DK171394B1

DK171394B1 - Support or carrier in solid phase, immobilized enzyme in solid phase on such a support, ligand-linked chromatography affinity matrix and method for separating or removing a substance from solution using the support

Info

Publication number: DK171394B1
Application number: DK305789A
Authority: DK
Inventors: Laura J Crane; Sunil V Kakodkar
Original assignee: Mallinckrodt Baker Inc
Priority date: 1989-06-20
Filing date: 1989-06-20
Publication date: 1996-10-14
Also published as: DK305789A; DK305789D0

Description

DK 171394 B1DK 171394 B1

Den foreliggende opfindelse angår en understøtning eller bærer i fast fase på basis af et materiale udvalgt fra gruppen bestående af (a) en understøtning med den almene formel 5 Silica-PrSi-PEI—f-R)x i hvilken Silica-PrSi-PEI er en kovalent bundet, ikke-tvær-bundet polyethylenimin-bundet-fast fase-understøtning, der er reaktionsproduktet af (1) a) partikelformig silicagel med en gennemsnitlig partikeldiameter på fra 1 til 200 μτη og 10 en gennemsnitlig porestørrelse på fra 0 til 100 nm, eller b) partikelformigt glas med kontrolleret porestørrelse med en gennemsnitlig partikeldiameter på fra 1 til 200 μιη og en gennemsnitlig porestørrelse på fra 0 til ca. 100 nm, med (2) polyethyleniminpropyl-trimethoxy-silan med en gennemsnit-15 lig molekylvægt på fra 400 til 1800, og (b) det svagt sure carboxylerede produkt af den faste Silica-PrSi-PEI-understøtning med et dibasisk syreanhydrid, hvorhos det carboxylerede produkt indeholder fra 0,3 til 1,2 carboxyl-milliækvivalenter pr. g, et immobiliseret enzym 20 i fast fase, en ligand-bundet chromatografiaffinitetsmatrix samt en fremgangsmåde til adskillelse eller rensning af et stof fra opløsning ved binding af stoffet i opløsning med en affinitetsmatrix, der har en ligand fra stoffet kovalent bundet til affinitetsmatrixen.The present invention relates to a solid phase support or carrier based on a material selected from the group consisting of (a) a support of the general formula Silica-PrSi-PEI-fR) xi in which Silica-PrSi-PEI is a covalently bonded , non-crosslinked polyethyleneimine bonded solid phase support, which is the reaction product of (1) a) particulate silica gel having an average particle diameter of from 1 to 200 μτη and an average pore size of from 0 to 100 nm, or b ) controlled pore size particulate glass having an average particle diameter of from 1 to 200 μιη and an average pore size of from 0 to approx. 100 nm, with (2) polyethyleneimine propyl trimethoxy silane having an average molecular weight of from 400 to 1800, and (b) the slightly acidic carboxylated product of the solid Silica-PrSi-PEI support with a dibasic acid anhydride, wherein the carboxylated product contains from 0.3 to 1.2 carboxyl milliequivalents per g, a solid phase immobilized enzyme 20, a ligand-bound chromatography affinity matrix, and a method for separating or purifying a substance from solution by binding the substance in solution with an affinity matrix having a ligand from the substance covalently bound to the affinity matrix.

25 Affinitetschromatografi er en adskillelses- og rens ningsteknik, der er baseret på en enestående og fundamental biologisk egenskab hos biologiske molekyler, nemlig deres selektive, specifikke høj-affinitetsgenkendelse af og reversible molekylære interaktion med andre molekyler. De fleste 30 biologiske makromolekyler har et substrat eller funktionelt bindingssted for en specifik ligand eller et effektormole-kyle, der selv kan være et andet protein.Affinity chromatography is a separation and purification technique based on a unique and fundamental biological property of biological molecules, namely their selective, specific high-affinity recognition of and reversible molecular interaction with other molecules. Most 30 biological macromolecules have a substrate or functional binding site for a specific ligand or effector molecule which itself may be another protein.

I almindelighed er en specifik ligand kovalent bundet til en fast understøtnings- eller bærermatrix. En prøve 35 indeholdende det biologiske molekyle, der specifikt vil bindes (absorberes) til den immobiliserede ligand, bringes DK 171394 B1 2 i kontakt med den immobiliserede ligand. Efter at uabsorbe-rede og forurenende molekyler er fjernet, elueres det specifikt bundne molekyle fra den faste bærer ved afbrydelse af den specifikt bundne molekyle-ligand-interaktion ved 5 hjælp af én af flere metoder, for eksempel ved ændring af ionstyrken eller pH-værdien af elueringspuffere.In general, a specific ligand is covalently bound to a solid support or carrier matrix. A sample 35 containing the biological molecule that will specifically bind (absorb) to the immobilized ligand is contacted with the immobilized ligand. After removing unabsorbed and contaminant molecules, the specifically bound molecule is eluted from the solid support by interrupting the specifically bound molecule-ligand interaction by one of several methods, for example, by changing the ionic strength or pH value. of elution buffers.

Ved denne metode kan immobiliserede lægemidler, vitaminer, peptider, hormoner og lignende anvendes til isolering af tilsvarende receptorer eller transportproteiner. Immobi le) liseret protein kan tjene til isolering af andre komplementære eller dermed samvirkende proteiner. Ligeledes kan en sådan fremgangsmåde anvendes til adskillelse af partikel-formede biologiske stoffer, for eksempel cellemembraner og selv intakte celler, der bærer specifikke receptorer. An-15 vendeisen af en sådan metode er også nyttig til rensning af polynucleotider, antigener, antistoffer, vira, enzymer og .lignende. Desuden har sådanne affinitets-bærermatrixer på fast basis været anvendt til immobilisering af enzymer til anvendelse ved reaktioner som katalysatorer og lignende.In this method, immobilized drugs, vitamins, peptides, hormones and the like can be used to isolate corresponding receptors or transport proteins. Immobilized protein may serve to isolate other complementary or thus interacting proteins. Likewise, such a method can be used to separate particulate biological agents, for example, cell membranes and even intact cells carrying specific receptors. The application of such a method is also useful for the purification of polynucleotides, antigens, antibodies, viruses, enzymes and the like. In addition, such solid affinity support matrices have been used to immobilize enzymes for use in reactions such as catalysts and the like.

20 Hidtil har de i størst udstrækning anvendte faste matrixer til affinitetschromatografi været polysaccharid-baserede matrixer, for eksempel agarose, cellulose og tværbundet dextrin, selvom man også anvendt tværbundne polyacryl-amider og siliciumdioxid eller mikroporøse glasperler.To date, the most commonly used solid matrices for affinity chromatography have been polysaccharide-based matrices, for example agarose, cellulose and cross-linked dextrin, although cross-linked polyacrylamides and silica or microporous glass beads have also been used.

25 Med de fleste sådanne faste matrixer, og navnlig med faste matrixer på basis af siliciumdioxid, er det faste basismateriale imidlertid ikke tilstrækkeligt afskærmet fra molekylerne i den biologiske prøve, der har interesse, hvilket har til følge, at der forekommer ikke-specifik bind-30 ing, hvilket viser sig ved absorption med andre materialer end den immobiliserede ligands specifikke bindingspartner.However, with most such solid matrices, and in particular solid silica-based matrices, the solid base material is not sufficiently shielded from the molecules of the biological sample of interest, resulting in non-specific binding. 30, which is evidenced by absorption with materials other than the specific binding partner of the immobilized ligand.

Med de faste matrixer på basis af siliciumdioxid, der tidligere har været anvendt, forekommer der kraftig hydrogenbinding og absorption ved silanolgrupper, hvilket er afbry-35 dende for processen. Endvidere forårsager de vandige opløsninger, der skal anvendes sammen med proteinerne eller andet DK 171394 B1 3 biologisk materiale, der har interesse, uønsket hydrolyse af siliciumdioxid-matrixerne, hvorved der udludes uønsket materiale fra matrixerne, hvilket resulterer i instabile matrixer.With the silicon dioxide solid matrices previously used, there is strong hydrogen bonding and absorption by silanol groups, which is disruptive to the process. Furthermore, the aqueous solutions to be used with the proteins or other biological material of interest cause undesirable hydrolysis of the silica matrices, thereby eliminating undesirable material from the matrices, resulting in unstable matrices.

5 Med de tidligere anvendte faste understøtningsmatrix er, der har været benyttet ved affinitetschromatografi, kunne man i almindelighed kun anvende sådanne understøtninger i 200-300 timer i vandige opløsninger, og endog mindre i sure eller alkaliske omgivelser. Endvidere kunne sådanne 10 kendte matrixer ikke tåle nogen udsættelse for 0,1 M natriumhydroxid .With the previously used solid support matrix used in affinity chromatography, such supports could generally only be used for 200-300 hours in aqueous solutions, and even less in acidic or alkaline environments. Furthermore, such known 10 matrices could not withstand exposure to 0.1 M sodium hydroxide.

Udviklingen af affinitetschromatografi er således i vid udstrækning blevet inhiberet af disse ulemper ved de hidtil anvendte faste bærermatrixer, navnlig på grund af 15 absorptionen af ikke-specifikke materialer og matrixernes instabilitet .Thus, the development of affinity chromatography has been largely inhibited by these disadvantages of the solid support matrices used so far, in particular due to the absorption of non-specific materials and the instability of the matrices.

Det er derfor ønskeligt, at der tilvejebringes faste bærermatrixer til affinitetschromatografi, der eliminerer eller i væsentlig grad formindsker risikoen for ikke-speci-20 fik binding, og som er stabile under en række forskellige pH-betingelser og i vandige opløsninger.It is therefore desirable to provide solid support matrices for affinity chromatography that eliminate or substantially reduce the risk of non-specific binding and are stable under a variety of pH conditions and in aqueous solutions.

Det har nu vist sig, at specielle, kovalent bundne, ikke-tværbundne polyethylenimin-siliciumdioxid-baserede understøtninger i fast fase tilvejebringer affinitetschrom-25 atografi-matrixer, der har i høj grad forbedret stabilitet i vandigt, surt og alkalisk miljø og i væsentlig grad formindsker eller praktisk taget eliminerer absorptionen af ikke-specifikke molekyler. De kovalent bundne, ikke-tværbundne polyethylenimin-silicabaserede faste understøtninger, ^0 der anvendes til tilvejebringelse af affinitetschromatografi-matrixerne ifølge den foreliggende opfindelse, er reaktionsprodukterne af polyethyleniminpropyl-trimethoxy-silan med partikelformig siliciumdioxidgel eller partikelformigt glas med reguleret porestørrelse og betegnes med formlen 35 Silica-PrSi-PEI.It has now been found that special, covalently bonded, non-crosslinked solid phase polyethyleneimine silica-based supports provide affinity chromatography matrices which have greatly improved stability in the aqueous, acidic and alkaline environment and substantially decreases or virtually eliminates the absorption of non-specific molecules. The covalently bonded, non-crosslinked polyethyleneimine silica based solid supports used to provide the affinity chromatography matrices of the present invention are the reaction products of polyethyleneimine propyl trimethoxy silane with particulate silica gel or controlled pore size particulate glass silica PrSi PEI.

Betegnelsen "Silica-PrSi-PEI" anvendes her i betydningen den DK 171394 B1 4 kovalent bundne, ikke-tværbundne polyethylenimin-bundne fase faste understøtning, der er reaktionsproduktet af (l) enten a) partikelformig siliciumdioxidgel med en gennemsnitlig partikeldiameter fra 1 til 200, fortrinvis fra 3 til 70 /zm, 5 og en gennemsnitlig porestørrelse på fra 0 til 100, fortrinsvis fra 5 til 100 nm, eller b) partikelformigt glas med reguleret porestørrelse med en gennemsnitlig partikeldiameter på 1 til 200 μπι og en gennemsnitlig porestørrelse på fra 0, fortrinvis ca. 4, til 100 nm, med (2) polyethy-10 leniminpropyl-trimethoxy-silan med en gennemsnitlig molekylvægt på fra 400 til 1800, eller det svagt sure carboxylerede produkt deraf med et dibasisk syreanhydrid, idet det carboxylerede produkt indeholder fra 0,3 til 1,2 carboxyl-milliækvivalenter pr. gram. Sådanne Silica-PrSi-PEI-produkter, 15 deres carboxylerede derivater og deres fremstilling er beskrevet i US patentskrift nr. 4.540.486. Sådanne produkter er løbende tilgængelige fra J.T. Baker Inc. som Bakerbond®-søjlechromatograf imatrixer.The term "Silica-PrSi-PEI" is used herein to mean the covalently bonded, non-crosslinked polyethyleneimine bonded phase solid support which is the reaction product of either (a) either a) particulate silica gel having an average particle diameter of 1 to 200 , preferably from 3 to 70 µm, 5 and an average pore size of from 0 to 100, preferably from 5 to 100 nm, or b) a controlled pore size particle glass having an average particle diameter of 1 to 200 µπι and an average pore size of from 0, preferably approx. 4, to 100 nm, with (2) polyethyleneimine propyl trimethoxy silane having an average molecular weight of from 400 to 1800, or the slightly acidic carboxylated product thereof with a dibasic acid anhydride, the carboxylated product containing from 0.3 to 1.2 carboxylic milliequivalents per gram. Such Silica-PrSi-PEI products, their carboxylated derivatives and their preparation are described in U.S. Patent No. 4,540,486. Such products are continuously available from J.T. Baker Inc. as Bakerbond® column chromatograph imatrixes.

Affinitetschromatografi-matrixerne ifølge opfindelsen 20 er til forskel fra de fra det nævnte US patent skrift nr. 4.540.486 kendte produkter derivatiserede Silica-PrSi-PEI-matrixer, i hvilke de primære og sekundære amoinogrupper i polyethylenimin-delen er omsat med en reaktiv molekyldel, der er i stand at danne kovalente bindinger med ligander 25 under ikke-denaturerende betingelser og er stabil under vandige hydrolytiske pufferbetingelser. De derivatiserede matrixer udviser i forhold til de ovennævnte, kendte matrixer stærkt forbedret stabilitet i vandigt, surt eller alkalisk miljø, og de formindsker væsentligt eller eliminerer ab-30 sorptionen af ikke-specifikke molekyler.The affinity chromatography matrices of the invention 20, unlike the products known from the aforementioned U.S. Patent No. 4,540,486, are derivatized Silica-PrSi-PEI matrices in which the primary and secondary amino-groups of the polyethylenimine moiety are reacted with a reactive moiety. which is capable of forming covalent bonds with ligands 25 under non-denaturing conditions and is stable under aqueous hydrolytic buffer conditions. The derivatized matrices exhibit markedly improved stability in the aqueous, acidic or alkaline environment relative to the aforementioned known matrices and substantially reduce or eliminate the absorption of non-specific molecules.

Understøtningen eller bæreren ifølge opfindelsen er således ejendommelig ved, at R i tilfælde af den ikke-svagt sure carboxylerede understøtning er resten af enhver kemisk reaktiv molekyldel, der er i stand til at undergå nucleophil 35 substitution ved to særskilte steder, således at R bliver kovalent bundet til de primære eller sekundære aminogrupper DK 171394 B1 5 af den anvendte PEI ved ét sådant sted, medens det andet sted er tilgængeligt og reaktivt for påfølgende nucleophil substitution under ikke-denaturerende betingelser med en affinitetschromatografi-ligand til dannelse af en anden 5 kovalent binding, der er stabil under vandige hydrolytiske pufferbetingelser, og x er et positivt helt tal mindre end eller lig med det totale antal af primære eller sekundære aminogrupper i PEI-molekyldelen, og i tilfælde af den svagt sure caboxylerede understøtning er R resten af enhver kemisk 10 reaktiv molekyldel, der er i stand til at lette nucleophil erstatning af carboxyl-hydroxylet til dannelse af en kovalent binding ved carboxyl-carbonatornet, hvorved der tilvejebringes et tilstrækkeligt elektrofilt sted, således at det let kan fortrænges med carboxyl-carbonatomet med en nucleophil funk-15 tionel gruppe på en affinitetschromatografi-ligand, og x er et positivt helt tal mindre end eller lig med det totale antal carboxylgrupper i den carboxylerede PEI-molekyldel.Thus, the support or carrier of the invention is characterized in that, in the case of the non-weakly acidic carboxylated support, R is the remainder of any chemically reactive moiety capable of undergoing nucleophilic substitution at two distinct sites such that R becomes covalent. bound to the primary or secondary amino groups of the PEI used at one such site, while the other site is available and reactive for subsequent nucleophilic substitution under non-denaturing conditions with an affinity chromatography ligand to form another covalent bond , which is stable under aqueous hydrolytic buffer conditions, and x is a positive integer less than or equal to the total number of primary or secondary amino groups in the PEI molecule, and in the case of the weakly acidic caboxylated support, R is the remainder of any chemical 10. reactive molecular moiety capable of facilitating nucleophilic replacement of carboxy the 1-hydroxyl to form a covalent bond at the carboxyl carbonator, thereby providing a sufficient electrophilic site to be readily displaced by the carboxyl carbon atom with a nucleophilic functional group on an affinity chromatography ligand, and x is a positive integer less than or equal to the total number of carboxyl groups in the carboxylated PEI moiety.

Som eksempler på R-rester af molekyldele, der er reaktive med de primære og sekundære amino- eller carboxylgrupper i 20 PEI-molekyldelen, og som opfylder de øvrige ovennævnte betingelser, kan der nævnes følgende rester: O Cl II /N <. ,0 «CH-CH^CH-jCHpCH , —<Γ O N , -CH2-^—^ , 25 2 2 2 \n —<As examples of R residues of molecular moieties that are reactive with the primary and secondary amino or carboxyl groups of the 20 PEI molecular moiety and which fulfill the other above conditions, the following residues may be mentioned: O Cl II / N <. , 0 «CH-CH ^ CH-jCHpCH, - <Γ O N, -CH2 - ^ - ^, 25 2 2 2 \ n - <

Cl OH 1 -CH2-Ah-CH2-0-(CH2)4-0-CH2-^-^ r -c-o<5>no2 , 30 o o ° oCl OH 1 -CH2-Ah-CH2-0- (CH2) 4-0-CH2 - ^ - ^ r -c-o <5> no2, 30 o o ° o

Il II /="» II }— -CH2-C-0-C2H5 , -C-N^ I / -ch2ch2-c-o-n^ o 35 o -'•{0)-VBFr °9 -CH2-{O^N2+BF4-, DK 171394 B1 6 som er afledt af henholdsvis glutaraldehyd, cyanursyrechlo-rid, epichlorhydrin, 1,4-butandiol-diglycidylether, p-nitro-phenyl-chlorformiat, ethylchloracetat, 1,1'-carbonyldiimi- dazol, diazoteret p-nitrobenzaldehyd-fluoboratsalt og dia-5 zoteret p-nitrobenzoylchlorid-fluoboratsalt.II II - = - »II} - -CH2-C-O-C2H5, -CN ^ I / -ch2ch2-con ^ o 35 o - '• {O) -VBFr ° 9 -CH2- {O ^ N2 + BF4 -, DK 171394 B1 6 derived from glutaraldehyde, cyanuric acid chloride, epichlorohydrin, 1,4-butanediol diglycidyl ether, p-nitro-phenyl chloroformate, ethyl chloroacetate, 1,1'-carbonyldiimidazole, diazotized p-nitrobenzalde, respectively. -fluoborate salt and diazotized p-nitrobenzoyl chloride fluoborate salt.

Det immobiliserede enzym i fast fase ifølge opfindelsen er ejendommeligt ved det i krav 4's kendetegnende del angivne, og den ligand-bundne chromatografiaffinitetsmatrix ifølge opfindelsen er ejendommelig ved det i krav 5's ken-10 detegnende del angivne.The solid phase immobilized enzyme of the invention is characterized by the characterizing portion of claim 4, and the ligand-bound chromatography affinity matrix of the invention is characterized by the characterizing portion of claim 5.

Fremgangsmåden ifølge opfindelsen til adskillelse eller rensning af et stof fra opløsning som ovenfor nævnt er ejendommelig ved det i krav 6's kendetegnende del angivne.The method of the invention for separating or purifying a substance from solution as mentioned above is characterized by the characterizing part of claim 6.

Affinitetsmatrixerne ifølge opfindelsen kan anvendes 15 til binding til enhver ligand, der bindes kovalent til affinitetsmatrixen. Affinitetsmatrixen er særlig anvendelig til reaktion med ligander, der har reaktive aminogrupper, omend den også er ganske anvendelig til reaktion med ligander, der har andre reaktive grupper, for eksempel ligander 20 indeholdende reaktive hydroxyl- og sulfhydrylgrupper. Affinitetsmatrixerne ifølge opfindelsen reagerer let med sådanne reaktive grupper af et protein, et enzym eller en anden sådan ligand, til dannelse af den immobiliserede ligand på affinitetsmatrixen. Som eksempler på ligander indehold-25 ende sådanne reaktive grupper, der kan immobiliseres på affinitetsmatrixerne ifølge opfindelsen ved kovalent binding dertil, kan der nævnes antigener, antistoffer, enzymer, inhibitorer, cofaktorer, hormoner, vitaminer, toxiner, vækstfaktorer, glycokonjugater, lectiner, nucleinsyrer og 30 proteiner, der er kendte i teknikken, for eksempel som beskrevet i I. Parikh et al., Affinity Chromatography, C&EN, 17-24,32, 26. august 1985. De ligandbundne affinitetsmatrixer ifølge opfindelsen anvendes til rensning eller adskillelse af stoffer, for eksempel proteiner fra opløsninger indehold-35 ende sådanne stoffer, ved binding eller adsorption af stoffet i opløsning med en affinitetsmatrix ifølge opfindelsen, der DK 171394 B1 7 har en ligand kovalent bundet til affinitetsmatrixen. Blandt sådanne stoffer, der skal adskilles eller renses, kan der for eksempel nævnes enzymer, receptorer, antistoffer, antigener og nucleinsyrer.The affinity matrices of the invention can be used to bind to any ligand covalently attached to the affinity matrix. The affinity matrix is particularly useful for reaction with ligands having reactive amino groups, although it is also quite useful for reacting with ligands having other reactive groups, for example ligands 20 containing reactive hydroxyl and sulfhydryl groups. The affinity matrices of the invention readily react with such reactive groups of a protein, enzyme or other such ligand to form the immobilized ligand on the affinity matrix. As examples of ligands containing such reactive groups which can be immobilized on the affinity matrices of the invention by covalently binding thereto, there may be mentioned antigens, antibodies, enzymes, inhibitors, cofactors, hormones, vitamins, toxins, growth factors, glycoconjugates, lectins, nucleic acids. and 30 proteins known in the art, for example, as described in I. Parikh et al., Affinity Chromatography, C&EN, 17-24,32, August 26, 1985. The ligand-bound affinity matrices of the invention are used for purification or separation of substances. , for example, proteins from solutions containing such substances, by binding or adsorption of the substance in solution with an affinity matrix according to the invention, which has a ligand covalently linked to the affinity matrix. Among such substances to be separated or purified may be mentioned, for example, enzymes, receptors, antibodies, antigens and nucleic acids.

5 Affinitetsmatixerne ifølge opfindelsen kan desuden anvendes til immobilisering af enzymer, enten som ligander til affinitetschromatografi som nævnt i det foregående, eller som immobiliserede enzymer til anvendelse som reaktionskatalysatorer. Sådanne immobiliserede enzymer, der er 10 immobiliseret på de her omhandlede affinitetsmatrixer, er i høj grad aktive og stabile katalysatorer og bibeholder deres ønskede specificitet. Sådanne immobiliserede enzymkatalysatorer kan genanvendes, tillader kontinuerlige reaktioner, tilvejebringer en bedre reaktionskontrol og resulterer i 15 højere renhed og udbytte af produkter. Endvidere vil sådanne immobiliserede enzymkatalysatorer i al almindelighed resultere i mindre forurening på grund af formindskelse eller elimination af tabet af enzymkatalysator. Anvendelsen af sådanne immobiliserede enzymer finder udstrakt brug i en lang række 20 enzym-katalyserede reaktioner, for eksempel ved fremgangsmåder til fremstilling af L-aminosyrer.In addition, the affinity matrices of the invention can be used to immobilize enzymes, either as affinity chromatography ligands as mentioned above, or as immobilized enzymes for use as reaction catalysts. Such immobilized enzymes immobilized on the affinity matrices herein are highly active and stable catalysts and retain their desired specificity. Such immobilized enzyme catalysts can be recycled, allow continuous reactions, provide better reaction control and result in higher purity and yield of products. Furthermore, such immobilized enzyme catalysts will generally result in less pollution due to reduction or elimination of the loss of enzyme catalyst. The use of such immobilized enzymes finds widespread use in a wide variety of 20 enzyme-catalyzed reactions, for example in methods of preparing L-amino acids.

Eksempel 1 25 Til 25,0 g Silica-PrSi-PEI (40 μτη, 2,8% N, 0,05 meq) i en reaktionsbeholder sættes der 13,0 g (0,128 mol) tri-ethylamin sammen med 100 ml chlorform, og der tilsættes 27,8 g (0,115 ml) cyanursyrechlorid ved ca. 20°C. Reaktionen er exoterm. Der tilsættes yderligere 200 mol chloroform, og 30 blandingen omrøres ved ca. 20°C i ca. 5 1/2 time, filtreres, vaskes 3 gange med 100 ml chlorform pr. gang og tørres ved ca. 80°C i ca. 4 timer. Udbyttet er 26,2 g.Example 1 To 25.0 g of Silica-PrSi-PEI (40 μτη, 2.8% N, 0.05 meq) in a reaction vessel 13.0 g (0.128 mol) of triethylamine are added with 100 ml of chloroform, and 27.8 g (0.115 ml) of cyanuric acid chloride are added at ca. 20 ° C. The reaction is exothermic. An additional 200 moles of chloroform are added and the mixture is stirred at ca. 20 ° C for approx. 5 1/2 hours, filter, wash 3 times with 100 ml of chloroform per ml. walking and drying at approx. 80 ° C for approx. 4 hours. The yield is 26.2 g.

Analyse: C = 10,74%, H = 2,11%, N = 6,32%, Cl = 5,52%.Analysis: C = 10.74%, H = 2.11%, N = 6.32%, Cl = 5.52%.

35 DK 171394 Bl 8DK 171394 Pg 8

Eksempel 2 300 ml chloroform anbringes i en reaktionsbeholder, der er udstyret med et termometer, en omrører og en kondensator, og der afkøles til 0-5°C ved hjælp af is og salt.Example 2 300 ml of chloroform is placed in a reaction vessel equipped with a thermometer, stirrer and condenser and cooled to 0-5 ° C by ice and salt.

5 Der tilsættes 27,6 g (0,15 mol) cyanursyrechlorid i løbet af 20 min., idet temperaturen holdes mellem 0 og 5°C. Dernæst tilsættes der på én gang 15,0 g (0,15 mol) triethylamin, og suspensionen bliver gul, og temperaturen stiger til ca. 25°C. Opløsningen afkøles derpå igen til 0-5°C, og der sættes 10 25,0 g Silica-PrSi-PEI (40 μπι, 2,8% N, 0,05 meq) til små portioner i et tidsrum på ca. 20 min., idet temperaturen holdes på 0-5 °C. Reaktionsblandingen omrøres ved denne temperatur i 30 min., hvorefter isbadet fjernes, og blandingen omrøres i ca. 22 timer, filtreres, vaskes 3 gange med 15 100 ml chloroform pr. gang og tørres dernæst ved ca. 0°C i ca. 4 timer. Udbyttet er 25,4 g.27.6 g (0.15 mole) of cyanuric acid chloride are added over 20 minutes, keeping the temperature between 0 and 5 ° C. Then, at the same time, 15.0 g (0.15 mole) of triethylamine is added and the suspension turns yellow and the temperature rises to approx. 25 ° C. The solution is then cooled again to 0-5 ° C, and 10 25.0 g of Silica-PrSi-PEI (40 μπι, 2.8% N, 0.05 meq) is added to small portions for a period of approx. 20 minutes, keeping the temperature at 0-5 ° C. The reaction mixture is stirred at this temperature for 30 minutes, then the ice bath is removed and the mixture is stirred for approx. 22 hours, filtered, washed 3 times with 100 ml of chloroform per ml. and then dried at approx. 0 ° C for approx. 4 hours. The yield is 25.4 g.

Analyse: C = 10,22%, H = 2,12%, N = 5,78%, Cl = 5,59%.Analysis: C = 10.22%, H = 2.12%, N = 5.78%, Cl = 5.59%.

Eksempel 3 20 25 g Silica-PrSi-PEI (40 μπι, 2,8% N, 0,05 meq) suspen deres i 100 ml chloroform, hvortil der sættes 15,0 g ( 0,15 mol) triethylamin. 28,0 g triazin opslæmmes med 200 ml chloroform og overføres til reaktionsbeholderen, hvorved der fremkommer en særdeles exoterm reaktion. Blandingen omrøres 25 i ca. 5 1/2 time ved ca. 20 °C, filtreres, vaskes 3 gange med 100 ml chloroform pr. gang og tørres ved ca, 80 °C i ca. 4 timer. Udbyttet er 26 g.EXAMPLE 3 25 g of Silica-PrSi-PEI (40 μπι, 2.8% N, 0.05 meq) were suspended in 100 ml of chloroform to which 15.0 g (0.15 mol) of triethylamine was added. 28.0 g of triazine is slurried with 200 ml of chloroform and transferred to the reaction vessel to produce a highly exothermic reaction. The mixture is stirred for approx. 5 1/2 hours at approx. 20 ° C, filtered, washed 3 times with 100 ml of chloroform per ml. and dried at about 80 ° C for approx. 4 hours. The yield is 26 g.

Analyse: C = 10,95%, H = 2,12%, N = 5,96%, Cl = 5,63%.Analysis: C = 10.95%, H = 2.12%, N = 5.96%, Cl = 5.63%.

30 Eksempel 4 I en reaktionsbeholder suspenderes 50,0 g Silica-PrSi-PEI (40 μπι, 2,8% N, 0,05 meq) i 200 ml chloroform, hvortil der sættes 29,0 g triethylamin. Der tilsætte gradvis 20,0 g cyanursyrechlorid, og resten af cyanursyrechloridet 35 opslæmmes med 200 ml chloroform og overføres til reaktionsbeholderen, idet der foregår en meget exoterm reaktion.Example 4 In a reaction vessel, 50.0 g of Silica-PrSi-PEI (40 μπι, 2.8% N, 0.05 meq) is suspended in 200 ml of chloroform to which 29.0 g of triethylamine is added. Gradually, 20.0 g of cyanuric acid chloride is added and the rest of the cyanuric acid chloride 35 is slurried with 200 ml of chloroform and transferred to the reaction vessel, undergoing a very exothermic reaction.

DK 171394 B1 9DK 171394 B1 9

Blandingen omrøres i ca. 5 1/2 time ved ca. 20 °C, filtreres, vaskes 3 gange med 200 ml. chloroform pr. gang og tørres ved ca. 80°C i ca. 4 timer. Udbyttet er 51 g.The mixture is stirred for approx. 5 1/2 hours at approx. 20 ° C, filtered, washed 3 times with 200 ml. chloroform per walking and drying at approx. 80 ° C for approx. 4 hours. The yield is 51 g.

Analyse: C = 10,71%, H =2,14%, N - 5,88%, Cl = 5.33%.Analysis: C = 10.71%, H = 2.14%, N - 5.88%, Cl = 5.33%.

55

Eksempel 5 400 ml chloroform suspenderes i en reaktionsbeholder, der er udstyret med en kondensator, omrører og et termometer og nedkøles til 0-5°C ved hjælp af is og salt. 56 g ( 0,29 10 mol) cyanursyrechlorid sættes til den afkølede chloroformopløsning i løbet af 15-20 min., idet temperaturen holdes på 0-5°C. 36 g (0,35 mol) triethylamin i 50 ml chloroform tilsættes dråbevis i løbet af ca. 30 min., idet temperaturen holdes under 10°C. Opløsningen bliver gul, efter at tilsæt-15 ningen af triethylamin er fulstændig. Blandingen afkøles til ca. 0-5°C, og der tilsættes 50,0 g Silica-PrSi-PEI-tria-zin, fremstillet ifølge eksempel 4, idet temperaturen holdes på under ca. 10°C. Tilsætningen af silica er fuldstændig i løbet af ca. 10 min. Reaktionsblandingen omrøres ved denne 20 temperatur i ca. 10 min., hvorefter isbadet fjernes, og blandingen omrøres ved ca. 20°C i ca. 18 1/2 time, filtreres, vaskes med 4 x 250 ml chloroform og tørres ved 80°C i ca. 4 timer. Udbyttet er 49 g.Example 5 400 ml of chloroform is suspended in a reaction vessel equipped with a condenser, stirrer and a thermometer and cooled to 0-5 ° C by ice and salt. 56 g (0.29 10 moles) of cyanuric acid chloride are added to the cooled chloroform solution over 15-20 minutes, keeping the temperature at 0-5 ° C. 36 g (0.35 mol) of triethylamine in 50 ml of chloroform are added dropwise over approx. 30 minutes, keeping the temperature below 10 ° C. The solution turns yellow after the addition of triethylamine is complete. The mixture is cooled to ca. 0-5 ° C and 50.0 g of Silica-PrSi-PEI-triazine, prepared according to Example 4, are added, keeping the temperature below ca. 10 ° C. The addition of silica is complete within approx. 10 min. The reaction mixture is stirred at this temperature for approx. 10 minutes, after which the ice bath is removed and the mixture is stirred at ca. 20 ° C for approx. 18 1/2 hours, filter, wash with 4 x 250 ml of chloroform and dry at 80 ° C for approx. 4 hours. The yield is 49 g.

Analyse: C = 11,3%, H = 2,25%, N = 6,31%, Cl = 4,86%.Analysis: C = 11.3%, H = 2.25%, N = 6.31%, Cl = 4.86%.

2525

Eksempel 6 I en reaktionsbeholder blandes 250 g Silica-PrSi-PEI (40 μτα, 2,8% N, 0,05 meq) og 100 ml 25% glutaraldehydopløs-ning. Blandingen holdes på en omryster i ca. 4 timer ved ca.Example 6 In a reaction vessel, 250 g of Silica-PrSi-PEI (40 μτα, 2.8% N, 0.05 meq) and 100 ml of 25% glutaraldehyde solution are mixed. The mixture is kept on a shaker for approx. 4 hours at approx.

30 20°C. Blandingen bliver lysebrun, filtreres efter ca. 4 timers forløb, vaskes med 3 x 100 ml afioniseret vand, 3 x 100 ml methanol og 3 x 100 ml acetone, og tørres ved 20 °C i en vakuumovn natten over til konstant vægt. Udbyttet er 26 g.30 ° C. The mixture turns light brown, filtered after approx. For 4 hours, wash with 3 x 100 ml deionized water, 3 x 100 ml methanol and 3 x 100 ml acetone, and dry at 20 ° C in a vacuum oven overnight to constant weight. The yield is 26 g.

35 Analyse: C = 15,43%, H = 2,48%, N = 2,34%.Analysis: C = 15.43%, H = 2.48%, N = 2.34%.

DK 171394 B1 10DK 171394 B1 10

Eksempel 7 25 g Silica-PrSi-PEI (40 μπι, 2,8% N, 0,05 meq) anbringes i en 500 ml rundbundet kolbe, og der tilsættes 5,1 g (0,05 ml) triethylamin i 100 ml tetrahydrofuran, efter-5 fulgt af 10,15 g (0,05 mol) p-nitrophenyl-chlorformiat og yderligere 150 ml tetrahydrofuran. Blandingen holdes på en omryster i ca. 16 timer ved ca. 20°C, hvorefter den filtreres, vaskes med 4 x 125 ml chloroform og tørres ved ca. 80°C i ca. 4 timer, Udbyttet er 26 g.Example 7 25 g of Silica-PrSi-PEI (40 μπι, 2.8% N, 0.05 meq) is placed in a 500 ml round bottom flask and 5.1 g (0.05 ml) of triethylamine is added in 100 ml of tetrahydrofuran , followed by 10.15 g (0.05 mol) of p-nitrophenyl chloroformate and an additional 150 ml of tetrahydrofuran. The mixture is kept on a shaker for approx. 16 hours at approx. 20 ° C, after which it is filtered, washed with 4 x 125 ml of chloroform and dried at ca. 80 ° C for approx. 4 hours, The yield is 26 g.

10 Analyse: C = 9,73%, H = 2,51%, N = 3,26%.Analysis: C = 9.73%, H = 2.51%, N = 3.26%.

Eksempel 8 25 g Silica-PrSi-PEI (40 μπι, 2,8% N, 0,05 meq) suspenderes i 250 ml tetrahydrofuran, og der tilsættes 10,12 g 15 (0,1 mol) triethylamin, efterfulgt af 9,32 g (0,01 mol) epi- chlorhydrin. Blandingen holdes på et omrysteapparat i ca.Example 8 25 g of Silica-PrSi-PEI (40 μπι, 2.8% N, 0.05 meq) are suspended in 250 ml of tetrahydrofuran and 10.12 g of (0.1 mole) triethylamine is added followed by 9 32 g (0.01 mole) of epichlorohydrin. The mixture is kept on a shaker for approx.

19 timer ved 20°C, hvorefter den filtreres, vaskes med 2 x 250 ml tetrahydrofuran, 2 x 250 ml chloroform og 2 x 250 ml acetone og tørres ved ca. 80°C i 4 timer. Udbyttet er 25,5 20 g.19 hours at 20 ° C, after which it is filtered, washed with 2 x 250 ml tetrahydrofuran, 2 x 250 ml chloroform and 2 x 250 ml acetone and dried at ca. 80 ° C for 4 hours. The yield is 25.5 20 g.

Analyse: C = 7,84%, H = 1,61%, N = 2,63%.Analysis: C = 7.84%, H = 1.61%, N = 2.63%.

Eksempel 9 I en reaktionsbeholder suspenderes 25 g Silica-PrSi-25 PEI (40 μπι, 2,68% N, 0,05 meq) i 250 ml tetrahydrofuran, og der tilsættes 11,25 g (0,055 mol) 1,4-butandiol-glycidyl-ether. Blandingen anbringes på et rysteapparat i ca. 8 timer ved ca. 20°C, hvorefter den filtreres, vaskes med 3 x 100 ml metanol og tørres ved ca. 80°C i ca. 4 timer.Example 9 In a reaction vessel, 25 g of Silica-PrSi-25 PEI (40 μπι, 2.68% N, 0.05 meq) is suspended in 250 ml of tetrahydrofuran and 11.25 g (0.055 mole) of 1,4-butanediol is added. -glycidyl ether. The mixture is placed on a shaker for approx. 8 hours at approx. 20 ° C, after which it is filtered, washed with 3 x 100 ml methanol and dried at ca. 80 ° C for approx. 4 hours.

30 Analyse: C =7,09%, H = 1,62%, N = 2,36%.Analysis: C = 7.09%, H = 1.62%, N = 2.36%.

Eksempel 10Example 10

Eksempel 9 gentages, med den undtagelse, at produktet vaskes med 3 x 100 ml acetone og 3 x 100 ml diethylether.Example 9 is repeated except that the product is washed with 3 x 100 ml acetone and 3 x 100 ml diethyl ether.

35 Analyse: C = 7,13%, H = 1,61%, N = 2,75%.Analysis: C = 7.13%, H = 1.61%, N = 2.75%.

DK 171394 B1 11DK 171394 B1 11

Eksempel li 10 g (0,061 ml) 1,1'- carbonyldiimidazol suspenderes i 150 ml acetone, og suspensionen sættes til 25 g Silica-PrSi-PEI (40 μιη, 2,8 N, 0,05 meq) i en reaktionsbeholder. Der 5 sættes yderligere 100 ml acetone til reaktionsbeholderen, og den holdes på en roterende filmfordamper ca. 18 timer ved ca. 30°C. Produktet skilles fra ved filtrering, vaskes med 3 x 250 ml acetone og 1 x 250 ml diethylether og tørres ved ca. 80°C i ca. 4 timer. Udbyttet er 26 g.Example 1 10 g (0.061 ml) of 1,1'-carbonyl diimidazole is suspended in 150 ml of acetone and the suspension is added to 25 g of Silica-PrSi-PEI (40 μιη, 2.8 N, 0.05 meq) in a reaction vessel. An additional 100 ml of acetone is added to the reaction vessel and held on a rotary film evaporator for approx. 18 hours at approx. 30 ° C. The product is separated by filtration, washed with 3 x 250 ml acetone and 1 x 250 ml diethyl ether and dried at ca. 80 ° C for approx. 4 hours. The yield is 26 g.

10 Analyse: C = 8,99%, H = 1,72%, N = 3,98%.Analysis: C = 8.99%, H = 1.72%, N = 3.98%.

Eksempel 12 25 g Silica-PrSi-PEI (C = 5,92%, H = 2,82%, 0,05 meq) suspenderes i 125 ml tetrahydrofuran, og der tilsættes 15 5,05 g (0,05 mol) triethylamin, og reaktionsblandingen om røres ved ca. 20°C i ca. 10 min., hvorefter der tilsættes 6,12 g ( 0,05 mol) ethylchloracetat efterfulgt af 125 ml tetrahydrofuran. Reaktionsblandingen omrøres ved ca. 20°C på et rysteapparat i ca. 24 timer, hvorefter den filtreres, 20 vaskes med 2 x 250 ml chloroform, 2 x 250 ml acetone og tørres ved ca. 80°C i ca. 4 timer.Example 12 25 g of Silica-PrSi-PEI (C = 5.92%, H = 2.82%, 0.05 meq) are suspended in 125 ml of tetrahydrofuran and 5.05 g (0.05 mole) of triethylamine are added. and the reaction mixture is stirred at ca. 20 ° C for approx. 10 minutes, then add 6.12 g (0.05 mole) of ethyl chloroacetate followed by 125 ml of tetrahydrofuran. The reaction mixture is stirred at ca. 20 ° C on a shaker for approx. 24 hours, after which it is filtered, 20 washed with 2 x 250 ml of chloroform, 2 x 250 ml of acetone and dried at ca. 80 ° C for approx. 4 hours.

Analyse: C = 6,61%, H - 1,67%, N = 2,80%.Analysis: C = 6.61%, H - 1.67%, N = 2.80%.

Eksempel 13 25 2,5 g (0,013 ml) 1-(3- dimethylaminopropyl)-3-ethyl- -carbodiimid-hydrochlorid opløses i 50 ml af en blanding af acetone og 2-propanol i forholdet 1:1. 1,3 g (0,011 mol) N-hydroxsuccinimid suspenderes i 100 ml acetone og holdes på et rysteapparat (dette stof går ikke fuldstændigt i op-30 løsning). 25 g succinoyleret Silica-PrSi-PEI (C = 11,89%, H = 1,86%, N = 2,69%, carboxylgruppe = 0,95 meq/g) anbringes i en reaktionsbeholder, og opløsningen af 1-(3-dimethylamino-propyl) -3-ethyl-carbodiimid-hydrochlorid i acetone/2-propanol tilsættes. Reaktionsbeholderen omrystes i ca. 5 min., hvor-35 efter opløsningen af N-hydroxy-succinimid i acetone tilsættes, efterfulgt af 25 ml 2-propanol. Reaktionsbeholderen DK 171394 B1 12 holdes på et rysteapparat i ca. 22 timer, hvorefter produktet skilles fra ved filtrering, vaskses med 2 x 250 ml isoprop-anol, 2 x 250 ml acetone og tørres ved ca. 80°C i ca. 4 timer. Udbyttet er 26,4 g.Example 13 2.5 g (0.013 ml) of 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride are dissolved in 50 ml of a mixture of acetone and 2-propanol in a 1: 1 ratio. 1.3 g (0.011 mol) of N-hydroxsuccinimide is suspended in 100 ml of acetone and kept on a shaker (this substance does not completely dissolve). 25 g of succinoylated Silica-PrSi-PEI (C = 11.89%, H = 1.86%, N = 2.69%, carboxyl group = 0.95 meq / g) are placed in a reaction vessel and the solution of 1- ( 3-Dimethylamino-propyl) -3-ethyl-carbodiimide hydrochloride in acetone / 2-propanol is added. The reaction vessel is shaken for approx. 5 minutes, after which the solution of N-hydroxy-succinimide in acetone is added, followed by 25 ml of 2-propanol. The reaction vessel DK 171394 B1 12 is kept on a shaker for approx. 22 hours, after which the product is separated by filtration, washed with 2 x 250 ml isopropanol, 2 x 250 ml acetone and dried at ca. 80 ° C for approx. 4 hours. The yield is 26.4 g.

5 Analyse: C = 13,60%, H = 2,14%, N = 3,38%.Analysis: C = 13.60%, H = 2.14%, N = 3.38%.

Eksempel 14Example 14

Fremstilling af diazo-affinitetschromatografi-matrixer ifølge opfindelsen kan ske ifølge de i det følgende angivne 10 metoder.Preparation of diazo-affinity chromatography matrices according to the invention can be carried out according to the following methods.

A) Silica-PrSi-PEI omsættes med p-nitrobenzaldehyd i methanol, og det fremkomne produkt omsættes med natriumborhydrid i metanol. Produktet suspenderes i natriumdithionitopløsning ved ca. 60°C. Det fremkomne produkt omsættes med iskold 15 HBF4, og produktet skilles fra ved filtrering, vaskes med isvand og tørres under vakuum, hvorved man får det ønskede diazonium-fluoborat produkt:A) Silica-PrSi-PEI is reacted with p-nitrobenzaldehyde in methanol and the resulting product is reacted with sodium borohydride in methanol. The product is suspended in sodium dithionite solution at ca. 60 ° C. The resulting product is reacted with ice-cold HBF4 and the product is separated by filtration, washed with ice-water and dried under vacuum to give the desired diazonium fluoborate product:

Silica-PrSi-PEI—f-NH-CH2 2+BF4”)x - 20 B) Silica-PrSi-PEI omsættes med p-nitrobenzoylchlorid i tetrahydrofuran og i triethylamin, og det dannede produkt suspenderes i en natriumdithionitopløsning, efterfulgt af behandling af reaktionsproduktet med iskold HBF4, filtrering, vask med isvand og tørring af produktet under vakuum, hvorved 25 man får det ønskede diazonium-fluoborat-produkt: 0Silica-PrSi-PEI-f-NH-CH2 2 + BF4) x - 20 B) Silica-PrSi-PEI is reacted with p-nitrobenzoyl chloride in tetrahydrofuran and in triethylamine, and the resulting product is suspended in a sodium dithionite solution, followed by treatment with the reaction product with ice cold HBF 4, filtration, washing with ice water and drying the product in vacuo to give the desired diazonium fluoborate product: 0

Silica-PrSi-PEI-f-NH-C-^0^-N2+BF4“)x ·Silica-PrSi-PEI-f-NH-C-O₂-N 2 + BF 4

Eksempel 15 30 10 g glutaraldehyd-PEI-silicagel-affinitetsmatrix, fremstillet ifølge eksempel 6, vaskes med 1,0 molær phosphat (pH = 8,0), indtil pH-værdien af effluenten er lig med pH-værdien af vaskepufferen (ækvilibreringspufferen) . Der sættes 20,0 ml 25 mg/ml- Human-IgG opløsning (i 0,1 molær phos-35 phatpuffer, pH = 0,8) til affinitetsmatrixen. Der anvendes en koncentreret proteinopløsning for at holde reaktionsrum- DK 171394 B1 13 fanget lille, og reaktionen får lov af foregå natten over ved 4 °C. Affinitetsmatrixen vaskes med følgende rækkefølge af puffere til fjernelse af ikke-kovalent bundet materiale: 0,1 molær phosphat, 0,01 molær phosphat + 1,0 molær NaCl, 5 0,1 molær phosphat. pH-værdien af alle pufferne indstilles på 8,0. Affinitetsmatrixen vaskes derpå med 0,2 molær etanol-amin ( pH = 8,0), til dækning af ikke-omsatte steder, i 1-3 timer ved 4°C. Affinitetsmatrixen ækvilibreres derpå med 0,1 molær phosphat, pH = 7,0. Affinitetsmatrixen opbevares 10 ved 4°C, og der tilsættes o,l% natriumazid som konseverings-middel. 96% af immonoglobulin-proteinet immobiliseres som bestemt ved analyse ved HPLC i omvendt fase, idet mængden af immonoglobulin-protein forbliver i opløsningen. Immobili-seret antistof er immunologisk aktivt som bestemt ved reten-15 tion af dets specifikke antigen somatrotropin, når dette peptid i opløsning ledes over en pakket kolonne af antistof-affinitetsmatrixen. Strukturelt ubeslægtede peptider tilbageholdes ikke af kolonnen.Example 15 30 g of glutaraldehyde PEI silica gel affinity matrix prepared according to Example 6 is washed with 1.0 molar phosphate (pH = 8.0) until the pH of the effluent is equal to the pH of the wash buffer (equilibration buffer). . 20.0 ml of 25 mg / ml Human IgG solution (in 0.1 molar phosphate buffer, pH = 0.8) is added to the affinity matrix. A concentrated protein solution is used to keep the reaction chamber trapped small and the reaction is allowed to proceed overnight at 4 ° C. The affinity matrix is washed with the following order of buffers to remove non-covalently bound material: 0.1 molar phosphate, 0.01 molar phosphate + 1.0 molar NaCl, 0.1 molar phosphate. The pH of all the buffers is set to 8.0. The affinity matrix is then washed with 0.2 molar ethanolamine (pH = 8.0), to cover unreacted sites, for 1-3 hours at 4 ° C. The affinity matrix is then equilibrated with 0.1 molar phosphate, pH = 7.0. The affinity matrix is stored at 4 ° C and 0.1% sodium azide is added as a preservative. 96% of the immunoglobulin protein is immobilized as determined by reverse phase HPLC analysis, with the amount of immunoglobulin protein remaining in the solution. Immobilized antibody is immunologically active as determined by retention of its specific antigen somatrotropin when this peptide is dissolved in solution over a packed column of the antibody affinity matrix. Structurally unrelated peptides are not retained by the column.

20 Eksempel 16 5 g glutaraldehyd-PEI-silicagel-affinitetsmatrix, der er fremstillet ifølge eksempel 6, vaskes med 0,1 molær acetat (pH = 8,7), indtil effluentens pH-værdi er lig med pH-værdien af vaskepufferen (ækvilibreringspufferen). 10 ml 25 chymotrypsin-opløsning med 30 mg pr. ml. i 0,1 molær acetatpuffer, pH-værdi = 8,7, sættes til affinitetsmatrixen. Der anvendes en koncentreret proteinopløsning for at holde reaktionsrumfanget lille, og reaktionen får lov at foregå natten over ved 5°C. Affinitetsmatrixen vaskes med 0,1 molær acetat-30 puffer med en pH-værdi på 7,0 til fjernelse af ikke-kovalent bundet materiale. Affinitetsmatrixen opbevares tørt ved 4°C eller i en blanding af acetatpuffer med en pH-værdi på 7,0 og 2-propanol i forholdet 9:1. 90% af enzymet immobiliseres som bestemt ved analyse ved HPLC i omvendt fase af den mængde 35 enzym-protein, der bliver tilbage i opløsning. Det immobili-serede enzym pakkes i en glaskolonne og anvendes som en DK 171394 B1 14 chromatografi-matrix til chromatografering af amino-syrer og aminosyrederivater.Example 16 5 g of glutaraldehyde PEI silica gel affinity matrix prepared according to Example 6 is washed with 0.1 molar acetate (pH = 8.7) until the pH of the effluent is equal to the pH of the wash buffer (equilibration buffer ). 10 ml of 25 chymotrypsin solution at 30 mg per ml. ml. in 0.1 molar acetate buffer, pH = 8.7 is added to the affinity matrix. A concentrated protein solution is used to keep the reaction volume small and the reaction is allowed to proceed overnight at 5 ° C. The affinity matrix is washed with 0.1 molar acetate buffer with a pH of 7.0 to remove non-covalently bound material. The affinity matrix is stored dry at 4 ° C or in a mixture of acetate buffer having a pH of 7.0 and 2-propanol in the ratio of 9: 1. 90% of the enzyme is immobilized as determined by reverse-phase HPLC analysis of the amount of 35 enzyme protein remaining in solution. The immobilized enzyme is packed in a glass column and used as a DK 171394 B1 14 chromatography matrix for chromatography of amino acids and amino acid derivatives.

Eksempel 17 5 50 ml glutaraldehyd-PEI-silicagel-affinitetsmatrix, der er fremstillet ifølge eksempel 6, vaskes med 0,05-01 molær phosphat (pH = 7,5-8,5), indtil effluentens pH-værdi er lig med pH-værdien af vaskepufferen (ækvilibreringspuf-fer). Der sættes 2,0 ml galactoseoxidase-opløsning med 0,5 10 mg pr. ml (i 0,05-0,1 molær phosphat- eller boratpuffer, pH = 7,5-8,5) til affinitetsmatrixen. Der anvendes koncentreret proteinopløsning for at holde reaktionsrumfanget lille, og reaktionen får lov at forløbe natten over ved 4°C. Affinitetsmatrixen vaskes med følgende rækkefølge af puffere til 15 fjernelse af ikke-kovalent bundet materiale: 0,1 molær phosphat, 0,1 molær phosphat + 1,0 molær NaCl, og 0,1 molær phosphat, idet pH-værdien for alle puffere indstilles på 8,05. Affinitetsmatrixen vaskes derpå med 0,2 molær ethanol-amin eller tris (pH = 8,0) til dækning af ikke-omsatte ste-20 der, i 1-3 timer ved 4°C. Affinitetsmatrixen ækvilibreres derpå med 0,1 molær phosphat, pH = 7,0. Affinitetsmatrixen opbevares ved 4°C. 98% af enzymet er immobiliseret, som bestemt ved analyse ved HPLC i omvendt fase af den mængde enzym-protein, der bliver tilbage i opløsning. Immobiliseret 25 enzym er aktivt som bestemt ved en kolorimetrisk oxidationsreduktions- reakt ion under anvendelse af o-dianisidin.Example 17 50 ml of glutaraldehyde PEI silica gel affinity matrix prepared according to Example 6 is washed with 0.05-01 molar phosphate (pH = 7.5-8.5) until the pH of the effluent is equal to pH value of the wash buffer (equilibration buffer). Add 2.0 ml of galactose oxidase solution at 0.5 10 mg per ml. ml (in 0.05-0.1 molar phosphate or borate buffer, pH = 7.5-8.5) to the affinity matrix. Concentrated protein solution is used to keep the reaction volume small and the reaction is allowed to proceed overnight at 4 ° C. The affinity matrix is washed with the following order of buffers to remove non-covalently bound material: 0.1 molar phosphate, 0.1 molar phosphate + 1.0 molar NaCl, and 0.1 molar phosphate, adjusting the pH of all buffers at 8.05. The affinity matrix is then washed with 0.2 molar ethanol-amine or tris (pH = 8.0) to cover unreacted sites, for 1-3 hours at 4 ° C. The affinity matrix is then equilibrated with 0.1 molar phosphate, pH = 7.0. The affinity matrix is stored at 4 ° C. 98% of the enzyme is immobilized, as determined by reverse-phase HPLC analysis of the amount of enzyme protein remaining in solution. Immobilized enzyme is active as determined by a colorimetric oxidation-reduction reaction using o-dianisidine.

Eksempel 18Example 18

Triazin-PEI-Silicagel-affinitetsmatrix, fremstillet 30 ifølge eksempel 3, vaskes med 0,1 molær phosphat (pH = 7,5), indtil effluentens pH-værdi er ækvilibreret til pH-værdien af vaskepufferen. Der sættes 2,5 ml peroxidaseopløsning med 1 mg pr. ml (i 0,1 molær phosphatpuffer, pH = 7,5) til affi-nitetsmatrixen, og blandingen får lov reagere ved stuetem-35 peratur i 4 timer. Affinitetsmatrixen vaskes med følgende rækkefølge af puffere til fjernelse af ikke-kovalent bundet DK 171394 B1 15 materiale: 0,1 molær phosphat, 0,1 molær phosphat + 1,0 molær NaCl, og 0,1 molær phosphat. Der anvendes en pH-værdi på 8,0 for alle puffere. Affinitetsmatrixen vaskes derpå med 0,2 molær ethanolamin (pH =8,0) i 1-3 timer ved stue-5 temperatur til dækning af ikke-omsatte steder. Derefter ækvilibreres affinitetsmatrixen med 0,1 molær phosphat med en pH-værdi på 7,0. Affinitetsmatrixen opbevares ved 4°C. Immobiliseringsprocessen følges ved styring af absorbansen ved 280 nm af den ovenstående væske fra reaktionssuspen-10 sionen. 54% af enzymet i opløsning absorberes. Det immobili-serede enzym udviser 55% af akviteten for en ækvivalent mængde af opløseligt enzym.Triazine PEI-Silica gel affinity matrix prepared according to Example 3 is washed with 0.1 molar phosphate (pH = 7.5) until the pH of the effluent is equilibrated to the pH of the wash buffer. Add 2.5 ml of peroxidase solution at 1 mg per ml. ml (in 0.1 molar phosphate buffer, pH = 7.5) to the affinity matrix and the mixture is allowed to react at room temperature for 4 hours. The affinity matrix is washed with the following order of buffers to remove non-covalently bound material: 0.1 molar phosphate, 0.1 molar phosphate + 1.0 molar NaCl, and 0.1 molar phosphate. A pH of 8.0 is used for all buffers. The affinity matrix is then washed with 0.2 molar ethanolamine (pH = 8.0) for 1-3 hours at room temperature to cover unreacted sites. Then, the affinity matrix is equilibrated with 0.1 molar phosphate with a pH of 7.0. The affinity matrix is stored at 4 ° C. The immobilization process is followed by controlling the absorbance at 280 nm of the above liquid from the reaction suspension. 54% of the enzyme in solution is absorbed. The immobilized enzyme exhibits 55% of the quality of an equivalent amount of soluble enzyme.

Eksempel 19 15 50 mg glutaraldehyd-PEI-silicagel-affinitetsmatrix vaskes grundigt med 0,1 molær phosphatpuffer, pH = 8, og ækvilibreres i den samme puffer. Der sættes 2,5 ml af en opløsning af β-galactosidase med 0,8 mg pr. ml til affini-tetsmatrix-suspensionen, og immobiliseringshastigheden følges 20 ved styring af absorbansen af den ovenstående væske ved 280 nm. Efter 20 timer ved 4°C er 73% af enzymet bundet til matrixen og udviser 40% af den specifikke aktivitet for opløseligt enzym.Example 19 50 mg of glutaraldehyde PEI silica gel affinity matrix is thoroughly washed with 0.1 molar phosphate buffer, pH = 8, and equilibrated in the same buffer. Add 2.5 ml of a solution of β-galactosidase at 0.8 mg per ml. to the affinity matrix suspension, and the rate of immobilization is followed by controlling the absorbance of the supernatant at 280 nm. After 20 hours at 4 ° C, 73% of the enzyme is bound to the matrix and exhibits 40% of the specific soluble enzyme activity.

Claims

1. Solid phase support or support on the basis of a material selected from the group consisting of (a) a support of the general formula

Silica-PrSi-PEI - (- R) xi which Silica-PrSi-PEI is a covalently bonded, non-crosslinked polyethyleneimine bonded solid phase support that is the reaction product of (1) a) particulate silica gel with a average particle diameter of from 1 to 200 μτη and an average pore size of from 0 to 100 nm; or b) controlled pore particulate glass with an average particle diameter of from 1 to 200 μιη and an average pore size of from 0 to ca. 100 nm, with (2) polyethyleneimine propyl trimethoxy silane having an average molecular weight of from 400 to 1800, and (b) the slightly acidic carboxylated product of the solid Silica-PrSi-PEI support with a dibasic acid anhydride, wherein the carboxylated product contains from 0.3 to 1.2 carboxyl milliequivalents per g, 20 characterized in that, in the case of the non-weakly acidic carboxylated support, R is the remainder of any chemically reactive moiety capable of undergoing nucleophilic substitution at two distinct sites such that R becomes covalently linked to the primary or secondary 25 amino groups of the PEI used at one such site, while the other site is available and reactive for subsequent nucleophilic substitution under non-denaturing conditions with an affinity chromatography ligand to form another covalent bond which is stable under aqueous hydrol-3 X is a positive integer less than or equal to the total number of primary or secondary amino groups in the PEI molecular moiety, and in the case of the weakly acidic caboxylated support, R is the remainder of any chemically reactive moiety present in capable of facilitating nucleophilic substitution of the carboxyl hydroxyl to form a covalent bond by carboxyl carbon the atom, thereby providing a sufficiently electrophilic site to be readily displaced by the carboxyl carbon atom with a nucleophilic functional group on an affinity chromatography ligand, and x is a positive integer less than or equal to the total number of carboxyl groups in the carboxylated PEI molecule moiety.

Solid phase support or support according to claim 1, characterized in that the silica gel has an average particle diameter of from 3 to 70 μτη and an average pore size of from 5 to 100 nm. Solid phase support or support according to claim 1 or 2, characterized in that R is selected from the groups consisting of o Cl II - C, o 15 = ch-ch 2ch 2ch 2ch, - <TO n, -ch 2 - ^ - ^ , Cl OH o T / ° \ ii / τγ \ -CH 2 -CH-CH 2 -O- (CH 2) 4-O-CH 2 - ^ - ^, -C-O-Mj> NO 2, 0 0 0 ° O II II II} -1 -CH2-C-O-C2H5, -CN ^ _ I, -ch2ch2-con ^ é— 25 ~ O -c / o) -N2 + BF4- and -CH2 ^ O> K2 + 3F4-.

4. Solid phase immobilized enzyme, known as the enzyme is immobile in series t on a solid phase support of an affinity chromatography matrix, the solid phase support being as defined in any of the preceding claims.

The ligand-bound chromatography affinity matrix, characterized in that the affinity matrix selected from a carrier (a) and a product (b) as defined in any of claims 1-3.

A method for separating or purifying a substance from solution by binding the substance in solution with an affinity matrix having a ligand from the substance covalently bonded to the affinity matrix, characterized in that as an affinity matrix a solid support or support is used. phase according to any one of claims 1-3. 10 15 20 25 30 35