DD289289A5 - METHOD FOR PRODUCING HYDROPHILIC POLYMER TRAINERS WITH AMINE OXIDE GROUPS - Google Patents

Abstract

The invention relates to a process for the preparation of hydrophilic polymeric supports with amine oxide groups based on crosslinked acrylonitrile-vinyl acetate copolymers. The copolymers are reacted with polyalkylene diamines, at the same time the polyvinyl acetate portion is saponified to polyvinyl alcohol. The tertiary amino group is then oxidized to the amine oxide by treatment with hydrogen peroxide. The carriers can be used for the chromatographic separation of biological cells or proteins. {Hydrophilic polymeric carriers; amino groups; Acrylonitrile-vinyl acetate copolymers; polyalkylenediamines; chromatographic separation}

Description

It has been found that the object can be achieved if porous copolymers of acrylonitrile and vinyl acetate and a crosslinker are prepared, which are then reacted with a polyalkylonediamine of the formula

η = 2-8 "

R, = H or alkyl group, such as CHj or CjHB-

R ₂ = alkyl group

are reacted and the reaction product is then oxidized to the amine oxide. The reaction with the polyalkylenediamine must be controlled so that the polyacrylonitrile in the copolymer reacts only partially with the amine and the Polyvinylacetatanteil is saponified simultaneously to polyvinyl alcohol. After the reaction with hydrogen peroxide, a carrier with the following structural elements thus results (without consideration of the crosslinker):

-CH ₂ -CH- -CH ₂ -PH- -CH ₂ -CH-

C? ⁰ ^ ¹ (CH) $ '^ ² 0 ^{Q 0H}

The matrix of the support thus has pronounced hydrophilic properties.

The starting point for the solution according to the invention was the finding uaß suitable porous cross-linked copolymers of acrylonitrile and vinyl acetate whose polyvinyl acetate portion was saponified to polyvinyl alcohol, barely have non-specific adsorption of cells and proteins. This property is maintained when the acrylonitrile is partially reacted with a polyalkylene diamine and the reaction product then oxidized with hydrogen peroxide to the amine oxide

Surprisingly, it has been found that even with the use of a polyvinylaromatic crosslinker such as divinylbenzene, the support has no increased unspecific adsorption, although, for example, styrene-divinylbenzene copolymers show pronounced hydrophobic interaction with biological macromolecules which are unspecific in nature.

As crosslinkers for the polymers according to the invention it is thus possible to use all polyvinyl compounds which copolymerize with acrylonitrile and vinyl acetate. In addition to the aromatic polyvinyl compounds, such as divinylbenzene, it is preferred to use derivatives of methacrylic acid, such as ethylene glycol dimethacrylate or trimethylolpropane trimethacrylate, and also derivatives of acrylic acid, such as triacryolyl-S-trihydrotriazine. However, it is also possible to use mixtures of these crosslinkers.

The amount of crosslinker can be varied within wide limits. Preference is given to 1-20Ma .-%, based on the polymerizable phase used.

The porosity of the matrix can be produced in a known manner by addition of precipitants and / or swelling agents during the polymerization, which are inert. Have proven useful as precipitating aliphatic hydrocarbons, such as. As gasoline mixtures or longer-chain alcohols, such as. B. n-octyl alcohol and as swelling agent aromatic hydrocarbons such. B. toluene.

The amount of inerting agent used is preferably 10 to 60% by mass, based on the total oil phase of monomers and inerting agent. The porosity of the copolymer can be controlled within wide limits by the nature and amount of the inert agent and by the Vernetzeranteil.

By the proportions of the comonomers acrylonitrile and vinyl acetate used, the hydrophilicity of the matrix and the capacity of the fun 'Onellen amine oxide groups of the carrier can be varied. The polymerizable phase preferably contains 20-89% by weight of acrylonitrile, 10-60% by weight of vinyl acetate and 1-20% by weight of crosslinker. In the case of the use of technical divinylbenzene occurs as a further comonomer ethylstyrene. The polymerization is carried out by the suspension polymerization method in a known manner. For example, cellulose derivatives can be used as suspension stabilizers. The polymerization process is controlled so that polymer beads having a diameter of 0.1-0.3 mm are obtained.

As polyalkylenediamines for the reaction of the copolymer are preferably the compounds 2-dimethylamino-1-ethylamine or

2-diethylamino-1-ethylamine or

3-dimethylamino-1-propylamine or

3-diethylamino-1-propylamine for use.

The reaction temperatures are preferably 90-140 ° C adjusted. The reaction time is dependent on porosity, degree of crosslinking and amine used at 2-20 hours.

Reaction temperature and time are chosen so that the acrylonitrile in the copolymer is reacted only a maximum of 95%, ie in the infrared spectrum in each case the C = N valence vibration at the wavenumber of about 2 200cm " ^{1 is} still clearly detectable.

To complete the hydrolytic reactions occurring in the treatment with the polyalkylenediamine, the resin is washed extensively with water until neutral.

The reaction of the ternary amino groups of the resin, which behaves like a weakly basic ion exchanger, to give the amine oxide takes place according to the instructions given in PS DD 131.859. Here, the resin at room temperature with approx.

Treated 30% hydrogen peroxide solution in the presence of physiological saline and sodium hydroxide solution.

Due to the good hydrodynamic properties in separation columns and the extremely low unspecific adsorption of proteins and biological cells, the polymer according to the invention makes it possible to carry out protein and cell separations by the method of patent specification DD 131.721 with significantly increased selectivity and yield.

Thus, it is possible, for example, pre-purified glucose oxidase from Penicillium notatum with a specific activity of 180 bis

200 U / mg of protein by means of this method in conjunction with the polymer according to the invention to increase to a specific activity of 350 to 400 U / mg protein and thus to obtain an electrophoretically uniform enzyme.

It also succeeds with this method in conjunction with the polymer according to the invention, one after morphological Evaluation of complete separation of blast cells and lymphocytes from human blood in Chronic Myelogenous Blast-stage leukemia. In the following examples, the invention will be explained in more detail. Ausführungsbelsplele example 1 polymerization In a plane ground cup 1.51 dist. Submitted H ₂ O and this 2g M hylzellulose dissolved with stirring. To the aqueous phase, the oil phase, consisting of 170 g of acrylonitrile, 90 g of vinyl acetate, 40 g techn. Divinylbenzene (about 50%), 1 g Porofor N and 200g toluene are added and by controlling the stirring speed a droplet size of 0.1-0.3mm

set. The polymerization is carried out at 60 ⁰ C and the copolymer is then cured for 8 hrs. At 85 ° C.

Subsequently, the copolymer is washed with water and after expelling the inert solvent toluene (by Treatment with steam) dried.

aminolysis

75 g of the dried copolymer are in a sulphonation with 600ml of 3-dimethylamino-1-propylamine, 9E% ig, 8 hr.

stirred at 11C ⁰ C. The excess amine is then aspirated and the resin with distilled water to the

Washed neutrality. 350 ml weakly basic ion exchangers with the following characteristics are obtained: Total weight capacity: 2.85 mmol / g dry resin in CT form Water content: 78.0Ma .-%. ammoxidation

100 ml of the salt swollen in physiological saline are stirred at room temperature with 100 ml of physiological saline and 100 ml of 30% hydrogen peroxide solution and titrated with 0.1 N to 0.2 N sodium hydroxide solution to a pH of about 11. After 25 minutes, the supernatant is filtered off with suction and the reaction is repeated in the same order. Subsequently, the resin is washed with distilled water to neutrality. According to IR spectroscopic findings and acid / base titration, the resin is fully converted according to this provision into the amine oxide form. The resin volume and the anion exchange capacity remain unchanged.

Example 2 For the aqueous phase prepared according to Example 1, the oil phase consisting of acrylonitrile, vinyl acetate, divinylbenzene, Ethylene glycol dimethacrylate and gasoline mixture and 1 g Porofor N added and as described in Example 1

polymerized. 25 g of the dried copolymer are stirred for 8 hours at 130 ° C. with 200 ml of 3-diethylamino-1-propylamine.

100 ml weakly basic anion exchangers with the following characteristics are obtained: Total weight capacity: 2.73 mmol / g dry resin in the Cl ~ form Water content: 72.5Ma .-% The amine oxidation is carried out according to the procedure given in Example 1. Example 3 Purification of glucose oxidase (Penicillium notatum) To the suitability of the polymer according to the invention with amine oxide groups according to Example 1 for protein separation after To demonstrate PS DD 131,721, a pre-purified enzyme solution of about 20,000 units of glucose oxidase to a Chromatography column (1.5 cm x 20.0 cm) applied with the polymer of the invention and foreign proteins in a Flow rate of 30 ml / h with 0.01 M Na acetate buffer, pH 5.5. Elution of the glucose oxidase was with 10 mM Phosphate buffer, pH 7.5.0.3m NaCl made. The glucose oxidase appears as a sharp peak in the eluate. The specific one Product activity increased from 180 to 200 U / mg protein to 350 to 400 U / mg protein. The purified in this way Glucose oxidase is electrophoretically uniform. Examples Separation of Blast Cells and Lymphocyte from Human Blood in Chronic Myeloid Leukemia (CML) Im Blast Stadium To the suitability of the polymer according to the invention with amine oxide groups according to Example 1 for cell separation after PS DD 131.721 to demonstrate, was the clinically still insufficiently solved task of the separation of Blast cells from blood / bone marrow mixtures examined. 5ml of blast stage human CML blood was loaded on a chromatography column (3.5cm x 5.5cm) with the

polymer and the blast cells at a flow rate of 2 ml / min with 34 m M HEPES-buffered Eagle MEM medium, pH 5.6, entfornt. The elution of the lymphocytes was the same with medium

Composition, but made pH 8.0. The cytomorphological evaluation of the separated cell fractions gives the following picture:

Control I.Fraction 2. Fraction (%) (%) (%)

Myeloblasts 64 78 Lymphocytes 11 1 100

other cells 35 21 -

(without erythrocytes)

The yield with respect to the lymphocytes (2nd fraction) is about b0%.

Claims (3)

1. worship for the production of hydrophilic polymeric carriers with amine oxide groups, characterized in that from acrylonitrile, vinyl acetate and a crosslinker in the presence of a precipitant and / or swelling agent for the copolymer by the process of suspension polymerization, a copolymer having a porous structure and a particle size of 0 , 1-0.3mm, this copolymer with an α, ω-polyalkylenediamine of the formula H oo \ / "= R ₁ = HoderAlkylgruppe ^N "( ^CH 2) n" ^N \ R ₂ = alkyl group _R / R ₂ η = 2-8 treated at temperatures of 90 to 140 ⁰ C and then the tertiary amino group of the resulting weakly basic ion exchanger is hydrogenated with hydrogen peroxide to the amine oxide. 2. The method according to claim 1, characterized in that divinylbenzene or derivatives of acrylic or methylacrylic acid or mixtures of these crosslinkers are used as crosslinkers. 3. The method according to claim 1 and 2, characterized in that in the treatment of the copolymer with the polyalkylenediamine, the acrylonitrile only partially, up to a maximum of 95 Ma .-%, aminolysed and the vinyl acetate is saponified simultaneously to the polyvinyl alcohol. Field of application of the invention The invention relates to a process for the preparation of hydrophilic polymeric carriers with amine oxide groups based on crosslinked copolymers. These carriers can be used for the chromatographic separation of biological cells or proteins. Areas of application include the fractionation of blood and bone marrow cells in clinical transplantation and transfusiology, the selection of cells with special metabolic activities and the purification of proteins from cell culture supernatants in biotechnology. Characteristic of the known state of the art The patent DD 131.859 describes the preparation of water-insoluble polymeric supports with N-oxide groups based on natural macromolecules. The term "natural macromolecules" encompasses polysaccharides such as cellulose or agarose, polysaccharide derivatives such as crosslinked agarose or dextran vern which have been crosslinked with epichlorohydrin To introduce the amine oxide groups, these macromolecules must carry tertiary amino groups, for example diethylamino groups, which are then treated by treatment with However, these known carriers have a number of unfavorable properties, such as being susceptible to microwave and enzymatic degradation, and having unsatisfactory hydrodynamic properties in conventional columnar methods However, separation of cells and proteins based on interaction with the functional amine oxide groups of the carriers has been shown to be nonspecific adsorption of the species to be separated on the matrix of natural macromolecules Sorption of the cells or proteins to be separated on the matrix impair the selectivity of the separation and significantly reduce the achievable yield of separate cells or proteins. Similarly, so-called double or tandem groups, which are unavoidable in the introduction of tertiary amino groups into natural macromolecules, are effective. This subsequent reaction of a tertiary amino group with another tertiary amino group converts the former to a positively charged quaternary amino group which undergoes undesirable ionic relationships with the cells or proteins to be separated. Object of the invention The aim of the invention is therefore a process for the preparation of such polymeric carriers, which no longer have these disadvantageous properties. Explanation of the essence of the invention The object of the invention is to provide a process for the preparation of a polymeric support by not starting from natural macromolecules, but by the copolymerization of suitable vinyl compounds by the suspension polymerization method builds up a support matrix. In this matrix tertiary amino groups are introduced, which are then oxidized in a known manner to the amine oxide.