RU2198668C2 - Method of preparing gamma-globulin solution designated for intravenous administration and preparation preparing by this method - Google Patents

Abstract

FIELD: medicine, preparative biochemistry. SUBSTANCE: invention relates to method of preparing gamma- globulin solution free of contaminating viruses designated for intravenous administration. Method involves stages of heat treatment at temperature from 50 C to 70 C for 10-100 h for inactivation of virus in the presence of stabilizing agent and fractionation of crude gamma-globulin solution with polyethylene glycol, treatment of gamma-globulin by ion-exchange resin followed by treatment of purified gamma-globulin with solvent-detergent for the further inactivation of viruses. Invention provides the development of combined multistage commercial process for preparing gamma-globulin that involves removal of denatured protein formed in heat sterilization. EFFECT: improved method of preparing, enhanced purity of preparation. 8 cl, 2 ex

Description

 The invention relates to a multi-stage commercial process for the production of immune serum globulin for intravenous administration containing IgG (g-globulin) as the main ingredient.

 The prior art various processes for obtaining solutions of g-globulin for intravenous administration, the source material for which are fractions of human plasma (fractions of Cohn). Some of the Cohn fractions contain higher g-globulin titers than others. Typically, the starting material for preparing g-globulin solutions is the Cohn II fraction or the Cohn II + III fraction.

 In the processes known from the prior art, various separation and sterilization techniques are used, modifications are constantly introduced in order to increase the purity of the final product, its safety and overall yield.

 Many commercial processes use a solvent / detergent treatment step or a heat treatment step to inactivate viruses. Today, multistage processes are not known for which the paste of Cohn II fraction or paste II + III would serve as the starting material and which would include two different virus inactivation procedures as part of an efficient process for the production of g-globulin with a high yield.

 In US patent 5151499 Kameyama et al. describes the process of obtaining protein compositions, according to which the protein composition is subjected to inactivation of enveloped viruses by treating the protein composition with a solvent / detergent and inactivation of envelope viruses by heat treatment of the protein composition. The patent states that the solvent / detergent treatment step is preferably carried out first in the presence of a protease inhibitor, and then heat treatment is carried out. When heat treatment is carried out in the liquid phase, the protein is isolated from the solvent / detergent mixture by adsorption on an ion exchange column prior to heat treatment. Heat treatment in the liquid phase can be carried out in the presence of sugar, sugar alcohol or amino acids as a stabilizer. While the '644 patent lists numerous source protein compositions, the preparation examples cited are limited to Factor IX, thrombin, fibrinogen and fibronectin. Removal of the denatured protein formed during the heat treatment step is not considered.

 In some prior art processes for the preparation of g-globulin solutions intended for intravenous administration, the introduction of heat treatment in the liquid phase, carried out in the presence of sorbitol as a heat stabilizer, is described as a stage of a multi-stage purification procedure, the starting material for which is a paste of Cohn II + III fractions . U.S. Pat. No. 4,876,088 to Hirao et al. the Cohn II + III fraction is subjected to fractionation with polyethylene glycol (hereinafter PEG) (first 8% w / v PEG, then 12% w / v PEG), then ion exchange chromatography (DEAE-Sephadex), and the antibodies of the human blood group are removed before heat treatment in the liquid phase in the presence of sorbitol as a protein stabilizer. On the other hand, in example 1 of US patent 4876088 Hirao et al. describe the preparation of a g-globulin solution for intravenous administration of fraction II + III from a paste. In this case, the paste is suspended in water, the pH is adjusted to 5.5 and centrifuged, after which the supernatant is subjected to heat treatment to inactivate viruses in the presence of 33% w / v sorbitol. This is followed by PEG fractionation (6% / 12%), in which the denatured protein is removed, and other purification steps, including DEAE-Sephadex ion exchange chromatography.

 The aim of the present invention was to develop a unifying, multi-stage commercial process for producing a highly purified g-globulin immune solution based on the Kohn fractionation process.

 Another objective of the present invention was to obtain a very pure, intended for intravenous administration of a solution of g-globulin, free of both enveloped viruses and envelope-free viruses, including all temperature-sensitive viruses.

 Another objective of the present invention was the creation of a commercial process for the production of g-globulin, which would include the removal to the second stage of inactivation of viruses of any denatured protein formed during thermal sterilization.

 The above and other objectives that are obvious to a qualified specialist, were achieved in the present invention, according to which alcohol fractions of Kohn, which can be partially purified, but enriched with g-globulin, are subjected to heat treatment in an aqueous medium in the presence of a heat stabilizer to inactivate viruses, then PEG fractionation and a second inactivation of viruses are carried out in the presence of a solvent or, preferably, a solvent / detergent mixture to destroy enveloped viruses, after which uet removing the solvent or mixture of solvent / detergent.

 According to a preferred embodiment of the present invention, sorbitol is a thermal stabilizer and trialkyl phosphate is used as a solvent.

 According to another preferred embodiment of the present invention, denatured products resulting from the inactivation of viruses by heat treatment are removed by PEG fractionation prior to the second viral inactivation, thereby obtaining further purified pure, warmed g-globulin.

 According to another preferred embodiment of the present invention, any particles present are removed prior to the solvent / detergent treatment step.

 According to another embodiment of the invention, g-globulin sterilized by heat and a solvent / detergent is prepared, suitable for intravenous administration.

 Fractions containing immunoglobulins are used as starting material. These fractions do not have to be human serum fractions, but must contain immunoglobulins. Specific examples of such fractions containing immunoglobulins are fraction II + III and fraction II obtained by Cohn fractionation with ethanol, as well as a paste of equivalent fractions containing immunoglobulins. Other starting materials are fractions I + II + III and fraction II + IIIw. The starting material may contain impurities such as human blood group antibodies, plasminogen, plasmin, kallikrein, prekallikrein activator, polymers IgM, IgA, IgG (referred to as "aggregates"), etc.

 The preferred starting material is the Cohn fraction II or the Cohn fraction II + III. When a paste of fractions II + III is used, it is recommended to subject it to a preliminary washing procedure to obtain fraction II + IIIw, which is used in the process according to the present invention. Fraction II + IIIw is a precipitate of fraction II + III washed with a solution of disodium phosphate.

Fraction II + IIIw can be obtained by dissolving the precipitate of fraction II + III in cold water for injection in a ratio of about 1 kg of paste II + III to 20 volumes of water. To solubilize lipids, lipoproteins and albumin, sodium phosphate solution is added to a final concentration of 0.003 M sodium phosphate. Cold ethanol is then added to a final concentration of about 20%. With the addition of alcohol, the temperature is gradually reduced to -5 ± 1 ^o C, and the pH is maintained at 7.2 ± 0.1, for example, by the addition of acetate buffer or dilute hydrochloric acid. The resulting precipitate of fraction II + IIIw is isolated by centrifugation and / or filtration at a temperature of -5 ± 1 ^o C.

According to the present invention, various preliminary steps of purification and / or reduction of the number of aggregates can be carried out prior to the first stage of virus inactivation. For example, when a paste of fractions II + III is used, containing about 20% alcohol and more than 70% IgG, it can be suspended in from 3 to 10 volumes, preferably from 3 to 5 volumes of cold water at at a temperature of about 0-5 ^{° C.} and with a pH adjusted to a value in the range of 4.5-6.0, preferably in the range of 5.0-5.5, using an acetate buffer with a pH of 4.0 or hydrochloric acid. The mixture is stirred for two to 15 hours so that all g-globulin goes into solution. Then, undissolved proteins such as albumin and a-globulins can be removed by centrifugation and / or filtration.

 If various Cohn fractions are used as starting material, the initial step or steps of the process can be appropriately selected so as to obtain fractions with a high IgG content. For example, when the Cohn II fraction (containing more than 95% IgG) is separated from the Cohn III fraction, the initial treatment of fraction II can be carried out at an acidic pH of from 3.2 to 5.0, preferably from 3.8 to 4.2, as described Uemura et al., In US Pat. No. 4,371,520, in order to destroy immunoglobulin aggregates present among immunoglobulin monomers and dimers, since the aggregates are known to have anti-complementary activity (ACA).

 Alternatively, when a Cohn II + III fraction is used as starting material, according to US Pat. No. 4,371,520, low pH treatment can be carried out as an additional step after the initial purification step described above and before virus inactivation by heat treatment.

 During the thermal sterilization step, the immunoglobulin protein is dissolved in water, or if it is an aqueous mixture, for example, the filtrate obtained after the partial purification described above, it can be used as is and sugar, sugar alcohol or amino acids can be added to it as a heat stabilizer . The thermal stabilizer is preferably sucrose, maltose, sorbitol or mannitol, most preferably sorbitol. Sugar or sugar alcohol is added to the immunoglobulin solution in powder form or mixed with a small amount of water before addition and its concentration in the solution is adjusted to 10 to 50 (w / v)% to saturation. At this point, the aqueous immunoglobulin solution contains a sufficient amount of water, so that the concentration of the total protein in the solution is from 1 to 6%, which corresponds to the usual starting material of fractions II + III, which contains about 300 g of protein per kilogram of paste.

After adding a heat stabilizer, the mixture is heated to a temperature of 50-70 ^o C for 10-100 hours, preferably up to 60 ^o C for 10-20 hours to inactivate temperature-sensitive viruses. The heat treatment step not only inactivates viruses, but, due to its denaturing effect, can selectively reduce the amount of individual undesirable proteins, usually associated with Cohn II + III fractions, such as precallicrein, plasmin, plasminogen and IgA.

After heat treatment, cold water is added in such an amount as to bring the protein concentration from 0.3 to 2.0%. The solution is cooled to 0-2 ^o C.

 Then carry out the fractionation of the heat-treated solution of PEG. PEG fractionation is a process well known in the art for the purification of immunologins, i.e. to separate the desired monomers and IgG dimers from aggregates of IgG and other impurities naturally occurring in the initial fractions of plasma proteins. However, in the described process, PEG fractionation also makes it possible to separate the desired IgG monomers and dimers from the unwanted denatured protein products formed during the heat treatment. These denatured protein products include denatured prekallikrein, plasminogen, plasmin, IgA, IgM and aggregates.

 Any PEG fractionation procedure known in the art may be used. Typically, two-stage PEG fractionation is performed. At the first stage of PEG fractionation, the concentration of PEG and pH is chosen so that the desired IgG monomers and dimers remain in solution, and undesirable proteins such as aggregates are precipitated from the solution. After centrifugation and / or filtration, the concentration of PEG is increased and the pH adjusted to precipitate the desired IgG monomers and dimers.

For example, the first step of PEG fractionation can be carried out at a pH of from 5.0 to 7.5, preferably in the range of pH values from 6.5 to 7.5, if a paste of fractions II + IIIw is used as the starting material, and preferably in the range of values pH from 5.5 to 6.0, if the paste of fractions II + IIIw is used as the starting material, and the concentration of PEG varies from 4-8%, preferably 4-6%, if the paste of fractions II + IIIw is used as the starting material , or 6-8%, if a paste of fractions II + III is used as the starting material. While maintaining the temperature at about 0-2 ^{° C., the} first step of PEG fractionation takes from 1 to 8 hours, after which the precipitate is removed as described above.

 Then the pH of the filtrate was adjusted to a value of 8.0-9.0, preferably from 8.5 to 8.9, and another portion of PEG was added to a final concentration of 10 to 15%, preferably about 12%. The resulting precipitate, which is purified immunoglobulin, is removed by filtration and / or centrifugation.

 Further details of PEG fractionation procedures applicable to the practice of the present invention can be found in the aforementioned US Pat. Nos. 4,867,088 to Hirao et al. And 4,845,199 to Hirao et al.

 The final necessary step of the process disclosed in the present invention is the second step of virus inactivation using a solvent or a solvent / detergent mixture. As described below, the subsequent cleaning steps, in particular using ion exchange resins, can be carried out both before and / or after treatment with a solvent / detergent. Of particular benefit is the procedure that combines anion exchange treatment before virus inactivation with solvent / detergent and cation exchange treatment after virus inactivation with solvent / detergent. During this procedure, some unwanted protein materials (such as prekallikrein activator, IgA, IgM and albumin) found in human plasma can be removed from IgG using an anion exchanger, and then these materials (prekallikrein activator, IgA, IgM, albumin and PEG) as well as residual reagents used for treatment with a solvent / detergent, can be removed by cation exchange chromatography.

 If this was not done at earlier stages of the process, then the solution intended for treatment with a solvent / detergent should be freed from any particles, which may include denatured protein. Therefore, it is preferable to filter the solution through a 1 micron or thinner filter before adding solvent / detergent. It will also reduce the likelihood of viruses entering large particles where they could avoid contact with solvent / detergent.

 Currently, the most preferred solvent for inactivating enveloped viruses is trialkyl phosphate. The trialkyl phosphate used in the practice of the present invention is not subject to any particular restrictions, but it is preferable to use tri- (n-butyl) phosphate ("TNBP"). Other useful trialkyl phosphates are tri- (ter-butyl) phosphate, tri- (n-hexyl) phosphate, tri- (2-ethylhexyl) phosphate, etc. A mixture of two or more different trialkyl phosphates is possible.

 Trialkyl phosphate is used in concentrations from 0.01 to 10 (w / v)%, preferably from 0.1 to 3 (w / v)%.

 Trialkyl phosphate can be used with or without a detergent or surfactant. It is preferable to use trialkyl phosphate in combination with a detergent. The functions of the detergent are to enhance the contact of the viruses contained in the immunoglobulin composition with trialkyl phosphate.

 Examples of detergents include polyoxyethylene fatty acid derivatives, partial esters of anhydrous sorbitol, such as Polysorbate 80 (Trade name: Tween 80, etc.) and Polysorbate 20 (Trade name: Tween 20, etc.); and non-ionic agents such as ethoxylated alkyl phenol (Trade Name: Triton X100, etc.), as well as other surfactants and detergents such as zwitterionic detergents, etc.

 When using a detergent, it should not be added in critical quantities, for example, it can be used in concentrations from 0.001 to 10%, preferably from 0.01 to 3%.

According to the present invention, the treatment of the immunoglobulin-containing composition with trialkyl phosphate is carried out at a temperature of from 20 to 35 ^° C, preferably from 25 to 30 ^° C, for more than one hour, preferably from 5 to 8 hours, most preferably from 6 to 7 hours

 During treatment with trialkylphosphate, immunoglobulin is in the aqueous medium in the form of a solution with a protein concentration of 3 to 8%.

If the anion exchange treatment has not been carried out before the treatment with the solvent / detergent, then it can be carried out with the already treated solvent / detergent immunoglobulin. Preferably, at least the cationic treatment is carried out with the treated solvent / detergent immunoglobulin. The ion exchange treatment is carried out with immunoglobulin dissolved in an aqueous solvent, usually with a pH of about 5-8, with a sufficiently low ionic strength for maximum IgG adsorption. The protein concentration is usually in the range of 1-15 (w / v)%, more preferably in the range of 3-10 (w / v)%. The ion exchanger is balanced with the same aqueous solvent in which the immunoglobulin is dissolved. You can apply both continuous and batch (batch) process. For example, when performing a batch anion exchange treatment, an immunoglobulin solution is mixed with an anion exchanger in an amount of 10 to 100 ml per ml of a pre-treated anion exchanger (for example, 1 g of DEAE Sephadex A-50 resin swells up to about 20 grams of wet weight in a 0.4% sodium chloride solution ), stirring the mixture at 0-5 ^o C for 0.5-5 hours, followed by filtration or centrifugation from 6000 to 8,000 rpm for 10-30 minutes to obtain a supernatant. In a continuous process, an immunoglobulin solution is passed through a column with an anion exchanger at a rate of 10 to 100 ml per ml of anion exchanger and an unbound fraction is collected.

 The anion exchanger used, for example, contains anion exchange groups bound to an insoluble carrier. Anion exchange groups include diethylaminoethyl (DEAE), a quaternary aminoethyl group (QAE), etc., and insoluble carriers include agarose, cellulose, dextran, polyacrylamide, etc.

Suitable cation exchangers include carboxymethyl Sephadex (CM-Sephadex), CM-cellulose, SP-Sephadex, CM-Sepharose and S-Sepharose. 1 ml of the pre-treated cation exchanger (for example, 1 g of DEAE Sephadex C-50 resin swells to approximately 30-35 g of wet weight in a 0.4% sodium chloride solution) is mixed with 0.5-5.0 ml of an immunoglobulin solution and stirred at 0-5 ^o С for 1 to 6 hours. The suspension is centrifuged or filtered to separate the adsorbed IgG resin. A continuous process may also be used.

 In the cation exchange treatment under the described conditions, IgG is adsorbed and, after washing the protein-adsorbed cation exchange resin, IgG can be eluted, for example, with a 1.4 N sodium chloride solution.

 After carrying out the above process steps, IgG is clarified, diafiltered and concentrated to the required degree. If necessary, a stabilizer such as D-sorbitol can be added and final concentrations adjusted to obtain a solution containing about 100 mg / ml IgG and about 50 mg / ml D-sorbitol with a pH of about 5.4. This solution is then sterilized by filtration through a sterilized bacterium-retaining filter and ampouled.

 The following examples are provided to illustrate the invention and do not limit its scope.

 If necessary, other immunoglobulin purification procedures can be combined with the process described above. For example, the bentonite clarification step can be used, which is useful for reducing the amount of kallikrein and prekallikrein activator. The following example is an illustration.

Example 1. g globulin, subjected to heat treatment and treatment with solvent / detergent
685 g of the paste of fraction II + IIIw are suspended in 11.9 kg of cold water. A solution of sodium acetate trihydrate was added to the suspension to a final concentration of about 0.04 M to selectively solubilize IgG. After stirring for 15 minutes, the pH of the suspension was adjusted to 4.8 with acetate buffer at pH 4.0. Cold alcohol (95%) was added to the suspension to a final concentration of 17%. During the addition of alcohol, the temperature of the suspension is gradually reduced to -6 ^° C. To the suspension, 303 g of Celite 535 (Celite Corporation) is added as a filtering agent to a final concentration of about 2%. After stirring for 1 hour, Celite and fraction III paste containing unnecessary proteins such as plasmin, plasminogen, IgA and IgM are removed by filtration using a filter press. The filtrate is then clarified by passing through filters of 0.45 μm and 0.2 μm.

The pH of the clarified solution of fraction II + III was adjusted to 4.0 by adding 0.1 N hydrochloric acid and concentrated by ultrafiltration to a volume of 3.4 L (1/5 of the initial volume). Cold water is added to the concentrated solution in an amount equal to the volume removed during the first ultrafiltration, and the solution is again subjected to ultrafiltration to 1/5 of the original volume. At this stage, the protein concentration in the solution is about 2%. The solution is concentrated to about 4.0% protein and diafiltered against cold water until the conductivity of the solution drops below 300 μS / cm, which helps to prevent aggregation and reduce denaturation during heat treatment. The solution is again concentrated to 8.8% protein. D-sorbitol is added to the solution to a final concentration of about 33%. After stirring for 30 minutes, the pH of the sorbitol-containing solution was adjusted to 5.5 by the addition of 0.5 N sodium hydroxide. Then the solution is heated for 10 hours at 60 ^o C. After heat treatment, 3 volumes of cold water are added to the solution and the diluted solution is cooled to 0-2 ^o C.

 The pH of the solution was adjusted to 6.9 with 0.25 N sodium hydroxide and 50% polyethylene glycol (PEG) 3350 was added to the solution to a final concentration of 4%. The concentration of sodium chloride in the solution was adjusted to 8 mM to facilitate the precipitation of impurities and aggregates at a pH of 6.9. The precipitate formed is removed by filtration. The pH of the filtrate was adjusted to 4.8 by adding 0.1 N hydrochloric acid and bentonite with a final concentration of about 0.25% was added. The pH of the suspension of bentonite is adjusted to 5.2 and the suspension is filtered to remove bentonite. The pH of the filtrate was adjusted to 8.5 by adding 0.25 N sodium hydroxide and 50% polyethylene glycol (PEG) 3350 was added to the solution to a final concentration of 12%. The resulting precipitate (purified immune globulin) is removed by centrifugation.

 About 175 g of the paste purified as described above immunoglobulin suspended in 750 g of a 0.3% solution of sodium chloride. The pH of the suspension was adjusted to 5.5 and the suspension was stirred for 2.5 hours.

 To the solution was added 62 g of a pre-balanced (0.3% sodium chloride solution with a pH of 5.5) SM-Sephadex A-50 resin and the suspension was stirred for 2 hours. The suspension was then filtered to remove the resin. The concentration of sodium chloride was adjusted to 0.4% and a mixture of tri-n-butyl phosphate (TNBP) and Polysorbate 80 was added to the filtrate, so that the final concentration of TNBP was 0.3%, and Polysorbate 80 -1%. After overnight incubation, the pH of the solution was adjusted to 5.8 and 860 g of a pre-balanced (0.4% sodium chloride solution with a pH of 5.8) SM-Sephadex C-50 resin was added. After stirring for 2 hours, the suspension is filtered. After washing the resin SM-Sephadex with 0.3% sodium chloride three times, the adsorbed IgG was eluted with 1.4 N sodium chloride. The eluate is clarified and diafiltered and concentrated. D-sorbitol is added and the final concentration is adjusted to obtain a solution with a concentration of 100 mg / ml IgG and about 50 mg / ml D-sorbitol with a pH of 5.4. It is then sterilized by filtration through a sterilized bacterium-retaining filter and is ampouled.

 The intermediate bentonite treatment step described in this example is useful for reducing the presence of antihypertensive enzymes such as kallikrein and prekallikrein activator (see table 1).

Example 2. g globulin, subjected to heat treatment and treatment with solvent / detergent
One (1) kg of paste of fraction II + III is suspended in 4.5 kg of cold water at 0-2 ^° C. After stirring for 1 h, the pH of the suspension is adjusted to 5.0 by adding 1N hydrochloric acid. After stirring for 3 hours at pH 5.0, the precipitate was removed by centrifugation. D-sorbitol is added to the centrifugate to a final concentration of 33% and the mixture is stirred for 1 hour. The pH of the suspension is adjusted to 5.5 and then it is heated at 60 ^{° C.} for 10 hours. After heat treatment, 3 volumes of cold water and a diluted solution are added to the solution. cooled to 0-2 ^o C. Add a 50% solution of PEG 3350 to a final concentration of PEG 6%. The pH of the PEG-containing suspension was adjusted to 5.7 by adding 0.5 N sodium hydroxide and the suspension was stirred for 2 hours. Acid washed Celite 535 was added to a final concentration of 1.5% and the suspension was stirred for 1 hour. The precipitate was removed together with celite by filtration. The pH of the filtrate was adjusted to 8.8 by the addition of 0.5 N sodium hydroxide, the concentration of PEG was adjusted to 12% by the addition of a 50% PEG solution. The pH of the suspension was adjusted to 8.8, the suspension was stirred for 1 h and filtered, collecting a paste of purified immunoglobulin. About 251 g of purified immunoglobulin paste was obtained.

251 g of purified immunoglobulin paste prepared as described above are suspended in 1.4 kg of a 0.3% sodium chloride solution. After stirring for 1 h, the pH of the suspension was adjusted to 6.0 by the addition of 5% acetic acid. After the paste is completely dissolved, 104 g of a pre-balanced (0.3% sodium chloride solution with a pH of 6.0) SM-Sephadex A-50 resin are added and the solution is stirred for 2 hours. The resin is removed by filtration. Then the filtrate is clarified by filtration through a 0.2 μm filter. The concentration of sodium chloride in the clarified solution is adjusted to 0.4%. Then a mixture of tri-n-butyl phosphate (TNBP) and Polysorbate 80 is added to the solution so that the final concentration of TNBP is 0.3% and Polysorbate 80 is 1%. The solution was incubated at 27 ^o C for 1 h and left overnight in the refrigerator at 4 ^o C. The next day, the pH of the solution was adjusted to 5.8 and 1.8 kg of pre-balanced (0.4% sodium chloride solution with pH 5 were added). , 8) SM-Sephadex S-50 resin. After stirring for 2 hours, the resin was removed by filtration. After washing the resin SM-Sephadex with 0.3% sodium chloride three times, the adsorbed IgG was eluted with 1.4 N sodium chloride. The eluate is clarified and diafiltered and concentrated. D-sorbitol is added and the final concentration is adjusted to obtain a solution with a concentration of 100 mg / ml IgG and about 50 mg / ml D-sorbitol. The solution was divided into two portions, portion A and portion B. The pH of portion A was adjusted to 5.4, and the pH of portion B was adjusted to 4.3. Then, the solutions of portions A and B are individually sterilized by filtration through sterilized bacteria-retaining filters and ampouled (see Table 2).

 For a qualified specialist obvious possible variations of the invention.

Claims (8)

1. A method of obtaining a gamma globulin solution intended for intravenous administration, comprising: a) heat treatment of a crude gamma globulin solution to inactivate heat-sensitive viruses at a temperature of from 50 to 70 ^o C for 10-100 hours in the presence of a thermal stabilizer, as which use sugar or sugar alcohol, or an amino acid; b) fractionation of the heat-treated gamma-globulin solution with polyethylene glycol to obtain a purified gamma-globulin solution, while polyethylene glycol fractionation is carried out in at least two stages, impurities are removed as precipitate in the first fractionation step, and gamma globulin is isolated in the form of precipitate on the second stage of fractionation with polyethylene glycol; c) treating the gamma globulin solution with an anion exchange resin; d) processing the purified gamma globulin solution with an organic solvent to inactivate enveloped viruses; and then e) treating the gamma globulin solution with a cation exchange resin.  2. The method according to claim 1, characterized in that the crude gamma globulin solution is a Cohn fraction I + II + III, a Cohn fraction II + III, a Cohn fraction II + IIIw, or a Cohn fraction II.  3. The method according to claim 1, characterized in that the crude gamma globulin solution is subjected to at least one purification step prior to the heat treatment step. 4. The method according to claim 1, characterized in that the heat treatment step is carried out at a temperature of about 60 ^o C for about 10 hours  5. The method according to claim 1, characterized in that the trialkyl phosphate is used as the organic solvent used in step (d).  6. The method according to claim 1, characterized in that the organic solvent contains a detergent.  7. The method according to claim 1, characterized in that after the first stage of fractionation with polyethylene glycol, the step of clarification with bentonite is carried out.  8. A solution of gamma globulin, intended for intravenous administration, obtained by the method according to claim 1.

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