WO1989008705A1 - Supports pour proteines et acides amines - Google Patents

Supports pour proteines et acides amines Download PDF

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Publication number
WO1989008705A1
WO1989008705A1 PCT/GB1989/000244 GB8900244W WO8908705A1 WO 1989008705 A1 WO1989008705 A1 WO 1989008705A1 GB 8900244 W GB8900244 W GB 8900244W WO 8908705 A1 WO8908705 A1 WO 8908705A1
Authority
WO
WIPO (PCT)
Prior art keywords
support matrix
matrix according
support
inorganic material
rigid
Prior art date
Application number
PCT/GB1989/000244
Other languages
English (en)
Inventor
Eric Robinson
Original Assignee
Eric Robinson
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 Eric Robinson filed Critical Eric Robinson
Publication of WO1989008705A1 publication Critical patent/WO1989008705A1/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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

  • the present invention relates to new compositions of matter upon which to reversibly or irreversibly attach amino acids, nuceic acids or proteins.
  • pore size or pore diameter of porous supports is specified generally in the range extending up to 1000 Angstroms, though U.K. Patent 4,141,857 discloses a support matrix possessing pore diameters from 100 to about 55,000 Angstroms.
  • the object of the present invention is to provide an improved support for biologically active molecules which has good mechanical properties and largely maintains or improves the activity and stability of the molecules supported thereon. - 2 -
  • a support matrix upon which to reversibly or irreversibly fix amino acids, nucleic acids or proteins being a composite comprising a rigid macroporous inorganic material, in the form of an aggregation of microparticles or with a cellular structure, having a pore volume of at least 0.4 ml/ml made up of interconnecting pores having diameters of between 0.05 and 50 micrometres, and an organic polymer bearing hydroxyl, amino or carboxyl groups.
  • the porous inorganic material is suitably an aggregation of micro ⁇ particles of silica such as those prepared according to my copending U.K. Application No. 8702285 or of microparticles of alumina, silica alumina, an aluminosilicate or titania, or a cellular structure such as pumice, a porous ceramic made by foaming or a cellular silica.
  • alumina silica alumina
  • an aluminosilicate or titania or a cellular structure
  • a porous ceramic made by foaming or a cellular silica.
  • the material is of a cellular structure it must contain a proportion of open cells allowing access to at least 40% of the volume of the material.
  • Aggregates of microparticles may be held together by for example fusion at high temperature or by adhesives such as colloidal alumina.
  • the microparticles will suitably be between 0.1 and 100 micrometres across.
  • the rigid inorganic material will have a surface micro- porosity to improve adhesion between elements of the composite.
  • the rigid inorganic material will have a pore volume of between 0.5 and 0.9 ml/ml made up of pores with diameters between 0.5 and 10 micrometres.
  • the rigid inorganic material will comprise between 60% and 99% of the weight of the composite.
  • organic polymers for use in the present invention include cellulose, derivatives of cellulose such as carboxymethyl cellulose and aminoethyl cellulose, dextran, derivatives of dextran such as diethyla inoethyl dextran, alginic acid, salts of alginic acid, - 3 -
  • the polymer may be cross-linked with formaldehyde or glutaraldehyde.
  • the material of the invention has numerous uses in supporting biologically active molecules for example proteins, and specifically enzymes, antigens, antibodies or nucleic acids or polypeptides or amino acids for peptide synthesis.
  • the material of the invention is particularly useful for the support of molecules which are required to interact with macromolecules in solution.
  • the pore shape, volume and size are appropriate to unobstructed diffusion of macromolecules within the structure permitting much higher reaction rates than has been possible hitherto.
  • the material of the invention is also particularly suitable for the support of enzymes which act on water-insoluble molecules in a reaction involving water, as in a hydrolysis reaction for example the hydrolysis of oils and fats.
  • a hydrolysis reaction for example the hydrolysis of oils and fats.
  • Sufficient water to perform the hydrolysis can be held in at least part of the pore volume of the hydrophilic support. This overcomes a difficult problem in the use of immobilised enzymes such as upases and avoids the use of troublesome emulsions.
  • the size of the support particle may be varied to suit the conditions of use, and will generally be from 300 to 2000 micrometres in dia ⁇ meter.
  • the large pores facilitate mass transfer within the particle.
  • large particles may be used with the advantages of easier handling and low pressure drop within fixed beds of material. High fluid flow can be obtained at low pressures through stable packed beds of the material.
  • the density of the composite support may be varied and depends largely - 4 -
  • the material of the invention may take the form of irregular particles, extrusions, tablets, balls, beads or a coating on rods, sheets or other carriers.
  • the support is particularly suitable for use in solid-phase assays involving immobilised antibodies.
  • particles of aggregated silica microspheres prepared according to my copending U.K. Application No. 8702285 with a pore volume of 0.75 ml/ml and pores between 1 and 20 micrometres are sieved to retain those between 1 and 2 mm. After heating at 110°C for 2 hours these are added to a solution of chitosan of up to 2% concentration in formic acid of up to 2% v/v. Excess chitosan is removed by vacuum filtration and the material is added to a stirred solution of ammonium hydroxide of up to 5% v/v, to insolubilise the chitosan.
  • the support material thus prepared is dried at between 60° and 110°C.
  • the chitosan may be cross-linked using for example a solution of glutaraldehyde of 0.5 to 5% concentration after which the support is ready for application of the biologically active material which may be attached by adsorption or covalent attachment.
  • a cellular silica with a pore volume of 0.90 ml/ml and pore diameter from 5 to 50 micro ⁇ metres is admixed with a 6% chitosan solution and extruded through an oriface to form cylindrical support elements. These are broken to short lengths after drying.
  • the polymer may be cross-linked as before and is ready for attachment of amino acids or proteins.
  • aggregates of silica micro ⁇ spheres in the size range 10 to 200 micrometres are suspended in a - 5
  • Rods which may be of any rigid material such as a thermoplastic or thermosetting polymer and may be for example from 4 to 20 cm in length and from 2 to 10 mm in diameter but may be of any convenient length or diameter are dipped in the suspension such that from 1 to 10 mm of one end of each rod is coated. After drying the coated tip may be recoated to increase the thickness of the coating. After drying the coating may be cross- linked as before. The coating of composite support is then ready for attachment of biologically active molecules.
  • the supported dextranase was tested for activity in comparison to the free enzyme at equivalent loading by measuring the reducing sugar produced from a 1% solution of dextran at 40°C.
  • Example 2 62 ml of supported dextranase prepared as in Example 1 was packed in a tubular reactor through which a sucrose solution (15° Brix) containing 10 g/1 dextran was passed at 40°C. The residual dextran in the effluent from the reactor was measured by Haze Analysis.
  • Example 1 A sample of the support as prepared in Example 1 was contacted with a solution containing 2 g Dextranase DN50L (Novo Industri A/S) per 10 g support for 24 hours. After washing the supported enzyme was tested for activity at 60°C using a 10 g/1 dextran solution at pH 7. Haze analysis showed that the dextran was completely hydrolysed in 8 minutes.
  • Dextranase DN50L Novo Industri A/S
  • Example 4 A sample of the support as prepared in Example 4 was contacted with 0.037 g of Lipase P (A ano International Enzyme Co.) per gram support for 24h. After washing 1.53 g of water was left in the pores of each gram of support. This was added to olive oil (4 g oil/g support) at 40°C. After stirring for 8 hours 38.1% of the olive oil had been hydrolysed to fatty acids.
  • Lipase P A sample of the support as prepared in Example 4 was contacted with 0.037 g of Lipase P (A ano International Enzyme Co.) per gram support for 24h. After washing 1.53 g of water was left in the pores of each gram of support. This was added to olive oil (4 g oil/g support) at 40°C. After stirring for 8 hours 38.1% of the olive oil had been hydrolysed to fatty acids.
  • silica microsphere aggregates with a pore volume of 0.65 ml/ml, an average pore diameter of 2 micrometres and a particle size range of 20 - 150 micrometres was mixed with 30 ml of a 2% solution of chitosan.
  • Polystyrene rods 8 cm long and 3 mm diameter were dipped into the suspension such that the lower 6 mm was coated by the suspension. These were dried and recoated. When dry these were dipped into a solution containing 1 g urease, 100,000 units, and 0.1 g bromthymol blue in 100 ml and dried.
  • the rods could now be used as a rapid indicator of urea.
  • the enzyme support coating turned blue/green.
  • Example 4 40 g of the silica used in Example 4 was mixed with 150 ml of a 5% cellulose xanthate solution and extruded through a 500 micrometre oriface into a 0.5% acetic acid solution. The insoluble support was well washed, and 0.5 g was contacted with 500 units of amylase. When placed in a solution of starch the supported enzyme degraded the starch into sugars within 1 hour at 15°C.
  • Example 4 50 ml of the silica used in Example 4 was mixed with 100 ml of a 4% solution of sodium alginate and dripped through a 1.5 mm oriface into a solution of calcium nitrate acidified with nitric acid. The beads formed were washed, and cross linked with a 1% solution of glutaraldehyde. After washing these beads could be used to bind soluble protein and nucleic acid antigens for use in immunoassays.
  • Example 8 7 g of the silica aggregates used in Example 8 were mixed with 20 ml of 16% gelatin and extruded through a heated 400 micrometre oriface. This was cross-linked with 40 ml of 1% glutaraldehyde and washed. The support was treated with a solution of lactase and the supported enzyme used for the hydrolysis of lactose in milk.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Les supports matriciels pour acides aminés, acides nucléiques et protéines sont des composites comprenant un matériau rigide macroporeux inorganique ayant un volume des pores d'au moins 0,4 ml/ml composé de pores interconnectés de 0,05 à 50 micromètres de diamètre et d'un polymère organique comportant des hydroxyles, des amino et des carboxyles. Le support matriciel est particulièrement utile pour soutenir des molécules biologiquement actives qui doivent être mises en interaction avec des macromolécules en solution ou avec des molécules insolubles dans l'eau dans une réaction où intervient l'eau, par exemple dans l'hydrolyse des huiles et des matières grasses.
PCT/GB1989/000244 1988-03-19 1989-03-10 Supports pour proteines et acides amines WO1989008705A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB888806571A GB8806571D0 (en) 1988-03-19 1988-03-19 Supports for proteins & amino acids
GB8806571 1988-03-22

Publications (1)

Publication Number Publication Date
WO1989008705A1 true WO1989008705A1 (fr) 1989-09-21

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Application Number Title Priority Date Filing Date
PCT/GB1989/000244 WO1989008705A1 (fr) 1988-03-19 1989-03-10 Supports pour proteines et acides amines

Country Status (2)

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GB (1) GB8806571D0 (fr)
WO (1) WO1989008705A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000021658A2 (fr) * 1998-10-14 2000-04-20 Millennium Pharmaceuticlas Limited Dispositif poreux
EP1683873A1 (fr) * 2005-01-20 2006-07-26 Samsung Electronics Co., Ltd. Méthode permettant d'enlever un inhibiteur d'amplification d'acides nucléiques d'un échantillon et système de Micro-PCR
US8022013B2 (en) 2003-08-29 2011-09-20 Illumina, Inc. Method of forming and using solid-phase support
US9073033B2 (en) 2010-01-19 2015-07-07 Illumina, Inc. Methods and compositions for processing chemical reactions

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986007345A1 (fr) * 1985-06-12 1986-12-18 Eric Robinson Silices structurees
WO1987002703A1 (fr) * 1985-10-22 1987-05-07 National Research Development Corporation Microporteur pour culture cellulaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986007345A1 (fr) * 1985-06-12 1986-12-18 Eric Robinson Silices structurees
WO1987002703A1 (fr) * 1985-10-22 1987-05-07 National Research Development Corporation Microporteur pour culture cellulaire

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000021658A2 (fr) * 1998-10-14 2000-04-20 Millennium Pharmaceuticlas Limited Dispositif poreux
WO2000021658A3 (fr) * 1998-10-14 2001-02-01 Cambridge Discovery Chemistry Dispositif poreux
US8022013B2 (en) 2003-08-29 2011-09-20 Illumina, Inc. Method of forming and using solid-phase support
US8912130B2 (en) 2003-08-29 2014-12-16 Illumina, Inc. Methods of forming and using a solid-phase support
EP1683873A1 (fr) * 2005-01-20 2006-07-26 Samsung Electronics Co., Ltd. Méthode permettant d'enlever un inhibiteur d'amplification d'acides nucléiques d'un échantillon et système de Micro-PCR
US9073033B2 (en) 2010-01-19 2015-07-07 Illumina, Inc. Methods and compositions for processing chemical reactions
US9649614B2 (en) 2010-01-19 2017-05-16 Illumina, Inc. Methods and compositions for processing chemical reactions

Also Published As

Publication number Publication date
GB8806571D0 (en) 1988-04-20

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