DE4113602A1 - Highly selective endotoxin adsorber - consists of bead-like water swollen cellulose prod. contg. polyethylene-imine as the functional ligand - Google Patents

Abstract

An endotoxin adsorber is based on a porous bead-like polysaccharide matrix consisting of a water-swollen cellulose prod. contg. as functional ligand, 0.1-25 wt.% polyethylimine (based) on the adsorber dry mass). Pref. the matrix is obtd. by binding 0.1-25 wt.% polyethyleneimine (based on the adsorber dry mass) on a porous bead-like cellulose prod. by treating the water swollen cellulose prod. with an aq. polyethylene imine soln. at 0-50 deg. C (for 1 min. to 6 hrs., removing non-bonded polyethylene imine and treating the prod. with water and/or 0.5-5 wt.% saline. The cellulose prod. is unmodified regenerate cellulose and the polyethylene imine is adsorptively bound. Alternatively, the cellulose prod. is an anionic cellulose deriv. contg. carboxyl, sulphate or phosphate gps. and the polyethylene imine is ionically bound. USE/ADVANTAGE - The adsorber can be used in the selective removal of endotoxins from aq. solns. blood plasma or full blood, and can be used in medicine and in pharmacy. The adsorber has better properties than previously used adsorbers. It has high bonding capacity and selectivity has good mechanical properties and is simple and economical to prepare. It can be autoclaved and, on account of its good mechanical stability, can be handled without abrasion. When used in columns or plasmaperfusion capsules, it shows stable run behaviour and can be regenerated and used many times

Description

The invention relates to an adsorber for removing Endotoxins from aqueous solutions, blood plasma and whole blood as well a process for its manufacture. Areas of application are in front especially medicine and pharmacy.

Endotoxins are predominantly complex compounds Liposaccharide structure that is gram-negative in the outer cell wall Bacteria are anchored and when they decay into the surrounding Medium can be released. Because of their pyrogenic and they are antigenic effect u. a. significantly responsible for the Fever reaction, for the emergence of the disseminated intravascular Coagulation, the Sanarelli-Shwartzman phenomenon, the septic Shocks and the coma hepaticum. Especially with liver failures the human body is very sensitive to endotoxins, where 0.2 ng / kf KM are febrile and higher concentrations lead to death in 80%.

A way of treating such sick patients is to remove the endotoxins from the bloodstream. This can be done either by plasmapheresis or plasma exchange or through the use of suitable endotoxin-binding adsorbents respectively. Disadvantages of plasmapheresis or plasma exchange are e.g. the relatively low effectiveness, which is stronger here taking into account the risk of infection. Sit down as alternatives endotoxin adsorbers. Most common are adsorbers based on activated carbon / A. S. Pegues et al. a .: Int. J. Artif. Organs 2 (1979) 153, which, however, are not selective. Adsorbers would have much better selectivity a polymeric carrier material and antibodies attached to it consist. So far, however, it has only been possible to use antibodies monoclonal absolute serospecificity was achieved, however not yet, monoclonal antibodies against endotoxins in general  to produce - K. T. CHONG: J. Infect. Dis. 156/5 (1978) 713; H.E. FARELLY: J. Med. Microbiol. 23 (1987) 1; A. FOMSGAARD: The Lancet Feb. 28 (1987) 514; M.C. LUC: hepat. 8 (1987) 117; K. SAUKKONEN. J. Infect. Dis. 158: 209 (1988); E. J. ZIEGLER. New Engl. J. Med. 307 (1982) 1225. The high cost is disadvantageous here for the production of the antibodies and the instability of the products with the usual sterilization processes as well as the technical to name complex production.

The disadvantage of the high cost also applies to adsorbers from a pearl-shaped polysaccharide matrix (agarose, Dextran, cellulose exist, attached to the polymyxin-B as a ligant was coupled - M. UMEDA u. a .: Biomat., Art. Cells, Art. Org. 18 (1990) 4, 491-497.

Polysaccharide adsorbers, which are more economical to produce as an endotoxin-binding functional group diethylaminoethyl groups - US-PS 38 97 309 - or other nitrogen-containing groups - US-PS 43 81 239 - use. Disadvantages of these adsorbers result resulting from a sometimes unfavorable blood tolerance. Farther Endotoxin adsorbers are described using amines - EP-PS 03 08 239, EP-PS 03 33 474 - and of agarose coupled protamine - I. M. HELANDER: Feb. 1987, 51-55. - Here too meet the disadvantages of unfavorable selectivity and too low Effectiveness of adsorbers too. The manufacturing process for the The adsorbers listed are based on the activated carbon in the all essential that a suitable polymeric carrier - usually a pearl-shaped polysaccharide matrix - first of all Introduction of reactive groups activated and then with covalently coupled to the ligand capable of endotoxin binding becomes. This procedure has disadvantages in that the previous one beaten ligands either very costly and technically have to be elaborately manufactured or not the desired ones Have binding and compatibility properties.

The carrier matrix used also often results Disadvantages, as these T. not the desired mechanical Stability or sufficient porosity.

The aim of the invention was therefore to provide an adsorber for binding Endotoxins from blood plasma, whole blood or aqueous solutions too develop, which has improved properties, as well as a economically more favorable method for producing such To show adsorbers.

The invention has for its object an endotoxin adsorber to develop, which is characterized by a high binding capacity and Selectivity as well as good mechanical properties and simple Manufacturability distinguishes.

According to the invention the object is achieved in that Endotoxin adsorber porous carrier based on cellulose products used, the polyethyleneimine bound as a ligand contain. The polyethyleneimine can be adsorptive, ionic or covalently on the inner and outer surface of the porous Pearl cellulose material bound. The adsorber is still characterized in that the amount of polyethyleneimine in End product 1 to 25 mass%, based on the dry mass of the Adsorbers, corresponding to a nitrogen content of 0.3 to 8 mass%. As a porous cellulose material Unmodified regenerated cellulose beads according to the invention Particle sizes from 0.1 to 5 mm anionically modified, in particular containing carboxyl, sulfate or phosphate groups Pearl cellulose products or reactive functional groups, such as e.g. B. containing aldehyde groups or epoxy groups Pearl cellulose products used. The application of the endotoxin adsorbers takes place in the water-moist, swollen state, whereby the Water content of the adsorber 50 to 95% by mass, preferably 80 to 90% by mass.

The adsorbers according to the invention are produced in simply by the fact that the porous, pearl-shaped Cellulose products with a particle size of 0.1 to 5 mm an aqueous 0.1 to 20 mass% solution of polyethyleneimine Treated at 0 to 50 ° C for 1 min to 6 h and then excess or unbound polyethylene imine Aspirate and wash with water or physiological NaCl solution Will get removed.

Experiments have shown that the adsorptively or ionically bound Polyethyleneimine buffered by water, NaCl solution or other aqueous solutions no longer from the adsorber according to the invention can be removed. In addition, these products have a good one Selectivity to endotoxins and only minor disadvantages  regarding blood tolerance. Plasma proteins, lipids and Platelets are only adsorbed to a reasonable extent. The Adsorber can be beneficial without affecting its Autoclave properties and due to its good mechanical Treat stability without abrasion. When used in columns or plasma perfusion capsules, it shows stable running behavior. It can be regenerated and used several times.

The adsorber according to the invention was treated with aqueous endotoxin Solutions and plasma both in the batch process and under Circulatory conditions tested. The following examples serve for a more detailed description of the invention.

Example 1

100 g macroporous, water-moist pearl cellulose with one Cellulose content of 10% by mass and an average Particle diameters of 0.2 mm are mixed with 200 ml of a 10% by mass Polyethyleneimine solution mixed and at 60 min Room temperature stirred. Then the treated one Aspirated pearl cellulose on a frit and 5 times with 200 ml each 1% NaCl solution and washed 5 times with 200 ml water. The adsorber obtained had a nitrogen content of 0.8% by mass on, based on the dry adsorber mass. To determine the Endotoxin binding, the adsorber was first activated as follows: 2 g of the adsorber were mixed with 10 ml NaOH in one concentration of 500 mmol / l incubated for 20 min and then with water rinsed until a neutral pH value has been reached, then were the same 2 g of the adsorber with 10 ml of HCl in one Concentration of 500 mmol / l incubated for 20 min and then with Wash water until a neutral pH is reached it was then rinsed 5 times with a pH-neutral buffer at. The reaction temperature was constant at 21 ° C.

In the experiments on endotoxin adsorption, both in batch and a ratio of adsorber to endotoxin in the circulation solution of 1:10 selected. The reaction temperature was a constant 37 ° C.

The adsorber described above already had incubation after 30 min with the endotoxin-containing solution from the initial concentration of  100 000 pg / ml solution 98 350 pg / ml bound adsorptively. To the quantitative endotoxin detection, the commercially available Limulus Amoebocyte lysate assay used.

Example 2

100 g water-moist pearl cellulose with a cellulose content of 15% by mass and an average particle diameter of 2.5 mm treated and washed with polyethyleneimine as in Example 1. The Nitrogen content in the resulting sample was 2.6 mass%, based on the dry matter.

The adsorbers were examined for endotoxin binding as in Example 1 described. After a reaction time of 90 min Initial concentration of 100,000 pg / ml solution 99,000 pg / ml bound.

Example 3

100 g aspirated, water-moist pearl cellulose with a Carboxyl content of 1.2 meq / g dry substance, a water content of 92% by mass and an average particle size of 0.2 mm mixed with 200 ml of a 5% by weight polyethyleneimine solution and Stirred for 360 min at room temperature. Then the product washed and as described in Example 1 Endotoxin binding examined. The dry matter Nitrogen content was 5.8 mass%. During the tests for Endotoxin adsorption was found after only 15 min Incubation time at an initial concentration of 100,000 pg / ml an adsorption of 99 240 pg / ml.

Example 4

100 ml aspirated, water-moist pearl cellulose with a phosphate content of 95% by mass and an average particle size of 0.25 mm are treated as in Example 1 with polyethyleneimine and on Endotoxin binding examined. The dry matter Nitrogen content was 8.7% by mass. At a  Initial concentration of the endotoxin-containing solution of 100,000 pg / ml 99 440 pg / ml were adsorbed after a reaction time of 90 min.

Example 5

100 ml of aspirated, water-moist pearl cellulose with a Sulphate content of 1.5 meq / g dry matter, a water content of 92 mass% and an average particle size of 0.2 mm are like described in Example 1 treated with polyethyleneimine and examined. The nitrogen content based on the dry matter was 4.25 mass%. When testing for endotoxin adsorption at an initial concentration of 100,000 pg / ml solution 15 min reaction time 99 850 pg / ml adsorptively bound.

Example 6

100 ml aspirated, water-moist cross-linked, pearl-shaped carboxy methyl cellulose with a degree of substitution of 0.7 and one average particle size of 3 mm is as in Example 1 Polyethyleneimine treated and examined for endotoxin binding. The nitrogen content based on the dry matter was 8.9 mass%. At an initial concentration of 100,000 pg / ml solution 96,000 pg / ml were adsorbed on edotoxins in 90 min.

Example 7

100 ml of aspirated, water-moist pearl cellulose with a Dialdehyde content of 20 µmol / ml sedimented material and one average particle size of 0.8 mm with a 20% by mass Polyethyleneimine solution stirred at 50 ° C for 10 min. Then will the product is suctioned off, with 5% NaCl solution and with water Instructions washed and examined for endotoxin binding. The on the dry matter nitrogen content was 8.8%.

At an initial concentration of 100,000 pg / ml endotoxin content 99 100 pg / ml were adsorbed in 15 min.

Claims (12)

1. Endotoxin adsorber based on a porous, pearl-shaped polysaccharide matrix and a functional ligand, characterized in that the porous, pearl-shaped polysaccharide matrix is a water-swollen cellulose, based on which as a ligand 0.1 to 25 mass% polyethylimine is bound to the dry adsorber mass. 2. Endotoxin adsorber based on a porous, pearl-shaped Polysaccharide matrix and a functional ligand, obtainable by binding 0.1 to 25% by mass of polyethyleneimine, based on the dry adsorber mass a porous, pearl-shaped cellulose product by treatment of the water-swollen cellulose product with an aqueous Polyethyleneimine solution at 0 to 50 ° C within 1 min to 6 h, subsequent removal of unbound polyethyleneimine and treating the product with water and / or 0.5 to 5 mass% saline. 3. Endotoxin adsorber according to claim 1 and 2, characterized records that the cellulose product unmodified regenerate is cellulose and the polyethyleneimine is adsorptively bound. 4. Endotoxin adsorber according to claim 1 and 2, characterized records that the cellulose product is an anionic cellulose is derivative, which carboxyl, sulfate or phosphate groups contains, and the polyethyleneimine is ionically bound. 5. Endotoxin adsorber according to claim 1 and 2, characterized records that the polyethyleneimine has reactive, functional groups, especially dialdehyde and epoxies groups is coupled to the cellulose matrix. 6. Endotoxin adsorber according to claims 1 to 5, characterized characterizes that the size of the beads 0.1 to 5 mm, is preferably 0.5 to 3 mm, and the adsorber 50  contains up to 95%, preferably 80 to 90%, water. 7. Process for the separation of endotoxins from endotoxin aqueous liquids, blood plasma or whole blood by contacting the liquid with an adsorber Cellulose base, characterized in that the endotoxin containing liquid with a 50 to 95% water the pearl-shaped cellulose product, which contains 0.1 to 25 mass% Polyethyleneimine, based on the dry adsorber mass, be load and has an average particle diameter of 0.1 to 5 mm, in contact at 20 to 37 ° C for 1 min to 3 h brought and the endotoxin-free liquid from the Adsorber is separated. 8. Process for the preparation of an endotoxin adsorber Implementation of a porous, pearl-shaped polysaccharide matrix with a functional ligand, characterized in that porous, pearl-shaped cellulose products with an aqueous 0.1 to 20 mass% solution of polyethyleneimine 1 min to 6 hours at 0 to 50 ° C and then untreated bound polyethyleneimine by suction and washing with Water and / or 0.5 to 5% by mass saline solution is removed. 9. The method according to claim 8, characterized in that as pearl-shaped cellulose product macroporous regenerated cellulose pearls with a particle size of 0.1 to 5 mm and a Water content of 50 to 95% by mass can be used. 10. The method according to claim 8, characterized in that as pearl cellulose product anionic pearl cellulose derivatives can be used with carboxyl, sulfate or phosphate groups. 11. The method according to claim 8, characterized in that as pearl-shaped cellulose product cellulose pearls are used, which have reactive aldehyde or epoxy groups. 12. Use of the adsorber according to one of claims 1 to 11 for selective removal of endotoxins from aqueous solutions, Blood plasma or whole blood.