Method for purifying GBS toxin/CM101

• US 6,407,069 B1
• Filed: 03/30/1999
• Issued: 06/18/2002
• Est. Priority Date: 09/30/1996
• Status: Expired due to Term

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Hellerqvist, Carl G.
• Primary Examiner(s): Prats, Francisco
• Application Number:
  US09/269,634
• Time in Patent Office: 1,176 Days
• Field of Search: 514/23, 514/25, 514/42, 514/43, 514/54, 514/57, 514/60, 435/252.1, 435/253.4, 435/72, 435/73, 536/6, 424/244.1
• US Class Current: 514/23
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12P 1/04 by using bacteria
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12R 2001/46 Streptococcus ; Enterococcus; Lactococcus

Abstract

A method for purifying a polysaccharide from group B β-hemolytic Streptococcus (GBS) bacteria includes contacting a bacterial fermentation stock with a hydrophobic interaction chromatography (HIC) resin. Additional steps may include a phenol/saline extraction and an ion exchange chromatography. The method results in a product having very high purity. The product of the purification provides a composition which is highly useful in both research and therapeutic settings.

Claims

1. A method of purifying a polysaccharide toxin from group B β

-hemolytic Streptococcus (GBS) bacteria, which method comprises;

(a) contacting an aqueous mixture containing the toxin with a hydrophobic interaction chromatography (HIC) resin,

(b) separating the toxin from the HIC resin,

(c) containing the toxin with an ion-exchange resin, and

(d) separating the toxin from the ion-exchange resin,

wherein the toxin resulting from the step of separating the toxin from the ion-exchange resin is at least approximately 95% pure.

2. A polysaccharide toxin from group B β

-hemolytic Streptococcus (GBS) bacteria purified by a method which comprises;

(a) contacting an aqueous mixture containing the toxin with a hydrophobic interaction chromatography (HIC) resin, and

(b) separating the toxin from the HIC resin,

wherein the toxin is greater than 60% pure after steps (a) and (b).

3. The toxin of claim 2 wherein the toxin is at least approximately 90% pure.

4. The toxin of claim 3 wherein the is at least approximately 95% pure.

5. The toxin of claim 2 further comprising sugar residues of mannose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine in a molar ratio of (0.2-1 mannose):

(2.5-3.5 galactose);

(0.5-1 glucose);

1 N-acetyl glucosamine);

(0.5-1 N-acetyl galactosamine).

6. The toxin of claim 5 wherein the sugar residues of mannose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine are present in an approximate molar ratio of (1 mannose):

(3 galactose);

(1 glucose);

(1 N-acetyl glucosamine);

(1 N-acetyl galactosamine).

7. The toxin of claim 2 having a molecular weight of approximately 300,000 Daltons as measured by gel filtration chromatography in non-denaturing conditions.

8. The toxin of claim 2 characterized in that the toxin elutes in a symmetrical peak measured at 203 nm absorbance at approximately 16 minutes from time zero when analyzed by HPLC in a 10% acetonitrile buffer with a flow rate of approximately 0.3 ml/minute.

9. The toxin of claim 2 characterized in that the toxin elutes in a symmetrical peak measured at 203 nm absorbance at approximately 24 minutes from time zero when analyzed by HPLC in a 10 mM phosphate buffer, pH 8.4.

10. The toxin of claim 2 having a specific activity approximately two to three times greater than the specific activity of a GBS toxin that has not had contact with an HIC resin as measured by an assay for increased pulmonary arterial pressure in sheep.

11. A composition consisting essentially of polysaccharide toxin from group B β

-hemolytic Streptococcus (GBS) bacteria, wherein the toxin is greater than 60% pure.

12. The composition of claim 11 wherein the toxin is at least approximately 90% pure.

13. The composition of claim 12 wherein the toxin is at least approximately 95% pure.

14. The composition of claim 11 wherein the toxin further comprises sugar residues of mannose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine in a molar ratio of (0.2-1 mannose):

(2.5-3.5 galactose);

(0.5 -1 galactosamine in a glucosamine);

(0.5-1 N-acetyl galactosamine).

15. The composition of claim 14 wherein the sugar residues of mannose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine are present in an approximate molar ratio of (1 mannose):

(3 galactose);

(1 glucose);

(1 N-acetyl glucosamine);

(1 N-acetyl galactosamine).

16. The composition of claim 11 wherein the toxin has a molecular weight of approximately 300,000 Daltons as measured by gel filtration chromatography in non-denaturing conditions.

17. The composition of claim 11 wherein the toxin elutes in a symmetrical peak measured at 203 nm absorbance at approximately 16 minutes from time zero when analyzed by HPLC in a 10% acetonitrile buffer with a flow rate of approximately 0.3 ml/minute.

18. The composition of claim 11 wherein the toxin elutes in a symmetrical peak measured at 203 nm absorbance at approximately 24 minutes from time zero when analyzed by HPLC in a 10 nM phosophate buffer, pH 8.4.

19. A pharmaceutical composition comprising polysaccharide toxin from group B β

-hemolytic Streptococcus (GBS) bacteria and a pharmaceutically acceptable carrier, wherein the toxin is greater than 60% pure.

20. A method of treating a patient with a tumor comprising administering the pharmaceutical composition of claim 19 to the patient.

21. A polysaccharide toxin from group B β

-hemolytic Streptococcus (GBS) bacteria, wherein the toxin is greater than 40% pure and the toxin comprises sugar residues of mannose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine are present in a molar ratio of (0.2-1 mannose);

(2.5-3.5 galactose);

(0.5-1 glucose);

(1 N-acetyl glucosamine);

(0.5-1 N-acetyl galactosamine).