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WO1992007254A2 - Utilisation de substances a ponts bisulfures - Google Patents

Utilisation de substances a ponts bisulfures Download PDF

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Publication number
WO1992007254A2
WO1992007254A2 PCT/EP1991/001877 EP9101877W WO9207254A2 WO 1992007254 A2 WO1992007254 A2 WO 1992007254A2 EP 9101877 W EP9101877 W EP 9101877W WO 9207254 A2 WO9207254 A2 WO 9207254A2
Authority
WO
WIPO (PCT)
Prior art keywords
substances
antibodies
oxidation
antigen
pbs
Prior art date
Application number
PCT/EP1991/001877
Other languages
German (de)
English (en)
Other versions
WO1992007254A3 (fr
Inventor
Kurt Lang
Thomas Subkowski
Juergen Schweden
Original Assignee
Basf Aktiengesellschaft
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.)
Filing date
Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1992007254A2 publication Critical patent/WO1992007254A2/fr
Publication of WO1992007254A3 publication Critical patent/WO1992007254A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to new applications of disulfide-bridged substances.
  • S-S bridges Most proteins and many peptides contain cysteine residues that can be linked by S-S bridges. These S-S bridges can be both intra- and intermolecular. If they are intermolecular, two or more protein molecules can be connected to one another.
  • Antibodies and suitable as calibration substances for the determination of molecular weights of higher molecular substances are provided.
  • the present invention is particularly suitable for those proteins or peptides which, in the native state, where the S-S bridges are generally intermolecular, are not immunogenic or are only extremely weak.
  • hirudin intermolecularly linked via S-S bridges is preferred for the production of antibodies against hirudin.
  • the starting point is the SH proteins or SH peptides, which e.g. in an aqueous buffer at pH values of 5 to 10, preferably 7.5 to 9, in a known manner using oxidizing agents such as oxygen or hydrogen peroxide to give S-S-di- or polymers.
  • the reaction is generally carried out at a temperature of 4 ° C to 45 ° C.
  • the optimal reaction temperature should be determined for each protein in a test range. Denatured and reduced proteins and peptides are particularly suitable for the production of intermolecular disulfide bridges.
  • the degree of polymerization can be determined by varying the concentration of SH protein or oxidizing agent in the oxi set dations approach. The degree of polymerization increases with increasing concentration of protein or peptide or oxidizing agent.
  • the reaction can e.g. by dilution, separation of the reaction components (e.g. gel filtration, precipitation, extraction, dialysis) or by acidification to pH values less than 5.0.
  • separation of the reaction components e.g. gel filtration, precipitation, extraction, dialysis
  • acidification to pH values less than 5.0.
  • non-proteinogenic substances with at least 2 SH groups can also be oligomerized in the same way and used according to the invention.
  • SH groups can subsequently be introduced by derivatizing other functional groups.
  • Compounds containing amino groups can e.g. by 2-iminothiolane, N-succinimidyl-S-acetylthioacetate (SATA) or N-succinimidyl-3'-2'-pyridyldithio-propionate (SPDP) to form SH-containing compounds.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3'-2'-pyridyldithio-propionate
  • the degree of polymerization should be such that the molecular weight range of the polymers includes the molecular weight of the substance to be examined.
  • the highest possible degree of polymerization should be achieved for the induction of antibodies.
  • polymerized proteins especially those with a
  • Aprotinin was dissolved to a concentration of 100 mg / ml in 6 M guanidinium chloride (GdmCl), 0.2 M Tris / HCl, 0.4 M dithiothreitol (DTT), pH 8.5 and incubated at 37 ° C. for 2 h.
  • the oxidation was initiated by adding 20% H 2 O 2 to a concentration of 1% in the aprotinin solution with constant stirring. After 30 min, the oxidation solution was diluted 10-fold by the addition of 20 mM Tris / HCl, pH 8.6 and then dialyzed against the same buffer.
  • the oxidation products are cross-linked oligomers of denatured aprotinin through intermolecular disulfide bridges.
  • Ribonuclease A was dissolved in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 to a concentration of 100 mg / ml and incubated for 2 hours.
  • the oxidation was carried out by adding 20% H 2 O 2 to a concentration of 1% to the ribonuclease solution with stirring. After 30 min, the oxidation solution was diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer.
  • the oxidation products are predominantly cross-linked ribonuclease oligomers via intermolecular disulfide bridges.
  • game 3
  • Desulfatohirudin produced by technical means was dissolved at 60 or 90 mg / ml in 6 M GdmCl, 0.2 M Tris / HCl, 0.4 M DTT, pH 8.7 and incubated at 37 ° C. for 2 h.
  • the oxidation was then carried out by adding 20% H2O2 to a concentration of 1% in the hirudin solution, with stirring. After 30 minutes, the oxidation batch was 1:10 diluted with water or with 20 mM Tris / HCl, pH 8.6 and dialyzed against water or the latter buffer.
  • the oxidation product consists predominantly of oligomers of denatured hirudin which are crosslinked via intermolecular disulfide bridges.
  • the oxidation products of the denatured and reduced proteins aprotinin, ribonuclease A and desulfatohirudin prepared in Examples 1, 2 and 3 were after dilution to 10 mg protein / ml in 20 mM Tris / HCl, pH 8.6 and dialysis against the same buffer SDS gel electrophoresis separated.
  • the oxidation products are primarily oligomers crosslinked via disulfide bridges, which differ in their molecular weight by two or more times the molecular weight of the respective monomeric protein (ribonuclease A: 13700 D; hirudin: 6960 D; aprotinin: 6150 D).
  • the semi-logarithmic plot of the molecular weights of the various oligomers as a function of the distance traveled in the gel shows that there is a linear dependence between these parameters over a wide range of molecular weights.
  • hirudin or desulfatohirudin is very suitable for therapeutic use as an anticoagulant.
  • antibodies are required for the sensitive detection of hirudin in the serum of treated patients by means of an ELISA specific for hirudin.
  • the induction of antibodies against hirudin has been problematic due to the low immunogenicity of hirudin.
  • Spinner et al. Thrombosis Research, 51, 617 (1988) antibodies to hirudin can only be produced in 2 of 11 sheep.
  • microtiter plates from Falcon were first coated with the antigen (oxidized desulfatohirudin or native desulfatohirudin) (2 h, 37 ° C., 5 ⁇ g / l 50 mM sodium carbonate buffer, pH 9.0). Unspecific binding sites on the microtiter plate were saturated by subsequent incubation with 3% BSA / PBS. The mixture was then incubated with various dilutions of the antiserum for 2 h and then the bound
  • antigen oxidized desulfatohirudin or native desulfatohirudin
  • Big-ET prepared by solid phase peptide synthesis was dissolved in 6 M GdmCl; 0.2 M Tris / HCL; 0.4 DTT; pH 8.7 dissolved to a concentration of 50 mg / ml and incubated for 2 hours at 37 ° C.
  • the oxidation was carried out by adding 20% H2O2 to a concentration of 1% with stirring. After 30 minutes the
  • Oxidation solution diluted 1:10 with 20 mM Tris / HCl and dialyzed against the same buffer.
  • the dialysis tube had an exclusion volume of 5000 Da, so that only cross-linked oligomers were retained intermolecularly by disulfide bridges and the by-products (intramolecular disulfide bridges) are separated.
  • the spleen was removed on day 45. Fusion and selection of the anti-big-ET secreting hybridomas is carried out according to the standard rules described (monoclonal antibodies: principles and practice; James W. Goding; Academic Press 1983).
  • Amiopterin was at a concentration of 20 mg / ml 50 mM triethanolamine pH 8.0; 10 mM DTT dissolved and with 2-iminothiolane (Final concentration 11.5 mg / ml) reacted for 4 hours at room temperature.
  • the SH groups of the aminopterin modified on the amino groups were oxidized by adding 10% H2O2 to a final concentration of 0.5%. After 30 minutes, the solution was diluted 1:10 with 50 mM triethanolamine pH 8.0 and dialyzed against the same buffer (exclusion volume of the dialysis membrane: 1000 Da).

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Endocrinology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Vascular Medicine (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne l'utilisation de substances reliées au niveau intermoléculaire par des ponts S-S afin de produire des anticorps et l'utilisation de ces substances comme substances d'étalonnage lors de la détermination de poids moléculaires.
PCT/EP1991/001877 1990-10-10 1991-10-01 Utilisation de substances a ponts bisulfures WO1992007254A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4032127.4 1990-10-10
DE19904032127 DE4032127A1 (de) 1990-10-10 1990-10-10 Verwendung von disulfidverbrueckten proteinen und peptiden

Publications (2)

Publication Number Publication Date
WO1992007254A2 true WO1992007254A2 (fr) 1992-04-30
WO1992007254A3 WO1992007254A3 (fr) 1992-06-11

Family

ID=6415997

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1991/001877 WO1992007254A2 (fr) 1990-10-10 1991-10-01 Utilisation de substances a ponts bisulfures

Country Status (2)

Country Link
DE (1) DE4032127A1 (fr)
WO (1) WO1992007254A2 (fr)

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4507233A (en) * 1981-04-22 1985-03-26 Oriental Yeast Co., Ltd. Colored molecular weight marker
US4713366A (en) * 1985-12-04 1987-12-15 The Ohio State University Research Foundation Antigenic modification of polypeptides
US5128319A (en) * 1987-08-28 1992-07-07 Board Of Regents, The University Of Texas System Prophylaxis and therapy of acquired immunodeficiency syndrome
JP2648855B2 (ja) * 1988-03-15 1997-09-03 大日本製薬株式会社 ペプチド、その抗体およびエンドセリンの定量
DK0380443T4 (da) * 1989-01-25 1999-11-29 Novartis Ag Monoklonale antistoffer specifikke for hirudin
CA2008122A1 (fr) * 1989-01-26 1990-07-26 Tetsuro Okabe Compose pharmaceutique pour traiter les maladies causees par la vasoconstriction
JP2750738B2 (ja) * 1989-06-13 1998-05-13 第一化学薬品株式会社 分子量マーカー
DE69129207T2 (de) * 1990-01-16 1998-10-08 Orgenics Ltd Peptide, von Virus-HIV-Hüllen-Glycoproteinen abstammend, deren Verwendung zum Nachweis einer Infektion dieser Viren und für die Impfung gegen AIDS

Also Published As

Publication number Publication date
WO1992007254A3 (fr) 1992-06-11
DE4032127A1 (de) 1992-04-16

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