Claims
- 1. A process for obtaining oligosaccharides and oligosaccharide fractions having activity against activated factor X of blood, as determined by the Yin-Wessler titer, (anti-Xa activity) comprising the steps of:
- contacting heparin or a heparin fraction possessing anticoagulant activity and comprised of polysaccharide chains of molecular weight ranging from about 2,000 to about 50,000, with an agent capable of depolymerizing or fragmenting the heparin polysaccharide chains, under conditions adjusted to obtain a mixture which contains an oligosaccharide fraction of reduced molecular weight composed of fragments or chains constituted by no more than 8 saccharide moieties having said anti-Xa activity and including a sequence of less than 8 saccharide moieties, which sequence has binding affinity for AT III (antithrombin III) and is responsible for the specific anti-Xa activity of said oligosaccharide fraction,
- separating at least the major part of said oligosaccharide fraction by (a) contacting the mixture with AT III to retain at least the major part of said oligosaccharide fraction having said anti-Xa activity and the binding affinity for AT III, (b) eliminating from the mixture the material not retained by AT III and (c) separating said oligosaccharide fraction from said AT III.
- 2. A process according to claim 1 wherein heparin is depolymerized by a chemical process which splits the heparinic chains between an N-sulfate glucosamine unit and the following uronic acid unit.
- 3. A process according to claim 2 wherein heparin is depolymerized with nitrous acid.
- 4. A process according to claim 3 wherein the heparin is depolymerized with nitrous acid in an aqueous medium at a pH of about 2 to 4 at about ambient temperature.
- 5. A process according to claim 1 wherein heparin is depolymerized by an enzymatic process which cleaves the heparinic chain between the anomeric carbon of an N-sulfate glucosamine residue and the following uronic acid unit.
- 6. A process according to claim 5 wherein heparin is depolymerized with bacterial heparinase at a pH of about 6 to 8 and at about ambient temperature.
- 7. A process according to claim 1 wherein heparin is depolymerized with purified bacterial heparinase originating from Flavobacterium heparinum.
- 8. A process according to claim 1, 2 or 5 wherein the separation of the major part of said oligosaccharide fraction from said mixture which contains an oligosaccharide of reduced molecular weight is carried out by affinity chromatography on a column containing bound AT III.
- 9. A process according to claim 8 wherein the column is equilibrated with a buffer having an ionic strength of about 0.1 M to about 0.2 M, and the separation is carried out on said column at a pH of 6 to 8, and wherein the components of the mixture having little or no affinity for AT III are eliminated by rinsing with a buffer solution.
- 10. A process according to claim 1, 2 or 5 and further comprising recovering the oligosaccharide fraction retained by said AT III by eluting said retained oligosaccharide fraction with a buffer having an ionic strength sufficient to separate said oligosaccharide fraction from said AT III, said buffer being selected among those which do not interfere with subsequent recovery steps including alcohol precipitation of the oligosaccharides contained in said fraction.
- 11. A process according to claim 1, 2 or 5 further comprising fractionating the mixture of oligosaccharides retained by said AT III.
- 12. The process according to claim 11 wherein said fractionation is carried out by gel permeation eluting first the larger molecules, then the smaller molecules are eluted and those fractions which have a Yin-Wessler titer and a USP titer which are in a ratio of at least 30 are recovered and the recovery is continued until all fractions which exhibit anti-Xa activity have been eluted and recovered.
- 13. The process of claim 12 wherein those fractons which have a Yin-Wessler titer and a USP titer in a ratio of at least 100 are recovered.
- 14. A process according to claim 1, 2 or 5 wherein before said mixture is contacted with AT III, separating the oligosaccharides having more than 8 saccharide moieties are from the mixture.
- 15. The process of claim 14 wherein oligosaccharides having more than 8 saccharide moieties are separated from the mixture by gel filtration.
Priority Claims (5)
Number |
Date |
Country |
Kind |
7934673 |
Oct 1979 |
GBX |
|
8000443 |
Jan 1980 |
GBX |
|
8021749 |
Jul 1980 |
GBX |
|
8021750 |
Jul 1980 |
GBX |
|
8029697 |
Sep 1980 |
GBX |
|
Parent Case Info
This application is a continuation-in-part of patent application Ser. No. 091,164, filed Nov. 5, 1979, which application has been abandoned in favor of pending application Ser. No. 204,505 filed Nov. 6, 1980.
US Referenced Citations (6)
Foreign Referenced Citations (1)
Number |
Date |
Country |
2002406 |
Feb 1979 |
GBX |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
91164 |
Nov 1979 |
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