Method for purifying GBS toxin/CM101

• US 5,939,396 A
• Filed: 02/24/1998
• Issued: 08/17/1999
• 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): Witz, Jean C.
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US09/028,972
• Time in Patent Office: 539 Days
• Field of Search: 435/72, 435/73, 435/253.4, 514/23, 514/25, 514/42, 514/43, 514/54, 514/57, 514/60, 530/412, 530/414, 530/415, 530/416, 536/6
• 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; and

(b) separating the toxin from the HIC resin.

2. The method of claim 1, wherein the purity of the toxin resulting from the HIC resin contacting step is increased approximately 100-500 fold relative to the purity of the toxin in the aqueous mixture.

3. The method of claim 1, wherein the toxin resulting from the step of separating the toxin from the HIC resin is greater than 40% pure.

4. The method of claim 1, further comprising extracting the toxin of step (b) with an aqueous phenol mixture to form an aqueous phase including the toxin.

5. The method of claim 4, wherein the toxin resulting from the step of extracting the toxin with an aqueous phenol mixture is at least approximately 60% pure.

6. The method of claim 5, wherein the toxin resulting from the step of extracting the toxin with an aqueous phenol mixture is at least approximately 90% pure.

7. The method of claim 4, wherein the toxin resulting from the step of extracting the toxin with an aqueous phenol mixture is at least approximately 60% pure.

8. The method of claim 7, wherein the toxin resulting from the step of extracting the toxin with an aqueous phenol mixture is at least approximately 90% pure.

9. The method of claim 1, wherein the HIC resin further comprises a resin having hydrophobic groups selected from the group consisting of alkyl, alkoxy, and aryl groups.

10. The method of claim 9, wherein the hydrophobic groups are alkyl groups having in the range of 2 to 12 carbons.

11. The method of claim 10, wherein the hydrophobic groups are butyl groups.

12. The method of claim 11, wherein the hydrophobic groups are normal butyl groups.

13. The method of claim 9, wherein the hydrophobic groups are phenyl groups.

14. The method of claim 1, further comprising:

(c) contacting the toxin from step (b) with an ion-exchange resin; and

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

15. The method of claim 14, wherein the toxin resulting from the step of separating the toxin from the ion-exchange resin is at least approximately 60% pure.

16. The method of claim 15, wherein the toxin resulting from the step of separating the toxin from the ion-exchange resin is at least approximately 90% pure.

17. The method of claim 1, further comprising after the step of separating the toxin from the HIC resin:

(c) extracting the toxin with an aqueous phenol mixture to form an aqueous phase including the toxin,

(d) contacting the toxin of step (c) with an ion-exchange resin; and

(e) separating the toxin from the ion-exchange resin.

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

-hemolytic Streptococcus (GBS) bacteria comprising;

(1) applying a source of GBS bacterial toxin to a hydrophic interaction chromatography (HIC) column,

(2) eluting the HIC column to obtain an HIC eluate including the toxin,

(3) extracting the HIC eluate with a combination of phenol and an aqueous solution to form an aqueous phase including the toxin,

(4) applying the aqueous phase to an ion exchange column, and

(5) eluting the ion exchange column to obtain an ion exchange eluate,

wherein the ion exchange eluate comprises a substantially pure toxin.

19. The method of claim 18 wherein the substantially pure toxin is CM101.

20. The method of claim 18 wherein the substantially pure toxin is at least approximately 60% pure.

21. The method of claim 20 wherein the substantially pure toxin is at least approximately 95% pure.