FI60216B - BIOLOGICALLY ACTIVE VID AFFINITETSREAKTIONER ANVAENDBART ADSORBTIONSMEDEL - Google Patents

Description

ΓΒΊ m | ANNOUNCEMENT, noi, J®a lBJ (11) utlAggningsskrift 6021 6 C ¢ 45} P.rteniti my'inn: · tty 10 1C 19-31 Patent meddelat (51) K ».ik? / I« .a .3 c 07 G 7/00, C 12 N 11/08, G 01 N 33/48 FINLAND — FINLAND <») Patent application - Patentana5knln | 751299 (22) Hakemlspllv · - AmMutlngtdag 30. OU. 75 (23) AlkupUvi — Giltlghutsdtg 30.0 ^ .75 (41) Tulkit lulkisektl - Bllvit offmtllg 07.11.75 _ _. . . , (44) Date of issue and date of issue. - ""

Patent and registration authorities in the United Kingdom and the United Kingdom of Great Britain and Northern Ireland 31.08.8l (32) (33) (31) Pyydetty etuoikeus — Begird prlorltet 06.05.71 *

Federal Republic of Germany (DE) P 21 * 21709.8 (71) Behring-wsrke Aktiengesellschaft, Marburg / Lahn, Federal Republic of Germany (DE) (72) Rudolf Schmidtberger, Marburg-Marbach, Heinrich Huemer

Taunus, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (7U) Oy Kolster Ab (5U) Biologically active adsorbent for affinity reactions - Biologiskt aktivt, vid affinitetsreaktioner användbart adsorption medel

The present invention relates to an adsorbent for use in biologically active finite reactions, which consists of a water-insoluble carrier and a biologically active substance chemically bound thereto.

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 complexation capacity of the carrier-bound substance and the other substance in the mixture, the substance in question can be removed from the mixture and, if desired, subsequently isolated 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, 6021 6 water-insoluble enzymes are available as reagents, which can be used several times. Because enzymes are characterized by their affinity not only for substrates but also for specific inhibitors, the preparation of enzyme inhibitors has proven to be particularly advantageous in the case of carrier-bound enzymes by finite chromatography. On the other hand, the binding of inhibitors to water-insoluble matrices allows the preparative preparation of the corresponding enzymes.

Antigens or antibodies bind as so-called immunoadsorbents to water-insoluble matrices and thus allow the isolation of the corresponding antibodies or antigens.

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

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 matrices, it is preferable to use substances whose properties include, in addition to insolubility in aqueous systems, the lowest possible non-specific adsorption capacity. In addition, the hydrophobic, hydrophilic and ionic interaction between the matrix and the reactant of the biologically active substance should not occur and in any case should not form bonds with substances which are not reactants of the biologically active substance.

The matrices used so far as carriers for biologically active substances 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 the α k active substance is bound via a covalent bond. The latter include vinyl polymers such as polyacrylic acids, polyacrylic acid amides and polystyrene containing 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 matrices and biologically active substances, the use of carbohydrates, especially those of 1 μl, dextran, starch, agar or their derivatives as matrices in aqueous systems has reached the widest prevalence, although these carboxyl substances therefore found to be disruptive. In addition, the thermal and chemical stability of these substances is rather low.

It has now been found that the disadvantages of carbohydrates used as matrices in connection with affinity-dependent reactions can be counteracted if polyhydroxymethyls are used as matrices.

The adsorbent 1 1 e according to the invention is characterized in that the water-insoluble 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 plasma protein, and the biologically active substance is optionally bound to the carrier. via a cationic organic compound.

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 are explained in m.m. in N.D. Field and J.R. Schaefgen, J. Polymer Sci., Vol. 58, 533-5 ^ 3 (1962), and G. Smets and K. Hayashi, J. Polymer Sci., Vol. 29, 257-27 ^ (1958). It can be prepared by base or acid hydrolysis from polyvinyl carbonate. Polyhydroxymethylene can be considered as a derivative of polyvinyl carbonate. Binding of the biologically active substance to the polyhydroxymethylene can be effected, on the one hand, by means which, in a known manner, bind the biologically active substance to the hydrogen-hard Dox, on the other hand, by reacting the polyvinyl carbonate with polycarbene. via an amino reaction of the carbonyl ring with a primary amine, for example hexamethylenediamide, resulting in a spacer or a biologically active substance-substituted polyvinylene carbonate via a urethane bond, followed by saponification of the remaining cycloocarbon groups with hydroxyl. 6021 6 r _ I- I I + r-nh2 III »0-.0 Λ 0 0 OH 0 ...

x \ / j saponification »-> g f- ° -►

0 n 0 HH

J i _ - polyvinylene carbonate --- ~ CH - CHl — CH CH -----

I I I I

OH OH OH 0, C = o

n-1 I

J NH

R

From the point of view of the use of the products, it is particularly advantageous that the biologically active substances are not allowed to react directly with the polyhydroxymethylene, but are allowed to bind to the matrix by covalent bonds via a so-called spacer.

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

Several generally known reactions can be found for the incorporation of biologically active substances into a carrier of a hard enzyme:

A particularly simple method is based on the activation of the hydroxyl groups of polyhydroxymethylene by means of cyanogen halides, mainly bromocyanyl 11a, and the reaction of biologically active substances containing amino groups via these activated groups.

Another method is based on reacting a polyhydroxymethylene, such as a polysaccharide, with a variant of an azide method (Curtius) with a carbonic acid such as chloroacetic acid in an alkaline environment, esterifying the corresponding acid with an alcohol, then converting the ester to the hydrazide, and finally the azide is incorporated into a poly (5 6021 6) hydroxymethylene matrix.

The biologically active agent can also be incorporated into the polyhydroxymethylene matrix by first acylating the hydroxyl groups with bromine cetyl bromide and then alkylating the amino group of the biologically active agent.

Corresponding reactions can be effected, for example, by reacting the hydroxyl groups of the support with reactive triazines, whereby some of the reactive groups of the triazine react with the polyhydroxymethylene compound and some with the amino group of the biologically active substance.

Diazotizable aromatic amines, which on the one hand can be bound 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 thereof.

Acylamino derivatives containing arylamino groups and sulfuric acid half-esters of β-hydroxyethyls can be reacted with the 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 epihydrogens or polyepoxides, for example epichlorohydrin or bisepoxide.

There are other methods for forming a hard bond between the hydroxyl groups of po 1 hydroxymethylene and the amino groups of a biologically active substance, such as the known reaction via complexing metal compounds, e.g. titanium compounds.

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 substance, a spacer can also be incorporated into a matrix if it has an amino group as one of the reactive groups. The incorporation of a spacer offers the possibility of incorporating a biologically active substance in other ways: for example, in the case where the matrix bound spacer has a free carboxyl group, this carboxyl group can be activated, for example, by a carbodiimide compound, which can then form an amide 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 isonium-containing derivatives of arylamino groups or sulfuric acid derivatives of N-hydroxyethylisulfones containing arylamino groups can be used to extend the spacer to arylamino groups, which are then di-azotated to known reactants, e.g. f -, - ^ - 1--1-

OH 0 OH

„L • Λ n r // \ / r- \ 0 NH- (CH2) 6-NH-CH2-CH2-SojO-NH2

Due to their specific adsorption properties, carrier-bound biologically active substances have a wide range of applications in affinity chromatography. Carriers bound in nature. Highly pure enzyme products can be prepared with the aid of synthetic enzyme inhibitors, and in turn, enzymes bound to carriers have been found to be remarkably well suited, in particular for the production or production of natural enzyme inhibitors. Carrier - bound water - insoluble antigens are used to isolate the relevant antibodies, thus obtained free of other serum components and free of other antigens. Non-precipitated antibodies as well as substances that cannot be precipitated due to their low serum concentration can also be isolated by affinity chromatography, and the assay can also be performed quantitatively.

In order to illustrate the invention, the following examples have shown that the polyhydroxymethylene support is suitable for the binding of a wide variety of biologically active substances and that polyhydroxymethylene can be suitably substituted for the binding of virtually any desired biologically active substance.

In the form of an example, it is further shown to which matrix-bound biologically active substances can be used.

Example 1. Polyhydroxymethylene-1-ethanustoxide compound Preparation of a matrix from (CO-amino-n-hexyl) -sulated 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 with stirring and the internal temperature at 15 ° C for 6 minutes by slow dropwise addition of 2- n soda lye. It is then immediately filtered, washed well with ice water and pressed into a cake. The polyhydroxymethylene thus activated, still moistened with water, is added to a well-stirred, room temperature, concentrated HCl solution adjusted to pH 8.5 with 20 g of hexamethylenediamine in 500 ml of H2O, the total volume being adjusted by adding water. To 1 liter, the pH is kept constant 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 nitrogen content of the substituted sample of the substituted polyhydroxymethylene of the (N, N-amino) group was 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 a 0.5 M solution of sodium phosphate at pH 10. The suspension is heated with stirring at 50 ° C and 5 g of 1-aminobenzene-k- / 3 "hydroxyethyl sulfonic acid side ester. After adjusting the pH to 10 with 2M NaOH, the batch is stirred for one hour at 50 [deg.] C. The suction is then filtered through a filter and washed. using 2 L of water, 2 L of acetone, and 2 L of 0.2 M HCl. To diazotize the polylhydroxymethylene-bonded arylamine groups, the filter residue is suspended in 200 mL of 0.2 M HCl, cooled to 2 ° C, and after stirring, add 1 M NaNO4 solution until a slight excess of nitrite is found 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 its then 200 m 1 of 11-m sodium phosphate with a pH of 7.5 and a temperature of 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 (1260 Lf / mg N) in 0.2 molar sodium phosphate, pH 7.5 and 2 ° C, and stirred 20 hours at 5 ° C. The unbound parts of the toxoid are washed off with suction filter water in 1 liter of 1 mol NaCl-1 solution.

8 60216

Use of the Polyhydroxymethylene Tetanus Toxoid Compound in the Preparation of Tetanus Antitoxin 10 g of the product of Example 1 are suspended in 150 ml of a 0.15 molar solution of NaC 1 to 1 to which 1/15 molar sodium chloride has been added ( PBS ") pH 7.2, and applied to a column 3 cm in diameter and 10 cm high. The column is loaded with 20 ml of teta - horse serum, 1650 IU (International Units) of tetanus toxin / m 1, which is then washed with 500 ml of 1: 11 PBS pH 7.2. Antibody e 1uation is performed with 0.5 M glycine / HCl buffer at pH 2.5. After neutralization with 2 to 1 N N a 0 H: 1 and subsequent centrifugation, the eluate contains a total of 2 0 mg of protein and 12,300 IU of tetanus antitoxin, which is determined by means of a protection test in mice.

Example 2. Polyhydroxymethylene-aminobenzamide compound Preparation of a matrix from (N-carboxy-n-pentyl) -substituted polyhydroxymethylene 20 g of polyhydroxymethylene are added according to Example 1 after activation of B r CN: 1 a to an aqueous solution of 25 g of aminocaproic acid and adjusted to pH 8.5 with dilute sodium hydroxide solution and a total volume of 1 liter. Stirring is continued for a further 5 hours at room temperature at pH 8.5 and filtered and washed well with water. The nitrogen content of the dried sample taken from the substituted polyhydroxymethylene of the (Co-carboxy-n-pentyl) group 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 morpholinoethanesulfonic acid and 1 g of 1-ethyl-3 '(3-dimethylaminopropyl) carbodiimide is added. hydrok1 oridia. Add 2-m H Cl until the pH is 0.8. To the batch is added with stirring 1 g of m-aminobenzamide. The suspension is stirred with pH adjustment for one hour and the adsorbent is then transferred to a chromatographic column 1 cm in diameter. The adsorbent is rinsed with 200 ml of 0.05-M tris-hydroxymethylaminomethane-HCl buffer (pH 8.0) to which 0.5 mol / l KCl (Tris-KCl) has been added.

Use of polyhydroxymethylene aminobenzamide ini in the preparation of thrombin 100 g of crude bovine thrombin obtained in bovine solution are dissolved in 5 ml of a 0,05 M tris / 0,5 M KCl solution at pH 8,0 and placed in the pretreatment as described above. column. The column is then rinsed with 20 0 m 1: 1 1 a tris - KC1 buffer. Thrombin bound to insolubilized 9,60216 m-aminobenzamide is cleaved from this 100 m 1: 1 1a solution of 0.05 moles of m-aminobenzamide in tris-KCl buffer.

The protein-containing fractions are pooled and run in Tris-KCl buffer through a Sephadex G-25 column (1 x 30 cm). A portion of the first protein-containing column eluate was detected by Chase and Shaw, Biochem. 8, 1969, page 2212, containing 78% of the activity of the starting material.

Example 3 · Polyhydroxy'methylene-pepsin preparation

Preparation of the matrix from (Ci) -amino-n-substituted polyhydroxymethylene from 8.0 g of dimethylformamide / methanol recombined polyvinyl carbonate (preparation: see ND Field and JR Schaefgen , J. Polymer Sci., Vol 58 (1 962) 534) is suspended in a solution of 7.5 g of hexamethylenediamine in 18 m of methanol at room temperature and stirred for 48 hours at room temperature, filtered, washed with methanol and kept for 4 days at room temperature suspended in a solution of 10 g of sodium methylate in 300 ml of 96-¾ methanol, washed with methanol and then very efficiently with water. The nitrogen content of the substituted, dried polyhydroxymethylene adsorbent sample via the spacer containing six CF 2 groups was 5.7 I.

Binding of pepsiin to the support 20 g of (Co-amino-n-hexyl) -substituted po 1 y-hydroxymethylene prepared according to Example 3 are suspended in 400 ml: to 0.2 M sodium phosphate (pH 10). 40 ml of 25-¾ glutaraldehyde solution are added to the suspension with stirring. After stirring for 2 hours at room temperature, it is filtered through a suction filter and the filter residue is washed 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 Anson1, in 0.2 ml of sodium acetate (pH 4.0) and stirred for 15 hours at 6 ° C. The product is then washed 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-Pepsi ini 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 activity assay according to Anson. The suspension contains a total of 2 1 0 00 IU of pepsin 10 6 0 21 6 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 ¾ solution of 30 g of human immunoglobulin G (I g G) in physiological NaC 1-1 l (pH k , 1). The cleavage batch is stirred for 2 ** hours at 37 ° C. After filtering off the pepsin bound to the support, 1250 ml of saturated ammonium sulfate solution are added to the filtrate. The precipitate formed is then centrifuged apart; it contains the immunoglobulin n F (a b) ^ “part n with its antibody activities. The filtrate is discarded.

Example k. Preparation of Polyhydroxymethylene-Axamic Acid Compound 10 g of polyhydroxymethylene is activated with BrCN as described in Example 1 and reacted with 5 g of tyramine dissolved in 150 ml of 0.1 M sodium hydrogen carbonate. At 5 ° C for 60 minutes with stirring. The unbound portion of tyramine is filtered off with suction and washed with 1 liter of 0.1 M sodium phosphate buffer, pH 7.0 and 5 ° C. The filter residue is suspended in 150 ml of a solution of 1 g of diazotized p-aminophenylxamic 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 acetic acid buffer, pH 5.5, to which 2 mmol of CaCl 2 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 k is resuspended in said setting buffer and transferred to a chromatographic apparatus 2 cm in diameter. Pass through this column 1 liter of Vibrio cho1 erae-vi 1je 1 mä filtrate with 800 IU neuraminidia / m 1, first adjusted to pH 5.5 with 2 M acetic acid and to which CaCl 2 has been added, and EDTA until their concentrations are 2 mmol and 0.2 mmol. Unbound proteins are washed away with 500 m of 1:11 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 collecting 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 2N 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 and 11.

This pH is maintained for a further hour by the addition of N a 0 H. 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 with hexamethylenediamine without water.

c) The product obtained in 5b) is stirred for 1 hour with 50 g of 1-aminobenzene-4- [3-hydroxyethylsulphosulphuric acid ester at 55 ° C and pH 10. The solid is then filtered off, washed with water, acetone and water. again with water.

d) 10 g of the product obtained in 5c) are washed with a suction filter with 200 ml of 0.1 N HCl and then suspended in 300 ml of 0.5 N HCl. A 0.1 N solution of NaNCl 2 is added to the suspension with stirring at 0-4 ° C until a small amount of nitrite-y is present in the KJ starch paper. After 10 minutes, filter using a suction filter, and the residue is washed with ice water and then with 0.15 M sodium phosphate buffer (pH 7.5) at 0-4 ° C.

e) 0.75 g of albimine are dissolved in 250 ml of phosphate buffer pH 7.5 and 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 Na a Cl and phosphate buffered saline (PBS, 0.9 ¾ aqueous NaCl with 1/15 mol Na ^ HPO ^ -KH PO ^ buffer (pH 7.2).

The albumin content of the filtrate and the wash water is determined by the radial immuno diffusion method. 75 mg of albumin bind to 1 g of the carrier thus prepared.

f) Dissolve 2.4 g of 1 g of M in 250 ml of phosphate buffer pH 7.5 and cool 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 further extended to 11 votes as described in Example 5 a) b).

b) 10 g of the carrier thus prepared are succinylated for 4 hours at 10 ° C and pH 6 using 5 g of succinic anhydride suspended in 200 any 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 at 5 ° C, with 2.5 g of N-cycloxene over - N 1 - (- (Names yy 1 imorphol ii no) - e) i) -carbodiimide p-toluenesulfonate, filtered off and washed rapidly with ice water.

c) 1 g of 1 g of G is dissolved in 250 of any phosphate buffer pH 7.5 and mixed with the carrier prepared according to 5f) for 24 hours at 4 ° C. After filtration, the product is washed with 1 M brine and PBS-1 solution 11a (see Example 5).

85 mg of IgG is bound (by covalent bonding) 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 pH of 10.

It is then filtered and washed with water.

(b) To 10 g of the product prepared in accordance with 7a) is added 2.5 g of N-cyclohexyl-N 1 - ((N 1 -methylimorpholinyl) -ethyl) -carbodiimide-p-toluene-su1 phonate and after 5 minutes, at a pH of 5, 2 g of N-hydroxysuccinimide are added. 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 saline solution and PBS-1 solution 11a (see Example 5) · 50 g of albumin is bound (by covalent bonds) to 1 g of support.

Example 8 10 g of polyhydroxymethylene are activated with Epichlorohydrin, reacted with hexamethylenediamine and further extended to 11 as described in Example 5 a) b).

a) 10 g of the carrier thus prepared are suspended in 100 ml of water and reacted with 500 ml of 25 ¾ g of 1out of 1dehyde. After stirring for about an hour, the solid is filtered off and washed with water or PBS 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-6021 6 L 3 solution and PBS-1 solution (see Example 5).

Example 9

Direct reaction with epichlorohydrin activated polyhydroxymethylene.

a) 10 g of polyhydroxymethylene are activated as described in Example 5a) in 50 ml of 1: 1 l of epichlorohydrin. After filtration, the product is washed rapidly with water, acetone, water and finally with PBS (see Example 5) · b) 0.5 g of albumin is dissolved in 200 ml of PBS (see Example 5) and stirred for 60 hours at k ° C. with a product manufactured in accordance with point 9a). After filtration, the carrier-bound protein is washed with 1 M saline and PBS-1 solution (see Example 5).

1 g of carrier binds 5 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 3 days at 25 ° C with 30 g of diethylene-glycoligl-ethyl ester, the product is filtered off with suction , washed well and added to 0.5 g of human 1 Bumi 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 PBS-1 (see Example 5).

1 g of carrier binds 35 mg of 1Bumin.

Example 11 10 g of polyhydroxymethylene are reacted as described in Example 10 with 30 g of g of lysidyltris (g of lysidyl ether), the mixture is poured into 0.5 g of immunoglobulin G (I g G) 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 saline and PBS-1 solution (see Example 5) · 1 g of carrier binds 50 mg of 1 g of G.