Method of covalently binding biologically active organic substances to polymeric substances

• US 4,415,665 A
• Filed: 12/01/1981
• Issued: 11/15/1983
• Est. Priority Date: 12/12/1980
• Status: Expired due to Term

• Current Assignee: Pharmacia AB A Company of
• Original Assignee: Pharmacia Fine Chemicals, AB (Danaher Corporation)
• Inventors: Nilsson, Kurt G. I., Mosbach, Klaus H.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/326,332
• Time in Patent Office: 714 Days
• Field of Search: 435/174, 435/176, 435/178, 435/179, 435/180, 435/181, 435/182
• US Class Current: 435/179
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• CPC Class Codes: B01J 20/262 obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds per se C08G
• CPC Class Codes: B01J 20/267 Cross-linked polymers
• CPC Class Codes: B01J 20/28047 Gels
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: B01J 20/3071 Washing or leaching
• CPC Class Codes: B01J 20/3092 Packing of a container, e.g. packing a cartridge or column of chromatography columns B01D15/206
• CPC Class Codes: B01J 20/3204 Inorganic carriers, supports or substrates
• CPC Class Codes: B01J 20/3212 consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3217 Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3246 having a well defined chemical structure
• CPC Class Codes: B01J 20/3248 the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
• CPC Class Codes: B01J 20/3251 comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
• CPC Class Codes: B01J 20/3253 comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/3425 of sorbents or filter aids comprising organic materials
• CPC Class Codes: B01J 20/345 using a particular desorbing compound or mixture elution or regeneration of stationary phases in liquid chromatography B01D15/08
• CPC Class Codes: B01J 20/3475 : in the liquid phase
• CPC Class Codes: B01J 2220/54 : Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: B01J 2220/58 : Use in a single column
• CPC Class Codes: C12N 11/10 : the carrier being a carbohydrate
• CPC Class Codes: C12N 11/12 : Cellulose or derivatives thereof
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/548 : Carbohydrates, e.g. dextran

Abstract

The invention relates to a method of coupling organic ligands to a polymeric carrier. The organic ligand contains at least one primary or secondary amino group, at least one thiol group and/or at least one aromatic hydroxy group, and the polymeric carrier contains at least one hydroxy group. According to the invention the coupling is performed by forming a reactive derivative of the polymeric carrier by treatment with a sulfonyl halogenid, and reacting the formed reactive derivative with the organic ligand, which is thereby bonded directly to a carbon atom in the polymeric carrier. The polymeric carrier can, for example, be a possibly cross-linked polysaccharide, and the organic ligand is preferably a biological material such as a protein. The coupling product can, for example, be used for immunologic determinations, for affinity chromatography, etc. The coupling method can be performed under mild conditions not damaging sensitive organic ligands.

Claims

1. A method of covalently binding

(a) a biologically active organic substance that contains at least one substituent selected from the group consisting of

primary and secondary amino groups,

thiol groups, and

aromatic hydroxy groups directly to a

(b) polymeric substance (PM) containing at least one hydroxy group, comprising the steps of:

(1) first forming a reactive derivative PM-O-SO₂ -R by reacting

(i) an organic sulfonyl halogenide of the formula Hal-SO₂ -R, with

(ii) a polymeric substance (PM) containing at least one hydroxy group,

wherein R is an organic group and Hal is halogen, and

wherein said at least one reacting hydroxy group is bonded to carbon atoms in the polymeric substance (PM), and then

(2) reacting said reactive derivative PM-O-SO₂ -R directly with the biologically organic substance as set forth in (a).

2. A method according to claim 1 wherein said polymeric substance (PM) is a polysaccharide or a derivative thereof.

3. A method according to claim 1 wherein said polymeric substance (PM) is cross-linked.

4. A method according to claim 1 wherein said biologically active organic substance of (a) exhibits biospecific affinity to another substance.

5. A method according to claim 1 wherein said biologically active organic substance of (a) is a polypeptide or a protein.

6. A method according to claim 1 wherein said biologically active organic substance (a) is selected from the group consisting of enzymes, antibodies, antigens, nucleotides, polynucleotides and haptens.

7. A method according to claim 1 wherein said polymeric substance is polysaccharide or polysaccharide containing materials.

8. A method according to claim 1 wherein said polymeric substance is agarose or cross-linked agarose.

9. A method according to claim 1 wherein said polymeric substance is dextran or cross-linked dextran.

10. A method according to claim 1 wherein said polymeric substance is cellulose.

11. A method according to claim 1 wherein said polymeric substance is silica particles containing at least one hydroxy group bonded thereto.

12. A method according to claim 1 wherein said sulfonyl halogenide is selected from the group consisting of

p-toluensulfonyl chloride and

2,2,2-trifluoroethane sulfonylchloride.

13. A method according to claim 1 wherein said biologically organic substance is selected from the group consisting of alcohol dehydrogenase, trypsin, protein A from Staphylococcus aureus and N⁶ -(6-aminohexyl)-adenosine-5'"'"'-monophosphate.