WO2012031916A1 - Procédé de réduction de la teneur en endotoxines d'un matériau contenant du collagène - Google Patents

Procédé de réduction de la teneur en endotoxines d'un matériau contenant du collagène Download PDF

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Publication number
WO2012031916A1
WO2012031916A1 PCT/EP2011/064753 EP2011064753W WO2012031916A1 WO 2012031916 A1 WO2012031916 A1 WO 2012031916A1 EP 2011064753 W EP2011064753 W EP 2011064753W WO 2012031916 A1 WO2012031916 A1 WO 2012031916A1
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WO
WIPO (PCT)
Prior art keywords
collagen
containing material
acid
treatment
treating
Prior art date
Application number
PCT/EP2011/064753
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German (de)
English (en)
Inventor
Ralf PÖRSCHKE
Original Assignee
Gelita Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gelita Ag filed Critical Gelita Ag
Publication of WO2012031916A1 publication Critical patent/WO2012031916A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather

Definitions

  • 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.
  • collagen-containing materials 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).
  • the present invention is therefore based on the object to overcome the disadvantages of the methods described above.
  • 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.%.
  • bone, cartilage, hides or tendons of mammals, poultry or fish may be used as collagenous materials (e.g., bovine cleft or pork rind).
  • 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).
  • this acid treatment alone is not suitable for lowering the endotoxin content of ossein to an acceptable level, especially for medicinal use of collagen.
  • the collagen-containing material has a relatively small particle size.
  • 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.
  • 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).
  • 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.
  • 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.
  • an organic solvent such as sodium hydroxide or potassium hydroxide.
  • a strong liquor is preferably used, such as sodium hydroxide or potassium hydroxide.
  • calcium hydroxide are used.
  • 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.
  • 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.
  • a strong caustic such as sodium hydroxide.
  • the treatment can also over a much longer period, eg. B. up to 55 days, be extended.
  • 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.
  • a strong acid is preferably used, such 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • elevated temperatures preferably at a temperature of about 100 to about 180 ° C.
  • 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.
  • other drying methods known from the prior art can also be used are, such.
  • 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.
  • the collagen-containing material is already comminuted to the desired particle size before drying.
  • z. B collagen particles are produced with a mean diameter in the swollen state of less than about 100 ⁇ .
  • 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.
  • 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.
  • 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.
  • 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.
  • a preferred application is the use of collagen powder as a hemostat.
  • 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.
  • 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.
  • the ossein was washed for a further 2.5 hours with 29 l of 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 ⁇ further ground. The particle size was determined by sieve analysis.
  • the endotoxin content of the raw material used is typically in the range of several thousand EU / g.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Materials Engineering (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de réduction de la teneur en endotoxines d'un matériau contenant du collagène, comprenant : le traitement du matériau contenant du collagène à l'aide d'une lessive alcaline ; le traitement du matériau contenant du collagène à l'aide d'un acide ; et le traitement du matériau contenant du collagène à l'aide d'un agent oxydant.
PCT/EP2011/064753 2010-09-10 2011-08-26 Procédé de réduction de la teneur en endotoxines d'un matériau contenant du collagène WO2012031916A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010037469.5 2010-09-10
DE201010037469 DE102010037469A1 (de) 2010-09-10 2010-09-10 Verfahren zur Reduzierung des Endotoxingehalts in einem kollagenhaltigen Material

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WO2012031916A1 true WO2012031916A1 (fr) 2012-03-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016085345A1 (fr) 2014-11-26 2016-06-02 Rousselot B.V. Purification de gélatine

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414531B (zh) * 2022-09-20 2024-06-04 上海亚朋生物技术有限公司 一种同种异体骨的脂肪及内毒素去除方法

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4374063A (en) * 1981-09-28 1983-02-15 General Foods Corporation Process for the preparation and purification of gelatin and pyrogen-free gelatin so prepared
DE3203957A1 (de) * 1982-02-05 1983-08-18 Chemokol Gesellschaft zur Entwicklung von Kollagenprodukten, 5190 Stolberg Verfahren zur herstellung von feinporigen kollagenschwaemmen
JPH07138809A (ja) 1993-11-10 1995-05-30 Mitsubishi Rayon Co Ltd コラーゲン繊維の製造方法
WO1999013902A1 (fr) * 1997-09-16 1999-03-25 Integra Lifesciences Corporation Produit a base de collagene favorisant la croissance de tissu dural ou meninge
DE10060643A1 (de) * 1999-12-07 2001-06-13 Geistlich Soehne Ag Verfahren zur Herstellung einer Kollagenmembran aus Schweinehaut
EP1153622A1 (fr) * 2000-05-12 2001-11-14 Johnson & Johnson Medical Ltd. Matériaux composites lyophilisés et leur procédé de préparation
JP2004300077A (ja) 2003-03-31 2004-10-28 Nitta Gelatin Inc コラーゲンタンパク質からのエンドトキシン除去方法
JP2007211170A (ja) 2006-02-10 2007-08-23 Air Water Inc 低エンドトキシン化ゼラチンの製造方法
EP1941916A1 (fr) * 2005-10-27 2008-07-09 Savaschuk, Dmitry Alekseevich Procede de fabrication de materiaux biologiques a partir de tissus osseux et materiau d'osteoplastie et d'ingenierie de tissus obtenu au moyen de ce procede
JP2009173678A (ja) 2009-04-30 2009-08-06 Nitta Gelatin Inc コラーゲンタンパク質からのエンドトキシン除去方法
DE102008036576A1 (de) 2008-07-31 2010-02-04 Gelita Ag Partikel aus Kollagenmaterial und Verfahren zur Herstellung

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4374063A (en) * 1981-09-28 1983-02-15 General Foods Corporation Process for the preparation and purification of gelatin and pyrogen-free gelatin so prepared
DE3203957A1 (de) * 1982-02-05 1983-08-18 Chemokol Gesellschaft zur Entwicklung von Kollagenprodukten, 5190 Stolberg Verfahren zur herstellung von feinporigen kollagenschwaemmen
JPH07138809A (ja) 1993-11-10 1995-05-30 Mitsubishi Rayon Co Ltd コラーゲン繊維の製造方法
WO1999013902A1 (fr) * 1997-09-16 1999-03-25 Integra Lifesciences Corporation Produit a base de collagene favorisant la croissance de tissu dural ou meninge
DE10060643A1 (de) * 1999-12-07 2001-06-13 Geistlich Soehne Ag Verfahren zur Herstellung einer Kollagenmembran aus Schweinehaut
EP1153622A1 (fr) * 2000-05-12 2001-11-14 Johnson & Johnson Medical Ltd. Matériaux composites lyophilisés et leur procédé de préparation
JP2004300077A (ja) 2003-03-31 2004-10-28 Nitta Gelatin Inc コラーゲンタンパク質からのエンドトキシン除去方法
EP1941916A1 (fr) * 2005-10-27 2008-07-09 Savaschuk, Dmitry Alekseevich Procede de fabrication de materiaux biologiques a partir de tissus osseux et materiau d'osteoplastie et d'ingenierie de tissus obtenu au moyen de ce procede
JP2007211170A (ja) 2006-02-10 2007-08-23 Air Water Inc 低エンドトキシン化ゼラチンの製造方法
DE102008036576A1 (de) 2008-07-31 2010-02-04 Gelita Ag Partikel aus Kollagenmaterial und Verfahren zur Herstellung
JP2009173678A (ja) 2009-04-30 2009-08-06 Nitta Gelatin Inc コラーゲンタンパク質からのエンドトキシン除去方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016085345A1 (fr) 2014-11-26 2016-06-02 Rousselot B.V. Purification de gélatine
NL2013880B1 (en) * 2014-11-26 2016-10-11 Rousselot B V Gelatin purification.
US10155805B2 (en) 2014-11-26 2018-12-18 Rousselot B.V. Gelatin purification

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