Methods of making a chitosan product having an ultra-low endotoxin concentration and the ultra-low endotoxin chitosan product derived therefrom and method of accurately determining inflammatory and anti-inflammatory cellular response to such materials

• US 20080248508A1
• Filed: 11/13/2007
• Published: 10/09/2008
• Est. Priority Date: 08/17/2006
• Status: Abandoned Application

• Current Assignee: Synedgen, Inc.
• Original Assignee: Synedgen, Inc.
• Inventors: Wiesmann, William P., Baker, Shenda
• Application Number:
  US11/985,057
• Publication Number:
  US 20080248508A1
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: C08B 37/0003 General processes for their isolation or fractionation, e.g. purification or extraction from biomass
• CPC Class Codes: C08B 37/003 Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• CPC Class Codes: G01N 2333/525 Tumor necrosis factor [TNF]
• CPC Class Codes: G01N 2333/5412 IL-6
• CPC Class Codes: G01N 2333/545 IL-1
• CPC Class Codes: G01N 2400/28 Chitin, chitosan
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Chitosan is a natural product having wide range of applications in the food and cosmetic industries. Food and commodity grade chitosan are laden with pyrogens, such as endotoxins and proteins which limit its applicability in the biological and medical arenas, as minute amounts of endotoxins may induce adaptive and innate responses when contacted with mammalian tissue, pharmaceuticals and biomedical devices. Due to chitosan'"'"'s ability to avidly bind endotoxin and other pyrogens, they are difficult to remove. The present invention is directed to methods for purifying chitosan from shells, food and commodity grade chitosan into ultra-pure, low endotoxin chitosan having biological and medical applicability. Additionally, the present invention is also directed to a method of determining the pyrogenicity of the ultra-pure low endotoxin chitosan.

Claims

1. A process for making ultrapure, low-endotoxin chitosan comprising:

(a) utilizing sterile, pyrogen-free labware, reagents and materials in strict endotoxin-free, sterile environment;

(b) swelling chitosan having endotoxins, for up to 24 hours;

(c) dissolving ratios equivalent to 1 kg/25 L to 1.5 kg/25 L of said chitosan in 0.01M to 4.0 M of a hydroxide base, forming a chitosan base solution and continuously stirring said chitosan base solution

(d) heating said base solution between 60°

C. and 100°

C. for 45 minutes to 4 hours and continuously stirring said heated base solution;

(e) rinsing said chitosan solution with up to 10×

volume of ultra-pure pyrogen-free water and removing endotoxin fragments and residual endotoxin;

(f) neutralizing said solution to a pH between 6.8 and 7.5;

(g) forming a ultra-pure low endotoxin chitosan slurry;

(h) transferring said slurry to a endotoxin-free closed system;

(i) removing excess water from said slurry and forming said ultra-pure, low-endotoxin chitosan having an endotoxin concentration between 100 EU/g and 20 EU/g;

(j) retaining molecular weight integrity of said chitosan at 30 kDa or greater and between 75% and 90% of said chitosan having endotoxins; and

(k) storing said chitosan in endotoxin-free storage containers in an endotoxin-free environment with sterile, endotoxin-free connects.

2. A process as recited in claim 1 wherein said chitosan having endotoxins is selected from a group consisting of food grade chitosan and commodity grade chitosan.

3. A process as recited in claim 2 wherein said base is NaOH.

4. A process as recited in claim 3 and further comprising processing said chitosan solution for 1 hour at 90°

C. in 1.0 M NaOH.

5. A process as recited in claim 4 and further comprising removing said water by a process selected from the group consisting of filtration, aspiration or decanting.

6. A process as recited in claim 5, wherein said low endotoxin chitosan having an endotoxin concentration of 20 EU/g or less.

7. A process as recited in claim 6 and further comprising utilizing pyrogen-free water for swelling said chitosan.

8. A process as recited in claim 6 and further comprising utilizing an ultrapure 10% methanol solution.

9. An ultrapure low endotoxin chitosan product formed from a high endotoxin chitosan having a molecular weight of 30 kDa or greater, said low endotoxin chitosan having an endotoxin concentration between 100 EU/g and 20 EU/g and a molecular weight between 75% and 90% of said high endotoxin chitosan molecular weight.

10. A chitosan product as recited in claim 9, wherein said endotoxin concentration is 20 EU/g or less.

11. A chitosan product as recited in claim 10, wherein said chitosan is a paste, housed in an endotoxin-free, sterile storage environment.

12. A chitosan product as recited in claim 10, wherein said chitosan is available in an aqueous solution, housed in an endotoxin-free, sterile storage environment.

13. A process for making ultra-pure, low endotoxin chitosan comprising:

(a) utilizing sterile, pyrogen-free labware, materials and reagents;

(b) utilizing an endotoxin-free, sterile processing environment;

(c) processing starting materials so as to form chitin;

(d) deacetylizing and deproteinating said chitin with a strong base and forming said ultra-pure, low endotoxin chitosan having an endotoxin concentration between 100 EU/g and 20 EU/g; and

(e) packaging, handling and storing said low endotoxin chitosan in an endotoxin-free, sterile environment

14. A process as recited in claim 13 wherein said endotoxin concentration of said low endotoxin chitosan is 20 EU/g or less and said molecular weight of said chitosan is 30 kDa or greater.

15. A process for determining pyrogenicity of chitosan

(a) utilizing sterile and pyrogen-free materials, reagents and labware in a sterile, endotoxin-free environment;

(b) growing and priming human dendritic cells in standard media utilizing pyrogen-free serum;

(c) delivering between 2 μ

g/mL to 20 mg/mL of chitosan to equivalent wells of said dendritic cells;

(d) utilizing food grade chitosan as control and validating said cell positive response to said chitosan having endotoxins;

(e) bracketing said response with known pyrogens;

(f) periodically testing media from said wells for inflammatory and anti-inflammatory cytokine response;

(g) quantifying mass of said media utilizing ELISA plates of cytokine antibodies;

(h) retaining said wells so as to develop a pyrogen response; and

(i) detecting said pyrogen response in said media.

16. A process as recited in claim 15, wherein said chitosan concentration is 2 mg/mL or less.

17. A process as recited in claim 16, wherein said periodic testing occurs at 1 to 24 hours.

18. A process as recited in claim 17, wherein said period testing occurs at 1 hour for IL-6, IL-1β

and TNF-α

.

19. A process as recited in claim 18, further comprising utilizing cell lines for two to three months and priming said cells with a primer selected from the group consisting of calcitriol and interferon-γ

.

20. A process as recited in claim 19 wherein said cells are primary cells.

21. A process as recited in claim 20, wherein said periodic testing includes testing for IL-6, IL-1β

, TNF-α

, IL-10, IL-4.

22. A process as recited 21 in claim wherein said chitosan comprises chitosan derivatives.