WO2000008463A2 - Immunoadsorbant permettant d'eliminer des endotoxines de liquides, procede de preparation et d'utilisation dudit immunoadsorbant - Google Patents

Immunoadsorbant permettant d'eliminer des endotoxines de liquides, procede de preparation et d'utilisation dudit immunoadsorbant 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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WO
WIPO (PCT)
Prior art keywords
endotoxins
immunoadsorber
antiköφer
lps
antibodies
Prior art date
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PCT/DE1999/002486
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German (de)
English (en)
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WO2000008463A3 (fr
Inventor
Hans-Werner Heinrich
Udo Meyer
Andreas Hlinak
Peter Kruschke
Mirko Sasse
Original Assignee
Bioserv Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Bioserv Ag filed Critical Bioserv Ag
Priority to AU64632/99A priority Critical patent/AU6463299A/en
Publication of WO2000008463A2 publication Critical patent/WO2000008463A2/fr
Publication of WO2000008463A3 publication Critical patent/WO2000008463A3/fr

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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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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Peptides Or Proteins (AREA)
  • External Artificial Organs (AREA)

Abstract

Immunoadsorbant permettant d'éliminer spécifiquement des endotoxines bactériennes de liquides, de solutions aqueuses, cristallines et/ou colloïdales, d'émulsions et de liquides corporels (tels que par exemple le sang, le plasma, la lymphe, le liquide céphalo-rachidien) et/ou de liquides obtenus à partir de liquides corporels (tels que par exemple le sérum ou le dialysat). Les endotoxines sont liées (in vitro, ex vivo ou in situ) à des anticorps anti-endotoxines aviaires et donc éliminés du liquide. Un procédé de préparation dudit immunoadsorbant est également décrit. La présente invention peut être utilisée dans les domaines suivants: médecine, médecine vétérinaire, techniques biomédicales, médecine dentaire, pharmacie, recherche biologique, pharmaceutique et médicale, fabrication des denrées alimentaires et éventuellement environnement.
PCT/DE1999/002486 1998-08-05 1999-08-05 Immunoadsorbant permettant d'eliminer des endotoxines de liquides, procede de preparation et d'utilisation dudit immunoadsorbant WO2000008463A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64632/99A AU6463299A (en) 1998-08-05 1999-08-05 Immunoadsorber for removing endotoxins from fluids, method for the production and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19839429 1998-08-05
DE19839429.2 1998-08-05

Publications (2)

Publication Number Publication Date
WO2000008463A2 true WO2000008463A2 (fr) 2000-02-17
WO2000008463A3 WO2000008463A3 (fr) 2000-04-20

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AU (1) AU6463299A (fr)
DE (1) DE19938394A1 (fr)
WO (1) WO2000008463A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7585620B2 (en) 2002-06-24 2009-09-08 Profos Ag Method for identifying and for extracting endotoxin
US8003313B2 (en) 2005-01-21 2011-08-23 Hyglos Invest Gmbh Method for detecting and removing endotoxin
RU2489172C1 (ru) * 2012-06-22 2013-08-10 Дятленко Оксана Валерьевна Способ проведения лечебного дискретного плазмафереза с экстракарпоральной модификацией эритроцитов и лейкоцитов индукторами интерферона, антиоксидантами и протекторами клеток
WO2021063708A1 (fr) 2019-09-30 2021-04-08 Hemotune Ag Ensemble pour le traitement extracorporel de fluides corporels

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10258944A1 (de) 2002-12-17 2004-07-01 B. Braun Medizintechnologie Gmbh Vorrichtung zur Entfernung von bakteriellen Lipopolysacchariden oder/und Lipoteichonsäuren aus proteinhaltigen Flüssigkeiten sowie deren Verwendung zur Behandlung von Sepsis
ATE356643T1 (de) * 2004-06-03 2007-04-15 Braun Medizintechnologie Gmbh Vorrichtung zur entfernung von bakteriellen lipopolysacchariden oder/und lipoteichonsäuren aus proteinhaltigen flüssigkeiten sowie verwendung zur herstellung eines mittels zur entfernung dieser stoffe
EP3477300A1 (fr) 2017-10-25 2019-05-01 Euroimmun Medizinische Labordiagnostika AG Immunoadsorbant à base de cellulose

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4113602A1 (de) * 1991-04-23 1992-10-29 Falkenhagen Dieter Dr Sc Med Endotoxinadsorber und verfahren zu seiner herstellung
DE19653669A1 (de) * 1996-12-13 1998-06-18 Affina Immuntechnik Gmbh Adsorptionsmatrix, ihre Herstellung und ihre Verwendung zur Entfernung von Immunglobulinen aus biologischen Flüssigkeiten

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0634633A (ja) * 1992-07-14 1994-02-10 Toyobo Co Ltd 鶏卵抗体固定化担体およびその製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4113602A1 (de) * 1991-04-23 1992-10-29 Falkenhagen Dieter Dr Sc Med Endotoxinadsorber und verfahren zu seiner herstellung
DE19653669A1 (de) * 1996-12-13 1998-06-18 Affina Immuntechnik Gmbh Adsorptionsmatrix, ihre Herstellung und ihre Verwendung zur Entfernung von Immunglobulinen aus biologischen Flüssigkeiten

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 120, no. 19, 9. Mai 1994 (1994-05-09) Columbus, Ohio, US; abstract no. 239646, XP002129937 & PATENT ABSTRACTS OF JAPAN vol. 18, no. 253 (P-1737), 10. Februar 1994 (1994-02-10) & JP 06 034633 A (TOYOBO COMPANY LTD) *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US8329393B2 (en) 2005-01-21 2012-12-11 Hyglos Invest Gmbh Method for detecting and removing endotoxin
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 (ru) * 2012-06-22 2013-08-10 Дятленко Оксана Валерьевна Способ проведения лечебного дискретного плазмафереза с экстракарпоральной модификацией эритроцитов и лейкоцитов индукторами интерферона, антиоксидантами и протекторами клеток
WO2021063708A1 (fr) 2019-09-30 2021-04-08 Hemotune Ag Ensemble pour le traitement extracorporel de fluides corporels

Also Published As

Publication number Publication date
AU6463299A (en) 2000-02-28
WO2000008463A3 (fr) 2000-04-20
DE19938394A1 (de) 2000-02-24

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