WO2012031916A1

WO2012031916A1 - Method for reducing the endotoxin content of a collagen-containing material

Info

Publication number: WO2012031916A1
Application number: PCT/EP2011/064753
Authority: WO
Inventors: Ralf PÖRSCHKE
Original assignee: Gelita Ag
Priority date: 2010-09-10
Filing date: 2011-08-26
Publication date: 2012-03-15
Also published as: DE102010037469A1

Abstract

The present invention relates to a method for reducing the endotoxin content of a collagen-containing material, comprising the following steps: - treating the collagen-containing material with aqueous alkali; - treating the collagen-containing material with acid; and - treating the collagen-containing material with an oxidizing agent.

Description

Method for reducing endotoxin content in one

collagenous material

The present invention relates to a method for reducing endotoxin content in a collagen-containing material.

Collagen-containing materials obtained as animal raw materials from bone or connective tissue are more or less contaminated with endotoxins. Endotoxins are primarily lipopolysaccharides from the outer cell membrane of Gram-negative bacteria or blue-green algae, which are continuously released by these microorganisms, but are liberated in their lysis. Endotoxins are among the pyrogens, i. In contact with the mucous membranes, but especially when they pass into the blood, they can cause fever in humans and may even lead to anaphylactic shock or death.

Because of these properties, it is desirable to reduce as much as possible the endotoxin content of collagen-containing materials, both when intended for use in the food or cosmetics industries, and more particularly in pharmaceutical or medical applications where the collagen-containing material is directly associated with the bloodstream comes in contact (for example, collagen powder can be used as a hemostat due to its swelling capacity).

Various methods for reducing the endotoxin content of collagen have already been described in the prior art. For example, it is proposed in JP 2004-300077 A and JP 2009-173678 A to treat fibrillated or dispersed collagen with a basic solution of alcohol and / or acetone. According to JP 07-138809 A, endotoxins can be removed from the solubilized collagen solution by being absorbed by a polyolefin. However, such methods have the disadvantage of being limited to the treatment of collagen which has been converted to a soluble or dispersible form. In the context of the present invention, however, particular interest is given to native collagen or collagen-containing material, which is present as an insoluble, particulate solid, since it is precisely for some medical applications in which a low endotoxin content is required that insoluble collagen is used. In addition, the processes described above can hardly be implemented on an industrial scale, since the required amounts of organic solvents or absorber materials would cause disproportionate costs. The use of combustible organic solvents such as alcohol would also make high demands on the safety technology.

Methods are also known for processing collagen into gelatin with a low endotoxin content. Thus, it is proposed in JP 2007-211170 A to inactivate the endotoxins in fish collagen by heating at a pH of 5 to 9. Under these conditions, however, the at least partial denaturation of the collagen into soluble gelatin also takes place, so that this and similar processes for the preparation of insoluble collagen with a low endotoxin content are likewise unsuitable. The same applies to processes such as ultrafiltration or reverse osmosis.

The present invention is therefore based on the object to overcome the disadvantages of the methods described above.

This object is achieved according to the invention in the method mentioned above in that the method comprises the following steps:

Treating the collagenous material with caustic;

Treating the collagenous material with acid; and

Treating the collagen-containing material with an oxidizing agent. It has surprisingly been found that by using these method steps, the endotoxin content of collagen-containing material can be significantly reduced, in particular to values below about 10 EU / g (according to the LAL test described below). The method according to the invention can be carried out relatively simply and inexpensively and is thus particularly suitable also for the industrial scale, ie for the processing of batches of collagen-containing material in the order of magnitude of approximately 30 to 50 tons.

The collagenous material according to the present invention comprises natural collagen, i. insoluble collagen, which is not or only slightly denatured or hydrolyzed. The material may contain different levels of collagen, the material usually being predominantly proportioned, i. to more than 50% by weight, consists of collagen. However, the collagen content is preferably much higher, z. B. at over 90 wt.%.

In particular, bone, cartilage, hides or tendons of mammals, poultry or fish may be used as collagenous materials (e.g., bovine cleft or pork rind). However, the collagen-containing material preferably comprises ossein, which is prepared by degreasing and demineralizing bones and contains a particularly high proportion of collagen (typically more than 95% by weight of the dry matter).

The usual production method for ossein comprises an acid treatment of the bones called maceration over a period of about 2 to 8 days at room temperature (<25 ° C). However, this acid treatment alone is not suitable for lowering the endotoxin content of ossein to an acceptable level, especially for medicinal use of collagen.

For carrying out the method according to the invention, it is favorable if the collagen-containing material has a relatively small particle size. at For example, using ossein may have particle sizes in the range of about 4 to about 12 mm.

The method according to the invention is not restricted to a particular sequence of the above-mentioned method steps, ie treating the collagen-containing material with alkali can be carried out both before and after treatment with acid. However, it is particularly favorable if the treatment with acid takes place after treatment with alkali, since in this order a particularly effective reduction of the endotoxin content can be achieved.

In a preferred embodiment of the method, the treatment with an oxidizing agent takes place during and / or after treatment with acid. The simultaneous use of acid and oxidant enables an efficient process and is particularly suitable for oxidants that are stable in an acidic environment (eg hydrogen peroxide).

According to a particularly preferred embodiment of the invention, the steps are carried out under such conditions that the collagen-containing material is not or not completely denatured by the method or converted into a soluble form. Thus, the inventive method is also suitable for the preparation of insoluble collagen with a low endotoxin content. For this it is advantageous if all steps of the process are carried out at a temperature of about 25 ° C or less, i. at room temperature, since denaturation of the collagen usually only occurs at higher temperatures, in mammalian collagen above 30 ° C.

The method according to the invention preferably does not involve treatment of the collagen-containing material with an organic solvent. As a result, the safety requirements and the cost of the process can be reduced, which is particularly important in the processing of collagen-containing materials on an industrial scale weight. For the treatment of the collagen-containing material with alkali, a strong liquor is preferably used, such as sodium hydroxide or potassium hydroxide. Alternatively, but also weaker alkalis, z. As calcium hydroxide, are used.

Conveniently, the liquor is used as an aqueous solution having a concentration of about 0.5 to about 1.5 mol / l, in particular from about 0.7 to about 1.2 mol / l. The treatment of the collagen-containing material with the liquor can be carried out batchwise (eg in a container with a stirrer) or, in particular for larger batches, in a circulation process (eg in a container with a sieve bottom, the liquor being repeated placed on the collagen-containing material and stripped below the sieve). Batch and circulation procedures can also be combined.

Preferably, the collagen-containing material is treated with caustic for about 12 to about 72 hours, more preferably about 24 to about 48 hours, especially when using a strong caustic such as sodium hydroxide. When using a weaker alkali such as calcium hydroxide, however, the treatment can also over a much longer period, eg. B. up to 55 days, be extended.

It is advantageous if the collagen-containing material is washed with water between the treatment with alkali and the treatment with acid. This will, if z. B. the lye treatment is carried out as a first step, the liquor before acid addition largely removed or at least diluted so that an undesirable increase in temperature by an exothermic neutralization reaction can be avoided.

For the treatment of the collagen-containing material with acid, a strong acid is preferably used, such as. As sulfuric acid, phosphoric acid, hydrochloric acid or mixtures thereof, with sulfuric acid is particularly preferred. The acid is preferably used as an aqueous solution with a concentra- tion of about 0.1 to about 0.5 mol / l, in particular from about 0.2 to about 0.4 mol / l used.

As already described in connection with the lye treatment, the treatment of the collagen-containing material with the acid can be carried out in a batch process or in a circulation process, wherein in particular for large batches, the technical realization of a circulation process is more favorable.

Furthermore, it is favorable if the concentration of the acid is continuously increased in order to avoid a local acidification of the material and a concomitant hydrolysis. A gradual metered addition of the acid can be carried out both in a batch and in a circulation process.

The duration of the acid treatment of the collagen-containing material is preferably about 6 to about 14 hours, especially about 8 to about 12 hours.

The treatment of the collagen-containing material with the oxidizing agent can, as already mentioned above, conveniently take place simultaneously with the acid treatment, for. By adding the oxidant after completion of the acid addition. The oxidizing agent preferably comprises hydrogen peroxide, which is relatively stable under acidic conditions.

The oxidizing agent is preferably used in an aqueous solution having a concentration of about 800 to about 3,200 ppm, in particular from about 1,200 to about 2,000 ppm.

After the final treatment with acid (or after the final lye treatment, if the acid treatment takes place as a first step), the collagen-containing material is preferably washed with water to the acid (or liquor) prior to further processing and / or storage of the collagen-containing material remove. A particularly preferred embodiment of the invention relates to a method for reducing the endotoxin content in ossein, the method comprising the following steps in the order given: a) treating the ossein with an aqueous sodium hydroxide solution; b) washing the ossein with water;

c) treating the ossein with an aqueous sulfuric acid solution with the addition of hydrogen peroxide; and

d) washing the ossein with water.

The amounts of liquor, acid and wash water, each of which is used in proportion to the amount of the collagen-containing material, can be adjusted depending on the nature of the process and the apparatus used. Typically, e.g. each about 0.5 to about 0.8 liters of liquid per kilogram of the collagen-containing material used. In a circulation process, it is favorable if the flow rate is in the range of about 0.3 to about 0.4 l / h »kg.

After carrying out the method steps described above, the collagen-containing material is in the form of an aqueous slurry or after removal of the liquid medium in a moist state. Since the material is not storable in this state, it is preferred if the method according to the invention further comprises a subsequent drying of the collagen-containing material.

The drying of the collagen-containing material can be carried out at elevated temperatures, preferably at a temperature of about 100 to about 180 ° C. In contrast to a treatment of collagen in an aqueous acidic or alkaline medium, high temperatures during drying, which are only for a short time, do not lead to hydrolysis of the collagen. A preferred drying method in the present invention is contact adhesive layer cylinder drying. However, other drying methods known from the prior art can also be used are, such. B. convection drying, spray drying, freeze-drying or vacuum belt drying.

In a further preferred embodiment of the invention, the collagen-containing material is comminuted before and / or after drying. A comminution of the material before drying may be required in order to carry out the drying process efficiently if the collagen-containing material does not already have a sufficiently small particle size before carrying out the process according to the invention. So it is z. B. advantageous to crush the collagen-containing material to a particle size of less than about 0.5 mm before a contact adhesive layer cylinder drying, so that the material can be applied without the aid of water as a subsidy to the cylinder and the film formation and drying on the cylinder trouble-free expires. After drying the collagen-containing material, it may optionally be further ground to obtain a collagen powder having the desired particle size. Such powders can be used in the food, cosmetics and pharmaceutical industries.

For certain applications, it has also proved to be particularly advantageous if the collagen-containing material is already comminuted to the desired particle size before drying. By a Hochdruckhomogenisa- tion of the material in suspension, which may optionally precede a wet grinding, z. B. collagen particles are produced with a mean diameter in the swollen state of less than about 100 μιη. A corresponding method is described in the published patent application DE 10 2008 036 576 AI. Such particles may i.a. used as a fat substitute in foods to simulate the creamy mouthfeel of 01-in-water emulsions.

The present invention further provides a collagenous material having an endotoxin content of less than about 10 EU / g (endotoxin units / g) prepared by the method described above. Determination of endotoxin content refers to the most sensitive currently available detection method for endotoxins, the LAL (Limulus Amoebocyte Lysate Test) described in the sixth edition of the European Pharmacopoeia (Ph. Eur. This is based on the activation of the coagulation cascade by endotoxins in an amebocyte lysate derived from horseshoe crabs.

The limit value of 10 EU / g of the collagen-containing material corresponds approximately to the lowest value which can still be reproducibly determined for an insoluble material with the aid of the LAL test. The collagenous material according to the present invention may thus be termed essentially endotoxin free. In other words, the endotoxin content can be reduced to such an extent by the method according to the invention that detection of endotoxins with the available methods is practically no longer possible.

Finally, the present invention also relates to the use of the above-described collagen-containing material for the production of pharmaceutical or cosmetic preparations or for the production of foods. In the pharmaceutical field, a preferred application is the use of collagen powder as a hemostat.

Finally, the collagen-containing material according to the invention can also be used as raw material for the production of essentially endotoxin-free gelatin, which likewise has a broad pharmaceutical spectrum of application. The use of collagen, which is endotoxin-free or has an extremely low endotoxin content, is one of several parameters that must be considered in addition to a corresponding process control in the production of endotoxin-free gelatin.

These and other advantages of the invention will be explained in more detail with reference to the following embodiment. example

The collagen-containing starting material used was 45 kg of porcine bone ossein having a particle size in the range of 0.4 to 12 mm. This was slurried in a container with sieve bottom, outlet valve under the sieve bottom and agitator in 29 l of water. The stirring speed was about 30 rpm during the entire process.

To the batch was added 1.7 L of a 50% by weight aqueous sodium hydroxide solution over a period of 2.5 hours, so that the final concentration of the caustic soda was about 0.7 mol / L. After stirring for 36 hours, the sodium hydroxide solution was displaced by 29 l of water over a period of 2.5 hours.

Following this lye treatment, the ossein was washed for a further 2.5 hours with 29 l of water.

Within 2.5 hours, the addition of 1.4 l of a 30% strength by volume aqueous sulfuric acid solution, corresponding to a final concentration of sulfuric acid of about 0.26 mol / l, was carried out. After addition of the sulfuric acid, the addition of 116 ml of a 35 wt.% Hydrogen peroxide solution was carried out within 15 min (final concentration about 1330 ppm). It was stirred for an additional 7.5 hours.

After treatment of the collagen-containing material with acid and an oxidizing agent, it was washed for 4 hours at a water flow rate of 12 l / h.

After draining the wash water, the ossein was subjected to a two-stage comminution process, initially wet milling to reduce the average particle size to a value of about 0.26 mm. The resulting suspension was dried by means of a contact adhesive layer cylinder drying at about 160 ° C. surface temperature and the dried film to a collagen powder with a mean particle size of about 110 μιη further ground. The particle size was determined by sieve analysis.

As an alternative to dry grinding, comminution by means of high-pressure homogenization, as described in DE 10 2008 036 576 A1, can also be carried out.

The determination of the endotoxin content of the collagen powder prepared in this way from ossein according to the LAL test (Ph. Eur. 6) was carried out at a dilution of 1:50 (in the form of a suspension) and resulted in an endotoxin content of less than 10 EU / g , This value corresponds approximately to the detection limit of the test at this dilution, whereby the performance of the test at a lower dilution due to the high swelling capacity of the collagen powder is no longer practicable.

In comparison, the endotoxin content of the raw material used is typically in the range of several thousand EU / g.

This example demonstrates that the method of the present invention allows for a drastic reduction in endotoxin content in collagen-containing materials and thus is suitable for producing a substantially endotoxin-free collagen-containing material. It can be used inexpensively and with relatively little effort even on an industrial scale, since it does not require the use of organic solvents and / or special absorbers.

Claims

Patent claims

A method of reducing endotoxin content in a collagen-containing material, comprising the following steps:

- Treating the collagen-containing material with alkali;

Treating the collagen-containing material with acid; and

Treating the collagen-containing material with an oxidizing agent.

2. The method of claim 1, wherein the collagen-containing material comprises ossein.

3. The method of claim 1 or 2, wherein the treatment with acid takes place after treatment with alkali.

4. The method according to any one of the preceding claims, wherein the treatment is carried out with an oxidizing agent during and / or after treatment with acid.

A method according to any one of the preceding claims, wherein the steps are carried out under conditions such that the collagenous material is not or not completely denatured or converted to a soluble form by the method.

6. The method of claim 5, wherein all steps of the method are performed at a temperature of about 25 ° C or less.

A method according to any one of the preceding claims, wherein the method does not involve treatment of the collagen-containing material with an organic solvent.

8. The method according to any one of the preceding claims, wherein the liquor as an aqueous solution with a concentration of about 0.5 to about 1.5 mol / l, in particular from about 0.7 to about 1.2 mol / l , is used.

9. The method according to any one of the preceding claims, wherein the collagen-containing material about 12 to about 72 hours, in particular about 24 to about 48 hours, treated with alkali.

10. A method according to any one of the preceding claims, wherein the collagen-containing material is washed with water between treating with caustic and treating with acid.

11. The method according to any one of the preceding claims, wherein the acid as an aqueous solution having a concentration of about 0.1 to about 0.5 mol / l, in particular from about 0.2 to about 0.4 mol / l , is used.

12. The method according to any one of the preceding claims, wherein the concentration of the acid is continuously increased.

13. The method according to any one of the preceding claims, wherein the collagen-containing material for about 6 to about 14 hours, in particular about 8 to about 12 hours, is treated with acid.

14. The method according to any one of the preceding claims, wherein the oxidizing agent comprises hydrogen peroxide.

15. The method according to any one of the preceding claims, wherein the oxidation agent is used in an aqueous solution with a concentration of about 800 to about 3,200 ppm, in particular from about 1,200 to about 2,000 ppm.

16. The method according to any one of the preceding claims, further comprising a subsequent drying of the collagen-containing material.

17. The method of claim 16, wherein the drying is carried out at a temperature of about 100 to about 180 ° C.

18. The method of claim 16 or 17, wherein the collagen-containing material is comminuted before and / or after drying.

19. Collagen-containing material with an endotoxin content of less than about

10 EU / g prepared according to the method of any one of the preceding claims.

20. Use of a collagen-containing material according to claim 19 for the production of pharmaceutical or cosmetic preparations, for the production of food or as raw material for the production of substantially endotoxin-free gelatin.