WO2003004536A2

WO2003004536A2 - Method for producing cross-linked polysaccharides and use thereof as stationary phases in separation processes

Info

Publication number: WO2003004536A2
Application number: PCT/EP2002/007236
Authority: WO
Inventors: Joachim Teller; Jutta Nagel; Michael Frank; Fritz Westphal
Original assignee: Micromod Partikeltechnologie Gmbh
Priority date: 2001-07-03
Filing date: 2002-07-01
Publication date: 2003-01-16
Also published as: WO2003004536A3; DE10132131A1

Abstract

The invention relates to a method for producing cross-linked particles which are used as stationary phases in separation processes. The inventive cross-linking strategy is based on a structured primary cross-linking with reactive nanoparticles and the use of a further molecular cross-linking agent. For areas of application which require inter alia a visualization or the magnetic mobility of cross-linked polysaccharide substances, corresponding coloured, fluorescent or magnetizable nanoparticles can be used as particular cross-linking agents. Said cross-linking is, however, of particular interest with regard to the production of spherical particles having a diameter of between 1 νm and 1 mm and the use of agarose as a matrix material.

Description

Process for the production of cross-linked polysaccharides and their use as stationary phases in separation processes

The invention relates to a process for the preparation of crosslinked polysaccharides which can be used in the form of spherical particles as stationary phases in separation processes.

Cross-linked polysaccharides in the form of spherical particles already have a number of established technical, biotechnological and biomedical applications. In particular, particles whose matrix consists of cellulose, agarose or dextran are considered bioinert and are therefore unmodified, activated or functionalized for various chromatographic processes for the separation of predominantly biological-chemical structures. Affinity chromatography has the largest share. Such methods are used to separate or purify peptides and proteins, antibodies, immunoglobulins and enzymes, biochemically relevant polysaccharides and lipids, steroids, nucleic acids and related structures as well as differently structured toxins. Biomedical applications of such particles are in the fields of dialysis or apheresis or are practiced in methods for extracorporeal detoxication. Furthermore, gel filtration, gel chromatography and ion exchange chromatography as well as related processes of desalination or decontamination of heavy metals or radionuclides are important technical fields of application for cross-linked polysaccharide particles.

Methods for stabilizing polysaccharide beads by crosslinking have been known for some time and have been constantly improved or refined, in particular in relation to the application. The subsequent crosslinking of preformed agarose beads with epichlorohydrin is based on GHETIE (US 3,507,851), while PORATH et al. (US 3 959 251) various bifunctional compounds were used as crosslinkers under reducing conditions. The subsequent crosslinking with two bifunctional compounds of different chain lengths in successive, separate chemical reactions is described by PERNEMALM et al. (US 4,665,164). A crosslinking strategy of LINDGREN (US Pat. No. 4,973,683), which is based on the polymerization of successively introduced activated alkene groups, preferably allyl groups, is comparable with this. Bead synthesis according to BERG (WO 9 738 018) also proceeds in several stages and optionally with two different crosslinkers, in which the potential crosslinker does exist before the actual particle formation However, the function of the crosslinker is only introduced through further chemical reactions after the actual bead synthesis.

The following crosslinkers have also been used for special applications: polyfunctional acylation reagents (PERRIER et al: US Pat. No. 6,132,750), hydrophilic polyalkylene glycol derivatives (WEISSLEDER et al .: US Pat. No. 5,514,379), organopolysiloxanes (YAMAZAKI et al .: US 5,658,849) and titanium or zirconium compounds (COTTRELL et al .: US 5,532,350).

The porosity of polysaccharide particles, in particular that of agarose beads, is essentially checked via the type of crosslinking or mediated by the chemical structure of the crosslinking agent used. LARSSON (US 5 723 601) claims a bead structure with diffusion and flow pores and HJERTEN et al. (US 5 135 050) a particle design with reduced porosity.

One possibility of at least partially compensating for the reduction in the number of OH groups (or generally functional groups) which is inevitably associated with the chemical crosslinking of polysaccharides is described by HJERTEN in US Pat. No. 4,591,640. The vinyl substituents of divinyl sulfone that are not fixed by crosslinking are deactivated by adding other mono- or polysaccharides. In addition to influencing the physicochemical behavior of polysaccharide particles through the crosslinking, processes have also become known which include the inclusion of further particulate materials in the polysaccharide matrix. In this sense, CALRSSON et al. (WO 9 218 237) polysaccharide beads with incorporated glass or silicon dioxide (quartz) particles. The common feature of all crosslinking strategies from the patents cited above is that, regardless of whether one or two crosslinking steps are carried out, the chemical crosslinking does not take place during particle formation, but in technologically subordinate process stages. The inclusion of further particulate materials in polysaccharide beads takes place during the bead formation, but is a chemically non-reactive process which consequently does not lead to the formation of covalent bonds between the material components. Since the time of a primary crosslinking has a significant influence on the pore structure of the polysaccharide particles and thus on their mechanical and flow properties, crosslinking or primary crosslinking should lead to advantageous physico-chemical properties of the polysaccharide particles thus generated even during particle formation. The present invention was therefore based on the object of making crosslinked polysaccharides accessible which are already structurally stabilized in the primary crosslinking. The method should be designed in such a way that it can be used in particular for spherical polysaccharide particles in the size range from d = 1 μm to d = 1 mm without restricting their reactivity and • their activatability or functionalization. Given the abundance of the available crosslinking agents, the activating and functionalizing reagents as well as other functionalities (e.g. fluorescence, magnetizability), particular importance should be attached to the extensive technological uniformity of the synthetic steps.

This object is achieved in that nanoparticles which have reactive chemical sequences on the surface are used as a structuring primary crosslinker and a further molecular crosslinker for crosslinking the polysaccharides. The type of crosslinking according to the invention can in principle be used for all types of polysaccharides, in particular that relates to dextran, chitosan, alginic acid, cellulose, starch and agarose, which can optionally be substituted or otherwise modified. However, this networking is of particular interest in connection with the production of spherical particles with diameters between 1 μm and 1 mm and the use of agarose as the matrix material.

The nanoparticles with chemically reactive surfaces used for crosslinking are not limited by the diameter, the particle matrix or their surface chemistry. It has proven to be advantageous to use nanoparticles with diameters between 1 and 800 nm. Both inorganic macromolecular structures, such as polysilicic acid or titanium dioxide, and natural or synthetic organic macromolecular substances, for example polyvinyl compounds, polyesters or amides or silicones, can be used in the matrix materials thereof. It is also possible to use composite materials for the matrix of the crosslinking nanoparticles. Appropriate colored, fluorescent or magnetizable nanoparticles can be used as particulate crosslinkers for special fields of application, which also require visualization or the magnetic mobility of the crosslinked polysaccharide materials. Suitable particle types, which may have to be subjected to a further chemical surface modification, are described by TELLER et al. (EP 1 036 763) or by GRÜTTNER et al. (J. Magn. & Magn. Mater. 194, 8 (1999)).

The nanoparticles, which act as particulate crosslinkers, have reactive sequences on the surface which can add or from the hydroxyl groups of the polysaccharide these are substituted. These are, for example, heterocumule sequences, such as isocyanates or isothiocyanates, activated alkene structures, for example with vinylsulfonyl residues or ring sequences from the field of small carbocycles or heterocycles, such as epoxides or aziridines. Substitutable sequences have haloalkyl groups, acyl halides or hetero-analogous structures derived therefrom, such as 4,6-dichloro-1,3,5-triazinyl units.

With regard to the regeneration of OH groups bound by crosslinking as ethers or esters, it is advantageous to use particulate crosslinkers which have reactive epoxide sequences, since new hydroxyl groups are generated by ring opening or hydrolysis.

The molecular crosslinkers used according to the invention are bifunctional or polyfunctional compounds which have chain lengths of 2 to 12 atoms between the binding sites. The bi- or polyfunctional compounds contain reactive sequences as have already been described for the particulate crosslinkers. It is not necessary, but has proven to be advantageous if the molecular crosslinkers have the same functional groups as the reactive nanoparticles. On the other hand, the structural analogy between the matrix of the particulate crosslinkers and sequences of the molecular crosslinkers can also have a favorable effect. This applies, for example, to the combination of polysilicic acid nanoparticles with epoxypropyl end groups on the surface with bis (epoxypropyloxy) silyl or bis or tris (epoxypropyloxy) disiloxanes as molecular crosslinkers, which are also structurally derived from silica. The crosslinked polysaccharides according to the invention can be further functionalized chemically by methods or synthetic methods known per se. This applies both to the introduction of reactive or affine groups, for example chloroacetyl, mercaptoacetyl, diethylaminoethyl or benzene boronic acid groups, the treatment by various activation methods, such as cyanogen bromide or active ester method (for example ONB carbonate activation) ) as well as the functionalization with, for example, antibodies or proteins.

The present method thus offers the possibility of producing crosslinked polysaccharides which contain both a particulate and a molecular crosslinker. Polysaccharides crosslinked in this way can be used in the form of particles (beads) with diameters in the range from 1 μm to 1 mm as stationary phases in separation processes. Such separation processes are used in chemistry and biochemistry, biotechnology and other life science technologies, as well as biomedicine. When used as stationary phases, the particles produced according to the invention can be used in all customary media, solvents and buffers. For example, for immunological or cell biological applications, the beads can be sterilized using conventional methods.

In some cases it has proven to be useful or necessary to provide such particles that function as stationary phases with further functional features. Depending on the separation task, these stationary phases can carry different reactive or affine groups on their surface or such structures are generated or converted during their use.

Furthermore, such stationary phases can be equipped with visualizable or mobile properties according to the separation task by using colored, fluorescent or magnetizable nanoparticles as crosslinkers in their manufacture. In the case of their visual properties, it is also possible to bind corresponding dyes or fluorescent dyes to the matrix of the stationary phases by known processes via addition or substitution reactions.

The invention is to be explained in more detail with reference to the following examples, without being restricted thereto.

example 1

lg reactive nanoparticles (d = 200nm, matrix: silicate, surface: terminal epoxy groups, product: sicastar® 43-08-202, micromod particle technology GmbH) together with 95g agarose (HSB-LV 2500, Bio Whittaker Molecular Applications APS) and 1900 ml of deionized water mixed with stirring at 200 rpm.

The reaction of the agarose with the crosslinking agent particles is carried out by stirring the reaction mixture for eight hours at a temperature of 50 ° C. and increasing the temperature to 80 ° C. for a further hour.

For the particle synthesis, 9.41 oil (peanut oil, Henry Laotte GmbH) thermostatted to 85 ° C. is introduced and the agarose suspension is metered into the preheated oil with stirring at 700 rpm using a thin jet.

The emulsion is then cooled to <40 ° C. and a suspension of agarose particles is formed with a maximum size distribution of around 100 μm. After the oil has been suctioned off, the crude product is washed with petroleum ether (101), with ethanol (91), with a 50% (v / v) ethanol / water mixture (51) and finally with deionized water (17, 51).

For crosslinking with the molecular crosslinker, 100 ml of crude agarose gel are first diluted with 100 ml of ultrapure water containing 0.665 g of sodium hydroxide and stirred at 50 rpm for 1 hour. After adding 0.25 g of sodium borohydride, stirring is continued for 1 hour and the water is then suctioned off. The gel particles are taken up in 100 ml of acetone, mixed with 10 g of a mixture of bis (glycidyloxy) propanol and tris (glycidyloxy) propane (Glycidether 100, Carl Roth GmbH & Co KG) and stirred for 2 hours at 150 rpm and room temperature. The reaction is continued for a further 8 hours at 40 ° C. After filtration, the particles are washed with acetone (100ml) and three times with ultrapure water (200ml each). The gel particles obtained in this way have the following properties:

Diameter: 100 μm

Acid-base resistance: pH 2-12

Temperature resistance:> 100 ° C

Linear flow rate at lbar 15ml column height: 180ml / min

Kd for M = 43,000 0.60

Coupling capacity with cibacron blue: 2.5 mg / ml gel

Examples 2 to 19

Examples 2 to 19 are carried out analogously to Example 1

DCT = 2,4-dichloro-1, 3,5-triazinyl groups) *** micromod particle technology GmbH A = 10g Glycidether 100, Carl Roth GmbH) * Abs .: 569nm, Em.:585 nm) **** Sigma Aldrich Chemie GmbH B = 10g 1,3-bis (3-glycidyloxypropyl) tetramethyldisiloxane) *) ** Abs .: 485nm, em .: 510nm C = 4g trichloroisocyanuric acid) ****

Claims

claims

1. A process for the production of crosslinked polysaccharides, characterized in that nanoparticles which have reactive chemical sequences on the surface and a molecular crosslinker are used as the crosslinker.

2. The method according to claim 1, characterized in that the crosslinked polysaccharides are spherical particles in the size range from 1 μm to 1 mm.

3. Verfaliren according to claims 1 and 2, characterized in that it is agarose in the crosslinked polysaccharides.

4. Process according to claims 1 to 3, characterized in that nanoparticles consisting of inorganic macromolecular substances, for example polysilicic acid or titanium dioxide, are used as crosslinking agents.

5. The method according to claims 1 to 3, characterized in that nanoparticles consisting of natural or synthetic organic macromolecular substances, for example polyvinyl compounds, polyesters or amides or silicones, are used as crosslinkers.

6. The method according to claims 1 to 3, germinated characterized in that nanoparticles consisting of composite materials are used as crosslinking agents.

7. The method according to claims 1 to 3, characterized in that colored, fluorescent or magnetizable nanoparticles are used as crosslinkers.

8. Verfaliren according to claims 1 to 7, characterized in that it is in the reactive chemical sequences on the surface of the nanoparticles to functional groups that add or are substituted by hydroxyl groups.

9. The method according to claims 1 to 8, characterized in that the reactive chemical sequences on the surface of the nanoparticles are epoxy groups.

10. The method according to claims 1 to 9, characterized in that the molecular crosslinkers are bi- or polyfunctional compounds which have chain lengths of 2 to 12 atoms between the binding sites.

11. The method according to claims 1 to 10, characterized in that the molecular crosslinkers have the same functional groups as the reactive nanoparticles.

12. The method according to claims 1 to 11, characterized in that the matrix of the reactive nanoparticles and the molecular crosslinkers are derived structurally from the silica.

13. Stationary phases in separation processes, characterized in that they are crosslinked polysaccharides in the size range from 1 μm to 1 mm, nanoparticles which have reactive chemical sequences and molecular crosslinkers being used as crosslinkers.

14. Stationary phases in separation processes according to claim 13, characterized in that chemically functionalized, colored, fluorescent or magnetizable nanoparticles are used as crosslinkers.

15. Stationary phases in separation processes according to claims 13 and 14, characterized in that the polysaccharide contains further functional groups or sequences.

16. Stationary phases in separation processes according to claims 13 to 15, characterized in that the polysaccharide is agarose.