Process for recovering heparinic oligosaccharides with an affinity for cell growth factors

Claim 2

A process for obtaining high anionic strength heparinic oligosaccharides from a mixture of heparinic glycosaminoglycans, which mixture of heparinic glycosaminoglycans is substantially free of non-heparin components and is produced by the depolymerization of heparin, comprising the steps of: contacting the mixture of glycosaminoglycans with a cationic or anionic cell growth factor to bind said oligosaccharide to the growth factor; and then eluting said heparinic oligosaccharides from the growth factor.

Claim 3

The process of claim 1, wherein the growth factor is anionic and is bound to a support.

Claim 4

The process of claim 2, wherein the support is an agarose support.

Claim 5

The process of claim 1, wherein the mixture of glycosaminoglycans contains chains of saccharide residues which do not have a binding site for AT III.

Claim 6

The process of claim 4, wherein the mixture of glycosaminoglycans contains chains containing a maximum of 14 saccharide residues selected from the group consisting of chains with an α,β- unsaturated uronic acid residue at a non-reducing end and chains with a residue possessing a 2,5 anhydromanno structure at a reducing end.

Claim 7

The process of claim 1, wherein the mixture of glycosaminoglycans is loaded on a chromatographic column containing a cationic or anionic growth factor fixed to an agarose support; the column being equilibrated with a buffer having an ionic strength corresponding to that of 0.2M NaCl; and then, the oligosaccharide retained on the support is eluted from the column with a buffer having an ionic strength corresponding to that of 1M to 2M NaCl.

Claim 8

The process of claim 6, wherein the mixture of glycosaminoglycans is homogeneous with regard to the molecular weight of the glycosaminoglycans.

Claim 9

The process of claim 1, wherein the mixture of glycosaminoglycans is subjected to ion exchange chromatography on a strongly basic anion exchange column of the quaternary ammonium type and an ionic strength gradient is set up in the elution buffer by varying the concentration of NaCl in the buffer from 0.5±0.1M to 1 to 2M, and the most anionic fractions are recovered.

Claim 10

The process of claim 8, wherein the mixture of glycosaminoglycans is obtained by depolymerization of heparin, followed by alcoholic fractionation in order to separate chains endowed with an antithrombin III affinity from chains lacking said affinity.

Claim 11

The process of claim 1 wherein a heparinic hexasaccharide is obtained in a fraction eluted in a volume between 32L and 36L of eluant from a gel filtration column wherein: 60 g of precipitate, obtained by alcoholic precipitation of a nitrous acid depolymerization mixture of heparin, was taken up in 500 ml of 0.5M NaCl solution and loaded on to a 100 cm×25 cm column containing 45 liters of agarose-acrylamide, equilibrated with a 0.5M NaCl buffer, pH 6.0, and elution was carried out at a flow rate of 1500 ml/hour; a product obtained was separated by affinity chromatography on anionic FGF SEPHAROSE® agarose gel using a 2.5 cm×6.5 cm column containing 30 ml of anionic FGF-SEPHAROSE® agarose gel and equilibrated with 0.01M Tris-HCL buffer, pH 7.4, containing 0.2M NaCl; 300 mg of the product in a solution of 60 ml of the Tris-HCl buffer was loaded on to the column and elution was carried out with this Tris-HCl buffer adjusted to 1M NaCl; and the hexasaccharide was recovered from a fraction collected by precipitation with ethanol.

Claim 12

The process of claim 1 wherein a heparinic octasaccharide is obtained in a fraction eluted in a volume between 28 L and 32L of eluant from a gel filtration column wherein: 60 g of precipitate, obtained by alcoholic precipitation of a nitrous acid depolymerization mixture of heparin, was taken up in 500 ml of 0.5M NaCl and loaded on to a 100 cm×25 cm column containing 45 liters of agarose-acrylamide and equilibrated with a 0.5M NaCl buffer, pH 6.0, and elution was carried out at a flow rate of 1500 ml/hour; a product obtained was separated by affinity chromatography on anionic FGF SEPHAROSE® agarose gel using a 2.5 cm×6.5 cm column containing 30 ml of anionic FGF SEPHAROSE® agarose gel and equilibrated with a 0.01M Tris-HCL buffer, pH 7.4, containing 0.2M NaCl; 300 mg of the product dissolved in 60 ml of the Tris-HCl buffer was loaded on to the column and elution was carried out with the Tris-HCl buffer adjusted to 1M NaCl; and the octasaccharide was recovered from a fraction collected by precipitation with ethanol.

Claim 13

The process of claim 1 wherein a heparinic decassacharide is obtained in a fraction eluted in a volume between 25L and 28 L of eluant from a gel filtration column wherein: 60 g of precipitate, obtained by alcoholic precipitation of a nitrous acid depolymerization mixture of heparin, was taken up in 500 ml of 0.5M NaCl solution and loaded on to a 100 cm×25 cm column containing 45 liters of agarose-acrylamide, equilibrated with a 0.5M NaCl buffer, pH 6.0, and elution was carried out at a flow rate of 1500 ml/hour: and a product obtained was separated by affinity chromatography on anionic FGF SEPHAROSE® agarose gel using a 2.5 cm×6.5 cm column containing 30 ml of anionic FGF-SEPHAROSE® agarose gel and equilibrated with 0.01M Tris-HCL buffer, pH 7.4, containing 0.2M NaCl; 300 mg of the product in a solution of 60 ml of the Tris-HCl buffer was loaded to the column and elution was carried out with the Tris-HCl buffer adjusted to 1M NaCl; and the decasaccharide was recovered from a fraction collected by precipitation with ethanol.