SE407333B - PROCEDURE FOR PURIFICATION OF GAMMA-GLOBULIN, SPECIAL ANTILYMPHOCATE-GAMMA-GLOBULIN - Google Patents

Description

7209600-1 'in ALG is usually absorbed and in most cases removed from ALG by contacting the gamma globulin with human erythrocytes or their stroma, human platelets or their stroma and / or being absorbed with human serum or plasma. Not only are undesirable antibodies that are immunologically distinct from the anti-human lymphocyte gamma globulin inactivated in this manner, but also antibodies that are cross-reactive with various human blood antigens are removed in this manner.

Although this type of procedure is theoretically simple, a number of significant problems arise in practice. When serum or plasma is used to absorb contaminating antibodies, the antigen-antibody complex usually does not precipitate. Furthermore, there are no practical aids for separating gamma globulin in the absorbing plasma or serum from ALG.

Thus, gamma globulin from the absorbent plasma or serum contaminates the final product. Consequently, not only ALG is administered to the patient but also large amounts of unnecessary and possibly antigenic proteins. In addition to the body's difficulty in metabolizing large amounts of extra protein, the administration of such mixed proteins appears to cause immune complex type diseases in some cases. A further disadvantage of using human whole blood serum or plasma for absorption and undesirable antibodies are the time consuming tests required to determine the amount of residual contaminating proteins in ALG.

According to another prior art purification step for ALG, erythrocyte stroma is used to absorb antihuman erythrocyte antibodies from gamma globulin. Before ALG is contacted with erythrocyte stroma, the contaminating stroma-dissolving medium and hemoglobin, i.e. menstruum, first removed from the stroma. This is usually done by separating the stroma from the contaminating liquid by centrifugation and washing. However, great difficulties are associated with this, due to the fact that stroma, especially stroma from old blood, cannot be easily separated from the menstrual cycle.

These problems and other problems mentioned below are solved by the method according to the present invention. In accordance with the present invention, there has been discovered a process for purifying gamma globulin from heterologous antigenic material, which is formed by immunization with a heterologous antigenic material, which antigenic material is branched with antigenic blood components derived from the same species as the antigenic material. , the antibodies directed against impurities in the antigenic material in the gamma-globulin solution being removed by contacting the solution with plasma proteins and preferably also erythrocytes or erythrocyte stroma, characterized in that a) a preferably acrinol-containing gamma-globulin solution was treated a suspension of acrinol-precipitated blood plasma protein, substantially free of gamma globulin and derived from the same species as the antigenic material, wherein contaminated antibodies to antigenic plasma protein components are adsorbed on the precipitated plasma protein and b) the solution of the purified gamma-purified gamma from the u precipitated material.

Although the invention is shown in detail with respect to human anti-human lymphocyte gamma globulin, the invention is of course not limited thereto. Other suitable antigenic materials include, for example, carcinoembryonic tissue. When the antigenic material is lymphocytic, suitable sources include spleen lymphocytes, thymocytes, blood lymphocytes, pulmonary vascular lymphocytes and cultured human lymphocytes, preferably thymocytes. The animals from which the antigen material is taken include chimpanzees, monkeys, rabbits, rats, dogs, pigs, etc., but humans are usually preferred. The animals whose gamma globulin is allowed to react with the antigenic material include horse, goat, cow, sheep, rabbit, chimpanzee, monkey, rat, with horse being preferred.

When the antigenic material is lymphocytes, the gamma globulin has immunosuppressive effects when administered to animals of the species from which the antigenic material is taken. Gamma globulin, for example, is useful in facilitating the survival of homologous and heterologous organs and skin grafts, which include kidney, liver, heart, lung, bone marrow and human skin and similar grafts in animals, such as chimpanzees and monkeys.

Gamma globulin can essentially be separated from blood serum or plasma. both of which can be used for the purposes set forth in the description and claims in various conventional ways. Examples of reagents which provide this separation include neutral salts, including sulfates, thiosulfates, phosphates, halogen salts of alkali metals, ammonium or magnesium; metal ions used in suitable concentrations, non-ionizable high molecular weight polymers, polyanions, water-soluble organic solvents, e.g. ethanol, methanol, isopropanol, organic d.?2n9soo+1 4 cations, e.g. acrinol and other acridines including trypaflavin, 5-aminoacridine (Clinica Chimíca Acta § (1961) 782-793). The critical factor in all of these methods is that the gamma globulin has been substantially separated from the mass of serum proteins and thus cannot contaminate the heterologously induced gamma globulin when solid plasma protein is used to absorb undesirable antibodies. The process can be exemplified with respect to acrinol, which has the additional advantage of effectively separating gamma globulin from whole blood as well as from serum or plasma. This property is especially useful in the separation of gamma globulin from whole blood from cattle - while erythrocytes from cattle do not separate as rapidly as human erythrocytes.

Blood plasma from animal species, used as the source of the antigen, is treated with an aqueous solution of acrinol, containing from about 0.1% to about 5% acrinol, to precipitate blood proteins other than gamma globulin. These proteins include albumin, alpha- and beta-globulin, etc. The plasma is at its normal pH of about 7-8, but it can be adjusted to about 7.6 by the addition of suitable reagent, such as 1N sodium hydroxide, 0.8M sodium bicarbonate or 1M sodium carbonate or 1N HCl or acetic acid. The precipitation usually takes place at about 25 ° C, although higher and lower temperatures may be used.

From 3.5 to 5 volumes, preferably about 4 volumes of 4% aqueous acrinol solution are used for each volume of plasma. The precipitate is allowed to complete by sedimentation and flocculation after the liquid has been allowed to stand, preferably at a temperature of about 400 from about 15 minutes until overnight and longer. The precipitate is separated from the supernatant containing gamma globulin, preferably by filtration, decantation or centrifugation. The precipitate containing acrinol-insoluble blood plasma protein is washed with a 0.1 - 5% aqueous solution of acrinol. The washed precipitate is then transferred to a solution of gamma globulin activated against antigen from a heterologous source and stirred for a few minutes to several hours at about 4 ° C to about 40 ° C, preferably about 25 ° C so that the smiplasmic protein antibodies. the gamma globulin solution is absorbed on the solid plasma protein. A sufficient amount of gamma-globulin 7 separating agent is kept in suspension so that a multiphase absorption state is present. The solid plasma protein with absorbed antiplasmic protein antibodies is separated, e.g. by decantation, filtration or centrifugation. The absorption process can be repeated as many times as 5 5209600-1 years necessary to reduce dangaèn 'föfóšénaaë antibodies tili: snake host level. The amount of solid plasma protein obtained from 0.01 to 5 or more liters of plasma was used to absorb the antiplasmic protein antibodies from heterologously reacted antimony lymphocyte gamma globulin obtained from one liter of plasma. This method of absorption has the additional advantage when acrinol is used as a gamma-globulin separating agent that hepatitis virus appears to precipitate together with plasma protein, thereby reducing the possibility of hepatitis virus contamination of the final product.

The gamma globulin which reacts to antigenic material can be practically produced in a manner known per se. One type of animal, preferably a horse, is inoculated, for example, with lymphoid tissue from different types of animals and humans. The lymphocytes are prepared in a manner known per se, for example by graded zonal centrifugation, to obtain separation of lymphocytes and blood cells from granulocytes and erythrocytes. Thereafter, isopycnic zone sedimentation can be used to separate the lymphocytes from the blood cells.

Although a satisfactorily pure population of lymphocytes is obtained in this way, the resulting lymphocyte preparation is still contaminated with various blood elements, the most important of which are human erythrocytes or other antigenic blood components. The animal is then inoculated with the lymphocyte preparation. After sufficient time, the animal is drained and the gamma globulin fraction is isolated by procedures previously described.

As previously mentioned, in the preparation of a purified gamma globulin for internal use, it is also desirable to remove anti-erythrocyte antibodies from the gamma globulin. This is conveniently done by contacting the gamma globulin solution with erythrocytes or preferably with erythrocyte stroma. The production of stromat for contact with the gamma globulin takes place in a manner known per se, with the exception of a new improvement of one of the isolation steps, which is described below.

After dissolution of the erythrocytes and before the gamma globulin is contacted with the stroma, the menstrual cycle must be separated from the stroma as effectively as possible. The addition of small amounts of acrinol to the dissolved erythrocytes facilitates the separation of the stroma from the menstrual cycle and centrifugations at relatively low speeds of 2000 rpm for 10-15 minutes are sufficient for separation compared to 10 or more washes and centrifugations at 1,000 rpm during more than 30 minutes, previously used. Satisfactory separation has in reality often been obtained by the present improvement only by allowing the material to separate overnight. vzoesoo-1 in the cold, for example, followed by decantation. Stromat is satisfactorily free of hemoglobin when the characteristic color of the suspended fluid has completely disappeared or been determined spectrophotometrically at 555 nanometers. The amount of acrinol used varies from about 1 to about 5 g / l of collected erythrocytes, preferably about 3-5 s / 1.

The preparatory steps in the production of stroma are known per se. To the erythrocyte precipitate is added an equal volume of 0.0% NaCl solution. The suspension is then mixed and centrifuged, followed by removal of the yellow film. The washed erythrocytes are then dissolved by a standardized lysis procedure, for example digitonin, water, freezing and thawing. Stromat is separated from the suspension by acrinol in the manner previously shown. It can further be pointed out that the choice of an acrinol solution of suitable tonicity enables the erythrocyte stroma to be dissolved and separated by a single reagent, thereby avoiding a separate lysis step.

Stromat is then added to the solution of gamma globulin, which has been reacted with heterologous antigen so that absorption of the anti-erythrocyte antibodies by stroma can be obtained. The absorption is performed as many times as necessary to absorb the anti-erythrocyte antibodies.

The amount of stroma obtained from 0.01 to 5 or more liters of collected erythrocytes was used to absorb the anti-erythrocyte antibodies from heterologously reacted gamma globulin, obtained from 1 liter of plasma.

Multiphase absorption is maintained with an appropriate amount of gamma-globulin separating agent.

Stroma absorption can be performed before, during or after plasma protein absorption.

The following examples illustrate the process of the present invention.

The examples are not intended to be limiting but serve only as an example of the method according to the invention.

Example 1 Preparation of anti-human lymphocyte-gamma-globulin human lymphocytes were prepared in any suitable known manner and a horse is injected with these lymphocytes. When the desired antibody titer is reached, the horse is drained and the serum is separated from the whole blood and the pH of the serum is adjusted to around 7.6 by adding 0.8 M sodium bicarbonate. An aqueous solution containing O.d.% 7 1209600-1 acrinol is slowly added to the serum with stirring to precipitate blood proteins other than gamma glohulin. An excess of acrinol solution was used to ensure complete precipitation.

The precipitate was allowed to settle overnight at a temperature of about D-400. The supernatant containing gamma globulin was decanted from the precipitate.

Example 2 Absorption of Human Anti-Plasma Protein Antibodies by Health Serum in a Properly Known Way To an equine anti-human lymphocyte gamma globulin solution prepared according to Example 1, an equal volume of human health serum was added. This health compartment inactivated the antibodies to human blood protein in the equine anti-lymphocyte gamma globulin. However, the human gamma globulin mixes with the equine anti-human lymphocyte gamma globulin and these two can only be separated with great difficulty if they can be separated at all.

Example 3 Absorption of human plasma antiplasmic protein antibodies A sufficient amount of an aqueous solution containing about 0.4% acrinol was slowly added to human plasma, the pH of which is adjusted to about 7.6 with 0.8 M sodium bicarbonate. with stirring to obtain a precipitate of the acrinol-insoluble proteins. The resulting precipitate was separated from the supernatant by centrifugation. Solid plasma protein obtained from 0.2 l of human plasma was added to a solution of ALG, obtained from 1 liter of plasma prepared as in Example 1. The resulting suspension was stirred continuously for 3 hours at room temperature and cooled and stored at 400 overnight. The supernatant containing ALG, purified from contaminating human anti-plasma protein antibodies, was then separated by dentrifugation at 2000 rpm for 10 minutes.

The ALG thus obtained was essentially unpolluted with harmful human blood plasma proteins or soluble antigen-antibody complexes.

Example 4 Absorption of human anti-erythrocyte antibodies in a manner known per se n To a volume of collected human erythrocytes was added an equal volume of 0.9% M NaCl aqueous solution. This solution was mixed and centrifuged at 5,000 rpm for 30 minutes at 200 rpm

Claims (3)

1 1209600-1 ~ 8 the supernatant and the yellow membrane were discarded. To each liter of collected erythrocytes was added 10 volumes of distilled water and the cells were dissolved after standing for 30 minutes. Stromat was washed and centrifuged 10 times at 3000 rpm to reduce the hemoglobin to a satisfactory level. A certain amount of stroma was lost during the washing process. Current from 0.1 volume of collected electrocytes was added to ALG obtained from 1 liter of plasma prepared according to Example 1 and stirred for 3 hours at 2 ”. The stroma was separated from the ALG solution by centrifugation and the supernatant was treated with additional stroma twice to reduce the anti-erythrocyte titer to an acceptable level. Example 5 Absorption of human anti-erythrocyte antibodies from ALG with stroma prepared by the addition of acrinol The procedure of Example 4 was repeated to the point where the erythrocytes were dissolved with distilled water. At this point, the dissolved erythrocyte solution was diluted with 2% volume of water calculated on the original erythrocyte volume. 5 grams of acrinol for each liter of originally collected erythrocyte volume was added. This suspension was stirred for 1 hour at room temperature and placed in a refrigerator overnight. The suspension was centrifuged at 2000 rpm for 10 minutes and the ALG solution was separated from the stromat. Two additional stroma absorptions were performed in the above manner to reduce the anti-erythrocyte titer to a desired level. CLAIMS '> A process for the purification of X 'globulin, which is formed by immunization with a heterologous antigenic material, which antigenic material is branched with antigenic blood components derived from the same species as the antigenic material, wherein they are against impurities in the antigenic material. the directed antibodies in the 5 "globulin solution are removed by contacting the solution with plasma proteins and preferably also erythrocytes or erythrocyte stroma, characterized in that a) a preferably acrinol-containing 5 'globulin solution is treated with a suspension of blood acrinol. from B 'globulin and derived from the same species as the antigenic material, wherein contaminated antibodies to antigenic plasma protein components are adsorbed on the precipitated plasma protein and b) separating the solution of the purified K-globulin from the precipitated material. A method according to claim 1, characterized in that the _J'-glohulin to be purified is derived from horses which have been immunized with human blood lymphocytes. 7 f 7209600-1 'REFERRED TO PUBLICATIONS: Sweden patent application 1,480 / 72 German tooth 2,062,000 Other publications American medical association. Chicago. Journal. 210 (1969), pp. 1059-1060. Progress in surgery. 7 (1969), pp. 148-149. Immunochemistry. 6 (1969), p. 5 3. Behríngwerk means of communication. 51 (1972), pp. 93-951