EP1007201A1 - Materiaux pour chromatographie, procede de preparation et utilisation de ces materiaux - Google Patents

Materiaux pour chromatographie, procede de preparation et utilisation de ces materiaux

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Publication number
EP1007201A1
EP1007201A1 EP98928815A EP98928815A EP1007201A1 EP 1007201 A1 EP1007201 A1 EP 1007201A1 EP 98928815 A EP98928815 A EP 98928815A EP 98928815 A EP98928815 A EP 98928815A EP 1007201 A1 EP1007201 A1 EP 1007201A1
Authority
EP
European Patent Office
Prior art keywords
groups
base matrix
ion exchange
vinyl
gel
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.)
Withdrawn
Application number
EP98928815A
Other languages
German (de)
English (en)
Inventor
Dag LINDSTRÖM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Sweden AB
Original Assignee
Amersham Pharmacia Biotech AB
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Amersham Pharmacia Biotech AB filed Critical Amersham Pharmacia Biotech AB
Publication of EP1007201A1 publication Critical patent/EP1007201A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C08F261/00—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00
    • C08F261/02—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00 on to polymers of unsaturated alcohols
    • C08F261/04—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00 on to polymers of unsaturated alcohols on to polymers of vinyl alcohol
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/291—Gel sorbents
    • 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
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions 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
    • B01J20/3231—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
    • B01J20/327—Polymers obtained by reactions 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
    • B01J20/3231—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
    • 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
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • 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
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/20—Anion exchangers for chromatographic processes
    • 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
    • C08F263/00—Macromolecular compounds obtained by polymerising monomers on to polymers of esters of unsaturated alcohols with saturated acids as defined in group C08F18/00
    • C08F263/02—Macromolecular compounds obtained by polymerising monomers on to polymers of esters of unsaturated alcohols with saturated acids as defined in group C08F18/00 on to polymers of vinyl esters with monocarboxylic acids
    • C08F263/04—Macromolecular compounds obtained by polymerising monomers on to polymers of esters of unsaturated alcohols with saturated acids as defined in group C08F18/00 on to polymers of vinyl esters with monocarboxylic acids on to polymers of vinyl acetate
    • 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
    • C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
    • C08F290/08—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups on to polymers modified by introduction of unsaturated side groups
    • C08F290/10—Polymers provided for in subclass C08B

Definitions

  • the present invention relates to products which are useful in chromatographic separation processes based on ion exchange.
  • the products comprise a base matrix consisting of a copolymer of low molecular weight vinyl monomers and water soluble macromolecules carrying vinylic groups on the polymer chain, which base matrix is modified with ion exchange groups.
  • the invention also relates to a process for the preparation of the new products and to the use of the products in separation processes.
  • Chromatographic processes for separation based on reversible adsorption/binding of a target substance to ion exchange groups attached to a porous base matrix in particle form are well known and extensively used. Such processes and materials are used both for analytical and preparative work in different fields, for example within the biochemical field for separation of proteins, nucleic acids etc.
  • the base matrix material which most often is in the form of spherical particles, can be inorganic, for example of glass or silica, or based on different synthetic or natural organic polymers.
  • Chromatography materials for ion exchange based separation processes should fulfil several important requirements, such as a) high capacity, b) high recovery, c) rapid kinetics during adsorption and desorption (this gives the possibility of utilizing high flow rates and short beds/columns, i.e. short contact time) , and d) permit adsorption of target substances, such as proteins, at one ion strength and desorption at a higher ion strength or by change of pH possibly in combination with a lower ion strength.
  • the materials should of course also have a suitable porosity and be physically and chemically stable and it should be possible to produce them by a technically comparatively simple process and at a reasonable cost.
  • a chromatographic material modified in this manner is for example disclosed in an article by Janzen, R. et al in Journal of Chromatography, 522 (1990) 77-93, which i.a. relates to materials prepared by subjecting pretreated glass beads and silica to a three step procedure .
  • products which comprise a porous base matrix in particle form made up from a copolymer of a vinyl derivative of one or more polyhydroxy polymers and one or more low molecular weight vinyl compounds, which base matrix has ion exchange groups bound to flexible polymeric parts of the vinyl substituted polyhydroxy polymer used extending into the pore volume are extremely good ion exchange chromatography materials.
  • the products according to the invention can be produced by a technically and economically advantageous process, since the flexible polymeric structures (extenders) already are an integrated part of the base matrix. Thus no extra steps are required to introduce these parts of the product.
  • the present invention thus relates to novel products useful in chromatography processes as defined in the appended claims .
  • the “base matrix” of the products according to the invention is made up from “polyhydroxy polymers” carrying vinyl substituents which polymers have been copolymerised with at least one vinyl compound, such as a divinyl compound.
  • polyhydroxy polymers is herein intended synthetic and natural polymers carrying a plurality of hydroxy groups.
  • the polyhydroxy polymers are water soluble and before copolymerisation they usually have weight average molecular weights of from 3000 and up to about 10 000 000 Dalton.
  • synthetic such polymers can be mentioned polyvinyl alcohol and partially hydrolysed polyvinyl acetate and as examples of natural polymers can be mentioned polysaccharides such as starch and cellulose which have been modified to suitable water solubility and agarose and dextran. Dextran is the preferred polyhydroxy polymer. It is an important feature that the polyhydroxy polymer from which the base matrix is formed carries vinyl substituents.
  • substituents include vinyl, allyl, methallyl, 3-allyloxy-2-hydroxypropyl and 2- chloroallyl.
  • the most preferred vinyl substituents are allyl groups and 3-allyloxy-2-hydroxypropyl groups.
  • the degree of vinyl substitution is suitably from 0.05 to 2 mmol/g polyhydroxy polymer and preferably from 0.1 to 1.5 mmol/g polyhydroxy polymer.
  • the vinyl substituted polyhydroxy polymer is copolymerised with at least one vinyl compound such as a divinyl compound or a mixture of divinyl and monovinyl compounds to give a base matrix in the form of a porous, rigid gel.
  • the divinyl compound is of low molecular weight and as examples of suitable divinyl compounds can be mentioned N, N ' -methylene- bisacrylamide, divinyl ketone and divinyl sulphone . N,N'- methylene-bisacrylamide is especially preferred.
  • the molecular weight shall typically be below 2000 dalton more often below 1000 dalton and contain no polyhydroxy polymer structure.
  • the copolymerisation is carried out according to known free- radical polymerisation techniques under suitable conditions. Usually an amount of vinyl substituted poly-hydroxy polymer in the range of 20 to 80% by weight is copolymerised with from 20 to 80% by weight of divinyl compound and 0 to 40% by weight of monovinyl compound.
  • the rigid gel obtained by the copolymerisation may be disintegrated to particles of desired size for the intended use but it is, of course, preferred to carry out the copolymerisation in such a manner that substantially spherical particles, beads, are formed directly by the utilized copolymerisation process. Such bead polymerisation processes are well known to the man skilled in the art.
  • the base matrix of the products of the present invention is most preferably based on vinyl substituted dextran and in particular dextran with allyl substituents which has been copolymerised with N, N ' -methylene-bisacrylamide .
  • Base matrices of this type are commercially available under the trademark Sephacryl® (Pharmacia Biotech AB, Sweden) which products are designed for size exclusion chromatography and have excellent properties with regard to porosity, chemical and physical stability etc. These base matrices and processes for their preparation are disclosed in US-A- , 094 , 833, which is incorporated herein by reference.
  • Base matrices based on other vinyl substituted polyhydroxy polymers than dextran can for example be prepared as disclosed in US-A-4 , 094 , 833.
  • the base matrix is in particle form, preferably in the form of beads, and usually has an average particle diameter within the range of from 10 to 1000 ⁇ m, preferably within the range of from 20 to 700 ⁇ m.
  • base matrices prepared from a vinyl substituted polyhydroxy polymer and a divinyl compound as a comonomer will be built up as a porous, rigid, network structure which as integrated parts comprise flexible polymeric parts of the starting polyhydroxy polymer, which have not been copolymerised into the network of the base matrix but extend from the pore surfaces of the base matrix into the pore volume.
  • the flexible polymeric parts are thus in fact part of the base matrix, derived from the original starting polyhydroxy polymer and integrated with the rigid base matrix by being attached to this at one or more points and the flexible parts will be substantially non-cross-linked.
  • the described flexible polymeric parts which extend into the pore volume like arms or branches, are herein referred to as "extenders".
  • the present invention thus offers the possibility of an extremely simple preparation method for chromatography materials by direct bonding of ion exchange groups to existing extenders in a base matrix based on vinyl substituted polyhydroxy polymers copolymerised with a divinyl compound. Thus special steps such as surface activation and/or other coupling steps to attach extenders to a base matrix are avoided.
  • ion exchange groups as the term is used herein is intended ligands or groups which have the capacity of attracting and reversibly adsorbing target substances that carry a charge of the opposite sign as ion exchange group on the support, for instance various types of nucleic acids, proteins, acids, amines etc.
  • Typical ion exchange groups are: Positively charged groups, e.g. protonated forms of primary, secondary and tertiary amino groups, and quaternary amino groups (anion exchanging groups) , and negatively charged groups: e.g.
  • the ion exchange groups can be chemically bonded to the base matrix by methods known to the man skilled in the art for attaching this type of groups to matrices exhibiting hydroxy groups. Some of the methods are disclosed in the experimental part. So called linker arms are typically introduced between the ion exchange groups (charged groups) and the extender.
  • the ion exchange groups in the chromatographic/separation material of the present invention are of the same kind as those expressly discussed above.
  • preferred ion exchange groups can be mentioned the protonated forms of diethylamino and trimethylamino, carboxy and sulfo groups, all of which being linked to the gel by the appropriate linker arm.
  • Popular linker arms comprise alkane or hydroxy alkane structures typically being interrupted by ether linkages and suitably having from 1 to 20 carbon atoms with preference for 1 to 6 carbon atoms.
  • a suitable ion exchange group can be mention the protonated form of 2- [tris (hydroxymethyl) amino] - 1-hydroxyethyl and of 2- [tris (hydroxymethyl) amino] - .
  • the products of the invention can be either strong or weak ion exchangers .
  • novel products of the invention have ion exchange groups bonded to hydroxyl groups on the polyhydroxy polymer in the rigid matrix and especially on the extenders, which are parts of the polyhydroxy polymer of the base matrix. They may have a total degree of substitution with respect to ion exchange groups (or ion exchange capacity) in the interval from 10 to 500 with preference for 100 to 350 ⁇ mol/ml gel.
  • the products will of course have some ion exchange groups attached to hydroxyl groups in the rigid base matrix, both on the outer particle surfaces and the pore surfaces, but the ion exchange groups will predominantly be attached to the extenders.
  • the products of the present invention fulfil the requirements for chromatography materials mentioned earlier in this specification to a very high extent and in particular they show a very high dynamic binding capacity for target substances, such as proteins, that are prone to be adsorbed by matrices carrying positively and/or negatively charged groups.
  • the dynamic binding capacity can be said to be a measure of the obtainable productivity.
  • the fact that the groups are bonded to extenders give an extremely rapid adsorption or binding of target molecules.
  • the products of the present invention are advantageously and preferably prepared by direct introduction/bonding of ion exchange groups to existing extenders in a base matrix based on vinyl substituted polyhydroxy polymers copolymerised with a vinyl compound, such as a divinyl compound, to a degree of substitution of ion exchange groups in the range of from 10 to 500 ⁇ mol/ml gel. It might also be possible to produce the products of the invention by utilizing as starting material for copolymerisation with vinyl compounds a vinyl substituted polyhydroxy polymer exhibiting the ion exchange groups.
  • the chromatography products of the present invention can be used in the different modes of separation/chromatography processes, such as in packed beds, fluidized beds or stirred suspensions according to per se well known techniques.
  • the products of the invention can function for separation of compounds of various molecular weights and types. Some examples are macromolecules, e.g. with molecular weights of from about 5000 Dalton, such as polysaccharides, proteins/polypeptides and nucleic acids and synthetic water soluble polymers. Also substances with molecular weights lower than 5000 may be separated by use of the present products.
  • the products of the invention can for example be used in treatment of processed and unprocessed supernatants/cul- ture media from fermentors and other cell culture vessels, serum, plasma, beverages etc. Either the target substance which is adsorbed or the sample from which it is adsorbed is then further processed.
  • the products of the invention are further preferably used in treatment/separation of macromolecules, as above, and especially for adsorption/binding of proteins.
  • the base matrix beads from Example 1 were drained by suction on a glass filter, and 100.0 g of beads were added to a reaction vessel.
  • 4.0 g sodium hydroxide and 0.1 g sodium borohydride were stirred with 20 ml distilled water to a clear solution and charged to the reaction vessel.
  • 90 ml gly- cidyltrimethylammonium chloride (GMAC) was pumped into the reaction vessel in 2 hours. The temperature was kept at 25°C and the reaction continued during the night (18 hours). The product was neutralised with acetic acid and washed with distilled water.
  • the amount of ion exchanger groups was determined by the following method. 1 to 3 ml of the beads were sedimented in a PD-10 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and the exact volume was determined. The column was eluted with 10 ml of 0.5 M hydrochloric acid and washed with 1 mM hydrochloric acid. The gel was transferred to a titration cup with 10 ml of distilled water and 1 drop of concentrated nitric acid was added. Titration was finally done with 0,1 M silver nitrate (Mettler titrator) . The result was 169 ⁇ mol ClVml gel.
  • An ion exchanger was prepared in a manner analogous with Example 2 but with 20 ml of GMAC.
  • the ion capacity of the product was 72 ⁇ mol ClVml gel.
  • An ion exchanger was prepared in a manner analogous with Example 2 but with 40 ml of GMAC.
  • the ion capacity was 116 ⁇ mol ClVml gel.
  • An ion exchanger was prepared in a manner analogous with Example 2 but with 200 ml of GMAC.
  • the ion capacity was 220 ⁇ mol Cl ⁇ /ml gel.
  • the amount of sulfonate groups bound to the beads was determined by the following method. A sample consisting of 1
  • Example 7 Two-step synthesis of a sulphopropyl cation exchanger by bisulphite coupling to vinyl groups remaining after polymerisation and subsequent allylation and a second bisulphite coupling.
  • Example 6 In a manner analogous with Example 6 a bisulphite coupling to the base matrix (Ex. 1) was done. The content of sulfonate groups (S0 3 H) was 141 ⁇ mol/ml gel. Allylation of the resulting beads was done in the following way. 100.0 g beads drained by suction on a glass filter and containing 141 ⁇ mol S0 3 H/ml were charged to the reaction vessel. 4.0 g sodium hydroxide. 0.4 g sodium borohydride, 13.0 g sodium sulphate and 50 ml distilled water were stirred to a clear solution and added to the reaction vessel. The temperature was 35°C and finally 40 ml allylglycidylether was added during stirring.
  • the amount of allyl groups was determined by the following method. The volume of the sample was measured in a PD-10 column. The beads were transferred with 10 ml of distilled water to a 100 ml filter flask, and during stirring bromide water was added until a permanent yellow colour remained. Then vacuum was applied under stirring until the mixture was colourless. The sample was now transferred to a titration cup with 10 ml of distilled water and 1 drop of concentrated nitric acid was added. Titration was done with 0,1 M silver nitrate and the result was 151 ⁇ mol allyl groups per ml of gel .
  • Example 9 Synthesis of a diethylaminoethyl anion exchanger .
  • An anion exchanger was prepared in a manner analogous with Example 8 but with 20.0 g 2-diethylaminoethyl chloride hydrochloride and 25°C during the reaction.
  • the ion capacity was 75 ⁇ mol ClVml gel.
  • An anion exchanger was prepared in a manner analogous with Example 8 but with 20.0 g 2-diethylaminoethyl chloride hydrochloride and 35°C during the reaction.
  • the ion capacity was 121 ⁇ mol Cl /ml gel.
  • the chromatographic equipment used was FPLC® System equipped with the controller unit LCC-501 plus with the software FPLC director®, two P-500 pumps, one MV-7 and two MV-8 valves and the monitors Monitor UV-M and Conductivity Monitor.
  • Buffer A 50 mM tris, pH 8.0 (anion exchangers)
  • Buffer B 50 mM tris, 1 M NaCI, pH 8.0 (anion exchangers) 50 mM glycine, 0.5 M NaCI, pH 9.0 (cation exchangers)
  • the ion exchangers were packed with suction in HR 5/5 columns and washed with at least 10 volumes of buffer A (buf- fer without salt), with a flow rate of 1200 cm/hour. About 1 ml of gel was packed in a column.
  • Sepharose ® Fast Flow is a chromatographic support consisting of porous, spherical particles of highly cross-linked agarose (from Amersham
  • the flow rate in the test in which the dynamic binding capacity (Q B, ⁇ 0% ) was determined was 300 cm/h.
  • Q B/10% represents the amount of protein fed to the column, when A 280 in the eluate is 10 % of A 280 in the protein solution fed to the column.
  • a 280 stands for the absorbance of UV-light at the wave length of 280 mm.
  • test protein was dissolved in buffer A (0,2 %), and the ion exchanger was saturated with protein during 3 hours. Then the ion exchanger was washed with A-buffer during 1 hour and finally the test protein was eluted during 1 hour with buffer
  • Anion exchangers were tested with bovine serum albumin (BSA) and the buffer was 50 mM tris pH 8.0.
  • the albumin was eluted from the ion exchanger with 1 M sodium chloride in the buffer .
  • Cationic exchangers were tested with lysozyme and the buffer 50 mM glycine pH 9.0. Lysozyme was eluted from the ion exchanger with 0.5 M sodium chloride in the buffer.
  • Quaternary ammonium ion exchanger Quaternary ammonium ion exchanger.
  • SP ion exchangers were in the size of 40-160 ⁇ m.
  • the base matrix was sieved using 40 and 160 ⁇ m sieves.
  • the product was modified to a quaternary ion exchanger analogous to Example 2.
  • the gel was packed at a flow rate of 1 ml/min in an HR 10/30 column (Amersham Pharmacia Biotech AB, Sweden) .
  • the testing was done by increasing the flow rate through the column, keeping the flow rate constant for a fixed period of time at each step.
  • the pressure drop over the system was recorded using the built-in recorder in FPLC System.
  • the collapse point of Sepharose Fast Flow was reached at around 16 ml/min.
  • the collapse point for the ion exchanger prepared according to the invention had not been reached at flow rate of 20 ml/min.
  • a quaternary ion exchanger and a diethylaminoethyl anion exchanger prepared as described in Example 2 and in Example 8 respectively were in two separate sets of experiments used as substitutes for Q Sepharose ® Fast Flow in a process for purification of IgG from blood plasma.
  • the ion exchangers prepared according to the invention could be loaded with three times as much of protein as Q Sepharose ® Fast Flow with retained high purity of the final IgG product.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Graft Or Block Polymers (AREA)

Abstract

Cette invention concerne des produits comprenant une matrice de base qui se compose, d'une part, d'un copolymère à base de monomères de vinyle d'un faible poids moléculaire et, d'autre part, d'un polymère de polyhydroxy, tel que du dextran, comprenant des groupes vinyliques sur la chaîne du polymère. Cette matrice de base est en outre modifiée à l'aide de groupes d'échange ionique. Ces produits peuvent être obtenus par liaison directe des groupes d'échange ionique, et peuvent être utilisés lors de processus de séparation chromatographique qui se fondent sur des principes d'échange ionique.
EP98928815A 1997-06-24 1998-06-22 Materiaux pour chromatographie, procede de preparation et utilisation de ces materiaux Withdrawn EP1007201A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9702405A SE9702405D0 (sv) 1997-06-24 1997-06-24 Chromatography materials, a process for their preparation and use of the materials
SE9702405 1997-06-24
PCT/SE1998/001213 WO1998058732A1 (fr) 1997-06-24 1998-06-22 Materiaux pour chromatographie, procede de preparation et utilisation de ces materiaux

Publications (1)

Publication Number Publication Date
EP1007201A1 true EP1007201A1 (fr) 2000-06-14

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EP98928815A Withdrawn EP1007201A1 (fr) 1997-06-24 1998-06-22 Materiaux pour chromatographie, procede de preparation et utilisation de ces materiaux

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SE (1) SE9702405D0 (fr)
WO (1) WO1998058732A1 (fr)

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US7700743B2 (en) 2003-07-22 2010-04-20 Millipore Corporation Immobilised affinity medium and its use in separation
EP1808697A1 (fr) * 2006-01-13 2007-07-18 Novartis Vaccines and Diagnostics, Inc. Utilisation d'une matrice d'échange d'ion pour déterminer la concentration de particules d'un virus et/ou d'antigènes d'un virus

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Publication number Priority date Publication date Assignee Title
SE420838B (sv) * 1975-12-12 1981-11-02 Pharmacia Fine Chemicals Ab Dextranderivatgel i partikelform for separationsendamal
DE3211309A1 (de) * 1982-03-26 1983-09-29 Metin Dipl.-Ing. 6100 Darmstadt Colpan Chromatographisches verfahren zur isolierung von makromolekuelen
GB8516570D0 (en) * 1985-07-01 1985-08-07 Common Services Agency For The Coupling reaction
AU722196B2 (en) * 1995-08-30 2000-07-27 Genzyme Corporation Chromatographic purification of adenovirus and AAV

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Title
See references of WO9858732A1 *

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WO1998058732A1 (fr) 1998-12-30
SE9702405D0 (sv) 1997-06-24

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