Process for producing low endotoxin chitosan

Abstract

The present invention relates to a process for producing a low endotoxin alkali chitosan, chitin, chitosan derivative or chitin derivative, and also to a process for producing low endotoxin neutral chitosan, chitosan salt and chitosan derivatives, and to the products of such processes. The process comprises contacting chitosan, chitin, chitosan derivative or chitin derivative with an alkali solution having a concentration of less than 0.25M to form a mixture; leaving the mixture for a period of less than 12 hours and optionally drying the mixture. The low endotoxin alkali chitosan may be used in the manufacture of other useful chitosan based products.

Claims

1. A process for producing a low endotoxin alkali chitosan, chitin or a derivative thereof having an endotoxin concentration of less than 50 EU/g, the process comprising the steps of: (a) contacting chitosan, chitin, a chitosan derivative or a chitin derivative with an alkali solution having a concentration of from 0.01M to 0.2M to form a mixture, wherein the chitosan derivative or chitin derivative is selected from the group consisting of carboxymethyl chitosan, hydroxyl butyl chitin, N-acyl chitosan, O-acyl chitosan, N-alkyl chitosan, O-alkyl chitosan, N-alkylidene chitosan, O-sulfonyl chitosan, sulfated chitosan, phosphorylated chitosan, nitrated chitosan, alkalichitin, alkalichitosan, and metal chelates with chitosan; and (b) leaving the mixture for a period of less than 12 hours; and then (c) drying the mixture.

2. The process as claimed in claim 1, wherein the concentration of the alkali solution is about 0.1M.

3. The process as claimed in claim 1, wherein the alkali solution comprises an alkali or alkaline earth component selected from the group consisting of metal hydroxides, metal carbonates, metal bisulphites, metal persilicates, conjugate bases, ammonium hydroxide, and combinations thereof, wherein the metal is optionally selected from the group consisting of sodium, potassium, calcium, and magnesium, and wherein the alkali component is selected from the group consisting of sodium hydroxide, potassium hydroxide and sodium carbonate.

4. The process as claimed in claim 1, wherein the alkali solution is sprayed onto the chitosan, chitin, chitosan derivative or chitin derivative.

5. The process as claimed in claim 1, wherein the mixture is left for a period of less than ten hours.

6. The process as claimed in claim 1, wherein a preservative is added to the mixture of step (a), wherein the preservative is optionally selected from silver ions, zinc ions, chlorohexadine, or combinations thereof.

7. The process for producing a low endotoxin neutral chitosan, a chitosan salt or a chitosan derivative having an endotoxin concentration of less than 50 EU/g comprising the step of contacting the product of the process of claim 1 with an acid.

8. The process as claimed in claim 7, wherein the step of contacting the alkali chitosan with an acid is performed before a drying step (c); and wherein the acid is optionally sprayed onto the alkali chitosan or the alkali chitosan is mixed with the acid.

9. The process as claimed in claim 7, wherein the acid is selected from the group consisting of: organic acids optionally selected from the group consisting of acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid, lactic acid and combinations thereof; carboxylic acids; fatty acids optionally selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and combinations thereof; amino acids optionally selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline, taurine, and combinations thereof; lewis acids; monoprotic acids, diprotic acids, and mineral acids; polyprotic acids; and nucleic acids.

10. The process as claimed in claim 7, wherein the acid has a concentration of about 1M.

11. The process as claimed in claim 7, wherein the acid is present as an acid liquor comprising the acid and a non-solvent optionally selected from the group consisting of ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, 80:20 mixture of ethanol:water, and mixtures thereof.

12. The process as claimed in claim 11, wherein the ratio of chitosan to acid liquor is from about 5:1 to about 1:5.

13. The process as claimed in claim 7, further comprising the step of drying the reaction product.

14. The process as claimed in claim 12, wherein the alkali chitosan is mixed with the acid for about 5 minutes.

15. The process as claimed in claim 1, wherein the chitosan, chitin, chitosan derivative or chitin derivative is mixed with the alkali solution.

16. The process as claimed in claim 9, wherein the mineral acid is selected from the group consisting of hydrochloric acid, sulphuric acid, nitric acid and combinations thereof.

17. The process as claimed in claim 1, wherein neither step of the process involves the use of endotoxin-free equipment.

18. The process as claimed in claim 1, wherein the quantity of chitosan, chitin, chitosan derivative or chitin derivative to alkali solution is from 1:10 to 10:1.

19. The process as claimed in claim 1, wherein neither step of the process involves a washing step, a rinsing step, use of a surfactant or phase transfer agents, and/or the use of endotoxin free water.

20. The process as recited in claim 1, whereby the viscosity of the low endotoxin alkali chitosan, chitin, or derivative thereof is reduced by less than 25%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the present invention will now be described further in the following non-limiting examples with reference to the accompanying drawing in which:

(2) FIG. 1 is a graph displaying the effect of different concentrations of acid salt by-product on the viscosity of a chitosan product in the different media.

DETAILED DESCRIPTION

(3) Endotoxin Testing

(4) 1. Make up USP (United States Pharmacopia) extraction solution as detailed in USP for chitosan endotoxin testing (4.6 ml of 1M HCl and 45.4 ml endotoxin free water); 2. Extract by adding 0.1 g of the test chitosan product to 9.9 ml of USP extraction solution and leave for 48 hours at 37° C.; 3. After 48 hours, dilute 100 μl of the extract in 0.9 ml of endotoxin free water; and 4. Mix 100 μl of the above in 100 μl of Endotoxin Specific (ES) buffer provided by Charles River.

(5) The resulting extract is tested using an Endosafe®-PTS™ handheld spectrophotometer that utilises FDA-licensed disposable cartridges. The extract process uses a 2000× dilution and a minimum test limit detection of 10 EU/g.

EXAMPLES

Example 1

(6) 50 g of chitosan was mixed with 50 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan crumb was dried immediately in a fluid bed drier at 40° C.

(7) Initial Endotoxin of raw chitosan: 64.8 EU/g

(8) Dry treated alkali Chitosan: 16.3 EU/g

Example 2

(9) 50 g of chitosan was mixed with 50 g 0.05M NaOH for 10 mins. The resulting wet alkali chitosan crumb was dried immediately in a fluid bed drier at 40° C.

(10) Initial Endotoxin of raw chitosan: 64.8 EU/g

(11) Dry treated alkali Chitosan: 20.0 EU/g

Example 3

(12) 50 g of chitosan was mixed with 50 g 0.01M NaOH for 10 mins. The resulting wet alkali chitosan crumb was dried immediately in a fluid bed drier at 40° C.

(13) Initial Endotoxin of raw chitosan: 64.8 EU/g

(14) Dry treated alkali Chitosan: <30 EU/g

Example 4

(15) 50 g of chitosan was mixed with 50 g 0.1M NaOH for 8 hrs. The resulting wet alkali chitosan crumb was dried immediately in a fluid bed drier at 40° C.

(16) Initial Endotoxin of raw chitosan: 64.8 EU/g

(17) Dry treated alkali Chitosan: 23.4 EU/g

(18) The process can be scaled up and used to make larger batch sizes.

(19) The process can be used on chitosan in different physical forms such as a chitosan fibre or chitosan fabric.

(20) The process can also utilise a different base to sodium hydroxide, such as potassium hydroxide for example.

(21) Examples 1-4 relate to the production of low endotoxin alkali chitosan. This low endotoxin alkali chitosan can subsequently be used as a raw material to make other chitosan based products. For example alkali chitosan can be neutralised to pH 7 to form a neutral chitosan by adding a low level of an appropriate acid that would react with the base to make a biocompatible salt. For example, if sodium hydroxide is used in the basic solution, it can be neutralised by the addition of hydrochloric acid. The product would contain a low amount of residual sodium chloride.

(22) The low endotoxin alkali chitosan formed in Examples 1-4 can also be used to make a low endotoxin water soluble chitosan salt or other chitosan derivatives. Beneficially, this can be achieved without the need for a sterile environment, without the use of large quantities of expensive endotoxin free water and without the need for rinsing or washing. For example, a low endotoxin alkali chitosan can be reacted with a greater level of an appropriate acid. A small portion of the acid will react with the base to make a biocompatible salt.

(23) In another example, low endotoxin alkali chitosan can also be used as a raw material for the manufacture of low endotoxin chitosan derivatives, such as carboxy methyl chitosan.

(24) Effect of Acid Salt on Viscosity

(25) Reacting the low endotoxin alkali chitosan with acid, to produce either a neutral pH chitosan or a chitosan salt, produces an acid salt by-product. The presence of this by-product can affect the performance of the chitosan product. For example, the level of by-product can affect the viscosity of a chitosan product in saline.

(26) Referring to FIG. 1, there is shown the results of adding sodium lactate to saline in different concentrations, and the resulting effect of this on the viscosity of a 2 g sample of the current market-available chitosan product, CELOX®, in a 20 g solution of the different media after three minutes.

(27) The base media was saline from body fluids, to which different levels of sodium lactate were added. The sodium lactate represented the by-product of the reaction between sodium hydroxide and lactic acid.

(28) The results are set out in Table 1 and FIG. 1.

(29) TABLE-US-00001 TABLE 1 Viscosity Concentration Test 1 Test 2 Test 3 Average Saline 55000 62000 59000 58667 1.5% 54000 52000 61000 55667 2.5% 50000 43000 39000 44000 5.0% 35000 51000 31000 39000 7.5% 35000 34000 38000 35667 10.0% 37000 32000 36000 35000

(30) It is clear from FIG. 1 that as the added salt level increases, the viscosity of the CELOX® in the media drops. It is therefore beneficial for there to be only a low level of residual salt by-product resulting in the chitosan products of the present invention.

(31) Effect of Low Concentration Alkali Solution on Viscosity

(32) The low endotoxin alkali chitosan of the present invention can be tested to demonstrate the effect of the treatment with acid on the viscosity of the chitosan polymer, considered to be a measure of molecular weight. The test comprises the following method steps: a) weigh out 5 g of low endotoxin alkali chitosan granules; b) weigh out 4.95 g of acetic acid in 600 ml beaker; c) add 490.05 g deionised water to the beaker to make up 495 g of a 1% solution of acetic acid; d) place the beaker on stirrer plate and turn on stir (increase as the viscosity of the solution increases; e) add the chitosan granules to the acetic acid solution; f) check the solution regularly until all the granules have dissolved and increase stirring level as the viscosity of the solution increases, if required; g) leave the solution for a total of 24 hours, measured from the time the chitosan granules were introduced into the acetic acid solution; h) attach a spindle 64 to a Brookfield Viscometer i) set the spindle to 10 rpm; j) insert the spindle into the solution to the mark on the spindle and turn the viscometer on and allow to stabilise; k) record the viscosity (cPs) at selected time intervals.
Effect of Lowering the Concentration Alkali Solution

(33) The effect of using a lower concentration of alkali solution in the process of the present invention can be tested in three experiments, focussing on (1) the percentage penetrability of saline into a test sample; (2) the time period to blood clotting; and (3) the percentage haemostasis in epigastric sever in-vivo models.

(34) The general test method for (1) the percentage penetrability into saline is as follows: 5 mls of distilled water is added to a test tube. A drop of red food dye is added to the water. 3 g of sample haemostatic powder is gently tipped on top of the water such that a layer is formed. After 1 minute, the distance traveled by the water into the haemostatic powder is measured and recorded as percentage penetration.

(35) The general test method for (2) the time period to blood clotting is as follows: 0.75 g of sample haemostatic powder is added to a test tube, to which 5 ml of heparinised rabbit blood is added. The test tube is then inverted and the time taken to fully clot the blood into a gel mass recorded.

(36) The general test method for (3) the percentage haemostasis in epigastric sever in-vivo models is as follows: a 3-5 cm sever is made in the epigastric artery of a swine model (non-heparinised). The haemostatic material in granular form is applied and a 1 minute compression applied. If re-bleeding occurs, a further 1 minute compression is undertaken.

(37) It is of course to be understood that the present invention is not intended to be restricted to the foregoing examples which are described by way of example only.