HK1074252B - Method for identifying and extracting endotoxin - Google Patents

Description

The present invention relates to a method for detecting and enriching endotoxins from a sample.

Endotoxin (ET) refers to a family of lipopolysaccharides that, together with proteins and phospholipids, form the outer cell wall of Gram-negative bacteria. Endotoxins occur exclusively in this group of bacteria and play an important role in the organization, stability and barrier function of the outer membrane.

All endotoxin variants consist of a heteropolysaccharide covalently bound to lipid A (Holst, O., 1999, Chemical structure of the core region of lipopolysaccharides. In: Endotoxin in health and disease (Brade, H., Morrison, D.C., Opal, S., Vogel, S. eds.), Marcel Dekker Inc. New York). Lipid A anchors the endotoxin in the outer bacterial membrane. The heteropolysaccharide, consisting of a herzoligosaccharide and the O-antigen, shows up in the surrounding solution and determines the serological identity of the bacterium. The O-antigen consists of repetitive O-ligosaccharide units whose composition is derived from this gene (see here, et al., Chloroplastin is the supracartilaginous of 2-Hesoligosaccharide (DO-3) and L-Dioxyl-Hesoligosaccharide (DO-3).

In addition, the phosphate groups at lipid A and the heart region can be variablely substituted with arabinose, ethanolamine, and phosphate. Individual saccharide building blocks of the O-antigen are accelerated, sialyzed, or glycosylated. The O-antigen also varies in the number of heterogeneities, which is why the population of each bacterium has a heterogeneous lipidic acid (E.P.E.P., 1980), and the length of the polychlorinated biochemical lipidic acid (E.P.E.P., 1980), which is a heterogeneous lipidic acid (E.P.E.P., 1980), and the length of the polychlorinated biochemical lipidic acid (E.P.E.P., 1980).

Endotoxins are biomolecules that can be found in almost all aqueous solutions without proper precautions. Endotoxins can lead to sepsis, a severe immune system malfunction, in humans and animals. Therefore, for example, when manufacturing pharmaceutical proteins, contamination with endotoxin must be accurately detected and subsequently completely removed. Endotoxin is a problem in genetically engineered drugs, gene therapeutics or substances injected into humans or animals (e.g. veterinary treatment or in animal experiments).

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The first two methods are very expensive (see Competitive Endotoxin Testing) and the high demand for test animals and blood from the very rare arrowtail cancer is a concern, not least for animal welfare reasons. The LAL test can be miniaturised and automated, but has huge disadvantages in application due to the low stability of the components. Once an open LAL solution has to be directly processed and used because the components aggregate within a few hours.

Overall, there is no easy to manage and cost-effective method for endotoxin detection and the methods currently used have a number of disadvantages, and there is therefore a need for a method that circumvents these disadvantages.

There are a number of methods for enriching endotoxins from biological solutions in general, but no generally applicable standard methods exist for proteins in particular. The methods used are adapted to the specific properties of the protein and the corresponding production process of the protein. There are various ways of enriching endotoxins, each with its own advantages and disadvantages.

Ultrafiltration (Petsch, D. & Anspach, F.B., 2000, J. Biotechnol. 76, 97-119 et seq.) is used for endotoxin enrichment from water and solutions with low molecular weight components such as salts, sugars and antibiotics, but is not suitable for high molecular weight proteins or DNA.

The two-stage extraction (e.g. WO 0166718, Merck) is intended to separate water-soluble proteins and DNA from endotoxin, but causes detergent residues in the purified product.

An anion exchanger (DEAE) method (e.g. US 5990301, Qiagen; WO 9414837, Enzon) is also used for the enrichment of endotoxins from DNA and basic proteins, but this requires a low ionic strength (< 50 mM NaCl) and results in protein co-adsorption on acid proteins.

Another method for enriching endotoxins from DNA and proteins (e.g. BSA, myoglobin, gamma globulin, cytochrome C) is affinity adsorption (e.g. polymyxin B, histamine, histidine, polylysin) e.g. GB 2192633 (Hammersmith Hospital), which is however toxic in the case of polymyxin B and may lead to protein co-adsorption at low ionic strengths.

In addition, immuno-affinity chromatography is used, where specificity for certain endotoxins can only be achieved by expensive antibodies (US 5179018, Centocor; WO 0008463, Bioserv) to cardiac oligosaccharide.

Furthermore, the S3delta peptide (WO 0127289) of factor C (a constituent of the LAL test) (WO 9915676 both National University of Singapore) is used on proteins (e.g. BSA, chemotrypsinogen) but this method has low efficiency at high ionic strengths and high manufacturing costs (production in insect cell culture). EP1399551, published under WO 03/000888, is the Article 54 (3) EPC technique and publishes a method for the removal of endotoxins from a sample by bacteriophage proteins.

In the pharmaceutical industry, three main methods are used for protein solutions adapted to the properties of the target proteins: Anion exchange chromatographyReversed phase chromatography; this has the disadvantage of not being equally suitable for all proteins, - particularly for hydrophobic proteins. In addition, this method is very time-consuming.RemTox (Fa. millipore): This method has the disadvantage of, in addition to a very long incubation period, a high non-specific binding percentage and often insufficient protein recovery.

Gross endotoxin depletion of proteins to a value of up to 10 EU/ml is possible in many cases with existing methods. However, the remaining endotoxin concentration is still toxic. Further depletion (= refinement) is therefore recommended or required depending on the dose of the protein in medical use, the European Pharmacopoeia (e.g. 5 EU/kg body weight and hour in intravenous applications) and the FDA. However, this refinement is often not satisfactorily ensured with existing methods.

Therefore, the purpose of the invention is to provide a method that can detect endotoxins in samples. The purpose of the invention is also to provide a method that can remove endotoxins from aqueous solutions.

The tasks are solved by the subject-matter defined in the claims.

The following figures explain the invention.

The following table shows the chemical structure of the endotoxin from E. coli 0111:B4 in a schematic. Hep = L-glycerol-D-mano-heptose; Gal = galactose; Glc = glucose; KDO = 2-keto-3-deoxychoic acid; NGa = N-acetyl-galactosamine; NGc = N-acetylglucosamine.

(A) Endotoxin removal from protein solutions: bovine serum albumin (BSA), carbonic anhydrase (CA) and lysozyme (Lys) were incubated on the column for 1 h and then eluted with buffer. The endotoxin concentration before and after the column was measured with the LAL test and the percentage reduction calculated. (B) Protein recovery: The protein concentrations of the starting solutions and fractions after the column were determined by absorption measurement at 280 nm and the percentage protein recovery was determined.

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Figure 4 shows the results of experiments with biotinyl p12 bound to magnetic beads by streptavidin. (A) Endotoxin enrichment from buffers (20 mM Hepes, 150 mM NaCl, pH 7.5) and protein solutions was determined by LAL test. (B) For protein solutions, protein recovery was determined by absorption measurements. The beads were separated from the solution by a magnetic separator.

Figure 5 shows the results of the endotoxin removal with p12 which was immobilized via biotin-streptavidin interactions on agarose beads. The separation of the immobilized p12 was done by centrifugation. The endotoxin removal from buffer (20 mM Tris, 150 mM NaCl, pH 8.0) and BSA solutions was determined from the endotoxin concentrations of the starting solution and residue.

The binding is to endotoxin of E. coli D21f1 which has been immobilized on a hydrophobic HPA chip. The injection of p12 and EDTA (5 mM) is marked by bars across the puff. Curves: 20 mM Tris, 150 mM NaCl, pH 8.0. (B) Equilibrium residual values for the binding of p12 to immobilised endotoxin (__) were measured at approximately 600 ppm after the start of the p12 injection and the associated p12K concentration was strongly associated with the immobilization of the P12K on the internal membrane of the heart (R1212+P12K12).

Figure 7 shows the schematic structure of the endotoxin heart region of different E. coli mutants.

Figure 8 shows the result of an endotoxin depletion by chromatographic column flow method. E means balance buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5), A means wash buffer A (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5), B means dilution buffer B (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5), C means regeneration buffer C (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, 0.005% NaDOC, pH 7.5), S means concentration of column protein and excretion buffer respectively. BSA means RSA. This means EU (End-Element) units. The final injection factor (F) was approximately 5 ml. The final injection was carried out in the first 15 ml (Figure 2 and 5 ml) of water and was then injected into the final 2 ml (Figure 2) of B.

The experiment was performed in parallel on 3 columns. Prior to the sample order, 1 ml of the balancing buffer (20 mM Hebex, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5) was collected in each column, followed by the un-directed injection of NHS-activated sepharose 4 FastFlow (Amersham Biosciences, Uppsala, Sweden) (8 mg p12/ 1 ml Sepharose) and 3 columns of 2 ml column volume each. The experiment was performed in parallel on 3 columns.

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The present invention relates to a method for detecting endotoxin, including the steps: (b) Evidence of endotoxin bound to bacteriophage tail proteins.

Preferably, the invention relates to a method where the detection is performed by spectroscopic methods, e.g. fluorescence emission, fluorescence polarization, absorption or circular dichroism, or by capacity measurement, e.g. electrical signals, or indirectly by competence detection.

If necessary, after step (a) and before step (b), an additional step (a) is introduced to separate the bacteriophage tail protein endotoxin complex from the sample.

The present invention also relates to a method for the removal of endotoxin from a sample, comprising the steps: (a) Incubation or exposure of a sample to non-specific or targeted bacteriophage tail proteins immobilized on a solid medium;

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The bacteriophage tail protein may be a naturally occurring or a molecular biological or biochemically modified one. The bacteriophage tail protein may be genetically and/or biochemically modified for various reasons. However, not only the naturally occurring bacteriophage tail proteins but also their variants may be used for the purposes of the invention. Variants, for the purposes of the present invention, means that the bacteriophage proteins have an altered amino acid sequence. These may be obtained by screening the naturally occurring variants, or by random mutagenesis or targeted mutagenesis, but also by targeted chemical modification. The bacteriophage proteins used for the invention may be adapted to their specific properties by a specific structure or mutagens.This binding to the carrier can be fixed, e.g. covalent, or via specific or non-specific biotinylation, but can also be reversible, e.g. via a reducing disulfide bridge. Furthermore, a modification can increase stability. Molecular biological or chemical mutagenesis introduces mutations, which can be amino acid additions, deletions, substitutions or chemical modifications. These mutations can be a change in the amino acid sequence in the binding region of the bacteriophage tail proteins, with the aim of adapting the specificity and binding affinity to requirements, e.g. to increase the binding of the bacterial phagocytes to the endoproteins or to increase the reliability of the protein, or to perform a genetic test or to improve the retention of the phagocytes.In addition, a genetic or chemical modification of phage proteins may be performed to adapt existing physical properties of the protein, such as solubility, thermostability, etc., within the meaning of the method of the invention.

The work on the three-dimensional structure of T4 p12 had shown that at elevated temperature proteolytic fragments of 33 kDa and 45 kDa can be produced, which are shortened N- and C-terminal (33 kDa) or only N-terminal (45 kDa).

The modification may also be specifically intended to allow direct detection, e.g. by measuring tryptophan fluorescence. For example, P12 quen has five tryptophan residues. The fluorescence spectrum of the native protein suggests that these residues are largely insoluble. A variety of scientific studies have shown that almost always aromatic amino acids are involved in the binding of sugar residues, as they are also found in endotoxin. The binding of sugar residues to proteins can be tracked by a tryptophan fluorescence, or, if necessary, also by a change in the fluorescence oximeter.Err1:Expecting ',' delimiter: line 1 column 545 (char 544)Subsequently, a targeted exchange of one of the six tyrosines in the C-terminal region for a tryptophan residue significantly increases the intensity of the measurable signal to obtain signal differences attractive for the development of an endotoxin detection kit.

The use of bacteriophage tail proteins depends on which endotoxins are to be detected or cleared. A large number of known bacteriophages are already available for most of the bacteria described so far and can be used for the methods of the present invention. The phages and the corresponding host bacteria are available from, inter alia, the following stock collections: ATCC (USA), DSMZ (Germany), UKNCC (UK), NCCB (Netherlands) and MAFF (Japan).

The bacteriophage tail proteins are preferably derived for the methods of the invention from bacteriophages whose host bacteria are of medically or biotechnologically relevant importance, such as E. coli, used in the production of recombinant proteins or nucleic acids for gene therapy. In particular, bacteriophage tail proteins that bind to highly conserved areas of endotoxin, such as the heart region or lipid A, are preferred. In particular, p12 and p12-like bacteriophage tail proteins can be used when a combination of endotoxin contaminants from different host bacteria is present.

The detection or depletion of endotoxin in or from a sample is achieved by binding endotoxin to the bacteriophage tail proteins. This binding can be demonstrated, for example, by direct measurement by spectroscopic methods, e.g. fluorescence emission, fluorescence polarization, absorption or circular dichroism. In addition, the binding can be made visible by electrical signals, e.g. a capacity measurement. Furthermore, the binding of endotoxin to the bacteriophage tail proteins can also be demonstrated indirectly by displacement experiments.

For the purpose of the present invention, the bacteriophage tail proteins may be coupled to suitable carrier structures, e.g. magnetic particles, agarose particles, microtiter plates, filter materials or flow cell comb, if necessary to separate the bacteriophage tail protein endotoxin complexes from the sample (indirect demonstration). The carrier structures may be e.g. polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, silicon or agarose. The coupling may be achieved e.g. by adsorption or covalent bonding.

The method of enrichment of the invention involves the attachment of bacteriophage tail proteins to solid media, which can be used to make materials for chromatography columns (e.g.

The following substances are to be classified as 'separated materials', 'filtration media', 'glass particles', 'magnetic particles', 'centrifugation or sedimentation materials' (e.g. agarose particles).

The functional coupling is important, i.e. tail proteins have structures accessible to endotoxin despite binding to the carrier material.

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Cysteine coupling is possible with any bifunctional crosslinker with NH and SH reactive groups, with or without intermediate spacers, e.g. 11-Malcimidoundecanoic acid sulfo-NHS or Succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-amido]caproates. If spacers are not available, 8-12 C-atom spacers with a final NH group can be inserted. Preferably, cysteine coupling is achieved by specific biotinylation of the cysteine by e.g. EZ-Link-PEO-maleimide activated biotin (Pierce).

However, this binding can be dissolved by the addition of appropriate chelators such as EDTA or EGTA. Preferred for binding are Ca2+ concentrations in the range of about 0,1 μM to about 100 mM, particularly in the range of about 0,1 μM to about 10 mM, particularly in the range of about 0,1 μM to about 1 mM, and furthermore particularly in the range of about 10 μM to about 1 mM.Mg2+ concentrations above 10 mM worsen the binding of endotoxin to p12, which is reflected in an increase in the dissociation constant. Without the addition of Mg2+, a Kd value of 50 nM is obtained and in a buffer with 10 mM Mg2+ a Kd value of 1 μM was measured. Zinc showed an even stronger inhibitory effect. 1 mM Zn increases the Kd value to 10 μM. A setting of the concentration of two ions or other ions (e.g. Cu2+, A2+, Zn2+, Fe2+, Ca2+, Ba2+, C2+, Mg2+, Fe2+, C2+) can provide an optimal range for binding by substances such as HTA,NTA or generally chelators/buffers (ADA: N-[2-acetamido]-2-iminodiacetic acid; 5-AMP: adenosine-5'-monophosphate; ADP: adenosine-5'-diphosphate; ATP: adenosine-5'-triphosphate; Bapta: 1,2-bis ((2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid; citrate: citric acid; EDTA: ethylene diamine tetraacetic acid; EGTA: ethylene glycol-bis ((β-aminoethyl ether) N,N,N',N'-ethylene diamine tetraacetic acid; HEDTA: N-hydroxyethylene diamine tetraacetic acid; NTA: nitritrile acetic acid; SO4. sulphate) which are available as two buffers that can be used for Iodine.

The methods of the invention may therefore also include washing steps. Depending on whether direct or indirect detection or enrichment requires separation of sample and bacteriophage tail protein, washing steps may be incorporated. Since Ca2+ or other metal ions (e.g. Mg2+) are essential for binding, the binding of endotoxin to e.g. p12 can be dissolved by appropriate washing steps. Depending on whether the endotoxin is to remain bound to the bacteriophage tail protein, e.g. p12, the endotoxin is to be washed with EDTA-free buffer if the binding is to be dissolved with EDTA-containing buffer, with EDTA concentrations in the range of at least 0.05 mM to 10 mM, preferably in the range of 2 to 5 mM.

The separation is carried out after incubation of the sample with the appropriate carrier material coupled with the bacteriophage tail proteins for about 5 to 60 min or about 30 to 180 min or overnight if necessary. For this purpose, the sample is e.g. eluted from the chromatography column, or filtered, or the relevant particles are decentrifuged or deposited, or separated magnetically by applying a magnetic field. The separation in the batch process described here, i.e. pre-incubation of the sample and the corresponding bacteriophage tail proteins coupled with the carrier materials, can be useful, especially at very low endotoxin concentrations.

The flow rate depends on the volume and geometry of the column. The flow rate also depends on the volume and endotoxin content of the sample, in order to achieve efficient flow through a long-term contact between column and endotoxin even at low endotoxin concentrations. The contact time is the time it takes for the sample to descend to the column until it drains out.

The separation step can be used, for example, in the enrichment process to regenerate the bacteriophage tail proteins coupled to the solid carrier, which allows the solid carrier, e.g. a matrix in a chromatography column, to be reused. Regeneration is achieved by removing the bound endotoxin by means of an appropriate regenerative buffer containing EDTA or a chelator. EDTA is preferred at a concentration of more than 2 mM EDTA, especially more than 10 mM EDTA.

Since ionic interactions are always influenced by changes in ionic strength, increases or decreases in other salts in solution, such as NaCl or KCl, can also affect the binding of endotoxin to the bacteriophage tail proteins.

To make the binding directly or indirectly visible in the detection process, the protein can also be modified molecularly or biochemically to enable or improve the measurement. To make a binding of an endotoxin, e.g. to p12, directly visible, a molecular biological exchange of tyrosine residues for tryptophan can be performed. To reduce the signal background, it may be necessary to exchange the originally contained tryptophanes for tyrosine.In order to measure even in protein-containing solutions, p12 can be additionally modified after tryptophan introduction.In this process, the endotoxin residues can be determined by Koshland reaction (2-5-hydroxy-marmorphol) and their spectral properties in terms of endotoxin concentration can be altered.The endotoxin can be released from the endotoxin (B12), and its chemical concentration can be determined by the fluorescent fluorescent.

The method of the present invention allows the detection and removal of endotoxin from and in all aqueous solutions, which may include proteins, plasmid DNA, genomic DNA, RNA, protein nucleic acid complexes such as phages or viruses, saccharides, vaccines, medicinal products, dialysis buffers, salts or other substances contaminated by endotoxin binding.

The production of nucleic acid, including the sequence of the bacteriophage protein and the tagging and the production of the expression product are the stand-alone technique and do not need to be explained here separately. Another aspect of the invention is the nucleic acid sequence, which is coded by a bacterial protein, specifically the histophage protein or histophage protein, and is also responsible for the identification of all other bacterial proteins, however, the histophage protein or histophage protein is also involved in the discovery of the protein.

Another aspect of the invention is bacteriophage proteins with a tag that has a surface-exposed cysteine for specific targeted biotinylation, e.g. the tags according to SEQ ID NO: 5, 6 and 7. An example of a p12 tag is the amino acid sequence listed in SEQ ID NO: 8.

The methods of the invention offer advantages over the methods of detection and purification of endotoxin and endotoxin in the performance of their respective applications, and the production of antibodies against LPS herzoligosaccharides is very difficult, which makes antibody-based methods very expensive.

The following examples illustrate the invention and are not intended to be taken as limiting; unless otherwise stated, standard molecular biology methods have been used, as described e.g. by Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

1. glassware, plasticware and buffers

For the endotoxin removal, all glass vessels were de-pyrogenised by baking at 200°C (4h) and only pyrogen-free plastic materials (e.g. pipette tips, microtiter plates) were used. Other non-heat-resistant devices or vessels were either treated with 3% hydrogen peroxide or washed with 1% sodium deoxycholate. They were then rinsed with endotoxin-free water. The buffers were made from largely endotoxin-free buffer substances (sigma) and treated with endotoxin-free water. Salts such as NaCl, which can be heated to 200°C, were excavated (200°C, 4h).

Endotoxin detection by LAL test

Endotoxin controls were performed using a chromogenic LAL test (Limulus-Amebocyte-Lysate Test, Charles-River Endosafe, Charleston, USA) as specified by the manufacturer. Endotoxin standards (Charles-River Endosafe, Charleston, USA) in the range of 0.005-50 and 0.02-50 EU/ml respectively were used for concentration determination. Absorption measurements were made at 405 nm in a tempered micro-titration plate reader (Genios, Tecan GmbH).

Western blot to p12 proof

The detection of p12 in the residue of bead-treated samples and in the affinity chromatography fractions was carried out by Western Blots. Some of the proteins were concentrated by NaDOC/TCA precipitation (sodium deoxycholate/tetrachloroacetate). The samples were then electrophoretically separated on 12% SDS gels and transferred to PVDF membranes (immobilion, millipore). The membranes were washed with PBS for 30 min, blocked with 5% milk chloride (1 cl) and then incubated with polyklonol-NB-p12 antibodies (1 h, dilution: 1: 1000). After incubation with a phosphate ion igase secondary conjugate (PIPG-B) antibodies (5 BCG/TIPG-B) the development of the antibodies was successful.

Endotoxin cleaning

The endotoxin purification was carried out according to the prescriptions of Galanos, C., Lüderitz, O. & Westphal, O. 1969, Europ. J. Biochem. 9, 245-249.

Example 5: Specific coupling of p12 to immobilized iodoacetyl residues:

To achieve a directed binding of p12 to the surface, the amino acid serine was replaced by cysteine at position 3 of the strep tag according to SEQ ID NO:5 as in example 12 and the protein was immobilized via iodoacetyl residues, which preferably bind free sulfhydryl residues.

1 ml of sulfolink coupling gel (Pierce) was poured, washed with 6 ml of 1% sodium deoxycholate and balanced with 6 ml of coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5), then 1 ml of p12S3C (= N-StrepS3Cp12) (1-1.5 mg/ ml in coupling buffer) was injected, the column was lightly shaken for 15 min, incubated for another 30 min at room temperature without shaking, and again 1 ml of p12S3C was injected and the incubation steps were repeated. This coupling of p12S3C cells (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5) was repeated 4 times and then the columns were washed with 6 ml of water. The collected p12S3C and p12S3C concentrations were determined by measuring the pH of the water at 150 mL and the pH of the water at 8 mL.

The ability of this gel to remove endotoxin from protein solutions was tested with BSA (2-4 mg/ml), carbon anhydrase (1-2 mg/ml) and lysozyme (3-4 mg/ml). BSA and lysozyme solutions were spiked with endotoxin from E. coli 055:B5 (Charles-River Endosafe, Charleston, USA) or E. coli HMS 174 (100-1000 EU/ml), while the carbon anhydrase was not infused with additional endotoxin. In addition, 0.5 ml of protein solution was given to each of the columns, incubated for 1 hour at room temperature and then washed the columns with pustules. The columns were fractionally collected and removed by endotoxins and removed by the columns of a chromosome of LAL-T (Charles-River Endosafe, USA) by means of a 150 mL pH measurement. The final pH of the columns could not be determined by the final pH measurements, which was approximately 280 pM (9.99 pM). The columns were then removed by means of a 150 mL of the chromosome of a single protein (LAL-T-T-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-

Example 6: Non-specific coupling of p12 to NHS activated carrier material:

N-hydroxysuccinimide (NHS) is displaced from compounds by primary amino acids and is therefore used to bind proteins to surfaces. NHS-activated sepharose columns (HiTrap NHS-activated HP, 1 ml, Amersham Pharmacia-Biotech) were first washed with 6 ml of 1 mM hydrochloric acid at room temperature in 10-15 ml p12S3C (1.0-3.5 mg/ ml) in 0.2 M NaHCO3, 0.5 M Na, pH 8.3 and then pumped circularly through the column (flow rate 0.8/ min). After 60 min, the column fraction was dissolved and the columns were replenished with 6 mM of buffer. These fractions were then dissolved by dissolving the corresponding pH of the solution in HiTrap Amphetamine (0.5 mL) or 0.12 mL (0.5 mL) and then measured at the manufacturer's pH, respectively, with 6 mg/ mL and 8 mL (0.5 mL) of NaCl. The pH of the column was measured at the manufacturer's pH, and the pH was measured at the manufacturer's pH, respectively, at 150 mL and 0.5 mL (0.5 mL).

The lysozyme was fractionally collected and the endotoxin content before and after the column reactions was re-determined by a chromogenic LAL test (Charles-River Column, Charleston, USA). The endotoxin was also detected by absorption of protein at 280 nm. The endotoxin was removed at 280 nm. The endotoxin was detected at 85 mF. 90% of the final solution was removed from the column, and the pH of the column was determined by measuring the pH of the column at 612 mF. The final solution was removed from the column at 85 mF. 90% of the final solution was removed from the column at 612 mF. The endotoxin was detected at 612 mF. The final reaction was detected by measuring the pH of the column at 512 mF. The final solution was removed from the column at 85 mF.

Example 7: Directed coupling of p12 to via diamminoethane and N-succinimidyl iodoacetate (SIA) as spacer to NHS-activated carrier column.

To achieve a directed binding to the chromatography carrier material, a bifunctional linker was bound to an NHS-activated surface that allows coupling of p12S3C via its free cysteine and iodoacetyl residues of the bifunctional linker.

After blocking excess NHS groups with ethanolamine (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing (0.1 M acetate, 0.5 M NaCl, pH 4.0) of the column, the column was washed with 6 ml of ice-cold 1 mM hydrochloric acid, then 1 ml of ethylene glycol (10 mg/ ml in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) was injected and the column incubated at room temperature for 30 min. After blocking excess NHS groups with ethanolamine (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing (0.1 M acetate, 0.5 M NaCl, pH 4.0) of the column, the column was injected with 6 ml of boric acid (50 mM columns of natriuric acid, 150 m NaCl, 5 m MTA, pH 8.3) followed by 30 ml of 10 mL of N-acetyl sulphate (50 mL of B12-acetyl sulphate) in a solution of 150 ml of water (5 mL of Tricloid-iod-iod-iod-iod-iod-iod-iodine, 10 mL, or 1 mL of B12-acethanol) at room temperature, the colum was in equilibrium with 1 ml of 150 ml of B12 mL of B12 mL, or 1 mL of B12 mL of B12 mL, and then the colum was injected at room temperature (1 mL, pH 1.50 mL, pH 1.5); the colum was in equilibrium with 1 mL was in equilibrium with the colum.

The lysozyme solutions were sprayed with E. coli HMS 174 endotoxin (~500 EU/ml). 0.5 ml of protein solution was applied to the column, incubated at room temperature for 1 hour and then washed with buffer. The lysozyme was fractionally whitewashed and the endotoxin content was re-examined before and after column action by a chromogenic LAL test (Charles-River Endozyme, Charleston, USA). The protein was also determined by measurements of 280 nm. The final pH of the solution was determined at 280 mF. The final pH of the solution was determined at 6 to 7 pM. The final pH of the solution was determined at 120 mF. The final pH of the solution was determined at 6 to 7 pM. The resulting polymer was obtained from a solution of 5 to 7 pM. The final pH of the solution was determined at 6 to 12 pM. The final pH of the solution was determined at 6 to 12 pM. The final pH of the solution was determined at 6 to 7 pM. The final pH of the solution was determined at 6 to 12 pM. The final pH of the solution was determined at 6 to 7 pM. The final pH of the solution was determined at 6 to 12 pM. The final pH of the solution was determined at 6 to 7 pM. The final pH of the solution was measured at 6 to 7 to 12 pM. The final pH of the solution was measured at 6 to 7 to 12 mF was determined at 6 m. The final pH of the solution was measured at 6 to 12 mF was measured at 6 to 12 m. The final pH of the solution at 6 to 12 m. The final pH of the solution was measured at 6 to 12 m. The final pH of the solution was measured at 6 to 12 m. The final pH of the solution was measured at 6 to 12 m. The final pH of the solution was measured at 6 to 12 m. The final pH of the solution was between 6 to 12 m. The solution at 6 to 12 m. The final pH of the solution at 6 to 12 m. The final solution was measured at 6 m. The final pH of the solution was measured at 6 to 12 m. The final pH of the solution was 6 to 12 m. The solution at 6 m.

Example 8: Removal of endotoxin from a BSA solution in the flow process

The resulting 1 m1 chromatography column was balanced at a flow rate of 1 ml/ min with 10 ml of Buffer A (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM CaCl2). Subsequently, 4 ml of BSA solution (11.5 mg BSA (Carl Roth GmbH, Germany) / EDC A) was applied (injection: I) and the column (Limestone) was covalently immobilized. The column was then measured with 15 mL of Puffer A at a flow rate of 1 ml/ min. The final concentration of the fluorinated water was determined at between 7.5% and 7.5% of the pH of the product. The final concentration of the fluorinated water was determined with a pH of 7 to 10%, with a final test of 6 to 10 ml of Benzodiazepine (Metazolamine, Benzodiazepine, Benzodiazepine, and Benzodiazepine) and a final measurement of 2 to 5 ml of Benzodiazepine (Metazolamine, Benzodiazepine, and Benzodiazepine).

Example 9: Removal of low levels of endotoxin from buffers by non-specifically coupled p12.

20 ml NHS-activated sepharose 4 FastFlow (Amersham Biosciences) was first washed with ice-cold hydrochloric acid and then incubated with 292 mg p12 (7 mg/ml in 25 mM Citrate pH 7.0) for 4 hours under shaking at room temperature. The sepharose was then washed with 7 x 80 ml 5 mM Citrate pH 2.0 and 1 ml of the wash fractions were dialysed against 5 mM Citrate pH 2.0 each. These dialysates were used to quantify the excess p12 in the wash fractions by absorption measurement at 280 nm. A loading density of 8.7 p12 per 1 ml sephase was determined. Untreated NHS residues were injected with 12 h of sepharose with 1 mg T2C and these were converted to 20% ethanol by volume at pH 8.0.

In 3 parallel trials, 4 ml of endotoxin solution (S) were applied to each column (see Fig. 9). The endotoxin solution consisted of endotoxin from E. coli O55:B5 (Charles-River Endosafe, Charleston, USA) in a balancing buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5).

The columns were first flushed with 12 ml of regeneration buffer (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5) and then with 12 ml of balancing buffer.

The endotoxin solution was applied to the columns (I) and fractions of 5 ml and 2 ml were collected. The column was then regenerated with 4 ml of regeneration buffer (B). No endotoxin was detected in the flow fractions, i.e. the endotoxin contamination was completely removed in all three experiments.

Example 10: non-specific coupling of biotinyl p12 to magnetic streptavidin beads.

NHS-activated biotin binds to primary amino residues of p12 and then 50 μl of biotin-injected p12 (1 mg/ ml) were administered to 1 ml of Streptavidin Beads (MagPrep Streptavidin Beads, Merck), shaken for 2 h at room temperature and then the excess p12 was removed by 12 quadruple doses of 1.5 mL 20 mL Tris, 10 mL EDTA, 7.5 pH.

The endotoxin was removed by spraying with 5 EU/ml (endotoxin from E. coli O55:B5, Charles-River Endosafe, Charleston, USA) of the buffer, 150 mM NaCl, 150 mM NaCl, pH 7.5 and protein solutions (0.1 mg/ml BSA, 0.1 mg/ml Lysozyme, 0.1 mg/ml Carbon Anhydrase in 20 mM Hepes, 150 mM NaCI, pH 7.5). The carbon anhydrase solution contained approximately 1 EU/ml. 200 μl of the buffer and protein solution were administered by 25 μl magnetic beads with immobilized p12 and mixed and incubated at room temperature. The solution was removed at 30 min. The unbiased solution was determined to be 57% complete (99%, 87%, and 49,9%) removed from the final solution, with the final test containing a sample of L-L-carbotoxin and a protein of approximately 87%, and the final result was determined by incubation and analysis (Base: 99% and 99%).

Example 11: non-specific coupling of biotinyllated p12 to immobilized streptavidin.

P12 (3 mg/ ml in PBS, 0.05% Tween20) was incubated with sulfo-NHS-LC-LC biotin (Pierce) at a ratio of 1:10 to 1:20 for one hour at RT and then dialysed against buffers (e. g. PBS or 20 mM Hepes, 150 mM NaCl 5 mM EDTA, pH 7.5) where NHS-activated biotin binds to primary amino residues of p12.

The endotoxin removal was tested with buffers (20 mM Tris, 150 mM NaCl, pH 8.0) and BSA (0.5 mg/ml in 20 mM Tris, 150 mM NaCl, pH 8.0). Each 1 ml of buffer or BSA solution was pickled with 10 EU/ml, 50 μl p12 agarose was added, agitated at room temperature for 1 hour. The p12 agarose was then decentrifuged and the endotoxin and protein concentration in the residue measured. 99% of the buffer was removed and 86% of the BSA solution (Fig. 5) was recovered with 90% BSA.

Example 12: Tests for p12 endotoxin binding by surface plasmon resonance measurements

Err1:Expecting ',' delimiter: line 1 column 500 (char 499) Tabelle 1: Dissoziationskonstanten von Endotoxin an p12 in Abhängigkeit von dem pH-Wert der Lösung.

6,00	3,09E-07
7,50	6,85E-08
8,00	5,86E-08
8,50	7, 86 E-08
9,00	3,29E-08
10,00	1,55E-07

Err1:Expecting ',' delimiter: line 1 column 413 (char 412)

Example 13: Recombinant p12 constructs

Other Construction of p12 with N-terminal strep tag (N-Strep-p12): The nucleotide sequence for the strep tag (US patent 5,506,121) was inserted at the 5' end of the T4p12 gene by PCR, for which a primer (5'-GAA GGA ACT AGT CAT ATGGCT AGC TGG AGC CAC CCG CAG TTC GAA AGA GCCT AAT AAT AAT TAT CAA CAC GTT-3' (SEQ ID:1) containing the nucleotide sequence of the strep tag at its 5' end (the end of the sequence) and a residue interface (the end of the sequence) was constructed for the 5' end of the p12 gene, which can be inserted into the right-mid-plate of the 3' expression of the p12-strand.The PCR was performed with 40 cycles (1 min 95°C, 1 min 45°C and 1 min 72°C). The PCR approach was performed by cutting the restriction endonucleases PNSI and DNA BamHI and inserting the desired fragment after size fractionation via an agarose gel and elution from the gel into the NdeI and BamHI sites of the expression plasmid pET21aes. The sequence of the N-Sp12Gpsection was further verified for accuracy by steps such as the PNSP1257 step, which was performed in the Burda-N-Sp12Gpsection.The plasmid pNS-T4p12p57 was then transformed into the expression strain BL21 (DE3). Inserting an N-terminal cysteine residue into N-Strep-p12 (N-Strep-S3C-p12 and N-Strep-S14C-p12): The insertion of an N-terminal cysteine residue was carried out as described in point 1, with two new primers designed for the 5' end. For N-Strep-S3C-p12 the 5' primer GAA GGA ACT AGT CAT ATGGCT TGT TGG AGC CAC CC CAG TTC GAA GAA GGC AGT AAT AAT AAT CAA CAA CAA CAA CAA CAA CAA GTT-3' (GGQ ID: 3) and for N-Strep-S14C-p12 the 5' primer GAA GAT CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAA CAOther The E. coli strain BL21(DE3) was purified with the plasmid pNS-T4p12p57 in 2 1 shaking cultures (LB medium with ampicillin 100 μg/ml) to an OD600 of 0.5-0.7 at 37°C and the expression of the N-Strep-p12 protein was induced by the addition of 1 mM IPTG (isopropyl-β-thiogalactopyranoside). After incubation at 37°C for 4 h, the cells were harvested.Err1:Expecting ',' delimiter: line 1 column 332 (char 331)This cleans approximately 100 mg of N-Strep T4p12 from 10 litres of culture.

Name	Sequenz des Tag
Nstrep-p12	MASWSHPQFEKGAS	SEQ ID NO: 5
Nstrep-p12-S3C		SEQ ID NO: 6
Nstrep-p12-S14C		SEQ ID NO: 7

The following is the list of the Member States:

The method of detection and removal of endotoxin is described in the following sections: The following is the list of substances which are to be classified as 'chemical' in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: The following shall be reported in the table of the Annex to Implementing Regulation (EU) No 540/2011 for the following substances: The following is a list of the substances which are to be classified as 'chemical' in Annex I to Regulation (EC) No 1907/2006 of the European Parliament and of the Council: The following is the list of the substances which are to be classified in the additive: The following is the list of substances which are to be classified in the additive: The following is the list of substances which are to be classified in the additive: The following is a list of the active substances in the active substance: Other

Claims (14)

1. A method for detecting endotoxin, comprising the steps:

   a) incubating a sample with a protein occurring in bacteriophages and being able to bind components of cell membranes,

   b) detecting endotoxin bonded to bacteriophage proteins.
2. The method according to claim 1, further comprising after step a) and prior to step b) the additional step of

   a') separating the bacteriophage protein-endotoxin complexes from the sample.
3. The method according to one of the claims 1 to 2, the detection being implemented by means of spectroscopic methods.
4. A method for removing endotoxin from a sample, comprising the steps:

   a) incubating a sample with or bringing a sample in contact with proteins occurring in bacteriophages and being able to bind components of cell membranes, the proteins being immobilised on a permanent carrier, non specifically or directed, and wherein the Ca²⁺ concentration in the incubation being 0.1 µM to 10 mM and/or the Mg²⁺ concentration being 0.1 µM to 10 mM.

   b) separating the bacteriophage protein-endotoxin complex from the sample.
5. The method according to claim 4, the steps a) and b) being implemented in a chromatography column throughflow method.
6. The method according to claim 4, the permanent carrier being filtration media, glass particles, magnetic particles, centrifugation materials, sedimentation materials or filling materials for chromatography columns.
7. The method according to claim 4 to 6, the bacteriophage proteins being immobilised on the permanent carrier via coupling groups.
8. The method according to claim 7, the coupling group being a lectin, receptor or anticalin.
9. The method according to claim 7, the coupling group being a streptavidin or avidin and the bacteriophage proteins being coupled with biotin or a Strep-tag.
10. The method according to claim 4 to 6, the bacteriophage proteins being immobilised on the permanent carrier covalently via chemical bonds.
11. The method according to anyone of the preceding claims, the bacteriophage protein having a Strep-tag or a His-tag.
12. The method according to claim 11, the tag having an amino acid sequence according to SEQ ID NO. 5, 6 or 7.
13. The method according claim 11 or 12, the p12 protein of the phage T4 being used as bacteriophage protein.
14. The method according to anyone of the claims 1 to 3, marked endotoxin being displaced from the bond with a bacteriophage protein and the marked endotoxin being subsequently detected.