CA2251050C - Process for the production of a porous cross-linked polysaccharide gel and its use as a gel filtration media and in chromatography - Google Patents

Abstract

A process for the production of a porous cross-linked polysaccharide gel and a gel obtainable by the following steps: a) preparing a solution or dispersion of the polysaccharide; b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a); c) reacting hydroxylgroups of the polysaccharide with the active site of the cross-linking agent;
d) forming a polysaccharide gel; e) activating the inactive site of the cross-linking agent; f) reacting the activated site from step e) with hydroxylgroups of the polysaccharide gel, whereby cross-linking of the gel takes place. The cross-linked polysaccharide gel obtained can further be cross-linked by conventional methods, one or several times.

Description

PROCESS FOR THE PRODUCTION OF A POROUS CROSS-LMKED POLYSACCHARIDE GEL

The present invention relates to a process for the pro-duction of a cross-linked polysaccharide gel with improved qualities and a gel obtainable by the process and use thereof. More precisely the invention refers to a new metrod of cross-linking, in which a bifunctional cross-linking agent is introduced into the polysaccharide solu-tion before emulsion and gel formation.
Chromatographic methods are commonly used for separa-tior_ and purification of molecules such as proteins, nu-clei~ acids, polysaccharides etc. A wide variety of separa-tion media is available, both inorganic material as well as synthetic polymers and polysaccharides.
Gel matrices of polysaccharides have long been used as separation media due to their good qualities and such ma-trices are now standard equipment in biochemistry laborato-ries. The polysaccharides are inert to biomolecules under a wide range of conditions. The polysaccharides are natural materials and as such are approved of by authorities (such as the Food and Drug Administration (FDA)in USA) for many fields of application. When using chromatographic separa-tion methods, there can be left traces of the separation medium left in the separated product. When polysaccharides are used, as separation medium, this is harmless, as the material is not toxic.
Generally, chromatographic separations are carried out in columns packed with the separation matrix in form of particulate beads. Separation media of a fast kinetics with rapid flow rates results in a high productivity and may be achieved by a reduction of the particle size. However, sma-~1 beads result in a higher back pressure due to the 3~ narrowing of the convective flow channels between the par-ticles in a packed bed. To be able to separate large mole-cules the particles should have large pores, but large pores may result= in a weakened structure of the particles. As th=

2 polysaccharides are soft materials the particles may easily collapse, especially at high flow rates. Thus, there is a demand on methods of manufacturing more stable polysaccha-ride particles. It is well known to increase the stability of polysaccharide particles by cross-linking the polymer.
The cross-linking process stabilises the polysaccharide gel matrices by chemically binding the polymer chains with each other at their respective free hydroxyl groups. The cross-linking takes place between the hydroxyl and the functional l0 groups of the cross-linkers. This affects the particle ri-gidity, but to a lesser extent or not at all the size of the pores. There are several patents and articles disclos-ing different cross-linking methods. Well known cross-linking agents are epichlorohydrin, bis-epoxides, divinyl sulphone.
In EP 203 049 it was found that the rigidity of the polysaccharides was considerably improved when the cross-linking agent used was monofunctional but also contained an additional masked functional group that could be activated later. The cross-linking was made in two steps. First the polysaccharide was derivatized with the monofunctional group. Then, in a next step the masked group was activated and made to react with the hydroxyl groups of the polysac-charide. In this manner the length of the cross-linking was controlled and the desired rigidity obtained.
The common characteristic for the state of the art methods is that the cross-linking is made on the polysac-charide polymer after the formation of the gel particles.
Thus, the cross-linking is made on the ready made struc-ture. Particles of e.g. agarose are prepared by dissolving the agarose in water by heating. The hot water solution is then emulsified to form spherical particles in an organic solvent such as toluene. The particles are precipitated af-ter cooling. The particles are then cross-linked. By vary-ing the concentration of the agarose solution, different pore sizes can be obtained. The lower the concentration of the agarose solution the larger pores are obtained.

3 The object of the present invention was to obtain an improved process for the production of a cross-linked poly-saccharide gel.
A further object of the invention was to produce rigid polysaccharide gel particles with improved capability to withstand high flow rates/back pressures, but with retained separation qualities.
The objects of the invention are achieved by the proc-ess and the polysaccharide gel as claimed in the claims.
l0 According to the invention a process for the production of a porous cross-linked polysaccharide gel is obtained, which process is characterized by the following steps:
a) preparing a solution or dispersion of the polysac-charide, b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dis-persion from step a), c) reacting hydroxyl groups of the polysaccharide with the active site of the cross-linking agent, d) forming a polysaccharide gel, e) activating the inactive site of the cross-linking agent, f)reacting the activated site from step e) with hy-droxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.
According to a further aspect of the invention a porous cross-linked polysaccharide gel is obtainable by the fol-lowing steps:
a) preparing a solution or dispersion of the polysac-charide, b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dis-persion from step a), - c) reacting hydroxyl groups of the polysaccharide with the active site of the cross-linking agent, d) forming a polysaccharide gel, e) activating the inactive site of the cross-linking agent,

4 f) reacting the activated site from step e) with hydroxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.
According to another aspect of the invention, there is provided use of porous, cross-linked polysaccharide gel as described herein as a gel filtration medium, in affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography chelate chromatography, covalent chromatography.
With the present invention it was surprisingly found that gels with increased pressure/flow capacities of more than 300% could be obtained, compared with known gels.
It was possible to manufacture highly rigid gel particles also with small particle diameters (about 10 Vim).
According to the new method of the invention the cross-linking agent, is introduced into the polysaccharide solution or dispersion before the gel formation. The cross-linking agent is a bifunctional agent with one active site and one inactive site. When added to the polysaccharide solution or dispersion the active site of the agent is allowed to react with the hydroxyl groups of the polysaccharide. Thereby the cross-linking agent is chemically bound to the polymer chains before the gel formation process is started. In this manner an internal cross-linking agent is introduced into the polysaccharide.
In the first step of the process a solution or dispersion of the polysaccharide is formed. Solvents or dispersing agents commonly used together with polysaccharides can be used as acetone, acetonitrile, dimethyl sulphoxide, dimethylformamide, pyridine, sec. and ' 30433-25 4a tert. alcohols, such as isopropanol, etc. However, according to a preferred embodiment of the invention an aqueous solution of the polysaccharide is used.
After the introduction of the cross-linking agent a gel is formed of the polysaccharide. If water has not been used as the solvent, the solvent or dispersing agent is then disposed of and the polysaccharide is dissolved in water. The gel is formed by emulsifying the water solution in an organic solvent such as toluene or heptane. Then, the inactive site of the cross-linking agent is activated and reacted with hydroxyl groups of the polysaccharide, whereby the gel is cross-linked.
The cross-linked gel can be further cross-linked by conventional methods as known by the state of the art.
This further cross-linking can be made one or several times de-pending on how rigid particles that are required. The con-ventional cross-linking can also be made on the gel from step d) before or at the same time as activating and react-

5 ing of the inactive site of the cross-linking agent in steps e) and f). In a further embodiment of the invention steps b) and c) can be repeated one or several times after steps d) or e) in order to add more cross-linking agent be-fore performing steps e) and f) or step f).
The bifunctional cross-linking agent used according to the invention comprises one active site and one inactive.
With active site is meant all groups capable of reaction with the hydroxyl groups of the polysaccharide. Examples of such groups are halides, epoxides, methylol groups. The in-active site is a group which does not react under the reac-tion conditions for the reactive site but can later on be activated to react with the hydroxyl groups of the polysac-charide. Groups containing double bonds such as allyl, vi-nyl, acryl groups are suitable. The group connecting the 2o active and inactive site is not essential, it should how-ever, lack binding activity and not be too long. Preferable cross-linking agents are allylglycidyl ether and allylhal-ides, preferably allylbromide, but it is also possible to use e.g. N-methylol acrylamide, vinyl benzylchloride, cin-namoyl chloride. The reactions between the hydroxyl groups and the active site and the activated inactive site, as well as the activation of this site, is well known chemis-try per see.
The reaction with the bifunctional cross-linking agent could be illustrated on agarose (AG) with the following re-action formulae:

6 Reaction with the active site of allylglycidyl ether:
NaOH
AG-OH + CHZ=CH-CHz-O-CH2-C~-~~ -~ AG-O-CHZ_~H-CHI-O-CHz-CH=CHZ
O OH
Activation and reaction of inactive site:
pH=7 AG-O-CHZ_CH-CHz-O-CH,-CH=CH2 + Br2/Hz0 -> AG-O-CHZ_ -CHZ-O-CHz C CH + HBr ~H ~H ~H l~r NaOH
AG-O-CHZ_CH-CHI-O-CHZ-CH-CHz -> AG-O-CHz.CH-CHz-O-CH2-CH-CHz + HBr bH dH ~r bH
LS
AG-O-CHz_CH-CHz-O-CHz-CH-CHZ+OH-AG-~ AG-O-CHz_CH-CHz-O-CHZ-CH-CH2-O-AG
OH ~ bH bH
The further cross-linking by conventional methods can be obtained by any of the known cross-linking agents. Suit-able compounds are one or several from the group of epiha-lohydrin, bis-epoxides, divinylsulphone, allylglycidyl ether and dibromopropan-1-ol. Thus, the conventional cross-linking can be made with the same cross-linker as in the internal first cross-linking step or with another cross-linker or with a mixture of cross-linkers.
The process according to the invention can be used on all type of polysaccharides such as agarose, agarose de-rivatives, starch derivatives, dextran or derivatives thereof, alginate, carrageenan. However, agarose is the preferred one.
The gel matrix according to the invention is preferably prepared as particles. The manufacture of the gel is made with well known methods. Agarose for example, is dissolved in water by heating the solution above the gel point for agarose. The cross-linking agent is added to the hot aque-ous agarose solution and the active site of the agent is allowed to react with the hydroxyl groups of the agarose.

7 The agarose solution is then emulsified in an organic sol-vent such as toluene. The gel particles are precipitated by cooling. Thereafter, the inactive site of the cross-linking agent is activated and reacted with hydroxyl groups of the agarose particles, whereby the gel is cross-linked.
The size of the particles is determined by the stirring speed when emulsifying the agarose solution. The final re-quired particle size is obtained by sieving. The pore sizes are regulated e.g. by varying the polysaccharide concentra-l0 tion. The process according to invention can be used to manufacture polysaccharide particles with conventional di-ameters and pore sizes. For the production of agarose par-ticles the concentration suitably is from 0.5 - 20 % by weight, preferably from 1 - 12 o by weight. The particle diameters can be from 1 mm - 1 Vim, preferably from 500 ~tm -1 Vim, most preferably 200 ~m - 1 Vim.
With the invention it is possible to produce highly rigid polysaccharide particles. The major parameter that influences the rigidity is the amount of added cross-linker, even if also the polysaccharide concentration has a significance for the rigidity and not only for the pore size as mentioned above. To obtain rigid particles the cross-linker concentration should preferably be within the range 30 - 80 ~mols/g gel, most preferably 45 - 60 ~mols/g gel. A concentration lower than 30 ~.mols/g tends to give gels with relatively low pressure/flow capacities. A con-centration above 80 ~.mols/g can result in gels which shrink to much to be acceptable.
The porous cross-linked polysaccharide gel according to the invention can be used as a gel filtration medium in which the molecules are separated according to differences in their size. They can also be used, after modification, in different types of affinity chromatography. The gel can be substituted with a lot of different groups in per see known manners. Among such groups can be mentioned:
1. Positively charged groups (primary, secondary, ter-tiary or quaternary amino groups),

8 PCT/SE97/00578 2. Negatively charged groups (e. g. carboxy, phosphonic acid, sulphonic acid etc.) 3. Amphoteric groups 4. Groups with specific affinity (e. g. such as between IgG-binding protein (protein A, G, L etc.) and IgG, lectin and carbohydrate, antigen/hapten and antibody, (strept)avidin and biotin, 5. Complementary nucleic acids/oligonucleotides, 6. Groups with pi-electron systems l0 7. Chelating groups 8. Hydrophobic groups.
With these groups the matrix can then be used in affin-ity chromatography, ion exchange chromatography, hydropho-bic interaction chromatography, reversed phase chromatogra-phy, covalent chromatography etc.
The invention will now be illustrated by the following examples which however are not intended to limit the inven-tion. With parts and percent are meant parts by weight and percent by weight if not stated explicitly.
Example 1:
Preparation of agarose solution An agarose solution is prepared in a batch reactor by add-ing 7g agarose to 100m1 distilled water under stirring for 2h at 95°C.
After 2h of reaction the solution is cooled to 70°C and lml NaOH 45o and 1,67m1 allylglycidyl ether (AGE) are added to the agarose slurry. The reaction continues for 2h under stirring at 70°C. The solution is then neutralised with 0,15m1 60% acetic acid and HC1 (pH=7-8).
Emulsion media The emulsion media is made in an emulsion reactor by adding 5,3g ethyl cellulose (N-50 emulsifier} to 117m1 toluene under stirring at 60°C (the dissolving of N-50 in toluene takes approximately 2h).
Transfer of the agarose solution to the emulsion reactor The agarose solution is transferred to the emulsion me-dia. The stirring is regulated to 130 rpm. Agarose gel par-

9 tic-.yes are thereby formed and their size can be controlled by variation of the rotation speed of the stirrer and the addition of extra N-50.
The desired maximal particle size is 150 ~.~m. If the gel particles are too large the rotation speed can be increased up to 220 rpm and extra N-50 can be added. The maximal par-ticle size is controlled by taking samples approximately every 10 min., which are analysed in a microscope with a built-in size graduation.
to Once the 150 ~m are reached, the solution is cooled down.
The cooling process The solution is cooled from 60°C to <25°C in approxi-mately 30 min.
Gel washing process The gel particles are washed under stirring with 11 ethanol 99,50 which is decanted. The gel is then washed on a nutsch with 4x11 ethanol 99,50 and 4x11 distilled water.
Activating of inactive site of allylglycidyl ether and cross-linking of the agarose = Cross-linking no.l Hromination lOg NaAc (sodium acetate) are added to a reactor con-taining a solution of 100m1 gel and 200m1 distilled water under stirring. After S min. bromine-water (Br2/H20) is added to the solution until a dark yellow colour is ob-tained and maintained for over 1 min. The reaction contin-ues for approximately 15 min. thereafter sodium formiate is added, giving the gel a white colour.
The gel is washed with 3x11 distilled water.
Cross-linking 5g Na2S04 are added to a reactor containing a solution of ~OOml brominated gel and 100m1 distilled water under stirring. After 15 min. lOml NaOH 45o and 0,3g NaBH4 are added to the solution. The reaction continues for 3h and then the temperature of the solution is increased to 40°C
and the reaction goes on for 16h.

The gel is washed with distilled water until the pH=7.
Further cross-linking with a conventional method =
Cross-linking no. 2 5 45,38 Na2S04 are added to a reactor containing a solu-tion of 100m1 AGE cross-linked gel and 33,3m1 distilled wa-ter (75o gel slurry) under stirring. The reaction tempera-ture is increased to 50°C and after 2h, 1,33m1 45a NaOH and 0,4g NaBH4 are added to the solution as well as 9,33m1 NaOH
l0 45% and lOml epichlorhydrin {ECH), which are added during a period of 6-8h. The reaction continues over night (ca.
16h). The gel is washed with distilled water and 60% acetic acid is added to obtain a pH=5-6.
The gel is then sieved to the desired particle size in-tervals (40-100 ~tm) .
Example 2:
In this example particles are prepared, which are cross-linked twice by conventional methods after the cross-linking according to the invention. Thus, particles pre-pared according to example 1 were cross-linked with Cross-linking no. 2 method:
1188 Na2S04 are added under stirring to a solution of 260m1 ECH cross-linked gel and 87m1 distilled water. The temperature is slowly increased to 50°C and after 2 hours 3,5m1 NaOH 45% and 0,358 NaBH4 are added, while 24m1 NaOH
45% and 26m1 ECH are slowly pumped (6-8 hours) into the so-lution. The reaction is kept for 16 hours and then the gel is washed with distilled water (and 0,6m1 acetic acid) un-til pH~5-6.
The gel is then sieved and tested.
Example 3:
As a comparable example agarose gel matrices allylated af-ter the emulsion process are prepared. A synthesis method similar to the synthesis method described in example 1 for allylation before emulsion is used. These products have the same allyl concentration as those produced with the newly developed technique. The alternative synthesis method used to produce these gels consists of the following steps:
Emulsion 28g agarose are added to 400m1 distilled water and heated to 95°C for 2 hours. Thereafter the solution is cooled to 70°C and transferred to a 60°C warm solution con-taining 470m1 toluene and 35g N-50 (emulsifier). After ap-proximately 45min. stirring an average particle size of 150 ~tm is obtained. The solution is cooled for about 30 min. to 22°C and washed with absolute ethanol (4x21) and distilled water (4x21) .
Allylation:
33,5g Na2S04 and 1,8g NaBH4 are added to a solution containing 355m1 agarose gel and 106,5m1 distilled water.
This solution is stirred for 5 min. at a temperature of 30°C and then 25m1 AGE and 71m1 NaOH 45o are slowly pumped (6-8 hours) to the solution. The stirring continues for 16 hours at the same temperature. The allylated gel is washed with distilled water (3x21).
Bromination:
36g sodium acetate is added to a solution of 360m1 al-lylated gel and 720m1 distilled water. After 5 min. stir-ring, 146m1 Br2/H20 is added to the solution and the reac-tion is run for 15 min. Then 0,5g sodium formate is added and the gel obtained a white colour. The gel is washed with 3x2 gel volume of distilled water.
Cross-linking no. 1:
18g Na2S04 is added to a solution of 360m1 brominated gel and 360m1 distilled water. The mixture is kept under stirring and after 15 min. 72m1 NaOH 45o is pumped (for 30 min.) into the solution together with 1,088 NaBH4. After 3 hours the temperature is increased to 40°C and the reaction is held for 16 hours. The gel is then washed with distilled water until pH is about 7.
Cross-linking no. 2:
156,48 Na2S04 is added to a solution containing 345m1 cross-linked gel and 115m1 distilled water. The solution is kept under stirring and slowly heated to 50°C. After 2 WO 97!38018 PCTlSE97/00578 hours 4,6m1 NaOH 45o and 0,468 NaBH4 are added to the mix-ture while 32,2m1 NaOH 45o and 34,5m1 ECH are pumped for 6-8 hours to the solution. The reaction continues for 16 hours and then the gel is washed with distilled water (and 0,6m1 acetic acid} until pH is about 5-6.
The gel is sieved and tested.
Example 4:
Extra allylation of an allylated agarose gel matrix after cross-linking no. 1 During the allylation process of the agarose solution, the bifunctional AGE molecule binds to the agarose polymer chains with its active site leaving its inactive site free.
The free site is first brominated and during cross-linking no. 1 it is epoxidized with NaOH, enabling the AGE molecule to bind to a second agarose polymer chain. The alternative synthesis method here aims at coupling more AGE to the polymer chains by repeating the allylation process one more time before starting the first cross-linking as it is ex-plained experimentally in the following steps:
Bromination:
35g sodium acetate is added to a solution of 350m1 al-lylated gel, prepared according to example 1, and 700m1 distilled water. The mixture is kept under stirring and af-ter 5 min. 160m1 Br2/H20 are added. The reaction continues for 15 min. and then 0,5g sodium formate is added. The gel colour changes to white and the gel is washed with dis-tilled water (3x11).
Allylation:
17,58 Na2S04, 0,5g NaBH4 and 35m1 NaOH 45o are added to a solution of 350m1 brominated gel and 175m1 H20. The mix-ture is held under stirring 40°C and after 1 hour 20m1 AGE
are added. The reaction continues for 16 hours after which the gel is washed with distilled water until pH is about 7.
Bromination:
38,5g sodium acetate are added to a stirred solution containing 385m1 gel allylated twice and 770m1 distilled water. After 5 min. 85m1 Br2/H20 are added and the reaction is kept for 15 min. and then 0,5g sodium formate are added givi.~.g the gel a white colour. The gel is then washed with distilled water (3x11).
Cross-linking no. 1:
19,259 Na2S04 are added to a stirred mixture containing 385m1 of the above mentioned brominated gel and 385m1 dis-tilled water. After 15 min. 38,5m1 NaOH 45o is pumped to the solution (pumping period = 30 min.) along with 1,169 NaBH4. The reaction continues for 45 min. before the tem-perature is increased to 40°C. The solution is kept under l0 stirring for 16 hours. The gel is then washed with dis-tilled water until pH is about 7.
Cross-linking no. 2:
174,49 Na2S04 are added to a stirred solution of 385m1 cross-linked gel and 128m1 distilled water. The mixture is slowly heated to 50°C and after 2 hours 5,13m1 NaOH 45% and 0,518 NaBH4 are added to the solution while 36m1 NaOH 45%
and 38,5m1 ECH are slowly pumped (6-8 hours)into the reac-tor. The reaction continues for 16 hours, and then the gel is washed with distilled water (and 0,6m1 acetic acid) un-til pH=5-6.
The gel is sieved and tested.
Examble 5:
Second cross-linking before the first cross-linking In this synthesis the ECH cross-linking is performed before cross-linking no. 1. The experimental procedure is explained in the following steps.
Cross-linking no. 2:
An allylated gel is produced according to the method described in example 1. 158,89 Na2S04 are added to a solu-tion containing 350m1 of this allylated gel and 117m1 dis-tilled water. The mixture is stirred and slowly heated to 50°C and after 2 hours 4,67m1 NaOH 45o and 0,679 NaBH4 are added to the solution while 32,7m1 NaOH 45% and 35m1 ECH
are slowly pumped (6-8 hours) into the reactor. The reac-tion is held for 16 hours and then the gel is washed with distilled water until pH is about 5-6.

WO 97!38018 PCT/SE97/00578 1.1 Bromination:
20,58 sodium acetate are added to a solution prepared from 205m1 ECH-cross-linked gel and 410m1 distilled water.
The mixture is stirred and after 5 min. 70m1 Br2/H20 are added and after 15 min. reaction time 0.5g sodium formate are added and the gel becomes white. The gel is then washed with distilled water (3x11).
Cross-linking no. l:
l0 10,25g Na2S04 are added to a solution containing 205m1 brominated gel and 205m1 distilled water. The mixture is stirred for 15 min. and then 20,5m1 NaOH 45% and 0,618 NaBHa_ are added. After 3 hours reaction time the tempera-ture is increased to 40°C and the solution is kept under stirring for another 16 hours. The gel is then washed with distilled water until pH is about 5-6.
The gel is sieved and tested.
The gels prepared in the examples were analysed with respect to ally! concentration, the pressure/flow capaci-ties, KaV values (KaV is an accepted definition of the relative pore size) and particle size distribution accord-ingly:
Ally! concentration analysis The ally! concentration was analysed on a Mettler DL40GP Memo Titrator with 0.1 M AgN03 according to standard methods.
The pressure/flow capacities analysis Instrument: XK-16/40 column HR 10/30 column FPLCdirectorT'~ system control unit (max. pres-sure/flow output:26-30 bar or 200-210 ml/min):
a Pharmacia Biotech Controller LCC-501 Plus and two Pharmacia Biotech Pump P-6000 and PC-unit with built-in interface.
Method: The testing of pressure/flow resistance of a gel matrix depends on the gel average bead size and the column packing process. The gel beads produced have two av-erage size intervals a) 8-12 ~.m b) 40-100 ~Cm.

1~
Packing:
a) The column packing process for gel beads with aver-age size 8-12 ~.m is made in a HR 10/30 column with a series of pressure/time variations in the same manner as for Kav tests (see next paragraph).
b) The column packing process for gel beads with aver-age size a0-100 /cm is a free sedimentation process made in a XK 16/40 column with a bed height of 31 ~ 1 cm.
Pressure/flow test:
a) Tests on the HR-10/30 column are made by increasing the flow 0,1 ml/min every 5 min and reading the back pres-sure variation every 5 min.
b) Tests on the XK-16/40 column are made by increasing the flow 1 or 5 ml/min every 5 min and reading the back pressure variation every 5 min.
The Kav-values determination analysis Instrument: Pharmacia UVM-detector Pharmacia Biotech FPLCdirectorT'~ system control 2 channel recorder (plotting unit) Method: The determination of the Kav-values of the gel matrix, results in an estimation of the pore size of the gel beads. The determination is made on the final product (the cross-linked gel). It is performed by graphic inter-pretation of the retardation time of several proteins, which have been injected into a column containing gel ma-trix.
Packing:
The final product is packed in a HR 10/30 column under a pressure of 17 bar. The packing solvent used is a solu-tion with the following composition:
60 g HAc + 1 g TweenT'~ 20 per 1000 ml To pack the column 30 g gel is dispersed in 30 g pack-ing solvent.
The gel matrix is first packed under a pressure of 6 bars for 50 min then under 17 bars under 10 min.
Protein injection and Kav determination:
The Kav-values are determined for four proteins:

~ Thyroglobulin (MW = 669000 g/mol) ~ Ferritin (MW = 440000 g/mol) ~ BSA (MW = 67000 g/mol) ~ R-nase (MW = 13700 g/mol) These proteins are injected into the column one or two at the time (to prevent overlaps to occur).
The eluent media used during this procedure is a buffer solution with pH = 7,2 and the following composition:
~ 50 mM Sodium dihydrophosphate (NaH2P04 x 2H20) ~ 150 mM Sodium chloride (NaCl) ~ 0,02 % Sodium azide (NaN3) To determine the Kav-values it is necessary to know the volume occupied by the void (volume around the agarose beads) which is done by injecting blue dextran into the column .
The obtained plots are interpreted and the data is ana-lysed with a PC which calculates the desired Kav-values.
The particle size distribution analysis:
The mean particle size distribution (d50v)was performed with a Coulter Multisizer.
The results from examples 1 - 5 are compared with a standard agarose gel (Sepharose~6FF) and presented in the following tables:
Table la:
Gel Allyl d50v Pressure/Flow test no. conc. Max Max Pressure flow Pres. In-crease [~Cmol/g [~,m] [cm/h] [bar] >1 bar* >1, 5 gel ] bar*

Seph.6FF - 91.1 1050 6.00 2.25 3.50 Example 53 74.9 3450 17.25 12.00 14.25 Example 53 74.9 4500 17.25 12.90 14.10 Example 45 71.4 1500 13.00 4.00 5.25 Example 46 77.2 >6000** 12.90 >12.90 >12.90 Example 46 83.7 5250 18.80 13.90 15.10 * Pressure for which the pressure increase is over 1 or 1.5 bar when the flow is increased by 150 cm/h every 5 min.
** ?6000 cm/h indicates the maximal flow the instrument is capable of delivering. The maximal f low capacity of the gel lies above this value.
Table 1b:
Gel Allyl Kav no. conc. Thyro Ferritin BSA R-vase [~mol/g gel ]

Seph.6FF - 0.37 0.48 0.64 0.81 Example 53 0.34 0.48 0.64 0.82 Example 53 0.33 0.48 0.64 0.80 Example 45 0.28 0.44 0.61 0.80 Example 46 0.28 0.43 0.56 0.70 Example 46 0.27 0.42 0.58 0.74 Example 6:
In this example the method according to the invention was used to prepare highly rigid agarose gel beads with 8.1 w/v o agarose. The process according to example 2 was re-peated but when preparing the agarose solution 8.1 g aga-rose per 100 ml water was used. The gel was sieved and two fractions with two different particle sizes were obtained, Example 6a and 6b. A further particle size, Example 6c was produced according the same manner. The gel beads were tested in the same way as mentioned above. The prepared 2o particles were compared with conventional particles with an agarose content of 8.1 w/v % (Superose~6 from Pharmacia).
The test results are put together in tables 2a and 2b.

Table 2a:
Gel Allyl d50v Pressure/Flow test no. conc. Max flow Max Pres [~.mol/g [um] [ml/min] [bar]
gel]

Superose - 13.2 0,9 15 Superose - 14.6 1,2 17 Example 6a 59 9.2 >_1.7_ >_26*

Example 6b 59 12.6 >_2.5 26*
>

Example 6c 57 11.2 >_2.5 >_26*

* 26 bar corresponds to the maximal pressure the instrument is capable of delivering. The maximal pressure and flow values obtained above are expected to be higher.
Table 2b:
Gel Allyl d50v Kav no. conc. Thyro Ferritin BSA R-vase [~mol/g [um]
gel]

Superose - 13.2 0.29 0.40 0.54 0.68 Superose - 14.6 0.36 - 0.60 -Example 6a 59 9.2 0.24 0.37 0.50 0.68 Example 6b 59 12.6 0.26 0.39 0.52 0.68 Example 6c 57 11.2 0.21 0.34 0.47 0.63 In the following examples gels with different agarose content were produced:
Example 7:
The method according to example 1 was repeated but with different agarose contents. The result is disclosed in ta-ble 3a and 3b below. Example 7a with 7 % agarose is identi-2o cal with example 1.

Example 8:
In this example different agarose contents were used and the method according to example 5 was used. The result is disclosed in the tables below.
Table 3a:
Gel Agarose d50v Pressure/Flow test (w/v Max Max Pressure %] In-flow Pres. crease [~cm] [cm/h] [bar] >1 bar* >1, S

bar*

Seph.4FF 4 98.6 480 1.8 -Seph.6FF 6 91.1 1050 6.0 2.25 3.5 Example 7a 7 74.9 3450 17.25 12.0 14.25 Example 7b 4 84.0 2250 9.00 5.45 6.60 Example 8a 7 78.4 >_6000** 19.90 >19.90 >19.90 Example 8b 5 61.7 3150 16.80 9.55 12.30 Example 8c 4 - 2700 9.50 6.25 7.40 Example 8d 3 87.5 1800 4.5 >4.5 >4.5 * Pressure for which the pressure increase is over 1 or l0 1.5 bar when the flow is increased by 150 cm/h every 5 min.
** ?6000 cm/h indicates the maximal flow the instrument is capable of delivering. The maximal flow capacity of the gel lies above this value.
15 Table 3b:
Gel Agarose 1 Kav (w/v ~] Thyro Ferritin BSA R-nase Seph.4FF 4 0.57 0.66 0.76 0.87 Seph.4FF 6 0.37 0.48 0.64 0.81 Example 7a 7 0.34 0.48 0.64 0.82 Example 7b 4 0.53 0.63 0.76 0.87 Example 8a 7 0.26 0.43 0.57 0.73 Example 8b 5 0.36 0.51 0.63 0.77 Example 8c 4 0.56 0.64 0.78 0.88 Example 8d 3 0.67 0.77 0.84 0.93 Conclusion:
From the tables it can be seen that the use of the new cross-linking method according to the invention results in 5 gels capable of withstanding more than three times higher flow than conventional gel particles or particles prepared according to known methods (Example 3), though the gels have similar Kay values.
The excellent behaviour of the gels of the invention l0 can also be illustrated as in figures 1 - 3. In the fig ures:
Figure 1 is a plot of the flow against the back pres-sure of examples 1 - 5 and comparable compound.
Figure 2 is a plot of the flow against the back of ex-15 amples 6a, 6b, 6c and comparable compound.
Figure 3a is a plot of the Kav values against the maxi-mal flow for examples 7a, 7b, 8a, 8b, 8c, 8d and comparable compounds.
Figure 3b is a plot of the flow against the back pres-to sure for the same examples as in figure 3a.
From the figures it is evident that for the state of the art particles the back pressure raises quickly when the flow increases above a moderate value, which indicates col-lapse of the particles if the flow is increased too much.
However, the pressure/flow plots for the gels according to the invention show a much lower inclination, indicating that the back pressure only raises slowly when the flow is increased.
In figure 3a the Kav values for the gel matrices ac-cording to examples 7a,b and 8a,b,c,d has been plotted against the maximal flow. It is readily seen from the dia-gram that the maximal tolerated flow is increased by 300 0 for matrices produced according to example 7 or example 8, idenpendently from the agarose w/v percentage. However, the agarose w/v percentage has an important impact on the gel beads pore size, which is expressed by the Kav values, as they increase with the reducing amount of agarose in the gel beads.

Claims (21)

CLAIMS:

1. A process for the production of a porous cross-linked polysaccharide gel, comprising the following steps:
a) preparing a solution or dispersion of the polysaccharide, b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a), c) reacting hydroxylgroups of the polysaccharide with the active site of the cross-linking agent, d) forming a polysaccharide gel, e) activating the inactive site of the cross-linking agent, f) reacting the activated site from step e) with hydroxylgroups of the polysaccharide gel, whereby cross-linking of the gel takes place.

2. The process according to claim 1, wherein the cross-linked polysaccharide gel obtained is further cross-linked, one or more times.

3. The process according to claim 1 or 2, wherein the gel from step d) is cross-linked before performing steps e) and f).

4. The process according to claim 1 or 2, wherein the gel from step d) is cross-linked at the same time as performing steps e) and f).

5. The process according to claim 1 or 2, wherein steps b) and c) are repeated one or more times after step d) before performing steps e) and f) or after step e) before performing step f).

6. The process according to any one of claims 1 to 5, wherein step a) comprises preparing an aqueous solution of the polysaccharide.

7. The process according to claim 6, wherein step d) comprises emulsifying the aqueous solution of the polysaccharide from step c) in an organic solvent to form particles.

8. The process according to any one of claims 1 to 7, wherein the bifunctional cross-linking agent is allylglycidyl ether or allylbromide.

9. The process according to any one of claims 2 to 5, wherein the further cross-linking is obtained by one or more compounds selected from epihalohydrin, bis-epoxides, divinylsulphon, allylglycidyl ether and dibromopropan-1-ol.

10. The process according to any one of claims 1 to 9, wherein the polysaccharide is agarose.

11. A porous, cross-linked polysaccharide gel obtained by the following steps:
a) preparing a solution or dispersion of the polysaccharide, b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a), c) reacting hydroxyl groups of the polysaccharide with the active site of the cross-linking agent, d) forming a polysaccharide gel, e) activating the inactive site of the cross-linking agent, f) reacting the activated site from step e) with hydroxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.

12. The polysaccharide gel according to claim 11, wherein the cross-linking polysaccharide gel obtained is further cross-linked, one or more times.

13. The polysaccharide gel according to claim 11 or 12, wherein the gel from step d) is cross-linked before performing steps e) and f).

14. The polysaccharide gel according to claim 11 or 12, wherein the gel from step d) is cross-linked at the same time as performing steps e) and f).

15. The polysaccharide gel according to claim 1.1 or 12, wherein steps b) and c) are repeated one or more times after step d) before performing steps e) and f) or after step e) before performing step f).

16. The polysaccharide gel according to any one of claims 11 to 15, wherein an aqueous solution is prepared of the polysaccharide in step a).

17. The polysaccharide gel according to claim 16, wherein the gel is formed by emulsifying the aqueous solution from step a) to form particles, in an organic solvent.

18. The polysaccharide gel according to any one of claims 11 to 17, wherein the bifunctional cross-linking agent is allylglycidyl ether or allylhalide.

19. The polysaccharide gel according to claim 12, wherein the cross-linked polysaccharide gel obtained is further cross-linked, by one or several compounds from any of epihalohydrin, bis-epoxides, divinylsulphon, allylglycidyl ether and dibromopropan-1-ol.

20. The polysaccharide gel according to any one of claims 11 to 19, wherein polysaccharide is agarose.

21. Use of porous, cross-linked polysaccharide gel according to any one of claims 11 to 20 as a gel filtration medium, in affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography chelate chromatography, covalent chromatography.