WO2000016897A1

WO2000016897A1 - Microfiltration filter layer for separating endotoxins and the use of said microfiltration filter layer

Info

Publication number: WO2000016897A1
Application number: PCT/EP1998/005974
Authority: WO
Inventors: Birger Anspach; Dagmar Petsch; Thomas Beeskow; Wolf-Dieter Deckwer
Original assignee: GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Priority date: 1997-09-17
Filing date: 1998-09-18
Publication date: 2000-03-30
Also published as: DE19740770A1

Abstract

The invention relates to a microfiltration filter layer for separating endotoxins from liquid media, especially water, protein solutions or parenteral fluids characterized by covalently bonded ligands for endotoxins, wherein the ligands are supported by a polymer applied on the filter layer material.

Description

Microfiltration filter layer for the separation of endotoxins and their use

The invention relates to a microfiltration filter layer for separating endotoxins from liquid media and to the use of this microfiltration filter layer.

Endotoxins are ipopolysaccharides from the outer cell filter layer of Gram-negative bacteria, which act as pyrogens. Due to the ubiquity of bacteria, endotoxins are also ubiguitary. In contrast to bacteria, however, they cannot be removed or rendered harmless by standard methods such as sterile filtration or autoclaving (1). For this reason, sterile is not synonymous with endotoxin-free. The presence of endotoxins in injection or infusion solutions (parenterals) is particularly critical, since they are administered intravenously and have a febrile effect in amounts of 1 ng per kg body weight. The symptoms range from a correspondingly high dose (e.g. due to large-volume parenterals) to severe shock and death (2, 3)). Therefore almost all pharmacopoeias write alongside the sterility, strict endotoxin limits: z. B. 0.2 EU per mg chloramphenicol for injection or only 0.003 EU per heparin unit (4). In practice, meeting these requirements presents some difficulties. In particular, the production of biological drugs cannot be endotoxin-free in all steps. The main endotoxin sources are:

- Raw materials such as plasma or tissue, which can already be contaminated with bacteria.

- In the case of recombinant products, the entry of host-specific endotoxins can be expected.

- Bacterial contamination of devices, filters or auxiliary materials during manufacture.

The heat decontamination (30 minutes at 250 ° C) that is usual for thermostable active ingredients is just as unsuitable for the preparations as treatment with acids, alkalis or strongly oxidizing agents (H2O2) (1).

When using activated carbon there are often noticeable product losses, so that their use remains limited to water treatment (5, 6).

Ultrafiltration has become very popular as a very gentle method in the field of endotoxin removal. In general, cut-offs of 10,000 or 5,000 are used to effectively separate monomeric components (MW approx. 14,000) that are present in addition to high-molecular aggregates (up to several million molecular weights). Nonetheless, problems with low molecular weight fragments that also have a pyrogenic effect occur (eg Lipid A). This applies above all to hemodialysis. Although the dialysis buffers are ultrafiltered, z. B In the United States, 400,000 hemodialysis patients have septic symptoms annually (7). - The need for small cut-offs limits the use of ultrafiltration to the decontamination of small molecules (8).

In the case of high molecular weight products such as pharmaceutical proteins, albumin preparations or heparin, great difficulties still exist: If endotoxin contamination occurs in these preparations, the only option currently available is reprocessing in order to comply with the regulations of the FDA, USP or EP become .

In order to avoid this time-consuming process, but still lead the product to its intended use, the possibility was examined to selectively decontaminate contaminated products using chromatographic sorbents with endotoxin-specific ligands. This also did not bring the desired success: Affinity sorbents with His, brain, PMB as ligands described so far proved to be unsuitable despite high to very high association constants at high endotoxin input concentrations (9). In addition, competing protein adsorption was observed in the presence of proteins, which led to reduced endotoxin depletion rates and sometimes high protein losses (especially with acidic proteins such as BSA).

literature

(1) S.K. Sharma

Endotoxin detection and elimination in biotechnology Biotechnol. Appl. Biochem. 8: 5-22 (1986)

(2) N. Haeffner-Cavaillon, J.M. Cavaillon, L. Szabό

Cellular receptors for endotoxin

Handbook of Endotoxins, Vol. 3: Biology of Endotoxins,

1-24

Elsevier Science Publishers B.V. (1985)

(3) D.C. Morrison, J.L. Ryan

Endotoxins and disease mechanisms Ann. Rev: Med. 38 (1987), 417-32

(4) USP XXII Suppl. 5 (Nov. 1991) (5) KC Hou, R. Zaniewski

Depyrogenation by endotoxin removal with positively charged depth filter cartridge

J. Parenteral Sei. Tech., Vol. 44, No. 4: 204-209 (1990)

(6) CUNO Newsletter for Pharmaceuticals (Oct. 1995), p. 3

(7) B.P. Smollich, D. Falkenhagen, J. Schneidewind, S. Mitzner, H. Klinkmann

Importance of endotoxins in high-flux dialysis nephrol. Dial. Transplant 3 (Suppl.) (1991) 83-85

(8) E. Flindt

Pyrogen removal using ultrafiltration Memoscript CONCEPT symposium "Pyrogene II" (June 1983), p. 54-60

(9) F.B. Anspach, O. Hillbeck

Removal of endotoxins by affinity sorbents J. Chromatogr. A 711 (1995), 81-92

According to the invention, this prior art can be improved by a microfiltration filter layer for separating endotoxins from liquid media, in particular water, protein solutions or parenterals, the microfiltration filter layer being characterized by covalently bound ligands for endotoxins, the ligands being carried by a polymer which is applied to the filter layer.

For filter layer technology and filter layer production, reference can be made to C. Dickenson, Filters and Filtration Handbook, Elsevier Science Publishers, Oxford 1992, and Ho & Sirkar (Editor), Membrane Handbook, Verlag van Nostrand Reinhold, New York, 1992. In particular, the filter layer materials can be depth filters.

The covalently bound ligands can be (a) an endotoxin-specific ligand, preferably histamine, histidine, polyethyleneimine, poly-L-lysine or polyxin

B and / or (b) act as a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycholate.

The filter layer material for the microfiltration filter layer according to the invention can be polysaccharides, e.g. Cellulose, in particular regenerated and microcrystalline cellulose and derivatives, in particular cellulose acetate, agarose and derivatives, crosslinked dextran and derivatives, chitosan and derivatives,

Synthetic organic polymers, e.g. Polyacrylonitrile, polysulfone, polyamide, in particular nylon, polyvinyl alcohol, polyethylene vinyl alcohol, polystyrene and polyacrylates and their derivatives,

Inorganic materials, e.g. Trade silica gel, glass and ceramic carriers and their derivatives.

The materials can be fibrous or particulate / spherical or irregularly shaped with a porous or non-porous structure, such as those used for chromatographic supports; parts of other structures can also be used, e.g. Fragments or specially cut hollow fiber membranes, broken particles or pearls. Preferably the materials should be water insoluble.

The size of the base materials is chosen so that the structure to be produced has a pore diameter in the layer as is customary for microfiltration, in particular between 0.1 and 10 μm.

The polymer which is applied to the microfiltration filter layer according to the invention can be a hydrophilic The polymers act, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose.

This hydrophilic polymer can itself be water-soluble, water-swellable or water-insoluble.

The polymer can be carried by the microfiltration filter layer according to the invention with the aid of a spacer. The covalently bound ligands can also be carried by a spacer. These spacers can be spacers derived from bisoxirane, glutardialdehyde, epihalohydrin or diisocyanate, if appropriate after oxidative activation.

For activation and immobilization chemistry, which also deals with endotoxin-specific ligands and spacers, reference is made, for example, to Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992.

According to the invention, microfiltration filter layers are therefore provided which are surface-modified in a suitable manner and remove endotoxins from water and aqueous solutions (buffers, protein solutions). The surface modification can consist in the application of a bifunctional covalently bound spacer, which is reacted with a hydrophilic polymer, whereby non-specific interactions of the filter layer, especially with proteins, are reduced. The covalently bound hydrophilic polymer can be reacted with endotoxin-specific ligands, optionally via a further spacer. For the principle of surface modification of the filter layer, see Figure 1.

The endotoxin depletion success in the presence of proteins may prove to be dependent on the net charge of the proteins. By optimizing the conditions (pH value), acidic proteins (such as BSA and mouse IgG 1) can be completely decontaminated without any significant loss of protein. In the case of basic proteins (such as lysozyme and bFGF), high depletions can also be achieved.

Polymer-coated microfiltration filter layers according to the invention with covalently bound endotoxin-specific ligands can remove endotoxins in one pass, even from highly contaminated solutions (6000 EU ml ^-1 ).

In principle, the filter layers are constructed in accordance with FIG. 1. A hydrophilic polymer is first applied via a spacer, which is then reacted further, possibly via a spacer, with endotoxin-specific ligands. The following are particularly suitable as filter layer materials:

- polysaccharides, such as

Cellulose, in particular regenerated and microcrystalline cellulose and derivatives, in particular cellulose acetate,

- agarose and derivatives, cross-linked dextran and derivatives,

- chitosan and derivatives,

- Synthetic organic polymers such as

- Polyacrylonitrile and its derivatives

- Polysulfone and its derivatives

- Polyamide, especially nylon, and its derivatives

- Polyvinyl alcohol and its derivatives

- Polyethylene vinyl alcohol and its derivatives

- polystyrene and its derivatives and

Polyacrylates and their derivatives,

- Inorganic materials like

- silica gel and its derivatives

- glass and

- ceramic supports. Reactive bifunctional compounds are suitable as spacers. The following are particularly suitable:

Bisoxiran Glutardialdehyde Epihalohydrine Diisocyanate

To activate the vicinal diol bond formed when bisoxirane and epihalohydrin are used, oxidation by periodate can optionally be used, an aldehyde group being formed. The spacer bound to the filter layer is further reacted with hydrophilic polymers. As such, the following are preferred:

Dextran

Polyvinyl alcohol (PVA)

Modified celluloses, especially hydroxyethyl cellulose (HEC)

The further reaction takes place either directly with the endotoxin-specific ligand or again via the mediation of one of the spacers mentioned above, if appropriate after its oxidative activation. The following act as endotoxin-specific ligands (see list of abbreviations): DAH, Him, His, PEI, PLL, PMB. The usually non-endotoxin-specific ligands, such as DEAE and DOC, also proved to be highly specific in the filter layer configuration with a high recovery of the proteins.

The performance of the developed filter layers is evident from the examples given. Endotoxin removal can almost always be said to be complete. It is usually below 1 EU ml ^-1 , often below the detection limit with the LAL test. No endotoxin depletion was obtained on control filter layers (nylon without modification and with applied hydrophilic polymer with or without spacer) without endotoxin-specific ligands.

The new filter layers can be used to remove endotoxins from water and parenterals. Good results are also achieved in the presence of proteins. In the case of basic proteins, however, it must be taken into account that interactions of the proteins with the endotoxin can occur, which can lead to endotoxic masking. Protein-bound endotoxin cannot be clearly identified with the LAL test. In this context it should be mentioned that it has not been finally clarified whether protein-bound endotoxin is still toxic.

The filter layers according to the invention can be used in a variety of ways.

Medical-pharmaceutical area:

- hemodialysis.

- Safe infusion and injection solutions

(Parenterals).

- Safe diagnostics (e.g. antibodies). In biotechnology:

- Manufacture of pharmaceutical products.

- Endotoxin depletion in process water and raw materials.

- Decontamination of products (processing costs are eliminated).

Methods

1. Production of the filter layers

Hydrophilic polymers, in particular dextran, polyvinyl alcohol and hydroxyethyl cellulose, are covalently bound to microfiltration filter layers. In the next step, endotoxin-specific ligands are immobilized on the applied polymers. Figure 1 illustrates the structure of a filter layer material based on nylon.

1.1. Coating a filter layer using the example of dextran

Microfiltration filter layers, e.g. based on nylon hollow fibers or cellulose fibers, are first activated with bisoxirane:

Such layers based on nylon hollow fibers are incubated for 16 hours at 80 ° C. in a reaction solution of 1 ml 25 M sodium carbonate (pH 11), 1 ml ethanol (96%) and 9 ml bisoxirane (FIG. 2a) .

Such layers based on cellulose fibers are shaken for three hours at room temperature in a mixture of 100 mg sodium borohydride in 15 ml 2 M sodium hydroxide solution, 25 ml water and 5 ml bisoxirane.

After thorough washing, both filter layers are then incubated for 15 minutes at room temperature with 5 ml of a 20 percent dextran 40,000 solution (pH 11) (see Figure 2b using the example of nylon). The filter layers are then dried at 120 ° C. for 14 hours. In order to remove non-specifically bound dextran, the filter layers are washed three times with 0.1 M sodium hydroxide solution and then three times with water.

As FIG. 3 shows, the filter layers on the basis of coated filter layer materials are characterized by significantly more unspecific interactions - expressed by the amount of hemoglobin adsorbed.

FIG. 3 also shows that a simple dextran coating cannot achieve the same effect as with PVA and HEC. A second layer can be used to achieve a further improvement, while a third layer has only minor effects. Dextran was therefore always used as a double coating.

1.2. Immobilization of endotoxin-specific ligands

The PLL, PMB and PEI ligands are either immobilized directly on the periodate-activated coating polymers or after incorporation of a periodate-oxidizable spacer (bisoxirane). The procedure is shown by way of example in FIG. 4. DEAE is coupled directly to the matrices without a spacer, the other low-molecular ligands were bound via epibromohydrin.

1.2.1. PEI immobilization via bisoxirane

For activation, the filter layers coated with hydrophilic polymers are incubated for three hours at room temperature in a mixture of 100 mg sodium borohydride, 5 ml bisoxirane and 45 ml 1 M sodium hydroxide solution. After hydrolysis of the free oxirane ring (30 minutes incubation at pH 2.5) and periodate oxidation of the vicinal diol obtained (90 minutes incubation in 0.2 M sodium periodate), the filter layers are washed with a solution of 0.5 g PEI (MW 50000) in 0.1 M phosphate buffer, which is adjusted to pH 8, at room temperature, so that the structure shown in FIG. 1 results. Finally, it is washed with a 1 molar sodium chloride solution and water.

1.2.2. Histidine immobilization

Histidine is immobilized on epibromohydrin-activated coated filter layers via DAH. Epibromohydrin activation is carried out as described for bisoxiran. Immobilized DAH is activated by reaction for 8 minutes with a mixture of 5 ml epibromohydrin and 5 ml 4 M sodium hydroxide solution at 90 ° C and immediately reacted with L-histidine at 80 ° C (0.5 g L-histidine in 20 ml water, pH 12). The finished filter layer is washed with 1 M sodium chloride and water.

Corresponding regulations are applied for the coating with other polymers and the covalent binding with the other endotoxin-specific ligands.

2. Another method for producing the filter layers

As an alternative to the method mentioned under 1., the filter layer materials can first be chemically modified and then the structure of the filter layer can be produced by washing the filter layer materials onto a suitable base body.

For this purpose, hydrophilic polymers, in particular dextran, polyvinyl alcohol and hydroxyethyl cellulose, are covalently bound to the filter layer materials. In the next step, endotoxin-specific ligands are immobilized on the applied polymers. The structure of the filter layer materials is also shown in FIG. 1.

2.1 Coating a filter layer material using the example of Sepharose 4B and dextran

Sepharose 4B was activated with bisoxirane according to a protocol by Sundberg and Porath (J. Chromatogr. 90, 1974, 87). In contrast to the regulation, the reaction time was reduced to 2 hours in order to ensure minimal loss of oxirane groups. For this purpose, 40 ml of a Sepharose 4B suspension with 20 ml of a 0.5 molar NaOH solution, 8 ml of bisoxirane and 40 mg of sodium borohydride were combined in a flask and shaken at 40 ° C. over a period of 2 hours. The activated Sepharose was then suctioned off and washed several times with water. see. The activated gel was combined with the same volume of a reaction solution which consisted of 20% dextran with an average molecular weight of 40,000 and 0.2% sodium borohydride in a 0.025 molar carbonate buffer and was adjusted to pH 11. This solution was shaken at room temperature for 60 minutes. The coated Sepharose was separated from the dextran solution by filtration and then incubated at 80 ° C. for a period of 24 hours.

2.2 Immobilization of endotoxin-specific ligands

The PLL, PMB and PEI ligands were either immobilized directly on the periodate-activated coating polymers or after incorporation of a periodate-oxidizable spacer (bisoxirane). DEAE was coupled directly to the matrices without a spacer, the other low-molecular ligands were bound via epibromohydrin.

2.2.1 Immobilization of PEI over Bisoxiran

The activation of the dextran-coated Sepharose with bisoxiran was carried out according to Sundberg and Porath, as described in 2.1. After hydrolysis of the free oxirane ring (30 minutes incubation at pH 2.5) and periodate oxidation of the vicinal diol obtained (90 minutes incubation in 0.2 M sodium periodate), the filter layer materials were mixed for two hours at room temperature with a solution which was adjusted to pH 8 and was composed of 0.5 g PEI with an average molecular weight of 50,000 and 10 ml of a 0.1 molar phosphate buffer. Finally, it was washed with 1 M sodium chloride solution and water. The mixing ratios of solids and reaction solution were chosen in each case so that a shaken suspension was formed.

2.2.2. Immobilization of histidine

Histidine was immobilized on epibromohydrin-activated coated filter layer materials via DAH. Epibromohydrin activation was carried out as described for bisoxiran. Real estate dated DAH was activated by reaction for 8 minutes with a mixture of 5 ml epibromohydrin and 5 ml 4 M sodium hydroxide solution at 90 ° C and immediately reacted with L-histidine at 80 ° C (0.5 g L-histidine in 20 ml water, pH 12). The finished filter sheet material was washed with 1 M sodium chloride and water. Here too, the mixing ratios of solids and reaction solutions were chosen so that shakable suspensions were formed.

Corresponding regulations are applied for the coating with other polymers and other filter layer materials as well as for the covalent binding with other endotoxin-specific ligands.

3. Depletion experiments

All studies on the adsorption behavior of endotoxins on the filter layers were carried out at room temperature in dead-end mode.

One filter layer disk each was fixed to the bottom of an ultrafiltration cell (filter layer area 13.4 cm ² ) and washed with 30% ethanolic 0.1 M sodium hydroxide solution, 1.5 M sodium chloride solution and pyrogen-free water in order to remove traces of endotoxin. After equilibration of the filter layer, 20 ml of contaminated solution were filtered through the filter layer at a flow rate of 2 ml / min. The filtrate was collected and examined in the LAL test.

4. Endotoxin test

A chromogenic Limulus amebocyte lysate test (LAL test) was used to quantify endotoxin in the starting solutions and filtrates. This test is based on the fact that endotoxin induces the release of the chromogen p-nitroaniline, with a linear between the released amount of p-nitroaniline and the present endotoxin concentration in the range 0 to 1.2 EU / ml Connection exists. From the photometric determination of p-nitroaniline, the endotoxin concentration of the samples can be determined using a calibration line (standard endotoxin E. coli 0111: B4).

The LAL test was introduced in Europe in 1985 by the European Pharmacopoeia Commission for testing for endotoxins and has also replaced the rabbit test in the monograph "Water for Injection Purposes" since 1989.

Examples of use

1. (Fig. 5) depletion from highly loaded buffer solutions Feed: 20 ml 20 mM phosphate buffer (pH 7) mixed with 6000 EU / ml

The filter layers marked with -d represent filter layers in which the incorporation of a spacer has been dispensed with.

2. (Fig. 6 to 7) depletion from endotoxin-enriched BSA solutions

Feed: 20 ml 20 mM phosphate buffer (pH 4.66) mixed with 1 mg / ml BSA and 6610 EU / ml

Protein recovery BSA

3. (Fig. 8) depletion from commercial BSA

Feed: 20 ml 20 mM phosphate buffer (pH 4.66) with 1 mg / ml BSA endotoxin concentration 65 EU / ml

4. (Fig. 9 to 10) depletion from commercial lysozyme

Feed: 20 ml 20 mM phosphate buffer (pH 7) with 1 mg / ml lysozyme - .16 _c -

Endotoxin concentration 134 EU / ml

Protein recovery lysozyme

5. (Fig. 11 to 12) depletion from MAX 16 H 5

Feed: 20 ml 20 mM phosphate buffer (pH 5.5) with 3 mg / ml protein endotoxin concentration 62.5 EU / ml

Protein recovery IgG

6. Depletion from already purified bFGF feed: 5 ml bFGF, which contained 9 EU / ml

The depletion process of a PEI filter layer was examined. 0.202 EU / ml was still detectable in the filtrate.

7. Depletion from Milli-Q water that has been treated with endotoxin

Feed: 1 1 water mixed with 270 EU / ml

A PEI and a DAHHis filter layer were used for depletion. PEI filtrate: <0.015 EU / ml DAHHis filtrate: 0.07 EU / ml

Claims

claims

1. Microfiltration filter layer for separating endotoxins from liquid media, in particular water, protein solutions or parenterals, characterized by covalently bound ligands for endotoxins, the ligands being carried by a polymer which is applied to the filter layer material.

2. Microfiltration filter layer according to claim 1, characterized by

(a) an endotoxin-specific ligand, preferably histamine, histidine, polyethyleneimine, poly-L-lysine or polymyxin B and / or (b) a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycholate.

3. microfiltration filter layer according to claim 1 or 2, characterized by polysaccharides, e.g. Cellulose, in particular regenerated and microcrystalline cellulose and derivatives, in particular cellulose acetate, agarose and derivatives, crosslinked dextran and derivatives, chitosan and derivatives,

Inorganic materials, e.g. Silica gel, glass and ceramic supports as well as their derivatives as filter layer material.

4. microfiltration filter layer according to one of the preceding claims, characterized by a hydrophilic polymer, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose.

5. microfiltration filter layer according to claim 4, characterized in that the hydrophilic polymer is itself water-soluble or water-insoluble.

6. Microfiltration filter layer according to one of the preceding claims, characterized in that the polymer is carried by the filter layer with the aid of a spacer.

7. Microfiltration filter layer according to claim 6, characterized by a spacer derived from bisoxirane, glutardialdehyde, epihalohydrin or diisocyanate.

8. Microfiltration filter layer according to one of the preceding claims, characterized in that the ligands are carried by the polymer with the aid of a spacer.

9. Microfiltration filter layer according to claim 8, characterized by a spacer derived from bisoxirane, glutardialdehyde, epihalohydrin or diisocyanate, optionally after oxidative activation.

10. Use of a microfiltration filter layer according to one of the preceding claims for removing endotoxins from liquid media, in particular from water, protein solutions or parenterals.