CS256654B1 - A method of preparing a specific anti-tetanus immunoglobulin from normal polyvalent human plasma or a suitable interphase thereof - Google Patents

Abstract

The solution concerns the preparation of a specific anti-tetanus human immunoglobulin from blood plasma, serum or a suitable fraction containing a polyvalent composition of immunoglobulins. Enrichment of immunoglobulin with specific anti-tetanus antibodies is based on the principle of immunoadsorption and desorption. The immunosorbent is prepared by immobilizing a tetanus non-toxic antigen on a solid porous carrier of the type of spherical cellulose or glycomethacrylate of domestic production. The prepared immunosorbent is brought into contact with a solution containing a polyvalent immunoglobulin under conditions under which immunosorption occurs. After separation of the uncaptured portion of proteins, desorption is performed by the combined effect of a concentrated solution of magnesium chloride and dioxane so as to avoid irreversible changes in the structure of the immunoglobulin molecule. From the eluate thus obtained, after separation of excess salts and dioxane, lyophilized immunoglobulin with a several-fold increased content of tetanus antibodies, suitable for the preparation of an injection form, can be prepared.

Description

(54) A method for preparing a specific anti-tetanus immunoglobulin from normal polyvalent human plasma or an appropriate intermediate fraction thereof

The invention relates to the preparation of a specific anti-tetanus human immunoglubulin from blood plasma, serum or a suitable fraction containing a polyvalent composition of immunoglobulins. The enrichment of immunoglobulins with specific anti-tetanus antibodies is based on the principle of immunoadsorption and desorption. The immunosorbent is prepared by immobilizing tetanus non-toxic antigen onto a solid porous carrier of the spherical cellulose or glycomethacrylate type of domestic manufacture.

The prepared immunosorbent is contacted with a solution containing a polyvalent immunoglobulin under immunosorption conditions. After separation of the non-retained protein fraction, desorption is performed by the combined action of a concentrated magnesium chloride / dioxane solution so as to avoid irreversible changes in the structure of the immunoglobulin molecule. From the eluate thus obtained, after separation of the excess salts and dioxane, a lyophilized immunoglobulin with a multiply increased content of tetanus antibodies can be prepared, suitable for the preparation of an injectable form.

The invention relates to the preparation of a specific human anti-tetanus immunoglobulin by immunoadsorption and elution using domestic raw materials and materials by a process which makes it possible to obtain it in a fully native form suitable for solution for injection with an increased content of anti-tetanus antibodies.

Injection solutions of primitive specific immunoglobulins, i.e. solutions with an increased content of antibodies to selected bacterial or viral antigens, are being prepared by a number of manufacturers. In Czechoslovakia are prepared some species in the Ostavu serum vaccines under the trade names TEGA, RHEGA, HEPAGA. Interest in these preparations is steadily increasing. However, their preparation is limited by the number of selected plasma donors having an increased content of the desired antibodies. The anti-tetanus human gamma globulin (Gamma-globulinum antitetanicum humanum) is prepared in the Czechoslovak Socialist Republic of Ostrava by serum fraction and vaccines by ethanol fractionation of human blood plasma from selected donors. The final solution for injection contains 10% to 16% protein, with a 2 ml ampoule of this solution containing at least 200 µl (International Units) of a specific anti-tetanus immunoglobulin (Corporate Materials ÚSOL, Prague). The possibility of preparing specifically enriched immunoglobulin from normal human plasma based on immunoaffinity chromatography has so far encountered a number of problems consisting in the selection of a suitable solid carrier, antigens and reagent to release a substantially solid antigen-antibody complex without irreversibly altering the structure of the bound immunoglobulin molecule.

Until this time, solutions having extreme pH values of 1 to 3 or 10 to 12.5 or concentrated urea or thiocyanate / M solutions have been used for desorption in immunoaffinity chromatography. Frenčik, B. Šárka: Biochemical Laboratory Methods /, / C. R. Lowe, P. D.

G. Dean: Affinity Chromatography /, leading to protein denaturation. The release of bound immunoglobulin by means of concentrated solutions of salts of a monovalent or divalent metal halide is only partial / S in the present case. Avrameas, T. Tornynk, Biochem. J. 102: 37b (1967).

Also, solutions of organic solvents alone, such as ethylene glycol and 1,4-dioxane, do not give satisfactory elution results. Despite the change in temperature during sorption and desorption, satisfactory results cannot be obtained. K. Anderson et al., J. Immunol. Methods 25, 375 (1979)]. In our conditions, it is also necessary to pay attention to the selection of a suitable solid carrier, because foreign materials are not available for a given purpose for wider use.

The proposed method for the preparation of specific human immunoglobulin by immunoaffinity adsorption and elution solves these drawbacks by attaching to a porous spherical cellulose or porous glycomethacrylate carrier by a chemical bond tetanus anatoxin or a mixture of anatoxins, after which the tetanus immunosorbent thus prepared is washed with pH 8 to 8.5, contacted batchwise or by column flow with a solution containing normal polyvalent immunoglobulin pH 8.5 to 6.5 in the cold for 6 to 48 hours, after which the non-captured protein solution is separated, immunosorbent with specific immunoglobulin bound is washed with 0.2 to 0.9% sodium chloride solution and the immunochemically bound immunoglobulin is desorbed at normal temperature with an aqueous 20% to 40% magnesium chloride solution containing 5% 1,4 dioxane or 1% to 2% glucose or maltose as a stabilizer, whereupon the thus obtained dec the flow of specifically enriched immunoglobulin frees undesirable amounts of salts and organic solvent by dialysis or diafiltration and is freeze-dried.

The proposed process for the preparation of an immunoglobulin enriched for specific anti-tetanus antibodies utilizes the advantageous properties of solid carriers of pearl cellulose or glycomethacrylate, both of domestic production / M. Benes et al .: Cs. AO 229 010 (H). Tláskalová: Immunochemistry 12, 801 (1975). Furthermore, non-toxic and non-infectious antigens produced in the Czechoslovak Socialist Republic by the Institute of Serums and Vaccines are used for the preparation of animal vaccines. The advantage of this anatoxin is that it contains a small amount of undesirable foreign ballast proteins.

As the source of polyvalent immunoglobulin, the immunoglobulin fraction or intermediate fraction resulting from the fractionation of human blood plasma can be used according to the Cohn / E method. J. Cofon et al .:

J, Amer. Chem. Soc. 72, 465 (1950). Preferably, immunoglobulin prepared from surrogate raw materials can be used.

The non-retained portion of the polyvalent immunoglobulin is further processed in a known manner to dried immunoglobulin and further to a Norga solution for injection. The elution of immunochemically coupled immunoglobulin is accomplished with a combined solution of magnesium chloride, dioxane and sugar under conditions that do not denature and lose the activity of the released immunoglobulin in a good yield. The specifically enriched immunoglobulin thus obtained is suitable for the preparation of a dosage form. The proposed procedure is technologically easy to carry out and not demanding on equipment. Immunosorbent can be regenerated repeatedly. Similarly, a variety of specific immunoglobulins can be prepared from a polyvalent immunoglobulin using a variety of immunosorbents with an appropriate coupled antigen.

Example 1

20 ml of 0.3 M hydrochloric acid and 0.08 g of sodium nitrite are added in the cold to 10 ml of a spherical substituted cellulose carrier Ostsorb AV. The suspension was stirred in the cold for 30 minutes. The liquid is then filtered off and the diazotized support is washed with cold water.

To the activated carrier is added 30 ml of a tetanus anatoxin solution, which contains about 0.2% increments, and which has been adjusted to a pH of 8.0 to 8.5.

The suspension is stirred for 30 minutes. Under these conditions, binding coupling occurs with the protein component of tetanus anatoxin. The liquid is filtered off, the immunosorbent is washed, the remaining free functional groups are deactivated with a 0.2 M glycine solution at pH 10 and washed with a borate 0.05 M buffer pH 8.4, a batch of 120 ml of a 8% polyvalent immunoglobulin solution prepared with ethanol is added. fractionation. The suspension is kept in contact at +4 ° C with occasional stirring for 16 hours at which time the immobilized antigen-antibody reaction is immunochemical. The non-captured protein solution was separated and the immunosorbent was washed with 0.2% sodium chloride solution. The non-captured protein solution can be further processed as necessary into a normal injectable solution of immunoglobulins in a known manner.

A chromatographic column is prepared from the washed immunosorbent with specific immunoglobulin bound and desorbed with a solution of 40% magnesium chloride, 5% dioxane and 2% glucose at normal temperature. The eluate containing the specific anti-tetanus immunoglobulin is freed from salts and dioxane by dialysis and freeze-dried. A solution for injection is prepared from dried immunoglobulin with an increased content of anti-tetanus antibodies so that 2 ml of this solution contains at least 200 µl.

Example 2

To 10 g of wet, i.e. 3.5 g of dry, glycol methacrylate carrier Separon R Herna 1000 E, add 15 ml of tetanus anatoxin diluted with 25 ml of 0.05 M borate buffer pH 8.3 and adjust the pH of the solution to 8, 0. 10.5 g of ammonium sulfate are added to the suspension. After dissolution of the salt, the suspension is left in contact in the cold for 24 hours with occasional stirring. The liquid was then separated by decantation, the immunosorbent was washed, free functional groups were deactivated by the addition of 20 ml of 0.1 M perchloric acid, and the immunosorbent was washed with 0.05 M borate buffer pH 8.3.

A chromatographic column is prepared from the washed immunosorbent, through which a 5% protein solution containing predominantly the immunoglobulin fraction is passed slowly, optionally repeatedly, in a total amount of 150 ml. During this operation, an immunochemical reaction occurs between the immobilized antigen and the antibody. The non-captured protein solution is then further processed as desired in a known manner. The specific immunoglobulin bound immunosorbent column is washed with 0.9% sodium chloride solution and desorption is performed with 20% magnesium chloride solution, 5% dioxane and 2% maltose at normal temperature. The eluate obtained is worked up as described in Example 1.

Claims (1)

SUBJECT OF THE INVENTION Process for preparing human specific immunoglobulin from normal, polyvalent human plasma or a suitable fraction thereof comprising immunoglobulins by immunoadsorption and elution, characterized in that a tetanus anatoxin or a mixture of anatoxins is chemically coupled to a porous spherical cellulose or porous glycomethacrylate carrier. tetanus immunosorbent after washing with 0.5 M borate buffer pH 8.0 to 8.5, contacted batchwise or by column flow with a solution containing normal polyvalent immunoglobulin at pH 6.0 to 8.5 at 1-8 ° C for for 6 to 48 hours, after which the non-captured protein solution is separated, the specific immunoglobulin-weighted immunosorbent is washed with 0.2 to 0.9% sodium chloride solution and the immunochemically bound immunoglobulin is desorbed with an aqueous 20% to 40% magnesium chloride solution which further contains 5% 1,4-dioxane or 1% to 2% glucose or maltose as a stabilizer, after which the solution of specifically enriched immunoglobulin is freed from undesirable amounts of salts and organic solvent by dialysis or diafiltration and dried by lyophilization.