Title of Invention

PROCESS FOR PRODUCTION OF IMMUNOGLOBULIN G AND IMMUNOGLOBULIN PRODUCT OBTAINED THEREBY

Abstract

1. A process for purifying Immunoglobulin G (IgG), from 3 crude immunoglobulin-contalning plasma protein fraction, which process comprises the steps of: (a) preparing an aqueous suspension of the cmde immunoglobulln-containing plasma protein fraction; (b) adding a water soluble, substantially non-denaturating protein precipitant to ttie said suspension of step (a) in an amount sufficient to cause precipitation of a iiigh proportion of non-immunoglobulin G proteins, aggre¬gated immunoglobulins and particles including potentially infectious particles such as vims particles, without causing substantial precipitation of monomeric immunoglobulin G, thereby forming a mixture of a solid precipitate and a liquid supematant;

Full Text

PROCESS FOR PRODUCING IMMUNOGLOBULINS FOR tNTRAVENOl^S ADMINI¬STRATION AND OTHER IMMUNOGLOBULIN PRODUCTS FIELD OF THE INVENTION The present invention relates to a process for purifying immunoglobulins, i.e, immuno¬globulin G (IgG), from crude plasma or from a crude plasma protein fraction. The invention also relates to an immunoglobulin product and to the use of such an immunoglobulin prod¬uct for medical purposes. BACKGROUND OF THE INVENTION Human normai immunoglobulin (HNI) for use in the prevention and treatment of a number of infectious diseases was introduced in the late 1940's. HNI prepared by the cold ethanol fractionation method according to 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 the modification made by Kistler and Nitschmann (Kistler P and Nitschmann HS, (1952), Vox Sang, 7, 414-424) proved to be both efficient and safe against the transmission of vi¬rus infection when administered subcutaneously or intramuscularly. Congenital or acquired total or partial lack of immunoglobulin (primary and secondary im¬munodeficiency syndrome, respectively) manifests itself through frequent ordinary and se¬rious infections, especially of a bacterial nature. The prevention of such infections was pre¬viously achieved by repeated intramuscular or sut)cutaneous injections of large amounts of HNI for up to several times a week as a life-lasting treatment, which is very painful when the medicament is given intramuscularly. In the early sixties, administration of HNI by the intravenous route was therefore attempted. Trials showed that about 5% of healthy volunteers and about 95% of patients with an im¬munoglobulin deficiency developed immediate adverse effects varying from dyspnoea to circulatory shock and being of such serious nature that the intravenous administration of HNI had to be abandoned. The reason for the adverse effects mentioned above turned out to be aggregates of immu¬noglobulins which, among other effects, strongly activated the complement system. This was in particular seen in patients lacking immunoglobulins. Especially serious adverse ef- fects of an anaphylactic nature couid be seen in patients who developed antibodies to IgA. Consequently, methods of avoiding aggregate formation and/or eliminating these aggre¬gates during the preparation process were developed, and some twenty years ago the first generation of an immunoglobulin for intravenous administration (IVIG) was tested and found suitable. The original purpose of an IVIG was to alleviate infectious episodes in patients with a con¬genital or acquired total or partial lack of immunoglobulins and to eliminate discomfort in connection with the administration of HNI. Another advantage of IVIG is that large doses of immunoglobulin can be given within a short time, and by this it is possible to obtain suffi¬ciently high blood concentrations very quickly. Especially when treating serious bacterial ■ infections it is of importance to establish high concentrations at sites of infections quickly. in recent years, IVIG has furthermore proved to be efficient in other serious diseases, the treatment of which can otherwise be difficult, e.g. haemorrhages caused by the disappear¬ance of the blood platelets on an immunological basis, idiopathic thrombocytopenic pur¬pura (ITP), in some rare diseases such as Kawasaki's syndrome and a number of autoim¬mune diseases such as polyradiculitis (Guillain Barre's syndrome). Other diseases the treatment of which has been difficult to the present day are currently being subjected to clinical trials with IVIG. The mechanism of action in these diseases has only partly been clarified. The effect is supposed to be related to so-called immunomodulating properties of !gG, e.g. a blockage of Fcy-receptors on phagocytic cells, increased metabolism of IgG. downregulation of the production of cytokines, and interference with a supposed network of idiotypes/anti-idiotypes, especially relevant for the neutralization of autoimmune reactivity. The first generation of IVIG was prepared by pepsin cleavage of the starting materiar(Cohn fraction It), the purpose of the cleavage being removal of immunoglobulin aggregates. No column chromatography steps were included in the process. The product had to be freeze-dried in order to remain stable for a reasonable period of time and was dissolved immedi¬ately prior to use. The starting material for the IVIG was HNI which bad proved to be safe with respect to the transmission of viruses when used for intramuscular injection. Hence, IVIG was considered to be just as safe. After several years of clinical use, however, IVIG products from some manufacturers were surprisingly shown to cause transfer of hepatitis C virus infection. studies to elucidate the fate of viruses during the production of HN! showgd that the re¬moval of virus in the fractionation process from plasma to HNl is modest. The safety of HNI for intramuscular use is liltely to be due to the fact that it contains protective immunoglobu¬lins. In combination with the modest volume injected and the intramuscular route of admini¬stration, these protective immunoglobulins can neutralize and render common viruses in plasma non-infectious. Especially when large doses of immunoglobulin are given intrave¬nously, virus infections may occur as demonstrated in the early 1990's. Therefore, it was recognised that the production processes should comprise one or more well-defined virus-inactivation and/or removal steps. A second generation of IVIG based on uncleaved and unmodified immunoglobulin mole¬cules with low anticomplementary activity and higher stability was introduced in the mid-eighties, but still in the form of a freeze-dried product. This IVIG was purified by several chromatography steps. Products of that kind presently dominate the marlcet for iVlG. The first and second generations of IVIG thus appear as freeze-dried powders which are dis¬solved immediately prior to use. Dissolution of freeze-dried IVIG is slow {up to 30 minutes for one via!). Several portions often have to be dissolved for one patient. As it is of high priority for the users to have an IVIG in a solution ready for use, liquid products have been introduced on the market. More importantly, there is still a need for improvement of the production process in order to ob¬tain a highly purified, stable and fully native IVIG preparation with higher clinical efficacy and less adverse drug reactions. A further developed and improved process for purifying IgG from crude plasma or a plasma protein fraction for a virus-safe, liquid IVIG product is thus needed. Finally, the process should be designed in such a way that it can be used in a large scale production. The purification process described in the present application leads to a liquid immuno¬globulin product for intravenous administration which can be characterized as a highly puri¬fied, fully native, biologically active, double virus-inactivated, and stable new generation of MG, which does not contain any detergent, polyethylene glycol (PEG) or albumin as a stabilizer. SUMMARY OF THE INVENTION The present invention relates to an in:iproved purification procedure and an improved liquid immunoglobulin product which, inter alia, can be administered intravenously. An immunoglobulin product obtained by the method of the present invention could be called a third generation IVIG. The process is characterized by the following conditions for fractionation: pepsin cleavage is avoided, aggregates and particles are removed by pre¬cipitation {a process step validated to function as a virus removal step), further purification is achieved by column chromatographic ion exchange methods, S/D treatment is intro¬duced as a virus-inactivating step, and the preparation is formulated as a liquid product. Due to the improved purity of the immunoglobulin product obtainable by the process of the invention as compared to the prior art products, the addition of stabilizers such as a non-ionic detergent, PEG or albumin is not necessary in order to avoid aggregation of IgG dur¬ing storage of the IVIG as a liquid product. The product obtainable by the process of the invention has a higher quality than the prior art products and provides improved clinical ef¬fects, and unwanted adverse effects are virtually absent. DETAILED DISCLOSURE OF THE INVENTION The present invention relates to a process for purifying immunoglobulins, i.e. IgG, from crude plasma or an immunoglobulin-containing plasma protein fraction, which process comprises the steps of: (a) preparing an aqueous suspension of the crude immunoglobulin-containing plasma protein fraction; (b) adding a water soluble, substantially non-denaturating protein precipitant to said sus¬pension of step (a) in an amount sufficient to cause precipitation of a high proportion of non-immunoglobulin G proteins, aggregated immunoglobulins and particles includ¬ing potentially infectious particles such as virus particles, without causing substantial precipitation of monomeric immunoglobulin G, thereby forming a mixture of a solid precipitate and a liquid supernatant; (c) recovering a clarified immunoglobulin G-containing supernatant from the mixture of step (b); (d) applying the clarified immunoglobulin G-containing supernatant of step (c) to an an¬ion exchange resin and subsequently a cation exchange resin; (e) washing out protein contaminants and the protein precipitant from the cation ex¬change resin with a buffer having a pH and ionic strength sufficient to remove the contaminants from the resin without causing substantial elution of immunoglobuiin G; (f) eluting immunoglobulin G from the cation exchange resin with a substantially non-denaturating buffer having a pH value and ionic strength sufficient to cause efficient elution of the immunoglobulin G, thereby recovering an immunoglobulin G-containing eluate; (g) perfomiing a dia/uitrafiltration on the immunoglobulin G-containing eluate of step (f) to concentrate and/or dialyse the eluate and optionally adding a stabilizing agent; (h) adding a virucidal amount of virus-inactivating agent to the immunoglobulin G-con¬taining dia/ultrafiltrated and optionally stabilized fraction of step (g) resulting in a sub¬stantially vinjs-safe immunoglobulin G-containing solution; (i) applying the immunoglobulin G-conlaining solution of step (h) to an anion exchange resin and subsequently to a cation exchange resin; 0) washing the cation exchange resin of step (i) with a buffer having a pH and ionic strength sufficient to wash out the protein contaminants and the virus-inactivating agent from the resin without causing substantial elution of immunoglobulin G; (k) eluting immunoglobulin G from the cation exchange resin of step (j) with a substarv-tially non-denaturating buffer having a pH and tonic strength sufficient to cause effi¬cient elution of the immunoglobulin G, thereby recovering an immunoglobulin G-con¬taining eluate; and (!) subjecting the immunoglobulin G-containing etuate of step (k) to dia/ullrafiltration to lower the ionic strength and concentrate the immunoglobulin G of the solution, and adjusting the osmolality by adding a saccharide. The starting material of the present purification process can be crude plasma, but is advan¬tageously an immunoglobuiin-containing crude plasma protein fraction. The starting mate¬rial for the purification process can be normal human plasma or may originate from donors with high titers of specific antibodies, e.g. hyperimmune plasma. In the present specifica¬tion, the term "immunoglobuiin-containing plasma fraction" is to encompass all possible starting materials for the present process, e.g. cryoprecipitate-free plasma or cryoprecipi-tate-free plasma from which various plasma proteins, such as Factor iX and Antithrombin, have been removed, different Cohn fractions, and fractions obtained through precipitation procedures by PEG (Poison et at., (1964), Biochem Biophys Acta, 82, 463-475; Poison and Ruiz-Bravo, (1972) Vox Sang, 23,107-118) or by ammonium sulphate. In a prefen-ed em¬bodiment, the plasma protein fraction is Cohn fractions II and III, but Cohn fraction II, or Cohn fractions 1, II and III can be used as well. The different Cohn fractions are preferably prepared from plasma by a standard Cohn-fractionation method essentially as modified by Kistler-Nitschmann. In addition to immunoglobulins, the Cohn fractions contain e.g. fibrino¬gen, a-globulins and p-globulins, including various lipoproteins, which should preferably be removed during the subsequent purification process. Filter aid may or may not be present depending on the isolation method used to obtain the Cohn fractions (i.e. centrifugation or filtration). The first step of the process according to the invention involves preparing an aqueous sus¬pension of an immunoglobuiin-containing plasma protein fraction, wherein the IgG concen¬tration in the suspension is sufficiently high so that, during the following precipitation step, a major proportion of the non-1 gG-proteins, especially those of higher molecular weight, the aggregated immunoglobulins and other aggregated proteins as well as potentially infec¬tious particles precipitate without substantial precipitation of monomeric IgG. This is gen¬erally achieved if the concentration of the IgG in the buffered and filtered suspension is at least about 4 g/i before the addition of the precipitant. It should be taken into consideration that the influence of the protein concentration as well as pH and temperature of the sus¬pension on the precipitation depends on the precipitant chosen. it is preferred that the plasma protein fraction is suspended in water and/or buffer at a sut>-stantialiy non-denaturating temperature and pH. The term "substantially non-denaturattng" implies that the condition to which the temi refers does not cause substaritTal irreversible loss of functional activity of the IgG molecules, e.g. loss of antigen binding activity and/or loss of biological Fc-function (see Example 2). Advantageously, the plasma protein fraction is suspended in water acidified with at least one non-denaturating buffer system at volumes of from 6 to 9, preferably from 7 to 8, times that of the plasma protein fraction. The pH of the immunoglobulin-containing suspension is preferably maintained at a pH below 6, such as within the range of 4.0-6.0, preferably 5.1-5.7, most preferably about 5,4, in order to ensure optimal solubility of the immunoglobulin and to ensure optimaf effect of the subsequent PEG precipifafion step. Any suitable acidic buffer can be used, but the buffer system preferably contains at least one of the following buffers and acids: sodium phosphate, sodium acetate, acetic acid, HCl. Persons skilled in the art will appreciate that numerous other buffers can be used. The immunogSobulin suspension is preferably maintained at a cold temperature, inter alia in order to prevent substantial protein denaturalion and to minimize protease activity. The immunoglobulin suspension and water as well as the buffer system added preferably have the same temperature within the range of 0-12°C, preferably O-S^C, most preferably 1-4'C. The suspension of an ethanol precipitated paste contains relatively large amounts of ag¬gregated protein materia). Optionally, the immunoglobulin-containing suspension is filtered in order to remove e.g. large aggregates, filter aid, if present, and residual non-dissolved paste. The filtration is preferably performed by means of depth filters, e.g. C150 AF, AF 2000 or AF 1000 (Schenk), 30LA (Cuno) or similar filters. The removal of aggregates, filter aid, if present, and residual non-dissolved protein material could also be carried out by centrifugation. At least one water-soluble, substantially non-denaturating protein precipitant is added to the immunoglobulin-containing filtered suspension in an amount sufficient to cause precipi¬tation of a high proportion of high molecular weight proteins, lipoproteins, aggregated pro¬teins, among these aggregated immunoglobulins. Other particulate material, such as po¬tentially infectious particles, e.g. virus particles, are also precipitated without causing sub¬stantial precipitation of monomericlgG. The term "infectious particles" in the present con¬text comprises e.g. virus particles (such as hepatitis vinjses, H1V1 and HIV2) and bacteria. Substantially non-denaturating, water-soluble protein precipitants are well known in the field of protein purification. Such precipitants are used for protein fractionation, resulting in partial purificalion of 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 sulphate. Those skilled in the art will appreciate that several other non-denaturating water soluble precipitants may be used as alternative means for the pre¬cipitation. The term "adding a protein precipitant" and variants of that term implies the addi¬tion of one or more types of protein precipitation agents. A preferred precipitant is the organic agent PEG, particularly PEG within the molecular weight range of 3000-8000 Da, such as PEG 3350. PEG 4000, PEG 5000, and especially PEG 6000 (the numbers of these specific PEG compounds represent their average mole¬cular weight). The advantage of using PEG as a precipitant is that PEG is non-ionic and has protein stabilizing properties, e.g. PEG in low concentration is well known as a stabi¬lizer of IVIG products. The precipitation step also functions as a virus-removal step. PEG concentrates and precipitates the viruses irrespective of the species, size, and surface coating of these. A given amount of protein precipitant is added to the filtrated suspension to precipitate the majority of high molecular weight and aggregated proteins and particles, without a sub¬stantial precipitation of monomeric IgG, forming a clear supernatant solution. The protein precipitant may be added as a solid powder or a concentrated solution. For PEG as precipitant a general rule applies that the higher the molecular weight of the compound, the tower the concentration of PEG is needed to cause protein to precipitate. When PEG 3350, PEG 4000 or preferably PEG 6000 is used, the concentration of the pre¬cipitant in the filtrated suspension is advantageously within the range of 3-15% by weight, such as 4-10% (such as about 5%, 6%, 7%, 8%, 9%, 10%), wherein 6% is most prefen-ed. In the precipitation step, the precipitation process is allowed to proceed at least until equi-Irbrium is reached between the solid and the liquid phase, e.g. usually for at least two hours, such as from about 2 hours to about 12 hours, preferably about 4 hours. Throughout the precipitation the suspension is preferably maintained at a low temperature (e.g. less than about 12°C, such as less than about 10°C, preferably between 2°C and 8'^C). The most suitable temperature depends on the identity of the protein pffectpitant. After completion of the protein precipifation, a clarified supernatant containing IgG almost exclusively in a monomeric form is recovered from the mixture of solid precipitate and liquid supernatant resulting from the precipitation. The recovery can be performed by conven¬tional techniques for separating liquid from solid phase, such as centrifugation and/or filtra¬tion. Preferably, a flow-through centrifuge (e.g. Westfalia) with 1000-5000 g force is used. Optionally, the recovered, clarified, IgG-containing supernatant is depth filtered to remove larger particles and aggregates. This is optionally followed by sterile filtration performed by use of a conventional sterilization filter (such as a 0.22 ^im filter from Millipore or Sartorius), which eliminates e.g. bacteria from the solution. The clarified and optionally filtrated IgG-containing supernatant is subjected to at least one step, such as two steps, but optionally more steps of anion and cation exchange chroma¬tography in order to remove a substantial proportion of the remaining non-IgG contami¬nants, e.g. IgA. albumin as well as aggregates. In a preferred embodiment, the clarified and optionally filtrated IgG-containing supernatant is applied to an anion exchange resin and subsequently a cation exchange resin packed in two columns of appropriate dimen¬sions. When performing the ion exchange chromatography steps for the purification of IgG, it is preferred that the conditions, e.g. the pH and ionic strength, are chosen in such a way that a major portion of the contaminants (e.g. non-IgG proteins such as IgA, transferrin, albu¬min, and aggregates) in the applied solution binds to the anion exchange resin, whereas substantially no IgG adsorbs to the anion exchange resin. With respect to the subsequent cation exchange chromatography, the preferred conditions chosen result in binding of sub¬stantially all of the IgG molecules present in the solution applied to the cation exchange resin. Protein contaminants not adsorbed to the anion exchange resin and the precipitation agent are removed in the subsequent washing of the cation exchange resin. In a preferred embodiment of the present process, the anion exchange resin and the cation exchange resin are connected in series. In the present context, the term "connected in se¬ries", when used in connection with the ion exchange resins, means that the proteins passing through the anion exchange resin are loaded directly onto the cation exchange resin with no change of buffer or other conditions. Several reasons make it advantageous that the anion exchange and caTTon exchange chromatography is carried out in one step using two serially connected chromatography columns, instead of two independent chromatography steps, e.g. with different buffer com¬positions. The use of two serially connected chromatography columns makes the operation more practical, e.g. there is no need for an intermediary step of collecting the IgG-contain-ing fraction between the two ion exchange chromatographic methods, for possibly adjust¬ing pH and ionic strength. In addition the buffer flow is applied to both of the columns at the same time, and the two columns are equilibrated with the same buffer. However, it is con¬templated that it is also possible to perfonn the chromatography step in two steps, i.e. the anion exchange resin and cation exchange resin are not connected in series. Performing the chromatography in two steps would though, as mentioned above, be more laborious compared to keeping the ion exchange resins connected in series. It is presently contemplated that the high degree of purity, the high content of IgG mono¬mers and dimers and the low content of IgA in the IVIG product of the invention are partly due to the use of two serially connected chromatography columns. As will be known by the person skilled in the art, ion exchangers may t>e based on various materials with respect to the matrix as well as to the attached charged groups. For exam¬ple, the following matrices may be used, in which the materials mentioned may be more or less crosslinked: agarose based (such as Sepharose CL-6B®, Sepharose Fast Flow® and Sepharose High Performance®), cellulose based (such as DEAE Sephace!®), dextran based (such as Sephadex®), silica based and synthetic polymer based. For the anion ex¬change resin, the charged groups which are covalentty attached to the matrix may e.g. be diethylaminoethyl (DEAE), quaternary aminoetbyl (QAE), and/or quaternary ammonium (Q). For the cation exchange resin, the charged groups which are covalently attached to the matrix may e.g. be carboxymethyl (CM), sulphopropyl (SP) and/or methyl sulphonate (S). In a preferred embodiment of the present process, the anion exchange resin employed is DEAE Sepharose Fast Flow®, but other anion exchangers can be used. A preferred ca¬tion exchange resin is CM Sepharose Fast Flow®, but other cation exchangers can be used. The appropriate volume of resin used when packed into an ion exchange chromatography column 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 e.g. the amount of IgG in the applied solu¬tion and the binding capacity of the resin used. Before performing an ion exchange chromatography, the ion exchange resin is preferably equilibrated with a buffer which allows the resin to bind its counterions. Preferably, the an¬ion and cation exchange resins are equilibrated with the same buffer, as this facilitates the process since then only one buffer has to be made and used. If, for instance, the chosen anion exchange resin is DEAE Sepharose FF® and the cation exchange resin CM Sepharose FF© and the columns are connected in series, then the columns are advantageously both equilibrated with a non-denaturating acidic buffer having about the same pH and ionic strength as the IgG solution to be loaded. Any of a variety of buffers are suitable for the equilibration of the ion exchange columns, e.g. sodium acetate, sodium phosphate, tris(hydroxymethyl)amino-methane. Persons skilled in the artv/ill ap¬preciate that numerous other buffers may be used for the equilibration as long as the pH and conductivity are about the same as for the applied IgG solution. A preferred buffer for (he equilibration of the anion exchange column and cation exchange column when con¬nected in series is a sodium acetate buffer having a sodium acetate concentration within the range of 5-25 mM, such as within the range of 10-20 mM, preferably about 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, such as within the range of 5.4-5.9, preferably about 5.7. The conductivity is within the range of 1.0-1,4 mS/cm, preferably about 1.2mS/cm. Suitable acetate buffers may be prepared from sodium acetate trihydrate and glacial acetic acid. Prior to loading the clarified and optionally filtrated IgG-containing supernatant onto the ion exchange columns, the buffer concentration and pH of said supernatant are preferably ad¬justed, if necessary, to values substantially equivalent to the concentration and the pH of the employed equilibration buffer. After loading the IgG-containing supernatant onto the columns in series, the columns are preferably washed (the initial washing) with one column volume of a washing buffer in or¬der to ensure that the IgG-containing solution is quantitatively transferred from the anion exchange column to the cation exchange column. Subsequently, the anion exchange and the cation exchange columns are disconnected, and the cation exchange column is pref¬erably washed in order to remove protein contaminants from the resin with a buffer having a pH and ionic strength sufficient to elule substantially all of the contaminants from the ca¬tion exchange resin without causing substantial elution of IgG. The initial washing is advantageously performed by using the equilibratiDn buffer, even though other buffers with a similar concentration and pH-value may be used for the wash¬ing. It is prefen^d that an acetate buffer is used for washing out contaminants from the ca¬tion exchange resin. The pH of the buffer could be from 5.0 to 6.0, such as within the range of 5.2-5.8, such as about 5.4. The etution of the IgG from the cation exchange resin is preferably performed vi/ith a sub¬stantially non-denaturating buffer having a pH and ionic strength sufficient to cause efficient elution of the IgG, thereby recovering an IgG-containing etuate. In this context, efficient elution means that at least 75%, such as at least 80%, e.g. at least 85%, of the IgG pro¬teins loaded onto the anion and cation exchange resins in series are eluted from the cation exchange resin. The elution is advantageously carried out as 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 5.0-6.0, such as 5.2-5.8, preferably about 5.4, and a concentration within the range of 5-40 mM, such as within the range of 10-25 mM, preferably about 15 mM. It is preferred that the salt concentration of the eluting buffer is sufficiently high to displace the IgG from the resin. However, it is contemplated that an increase in pH and a lower salt concentration can be used to elute the IgG from the resin. In a preferred embodiment of the present process, the elution is conducted as a continuous salt gradient elution with sodium chloride concentrations within the range of 50-500 mM, preferably from about 125 mM to about 350 mM sodium chloride. The elution can also be performed by step gradient elution. It is contemplated that the elu¬tion could also be performed as a constant salt elution, in which the elution buffer applied to the cation exchange column has only one single salt concentration in contrast to the gradient elution. If a constant salt elution is performed, the concentration of salt may ad¬vantageously be within the range of from about 350 mM to about 500 mM sodium chloride. The advantage of the gradient elution compared to the constant salt elution is that the elu¬tion 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 is critical to the stability of IgG. The elution buffer may further comprise a protein stabilizing agent such as those mentioned below. Various other suitable buffer systems may be used for eluting the IgG, as wilt be appreciated by those skilled in the art Preferably, at least one protein stabilizing agent is applied to the IgG fraction immediately after or during the elution. Protein stabilizing agents are known to those skilled in the art and include e.g. different sugar alcohols and saccharides (such as sorbitol, mannose, glu¬cose, trehalose, maltose), proteins (such as albumin), amino acids (such as lysine, glycine) and organic agents (such as PEG). Advantageously, the intermediary stabilizer chosen may be one that can be removed from the IgG-containJng solution in the subsequent steps. The suitable concentration of the protein stabilizing agent in the IgG-containing solution depends on the specific agent employed. In one preferred embodiment, the stabilizing agent is sorbitol, preferably at a final concentration within the range of 2-15% (w/v) sorbitol, e.g. about 2.5%. Subsequent to elution from the cation exchange column, the eluate is preferably desali¬nated (i.e. dialysed) and advantageously concentrated. The change of buffer and the con¬centration of IgG can be performed by a combined dia/ultrafittration process. The term "dia/uttrafiltration" means that the dialysis and concentration by diafiltration and ultrafiltra¬tion, respectively, are performed in one step. It is contemplated that the diafiltration and ultrafiltration may be performed as two separate steps. However, in order to prevent un¬necessary loss of the product, it is presently preferred to perform the dialysis and concen¬tration by the methods of diafiltration and ultrafiltration in one step. The membranes employed for the dia/uttrafiltration advantageously have a nominal weight cutoff within the range of 10,000-100,000 Da. A prefen^ed membrane type for the present process is a polysutfone membrane with a nominal weight cutoff of 30,000 Da, obtained from Millipore. Other ultrafiltration membranes of comparable material and porosity may be employed. The extent of concentration may vary considerably. The solution is concentrated from about 10 g/l IgG to about 100 g/1, preferably to about 50 g/l. Fojlowing the concentration, the IgG concentrate is advantageously dialysed against a buffer with low ionic strength. Besides removing salt ions, this step also removes contaminants of low molecular weight from the solution and provides a means for buffer exchange for the next purification step. A preferred buffer for the diafiltration is 15 mM sodium acetate, pH 5.4 containing 2.5% (wA^) sorbitol. The exchange of buffer is continued until the conductivity of the ultrafittrated solu¬tion is reduced to a value less than about 1.5 mS/cm, preferably less than about 1.3 mS/cm. During the dia/uttrafiltration, the pH is preferably kept within the range of 4.0-6.0. preferably 5.1-5.7, most preferably at about 5.4. After dia/ultrafiltration, the concentration of the protein stabilizing agent is advantageously adjusted in the solution, if necessary, to the final optimal concentration characteristic for the specific protein stabilizing agent employed. If sorbitol is used, the sorbitol concentration is preferably adjusted to about 10% by weight. It is preferred that the stabilized solution is filtered with a filler with a pore diameter within the range of 0.2-1.0 fim, preferably about 0.45 [am, in order to remove aggregates before the next step. At this stage the IgG-containing solution appears as a clear solution of an appropriate volume with a high stability as a combined result of the high purity, the low ionic strength, the acidic pH, the relatively high concentration of IgG and the stabilizer added. In the production process of the IVIG product, at least two defined and validated virus re¬moval and inactivation steps are presently incorporated, these steps preferably being pre¬cipitation with PEG as a general virus-removal step and an S/D treatment as a virus-inacti¬vating step towards lipid enveloped viruses. Besides the stringent requirements to virus safety of the starting material, according to international guidelines, and the well known vi¬rus reducing capacity of a multistep purification process, the incorporation of two inde¬pendent virus reduction steps being active against both enveloped and non-enveloped vi¬ruses, the medicament of the present invention is substantially virus-safe. Infectious lipid enveloped viruses that may still be present in the IgG-containing solution are preferably inactivated at this stage of the process by addition of a virucidal amount of virus-inactivating agent to the IgG-containing solution. A "virucidal amount" of virus-inacti¬vating agent is intended to denote an amount giving rise to a solution in which the virus particles are rendered substantially non-infectious and by this a "virus-safe IgG-containing solution" as defined in the art. Such "vimcidal aniount" will depend on the virus-inactivating agent employed as well as the conditions such as incubation time, pH, temperature, con¬tent of lipids, and protein concentration. The term Virus-inaclivating agent" is intended to denote such an agent or a method which can l>e used in order to inactivate lipid enveloped viruses as well as non-lipid enveloped viruses. The term "virus-inactivating agent" is to be understood as encompassing both a combination of such agents and/or methods, whenever that is appropriate, as well as only one type of such agent or method. Preferred vims-inactivating agents are detergents and/or solvents, most preferably deter¬gent-solvent mixtures. It is to be understood that the virus inactivating agent is optionally a mixture of one or more detergents with one or more solvents. Solvent/detergent (S/D) treatment is a widely used step for inactivating lipid enveloped viruses (e.g. HIV1 and HIV2, hepatitis type C and non A-B-C, HTLV 1 and 2, the herpes virus family, including CMV and Epstein Barr virus) in blood products, A wide variety of detergents and solvents can be used for virus inactivation. The detergent may be selected from the group consisting of. non-ionic and ionic detergents and is selected to be substantially non-denaturating. Prefer¬ably, a non-ionic detergent is used as it facilitates the subsequent elimination of the deter¬gent from the IgG preparation by the subsequent step. Suitable detergents are described, e.g. by Shanbrom et al.. in US Patent 4,314,997. and US Patent 4,315,919. Preferred de¬tergents are those sold under the trademarks Triton X-100 and Tween 80. Preferred sol¬vents for use in virus-inactivating agents are di- or thalkylphosphates as described e.g. by Neurath and Horowitz in US Patent 4,764,369. A prefen-ed solvent is tri(n-butyl)phosphale (TNBP). An especially preferred vinjs-inactivating agent for the practice of the present in¬vention is a mixture of TNBP and Tween 80, but, alternatively, other combinations can be used. The preferred mixture is added in such a volume that the concentration of TNBP in the IgG-containing solution is within the range of 0.2-0.6% by weight, preferably at a con¬centration of about 0.3% by weight. The concentration of Tween 80 in the IgG-containing solution is within the range of 0.8-1.5% by weight, preferably at a concentration of about 1% by weight. The virus-inactivatton step is conducted under conditions inactivating enveloped viruses resulting in a substantially virus-safe IgG-containing solution. In general, such conditions include a temperature of 4-30°C, such as 19-28°C, 23-27'C, preferably about 25'C, and an incubation time found to be effective by validation studies. Generally, an incubation time of 1-24 hours is sufficient, preferably 4-12 hours, such as about 6 hours, to ensure sufficient virus inactivation. However, the appropriate conditions (temperature and incubation times) depend on the virus-inactivating agent employed, pH, and the protein concentration and lipid content of the solution. . It is contemplated that other methods for removal of or inactivating virus-can also be em- -ployed to produce a virus-safe product, such as the addition of methylene blue with subse¬quent inactivafion by radiation with ultraviolet light or nanofiltration. After the solvent/detergent treatment, the solution is advantageously diluted with buffer. Optionally, the substantially virus-safe IgG-containing solution is filtered, preferably through a depth filter as described previously in an earlier step of the present process and/or through a sterile filter. After viruS'inactivation and preferably filtration, ion exchange chromatography is performed in order to remove the virus-inactivating agent and protein contaminants. This step is pref¬erably performed as already described for the previous ion-exchange chromatography step in the present process, with the exceptions that the volume of the anion exchange resin is about half that of the cation exchange resin and that the washing before elution of IgG is more extensive, at least six column volumes of buffer are used. Additionally, in a preferred embodiment of the invention, the equilibration buffer is sodium acetate with a concentration within the range of about 5-25 mM, preferably 15 mM, and a pH within the range of about 5.0-5.8, preferably 5.4. As mentioned previously, the sodium acetate content and the pH of the IgG-containing solution are preferably adjusted to the same concentration and pH as the equilibration buffer. Additionally, in a preferred embodiment of the invention, a protein stabilizing agent, preferably maltose, is added to the recovered eluate to a final concentra¬tion within the range of 1-5%, preferably about 2.5% by weight. The preferred method of eliminating the viais-inactivating agent is by ion exchange chro¬matography. However, other methods, such as oil extraction and alternative chroma¬tographic methods, are contemplated to be useful. The appropriate method depends on the virus-inactivating agent employed. Removal of solvent/detergent may thus be achieved by binding the IgG to a resin and, subsequently, a thorough washing out of the inactivating agent with buffer. Cation exchange chromatography is a usable method. In a preferred embodiment of the present invention, anion exchange chromatography is also performed in addition to the cation exchange chromatography in order to improve the quality and overall purity of the final product of the present process. After the ion exchange chromatography step, the IgG-containing eluate is preferably dial-ysed and concentrated; hereby the content of remaining smaller protein components is also effectively reduced. Advantageously, this can be performed by dia/uftrafiltration as de- scribed previously. The buffer employed for the diafillration is sodium acetale, preferably at a concentration from about 4 to 10 mM, preferably 7.5 mM, and at a pH within the range from about 4.0 to 6.0, preferably about 5.1-5.7, such as about 5.4. Alternatively, other buf¬fers such as sodium phosphate or acids can be used for the diafiltration. The diafiltratlon continues until the conductivity is less than or equal to 1 mS/cm. Optionally, the IgG-con-taining solution is further sterile filtered. If desired, the purified IgG-containing solution which is substantially free from the virus-in¬activating agent is subjected to further treatments for the purpose of making it suitable for formulation as a liquid product to be used e.g. intravenously, subcutaneousty, or intramus¬cularly. From a practical point of view it is preferred that the content of the liquid formulation of the immunoglobulin product is the same for storage as for use. The final concentration of IgG in the product is preferably within the range of 0.25-20% by weight (corresponding to 2.5-200 g of IgG/I), such as about 1-20% by weight, i.e. about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%. It is known that a high protein concentration results in a higher stability of IgG. On the other hand, a high IgG concentration means that the maximum infusion rate when administering IgG intravenously to the patient has to be quite low as transfusion problems, due to the high osmotic pressure of the product, have to be avoided. A presently recommended con¬centration for intravenous administration by European Pharmacopoeia (Ph.Eur.) is 5% (w/v). On the other hand, a quite concentrated product (e.g. 10% or above) is advantage¬ous for intramuscular or subcutaneous injections. Although not preferred, it is evident that the products obtainable by the various process steps of the invention can also be used as e.g. freeze-dried products instead of as liquid formulations, although this is less favourable compared to the use of the immunoglobulin products as instant liquid formulations. The tatter embodiment will be described in more detail in the following. Liquid immunoglobulin products are most stable at an ionic strength markedly lower than that of plasma, i.e. the conductivity is preferably less than 1.0 mS/cm, preferably about 0.8 mS/cm. The pH has an impact on the stability of IgG and on the infusion rate. Liquid immunoglobu¬lin products are most stable under acidic conditions, i.e. below the isoelectric point of IgG, pH 6,4-8.5. The closer the pH value is to the physiological pH value (7.1-7,3), the higher infusion rate can be employed. As a consequence of the stability required, the pH of the immunoglobulin product of the invention will preferably be within the range of 5.1-5.7, such as between 5.2 and 5.6, such as about 5.4. Furthermore, the immunoglobulin product may comprise protein stabilizing agents as de¬scribed previously. In addition to sugar alcohols and saccharides (such as sorbitol, man-nose, glucose, trehalose, maltose), also proteins (such as albumin), amino acids (such as lysine, glycine) and organic agents (such as PEG and Tween 80) may be used as well as stabilizers. The suitable concentration of the stabilizing agent in the IgG-containing solution depends on the specific agent employed as described previously. The purified IgG solution is adjusted if necessary in order to obtain a stable and isotonic solution. The temi "isotonic solution" is intended to denote that the solution has the same osmotic pressure as in plasma. As mentioned above, the ionic strength is markedly lower in the immunoglobulin product of the invention as a liquid formulation than in plasma. For that reason it is preferred that mono- or disaccharides are used to increase the osmolality of the solution since this does not affect the ionic strength. In a preferred embodiment of the present invention, maltose is added at a concentration ensuring that the solution is isotonic and, at the same time, maltose functions as an immunoglobulin-stabilizing agent. This is preferably performed by addition of maltose to a final concentration within the range of about 5% to 15% (w/v), preferably 10% (w/v); other saccharides, such as mannose and glucose, can alternafively be used. The preferred final conditions for the immunoglobulin product are a compromise between stability and physiologically acceptable conditions with respect to e.g. pH, ionic strength and tonicity. Furthermore, the immunoglobulin product has to comply with the requirements of quality control tests, as specified in Monograph No. 918, Ph. Eur., 1997. The main advantages of the product obtainable by the method of the invention are that, when formulated as a liquid preparation, the product is a combination of a liquid, ready-for-use product which, at the same time, is very stable, highly purified, has a largely normal distribution of IgG subclasses and has an extremely low !gA content as well as a low IgM content, and retained antibody activity and biological activity shown by the Fc function. Moreover, it contains essentially no aggregates of immunoglobulins and/or other plasma proteins measured as polymers higher than dtmers and has a low anticomplementary ac¬tivity, and it has a very high content of IgG monomers and dimers. Monomeric IgG consti¬tutes at least 90%, which is considered to be ideal. Due to the high stability it is possible to avoid the addition of other stabilizers, such as albumin, glycine, detergent, or PEG. Finally, the product is virus-safe as the process comprises well-defined and validated virus-reduc¬tion steps aimed at removing and/or inactivating both lipid enveloped and non-lipid envel¬oped viruses. The aim of validating a production step as a virus reduction step is to provide evidence that the production process will effectively inactivate/remove viruses which are either known to contaminate the starting materials, or which could conceivably do so. Validation studies involve the deliberate addition of a virus prior to the production steps to be validated and measuring the extent of its removal/inactivation after the production step or steps. GMP restraints prevent the deliberate introduction of any virus into the production facilrties. Therefore, the validation should be conducted in a separate laboratory equipped for virological work on a scaled-down version of the production step and perfomied by staff with viroiogicaf expertise in conjunction with the production engineers. The amount of virus added to the starting material for the production step which is to be validated should be as high as possible in order to determine the capacity of the production step to inactivate/remove viruses adequately. However, the virus spike should be added such that the composition of the production materia! is not significantly altered. Preferably the volume of the virus spike will be equal to or less than 10%. Quantitative infectivity assays should be performed according to the principles of GLP and may involve plaque formation, detection of other cytopalhic effects such as syncytia or foci formation, end point titration (eg., TCID50 assays), detection of virus antigen synthesis or other methods. The method should have adequate sensitivity and reproducibility and should be performed with sufficient replicates and controls to ensure adequate statistical I accuracy of the results. Typically, a process step is challenged with 6 logs of virus, and if a reduction in the order of 4 logs or more is acquired, it is indicative of a clear effect with the particular test virus under investigation. Similarly, a reduction in the order of 4.5 logs, 5 logs, or even 5.5 logs, is > indicative of a clear effect Wflth the particular test vinjs under investigation, and the step can be classified as a validated virus reduction step The vims validation studies should be performed with viruses resembling those vi/htch may contaminate the product as closely as possible and secondly to represent as wide a range of physico-chemical properties as possible in order to test the ability of the system to eliminate viruses in general. The virus validation studies should t>e performed in accordance with the CPMP Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses (CPMP/BWP/268/95) and Note for Guidance on Plasma Derived Medicinal Products (CPMP/BWP/269/95). The validation studies of the present process are presented in example 5. The product of the invention is more than 95% pure, preferably above 98%. The high de¬gree of purity is, inter alia, due to the fact that the product of the invention is obtained by at least one, preferably two, optionally serially connected anion-cation exchange chromatog¬raphy steps. It is noteworthy in this context that it has been possible to obtain a high yield in spite of the number of process steps employed, in production scale of at least 3.5 g of IgG protein per kg of fresh frozen plasma. The comparative studies which have been carried out (Example 2) have shown that the immunoglobulin product obtainable by the process of the invention has ideal functional properties, such as prominent antigen binding activities and a high Fc function. The pres¬ently preferred medicament developed by the present Inventors is a 5% by weight immu¬noglobulin solution. Stability tests have so far indicated stability at 4'C storage for more than one year, i.e. that the immunoglobulin product is devoid of aggregate formation or fragmentation of immunoglobulins G, loss of the desired biofogical activity, or increase of undesired activities, e.g. anticomplementary activity and prekallikrein activity as measured in vitro. Based on the present invention, it is possible to obtain an IgG product that is more than 95%, such as at least 96%, or at least 97%, e.g. at least 98%, preferably at least 99%, more preferably at least 99.5% pure. The IgG product should contain less than 6 mg of IgA/l, such as less than 4 mg of IgA/l, preferably less than 3 mg of IgA/l, more preferably less than 2 mg of IgA/!. It should be stressed that other products contain stabilizers in the form of a detergent, PEG, or albumin. In a prefened embodiment, the product of the present invention does not contain any of said stabilizers, instead a well-tolerated saccharide has been chosen. The product of the present invention has, as one of its characteristics, 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, such as less than 1%, e.g. less than 0.5%, or less than 0.25% polymers and aggregates. The content of IgG monomers and dimers is at least 95%, such as at least 96%, or at least 97%, e.g. at least 98%, preferably at least 98,5%, or 99%. The content of monomehc IgG is at least 90% in the product. Trials have shown clinical effect of the product of the present invention comparable to reg¬istered IVIG products. The product has been well-tolerated by the patients, and the turnover time of the immunoglobulins in circulation has been determined to be four weeks. In the present trials, the immunomodulating effect of IVIG, SSI has been shown to be convincing (data are presented in example 3). The indications for IVIG are primary hypo/agammaglobulinaemia including common variable immunodeficiency. Wiskott-Aldrich syndrome and severe combined immunodeficiency (SCID), secondary hypo/agammaglobulinaemia in patients wJlh chronic lymphatic leukaemia (CLL) and multiple myeloma, children with AIDS and bacterial infections, acute and chronic idiopathic thrombocytopenic purpura (ITP), allogenic bone marrow transplantation (BMT), Kawasaki's disease, and Guillan-Barre's syndrome. Neurology: Chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motoric neuropathy, multiple sclerosis, Myasthenia Gravis, Eaton-Lambert's syndrome. Opticus Neuritis, epilepsy. Gynaecology: Abortus habltualis, primary antiphospholipid syndrome. Rheumatology; Rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, vasculitis, Wegner's granulomatosis, Sjogren's syndrome, juvenile rheumatoid arthritis. Haematology: Autoimmune neutropenia, autoimmune haemolyttc anaemia, neutropenia. Gastrointestinal: Crohn's disease, colitic ulcerous, coeliac disease. Others: Asthma, septic shock syndrome, chronic fatigue syndrome, psori^is, toxic shock -syndrome, diabetes, sinuitis, dilated cardiomyopathy, endocarditis, atherosclerosis, adults with AIDS and bactehal infections. Apart from the mentioned indications for treatment with IVIG products, several severe autoimmune diseases, which commonly respond to cortico-steroid and immunosuppressive therapy, are considered target conditions for therapy wifh the product of the present inven¬tion. Among these are several neurological diseases such as polyradiculitis, and some im¬mune-mediated peripheral polyneuropathies, but also some chronic inflammatory rheu-' matic and vascu}ar conditions such as systemic vasculitis involving small vessels, poly¬myositis, 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 of IgG metabolism. '} The invention is further illustrated by the following examples, which are not intended to be limiting. EXAMPLES Example 1 PROCESS STEPS IN THE PURIFICATION OF IMMUNOGLOBULIN (with the exception of step 5, all steps are carried out at StS^C) Step 1: Preparation of Cohn fraction II + III paste: Cohn fraction II + III paste is prepared from human plasma by the standard Cohn-frac-tionation 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). The ethanol precipitation is initiated after the cryoprecipitate has been removed and, if desired, after adsorption of certain plasma proteins (such as Factor IX and Antithrombin) to e.g. an ion exchange material and/or a Heparin Sepharose® matrix. The exact conditions (pH, ethanol concentration, temperature, protein concentration) for obtaining the fraction 11-111 paste appear from the figure at page 266 in Harns JR (ed), Blood Separation and Plasma Fractionation, Wiley-Liss, New York, 1991. The paste is isolated on a filter press by adding filter aid prior to filtration. Step 2; Extraclfon of jmmunoqlobulins from Cohn fraction 1) + II) paste: From 140 kg effraction II + III paste including 30 kg of filter aid (Schenk, Germany) (corre¬sponding to a starling volume of plasma of about 1150 kg), extraction is accomplished by first adding 525 kg of 2,33 mM sodium phosphate/acetate buffer, pH 4.0, with slow stirring for about 1.5 hours, followed by 2 consecutive additions of 350 kg of water for injection (WFI) with stirring for about 1.5 hours after each addition. Finally, about 280 kg of 21.5 mM sodium phosphate/acetate, pH 7.0, are added, thereby adjusting the pH of the suspension to 5.4. I The suspension is filtered through a depth filter (C-150AF, Schenk, Germany). The filtrate contains, among other proteins, the immunoglobulins: Step 3: Precipitation of protein aggregates and removal of virus by PEG 6000: PEG 6000 (Merck, Germany) is added to the filtrate of step 2 to a final concentration of 6% I by weight. After precipitation for 4 hours, the PEG suspension is centrifuged to clarity in a flow-through centrifuge (Westfaiia BKA28, Germany) and is depth filtered fSOLA and 90LA.. Cuno, France) and subsequently sterile filtered through a 0.22 ^m filter (Durapore, Milli¬pore, U.S.A.). The filtered PEG supernatant is buffer-adjusted by adding 1 part of a 0.45 M sodium acetate buffer, pH 5.7, to 29 parts of supernatant to reach a pH of 5.7, Step 4: Purification by serial anion and cation exchange chromatography (I): Two chromatography columns are packed with 56 I of DEAE Sepharose FF© {Pharmacia Biotech, Sweden) and 56 I of CM Sepharose FF® (Pharmacia Biotech. Sweden), respec¬tively. The columns are connected in series so that the liquid fiow first passes through the DEAE Sepharose resin and, subsequently, through the CM Sepharose resin. The column resins are equilibrated with 15 mM sodium acetate buffer, pH 5.7. Then, the solution from step 3 is applied to the two columns in series. During the ion exchange chromatography, most contaminating proteins in the applied solu¬tion bind to the DEAE Sepharose resin. Whereas IgG runs through without binding to the DEAE Sepharose resin, IgG binds to the CM Sepharose resin when the solution migrates through it. After application of the solution, and washing with one column volume of equili¬bration buffer, the DEAE column is disconnected from the CM column. Then the CM col¬umn is washed with three column volumes of 15 mM sodium acetate buffer, pH 5.4, then IgG is eluted with a gradient of NaCI from 125 mM to 350 mM NaCI, 15 mM sodium ace¬tate, pH 5.4. The eluted IgG fraction is collected in sorbitol to a final concentration of 2.5% by weight. Step 5: Solvent/detergent (S/D) treatment of the IgG fraction: The eluted IgG fraction is concentrated and desalted by ultra/diafiltralion to a concentration of approximately 50 g of IgG/litre. The employed membrane is a polysulfone membrane, nominal weight cutoff of 30 kDa (Millipore). The diafiltration is performed against a buffer of 15 mM sodium acetate, pH 5.4, containing 2.5% by weight of sorbitol and is continued unfil the conductivity is less than 1.4 mS/cm. The IgG content of the solution is determined spectrophotometrically by measuring at 280 nm (A2BO)- The sorbitol concentration is ad¬justed to 10% by weight and the solution is filtered through a 0.45 pm filter (Pall Corpora¬tion, UK). Tween 80 and TNBP are then added to a final concentration of 1 % and 0.3% by weight, respectively, for subsequent S^ treatment. The S/D treatment proceeds for at least 6 hours at 25'C. step 6: Removal of S/D by ion exchange chromatography (If): Two serially connected columns packed with 28 I of DEAE and 56 I of CM Sepharose FF, respectively, are equilibrated with 15 mM sodium acetate, pH 5A. The S/D-treafed IgG fraction from step 5 is diluted with 5 parts of 15 mM acetate buffer, pH 5.4, filtered through a depth filter (Cuno 90 LA) and subsequently sterile filtered (Sartobran, Sartorius), and ap¬plied to the two columns connected in series. The ion exchange chromatography and ^e subsequent elution of IgG from the CM column are carried out essentially as described in step 4, except that the CM column is extensively washed with 6 column volumes of buffer to remove agents from the S/D treatment. The eluted IgG fraction is collected in maltose (Merck, Germany) to a final concentration of 2-5% by weight. Step 7: Final concentration and formulation of immunoglobulin for intravenous use: The eluted IgG fraction from step 5 is subjected to ultrafiltration and desalting by diafiltra-tion against 7.5 mM sodium acetate containing 2.5 % by weight of maltose, pH 5.4 to a fi¬nal conductivity of less than 1 mS/cm. The employed membrane is a polysutfone mem¬brane with a 100 kDa nominal weight cutoff allowing proteins with lower molecular weight to be eliminated. The final concentration of IgG is adjusted to 50 g/Iitre, and the maltose is adjusted to a final concentration of 10% (w/v). The maltose-adjusted final preparation is filtered through a sterile filter (Sartopure GF 2, Sartorius), and filled asepticalty. Example 2 ~ Purity (protein composition) Phamiacopoeia purity requirements for an iVIG-preparation is at least 95% IgG, that is not more than 5% non-lgG-contaminating proteins present. Purity is regarded as being of very high importance for several reasons. From a rational point of view, only the protein w^hich carries the desired function should be present, and other contaminating proteins may be potentially harmful, e.g. cause unwanted adverse effects and/or influence the stability of the product. Purity can e.g. be analyzed by an electrophoretic technique as described in detail in Ph. Eur., 1997, pages 964-965, where proteins are separated in a cellulose acetate gel. For practical purposes, however, an agarose gel is used. After electrophoresis, ttie gel is fixed, dried, and stained. Protein bands are finally monitored by scanning. If appears fi-om the table above that the product of the invenlion is virtually pure (99.8%). Albumin The albumin content was analyzed by crossed immuno-electrophoresis essentially as de¬scribed by C.B. Laurell (Anal Biochem (1965), 10, 358-361). 5 p] of product was analyzed against anti-human albumin antibodies (DAKO A/S, Denmark, No. A0001 (1/100)). Due to the high purity no albumin was detectable in the analyzed product of the invention. I Content of IgG monomers and dimers The content of IgG monomers and dimers can be analyzed by get permeation chromatog¬raphy, and monitored from the chromatogram by integration of the areas of the monomer and of the dimer peak, cf. Ph.Eur. The results of the various analyses are listed In the table > above from which it appears that the sum of the monomer + dimer areas constitutes 99.3% of the total area of the chromatogram (from this monomeric IgG constitutes 92%) for the product of the invention. Content of polymers and aggregates 3 The presence of polymers and aggregates is known to be the cause of serious adverse effects, often influenza-like symptoms. Because of the very high degree of purity reached by the rather gentle production process, the immunoglobulin product obtainable by the pro¬cess of the invention is largely free of polymers and aggregates. olymers can be analyzed by gel permeation chromatography, and any"prolein peaks with jtention times shorter than the retention time for dimeric IgG are considered polymeric as escribed in Ph.Eur. iccording to PhEur. and other guidelines, the content of protein aggregates should pref-rably be less than 3%. The product of the present process contains no measurable aggre-lates and is thus considered to contain less than 0.1% polymers and aggregates. ^nti-complementarv activity (ACA) and prekallikrein activator activity (PKA) \CA and PKA are measured as described in Ph.Eur. \CA should preferably be as low as possible. According to Ph.Eur. the complement con¬sumption should be less than or equal to 50%. The complement consumption of the meas¬ured sample of the product of the invention is about 30%, i.e. comparable to that of the other products analyzed. It should be noted thai the presence of albumin tends to suppress complement consumption (inventor's observation). PKA, if present in substantial amounts, is essential for the hypotensive adverse effect of the product. Therefore, PKA should preferably be as low as possible in an immunoglobulin product. According to Ph.Eur. it should be Haemaqqlutinins The IgM fraction of plasma immunoglobulins comprises the haemagglutinins, that is anti¬bodies against blood type A and B antigens. The presence of such antibodi'es may cause unwanted adverse effects due to a possible haemolylic reaction if the recipient carries blood types A and/or B. According to Pharmacopoeia requirements, the content of haemagglutinins must be lower than that causing agglutination of A/B erythrocytes in a dilution 1:64 of the immunoglobulin product. All the products analyzed fulfil this requirement. Fc-functiori tetained antigen binding activities are essential for (he biological functions of the IVIG. "his is also true for the immunomodulating activities. On the other hand, a retained Fc-unction is essential for the effect of IVIG on various phagocytic cells and activation of the ;omplement system. Fc-function can be demonstrated using various techniques, but an accepted methodology described in Ph. Eur. measures the complement-activating potential 3f antibodies in the preparation against rubella antigen. Activity is compared to that of a Diological reference preparation (BRP, Ph.Eur.) of immunoglobulins set to be 100%. The product complies with the test if the relative activity is more than 60% of the reference preparaljon, II appears that the Fc-function of the product of the invention is very well pre¬served, particularly in comparison with the other liquid product 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 as described by A. Ingild (Scand J Immunol, (1983), 17, 41). The con¬centrations are determined by use of a WHO reference serum (67/97). It is required that the subclass distribution should be within the range of normal human plasma with median concentrations in the range of 3,7-10.2 glgG1/l serum, 1.1 - 5.9 g lgG2/l serum, 0.15 - 1.3 g lgG3/l serum, and 0.06 - 19 g JgG4/l serum (R. Djurup et a), Scand J. Clin Lab Invest 48, 77-83). Thus, the subclass distribution of all the products is acceptable. IqA-content The presence of IgA is known to potentially cause sensibilisation of IgA-deftcient recipients. If an IgA-deficient patient receives an IgA-containing immunoglobulin preparation, IgA may be considered as a foreign antigen, and the result may be the induction of antibodies against IgA in the recipient. The next time an IgA-containing preparation is infused to the patient, an anaphylactic reaction may be provoked. It is therefore essential that an immu¬noglobulin preparation contains as little IgA as possible. IgA in an IVIG product can be monitored using an ELISA-technique, e.g; where a polyclonal anti-igA is used to capture IgA, and a labelled anti-lgA is used for the detection of bound IgA. Standards are con¬structed 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 nrg of IgA/l which is a considerably lower IgA-content than that of the other analyzed liquid product. The physico-chemical similarities behween IgG and IgA make it difficult to separate these im¬munoglobulins during a purification process. However, the two anion/calion exchange chro¬matography steps in the process reduce the IgA-content to a very low level. IqM-content IgM in an Ig-preparation can be monitored using an ELISA-technique, e.g. where a poly-clona) anti-lgM is used to capture IgM, and a labelled anti-IgM is used for detection. Stan¬dards are constructed by dilutions of a calibrator (No. X908, DAKO A/S, Denmark) with a declared IgM-content. It appears from the table that the tgM-content of the product of the invention is very low and markedly lower than that of the other liquid product. Tween 80. TNBP and PEG Tween 80, TNBP and PEG are measured by standard procedures. In general, the content of these additives should be as low as possible. eH pH of the analyzed liquid products is acidic, pH 5.6-5.7, whereas the analyzed lyophiiized products are neutral after dissolution, with a pH of 6.7. Total protein concentration According to Ph.Eur. the protein concentration should be at least 50 g/l ±10%; all the prod¬ucts fulfil (his requirement. The protein concentration is measured by the method of Kjel-dahl. Maltose and glucose stabilizers Saccharides are commonly used stabilizers of immunoglobulin products, they have good stabilizing properties and are quickly excreted. The content of maltose, sucrose, and glu¬cose is determined by use of a commercial kit (Boehringer Mannheim, Germany) with maltose as a reference. It appears that the two lyophiiized products stabilized by albumin and albumin as well as PEG, respectively, also contain a saccharide stabilizer in concentrations from about 15 mg/mi to 20 mg/ml. The product of the invention and the other liquid product are very equally stabilized, i.e. with about 9%, 88 mg/ml and 92 mg/ml, of maltose, ^y regarding the content of polymers and aggregates as a parameter of stability, the product of the invention has a higher stability than the other liquid product analyzed, although their formulations ap¬pear very similar. Example 3 RESULTS FROM CLINICAL TRIALS The clinical studies of the product of the present invention, also referred to as IVIG, SSI, are carried out in accordance with ICH and CPMP/388/95 guidelines. Pharmacokinetics, effect and safety have been examined. The clinical trials have so far included four groups of patients; patients with primary immunodeficiency syndrome (15 pa¬tients), secondary immunodeficiency syndrome (6 patients), idiopathic thrombocytopenic purpura (15 patients) and patients with chronic inflammatory demyelinating polyneuropa-thia (5 patients). Patients with primary immunodeficiency syndrome or secondary immunodeficiency syndrome were treated with 0.2-0.4 g/kg with intervals of 2-5 weeks. Patients with idiopathic thrombocytopenic purpura were treated with 400 mg/kg per day for five days or with 1000 mg/kg per day for two days. For safety measures serum-transaminases, senjm-creatinine and virus markers have been determined in ail patients. Five patients with idiopathic thrombocytopenic purpura have been followed for virus, kidney and liver safety markes for up to a total of 24 weeks. Pharmacokinetics J'A was measured to 30,5 days (median). This is in accordance with results of other IVIG medicaments. Effect For patients with primary and secondary immunodeficiency syndrome, days lost through sickness, hospitalisations, days with antibiotics, days with fever and the number of pneu¬monias were registered retrospectively for a 6-month period during which the patients had jeen treated with other registered IVIG medicaments. In the following 6 rfTonths during ivhich the patients were treated with Immunoglobulin SSI, liquid, the same parameters were registered. rhe conctusion is that Immunoglobulin SSI, liquid is just as effective as other IVIG compo¬sitions 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 raised from 50 x 10^/L after the treatment. The increase in the platelet count and the duration of the remission in the individual patient were on the same level as after administration of the same dose of other IVIG medicaments, in the cases where comparison was possible. One patient receiving IVIG for the first time was refractory to the test drug. Such a reaction to IVIG is not infre¬quent, and thus not surprising. Details of the rise of platelets and the duraiion of the rise are under way. The conclusion is that Immunoglobulin SSI, liquid is just as effective as other IVIG me¬dicaments in the treatment of low platelet count in patients with chronic idiopathic throm-bogenic purpura. According to clinicians, and patients suffering from chronic inflammatory demyelinattng polyneuropalhia, the IVIG, SSI has shown identical efficacy to the IVIG administrered prior to the trial. IVIG, SSl was tolerated by the patients equally well as other IVIG products were tolerated by the patients. Safely Apart from one severe adverse event, splenectomia assessed by the investigator to have no relation to test drug, only minor adverse events have been registered. These adverse effects were mainly headache fever, and vomiting. So far, there have been no reports on abnormal vital signs during infusions of IVIG, SS!. No viral seroconversions have been registered. There have been no reports on kidney or liver damages or cases of anaphylactic shocks. The clinical studies show that Immunoglobulin SSI, liquid is well tolerated. The frequency of ' side effects, degree and species does not deviate from experiences with other IVIG me¬dicaments. Example A RESULTS FROM STABILITY STUDY FOR IVIG LIQUID In order to lest if the liquid IVIG product is stable over time, a Real time Real conditions study of stability was conducted. A total of 4 consecutive batches (250 ml of each sample) of the IVIG product were involved in the study and stored at between 2°C - B°C for at least 12 months. Samples from the four batches were analyzed at time zero, 6 month at storage and 12 months at storage. The results of the study are presented below as means of 4 batches. M\ the above mentioned tests were carried out in accordance with Ph.EuTI and as de¬scribed in Example 2. The observation that the content of monomers and dimers is constant over a period of 12 months indicates that polymers are not formed in the sample. The presence of immunoglobulin polymers is l^nown, among others, to be the cause of serious adverse effects, often influenza-like symptoms. Because of the very high stability of the immunoglobulin product obtainable by the process of the invention, the product is largely free of polymers and aggregates even after a long period of storage. No increase in ACA is observed over time, although batches expressing rather high ACA deliberately have been included in this stability study. If an increase in ACA was observed, it might indicate that aggregates were being formed during storage. Thus, the constant ACA over time indicates that no aggregates are being formed. The results further indicate that no prekallikrein activator activity has developed during storage of the product, as the PKA activity does not increase. It should be noted, however, that the values measured are below the lower quantification level. The measure of Fc-funclion indicates that the presence of intact functional IgG is main¬tained during storage. Thus, no proteases are present in the samples, as they would have degraded the proteins and thereby lowered the Fc-function. Denaturafion of IgG molecules has neither taken place as this would have decreased antigen binding activity. As it will be known by the person skilled in the art, there might be difference in the stability of the various subclasses of IgG. As can be seen from the present results, all subclasses are maintained during storage indicating that the product is stable. This is further supported by the finding that the protein composition of IgG in the samples with approximately the same total protein concentration is almost unchanged overtime, indicating that there is no overall degradation of IgG. I.e. the product of the present invention is stable and can be stored at least for 12 months at 2-8°C without significant changes of characteristics, and by this efficacy and safety is demonstrated. Example 5 VALIDATED VIRUS REDUCTION STEPS IN THE PRESENT PROCESS OF IVIG VIRUS REMOVAL BY A PARTITIONING STEP Precipitation of virus present in the immunogloblulin solution by polyethylene glycol Virus validation studies have been performed employing two small non-enveloped viruses, the following virus reductions were obtained: removal of 6.3 togio of Hepatitis A Virus (HAV) removal of 7.2 logio of Polio Virus Virus validation studies have been performed employing two enveloped viruses, the following virus reductions were obtained: removal of 7.6 logic of HIV removal of 7.5 logio of BVDV VIRUS INACTIVATION BY A S/D TREATMENT STEP Treatment of the immunogtobulin solution with 1 % Tween 80 + 0.3% TNBP, at 25'C for > 6 hours. Virus validation studies have been performed employing four enveloped viruses, the following virus reductions were obtained: inactivation of 7.4 logn, of HIV inactivation of 5.3 logm of Sindbis Virus inactivation of 4.1 log,o of BVDV inactivation of 5.1 logio of PRV A total of 8 validation studies have been performed on two different steps in the process of the present invention. The PEG precipitation step has been validated as a virus removal step employing four different viruses, two small non-enveloped viruses HAV and Polio virus, and two enveloped viruses HiV and BVDV as model for Hepatitis C Virus. These studies showed that all four viruses were efTiciently removed by PEG precipitation. The PEG precipitation step is Ihierefore validated as an efficient vims removal step. The S/D treatment has validated employing four different enveloped viruses. From the data of the validation studies it appears that the S/D treatment step efficiently inactivated all four viruses. The S/D treatment step is therefore validated as an efficient virus inactivation step. Both vims reduction steps in the IVIG process, removal by PEG precipitation and inactivation by S/D treatment, have been validated efficiently to remove and inactivate four different viruses each. The cumulative reduction factors of HIV and BVDVIn the process are 15 and 11.6, respectively. By this the product of the present process can be regarded as virus safe. CLAIMS 1. A process for purifying immuncgkA>urin G (IgG), from a cajde immunoglobuSin-containirtg plasma protein fraction, which process comprises the steps of: (a) preparing an aqueous suspension of the caitJe immunoglobulin-containing plasma protein fraction; (b) adding a water soluble, substantially non-denaturating protein precipitant lo the said I suspension of step (a) in an amour^l sufficient to cause precipitation of a high proportion of non-immunoglobulin G proteins, aggregated immunogloburins and particles including potentiatty Infecttoua particles such as virus particles, without causing substantial precipitation of monomeric immunoglobulin G, thereby forming a mixture of a solid precipitate and a liquid supemalani; (c) recovering a clarified immunoglobuiin G-conlaining supernatant from the mixture of step (b): (d) applying the clarified immunoglobulin G-containing supernatant of step (c) to an arv 1 . ion exchange resin and subsequenyy a cation exchange lestn, wherein the anion exchange resin and the cation exchange resin are connected in series and wherein the buffer used for ths anion exchange chromatography and the cation exchange chromatography is the same buffer, the pH of said same buffer is below 6.0. (e) washing out protein contaminants and the protein precipitant from the cation ex¬change resin of step (d) with a buffer having a pH and ionic strength sufficient to re- / move Ihe contaminants from the resin without causing substantial elulion of immunoglobulin G; (f) eluting immunoglc^ulin G from the cation exchange resin of step (e) with a sutwtan-tially non-denaturating buffer having a pH and ionic strength suflident to cause effi- ^ dent elution of the immunoglobulin G, thereby recovering an immunoglobulin G-containing eluate; (g) performing a dia/ultrafiftration on ttie immunoglobulin G-conlaining eluate of step (f) to concentrate and/or diatyse the eluats, and optionally adding a stabilizing agent; (h) adding a virucidal amount of virus-inactivating agent to the immunoglobulin G-con-taining dia/ultrafritrated and opfionally stabilized fraction of step (g) resulting in a substantially vinjs-safe immunoglobulin G-containing solution; (i) applying the immunoglobutin G-containjng solution of step (h) to an anion exciiange resin and subsequently to a cation exchange resin; (j) washing the cation exchange resin of step (1) with a buffer having a pH and ionic V strength sufficient to wash out the protein contaminants and the virus-inactivating agent from the resin withoU causing substantial etution of immunoglobulin G; (k) eluling immunoglobulin G from the cation exchange resin of step (j) with a sijbstan-tially non-denaluralitv] bt ifferJiaviBQ a pH and ionic strer^gth sufficient to cause effi¬cient elution of the immunoglobulin G, ihereby recovering an immunogloburLT G-conlaining eluate; and (l) subjecting Ilia immunoglobulin G-containing eluate of step (k) to dta/utlrafiltralion to lower the ionic strength and concentrate immunoglobulin G of the solution, and ad- justing the osmolality by adding a saccharide. 2. A process according to claim 1, wherein the immunoglobulin G-containing plasma pro¬tein fraction is selected from the group consisting of Cohn fraction It; Cohn fractions II and III; and Cohn fractions I. II and III. 3. A process according to claim 1 or 2. v^hsrein the suspension in step (a) !s maintained at a temperature from (TC to 12°C and the suspension in step (a) is maintained at a pH below 6. 4. A process according to any of the preceding claims, wherein the protein precipitani in step (b) is selected from the group consisting of polyethylene glycol- (PEG), caprylic acid, and ammonium sulphate. 5. A process according to daim 4. wher«h the protein predpilant is selected from Iha group consisting of PEG within the molecular weight range 3000-8000 Da, such as PEG 3350. PEG 4000, PEG 5000, and PEG 6000. 6. A process according to any of the preceding claims, wherein the aruon exchange resin and the cation exchange rosin m step (i) are connected in series. (7-yv process according to claim 6, wherein the buffer used for the anion exchange chro-fTiatography and the cation exchange chromatography is the same buffer, and the pH of said same buffer is below 6.0. 8. A process according to any of the preceding claiins. wherein the anion exchange reS'n in step (d) and/or step (t) contains diethylaminoethyf groups and/or the cation exchange resin in step (d) and/or step (i) contains carboxymethyl groups, the resins preferably being DEAE Sepharose FF® and CM Sepharose FF®. 9. A process according to any of Ihe preceding claims, whorein Ihe buffer used throughout steps (b) to (!) is an acetate buffer, such as an acetate buffer with a pH of 5.0-6.0 and having a molarity of 5-25 mM. 10. A pnDcess according to any of the preceding daims, wherein the virus-inactivating agent in step (h) is a mixture DI al least one norvionic or ionic detergent and at least one solvent. 11. A process according to any of the preceding claims, wherein the virus-inactivating agent in step (h) is a mixture of at least one subslantially non-denaturating detergent and at least one tri{lower aikyi) phosphate solvent. (\2Jkn immur)oglobuHn_£roduct^which is obtainable by the process according to any of claims 1-11. 13,1 An immunoglobulin product which is obtainable by the process according to any of rlaims 6-8. /■ -I 14. An immunoglobulin product having the following characterietics: a) a purity of more than 98%, bl a content of IgG monomers and dimers of more than 98.5%, c) a content of IgA less than 4 mg of IgA/l, and d) a content of !gG1, lgG2. lgG3 and I9G4. 'i^jM immunt>globuiin product according I0 claim 14 which does not comprise detergent, ~^£G or altjumin as a stab^iizer. [ 'lO.Wn immunoglobulin product according to claim 14 or 15 which contains less than 3 n^g/llgA. , IJt An immunoglobuVm product according to any of claims 14-16 which contains between 55 and 65% IgGI, between 30 and 40% lgG2, between 2 and 5% lgG3, and between 1 and 4% lgG4. r^/ )An immunoglobulin product according to any of claims 14-17 which contains less than 0,5% polymers and aggregates. 19. A liquid immunoglobulin product according to any of claims 14-18. 20. An immunoglobulin product according lo any of claims 14-19 for instant intravenous adm'mtBtration. 21. An immunoglobulin product according lo any of daims 14-20 for use tn medicine. 22. Use of an immunoglobulin product according to any of clainis 14-21 for the preparation of a medicament for tfia treatment of a mamma! with PID (Primary Immune Deficiency), S)D (Secondary Immune Deficiency), ITP (Idiopathic Thrombocytopenic Purpura), potyradiculilis, peripheral polyneuropathies. KawasakPs disease, polymyositis, severe chronic autoimmune diseases. Chronic infjannmatory demyelinating polyneuropathy (CIDP), multifocal motoric neuropathy, multiple sclerosis. Myasthenia Giaivis, Eaton-Lamtjert's syndrome, Opticus Meuritis, epilepsy. Abortus habitualis. primary antiphospholipHd syndrome. Rheumatoid arthritis, systemic lupus erythematosus, systemic sderodemna, vasculitis, Wegner's granulomatosis, Sjogren's syndrome, juvenile rheumatoid arthritis. Autoimmune neutropenia, autoimmune haemolytlc anaemia, rieutrapenia, Crohn's disease, colHic ulcerous, coeliac disease. Asthma, septic shock syndrome, chronic fatigue syndrome, psoriasis, toxic shock syndrome, diiabetes, stnuitis, dilated cardiomyopathy, endocarditis, atherosclerosis, and adults wth AIDS and bacterial infections. 23. Us9 according to claim 22, wherein the mammal is a human being. 24. A process for puritying immunoglobulin G substantially as hereinbefore described. 25. An immunoglobulin product substantially as hereinbefore described.

Documents:

in-pct-2000-0799-che abstract-duplicate.pdf

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in-pct-2000-0799-che claims.pdf

in-pct-2000-0799-che correspondence-others.pdf

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in-pct-2000-0799-che description(complete)-duplicate.pdf

in-pct-2000-0799-che description(complete).pdf

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in-pct-2000-0799-che form-26.pdf

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in-pct-2000-0799-che pct search report.pdf

in-pct-2000-0799-che pct.pdf

in-pct-2000-0799-che petition.pdf