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US-20260125419-A1 - PURIFICATION METHODS

US20260125419A1US 20260125419 A1US20260125419 A1US 20260125419A1US-20260125419-A1

Abstract

This invention relates to methods for purifying type II collagen from cartilage tissue. The present invention also relates to a type II collagen composition, and the use of the type II collagen composition as a dietary supplement or treatment for cartilage-related disorders. Methods of treating or preventing cartilage-related disorders are also provided.

Inventors

  • Mathew Hoani CUMMING

Assignees

  • THE NEW ZEALAND INSTITUTE FOR PLANT AND FOOD RESEARCH LIMITED

Dates

Publication Date
20260507
Application Date
20231004
Priority Date
20221004

Claims (20)

  1. 1 . A method for purifying type II collagen from cartilage tissue, said method comprising: i) providing a sample of cartilage tissue; ii) contacting the sample with a salt solution under conditions that allow at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from the group consisting of proteoglycan, glycosaminoglycan, and calcium salt; and iii) removing the salt solution and the at least one component of the cartilage tissue from the sample; wherein step iii) removes at least about 25% of the glycosaminoglycan from the sample; and wherein, following step iii), the sample comprises type II collagen of which at least about 50% (w/w) is undenatured type II collagen.
  2. 2 . The method of claim 1 , wherein step iii) removes at least about 35% of the glycosaminoglycan from the sample, preferably at least about 45%.
  3. 3 . The method of claim 1 wherein, following step iii), the sample comprises type II collagen of which at least about 70% (w/w) is undenatured type II collagen).
  4. 4 . The method of claim 1 , wherein the sample after step iii) has a solubility of less than about 80%.
  5. 5 . The method of claim 1 , wherein the salt solution has a pH of about 3 to about 10.
  6. 6 . The method of claim 1 , wherein the salt solution is a sulfate salt solution or chloride salt solution.
  7. 7 . The method of claim 6 , wherein the salt is sodium sulfate or magnesium sulfate.
  8. 8 . The method of claim 1 , wherein the salt solution has a sulfate or chloride concentration of about 0.4 M to about 2 M.
  9. 9 - 11 . (canceled)
  10. 12 . The method of claim 1 , wherein sample of cartilage tissue has been obtained from: a. a bony fish; or b. a cartilaginous fish.
  11. 13 . (canceled)
  12. 14 . The method of claim 12 , wherein the sample of cartilage tissue has been obtained from a cartilaginous fish, and wherein prior to step ii) the sample of cartilage tissue is subjected to a demineralisation treatment comprising contacting the sample of cartilage tissue with a solution comprising EDTA, hydrochloric acid, phosphoric acid, acetic acid, lactic acid, or any combination of any two or more of these.
  13. 15 . The method of claim 1 , wherein the sample of cartilage tissue has been obtained from a chicken, a domestic cow, or a sheep.
  14. 16 . The method of claim 12 , wherein the component dissolved by the salt solution is a glycosaminoglycan.
  15. 17 . The method of claim 14 , wherein the component dissolved by the salt solution is calcium salt, for example calcium phosphate.
  16. 18 . The method of claim 1 , wherein step iii) removes about 10% to about 100% of the proteoglycan, and/or calcium salt from the sample.
  17. 19 . The method of claim 1 , wherein the percentage (w/w) of type II collagen in the sample is about 10% to about 100% greater than the percentage (w/w) of type II collagen in the sample before step iii), and the percentage (w/w) of undenatured type II collagen in the sample is about 20% to about 100% greater than the percentage (w/w) of undenatured type II collagen in the sample before step iii).
  18. 20 . A type II collagen composition obtained by the method of claim 1 .
  19. 21 . The composition of claim 20 comprising a pharmaceutically acceptable excipient.
  20. 22 . The composition of claim 21 wherein the composition is a medicament or a dietary supplement.

Description

FIELD OF THE INVENTION The present invention relates to methods for purifying type II collagen from cartilage tissue. The present invention also relates to a type II collagen composition, and the use of the type II collagen composition as a dietary supplement or treatment for cartilage-related disorders. BACKGROUND OF THE INVENTION Type II collagen has shown promising beneficial effects on human health such as improving joint function and reducing joint inflammation (Harris et al., J. Diet Suppl. 2021, 1:1-16). It has also shown promise as a treatment for cartilage-related disorders, such as osteoarthrosis and rheumatoid arthritis (Scarpellini et al., J Orthop Traumatol. 2008; 9(2): 81-7, and Crowley et al., Int J Med Sci. 2009; 9; 6(6): 312-21). The beneficial effects of type II collagen are believed to result from the binding of tolerising epitopes present in undenatured type II collagen to Peyer's patches in the intestine. Through these interactions, the type II collagen is believed to elicit an immune response that reduces joint inflammation and discomfort (Harris et al., J. Diet Suppl. 2021, 1:1-16). Examples of type II collagen products that are commercially available and reported to contain undenatured type II collagen include Collavant n2® (previously known as b-2Cool®, Bioberica, S.A.U.; Barcelona, Spain) and UC-II® (Lonza Consumer Health Inc, USA). Both products are reported to be derived from chicken sternal cartilage. Common methods for obtaining type II collagen from cartilage tissue involve washing, drying and grinding cartilage tissue followed by treatment with guanidinium hydrochloride and/or enzymatic digestion (for example with trypsin, pepsin and/or elastase) to remove proteoglycans and glycosaminoglycans, and solubilise type II collagen. The cartilage tissue is typically obtained from livestock animals or poultry. Treatment of cartilage tissue with guanidinium hydrochloride and/or enzymatic digestion can lead to denaturation and/or hydrolysis of the collagen in the cartilage. As a result, type II collagen products prepared by such methods often contain low levels of undenatured type II collagen. Thus, there remains a need for methods suitable for obtaining type II collagen, in particular methods for obtaining undenatured type II collagen suitable for use in pharmaceuticals and dietary supplements. It is also an object of the present invention to provide an improved or alternative method or composition, and/or to at least provide the public with a useful choice. SUMMARY OF THE INVENTION The present invention provides a method for purifying type II collagen from cartilage tissue, said method comprising: i) providing a sample of cartilage tissue;ii) contacting the sample with a salt solution under conditions that allow at least one component of the cartilage tissue to dissolve in the salt solution, wherein the at least one component of the cartilage tissue is selected from the group consisting of proteoglycan, glycosaminoglycan, and calcium salt (e.g. calcium phosphate); andiii) removing the salt solution and the at least one component of the cartilage tissue from the sample;wherein step iii) removes at least about 25% of the glycosaminoglycan from the sample; and wherein, following step iii), the sample comprises type II collagen of which at least about 50% (w/w) is undenatured type II collagen. The sample provided following step iii) of the method of the invention is referred to herein as a type II collagen composition. The present inventors have found that the type II collagen composition obtained from the method of the invention comprises undenatured type II collagen at a level and purity that make it particularly suitable for use as a medicament or dietary supplement. The present inventors have also found that the method of the invention is particularly effective at purifying undenatured type II collagen from fish cartilage, and particularly fish cartilage from fish parts generally discarded during commercial processing of fish for consumption. This feature of the present invention enables the production of low-cost undenatured type II collagen products with a limited impact on the environment. For the avoidance of doubt, the term “undenatured type II collagen” as used herein refers to type II collagen that retains its native triple helix structure. This contrasts with denatured type II collagen, which has lost its native triple helix structure. An example of denatured type II collagen is type II collagen that has been hydrolyzed into peptide fragments (often referred to as collagen hydrolysates). Undenatured type II collagen typically has a molecular weight of about 300 kDa, whereas the molecular weight of collagen hydrolysates ranges from about 2 kDa to about 9 kDa. Typically, undenatured type II collagen is insoluble in aqueous solutions whereas denatured type II collagen is soluble in aqueous solutions. The term “type II collagen” as used herein is to be understood as referring to