CA1069070A - Water insoluble biologically active compounds and process for their manufacture - Google Patents
Water insoluble biologically active compounds and process for their manufactureInfo
- Publication number
- CA1069070A CA1069070A CA266,092A CA266092A CA1069070A CA 1069070 A CA1069070 A CA 1069070A CA 266092 A CA266092 A CA 266092A CA 1069070 A CA1069070 A CA 1069070A
- Authority
- CA
- Canada
- Prior art keywords
- copolymer
- biologically active
- active substance
- groups
- vinylene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—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
- C12N11/087—Acrylic polymers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/44—Antibodies bound to carriers
-
- 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
- C08F8/12—Hydrolysis
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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
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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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
- C08F8/34—Introducing sulfur atoms or sulfur-containing groups
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—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
- C12N11/091—Phenol resins; Amino resins
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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
- C08F2800/00—Copolymer characterised by the proportions of the comonomers expressed
- C08F2800/20—Copolymer characterised by the proportions of the comonomers expressed as weight or mass percentages
-
- 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
- C08F2810/00—Chemical modification of a polymer
- C08F2810/50—Chemical modification of a polymer wherein the polymer is a copolymer and the modification is taking place only on one or more of the monomers present in minority
Abstract
Abstract of the Disclosure:
The present invention relates to water insoluble biologically active compounds in which a copolymer of vinylene glycol is chemically bound to a biologically active substance, a process for their manufacture and their use, preferably in the affinity chromatography.
The present invention relates to water insoluble biologically active compounds in which a copolymer of vinylene glycol is chemically bound to a biologically active substance, a process for their manufacture and their use, preferably in the affinity chromatography.
Description
The present il1vention relates to water insoluble biologi~
cally active compounds a~d to a process fo~ their manufacture.
Object of the invention are new compounc~s of ~iologically active subst~nces and water-insoluble high-mol.ecular weight 5 compounds, a process for their manufacture and their use pre-ferably in the affinity chromatography.
In the recent years, a new technique within.the biochemical working methods increasingly gained in use. Its primary charac-teristic is the use of the af-finity o carrier-bouncl, biological-ly active substances for selec-tive reactions.
. Through a specific complex formation of the carrier-bound substance with a second substance in a mixture, this substance : can be eliminated from the mixture and if des~red, suhsequently isolated by desorption.
Carrier-bound enzymes have the advantage that they allow the transformation of a substance in continuous processes and the reaction products obtained to be free oE enzymes.
In the bioch.emi~al, enzymatic analysis, carrier-bound water-insoluble enzymes can repea-tedly be used as reactants. I
Because the enzymes have af~inity not only to the substra- .
tes, but also to the specific inhibitors, the affinity chroma-tograph.y on carrier-bound enzymes proved to be especial.ly fa-vorable for the obtention of enzyme inhibi.tors, On the other hand, i:nh.ibitor$ ~ound to water-insoluble matrices permit the preparative obtention of the corresponding enzymes, As so~called i~mune adsorbents, antigens or anti~odies are bound to water-:insoluble matrices wherea~ter the correspond-ing antibodies or antigens can be isolated~
cally active compounds a~d to a process fo~ their manufacture.
Object of the invention are new compounc~s of ~iologically active subst~nces and water-insoluble high-mol.ecular weight 5 compounds, a process for their manufacture and their use pre-ferably in the affinity chromatography.
In the recent years, a new technique within.the biochemical working methods increasingly gained in use. Its primary charac-teristic is the use of the af-finity o carrier-bouncl, biological-ly active substances for selec-tive reactions.
. Through a specific complex formation of the carrier-bound substance with a second substance in a mixture, this substance : can be eliminated from the mixture and if des~red, suhsequently isolated by desorption.
Carrier-bound enzymes have the advantage that they allow the transformation of a substance in continuous processes and the reaction products obtained to be free oE enzymes.
In the bioch.emi~al, enzymatic analysis, carrier-bound water-insoluble enzymes can repea-tedly be used as reactants. I
Because the enzymes have af~inity not only to the substra- .
tes, but also to the specific inhibitors, the affinity chroma-tograph.y on carrier-bound enzymes proved to be especial.ly fa-vorable for the obtention of enzyme inhibi.tors, On the other hand, i:nh.ibitor$ ~ound to water-insoluble matrices permit the preparative obtention of the corresponding enzymes, As so~called i~mune adsorbents, antigens or anti~odies are bound to water-:insoluble matrices wherea~ter the correspond-ing antibodies or antigens can be isolated~
2~ Biologically active substances in the $ense of the inven~
2 _ ~
. . ' - '' ' '' ' : "' " ' . , ~.
,, ~ ' ' ~ ' ,, !
' ~ 6~ HOE 75/B 014 .
tion are natural ancl synthetic substances acting ln VlVO and in vitro, which b~long ~o the wide range of enzymes, activa-___ tors, inhibitors, antigens or antibodies, vitamins and hormons.
These biologically active substances representing the active principles of the water-insoluble biologicàlly active compounds of -the invention are called effectors.
Most of the carrier-bound efEectors so far described are essentially more s-table than the correspondiny biologically active substances in solution.
Suitable carrier materials, so-called matrices, are ad-vantageously substances that are insoluble in aqueo~s systems and exhibit an unspecific adsorption as low as possible. To this effect, hydrophobic, hydrophi]ic and ionic in-teractions between the matrix and the reactant of the effector should as largely as possible be avoided. A bond with substances to the effector, which ~s undesired, for example of those which are no specific reactants of the effector, should be excluded.
The matrices so far used as carriers for biologically ac-tive substances are on the one hand those that bind the effec-tors by physical adsorption, for example polystyrene, active charcoal and glass beads, and, on the other hand, those that form a covalent bond with the effectors, for example vinyl polymers as homo- and ~opolymers, for example polyacrylic acids, polyacrylic acid amides and amino-, carboxy- or sulfonyl-substi-tuted polystyrene, cellulose and its derivatives and natural and synthetic polypeptides and proteins. Because of the equi-librated interaction be~ween the matrix and the effector, car-hohydrates, especially cellulose, dextrane, starch, ayar and 29 their derivatives, are widely used as matrices in aqueous sy-.
. ~
HOE 7~s 014 s~
stems, even ~hou~h ~he carboxyl yroups frequently contained in these natural substances are considered to be -troublesome be~
cause oE their unspecific affinities. Moreover, these substan-ces have a relativel~ poor thermal and chemical stability.
Many of these disadvankages could be eliminated by using polyvinylene glycol, a synthe-tic polymer, as a matrix. Poly-~inylene glycol, also called polyhydroxymethylene, is prepared by acid or basic hydrolysis from polyvinylene carbonate. Each carbon atom bears a hydroxyl group and a hydrogen atom. The uninterrupted -C-C-bond gives the carrler an especially high stability.
Now~ it was found that instead of polyvinylene glycol vinylene g]ycol copolymers are advantageously used as carrier matrix. By incorporating sui-table comonomer units into poly-vinylene carbonate by polymerization, the physical and chemi-- cal propert~es of the polyvinylene glycol prepared therefrom can be varied to a large extent.
So, for exampl~, ~he hydrophilic property or "swelling"
of the polyvinylene glttcol can be modified by the incorpora-tion of hydrophobic monomers, for example ethylene, vinyl chloride or styrene into the polyvinylene carbonate by poly-meriæation. By the copolymerization of vinyl acetate, a vinylene glycol group is replaced in the saponified end pro-duct by a vinyl alcohol group, which increases the swelling property in an aqueous medium.
Comonomers, for example diethylene glycol divinyl ether and divinyl benzene enable the cross-linking and thus the improvernent of the mechanical properties of the carrier. By 2g incorporating electrophilic functions, for example oxirane ", .
: . , , . ,, , . ,- :
- : ~, -. , :' . :. . '. ' ' ~ 7 0 groups ~epo~i groups) ~rom vinylglycidyl ether comonomers by polymer~zation, the matrix can directly be reac~ed with nucleo-philic functions (-NH2, -OH etc.). Carboxyl groups can be in-troduced through acrylic acid ester comonomers. When the copolymerization is completed, the ester groups are saponified to carboxyl groups. The in-troduction of -NH2 or -COOH groups allows the activation by means oE carbodilmides, isoxazolium salts, glutar aldehydes etc. Copolymers of vinylene glycol/
vinyl alcohol have a higher specific surface and so an increas-ed binding capaclty for, e.g. proteins on the surface of the polymer powder compared to a vinylene ylycol homopolymer.
The presen-t invention provides biologically active com-pounds from a preferably water-insoluble copolymer of the poly-vinylene glycol and biologically active substance bound there-to with the maintenance of its biological activity. In these compounds, the carrier matrix is a polyvinylene glycol copoly-mer containing up to 45 %, preferably 5 - 20 ~, of a comonomer.
The biologically active substancesare enzymes, activators, in-hibitors, antigens or antibodies, other plasma pro-teins, blood ~0 group substances, phythemagglutinines, antibiotics, vitamins - or hormons, peptides or amino acids or synthetic effectors.
The polymer carrier and the biologically active substance are bound to each other either direetly or via a side chain (Spaeer) in a eovalent bond.
To bind the biologically active compounds via a side c~ain (Spacer) is advantageous when the molecular weights of their affinity partners are very diferent, or when in a bio-speeific process proteins having a very high molecular weight 29 or those comprising several sub-units take part. Spaeers are ~06~7~ HOE 75/B 014 l, side-chains of a certain lengths anchored to the polymer matrix which can be obtained by polymerization, by addition or incorpora-tion (linkage of bi- or polyfunctional compounds - for example tripeptide - to the matrix) or by incorporation of functional groups and stepwise prolongation.
Object of the invention are furthermore processes for the covalent bond of the biologically active substances to the carrler .
For this purpose, a series of generally known reactions is available.
They firstly concern the processes for the manufacture of the polyvinylene glycol copolymer. These processes are charac-terized in that vinylene carbonate is polymerized with comonomers of the general formulae:
/ Rl / 1 .
; H2C=C H C=C H C=C
\ COOR3 O-CO-CH Cl ~Rl /Rl / Rl H2C=C ~ ~ ~ H2C=C CH=CH2 (0CH2-CH2) O- CH=CH2 / 1 ~R
H2C=C~ /C~2 H2C=C
N (CH2)n (CH2)n~C\H /cH2 oc _ l O
~Rl ~R
H2C=C ~ H C-C
O-CH-CH ~CH-CIH2 O ' /
...... .
, . : . : , . . .
:::
. .
:. .....
. .
~B 0 14 9t)7~
. f in which R1 is hydrogen, methyl, ethyl, R 3 i5 methyl, ethyl, propyl and n is a whole number be-tween 1 and 4.
The polymerization of the polyvinylene carbonate occurs with the use of 45 % of cornonomers, pre~erably 5 ~ 20 %~
~he biologically active compound can ~e prepared, for ex-ample by introducing an electrophilic group eithér into the biologically active substance or into the polyvinylene glycol copolymer through which the b.iolo~ically active substance re- :~
acts with the polyvinylene glycol copolymers. The introduction of the electrophilic groups into the carrier matrix occurs either by copolymerizing vinylene carbonate with comonomers containing electrophilic groups or by reacting the carrier matrix with low molecular weight compounds carrying activating electropnilic groups.
Generally, the chemical coupling of a reactant to the carrier ma-trix is simple when the one side carries the nucleo-phi.lic functions and the other side the electrophilic functions.
Reactive, nucleophilic functions are above all amino, sulfhydryl and hydroxyl groups, w~ich are generally already contained : 20 either in the matrix o- in the reactan-t. Electrophilic func~
tions have to be introduced. To this effect, the carboxyl groups can be transformed into acid halides, acid azides, acid anhydrides, imidazolides or directly be act.ivated with carbodiimides. Reactive electrophilic groups are also isocy-25 . anate, ~sothiocyanate~ diazonium groups or cyclic imidocarbo nate esters. Especially suitahle is the introduction of re-act.ive yroups by mean.s of copolymerization.
By the introduction of monomers having epoxy groups, for 29 example epoxyalkylvinyl ethers, in~o the polyvinyl carbona~e ~ 7 ~
~ HOE 75/B 01~
by polymeriæatlon ~nd because o~ the very big differences in the saponiEication speed between cyclic carbonate and epoxy groups, it is eas~ to obtain a polyvinylene glycol matrix hav-in~ reactive epoxy groups which can be re~cted with nucleo-philic functions. So~ the direct bond of a biologically ac-tive substance and/or the introduction (prolongation) of a side chain (Spacer) is possible.
The further advantage of the use of copolymers is the possibility to introduce directly by polymerization electro-philic functions which allow a reaction accordiny to known me-thods.
The copolym~rization of viny].ene carbonate with ethyl acrylate and Eollowi.ng saponification of the polymers gives a matrix having -OH- a~d -COOH-~unctions. The carboxyl groups can be activated by means of carbodi.imides, whereupon an amide .-linkage to the amino groups of the effector occurs. Carboxyl groups further allow the formation of amide ~onds by means of the isoxazolium salts suggested by Woodward.
A fur-ther method is the coupling of biologically active compounds to monomerscontaining carboxyl groups by means of N-ethoxycarbonyl-2-ethoxy 1,2-dihydroqu.inoline (EEDQ).
The carboxyl groups can be esterified according to known methods, the ester then be converted into hydrazide and the azicle resulting therefrom be linked via the amino group o the 25. effector protein to the polyvinylene glycol matrix~
When the comonomer polymerized into the matrix contains amino groups, which can be set free by following reactions, arylamino groups can be introduced by means of vinylsulfon 29 derivatives containing arylamino groups or of sulfuric acid - ' , ... .. . . .
: -- .; , ': , ~,. - , -- . -, -. . ..
- ~ .
~ ~Q69~0 HOE 75/~ 014 esters of ~-hydroxyethyl sulfones. Then the aryl amino groups can be diazotized according to known methods and bound to reactive groups of an effector, for example _ _ H H I H H ~ ¦
...---C--C---C--C--C--C--...
. OH 1H n 1H H O 1 - HO-CH
1H2-- NH- (CH2) 6-NH CH2-SO2-~3-N~2 In this way, ~ prolongation of a Spacer is possible.
Proteins can also be bound via the amino groups by means of glutardialdehyde.
The linkage of the effector to the matrix after an acti-vation of the hydroxyl or amino groups of the polyvinylene-glycol copolymer is easy using cyano halides, preferably bromocyane and effecting a following reaction of the biologically active effec-tors containing the amino groups through these activated groups.
The effector can also be bound to the polyvinylene glycol or polyvinylene glycol copolymer matrix by acylating the hydroxyl groups with bromoacetyl bromide, followed by the aklyla~
tion of the amino group of the effector.
Similar courses of the reaction can be achieved, for example, by reacting the hydroxyl groups of the carrier with re-active triazines, one part of the reactive groups of the triazine reacting with the polyvinyleneglycol compound, another one with the amino groups of the effector.
~Qgj9~70 Diazo-tiza~le aromatic amines which can react through a further reactive group with the hydroxyl groups of the carrier - permit on the other hand the coupling with capable activated amino acids, for exanlple the tyrosine or histidine radicals of the protein effector.
Vinylsulfone derivatives con-taininy arylami~o groups and sulfuric acid semi esters of B-hydroxylethyl sulfones can be reacted wi-th the hydroxyl groups of the carrier. They allow -the effector to be bound according to the precited diazoti-sation reaction.
Especially stable ether bonds are obtained in the reac-tion of the hydroxyl groups of the polyvinyleneglycol copoly-mer with epoxides which dqhot form ions and which contain at least two reactive groups, such as the epihalohydrines or the polyepoxides, ~or exa~ple epichlorohydrine or bisepoxides.
A further mode of modification is the introduction by poly-merization of comomomers into the polyvinylene carbonate chain (for example of vinylp~rrolidone) followed by the partial re-action of the cyclocarbonate rings of the polyvinylene carbo-nate with an amine, for example hexamethylene diamine, to give a polyvinylene carbonate copolymer substituted by a Spacer or an effector through an urethane compound. The residual cyclo-cdrbonate groups are then saponified to h~droxyl groups:
-_ ~o _ .. . . . . ..
~ " :
:1~69070 - fH-CH2--CH--CH ... -CE~-CH2---CH--C~fH--CH~
N O P +R-NH N~ O O OH o H2\ / c 2 / \ o 11 C = o Verseifung ~,~
H2 H2 nH2 H2 r -1 ~H
PolyvinylencarbOnat - CH-CH ---CH--CH --CH--CH-- ...
t 2 l l l l N OH OH OH O
H / \~ O _ _ C - O
R
Beside the cited examples of methods for the production of the covalent bond between the carrier matrix and the protein effector or other effectors there are further methods which lead to the reaction of the hydroxyl groups or of given groups of the copolymer with an effector so giving rise to the covalent bond between them, for example the known reaction with complex-forming metal compounds, for example titanium compounds. All these methods are known to those skilled in the art.
When low-molecular weight, biologically active compounds are to be bound, it is aavantageous not to activate the carrier but the efector.
Principally, all the methods can be used which are known in the macromolecular chemistry for the modification of synthetic or natural macromolecular compounds.
I-IOE 7~B 01 2 11~9~7~ :
The polyvinyle~e glycol copolymers are dlstinguished be-side their chemical and thermal stability by advantageous properties for technical procedures which makes them superior to the carrier matrices on the basis of natural carbohydrates previously deemed to be optimum. For example, they can be pre-pared in the form of fibers, threads, foils or spheric partic-- les, so that the most suitable form can be choosen depend~ng on the intended application purpose of the effector to be bound thereto. Preferably the copolymers are in the form of finely divided powders ~aving a high specific surace.
A further advantage of the polyvinyleneglycol copolymers over the known carrier materials is the con~rollability on the industrial scale of the dimension of the surface accessib]e to the linkage of the eEfectors or the Spacers.
The biologically active compounds according to the in-vention can be used for most of the methods which have become known so far for other effectors bound to hydrophilic, water-insoluble carriers. So, enzymes can be made water-insoluble .
The i:nsoluble enzymes are increasingly used for the determina-2a tion of substrates in analysis automatons and as so-called enzyme electrodes. Because of the higher sta~ility, a series of carrier bound enzymes suits for the enzymatic reactions on a technical scale.
Because of their property as specific adsorbents, carrier-bound, biologically active substances have found ~ide applica-tion in the affinity chromatography. Carrier bound, natural or synthetic enzyme inhibitors enable the high-grade purifi-cation of enzymes, while the enzymes proved to be excellently 29 suitable effectors especially for the obtention o natural en-_ 12 ~
~ 3~
z~me inhibitors from crude extrac-ts. Carrier-bound water--inso-luble anti~ens are used for the isola~Dn of -the adherent anti-bodies which are thus obtained in a state free of other serum constituents and antiyens. In the affinity chromatography, antibodies incapable of being precipitated and those which cannot be precipitated because of their low concentration in the serum can be isolated and be determined quantita-tivelyO
The following Examples illustrate -the invention:
E X A M P L E S:
~anufactu~e o . olymers The vinylene carbonate used in the preparation of the co-polymers (CP) was boiled under reElux,-before its use, for an hour over sodium boron hydride (100 parts by weight of vinylene carbonate to 2 parts by weight of NaBH4) under a pressure of 33 mm, distilled at 75/33mm over a 50 cm long silver-sleeve column filled with ~aschig glass rings and used as straight as possible for the copolymerization. Also, the comonomers used were purified before by distillation.
E X A M P L E 1:
.. . . ... ..
Copolymer o~ vin~lene glycol/viny~ alcohol from saponified copolymer of vinylene carbo _ e/vinyl acetate a) 0.05 Part by weiyht of azobisisobutyronitrile are dissolv-ed under nitroyen in 9 parts by weight oE vinylene carbo-nate and I part by weight O:e vinyl acetate. The monomer mixture is Eilled under nitrogen in a flattened screw cap aluminum tube (4 mm thick, 60 mm large, 150 mm long) (total volume o~ the mixture filled in: about 35 ml), sealed under N2 and hung into a 50 C hot water bath duriny 48 29 hours. Hard, brittle plastic plates are obtained which .
: , , , ' . . .
`` HOE 75/B 014 3V~
, .
are preco~minu-ted in a crushing mill. Then, the granules - are heated under a pressure of 1 - 2 mm for 5 hours at 120 C, to separate the part of the monomer which has not been polymerized. The demonomerized granules are urther ground in a mill to get a grain size of C 0.1 mm, introduc-ed into 500 parts by weight o 0.5 N sodium hydroxide solu-tion, mixecl at 20 C during 1 hour by means of a high--spee~
stirrer, whereupon the carbonate and acetyl groups are saponi~ied. The water-insoluble CP-vinylene /glycol/vinylalco-hol is suction-Eiltered, thoroughly freed from inorganic salts with water and lyophilised.
~leld: 4.7 parts by weight of CP-vinylene ylycol/vinyl al-cohol having a specific surace measured according to BET of 34 m g 1.
IS b) Process as described under 1a), using instead 7 parts by weight of vinylenecarbonate to 3 parts by weight of vinyl acetate~
Yield: 4.1 parts by weight oE CP-vinylene glycol/vinyl alcohol having a specific surface of the lyophilised poly-mer powder of 62.5 m2g 1.
c) Comparison test as described~der1a), differing in that 10 parts by weight of vinylene carbonate and no comonomer are used.
Yield: 4~7 par-ts by weight oE homopolymer of vinylene glycol having a speciEic surface o the lyophilised poly-mer powder of 20 5 m2g 1.
_ X A M P L E 2:
Copolymer of vinylene glycol/vinyl~ly~idvl ether 29 A mixture o;f 7 parts by weight of vinylene carbonate, 3 - 14 _ - .. - ' ' . ' ' "
,, .: . ' , : : ' , -"' " , ' ' ' ' . . ' ' ' ~' ' ' .. . .
.
., , , ~ , ,. . :
. - . . . . .. . .
1~ 7~
parts by w~ight of vinylglycidyl ether and 0.1 part by weight of azobi~lsobu-tyronitrile is heated to 50 C for 3 days under- -: N2 in flattened aluminum tubes as described under 1a). As des cribed under 1a) the plastic plates obtai.ned are co~ninuted, demonomerized~yround to a yrain size of ~ 0.1 mm, suspended in 500 parts by weight of ice cold O.S ~ NaOH us'ing a high-speed stirrer during 30 minutes, the saponified polymer is immediately centrifuyed off, suspended in ice-water, neutralis - ed to pH 7 with 2 N H2S04 while cooling with ice, suction--fil-tered, washed w.ith ice water and lyophilised~
Yield: 3.9 parts by weight of CP-vinylene glycol/vinylglycidyl ether containing 1.3 milliequivalent of oxirane groups/g.
(determined according to "Prak-tlkum der makromolekularen orga~
nischen Chemie", page 221, by Braun, D.l Cherdron, H.~Kern, W. Huthig Verlag, E~eidelberg 1966).
Aminosubstituted copolymer-carrier mater.ial
2 _ ~
. . ' - '' ' '' ' : "' " ' . , ~.
,, ~ ' ' ~ ' ,, !
' ~ 6~ HOE 75/B 014 .
tion are natural ancl synthetic substances acting ln VlVO and in vitro, which b~long ~o the wide range of enzymes, activa-___ tors, inhibitors, antigens or antibodies, vitamins and hormons.
These biologically active substances representing the active principles of the water-insoluble biologicàlly active compounds of -the invention are called effectors.
Most of the carrier-bound efEectors so far described are essentially more s-table than the correspondiny biologically active substances in solution.
Suitable carrier materials, so-called matrices, are ad-vantageously substances that are insoluble in aqueo~s systems and exhibit an unspecific adsorption as low as possible. To this effect, hydrophobic, hydrophi]ic and ionic in-teractions between the matrix and the reactant of the effector should as largely as possible be avoided. A bond with substances to the effector, which ~s undesired, for example of those which are no specific reactants of the effector, should be excluded.
The matrices so far used as carriers for biologically ac-tive substances are on the one hand those that bind the effec-tors by physical adsorption, for example polystyrene, active charcoal and glass beads, and, on the other hand, those that form a covalent bond with the effectors, for example vinyl polymers as homo- and ~opolymers, for example polyacrylic acids, polyacrylic acid amides and amino-, carboxy- or sulfonyl-substi-tuted polystyrene, cellulose and its derivatives and natural and synthetic polypeptides and proteins. Because of the equi-librated interaction be~ween the matrix and the effector, car-hohydrates, especially cellulose, dextrane, starch, ayar and 29 their derivatives, are widely used as matrices in aqueous sy-.
. ~
HOE 7~s 014 s~
stems, even ~hou~h ~he carboxyl yroups frequently contained in these natural substances are considered to be -troublesome be~
cause oE their unspecific affinities. Moreover, these substan-ces have a relativel~ poor thermal and chemical stability.
Many of these disadvankages could be eliminated by using polyvinylene glycol, a synthe-tic polymer, as a matrix. Poly-~inylene glycol, also called polyhydroxymethylene, is prepared by acid or basic hydrolysis from polyvinylene carbonate. Each carbon atom bears a hydroxyl group and a hydrogen atom. The uninterrupted -C-C-bond gives the carrler an especially high stability.
Now~ it was found that instead of polyvinylene glycol vinylene g]ycol copolymers are advantageously used as carrier matrix. By incorporating sui-table comonomer units into poly-vinylene carbonate by polymerization, the physical and chemi-- cal propert~es of the polyvinylene glycol prepared therefrom can be varied to a large extent.
So, for exampl~, ~he hydrophilic property or "swelling"
of the polyvinylene glttcol can be modified by the incorpora-tion of hydrophobic monomers, for example ethylene, vinyl chloride or styrene into the polyvinylene carbonate by poly-meriæation. By the copolymerization of vinyl acetate, a vinylene glycol group is replaced in the saponified end pro-duct by a vinyl alcohol group, which increases the swelling property in an aqueous medium.
Comonomers, for example diethylene glycol divinyl ether and divinyl benzene enable the cross-linking and thus the improvernent of the mechanical properties of the carrier. By 2g incorporating electrophilic functions, for example oxirane ", .
: . , , . ,, , . ,- :
- : ~, -. , :' . :. . '. ' ' ~ 7 0 groups ~epo~i groups) ~rom vinylglycidyl ether comonomers by polymer~zation, the matrix can directly be reac~ed with nucleo-philic functions (-NH2, -OH etc.). Carboxyl groups can be in-troduced through acrylic acid ester comonomers. When the copolymerization is completed, the ester groups are saponified to carboxyl groups. The in-troduction of -NH2 or -COOH groups allows the activation by means oE carbodilmides, isoxazolium salts, glutar aldehydes etc. Copolymers of vinylene glycol/
vinyl alcohol have a higher specific surface and so an increas-ed binding capaclty for, e.g. proteins on the surface of the polymer powder compared to a vinylene ylycol homopolymer.
The presen-t invention provides biologically active com-pounds from a preferably water-insoluble copolymer of the poly-vinylene glycol and biologically active substance bound there-to with the maintenance of its biological activity. In these compounds, the carrier matrix is a polyvinylene glycol copoly-mer containing up to 45 %, preferably 5 - 20 ~, of a comonomer.
The biologically active substancesare enzymes, activators, in-hibitors, antigens or antibodies, other plasma pro-teins, blood ~0 group substances, phythemagglutinines, antibiotics, vitamins - or hormons, peptides or amino acids or synthetic effectors.
The polymer carrier and the biologically active substance are bound to each other either direetly or via a side chain (Spaeer) in a eovalent bond.
To bind the biologically active compounds via a side c~ain (Spacer) is advantageous when the molecular weights of their affinity partners are very diferent, or when in a bio-speeific process proteins having a very high molecular weight 29 or those comprising several sub-units take part. Spaeers are ~06~7~ HOE 75/B 014 l, side-chains of a certain lengths anchored to the polymer matrix which can be obtained by polymerization, by addition or incorpora-tion (linkage of bi- or polyfunctional compounds - for example tripeptide - to the matrix) or by incorporation of functional groups and stepwise prolongation.
Object of the invention are furthermore processes for the covalent bond of the biologically active substances to the carrler .
For this purpose, a series of generally known reactions is available.
They firstly concern the processes for the manufacture of the polyvinylene glycol copolymer. These processes are charac-terized in that vinylene carbonate is polymerized with comonomers of the general formulae:
/ Rl / 1 .
; H2C=C H C=C H C=C
\ COOR3 O-CO-CH Cl ~Rl /Rl / Rl H2C=C ~ ~ ~ H2C=C CH=CH2 (0CH2-CH2) O- CH=CH2 / 1 ~R
H2C=C~ /C~2 H2C=C
N (CH2)n (CH2)n~C\H /cH2 oc _ l O
~Rl ~R
H2C=C ~ H C-C
O-CH-CH ~CH-CIH2 O ' /
...... .
, . : . : , . . .
:::
. .
:. .....
. .
~B 0 14 9t)7~
. f in which R1 is hydrogen, methyl, ethyl, R 3 i5 methyl, ethyl, propyl and n is a whole number be-tween 1 and 4.
The polymerization of the polyvinylene carbonate occurs with the use of 45 % of cornonomers, pre~erably 5 ~ 20 %~
~he biologically active compound can ~e prepared, for ex-ample by introducing an electrophilic group eithér into the biologically active substance or into the polyvinylene glycol copolymer through which the b.iolo~ically active substance re- :~
acts with the polyvinylene glycol copolymers. The introduction of the electrophilic groups into the carrier matrix occurs either by copolymerizing vinylene carbonate with comonomers containing electrophilic groups or by reacting the carrier matrix with low molecular weight compounds carrying activating electropnilic groups.
Generally, the chemical coupling of a reactant to the carrier ma-trix is simple when the one side carries the nucleo-phi.lic functions and the other side the electrophilic functions.
Reactive, nucleophilic functions are above all amino, sulfhydryl and hydroxyl groups, w~ich are generally already contained : 20 either in the matrix o- in the reactan-t. Electrophilic func~
tions have to be introduced. To this effect, the carboxyl groups can be transformed into acid halides, acid azides, acid anhydrides, imidazolides or directly be act.ivated with carbodiimides. Reactive electrophilic groups are also isocy-25 . anate, ~sothiocyanate~ diazonium groups or cyclic imidocarbo nate esters. Especially suitahle is the introduction of re-act.ive yroups by mean.s of copolymerization.
By the introduction of monomers having epoxy groups, for 29 example epoxyalkylvinyl ethers, in~o the polyvinyl carbona~e ~ 7 ~
~ HOE 75/B 01~
by polymeriæatlon ~nd because o~ the very big differences in the saponiEication speed between cyclic carbonate and epoxy groups, it is eas~ to obtain a polyvinylene glycol matrix hav-in~ reactive epoxy groups which can be re~cted with nucleo-philic functions. So~ the direct bond of a biologically ac-tive substance and/or the introduction (prolongation) of a side chain (Spacer) is possible.
The further advantage of the use of copolymers is the possibility to introduce directly by polymerization electro-philic functions which allow a reaction accordiny to known me-thods.
The copolym~rization of viny].ene carbonate with ethyl acrylate and Eollowi.ng saponification of the polymers gives a matrix having -OH- a~d -COOH-~unctions. The carboxyl groups can be activated by means of carbodi.imides, whereupon an amide .-linkage to the amino groups of the effector occurs. Carboxyl groups further allow the formation of amide ~onds by means of the isoxazolium salts suggested by Woodward.
A fur-ther method is the coupling of biologically active compounds to monomerscontaining carboxyl groups by means of N-ethoxycarbonyl-2-ethoxy 1,2-dihydroqu.inoline (EEDQ).
The carboxyl groups can be esterified according to known methods, the ester then be converted into hydrazide and the azicle resulting therefrom be linked via the amino group o the 25. effector protein to the polyvinylene glycol matrix~
When the comonomer polymerized into the matrix contains amino groups, which can be set free by following reactions, arylamino groups can be introduced by means of vinylsulfon 29 derivatives containing arylamino groups or of sulfuric acid - ' , ... .. . . .
: -- .; , ': , ~,. - , -- . -, -. . ..
- ~ .
~ ~Q69~0 HOE 75/~ 014 esters of ~-hydroxyethyl sulfones. Then the aryl amino groups can be diazotized according to known methods and bound to reactive groups of an effector, for example _ _ H H I H H ~ ¦
...---C--C---C--C--C--C--...
. OH 1H n 1H H O 1 - HO-CH
1H2-- NH- (CH2) 6-NH CH2-SO2-~3-N~2 In this way, ~ prolongation of a Spacer is possible.
Proteins can also be bound via the amino groups by means of glutardialdehyde.
The linkage of the effector to the matrix after an acti-vation of the hydroxyl or amino groups of the polyvinylene-glycol copolymer is easy using cyano halides, preferably bromocyane and effecting a following reaction of the biologically active effec-tors containing the amino groups through these activated groups.
The effector can also be bound to the polyvinylene glycol or polyvinylene glycol copolymer matrix by acylating the hydroxyl groups with bromoacetyl bromide, followed by the aklyla~
tion of the amino group of the effector.
Similar courses of the reaction can be achieved, for example, by reacting the hydroxyl groups of the carrier with re-active triazines, one part of the reactive groups of the triazine reacting with the polyvinyleneglycol compound, another one with the amino groups of the effector.
~Qgj9~70 Diazo-tiza~le aromatic amines which can react through a further reactive group with the hydroxyl groups of the carrier - permit on the other hand the coupling with capable activated amino acids, for exanlple the tyrosine or histidine radicals of the protein effector.
Vinylsulfone derivatives con-taininy arylami~o groups and sulfuric acid semi esters of B-hydroxylethyl sulfones can be reacted wi-th the hydroxyl groups of the carrier. They allow -the effector to be bound according to the precited diazoti-sation reaction.
Especially stable ether bonds are obtained in the reac-tion of the hydroxyl groups of the polyvinyleneglycol copoly-mer with epoxides which dqhot form ions and which contain at least two reactive groups, such as the epihalohydrines or the polyepoxides, ~or exa~ple epichlorohydrine or bisepoxides.
A further mode of modification is the introduction by poly-merization of comomomers into the polyvinylene carbonate chain (for example of vinylp~rrolidone) followed by the partial re-action of the cyclocarbonate rings of the polyvinylene carbo-nate with an amine, for example hexamethylene diamine, to give a polyvinylene carbonate copolymer substituted by a Spacer or an effector through an urethane compound. The residual cyclo-cdrbonate groups are then saponified to h~droxyl groups:
-_ ~o _ .. . . . . ..
~ " :
:1~69070 - fH-CH2--CH--CH ... -CE~-CH2---CH--C~fH--CH~
N O P +R-NH N~ O O OH o H2\ / c 2 / \ o 11 C = o Verseifung ~,~
H2 H2 nH2 H2 r -1 ~H
PolyvinylencarbOnat - CH-CH ---CH--CH --CH--CH-- ...
t 2 l l l l N OH OH OH O
H / \~ O _ _ C - O
R
Beside the cited examples of methods for the production of the covalent bond between the carrier matrix and the protein effector or other effectors there are further methods which lead to the reaction of the hydroxyl groups or of given groups of the copolymer with an effector so giving rise to the covalent bond between them, for example the known reaction with complex-forming metal compounds, for example titanium compounds. All these methods are known to those skilled in the art.
When low-molecular weight, biologically active compounds are to be bound, it is aavantageous not to activate the carrier but the efector.
Principally, all the methods can be used which are known in the macromolecular chemistry for the modification of synthetic or natural macromolecular compounds.
I-IOE 7~B 01 2 11~9~7~ :
The polyvinyle~e glycol copolymers are dlstinguished be-side their chemical and thermal stability by advantageous properties for technical procedures which makes them superior to the carrier matrices on the basis of natural carbohydrates previously deemed to be optimum. For example, they can be pre-pared in the form of fibers, threads, foils or spheric partic-- les, so that the most suitable form can be choosen depend~ng on the intended application purpose of the effector to be bound thereto. Preferably the copolymers are in the form of finely divided powders ~aving a high specific surace.
A further advantage of the polyvinyleneglycol copolymers over the known carrier materials is the con~rollability on the industrial scale of the dimension of the surface accessib]e to the linkage of the eEfectors or the Spacers.
The biologically active compounds according to the in-vention can be used for most of the methods which have become known so far for other effectors bound to hydrophilic, water-insoluble carriers. So, enzymes can be made water-insoluble .
The i:nsoluble enzymes are increasingly used for the determina-2a tion of substrates in analysis automatons and as so-called enzyme electrodes. Because of the higher sta~ility, a series of carrier bound enzymes suits for the enzymatic reactions on a technical scale.
Because of their property as specific adsorbents, carrier-bound, biologically active substances have found ~ide applica-tion in the affinity chromatography. Carrier bound, natural or synthetic enzyme inhibitors enable the high-grade purifi-cation of enzymes, while the enzymes proved to be excellently 29 suitable effectors especially for the obtention o natural en-_ 12 ~
~ 3~
z~me inhibitors from crude extrac-ts. Carrier-bound water--inso-luble anti~ens are used for the isola~Dn of -the adherent anti-bodies which are thus obtained in a state free of other serum constituents and antiyens. In the affinity chromatography, antibodies incapable of being precipitated and those which cannot be precipitated because of their low concentration in the serum can be isolated and be determined quantita-tivelyO
The following Examples illustrate -the invention:
E X A M P L E S:
~anufactu~e o . olymers The vinylene carbonate used in the preparation of the co-polymers (CP) was boiled under reElux,-before its use, for an hour over sodium boron hydride (100 parts by weight of vinylene carbonate to 2 parts by weight of NaBH4) under a pressure of 33 mm, distilled at 75/33mm over a 50 cm long silver-sleeve column filled with ~aschig glass rings and used as straight as possible for the copolymerization. Also, the comonomers used were purified before by distillation.
E X A M P L E 1:
.. . . ... ..
Copolymer o~ vin~lene glycol/viny~ alcohol from saponified copolymer of vinylene carbo _ e/vinyl acetate a) 0.05 Part by weiyht of azobisisobutyronitrile are dissolv-ed under nitroyen in 9 parts by weight oE vinylene carbo-nate and I part by weight O:e vinyl acetate. The monomer mixture is Eilled under nitrogen in a flattened screw cap aluminum tube (4 mm thick, 60 mm large, 150 mm long) (total volume o~ the mixture filled in: about 35 ml), sealed under N2 and hung into a 50 C hot water bath duriny 48 29 hours. Hard, brittle plastic plates are obtained which .
: , , , ' . . .
`` HOE 75/B 014 3V~
, .
are preco~minu-ted in a crushing mill. Then, the granules - are heated under a pressure of 1 - 2 mm for 5 hours at 120 C, to separate the part of the monomer which has not been polymerized. The demonomerized granules are urther ground in a mill to get a grain size of C 0.1 mm, introduc-ed into 500 parts by weight o 0.5 N sodium hydroxide solu-tion, mixecl at 20 C during 1 hour by means of a high--spee~
stirrer, whereupon the carbonate and acetyl groups are saponi~ied. The water-insoluble CP-vinylene /glycol/vinylalco-hol is suction-Eiltered, thoroughly freed from inorganic salts with water and lyophilised.
~leld: 4.7 parts by weight of CP-vinylene ylycol/vinyl al-cohol having a specific surace measured according to BET of 34 m g 1.
IS b) Process as described under 1a), using instead 7 parts by weight of vinylenecarbonate to 3 parts by weight of vinyl acetate~
Yield: 4.1 parts by weight oE CP-vinylene glycol/vinyl alcohol having a specific surface of the lyophilised poly-mer powder of 62.5 m2g 1.
c) Comparison test as described~der1a), differing in that 10 parts by weight of vinylene carbonate and no comonomer are used.
Yield: 4~7 par-ts by weight oE homopolymer of vinylene glycol having a speciEic surface o the lyophilised poly-mer powder of 20 5 m2g 1.
_ X A M P L E 2:
Copolymer of vinylene glycol/vinyl~ly~idvl ether 29 A mixture o;f 7 parts by weight of vinylene carbonate, 3 - 14 _ - .. - ' ' . ' ' "
,, .: . ' , : : ' , -"' " , ' ' ' ' . . ' ' ' ~' ' ' .. . .
.
., , , ~ , ,. . :
. - . . . . .. . .
1~ 7~
parts by w~ight of vinylglycidyl ether and 0.1 part by weight of azobi~lsobu-tyronitrile is heated to 50 C for 3 days under- -: N2 in flattened aluminum tubes as described under 1a). As des cribed under 1a) the plastic plates obtai.ned are co~ninuted, demonomerized~yround to a yrain size of ~ 0.1 mm, suspended in 500 parts by weight of ice cold O.S ~ NaOH us'ing a high-speed stirrer during 30 minutes, the saponified polymer is immediately centrifuyed off, suspended in ice-water, neutralis - ed to pH 7 with 2 N H2S04 while cooling with ice, suction--fil-tered, washed w.ith ice water and lyophilised~
Yield: 3.9 parts by weight of CP-vinylene glycol/vinylglycidyl ether containing 1.3 milliequivalent of oxirane groups/g.
(determined according to "Prak-tlkum der makromolekularen orga~
nischen Chemie", page 221, by Braun, D.l Cherdron, H.~Kern, W. Huthig Verlag, E~eidelberg 1966).
Aminosubstituted copolymer-carrier mater.ial
3.9 parts by weight o CP-vinylene glycol/vinylglycidyl ether (of example 2) are suspended ~n 100 parts by weight of ~2 by means of the Ultra-Turrax stirrer and 10 ml of 30 %
ammonia are added. The mixture is stirred at 50 C for again 10 hours by rneans of a small magnetic stirrer, suction-filter-ed and thoroughly washed with water and lyophilised.
Yield: 3.~ part~ by weight of copolymer-carrier ma-terial con-taining 0.9 m o~ equivalent amino groups/g in the form of 3-amino-2-hydroxy-propyl groups (obtained by reacting ammonia with glycidyl groups~
... , , . : , . ~ . . . , - . .
,, : .
.: ., . : .
-: . : ..... .: : .
: . .. ~ . . ~ -- HOE 75/B _1~
~069~0 E X A kl P L E 4:
CP-vinylen~ y~ol/ac~lic acid ~s described in Example 1a), 9 parts by weight of vinylene carbonate, 1 part by weight of acrylic ~cid ethyl ester and 0.05 part by weight of ~obisisobutyronitrile are polymerized under N2, ~emonomerized and ground to a grain size of ~ 0.1 mm, saponified, suction-filtered, washed and lyophilised.
Yield: 5 parts by weight of hydrophilic polymer powder contain-ing 1.1 milliequivalen-t of carboxyl groups per gramme o~ carrier having a specific surface according to BET of 27.2 m g ... .. .. _ . ._ ._ .
Polyvinylene gly~ol substituted by ~ -amino~
5 parts by weight of CP-vinylene glycol/acrylic acid (Example 4) are suspended in 100 ml of H2O by means of the Ultra-Turrax stirrer, cooled to 5 C, 2.5 g o~ N-cyclohexyl-N'-~N-methylmorpholino)-ethyl-carbodiimide-p-toluene-sulfonate are added, the mixture is stirred for 30 minutes at pH~and at 5 C, filtered off an~ rapidly washed with water. The activated carrier is then immediately suspended in a solution of 5 g of hexamet~ylene diamine in 100 ml of water adjusted to pH 7.5 with 2 N HCl and cooled to 5 C and fur-ther stirred under these conditions using a magnetic stirrer, suction-filtered, thorough-ly washed with water and lyophilized.
Yield: 4.5 parts by weight of polyvinylene glycol substituted by ~ -aminohexyl groups containing 0.7 milliequivalent of aminogroups per gramme of carrier.
.
HOE 7~ 14 r70 E X A M P r.. E 6:
Reaction of CP-vinylene qlycol/ vinyl alcohol of Example la) clf~c su face accordin~ to B~T_f 3~ m g a) wl-th epiehlorohydrine:
50 g of a CP-vinylene g:Lycol/vinyl alcohol prepared according to Example la) are suspended i~ 1 liter of 2 N NaOH, ~50 ml of epichlorohydrine are added to the suspension and the mixture is stirred at 5S - 60 C for 2 hours. After a short time, the pH of the suspension drops to 10 - 11. By adding NaOH this pH value is main-tained for another hour. After a reaetion time of 2 hours, the solid substance is suetion-filtered, washed wi-th water, aeetone ancl finally again with water.
b) with hexamethylene diamine:
50 g of hexamethylene diamine are dissolved in 1.5 1 of water and HCl is added to reach pH 10~ The CP-vinylene glycol/vinyl aleohol aetiva-ted aeeording to 6a is adaed to the solut~on and stirred at 50 - 55 C for 6 hours. Then, the product is suetion-filtered and washed with water until free of hexamethylene diamine.
e) with 1-aminohenzene-4-~-hydroxyethylsulfonsulfurie aeid ester:
The produet obt~ned aeeording to 6b) is stirred at 55 C and at pH 10 for an houx with 50 g of 1-aminobenzene-
ammonia are added. The mixture is stirred at 50 C for again 10 hours by rneans of a small magnetic stirrer, suction-filter-ed and thoroughly washed with water and lyophilised.
Yield: 3.~ part~ by weight of copolymer-carrier ma-terial con-taining 0.9 m o~ equivalent amino groups/g in the form of 3-amino-2-hydroxy-propyl groups (obtained by reacting ammonia with glycidyl groups~
... , , . : , . ~ . . . , - . .
,, : .
.: ., . : .
-: . : ..... .: : .
: . .. ~ . . ~ -- HOE 75/B _1~
~069~0 E X A kl P L E 4:
CP-vinylen~ y~ol/ac~lic acid ~s described in Example 1a), 9 parts by weight of vinylene carbonate, 1 part by weight of acrylic ~cid ethyl ester and 0.05 part by weight of ~obisisobutyronitrile are polymerized under N2, ~emonomerized and ground to a grain size of ~ 0.1 mm, saponified, suction-filtered, washed and lyophilised.
Yield: 5 parts by weight of hydrophilic polymer powder contain-ing 1.1 milliequivalen-t of carboxyl groups per gramme o~ carrier having a specific surface according to BET of 27.2 m g ... .. .. _ . ._ ._ .
Polyvinylene gly~ol substituted by ~ -amino~
5 parts by weight of CP-vinylene glycol/acrylic acid (Example 4) are suspended in 100 ml of H2O by means of the Ultra-Turrax stirrer, cooled to 5 C, 2.5 g o~ N-cyclohexyl-N'-~N-methylmorpholino)-ethyl-carbodiimide-p-toluene-sulfonate are added, the mixture is stirred for 30 minutes at pH~and at 5 C, filtered off an~ rapidly washed with water. The activated carrier is then immediately suspended in a solution of 5 g of hexamet~ylene diamine in 100 ml of water adjusted to pH 7.5 with 2 N HCl and cooled to 5 C and fur-ther stirred under these conditions using a magnetic stirrer, suction-filtered, thorough-ly washed with water and lyophilized.
Yield: 4.5 parts by weight of polyvinylene glycol substituted by ~ -aminohexyl groups containing 0.7 milliequivalent of aminogroups per gramme of carrier.
.
HOE 7~ 14 r70 E X A M P r.. E 6:
Reaction of CP-vinylene qlycol/ vinyl alcohol of Example la) clf~c su face accordin~ to B~T_f 3~ m g a) wl-th epiehlorohydrine:
50 g of a CP-vinylene g:Lycol/vinyl alcohol prepared according to Example la) are suspended i~ 1 liter of 2 N NaOH, ~50 ml of epichlorohydrine are added to the suspension and the mixture is stirred at 5S - 60 C for 2 hours. After a short time, the pH of the suspension drops to 10 - 11. By adding NaOH this pH value is main-tained for another hour. After a reaetion time of 2 hours, the solid substance is suetion-filtered, washed wi-th water, aeetone ancl finally again with water.
b) with hexamethylene diamine:
50 g of hexamethylene diamine are dissolved in 1.5 1 of water and HCl is added to reach pH 10~ The CP-vinylene glycol/vinyl aleohol aetiva-ted aeeording to 6a is adaed to the solut~on and stirred at 50 - 55 C for 6 hours. Then, the product is suetion-filtered and washed with water until free of hexamethylene diamine.
e) with 1-aminohenzene-4-~-hydroxyethylsulfonsulfurie aeid ester:
The produet obt~ned aeeording to 6b) is stirred at 55 C and at pH 10 for an houx with 50 g of 1-aminobenzene-
4-~-hydroxyethylsulfonsulfuric aeid es-ter. Then, the solid substanee is filtered off~ washed with water, aeetone and again with water.
d) Diazotizat;on:
29 10 g of the produet obta~ned aeeording to Example 6e) i~
, -: . , , :
. . .
. . - . ::: : , : : : ::
: , 0~
~91~
are ~1ashed with 200 ml of 0.1NHCl on the suction -filter and then suspended in 300 ml of 0.5 N HCl. 0.1 N NaNO2-solution is addecl to the suspension a-t 0 - 4 C while stir-: ring un-til a sli~ht nitrite excess is stated by means of potassium .iodide starch paper in the diazotation. After 10 minu-tes, the mixture is suction-filtered and the residue is washed with ice water and then with 0.15 M sodium phos-phate buffer at pH 7.5 at 0 - 4 C.
e) Covalent bond with protein:
0.8 g of ~bumin are dissolved in 350 ml of phos-phate buffer at pH 7.5, cooled to 4 C and the product prepared under 6d) is added. The suspension is stirred a-t 4 C :Eor 20 houxs, filtered off and the solid substance is washed with 1 M NaCl and phosphate-buffered sodium chloride solution ~PBS) (aqueous 0.9 % NaCl-solution having a content of 1/15 mol of Na2HPO4-lCH2PO4 buffer of pH 7.2).
The filtrate and wash.ing liquors are tested for albumin according to the method of the radial immuno diffusion. 60 mg of albumin are bound to 1 g of the carrier so prepared.
: 20 E X A M P L E 7:
_ _ _ _ _ Reaction of CP-viny~ene qlycol/vinyl alcohol oE Example 1b havinq ~ ecif_c surface accorclirl to BET of 62.5 ~2q 1 According to the reactions clescribed under 6a to e, 80 mg - of albumin can be bound to 1 g of activated carrier quan-ti-tatively.
E X A M P L E 8: ;
. . .~ :
Reaction of the homopoly~mer viny_ene cll~col of exam~e 1c havl.ng a specific surface accorcling to BET of 20.5 m g 1 29 Acco.rding -to the reactions described under 6a) to e) 45 mg , . - . - ..
. . : - . : :
,' : : . `
.
: . : . .
- : , ::
1~9~70 HOE 75/B 01 4 of albuml.n can qu~ntitativ~ly be bound to 1 g of acti.vated carrier.
E x A ~I P L E 9 Vin~lene c.J~col/v.inylpyrrolido e coeol~mer substi-ut~
C~ -aminohexyl ~roups 0.05 par-t by weight of azobisisobu-tyroni~rile : are dissolved under nitroyen in 9 parts by weight of vinylene car-bonate and 1 part by weight of vinylpyrrolidone and, as des-cr;.bed in Example 1a), polymerized and worked up.
After demonomerization, the granules are dissolved to give a 12 % by wei.ght dimethylformamide solution and this soltution is introduced through a nozzle lnto a methanol pre-.
cipitation bath under a pressure of 15 atmg. A copolymer of vinylene carbonate and vinylpyrrolidone precipitates in Ei-brillate form, it is suction-fi~ered, washed with methanol and resuspended in 200 parts by weight oE methanoL 6 parts by weight of hexamethylen~ diamine, dissolved in 50 parts by weight of me-thanol are added, the suspension is stirred for 2 days at room temperature, suction-filtered and washecl wi-th methanol. The washed filter resi.due is then suspended in a solution of ~0 parts by weight of sodi.um methylate in 300 parts by weight of 96 % methanol during 4 days at room tem-perature, w~shed wlth methanol ancl then very thoroughly with wate:r and lyophilised.
Yield: 5.0 parts by weight of a copolymer of ~inylene glycol and vinylpyrrolidone substituted by ~-aminohexyl groups through.
urethane bonds containing 1.6 m of equivalent NH2-groups/g of carrier, - : , : . ................... .
: ` :: : . ' :: , H0~ 0 14 E X A M P L E 10:
Linka~e of IqG (_mmuno~Llobulln a) to the copolymer substituted ~-amin h~y~_~roups of ~xample 9 by means f succinic acid anhydride and wa-ter-soluble carbodlimide
d) Diazotizat;on:
29 10 g of the produet obta~ned aeeording to Example 6e) i~
, -: . , , :
. . .
. . - . ::: : , : : : ::
: , 0~
~91~
are ~1ashed with 200 ml of 0.1NHCl on the suction -filter and then suspended in 300 ml of 0.5 N HCl. 0.1 N NaNO2-solution is addecl to the suspension a-t 0 - 4 C while stir-: ring un-til a sli~ht nitrite excess is stated by means of potassium .iodide starch paper in the diazotation. After 10 minu-tes, the mixture is suction-filtered and the residue is washed with ice water and then with 0.15 M sodium phos-phate buffer at pH 7.5 at 0 - 4 C.
e) Covalent bond with protein:
0.8 g of ~bumin are dissolved in 350 ml of phos-phate buffer at pH 7.5, cooled to 4 C and the product prepared under 6d) is added. The suspension is stirred a-t 4 C :Eor 20 houxs, filtered off and the solid substance is washed with 1 M NaCl and phosphate-buffered sodium chloride solution ~PBS) (aqueous 0.9 % NaCl-solution having a content of 1/15 mol of Na2HPO4-lCH2PO4 buffer of pH 7.2).
The filtrate and wash.ing liquors are tested for albumin according to the method of the radial immuno diffusion. 60 mg of albumin are bound to 1 g of the carrier so prepared.
: 20 E X A M P L E 7:
_ _ _ _ _ Reaction of CP-viny~ene qlycol/vinyl alcohol oE Example 1b havinq ~ ecif_c surface accorclirl to BET of 62.5 ~2q 1 According to the reactions clescribed under 6a to e, 80 mg - of albumin can be bound to 1 g of activated carrier quan-ti-tatively.
E X A M P L E 8: ;
. . .~ :
Reaction of the homopoly~mer viny_ene cll~col of exam~e 1c havl.ng a specific surface accorcling to BET of 20.5 m g 1 29 Acco.rding -to the reactions described under 6a) to e) 45 mg , . - . - ..
. . : - . : :
,' : : . `
.
: . : . .
- : , ::
1~9~70 HOE 75/B 01 4 of albuml.n can qu~ntitativ~ly be bound to 1 g of acti.vated carrier.
E x A ~I P L E 9 Vin~lene c.J~col/v.inylpyrrolido e coeol~mer substi-ut~
C~ -aminohexyl ~roups 0.05 par-t by weight of azobisisobu-tyroni~rile : are dissolved under nitroyen in 9 parts by weight of vinylene car-bonate and 1 part by weight of vinylpyrrolidone and, as des-cr;.bed in Example 1a), polymerized and worked up.
After demonomerization, the granules are dissolved to give a 12 % by wei.ght dimethylformamide solution and this soltution is introduced through a nozzle lnto a methanol pre-.
cipitation bath under a pressure of 15 atmg. A copolymer of vinylene carbonate and vinylpyrrolidone precipitates in Ei-brillate form, it is suction-fi~ered, washed with methanol and resuspended in 200 parts by weight oE methanoL 6 parts by weight of hexamethylen~ diamine, dissolved in 50 parts by weight of me-thanol are added, the suspension is stirred for 2 days at room temperature, suction-filtered and washecl wi-th methanol. The washed filter resi.due is then suspended in a solution of ~0 parts by weight of sodi.um methylate in 300 parts by weight of 96 % methanol during 4 days at room tem-perature, w~shed wlth methanol ancl then very thoroughly with wate:r and lyophilised.
Yield: 5.0 parts by weight of a copolymer of ~inylene glycol and vinylpyrrolidone substituted by ~-aminohexyl groups through.
urethane bonds containing 1.6 m of equivalent NH2-groups/g of carrier, - : , : . ................... .
: ` :: : . ' :: , H0~ 0 14 E X A M P L E 10:
Linka~e of IqG (_mmuno~Llobulln a) to the copolymer substituted ~-amin h~y~_~roups of ~xample 9 by means f succinic acid anhydride and wa-ter-soluble carbodlimide
5 parts by weight of the CJ -aminohexyl-subs-ti-tuted carrier prepared according to Example 9 are succinoylated at 10 C and a-t pH G for 4 hours with 2.5 parts by weight of succinic acid anhydride which are suspended in 200 parts by weiyht of water.
The pH is adjusted with 2 N NaOHO AEter washing -the solid sub-stance with wa-ter, the product is stirred with 1.25 parts by weight of N-cyclohexyl-N'-(N-methyl-morpholino)-ethyl)-carbodi-imide-p-toluene-sulfonate at pH 5 and 5 C for 30 minutes, fil-tered off and rapldly washed with ice water.
0.5 part by weight of IgG are dissolved in 150 parts by weiyht of phosphate buffer of pH 7.5 and stirred with the ac-tivated carrier for 24 hours at 4 C. After filtration, the product i$ washed with 1 M sodium chloride salt and with PBS.
75 mg of IgG are linked to 1 g of carrier in a covalent bond.
E X A M P L E ~1:
Copo~y~r of vinylene qlycol/diethyl_n~_~lycol divinyl ether 0.1 part by weight of azob~sisobutyronitrlLe are dissolved under nitrogen in 9 parts by weight oE vinylene carbonate and - 1 part by weight of diethylene glycol dlvinyl ether ànd heat-ed as described in the Example 1a) in flat aluminum tubes under N2 to 50 C duriny 3 days and polymerized. After working-up and saponification as described under 1a), 3.5 parts by weight of the CP-vinylene glycol/diethylene glycol divinyl ether are 29 obtained.
,.. .- . . . , ' . ' , ~ ' .
HOE; 75/B O 14 9~)70 E X A M P I. E 12:
~E~Xmer o-f vin~le _ ~y~ N-acryloylaminoaceta]deh~
methyl acetal a) 0.05 part by weight of azobisisobutyronitri]e are dissolved under nitroyen in 9 parts by weight of vinylene carbonate and 1 part ~y weight of N-acyloylaminoacetaldehyde-dlmethyl~
acetal. The monomer mixture is polymerized as described under la), worked-up and saponified to give the 4.1 parts by weight of the copolymer o-F N-acryloyl-aminoacetaldehyde-dimethyl-acetal and vinylene glycol.
10 parts by wei~ht of the copolymer of N-acryloylamino-acetaldehyde-dimethylacetal and vinylene glycol were stlrred in 100 parts by weight of 1 N ~ICl for 4 - 5 hours. The activated carrier was washed with water and phosphate - 15 buffer of p~ 7.5.
b~ 0.5 g of albumin was dissolved in 200 ml of PBS and stirred with the product prepared under a) at 4 C during 14 hours.
After filtration`the carrier-bound protein was washed with 1 M of sodium chloride salt and with PBS.
1 g of the carrier so prepared binds 40 mg ol albumin.
: ` . : ,, ' . . ;. . .
The pH is adjusted with 2 N NaOHO AEter washing -the solid sub-stance with wa-ter, the product is stirred with 1.25 parts by weight of N-cyclohexyl-N'-(N-methyl-morpholino)-ethyl)-carbodi-imide-p-toluene-sulfonate at pH 5 and 5 C for 30 minutes, fil-tered off and rapldly washed with ice water.
0.5 part by weight of IgG are dissolved in 150 parts by weiyht of phosphate buffer of pH 7.5 and stirred with the ac-tivated carrier for 24 hours at 4 C. After filtration, the product i$ washed with 1 M sodium chloride salt and with PBS.
75 mg of IgG are linked to 1 g of carrier in a covalent bond.
E X A M P L E ~1:
Copo~y~r of vinylene qlycol/diethyl_n~_~lycol divinyl ether 0.1 part by weight of azob~sisobutyronitrlLe are dissolved under nitrogen in 9 parts by weight oE vinylene carbonate and - 1 part by weight of diethylene glycol dlvinyl ether ànd heat-ed as described in the Example 1a) in flat aluminum tubes under N2 to 50 C duriny 3 days and polymerized. After working-up and saponification as described under 1a), 3.5 parts by weight of the CP-vinylene glycol/diethylene glycol divinyl ether are 29 obtained.
,.. .- . . . , ' . ' , ~ ' .
HOE; 75/B O 14 9~)70 E X A M P I. E 12:
~E~Xmer o-f vin~le _ ~y~ N-acryloylaminoaceta]deh~
methyl acetal a) 0.05 part by weight of azobisisobutyronitri]e are dissolved under nitroyen in 9 parts by weight of vinylene carbonate and 1 part ~y weight of N-acyloylaminoacetaldehyde-dlmethyl~
acetal. The monomer mixture is polymerized as described under la), worked-up and saponified to give the 4.1 parts by weight of the copolymer o-F N-acryloyl-aminoacetaldehyde-dimethyl-acetal and vinylene glycol.
10 parts by wei~ht of the copolymer of N-acryloylamino-acetaldehyde-dimethylacetal and vinylene glycol were stlrred in 100 parts by weight of 1 N ~ICl for 4 - 5 hours. The activated carrier was washed with water and phosphate - 15 buffer of p~ 7.5.
b~ 0.5 g of albumin was dissolved in 200 ml of PBS and stirred with the product prepared under a) at 4 C during 14 hours.
After filtration`the carrier-bound protein was washed with 1 M of sodium chloride salt and with PBS.
1 g of the carrier so prepared binds 40 mg ol albumin.
: ` . : ,, ' . . ;. . .
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a copolymer of vinylene glycol and a biologically active substance chemically bound thereto in which (A) a copolymer of the vinylene carbonate is reacted with a biologically active substance and the cyclocarbonate groups still present are subsequently converted into hydroxy groups, or (B) the cyclocarbonate groups of a copolymer of the vinylene carbonate are converted into hydroxy groups and (a) the electrophilic groups contained in or introduced into the copolymer are reacted with a biologically active substance, or (b) the hydroxy groups are (1) reacted with a compound containing an electro-philic group and then with a biologically active substance, or (2) reacted with a biologically active substance carrying electrophilic groups.
2. A process as claimed in claim 1 in which the copolymer is water-insoluble.
3. A process as claimed in claim 1 in which the copolymer contains at least 55% of monomer units of the vinylene glycol and at most 45% of copolymer units of at least one compound of the general formulae wherein R1 is hydrogen, methyl, ethyl, R3 is methyl, ethyl, propyl and n is a whole number between 1 and 4.
4. A compound of a copolymer of vinylene glycol and a biologically active substance chemically bound thereto, whenever obtained according to a process as claimed in claim 1, claim 2 or claim 3 or by an obvious chemical equivalent thereof.
5. A process as claimed in claim 2 in which the biolo-gically active substance is an enzyme, an activator, an inhibitor, an antigen, an antibody, a plasma protein, a blood group sub-stance, a phythemagglutinine, an antibiotic, a vitamin, a hormone, a peptide, an amino acid or a natural or synthetic effector.
6. A process as claimed in claim 3 in which the comonomer units are present in an amount of 5 to 20%.
7. A process as claimed in claim 1 in which the reaction is carried out according to reaction (A).
8. A compound of the copolymer of vinylene glycol and a biologically active substance chemically bound thereto, whenever obtained according to a process as claimed in claim 4, claim 5 or claim 6 or by an obvious chemical equivalent thereof.
9. A process as claimed in claim 1 in which the reaction is carried out according to reaction (B) (a).
10. A process as claimed in claim 1 in which the reaction is carried out according to reaction (B) (b).
11. A compound of a copolymer of vinylene glycol and a biologically active substance chemically bound thereto, whenever obtained according to a process as claimed in claim 9 or claim 10 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2552510A DE2552510C3 (en) | 1975-11-22 | 1975-11-22 | Biologically active compounds and processes for their preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1069070A true CA1069070A (en) | 1980-01-01 |
Family
ID=5962419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA266,092A Expired CA1069070A (en) | 1975-11-22 | 1976-11-19 | Water insoluble biologically active compounds and process for their manufacture |
Country Status (20)
| Country | Link |
|---|---|
| JP (1) | JPS6048524B2 (en) |
| AT (1) | AT358275B (en) |
| AU (1) | AU513890B2 (en) |
| BE (1) | BE848601A (en) |
| CA (1) | CA1069070A (en) |
| CH (1) | CH636628A5 (en) |
| DE (1) | DE2552510C3 (en) |
| DK (1) | DK522976A (en) |
| ES (1) | ES453362A1 (en) |
| FI (1) | FI763314A (en) |
| FR (1) | FR2332287A1 (en) |
| GB (1) | GB1571182A (en) |
| IL (1) | IL50940A (en) |
| IT (1) | IT1121742B (en) |
| LU (1) | LU76243A1 (en) |
| NL (1) | NL7612771A (en) |
| NO (1) | NO763975L (en) |
| SE (1) | SE430066B (en) |
| YU (1) | YU283676A (en) |
| ZA (1) | ZA766942B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6929955B2 (en) | 1997-04-14 | 2005-08-16 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Interactive system for presenting and eliminating substances |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4210722A (en) * | 1977-11-14 | 1980-07-01 | Minnesota Mining And Manufacturing Company | Protein immobilizer |
| DE2757206A1 (en) * | 1977-12-22 | 1979-07-05 | Basf Ag | POLYAMIDACETAL |
| GB2043996B (en) * | 1978-12-27 | 1983-09-07 | Nihon Dempa Kogyo Co | Thickness shear quartz crystal oscillator |
| EP0015473A1 (en) * | 1979-02-28 | 1980-09-17 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Process for immobilizing cells |
| FR2450263A1 (en) * | 1979-02-28 | 1980-09-26 | Dow Chemical Co | Coupling protein to latex particles having epoxy gps. - for use in immunological analysis esp. pregnancy testing |
| DE3145082A1 (en) * | 1981-11-13 | 1983-05-19 | Behringwerke Ag, 3550 Marburg | "A LATEX, BIOLOGICALLY ACTIVE LATEX CONJUGATES AND METHOD FOR THEIR PRODUCTION" |
| JPS58209984A (en) * | 1982-05-28 | 1983-12-07 | Japan Synthetic Rubber Co Ltd | Carrier composed of granular polymer |
| DE3243591A1 (en) * | 1982-11-25 | 1984-05-30 | Hoechst Ag, 6230 Frankfurt | VINYLENE CARBONATE POLYMERYSATE, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
| DE3344912A1 (en) * | 1983-12-13 | 1985-06-20 | Hoechst Ag, 6230 Frankfurt | CROSSLINKED POLYMERISATES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
| SE8401437D0 (en) * | 1984-03-14 | 1984-03-14 | Sven Gothe | SURFACE MODIFIED PLASTIC SURFACES AND ITS APPLICATIONS FOR IMMUNOASSAYS (IA) |
| DE3413904A1 (en) * | 1984-04-13 | 1985-10-24 | Hoechst Ag, 6230 Frankfurt | POLYMERISATES BASED ON POLYVINYLENE CARBONATE AND / OR POLYHYDROXYMETHYLENE, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE |
| GB8423228D0 (en) * | 1984-09-14 | 1984-10-17 | Unilever Plc | Specific binding materials |
| US4780409A (en) * | 1985-05-02 | 1988-10-25 | Genetic Systems Corporation | Thermally induced phase separation immunoassay |
| DE3637421C1 (en) * | 1986-11-03 | 1987-10-29 | Nerbe Plus Ges Fuer Medizinisc | Reaction vessel for the blood coagulation reaction |
| DE3811042A1 (en) * | 1988-03-31 | 1989-10-19 | Merck Patent Gmbh | ION EXCHANGER |
| JP2001247738A (en) * | 2000-03-06 | 2001-09-11 | Unitika Chem Co Ltd | Polyvinyl alcohol-based resin composition and paper coating agent consisting essentially of the same |
| JP6405669B2 (en) * | 2014-03-31 | 2018-10-17 | 東ソー株式会社 | Novel polymer and cell culture substrate having the same |
| CN108264604B (en) * | 2016-12-30 | 2020-08-11 | 北京爱普聚合科技有限公司 | Dry fracturing fluid drag reduction thickener and preparation method thereof |
| EP3502274A1 (en) * | 2017-12-22 | 2019-06-26 | Attomol GmbH | Sample carrier and method of production |
| CN110504452B (en) * | 2019-09-04 | 2022-06-21 | 中国科学院青岛生物能源与过程研究所 | High-peel-strength polymer binder and application thereof in secondary lithium battery |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE810605A (en) * | 1973-03-01 | 1974-05-29 | ENZYMES AND OTHER PROTEINS LINKED TO POLYMERIC SUBSTRATES CONTAINING VINYLENE CARBONATE UNITS | |
| ES437153A1 (en) * | 1974-05-06 | 1977-04-16 | Marburgnlahn | Biologically active compounds and process for their manufacture |
-
1975
- 1975-11-22 DE DE2552510A patent/DE2552510C3/en not_active Expired
-
1976
- 1976-11-16 ES ES453362A patent/ES453362A1/en not_active Expired
- 1976-11-17 NL NL7612771A patent/NL7612771A/en not_active Application Discontinuation
- 1976-11-18 FI FI763314A patent/FI763314A/fi not_active Application Discontinuation
- 1976-11-18 CH CH1453676A patent/CH636628A5/en not_active IP Right Cessation
- 1976-11-19 ZA ZA766942A patent/ZA766942B/en unknown
- 1976-11-19 SE SE7612998A patent/SE430066B/en not_active IP Right Cessation
- 1976-11-19 AU AU19817/76A patent/AU513890B2/en not_active Expired
- 1976-11-19 IT IT7629576A patent/IT1121742B/en active
- 1976-11-19 AT AT863176A patent/AT358275B/en active
- 1976-11-19 DK DK522976A patent/DK522976A/en not_active Application Discontinuation
- 1976-11-19 LU LU76243A patent/LU76243A1/xx unknown
- 1976-11-19 NO NO763975A patent/NO763975L/no unknown
- 1976-11-19 YU YU02836/76A patent/YU283676A/en unknown
- 1976-11-19 CA CA266,092A patent/CA1069070A/en not_active Expired
- 1976-11-19 IL IL50940A patent/IL50940A/en unknown
- 1976-11-22 GB GB48603/76A patent/GB1571182A/en not_active Expired
- 1976-11-22 FR FR7635149A patent/FR2332287A1/en active Granted
- 1976-11-22 JP JP51139645A patent/JPS6048524B2/en not_active Expired
- 1976-11-22 BE BE172569A patent/BE848601A/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6929955B2 (en) | 1997-04-14 | 2005-08-16 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Interactive system for presenting and eliminating substances |
| US7494824B2 (en) | 1997-04-14 | 2009-02-24 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. V., Berlin | Interactive system for presenting and eliminating substances |
Also Published As
| Publication number | Publication date |
|---|---|
| AT358275B (en) | 1980-08-25 |
| NL7612771A (en) | 1977-05-24 |
| LU76243A1 (en) | 1977-06-07 |
| YU283676A (en) | 1982-06-30 |
| SE7612998L (en) | 1977-05-23 |
| ATA863176A (en) | 1980-01-15 |
| CH636628A5 (en) | 1983-06-15 |
| ES453362A1 (en) | 1977-11-16 |
| IL50940A0 (en) | 1977-01-31 |
| JPS6048524B2 (en) | 1985-10-28 |
| FR2332287A1 (en) | 1977-06-17 |
| DE2552510C3 (en) | 1981-02-19 |
| IL50940A (en) | 1980-03-31 |
| DK522976A (en) | 1977-05-23 |
| GB1571182A (en) | 1980-07-09 |
| AU513890B2 (en) | 1981-01-15 |
| FR2332287B1 (en) | 1982-11-12 |
| DE2552510A1 (en) | 1977-06-30 |
| IT1121742B (en) | 1986-04-23 |
| JPS5265596A (en) | 1977-05-31 |
| AU1981776A (en) | 1978-05-25 |
| DE2552510B2 (en) | 1980-06-04 |
| ZA766942B (en) | 1977-10-26 |
| FI763314A (en) | 1977-05-23 |
| BE848601A (en) | 1977-05-23 |
| SE430066B (en) | 1983-10-17 |
| NO763975L (en) | 1977-05-24 |
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