Process for producing low endotoxin chitosan

Abstract

The present invention relates to a process for producing a low endotoxin alkali chitosan, 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 with an alkali solution to form a mixture and leaving the mixture for at least about 12 hours. 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, having an endotoxin concentration of less than 50 EU/g, the process comprising the steps of: (a) contacting chitosan with an alkali solution having a concentration of from 0.01M to 0.2M to form a mixture; and (b) leaving the mixture for at least 12 hours.

2. The process as claimed in claim 1, wherein the process further comprises a step (c) of drying the mixture, wherein the drying step is optionally performed in an oven.

3. The process as claimed 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 and ammonium hydroxide; wherein the metal is optionally selected from sodium, potassium, calcium, or magnesium; and wherein the alkali component is optionally selected from sodium hydroxide, potassium hydroxide or sodium carbonate.

4. The process as claimed claim 1, wherein the alkali solution is sprayed onto the chitosan or the chitosan is mixed with the alkali solution.

5. The process as claimed in claim 1, wherein the mixture is left for at least 48 hours in step (b) and optionally for about two to four weeks.

6. The process as claimed in claim 1, wherein the mixture further comprises a preservative 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 comprising the step of contacting an alkali chitosan prepared by the process of claim 1 with an acid; wherein the chitosan derivative is selected from the group consisting of carboxymethyl chitosan, hydroxyethyl chitosan, acyl chitosan, alkyl chitosan, sulphonyl chitosan, phosphorylated chitosan, alkylidene chitosan, metal chelates, chitosan chloride, chitosan lactate, chitosan acetate, chitosan malate and chitosan gluconate.

8. The process as claimed in claim 7, wherein the process further comprises a step (c) of drying the mixture, the drying step is optionally performed in an oven; wherein the step of contacting the alkali chitosan with an acid is performed before 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, carboxylic acids, fatty acids, amino acids, lewis acids, monoprotic acids, diprotic acids, polyprotic acids, nucleic acids and mineral 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 ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, 80:20 mixture of ethanol:water or 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, wherein the drying step is optionally performed in an oven or by filtration of the product through an air dryer.

14. A low endotoxin neutral chitosan, a chitosan salt or a chitosan derivative obtainable by the process of claim 1.

15. The process as claimed in claim 1, wherein the quantity of alkali solution to chitosan is in the range of from 1 part chitosan to 10 parts alkali solution to 10 parts chitosan to 1 part alkali solution.

16. The process as claimed in claim 5, wherein the mixture is left in a clean container.

17. The process as claimed in claim 5, wherein the mixture is left under an inert atmosphere.

18. The process as claimed in claim 5, wherein the mixture is left in a clean container and under an inert atmosphere.

19. The process as claimed in claim 9, wherein the organic acid is selected from group consisting of acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid and lactic acid.

20. The process as claimed in claim 9, wherein the fatty acid is selected from the group consisting of myristoleic acid, palm itoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-Linolenic acid, arachidonic acid, eicosapentaenoic acid, eurcic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palm itic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid and cerotic acid.

21. The process as claimed in claim 9, wherein the amino acid is selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline and taurine.

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

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

24. The process as claimed in claim 12, wherein the ratio of chitosan to acid liquor is from about 5:1 to about 1:5 and the alkali chitosan is mixed with the acid for about 5 minutes.

25. A low endotoxin neutral chitosan produced by a process comprising the step of contacting the alkali chitosan of claim 1 with an acid; wherein the acid is selected from the group consisting of organic acids, carboxylic acids, fatty acids, lewis acids, monoprotic acids, diprotic acids, polyprotic acids, nucleic acids and mineral acids.

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;

(3) FIG. 2 is a graph showing the effect of the treatment with acid on the viscosity of the chitosan polymer.

(4) FIG. 3 is a graph showing the penetration of saline into two different haemostatic materials according to the present invention;

(5) FIG. 4 is a graph showing the time period for blood clotting of two different haemostatic materials according to the present invention;

(6) FIG. 5 is a graph showing the percentage haemostasis in epigastric sever tests of two different haemostatic materials according to the present invention.

DETAILED DESCRIPTION

Endotoxin Testing

(7) 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.

(8) 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

(9) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 1M NaOH for 30 mins. The resulting wet alkali chitosan crumb was left at room temperature for 48 hours. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 10.6 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 2

(10) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 0.5M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for 72 hours. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 38.3 EU/g

Example 3

(11) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 0.2M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 27.9 EU/g

Example 4

(12) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for 14 days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 12.7 EU/g

Example 5

(13) 50 g of Chitosan (Primex Iceland) was mixed with 100 g 0.2M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for two days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 35.7 EU/g

Example 6

(14) 50 g of Chitosan (Primex Iceland) was mixed with 100 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for two days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 49.3 EU/g

Example 7

(15) 50 g of Chitosan powder (Cognis, Germany) was mixed with 50 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 45.3 EU/g Dry treated alkali Chitosan: <10 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 8

(16) 50 g of Chitosan powder (Cognis, Germany) was mixed with 50 g 0.05M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 45.3 EU/g Dry treated alkali Chitosan: <10 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 9

(17) 50 g of Chitosan powder (Cognis, Germany) was mixed with 50 g 0.025M NaOH for 10 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in an oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 45.3 EU/g Dry treated alkali Chitosan: <13.5 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

(18) The process can also be scaled up and used to make larger batch sizes. Examples 10 and 11 were made in a Class 100,000 cleanroom (US FED STD 209E cleanroom standards) commonly used in medical device manufacture.

Example 10

(19) 3.5 kg of Chitosan powder (Primex Iceland) was mixed with 3.5 kg 0.1M NaOH for 30 mins. The resulting wet alkali chitosan crumb was left at room temperature for 14 days. It was then dried by filtering through an air drier at 40° C. Initial Endotoxin of raw chitosan: 288 EU/g Dry treated alkali Chitosan: 10.2 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 11

(20) 3.5 kg of Chitosan (Primex Iceland) powder was mixed with 3.5 kg 1M NaOH for 30 mins. The resulting wet alkali chitosan crumb was left at room temperature for 24 hours. It was then dried by filtering through an air drier at 40° C. Initial Endotoxin of raw chitosan: 288 EU/g Dry treated alkali Chitosan: 15.3 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

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

Example 12

(22) 10 g of Chitosan fibre (1.8 dtex×28 mm) was mixed with 10 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan fibre was left at room temperature for two days. It was then dried in a laboratory oven on a tray at 40° C. Initial Endotoxin of raw chitosan fibre: 88 EU/g Dry treated alkali Chitosan fibre: <10 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 13

(23) 5 g of Chitosan nonwoven fabric (60 gsm) was mixed with 5 g 0.1M NaOH for 10 mins. The resulting wet alkali chitosan fabric was left at room temperature for two days. It was then dried in a laboratory oven on a tray at 40° C. Initial Endotoxin of raw chitosan fabric: 401 EU/g Dry treated alkali Chitosan fabric: <78 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

(24) The following examples 14 and 15 utilise a different base to sodium hydroxide.

Example 14

(25) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 0.2M KOH (potassium hydroxide) for 30 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in a laboratory oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 14.4 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 15

(26) 50 g of Chitosan (Primex Iceland) was mixed with 50 g 0.5M Sodium Carbonate for 30 mins. The resulting wet alkali chitosan crumb was left at room temperature for seven days. It was then dried in a laboratory oven on a tray at 40° C. Initial Endotoxin of raw chitosan: 383 EU/g Dry treated alkali Chitosan: 25.8 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g.

(27) Examples 1-15 all 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.

Example 16

(28) 20 g of wet alkali chitosan crumb from Example 4 was weighed into a beaker. This contained 10 g of chitosan and 10 g of 0.1M NaOH. To neutralise the NaOH, 1 g of 1M HCl was required. This was mixed in a separate beaker with 9 g of ethanol. The acid liquid was then mixed into the wet alkali chitosan crumb and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40° C. It contained 0.29% sodium chloride.

Example 17

(29) 20 g of wet alkali chitosan crumb from Example 4 was weighed into a beaker. This contained 10 g of chitosan and 10 g of 0.1M NaOH. To neutralise the NaOH, 1 g of 1M acetic acid was required. This was mixed in a separate beaker with 9 g of ethanol. The acid liquid was then mixed into the wet alkali chitosan crumb and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40° C. It contained 0.5% sodium acetate.

(30) The low endotoxin alkali chitosan formed in Examples 1-15 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.

Example 18

(31) 3.1 kg of dried low endotoxin alkali chitosan from Example 10 was weighed into a stainless steel mixer in a Class 100,000 cleanroom. 3.6 kg of lactic acid was pre-mixed with 0.9 kg of endotoxin free water. This was sprayed on to the chitosan while the mixer was running. The resulting material was dried at 40° C. by filtering through an air dryer.

(32) The material was found to be completely water soluble. Initial Endotoxin of raw chitosan: 288 EU/g Dry treated alkali chitosan: 10.2 EU/g (Example 10) Dry water soluble chitosan: 13.8 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

Example 19

(33) 3.1 kg of dried low endotoxin alkali chitosan from Example 11 was weighed into a stainless steel mixer in a Class 100,000 cleanroom. 3.9 kg of lactic acid was pre-mixed with 0.9 kg of endotoxin free water. This was sprayed on to the chitosan while the mixer was running. The resulting material was dried at 40° C. by filtering through an air dryer.

(34) The material was found to be completely water soluble. Initial Endotoxin of raw chitosan: 288 EU/g Dry treated alkali chitosan: 15.3 EU/g (Example 4) Dry water soluble chitosan: 14.6 EU/g Control (0.1 g endotoxin free water used in extraction process instead of chitosan): <10 EU/g

(35) 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.

Example 20

(36) 20 g of wet alkali chitosan crumb from Example 1 was weighed into a beaker. This contained 10 g of chitosan and 10 g of 1M NaOH. In a separate beaker a mixture of 5 g of sodium chloroacetate and 5 g of water and 10 g of ethanol was prepared. The mixed liquid was then stirred into wet alkali chitosan crumb. Its temperature was then raised to 60° C. for 4 hours. The resulting mixture was washed three times with 10 g of ethanol to remove any residual sodium chloroacetate before being dried in a laboratory oven at 40° C.

(37) Effect of Acid Salt on Viscosity

(38) 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.

(39) 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.

(40) 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.

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

(42) 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

(43) 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.

(44) Effect of Low Concentration Alkali Solution on Viscosity

(45) The low endotoxin alkali chitosan produced in Example 10 was 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 followed the following method steps: a) weigh out 5 g of low endotoxin alkali chitosan granules produced in Example 10; 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.

(46) The results of the above described viscosity test are shown in Table 2 below and FIG. 2. For each Batch, an average was taken over three readings at each time interval.

(47) TABLE-US-00002 TABLE 2 Viscosity (cPs) Batch 1 (washed) Batch 2 (washed) Batch 3 (washed) Weeks Average STD Average STD Average STD 0 419.9 0.0 399.9 34.6 419.9 34.6 1 379.9 34.6 379.9 34.6 419.9 0.0 2 399.9 34.6 419.9 0.0 359.9 0.0 4 239.9 0.0 239.9 0.0 239.9 0.0 8 180.0 0.0 180.0 0.0 219.9 34.6 12 166.7 5.8 110.0 0.0 170.0 0.0 16 110.0 0.0 96.7 5.8 123.3 5.8 24 120.0 0.0 100.0 34.6 190.0 34.6

(48) It can be deduced from the viscosity measurements shown in FIG. 2 that the molecular weight of the low endotoxin alkali chitosan polymer prepared using a 0.1M sodium hydroxide solution is stable for weeks.

(49) Effect of Lowering the Concentration Alkali Solution

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

(51) Referring to FIG. 3, there is shown the results of the penetrability testing.

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

(53) As can be seen in FIG. 3, the haemostatic powder prepared by using 1.0M sodium hydroxide is less stable over time compared to the haemostatic powder prepared using 0.1M sodium hydroxide and the increasing penetration into the granules by the saline solution indicates a greater opportunity for blood to pass through the granules and not form the gel plug.

(54) Referring to FIG. 4, there is shown the results of the blood clotting testing.

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

(56) As can be seen in FIG. 4, the haemostatic powder prepared by using 1.0M sodium hydroxide takes longer to clot the blood than the haemostatic powder prepared by using 0.1M sodium hydroxide. The time period is approximately three times longer, which relates to lower haemostatic properties (data has shown that time periods greater than 180 seconds does not result in 100% haemostasis in a low pressure, medium volume in-vivo bleed model.

(57) Referring to FIG. 5, there is shown the results of the haemostasis testing.

(58) The general test method was as follows: a 3-5 cm sever was made in the epigastric artery of a swine model (non-heparinised). The haemostatic material in granular form was applied and a 1 minute compression applied. If re-bleeding occurred a further 1 minute compression was undertaken.

(59) As can be seen in FIG. 5, the haemostatic granules prepared by using 1.0M sodium hydroxide obtained haemostasis in 60% of the tests, compared to 100% with the haemostatic granules prepared by using 0.1M sodium hydroxide.

(60) The test results indicate that lower the concentration of alkali solution used in the preparation of the haemostatic material of the present invention, the better the material performs in penetrability, blood clotting and haemostasis.

(61) 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.