DE19740770A1 - Microfiltration layer for removing endotoxins from liquids - Google Patents

Abstract

A microfiltration filter layer, for removal of endotoxins from liquid media, includes covalently bonded ligands for endotoxins, carried by a polymer applied to the filter layer material.

Description

The invention relates to a microfiltration filter layer for Separation of endotoxins from liquid media and the Ver application of this microfiltration filter layer.

Endotoxins are lipopolysaccharides from the outer cell filter layer of Gram-negative bacteria, which act as pyrogens. Due to the ubiquity of bacteria, endotoxins are also ubiquitous. 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 pHarmacopöen prescribe strict endotoxin levels in addition to sterility: 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 medicinal products cannot be endotoxin-free in all steps. The main endotoxin sources are:

• - Raw materials such as plasma or tissue that already contain bacteria can be mined.
• - In the case of recombinant products, the entry of host-spe specific endotoxins.
• - Bacterial contamination of devices, filters or auxiliary substances fen during manufacture.

The usual heat decontamination for thermostable active ingredients (30 minutes at 250 ° C) is just as unsuitable for the preparations as treatment with acids, alkalis or strongly oxidizing agents (H ₂ O ₂ ) (1).

When using activated carbon there are often noticeable product losses most to be recorded, so that their application to water treatment horse riding remains limited (5, 6).

Ultrafiltration has a large population as a very gentle method achieved in the field of endotoxin removal. Here is generally worked with cut-offs of 10,000 or 5,000 to also effectively separate monomer components (MW approx. 14000), which in addition to high molecular weight aggregates (up to several million Molecular weight). Nonetheless, there are always pros cream with low molecular weight split pieces, which are also py act roe (e.g. Lipid A). This mainly affects hemo dialysis: Although the dialysis buffers are ultrafiltered, ent wrap z. For example, 400,000 hemodialysis patients annually in the United States septic symptoms (7). - The need for small cut-offs  also limits the use of ultrafiltration to De contamination of low molecular weight substances (8).

In the case of high molecular weight products such as pH protein, Albu Min supplements or heparin still exist in large quantities Difficulties: Fndotoxin contamination occurs in these preparations only the possibility of repro to meet the requirements of FDA, USP or EP become.

In order to avoid this complex process, the product nevertheless, to lead to its purpose, the possibility was ge checks contaminated products via chromatographic sorbents to decontaminate selectively with endotoxin-specific ligands. This also did not bring the desired success: So far described bene affinity sorbents with His, Him, PMB as ligands despite high to very high association constants high endotoxin concentrations as unsuitable (9).

In the presence of proteins, competing Pro Tin adsorption observed, leading to decreased endotoxin abrasion backup rates and sometimes high protein losses (especially with acidic proteins like BSA).

literature

• (1) SK Sharma
  Endotoxin detection and elimination in biotechnology Biotechnol. Appl. Biochem. 8: 5-22 (1986)
• (2) N. Haeffner-Cavaillon, JM Cavaillon, L. Szabó
  Cellular receptors for endotoxin Handbook of Endotoxins, Vol. 3: Biology of Endotoxins, 1-24 Elsevier Science Publishers BV (1985)
• (3) DC Morrison, JL 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 Sci. Tech., Vol. 44, No. 4: 204-209 (1990)
• (6) CUNO Newsletter for pHarmaceuticals (Oct. 1995), p. 3
• (7) BP 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), pp. 54-60
• (9) FB Anspach, O. Millbeck
  Removal of endotoxins by affinity sorbents J. Chromatogr. A 711 (1995), 81-92.

This prior art can be inventively by Microfiltration filter layer for the separation of endotoxins liquid media, especially water, protein solutions or Pa improve renteralien, the microfiltration filter layer 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 also filter layer production can be found on C. Dickenson, Filters and Filtration Handbook, Flsevier Science Publishers, Oxford 1992, and Ho & Sirkar (Editor), Membrane Handbook, Verlag van Nostrand Reinhold, New York, 1992. In particular, it can the filter layer materials are depth filters.

The covalently bound ligands can be

• (a) an endotoxin-specific ligand, preferably hist amine, histidine, polyethyleneimine, poly-L-lysine or polymyxin B and / or  
• (b) act as a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycho lat.

The filter layer material for the microfiltration filter layer according to the invention can be polysaccharides, e.g. B. 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, e.g. B. polyacrylonitrile, polysulphone, polyamide, in particular nylon, polyvinyl alcohol, polyethylene vinyl alcohol, polystyrene and polyacrylates and their derivatives,
Inorganic materials, e.g. B. act silica gel, glass and ceramic supports and their derivatives.

The materials can be fibrous or particulate, spherical or irregularly shaped with a porous or non-porous structure such as are used for chromatographic supports Come into play; parts of other structures can also be included be set, such as B. fragments or specially tailored Hollow fiber membranes, broken particles or pearls. Preferential the materials should be water-insoluble.

The size of the raw materials is chosen so that they are manufactured has a pore diameter in the layer, as it is used for microfiltration, especially between 0.1 and 10 µm.

In the case of the polymer based on the microfiltrati according to the invention onsfilterschicht is applied, it can be a hydropHi  The polymers act, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose.

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

The polymer can be removed from the microfiltration fil layer can be worn with the help of a spacer. Even the knockout valent bound ligands can be carried by a spacer the. These spacers can be spacers that are of bisoxirane, glutardialdehyde, epihalohydrin or diisocya derive nat, if necessary after oxidative activation.

For activation and immobilization chemistry, which is also available on endo toxin-specific ligands and spacers, for example point to Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992.

According to the invention, microfiltration filter layers are therefore seen which are surface-modified in a suitable manner and remove en dotoxins from water and aqueous solutions (buffers, protein solutions). The surface modification can consist of applying a bifunctional covalently bonded spacer which is reacted with a hydrophilic polymer, with unspecific interactions of the filter layer, especially with proteins, being 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 Fig. 1.

Endotoxin depletion success may vary in the presence of Proteins as dependent on the net charge of the proteins sen. By optimizing the conditions (pH value) acidic Proteins (such as BSA and mouse IgG 1) completely decontaminated  without any significant loss of protein. In the case basic proteins (such as lysozyme and bFGF) high depletions are also 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 loaded solutions (6000 EU ml ^-1 ).

In principle, the filter layers are constructed in accordance with FIG. 1. First, a hydrophilic polymer is 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, especially regenerated and microcrystalline Cellulose and derivatives, especially 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:

• Bisoxirane
• - glutardialdehyde
• - epihalohydrins
• - diisocyanates

To activate the vicinal diol bond formed when using bisoxirane and epihalohydrin, oxidation by periodate can optionally be used, an aldehyde group being formed. The spacer bonded 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 ligands or again through the mediation of a the above-mentioned spacer, if necessary after its oxidative acti crossing. Act as endotoxin-specific ligands (see list of Abbreviations): DAH, Him, His, PEI, PLL, PMB. Even the more common have non-endotoxin-specific ligands such as DEAE and DOC proved to be highly specific in the filter layer configuration with high protein recovery.

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.

On control filter layers (nylon without modification and with on brought hydrophilic polymer with or without spacer) without endo toxin-specific ligands were not depleted of endotoxins receive.

The new filter layers can be used for Endoto xin removal from water and parenterals. Even in the presence of Proteins have good results. In the case of basic pro However, one must take into account that interactions of the proteins with the endotoxin that can lead to a endotoxic masking. Protein-bound endo toxin cannot be clearly demonstrated with the LAL test. In this context it should be mentioned that ge It is clear whether protein-bound endotoxin is still toxic.

The filter layers according to the invention can be versatile be turned.

Medical-pharmaceutical sector:

• - hemodialysis.
• - Safe infusion and injection solutions (Parenterals)
• - Safe diagnostics (e.g. antibodies)

In biotechnology:

• - Manufacture of pHarma products.
• - Endotoxin depletion in process water and raw materials.
• - Decontamination of products (expenditure for processing ent falls)

Methods 1. Production of the filter layers

Hydrophilic polymers, especially 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. Fig. 1 illustrates the structure of a filter sheet material on the basis of nylon.

1.1. Coating a filter layer using the example of dextran

Microfiltration filter layers, e.g. B. 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 of 25 mM sodium carbonate (pH 11), 1 ml of ethanol (96%) and 9 ml of bisoxirane ( Fig. 2a) .

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

Both filter layers are then incubated for 15 minutes at room temperature after thorough washing with 5 ml of a 20 percent dextran 40,000 solution (pH 11) (see Fig. 2b using the example of nylon). The filter layers are then dried at 120 ° C. for 14 hours. 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 shown in FIG. 3, the filter layers are based on coated filter layer materials by significantly lower non-specific interactions - expressed by the adsorbed amount of hemoglobin.

From Fig. 3 also shows that with a simple Dex tran-coating, the same effect can not be achieved 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 ligands PLL, PMB and PEI 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 weight 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 50,000) in 0.1 M pHospHat 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 coated on DAH to epibromohydrin-activated Immobilized filter layers. Epibromohydrin activation will as described for bisoxiran. Immobilized DAH  is by 8 minutes reaction with a mixture of 5 ml Epi bromohydrin and 5 ml of 4 M sodium hydroxide solution activated at 90 ° C and so continued at 80 ° C with L-histidine (0.5 g L-histidine in 20 ml of water, pH 12). The finished filter layer is covered with 1 M natri umchloride and water washed.

Corresponding regulations are applied for the coating with other polymers and the covalent bond with the other en dotoxin-specific ligands.

2. Another method for producing the filter layers

As an alternative to the method mentioned under 1. can first the filter layer materials chemically modified and in the an then the structure of the filter layer by precoat the filter layer materials on a suitable base body getting produced.

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 on the filter layer materials is also shown by Fig. 1.

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

SepHarose 4B was made according to a regulation by Sundberg and Porath (J. Chromatogr. 90, 1974, 87) activated with bisoxirane. In Abän Change to the regulation, the reaction time was re to 2 hours induced to ensure minimal loss of oxirane groups put. For this purpose, 40 ml of a SepHarose 4B suspension were added 20 ml of a 0.5 molar NaOH solution, 8 ml of bisoxirane and 40 mg Sodium borohydride combined in one flask and at 40 ° C over egg shaken for a period of 2 hours. Then the activated SepHarose aspirated and washed several times with water  . The activated gel was treated with the same volume of one Reaction solution combined, which consists of 20% dextran with a medium Ren molecular weight of 40,000 and 0.2% sodium borohydride in egg nem 0.025 molar carbonate buffer existed and adjusted to pH 11 represents was. This solution was ge for 60 minutes at room temperature shakes. The coated SepHarose was from the dextran solution separated by filtration and then over a period of time incubated at 80 ° C for 24 hours.

2.2 Immobilization of endotoxin-specific ligands

The ligands PLL, PMB and PFI were either directly linked to the Per iodate-activated coating polymers or after incorporation of a Periodate-oxidizable spacers (bisoxirane) immobilized. DEAB was coupled directly to the matrices without spacers, the others low molecular weight ligands were bound via Bpibromhydrin.

2.2.1 Immobilization of PEI over Bisoxiran

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) were the filters layer materials for two hours at room temperature with a solvent solution, which is adjusted to pH 8 and made from 0.5 g PEI an average molecular weight of 50,000 and 10 ml of a 0.1- molar pHospHat buffer was composed. In conclusion washed with 1 M sodium chloride solution and water. The Wed ratios of solid and reaction solution in the individual reactions chosen so that a pour Telbare suspension was created.

2.2.2. Immobilization of histidine

Histidine was coated on epibromohydrin-activated via DAH Immobilized filter layer materials. Epibromohydrin activation tion was carried out as described for bisoxirane. Immobili  DAH was resolved by reacting with a mixture for 8 minutes of 5 ml epibromohydrin and 5 ml 4 M sodium hydroxide solution at 90 ° C acti fourth and immediately reacted at 80 ° C with L-histidine (0.5 g L-Hi stidine in 20 ml water, pH 12). The finished filter layer mat rial was washed with 1 M sodium chloride and water. Also here the mixing ratios of solid and reak tion solutions chosen so that shakeable suspensions are formed the.

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

3. Depletion experiments

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

Each filter sheet was fixed to the bottom of an ultrafiltration cell (filter sheet 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

For endotoxin quantification in the starting solutions and fil a chromogenic Limulus amebocyte lysate test (LAL- Test). This test is based on the fact that endotoxin Release of the chromogen p-nitroaniline induced, where between the released amount of p-nitroaniline and the present Endotoxin concentration in the range 0 to 1.2 EU / ml a linear  Connection exists. From the pHotometric p-nitroaniline Be can be tuned using a calibration line (standard endo toxin E. coli 0111: B4) on the endotoxin concentration of the Pro ben to be closed.

The LAL test was introduced in Europe in 1985 by the European Pharmacopoeia Commission for testing for endotoxins and since 1989 it has also been used in the monograph "Water for injections" ¹ the rabbit test.

Examples of use

• 1. ( Fig. 5) depletion from highly loaded buffer solutions
  Feed: 20 ml of 20 mM pHospHat buffer (pH 7) mixed with 6000 EU / ml
  The filter layers marked with -d represent filter layers in which no spacer has been incorporated.
• 2. ( Fig. 6 to 7) depletion from endotoxin-enriched BSA solutions
  Feed: 20 ml 20 mM pHospHat 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 pHospHat 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 pHospHat buffer (pH 7) with 1 mg / ml lysozyme
  Endotoxin concentration 134 EU / ml
  Protein recovery lysozyme
• 5. ( Fig. 11 to 12) depletion from MAX 16 H 5
  Feed: 20 ml 20 mM pHospHat buffer (pM 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 (10)

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, z. B. cellulose, in particular re-generated and microcrystalline cellulose and derivatives, in particular special cellulose acetate, agarose and derivatives, cross-linked dextran and derivatives, chitosan and derivatives,
Synthetic organic polymers, e.g. B. polyacrylonitrile, polysulphone, polyamide, in particular nylon, polyvinyl alcohol, polyethylene vinyl alcohol, polystyrene and polyacrylates and their derivatives,
Inorganic materials, e.g. B. silica gel, glass and ceramic supports and their derivatives as filter layer material. 4. Microfiltration filter layer according to one of the preceding Claims, characterized by a hydrophilic polymer, ins special dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose. 5. microfiltration filter layer according to claim 4, characterized ge indicates that the hydrophilic polymer is water-soluble per se or is insoluble in water. 6. Microfiltration filter layer according to one of the preceding Claims, characterized in that the polymer from the fil layer is worn with the help of a spacer. 7. microfiltration filter layer according to claim 6, characterized through one of bisoxirane, glutardialdehyde, epihalogeny in it or diisocyanate-derived spacer.   8. Microfiltration filter layer according to one of the preceding Claims, characterized in that the ligands from the Po lymeren can be worn with the help of a spacer. 9. microfiltration filter layer according to claim 8, characterized through one of bisoxirane, glutardialdehyde, epihalogeny in it or diisocyanate-derived spacer, optionally after oxidative activation. 10. Use of a microfiltration filter layer according to one of the preceding claims for removing endotoxins from liquid media, especially water, protein solutions or Parenterals.