EP1007201A1

EP1007201A1 - Chromatography materials, a process for their preparation and use of the materials

Info

Publication number: EP1007201A1
Application number: EP98928815A
Authority: EP
Inventors: Dag LINDSTRÖM
Original assignee: Amersham Pharmacia Biotech AB
Current assignee: Cytiva Sweden AB
Priority date: 1997-06-24
Filing date: 1998-06-22
Publication date: 2000-06-14
Also published as: SE9702405D0; WO1998058732A1

Abstract

Products comprising a base matrix consisting of a copolymer of low molecular weight vinyl monomers and a polyhydroxy polymer, for example dextran, carrying vinylic groups on the polymer chain, which base matrix is modified with ion exchange groups. The products, which can be prepared by direct bonding of ion exchange groups, are useful in chromatographic separation processes based on ion exchange principles.

Description

CHROMATOGRAPHY MATERIALS, A PROCESS FOR THEIR PREPARATION AND USE OF THE MATERIALS

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.

The above-mentioned chromatographic base matrices have been described in US-A-4 , 094 , 833 and one variant is sold under the name Sephacryl® (Amersham Pharmacia Biotech AB, Uppsala, Sweden). US-A- , 09 , 833 disclose that ion exchange groups can be linked to the part of the matrix derived from the lower molecular weight vinylic monomers. There are some publications disclosing that conventional affinity ligands linked to Sephacryl® via nucleophilic hydroxy groups of the base matrix have been used in affinity adsorptions based on specific interactions (e.g. Bisse et al, J. Chromatog. 575(2) (1992) 223-228; Bonde et al., Anal. Bioche . 200(1) (1992) 195-198; Algiman et al, J. Chromatog. 510 (1990) 165-175; Pepper, EP-A-209251) . 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. In several widely used commercial products for both standard chromatography, i.e. based on size exclusion principles, and chromatography based on ion exchange, natural, hydrophilic polymers, such as cellulose, agarose or dextran, are used as matrices, most often in cross-linked form to increase the physical stability.

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.

In comparison with chromatography materials having affinity groups including ion exchange groups bonded directly via simple ligand arms to a rigid base matrix, materials with increased capacity for proteins and other macromolecules have been developed through the binding of affinity groups to flexible polymeric structures ("extenders") which in turn are bonded to the surfaces of the pores of base matrix. The increase in capacity is believed primarily to depend on an increased or higher availability of the affinity groups for the target substance to be adsorbed than in conventional affinity supports. This increase in capacity can be obtained starting from a base matrix by binding the flexible extending polymeric structures to the base matrix and then binding the affinity structures to these extending parts. Further, at least one additional reaction step of activating the surface of the base matrix in order to bind the extending polymeric structures is usually required. 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 . According to the present invention it has been found that 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. They fulfil the above mentioned requirements and the products have a surprisingly high capacity for proteins. Further, 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. By "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. As examples of 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.

The vinyl substituent can in principle be any substituent containing the vinyl grouping CH₂=CR- in which R is hydrogen, methyl, halogen or cyano . Examples of 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. In order to be called low molecular weight vinyl compound, 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 μm, preferably within the range of from 20 to 700 μm.

It has been found that base matrices prepared from a vinyl substituted polyhydroxy polymer and a divinyl compound as a comonomer, e.g. as disclosed in US-A-4 , 094 , 833, 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.

By "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. carboxy groups (-COOVCOOH) , phosphonate/phosphonic acid groups (-P0₃ ^2"/-P0₃H^"/-P0₃H₂) , phosphate/phosphoric acid groups (-0-P0₃ ²V-0-P0₃H^"/-0-P0₃H₂) , sulphonate/sulphonic acid groups (-S0₂ ^~/-S0₂H; sulfo groups), (cation exchanging groups) . 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.

It is preferred that the ion exchange groups in the chromatographic/separation material of the present invention are of the same kind as those expressly discussed above. As examples of 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. As another example of 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 .

It is known to incorporate ion exchange groups in the base matrix material according to US-A-4 , 094 , 833 cited above. The only actual example shown in this reference is, however, a polymerisation of allyl substituted dextran with a divinyl compound and additionally a charged comonomer. In this case the charged comonomer will form part only of the rigid base matrix and not be attached to extenders and the high capacity observed when ion exchange groups are bound to extenders will not be obtained in this case.

The 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 μmol/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.

As mentioned above, 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 μmol/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.

It is particularly preferred to use the present products in separations based on packed bed chromatography with plug flow in order to take advantage of the excellent properties the products of the invention have. A specific advantage is here that a high capacity can be obtained with fairly large particles, which is advantageous with regard to flow rate. The products of the invention are further preferably used in treatment/separation of macromolecules, as above, and especially for adsorption/binding of proteins.

The invention is further illustrated in the following examples which, however, are not intended to limit the same. Parts and per cent relate to parts by weight and per cent by weight unless otherwise stated.

E X P E R I M E N T A L P A R T PART 1. Synthesis Example 1. Synthesis of a base matrix.

75 g of fine-grade chalk coated with stearate, 2.0 g anionic surfactant (organic phosphate ester) and 1000 ml heptane were charged in a round bottomed flask. The mixture was heated to 50°C.

35.0 g of allyldextran (Mw = 2,000,000 and degree of substitution 1.40 mmol allyl groups per g of dextran derivative) and 75.0 g of N, N ' -methylene-bisacrylamide were dissolved in a mixture of 250 ml of distilled water and 250 ml of methanol at 50°C. When a clear solution had been obtained, 0.5 g of ammonium persulphate was added. The water-methanol solution and heptane mixture were brought together and stirred for 10 minutes at 50°C in a nitrogen gas atmosphere. 0.36 ml of N, N, N ' , N ' -tetramethylethylenediamine was added and the reaction was continued for 2 hours at 50°C. Insoluble gel beads having a size of 40-300 μm were obtained. The product was washed with acetic acid, ethanol and distilled water. Subsequent to sedimentation, the yield was measured to 810 ml.

Example 2. Synthesis of a quaternary ammonium exchanger.

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 μmol ClVml gel.

5 Example 3. Synthesis of a quaternary ammonium exchanger.

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 μmol ClVml gel.

10 Example 4. Synthesis of a quaternary ammonium exchanger .

An ion exchanger was prepared in a manner analogous with Example 2 but with 40 ml of GMAC. The ion capacity was 116 μmol ClVml gel.

15 Example 5. Synthesis of a quaternary ammonium exchanger.

An ion exchanger was prepared in a manner analogous with Example 2 but with 200 ml of GMAC. The ion capacity was 220 μmol Cl^~/ml gel.

20 Example 6. Synthesis of a sulphopropyl cation exchanger .

100.0 g base matrix (Ex. 1) beads drained by suction on a glass filter were mixed with 26 ml distilled water and 15.0 g sodium bisulphite. Aqueous 45% sodium hydroxide was added during stirring until the pH was 6.8. The temperature was

25 25°C and air was bubbled into the reaction vessel through a capillary. The reaction continued during stirring overnight at 25°C. The product was washed with distilled water.

The amount of sulfonate groups bound to the beads was determined by the following method. A sample consisting of 1

30 to 3 ml of the beads was sedimented in a PD-10 column (Amersham Pharmacia Biotech AB, Sweden) and the exact volume of the beads was determined by measuring the height and diameter of the sediment in the column. The column was eluted with 10 ml of 0.5 M hydrochloric acid and washed with 1 mM hydrochloric acid. The beads were transferred to a titration cup with 10 ml of distilled water and titrated with 0.1 M sodium hydroxide (Mettler titrator) . The observed consumption of sodium hydroxide corresponds to the presence of 134 μmol S0₃ groups per ml gel.

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.

In a manner analogous with Example 6 a bisulphite coupling to the base matrix (Ex. 1) was done. The content of sulfonate groups (S0₃H) was 141 μmol/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 μmol S0₃H/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 reaction continued overnight and the product was neutralised with acetic acid and washed with distilled water. 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 μmol allyl groups per ml of gel .

Bisulphite coupling of the allylated beads above was done in a manner analogous with Example 6, and the content of S0₃H was 249 μmol/ml gel, determined as in example 6.

Example 8. Synthesis of a diethylaminoethyl anion exchanger.

21.0 g sodium sulphate, 6.0 g sodium hydroxide and 0.2 g sodium borohydride were stirred with 60 ml of water in a reaction vessel to a clear solution and 100.0 g of beads, drained by suction on a glass filter, of base matrix (Ex. 1) was added. 50.0 g of 2-diethylaminoethyl chloride hydrochloride (65 % solution ) was added and the mixture was stirred at 35°C. After one hour the pH was 6.2 and 6.0 g sodium hydroxide were charged. After 15 minutes the pH was 12.4 and additionally 2.0 g sodium hydroxide was added causing the pH to rise to 12.6. The reaction mixture was stirred overnight. The product was neutralised with acetic acid and washed with distilled water. The amount of ion exchanger groups was determined in a manner analogous with Example 2. The result was an ion capacity of 267 μmol ClVml 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 μmol ClVml gel.

Example 10. 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 35°C during the reaction. The ion capacity was 121 μmol Cl /ml gel.

PART 2. DETERMINATION OF THE DYNAMIC PROTEIN BINDING CAPACITIES, TOTAL PROTEIN CAPACITIES AND PROTEIN RECOVERIES OF THE ION EXCHANGERS.

Instruments .

All the instruments used were manufactured by Amersham Pharmacia Biotech AB, Sweden. 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.

Buffers

Buffer A 50 mM tris, pH 8.0 (anion exchangers)

50 mM glycine, pH 9.0 (cation 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)

Packing of the column.

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.

Comparison between ion exchangers .

One of the most used media in ion exchange chromatography of proteins is Sepharose^® Fast Flow. Sepharose^® Fast Flow is a chromatographic support consisting of porous, spherical particles of highly cross-linked agarose (from Amersham

Pharmacia Biotech AB, Sweden) . A comparison between the present ion exchangers and Sepharose^® Fast Flow ion exchangers was made as follows.

The flow rate in the test in which the dynamic binding capacity (Q_B,ι0%) was determined was 300 cm/h. Q_B/10% represents the amount of protein fed to the column, when A₂₈₀ in the eluate is 10 % of A₂₈₀ in the protein solution fed to the column. A₂₈₀ stands for the absorbance of UV-light at the wave length of 280 mm.

The 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

B.

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.

RESULTS .

Quaternary ammonium ion exchanger.

Ex . no Ion exchange Bead size Total BSA Recovery capacity (μm) (albumin/ capacity of protein

(μmol Cl^'/ml ge 1) ml gel) (mg/ml gel) ⁽%⁾

3 72 160-315 81 91 86.9

4 116 160-315 131 150 93.8

2 169 160-315 159 189 96.1

5 220 160-315 153 185 102

Q Sepharose^''

Fast Flow 180-250 40-160 26 90.2

The dynamic binding capacity Q_B_ ₁₀ of the ion exchanger of Example 2 is higher by a factor 6 ( = 159/26 ) than that of Q Sepharose^® Fast Flow, despite the difference in particle size .

Sulphopropyl ion exchanger.

Ex . no Titration with Q_B_ ₁₀. Total lysozyme Recovery

NaOH ( μmol lysozyme capacity (mg/ of protein OH/ml gel ) (mg/ml gel ) ml gel ) ( % )^'

6 134 182 183 98 . 7

7 249 167 230 103

SP Sepharose

Fast Flow 180-250 110 149 93 . 7

All The SP ion exchangers were in the size of 40-160 μm. The increase of Q_B;10% with the ion exchanger according to the invention was 167/110=1.5 as compared to SP Sepharose^® Fast Flow. Diethylaminoethyl ion exchanger

Ex . no Ion exchange ^B, 10. Total BSA Recovery capacity (μmol (albumin/ml capacity ( g/ of protein

ClVml gel) gel) ml gel) (%)

267 120 139 71.5

9 75 67 79 74.0

10 121 96 136 86.0 DEAE Sepharose¹

Fast Flow 110-160 39 94 86.7

All the DEAE ion exchangers were in the size of 40-160 μm. The increase of Q_Brl0% with the ion exchanger according to the invention in Example 8 was 120/39=3.1.

PART 3. Rigidity of the base matrix .

A comparison was made between the rigidity of Sepharose® Fast Flow and a quaternary ion exchanger prepared according to the invention. A fraction of a base matrix prepared in a manner analogous to Example 1 but with a smaller particle size was used as starting material. The base matrix was sieved using 40 and 160 μm 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.

For comparison the same experiment was done with Q Sepharose^® Fast Flow. The particle size is similar to that of the 40-160 μm fraction of the ion exchanger prepared according to the invention. The result was that the Q ion exchanger according to the invention can withstand higher flow rates than Sepharose® Fast Flow.

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.

PART 4. Chromatographic experiment

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.

In both cases 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.

Claims

1. A product suitable for use in chromatography processes comprising a porous, rigid base matrix gel in particle form, characterized in that the base matrix is made up from a vinyl substituted, water soluble polyhydroxy polymer which has been copolymerised with a low molecular weight vinyl compound, such as a divinyl compound, said base matrix having flexible polymeric parts of the polyhydroxy polymer attached to the base matrix and integrated with this, which flexible polymeric parts (extenders) extend into the pore volume and in that the hydroxy groups of the polymer are substituted with ion exchange groups to a total degree of substitution of from 10 to 500 ╬╝mol/ml gel.

2. A product according to claim 1, characterized in that the polyhydroxy polymer is selected from the group consisting of polyvinyl alcohol, partially hydrolysed polyvinyl acetate, water soluble modified starch or cellulose, agarose and dextran.

3. A product according to claim 2, characterized in that the polyhydroxy polymer is dextran.

. A product according to any of claims 1 to 3, characterized in that the vinyl substituent on the polyhydroxy polymer is an allyl group or a 3-allyloxy-2-hydroxypropyl group .

5. A product according to claim 1, characterized in that the vinyl compound is a divinyl compound which is N,N'- methylene-bisacrylamide .

6. A product according to anyone of claims 1-5, characterized in that the ion exchange groups are diethylamino, trimethylamino, carboxy or sulfo groups linked to the gel via alkane or hydroxy alkane groups, optionally containing ether linkages.

7. A product according to claim 1, characterized in that the base matrix is made up from a vinyl substituted dextran which has been copolymerised with a divinyl compound, said base matrix having extenders of the vinyl substituted dextran and in that the hydroxy groups of the dextran are substituted with ion exchange groups to a total degree of substitution of from 10 to 500 ╬╝mol/ml gel and in that the product is in the form of beads having an average diameter of from 10 to 1000 ╬╝m.

8. A product according to claim 7, characterized in that the vinyl substituent is an allyl group or a 3-allyloxy-2- hydroxypropyl group.

9. A product according to any one of claims 7-8, characterized in that the ion exchange groups are amino groups, such as diethylamino or trimethylamino; or carboxy; or sulfo groups linked to the gel via alkane or hydroxy alkane groups, optionally containing ether linkages .

10. A process for preparing a product as defined in any of claims 1 to 10, characterized in that a base matrix made up from a vinyl substituted, water soluble polyhydroxy polymer which has been copolymerised with a vinyl compound, such as a divinyl compound, said base matrix having flexible polymeric parts of the polyhydroxy polymer attached to the base matrix and integrated with this, which flexible polymeric parts (extenders) extend into the pore volume, is reacted with a compound introducing ion exchange groups to a total degree of 5 substitution in the interval from 10 to 500 ╬╝mol/ml gel.

11. A process for preparing a chromatography product, characterized in that a base matrix made up from vinyl substituted, preferably allyl or 3-allyloxy-2-

10 hydroxypropyl substituted, dextran which has been copolymerised with a divinyl compound, preferably N,N'- methylene-bisacrylamide, said base matrix having flexible polymeric parts of the dextran attached to the base matrix and integrated with this, which flexible polymeric

15 parts (extenders) extend into the pore volume, said base matrix with extenders having been prepared by copolymerisation of vinyl substituted, preferably allyl substituted, dextran and a divinyl compound, preferably N, N ' -methylene-bisacrylamide, is reacted with a compound

20 introducing ion exchange groups to a total degree of substitution in the interval from 10 to 500 ╬╝mol/ml gel.

12. A process according to anyone of claims 10-11 characterized in that the ion exchange groups are are

25 amino groups, such as diethylamino or trimethylamino; carboxy; or sulfo groups linked to the gel via alkane or hydroxy alkane groups, optionally containing ether linkages .

30 13. Use based on ion exchange principles of a product as defined in any of claims 1-9, as chromatographic separation material.

14. Use according to claim 13, for separation of macromolecules .

15. Use according to claim 14, for separation of proteins.