CS273487B1 - Macroporous polymer membranes for biopolymers' chromatographic separation - Google Patents

Abstract

The solution concerns macroporous polymerous membranes for chromatographic biopolymer separation.According to the solution they consist of macroporous copolymer of mono- and divinylic monomer, while the monovinylic monomer is glycidyl methacrylate in the amount of 5 to 80 percent by volume and the divinylic monomer is ethylene dimethacrylate, whose quantity in the overall amount of polymerised monomers ranges from 20 to 95 percent by volume, and on the inner surface, achieving in dry state area of 1 to 400 square meters per gram, they carry chemical function groups of the original monomer and/or their derivates, selected from a group consisting of epoxy, allyl, hydroxyl, amino, sulphonic, hydrogen sulphate, sulfhydryl, or alkyl group with a strand containing up to 18 C atoms.These macroporous polymerous membranes enable chromatographic separation of biopolymers at industrial rate and according to a preset program.

Description

The present invention relates to macroporous polymer membranes for the ehromatographic separation of biopolymers.

Although the issue of separation of macromolecules from their mixture with low- or high-molecular substances is currently very frequent in the literature and practice, and considerable success has been achieved, it has not yet been considered as solved. With the advancement of modern scientific and technical disciplines, such as biotechnology, the problem of efficient separation of the product produced from the reaction medium becomes a crucial criterion for the success of the solution. It is not usually a big problem to find a method of analytical identification of a substance or to determine its concentration. Problems, however, brings not yet fully resolved the issue of preparative industrial separation with high efficiency at a reasonable expenditure of labor, energy and capital costs of both, as well as material _r and with minimal environmental impact.

The most important for the application is the separation and isolation of biopolymers. This group of macromolecular substances includes oligo- and polypeptides, proteins, enzymes, lectins, antibodies, antigens, nucleic acids, polysaccharides. Separation of biopolymers from natural sources has been a common problem since the last century.

The original purification methods consisted mainly of the precipitation of proteins, for example, or their salting-out with neutral salts, which can be carried out with simultaneous pH changes and thus achieve multiple fractions in one step. Even so, the long way to obtain pure protein was crowned with success only in 1926, when Sumner isolated crystalline urease. However, even now, as will be shown later, the effect of ionic strength or pH in protein separation is reused.

In parallel, a method called its discovery by M. Cvett, chromatography (Ber. Deut. Botan. Ges. 24, 316, 1906), was also developed. However, the work of A. J.

P. Martina and R. L. M. Synge (Biochem. J. 35, 1358, 1941), which introduced the notion of a theoretical tray in liquid chromatography. It has also been reported that columns loaded with microparticles are particularly advantageous for the separation of macromolecules whose diffusion coefficients are very small. As the microparticles had not yet been discovered at that time, the High Performance Liquid Chromatography (HPLC) process was only implemented twenty years later.

The chromatography of the polymers was developed based on the knowledge of Peterson and Sober (J. firner. Chem. Soc. 76, 1711, 1954) that the proteins can be adsorbed onto the diethylaminoethyl cellulose derivative and then eluted sequentially with a solution with increasing ionic strength.

Later other cellulose derivatives, such as carboxymethyl, have also been used for the same purpose. In the late 1950s, Pharmacia Uppsala introduced cross-linked dextran exclusion chromatography (SEC) gels of proteins and nucleic acids in Sweden (Ger. Pat. 1292883, Brit. Pat. 974 054), where the separation was based on the availability of various proportions of porous gel structure for species with different size macromolecules. However, the low mechanical strength of the gel media did not allow the HPLC principle to be fully realized.

A breakthrough occurred when bacitracim fractionated on alkylsilane-derivatized silica gel microparticles (K. Tsuji, J.H. Robinson, J. Chromatogr. 112, 663, 1976). Using an acidic mobile phase containing an organic solvent, several structural forms of said peptide were separated by the so-called reverse phase chromatography (RPLC) method on a 30 cm column. Soon thereafter, work describing the use of silica gel for high performance size exclusion chromatography (SEC) and ion exchange chromatography (IELC) of proteins appeared, with almost quantitative yield and activity retention. Compared to gel materials, it is possible to use up to two orders of magnitude higher flow rates of the mobile phase in these cases, which significantly shortens the analysis times.

Macromolecules generally interact with the stationary phase in the ehromatographic column in various ways. This leads to the capacity factor defined in the isocratic elution, i.e. eluting using the same solvent as

EN 273 487 Bl ^k '= - * 0 ^ 0 where t _R is retention time of the analyte and TG dead volume of the column, is changing rapidly at slight modification of the eluent composition. At a certain composition, k 'is so high that the macromolecule practically does not move in the column. However, a small change in solvent causes a drop to a value close to zero and the macromolecule passes through the column without interacting with the column. The commonly reported and reported dependence of log k 'on the eluent composition is thus very steep, often almost vertical. However, it also follows that the length of the chromatographic column does not have a decisive influence on the quality of the separation in this case. Thus, very short columns can be used, containing only the necessary amount of sorbent needed to adsorb the split macromolecules in such an amount that the resolved substances can be detected.

It has been shown that in the chromatography of macromolecules, for example, by the RPLC method, the resolution function is proportional to 1/2.

Where the diffusion coefficient of the solute is d, the diameter of the particles loaded in the column and the tg the time over which the composition of the solvent mixture (gradient) changes. Since the first quantity is a characteristic constant of the separated substance, the quality of the separation can be improved by slowly changing the solvent composition or reducing the particle size of the column packing. In the first case, the time required for the separation of the mixture also increases, in the second case the pressure drop on the column increases and it is necessary to feed the eluent at very high pressure, which can reach several units up to tens of MPa.

These circumstances indicate that increasing the scale of chromatographic separation from analytical or laboratory can be a significant problem. Obviously, the use of bulky columns with a relatively large diameter can, even when using incompressible packings, entail difficulties manifesting in results that are not comparable to the previous ones. The so-called tailing of chromatographic peaks, or their overlap, often occurs. This may be due, for example, to the varying flow rate of the eluent at different locations in the column cross-section.

To avoid this, it is necessary to achieve a uniform horizontal queue speed through the column from the eluent inlet to the column outlet. This problem is discussed in US Pat. 3250058 and to solve it use splitters mounted inside the column. A method of influencing the flow of liquid in a column is also the subject of other patents (US 3539505, DOS 2036525, DOS 2224794, Jap. 73-68752) and allows to increase the scale of separation but to the detriment of the original simplicity.

In order to circumvent the complexity of the column design, some authors use interventions in the packing itself (US 3856681) or special methods of filling the column (US 4211656). Later, it has been shown that a good effect can be obtained if the fine particles of the sorbent on which the separation takes place are incorporated into a porous inert matrix having the shape of fibers. The fibrous mass is then packed into a column of special construction, in which there is much less pressure loss and zone wash (US 4384957, US 4512897, US 4496461, US 4604198).

As already indicated, the vast majority of packings of biopolymers separation columns are porous, whether inorganic (silica gel, glass) or organic polymers (styrene divinylbenzene, acrylate or methacrylate copolymers, etc.), usually in the form of spherical particles (FE Regnier, Chromatography) 24, 241 (1987). These particles are mainly produced by the suspension technique, and more recently, multi-stage dispersion methods (seeded polymerization) have emerged. Upon completion of the polymerization, a narrow sorbent size fraction must be recovered from the crude product, with few exceptions, since the quality of the packed column and hence its efficiency is strongly dependent on the particle size dispersion. Particle fractionation is very difficult and the HPLC usable particle fraction represents only a small fraction of the entire crude product.

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In view of the use of small diameter particles (5 to 10 µm), organic sorbents must also be mechanically sufficiently strong, which implies a relatively high crosslinker concentration in the polymerization batch. At the same time, the existing porosity requirement is solved by the synthesis of macroporous particles, ie particles characterized by porosity in the dry state as well as in possibly thermodynamically poor solvents (Author's Certificate No. CS 168268,

Brit 1512462, Canada 1049194). From a morphological point of view, macroporous polymers are characterized by a so-called globular structure, ie particles consist of interconnected submicroscopic spherical formations called globules. The specific surface area of the macroporous polymers is then actually the surface of these globules and the interstitial spaces therebetween are pores (see, for example, Z. Pelzbauer et al., J. Chromatogr. 171, 101, 1979). The globular structure of the porous particle resembles to a certain extent a spherical body filled with tiny as well as spherical globules. This notion is no longer distant from the conditions prevailing inside the packed chromatographic column, which however has the shape of a cylinder. If the column were packed with particles with a globular size (0.1 to 0.4 µm), the chances of realizing the chromatographic separation would be slight, since the pressure to be applied would be unrealistically high. Therefore, according to MS. No. 2,744,433, polymeric films having a thickness of 0.2 to 15 mm characterized in total volume by the same macroporous structure as the particles commonly used in different types of HPLC have been invented.

Electrodialysis membranes having only a macroporous polymer surface layer are the subject of US Pat. 3926864. After modification with ionogenic groups, such a surface has pronounced antifouling properties. However, since it is also necessary to achieve good electrodialysis properties, the polymerization is conducted so that the inner part of the membranes is microporous (gel). Therefore, these membranes cannot be used for separating polymers; they are suitable for water desalination, ion removal and the like.

The membranes according to the invention (foils, plates) have a shape close to the thin-layer chromatography plates, but the sorbent is deposited in a layer deposited on a solid non-porous substrate (glass, metal). However, the actual separation process, which includes chromatographic separation elements, utilizes a layer in the tangential direction, i.e. in a length that many times exceeds the particle size of the sorbent. The disadvantage of thin layers, which are loose or depressed, is also low resistance to mechanical damage. Thin layer chromatography is difficult to use for preparative work.

This overview of the prior art clearly illustrates that there is not yet a reliable and simple method for large-scale separation of polymers. It was therefore necessary, in principle, to find a novel solution which is the object of the present invention. It combines both the membranes themselves, characterized by the method of preparation consisting of polymerization and modification, and their use for the separation of biopolymers.

The subject of the present invention are macroporous polymer membranes for the chromatographic separation of biopolymers consisting of a macroporous copolymer of a mono- and divinylic monomer, wherein the monovinyl monomer is glycidyl methacrylate in an amount of 5 to 80% by volume and the divinyl monomer is ethylene dimethacrylate. 20 to 95% by volume of the total polymerized monomers, and on the inner surface, which, even in the dry state, is 1 to 400 m ² / g, carry the chemical functional groups of the original monomer and / or derivatives thereof selected from the group consisting of epoxy, allyl, hydroxyl, amine, sulfone, hydrogensulfate, sulfhydril, alkyl chains of up to 18 carbon atoms.

These membranes can be prepared by polymerizing a mixture of mono- and divinyl monomer in the presence of an inert porogenic solvent, or a solvent mixture initiated by a free-radical initiator, in heated form having one dimension (usually thickness) equal to the desired dimension of the final membrane. Any mixture of both types can be used. monomers and in this way vary the porous properties of the resulting membrane. The choice of monomers may also be wider.

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Azobisisobutyronitrile is used to initiate radical polymerization, but any radical initiator selected from the group of azo compounds, peroxides, hydroperoxides, redox systems and the like can be used.

An important part of the polymerization system is a porogenic solvent. Preferably cyclohexanol or a mixture thereof with dodecanol is used. Of course, other porogenic agents such as aliphatic or aromatic alcohols, esters, ethers, ketones, hydrocarbons, silicone oil, low molecular weight polymers, and others can be used.

The membrane containing epoxy groups in the side chains is generally not the final product and is chemically modified to modify the inner surface character.

By chemical modification, groups of, for example, allyl, amine, sulfonated, hydrogen sulfate, hydroxyl, sulfhydril, alkyl groups of up to 16 carbon atoms are bonded to the membrane surface.

Chromatographic separation of biopolymers using the membranes of the invention is accomplished, for example, by placing a sector of any size and shape obtained from a macroporous polymeric membrane on a support plate for position stability on any wall forming part of the chamber and allowing collection of liquid through the membrane. The chamber is filled with a polymer solution or polymer blend; which is passed through a membrane in which the polymer (s) is adsorbed under a pressure of less than 1 MPa. A solvent is introduced into the chamber through the pump in which the polymer (s) has been dissolved, and then the individual components of the adsorbed polymer were gradually redissolved and eluted from the membrane, thereby separating itself. The course of the separation is either detected only by a suitable device and the composition of the polymer or the polymer mixture is evaluated qualitatively or quantitatively, or the individual fractions are collected and the individual components of the split mixture are produced as individual (preparative separation).

Programmatic change of solvent properties causing gradual dissolution of the individual components of the mixtures (gradient) may relate to pH, ionic strength, organic solvent content, temperature and other variables.

Macroporous polymer membranes for separating polymers, especially biopolymers, and the process for separating them have a number of advantages over the prior art. Especially their preparation is very simple and is not limited in size. The membranes are characterized by sufficient mechanical stability and resistance to physical and chemical influences. The mechanical strength can be further increased if necessary by polymerizing the reinforcing component into the membrane in the process of its preparation. Because the membranes contain reactive groups, they can be easily chemically modified and functional groups can be introduced on their inner surface to alter their properties and thereby significantly extend their application. The actual separation of biopolymers, for example, then proceeds very quickly in comparison with the known solutions at a considerably higher load on the weight unit of the separating element compared to the load which is possible on the chromatographic columns. The pressure required to achieve the desired flow rate is one to two orders of magnitude lower than that of column methods of comparable efficiency. However, the essential advantage of the membranes and the method of their use according to the invention is the possibility to create theoretically unlimited large areas on which the polymer separation is carried out, and thus to realize the industrial process for obtaining individual substances. It is not necessary to obtain the required surface by increasing the size of one membrane and chamber, but it is also possible to combine the required number of smaller membranes and chambers into blocks fulfilling the same purpose.

Example 1

The mold, consisting of two metal plates in which the communicating channels are drilled, and a 1 mm square spacer made of silicone rubber, in which a 8 cm square square hole is cut and a hole allowing filling the mold space from the outside, is filled with polymerization a mixture consisting of 6 ml of glycidyl methacrylate,

CS 273487 B1 ml ethylene dimethacrylate, 0.12 g azobisisobutyronitrile, 16.2 ml cyclohexanol and 1.8 ml dodecanol. Water of 70 ° C for 8 hours is fed into the mold channels. After the polymerization is complete, the mold is allowed to cool and disassembled. The plate of the macroporous membrane is washed thoroughly with alcohol, water and again with alcohol and allowed to dry. A round target with a diameter of 10 mm (surface area approx. 300 mm?) Is punched from a square plate with a punch. The specific surface area of the membrane in the dry state is 62 m ² / g.

Examples 2 to 8

The mixture described in Example 1 is filled into the mold described in Table 1, the contents are polymerized and processed in an identical manner.

TABLE 1

Composition of polymerization mixture used for preparation of macroporous polymer membranes and their specific surface area

Example GMA ml EDMA ml AIBN G CyOH ml DoOH ml ^s g m ² / g 2 4.8 7.2 0.12 18 0 80 3 7.2 4.8 0.12 16.2 1,8 45 4 9.6 2.4 0.12 17.1 0.9 160 5 0.6 11.4 0.12 17.1 0.9 260 6 7.2 4.8 0.12 18 0 53 7 0.6 11.4 0.06 18 0 250 8 7.2 4.8 0.24 14.4 3.6 35

At the top of the table, GMA indicates glycidyl methacrylate, EDMA ethylenedimethacrylate, AIBN azobisisobutyronitrile, CyOH cyclohexanol, DoOH dodecanol and S dry surface area measured from thermal nitrogen desorption by the dynamic method.

Example 9

The macroporous membrane circular targets prepared according to Example 3 were left in a 0.01 mol / L solution of NaOH in butanol for 24 hours. After completion of the reaction, the targets are washed with ethanol, water and stored in a wet state for further use. Alkyl groups with 4 C-carbons are attached to their surface by ether bonding as evidenced by infrared spectra.

Examples 10 and 11

In the same manner as in the previous Example 9, the epoxide groups of the membrane were modified with octanol or octadecanol. In the latter case, the modification was carried out at a temperature of 65 ° C. The product according to IR analysis contains up to 0.2 mmol / g of attached alkyls having 8 and 18 C atoms, respectively.

Example 12

10 mm diameter circular targets punched from the membrane defined in Example 6 were immersed for 6 hours in a commercial 25% solution of ammonia in water and heated to reflux at 80 ° C. The targets were then washed with water until the reaction to ammonia disappeared.

Elemental analysis showed the presence of 1.8 mmol / g amino groups in the modified polymer.

Examples 13 to 20

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Examples 13 to 20

Using the same procedure as in Example 12, the membrane targets prepared by the method described in Example 1 were modified by reaction with the pure amine present in the flask or sealed ampoule. The reaction conditions and the content of groups bound to the polymer membrane are summarized in Table 2.

TABLE 2

Reaction conditions of macroporous membrane modification and content of bound groups in the product Example Amin Concentration of the solution in H ₂ 0 wt. Reaction Temperature time Content of mmol / g groups h C 13 dimethyl- 100 ALIGN! 3 60 2,0 14 2-hydroxyethyl- 100 ALIGN! 6 70 2.2 15 Dec bis-2-hydroxyethyl- 100 ALIGN! 6 70 2.1 16 octyl- 100 ALIGN! 12 70 0.4 17 ethyl- 50 6 80 1.6 18 ethylenedi 50 10 80 1.7 19 Dec trimethyl HCl 50 24 80 2.2 20 May tributy1 100 ALIGN! 24 80 0.1

Example 21

The circular targets punched from the polymeric macroporous membrane prepared according to Example 2 were immersed for 6 hours in 0.01 mol / L sulfuric acid and heated to 80 ° C to hydrolyze the epoxy group to two vicinal hydroxyls. The product can optionally be separated and used differently. After washing with water until the acidic reaction disappeared, the targets were immersed in a solution of 10.2 g of NaOH in 36 ml of water and the mixture cooled to 0 ° C. While stirring the liquid above the membranes, 24 g of propanesultone was added dropwise. After thirty minutes, the mixture was slowly warmed to 35 ° C and the reaction continued for a further 3 hours. After heating, the mixture was left at room temperature for 12 hours. The targets were washed with water, 0.5 M HCl and again with water until the acid reaction disappeared. 0.85 mmol / g of sulfonated groups were thus obtained on the surface of the polymer membrane.

Example 22

A 10 x 5 cm rectangle from a plate prepared analogously to Example 2, whose epoxy groups were hydrolyzed with sulfuric acid solution to the vicinal dihydroxy derivative as in Example 21, was washed with water and dried. The dry plate was immersed for 5 hours in an allylglycidyl ether-dioxane (1: 1 by volume) mixture containing 0.3 vol% boron trifluoride etherate and heated to 50 ° C. The allyl grafted product was then modified with a 30% solution of potassium thiosulfate in water at room temperature for 24 hours, and oxygen was pumped from the storage tank (bombs) every 10 minutes for 10 minutes. After washing with water, 0.4 mol / L HCl and again with water, acid-base titration revealed that the membrane contained 0.37 mmol / g sulfonated groups.

Example 23

A circular target of 20 cm diameter, cut from a 25 cm side square plate and 3 mm thickness was immersed in a glass bowl containing 100 ml of 1,2-dichloroethane and left for 20 hours. Then, at room temperature, 45 ml of sulfur trioxide monohydrate was slowly added while stirring the bowl. After one hour of reaction, it was taken up and washed with ethanol and water. Acid-base titration of the sample determined that the membrane contained 1.59 mmol / g hydrogen sulfate groups.

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Example 24

Round targets of 30 mm diameter punched from the plate prepared according to Example 4 were mixed with a 10% solution of sodium sulfide in water. The mixture was gently shaken for 12 hours at 25 ° C. The targets were washed with water until the odor of hydrogen sulfide had disappeared. The modified polymer contains 0.52 mmol / g sulfhydril groups.

Example 25

At the bottom of a 1 ml round-bottomed vessel, a target of about 1 cm diameter is placed from a 1 mm thick membrane prepared according to Example 1 and modified according to Example 9 so that when the vessel is filled with liquid all flow is exclusively through the membrane. The contents of the container are mixed with a propeller stirrer. The eluent is fed to the vial according to a defined gradient. Under the membrane located on a surface provided with a collection channel system ensuring perfect and uniform drainage of the eluent from each point of the membrane, a central drainage capillary outlet from which the eluent flowing (or a portion thereof) is routed to the detector and then optionally collected to recover separated materials. A solution containing 0.1 mg of ribonuclease, 0.1 mg of ovalbumin and 0.1 mg of chymotripsinogen in 0.02 mol / l phosphate buffer, pH 5.8, in which 2 mol of ammonium sulfate was dissolved, was introduced into the vessel. / 1. While stirring, the same buffer containing the ammonium sulphate in a decreasing amount of 0.5 ml / min was fed into the vessel at 0.1 MPa, and the eluent contained 1% of the original ammonium sulphate in 35 minutes . The effluent from the vial was fed to a Gilson OV detector and the detector response record (chromatogram) showing the separation is shown in Figure 1. 100% of all originally adsorbed proteins were eluted from the membrane.

Example 25

Using the same procedure and on the same apparatus as in Example 25, but with the target modified according to Example 15, the same protein mixture was split. The chromatogram is shown in Figure 2.

Example 27

On the same apparatus and under the same conditions as in Example 25, the same proteins were separated, but the amount of each of them in the solution submitted to the vial was 5 mg, 50 times higher. The same distribution as in Figure 1 was achieved.

Example 28

Under the conditions of Example 25 and on the same apparatus but with the membrane produced according to Example 21, a mixture consisting of 0.1 mg ribonuclease and 0.1 mg lysozyme dissolved in 0.02 mol / l phosphate buffer pH 6.8 was split. . Elution was performed at a flow rate of 1 ml / min. and a pressure of 0.1 MPa using an ionic strength gradient with the same buffer in which the sodium chloride content was increased such that after 15 minutes the salt content was 0.5 mol / l. The result of the division is shown in Figure 3.

Example 29

Under conditions identical to Example 28, the same mixture of ribonuclease and lysozyme was separated on the membrane prepared according to Example 26. The chromatogram is shown in Figure 4.

Example 30

A prism-shaped apparatus in which a 10 ml chamber was formed, the wall of which was formed by a rectangular membrane of 6 x 8 cm and a thickness of 1.5 mm prepared according to Examples 1 and 9, was subjected to separation of a mixture containing 150 mg of ribonuclease ,

100 mg ovalbumin and 150 mg chymotripsinogen. The eluent flow rate was 5 ml / min. at pressure

0.8 MPa. Using the same ionic strength gradient as in Example 1, the reaction was carried out

CS 273487 B1 8 elute and collect the eluent in 10 ml tubes. The fifth tube contained 5%, the sixth 85% and the seventh 10% ribonuclease, the eighth tube 15%, the ninth 75% and the tenth 10% ovalbumin and finally the twelfth 3%, the thirteenth 28%, the fourteenth 16 %, in the fifteenth 32% and in the sixteenth 20% chymotripsinogen.

Example 31

A mixture of 0.15 mg of mioglobin and 0.3 mg of ovalbumin was separated on the membrane and the apparatus of Example 25 except that acetonitrile was added to the decreasing ionic strength buffer at a concentration of 12% v / v in 20 minutes. pressure 0.25 MPa. The distribution is shown in Figure 5.

Claims (1)

SUBJECT OF THE INVENTION Macroporous polymeric membranes for the chromatographic separation of biopolymers, characterized in that they consist of a macroporous copolymer of mono- and divinyl monomer, wherein the monovinyl monomer is glycidyl methacrylate in an amount of 5-80% by volume and the divinyl monomer is ethylenedimethacrylate having a proportion of the total polymerized monomers 20 to 95% by volume, and on the inner surface, which, even in dry state, reaches 1 to 400 m ^a / g, carry the chemical functional groups of the original monomer and / or derivatives thereof selected from the group consisting of epoxy, allyl, hydroxyl , amine, sulfone, hydrogen sulfate, sulfhydril, alkyl having up to 18 carbon atoms.