The invention relates to sorbents for ion-exchange chromatography which are particularly suitable for chromatographic separations of biological substances (for example proteins, peptides or nucleic acids, as well as viruses).
In the chromatographic separation of biological substances, in particular in preparative processes, the sorbents have to have a plurality of properties; these include firstly good resistance to alkaline solutions, as are usual for the cleaning of separation devices (cleaning in place: CIP), and adequate mechanical stability in order that high flow rates can be achieved, and also adequate hydrophilicity in order that irreversible binding of the substances to be purified to the sorbent is avoided. Furthermore, a high binding capacity is also necessary in order that the separation processes can be carried out economically. The binding capacity should also be essentially retained at a high flow rate, i.e. not only the static binding capacity, but also the dynamic binding capacity should be high. If the sorbents consist of derivatised, for example grafted materials, essential properties of the base material must not be impaired by the derivatisation.
EP 0 337 144 discloses sorbents for ion-exchange chromatography. According to the teaching of EP 0 337 144, derivatised acrylamide monomers are grafted onto diol-substituted base materials, such as (meth)acrylate-crosslinked polymers or silica gel derivatives. Although the ion exchangers obtainable in this way exhibit good separation properties and good pressure stability, they have, however, only limited stability to alkaline solutions.
The object thus arises to provide sorbents for ion-exchange chromatography having improved properties, in particular improved alkali stability, and a good dynamic binding capacity.
The invention relates to alkali-stable, hydrophilic sorbents for ion-exchange chromatography obtainable by the following process steps:
The invention relates to processes for the preparation of alkali-stable, hydrophilic sorbents for ion-exchange chromatography having the above-mentioned process steps, and to the use of the sorbents obtainable in this way for the separation of at least two substances.
It has been found that ion exchangers having considerably improved properties are obtainable if base materials having a content of diol groups of greater than 1 mmol per gram of base material (dry weight) are used and the reaction in the graft polymerisation is carried out in such a way that a degree of grafting of from 0.5 to 2 mmol per gram of base material (dry weight) is achieved. Suitable base materials are hydrophilic, macroporous, highly crosslinked polymer materials, in particular crosslinked vinyl polymers comprising diol group-containing vinyl monomers and crosslinking monomers whose polymerisable groups are linked by carboxamide group-containing chains. These contain more than 1 mmol of diol groups per gram of base material, in particular from 2 to 6 mmol of diol groups/g. Preference is given to the use of porous bead copolymers comprising N,N-methylenebisacrylamide and 3-allyloxy-1,2-propanediol (1:0.6) (manufacturer Degussa/Röhm, Darmstadt). The base material used can also be polymers having amidic or ethereal crosslinking and ethereally or amidically bonded aliphatic hydroxyl groups on the surface or groups which can be chemically converted into the latter. The important factor is the presence of hydroxyl groups, preferably in the form of aliphatic diol groups, in adequate concentration on the surface of the base material. In the ideal case, all functional radicals of the monofunctional monomer component of the polymeric base material should be represented by aliphatic diol groups. However, proportions of at least 50% of these groups in the form of diol groups are also suitable. Methods for the determination of diol groups are known to the person skilled in the art. Particularly suitable base materials are disclosed in EP 0 482 339.
The base materials used in accordance with the invention are, in particular, in the form of (macro)porous bead polymers; however, it is also possible to employ nonporous bead polymers or polymers in another form, for example porous cylinders or mouldings having a different shape, or also membranes, filaments or woven fabrics.
Monomers are grafted onto these base materials, with the graft polymerisation, as explained in greater detail below, being controlled in such a way that degrees of grafting of from 0.5 to 2 mmol per gram of base material are obtained. If the upper limit of the degree of grafting is significantly exceeded, the resultant sorbent frequently has impaired mechanical properties. In accordance with the invention, ion exchangers are obtained which have improved properties, in particular an improved dynamic binding capacity, and very good alkali stability.
In accordance with the invention, the grafting is initiated by means of cerium(IV) ions (G. Mino and S. Kaizerman (1958) J. Polymer Science 31, 242–243; G. Mino et al. (1959) J. Polymer Science 38, 393–401). Further details are disclosed in EP 0 337 144. The reaction with cerium(IV) salts in strongly acidic aqueous solution generates free radicals on the base material, with diol groups apparently reacting more quickly than aliphatic hydroxyl groups. A free-radical chain reaction commences on the free radicals formed in this reaction, with the added monomers being incorporated into the chain. This chain is thus linear and linked to a monomer unit containing the aliphatic radical of the base material. The polymerisation is ended by termination reactions with participation of the cerium salts. For this reason, the (average) chain length can be influenced by the concentration ratios of the base material, of the initiator and of the monomers. Details in this respect are described below. Furthermore, uniform monomers or alternatively mixtures of different monomers can be employed; in the latter case, grafted copolymers are formed.
The reaction with cerium(IV) salts is also possible if the solvents used are mixtures comprising organic solvents in addition to water, where these solvents are miscible with water and contain no hydroxyl groups. Particular preference is given here to dioxane or tetrahydrofuran. The proportion of the organic solvent in the reaction batch is typically 5–80% by volume, in particular 20–50% by volume. This process variant enables grafting with monomers which are only sparingly soluble in water.
In order to achieve the degree of grafting of from 0.5 to 2 mmol per gram of base material which is preferred in accordance with the invention, the process parameters of concentration of the base material (suspension density), monomer concentration, initiator concentration, pH, time, temperature and addition of polymerisation inhibitors are varied. For a given suspension density of the base material to be grafted and thus a defined reactive surface area and accessible number of functional groups which can be oxidised by the cerium(IV) salt, the degree of grafting can be set principally by variation of the following parameters in the sense of the graft product according to the invention:
The following monomers, in particular, are preferred for the graft polymers according to the invention: N-(2-diethylaminoethyl)acrylamide, N-(2-di-methylaminoethyl)acrylamide, N-(2-trimethylammoniumethyl)acrylamide hydrochloride, N-(3-trimethylammoniumpropyl)acrylamide hydrochloride, 2-acrylamido-2-methyl-1-propanesulfonic acid, acrylic acid, acrylonitrile, and also 2-acrylamidoethanesulfonic acid, acrylamide and N,N-dimethylacrylamide.
Besides acrylic acid and acrylamide, N-substituted acrylamides in general are basically suitable for the graft polymers according to the invention so long as the amide nitrogen does not carry any substituents which contain functional groups which can be oxidised by cerium(IV) (for example hydroxyl or carboxyl groups). Furthermore, these monomers should contain ionic groups or groups which can be converted into ionic groups.
Graft polymers which contain, for example, acrylonitrile or acrylamide can be converted into ion exchangers containing carboxyl groups by polymer-analogous hydrolysis.
However, it may also be appropriate to add comonomers which do not contain ionic groups in order to regulate the charge density. Besides acryl-amide, N-(2-methoxyethyl)acrylamide and N-(2-ethoxyethyl)acrylamide, for example, are suitable for this purpose.
The corresponding methacrylic acid derivatives are likewise suitable.
It has been found that amide bonds, in particular N-alkyl-amide bonds, are very alkali-stable.
The sorbents according to the invention have significantly increased dynamic binding capacities compared with the sorbents as disclosed in EP 0 337 144. This has been made possible since the correlation between binding capacity and degree of grafting which results in a pronounced optimum was not recognised in the prior art. Furthermore, the sorbents according to the invention have significantly improved alkali stability.
The sorbents according to the invention can advantageously be used instead of the sorbents known hitherto, for example instead of sorbents as disclosed in EP 337 144. Thus, the sorbents according to the invention can be used for the separation of biopolymers, such as peptides, proteins or nucleic acids, but also for the separation and purification of viruses, cell organelles, prokaryotic or eukaryotic cells, and protein complexes.
Even without further comments, it is assumed that a person skilled in the art will be able to utilise the above description in its broadest scope. The preferred embodiments and examples should therefore merely be regarded as descriptive disclosure which is absolutely not limiting in any way.
The complete disclosure content of all applications, patents and publications mentioned above and below as well as of the corresponding application DE 101 49 256.1, filed on May 10, 2001, is incorporated into this application by way of reference.
Unless otherwise stated, the weight data for base materials relate to the dry weight for the determination of which the material is washed five times with an excess of acetone and subsequently dried to constant weight at 50° C. under reduced pressure.
Room temperature (R.T.) means from 15 to 30° C.
a) Monomer Solution:
The monomer solution is obtained by acrylation of 127 g (1.09 mol) of N,N-diethylenediamine, dissolved in 1.2 liters of water, at 0–5° C. To this end, 95 g (1.05 mol) of acryloyl chloride are added to the stirred amine solution at such a rate that the indicated temperature range is maintained during the reaction. When the addition of the acryloyl chloride is complete, the mixture is allowed to react for a further 30 minutes, and the solution is then adjusted to pH 2.0 using 65% nitric acid.
b) Grafting:
A mixture of 1 kg (moist weight) of the base material (hydrophilic, macroporous, highly crosslinked polymer material, copolymer of N,N-methylenebisacrylamide and 3-allyloxy-1,2-propanediol (1:0.6); manufacturer: Degussa/Röhm, Darmstadt, Germany) is added to the freshly prepared monomer solution in a temperature-controllable stirred reactor. The mixture of base material and monomer solution is stirred up to give a suspension, and water is added so that a total volume of 3 liters is obtained. The suspension is subsequently heated to 42° C. and blanketed with nitrogen, and a solution of 16.44 g of ammonium cerium(IV) nitrate (30 mmol) in 60 ml of 0.5 M nitric acid is added with further stirring.
After 12–18 hours at 42° C., the suspension is filtered or centrifuged, and the gel which remains is washed a number of times and successively with water, a mixture of 0.2M ascorbic acid/0.5M sulfuric acid, then again with water and with 1M NaOH and again with water until all the soluble constituents of the reaction mixture have been removed from the gel down to the limits of the detection sensitivity of standard analytical methods.
The finished product is stored in suspension with salt solution containing 20% of ethanol or, for example, in 0.1M NaOH at R.T.
Analyses:
The binding capacity for BSA is about 500 mg/g (based on the dry weight of the gel).
The degree of grafting is about 1 mmol/g (in monomer units, based on the dry weight), measured by acid/base titration of the immobilised DEAE groups.
The cation exchanger containing SO3 groups is prepared analogously to the description of Preparation Example A1, using 2-acrylamido-2-methyl-1-propanesulfonic acid as monomer instead of the monomer mentioned therein.
The preparation is repeated with various variants, giving various preparations of exchangers with different degrees of grafting. The respective degree of grafting is determined by elemental analysis of the sulfur content.
The anion exchanger containing TMAE groups is prepared analogously to the description of Preparation Example A1, using N-(2-trimethylammoniumethyl)acrylamide hydrochloride as monomer instead of the monomer mentioned therein.
Ion-exchanger material prepared in accordance with Example A1 is introduced into a SuperFormance® glass column (150×16 mm; depth of the sorbent bed 125 mm) and equilibrated with the application buffer (50 mM TRIS buffer, pH 8.3). A solution of bovine serum albumin (5 mg/ml) in this buffer is applied continuously (linear flow: 100 cm/h), and the elution diagram is measured by photometry at 280 nm. The capacity (125 mg/ml of gel) is determined from the breakthrough curve (10%).
The experiment is repeated with higher flow rates. The results are shown in
Ion-exchanger material prepared in accordance with Example A2 is introduced into a SuperFormance® glass column (150×16 mm; depth of the sorbent bed 100 mm) and equilibrated with the application buffer (20 mM phosphate buffer, pH 7.0). A solution of lysozyme (5 mg/ml) in this buffer is applied continuously (linear flow: 100 cm/h), and the elution diagram is measured by photometry at 280 nm. The capacity (110 mg of lysozyme/ml of gel) is determined from the breakthrough curve (10%).
The experiment is repeated with various preparations (different degree of grafting). The results are shown in
DEAE ion exchanger prepared in accordance with Example A1 is stored in aqueous NaOH solution (1 or 5 mol/l) at room temperature. After one year, the binding capacity had dropped by 5% (in 1 mol/l of NaOH) or 10% (in 5 mol/l of NaOH).
Number | Date | Country | Kind |
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101 49 256 | Oct 2001 | DE | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP02/10072 | 9/9/2002 | WO | 00 | 4/2/2004 |
Publishing Document | Publishing Date | Country | Kind |
---|---|---|---|
WO03/031062 | 4/17/2003 | WO | A |
Number | Name | Date | Kind |
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5453186 | Muller et al. | Sep 1995 | A |
Number | Date | Country |
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43 16 136 | Nov 1994 | DE |
0 337 144 | Oct 1989 | EP |
0 482 339 | Apr 1992 | EP |
96 14151 | May 1996 | WO |
Number | Date | Country | |
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20040266896 A1 | Dec 2004 | US |