FI61811B - ANALYSIS OF A BIOLOGICALLY ACTIVE PRODUCT IN THE FIELD OF A BIO-BASED PRODUCT AND A BIOLOGICALLY ACTIVE AEMNE SAOSOM ADSORPTIONSMEDEL FOER AFFINITETSKROMATOGRAFI - Google Patents

Description

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Patent and Registration Office (44) Date of issue and date of publication——

Patent and registration authorities Ansdkan utlagd och utl.skrlftan published 30.06.82 (32) (33) (31) Pyyd «« y atuolkaus — Begird prloritet 06.05 · 7 ^

Federal Republic of Germany Förbundsrepubliken Tyskland (DE) P 21 + 21789.8 (7 :) iH'hringwerke Aktiengesellschaft, Marburg / Lahn, Federal Republic of Germany-Forbund srepubliken Tyskland (DE) (73) Rudolf Sc.'hmidtberger, Marburg-Marbach, Heinrich Huemer Schwalbac'n /

Taunus, Federal Republic of Germany Tyskland (DE) (YM Oy Kolater Ab (5) 1) Use of a biologically active product consisting of a water-insoluble carrier and a biologically active substance as an adsorbent in affinity chromatography - Användning av en biologiskt active substance product bärare och et biologisktti ämne säsom adsorptionmedel för affinitetskomatografi (62) Separated from application 751299 (patent 60216) -Avdelad fran ansökan 751299 (patent 60216)

The invention relates to the use of a biologically active compound consisting of a water-insoluble carrier and a chemically bound biologically active substance as an adsorbent in affinity chromatography, which non-water carrier is polyhydroxymethylene, and which biologically active substance is an amino group or amino group-containing substance such as substance or other plasma protein, and which biologically active substance is bound to the carrier, possibly via a polyfunctional organic compound.

In recent years, more and more new techniques have emerged in the field of biochemical working methods, the primary feature of which is the exploitation of the affinity of biologically active substances bound to carriers in selectively feasible reactions.

Utilizing the specific complexing ability of the carrier-bound substance and the other substance in the mixture, the substance in question can be removed from the mixture and subsequently isolated, if desired, by desorption.

Thanks to the enzymes bound to the support, the advantage is obtained that the modifications of the substances can be carried out in preparative, continuous processes and the reaction products are recovered without enzymes.

In biochemical, enzymatic analysis, non-water-based enzymes are available as reagents and can be used several times. Because enzymes are characterized by affinity not only for substrates but also for specific inhibitors, the preparation of enzyme inhibitors has proven to be particularly advantageous with affinity chromatography on carrier-bound enzymes. On the other hand, the binding of inhibitors to water on unreaded carriers allows the preparative preparation of the corresponding enzymes.

Antigens or antibodies, as so-called immunoadsorbents, bind to water on innumerable carriers and thus make it possible to isolate the corresponding antibodies or antigens.

As described above, biologically active substances refer to in vivo and in vitro active naturally occurring and artificially prepared substances, which in the broadest sense may be referred to as enzymes, antigens or antibodies.

Most of the carrier-bound biologically active agents described so far are substantially better preserved than the corresponding agents in solution.

As carriers, so-called carriers, it is preferable to use only those substances whose properties include, in addition to insolubility in aqueous systems, the lowest possible non-specific adsorption capacity. Therefore, the hydrophobic, hydrophilic and ionic interaction between the carriers and the reactant of the biologically active substance should not occur with substances which are not reactants of the biologically active substance and in no case form bonds with the 6 6 1 8 ' 1 '1 carriers are e.g. those that bind by physical adsorption of the active ingredient; these include activated carbon and glass beads. The second group of carriers consists of those substances to which it binds via a covalent bond. The latter include vinyl polymers, e.g. polyacrylic acids, polyacrylic acid pyramids and polystyrene having an amino, carboxy or sulfonyl substituent, further cellulose and its derivatives, and finally naturally occurring and synthetic polypeptides and proteins. Due to the steady interaction between carriers and biologically active substances, the use of carbohydrates, especially cellulose, dextran, starch, agar or their derivatives as carriers in aqueous systems has reached the widest prevalence, although the carboxyl groups of many of these naturally occurring substances In addition, the thermal and chemical stability of these substances is relatively low.

It has now been found that the disadvantages of carbohydrates used as carriers in connection with affinity-dependent reactions can be counteracted if polyhydroxymethylene is used as the carrier.

The adsorbent of the invention is characterized in that the non-water carrier is polyhydroxymethylene, and the biologically active substance is an amino group or amino group-containing substance such as an enzyme, antigen or antibody or other pasma protein, and the biologically active substance is bound to the carrier by a possibly polyfunctional organic compound. through.

Polyhydroxymethylene is a synthetic polymer with a single carbon chain. Each carbon atom has a hydrogen atom and a hydroxyl group. Its manufacture and properties have been explained e.g.

N. D. Field, J.R. Schaefgen, J. Polymer Sci., Vol. 58, 533-543 (1962) and G. Smets, K. Hayashi, J. Polymer Sci., Vol. 29, 257-274 (1958). It can be prepared by base or acid hydrolysis from polyvinylene carbonate. Polyhydroxymethylene can be considered as a derivative of polyvinylene carbonate. Binding of the biologically active substance to polyhydroxymethylene can be achieved, on the one hand, by reacting the hydroxyl group of the biologically active substance carrier with covalent bonds, on the other hand by reacting the polyvinylene carbonate via polyvinylene carbonate substituted by a specer or a biologically active substance, the remaining cyclocarbonate groups of which are then saponified to hydroxyl groups: ---! CH-ClT ----- ---- CH-CH | —CH-CH ----- '1; f j! + R-NH ~ ' | OH 0 saponification 0 O O O l '> \ \ ^ I c = o! C 'C f i "i";

0 n! NH

'"- J; n-1 polyvinylene carbonate -Γ CH-CiT] -CH-CH ---- | I I> I f I OH OH; OH o;;; .o l In.— 1'

- - * NH

R

It is particularly advantageous for the use of the products that the biologically active substances are not allowed to react directly with the polyhydroxymethylene, but are allowed to bind to the support via covalent bonds via a so-called spacer.

Spacer substances are most often hydrocarbon-based and have a concentration of 1-2 nm. Thanks to the two reactive end groups, the spacer can react with both the carrier and the biologically active substance, so that both polyhydroxymethylene and polyvinylene carbonate can be used in the reaction with the carrier under suitable conditions.

Several commonly known reactions can be found for the incorporation of biologically active substances into a covalent dox carrier: 5,61811

A particularly simple method is based on the activation of the hydroxyl group of polyhydroxymethylene by means of cyanogen halides, mainly bromocyanine, and the reaction of biologically active substances containing amino groups via these activated groups.

Another method is based on reacting a polyhydroxymethylacin such as a polysaccharide with a halocarboxylic acid such as chloroacetic acid in an alkaline environment using a variation of the azide method (Curtius), the corresponding acid being esterified with an alcohol, then the ester converted to a hydrazide and finally the azido group-containing azide thus formed is incorporated into a polyhydroxymethylene matrix.

The biologically active agent can also be attached to a polyhydroxymethylene support by first acylating the hydroxyl groups with bromoacetyl bromide, followed by alkylation of the amino group of the biologically active agent.

Similar reactions are effected, for example, by reacting the hydroxyl groups of the carrier with reactive triazines, whereby some of the reactive groups of the triazine react with the polyhydroxymethylene compound, part with the amino group of the biologically active substance.

Diazotizable aromatic amines, which on the one hand can bind to the hydroxyl groups of the support via another reactive group, on the other hand, allow the formation of a bond with, for example, tyrosine or histidine residues of activated proteins capable of this.

Vinyl sulfone derivatives containing arylamino groups and sulfuric acid half-esters of β-hydroxyethyl sulfones can be reacted with hydroxyl groups of the support. They allow the biologically active substance to bind after the diazotization reaction described above.

A particularly strong ether bond is formed by reacting the hydroxyl groups of polyhydroxymethylene with non-ionic epoxides having at least 2 reactive groups, such as epihalohydrins or polyepoxides, for example epichlorohydrin or bisepoxide.

There are other methods for forming a covalent bond between the hydroxyl groups of polyhydroxymethylene and the amino groups of a biologically active substance, such as the known reaction via complexing metal compounds, e.g. titanium compounds.

6 6181 1

In addition to said chemical and thermal stability, polyhydroxymethylene has other excellent properties. Thus, it can be easily modified into a fiber, yarn, film or grain shape, so the shape can be selected to suit the application. The surface area of the polyhydroxymethylene is also easily adjustable.

According to the corresponding reaction mechanisms described above in connection with the incorporation of a biological agent, it can also be attached to a support if it has an amino group as one of the reactive groups. The incorporation of a spacer offers the possibility of attaching a biologically active substance in other ways, for example in the case where the spacer bound to the matrix has a free carboxyl group, this carboxyl group can be activated by, for example, carbodiimide compounds which can then form an AIDS bond with the biologically active substance. Carboxyl groups further allow the formation of amide bonds with the isoxazolium salts proposed by Woodward.

If a free amino group is available in the matrix-bound spacer, vinyl sulfone derivatives or sulfuric acid esters of β-hydroxyethyl sulfones containing aryl amino groups can be used to extend the spacer to arylamino groups, which are then diazotized by known methods and bound to the corresponding reactants of the biologically active compound.

I OH j 0 OH

σ ^ \ h- (ch2) 6-nh-ch2-ch2-so2- ^ ~ ^ - nh2

Due to their specific adsorption properties, biologically active substances bound to carriers have wide use in affinity chromatography. Highly pure enzyme products can be prepared with carrier-bound naturally occurring or synthetic enzyme inhibitors, and carrier-bound enzymes, in turn, have been found to be remarkably well suited, especially for the recovery of natural enzyme inhibitors from crude extracts. Carrier-bound water-insoluble antigens are used to isolate the relevant antibodies, which in this way 7,61811 are obtained free of other serum moieties and free of other antigens. Unprecipitated antibodies as well as substances which cannot be precipitated can also be isolated by affinity chromatography because their concentration in the serum is low and the assay can also be performed quantitatively.

To illustrate the invention, the following examples have shown that a polyhydroxymethylene carrier is suitable for binding a wide variety of biologically active substances, and that polyhydroxymethylene can be made suitable for binding virtually any desired biologically active substance by suitable substitution.

By way of example, it is further indicated to which matrix-bound biologically active substances can be used.

Example 1

Polyhydroxymethylene-tetanus toxoid compound Preparation of a matrix from (to-amino-n-hexyl) -substituted polyhydroxymethylene:

To a mixture of 20 g of polyhydroxymethylene suspended in 900 ml of water is added a solution of 20 g of BrCN in 50 ml of dimethylformamide and the pH is maintained under stirring and at an internal temperature of 15 ° C for 6 minutes by slow dropwise addition of 2- n soda liquor. It is then filtered immediately, washed well with ice water and pressed in half. The polyhydroxymethylene thus activated, still moistened with water, is added to a well-stirred solution of 20 g of hexamethylenediamine in 500 ml of H 2 O, adjusted to pH 8.5 with concentrated HCl, kept at room temperature, the total volume being adjusted to 1 liter by adding water. the value is kept continuously between 8.5 and 9.0 and stirring is continued for another 5 hours at room temperature. It is then filtered and washed well with water. The dried sample of the substituted polyhydroxymethylene of the (6) -amino-n-hexyl) group had a nitrogen content of 1.9%. Addition of Sanger's reagent to detect primary free amino groups yields intensely yellow stained products.

Binding of the tetanus antigen to the support 10 g of the support of Example 1 are suspended in 200 ml of 0.5 M sodium phosphate solution, pH 10. The suspension is heated with stirring at 50 ° C and 5 g of 1-aminobenzene-4- [3-hydroxy- etyylisulfonirikkihappopuoliesteriä. After adjusting the pH to 8,618,11 with 2-NaOH, the batch is stirred for one hour at 50 ° C. It is then filtered through a suction filter and washed with 2 l of water, 2 l of acetone and 2 l of 0.2 M HCl. To diazotize the arylamine groups attached to the polyhydroxymethylene, the filter residue is suspended in 200 ml of 0.2 M HCl, cooled to 2 ° C and 1 M NaNO 2 solution is added with stirring until a slight excess of nitrite is detected on KJ starch paper. After 10 minutes, filter through a suction filter and wash the filter residue with 1 liter of 2 ° C, 0.01 M aqueous amidosulphonic acid solution and then with 200 ml of 0.2 M sodium phosphate, pH 7.5 and temperature 2 ° C. For the coupling reaction, the product containing diazonium groups is suspended in 150 ml of a solution of 2 g of tetanus toxoid (1,260 Lf / mg N) in 0.2 M sodium phosphate, pH 7.5 and temperature 2 ° C, and stirred 20 hours at 5 ° C. The unbound parts of the toxoid are washed off with a suction filter with 1 liter of 1 M NaCl solution.

Use of polyhydroxymethylene tetanus toxoid

In the preparation of tetanus antitoxin, 10 g of the product of Example 1 are suspended in 150 ml of 0.15 M NaCl solution to which 1/15 mol / l sodium phosphate, pH 7.2, has been added and applied to a column 3 cm in diameter and 10 cm high. cm. 20 ml of tetanus horse serum, 1650 IU (International Units) of tetanus antitoxin / ml are applied to the column, which is then washed with 500 ml of sodium phosphate buffer solution, pH 7.2. The antibody is eluted with 0.5 M glycine / HCl buffer pH 2.5. After neutralization with 2-m NaOH and subsequent centrifugation, the eluate contains a total of 240 mg of protein and 12,300 IU of tetanus antitoxin, which is determined by a protective experiment in mice.

Example 2

Polyhydroxymethylene-aminobenzamidine compound Preparation of a matrix from (u-carboxy-n-benzyl) -substituted polyhydroxymethylene After activation with BrCN according to Example 1, 20 g of polyhydroxymethylene are added to an aqueous solution of 25 g of ε-aminocarboxylic acid at pH with sodium hydroxide solution to 8.5 and a total volume of 1 liter. The clarification is continued for a further 5 hours at room temperature at pH 8.5 and filtered to 9,618 ° C and washed well with water. The nitrogen content of the dried sample of the (61-carboxy-n-pentyl) group taken from the substituted polyhydroxymethylene was 0.86%.

Binding of aminobenzamidine to the support 2 g of the polyhydroxymethylene derivative prepared according to Example 2 are suspended in 25 ml of 0.1 m of morpholinoethanesulfonic acid and 1 g of 1-ethyl-3- (3-dimethylaminopropyl) -corboximide hydrochloride is added. Add 2 M HCl until the pH is 4.8. 1 g of m-aminobenzamidine is added to the batch with stirring. The suspension is stirred with pH adjustment for one hour and the adsorbent is then transferred to a chromatography column with a diameter of 1 cm. The adsorbent is rinsed with 200 ml of 0.05 M Tris-hydroxymethylaminomethane-HCl buffer (pH 8.0) to which 0.5 M KCl (Tris-KCl) has been added.

Use of Polyhydroxymethylene-Aminobenzamidine for the Preparation of Thrombin 100 mg of crude bovine thrombin obtained are dissolved in 5 ml of 0.05 M Tris / 0.5 M KCl solution, pH 8.0, and applied to a column pretreated as described above. The column is then rinsed with 200 ml of Tris-KCl buffer. Thrombin bound to insolubilized m-aminobenzamidine is cleaved from this in 100 ml of a solution of 0.05 m m-aminobenzamidine in Tris-KCl buffer.

The protein-containing fractions are pooled and run in Tris-KCl buffer through a Sephadex G-25 column (manufactured by Pharmacia, Uppsala, Sweden; column size 1 x 30 cm). The first portion of the column-containing column eluate was found in Chase and Shaw's Biochem. 8, 1969, p. 2212, containing 78% of the activity of the starting material.

Example 3

Polyhydroxymethylene-pepsin formulation Preparation of carrier from (to-amino-n-hexyl) -substituted polyhydroxymethylene: 8.0 g of polyvinylene carbonate reprecipitated from dimethylformamide / methanol (preparation: see ND Field, JP Schaefgen, J. Polymer). , Vol. 58, p. 534 (1962)), is suspended at room temperature in a solution of 7.5 g of hexamethylenediamine in 180 ml of methanol and stirred for 48 hours at room temperature, filtered, washed with methanol and kept for 4 days. 61 81 l suspended in a solution of 10 g of sodium methylate in 300 ml of 96% methanol, washed with methanol and then very efficiently with water, via a spacer containing six Ch 2 groups, the nitrogen content of the dried polyhydroxymethylene adrorbent sample substituted by the primary amino groups 5.7%. Binding of pepsin to the support 20 g of (-amino-n-hexyl) -substituted polyhydroxymethylene prepared according to Example 3 are suspended in 400 ml of 0.2 M sodium phosphate (pH 10). 40 ml of a 25% glutaraldehyde solution are added to the suspension with stirring. After stirring for 2 hours at room temperature, filter through a suction filter and wash the filter residue with 4 liters of 0.2 M sodium acetate buffer (pH 4.0). The reaction product is then added to 500 ml of a solution of 5 g of pepsin, a total of 500,000 IU, determined according to Anson, in 0.2 M sodium acetate, (pH 4.0) and stirred for 15 hours at 6 ° C. The product obtained is then washed with a suction filter with 5 liters of 0.1 M sodium acetate / acetic acid solution (pH 4.0) to which 1 mol / l NaCl has been added.

Use of Polyhydroxymethylene-Pepsin for Proteolytic Cleavage of Immunoglobulins The adsorbent obtained according to the above example is suspended in 0.1 M acetate buffer (pH 4.0) and subjected to an Anson activity assay. The suspension contains a total of 21,000 IU pepsin in bound and detectable form by this experiment. For proteolytic cleavage of human immunoglobulin G, the pepsin adsorbent is filtered off and the residue is suspended in a 2.5% solution of 30 g of human immunoglobulin G (igG) in physiological NaCl solution (pH 4.1). The cleavage batch is stirred for 24 hours at 37 ° C. After filtering off the pepsin bound to the support, 1,250 ml of saturated ammonium sulfate solution are added to the filtrate. The precipitate formed is then centrifuged apart; it contains an immunoglobin (F (ab) 2 ~ portion with its antibody activities. The filtrate is discarded.

Example 4

Preparation of Polyhydroxymethylene-Oxamic Acid 10 g of polyhydroxymethylene are activated as described in Example 1 with BrCN and reacted with 5 g of tyramine dissolved in 150 of any 0.1 M sodium hydrogen carbonate, n 6181 1 at 5 ° C. Stirring for 60 minutes. The unbound part of the tyramine is filtered off with suction and the filter residue is washed with 1 liter of 0.1 M sodium phosphate spray at pH 7.0 and 5 ° C. The filter residue is suspended in 150 ml of a solution of 1 g of diazotized p-aminophenyloxamic acid cooled to 5 ° C and stirred for 15 hours at 5 ° C. The adsorbent is washed on a suction filter with 1 liter of 0.05 M sodium acetate-acetic acid buffer (pH 5.5) to which 2 mmol of CaCl2 and 0.2 mmol of EDTA have been added.

Use of a polyhydroxymethylene oxamic acid compound in the manufacture of neuraminidase

The adsorbent obtained according to Example 4 is resuspended in said acetate buffer and transferred to a chromatography column 2 cm in diameter. Pass through this column 1 liter of Vibrio Cholorae culture filtrate containing 800 IU neuraminidase / ml, first adjusted to pH 5.5 with 2-m acetic acid and to which CaCl2 and EDTA have been added until their concentrations are 2 mmol / l resp. 0.2 mmol / l. Unbound proteins are washed off with 500 ml of acetate buffer.

Elution of the neuraminidase adsorbed on the carrier-bound inhibitor is accomplished by washing the column with 0.1 M sodium bicarbonate solution and pooling the fractions containing neuraminidase. They contain 86% of the activity used.

Example 5 a) Reaction of polyhydroxymethylene with epichlorohydrin 50 g of polyhydroxymethylene are suspended in 1 liter of 2-n

NaOH, 250 ml of epichlorohydrin are added and the mixture is stirred for 2 hours at 55-60 ° C. The pH of the suspension drops in a short time between 10-11. This pH is maintained for another hour by the addition of NaOH. After a reaction time of 2 hours, the solid is filtered off, washed with water, acetone and finally again with water.

b) 50 g of hexamethylenediamine are dissolved in 1.5 liters of water and HCl is added until the pH is 10. Polyhydroxymethylene activated according to a) is added to the solution and stirred for 6 hours at 50-55 ° C. The product is then filtered off and washed free of hexamethylenediamine with water.

c) The product obtained in 5b) is stirred for one hour with 50 g of 1-aminobenzene-4- [4-hydroxyethylsulphosulphuric acid ester at 55 ° C and pH 10. The solid is then filtered off, washed with water, acetone and again with water.

d) 10 g of the product obtained in 5) are washed with a suction filter in 200 ml of 0.1 N HCl and then suspended in 300 ml of 0.5 N HCl. A 0.1 N NaN 4 solution is added to the suspension with stirring at 0-4 ° C until a slight excess of nitrite is detected on KJ starch paper. After 10 minutes, filter using a suction filter and wash the residue with ice water followed by 0.15 M sodium phosphate buffer (pH 7.5) at 0-4 ° C.

e) 0.75 g of albumin is dissolved in 250 ml of phosphate buffer pH 7.5, cooled to 4 ° C and the product prepared in 5d) is added. The suspension is stirred for 20 hours at 4 ° C, then filtered and the solid is washed with 1 M NaCl and phosphate buffered saline (0.9% aqueous NaCl with 1/15 mol / l Na 2 HPO 4-KH 2 PO 4). buffer, with a pH of 7.2).

The albumin content of the filtrate and washings is determined by the radial immunodiffusion method. 75 mg of albumin bind to 1 g of the carrier thus prepared.

f) 2.4 g of IgM are dissolved in 250 ml of phosphate buffer pH 7.5 and cooled to 4 ° C. 10 g of the diazotized product according to 5d) are added and the suspension is stirred for 20 hours at 4 ° C. After filtration, the solid is washed with 1 M NaCl solution and PBS solution.

1 g of carrier prepared according to 5f) is bound by 240 mg

IgM.

Example 6 a) 10 g of polyhydroxymethylene are activated with epichlorohydrin, reacted with hexamethylenediamine and worked up as described in Example 5 a) and b).

b) 10 g of the carrier thus prepared are succinylated for 4 hours at 10 ° C and pH 6.5 using 5 g of succinic anhydride suspended in 200 ml of water. The pH is adjusted with 2N NaOH. After washing the solid with water, the product is stirred for 30 minutes at pH 5 and 5 ° C with 2.5 g of N-cyclohexyl-N '- (TN-methylmorpholino) -ethyl / -carbodiimide-p- toluene sulfonate, filtered off and washed rapidly with ice.

c) 1 g of IgG is dissolved in 250 ml of phosphate buffer, pH 7.5, and mixed with the support prepared according to 5f) for 24 hours at 4 ° C. After filtration, the product is washed with 1 M brine and phosphate buffer solution (see Example 5).

6181 1 13 85 g of IgG are bound (by covalent bonds) to 1 g of carrier.

Example 7 a) 20 g of the product prepared in 5a) are mixed with 20 g of aminocaproic acid for 6 hours at 50-55 ° C and a pI of 10. It is then filtered and washed with water.

b) To 10 g of the product prepared according to 7a) is added 2.5 g of N-cyclohexyl-N '- [(N-methylmorpholino) ethyl] 7-carbodimide-p-toluenesulfonate, and after 5 minutes the pH is added. 5.2 g of N-hydroxysuccinimide. After stirring for 5 hours at 24 ° C, the solid is filtered off and washed with water.

c) 0.5 g of albumin is dissolved in 200 ml of phosphate buffer and mixed for 24 hours at 4 ° C with the activated carrier prepared according to 7b). After filtration, the product is washed with 1 M brine and phosphate buffer solution (see Example 5).

50 mg of albumin is bound (by covalent bonds) to 1 g of carrier.

Example 8 10 g of PHM are activated with epichlorohydrin, reacted with hexamethylenediamine and worked up as described in Example 5 a) and b).

a) 10 g of the carrier thus prepared are suspended in 100 ml of water and reacted with 500 ml of 25% glutaraldehyde. After stirring for about an hour, the solid is filtered off and washed with water or solution (see Example 5).

b) 0.5 g of albumin is dissolved in 200 ml of phosphate buffer and mixed for 20 hours at 4 ° C with the support prepared according to 8a). After filtration, the product is washed with 1 M saline solution and phosphate buffer solution.

Example 9

Direct reaction with epichlorohydrin activated polyhydroxymethylene.

a) 10 g of polyhydroxymethylene are activated as described in Example 5a) in 50 ml of epichlorohydrin. After filtration, the product is washed rapidly with water, acetone, water and finally with phosphate buffer solution (see Example 5).

b) 0.5 g of albumin is dissolved in 200 ml of PBS solution (see Example 5) and stirred for 60 hours at 4 ° C with the product prepared in accordance with 9a1811. After filtration, the carrier-bound protein is washed with 1 M saline and phosphate buffer solution.

1 g of carrier binds 45 mg of albumin.

Example 10 10 g of water-insoluble polyhydroxymethylene at pH 9.5 (0.15 M Na bicarbonate / NaOH buffer) are stirred for three days at 25 ° C with 30 g of diethylene glycol diglycidyl ether, the product is filtered off with suction, washed well and added at 0 ° C. To 5 g of human albumin in 200 ml of 0.15 M phosphate buffer (pH = 8) and stirred for 60 hours at 10 ° C. After filtration, the carrier-bound protein is washed with 1 M saline and phosphate buffer. 1 g of carrier binds 35 mg of albumin.

Example 11 10 g of polyhydroxymethylene are reacted as described in Example 10 with 30 g of glycidyl tris (glycidyl ether), the mixture is poured into 0.5 g of immunoglobulin G (IgG) in 200 ml of 0.15 m phosphate buffer (pH = 7) and stirred for 60 hours at 4 ° C. After filtration, the product is washed with 1 M brine and sodium phosphate buffer solution. 1 g of carrier binds 50 mg of IgG.