CS247484B1 - Method of immunoglobulin preparation against human lymphocytes - Google Patents

Abstract

The present invention relates to the field of immunobiological production preparations for therapeutic purposes. A method of preparing an immunoglobulin is solved against human lymphocytes / thymocytes /. Listed immunoglobulins are immunosuppressive effects used in transmissions tissues and organs. The essence of the solution is a simple procedure isolating the immunoglobulin fraction from sera immunized animals based on precipitation reactions. At 18-22 ° C and pH 4.15 up to 4.25 occurs in the first stage of separation immunoglobulin solution from the ballast clot protein with caprylic acid. Ve in the second step, the immunoglobulin fraction is purified by ethanol precipitation at -6 to -10 ° C. The procedure remains native the character of the immunoglobulin molecule that have biological properties suitable for po required use of the preparation.

Description

The invention provides a method for preparing an immunoglobulin against human lymphocytes.

Immunosuppressive agents are used in surgical transfers of tissues and organs. Purified animal immunoglobulin against human lymphocytes has a number of advantages over other immunosuppressive agents or radiation for which it is used in transplantation.

The source of antibodies is the serum of animals immunized with human lymphocytes. A preparation prepared in the past from anti-lymphocyte serum did not show suitable immunosuppressive effects. Ammonium sulphate salting was used to isolate the immunoglobulin fraction, followed by adsorption of ballast proteins on the ion exchangers and final saturation or purification procedures using only ion exchangers.

An isolation method based on rivanol precipitation was also tested. The method was not suitable for industrial scale insulation; in the 1970s it was used in the USA - Stanford Univ. Hospital in California.

Because it is not a common commercial preparation - immunoglobulin against human thymocytes / can only be applied in special medical facilities dealing with the transfer of tissues and organs - its preparation is limited to a small number of manufacturers.

For production of small quantities, procedures based on the principles of affinity chromatography, ion exchange chromatography and sorption on specific sorbents are used. A common disadvantage of the cited processes is the high cost of the sorption materials, the applicability only to the processing of small volumes of serum and hence the high cost of the final preparation.

The proposed method for the isolation of immunoglobulin from the serum of animals immunized with human thymocytes, i.e. lymphocytes isolated from the thymus, has all the advantages of clotting procedures in terms of processed volume and since other animal serum serum proteins are unusable for therapeutic purposes, the whole isolation is simplified into two degrees - precipitation of ballast proteins with caprylic acid and precipitation of the immunoglobulin fraction at reduced temperature with ethanol.

The simplicity of the process and the low cost of the precipitating agents increase the efficiency of the process for use in larger volumes and guarantee high product yields at low cost. In contrast to the procedure described for the preparation of human immunoglobulin enriched for IgA and XgM, the proposed procedure is simplified by a purification step of calcium phosphate precipitation for a given preparation by suitably changing the reaction conditions in both isolation steps used.

The proposed method for the preparation of an immunoglobulin against human thymocytes involves two-step precipitation of serum proteins. In the first step, at 20 ° C, most serum proteins precipitate from the serum of animals immunized with human lymphocytes at pH 4.15 to 4.25 and at 10 to 22 ° C to a caprylic acid concentration of 7 to 8% v / v in acetate ions.

The ballast protein sediment is separated by centrifugation and filtration, leaving an immunoglobulin fraction in the solution which precipitates at -6 to -10 ° C with ethanol at a concentration of 25 to 30% by volume. The immunoglobulin sediment is dissolved in water containing glucose and glycine as a stabilizer. The aqueous solution is freeze-dried, dissolved in water, adjusted the active ingredient and stabilizer concentrations, and lyophilized again under sterile conditions after filling.

and

Example 1

The serum of rabbits immunized with human thymocytes stored at -20 ° C was thawed at +4 ° C and a pool of sera from animals # 62-69 was pooled. 9,800 ml of 0.06 M acetate buffer pH 3.53 prepared from redistilled pyrogen-free and sterile water was added. The pH of the diluted serum 4.51 was adjusted to 4.28 by the addition of 20 mL of concentrated acetic acid.

In a fractionation pot, serum was precipitated with 375 ml of caprylic acid p.a added over 30 minutes at 20 ° C. The suspension was stirred for 60 minutes and the sediment separated by centrifugation on a cuvette centrifuge cooled to +4 ° C. Spin time 6 minutes at 3000 rpm.

The slightly opalescent supernatant was filtered through filter paper. The pH of the supernatant 4.22 was adjusted to 5.05 by the addition of 79 ml of 5 M sodium hydroxide solution. 12 300 ml of the supernatant was cooled in a fractionation kettle to 0 ° C and precipitated at this temperature by the slow addition of 4,456 g of ethanol 96% by volume under -15 ° C. The temperature during precipitation of 0 ° C, after the addition of ethanol, was cooled to -9 ° C with stirring, and stirred for 16 hours.

The immunoglobulin fraction sediment was separated by centrifugation on a cuvette centrifuge at 3000 rpm. for 20 minutes at -8 ° C. The sediment was stirred in 1200 ml of ice-cold pyrogen-free water containing 40 g of glucose, 40 g of glycine and 10 g of sodium chloride and the solution was dispensed into 7 bottles of 500 ml of 200 ml each.

The solution was frozen on the bottle walls in a -50 ° C bath and dried under vacuum at a maximum drying temperature of 35 ° C. 129.3 g of lyophilisate was obtained, which contained 39.3 g of protein, corresponding to a yield of 8 g of pure protein from one liter of serum. The lyophilisate was dissolved in 1500 ml of pyrogen-free water, supplemented with 2.75 g of glycine and 3.3 g of sodium chloride and water to a volume of 1900 ml.

The pH of the solution 5.2 was adjusted to 6.74 by addition of 1.4 ml of 5M sodium hydroxide solution. Under semi-sterile conditions, the solution was filtered with a set of 140 mm diaphragm filters in the order of: prefilter, 1.2 µm, 0.8 µm, 0.45 µm, 0.22 µm, and under sterile conditions with a set of membrane filters of diameter 90 mm in sequence: filter, 0.45 and 0.22 ₍ / µm. The solution contained:

proteins 1.79 g / 100 ml glycine 2.05 g / 100 ml glucose 1.97 g / 100 ml sodium chloride 0.69 g / 100 ml PH 6.9

Electrophoretic purity of Ig 91.8% by weight, solution was sterile, harmless to guinea pigs and mice, pyrogenic values satisfactory / 0.0; 0.0; 0.4 ° C /, hemagglutinins 1:16, antibodies against basement membranes of glomeruli O, binding to human lymphocytes 80 to 84%, Inhibition of rosette formation 1: 500, cytotoxic text with human lymphocytes 1: 2 560, anti-serum antibodies proteins: IgG traces, thromboagglutinins and thrombolysins: negative, incomplete 1: 2.

The solution was dispensed under sterile conditions into 34 L 5 ml vials and lyophilized for 48 hours at a maximum temperature of 35 ° C. After closing the vials under vacuum and sealing the caps, a moisture content of 1.8 to 1.9% by weight was determined. and solubility in 30 sec. to a clear colorless solution. The preparation was applied at the Institute of Clinical and Experimental Medicine in Prague - Krč under the designation ATG Sevac 02/84.

In all parameters the preparation complied with the requirements for immunosuppressants based on immunoglobulins for transplantation purposes, which are still imported.

Example 2

Rabbit serum Immunized with human thymocytes stored at -20 ° C was thawed at + 4 ° C and group # 70-74 was pooled. Initial volume of 3,300 ml serum. 6,600 ml of 60 mM acetate buffer was added. pH 3.50 prepared from redistilled pyrogen-free and sterile water.

The pH of the diluted serum 4.48 was adjusted to 4.21 by the addition of 15 mL of concentrated acetic acid. In the fractionation pot, serum was precipitated with 250 ml of caprylic acid p.a. added over 15 minutes at 21 ° C. The suspension was stirred for 60 minutes and the sediment collected by centrifugation on a cuvette centrifuge cooled to +4 ° C.

Spin time 10 minutes at 2800 rpm. The opalescent solution was filtered through filter paper. The pH of the supernatant 4.20 was adjusted to 5.02 by the addition of 58 ml of 5M sodium hydroxide solution. 8 150 ml of the supernatant was cooled in a fractionation kettle to Q ° C and at this temperature was precipitated by the gradual addition of 2 955 g of ethanol 96% by volume supercooled to -15 ° C.

The temperature during the precipitation of 0 ° C, after the addition of ethanol, was cooled to -8 ° C with stirring and stirred for 18 hours. The sediment of the immunoglobulin fraction was separated by centrifugation on a cuvette centrifuge at 2900 rpm. for 20 minutes and at -10 ° C. The sediment was stirred in 850 ml of ice-cold pyrogen-free water containing 20 g of glucose, 20 g of glycine and 6 g of sodium chloride, and the solution was dispensed into 5 bottles of 500 ml of 200 ml each.

The solution was frozen on the bottle walls in a -50 ° C bath and dried under vacuum at a maximum drying temperature of 35 ° C. 71 g of lyophilisate were obtained, which contained 25 g of protein, corresponding to a yield of 7.5 g of pure protein from 1 liter of serum. The lyophilisate was dissolved in 1000 ml of pyrogen-free water and made up to a volume of 1100 ml.

The pH of the solution 5.44 was adjusted to 6.61 by the addition of 0.7 ml of 5 M sodium hydroxide solution. Under semi-sterile conditions, the solution was filtered with a set of 146 mm diaphragm filters in the order of: filter, 1.2 µm, 0.8 µm, 0.45 µm and 0.22 µm and under sterile conditions with a set of membrane filters of diameter 90 mm in order: a prefilter, 1.2 / um, 0.8 / um, about 45 .mu.m and about 22 _/ um. The solution contained:

proteins 2.06 g / 100 ml glycine 2.17 g / 100 ml glucose 1.92 g / 100 ml sodium chloride 0.70 g / 100 ml PH 6.68

Electrophoretic purity 92.4% by weight, solution was sterile, harmless to guinea pigs and mice, pyrogenic values satisfactory / 0.1; 0.0; Haemagglutinins 1:32 (anti A), respectively. 1:64 (anti B), antibodies against basal membranes of glomeruli negative, binding to human lymphocytes 88%, inhibition of rosette formation 1: 500, cytotoxic text with human lymphocytes 1: 2 560, antibodies against serum proteins in the region igG, thromboagglutinins and thrombolycins negative.

The solution was filled under sterile conditions into 186 vials and lyophilized for 48 hours at a maximum temperature of 35 ° C. After closing the vials under vacuum and sealing the caps, a residual moisture content of 1.7% by weight was determined. and solubility in 40 sec to a clear colorless solution. The preparation was applied at the Institute of Clinical and Experimental Medicine in Prague - Krč under the designation ATG Sevac 03/84.

The preparation in all parameters complied with the requirements for immunosuppressants based on immunoglobulins for transplantation purposes, which are still being imported.

Claims (1)

SUBJECT OF THE INVENTION Method for the preparation of immunoglobulin against human lymphocytes for immunosuppressive purposes in tissue and organ transmissions, characterized in that the fraction of immunoglobulins contained in sera of animals immunized with human lymphocytes, preferably lymphocytes isolated from thymes, is isolated by caprylic acid precipitation at pH 4.15 to 4, 25 and at a temperature of 18 to 22 ° C to a concentration of 7 to 8% v / v in an acetate ion medium, the ballast protein sediment is separated by centrifugation and optionally filtered and the immunoglobulin fraction is isolated from the clear supernatant by ethanol precipitation at -6 to -10 ° C. at a temperature of -8 ° C and at a final ethanol concentration of 25 to 30% vol.