EP0423938B1
Expired / Lapsed
EP
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Ligand-containing medium for chromatographic separation, process for preparing the medium, and use of the medium for isolating synthetic or natural molecules from a fluid mixture
Assignee: Rohm and Haas Co
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Filing: 1990-09-17
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Granted: 1994-03-02
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Expires: 2010-09-17
Abstract
Abstract Improved ligand-containing media, a method for preparing the media, and use of the media in the production of peptides, proteins, and the like, by chromatograptuc separation, are provided. More specifically, the media have permanently attached, via a covalent bond to an inert solid substrate, an avidin polypeptide ligand in the dissociated renatured form which reversibly binds to certain molecules such as proteins, peptide, nucleotides, oligonucleotides, and the like, and to other molecules which bind to avidin via biotinylation or by way of their secondary/tertiary micromolecular structures.
Inventors
- 👤 Ferdinand Carl Haase
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 11:23
CPC Classifications
B; B01; B01J; B01J20/00; B01J20/281; B01J20/286; B01D; B01D15/00; B01D15/08; B01D15/26; B01D15/38; B01D15/3804; B01D15/3823; B01J20/30; B01J20/32; B01J20/3202; B01J20/3206; B01J20/3208; B01J20/321; B01J20/3214; B01J20/3217; B01J20/3219; B01J20/3231; B01J20/3242; B01J20/3268; B01J20/3272; B01J20/3274; Y; Y10; Y10S; Y10S530/00; Y10S530/81; Y10S530/812; Y10S530/815; Y10S530/816
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
101 words
A ligand-containing medium for chromatographic adsorption, which comprises a crosslinked organic polymer substrate having covalently bonded thereto, through a chemically stable, non-hydrolyzable linking group, an avidin polypeptide ligand in the dissociated renatured form wherein more than 50% by weight of the avidin ligand is in a monomeric form which binds with biotin to form a complex with a dissociation constant of greater than 10⁻¹⁰ Molar and wherein the crosslinked organic polymer is capable of maintaining the avidin in the monomeric form, with the proviso that the ligand-containing medium does not include a spacer which is a substantially non-ionic, hydrophilic polymeric spacer.
Claim 2
Dependent Claim
35 words
A medium as claimed in claim 1, wherein the non-hydrolyzable linking group contains a carbon-nitrogen group or a sulfur-nitrogen group, for example, the non-hydrolyzable linking group is selected from -CH₂NH-, -C(O)NH-, -NHC(O)NH-, -C(O)NHNHC(O)NH- and -SO₂NH-.
Claim 3
Dependent Claim
43 words
A medium as claimed in claim 3, wherein the crosslinked organic polymer is a porous, adsorbent or ion exchange resin in particulate form, for example, an adsorbent or ion exchange resin in particulate form and derived primarily from an acrylic or styrenic monomer.
Claim 4
Independent Claim
36 words
A medium as claimed in any preceding claim, wherein the avidin affinity group is in a monomer form which binds with biotin to produce a complex with a dissociation constant of not less than 10⁻⁹ Molar.
Claim 5
Dependent Claim
38 words
An affinity chromatographic column, which comprises a tubular container with inlet and outlet means at opposite ends of the tube and having housed, within the tube, a ligand-containing medium as claimed in any of claims 1 to 4.
Claim 6
Dependent Claim
131 words
A process for preparing an improved ligand-containing medium, as claimed in any of claims 1 to 4, which comprises: (a) reacting, at a pH near neutrality, an effective amount of an avidin tetramer with functional groups of a crosslinked organic polymer substrate to form chemically stable, non-hydrolyzable imino linking groups between the substrate and the avidin until 1 to 3 covalent linkages are formed, with the polymer functional groups, for each avidin tetramer molecule; (b) separating the avidin-containing substrate and reducing the formed imino bond to the corresponding amine bond; (c) denaturing the avidin tetramer to form monomeric avidin linked to the chemically stable substrate; and (d) separating the denatured avidin from dissociated subunits of avidin and renaturing the avidin-containing substrate by removal of the denaturant to form the ligand-containing medium.
Claim 7
Dependent Claim
35 words
A process as claimed in claim 6 wherein: i) the pH of step (a) is from 6.5 to 8.5; and/or ii) the pH of the ligand-containing medium of step (d) is adjusted to near neutrality.
Claim 8
Independent Claim
81 words
A method for isolating synthetic or natural molecules from a fluid mixture containing the same wherein the molecules have an affinity for avidin, or may be biotinylated to have an affinity for avidin, or their recombinant versions having affinity for avidin, or biotinylated recombinant versions, which comprises passing the fluid mixture into intimate contact with a ligand- containing medium as claimed in any of claims 1 to 4 and subsequently eluting the adsorbed synthetic or natural molecules from the ligand-containing medium.
Claim 9
Dependent Claim
18 words
A method as claimed in claim 8, wherein the fluid mixture is an organic or aqueous liquid mixture.
Claim 10
Dependent Claim
31 words
A method as claimed in claim 8 or claim 9, wherein the synthetic or natural molecules consist of peptides, proteins, enzymes, nucleotides, oligonucleotides, or recombinant or biotinylated or recombinant-biotinylated versions thereof.
Claim 11
Independent Claim
32 words
A method as claimed in any of claims 8 to 10, wherein the fluid mixture, containing the synthetic or natural molecules, is contacted with the ligand-containing medium contained within a chromatographic column.
Claim 12
Independent Claim
41 words
A method as claimed in any of claims 8 to 10, wherein a liquid mixture comprising synthetic or natural molecules is separated by liquid affinity chromatography in a column comprising a medium as claimed in any of claims 1 to 4.
References Cited (Backward Citations)
No citation records indexed.
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