DE4234691A1 - Chromatographic support media with polymer coating - comprises porous solid coated with post reacted vinyl] chloroformate functional copolymer thin film for protein sepn. or purificn. - Google Patents

Abstract

The materials comprise a porous solid coated with a thin film of a homo- or copolymer having functional gps. of formula -O-CO-X, X = halogen or a cleavable gp., e.g. derived from N-hydroxy succinimide (NHS), a sulphonic acid eg. aminomethane sulphonic acid, an activated phenol or 4-aminobenzamidine. The polymer is pref. a copolymer of vinyl chloroformate (VCF) and a vinylic comonomer, esp. vinyl pyridine (VP), vinyl imidazole, N-vinyl pyrrolidone, N-vinyl morpholine, (dimethyl)acrylamide (DMA), N-vinyl methylacetamide or a 1-10C alkyl (meth)acrylate. The solid is dried, impregnated with a soln. of the polymer, washed with a solvent, and opt. reacted with NHS, a sulphonic acid or an activated phenol. USE - Used to separate or purify proteins or other biological substances.

Description

The invention relates to usable in the chromatography Carrier. The carrier comprises a porous solid, such as z. B. a mineral oxide on which a thin coating (film) is deposited from a polymer or copolymer, the Halogenoformate functions or derived functions carries, which consist of detachable groups, like one Succinimido, phenol, imidazole or thiosulfate residue.  

The invention further relates to the use of this Trä gers for the preparation of chromatography columns for the Separation or purification of proteins.

The present invention relates to a novel carrier for the preparation of columns, which is the separation of biological substances or their purification from them allow containing mixture consisting of a poly mers or copolymers which are on a porous solid, such as As a mineral oxide is deposited, a Verfah for its manufacture and its use in the Chromatography.

It is known that it is possible to different polymers on different which deposit mineral carriers for the production of columns for the separation of different substances. The deposition of synthetic polymers on silicon dioxide, alumina or titanium oxide has already been used in ver various publications. The use for it The neutral polymers are, for example, polyethylene glycol, polyvinylpyrrolidone, silicones, polyvinylimidazole, as described in FR-PS 25 98 512, or polydimethyl acrylamide, as described in FR-PS 26 31 849. In US PS 45 17 241 an inert carrier is described, on whose Surface of a polysuccinimide is fixed, which one che to modify it to use in the mix Liquid phase chromatography for the separation of Protei to adapt.

In EP 02 44 802 a modified silica be wrote the substituent with a hydroxyl group and having active derivatives of such substituents, by Exposure to a compound such as a chloroformi ats, a carboimidazole, a cyanobromide or a Sulfonyl chloride, has been modified. The production is an ethoxytrimethoxysilane on the silicon to fix dioxide, to heat it in an acidic environment  to prepare a diol which is converted to an aldehyde which then converts to a primary hydroxyl redu is graced.

In the patent EP 03 37 144 is a carrier for the separation of substances, such as proteins, described by one covalent bond carries fixed polymer and in the chro matography is usable.

In US-PS 43 32 694 is a porous carrier, such as. B. Silica, described on which one epoxy groups pointing polymer is adsorbed.

The present invention relates to a reactive Support intended for the manufacture of columns for the separation or purification of biological molecules and in particular of proteins by reducing their irreversible adsorption, resulting in improved separation assets, based on the usual products of the state the technology leads. The carrier according to the invention has a porous solid, such as. B. a mineral oxide, on a thin coating (film) of a polymer or Copolymer is deposited. This thin coating (film) may be adsorbed on the surface of the carrier or he may be fixed in a covalent manner. In addition, you can Crosslinking reactions with the reactive functions of the polymer or copolymer.

The invention also relates to a method for manufacturing a support of the type described above, the is determined for the separation and cleaning verschie dener substances, and its use in the chromato graphie for the separation and purification of proteins.

The carrier according to the invention is characterized by that the polymer or copolymer haloformate functions or derived functions that consist of  cleavable groups, such as the carbonate of N-hydro xysuccinimide or phenol.

The polymers or copolymers used according to the invention are preferably of one type, the recurring one contains the general formula:

in the X is a halogen atom such as chlorine, or a cleavable Group which is selected from a succinimide, Phenol, imidazole or thiosulfate radical and n for one integer between 10 and 10 000, preferably between 1000 and 5000, stands.

The term "cleavable group" used here stands for groups that follow the classic rules in one can split off (separate) nucleophilic reaction.

The polymer thus obtained is, for example, a vinylpoly chloroformate, a sodium polyvinyloxycarbonylthiosulfate or an N-hydroxysuccinimide polyvinyl carbonate.

The copolymerizable monomer may be a derivative having ethy lenischer unsaturation of the formula

CH₂ = CR₁R₂

in the mean
R _{1 is} a hydrogen atom or a methyl group and
R _{2 is} a hydrophilic group which carries one or more ester, amide, carbamate, epoxy, imidazole functions or also a silanol radical of the formula SiY ₃ , wherein Y represents a halogen atom or a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms (preferably a methoxy group) or an acetoxy or phenoxy group.

R ₂ may, for example, be a lower alkoxy group having 1 to 3 carbon atoms, such as methoxy, ethoxy or n-propoxy, a cyano group or a group of the formula COOR ₃ , in which R _{3 is} a lower alkyl group having 1 to 4 carbon atoms, such as a methyl - or ethyl group, or an epoxyalkyl group, such as. As an epoxyethyl or 2,3-Epoxypropylgruppe.

In addition, R ₁ and R ₂ together with the carbon atom they carry form a ring, such as. A pyrrolidinyl, imidazolyl or morpholinyl ring, or more generally, they can form any hydrophilic ring that is free of a reactive hydrogen atom.

The copolymerizable monomer may in particular be are selected from vinylpyridine, vinylimidazole, N-vinylpyr rolidone, N-vinylmorpholine, acrylamide, an alkyl substituent Acrylamide, such as dimethylacrylamide, N-vinylmethylace tamide and alkyl acrylates or methacrylates with a Chain with 1 to 10 carbon atoms.

The polymers and copolymers which can be used according to the invention can be prepared from vinyl haloformates by known processes, for example the process according to Meunier et al. ("Polymer", Vol. 23 (1982), p. 849). They must be prepared and used in dry aprotic solvents. These polymers and copolymers can be modified to adapt or improve their properties as a function of intended use and, for example, give active polycarbonates after the reaction of the above products with N-hydroxysuccinimide or phenols, which are preferably activated by one or more Activator groups, such as -NO ₂ or -F. The active polycarbonates thus obtained can react with nucleophilic organic compounds or with the nucleophilic groups of proteins in an aqueous medium.

The inventive method for producing the carrier consists of the previously dried porous solid to be impregnated with a solution of a polymer or copo lymers containing the chloroformate residues and / or abge contains guided functions, and then with the To rinse solvent. Preferably, the polymer or Copolymer in an increased concentration (more than 5 % By weight) in the solvent to give a good adsorp tion to ensure the porous solid. The ver solvents used may be compounds that are free of active hydrogen and nucleophilic functions. Examples of useful solvents are alkyl or Aryl halides, acetone, acetonitrile, tetrahydrofuran.

The porous solid is preferably a mineral oxide, such as Silica, alumina or titanium or zirconium dioxide. The invention preferably uses silicon dioxide. This oxide can be a prior treatment un be subjected to a reactive silane, for example, such as those in US-PS 44 15 631 be are written, d. H. those which are amine, hydroxy, epoxy, Wear ester or thiol groups. You can, for example a silica with a particle size distribution between about 1 μm and about 1 mm and having a pore diameter between about 2 and about 500 nm.  

Optionally, the deposited polymer or copoly mer are crosslinked by known methods, for example by treatment with an ethylenediamine solution.

In this way it is possible to Trä the invention ger to use chemical groups or compounds to fix what the subsequent separation of Protei NEN or hydrophilic polymers allow using a mechanism of exclusion by size, Ion exchange, hydrophobing, interactions with dyes or complexed metals or the Interactions with specific ligands (affinity or immunoaffinity chromatography).

The following examples are intended to illustrate the invention, without restricting it to it.

example 1 Polymerization of vinyl chloroformate

Vinyl chloroformate (VCF) was prepared according to the method described by Meunier et al. method described ("Polymer", Vol. 23 (1982), p. 849). polymerized.

A solution of 10% by weight VCF in dichloromethane is added Reflux of the solvent to a temperature of 35 ° C brought. The initiator is dicyclohexyl peroxydicarbo nat, in an amount of 1.2%, based on the Monomers added. The reaction medium is absorbed kept dry nitrogen. After 48 hours of reaction the polymer is precipitated by ether, then filtered off and under vacuum at ambient temperature for 12 hours dries.

The polymer is then characterized by infrared analysis (bands C = O at 1770 cm ^-1 and C-Cl at 680 cm ^-1 ) and elemental analysis (Cl: 32.8% (theory 33.3%) C: 34, 1% (theory 33.7%)).

Example 2 Copolymerization of vinyl chloroformate and N-vinyl pyrrole lidon (VP)

The two monomers are dissolved in dichloromethane in a Ge total concentration of 10 wt .-% and in a molar ratio VCF / VF of 25/75 solved.

The polymerization initiator is the azobisisobutyronitrile (AIBN) in a molar concentration of 1.5% on the monomers. The polymerization temperature is 35 ° C and the polymerization time is 48 h. The Reacti Onsmedium is kept under dry nitrogen. To In the polymerization, the copolymer is liberated with ether falls, filtered off and then dried.

The polymer is analyzed by infrared spectroscopy (band C = O of the chloroformate at 1770 cm ^-1 ). The elemental analysis yields 16% chloroformate units and 84% pyrrolidone units.

Example 3 Copolymerization of vinyl chloroformate with dimethyl acrylamide (DMA)

The working method is similar to that of the above said copolymer. DMA and VCF are dissolved in dichloromethane in a concentration of 10% by weight and in one molar DMA / VCF ratio of 40/60.

The radical polymerization is characterized by AIBN (molar Concentration 1.5%, based on the monomers) einei  tet. The polymerization time is only 3 h because of higher polymerization rate than in the above th case. The copolymer falls in the course of Polymerisa tion and is then dissolved in toluene. The copolymer is then precipitated with ether, then filtered off and dried under vacuum at ambient temperature for 12 H.

The characterization is carried out by infrared spectroscopy (band C = O of VCF at 1770 cm ^-1 and C = O of DMA at 1640 cm ^-1 ) and the elemental analysis shows a composition of 88 mol% DMA and 12 mol% VCF.

Example 4 Deposition of a homopolymer or copolymers on silica

Porous silica, for example Nucleosil of the company MACHEREI-NAGEL with a granulometry of 10 μm and egg The pore diameter of 30 nm is kept under vacuum for 12 hours dried at 150 ° C. The polymer (or copolymer) is in Dichloromethane dissolved in a concentration of 5 wt .-%. 1 g of silica is dissolved in 10 ml of the solution of the polymer (or copolymer) is suspended. The mixture becomes homogeneous Siert and degassed by stirring for 5 minutes with a UI traschallrührer, then the suspension for 4 h at Um stirred ambient temperature.

The impregnated silica is regenerated via Cellulose membranes in a filtration cell under stick fabric is filtered; the product is then resuspended and washed with dichloromethane. The silica becomes then dried under vacuum at ambient temperature kept in an inert atmosphere.  

The deposition of polymers can be detected by infrared spectroscopy performed on impregnated silica spheres and the appearance of the vibrational bands C = O at 1770 cm ^-1 corresponding to the VCF is observed.

The amount of the polymer or copolymer is determined by elements determined by tar analysis. The results are the following:

polymer adsorbed amount in mg / g VCF polymer 175 VCF / VP copolymer 150 VCF / DMA copolymer 130

Example 5 Chemical modification of chloroformate functions N-hydroxysuccinimide

The N-hydroxysuccinimide (NHS) is dissolved in dimethylformamide (DMF) dissolved in a concentration of 0.52 mol / l.

This solution is applied to a suspension of (after Stated method described) coated silica poured into dichloromethane. The suspension contains 20 g of Si licium dioxide per liter of solvent. You pour one Excess of 3 moles of NHS to 1 chloroformate unit and simultaneously gives a solution of pyridine in dichloromethane (0.33 mol / L) until an amount of 1.3 moles of pyridine per Mol NHS is present. The temperature is kept at 0 ° C and stir gently every 10 minutes. The silicon dioxide xid is then filtered off, with a dichloromethane than / DMF (50/50 by volume) mixture and with rinsed with pure dichloromethane, then the product is Vacuum dried at ambient temperature.  

The elemental analysis indicates a degree of conversion of the chloroformate functions of 95%. The spectroscopic infrared analysis shows the appearance of the C = O carbamate band at 1744 cm ^-1 .

Example 6 Reaction of Aminomethanesulfonic Acid (AMS) on a Reak silicon dioxide column

A chromatography column with a length of 5 cm and an inner diameter of 4 mm is with the Siliziumdio xid obtained by the method described in Example 5 above drive has been modified, filled.

An aqueous solution of aminomethanesulfonic acid (AMS) (NH ₂ -CH ₂ SO ₃ Na) is allowed to pass through the column at a concentration of 0.1 M which has been equilibrated with a 0.67 M phosphate buffer at pH 7.4 to run. The optical density at 260 nm is determined and it indicates the release of the NHS, which ceases when the reaction is complete. This experiment allows the calculation of the amount of released NHS which is 7.5 x 10 ^-5 moles.

For comparison, passing a solution shows at pH 7.4, which is free of AMS, that the release of the NHS proceeds after a much slower procedure than in the above case.

The measurement of the ionic capacity of the column with AMS fixed thereto was carried out by frontal analysis of a pyridine solution brought to pH 3 with citric acid. The capacity was found to be 5 x 10 ^-5 eq / g.

Example 7 Use of the AMS modified carrier for the tren protein

The modified according to the method of Example 6 above Carrier contains sulfonate functions, the cation exchange represent centers of excellence. This material may be for the Separation of proteins by an ion exchange method be used.

Figure 1 shows the example of the separation of two proteins (α-chymotrypsinogen and lysozyme) positively charged eluted at pH 3 by adding in portions a solution of NaCl of 1 M concentration in a column of length of 5 cm and an inner diameter of 4.2 mm (injection of proteins: 0.67 g / l, 50 μl, 10 mM citrate buffer) containing the carrier of Example 6 above.

The chromatogram shows that the α-chymotrypsinogen (1) and lysozyme are completely separated ( Figure 1).

Example 8 Use of the Modified Support for the Covalent Fi xation of protein

The modified carrier described in Example 5, the NHS carbonate functions will be as above Trap introduced into a chromatography column (height 8 cm; Diameter 4.2 mm).

An aqueous albumin solution (concentration <2 g / l) with a pH of 7.4 (0.67 M phosphate buffer) is determined by the Pillar run.  

The fluorescence (λ ex = 287 nm, λ em = 328 nm) of the effluent is determined and allows the determination of the Column fixed amount of albumin by frontal analysis; you finds a lot of 5 mg / g carrier.

To the presence of the fixed on the carrier albumin Demonstrate a connection like warfarin, with a Affinity injected for this protein. After the injection of the compound in different concentrations the chromatograms obtained are characteristic of one Affinity of Wafarin for Albumin. Similar experiments, the be carried out with a carrier using one of impregnated polymer described above, however was free of albumin, show no affinity of the carrier towards the warfarin molecule.

Example 9 Preparation of an affinity support for the purification of enzymes

A copolymer containing aminobenzamidine units becomes prepared by adding a VCF / VP (50/50, molar ratio) - Copolymer as described above with amino benzamidine (specific ligand of serine proteases) rea let it ferment

according to the following procedure:

• - 3.9 g of aminobenzamidine are dissolved in 200 ml of dimethylformamide Dissolved (DMF), the 8 ml of triethylamine (TEA) added who the.
• 1 g of silica coated with a copolymer, which contains chloroformate units, prepared as in Example 4 above, enters the above solution guided. The reaction is carried out at ambient temperature for 20 h performed.

The solid is then filtered off, then rinsed with DMF and then with a 50/50 (volume ratio) mixture of DMF and dichloromethane, then rinsed with pure dichloromethane and finally dried. The determination of the amount of benzamidine units is carried out by frontal analysis of a sodium nitrate solution. The increase in optical density at 280 nm after passing a certain volume of solution allows the calculation of the amount of original amidine centers. The number of fixed benzamidine units was thus found to be 3.7 x 10 ^-5 units / g of silica.

Example 10 Use of the benzamidine-containing carrier for the Af finitätschromatographie

The above carrier was used to purify a Tryp sin sample. A volume of 1 ml of trypsin solution, the be has been cleaned (T8128 Sigman 45IU per mg Pro tein), which contains a protein amount of 1.12 mg, becomes abandoned the vehicle, with a 10 mM Phosphate buffer solution at pH 7.4 (Eluant A) is eluted.

After elution of a fraction of the injected proteins in the form of a non-retained peak, a glycine buffer solution (50 mM, pH 2.8, NaCl 50 mM) is continuously applied to a column (Eluant B). Both peaks corresponding to the originally fixed trypsin occur. Only the second peak indicates enzymatic activity ( Figure 2) and contains 0.46 g of protein.

The exact measurement of enzymatic activity shows one Degree of purification of 4 (165 I.U. per mg protein). The Investigation of the influence of a competitor to the Li The trypsin was performed under preparation of chromatograms that eluted the injection of the Trypsin corresponded with an L-arginine solution in under different concentrations. The results obtained show the reduction of the retention volume of the Trypsin with an increasing concentration of L-arginine, what the affinity of the protein to the carrier be has.

In Fig. 2, the curve in solid lines corresponds to the injection of 1 ml of pure trypsin and the curve in dotted lines corresponds to the measurement of enzymatic activity of trypsin.

Claims (9)

A support for the production of chromatography columns having a porous solid on which is deposited a thin coating (film) of a polymer or copolymer, characterized in that the polymer or copolymer has haloformate functions or derived functions consist of cleavable groups has. 2. Vehicle according to claim 1, characterized in that the polymer or copolymer contains the repeating unit of the following general formula: in which mean:
X is a halogen atom such as chlorine, or a leaving group such as succinimido, phenol, imidazole or thiosulfate, and
n is an integer between 10 and 10 000. 3. A carrier according to any one of claims 1 and 2, characterized ge indicates that the polymer is selected from the Group Vinyl polychloroformate, sodium polyvinyloxycarbo nylthiosulfate and N-hydroxysuccinimide polyvinyl carbonate. 4. A carrier according to any one of claims 1 to 3, characterized in that the copolymer is obtained from a copolymerizable monomer of the formula CH ₂ = CHR ₁ R ₂ wherein
R _{1 is} a hydrogen atom or a methyl group and
R _{2 is} a hydrophilic group bearing one or more ester, amide, carbamate, epoxy, imidazole functions or a silanol radical of the formula SiY ₃ , wherein Y is a halogen atom or a hydrogen atom, or an alkoxy group having 1 to 4 carbon atoms or an acetoxy or phenoxy group. 5. A support according to claim 4, characterized in that R ₁ and R ₂ together with the carbon atoms which they tra gene, form a ring which is selected from the pyrro lidonyl, imidazolyl and Morpholinyl rings. 6. A carrier according to claim 4, characterized in that the copolymerizable monomer is selected from Group vinylpyridine, vinylimidazole, N-vinylpyrrolidone, N- Vinylmorpholine, acrylamide, N-vinylmethylacetamide and the Alkyl acrylates or methacrylates containing a chain with 1 have up to 10 carbon atoms. 7. A method for producing a carrier according to a of claims 1 to 6, characterized in that it This consists of removing the porous solid previously has been dried with a solution of a polymer or copolymer impregnated, the haloformate Funktio NEN, and then with the solvent flushes. 8. The method according to claim 7, characterized that the solvent is selected from the group Al kyl or aryl halides, acetone, acetonitrile and Tetrahy drofuran.   9. The method according to any one of claims 7 and 8, characterized characterized in that the polymer or copolymer is treated is with N-hydroxysuccinimide, a sulfonic acid or a activated phenol.