WO2000008463A2 - Immunoadsorber for removing endotoxins - Google Patents

Immunoadsorber for removing endotoxins Download PDF

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Publication number
WO2000008463A2
WO2000008463A2 PCT/DE1999/002486 DE9902486W WO0008463A2 WO 2000008463 A2 WO2000008463 A2 WO 2000008463A2 DE 9902486 W DE9902486 W DE 9902486W WO 0008463 A2 WO0008463 A2 WO 0008463A2
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endotoxins
immunoadsorber
antiköφer
lps
antibodies
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PCT/DE1999/002486
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German (de)
French (fr)
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WO2000008463A3 (en
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Hans-Werner Heinrich
Udo Meyer
Andreas Hlinak
Peter Kruschke
Mirko Sasse
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Bioserv Ag
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Publication of WO2000008463A3 publication Critical patent/WO2000008463A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • Immunoadsorbents for removing endotoxins from liquids process for their preparation and use
  • Endotoxins are complex lipopolysaccharide molecules (LPS) that occur in the outer cell wall of Gramnegattver bacteria and are released when they decay.
  • LPS lipopolysaccharide molecules
  • the LPS is bound to a serum protein (LBP) and subsequently taken up via LPS receptors (CD 14) of the monocytes / acrophages and dendritic cells, the CD14 + positive cells being activated and massively different cytokines (IL-1, TNFa, Release IL-6, IL-8).
  • LPS-induced cytokine release leads, supported by the bacteria-induced complement activation, to life-threatening fever (therefore the LPS molecules are also called pyrogens), diffuse intravascular blood coagulation with the release of further "shock factors” (PAF, prostaglandins), changes in vascular permeability consequent loss of fluid in the tissues and consecutive drop in blood pressure, circulatory collapse and hemorrhagic necrosis and finally over multiple organ failure to death.
  • Diseases in which bacterial endotoxemia plays a predominant role are gram-negative sepsis, septic shock, acute liver failure, acute pancreatitis and acute respiratory distress syndrome (ARDS), although these diseases are still characterized by an extraordinarily high letai '' are characterized (up to 80%).
  • the endotoxins do not only enter the body through a bacterial invasion, but can also be applied exogenously as "pyrogenic" contamination with infusion solutions, infusion sets, biomaterials and with genetically engineered (bacterial) pharmaceutical products.
  • endotoxin level does not exceed 0.5 ng / ml, if there is no destruction of active substances in solution and if none for endotoxin elimination and or - Inactivating substances remain in solution as bio-incompatible impurities.
  • a decisive disadvantage of all of these methods is the inadequate efficiency and lack of specificity of the elimination, which in part also contains others in the solutions because they act nonspecifically, eliminates substances or at least reduces their solution concentration.
  • the materials previously patented for endotoxin binding are also characterized in that an exchange between free and bound endotoxin cannot be ruled out, so that the elimination cannot fall below a limit value.
  • the inadequate specificity of the endotoxin binding can be overcome by using antibodies against LPS which are directed against the cross-species “core polysaccharides” and / or the lipid A portion of the endotoxin molecules, and are equipped with a sufficiently high avidity, used in a sufficiently high concentration, Efficiently and specifically remove endotoxin from the solutions presented, polyclonal antibodies which can be used for this purpose are usually obtained from mammals, and it is known to the person skilled in the art that this is limited in the case of LPS antibodies, since endotoxins are extremely toxic to mammals, and moreover they are have a low intensity and cross-species anti-LPS antibodies are only induced in traces.
  • Hybridoma cells that synthesize monoclonal anti-LPS antibodies were successfully established. So far, various obstacles have stood in the way of their extensive use. In earlier patents, the expected high specificity was recognized without a doubt (DE 4113602, DE 4209988), but primarily the high costs were criticized as disadvantageous. The large-scale use of monoclonal antibodies, which are obtained as ascites in mice and other small rodents, is also not feasible for animal welfare reasons, although these antibodies are most likely to be present in the necessary concentrations. Antibody production using hybridomas in cell culture primarily causes the high costs, since the antibodies have to be enriched (concentrated) and the cell culture requires absolute sterile conditions either under serum-free conditions or in LPS-free serum.
  • the result of an artificial immunization can be high specific Antibody concentrations in plasma and egg yolk can be achieved. If the antibody production is carried out using SPF chickens, both the required concentrations of antibodies and the necessary amount can also be produced inexpensively for medical use.
  • a disadvantage of a medical application of “mammal” antibodies is that the human complement system is activated in serum or plasma. Activated complement can, under certain circumstances, cause serious diseases. This property is bound to the Fc receptor of the immunoglobulins, so that either these antibodies can only be used as Fab fragments or the Fc part of the antibody is bound to a carrier material and thus biologically inactivated (DE 19653669) .At least the use of Fab fragments makes the process considerably more expensive.
  • the invention was therefore based on the object of developing immunoadsorbents for removing endotoxins from liquids which do not have the disadvantages of the prior art described.
  • An essential component of the immunoadsorbents according to the invention are specific avian immunoglobulins of the IgY type which, in addition to a surprisingly found extraordinary avidity and overall specificity for endotoxins, do not activate the human complement system.
  • the immunoadsorber according to the invention is particularly suitable for removing acid-sensitive and alkali-sensitive, heat-sensitive, radiation-sensitive solutions with or without serum from LPS. Furthermore, it is used in genetically engineered pharmaceuticals or similar products that use gram-negative E. coli strains as a production source and therefore always a potential contamination with LPS may have displayed. In addition, the use in cleaning solutions with low final concentrations of active ingredient should be particularly emphasized because of the high specificity and avidity of the antibodies.
  • the avian antibodies are preferably covalently but also adsorbently coupled to natural or synthetic carrier materials or bound to them via spacers or linkers in such a way that no antibody leakage (diffusion of the antibody after the washout phase) is detectable.
  • the carrier materials can be used as particles for batch-type cleaning, as columns for large-volume cleaning or in the form of membranes (hoses, hollow fibers).
  • LPS 25 ⁇ g / ml LPS (E. coli J5) is added to PBS.
  • 1 ml LPS-PBS is mixed with 1 ml IgY-Sepharose and incubated for 1 h at room temperature with gentle stirring. After this time, the content of LPS in the buffer and bound to the gel is determined after separation using polyacrylamide gel electrophoresis ( Figures 1 and 2). It can be seen that homologous LPS was removed quantitatively from the buffer.
  • LPS of the following species were dissolved in PBS at a concentration of 25 ⁇ g / ml each: E. coli J5, E. coli 0111: B4, Salmonella enteritidis, Shigella fiexnerie and Bordetella bronchiseptica.
  • each of LPS-PBS is mixed with 1 ml of IgY-Sepharose against LPS from E. coli J5 and incubated for 1 h at room temperature with gentle stirring. After this time, the content of LPS in the buffer and bound to the gel is determined after separation by means of polyacrylic gel electrophoresis. LPS of heterologous origin is also removed quantitatively from the buffer.
  • IgY gel 1 ml is placed in a mini column and equiibrated by alternative washing with acidic and neutral PBS.
  • LPS LPS
  • E.coli J5 1 ml of LPS (E.coli J5) was added to 10 ml of donor plasma.
  • the plasma treated in this way was passed 5 times over the column.
  • the LPS content in the plasma was determined with the Limulus test to be ⁇ 50 pg / ml.

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Abstract

The invention relates to an immunoadsorber for specific removal of bacterial endotoxins from fluids or aqueous, crystalline and/or colloidal solutions, emulsions and body fluids (e.g. blood, plasma, lymph, cerebrospinal fluid) and/or fluids (e.g. serum, dialysate) obtained from body fluids. The endotoxins are bonded (in vitro, ex vivo or in situ) to avian endotoxin antibodies and eliminated from said fluid. A method for the production of the inventive immunoadsorber is also disclosed. The invention can be used in medicine, veterinary medicine, biomedicine, biomedical techniques, dentistry, pharmacy, biomedical research, pharmaceutical research, medical research, food production and, optionally, environmental protection.

Description

Immunadsorber zur Entfernung von Endotoxinen aus Flüssigkeiten, Verfahren zu ihrer Herstellung und Verwendung Immunoadsorbents for removing endotoxins from liquids, process for their preparation and use
Endotoxine sind komplexe Lipopolysaccharidmoieküle (LPS), die in der äusseren Zθllwand gramnegattver Bakterien vorkommen und bei deren Zerfall freigesetzt werden. In vivo wird das LPS an ein Serumprotein (LBP) gebunden und nachfolgend via LPS-Rezeptoren (CD 14) der Monozyten/ akrophagen und dendritischen Zellen aufgenommen, wobei die CD14+ positiven Zellen aktiviert werden und massiv verschiedene Zytokine (IL-1, TNFa, IL-6, IL-8) freisetzen. Die LPS-induzierte Zytokinfreisetzung führt, unterstützt durch die Bakterien-induzierte Komplementaktivierung zu lebensbedrohendem Fieber (daher werden die LPS- Moleküle auch als Pyrogene bezeoichnet), diffuser intravasaler Blutgerinnung mit Freisetzung weiterer „Schockfaktoren'' (PAF, Prostaglandine), Änderung der Gefäßpermeabilität mit nachfolgendem Flüssigkeitsverlust in die Gewebe und konsekutivem Blutdruckabfall, Kreislaufzusammenbruch und hämorrhagischen Nekrosen und schlußendlich über ein Multiorganversagen zum Tod. Krankheitsbilder, bei denen die bakterielle Endotoxämie eine prädominante Rolle spielt, sind die gramnegative Sepsis, der septische Schock, das akute Leberversageπ, die akute Pankreatitis und das akute respiratorische Distress-Syndrαm (ARDS), wobei diese Erkrankungen auch heute noch durch eine außerordentlich hohe Letai'rtät (bis zu 80%) charakterisiert sind. Die Endotoxine gelangen jedoch nicht nur durch einen bakterielle Invasion in den Körper, sondern können auch als „pyrogene'' Verunreinigung mit Infusionslösungen, Infusionsbestecken, Biomaterialien und mit gentechnisch (bakteriell) erzeugten pharmazeutischen Produkten exogen appliziert werden.Endotoxins are complex lipopolysaccharide molecules (LPS) that occur in the outer cell wall of Gramnegattver bacteria and are released when they decay. In vivo, the LPS is bound to a serum protein (LBP) and subsequently taken up via LPS receptors (CD 14) of the monocytes / acrophages and dendritic cells, the CD14 + positive cells being activated and massively different cytokines (IL-1, TNFa, Release IL-6, IL-8). The LPS-induced cytokine release leads, supported by the bacteria-induced complement activation, to life-threatening fever (therefore the LPS molecules are also called pyrogens), diffuse intravascular blood coagulation with the release of further "shock factors" (PAF, prostaglandins), changes in vascular permeability consequent loss of fluid in the tissues and consecutive drop in blood pressure, circulatory collapse and hemorrhagic necrosis and finally over multiple organ failure to death. Diseases in which bacterial endotoxemia plays a predominant role are gram-negative sepsis, septic shock, acute liver failure, acute pancreatitis and acute respiratory distress syndrome (ARDS), although these diseases are still characterized by an extraordinarily high letai '' are characterized (up to 80%). However, the endotoxins do not only enter the body through a bacterial invasion, but can also be applied exogenously as "pyrogenic" contamination with infusion solutions, infusion sets, biomaterials and with genetically engineered (bacterial) pharmaceutical products.
Um Endotoxinverunreinigungen zu entfernen, werden Verfahren, wie die Hitzesterilisation, Dampfsterilisation, Bestrahlen mit γ-Strahlen, die Behandlung mit Säuren oder Laugen (z.B. US 3644175 und US 3659027), die Adsorption an uπspezifisch bindende Substanzen (wie Aktivkohle, lonenaustauschemarze, Polymyxin B, DEAE (US 3897309) oder andere stickstoffhaltige Gruppen (US 4381239), A ine (EP 0308239, EP 0333474, DE 19648954), Polyäthylenimin (DE 4209988), und Surfaktantmoleküle (US 4412985)) angewandt. Der größte Teil dieser Verfahren ist ausreichend, wenn der verbleibende Endotoxinspiegel 0,5 ng/ml nicht überschreitet, wenn keine Zerstörung von in Lösung vorhandenen Wirksubstanzen zu verzeichnen ist, und wenn keine für die Endotoxinelimination und oder - inaktivierung eingesetzten Substanzen als bioinkompatible Verunreinigung in Lösung verbleiben. Ein entscheidender Nachteil aller dieser Verfahren besteht in der unzureichenden Effizienz und fehlenden Spezifität der Elimination, die zum Teil auch andere in den Lösungen enthaltenen, weil unspezifisch wirkend, Substanzen eliminiert oder zumindest deren Lösungskonzentration reduziert. Die bisher für die Endotoxinbindung patentierten Materialien sind ausserdem dadurch charakterisiert, daß ein Austausch zwischen freiem und gebundenem Endotoxin nicht ausgeschlossen werden kann, so daß die Elimination einen Grenzwert nicht unterschreiten kann.In order to remove endotoxin impurities, processes such as heat sterilization, steam sterilization, irradiation with γ-rays, treatment with acids or alkalis (e.g. US 3644175 and US 3659027), adsorption on uπspecific binding substances (such as activated carbon, ion exchange mares, polymyxin B, DEAE (US 3897309) or other nitrogen-containing groups (US 4381239), A ine (EP 0308239, EP 0333474, DE 19648954), polyethylene imine (DE 4209988), and surfactant molecules (US 4412985)) were used. Most of these procedures are sufficient if the remaining endotoxin level does not exceed 0.5 ng / ml, if there is no destruction of active substances in solution and if none for endotoxin elimination and or - Inactivating substances remain in solution as bio-incompatible impurities. A decisive disadvantage of all of these methods is the inadequate efficiency and lack of specificity of the elimination, which in part also contains others in the solutions because they act nonspecifically, eliminates substances or at least reduces their solution concentration. The materials previously patented for endotoxin binding are also characterized in that an exchange between free and bound endotoxin cannot be ruled out, so that the elimination cannot fall below a limit value.
Die unzureichende Spezifität der Endotoxinbindung kann durch den Einsatz von Antikörpern gegen LPS überwunden werden, die gegen die speziesübergreifenden „Core Polysaccharide" und/oder den Lipid A-Anteil der Endotoxinmoleküle gerichtet sind, und mit ausreichend hoher Avidität ausgestattet, in ausreichend hoher Konzentration eingesetzt, Endotoxin effizient und spezifisch aus den vorgelegten Lösungen entfernen. Dafür einsetzbare polyklonale Antikörper werden üblicherweise von Säugetieren gewonnen. Dem Fachmann ist bekannt, daß dies im Falle von LPS- Antikörpern auf Grenzen stößt, da Endotoxine für Säuger außerordentlich toxisch sind, sie darüber hinaus auch nur eine geringe Aπtigenität aufweisen und speziesübergreifende anti-LPS-Antikörper nur in Spuren induziert werden.The inadequate specificity of the endotoxin binding can be overcome by using antibodies against LPS which are directed against the cross-species “core polysaccharides” and / or the lipid A portion of the endotoxin molecules, and are equipped with a sufficiently high avidity, used in a sufficiently high concentration, Efficiently and specifically remove endotoxin from the solutions presented, polyclonal antibodies which can be used for this purpose are usually obtained from mammals, and it is known to the person skilled in the art that this is limited in the case of LPS antibodies, since endotoxins are extremely toxic to mammals, and moreover they are have a low intensity and cross-species anti-LPS antibodies are only induced in traces.
Es gelang die Etablierung von Hybridomzellen, die monoklonale anti-LPS-Antikörper synthetisieren. Ihrem umfangreichen Einsatz standen bisher verschiedene Hindemisse im Wege. In früheren Patentschriften wurde zwar die zu erwartende hohe Spezifität zweifelsfrei anerkannt (DE 4113602, DE 4209988) aber vorrangig die hohen Kosten als nachteilig bemängelt. Der großtechnische Einsatz monokloπaler Antikörper, die als Ascites in Mäusen und anderen Kleinnagern gewonnen werden, ist auch aus Gründen des Tierschutzes nicht zu realisieren, obwohl diese Antikörper am ehesten in den notwendigen Konzentrationen vorliegen dürften. Die Antikörperproduktion mittel Hybridoms in Zellkultur bedingt vorrangig die hohen Kosten, da die Antikörper angereichert (konzentriert) werden müssen und die Zellkultur absolute Sterilbedingungen entweder unter serumfreien Bedingungen oder in LPS-freien Serum erfordert.Hybridoma cells that synthesize monoclonal anti-LPS antibodies were successfully established. So far, various obstacles have stood in the way of their extensive use. In earlier patents, the expected high specificity was recognized without a doubt (DE 4113602, DE 4209988), but primarily the high costs were criticized as disadvantageous. The large-scale use of monoclonal antibodies, which are obtained as ascites in mice and other small rodents, is also not feasible for animal welfare reasons, although these antibodies are most likely to be present in the necessary concentrations. Antibody production using hybridomas in cell culture primarily causes the high costs, since the antibodies have to be enriched (concentrated) and the cell culture requires absolute sterile conditions either under serum-free conditions or in LPS-free serum.
Erstaunlicherweise vertragen Vögel große Mengen von LPS ohne klinische Reaktion. Im Ergebnis einer künstlichen Immunisierung können hohe spezifische Antiköφerkonzentrationen im Plasma und Eidotter erzielt werden. Wird die Antikörpeφroduktion unter Verwendung von SPF-Hühnern durchgeführt, können sowohl die erforderlichen Konzentrationen an Antikörpern, als auch die notwendige Menge kostengünstig auch für eine medizinische Anwendung hergestellt werden.Amazingly, birds tolerate large amounts of LPS without a clinical response. The result of an artificial immunization can be high specific Antibody concentrations in plasma and egg yolk can be achieved. If the antibody production is carried out using SPF chickens, both the required concentrations of antibodies and the necessary amount can also be produced inexpensively for medical use.
Nachteilig für eine medizinische Anwendung von „Säugetier"-Antiköφern ist. daß in Serum oder Plasma die Aktivierung des menschlichen Komplementsystems erfolgt. Aktiviertes Komplement kann unter bestimmten Umständen schwere Erkrankungen hervorrufen. Diese Eigenschaft ist an den Fc-Rezeptor der Immunglobuline gebunden, so daß entweder diese Antikörper nur als Fab-Fragmente eingesetzt werden können oder der Fc-Teil des Antiköφers an ein Trägermaterial gebunden und damit biologisch inaktiviert wird (DE 19653669). Zumindest die Verwendung von Fab-Fragmeπten verteuert das Verfahren beträchtlich.A disadvantage of a medical application of “mammal” antibodies is that the human complement system is activated in serum or plasma. Activated complement can, under certain circumstances, cause serious diseases. This property is bound to the Fc receptor of the immunoglobulins, so that either these antibodies can only be used as Fab fragments or the Fc part of the antibody is bound to a carrier material and thus biologically inactivated (DE 19653669) .At least the use of Fab fragments makes the process considerably more expensive.
Der Erfindung lag deshalb die Aufgabe zugrunde, Immunadsorber zur Entfernung von Endotoxinen aus Flüssigkeiten zu entwickeln, die die Nachteile des beschriebenen Standes der Technik nicht aufweisen.The invention was therefore based on the object of developing immunoadsorbents for removing endotoxins from liquids which do not have the disadvantages of the prior art described.
Die Erfindung wird gemäß den Patentansprüchen 1, 7 und 9 realisiert. Die Unteransprüche sind Vorzugswarianten. Wesentlicher Bestandteil der erfindungsgemäßen Immunadsorber sind spezifische aviäre Immunglobuline vom Typ IgY, die neben einer überraschend festgestellten außerordentlichen Avidität und übergreifenden Spezifität zu Endotoxinen das menschliche Komplementsystem nicht aktivieren.The invention is implemented in accordance with patent claims 1, 7 and 9. The subclaims are preferred variants. An essential component of the immunoadsorbents according to the invention are specific avian immunoglobulins of the IgY type which, in addition to a surprisingly found extraordinary avidity and overall specificity for endotoxins, do not activate the human complement system.
Die beschriebene Serospezifität von monoklonaien Antikörpern gegen LPS, erlauben ausschließlich die sehr selektive Elimination definierter LPS-Moleküle. Durch die Verwendung von Antigenen gegen die speziesübergreifende „Core-Polysaccharide" und/oder den Lipid-A-Anteil des Endotoxinmoleküls bei der Immunisierung der Hühner werden polyklonale Antiköφer erzeugt, die erfind ungsgemäß eine breites Spektrum von LPS-Molekülen erfassen und somit den Nachteil, der für monokloπale Antiköper beschrieben worden ist, überwinden.The described serospecificity of monoclonal antibodies against LPS only allow the very selective elimination of defined LPS molecules. By using antigens against the cross-species “core polysaccharides” and / or the lipid A portion of the endotoxin molecule in the immunization of the chickens, polyclonal antibodies are generated which, according to the invention, cover a broad spectrum of LPS molecules and thus have the disadvantage of which has been described for monoclonal antibodies.
Der erfindungsgemäße Immunadsorber ist besonders geeignet, säure- und laugenempfindliche, hitzesensitive, strahlenempfindliche Lösungen mit oder ohne Serum von LPS zu befreien. Weiterhin ist der Einsatz bei gentechnisch hergestellten Pharmaka oder ähnlichen Produkten, die als Produktionsquelle gramnegative E. coli- Stämmβ verwenden und deshalb immer eine potentielle Verunreinigung mit LPS aufweisen können, angezeigt. Außerdem soll der Einsatz beim Reinigen von Lösungen mit schwachen Endkonzentrationen an Wirkstoff wegen der hohen Spezifität und Avidität der Antikörper besonders hervorgehoben werden.The immunoadsorber according to the invention is particularly suitable for removing acid-sensitive and alkali-sensitive, heat-sensitive, radiation-sensitive solutions with or without serum from LPS. Furthermore, it is used in genetically engineered pharmaceuticals or similar products that use gram-negative E. coli strains as a production source and therefore always a potential contamination with LPS may have displayed. In addition, the use in cleaning solutions with low final concentrations of active ingredient should be particularly emphasized because of the high specificity and avidity of the antibodies.
Die aviären Antiköφer werden vorzugsweise kovalent aber auch adsoφtiv an natürliche oder synthetische Trägermaterialien gekoppelt oder über Spacer oder Linker an diese so gebunden, daß kein Antikörperleakage (Abdiffundieren des Antikörpers nach der Auswaschphase) nachweisbar ist. Die Trägermaterialien können als Partikel für batch-type-Reiπiguπg, als Säulen für Größvolumenreinigung oder in Form von Membranen (Schläuche, Hohlfasern) eingesetzt werden.The avian antibodies are preferably covalently but also adsorbently coupled to natural or synthetic carrier materials or bound to them via spacers or linkers in such a way that no antibody leakage (diffusion of the antibody after the washout phase) is detectable. The carrier materials can be used as particles for batch-type cleaning, as columns for large-volume cleaning or in the form of membranes (hoses, hollow fibers).
AUSFÜHRUNGSBEISPIEL 1EMBODIMENT 1
Herstellung spezifischer IgY durch Immunisierung von Hühnern mit LPS: 10 Tage nach Aufstauung werden 4 Hühner mit je 100 μg LPS (Escherichia coli J5, Fa. Sigma) immunisiert. Das Antigen ist in 0,5 ml PBS gelöst und wird unmittelbar vor Immunisierung in 0,5 ml kompletten Freundschen Adjuvans dispergiert. Die Immunisierung erfolgt i.m. in 5 Depots. 3 Wochen nach der ersten Immunisierung werden die Hühner mit gleicher Antigendosis unter Verwendung inkompletten Freundschen Adjuvans erneut immunisiert. Nach weiteren 3 Wochen erfolgt eine zweite Boosterung. Ab 8. Woche nach Immunisierungsbeginn werden die Eier für die Gewinnung spezifischer IgY gesammelt. Dazu wird das Eidotter präpariert und die darin enthaltenen Antikörper nach mehreren Gefrier-Tau-Zyklen durch fraktionierte Fällung gewonnen. Pro Dotter können zwischen 20 und 50 mg IgY präpariert werden, 5 - 10 mg sind üblicherweise spezifische Antikörper.Production of specific IgY by immunizing chickens with LPS: 10 days after the build-up, 4 chickens are immunized with 100 μg LPS (Escherichia coli J5, Sigma). The antigen is dissolved in 0.5 ml PBS and is dispersed in 0.5 ml complete Freund's adjuvant immediately before immunization. The immunization is carried out i.m. in 5 depots. 3 weeks after the first immunization, the chickens are again immunized with the same antigen dose using incomplete Freund's adjuvant. After a further 3 weeks there is a second booster. From the 8th week after the start of immunization, the eggs are collected for the production of specific IgY. For this purpose, the egg yolk is prepared and the antibodies contained therein are obtained by fractional precipitation after several freeze-thaw cycles. Between 20 and 50 mg IgY can be prepared per yolk, 5 - 10 mg are usually specific antibodies.
Kopplung an CNBr-Sepharose (Fa. Pharmacia):Coupling to CNBr-Sepharose (Pharmacia):
10 mg spezifischer Antikörper werden nach Vorschrift des Herstellers an 5 ml CNBr- Sepharose gekoppelt.10 mg of specific antibodies are coupled to 5 ml of CNBr-Sepharose according to the manufacturer's instructions.
Entfernung von LPS aus Puffer (Batch-Verfahreπ):Removal of LPS from buffer (batch process):
PBS wird mit 25 μg/ml LPS (E. coli J5) versetzt. 1 ml LPS-PBS wird mit 1 ml IgY- Sepharose gemischt und für 1 h bei Raumtemperatur unter leichtem Rühren inkubiert. Nach dieser Zeit wird der Gehalt an LPS im Puffer und am Gel gebunden nach Auftrennung mittels Polyacrylamidgel-Elektrophorese bestimmt (Abbildungen 1 und 2). Es ist ersichtlich, daß homologes LPS quantitativ aus dem Puffer entfernt wurde.25 μg / ml LPS (E. coli J5) is added to PBS. 1 ml LPS-PBS is mixed with 1 ml IgY-Sepharose and incubated for 1 h at room temperature with gentle stirring. After this time, the content of LPS in the buffer and bound to the gel is determined after separation using polyacrylamide gel electrophoresis (Figures 1 and 2). It can be seen that homologous LPS was removed quantitatively from the buffer.
AUSFÜHRUNGSBEISPIEL 2EMBODIMENT 2
10 mg spezifische Antiköφer wurden nach Vorschrift des Herstellers an 5 ml Affi- Prep Hz Gel (Fa. BIO-RAD) gekoppelt.10 mg of specific antibodies were coupled to 5 ml of Affi-Prep Hz Gel (BIO-RAD) according to the manufacturer's instructions.
LPS folgender Spezies wurden in einer Konzentration von je 25 μg/mi in PBS gelöst: E. coli J5, E. coli 0111:B4, Salmonella enteritidis, Shigella fiexnerie und Bordetella bronchiseptica.LPS of the following species were dissolved in PBS at a concentration of 25 μg / ml each: E. coli J5, E. coli 0111: B4, Salmonella enteritidis, Shigella fiexnerie and Bordetella bronchiseptica.
Je 1 ml LPS-PBS wird mit 1 ml IgY-Sepharose gegen LPS von E.coli J5 gemischt und für 1 h bei Raumtemperatur unter leichtem Rühren inkubiert. Nach dieser Zeit wird der Gehalt an LPS im Puffer und am Gel gebunden nach Auftrennung mittels Polyacryla idgel-Elektrophorese bestimmt. Auch LPS heterologer Herkunft wird quantitativ aus dem Puffer entfernt.1 ml each of LPS-PBS is mixed with 1 ml of IgY-Sepharose against LPS from E. coli J5 and incubated for 1 h at room temperature with gentle stirring. After this time, the content of LPS in the buffer and bound to the gel is determined after separation by means of polyacrylic gel electrophoresis. LPS of heterologous origin is also removed quantitatively from the buffer.
AUSFÜHRUNGSBEISPIeL 3EXAMPLE 3
5 ml ONB-Sepharose 4FF (39 μmol/ml Gel) wird aus Aceton in Wasser übertragen und mit 5 ml IgY-Lösung (22 mg/ml) vermischt. Der pH wird auf 3,0 eingestellt und die Suspension für 1 h bei Raumtemperatur leicht bewegt. Nach dem Koppeln wird das Gel gewaschen und unter Verwendung von 1 M Ethanolamin in 0,1 m Boratpuffer geblockt. Nach erneutem Waschen wird das Gel für 3 d in Boratpuffer (pH 8,3) leicht bewegt. Nach alkalische Hydrolyse konnte eine gekoppelte Proteinkonzentration von 12 mg/ml Gel ermittelt werden.5 ml of ONB-Sepharose 4FF (39 μmol / ml gel) is transferred from acetone to water and mixed with 5 ml of IgY solution (22 mg / ml). The pH is adjusted to 3.0 and the suspension is gently agitated for 1 h at room temperature. After coupling, the gel is washed and blocked using 1 M ethanolamine in 0.1 M borate buffer. After washing again, the gel is gently agitated for 3 d in borate buffer (pH 8.3). After alkaline hydrolysis, a coupled protein concentration of 12 mg / ml gel could be determined.
1 ml IgY-Gel wird in eine Minisäule verbracht und durch alternatives Waschen mit saurem und neutralem PBS equiiibriert.1 ml of IgY gel is placed in a mini column and equiibrated by alternative washing with acidic and neutral PBS.
10 ml Spenderplasma wurden mit 1 μg LPS (E.coli J5) versetzt. Das so behandelte Plasma wurde 5 mal über die Säule geleitet. Nach dieser Behandlung wurde der LPS-Gehait im Plasma mit dem Limulus-Test mit < 50 pg/ml bestimmt. 1 ml of LPS (E.coli J5) was added to 10 ml of donor plasma. The plasma treated in this way was passed 5 times over the column. After this treatment, the LPS content in the plasma was determined with the Limulus test to be <50 pg / ml.

Claims

Patentansprüche: Claims:
1. Immunadsorber zur Entfernung von Endotoxinen aus Flüssigkeiten gekennzeichnet durch Trägermaterialien aus organischen oder synthetischen Polymeren mit gebundenen Antiköφem, die gegen Endotoxine gerichtet sind.1. Immunoadsorbent for removing endotoxins from liquids characterized by carrier materials made of organic or synthetic polymers with bound antibodies which are directed against endotoxins.
2. Immunadsorber nach Anspruch 1 , dadurch gekennzeichnet, daß die Aπtiköφer aviäre Antiköφer des Typs IgY sind.2. Immunoadsorber according to claim 1, characterized in that the Aπtiköφer are avian Antiköφer of the IgY type.
3. Immunadsorber nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Antiköφer überwiegend gegen die spezies übergreifende „Core Polysaccharide" und/oder den Lipid A-Anteil der Endotoxine gerichtet sind.3. Immunoadsorber according to claim 1 and 2, characterized in that the Antiköφer are mainly directed against the species-spanning "core polysaccharides" and / or the lipid A portion of the endotoxins.
4. Imunadsorber nach Anspruch 1 bis 3 dadurch gekennzeichnet, daß die Antiköφer kovaient oder adsorptiv an das Trägermateriaigebunden sind.4. Imunadsorber according to claim 1 to 3, characterized in that the Antiköφer covalently or adsorptively bound to the support material.
5. Immunadsorber nach Anspruch 1 bis 4,dadurch gekennzeichnet, daß die Antiköφer über Spacer oder Linker an das Trägermaterial fixiert sind.5. Immunoadsorber according to claim 1 to 4, characterized in that the Antiköφer are fixed to the carrier material via spacers or linkers.
6. Immunadsorber nach Anspruch 1 bis 5/dadurch gekennzeichnet, daß das Trägermaterial aus Membranen oder Partikeln aus z.B. Zellulosederivaten oder Acryiaten besteht.6. Immunoadsorber according to claim 1 to 5 / characterized in that the carrier material consists of membranes or particles of, for example, cellulose derivatives or acrylates.
7. Verfahren zur Herstellung von Immunadsorbern gemäß der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß an Trägermaterialien aus organischen oder synthetischen Polymeren, Antiköφer, die gegen Endotoxine gerichtet sind, kovaient oder adsoφtiv gekoppelt werden.7. A process for the preparation of immunoadsorbents according to claims 1 to 6, characterized in that antibodies, which are directed against endotoxins, are covalently or adsorptively coupled to carrier materials made of organic or synthetic polymers.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß die Antiköφer durch Immunisierung vorzugsweise von Kleinsäugern, wie Mäuse, Ratten oder Kaninchen oder Vögeln, wie z.B. Hühnern, mit dem entsprechenden Antigen hergestellt werden.8. The method according to claim 7, characterized in that the Antiköφer preferably by immunization of small mammals, such as mice, rats or rabbits or birds, such as. Chickens, with the appropriate antigen.
9. Verwendung von Immunadsorbern gemäß Ansprüchen 1 bis 6 als wirksamer Bestandteil einer Vorrichtung zur Entfernung von Endotoxinen aus Flüssigkeiten, wie Blutplasma oder pharmazeutischen Zubereitungen. 9. Use of immunoadsorbents according to claims 1 to 6 as an effective component of a device for removing endotoxins from liquids, such as blood plasma or pharmaceutical preparations.
0. Verwendung nach Anspruch 9, dadurch gekennzeichnet, daß die Immunadsorber für die Plasmapherese bei Patienten mit Endotoxämie und/oder Sepsis eingesetzt werden. 0. Use according to claim 9, characterized in that the immune adsorbers are used for plasmapheresis in patients with endotoxemia and / or sepsis.
PCT/DE1999/002486 1998-08-05 1999-08-05 Immunoadsorber for removing endotoxins WO2000008463A2 (en)

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US8003313B2 (en) 2005-01-21 2011-08-23 Hyglos Invest Gmbh Method for detecting and removing endotoxin
RU2489172C1 (en) * 2012-06-22 2013-08-10 Дятленко Оксана Валерьевна Method of therapeutic discrete plasmapheresis with extracorporeal erythroxyte and leukocyte modification with interferon inducers, antioxidants and cell protectors
WO2021063708A1 (en) 2019-09-30 2021-04-08 Hemotune Ag Assembly for extracorporeal treatment of body fluids

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DE10258944A1 (en) 2002-12-17 2004-07-01 B. Braun Medizintechnologie Gmbh Device for removing bacterial toxins from blood or plasma, useful for treating sepsis, also for analysis and diagnosis, includes hollow fiber material for selective binding of the toxins
ATE356643T1 (en) * 2004-06-03 2007-04-15 Braun Medizintechnologie Gmbh DEVICE FOR THE REMOVAL OF BACTERIAL LIPOPOLYSACCHARIDES AND/AND LIPOTEICHO ACIDS FROM PROTEIN-CONTAINING LIQUIDS AND USE FOR PRODUCING AN AGENT FOR THE REMOVAL OF THESE SUBSTANCES
EP3477300A1 (en) 2017-10-25 2019-05-01 Euroimmun Medizinische Labordiagnostika AG Cellulose-based immunoadsorber

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US7585620B2 (en) 2002-06-24 2009-09-08 Profos Ag Method for identifying and for extracting endotoxin
US7858299B2 (en) 2002-06-24 2010-12-28 Hyglos Invest Gmbh Method for detecting and for removing endotoxin
US8003313B2 (en) 2005-01-21 2011-08-23 Hyglos Invest Gmbh Method for detecting and removing endotoxin
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US8822641B2 (en) 2005-01-21 2014-09-02 Hyglos Invest Gmbh Method for detecting and removing endotoxin
US9229002B2 (en) 2005-01-21 2016-01-05 Hyglos Invest Gmbh Nucleic acids encoding bacteriophage tail proteins
RU2489172C1 (en) * 2012-06-22 2013-08-10 Дятленко Оксана Валерьевна Method of therapeutic discrete plasmapheresis with extracorporeal erythroxyte and leukocyte modification with interferon inducers, antioxidants and cell protectors
WO2021063708A1 (en) 2019-09-30 2021-04-08 Hemotune Ag Assembly for extracorporeal treatment of body fluids

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