MXPA00012230A - Process for producing immunoglobulins for intravenous administration and other immunoglobulin products - Google Patents

Abstract

The present invention relates to a process for purifying immunoglobulin G from a crude immunoglobulin-containing plasma protein fraction. Said process includes a number of steps of which the anion exchange chromatography and the cation exchange chromatography are preferably connected in series. An acetate buffer having a pH of about 5.0-6.0 and having a molarity of about 5-25 mM is preferably used throughout the purification process. The invention further comprises an immunoglobulin product which is obtainable by this process. The invention also relates to an immunoglobulin product which has a purity of more than 98%, has a content of IgG monomers and dimers of more than 98.5%, has a content of IgA less than 4 mg of IgA/l, and contains less than 0.5%polymers and aggregates. Said product does not comprise detergent, PEG or albumin as a stabilizer. The product is stable, virus-safe, liquid and ready for instant intravenous administration.

Description

PROCESS OF PRODUCTION OF IMMUNOGLOBÜLNAS FOR INTRAVENOUS ADMINISTRATION AND OTHER PRODUCTS OF IMMUNOGLOBULIN FIELD OF THE INVENTION The present invention relates to a process for purifying immunoglobulins, i.e., immunoglobulin G (IgG), from crude plasma or from a protein fraction of crude plasma. The present invention also relates to an immunoglobulin product and to the use of such an immunoglobulin product for medical purposes. BACKGROUND OF THE INVENTION Human nopales immunoglobulins (INH) for use in the prevention and treatment of a number of infectious diseases were introduced at the end of the 1940s. INHs prepared by the cold alcohol fractionation method in accordance with Cohn & Oncley (Cohn E ~ et al., (1946), J Am Chem Soc, 68, 459-475), (Oncley et al., (1949), J Am Chem Soc, 71,541-550) and subsequently also by modification made by Kistler and Nitsch ann (Kistler P and Nitschmann HS, (1952), Vox Sang, 7, 414-424) proved to be efficient and safe against the transmission of viral infections when administered subcutaneously or intramuscularly. The lack of congenital immunoglobulins or Ref: 125644 acquired total or partial (primary and secondary immunodeficiency syndrome, respectively) manifests itself through frequent and frequent serious infections, especially of bacterial nature. The prevention of such infections was previously achieved by repeated intramuscular or subcutaneous injections of large amounts of INH for up to several times a week as a lifelong treatment, which is very painful when the drug is administered intramuscularly. Therefore, at the beginning of 60 xs, the administration of INH was attempted intravenously. The studies showed that approximately 5% of healthy volunteers and approximately 95% of patients with immunoglobulin deficiency, developed immediate adverse effects ranging from dyspnea to circulatory shock and being of such a serious nature that the intravenous administration of INH had to be abandoned . The reason for the aforementioned adverse effects was due to aggregations of immunoglobulins which, among other effects, strongly activated the complement system. This was observed particularly in patients lacking immunoglobulins. Particularly serious adverse effects of anaphylactic nature could be observed in patients who developed antibodies against IgA. Consequently, methods were developed to avoid the formation of aggregates and / or eliminate these aggregates during the preparation process and about twenty years ago the first generation of immunoglobulins for intravenous administration (IVIG) was tested and found adequate. The original purpose of IVIG was to relieve infectious episodes in patients with complete or partial lack of congenital or acquired immunoglobulins, and to eliminate discomfort related to the administration of INH. Another advantage of IVIGs is that large doses of immunoglobulin can be administered in a short time, whereby it is possible to obtain sufficiently high blood concentrations very rapidly. Especially when treating serious bacterial infections, it is important to establish high concentrations at the sites of infection and quickly. In recent years, IVIGs proved to be efficient in other serious diseases whose treatment would otherwise be difficult, e.g. hemorrhages caused by the disappearance of immune-based blood platelets, idiopathic thrombocytopenic purpura (ITP), in some rare diseases such as Kawasaki syndrome and a number of autoimmune diseases such as polyradiculitis (Guilléiin-Barre syndrome). The treatment of other diseases that have been difficult to date, is undergoing clinical studies with IVIG. The mechanism of action of these diseases has only been partially clarified. The effect is supposed to be related to the so-called immunomodulatory properties of IgG, e.g. a blockage of Fc receptors? of phagocytic cells, an increase in IgG metabolism, the deregulation of cytokine production and interference with a putative network of idiotypes / anti-idiotypes, especially relevant for the neutralization of autoimmune reactivity. The first generation of IGIV was prepared by breaking Pepsin from raw material 8fraction II of Cohn), the purpose of which was to remove the aggregates of ip immunoglobulin. No column chromatography steps were included in the process. The product had to be lyophilized in order to remain stable for a reasonable period of time and dissolve immediately before use. The raw material for IVIG was INH that had proven to be safe with respect to virus transmission when administered by intramuscular injection. Thus, IVIGs were considered safe. After several years of clinical use, however, IVIG products from some manufacturers surprisingly demonstrated that they caused the transfer of the hepatitis C virus.

Studies to clarify the fate of the viruses during the production of INH, showed that the removal of the virus in the plasma fractionation process to INH is not very good. The safety of INHs for intramuscular use is likely due to the fact that it contains protective immunoglobulins. In combination with the modest volume injected and the intramuscular route of administration, these protective immunoglobulins could neutralize common viruses in non-infectious plasma. Viral infections can occur especially when large doses of immunoglobulins are administered intravenously, as was demonstrated at the beginning of the 1990s. Therefore, it was recognized that the production process should comprise one or more well-defined stages of inactivation. and / or virus removal. A second generation of IG3V based on unruptured and unmodified immunoglobulin molecules with low anticomplementary activity and high stability was introduced in the mid 80's, but still in the form of lyophilized product. These IVIG were purified by several chromatography steps. Products of this type currently dominate the IGIV market. 'The first and second generations of IGIV, therefore, appear in the form of lyophilized powders that dissolve immediately before use.

The solution of lyophilized IVIG is slow (up to 30 minutes for a bottle). Often several portions have to be dissolved for a patient. As it is a high priority for users to have an IVIG in a ready-to-use solution, liquid products have been introduced into the market. More importantly, there is still a need to improve the production processes in order to obtain a highly purified, stable and completely natural IGIV preparation, with greater clinical efficacy and fewer adverse reactions. Therefore, there is a need to develop improved processes for purifying IgG from crude plasma or from a plasma protein fraction to obtain a virus-safe liquid IGIV product. Finally, the process must be designed in such a way that it can be used in large-scale production. The purification process described in the present application, leads to obtain a liquid immunoglobulin product for intravenous administration which can be characterized as a new generation of highly purified, completely natural, biologically active, IVIV-inactivated, for virus and stable, IVIG. which does not contain any detergent, polyethylene glycol (PEG) or albumin as a stabilizer.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to an improved purification process and an improved immunoglobulin product which, inter alia, can be administered intravenously. An immunoglobulin product obtained by the method of the present invention can be referred to as third generation IGIV. The process is characterized by the following conditions for fractionation: the pepsin break is avoided, the aggregates and the particles are removed by precipitation (validated process stage to function as a virus removal stage), an additional purification is achieved by means of methods of ion exchange column chromatography, an S / D treatment is introduced as a virus inactivating step and the preparation is formulated as a liquid product. Due to the improved purity of the immunoglobulin product c_oe is obtained by the process of the present invention, in comparison with the products of the prior art, the addition of stabilizers such as non-ionic detergent, PEG or albumin with the i 'is not necessary. to avoid the aggregation of IgG during the storage of IVIG as a liquid product. The product obtained by the process of the present invention has a higher quality than the products of the prior art and provides better clinical effects and undesirable adverse effects are virtually absent. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process for purifying immunoglobulins, i.e. IgG, from crude plasma or from a plasma protein fraction containing immunoglobulins, wherein the process comprises the steps of: (a) preparing an aqueous suspension of the plasma protein fraction containing crude immunoglobulin; (b) adding a precipitant of soluble, substantially non-denaturing proteins, to the suspension of the step of part a) in an amount sufficient to cause the precipitation of a high proportion of non-immunoglobulin G proteins, aggregated immunoglobulins and potentially including particles infectious particles such as viral particles, without causing substantial precipitation of the monomeric immunoglobulin G, * thereby forming a mixture of a solid precipitate and a liquid supernatant; (c) recovering a clarified supernatant containing immunoglobulin G from the mixture of the step of part (b); (d) applying the clarified supernatant containing immunoglobulin G from the step of part (c) to an ion exchange resin and subsequently a cation exchange resin; (e) washing the protein contaminants and proteins precipitated from the cation exchange resin with a buffer solution having a pH and ionic strength sufficient to remove contaminants from the resin without causing substantial elution of the immunoglobulin G; (f) eluting the immunoglobulin G from the cation exchange resin with a substantially non-denaturing buffer having a pH value and ionic strength sufficient to cause efficient elution of the immunoglobulin G, thereby recovering an eluate containing immunoglobulin G; (g) performing a day / ultrafiltration of the immunoglobulin G-containing eluate of the step of part (f) to concentrate and / or dialyse the eluate and optionally add a stabilizing agent; (h) adding a virucidal amount of virus inactivating agent to the dialyzed / ultrafiltered fraction containing immunoglobulin G and optionally stabilized from the step of part (g), obtaining a substantially safe solution against virus containing immunoglobulin G; (i) applying the solution containing immunoglobulin G from the step of part (h) to an anion exchange resin and subsequently to a cation exchange resin; (j) washing the cation exchange resin of the step of part (i) with a buffer solution having a pH and ionic strength sufficient to wash the protein contaminants and virus quenching agent from the resin, without causing a substantial elution of immunoglobulin G; (k) eluting the immunoglobulin G from the cation exchange resin of the step of part (j) with a substantially non-denaturing buffer having a pH and sufficient ionic strength p > to cause efficient elution of immunoglobulin G, thereby recovering an eluate containing immunoglobulin G; and (1) subjecting the immunoglobulin G-containing eluate from step (k) to one day / ultrafiltration to decrease the ionic strength and concentrate the immunoglobulin 'G of the solution, and adjust the osmolarity by the addition of a saccharide The raw material of the purification process of the present invention may be crude plasma, but advantageously it is a crude plasma protein fraction containing immunoglobulins.The raw material for the purification process may be normal human plasma or it may originate from donors with high levels. Specific antibody titers, eg hyperimmune plasma In the present description, the term "immunoglobulin-containing plasma fraction" encompasses all possible raw materials for the present process, eg cryoprecipitate-free plasma or cryoprecipitate-free plasma from which Removed several plasma proteins such as factor IX and antithrombin, different fractions of C ohn and fractions obtained from the PEG precipitation procedure (Polson et al. (1964), Biochem Biophys Acta, 82, 463-475; Polson and Ruiz-Brazo, (1972)) Vos Sang, 23, 107-118) or with ammonium sulfate. In a preferred embodiment, the plasma protein fraction are fractions II and III of Cohn, but Cohn fraction II or Cohn fractions I, II and III can also be used. The different Cohn fractions are preferably prepared from plasma by the standard Cohn fractionation method, essentially as modified by Kistler-Nitschmann. In addition to the immunoglobulins, the Cohn fractions contain for example fibrinogen, alpha-globulins and beta-globulins, including several lipoproteins, which are preferably removed during the subsequent purification process. Filters may or may not be present, depending on the isolation method used to obtain the Cohn fractions (i.e., centrifugation or filtration). The first stage of the process according to the present invention includes preparing an aqueous suspension of a plasma protein fraction containing immunoglobulins, wherein the concentration of IgG in the suspension is sufficiently high so that, during the next stage of precipitation, a large proportion of non-IgG proteins, especially those of higher molecular weight, aggregated immunoglobulins and other aggregated proteins as well as potentially infectious particles, precipitate without the substantial, monomeric IgG precipitation. This is generally achieved if the concentration of the IgG in the regulated and filtered suspension is at least about 4 g / L before the addition of the precipitating agent. It should be taken into consideration that the influence of the protein concentration as well as the pH and the temperature of the suspension in the precipitation step depend on the selected precipitating agent. It is preferred that the plasma protein fraction is suspended in water and / or a buffer at a substantially non-denaturing temperature and pH. The term "substantially non-denaturing" implies that the conditions to which the term refers do not cause the irreversible substantial loss of the functional activity of the IgG molecules, eg, loss of antigen-binding activity and / or loss of activity. biological Fc function (see Example 2). Advantageously, the plasma protein fraction is suspended in acidified water with at least one non-denaturing pH regulator system at volumes of 6 to 9, preferably 7 to 8 times greater than that of the plasma protein fraction. The pH of the suspension containing immunoglobulins is preferably kept below 6, for example within the range of 4.0 to 6.0, preferably from 5.1 to 5.7, more preferably approximately 5.4, in order to ensure optimal solubility of the immunoglobulins and to ensure the optimal effect of the subsequent stage of PEG precipitation. Any suitable acidic buffer solution can be used, but the regulating system preferably contains at least one of the following regulators and acids: sodium phosphate, sodium acetate, acetic acid, HCl. Technicians in the field will see what many other regulatory solutions can be used. The immunoglobulin suspension is preferably kept at a cold temperature, inter alia in order to avoid substantial protein denaturation to minimize protease activity. The immunoglobulin suspension and water, as well as the added regulatory system, preferably have the same temperature within the range of 0 to 12 ° C, preferably 0 to 8 ° C, more preferably 1 to 4 ° C. The suspension of a pulp precipitated with ethanol contains relatively large amounts of added protein material. Optionally, the suspension containing immunoglobulins is filtered for the purpose of removal, e.g. the large aggregates, the filtration improver if any, and the residual paste not dissolved. The preferred filtration is carried out by means of depth filters, e.g. C 150 AF, AF 2000 or AF 1000 (Schenk), 30LA (Cuno) or similar filters. The removal of aggregates, filtration improvers if any and residual protein material not dissolved, it can also be carried out by centrifugation. At least one water-soluble, substantially non-denaturing protein precipitant is added to the filtered suspension containing immunoglobulins, in an amount sufficient to cause precipitation of a high proportion of the highest molecular weight proteins, lipoproteins, aggregated proteins, among these the immunoglobulins added.

Other particulate materials such as potentially infectious particles, e.g. Viral particles are also precipitated without causing substantial precipitation of the monomeric IgG. The term "infectious particles" as used in the present invention comprises e.g. virus particles (such as hepatitis viruses, HIV 1 and HIV 2) and bacteria. The substantially non-denaturing and water-soluble protein precipitants are well known in the field of protein purification. Such precipitants are used to fractionate proteins, resulting in partial purification of the proteins from suspensions. Suitable protein precipitants for use in the process of the present invention include various molecular weight forms of PEG, caprylic acid, and ammonium sulfate. Those skilled in the art will note that other water-soluble and non-denaturing precipitants can be used as alternative means for precipitation. The term "add a protein precipitant" and variants of that term mean the addition of one or more types of protein precipitation agents. A preferred precipitating agent is the organic agent PEG, particularly PEG within the molecular weight range of 3000 to 8000 Da, such as PEG 3350, PEG 4000, PEG 5000 and especially PEG 6000 (the numbers of these specific PEG compounds represent their weight average molecular). The advantage of using PEG as a precipitant is that PEG is non-ionic and has protein stabilizing properties, e.g., PEG at a low concentration is well known as stabilizers for IVIG products. The precipitation stage also works as a virus removal stage. PEG concentrates and precipitates viruses independently of the species, size and surface coating of the same. A given amount of protein precipitant is added to the filtered suspension to precipitate most of the high molecular weight proteins and aggregated proteins and particles, without substantial precipitation of the monomeric IgG, forming a clean supernatant solution. The protein precipitant can be added as a solid powder or in the form of a concentrated solution. For PEG as a precipitant, a general rule applies that the higher the molecular weight of the compound, the lower the concentration of PEG needed to cause the proteins to precipitate. When PEG 3350, PEG 4000 or "PEG 6000 preference" is used, the concentration of the precipitant in the filtered suspension advantageously is in the range of 3 to 15% by weight, for example 4 to 10% (for example about 5%). %, 6%, 7%, 8%, 9%, 10%), where 6% is the most preferred amount.In the precipitation stage, the precipitation process is allowed to proceed at least until the equilibrium is reached. the solid and liquid phase, eg, normally for at least 2 hours, for example from about 2 to about 12 hours, preferably about 4 hours.During the precipitation stage, the suspension is preferably kept at low temperature (eg less of about 12 ° C, for example less than about 10 ° C, preferably between 2 and 8 ° C.) The most suitable temperature depends on the identity of the protein precipitating agent.After concluding protein precipitation, a clarified supernatant q IgG contains almost exclusively in monomeric form, is recovered from the mixture of the solid precipitate and the liquid supernatant resulting from the precipitation. The recovery can be carried out by conventional techniques for separating liquids from solid phases, for example centrifugation and / or filtration. Preferably, a flow centrifuge (e.g., Westfalia) with a force of 1000 to 5000 g is used. Optionally, the supernatant containing clarified recovered IgG is filtered to remove large and aggregated particles. This is optionally followed by a sterile filtration performed by the use of a conventional sterilization filter (such as a 0.22 μm Millipore or Sartorius filter), which eliminates e.g. bacteria of the solution. The clarified and optionally filtered supernatant containing IgG, is subjected to at least one step, for example two steps, but optionally more steps of anionic and cation exchange chromatography, in order to remove a substantive proportion of the remaining non-IgG contaminants, for example IgA, albumin as well as aggregates. In a preferred embodiment, the clarified and optionally filtered supernatant containing IgG is applied to an anion exchange resin and subsequently to a cation exchange resin, packaged in two columns of appropriate dimensions When the ion exchange chromatography steps are performed for the IgG purification, it is preferred that the conditions, eg pH and ionic strength are selected in such a way that a large portion of the contaminants (eg non-IgG proteins such as IgA, transferrin, albumin and aggregates) in the solution bind to the anion exchange resin, while substantially no amount of igG is adsorbed to the anion exchange resin. With respect to the subsequent cation exchange chromatography, the selected preferred conditions result in the binding of substantially all of the IgG molecules present in the solution that was applied to the cation exchange resin. Protein contaminants not adsorbed to the anion exchange resin and the precipitating agent are removed in the subsequent washing of the cation exchange resin. In a preferred embodiment of the process of the present invention, the anion exchange resin and the cation exchange resin are connected in series. In the present context, the term "connects in series", when used with reference to ion exchange resins, means that the proteins that pass through the anion exchange resin are charged directly to the cation exchange resin, without change of regulatory solution or other conditions. There are several reasons that make it advantageous that anion exchange chromatography and cationic exchange chromatography are performed in a single step using two chromatography columns connected in series, instead of two independent chromatography steps, e.g. with different compositions of regulatory solution. The use of two chromatography columns connected in series makes the operation more practical, e.g. there is no need for an intermediate stage of collection of the fraction containing IgG between the two methods of ion exchange chromatography, to possibly adjust the pH and ionic strength. In addition, the flow of buffer solution is applied to both columns at the same time and the two columns are balanced with the same buffer. However, it is contemplated that it is also possible to perform the chromatography step in two steps, i.e. An anion exchange resin and the cation exchange resin are not connected in series. Performing the chromatography in two steps, as mentioned above, would be more laborious as compared to keeping the ion exchange resins connected in series. It is currently contemplated that the high degree of purity, the high content of IgG monomers and dimers and the low IgA content in the IGIV product of the present invention are partially due to the use of two chromatography columns connected in series. As will be apparent to a person skilled in the art, ion exchangers can be based on various materials with respect to the matrix as well as to the groups with attached charge. For example, the following matrices can be used, in which "the aforementioned materials can be more or less cross-linked: based on agarose (such as Sepharose CL-6B®, Sepharose Fast Flow® and Sepharose High Performance®), based in cellulose (such as DEAE Sephacel®), based on dextran (such as Sephadex®), based on silica and based on synthetic polymers. For the anion exchange resin, the charged groups that are covalently bound to the matrix, for example may be dimethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and / or quaternary ammonium (Q). For the cation exchange resin, charged groups that are covalently bound to the matrix, for example, can be carboxymethyl (CM), sulfopropyl (SP) and / or ethylsulphonate (S). In a preferred embodiment of the process of the present invention, the anion exchange resin used is DEAE Sepharose Fast Flow®, but other anion exchangers can be used. A preferred cation exchange resin is the CM Sepharose Fast Flow®, but other cation exchangers can also be used. The appropriate volume of resin used when packed in a column of ion exchange chromatography, is reflected by the dimensions of the column, i.e. the diameter of the column and the height of the resin, and varies depending on, for example, the amount of IgG in the applied solution and the binding capacity of the resin used. Prior to performing ion exchange chromatography, the ion exchange resin is preferably equilibrated with a buffer which allows the resin to bind with its counter-ions. Preferably, the anionic and cationic exchange resins are balanced with the same buffer, since this facilitates the process since only one has to be prepared and a buffer solution used. For example, if the selected anion exchange resin is DEAE Sepharose FF® and the cation exchange resin is CM Sepharose FF® and the columns are connected in series, then the columns are advantageously equilibrated with a non-denaturing acid buffer having approximately the same pH and ionic strength as the IgG solution to be charged. Any of a variety of buffer solutions for the equilibration of the ion exchange columns e.g., sodium acetate, sodium phosphate, tris (hydroxymethyl) aminomethane, is suitable. Those skilled in the art will note that many other regulatory solutions can be used to balance the column, as long as the pH and conductivity are approximately the same as for the applied IgG solution. A preferred buffer solution for balancing the anion exchange column and the cationic interconnect column when connected in series, is a sodium acetate buffer solution having a sodium acetate concentration in the range of 5 to 25 mM, for example within the range of 10 to 20 mM, preferably approximately 15 mM. It is preferred that the pH of the sodium acetate buffer used for equilibration is within the range of 5.0 to 6.0, for example within the range of 5.4 to 5.9, preferably about 5.7. The conductivity is within the range of 1.0 to 1.4 mS / cm, preferably approximately 1.2 mS / cm. Suitable acetate buffer solutions can be prepared with sodium acetate trihydrate and glacial acetic acid. Before loading the clarified and optionally filtered supernatant containing IgG into the ion exchange columns, the concentration of the buffer solution and the pH of the supernatant are preferably adjusted, if necessary, to values substantially equivalent to the concentration and pH of the the balancing regulating solution used. After loading the supernatant containing IgG into serial columns, the columns are preferably washed (initial wash) with a column volume of a wash buffer, in order to ensure that the IgG-containing solution is quantitatively transferred. from the anion exchange column to the cation exchange column. Subsequently, the anionic and cation exchange columns are disconnected and the cation exchange column is preferably washed for the purpose of removing the protein contaminants from the resin with a buffer having a pH and ionic strength sufficient to elute substantially all contaminants of the cation exchange resin, without causing a substantial elution of the IgG. The initial washing is advantageously carried out using the balancing buffer solution, although other buffer solutions with similar concentration and pH values can be used for washing. It is preferred that an acetates buffer be used to wash the contaminants from the cation exchange resin. The pH of the buffer solution may be from 5.0 to 6.0, for example it may be within the range of 5.2 to 5.8, for example about 5.4. The IgG elution of the cation exchange resin is preferably carried out with a substantially non-denaturing buffer having a pH and ionic strength sufficient to cause efficient elution of the IgG, thereby recovering an IgG-containing eluate. In this context, the term "efficient elution" means that at least 75%, for example 80%, e.g. at least 85% of the IgG proteins loaded in the anionic and cationic exchange resins in series are eluted from the cation exchange resin. The elution is advantageously carried out in the form of a gradient elution step. In the process of the present invention, the preferred buffer used is sodium acetate having a pH within the range of 5.0 to 6.0, for example 5.2 to 5.8, preferably about 5.4, and a concentration within the range of 5 to 40 mM , for example within the range of 10 to 25 mM, of Dreference approximately 15 mM. It is preferred that the salt concentration of the eluting buffer solution be sufficiently high to displace the IgG from the resin. However, it is contemplated that a lower pH and salt concentration can be used to elute the IgG from the resin. In a preferred embodiment of the process of the present invention, the elution is carried out in the form of a continuous salt gradient elution with sodium chloride concentrations within the range of 5 to 500 mM, preferably about 125 a 350 mM sodium chloride. Elution can also be carried out by step gradient elution. It is contemplated that the elution can also be performed in the form of a constant salt elution, in which the elution buffer applied to the cation exchange column has only one salt concentration, in contrast to the gradient elution. If a constant salt elution is performed, the salt concentration advantageously may be within the range of about 350 to about 500 mM sodium chloride. The advantage of gradient elution compared to constant salt elution is that elution is more effective with a salt gradient, but another advantage is that the eluate has a lower ionic strength, which is advantageous because A high ionic strength for the stability of IgG is critical. The elution buffer solution may further comprise a protein stabilizing agent, such as will be mentioned later. Various other buffer systems suitable for eluting IgG can be used, as will be observed by those skilled in the art. Preferably, at least one protein stabilizing person is applied to the IgG fraction immediately after or during elution. Protein stabilizing agents are known to those skilled in the art and include, for example, different sugar alcohols and saccharides (such as sorbitol, mannose, glucose, trehalose, maltose), proteins (such as albumin), amino acids ( such as lysine, glycine) and organic agents (such as PEG). Advantageously, the stabilizer selected may be one that can be removed from the solution containing IgG in the subsequent steps. The appropriate concentration of the protein stabilizing agent in the IgG-containing solution depends on the specific agent employed. In a preferred embodiment, the stabilizing agent is sorbitol, preferably at a final concentration within the range of 2 to 15% (w / v) of sorbitol, e.g. approximately 2.5%. After elution of the cation exchange column, the eluate is preferably desalted (i.e., dialyzed) and advantageously concentrated. The change of regulatory solution and the concentration of IgG can be done medieinte a combined process of dia / ultrafiltración. The term "dia / ultrafiltration" as used herein, means that dialysis and concentration by diafiltration and ultrafiltration, respectively, are carried out in a single step. It is contemplated that the diafiltration and the ultrafiltration are carried out in the form of two separate stages. However, in order to prevent unnecessary loss of product, it is currently preferred to perform dialysis and concentration by the diafiltration and ultrafiltration methods in a single step. The membranes used for day / ultrafiltration advantageously have a nominal weight cut within the range of 10,000 to 100,000 Da. One type of membrane preferred for the process of the present invention is a polysulfone membrane with a nominal weight cut of 30,000 Da, marketed by Millipore. Other comparable ultrafiltration membranes of materials and porosity can be employed. The degree of concentration can vary considerably. The solution is concentrated from about 10 g / L of IgG to about 100 g / L, preferably of about 50 g / L. After concentration, the IgG concentrate is advantageously dialyzed against a buffer solution of low ionic strength. In addition to removing the salt ions, this step also removes low molecular weight contaminants from the solution and provides a mechanism for the exchange of buffer solution for the next purification step. A preferred regulatory solution for diafiltration is 15 mM sodium acetate, pH 5.4 containing 2.5% (w / v) sorbitol. The exchange of buffer is continued until the conductivity of the ultrafiltered solution is reduced to a "value less than about 1.5 mS / cm, preferably less than about 1.3 mS / cm.During the day / ultrafiltration, the pH is preferably maintains within the range of 4.0 to 6.0, preferably from 5.1 to 5.7, more preferably to about 5.4.After the day / ultrafiltration, the concentration of the protein stabilizing agent advantageously adjusts in the solution, if necessary, to a concentration Optimal final characteristic for the specific protein stabilizing agent that was used If sorbitol is used, the concentration of sorbitol is preferably adjusted to approximately 10% by weight It is preferred that the stabilized solution is filtered with a filter having a diameter of pore within the range of 0.2 to 1.0 μm, preferably approximately 0.45 μm, in order to remove the aggregates before the Next step: In this step, the solution containing IgG has an appearance of transparent solution of appropriate volume with high stability as the combined result of high purity, low ionic strength, acidic pH, relatively high concentration of IgG and the added stabilizer. In the production process of the IGIV product, at least "two" defined and validated stages of virus removal and inactivation are currently incorporated, where these stages of preference are precipitation with PEG as a general virus removal step and a treatment with S / D as inactivating stage of viruses against virus enveloped in lipids. In addition, strict security requirements against viruses of raw materials, in accordance with international standards and the well-known virus reducing capacity of a multi-stage purification process, the incorporation of two independent viral reduction stages that are active against Wrapped and uninvolved viruses, the medicament of the present invention is substantially safe against viruses. Lipid-enveloped infectious viruses that may still be present in the IgG-containing solution are preferably inactivated at this stage of the process by the addition of a virucidal amount of a virus-inactivating agent to the solution containing IgG. The term a "virucidal amount" of the virus inactivating agent, as used herein, means an amount that gives rise to a solution in which the viral particles have substantially become non-infectious, obtaining a "solution containing IgG". virus safe "as defined in the art. Such "virucidal amount" will depend on the virus inactivating agent employed as well as conditions such as incubation time, pH, temperature, lipid content and protein concentration. The term "virus inactivating agent" as used herein, denotes an agent or method that can be used for the purpose of inactivating lipid-enveloped viruses as well as viruses enveloped in non-lipid materials. The term "virus inactivating agent" should be understood to encompass both a combination of such agents and / or methods, as appropriate, as well as a single type of such agents or methods. Preferred virus inactivating agents are detergents and / or solvents, preferably detergent / solvent mixtures. It should be understood that the virus inactivating agent optionally is a mixture of one or more detergents with one or more solvents. Solvent / detergent (S / D) treatment is a widely used step to inactivate lipid-enveloped viruses (eg HIVl and HIV2, hepatitis C and non-ABC, VLTH 1 and 2, the family of herpesviras, including CMV and HPV). Epstein-Barr) in blood products. A wide variety of detergents and solvents can be used for the inactivation of vi. The detergent may be selected from the group consisting of non-ionic and ionic detergents and selected to be substantially non-denaturing. Preferably a non-ionic detergent is used, since it facilitates the subsequent removal of the detergent from the IgG preparation in the subsequent steps. Suitable detergents are described, e.g. in Shanbrom et al in U.S. Patent Nos. 4,314,997 and U.S. 4,315,919. Preferred detergents are those sold commercially with the brands Triton X-100 and Tween 80. Preferred solvents for use in virus quenching agents are dialkyl phosphates or trialkyl phosphates such as those described for example by Neurath and Horowitz in the US Patent US 4,764,369. A preferred solvent is tri (n-butyl) phosphate (TNBP). A virus inactivating agent especially preferred for the practice of the present invention is a mixture of TNBP and Tween 80, but alternatively other combinations can be used. The preferred mixture is added in a volume such that the concentration of TNBP in the IgG-containing solution is within the range of 0.2 to 0.6% by weight, preferably at a concentration of approximately 0.3% by weight. The concentration of Tween 80 in the solution containing IgG is within the range of 0.8 to 1.5% by weight, preferably at a concentration of approximately 1% by weight. The virus inactivation step is carried out under conditions that inactivate enveloped viruses, resulting in a solution containing "IgG" substantially safe against virus. In general, such conditions include a temperature of 4 to 30 ° C, for example 19 to 28 ° C, 23 to 27 ° C, preferably approximately 25 ° C and an incubation time that is effective by means of studies. of validation. Generally, an incubation time of 1 to 24 hours, preferably 4 to 12 hours, for example about 6 hours, is sufficient to ensure sufficient virus inactivation. Nevertheless, the appropriate conditions (temperature and incubation times) depend on the virus inactivating agent used, the pH and the concentration of proteins and the lipid content of the solution. It is contemplated that other methods for virus removal or inactivation may also be employed to produce a virus safe product, such as the addition of methylene blue with subsequent inactivation by ultraviolet light or nanofiltration. After the treatment with the solvent / detergent, the solution is advantageously diluted with a buffer solution. Optionally, the solution containing substantially virus-safe IgG is filtered, preferably through a depth filter as previously described in a previous step of the process of the present invention and / or through a sterile filter. After inactivation of the virus and preferably filtration, ion exchange chromatography is carried out in order to remove the virus inactivating agent and the protein contaminants. This preferred step is carried out in the manner already described for the previous ion exchange chromatography step in the present process, with the exception that the volume of the anion exchange resin is about half that of the cation exchange resin. and that washing before eluting IgG is more extensive, using at least six column volumes of buffer. Additionally, in a preferred embodiment of the present invention, the balancing buffer is sodium acetate with a concentration within the range of about 5 to 25 mM, preferably 15 mM and a pH within the range of about 5.0 to 5.8, preference 5.4. As previously mentioned, the sodium acetate content and the pH of the IgG-containing solution are preferably adjusted to the same concentration and pH as the balancing buffer. Additionally, in a preferred embodiment of the present invention, a protein stabilizing agent, preferably maltose, is added to the recovered eluate to a final concentration within the range of 1 to 5%, preferably of "about 2.5% by weight. The preferred method for removing the virus inactivating agent is by ion exchange chromatography, but other methods such as oil extraction and alternative chromatographic methods are also contemplated.The appropriate method will depend on the virus inactivating agent employed. detergent, then, can be performed by attaching the IgG to a resin and subsequently, by washing the quenching agent well with a buffer solution.Cathion exchange chromatography is a useful method.In a preferred embodiment of the present invention, chromatography is also performed of anion exchange in addition to exchange chromatography ationic, in order to improve the quality and overall purity of the final product of the process of the present invention. After the ion exchange chromatography step, the eluate containing IgG is preferably dialyzed and concentrated; in this way the content of the remaining smaller protein components is also effectively reduced .. Advantageously, this can be carried out per day / ultrafiltration as already described above. The regulatory solution employed for "the" diafiltration is sodium acetate, preferably a concentration of about 4 to 10 mM, preferably 7.5 mM, and a pH in the range of about 4.0 to 6.0, preferably about 5.1 to 5.7, per example approximately 5.4. Alternatively, other buffer solutions such as sodium phosphate or acids for diafiltration may be used. The diafiltration continues until the conductivity is less than or equal to 1 mS / cm. Optionally, the solution containing IgG is sterilized by additional filtration. If desired, the solution containing purified IgG that is substantially free of the virus inactivating agent, is subjected to other treatments with the purpose of making it suitable for its formulation as a liquid product to be administered, for example intravenously, subcutaneously or intramuscularly. . From a practical point of view, it is preferred that the content of the liquid formulation of the immunoglobulin product be the same for storage as for use. The final concentration of IgG in the product of preference is in the range of 0.25 to 20% by weight (corresponding to an amount of 2.5 to 200 g of IgG / L), for example from about 1 to 20%, i.e. approximately 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%. It is known that a high concentration of proteins results in greater stability of the IgG. On the other hand, a high concentration of IgG means that the maximum infusion rate when the IgG is administered intravenously to the patient, has to be very low in order to decrease the transfusion problems due to the high osmotic pressure of the product. A concentration currently recommended for intravenous administration by the European Pharmacopoeia (Ph.Eur.) Is 5% (w / v). On the other hand, a highly concentrated product (e.g., 10% or greater) advantageously serves for intramuscular or subcutaneous injection. Although not preferred, it is evident that the products obtained by the various stages of the process of the present invention can also be used, for example, as lyophilized products instead of liquid formulations, although this is less favorable compared to the use of the immunoglobulin products in the form of ready-to-use liquid formulations. This last modality will be described in more detail later. The prod-. Liquid immunoglobulin compounds are more stable at a markedly lower ionic strength than plasma, i.e- the conductivity is preferably less than 1.0 mS / cm, preferably about 0.8 mS / cm. The pH caused an impact on the stability of the IgG and on the infusion rate. The liquid immunoglobulin products are more stable under acidic conditions, i.e. below the isoelectric point of the IgG, pH 6.4-8.5. The closer the pH value is to the physiological pH value (7.1-7.3), the higher the infusion rate can be used. As a consequence of the required stability, the pH of the immunoglobulin product of the present invention will preferably be within the range of 5.1 to 5.7, for example between 5.2 and 5.6, preferably about 5.4. In addition, the immunoglobulin product may comprise protein stabilizing agents as previously described. In addition to sugar alcohols and saccharides (such as sorbitol, mannose, glucose, trehalose, maltose), proteins (such as albumin), amino acids (such as lysine, glycine) and organic agents (such as PEG and Tween 80) can also be used. use as stabilizing agents. The appropriate concentration of the stabilizing agent in the solution containing IcrG, depends on the specific agent employed, as previously described. The purified IgG solution is adjusted, if necessary, in order to obtain a stable and isotonic solution. The term "isonic solution" as used herein, denotes that the solution has the same osmotic pressure as the plasma. As mentioned above, the ionic strength is markedly lower in the immunoglobulin product of the present invention in liquid formulation, than in the plasma. For this reason, it is preferred to use monosaccharides or disaccharides to increase the osmolarity of the solution, since these do not affect the ionic strength. In a preferred embodiment of the present invention, maltose is added at a concentration which ensures that the solution is isotonic and, at the same time, the maltose functions as an immunoglobulin stabilizing agent. This is preferably done by adding the maltose to a final concentration in the range of about 5 to 15% (w / v), preferably 10% (w / v); alternatively, other saccharides such as mannose and glucose can be used. The preferred end conditions for the immunoglobulin product are a compromise between stability and physiologically acceptable conditions with respect, for example, to pH, ionic strength and tonicity. In addition, the immunoglobulin product must meet the requirements of quality control tests, as specified in Monograph No. 918, Ph. Eur. 1997. The main advantages of the product obtained by the method of the present invention "are that, when formulated as a liquid preparation, the product is a combination of a liquid, a ready-to-use product which, upon At the same time, it is very stable, highly purified, has a very normal distribution of subclasses of IgG and has an extremely low IgA content, as well as a low IgM content, and retains the activity of antibodies and biological activity, which In addition, it essentially does not contain aggregates of immunoglobulins and / or other plasma proteins measured as polymers greater than dimers and has a low anticomplementary activity and has a very high content of monomers and dimers of IgG. less than 90%, which is considered ideal, due to the high stability, it is possible to avoid the addition of other stabilizing agents, such as albumen na, glycine, detergent or PEG. Finally, the product is virus-safe, since the process includes well-defined and validated virus reduction steps for the purpose of removing and / or inactivating both virus with lipid envelope and virus with non-lipid envelope. The purpose of validating a production stage as a virus reduction stage is to provide evidence that the production process will inactivate / effectively remove the viruses, which are known to contaminate raw materials or that could conceivably do so. Validation includes the deliberate addition of a virus before the production stages to be validated and measuring the extent of its removal / inactivation after the production stage or stages.The restrictions of Good Manufacturing Practices avoid the deliberate introduction of any virus To the production facilities, therefore, the validation must be carried out in a separate laboratory equipped for virological work in a scaled-down version of the production stage and must be carried out by personnel with virological experience in conjunction with production engineers The amount of virus added to the raw material for the pro stage duction that will be validated, should be as high as possible in order to determine the capacity of the production stage to inactivate / remove viruses adequately. However, the virus must be added in such a way that the composition of the production material is not significantly altered. Preferably, the virus inoculum volume will be equal to or less than 10%. Quantitative infectivity assays should be carried out in accordance with the GLP basics and may involve plaque formation, detection of other cytopathic effects such as syncytia or foci formation and endpoint titration (eg, TCID50 assays), detection of the synthesis of viral antigens or other methods. The method should have adequate sensitivity and reproducibility and should be performed with sufficient replication and controls to ensure adequate statistical accuracy of the results. Typically, a process step is challenged with 6 logarithms of viruses and if a reduction is achieved in the order of 4 logarithms or more, these are indicative of a clear effect with the particular test virus under investigation. Similarly, a reduction in the order of 4.5 logarithms, 5 logarithms or even 5.5 logarithms, is indicative of a clear effect with the particular test virus being investigated and this stage can be classified as a validated virus reduction stage . Virus validation studies should be carried out with viruses that resemble those that can contaminate the product as much as possible and, secondly, that represent as broad a range of physicochemical properties as possible, in order to Test the system's ability to eliminate viruses in general. Validation studies "of" viruses must be carried out in accordance with the CPMP Note for Guidelines on Virus Validation Studies: Design, Contribution and Interpretation of Studies that Validate Virus Inactivation and Removal (CPMP / BWP / 268 / 95) and Note for Guidelines on Medicinal Products Derived from Plasma (CPMP / BWP / 269/95). Validation studies of the process of the present invention are presented in Example 5. The product of the present invention has a purity greater than 95%, preferably greater than 98%. The degree of purity inter alia, is due to the fact that the product of the invention is obtained by at least one, preferably two, optionally connected in series, steps of anion exchange chromatography and cation exchange. It is worth noting in this context that it has been possible to obtain a high yield despite the number of processing steps employed, in production scale of at least 3.5 g of IgG protein per kg of fresh frozen plasma. The comparative studies that were carried out (Example 2) demonstrated that the immunoglobulin product obtained by the process of the present invention has ideal functional properties, such as a prominent antigen binding activity and a high Fc function. The currently preferred medicament developed by the present inventors is a 5% by weight immunoglobulin solution. Stability tests to date have indicated a stability at 4 ° C for more than a year, ie, that the immunoglobulin product is devoid of aggregate formation or fragmentation of immunoglobulins G, loss of desired biological activity or increase of undesirable activities, eg anticomplementary activity and prekalicyrein activity, as measured in vi tro. Based on the present invention, it is possible to obtain an IgG product having a purity greater than 95%, for example 96% or at least 97%, for example at least 98%, preferably at least 99%, more preferably when minus 99.5% purity. The IgG product should contain less than 6 mg of IgA / L, for example less than 4 mg of IgA / L, preferably less than 3 mg of IgA / L, more preferably less than 2 mg of IgA / L. It should be emphasized that other products contain stabilizers in the form of detergent, PEG or albumin. In a preferred embodiment, the product of the present invention does not contain any of said stabilizers, instead a well-tolerated saccharide has been selected. The product according to the present invention has a very low content of polymers and aggregates. In a preferred embodiment, the product of the present invention contains less than 1.5% polymers and aggregates, for example less than 1%, e.g. less than 0.5% or less than 0.25% of polymers and aggregates. The content of IgG monomers and dimers is at least 95%, for example at least 96% or at least 97%, e.g. at least 98%, preferably at least 98.5% or 99%. The content of monomeric IgG is at least 90% in the product. Some studies have demonstrated the clinical effect of the product in accordance with the present invention in comparison with the registered IGIV products. The product has been well tolerated by patients and the turnover time of immunoglobulins in the circulation has been determined to be four weeks. In the present studies, the immunomodulatory effect of IGIV, SSI has proved to be convincing (the data are presented in Example 3). The indications for IGIV are hypo / agammaglobulinemia including common variable immunodeficiency, Wiskott-Aldrich syndrome and severe combined immunodeficiency (SCID), secondary hypo / agammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) and multiple myeloma, children with AIDS and bacterial infections, chronic idiopathic thrombocytopenic purpura (ITP), allogeneic bone marrow transplantation (BMT), "Kawasaki disease" and "Guillan-Barré syndrome." Neurology: chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, syndrome of Eaton-Lambert, optic neuritis, epilepsy Gynecology: Abortus habitualis, primary antiphospholipid syndrome Rheumatology: rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, vasculitis, Wegner granulosum atosis, Sjocren's syndrome, juvenile rheumatoid arthritis Hematology: autoimmune neutropenia , hemolytic anemia autoimmune, neutropenia. Gastrointestinal: Crohn's disease, ulcerative colitis, celiac disease. Others: asthma, septic shock syndrome, chronic fatigue syndrome, psoriasis, toxic shock syndrome, diabetes, sinusitis, dilated cardiomyopathy, endocarditis, atherosclerosis, adults with AIDS and bacterial infections. Apart from the aforementioned indications for treatment with IVIG products, several severe autoimmune diseases which commonly respond to therapy with corticosteroids and immunosuppressants are considered as white disorders for therapy with the product of the present invention. Among these are several neurological diseases such as polyradiculitis and some peripheral polyneuropathies mediated by the immune system, but also some chronic inflammatory rheumatic disorders and vascular disorders such as systemic vasculitis involving small vessels, polymyositis and others. A different mode of action of the product of the present invention may be the elimination of infectious antigens in chronic infections and an increase in IgG metabolism. The present invention will be further illustrated with the following examples, which are not intended to be limiting. EXAMPLES EXAMPLE 1 STAGES OF PROCESS IN THE PURIFICATION OF IMMUNOGLOBULIN (with the exception of stage 5, all stages are carried out at 5 ± 3 ° C) Stage 1: Preparation of fraction II + III of Cohn in paste: The fraction Cohn II + III in paste is prepared from human plasma by the standard Cohn fractionation method (Cohn E., et al., (1946) J Am Chem Soc, 459-475) essentially as modified by Kistler -Nitschmann (Kistler P and Nitschmann HS, (1952), Vox Sang, 7, 414-424). Precipitation with ethanol is initiated after the cryoprecipitate has been removed and, if desired, after the adsorption of certain plasma proteins (such as factor IX and antithrombin) in for example an ion exchange material and / or a Heparin Sepharose® matrix. The exact conditions (pH, ethanol concentration, temperature, protein concentration) to obtain fraction II-III in paste appear in the figure on page 266 of Harns JR (ed), Blood Separation and Plasma Fractionation, Wiley-Liss, New York, 1991. The paste is isolated on a filter by adding a filter aid before filtration. Stage 2: Extraction of immunoglobulins from Cohn fraction II + III in pulp: From 140 kg of fraction II + III in pulp including 30 kg of filter aid (Schenk, Germany) (corresponding to an initial volume of plasma of approximately 1150 kg), the extraction is achieved by first adding 525 kg of sodium phosphate buffer / 2.33 mM acetate, pH 4.0, with slow stirring for approximately 1.5 hours, followed by two consecutive additions of 350 kg of injectable water (AI ) with shaking for 1.5 hours' after "each addition., approximately 280 kg of 21.5 mM sodium phosphate / acetate, pH 7.0, are added, thus adjusting the pH of the suspension to 5.4.

The suspension is filtered through a depth filter (C-150AF, Schenk, Germany). The filtrate contains, among other proteins, immunoglobulins. Step 3: Precipitation of protein aggregates and virus removal by PEG 6000: PEG 6000 (Merck, Germany) is added to the filtrate from step 2 at a final concentration of 6% by weight. After precipitation for 4 hours, the PEG suspension is centrifuged to clarity in a full flow centrifuge (Westfalia BKA28, Germany) and filtered thoroughly (50LA and 90LA, Cuno, France) and subsequently sterilized by filtration to through a 0.22 μm filter (Durapore, Millipore, USA). The filtered PEG supernatant is adjusted with a buffer solution by adding one part of the 0.45 M sodium acetate buffer solution, pH 5.7, to 29 parts of the supernatant, to achieve a pH of 5.7. Step 4: Purification by anion exchange chromatography and serial cation (I): Two chromatography columns are packed with 56 L of DEAE Sepharose FF® (Pharmacia Biotech, Sweden) and 56 L of CM Sepharose FF® (Pharmacia "Biotech, Sweden), respectively.The columns are connected in series so that the liquid flow first passes through the DAE Sepharose resin, subsequently through the CM Sepharose resin.The column resins are equilibrated with acetate buffer solution. 15 mM sodium, pH 5.7 Then, the solution from step 3 is applied to the two columns in series.At ion exchange chromatography, most of the contaminating proteins in the applied solution bind to the DEAE Sepharose resin. that the IgG passes through without binding to the DEAE Sepharose resin, the IgG binds to the CM Sepharose resin when the solution migrates through it, after the application of the solution and washing with a In the case of a balancing buffer column column, the DEAE column is disconnected from the CM column. Then, the CM column is washed with three column volumes of 15 mM sodium acetate buffer, pH 5.4, and then the IgG is eluted with a NaCl gradient of 125 to 350 mM NaCl, 15 mM sodium acetate, pH 5.4. The eluted IgG fraction is collected in sorbitol at a final concentration of 2.5% by weight. Step 5: Solvent / detergent treatment (S / D) of the IgG fraction: The IgG eluted fraction is concentrated and desalted by ultra / diafiltration to a concentration of approximately 50 g of IgG / liter. The membrane used is a polysulfone membrane, with a nominal weight cut-off of 30 Da (Millipore). The diafiltration is carried out against a buffer solution of 15 mM sodium acetate, pH 5.4, containing 2.5% by weight of sorbitol and is continued until the conductivity is less than 1.4 mS / cm. The IgG content of the solution is determined spectrophotometrically, measuring at 280 nm (A28o) • The sorbitol concentration is adjusted to 10% by weight and the solution is filtered through a 0.45 μm filter (Pall Corporation, GB). Then Tween 80 and TNBP are added to a final concentration of 1 and 0.3% by weight, respectively, for the subsequent S / D treatment. S / D treatment proceeds for at least 6 hours at 25 ° C. Step 6: Removal of S / D by ion exchange (II) chromatography: Two columns connected in series packed with 28 L DEAE and 56 L CM Sepharose FF®, respectively, are equilibrated with 15 mM sodium acetate, pH 5.4 . The fraction of IgG treated with S / D from step 5 is diluted with 5 parts of 15 mM acetate buffer solution, pH 5.4, filtered through a depth filter (Cuno 90 LA) and then sterilized by filtration ( Sartobran, Sartorius) and applies to the two columns connected in series. The ion exchange chromatography and the subsequent elution of the IgG from the CM column are carried out essentially in the manner described in step 4, except that the CM column is extensively washed with 6 column volumes of buffer to remove the agents of the S / D treatment. The eluted IgG fraction is collected in maltose (Merck, Germany) at a final concentration of 2.5% by weight. Step 7: Final concentration and formulation of the immunoglobulin for intravenous administration: The IgG fraction eluted from step 6 is subjected to ultrafiltration and desalted by diafiltration against 7.5 mM sodium acetate, containing 2.5% by weight of maltose, pH 5.4 to a final conductivity of less than 1 mS / cm. The membrane used is a polysulfone membrane with a nominal weight cut-off of 100 kDa, which allows the proteins of lower molecular weight s €They have been eliminated. The final IgG concentration is adjusted to 50 g / liter and the maltose is adjusted to a final concentration of 10% (w / v). The final maltose-adjusted preparation is filtered through a sterile filter (Sartopure GF 2, Sartorius) and aseptically filled into bottles.

EXAMPLE 2 RESULTS OF AN ANALYTICAL STUDY OF A PRODUCT OBTAINED BY THE PROCESS OF THE PRESENT, IN COMPARISON WITH OTHER IGIV PRODUCTS 1: Without correction for HSA; 2: declared by the producer; 3: corrected for the HSA peak; 4: used as a stabilizer. Purity (protein composition) The purity requirements of Pharmacopoeia for an IVIG preparation are at least 95% IgG; that is, no more than 5% of contaminating proteins that are not IgG present. Purity is considered of very high importance for several reasons. From a rational point of view, only the protein that is carrying the desired function must be present and other contaminating proteins can be potentially dangerous, e.g., causing undesirable adverse effects and / or influencing the stability of the product. The purity can be analyzed, for example, by an electrophoretic technique in the manner described in detail in the European Pharmacopoeia Ph. Eur., 1997, pages 964-965, wherein the proteins are separated in a cellulose acetate gel. However, for practical purposes an agarose gel is used. After electrophoresis, the gel is fixed, dried and stained. Finally, the protein bands are monitored by sweeping. From the above table it is observed that the product of the present invention is virtually pure (99.8%). Albumin The albumin content was analyzed by cross immunoelectrophoresis, essentially in the manner described by C.B. Laurell (anal Biochem (1965), 10, 358-361). 5 μL of the product against antibodies against albumin is analyzed (DAKO A / S, Denmark, No. A0001 (1/100)). Due to the high purity, albumin was not detectable in the analyzed product of the present invention. Content of monór. > The content of IgG monomers and dimers can be analyzed by gel permeation chromatography and monitored from the chromatogram by integrating the areas of the monomer and dimer peaks, cf. Ph. Eur. The results of the various analyzes are listed in the table above, from which it is observed that the sum of the monomer + dimer areas constitutes 99.3% of the total area of the chromatogram (starting from this, the IgG monomeric constitutes 92%) for the product of the present invention. Polymer content and aggregates The presence of polymers and aggregates is known to cause serious adverse effects, often similar to influenza symptoms. Due to the very high degree of purity achieved by the gentle production process, the immunoglobulin product obtained by the process of the present invention is substantially free of polymers and aggregates. The polymers can be analyzed by gel permeation chromatography and the protein peaks with retention times shorter than the retention time for the dimeric IgG, are considered as polymeric, in the manner described in the Ph. Eur. In accordance with the Ph. Eur. and other standards, the content of protein aggregates of "preference should be less than 3%." The product of the process of the present invention does not contain measurable amounts of aggregates and, therefore, is considered to have less 0.1% of polymers and aggregates Anti-complementary activity (AAC) and prekalicyrein activating activity (APK) The AAC and APK were measured in the manner described in the Ph. Eur. The AAC should preferably be as low as possible. According to the Ph. Eur, the complement consumption must be less than or equal to 50% The complement consumption of the measured sample of the product of the present invention is approximately 30%, ie it is comparable to that of the other products analyzed. It should be noted that the presence of albumin tends to suppress complement consumption (observation of the inventors). The APK, if present in substantial quantities, is essential for the adverse hypotensive effect of the product. Therefore, the APK should preferably be as low as possible in an immunoglobulin product. In accordance with the Ph. Eur., It should be <35 IU / ml when measured in the manner indicated in said reference. The APK of the product of the present invention, as well as that of the other products analyzed, is lower than the level of quantification of "the method", i.e., below 8.5 μL / ml. Hemagglutinins The IgM fraction of plasma immunoglobulins comprises hemagglutinins; that is, antibodies against blood type A and B antigens. The presence of such antibodies can cause undesirable side effects due to the possibility of haemolytic reaction if the recipient is carrying blood types A and / or B. In accordance with the requirements of Pharmacopoeia, the content of hemagglutinins should be lower than that which causes agglutination of A / B erythrocytes in a 1:64 dilution of the immunoglobulin product. All the products analyzed met this requirement. Fc function Retention of antigen binding activities is essential for the biological functions of IVIG. This also applies to immunomodulatory activity. On the other hand, the retention of the Fc function is essential for the effect of the IVIG on several phagocytic cells and the activation of the complement system. The Fc function can be demonstrated using several techniques, but a methodology accepted and described in Ph. Eur., Measures the complement activating potential of the antibodies in the preparations against rubella antigen. The activity is compared to that of a biological reference preparation (BRP, Ph. Eur.) Of immunoglobulins, established as 100%. The product meets the test and the relativistic activity is greater than 60% with respect to the reference preparation. It seems that the Fc function of the product of the present invention is very well conserved, particularly in comparison with the other liquid products analyzed, most likely due to the gentle purification process. Subclass distribution The distribution of IgG subclasses is measured by a standard Mancini immunodiffusion method, essentially in the manner described by A. Ingild (Scand J Immunol, (1983), 17, 41). The concentrations are determined using a WHO reference serum (67/97). It is required that the subclass distribution should be in the range of normal human plasma with median concentrations in the range of 3.7 to 10.2 g of IgGl / L of serum, from 1.1 to 5.9 g of IgG2 / L of serum, from 0.15 to 1.3 g of IgG3 / L of serum and 0.06 to 1.9 g of IgG4 / L of serum (R Djurup et al .. Scand J. Clin Lab Invest 48, 77-83). Thus, the distribution of subclasses of all products is acceptable.

IgA content It is known that the presence of IgA potentially causes sensitization of IgA-deficient receptors. If an IgA deficient patient receives an immunoglobulin preparation containing IgA, IgA can be considered as a foreign antigen and the result may be the induction of anti-IgA antibodies in the recipient. The next time an IgA-containing preparation is infused into the patient, an anaphylactic reaction may be caused. Therefore, it is essential that the immunoglobulin preparation contain as little IgA as possible. IgA in an IGIV product can be monitored using an ELISA technique, e.g. where an anti-IgA polyclonal antibody is used to capture the IgA and a labeled anti-IgA is used for the detection of IgA binding. Standards are constructed by dilutions of a calibrator (No. X908, DAKO A / S, Denmark) with a declared IgA content. The product of the process described in Example 1 contains less than 2 mg of IgA / L, which is a significantly lower IgA content than that of the other liquid products analyzed. The physicochemical similarities between IgG and IgA make it difficult to separate these immunoglobulins during a purification process. However, the two anion / cation exchange chromatographies in the "process reduce the IgA content to a very low level." IgM content In an Ig preparation, the IgM can be monitored using an ELISA technique, eg where uses a polyclonal anti-IgM antibody to capture the IgM and a labeled anti-IgM serum is used for detection Standards are constructed by dilutions of a calibrator (No. X908, DAKO A / S, Denmark) with a declared IgM content It can be seen from the table that the IgM content of the product of the present invention is very low and markedly lower than that of the other liquid products Tween 80, TNBP and PEG The Tween 80, TNBP and PEG are measured by the standard procedures In general, the content of these additives should be as low as possible ES The pH of the liquid products analyzed is acid, pH 5.6-5.7, while that of the lyophilized products analyzed is neutral after is of the dilution, with a pH of 6.7. Total concentration of proteins According to the Ph. Eur., The concentration of proteins must be at least 50 g / L ± 10%; All products comply with 'this requirement. The protein concentration is measured by the Kjeldahl method.

Maltose and glucose stabilizers Saccharides are commonly used as stabilizers of immunoglobulin products, have good stabilizing properties and are easily excreted. The content of maltose, sucrose and glucose is determined using a commercial package (Boehringer Mannheim, Germany) with maltose as reference. It appears that the two freeze-dried products stabilized with albumin and albumin as well as PEG, respectively, also contain a saccharide stabilizer at concentrations of about 15 mg / ml to 20 mg / ml. The product of the present invention and the other liquid product are stabilized in a very similar manner, e.g., with about 9%, 88 mg / ml and 92 mg / ml maltose. With respect to the content of polymers and aggregates as a stability parameter, the product of the present invention has greater stability than the other liquid product analyzed, although its formulations appear to be very similar. EXAMPLE 3 RESULTS OF CLINICAL STUDIES The clinical studies of the product of the present invention, also referred to as IGIV, SSI, were carried out in accordance with ICH and CPMP / 388/95 standards. The pharmacokinetics, effect and safety were examined. Clinical studies to date have included four groups of patients: patients with primary immunodeficiency syndrome (15 patients), secondary immunodeficiency syndrome (6 patients), idiopathic thrombocytopenic purpura (15 patients) and patients with chronic inflammatory demyelinating polyneuropathy (5 patients) ). Patients with primary or secondary immunodeficiency syndrome were treated with 0..2 to 0.4 g / kg, with intervals of 2 to 5 weeks. Patients with idiopathic thrombocytopenic purpura were treated with 400 mg (kg per day for five days or 1000 mg / kg per day for two days.) As a safety measure, serum transaase, serum creatinine, and viral markers were determined in all patients. Five patients with idiopathic thrombocytopenic purpura underwent follow-up of viral, renal and hepatic safety markers for up to a total of 24 weeks Pharmacokinetics The T? / 2 was measured at 30.5 days (median). The results of the other IGIV drugs Effect For patients with primary and secondary immunodeficiency syndrome, the days lost due to illness, hospitalizations, days under antibiotic therapy, days with fever and the number of pneumonias were registered for a period of time, respectively. 6 months, during which patients had been treated with other registered IVIG medications. 6 months during which patients were treated with the immunoglobulin SSI, liquid, the same parameters were recorded. The conclusion is that the immunoglobulin SSI, liquid, is as effective as the other IVIG compositions for the prophylaxis / prevention of infections in patients with primary and secondary immunodeficiency syndrome. In 80% of patients with idiopathic thrombocytopenic purpura, the number of platelets rose from <30 x 109 / L before treatment with liquid immunoglobulin SSI, a > 50 x 109 / L after the treatment. The increase in the platelet count and the duration of remission in the individual patients were at the same level as after the administration of the same dose of other IGIV drugs, in cases where the comparison was possible. A "patient who received IGIV for the first time was refractory to the drug test." Such a reaction to IGIV is not uncommon and, therefore, is not surprising.The details of platelet elevation and the duration of such elevation are The conclusion is that liquid SSI immunoglobulin is as effective as other IVIG drugs in the treatment of low platelet counts in patients with idiopathic thrombocytopenic purpura, in accordance with clinicians and patients suffering from chronic inflammatory demyelinating polyneuropathy. IVIG SSI has shown an identical efficacy to IVIG administered before the study, IVIG SSI was tolerated by patients as well as the other products IVIG were tolerated. Safety Apart from a serious adverse event, the splenectomy performed by the researcher determined that it was not related to the drug, only minor adverse events have been recorded. These adverse effects were mainly headache with fever and vomiting.

To date there have been no reports of abnormal vital signs during IGIV SSI infusions. No viral seroconversions have been registered. There have been no reports of liver or kidney damage or "cases of anaphylactic shock Clinical studies show that liquid SSI immunoglobulin is well tolerated.The frequency of side effects, degree and nature of the same does not deviate from the experience that has with other IGIV drugs EXAMPLE 4 RESULTS OF STABILITY STUDIES FOR LIQUID IGIVES In order to test whether the liquid IVIG product is stable over time, a stability study was conducted in Real and Real Time conditions. 4 consecutive lots (250 ml each sample) of the IGIV product in the study and stored at a temperature between 2 and 8 ° C for at least 12 months Samples from the four lots were analyzed at time zero, at 6 months and after 12 months of storage, the results of the study are presented below in the form of the average of the 4 lots.

All the aforementioned tests were carried out in accordance with Ph. Eur. And in the manner described in Example 2. The observation that the content of monomers and dimers is constant over a period of 12 months indicates that form polymers in the sample. The presence of immunoglobulin polymers is known, among other things, to cause serious adverse effects, often similar to influenza symptoms. Due to the very high stability of the immunoglobulin product obtained by the process of the present invention, the product is substantially free of polymers and aggregates, even after a long storage period. No increase in CAA was observed over time, although lots that deliberately express a high CAA have been included in this stability study. If an increase in AAC is observed, it could indicate that aggregates could be forming during storage. Thus, the constant AAC over time indicates that aggregates are not being formed. The results also indicate that prekallikrein activating activity was not developed during the storage of the product, since the APK activity was not increased. However, it should be noted that the measured values are below the minimum quantification level. The measurement of the Fc function indicates the presence of intact functional IgG that has been maintained during storage. Thus, there are no proteases present in the samples, since these would have degraded the proteins and therefore decreased the Fc function. There has also been no denaturing of IgG molecules, since this would have decreased antigen binding activity. As is known to those skilled in the art, there could be differences in the stability of the various classes of IgG. As can be seen in the present results, all subclasses were maintained during storage, indicating that the product is stable. This is further supported by the finding that the composition of IgG proteins in the samples with approximately the same concentration "of total proteins, was almost not altered over time, indicating that there is no general degradation of IgG - ie, the product of the present invention is stable and can be stored for at least 12 months at 2-8 ° C without significant changes in its characteristics and, by this, its efficacy and safety is demonstrated EXAMPLE 5 STAGES OF VIRUS REDUCTION VALIDATED IN THE PROCESS OF THE PRESENT OF IGIV REMOVAL OF VIRUSES THROUGH A PARTITION STAGE Precipitation of the viruses present in the immunoglobulin solution by polyethylene glycol. Virus validation studies have been conducted using two small non-enveloped viruses, obtaining the following virus reductions: removal of 6.3 logio of hepatitis A virus (HAV) removal of 7.2 logio of poliovirus. Virus validation studies were carried out using two enveloped viruses, obtaining the following virus reductions: removal of 7.6 logio of HIV removal of 7.5 logio of BVDV INACTIVATION OF VIRUSES BY A PROCESSING STAGE WITH S / D Treatment of the immunoglobulin solution with 1% Tween 80 + 0.3% TNBP, at 25 ° C during > 6 hours. Virus validation studies were carried out using 4 enveloped viruses, obtaining the following results of virus reductions: inactivation of 7.4 HIV logons inactivation of 5.3 logio of Sindbis virus inactivation of 4.1 log_.0 of BVDV inactivation of 5.1 logio of PRV conducted a total of 8 validation studies on two different stages in the process of the present invention. The PEG precipitation stage was validated as a virus removal step using 4 different viruses, two small non-enveloped viruses, VAH and poliovirus, and two enveloped viruses HIV and BVDV as a model for the hepatitis C virus. These studies demonstrated that all four viruses were efficiently removed by PEG precipitation. The PEG precipitation stage was therefore validated as an efficient virus removal step. Treatment with S / D was validated using four different enveloped viruses. From the data of the validation studies, it is observed that the stage of treatment with S / D efficiently inactivates the four viruses. Therefore, treatment with S / D was validated as an efficient stage of virus inactivation. Both stages "of" reduction in the IVIG process, removal by precipitation with PEG and inactivation by treatment with S / D, were validated, declaring that they are efficient to remove and inactivate four different viruses each. The cumulative reduction factors of HIV and BVDV in the process are 15 and 11.6, respectively. For all this, the product of the process of the present invention can be considered as virus safe. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (23)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. A process for purifying immunoglobulin G (IgG) from a crude plasma protein fraction containing immunoglobulins, characterized in that the process comprises the steps of: (a) preparing an aqueous suspension of the crude plasma protein fraction containing immunoglobulins; (b) adding a substantially non-denaturing, water-soluble protein precipitating agent to the suspension of the step of part (a) in an amount sufficient to cause precipitation of a high proportion of non-immunoglobulin G proteins, aggregated immunoglobulins and particles , including potentially infectious particles such as viral particles, without causing substantial precipitation of the monomeric R-immunoglobulin G, thereby forming a mixture of a solid precipitate and a liquid supernatant; (c) recovering a clarified supernatant containing immunoglobulin G from the mixture of the step of part (b); (d) applying the clarified supernatant containing immunoglobulin G from the step of part (c) to an anion exchange resin and subsequently to a cation exchange resin, wherein the anion exchange resin and the cation exchange resin are connected at series and where the regulatory solution used for anion exchange chromatography and for cation exchange chromatography is the same, the pH of said buffer being less than 6. 0; (e) washing the protein contaminants and protein precipitating agent from the cation exchange resin of the step (d) step with a buffer solution having a pH and ionic strength sufficient to remove contaminants from the resin without causing elution substantial immunoglobulin G; (f) eluting the immunoglobulin G from the cation exchange resin of the step of part (e) with a substantially non-denaturing buffer solution having a pH and ionic strength sufficient to cause efficient elution of immunoglobulin G, thereby recovering an eluate containing immunoglobulin G; (g) performing a day / ultrafiltration in the immunoglobulin G-containing eluate of the step of part (f) to concentrate and / or dialyze the eluate, and optionally add a stabilizing agent; (h) adding a virucidal amount of a virus inactivating agent to the fraction containing immunoglobulin G dia / ultrafiltered and optionally stabilized from the step of part (g), obtaining a solution containing immunoglobulin G substantially safe against virus; (i) applying the solution containing immunoglobulin G from the step of part (h) to an anion exchange resin and subsequently to a cation exchange resin; (j) washing the cation exchange resin of the step of part (i) with a buffer solution having a pH and ionic strength sufficient to wash the protein contaminants and virus quenching agent from the resin, without causing substantial circumvention of the immunoglobulin G; (k) eluting the immunoglobulin G from the cation exchange resin of the step of part (j) with a substantially non-denaturing buffer having a pH and ionic strength sufficient to cause efficient elution of immunoglobulin G, recovering from this way an eluate containing immunoglobulin G; and (1) subjecting the immunoglobulin G-containing eluate from step (k) to one day / ultrafiltration to decrease the ionic strength and concentrate the immunoglobulin G of the solution, and adjust the osmolarity by adding a saccharide.
2. 2. A process according to claim 1, characterized in that the plasma protein fraction containing immunoglobulin G is selected from the group consisting of Cohn II fraction; fractions of Cohn II and III and fractions of Cohn I, II and III.
3. A process according to any of claims 1 or 2, characterized in that the suspension of the stage of part (a) is maintained at a temperature between 0 and 12 ° C and the suspension of the stage of part (a) is maintains a pH less than 6.
4. 4. A process according to any of the preceding claims, characterized in that the protein precipitant of the step of part (b) is selected from the group consisting of polyethylene glycol (PEG), caprylic acid and ammonium sulphate.
5. 5. A process according to claim 4, characterized in that the protein precipitant is selected from the group consisting of PEG with a molecular weight in the range of 3000 to 8000 Da, such as PEG 3350, PEG 4000, PEG 5000 and PEG 6000. H.H.
6. A process according to any of the preceding claims characterized in that the anion exchange resin and the cation exchange resin of the stage of part (i) are connected in series.
7. 7. A process according to claim 6, characterized in that the buffer solution used for anion exchange chromatography and for cation exchange chromatography is the same, and the pH of said buffer is less than 6.0.
8. A process according to any of the preceding claims, characterized in that the anion exchange resin of the stage of part (d) and / or of the stage of part (i) contains diethylaminoethyl groups and / or the cation exchange resin of the stage of part (d) and / or of the stage of part (i) contains carboxymethyl groups, wherein the resins are preferably DEAE Sepharose FFR and CM Sepharose FFR.
9. 9. A process according to any of the preceding claims, characterized in that the , regulatory solution used in the step of items (b) to (1) is an acetate buffer solution, such as an acetate buffer with a pH of 5.0 to 6.0 and having a molarity of 5 to 25 mM.
10. A process according to any of the preceding claims, characterized in that the virus inactivating agent of the step of part (h) is a mixture of at least one ionic or nonionic detergent and at least one solvent.
11. 11. A process according to any of the preceding claims, characterized in that the virus inactivating agent of the step of part (h) is a mixture of at least one substantially non-denaturing detergent and at least one tri (lower alkyl) solvent -phosphate.
12. 12. An immunoglobulin product, characterized in that it is obtained by the process according to any of claims 1 to 11.
13. 13. An immunoglobulin product characterized in that it is obtained by the process according to any of claims 6 to 8.
14. 14. An immunoglobulin product characterized in that it has: a) a purity greater than 98%, b) an IgG monomer and dimer content greater than 98.5%, c) an IgA content less than 4 mg IgA / 1, and d) a content of IgGl, IgG2, IgG3 and IgG4.
15. 15. An immunoglobulin product according to claim 14, characterized in that it does not comprise detergent, PEG or albumin as stabilizers.
16. 16. An immunoglobulin product according to any of claims 14 or 15, characterized in that it contains less than 3 mg / l of IgA.
17. 17. An immunoglobulin product according to any of claims 14 to 16, characterized in that it contains between 55 and 65% of IgGl, between 30 and 40% of IgG2, between 2 and 5% of IgG3 and between 1 and 4% of IgG4.
18. 18. An immunoglobulin product according to any of claims 14 to 17, characterized in that it contains less than 0.5% polymers and aggregates.
19. 19. A liquid immunoglobulin product according to any of claims 14 to 18.
20. 20. An immunoglobulin product according to any of claims 14 to 19, ready for intravenous administration.
21. 21. An immunoglobulin product according to any of claims 14 to 20, characterized in that it is used in medicine.
22. 22. The use of an immunoglobulin product according to any of claims 14 to 21 for the preparation of a medicament for the treatment of a mammal suffering from PID (primary immunodeficiency), IDS (secondary immunodeficiency), ITP (thrombocytopenic purpura) idiopathic), polyradiculitis, peripheral polyneuropathies, Kawasaki disease, polymyositis, severe chronic autoimmune diseases, chronic inflammatory deineminating polyneuropathy (PDIC), multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, Eaton-Lambert syndrome, optic neuritis, epilepsy, abortus habitualis, primary antiphospholipid syndrome, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, vasculitis, Wegner's granulomatosis, Sjógren, juvenile rheumatoid arthritis, autoimmune neutropenia, autoimmune hemolytic anemia, neutropenia, Crohn's disease, ulcerative colitis, disease, septic shock syndrome, chronic fatigue syndrome, psoriasis, toxic shock syndrome, diabetes, sinusitis, dilated cardiomyopathy, endocarditis, atherosclerosis and adults with AIDS and bacterial infections.
23. 23. The use according to claim 22, characterized in that the mammal is a human being.