Methods of detecting glycosaminoglycans
View Patent ↗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.
1. A method of separating a factor VIII (FVIII) protein or a factor IX (FIX) protein from glycosaminoglycan during chromatography, the method comprising:
(i) subjecting a sample comprising one or more the FVIII or FIX proteins and glycosaminoglycan to a size-exclusion chromatography using a mobile phase having a pH of less than 3.0, and
(ii) collecting a mobile phase fraction comprising the FVIII or FIX proteins;
wherein the mobile phase comprises a salt;
wherein the mobile phase fraction does not include the glycosaminoglycan or includes only small amounts of the glycosaminoglycan, and
wherein the method does not include a use of fluorescent labeling.
2. The method of claim 1 , further comprising using a detection device to determine the amount of glycosaminoglycan in the mobile phase fraction.
3. The method of claim 2 , further comprising determining if the amount of glycosaminoglycan in the sample is above a threshold level.
4. The method of claim 3 , wherein the threshold level is 2 microgram/ml.
5. The method of claim 2 , wherein the detection device is a UV detector.
6. The method of claim 1 , wherein the sample is unpurified or partially purified.
7. The method of claim 1 , wherein the sample comprises a FVIII protein.
8. The method of claim 1 , wherein the sample comprises a FIX protein.
9. The method of claim 1 , wherein the glycosaminoglycan is negatively charged.
10. The method of claim 1 , wherein the glycosaminoglycan is dextran sulfate or heparin sulfate.
11. The method of claim 10 , wherein the glycosaminoglycan is dextran sulfate having a size of 6-8 kDa.
12. The method of claim 1 , wherein the size-exclusion chromatography packing material and mobile phase allow for secondary interactions.
13. The method of claim 12 , 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.
14. The method of claim 1 , wherein the size-exclusion chromatography is performed on a silica-based size-exclusion column.
15. The method of claim 1 , wherein the pH of the mobile phase is about 2.5.
16. The method of claim 15 , wherein the pH of the mobile phase is 2.5.
17. The method of claim 1 , wherein the salt is 200 mM NaCl.
18. The method of claim 1 , wherein the FVIII protein is Factor VIIIFc.
19. The method of claim 1 , wherein the FIX protein is Factor IXFc.