CA1063049A - Preparation of water insoluble immobilized bio-active compounds - Google Patents
Preparation of water insoluble immobilized bio-active compoundsInfo
- Publication number
- CA1063049A CA1063049A CA226,193A CA226193A CA1063049A CA 1063049 A CA1063049 A CA 1063049A CA 226193 A CA226193 A CA 226193A CA 1063049 A CA1063049 A CA 1063049A
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- Prior art keywords
- biologically active
- active substance
- water
- hydroxymethylene
- poly
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21005—Thrombin (3.4.21.5)
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- Peptides Or Proteins (AREA)
- Polymers With Sulfur, Phosphorus Or Metals In The Main Chain (AREA)
- Polyesters Or Polycarbonates (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
Abstract of the Disclosure:
A biologically active compound in which a water-insoluble poly(hydroxymethylene) is bound chemically to a biologically active substance directly or by way of a spacer while maintaining its biological activity, its manufacture and its use in affinity chromatography and for the preparation of antibodies as well as for the implementation of enzyme reactions.
A biologically active compound in which a water-insoluble poly(hydroxymethylene) is bound chemically to a biologically active substance directly or by way of a spacer while maintaining its biological activity, its manufacture and its use in affinity chromatography and for the preparation of antibodies as well as for the implementation of enzyme reactions.
Description
HOE 74/B 008 - Ma 187 The present invention relates to novel compounds of biologically active substances and water-insoluble high molecular weight compounds, a process for their manufacture and their use in the affinity chromatography.
In the last few years, a new technique has become more and more important within the biochemical operating methods, the primary feature of which consists in the utilization of the affinity of biologically active substances bound to a carrier to reactions that are to be carried out selectively.
By way of a specific complex formation of the carrier-bound substance with a second compound which is present in a mixture, it is possible to eliminate the respective substance from the mixture and, optionally, to isol&te the said sub-stance subsequently by deæorption.
Carrier-bound enzymes have the advantage that trans-formations of substances can be carried out in preparative continuous processe~ and that the reaction products are ob-tained free from enzymes.
In the biochemical enzymatic analysis, water-insoluble enzymes are available as reagents to be used several times.
Due to the characteristic feature of the enzymes to show an affinity not only towards the substrates, but also towards the specific inhibitors, the obtaining of enzyme inhlbitors by means of the affinity chromatography on enzymes bound to a carrier has been found to be particularly advantageous. On t~e other hand, the bond of inhibitors to water-insoluble macro- - -molecular resin networks permits the preparative production of ~ -the corresponding enzymes.
29 As so-called lmmunoadsorbents, antlgens or ant~bodies are ~; - 2 - ~ --~. . - . : - .. . . . . . .
.. . .... ,, . . . . . ~ . . - . . ~.. ; .. : . -.. . . .. . . . .. . . . . . . .
, . . ~ ,. . .
. - , . .
HOE 74/B 008 - Ma 187 1063~349 bound to water-insoluble macromolecular resin networks and permit subsequently the isolation of the corresponding anti-bodie~ and/or antigens.
By biologically active substances there are to be under-stood, according to the description given above, natural sub-stances and substances that have been artificislly made which are e~fective in vivo and in vitro, substances that can be designated, in the broadeæt sense, as enzymes, activators, inhibitors, antigens or antibodies, vitamins, and hormones.
These biologically active subætances may be termed "effectorsn, since they represent the active principles of the water-in-soluble systems.
Most of the effectors bound to a carrier that have been descr~bed 80 far are considerably more stable than the cor-1S responding substances in solution.
As carrier substances, so-called macromolecular resin networks, there are to be used advantageously only those sub-stances ~hich show, besides the insolubility in aqueous systems, an unspecific adsorption that is aæ low as possible. This means that hydrophobic, hydrophilic and ionic interactions between the macromolecular resin network and the reactant of the effector have to be largely prevented. A bond with sub- -~; stances which do not represent a reactant of the effector should --be excluded.
The resin networks used 80 far as carriers for biologically active substances can be æub-divided into those b~nding the effectors by physical adsorption - this group includes activated charcosl and glass beads -, and those whlch are bound 29 to one another with the effectors by a covalent bond.
. -, . .
. ~ , .
- ~ - - ,... , . - . . . .
.. . , . ~ ~ , ,. ,: , . ; , ~ HOE 74/B 008 - Ma 187 ' 1063~)49 The latter include vinyl polymers, for example, polyacrylic acids, polyacrylic acid amides and amino-, carboxy- or sulf-onyl-substituted polystyrene, moreover, cellulose and its derivatives and, finally, natural and synthetic polypeptides ~5 and proteins. Due to the balanced interaction between the resin network and the effector, the use of carbohydrates, in particular of cellulose, dextran, starch, agar and/or their derivatives, as network in aqueous systems has become more and more common, although the carboxylic groups that are frequently contained in these natural substances are often felt to be inconvenient, due to their unspecific reaction. Besides, these substances show a relatively poor thermal and chemical stability.
It has now been found that the disadvantages shown by the carbohydrate derivatives as ~etworks in reactions depending on affinity can be overcome if polyhydroxymethylene is used as resin network.
The present invention provides biologically active compounds which contain a water-insoluble poly(hydroxymethylene), to which a biologically active substance is bound, while main- -.
taining its biological activity. In these compounds, - --.
a) the carrier network is polyhydroxymethylene, b) the biologically active substances are substances such as enzymes, activators, inhibitors, antigens or antibodies, other plasmaproteins, blood group substances, phythem-agglutinins, antibiotics, vitamins or hormones, peptides or amino acids, or synthetically prepared efectors, c) polyhydroxymethylene and the biologically active substances are bound to one another by a covalent bond, direct or by :~ - 4 - :
.. . ~ .... . .... . .. . , ,, - , .. . . . . . . . .
' ' ''"'; ' "'' .' , ' " '."' ' ''' ' " '. '' ', ' ~' ' ' .', ' .' ~ " ' . ' , ' ' HOE 74/B 008 - Ma 187 1~63049 way of an intermediate member, a so-called "spacern.
Polyhydroxymethylene is a synthetic polymer with a con-tinuous carbon chain. Each carbon atom carries a hydrogen atom and a hydroxyl group. Its preparation and its properties have been de~cribed, inter alia, by N.D. Field, J.R. Schaefgen, J.Polymer Sci., vol. 58, 533-543 (1962) and G. Smets, K.Hayashi, J. Polymer Sci., vol.29, 257-274 (1958). It can be prepared by way of basic or acid hydrolysis from polyvinylene carbonate.
In a broader sense, the polyhydroxymethylene is to be under-stood as a derivative of polyvinylene carbonate. The bond of ; the effectoræ to the polyhydroxymethylene can be obtained, on one hand, by the measure used for the covalent bond of the effectors with the hydroxyl group, and on the other hand~ by reacting the polyvinylene carbonate by way of the equally kno~m am~nolytic reaction of a cyclocarbonte ring of the polyvinylene carbonate with a prlmary amine, for example, with hexamethylene diamine, to obtain a polyvinylene carbonate substituted by a ~pacer or effector over a urethane bond, and by subsequently saponifying the remaining cyclo-carbonate groups of said poly-vinylene carbonate to give hydroxyl groups:
._ -CH---C~ ~ CH - c~L CH - CH ~
l l ~ H2 l l OH 1 saponi-~C ~ --> ~ucf I fication ~ ~ o o Jn 1 ~H
polyvinylene carbonate H - ~ -~H-~
OH OH OH O
. _ 1l-1 C = o NH
' -.. ,,, ... ;
\ HOE 74/B 008 - Ma 187 It is especially advantageous for the application of the products if the biologically active effectors are not reacted directly with the polyhydroxymethylene, but are linked ~ to the network by a covalent bond by way of an intermediate member termed "arm" in the affinity chromatography or known by the term of "spacer" in British literature.
The spacer substances are in most cases hydrocarbon structures of a length of about 1 - 2 nm. ~wo functional end groups of the spacer permit in each case the reaction with the network as well as with the effector; in the reaction with the network, polyhydroxymethylene as well as polyvinylene carbonate may be used under appropriate conditions.
The present invention also provides a process for the covalent binding of the biologically active substances to the carrier.
To reach this goal, use can be made of a series of generally known reactions:
The binding of the effector to the network is effected in a particularly simple way a~fter an activation of the hydroxyl groups of the polyhydroxymethylene by means of cyanohalides, advantageously by bromocyanogen, and a subsequent reaction of the biologically active effectors containing amino groups by way of these activated groups.
Another process - which is a variant of the azide method described by Curtius - comprises reacting polyhydroxymethylene, instead of polysaccharides used by Curtiusj;with a halogeno-carboxylic acid, such as chloroacetic acid, in an alkaline medium, esterifying the corresponding acid with aloohol, subsff~ntly oonver~ the ester into the hydrazide, and finally binding the resulting HOE 74/B 008 - Ma 187 1063~49 azide with the amino group of the effector protein to the polyhydroxymethylene network.
Another possibility of binding the effector to a poly-hydroxymethylene network consists in the acylation of the hydroxyl groups w1th bromo-acetylbromide, followed by the alkylation of the amino group of the effector.
Similar reactions are obtained, for example, by reacting the hydroxyl groups of the carrier with reactive triazines;
in this çase, a part of the reactive groups of the triaz1ne is reacted with the polyhydroxymethylene compound, another part reacts-with amino groups of the effector.
Diazotizable aromati~ amines which may be linked, on one hand, to the hydroxyl groups of the carrier by way of a further reactive group, permit on the other hand the coupling with appropriate activated amino acids, for example, the tyrosine or histidine radicals of the protein effector.
Vinylsulfone derivatives containing arylamino groups and sulfuric acid semi-esteræ of B-hydroxyethylsulfones can be reacted with the hydroxyl groups of the carrier. They make the binding of the effector possible according to the above-mentioned diazotizing reaction.
A particularly stable ether bond is obtained in the reaction of the hydroxyl groups of the polyhydroxymethylene with epoxides which do not form ions and which contain at leaæt
In the last few years, a new technique has become more and more important within the biochemical operating methods, the primary feature of which consists in the utilization of the affinity of biologically active substances bound to a carrier to reactions that are to be carried out selectively.
By way of a specific complex formation of the carrier-bound substance with a second compound which is present in a mixture, it is possible to eliminate the respective substance from the mixture and, optionally, to isol&te the said sub-stance subsequently by deæorption.
Carrier-bound enzymes have the advantage that trans-formations of substances can be carried out in preparative continuous processe~ and that the reaction products are ob-tained free from enzymes.
In the biochemical enzymatic analysis, water-insoluble enzymes are available as reagents to be used several times.
Due to the characteristic feature of the enzymes to show an affinity not only towards the substrates, but also towards the specific inhibitors, the obtaining of enzyme inhlbitors by means of the affinity chromatography on enzymes bound to a carrier has been found to be particularly advantageous. On t~e other hand, the bond of inhibitors to water-insoluble macro- - -molecular resin networks permits the preparative production of ~ -the corresponding enzymes.
29 As so-called lmmunoadsorbents, antlgens or ant~bodies are ~; - 2 - ~ --~. . - . : - .. . . . . . .
.. . .... ,, . . . . . ~ . . - . . ~.. ; .. : . -.. . . .. . . . .. . . . . . . .
, . . ~ ,. . .
. - , . .
HOE 74/B 008 - Ma 187 1063~349 bound to water-insoluble macromolecular resin networks and permit subsequently the isolation of the corresponding anti-bodie~ and/or antigens.
By biologically active substances there are to be under-stood, according to the description given above, natural sub-stances and substances that have been artificislly made which are e~fective in vivo and in vitro, substances that can be designated, in the broadeæt sense, as enzymes, activators, inhibitors, antigens or antibodies, vitamins, and hormones.
These biologically active subætances may be termed "effectorsn, since they represent the active principles of the water-in-soluble systems.
Most of the effectors bound to a carrier that have been descr~bed 80 far are considerably more stable than the cor-1S responding substances in solution.
As carrier substances, so-called macromolecular resin networks, there are to be used advantageously only those sub-stances ~hich show, besides the insolubility in aqueous systems, an unspecific adsorption that is aæ low as possible. This means that hydrophobic, hydrophilic and ionic interactions between the macromolecular resin network and the reactant of the effector have to be largely prevented. A bond with sub- -~; stances which do not represent a reactant of the effector should --be excluded.
The resin networks used 80 far as carriers for biologically active substances can be æub-divided into those b~nding the effectors by physical adsorption - this group includes activated charcosl and glass beads -, and those whlch are bound 29 to one another with the effectors by a covalent bond.
. -, . .
. ~ , .
- ~ - - ,... , . - . . . .
.. . , . ~ ~ , ,. ,: , . ; , ~ HOE 74/B 008 - Ma 187 ' 1063~)49 The latter include vinyl polymers, for example, polyacrylic acids, polyacrylic acid amides and amino-, carboxy- or sulf-onyl-substituted polystyrene, moreover, cellulose and its derivatives and, finally, natural and synthetic polypeptides ~5 and proteins. Due to the balanced interaction between the resin network and the effector, the use of carbohydrates, in particular of cellulose, dextran, starch, agar and/or their derivatives, as network in aqueous systems has become more and more common, although the carboxylic groups that are frequently contained in these natural substances are often felt to be inconvenient, due to their unspecific reaction. Besides, these substances show a relatively poor thermal and chemical stability.
It has now been found that the disadvantages shown by the carbohydrate derivatives as ~etworks in reactions depending on affinity can be overcome if polyhydroxymethylene is used as resin network.
The present invention provides biologically active compounds which contain a water-insoluble poly(hydroxymethylene), to which a biologically active substance is bound, while main- -.
taining its biological activity. In these compounds, - --.
a) the carrier network is polyhydroxymethylene, b) the biologically active substances are substances such as enzymes, activators, inhibitors, antigens or antibodies, other plasmaproteins, blood group substances, phythem-agglutinins, antibiotics, vitamins or hormones, peptides or amino acids, or synthetically prepared efectors, c) polyhydroxymethylene and the biologically active substances are bound to one another by a covalent bond, direct or by :~ - 4 - :
.. . ~ .... . .... . .. . , ,, - , .. . . . . . . . .
' ' ''"'; ' "'' .' , ' " '."' ' ''' ' " '. '' ', ' ~' ' ' .', ' .' ~ " ' . ' , ' ' HOE 74/B 008 - Ma 187 1~63049 way of an intermediate member, a so-called "spacern.
Polyhydroxymethylene is a synthetic polymer with a con-tinuous carbon chain. Each carbon atom carries a hydrogen atom and a hydroxyl group. Its preparation and its properties have been de~cribed, inter alia, by N.D. Field, J.R. Schaefgen, J.Polymer Sci., vol. 58, 533-543 (1962) and G. Smets, K.Hayashi, J. Polymer Sci., vol.29, 257-274 (1958). It can be prepared by way of basic or acid hydrolysis from polyvinylene carbonate.
In a broader sense, the polyhydroxymethylene is to be under-stood as a derivative of polyvinylene carbonate. The bond of ; the effectoræ to the polyhydroxymethylene can be obtained, on one hand, by the measure used for the covalent bond of the effectors with the hydroxyl group, and on the other hand~ by reacting the polyvinylene carbonate by way of the equally kno~m am~nolytic reaction of a cyclocarbonte ring of the polyvinylene carbonate with a prlmary amine, for example, with hexamethylene diamine, to obtain a polyvinylene carbonate substituted by a ~pacer or effector over a urethane bond, and by subsequently saponifying the remaining cyclo-carbonate groups of said poly-vinylene carbonate to give hydroxyl groups:
._ -CH---C~ ~ CH - c~L CH - CH ~
l l ~ H2 l l OH 1 saponi-~C ~ --> ~ucf I fication ~ ~ o o Jn 1 ~H
polyvinylene carbonate H - ~ -~H-~
OH OH OH O
. _ 1l-1 C = o NH
' -.. ,,, ... ;
\ HOE 74/B 008 - Ma 187 It is especially advantageous for the application of the products if the biologically active effectors are not reacted directly with the polyhydroxymethylene, but are linked ~ to the network by a covalent bond by way of an intermediate member termed "arm" in the affinity chromatography or known by the term of "spacer" in British literature.
The spacer substances are in most cases hydrocarbon structures of a length of about 1 - 2 nm. ~wo functional end groups of the spacer permit in each case the reaction with the network as well as with the effector; in the reaction with the network, polyhydroxymethylene as well as polyvinylene carbonate may be used under appropriate conditions.
The present invention also provides a process for the covalent binding of the biologically active substances to the carrier.
To reach this goal, use can be made of a series of generally known reactions:
The binding of the effector to the network is effected in a particularly simple way a~fter an activation of the hydroxyl groups of the polyhydroxymethylene by means of cyanohalides, advantageously by bromocyanogen, and a subsequent reaction of the biologically active effectors containing amino groups by way of these activated groups.
Another process - which is a variant of the azide method described by Curtius - comprises reacting polyhydroxymethylene, instead of polysaccharides used by Curtiusj;with a halogeno-carboxylic acid, such as chloroacetic acid, in an alkaline medium, esterifying the corresponding acid with aloohol, subsff~ntly oonver~ the ester into the hydrazide, and finally binding the resulting HOE 74/B 008 - Ma 187 1063~49 azide with the amino group of the effector protein to the polyhydroxymethylene network.
Another possibility of binding the effector to a poly-hydroxymethylene network consists in the acylation of the hydroxyl groups w1th bromo-acetylbromide, followed by the alkylation of the amino group of the effector.
Similar reactions are obtained, for example, by reacting the hydroxyl groups of the carrier with reactive triazines;
in this çase, a part of the reactive groups of the triaz1ne is reacted with the polyhydroxymethylene compound, another part reacts-with amino groups of the effector.
Diazotizable aromati~ amines which may be linked, on one hand, to the hydroxyl groups of the carrier by way of a further reactive group, permit on the other hand the coupling with appropriate activated amino acids, for example, the tyrosine or histidine radicals of the protein effector.
Vinylsulfone derivatives containing arylamino groups and sulfuric acid semi-esteræ of B-hydroxyethylsulfones can be reacted with the hydroxyl groups of the carrier. They make the binding of the effector possible according to the above-mentioned diazotizing reaction.
A particularly stable ether bond is obtained in the reaction of the hydroxyl groups of the polyhydroxymethylene with epoxides which do not form ions and which contain at leaæt
2 reactive groups, such aæ the epihalohydrines or the poly-epoxides, for example, epichlorhydrin or bisepoxide.
Besides the processes mentioned above as examples for the preparation of the covalent bond between the carrier net-29 work and the protein effector or other effectors, there may be . - . - . - . - . . ., .. , ~
-,. . .
.
.
mentioned further methods which lead to the reaction of the hydroxyl groups of the polyhydroxymethylene on one hand, and to the reaction of determined groups of the effector on the other hand, thus forming a covalent bond between the two reactants, such as the know reaction with complex-forming metal compounds, for example, titanium compounds.
Besides by its above-mentioned chemical and thermal stability, the polyhydroxymethylene is marked by advantageous processing properties, thus becoming superior to the carrier networks on the basis of natural carbohydrates that had been considered optimal up to this time. Polyhydroxymethylene can be manufactured, for example, in the form of fibers, filaments, films or spherical particles or, perferably, powdered material, so that the most appropriate form may be chosen, depending on the purpose of application of the effector to be bound to it.
Another advantage of the polyhydroxymethylene as com-pared against the carrier materials according to the state of the art is to be seen in the fact that the surface which is accessible for the bond of the effectors and/or the spacers can be controlled during production.
According to the analogous reaction mechanisms as they have been described for the bond of the effector with the poly-hydroxymethylene network, the spacer substances can also be bound to the network, for example if the spacers carry an amino group as one of the functional groups. The introduction of a spacer also makes it possible, however, to introduce ~ additional reaction possibilities for the bond of the effector.
- In the case that the spacer bound to the network carries a-~ free carboxyl group, this carboxyl group can be activated, for _, - - ~ . , ~ - .
- : , . . : . , , , , -~ . , .: : ., . ' . . .. , : ; ., : :, ~
.
HOE 74/B 008 - Ma 187 .
1063~49 example, by carbodiimide compounds and can then form an amide bond with amino groups of the effector. Carboxyl groups also permit the formation of amide bonds by means of the isoxazolium salts introduced by Woodward.
If the spacer bound to the network carries an available free amino group, the introduction of arylamino groups in the extension of the spacer is possible, by means of vinylsulfone derivatives containing arylamino groups or sulfuric acid esters of B-hydroxyethylsulfones, said arylamino groups subsequently being diazotized according to known methods and then bond to correspondingly reactive groups of the effector, for example:
, . . . ..
r . n 0 NH- (CH2) 6-NH-CH2-CH2 s 2 ~ -NH2 .
... . ...
The biologically active compounds of the invention are suitable for most application processes that have so far been known for other effectors bound to hydrop~ilic water-insoluble carriers. This means that enzymes can be made water-insolub~e.
The insoluble enzymes are more and more used to deterDine sub-~trates in automatic analyzers and as so-called enzyme elec-trodes. Due to the increased stability, a series of carrier-bound enzymes is suitable for the implementation of enzymatic reactions on an industrial scale.
Carrier-bound biologically active substances are used on a large scale in the afflnity chromatography, due to their properties as specific adsorbents. By means of carrier-bo~d 29 natural or synthetic enzyme inhibitors, a high degree Or purity _ 9 _ .. . . .. . . . .. .. . .. . .. . ..
. . , ~ . , . . - , . . ~ . -- . . , . .. ~ . -. . . .. , . . . , . - - - . .. . . . . .. . . . .. . .
: . . . , ~ . .~, , , : . . ~ . - -HOE 74/B 008 - Ma 187 1~63~49 is obtained for enzymes, whereas the enzymes have been found to be extremely suitable as effectors particularly for the production of natural enzyme inhibitors from raw extracts.
Carrier-bound water-insoluble antigens are used for the iso- -lation of the corresponding antibodies, which are thus obtained free from other serum constituents and free from other antigens.
By means of affinity chromatography it is also possible to isolate those antibodies that cannot be precipitated, as well as those that exclude precipitation due to their low concentration - 10 in the serum, and also to determine them quantitatively.
The Examples given below in order to illustrate the in-vention show that a carrier prepared according to a single process is suitable for the bond of many different effectors and that the basic structure of polyhydroxymethylene can be ;~ 15 made suitable, by an appropriate substitution, for the bond of any desired effector.
Moreover, the Examples show how the effectors bound to the network can be used.
. \
'' \' ~1 . ' \ .
\
.' ' , \
\_ ~ - 10 -.
~, ' ' ~ . .. .
' .': , . ' .
:
- ' ~ , :
- ~ HOE 74/B 008 - Ma 187 1063~49 E X A M P L E ls Pol~hydrox~meth~lene-tetanu~ toxoid compound Preparation of the network from (~ -amino-n-hexyl~-substituted pol~h~droxymethvlene: -20 Grams of polyhydroxymethylene suspended in 900 ml of water were mixed with a solution of 20 g Or BrCN in 50 ml of --dimethylformamide and were maintained at a pH ~alue of 11.5 for 6 minutes at an internal temperature Or 15C by 810wly adding dropwise, while stirring, 2 n sodium hydroxide solu-tion. Subsequently the mixture was suction-filtered i~nediate-ly, was washed thoroughly with ice water and was squeezed to remo~e the remaining water. The polyhydroxymethylenc thus activated, which was still moist with water, was added to a well-stirred solution of 20 g of hexamethylene diamine in 500 ml of H20, which was maintained at room temperature and was ad~usted with concentrated HCl to a pH value of 8.5, the mixture was then made up with water to a total ~olume of 1 liter, while the pH value was constantly ad~usted between 8.5 and 9.0, and was stirred at room temperature for another 5 hours. SubsequQntly the reaction mixture was suction-fil-tered and was washed thoroughly with water. A dried sample of the polyhydroxymethylene substituted by (~ -amino-n-he~yl) group~ had a nitrogen content of 1.9~. By means of Sanger~
reagent for the detection of primary free amino groups, pro-ducts we~e ob$iained which had a ~trong yellow color shade.
Preparation of the carrier-bound effector Effeotors Tetanus antigen 10 Grams of the carrier of EY.ample 1 were suspended in - 29 200 ml of a 0.5 molar sodium pho~phate ~olution ha~ing a pH
'- - 11 -." ' ' . ~ .: : i . : . ~ . . - , :
: .,............ ; . . , : . ,. , ~ -. : .. ~ ' .~ : . :
. ; i , . ; ~.
HOE 74/B 008 - Ma 187 _,~
1063~49 value of 10. The suspension wag heated, while ~tirring, at 50 C and was mixed with 5 g Or l-aminobenzene-4-B-hydro~yethyl-sulrone-sulfuric acid-semi-ester. After ~he pH value had been corrected to 10 by means Or 2 molar NaOH, the reaction mixture was- stirred for 1 hour at 50C. Subsequently the mixture was suction-filtered by means of a strainer and was washed with 2 1 of water, 2 1 Or acetone and 2 1 of 0.2 molar HCl. ~or the diazotizing Or the arylamine groups introduced into the polyhydroxymethylene, the filter residue was su~-pended in 200 ml of 0.2 molar HCl, was cooled to 2C and was mixed, while stirring, with 1 molar NaN02 solution until a slight exces~ Or nitrite could be detected by means of KJ-starch paper. After 10 mimlteJ the reaction mixture was suction-filtered by way of a stra$ner, and the filter residue was washed with 1 1 Or an aqueous 0.01 molar amidosulfonic acid Or a temperature of ~C and then with 200 ml Or 0.2 mo-lar sodium phosphate of 2C and a pH ~alue of 7.5. For the coupling, the.product containing diazonium groups was sus-pended in 150 ml Or a solution of 2 g Or tetanus toxoide with a 1260 Lf/mg Or N in 0.2 molar sodium phosphate Or a p~ value of 7.5 and a temperature Or 2C, and was stlrred for 20 hour~ at 5C. Those portions of the toxo~de that had not been bound were washed out with 1 1 of 1 molar NaCl solution on a trainer.
; 25 AD~lication Or the compound of polvhydroxvmethylene-tetanus toxoide Preparation of tetanu~ antitoxin 10 Grams of the product described in Example 1 were ou~-29 pended in 150 ml Or o- 15 molar NaCl solution~ with the addi-:~ ,. .
~- ~ tion of M/15 sodium pho~phate, ("PBS") of a pH ~alue Or 7.2, ~` _ 12 -.; ~ ; ' .
". ' ~' ' ' .
~OE 74/~ 0~8 - Ma 187 and wero introduced into a column of a diameter of 3 cm and a height of 10 cm. 20 Millimeters-of tetanu~ horse serum with 1650 IU of tetanus antitoxin/ml were introduced into the co-lumn which was subsequently washed with 500 ml of PBS, pH
value 7.2. The elution of the antibodies was effected with 0.5 molar glycine/HCl buffer, pH value of 2.5. After neu-tralization with 2 molar NaOH and subsequent centrifuging, the eluate contained a total of 240 mg of protein and 12.300 IU of ~ ~*~in as was found in a protection test with mice.
E X A M P L E 2: -Pol~hvdroxymethylene-aminobenzamidine compound - Preparation of the network from (~ -carbox~-n-pentyl~ subst~-tuted_pol~h~droxv~eth~lene-20 Grams of polyhydroxymethylene activated with BrCN
according to Example 1 were added to an aqueous solution of 25 g Or ~-amino-caproic acid that had been adjusted to a pH
value Or 8.5 by mean~ of diluted sodium hydroxide solution, and the total volume of which had been made up to 1 liter.
The reaction mi~ture was then stirred for another 5 hours at room temperature and a pH ~alue of 8.5 and was suction-fil-tered, then the product was wa~hed thoroughly with ~ater.
dried sample of the polyhydroxymethylene substituted by (~ -carboxy-n-pentyl) groupq had a nitrogen content of o.86~.
Preparation of the carrier-bound effector Efrector: Aminobenzamidine 2 Grams of a polyhydroxymethylene derivati~e prepared aocording to ExamplQ 2 were ~uspended in 25 ml of 0.1 molar morpholino-ethane-sulfonic acid and wero mixed with 1 ~ of 29 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide-HCl. By ad-.
' ~ , . ' . ', , ' ' , ,' ~ , . ' . .
NOE 74/8 008 - Ma 187 . 1063049 ding 2 molar HCl while controlling the pH value, *he pH wa~
adjusted to 4.8. 1 Gram of m-aminobenzamidine was added to the reaction mixture while stirring. The su~pension was stirred for 1 hour while correcting the pH value, and the adsorbent was subsequently introduced into a chromatography column ha~ing a diameter Or 1 cm. The adsorbent was then rinsed with 200 ml of 0.05 molar trishydroxymethylaminomethane-HCl-buffer solution, pH of 8.0, with an addition of 0.5 molar KCl (tris-KCl).
Application of the compound of polyhvdroxvmethylene-aminobenz-amidine Preparation of thrombln ~C 3 ~ ~/s) (~C3 y~/5~
100 Milligrams of bovine raw thrombin~ were dissolved in 5 ml of tris-KCl~ and were introduced into the column which had been pr0pared as has been described a~ove. The column was subsequently rinsod with 200 ml of tris-KCl buffer solution.
The thrombin bound to the insolubilized m-aminob~nzamidine was separated from this compound with 100 ml Or a solution of 0.05 molar m-aminobenzamidine in tris-KCl buffer.
The fractions containing protein were combined and were pas~ed over a column of l x 30 cm of sephadex G-25 into a tris-KCl buffer solution. In the first peak of the column eluate - the peak containing the protein - 78% of the initial activity - could be detected by determining the activity according to Chase and Shaw, ~iochem. 8, 1969, page 2212.
:
_ 14 -.
r . - . . - . ' ' '. ' ' ~ ' ' ' ' '.' . ;' .
, ' , ~ .. . . ,; , ,.
,., ' ; ' ". "; ' ' '' ' '~ " ' ' ~ ; ' '' , ' ~ ' , ' ' ' , ' . ' ..
~ HOE 74/~ 008 - Ma 187 1063~49 E X A M P L E 3s Pol~hydroxymethylene-popsin compound Preparation of the net*ork from (~ -amino-n-hexyl)-substituted polyh~droxymethylenes . 8.0 Grams of polyvinylene carbonate dissolved and repreci-pitated from dimethylformamide and methanol, which product - had been prepared according to N.D. Field, J.R. Schaefgen, J. Polymer Sci., vol. 58, p. 534 (1962), were suspended at room temperature in a solution of 7.5 g of hexame~yle~e d~m~ in 180 ml of methanol and were stirred for ~8 houri at room tem-perature, then suction-filtered, washed with methanol and . :
suspended in a solution of 10 g Or sodium methylate in 300 ml of ~ethanol of 96~ strength for 4 days at room temperature, and were then washed with methanol and vory thoroughly with watër. A dried sample of the polyhydro~ymethylene adsorbent substituted by primary amino groups over a spacer of 6 CH2 groups had a nitrogen content of 5.7%.
Preparation of the carrier-bound effector B Effectors Pep8in ~C 3~
20 Gram~ of (~ -amino-n-hexyl)-substituted polyhydroxy-methylene prepared according to Example 3 were suspended in 400 ml of 0.2 molar sodium phosphate, pH ~alue of 10. 40 Hilli-liter~ of a glutardialdehyde solution o~ 25% strength were added to the suspension, while stirring. After having been 25. stirred for 2 hours at room temperature, the reaction mi~ture --wa~ filtered off by means Or a strainer, and the filter resi-due was washed with 4 liter~ of 0.2 molar sodium acetate buffer solution, pH value of 4.0, Subsequently the reaction product . ~C3~-4-~) 29 was introduced into 500 ml Or a solution Or 5 g.of pepsi~ with . - 15 -.
.:
,,. . .. -, , . : : , .: . . - ,, ,: . .
~OE 74~B 008 - Ma 187 a total of 500.000 units, determined according to Anson, ~n 0.2 molar sodium acetate, pH value of 4.0, and was then ~tirred for 15 hours at 6C. The product obtained was then washed on a ~trainer with 5 liters of 0.1 molar sodium acetate/
acetic acid, pH value of 4.0, and an addition Or 1 molar NaCl.
A~plication of th~ compound polyhvdroxymethylene-pePsin Proteolytic separation of immuno~lobulins .
The adsorbent of the present Examplè wa~ suspended in 0.1 molar acetate buffsr solution, pH value of 4.0, and the acti-vity was dermined according to Anson. The su~pension contained ~ EC ~ 4 /) B a total of 21.000 units of pepsin~in a bound state which could be detected by mean of thi~ te~t. For the proteolytic ~epara-tion of human immunoglobulin G, the pepsin adsorbent ~ ~
filtered off and the residue was suspended in a ~olution of 2.5% strength of 30 g of human IgG in a physiological NaCl solution, pH value of 4.1. The separation mixture was ~tirred ~or 24 hour~ at 37C. After elimination of the carrier-bound pepsin by filtration the filtrate was added to 1250 ml of ~aturated ammonium sulf~te solution. The precipitate obtained in this process was then removed by centrifuging; it contained ;~ the ~(ab)2 ~ragment of the immunoglobulin with the antibody activity of the latter. The residue liquid was re~ected.
, E X A M P_L E 4s ~re~aration of the com~ound Or ~olvhvdroxvmeth~lene-o~amic acid 10 Grams Or polyhydroxymethylene were activated with BrCN a~ has been described in Example 1 and were reacted with 5 g o~ tyramin dlssolved in 150 ml o~ 0.1 molar 30dium~ydro-29 geno-carbonate, at a temperature of 5C for 60 minutes, while "
.. . , ... ~ . . . ... ..
: . , .:, . . :
- - . ' .. . . . . . .. . . . .
. , ., .:
~ HOE 74/B 008 - Ma 187 stirring. Those portion~ of the tyr~min which had not been bound were suction-filtered by mean~ of a strainer, and the filter residue was washed with 1 1 of 0.1 molar sodiumpho~-phate buffer solution having a pH value of 7.0 and a tempera-ture of 5C. The filter residue was suspended in 150 ml of a solution of 1.0 g of diazotized p-aminophenyloxamic acid, the solution having been cooled to 5C, and the mixture was stirred for 15 hours at a temperature of 5C. The adsorbent wa~ washed on a s*rainer with 1 1 of 0.05 molar sodium ace-tate-acetic acid buffer solution, pH value of 5.5, with an addition of 2 mil~les of CaC12 and 0.2 mM of EDTA, ~pl~cation of_the comnound of nolvhvdroxvmethvlene-oxamic acid ~ . . .
o~ r ~-~r~ 3~l~
The adsorbent according to E~ample 4 was resuspended in 15 the above-mentioned acetate buffer solution and was introduced into a chromatography column having a diameter Or 2 cm. 1 Li-ter of a culture filtrate of Vibrio Cholerae with 800 IU of ~3-Z~I-I~) neuraminida~/ml were passed through this column,- the filtra-te having been adJusted beforehand with 2 molar acetic acid to a pH ~alue Or 5.5 and having been ~ixed with CaC12 and EDTA up to a concentration Or 2 millimoles and 0.2 milli-mole~ respec*ively. Tho~e proteins which had not been bound were ~ashed out by means Or 500 ml of acotate buffer solution.
The elution o f the neuraminidaoe adsorbed to the carrier-bound inhibitor was effected by washing the column with 0.1 molar sodium-~ydrogeno-carbonate solution and collecting the fraction~ containing active neuraminidase. They contain ~6 29 f the ~ctivity used.
1? :
.
.. ,. . "..., . ~ .. ;., .,.... ...., , -... .. . ... . . .. . . ., .... . . . , . ~ . ~
.. . . . . . , . ., . . . . . .. .. .. - . .
.
, . . . : . - . ~.
HOE 74/B 008 - Ma 187 E X A M P L E 5:
a)Reaction of polyhydroxymethylene with epichlorhydrin 50 Grams of polyhydroxymethylene were suspended in 1 1 of 2n NaOH, were mixed with 250 ml of epichlorhydrin and were then stirred for 2 hour~ at a temperature in the range Or from 55 to 60C. The pH value of the suspension was re-duced after a short time to 10 - 11. Ihis pH ~alue wa maintained for another hour by adding NaOH. After a reaction time of 2 hours, the solid matter was suction-flltered, and was washed with water, acetone and finally again with water.
. .
b) 50 Grams Or hexamethylene-diamine were dissol~ed in 1.5 1 of water and were mixed with HCl up to a pH value of 10. The poly~ydro~ymethylene activated under 5a) was added to the solution and was stirred for 6 hours at a temperature in the - range of from 50 to 55 C. Then the product was 6uction-fil-tered and was washed with water until it was free from hexa-methylene-diamine.
¢) The product obtained under Example 5b) was stirred with 50 g of 1-aminobenzene-4-B-hydroxyethyl-sulfone-sulfuric acid e~ter for 1 hour at 55 C and a pH ~alue o~ 10. Subsequently th0 ~olid matter was filtered ofr, and was then washed with water, acetone and again with water.
d) 10 Grams 0r the product obtained ac¢ording to 5 c) were ; - washed on a strainer with 200 ml Or 0.1 n HCl and were after-25- wards sus~ended in 300 ml Or 0.5 n HCl. At a temperature in the range Or ~rom 0 to 4C, the suspension was mixed, while Jtirring, with 0.1 n NaN0~ solution, until a Rlight excess ~--~ of nitrite could be detected in the diazotl~ing ~ith KJ-starch 29 pap~r. Af ter 10 minutes, the reaction mixturq was suction-_ 18 -' .. . . . , .. , .: .. ,, - , , . . . ~ : , -, HOE 74/B 008 - Ma 187 filtered by way of a ~trainer, and the residue was washed with ice water and subsequently with 0.15 molar sodium phos-phate buffer solution having a pH value of 7.5 and a tempera-ture of from 0 to 4C.
e) 0.75 Grams of albumin were dissolved in 250 ml of phos-phate buffer solution, pH value of 7.5, were then cooled to 4C, and the produot prepared under 5d) was added. The sus-pension was stirred at 4C for 20 hours, was then filtered off, and the so~id matter was washed with 1 molar NaCl and a phosphatebuffered solution Or sod-i-um chloride (P~S) (aque-ous NaCl solution of 0.9% strength with a content of 1/15 mo-les of Na2}3P04-KH2P04 buffer of a pH value of 7.2).
The filtrate and the washing lye~ were tested for albumin according to the method o~ the radia~ imm-~odif~usion. 75 Mil-~5 ligrams o~ albumin w~e-bound to 1 g of the carrier thus pre-- pared.
- r) 2.4 Grams of IgM were dissolved in 250 ml of phosphate bufrer solution, pH of 7.5, and were ~ooled to 4C. 10 Grams o~ the product diazotized according to 5d) were added, and the suspension was stirred at 4C *or 20 hours. A~ter filtra-tion the solid-matter was wsshed wlth 1 molar NaCl solution and wlth PBS.
1 Gram of the carrier prepared according to 5 f) was able to bind 240 mg of IgM.
~ ~ A ~ L 6 s a) A~ has bcen described in Example 5 a)b?, 10 g of poly-hydroxy~thyle~e were activated wi~h epichlorhydrin, were reacted with hexamothylel1e-diamiIla and wore worked up.
; 29 b) 10 Grams o~ the carrier thus prepared were`succinoylated - 19 ~
:
;,. . -, . .
HOE 74/B 008 - Ma 187 . .
1063~49 for 4 hours at 10 C and a pH ~alue of 6, with 5 g of succinic acid a~ydride suspended in 200 ml of water. The pH value was adjusted with 2 n NaOH. After washing the solid matter with water, the product was Rtirred at 5C for 30 minutes, at a pH value of 5, with 2.5 g of N-cyclohexyl-N'-(-(N-methyl-morpholino)-ethyl)~carbodiimide-p-toluene-sulfonate, was filtered off and was washed fast with ice water.
c) 1 Gram of IgG was dissolved in 250 ml of a phosphate buffer solution, pH value of 7.5, and wa~ stirred for 24 hours at 4C with the carrier prepared under 6 f). After filtration, the product wa~ washed out with 1 molar ~odium chlor~de and with PBS.
85 Milli~rams of IgG were bound in a covalent bond to 1 g of the carrier.
E X A M P L E 7s a) 20 Grams of the product prepared under 5 a) were stirred for 6 hours, at a temperature in the range of from 50 to 55 C
and at a pH value of 10, with 20 g of aminocaproic acid. Sub-~equently the product was suctlon-filtered and washed with water-b) 10 Grams of the product prepared'under 7 a) were mixed with 2.5 g Or N-cyclohesyl-N'-(N-methylmorpholino)-ethyl)-carb- -, odiimide-p-toluene-sulfonate, and after 5 minutes, 2 g of ~ N-hydroxysuccinimide were added which had a pH value of 5.
- 25 After having been stirred for 5 hours at 24C, the ~olid matt-r wao filtered off and was washed with water.
c) 0.5 Gram of albumin were dissolYed in 200 ml of phosphate buffer solution and were stirred for 24 hours, at a tempera-29 ture of 4C, with the activated carrier propared under ~ b).
.
- . ,: . , HOE ?4/B 00~ - Ma 187 After filtration, the product was washed out with 1 molar sodium chloride and with PBS.
- 50 Milligrams of albumin were bound in a covalent bond to 1 g of the carrier.
5 E X A M P L E 8:
10 Gram6 of polyhydroxymethylene were activated with epichlorhydrin as has been described ill Example 5 a) b), were reacted wlth hexaJmethylene-diamine and were worked up.
a) 10 Grams of the carrier thus prepared were suspended in 1Q- 100 ml of water and were reacted with 500 ml of glutardialde-hyd~ of 25% strength. After having been stir~cd for 1 hour~
the solid matter was flltered off and was washed with water and/or PBS.
b) 0.5 Gr~m of albumin were dissolved in 200 ml of a phos-p'.late buffer solution and were stirred for 20 hours at 4C
~ith the carrier prepared under 8 a). After filtration, ths product was washed with 1 molar sodium chloride and with PBS.
E X A M P L E 9s D~rect reaction of polyhydrox~ethylene activated with 20 - epichlorhydrins - -a) 10 Grams of polyhydroxymethylene were activated with 50 ml of epichlorhydrin as has been described in Example 5 a).
After filtration, the product was washed fast with water, acetone~water, and finally with PBS.
b) 0.5 Gram of albumin were dissolved in 200 ml of PBS and were stirred for 60 hours at 4C with the product prepared under 9 a). After filtration, the carrier-bound protein was washed with 1 molar sodium chlorlde and with PBS.
1 Gram of the carrier thus prepared was able to bind 45 mg of albumin. Ik . . ` .
~ . - 2~ -, ~- .
- ; . . . . ....
.. , . . . . - . , . . .: .
':~ ''. `.' ~- ........ ~:
'' . ;'' ' :
HOE 74/B 008 - ~a 187 .
10 Grams of water-insoluble polyhydroxymethylene were stirred at a pH value of 9.5 (0.15 molar sodium bicarbonate/
. NaOH buffer) for 3 days-- at 25C with 30 g of diethylene glycol-diglycidylether; the product was suction-filtered, was washed thoroughly, was added to 0.5 g of human albumen in 200 ml of 0.15 molar phosphate buffer having a pH
value of 8, then the reaction mixture was stirred for 60 hours at 10C. After filtration, the carrier-bound protein was washed with 1 molar sodium chloride solution and with "PBS".
- 1 Gram of the carrier thus prepared contained 35 mg of bound albumen.
E X A M P L E 11:
10 Grams of polyhydroxymethylene were reacted with 30 g f glycidyl-tris-(glycidylether), as it has been described in Example 10, were introduced into 0.5 g of immunoglobulin G
(IgG) in 200 ml of 0.15 molar phosphate buffer having a pH
value of 7.5, and the reaction mixture was stirred for 60 hours at 4C. After filtration, the product was washed with 1 molar sodium chloride solution and with "PBS".
1 Gram of the protein resin thus prepared contained 50 mg of bound IgG.
.
' .
. . _ 21 a - -, .,~ , , ~ . . . . ... .. .. . ,5,-, ,
Besides the processes mentioned above as examples for the preparation of the covalent bond between the carrier net-29 work and the protein effector or other effectors, there may be . - . - . - . - . . ., .. , ~
-,. . .
.
.
mentioned further methods which lead to the reaction of the hydroxyl groups of the polyhydroxymethylene on one hand, and to the reaction of determined groups of the effector on the other hand, thus forming a covalent bond between the two reactants, such as the know reaction with complex-forming metal compounds, for example, titanium compounds.
Besides by its above-mentioned chemical and thermal stability, the polyhydroxymethylene is marked by advantageous processing properties, thus becoming superior to the carrier networks on the basis of natural carbohydrates that had been considered optimal up to this time. Polyhydroxymethylene can be manufactured, for example, in the form of fibers, filaments, films or spherical particles or, perferably, powdered material, so that the most appropriate form may be chosen, depending on the purpose of application of the effector to be bound to it.
Another advantage of the polyhydroxymethylene as com-pared against the carrier materials according to the state of the art is to be seen in the fact that the surface which is accessible for the bond of the effectors and/or the spacers can be controlled during production.
According to the analogous reaction mechanisms as they have been described for the bond of the effector with the poly-hydroxymethylene network, the spacer substances can also be bound to the network, for example if the spacers carry an amino group as one of the functional groups. The introduction of a spacer also makes it possible, however, to introduce ~ additional reaction possibilities for the bond of the effector.
- In the case that the spacer bound to the network carries a-~ free carboxyl group, this carboxyl group can be activated, for _, - - ~ . , ~ - .
- : , . . : . , , , , -~ . , .: : ., . ' . . .. , : ; ., : :, ~
.
HOE 74/B 008 - Ma 187 .
1063~49 example, by carbodiimide compounds and can then form an amide bond with amino groups of the effector. Carboxyl groups also permit the formation of amide bonds by means of the isoxazolium salts introduced by Woodward.
If the spacer bound to the network carries an available free amino group, the introduction of arylamino groups in the extension of the spacer is possible, by means of vinylsulfone derivatives containing arylamino groups or sulfuric acid esters of B-hydroxyethylsulfones, said arylamino groups subsequently being diazotized according to known methods and then bond to correspondingly reactive groups of the effector, for example:
, . . . ..
r . n 0 NH- (CH2) 6-NH-CH2-CH2 s 2 ~ -NH2 .
... . ...
The biologically active compounds of the invention are suitable for most application processes that have so far been known for other effectors bound to hydrop~ilic water-insoluble carriers. This means that enzymes can be made water-insolub~e.
The insoluble enzymes are more and more used to deterDine sub-~trates in automatic analyzers and as so-called enzyme elec-trodes. Due to the increased stability, a series of carrier-bound enzymes is suitable for the implementation of enzymatic reactions on an industrial scale.
Carrier-bound biologically active substances are used on a large scale in the afflnity chromatography, due to their properties as specific adsorbents. By means of carrier-bo~d 29 natural or synthetic enzyme inhibitors, a high degree Or purity _ 9 _ .. . . .. . . . .. .. . .. . .. . ..
. . , ~ . , . . - , . . ~ . -- . . , . .. ~ . -. . . .. , . . . , . - - - . .. . . . . .. . . . .. . .
: . . . , ~ . .~, , , : . . ~ . - -HOE 74/B 008 - Ma 187 1~63~49 is obtained for enzymes, whereas the enzymes have been found to be extremely suitable as effectors particularly for the production of natural enzyme inhibitors from raw extracts.
Carrier-bound water-insoluble antigens are used for the iso- -lation of the corresponding antibodies, which are thus obtained free from other serum constituents and free from other antigens.
By means of affinity chromatography it is also possible to isolate those antibodies that cannot be precipitated, as well as those that exclude precipitation due to their low concentration - 10 in the serum, and also to determine them quantitatively.
The Examples given below in order to illustrate the in-vention show that a carrier prepared according to a single process is suitable for the bond of many different effectors and that the basic structure of polyhydroxymethylene can be ;~ 15 made suitable, by an appropriate substitution, for the bond of any desired effector.
Moreover, the Examples show how the effectors bound to the network can be used.
. \
'' \' ~1 . ' \ .
\
.' ' , \
\_ ~ - 10 -.
~, ' ' ~ . .. .
' .': , . ' .
:
- ' ~ , :
- ~ HOE 74/B 008 - Ma 187 1063~49 E X A M P L E ls Pol~hydrox~meth~lene-tetanu~ toxoid compound Preparation of the network from (~ -amino-n-hexyl~-substituted pol~h~droxymethvlene: -20 Grams of polyhydroxymethylene suspended in 900 ml of water were mixed with a solution of 20 g Or BrCN in 50 ml of --dimethylformamide and were maintained at a pH ~alue of 11.5 for 6 minutes at an internal temperature Or 15C by 810wly adding dropwise, while stirring, 2 n sodium hydroxide solu-tion. Subsequently the mixture was suction-filtered i~nediate-ly, was washed thoroughly with ice water and was squeezed to remo~e the remaining water. The polyhydroxymethylenc thus activated, which was still moist with water, was added to a well-stirred solution of 20 g of hexamethylene diamine in 500 ml of H20, which was maintained at room temperature and was ad~usted with concentrated HCl to a pH value of 8.5, the mixture was then made up with water to a total ~olume of 1 liter, while the pH value was constantly ad~usted between 8.5 and 9.0, and was stirred at room temperature for another 5 hours. SubsequQntly the reaction mixture was suction-fil-tered and was washed thoroughly with water. A dried sample of the polyhydroxymethylene substituted by (~ -amino-n-he~yl) group~ had a nitrogen content of 1.9~. By means of Sanger~
reagent for the detection of primary free amino groups, pro-ducts we~e ob$iained which had a ~trong yellow color shade.
Preparation of the carrier-bound effector Effeotors Tetanus antigen 10 Grams of the carrier of EY.ample 1 were suspended in - 29 200 ml of a 0.5 molar sodium pho~phate ~olution ha~ing a pH
'- - 11 -." ' ' . ~ .: : i . : . ~ . . - , :
: .,............ ; . . , : . ,. , ~ -. : .. ~ ' .~ : . :
. ; i , . ; ~.
HOE 74/B 008 - Ma 187 _,~
1063~49 value of 10. The suspension wag heated, while ~tirring, at 50 C and was mixed with 5 g Or l-aminobenzene-4-B-hydro~yethyl-sulrone-sulfuric acid-semi-ester. After ~he pH value had been corrected to 10 by means Or 2 molar NaOH, the reaction mixture was- stirred for 1 hour at 50C. Subsequently the mixture was suction-filtered by means of a strainer and was washed with 2 1 of water, 2 1 Or acetone and 2 1 of 0.2 molar HCl. ~or the diazotizing Or the arylamine groups introduced into the polyhydroxymethylene, the filter residue was su~-pended in 200 ml of 0.2 molar HCl, was cooled to 2C and was mixed, while stirring, with 1 molar NaN02 solution until a slight exces~ Or nitrite could be detected by means of KJ-starch paper. After 10 mimlteJ the reaction mixture was suction-filtered by way of a stra$ner, and the filter residue was washed with 1 1 Or an aqueous 0.01 molar amidosulfonic acid Or a temperature of ~C and then with 200 ml Or 0.2 mo-lar sodium phosphate of 2C and a pH ~alue of 7.5. For the coupling, the.product containing diazonium groups was sus-pended in 150 ml Or a solution of 2 g Or tetanus toxoide with a 1260 Lf/mg Or N in 0.2 molar sodium phosphate Or a p~ value of 7.5 and a temperature Or 2C, and was stlrred for 20 hour~ at 5C. Those portions of the toxo~de that had not been bound were washed out with 1 1 of 1 molar NaCl solution on a trainer.
; 25 AD~lication Or the compound of polvhydroxvmethylene-tetanus toxoide Preparation of tetanu~ antitoxin 10 Grams of the product described in Example 1 were ou~-29 pended in 150 ml Or o- 15 molar NaCl solution~ with the addi-:~ ,. .
~- ~ tion of M/15 sodium pho~phate, ("PBS") of a pH ~alue Or 7.2, ~` _ 12 -.; ~ ; ' .
". ' ~' ' ' .
~OE 74/~ 0~8 - Ma 187 and wero introduced into a column of a diameter of 3 cm and a height of 10 cm. 20 Millimeters-of tetanu~ horse serum with 1650 IU of tetanus antitoxin/ml were introduced into the co-lumn which was subsequently washed with 500 ml of PBS, pH
value 7.2. The elution of the antibodies was effected with 0.5 molar glycine/HCl buffer, pH value of 2.5. After neu-tralization with 2 molar NaOH and subsequent centrifuging, the eluate contained a total of 240 mg of protein and 12.300 IU of ~ ~*~in as was found in a protection test with mice.
E X A M P L E 2: -Pol~hvdroxymethylene-aminobenzamidine compound - Preparation of the network from (~ -carbox~-n-pentyl~ subst~-tuted_pol~h~droxv~eth~lene-20 Grams of polyhydroxymethylene activated with BrCN
according to Example 1 were added to an aqueous solution of 25 g Or ~-amino-caproic acid that had been adjusted to a pH
value Or 8.5 by mean~ of diluted sodium hydroxide solution, and the total volume of which had been made up to 1 liter.
The reaction mi~ture was then stirred for another 5 hours at room temperature and a pH ~alue of 8.5 and was suction-fil-tered, then the product was wa~hed thoroughly with ~ater.
dried sample of the polyhydroxymethylene substituted by (~ -carboxy-n-pentyl) groupq had a nitrogen content of o.86~.
Preparation of the carrier-bound effector Efrector: Aminobenzamidine 2 Grams of a polyhydroxymethylene derivati~e prepared aocording to ExamplQ 2 were ~uspended in 25 ml of 0.1 molar morpholino-ethane-sulfonic acid and wero mixed with 1 ~ of 29 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide-HCl. By ad-.
' ~ , . ' . ', , ' ' , ,' ~ , . ' . .
NOE 74/8 008 - Ma 187 . 1063049 ding 2 molar HCl while controlling the pH value, *he pH wa~
adjusted to 4.8. 1 Gram of m-aminobenzamidine was added to the reaction mixture while stirring. The su~pension was stirred for 1 hour while correcting the pH value, and the adsorbent was subsequently introduced into a chromatography column ha~ing a diameter Or 1 cm. The adsorbent was then rinsed with 200 ml of 0.05 molar trishydroxymethylaminomethane-HCl-buffer solution, pH of 8.0, with an addition of 0.5 molar KCl (tris-KCl).
Application of the compound of polyhvdroxvmethylene-aminobenz-amidine Preparation of thrombln ~C 3 ~ ~/s) (~C3 y~/5~
100 Milligrams of bovine raw thrombin~ were dissolved in 5 ml of tris-KCl~ and were introduced into the column which had been pr0pared as has been described a~ove. The column was subsequently rinsod with 200 ml of tris-KCl buffer solution.
The thrombin bound to the insolubilized m-aminob~nzamidine was separated from this compound with 100 ml Or a solution of 0.05 molar m-aminobenzamidine in tris-KCl buffer.
The fractions containing protein were combined and were pas~ed over a column of l x 30 cm of sephadex G-25 into a tris-KCl buffer solution. In the first peak of the column eluate - the peak containing the protein - 78% of the initial activity - could be detected by determining the activity according to Chase and Shaw, ~iochem. 8, 1969, page 2212.
:
_ 14 -.
r . - . . - . ' ' '. ' ' ~ ' ' ' ' '.' . ;' .
, ' , ~ .. . . ,; , ,.
,., ' ; ' ". "; ' ' '' ' '~ " ' ' ~ ; ' '' , ' ~ ' , ' ' ' , ' . ' ..
~ HOE 74/~ 008 - Ma 187 1063~49 E X A M P L E 3s Pol~hydroxymethylene-popsin compound Preparation of the net*ork from (~ -amino-n-hexyl)-substituted polyh~droxymethylenes . 8.0 Grams of polyvinylene carbonate dissolved and repreci-pitated from dimethylformamide and methanol, which product - had been prepared according to N.D. Field, J.R. Schaefgen, J. Polymer Sci., vol. 58, p. 534 (1962), were suspended at room temperature in a solution of 7.5 g of hexame~yle~e d~m~ in 180 ml of methanol and were stirred for ~8 houri at room tem-perature, then suction-filtered, washed with methanol and . :
suspended in a solution of 10 g Or sodium methylate in 300 ml of ~ethanol of 96~ strength for 4 days at room temperature, and were then washed with methanol and vory thoroughly with watër. A dried sample of the polyhydro~ymethylene adsorbent substituted by primary amino groups over a spacer of 6 CH2 groups had a nitrogen content of 5.7%.
Preparation of the carrier-bound effector B Effectors Pep8in ~C 3~
20 Gram~ of (~ -amino-n-hexyl)-substituted polyhydroxy-methylene prepared according to Example 3 were suspended in 400 ml of 0.2 molar sodium phosphate, pH ~alue of 10. 40 Hilli-liter~ of a glutardialdehyde solution o~ 25% strength were added to the suspension, while stirring. After having been 25. stirred for 2 hours at room temperature, the reaction mi~ture --wa~ filtered off by means Or a strainer, and the filter resi-due was washed with 4 liter~ of 0.2 molar sodium acetate buffer solution, pH value of 4.0, Subsequently the reaction product . ~C3~-4-~) 29 was introduced into 500 ml Or a solution Or 5 g.of pepsi~ with . - 15 -.
.:
,,. . .. -, , . : : , .: . . - ,, ,: . .
~OE 74~B 008 - Ma 187 a total of 500.000 units, determined according to Anson, ~n 0.2 molar sodium acetate, pH value of 4.0, and was then ~tirred for 15 hours at 6C. The product obtained was then washed on a ~trainer with 5 liters of 0.1 molar sodium acetate/
acetic acid, pH value of 4.0, and an addition Or 1 molar NaCl.
A~plication of th~ compound polyhvdroxymethylene-pePsin Proteolytic separation of immuno~lobulins .
The adsorbent of the present Examplè wa~ suspended in 0.1 molar acetate buffsr solution, pH value of 4.0, and the acti-vity was dermined according to Anson. The su~pension contained ~ EC ~ 4 /) B a total of 21.000 units of pepsin~in a bound state which could be detected by mean of thi~ te~t. For the proteolytic ~epara-tion of human immunoglobulin G, the pepsin adsorbent ~ ~
filtered off and the residue was suspended in a ~olution of 2.5% strength of 30 g of human IgG in a physiological NaCl solution, pH value of 4.1. The separation mixture was ~tirred ~or 24 hour~ at 37C. After elimination of the carrier-bound pepsin by filtration the filtrate was added to 1250 ml of ~aturated ammonium sulf~te solution. The precipitate obtained in this process was then removed by centrifuging; it contained ;~ the ~(ab)2 ~ragment of the immunoglobulin with the antibody activity of the latter. The residue liquid was re~ected.
, E X A M P_L E 4s ~re~aration of the com~ound Or ~olvhvdroxvmeth~lene-o~amic acid 10 Grams Or polyhydroxymethylene were activated with BrCN a~ has been described in Example 1 and were reacted with 5 g o~ tyramin dlssolved in 150 ml o~ 0.1 molar 30dium~ydro-29 geno-carbonate, at a temperature of 5C for 60 minutes, while "
.. . , ... ~ . . . ... ..
: . , .:, . . :
- - . ' .. . . . . . .. . . . .
. , ., .:
~ HOE 74/B 008 - Ma 187 stirring. Those portion~ of the tyr~min which had not been bound were suction-filtered by mean~ of a strainer, and the filter residue was washed with 1 1 of 0.1 molar sodiumpho~-phate buffer solution having a pH value of 7.0 and a tempera-ture of 5C. The filter residue was suspended in 150 ml of a solution of 1.0 g of diazotized p-aminophenyloxamic acid, the solution having been cooled to 5C, and the mixture was stirred for 15 hours at a temperature of 5C. The adsorbent wa~ washed on a s*rainer with 1 1 of 0.05 molar sodium ace-tate-acetic acid buffer solution, pH value of 5.5, with an addition of 2 mil~les of CaC12 and 0.2 mM of EDTA, ~pl~cation of_the comnound of nolvhvdroxvmethvlene-oxamic acid ~ . . .
o~ r ~-~r~ 3~l~
The adsorbent according to E~ample 4 was resuspended in 15 the above-mentioned acetate buffer solution and was introduced into a chromatography column having a diameter Or 2 cm. 1 Li-ter of a culture filtrate of Vibrio Cholerae with 800 IU of ~3-Z~I-I~) neuraminida~/ml were passed through this column,- the filtra-te having been adJusted beforehand with 2 molar acetic acid to a pH ~alue Or 5.5 and having been ~ixed with CaC12 and EDTA up to a concentration Or 2 millimoles and 0.2 milli-mole~ respec*ively. Tho~e proteins which had not been bound were ~ashed out by means Or 500 ml of acotate buffer solution.
The elution o f the neuraminidaoe adsorbed to the carrier-bound inhibitor was effected by washing the column with 0.1 molar sodium-~ydrogeno-carbonate solution and collecting the fraction~ containing active neuraminidase. They contain ~6 29 f the ~ctivity used.
1? :
.
.. ,. . "..., . ~ .. ;., .,.... ...., , -... .. . ... . . .. . . ., .... . . . , . ~ . ~
.. . . . . . , . ., . . . . . .. .. .. - . .
.
, . . . : . - . ~.
HOE 74/B 008 - Ma 187 E X A M P L E 5:
a)Reaction of polyhydroxymethylene with epichlorhydrin 50 Grams of polyhydroxymethylene were suspended in 1 1 of 2n NaOH, were mixed with 250 ml of epichlorhydrin and were then stirred for 2 hour~ at a temperature in the range Or from 55 to 60C. The pH value of the suspension was re-duced after a short time to 10 - 11. Ihis pH ~alue wa maintained for another hour by adding NaOH. After a reaction time of 2 hours, the solid matter was suction-flltered, and was washed with water, acetone and finally again with water.
. .
b) 50 Grams Or hexamethylene-diamine were dissol~ed in 1.5 1 of water and were mixed with HCl up to a pH value of 10. The poly~ydro~ymethylene activated under 5a) was added to the solution and was stirred for 6 hours at a temperature in the - range of from 50 to 55 C. Then the product was 6uction-fil-tered and was washed with water until it was free from hexa-methylene-diamine.
¢) The product obtained under Example 5b) was stirred with 50 g of 1-aminobenzene-4-B-hydroxyethyl-sulfone-sulfuric acid e~ter for 1 hour at 55 C and a pH ~alue o~ 10. Subsequently th0 ~olid matter was filtered ofr, and was then washed with water, acetone and again with water.
d) 10 Grams 0r the product obtained ac¢ording to 5 c) were ; - washed on a strainer with 200 ml Or 0.1 n HCl and were after-25- wards sus~ended in 300 ml Or 0.5 n HCl. At a temperature in the range Or ~rom 0 to 4C, the suspension was mixed, while Jtirring, with 0.1 n NaN0~ solution, until a Rlight excess ~--~ of nitrite could be detected in the diazotl~ing ~ith KJ-starch 29 pap~r. Af ter 10 minutes, the reaction mixturq was suction-_ 18 -' .. . . . , .. , .: .. ,, - , , . . . ~ : , -, HOE 74/B 008 - Ma 187 filtered by way of a ~trainer, and the residue was washed with ice water and subsequently with 0.15 molar sodium phos-phate buffer solution having a pH value of 7.5 and a tempera-ture of from 0 to 4C.
e) 0.75 Grams of albumin were dissolved in 250 ml of phos-phate buffer solution, pH value of 7.5, were then cooled to 4C, and the produot prepared under 5d) was added. The sus-pension was stirred at 4C for 20 hours, was then filtered off, and the so~id matter was washed with 1 molar NaCl and a phosphatebuffered solution Or sod-i-um chloride (P~S) (aque-ous NaCl solution of 0.9% strength with a content of 1/15 mo-les of Na2}3P04-KH2P04 buffer of a pH value of 7.2).
The filtrate and the washing lye~ were tested for albumin according to the method o~ the radia~ imm-~odif~usion. 75 Mil-~5 ligrams o~ albumin w~e-bound to 1 g of the carrier thus pre-- pared.
- r) 2.4 Grams of IgM were dissolved in 250 ml of phosphate bufrer solution, pH of 7.5, and were ~ooled to 4C. 10 Grams o~ the product diazotized according to 5d) were added, and the suspension was stirred at 4C *or 20 hours. A~ter filtra-tion the solid-matter was wsshed wlth 1 molar NaCl solution and wlth PBS.
1 Gram of the carrier prepared according to 5 f) was able to bind 240 mg of IgM.
~ ~ A ~ L 6 s a) A~ has bcen described in Example 5 a)b?, 10 g of poly-hydroxy~thyle~e were activated wi~h epichlorhydrin, were reacted with hexamothylel1e-diamiIla and wore worked up.
; 29 b) 10 Grams o~ the carrier thus prepared were`succinoylated - 19 ~
:
;,. . -, . .
HOE 74/B 008 - Ma 187 . .
1063~49 for 4 hours at 10 C and a pH ~alue of 6, with 5 g of succinic acid a~ydride suspended in 200 ml of water. The pH value was adjusted with 2 n NaOH. After washing the solid matter with water, the product was Rtirred at 5C for 30 minutes, at a pH value of 5, with 2.5 g of N-cyclohexyl-N'-(-(N-methyl-morpholino)-ethyl)~carbodiimide-p-toluene-sulfonate, was filtered off and was washed fast with ice water.
c) 1 Gram of IgG was dissolved in 250 ml of a phosphate buffer solution, pH value of 7.5, and wa~ stirred for 24 hours at 4C with the carrier prepared under 6 f). After filtration, the product wa~ washed out with 1 molar ~odium chlor~de and with PBS.
85 Milli~rams of IgG were bound in a covalent bond to 1 g of the carrier.
E X A M P L E 7s a) 20 Grams of the product prepared under 5 a) were stirred for 6 hours, at a temperature in the range of from 50 to 55 C
and at a pH value of 10, with 20 g of aminocaproic acid. Sub-~equently the product was suctlon-filtered and washed with water-b) 10 Grams of the product prepared'under 7 a) were mixed with 2.5 g Or N-cyclohesyl-N'-(N-methylmorpholino)-ethyl)-carb- -, odiimide-p-toluene-sulfonate, and after 5 minutes, 2 g of ~ N-hydroxysuccinimide were added which had a pH value of 5.
- 25 After having been stirred for 5 hours at 24C, the ~olid matt-r wao filtered off and was washed with water.
c) 0.5 Gram of albumin were dissolYed in 200 ml of phosphate buffer solution and were stirred for 24 hours, at a tempera-29 ture of 4C, with the activated carrier propared under ~ b).
.
- . ,: . , HOE ?4/B 00~ - Ma 187 After filtration, the product was washed out with 1 molar sodium chloride and with PBS.
- 50 Milligrams of albumin were bound in a covalent bond to 1 g of the carrier.
5 E X A M P L E 8:
10 Gram6 of polyhydroxymethylene were activated with epichlorhydrin as has been described ill Example 5 a) b), were reacted wlth hexaJmethylene-diamine and were worked up.
a) 10 Grams of the carrier thus prepared were suspended in 1Q- 100 ml of water and were reacted with 500 ml of glutardialde-hyd~ of 25% strength. After having been stir~cd for 1 hour~
the solid matter was flltered off and was washed with water and/or PBS.
b) 0.5 Gr~m of albumin were dissolved in 200 ml of a phos-p'.late buffer solution and were stirred for 20 hours at 4C
~ith the carrier prepared under 8 a). After filtration, ths product was washed with 1 molar sodium chloride and with PBS.
E X A M P L E 9s D~rect reaction of polyhydrox~ethylene activated with 20 - epichlorhydrins - -a) 10 Grams of polyhydroxymethylene were activated with 50 ml of epichlorhydrin as has been described in Example 5 a).
After filtration, the product was washed fast with water, acetone~water, and finally with PBS.
b) 0.5 Gram of albumin were dissolved in 200 ml of PBS and were stirred for 60 hours at 4C with the product prepared under 9 a). After filtration, the carrier-bound protein was washed with 1 molar sodium chlorlde and with PBS.
1 Gram of the carrier thus prepared was able to bind 45 mg of albumin. Ik . . ` .
~ . - 2~ -, ~- .
- ; . . . . ....
.. , . . . . - . , . . .: .
':~ ''. `.' ~- ........ ~:
'' . ;'' ' :
HOE 74/B 008 - ~a 187 .
10 Grams of water-insoluble polyhydroxymethylene were stirred at a pH value of 9.5 (0.15 molar sodium bicarbonate/
. NaOH buffer) for 3 days-- at 25C with 30 g of diethylene glycol-diglycidylether; the product was suction-filtered, was washed thoroughly, was added to 0.5 g of human albumen in 200 ml of 0.15 molar phosphate buffer having a pH
value of 8, then the reaction mixture was stirred for 60 hours at 10C. After filtration, the carrier-bound protein was washed with 1 molar sodium chloride solution and with "PBS".
- 1 Gram of the carrier thus prepared contained 35 mg of bound albumen.
E X A M P L E 11:
10 Grams of polyhydroxymethylene were reacted with 30 g f glycidyl-tris-(glycidylether), as it has been described in Example 10, were introduced into 0.5 g of immunoglobulin G
(IgG) in 200 ml of 0.15 molar phosphate buffer having a pH
value of 7.5, and the reaction mixture was stirred for 60 hours at 4C. After filtration, the product was washed with 1 molar sodium chloride solution and with "PBS".
1 Gram of the protein resin thus prepared contained 50 mg of bound IgG.
.
' .
. . _ 21 a - -, .,~ , , ~ . . . . ... .. .. . ,5,-, ,
Claims (15)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a biologically active compound consisting essentially of a water-insoluble poly(hydroxymethylene) having a biologically active substance bound chemically thereto, in which (a) a water-insoluble poly(hydroxymethylene) is reacted with a biologically active substance, or (b) a poly(vinylene-carbonate) is reacted with a biologically active substance and the remaining cyclo-carbonate groups are hydrolyzed to hydroxyl groups.
2. A process as claimed in claim 1 in which the poly (hydroxymethylene) or polyvinylidene carbonate is first reacted with a compound having at least two funtional groups and the resulting complex is reacted with a biologically active substance.
3. A process as claimed in claim 1 in which the biolog-ically active substance is selected from the group of enzymes, activators, inhibitors, antigens, antibodies, plasmaproteins, blood group substances, phythemagglutinins, anti-biotics, vitamins, hormones, peptides, amino acids or synthetically prepared effectors.
4. A biologically active compound as defined in claim 1, whenever obtained according to a process as claimed in claim 1 or claim 3 or by an obvious chemical equivalent thereof.
5. A process as claimed in claim 1 for the preparation of a biologically active compound consisting essentially of a water-insoluble poly(hydroxymethylene) having a biologically active substance directly bound chemically thereto, in which the poly(hydroxymethylene) is reacted directly with the biologically active substance.
6. A process as claimed in claim 5 in which the biologi-cally active substance is selected from the group of enzymes, activators, inhibitors, antigens, antibodies, plasmaproteins, blood group substances, phythemagglutinins, anti-biotics, vitamins, hormones, peptides, amino acids or synthetically pre-pared effectors.
7. A biologically active compound consisting essentially of a water-insoluble poly(hydroxymethylene) having a biologi-cally active substance directly bound chemically thereto, when-ever obtained according to a process as claimed in claim 5 or claim 6 or by an obvious chemical equivalent thereof.
8. A process as claimed in claim 2 for the preparation of a biologically active compound consisting essentially of a water-insoluble poly(hydroxymethylene) having a biologically active substance bound thereto by means of a compound having several functional groups, in which a water-insoluble poly-(hydroxymethylene) is reacted with a compound having several functional groups and with a biologically active substance.
9. A process as claimed in claim 8 in which the bio-logically active substance is selected from the group of enzymes, activators, inhibitors, antigens, antibodies, plasma-proteins, blood group substances, phythemagglutinins, anti-biotics, vitamins, hormones, peptides, amino acids, or syntheti-cally prepared effectors.
10. A biologically active compound consisting essentially of a water-insoluble poly(hydroxymethylene) having a biologi-cally active substance bound thereto by means of a compound having several functional groups, whenever obtained according to a process as claimed in claim 8 or claim 9 or by an obvious chemical equivalent thereof.
11. A process as claimed in claim 2 for the preparation of a biologically active compound as defined in claim 1 in which a poly(vinylene-carbonate) is reacted with a poly-functional compound or a synthetic effector, any remaining cyclocarbonate groups are hydrolized and the resultant product is further reacted with a biologically active compound.
12. A process as claimed in claim 9 in which the bio-logically active substances is selected from the group of enzymes, activators, inhibitors, antigens, antibodies, plasma-proteins, blood group substances, phythemagglutinins, anti-biotics, vitamins, hormones, peptides, amino acids, or synthe-tically prepared effectors.
13. A biologically active compound, whenever obtained according to a process as claimed in claim 9 or claim 10 or by an obvious chemical equivalent thereof.
14. A process as claimed in claim 1 in which the water-insoluble poly(hydroxymethylene) is a carrier matrix for the biologically active compound.
15. A biologically active compound, whenever obtained according to a process as claimed in claim 14 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19742421789 DE2421789C3 (en) | 1974-05-06 | Biologically active products and processes for their manufacture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1063049A true CA1063049A (en) | 1979-09-25 |
Family
ID=5914739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA226,193A Expired CA1063049A (en) | 1974-05-06 | 1975-05-02 | Preparation of water insoluble immobilized bio-active compounds |
Country Status (16)
| Country | Link |
|---|---|
| JP (1) | JPS599153B2 (en) |
| AT (1) | AT338417B (en) |
| BE (1) | BE828776A (en) |
| CA (1) | CA1063049A (en) |
| CH (1) | CH630410A5 (en) |
| DK (1) | DK196175A (en) |
| ES (1) | ES437153A1 (en) |
| FI (1) | FI60216C (en) |
| FR (1) | FR2270266B1 (en) |
| GB (1) | GB1512651A (en) |
| IE (1) | IE41040B1 (en) |
| IT (1) | IT1054620B (en) |
| LU (1) | LU72407A1 (en) |
| NL (1) | NL181782C (en) |
| NO (1) | NO144672C (en) |
| SE (2) | SE427086B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2552510C3 (en) * | 1975-11-22 | 1981-02-19 | Behringwerke Ag, 3550 Marburg | Biologically active compounds and processes for their preparation |
| EP2138575B8 (en) | 2002-02-20 | 2013-07-24 | Mersana Therapeutics, Inc. | Conjugates comprising a biodegradable polymer and uses therefor |
| JP6476934B2 (en) * | 2015-02-02 | 2019-03-06 | 東ソー株式会社 | Novel polymer and cell culture substrate having the same |
-
1975
- 1975-04-29 ES ES437153A patent/ES437153A1/en not_active Expired
- 1975-04-30 FI FI751299A patent/FI60216C/en not_active IP Right Cessation
- 1975-05-01 NL NLAANVRAGE7505152,A patent/NL181782C/en not_active IP Right Cessation
- 1975-05-02 IT IT22967/75A patent/IT1054620B/en active
- 1975-05-02 CA CA226,193A patent/CA1063049A/en not_active Expired
- 1975-05-05 DK DK196175A patent/DK196175A/en not_active Application Discontinuation
- 1975-05-05 IE IE996/75A patent/IE41040B1/en unknown
- 1975-05-05 CH CH575075A patent/CH630410A5/en not_active IP Right Cessation
- 1975-05-05 LU LU72407A patent/LU72407A1/xx unknown
- 1975-05-05 AT AT342275A patent/AT338417B/en not_active IP Right Cessation
- 1975-05-05 GB GB18761/75A patent/GB1512651A/en not_active Expired
- 1975-05-05 NO NO751599A patent/NO144672C/en unknown
- 1975-05-06 SE SE7505275A patent/SE427086B/en not_active IP Right Cessation
- 1975-05-06 FR FR7514177A patent/FR2270266B1/fr not_active Expired
- 1975-05-06 BE BE156099A patent/BE828776A/en not_active IP Right Cessation
- 1975-05-06 JP JP50054436A patent/JPS599153B2/en not_active Expired
-
1978
- 1978-05-16 SE SE7805544A patent/SE444947B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| FR2270266B1 (en) | 1979-03-16 |
| FI60216B (en) | 1981-08-31 |
| AU8081875A (en) | 1976-11-11 |
| JPS599153B2 (en) | 1984-02-29 |
| JPS50155679A (en) | 1975-12-16 |
| NL7505152A (en) | 1975-11-10 |
| NO144672B (en) | 1981-07-06 |
| NO751599L (en) | 1975-11-07 |
| FI60216C (en) | 1981-12-10 |
| IE41040L (en) | 1975-11-06 |
| FI751299A (en) | 1975-11-07 |
| DK196175A (en) | 1975-11-07 |
| DE2421789B2 (en) | 1976-10-07 |
| ES437153A1 (en) | 1977-04-16 |
| IE41040B1 (en) | 1979-10-10 |
| FR2270266A1 (en) | 1975-12-05 |
| NL181782C (en) | 1987-11-02 |
| SE7505275L (en) | 1975-11-07 |
| IT1054620B (en) | 1981-11-30 |
| GB1512651A (en) | 1978-06-01 |
| NL181782B (en) | 1987-06-01 |
| DE2421789A1 (en) | 1975-11-13 |
| BE828776A (en) | 1975-11-06 |
| ATA342275A (en) | 1976-12-15 |
| AT338417B (en) | 1977-08-25 |
| CH630410A5 (en) | 1982-06-15 |
| SE427086B (en) | 1983-03-07 |
| LU72407A1 (en) | 1977-02-09 |
| SE444947B (en) | 1986-05-20 |
| NO144672C (en) | 1981-10-14 |
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