DE4141302A1 - Protein irreversibly immobilised on macroporous cellulose@ beads - by reversible adsorption then crosslinking or physical inclusion, e.g. for conversion or sepn. processes - Google Patents

Protein irreversibly immobilised on macroporous cellulose@ beads - by reversible adsorption then crosslinking or physical inclusion, e.g. for conversion or sepn. processes

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DE4141302A1
DE4141302A1 DE19914141302 DE4141302A DE4141302A1 DE 4141302 A1 DE4141302 A1 DE 4141302A1 DE 19914141302 DE19914141302 DE 19914141302 DE 4141302 A DE4141302 A DE 4141302A DE 4141302 A1 DE4141302 A1 DE 4141302A1
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protein
carrier
buffer
pearl cellulose
cellulose
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Klaus Dr Pommerening
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INST MOLEKULARBIOLOGIE AK
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    • 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
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12—Cellulose or derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28095—Shape or type of pores, voids, channels, ducts
    • B01J20/28097—Shape or type of pores, voids, channels, ducts being coated, filled or plugged with specific compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • C07K17/12—Cellulose or derivatives thereof

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  • Peptides Or Proteins (AREA)

Abstract

Carrier-immobilised, biologically or physiologically active protein (I) comprises unmodified, macroporous cellulose beads on which (I) is immobilised, after reversible adsorption, by a chemical or physical process, i.e. an inclusion process or crosslinking via a multifunctional agent. Pref. (A) is glutaraldehyde used at a final concn. of up to 2.5% of the reaction mixt. and (I) is cytochrome c; haemoglobin; ferritie or lysozyme. USE/ADVANTAGE - The products are useful for conversion or sepn. processes in biological science, biotechnology and in the chemical and pharmaceutical industries. The method is simple; provides irreversible immobilisation (no gradual loss of (I) during use) independently of the type of medium, and crosslinking can be done under mild conditions which do not cause denaturation. No modification of the beads is required

Description

Die Erfindung betrifft trägerfixierte biologisch aktive oder physiologisch wirksame Proteine und Verfahren zu ihrer Her­ stellung. Der Einsatz erfolgt in Prozessen der Stoffwandlung und -trennung in den Biowissenschaften, der Biotechnologie, chemischen und pharmazeutischen Industrie und in anderen Ge­ bieten.The invention relates to carrier-fixed biologically active or physiologically active proteins and processes for their production position. They are used in processes of material conversion and separation in life sciences, biotechnology, chemical and pharmaceutical industries and other ge Offer.

Gegenwärtig ist eine Vielzahl von trägerfixierten biologisch aktiven oder physiologisch wirksamen Proteinen und Verfahren zu ihrer Herstellung bekannt.Currently, a variety of carrier-fixed are biological active or physiologically active proteins and methods known for their manufacture.

Die Proteinkomponente sind sowohl Enzyme als auch Antikörper, Antigene, Effektoren und Hormone.The protein components are both enzymes and antibodies, Antigens, effectors and hormones.

Als Trägerkomponente werden Biopolymere und synthetische or­ ganische Polymere sowie auch anorganische Träger genutzt. Zur Immobilisierung kommen sowohl physikalische Methoden (Ein­ schluß- und Adsorptionsverfahren) und chemische Methoden (kovalente Bindung, Quervernetzung) als auch eine biospezifi­ sche Komplexbildung zum Einsatz (Applied Biochemistry and Bioengineering, Vol. 1, Immobilized Enzyme Principles, Her­ ausgeber: L. B. Wingard, Jr., E. Katchalsky-Katzir, L. Gold­ stein, Academic Press, New York, San Francisco, London, 1976).Biopolymers and synthetic or ganic polymers as well as inorganic carriers used. For Immobilization come both physical methods (a closing and adsorption processes) and chemical methods (covalent binding, cross-linking) as well as a biospecific complex formation for use (Applied Biochemistry and Bioengineering, Vol. 1, Immobilized Enzyme Principles, Her issued by: L. B. Wingard, Jr., E. Katchalsky-Katzir, L. Gold stein, Academic Press, New York, San Francisco, London, 1976).

Die Zugänglichkeit und Bereitstellung verschiedener makropo­ röser sphärischer Celluloseträger führten wieder zu einem ge­ steigerten Interesse an diesem ökonomisch vorteilhaften Trä­ germaterial (P. Mohr and K. Pommerening "Affinity Chromato­ graphy, Practical and Theoretical Aspects", Herausgeber: Marcel Dekker, Inc., New York and Basel, 1985, S. 45, P. Ge­ meiner et al., Chem. Papers, 43, 805 (1989)).The accessibility and provision of various makropo röser spherical cellulose carrier again led to a ge increased interest in this economically advantageous trade germaterial (P. Mohr and K. Pommerening "Affinity Chromato graphy, Practical and Theoretical Aspects ", publisher: Marcel Dekker, Inc., New York and Basel, 1985, p. 45, P. Ge Meine et al., Chem. Papers, 43, 805 (1989)).

Die nach DD-PS 1 18 887 hergestellte makroporöse Perlcellulose ist ein leistungsfähiger Träger für die Stofftrennung und Stoffwandlung (H.-F. Boeden et al. D. Chromatogr. 552, 389-414 (1991)). Nach dem Stand der Technik muß die Perlcellulose jedoch für diesen Einsatz mit Hilfe häufig aufwendiger Ver­ fahren modifiziert werden. So erfolgt eine Adsorption eines Proteins erst nach Einbau ionischer, hydrophober oder bio­ affiner Gruppen in die Cellulosematrix.The macroporous pearl cellulose produced according to DD-PS 1 18 887 is a powerful carrier for material separation and Substance conversion (H.-F. Boeden et al. D. Chromatogr. 552, 389-414 (1991)). According to the prior art, the pearl cellulose  however for this use with the help of often complex Ver driving be modified. So an adsorption takes place Proteins only after incorporation of ionic, hydrophobic or bio affine groups in the cellulose matrix.

Die nach DD-PS 1 18 887 hergestellte unmodifizierte makroporö­ se Perlcellulose zeigt eine Wechselwirkung mit Proteinen, die z. T. zu einer deutlich verzögerten Elution der Proteine führt, so daß dieser Träger nicht als Molekularsieb in der Gelpermeation eingesetzt werden kann.The unmodified macroporoe manufactured according to DD-PS 1 18 887 Pearl cellulose interacts with proteins that e.g. T. to a significantly delayed elution of the proteins leads, so that this carrier is not as a molecular sieve in the Gel permeation can be used.

Aufgabe der Erfindung ist es, trägerfixierte biologisch akti­ ve oder physiologisch wirksame Proteine bereitzustellen und technisch einfache Verfahren zu ihrer Herstellung zu finden, die für die Prozesse der Stoffwandlung und -trennung einsetz­ bar sind.The object of the invention is to biologically acti fixed carrier to provide ve or physiologically active proteins and to find technically simple processes for their production, which are used for the processes of material conversion and separation are cash.

Überraschend wurde gefunden, daß sich einzelne Proteine unter ausgewählten und sehr unterschiedlichen Bedingungen quantita­ tiv an unmodifizierte makroporöse Perlcellulose adsorptiv binden. Diese Adsorption ist stark vom pH, von der Ionenstär­ ke und der Temperatur abhängig und voll reversibel. Eine Quervernetzung der Proteine mit multifunktionellen Agentien oder Schrumpfung der makroporösen Perlcellulose nach der Ad­ sorption des Proteins führt zu einer milieuunabhängigen ir­ reversiblen Immobilisierung. Die Erfindung wird gemäß der An­ sprüche 1, 7 und 8 realisiert, die Unteransprüche sind Vor­ zugsvarianten.It has surprisingly been found that individual proteins are found under selected and very different conditions quantita adsorptive to unmodified macroporous pearl cellulose tie. This adsorption is strong on pH, on ionic strength ke and temperature dependent and fully reversible. A Cross-linking of proteins with multifunctional agents or shrinkage of the macroporous pearl cellulose after the ad sorption of the protein leads to an environment-independent ir reversible immobilization. The invention is according to the An sayings 1, 7 and 8 realized, the subclaims are before train variants.

Erfindungsgemäß sind die trägerfixierten biologisch aktiven oder physiologisch wirksamen Proteine und Verfahren zu ihrer Herstellung dadurch gekennzeichnet, daß aus einer wäßrigen Lösung, die ein Protein oder Proteingemisch enthält, unter definierten und an das Protein angepaßten Bedingungen, insbe­ sondere des pH, der Ionenstärke und der Temperatur, das Pro­ tein zunächst reversibel adsorbiert und anschließend durch Zugabe eines multifunktionellen Agenz eine Quervernetzung er­ folgt. According to the invention, the carrier-fixed are biologically active or physiologically active proteins and methods for their Production characterized in that from an aqueous Solution containing a protein or protein mixture under defined and adapted to the protein conditions, esp special of pH, ionic strength and temperature, the pro initially reversibly adsorbed and then by Adding a multifunctional agent to cross-link it follows.  

Andererseits kann erfindungsgemäß das Verfahren auch so durchgeführt werden, daß Adsorption und Quervernetzung gleichzeitig erfolgen. Die unmodifizierte Perlcellulose wird zunächst mit dem Vernetzungsmittel inkubiert und nach Entfer­ nen des Überstandes anschließend mit der wäßrigen Lösung, die das Protein enthält, in Kontakt gebracht. Erfindungsgemäß er­ folgen Adsorptionsschritt und Quervernetzung unter milden Be­ dingungen, so daß keine Denaturierung infolge der Immobili­ sierungsschritte auftritt.On the other hand, the method can also be used according to the invention be carried out that adsorption and crosslinking done simultaneously. The unmodified pearl cellulose is first incubated with the crosslinking agent and after removal NEN of the supernatant then with the aqueous solution containing the protein. According to the invention follow the adsorption step and cross-linking under mild loading conditions so that no denaturation as a result of immobilization steps occur.

Der Adsorptionsschritt, die Quervernetzung und der Waschpro­ zeß erfolgen bei einer geringen Ionenstärke, vorzugsweise mit einer 0,01-0,1 M Pufferkonzentration, einem pH zwischen 6 und 9, einer Endkonzentration an Vernetzungsmittel bis zu 2,5%, vorzugsweise zwischen 0,5-1%, und einer Temperatur zwischen 0 und 35°C.The adsorption step, the crosslinking and the washing pro zeß occur with a low ionic strength, preferably with a 0.01-0.1 M buffer concentration, a pH between 6 and 9, a final concentration of crosslinking agent up to 2.5%, preferably between 0.5-1%, and a temperature between 0 and 35 ° C.

Eine andere Möglichkeit der irreversiblen Immobilisierung wird durch einen Schrumpfungsprozeß entsprechend DD-PS 2 12 266 realisiert. Erfindungsgemäß wird das an die makroporöse Perl­ cellulose reversibel adsorbierte Protein auf Temperaturen un­ ter 0°C abgekühlt, 12-24 Stunden im gefrorenen Zustand ge­ lagert und anschließend wieder aufgetaut.Another possibility of irreversible immobilization is by a shrinking process according to DD-PS 2 12 266th realized. According to the macroporous pearl cellulose reversibly adsorbed protein at temperatures and cooled to 0 ° C, frozen for 12-24 hours stored and then thawed again.

Die Erfindung hat den großen Vorteil, daß makroporöse Perl­ cellulose in unmodifizierter Form eingesetzt und eine verzö­ gerte Elution der Proteine vermieden werden kann. Der Erfin­ dungsgegenstand soll an einigen Beispielen erläutert werden, die die Erfindung jedoch nicht einschränken.The invention has the great advantage that macroporous pearl cellulose used in unmodified form and a delay Elution of the proteins can be avoided. The inventor The subject of the application will be explained using a few examples, which, however, do not limit the invention.

Beispiel 1example 1

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,01 M Boratpuffer, pH 9, wird mit 1 ml einer 25-100 µm Cytochrom C-Lösung (Pferd) im gleichen Puffer bei 0-30°C inkubiert. Nach Adsorption des Cytochrom C an der Perlcellu­ lose wird mit Glutaraldehyd bis zu einer Endkonzentration von 2,5%, vorzugsweise 0,5-1%, unter ständiger Durchmischung 4 h inkubiert. 1 ml of swollen macroporous pearl cellulose, equilibrated with 0.01 M borate buffer, pH 9, is mixed with 1 ml of a 25-100 µm Cytochrome C solution (horse) in the same buffer at 0-30 ° C incubated. After adsorption of cytochrome C on the Perlcellu is loosened with glutaraldehyde to a final concentration of 2.5%, preferably 0.5-1%, with constant mixing Incubated for 4 h.  

Nach Auswaschen mit einem 0,1 M Puffer, pH 7, sind 85-100% (0,35-1,3 µg Protein/ml Cellulose) des Cytochroms irrever­ sibel immobilisiert.After washing with a 0.1 M buffer, pH 7, 85-100% (0.35-1.3 µg protein / ml cellulose) of the cytochrome irreversible sibel immobilized.

Beispiel 2Example 2

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,01 M Boratpuffer, pH 9, wird mit Glutaraldehyd bis zu einer Endkonzentration von 2,5% inkubiert und durch Absaugen vom Überstand und anhaftender Flüssigkeit befreit. Anschließend wird die Perlcellulose mit 1 ml einer 450 µm Cytochrom C-Lö­ sung (Pferd) im gleichen Puffer bei 0-30°C unter ständiger Durchmischung 4 h inkubiert. Nach Auswaschen mit 0,1 M Puf­ fer, pH 7, sind 18% (1,14 mg/ml) Cytochrom C irreversibel immobilisiert.1 ml of swollen macroporous pearl cellulose, equilibrated with 0.01 M borate buffer, pH 9, is mixed with glutaraldehyde up to a Final concentration of 2.5% and aspirated from Free supernatant and adhering liquid. Subsequently the pearl cellulose with 1 ml of a 450 µm cytochrome C-Lö solution (horse) in the same buffer at 0-30 ° C under constant Mixing incubated for 4 h. After washing out with 0.1 M puf fer, pH 7, 18% (1.14 mg / ml) cytochrome C is irreversible immobilized.

Beispiel 3Example 3

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,01 M Boratpuffer, pH 9, wird mit 1 ml einer 25-100 µm Cytochrom C-Lösung (Pferd) im gleichen Puffer bei 2-30°C inkubiert. Nach Adsorption des Cytochrom C an die Perlcellu­ lose wird die Suspension unter 0°C abgekühlt, 12-24 Stunden bei -20°C im gefrorenen Zustand gelagert und anschließend wieder aufgetaut. Nach Auswaschen mit 0,1 M Phosphatpuffer, pH 7, sind 25-100% (0,35 mg Protein/ml Cellulose) irrever­ sibel immobilisiert.1 ml of swollen macroporous pearl cellulose, equilibrated with 0.01 M borate buffer, pH 9, is mixed with 1 ml of a 25-100 µm Cytochrome C solution (horse) in the same buffer at 2-30 ° C incubated. After adsorption of cytochrome C onto the Perlcellu the suspension is cooled loosely below 0 ° C. for 12-24 hours stored at -20 ° C in the frozen state and then thawed again. After washing out with 0.1 M phosphate buffer, pH 7, 25-100% (0.35 mg protein / ml cellulose) are irreversible sibel immobilized.

Beispiel 4Example 4

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,01 M Puffer, pH 7, wird mit Glutaraldehyd bis zu einer End­ konzentration von 2,5% inkubiert und durch kurzes Absaugen vom Überstand und anhaftender Flüssigkeit befreit. Anschlie­ ßend wird die Perlcellulose mit 1 ml einer 0,51 mM Methämo­ globin-Lösung (Mensch) im gleichen Puffer bei 0-30°C unter ständiger Durchmischung 4 h inkubiert. 1 ml of swollen macroporous pearl cellulose, equilibrated with 0.01 M buffer, pH 7, is brought to an end with glutaraldehyde concentration of 2.5% and briefly aspirated freed from the supernatant and adhering liquid. Then The pearl cellulose is eats with 1 ml of a 0.51 mM methemo globin solution (human) in the same buffer at 0-30 ° C below constant mixing incubated for 4 h.  

Nach intensivem Waschen mit 0,1 M Puffer, pH 7, sind bis zu 90% Methämoglobin irreversibel immobilisiert.After intensive washing with 0.1 M buffer, pH 7, are up to 90% irreversibly immobilized on methaemoglobin.

Beispiel 5Example 5

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,1 M Puffer, pH 7, wird mit 1 ml Ferritin-Lösung (Pferd) (0,37 mg Protein/ml) im gleichen Puffer bei 0-30°C inku­ biert. Nach Adsorption des Ferritins wird mit Glutaraldehyd bis zu einer Endkonzentration von 2,5%, vorzugsweise 0,5-1%, unter ständiger Durchmischung 5 h inkubiert.1 ml of swollen macroporous pearl cellulose, equilibrated with 0.1 M buffer, pH 7, is mixed with 1 ml ferritin solution (horse) (0.37 mg protein / ml) in the same buffer at 0-30 ° C incu beer. After adsorption of the ferritin with glutaraldehyde up to a final concentration of 2.5%, preferably 0.5-1%, incubated for 5 hours with constant mixing.

Nach Auswaschen mit 0,1 M Boratpuffer, pH 9, sind mehr als 95% Ferritin irreversibel immobilisiert.After washing with 0.1 M borate buffer, pH 9, more than 95% irreversibly immobilized ferritin.

Beispiel 6Example 6

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,1 M Phosphatpuffer, pH 7, wird mit 0,1 ml Ferritin-Lösung (Pferd) (0,16-0,37 mg Protein/ml) bei Raumtemperatur inku­ biert. Nach Adsorption des Ferritins wird die Suspension un­ ter 0°C abgekühlt, 12 bis 24 Stunden bei -20°C im gefrorenen Zustand gelagert und wieder aufgetaut.1 ml of swollen macroporous pearl cellulose, equilibrated with 0.1 M phosphate buffer, pH 7, is mixed with 0.1 ml ferritin solution (Horse) (0.16-0.37 mg protein / ml) at room temperature beer. After adsorption of the ferritin, the suspension is un cooled to 0 ° C, 12 to 24 hours at -20 ° C in the frozen Condition stored and thawed again.

Nach Auswaschen des nicht gebundenen Ferritins mit 0,1 M Boratpuffer, pH 9, sind 70-77% (0,1-0,4 mg Protein/ml Cellulose) irreversibel immobilisiert.After washing the unbound ferritin with 0.1 M Borate buffer, pH 9, is 70-77% (0.1-0.4 mg protein / ml Cellulose) irreversibly immobilized.

Beispiel 7Example 7

1 ml gequollene makroporöse Perlcellulose, äquilibriert mit 0,01 M Boratpuffer, pH 9, wird mit 1 ml Lysozym-Lösung (Hüh­ nereiweiß) (5,65 mg Protein/ml) im gleichen Puffer bei 22-24°C inkubiert. Nach Adsorption des Lysozyms wird mit Glutar­ aldehyd bis zu einer maximalen Endkonzentration von 1% unter ständiger Durchmischung 4 h inkubiert.1 ml of swollen macroporous pearl cellulose, equilibrated with 0.01 M borate buffer, pH 9, is mixed with 1 ml lysozyme solution (Hüh egg white) (5.65 mg protein / ml) in the same buffer at 22-24 ° C incubated. After adsorption of the lysozyme with Glutar aldehyde up to a maximum final concentration of 1% below constant mixing incubated for 4 h.

Nach Auswaschen mit 0,1 M Boratpuffer, pH 9, der 0,5 M NaCl enthält, sind 91,5% Lysozym (5,2 mg Protein/ml Perlcellulo­ se) irreversibel immobilisiert.After washing with 0.1 M borate buffer, pH 9, the 0.5 M NaCl contains, are 91.5% lysozyme (5.2 mg protein / ml pearl cellulo se) irreversibly immobilized.

Claims (16)

1. Trägerfixierte biologisch aktive oder physiologisch wirk­ same Proteine, gekennzeichnet durch unmodifizierte makro­ poröse Perlcellulose, an die ein Protein nach reversibler Adsorption durch einen chemischen oder physikalischen Pro­ zeß immobilisiert ist, der ein Einschlußverfahren oder eine Quervernetzung über ein multifunktionelles Agenz ist.1. carrier-fixed biologically active or physiologically active proteins, characterized by unmodified macro-porous pearl cellulose, to which a protein is immobilized after reversible adsorption by a chemical or physical process, which is an inclusion process or a crosslinking via a multifunctional agent. 2. Trägerfixierte Proteine nach Anspruch 1, dadurch gekenn­ zeichnet, daß das multifunktionelle Agenz vorzugsweise Glutaraldehyd mit einer Endkonzentration bis zu 2,5% im Reaktionsansatz ist.2. carrier-fixed proteins according to claim 1, characterized records that the multifunctional agent is preferred Glutaraldehyde with a final concentration up to 2.5% in Reaction approach is. 3. Trägerfixierte Proteine nach Anspruch 1 und 2, dadurch ge­ kennzeichnet, daß das Protein Cytochrom C ist.3. carrier-fixed proteins according to claim 1 and 2, characterized ge indicates that the protein is cytochrome C. 4. Trägerfixierte Proteine nach Anspruch 1 und 2, dadurch ge­ kennzeichnet, daß das Protein Hämoglobin ist.4. carrier-fixed proteins according to claim 1 and 2, characterized ge indicates that the protein is hemoglobin. 5. Trägerfixierte Proteine nach Anspruch 1 und 2, dadurch ge­ kennzeichnet, daß das Protein Ferritin ist.5. carrier-fixed proteins according to claim 1 and 2, characterized ge indicates that the protein is ferritin. 6. Trägerfixierte Proteine nach Anspruch 1 und 2, dadurch ge­ kennzeichnet, daß das Protein Lysozym ist.6. carrier-fixed proteins according to claim 1 and 2, characterized ge indicates that the protein is lysozyme. 7. Verfahren zur Herstellung trägerfixierter biologisch akti­ ver oder physiologisch wirksamer Proteine, dadurch gekenn­ zeichnet, daß aus einer wäßrigen Lösung, die ein Protein oder Proteingemisch enthält, ein Protein an unmodifizierte makroporöse Perlcellulose reversibel adsorptiv bei einem pH 6, einer Pufferkonzentration 0,1 M und einer Tempe­ ratur von 0-35°C gebunden wird und daß das reversibel adsorbierte Protein weiter durch
  • - ein multifunktionelles Agenz durch Quervernetzung an den Träger oder
  • - einen Einschluß in die Poren der Perlcellulose irrever­ sibel immobilisiert wird.
7. Process for the preparation of carrier-fixed biologically active or physiologically active proteins, characterized in that from an aqueous solution which contains a protein or protein mixture, a protein reversibly adsorptively at unmodified macroporous pearl cellulose at pH 6, a buffer concentration of 0.1M and a temperature of 0-35 ° C is bound and that the reversibly adsorbed protein continues through
  • - A multifunctional agent by cross-linking to the wearer or
  • - An inclusion in the pores of the pearl cellulose is irreversibly immobilized.
8. Verfahren zur Herstellung trägerfixierter biologisch akti­ ver oder physiologisch wirksamer Proteine, dadurch gekenn­ zeichnet, daß makroporöse Perlcellulose mit einem bifunk­ tionellen Agenz bis zu einer Endkonzentration von 2,5% inkubiert wird und nach Entfernen des Überstandes ein Protein unter Bedingungen seiner reversiblen Adsorption an die makroporöse Perlcellulose inkubiert wird.8. Process for the preparation of carrier-fixed biologically active substances ver or physiologically active proteins, characterized thereby records that macroporous pearl cellulose with a bifunk  tional agent up to a final concentration of 2.5% is incubated and after removing the supernatant Protein under the conditions of its reversible adsorption is incubated on the macroporous pearl cellulose. 9. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß als multifunktionelles Agenz Glutaraldehyd verwendet wird.9. The method according to claim 7, characterized in that used as a multifunctional agent glutaraldehyde becomes. 10. Verfahren nach Anspruch 7 und 9, dadurch gekennzeichnet, daß die Endkonzentration des multifunktionellen Agenz im Reaktionsansatz bis zu 2,5%, vorzugsweise 0,5-1,0%, beträgt.10. The method according to claim 7 and 9, characterized in that the final concentration of the multifunctional agent in Reaction batch up to 2.5%, preferably 0.5-1.0%, is. 11. Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß der Einschluß in die Poren der Perlcellulose durch eine irreversible Schrumpfung der Perlcellulose erfolgt.11. The method according to claim 7, characterized in that the inclusion in the pores of the pearl cellulose by a irreversible shrinkage of the pearl cellulose occurs. 12. Verfahren nach Anspruch 7 und 11, dadurch gekennzeichnet, daß die irreversible Schrumpfung der makroporösen Perl­ cellulose durch Abkühlen der wäßrigen Suspension auf Tem­ peraturen unter 0°C, Lagerung im gefrorenen Zustand zwi­ schen 12 und 24 Stunden und anschließendem Auftauen der Probe erfolgt.12. The method according to claim 7 and 11, characterized in that the irreversible shrinkage of the macroporous pearl cellulose by cooling the aqueous suspension to tem temperatures below 0 ° C, storage in the frozen state between between 12 and 24 hours and then thawing the Rehearsal is done. 13. Verfahren nach Anspruch 7 bis 12, dadurch gekennzeichnet, daß das Cytochrom C bei 0,01 M Puffer, pH 7-9 reversi­ bel adsorbiert wird.13. The method according to claim 7 to 12, characterized in that that the cytochrome C at 0.01 M buffer, pH 7-9 reversi is adsorbed. 14. Verfahren nach Anspruch 7 bis 12, dadurch gekennzeichnet, daß das Hämoglobin bei 0,01 M Puffer, pH 6-7 reversibel adsorbiert wird.14. The method according to claim 7 to 12, characterized in that the hemoglobin is reversible at 0.01 M buffer, pH 6-7 is adsorbed. 15. Verfahren nach Anspruch 7 bis 12, dadurch gekennzeichnet, daß das Ferritin bei 0,1 M Puffer, pH 7 reversibel adsor­ biert wird.15. The method according to claim 7 to 12, characterized in that the ferritin is reversibly adsorbed at 0.1 M buffer, pH 7 beers. 16. Verfahren nach Anspruch 7 bis 12, dadurch gekennzeichnet, daß das Lysozym bei 0,01 M Puffer, pH 9 reversibel adsor­ biert wird.16. The method according to claim 7 to 12, characterized in that the lysozyme is reversibly adsorbed at 0.01 M buffer, pH 9 beers.
DE19914141302 1991-12-14 1991-12-14 Protein irreversibly immobilised on macroporous cellulose@ beads - by reversible adsorption then crosslinking or physical inclusion, e.g. for conversion or sepn. processes Withdrawn DE4141302A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996030409A1 (en) * 1995-03-27 1996-10-03 Ústav Makromolekulární Chemie Akademie Ve^¿ C^¿Eské Republiky Method for immobilisation of proteins and polyelectrolytes on surfaces of solids
CN108486096A (en) * 2018-02-02 2018-09-04 东华大学 A kind of preparation method of the cellulose fixed lysozyme of magnetic base

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996030409A1 (en) * 1995-03-27 1996-10-03 Ústav Makromolekulární Chemie Akademie Ve^¿ C^¿Eské Republiky Method for immobilisation of proteins and polyelectrolytes on surfaces of solids
CN108486096A (en) * 2018-02-02 2018-09-04 东华大学 A kind of preparation method of the cellulose fixed lysozyme of magnetic base

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