MXPA00007605A - Device for producing polymer support materials in the form of porous polymer beads - Google Patents

Abstract

The invention relates to a method for producing a bead-shaped, cross-linked hydrophilic mixed polymer which actively bonds with ligands with nucleophilic groups, by inversely bead-polymerising a monomer phase. The invention also relates to polymer support materials with a high capacity for bonding with penicillin acylase and with a lower swelling number, and to their use.

Description

Apparatus for synthesis of polymer support materials in the form of porous polymer beads The invention relates to a process for synthesis, through reverse suspension polymerization of the monomer phase, of a crosslinked hydrophilic copolymer similar to a pearl which has an agglomerating activity towards the ligaments containing nucleophilic groups. The invention also relates to support polymer materials with a high agglutination capacity for penicillin amidase and a low swelling factor as well as the use thereof. Prior technology Porous polymer support materials for proteins, especially enzymes, are well known. There are applications in medicine, for example, in the division induced by enzymes of β-lactam antibiotics such as penicillin G for 6-aminopenicilinic acid (6-APA) by means of penicillin acylase (penicillin amidase). The important goals for development are primarily the highest possible load capacity, low swelling ability and the lowest possible residual solvent content. Halogenated solvents should, in principle, be avoided for synthesis.

German open patent application DE-OS 2237316 describes the process of the synthesis of bead-like crosslinked copolymers through radical polymerization of a monomer mixture containing a radical forming initiator and comprises a monomer having an agglutination activity towards the biological substances, a monomer with crosslinking and at least one additional comonomer, this monomer mixture is suspended as droplets and polymerized in a non-polar organic liquid. Aliphatic hydrocarbons, in particular, and especially with 8 and more C atoms, are suitable as the non-polar organic liquid. Mixtures of n-heptane and perchlorethylene are used in these examples. The ratio of the monomer phase to the organic dispersion medium can be in the range between 1: 1 and 1:10, but the ratio between 1: 1.5 and 1: 4 is preferred. German Patent DE A 3106456 describes an improved process compared to DE-OS 2237316 with regard to the binding capacity of polymer beads. Particularly high agglutination capacities are obtained for the proteins, especially for the penicillin acylase enzyme (penicillin amidase) when the support polymers contain . ^^ «« tJa.i. high contents of crosslinked monomers and when the monomer phase, formed from the monomers and the diluent, contains a solvent mixture as a diluent. Suitable mixtures can be, for example, water / methanol or formamide / methanol. The monomers and diluents are present in a ratio of about 1: 2.6. A mixture of n-hexane and perchlorethylene is used as the organic dispersion medium. In the examples, the ratio of the monomer phase to the organic dispersion medium is of the order of 1: 2.8. When the proportion of the crosslinked agent in the monomer mixture is 50% by weight and water / methanol is used as the diluent, it is possible to obtain support polymers with an agglutination capacity of up to 125 U / g, measured as the activity of the penicillin acylase. OBJECTIVES AND ACHIEVEMENTS The object of the invention is to provide an improved process for the synthesis of crosslinked copolymer-like pearls. It is also the intention to avoid the use of halogenated solvents in an organic dispersion medium and at the same time to achieve a binding capacity of at least 220 [U / g humidity] for the penicillin amidase enzyme (EC 3.5. 1.11) under standardized conditions (with lg loading of polymeric support material with 1530 units of penicillin amidase). In addition, the swelling of the polymer beads in water should not exceed 1.5, expressed as a swelling factor (wet ml / dry ml). The object was achieved through the process for synthesis, by inverse polymerization of a monomer phase pearl, of a hydrophilic, crosslinked copolymer similar to a bead having a binding activity towards the ligaments containing nucleophilic groups, whose monomer phase comprises monomers and a diluent, which contains as monomers: a) 5 to 40% by weight of hydrophilic monomers containing a vinyl group. which can overcome the radical polymerization and form at least 10% aqueous solutions at room temperature. b) 30 to 50% by weight of monomers containing a vinyl group and a further functional group, which can overcome the radical polymerization and in a polymer-like reaction with nucleophilic groups of ligaments, can form covalent bonds. c) 20 to 60% by weight of hydrophilic crosslinking monomers containing two or more polymerizable unsaturated groups of the ethylene type which can overcome the radical polymerization, provided that a), b) and c) add up to 100% by weight, using as a diluent a mixture of methanol and water in the ratio of 1: 1.0 to 1: 4.0, the monomer phase is dispersed as droplets in a dispersion medium comprising an organic solvent selected from aliphatic hydrocarbons with 5 to 7 carbon atoms. carbon, the ratio of the monomer phase to the dispersion medium being in the range of 1: 2.0 to 1: 4.0, and which in its form is subject to radical polymerization in the presence of a polymerization initiator and a protective colloid, with the proviso that the ratio of the monomers to the diluent are in the range of 1: 1.7 to 1: 2.4. Through the application of the inventive process it is possible to obtain an innovative support polymer material, which has a loading capacity for penicillin amidase of at least 220 [U / g moisture], resulting from the reaction of 1530 units of penicillin acylase with 1 g of polymeric support material, and exhibiting a swelling factor not greater than 1.5. It was not predictable that the definition of the various parameters of the process relative to each could lead to an agglutination capacity | ^^^^ d ^^ £ ^^ iuflHd | ^ a | ^^ a clearly higher for the enzyme of penicillin amidase and that at the same time, however, the swelling would decrease. It was also surprising that, through the application of the inventive process, the use of halogenated hydrocarbons such as pecloroethylene, which until now have been the most widely used compounds to equalize the phase densities, can be avoided by choosing the solvent organic like hydrocarbon aliphatic with 5 to 7 carbon atoms. Operation of the Invention Monomers In order to ensure that the monomer mixture is hydrophilic, it must comprise predominantly hydrophilic monomers. Hydrophilic monomers are monomers which form at least 10% of aqueous solutions at room temperature and preferably contain no ionic groups or groups which can be ionized. through the addition of acids or bases. The monomers a) comprise from 5 to 40% by weight, 8 to 35% by weight, especially 9 to 12% by weight of hydrophilic monomers containing a vinyl group, which can overcome the radical polymerization and form at least 10% aqueous solution to ^ aagg ^^ ambient temperature. Suitable as monomers are a) in particular acrylamide and / or methacrylamide, but methacrylamide is preferred. Additional examples are 5 hydroxyalkyl ethers or polymerizable carboxylic acids, such as hydroxyethyl acrylate and hydroxyethyl methacrylate or N-vinylpyrrolidone. The monomers b) comprise from 30 to 50% by weight, preferably from 35 to 45% by weight of monomers Containing a vinyl group and an additional functional group, preferably a group of ethylene oxide (epoxy group), which can overcome the radical polymerization and, in a reaction analogous to the polymerization, can form covalent bonds with the nucleophilic groups of the ligaments. The ethylene oxide groups in particular are suitable for bng ligaments while retaining their biological activity. The monomers b) are preferably glycidyl methacrylate and / or glycidyl allyl ester. Especially preferably, the two monomers are used in approximately equal proportions at the same time. The monomers c) comprise from 20 to 60% by weight, especially 25 to 55% by weight, HHj ^ «^ '< B is preferably from 40 to 55% by weight of crosslinked, hydrophilic monomers containing two or more polymerizable unsaturated groups of the ethylene type which can overcome the radical polymerization. The monomers c) are preferably N, N'-methylenebisacrylamide or N, N'-methylenebismethacrylamide, N, N'-methylenebismethacrylamide which is especially preferred. If necessary, it can also be used, from 0 to 10% by weight of a crosslinked monomer further containing two or more unsaturated polymerizable groups of the ethylene type and which can overcome the radical polymerization. Suitable are hydrophilic di (meth) acrylates such as polyethylene oxide di (meth) acrylates. 15 Monomers a), b) and c) add up to 100% by weight in all cases. Diluent The monomer phase comprises monomers a) to c), which are dissolved in a diluent, which must is a mixture of methanol and water in the ratio of 1: 1.0 to 1: 4.0. Especially favorable mixing ratios for methanol and water are in the range from 1: 1.2 to 1: 2.5, especially from 1: 1.3 to 1: 1.7. Ratio of monomers to diluent 25 The ratio of monomers to diluent is tiMittÉ ^ attn especially critical. It should be in the range of 1: 1.7 to 1: 2.4, especially preferred from 1.9 to 2.1. Dispersion Medium An organic solvent comprises an aliphatic hydrocarbon with 4 to 7 C atoms suitable as a dispersion medium. N-Heptane is preferred and cyclohexane is especially preferred. Ratio of the monomer phase to the dispersion medium The ratio of the monomer phase to the dispersion medium formed by the organic solvent should be in the range of 1: 2.0 to 1: 4.0, preferably from 1: 2.8 to 1: 3.3. Additional process conditions As additional constituents the suspended monomer phase contains polymerization initiators which are known, preferably sulfur-free initiators and especially preferably 4,4'-azobis- (4-valeric acid), as well as protective colloids (emulsifiers), as a copolymer comprising 95 parts of n-butyl methacrylate and 5 parts of 2-trimethylammoniomethyl methacrylate chloride with molecular weights (weight-average) in the range of 30,000 to 80,000.

The polymerization of beads (also known as suspension polymerization) is carried out in another manner in a known manner, for example by first introducing the dispersion medium and the protective colloid, then dispersing the monomer phase, which also contains an initiator, into stirring the organic phase at 40 to 60 ° C for example, and then heating at 60 to 70 ° C. The water / methanol mixture can be removed from the loop almost completely in the form of an azeotrope for a period of, for example, 6 hours. The mixture is allowed to react after about 3 to 5 hours and after cooling to room temperature. The resulting beads are sucked and vacuum dried for a period of, for example, 12 hours. Alternatively, the pearl polymers can also be filtered and washed with water. The drying is preferably carried out in a fluidized-bed dryer, since in this way the solvent residues can be removed particularly effectively. The polymer beads obtained (= polymer support material) have a size in the range of 50 to 500 μm, especially of 120 to 250 μm. By binding capacity it must be understood that the activity of the enzyme that can be achieved when the polymer support material is fully loaded with a * j¡á ^ | ^ ^^^^^^^^^^^^^^^^^^^^^ e = áßya ^ ^ specific enzyme. An important application of the inventive polymer support material is the division of penicillin G to 6-aminopenicilinic acid (6-APA) via penicillin amidase bonds of E. coli The agglutination capacity is expressed as penicillin amidase activity in units per g of support polymer beads [U / g moisture]. The binder capacity of the inventive support polymer beads in this measurement method is at least 220 [U / g moisture]. The swelling of the polymer beads in water is expressed by the swelling factor [wet ml / dry ml]. The inventive polymer beads exhibit a swelling factor that is not greater than 1.5. Use of the inventive support polymer materials The inventive support polymer materials can be used in stirred or flowing reactors for covalent bonding of ligaments by means of ethylene oxide groups they contain. This can be achieved, for example, through the addition of proteins, especially enzymes, of concentrated solutions through covalent bonding with retention of their biological activity. Peptides, amino acids, ß-lactam antibiotics, lipids, nucleoptics, polynucleotides, nucleophilic compounds with low molecular weight or metal-organic compounds that can also be reacted with the ethylene oxide groups of the support beads. The support beads are loaded with ligaments that can be used in methods known per se for the stereospecific synthesis of chiral substances such as amino acids (d-phenylalanine, p-hydroxy-d-phenylalanine, 1-tert-leucine) or pharmaceuticals as ibuprofen. These are also used as supports in the enzyme-induced cleavage of penicillin F to 6-aminopenicilinic acid (6-APA), from cephalosporin G to 7-aminodesacetoxycephalosporinic acid (7-ADCA) or from cephalosporin C to 7-aminocephalosporinic acid (7). -HERE). The procedure is described in the Annual Conference DECHEMA 1996 - Abstracts [in German], Vol. 1, DECHEMA e.V. Other applications are the specific enzyme-induced synthesis of amoxicillin and ampicillin or substrates as the split products above. An additional application comprises the synthesis of fine chemicals or basic products (such as malic acid) for chemical synthesis. Polymer beads can also be used in separation technology for adsorption chromatography or gel permeation chromatography. To achieve this specific adsorption, the polymer beads can be loaded with immunoglobulin fractions of antisera or with monoclonal antibodies. The use of the polymer support material loaded with enzymes or antibodies as adsorbents is an extracorporeal therapy, in which the pathogenic or toxic substances that are removed from all the blood, can be cited as an application additional. Examples: (The method of determination herein is already known to the person skilled in the polymer support materials technology, and will be described only for the purpose of determining) Determination of the agglutination capacity of penicillin amidase (= penicillin F acylase) of E. coli (EC 3.5.1.11) a) Covalent agglutination of penicillin amidase for the support polymer material lg of polymeric support material was added to 1530 units of penicillin amidase in 5 ml of 1 M sterile potassium phosphate buffer with a pH of 7.5 and incubated for 48 hours at 23 ° C. 25 Subsequently, the polymer beads were 13 placed in a sintered glass filter (porosity of 2 or 3) and, in a suction process, washed in the filter twice with deionized water and then twice with 0.1M of potassium phosphate buffer with a pH of 7.5 which contained 0.05% ethyl-4-hydroxybenzoate. The moisture weight of the resulting beads loaded with penicillin acylase was determined. b) Determination of binder capacity 250 to 300 mg of moisture was added to the polymeric support material (polymer beads) coupled with penicillin amidase to 20 ml of a 2% solution of penicillin F in 0.05 M potassium phosphate buffer with a pH of 7.5, which contains 0.05% ethyl-4- 15 hydroxybenzoate, and is maintained at 37 ° C. The released phenylacetic acid was titrated under stable stirring with 0.5 M NaOH with a constant pH of 7.8 for a period of 10 minutes, during which the consumption of NaOH was recorded. From that moment the polymer beads under a) were collected on a sintered glass filter through 20 ml suction elements with 0.05 ml of a potassium phosphate buffer with a pH of 7.5 containing 0.05% of ethyl-4-hydroxybenzoate, and measurement is repeated twice. 14 N ^ ¡^ c) Calculation of agglutination capacity The linear region of the measured curve (usually the region from 1 to 5 minutes) was used as a basis for the calculation and extrapolated at a 10-minute interval. The agglutination capacity was expressed in units of penicillin amidase per g of moisture of the polymeric support material / U / g humidity). One unit corresponds to one μmol of hydrolyzed penicillin F per minute (μmol / min); in this way 1 liter of 0.5 M NaOH is equivalent to 500 μmol of hydrolyzed G penicillin. (The water content of the polymer support material is approximately constant and therefore can be discarded). Examples 1 to 3 Common test conditions of Examples 1 to 3: In a two liter flask with stirring, the thermometer, water separator, reflux condenser and nitrogen inlet tube were placed in an organic solvent, 3 g of a copolymer comprising 95 parts of n-butyl methacrylate and 5 parts of 2-trimethylammoniomethyl methacrylate chloride as a protective colloid and 5 g of dry ice. Under stirring and nitrogen passage, a monomer phase comprising water and methanol in a ratio of 1: 1.5 as a diluent, plus 10 g of methacrylamide 20 g of allyl glycidyl ether 20 g were dispersed in an organic phase at 50 ° C. of glycidyl methacrylate, and 50 g of methylenebismethacrylamide plus 2 g of 4,4'-azobis-4-cyanovaleric acid (as a polymerization initiator), after which the contents were heated to a boiling point of 65 to 70 ° C. The mixture was incubated for 6 hours and then cooled to room temperature. The resulting polymer beads were sucked, washed and dried in a fluid bed dryer. From this, the agglutination capacity of the penicillin amidase [U / g humidity] and the swelling factor [ml moisture / dry ml] is determined. The main parameters of the test and the results of Examples 1 to 3 are presented in the following table

Claims (10)

CLAIMS 1. A process for synthesis, by inverse polymerization of beads of a monomer phase, of a crosslinked hydrophilic copolymer similar to a bead having an agglutination activity towards the ligaments containing nucleophilic groups, whose monomer phase comprises monomers as a diluent, which contains as monomers a) 5 to 40% by weight of hydrophilic monomers containing a vinyl group, which can overcome the radical polymerization and which form at least 10% of aqueous solutions at temperature. b) 30 to 50% by weight of monomers containing a vinyl group and a functional group, which can overcome the radial polymerization and, in a polymer-like reaction with the nucleophilic groups of the ligaments, which can form covalent bonds c) 20 to 60% by weight of crosslinked monomers containing two or more unsaturated polymerizable ethylene-type groups that can overcome radical polymerization, provided that a), b) and c) add up to 100% by weight, which uses as a diluent a mixture of methanol and water in a ratio of 1: 1.0 to 1: 4.0, the monomer phase is dispersed as droplets in a dispersion medium comprising an organic solvent selected from the aliphatic hydrocarbons with 5 to 7 carbon atoms, the ratio of the monomer phase to the dispersion medium is in the range of 1: 2.0 to 1: 4.0, and that in this form it is subject to radical polymerization in the presence of a polymerization initiator and a protein colloid. ctor, with the proviso that the ratio of monomers to the diluent is in the range of 1: 1.7 to 1: 2.4. 2. A process according to claim 1, characterized in that monomers are used: a) acrylamide and / or methacrylamide b) glycidyl methacrylate and / or allyl glycidyl ether c) methylenebisacrylamide or methylenebismethacrylamide. 3. A procedure according to the
1. Claim 1, characterized in that cyclohexane is used as the organic solvent. 4. A polymeric support material that can be synthesized through a process according to one or more of Claims 1 to 3, characterized in that it has an agglutination capacity for E. coli penicillin amidase of at least 220 [ U / g moisture], resulting from the reaction of 1530 units of penicillin amidase with 5 1 g of polymer support material, and exhibits a swelling factor of at most 1.5. 5. The use of a polymeric support material according to claim 4 for binding proteins. 6. The use of the polymer support material according to claim 5 for the agglutination of enzymes. 7. The use of polymeric support material according to Claim 5 for the 15 agglutination of antibodies. 8. The use of polymeric support material according to Claim 4 in chromatography. 9. The use of polymeric support material according to claim 4 for synthesis of pharmaceuticals. 10. The use of polymeric support material according to Claim 4 for stereospecific synthesis of chiral substances. 25 twenty