DD289203A5 - PROCESS FOR THE PRODUCTION OF ADSORBENTS FOR IMMUNE LOBULINS - Google Patents

Abstract

The invention relates to a process for the preparation of selective hydrophilic adsorbents with primary, secondary or tertiary amino groups. The adsorbents are prepared by copolymerization of acrylonitrile, vinyl acetate and a crosslinker and subsequent reaction with a polyalkylenepolyamine. They can be used to separate and / or recover class M immunoglobulins from aqueous solutions. {Adsorbent, selective; Immunoglobulin M; copolymers; acrylonitrile; vinyl acetate; crosslinking agents; polyalkylenepolyamine}

Description

Field of application of the invention

The invention relates to a process for the preparation of selective adsorbents for immunoglobulins of class M (IgM). These adsorbents can be used for the separation and / or recovery of IgM from aqueous solutions, in particular blood plasma or blood serum in medicine, medical technology and IgM Präpaiation.

Characterization of the known prior art

In the literature, immunoadsorbents are described which are suitable for binding or removing IgM from blood plasma or blood serum (Bensinger, WI et al .: "Immunoadsorption for removal of A and B blood group antibodies" New Engl. J. Med. 304, 160- 162 [1981]; Banned, AD et al .: "Immunoadsorption and renal transplantation in two patients with major ABO incompatibility." Transplantation 43, 909-911 [1987]; Osterwalder, B. et al.: Immunoadsorption for removal of anti-A and anti-B bloodgroup antibodies in ABO-incompatible bone marrow transplantation. "Blood 53, 379-390 [1986]).

This immunosorbents are used, which are prepared by coupling of corresponding antigens for IgM to Silikatg.anula. The "matching" IgMs then bind to these adsorbents, but the production of the adsorbents is complicated and complicated, so that they are expensive.

Furthermore, it is known that adsorbents on Dextranbasis with Anionenaustauschercharakter such. As DEAE-Sephadex, for the preparation of IgM can be used. However, these adsorbents show only a low selectivity for IgM, so that the preparation of pure IgM from blood serum in addition to binding to the adsorbents comprises three further steps. The known method for the preparation of IgM is therefore labor intensive and costly.

Object of the invention

The object of the invention is to provide a simpler and more cost-effective method for the production of immunororbents for IgM, wherein the adsorbents should have a high selectivity for these species.

Explanation of the essence of the invention

The object of the invention is to develop a process for the preparation of selective adsorbents for IgM based on plastic.

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 an α, ω-polyalkylenediamine of the formula

R ₁ = H odor alkyl group, such as CH ₃ or C ₂ H ₅ -

be implemented. 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. This produces a carrier with the following functional groups:

-CH ₂ -CH- -CH ₀ -CH- -CH ₂ -CH-

/ OH

C = N

The matrix of the support thus has pronounced hydrophilic properties. The starting material for the solution according to the invention was the finding that suitable porous cross-linked copolymers of Acrylonitrile and vinyl acetate whose polyvinyl acetate portion was saponified to polyvinyl alcohol, hardly a nonspecific Adsorption of bioactive materials. Surprisingly, it has been found that even when using a polyvinyl aromatic crosslinker, such as divinylbenzene, the Carrier has no non-specific adsorption, although, for example, styrene-divinylbenzene copolymers oine pronounced

show hydrophobic interaction with bioactive materials that are non-specific in nature.

As a crosslinker for the polymers according to the invention thus all polyvinyl compounds can be used with the Copolymerize acrylonitrile and vinyl acetate. In addition to the aromatic polyvinyl compounds, such as divinylbenzene, are

preferably derivatives of methacrylic acid such as ethylene glycol dimethacrylate or trimethylolpropane trimethacrylate and derivatives of acrylic acid such as triacryolyl-S-trihydrotriazine applied. However, mixtures of these crosslinkers can also be used.

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

Polyrnerisationsfähige phase used.

The porosity of the matrix can in a known manner by addition of precipitants and / or swelling agents in the polymerization, which

inert behavior, be generated. Have proven useful as precipitating aliphatic hydrocarbons, such as. As gasoline blends or longer chain alcohols, such as. B. n-octyl alcohol and as swelling agent aromatic hydrocarbons, such as. B. toluene.

The amount of the inert agent used is preferably 10 to 60% by mass, based on the total oil phase of monomers

and inert agents. The porosity of the copolymer can be controlled within wide limits by the type and amount of the inert agent and by the Varnetzeranteil.

By the proportions of the comonomers acrylonitrile and vinyl acetate used, the hydrophilicity of the matrix and the capacity

the functional amino group of the carrier can be varied. The polymerizable phase preferably contains 20-89% by mass

Acrylonitrile, 10-60Ma .-% vinyl acetate and 1-20Ma .-% crosslinker. In the case of the use of technical divinylbenzene occurs as

further monomer ethylstyrene. The polymerization is by the method of suspension polymerization in known

Manner performed. For example, cellulose derivatives can be used as suspension stabilizer. The Polymerization process is controlled so that polymer balls are obtained with a diameter of 0.1-0.3 mm. As polyalkylenediamines come to the implementation of the copolymer, starting from the general formula

<. R ₁

Ri = H or alkyl group, such as CHj or C ₂ Hs

the compounds of ethylenediamine to hexamethylenediamine and their alkyl derivatives, such as. For example, 3-dimethylamino-ipropylamine or 3-diethylamino-1-propylamine can be used. The resulting weakly basic anion exchangers thus have as functional groups primary, secondary or tertiary amino groups. As reaction temperatures 90-140 ⁰ C are preferably set. The reaction time is, depending on porosity, Verneuungsgrad and amine used, at 2-20 hours. Reaction temperature and time are chosen so that the acrylonitrile in the copolymer is only a maximum of 95% implemented, ie, in the infrared spectrum in each case the C ^ N valence vibration at the wavenumber of about 2200cm ~ ¹ , still clearly detectable.

To complete the hydrolytic reactions taking place in the treatment with the polyalkylenediamine, the resin is luagently washed with water until neutral or treated alternately with HCl / NaOH at room temperature and then likewise washed with water until neutral.

Before use, the adsorbents should be adjusted to pH values of 5.5 to 7.5. To remove the proteins from the blood serum or plasma, the adsorbents may be directly suspended therein or applied as a packing in a chromatography column. In both cases, the adsorbents are first several times with distilled water. washed and the washing liquid, ζ. B. by centrifugation, largely removed. They are then contacted with 0.1 M glycine-HCl buffer, pH 2.8 or with 0.01 M HCl, after removal of the acidic solutions with phosphate-buffered 0.15 M NaCl solution (PBS), pH 7, 4 washed several times until the adsorbent is equilibrated in the desired pH range. Before use, the PBS is removed as completely as possible and the residual moist adsorber substances with the protein-containing aqueous liquid, eg. As serum, brought into contact. This can be done in a simple manner by suspending the adsorbents in the serum and, with gentle shaking, for 10 to 120 minutes, e.g. incubated at room temperature or 37 ° C. This method can be used to prepare IgM. For this purpose, the adsorbents are washed after contact with serum with 0.15M NaCl solution and subsequent desorption with acidic solutions or higher concentrated salt solutions, pH 4.5 to 6.5, made. The eluates contain in addition to the high molecular weight IgM some plasma proteins with molecular weights below 200,000 daltons

such as the complement factors C3, C4 and C9, prealbumin, O ₁ and dj glycoprotein and ceruloplasmin. You can in a Nachreinigungsschritt, z. B. by gel chromatography on Sephadex G 200, are separated. The described method for IgM preparation represents a substantial simplification of the hitherto customary methods, since instead of the 4 process steps required according to the prior art, only 2 steps are required to obtain pure IgM. The decisive step here is the enrichment of the IgM on the adsorber produced according to the invention.

embodiments

example 1

polymerization

In a plane ground cup 1.51 dist. Submitted H ₂ O and herein 2g methyl cellulose dissolved with stirring.

To the aqueous phase, the oil phase, consisting of 170 g of acrylonitrile, 90 g of vinyl acetate, 40g techn. Divinylbenzene (about 50%), added 1 g Porofor N and 200g toluene and adjusted by controlling the stirring speed, a droplet size of 0.1-3.0mm. The polymerization is carried out at 60 ° C and the copolymer then 8 hours at 85 ⁰ C cured.

Subsequently, the copolymer is washed with water and dried after expelling the inert solvent toluene (by treatment with steam) and sieved to a particle size of 0.1-3.0 mm.

Example 2

Production of an adsorber with primary amino groups

00g copolymer, prepared according to Example 1, are mixed with 350 ml of 1,3-diaminopropane and the suspension 8 hrs. At 11O ⁰ C stirred.

The resin is mixed with dist. H ₂ O and then at room temperature twice in alternation with HCI / NaOH, 5%, treated.

After the last NaOH treatment, the adsorber is treated with dist. H ₂ O washed neutral.

There are obtained 170 ml adsorber with the following characteristics:

Anion exchange capacity (mmol / g dry adsorbent): 2.86

Water content (g / g dry adsorber): 3.0

Swelling (ml / g dry adsorber): 4.4

Example 3

Production of an adsorber with tertiary amino groups

75 g of copolymer prepared according to Example 1, with 600ml of 3-dimethylamino-i-propylamine 8h. stirred at 11O ⁰ C.

The resin is mixed with dist. H ₂ O and then at room temperature twice in alternation with HCI / NaOH, 5%, treated.

After the last NaOH treatment, the adsorber with. least. H ₂ O washed neutral.

There are obtained 350 ml of adsorber with the following characteristics:

Total weight capacity (mmol / g dry adsorber): 2.76

Water content (g / dry adsorber): 3.6

Swelling (ml / g dry adsorber): 7.4

Example 4

Protein amounts in the plasma after incubation with the adsorbers 1 and 2 .. based on the protein amounts before the adsorption of the plasma (= 100, indicated in%).

protein Adsorber 1 Adsorber 2 prealbumin 0 albumin 73 + 0 67 ± 6 α, glycoprotein 40 ± 6 - 0.2-glycoprotein 30 + 6 - haptoglobin 67 ± 1 53 ± 4 hemopexin 79 ± 7 88 ± 3 ceruloplasmin 0 0 a ₂ -macroglobulin 78 + 5 84 + 4 transferrin 86 + 5 81 ± 3 ß-lipoprotein 85 ± 3 80 ± 1 IgG 79 ± 5 84 ± 3 IgA 70 ± 4 72 + 4 IgM 17 + 0 20 ± 2 IgD 90 ± 10 90 ± 2 IgE 79 ± 3 85 ± 3 C, q 76 ± 4 76 ± 9 C4 0 - C3 32 ± 6 5 ± 0 C9 0 - C 1INA 26 ± 4 - C3-activator 85 ± 4 -

Adsorber 1: prepared according to Example 2 Adsorber 2: prepared according to Example 3

Claims (3)

1. A process for the preparation of adsorbents for immunoglobulins of class M, 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 0.1-0.3 mm is prepared and this then with an α, ω-polyalkylenepolyamine of the formula Ri = H or alkyl group η = 2-8 at temperatures of 90-140 ⁰ C is used. 2. The method according to claim 1, characterized in that are used as crosslinking agents for the copolymer divinylbenzene or derivatives of acrylic or methacrylic acid or mixtures of these crosslinkers. 3. The method according to claim 1-2, characterized in that in the treatment with the polyalkylenepolyamine the acrylonitrile only partially, up to a maximum of 95Ma .-%, aminolysiert and the vinyl acetate content while saponified to polyvinyl alcohol.