The method of  claim 12 , wherein the dextran sulfate has a size of 6-8 kDa. 
     
     
         14 . The method of  claim 1 , wherein the glycosaminoglycan is heparin sulfate. 
     
     
         15 . The method of  claim 2 , wherein the detection device is a UV detector. 
     
     
         16 . The method of  claim 1 , wherein the size-exclusion chromatography packing material and mobile phase allow for secondary interactions. 
     
     
         17 . The method of  claim 16 , wherein the size-exclusion chromatography packing material is silica-based, has a pore size of between 100-200 Angstrom, and a particle size of 5 microns or less. 
     
     
         18 . The method of  claim 1 , wherein the size-exclusion chromatography is performed on a silica-based size-exclusion column. 
     
     
         19 . The method of  claim 1 , wherein the pH of the mobile phase is 6.0 or lower. 
     
     
         20 . The method of  claim 1 , wherein the pH of the mobile phase is 5.0 or lower. 
     
     
         21 . The method of  claim 1 , wherein the pH of the mobile phase is 4.0 or lower. 
     
     
         22 . The method of  claim 1 , wherein the pH of the mobile phase is 3.0 or lower. 
     
     
         23 . The method of  claim 1 , wherein the pH of the mobile phase is 2.5. 
     
     
         24 . The method of  claim 1 , wherein the mobile phase comprises salt. 
     
     
         25 . The method of  claim 24 , wherein the salt is 200 mM NaCl.

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