Process for producing immunoglobulins for intravenous administration and other immunoglobulin products

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.

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 crude plasma
 protein fraction. The invention also relates to an immunoglobulin product
 and to the use of such an immunoglobulin product for medical purposes.
 BACKGROUND OF THE INVENTION
 Human normal 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 virus infection when administered subcutaneously or
 intramuscularly.
 Congenital or acquired total or partial lack of immunoglobulin (primary and
 secondary immunodeficiency syndrome, respectively) manifests itself
 through frequent ordinary and serious infections, especially of a
 bacterial nature. The prevention of such infections was previously
 achieved by repeated intramuscular or subcutaneous 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 immunoglobulin 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 immunoglobulins which, among other effects, strongly
 activated the complement system. This was in particular seen in patients
 lacking immunoglobulins. Especially serious adverse effects of an
 anaphylactic nature could be seen in patients who developed antibodies to
 IgA. Consequently, methods of avoiding aggregate formation and/or
 eliminating these aggregates 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 congenital 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 sufficiently 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 disappearance of the blood platelets on an
 immunological basis, idiopathic thrombocytopenic purpura (ITP), in some
 rare diseases such as Kawasaki's syndrome and a number of autoimmune
 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 IgG,
 e.g. a blockage of Fc.gamma.-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 material (Cohn fraction II), 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
 immediately prior to use.
 The starting material for the IVIG was HNI which had 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 HNI
 showed that the removal of virus in the fractionation process from plasma
 to HNI is modest. The safety of HNI for intramuscular use is likely to be
 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 can neutralize and render
 common viruses in plasma non-infectious. Especially when large doses of
 immunoglobulin are given intravenously, virus infections may occur as
 demonstrated in the early 1990's. Therefore, it was recognized 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 molecules 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 market for IVIG. The
 first and second generations of IVIG thus appear as freeze-dried powders
 which are dissolved immediately prior to use.
 Dissolution of freeze-dried IVIG is slow (up to 30 minutes for one vial).
 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 obtain 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 immunoglobulin product for intravenous administration which can be
 characterized as a highly purified, fully native, biologically active,
 double virus-inactivated, and stable new generation of IVIG, which does
 not contain any detergent, polyethylene glycol (PEG) or albumin as a
 stabilizer.
 SUMMARY OF THE INVENTION
 The present invention relates to an improved 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 precipitation (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
 introduced 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 during 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 effects, 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 suspension of step (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 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 anion exchange resin and subsequently a cation exchange resin;
 (e) washing out protein contaminants and the protein precipitant from the
 cation exchange resin with a buffer having a pH and ionic strength
 sufficient to remove the contaminants from the resin without causing
 substantial elution of immunoglobulin 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) performing a dia/ultrafiltration 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-containing dia/ultrafiltrated and optionally stabilized
 fraction of step (g) resulting in a substantially virus-safe
 immunoglobulin G-containing solution;
 (i) applying the immunoglobulin G-containing solution of step (h) to an
 anion exchange resin and subsequently to a cation exchange resin;
 (j) 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 substantially non-denaturating buffer having a pH and ionic
 strength sufficient to cause efficient elution of the immunoglobulin G,
 thereby recovering an immunoglobulin G-containing eluate; and
 (l) subjecting the immunoglobulin G-containing eluate of step (k) to
 dia/ultrafiltration 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 advantageously an immunoglobulin-containing crude plasma
 protein fraction. The starting material 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
 specification, the term "immunoglobulin-containing plasma fraction" is to
 encompass all possible starting materials for the present process, e.g.
 cryoprecipitate-free plasma or cryoprecipitate-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 al., (1964), Biochem Biophys
 Acta, 82, 463-475; Poison and Ruiz-Bravo, (1972) Vox Sang, 23, 107-118) or
 by ammonium sulphate. In a preferred embodiment, the plasma protein
 fraction is Cohn fractions II and III, but Cohn fraction II, or Cohn
 fractions I, 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. fibrinogen,
 .alpha.-globulins and .beta.-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 suspension of an immunoglobulin-containing plasma protein
 fraction, wherein the IgG concentration in the suspension is sufficiently
 high so that, during the following precipitation step, a major proportion
 of the non-IgG-proteins, especially those of higher molecular weight, the
 aggregated immunoglobulins and other aggregated proteins as well as
 potentially infectious particles precipitate without substantial
 precipitation of monomeric IgG. This is generally achieved if the
 concentration of the IgG in the buffered and filtered suspension is at
 least about 4 g/l 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 suspension 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 substantially non-denaturating temperature and pH. The
 term "substantially non-denaturating" implies that the condition to which
 the term refers does not cause substantial 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 optimal effect of the subsequent PEG
 precipitation 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, HCI. Persons skilled
 in the art will appreciate that numerous other buffers can be used.
 The immunoglobulin suspension is preferably maintained at a cold
 temperature, inter alia in order to prevent substantial protein
 denaturation 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.degree. C., preferably
 0-8.degree. C., most preferably 1-4.degree. C.
 The suspension of an ethanol precipitated paste contains relatively large
 amounts of aggregated protein material. 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 precipitation of a high proportion of
 high molecular weight proteins, lipoproteins, aggregated proteins, among
 these aggregated immunoglobulins. Other particulate material, such as
 potentially infectious particles, e.g. virus particles, are also
 precipitated without causing substantial precipitation of monomeric IgG.
 The term "infectious particles" in the present context comprises e.g.
 virus particles (such as hepatitis viruses, HIV1 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 purification 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 precipitation. The term "adding a protein
 precipitant" and variants of that term implies the addition 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 molecular 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
 stabilizer 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 substantial 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 lower 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 precipitant 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
 preferred. In the precipitation step, the precipitation process is allowed
 to proceed at least until equilibrium 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.degree. C., such as less than about 10.degree.
 C., preferably between 2.degree. C. and 8.degree. C.). The most suitable
 temperature depends on the identity of the protein precipitant.
 After completion of the protein precipitation, 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 conventional techniques
 for separating liquid from solid phase, such as centrifugation and/or
 filtration. 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 .mu.m 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 chromatography in order to remove a
 substantial proportion of the remaining non-IgG contaminants, 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 dimensions.
 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, albumin, 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 substantially 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 series", 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 cation
 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 compositions. 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-containing fraction between the two ion exchange chromatographic
 methods, for possibly adjusting 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
 contemplated that it is also possible to perform 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 monomers 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 be
 based on various materials with respect to the matrix as well as to the
 attached charged groups. For example, the following matrices may be used,
 in which the materials mentioned may be more or less crosslinked: agarose
 based (such as Sepharose CL-6B.RTM., Sepharose Fast Flow.RTM. and
 Sepharose High Performance.RTM.), cellulose based (such as DEAE
 Sephacel.RTM.), dextran based (such as Sephadex.RTM.), silica based and
 synthetic polymer based. For the anion exchange resin, the charged groups
 which are covalently attached to the matrix may e.g. be diethylaminoethyl
 (DEAE), quaternary aminoethyl (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.RTM., but other anion exchangers can be used. A preferred cation
 exchange resin is CM Sepharose Fast Flow.RTM., 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 solution 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 anion 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.RTM.
 and the cation exchange resin CM Sepharose FF.RTM. 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 art will appreciate 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 the
 equilibration of the anion exchange column and cation exchange column when
 connected 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 1.2
 mS/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 adjusted, 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 order 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 preferably washed in order to remove protein contaminants from the
 resin with a buffer having a pH and ionic strength sufficient to elute
 substantially all of the contaminants from the cation exchange resin
 without causing substantial elution of IgG.
 The initial washing is advantageously performed by using the equilibration
 buffer, even though other buffers with a similar concentration and
 pH-value may be used for the washing. It is preferred that an acetate
 buffer is used for washing out contaminants from the cation 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 elution of the IgG from the cation exchange resin is preferably
 performed with a substantially non-denaturating buffer having a pH and
 ionic strength sufficient to cause efficient elution of the IgG, thereby
 recovering an IgG-containing eluate. In this context, efficient elution
 means that at least 75%, such as at least 80%, e.g. at least 85%, of the
 IgG proteins 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 elution 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 advantageously 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 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 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 will 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, glucose,
 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-containing 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 desalinated (i.e. dialysed) and advantageously concentrated.
 The change of buffer and the concentration of IgG can be performed by a
 combined dia/ultrafiltration process. The term "dia/ultrafiltration" means
 that the dialysis and concentration by diafiltration and ultrafiltration,
 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 unnecessary loss of the product, it is
 presently preferred to perform the dialysis and concentration by the
 methods of diafiltration and ultrafiltration in one step.
 The membranes employed for the dia/ultrafiltration advantageously have a
 nominal weight cutoff within the range of 10,000-100,000 Da. A preferred
 membrane type for the present process is a polysulfone 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/l, preferably to about
 50 g/l. Following 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% (w/v) sorbitol. The exchange of
 buffer is continued until the conductivity of the ultrafiltrated solution
 is reduced to a value less than about 1.5 mS/cm, preferably less than
 about 1.3 mS/cm. During the dia/ultrafiltration, 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 filter with
 a pore diameter within the range of 0.2-1.0 .mu.m, preferably about 0.45
 .mu.m, 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 removal and inactivation steps are presently incorporated,
 these steps preferably being precipitation with PEG as a general
 virus-removal step and an S/D treatment as a virus-inactivating step
 towards lipid enveloped viruses. Besides the stringent requirements to
 virus safety of the starting material, according to international
 guidelines, and the well known virus reducing capacity of a multistep
 purification process, the incorporation of two independent virus reduction
 steps being active against both enveloped and non-enveloped viruses, 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-inactivating
 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
 "virucidal amount" will depend on the virus-inactivating agent employed as
 well as the conditions such as incubation time, pH, temperature, content
 of lipids, and protein concentration.
 The term "virus-inactivating agent" is intended to denote such an agent or
 a method which can be 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 virus-inactivating agents are detergents and/or solvents, most
 preferably detergent-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-denaturing. Preferably,
 a non-ionic detergent is used as it facilitates the subsequent elimination
 of the detergent from the IgG preparation by the subsequent step. Suitable
 detergents are described, e.g. by Shanbrom et al., in U.S. Pat. No.
 4,314,997, and U.S. Pat. No. 4,315,919. Preferred detergents are those
 sold under the trademarks Triton X-100 and Tween 80. Preferred solvents
 for use in virus-inactivating agents are di- or trialkylphosphates as
 described e.g. by Neurath and Horowitz in U.S. Pat. No. 4,764,369. A
 preferred solvent is tri(n-butyl)phosphate (TNBP). An especially preferred
 virus-inactivating agent 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 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 concentration 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-inactivation 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.degree. C., such as 19-28.degree. C., 23-27.degree. C., preferably
 about 25.degree. 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 employed to produce a virus-safe product, such as the addition
 of methylene blue with subsequent inactivation 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 preferably 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 concentration within the range of 1-5%, preferably about 2.5% by
 weight.
 The preferred method of eliminating the virus-inactivating agent is by ion
 exchange chromatography. However, other methods, such as oil extraction
 and alternative chromatographic 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 dialysed and concentrated; hereby the content of remaining
 smaller protein components is also effectively reduced. Advantageously,
 this can be performed by dia/ultrafiltration as described previously. The
 buffer employed for the diafiltration is sodium acetate, 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 buffers such as sodium phosphate or acids can be
 used for the diafiltration. The diafiltration continues until the
 conductivity is less than or equal to 1 mS/cm. Optionally, the
 IgG-containing 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, subcutaneously, or intramuscularly.
 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/l), 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 concentration 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 advantageous 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 latter 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 immunoglobulin 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 described previously. In addition to sugar alcohols and
 saccharides (such as sorbitol, mannose, 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 term "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 alternatively
 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 IgA 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 dimers and has a
 low anticomplementary activity, and it has a very high content of IgG
 monomers and dimers. Monomeric IgG constitues 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-reduction steps aimed at removing and/or
 inactivating both lipid enveloped and non-lipid enveloped 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 facilities. Therefore, the
 validation should be conducted in a separate laboratory equipped for
 virological work on a scaled-down version of the production step and
 performed by staff with virological 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 material 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
 cytopathic effects such as syncytia or foci formation, end point titration
 (eg., TCID.sub.50 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 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 with the particular test virus under
 investigation, and the step can be classified as a validated virus
 reduction step
 The virus validation studies should be performed with viruses resembling
 those which 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 be 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 degree 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 chromatography 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 presently preferred medicament
 developed by the present inventors is a 5% by weight immunoglobulin
 solution. Stability tests have so far indicated stability at 4.degree. 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 biological 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/I, such as less than
 4 mg of IgA/I, preferably less than 3 mg of IgA/I, more preferably less
 than 2 mg of IgA/I.
 It should be stressed that other products contain stabilizers in the form
 of a 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 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 monomeric IgG is at
 least 90% in the product.
 Trials have shown clinical effect of the product of the present invention
 comparable to registered 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 with 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 habitualis, primary antiphospholipid syndrome.
 Rheumatology: Rheumatoid arthritis, systemic lupus erythematosus, systemic
 scleroderma, vasculitis, Wegner's granulomatosis, Sjobgren's syndrome,
 juvenile rheumatoid arthritis.
 Haematology: Autoimmune neutropenia, autoimmune haemolytic anaemia,
 neutropenia.
 Gastrointestinal: Crohn's disease, colitic ulcerous, coeliac disease.
 Others: Asthma, septic shock syndrome, chronic fatigue syndrome, psoriasis,
 toxic shock syndrome, diabetes, sinuitis, dilated cardiomyopathy,
 endocarditis, atherosclerosis, adults with AIDS and bacterial 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 with the product of the present invention. Among
 these are several neurological diseases such as polyradiculitis, and some
 immune-mediated peripheral polyneuropathies, but also some chronic
 inflammatory rheumatic and vascular conditions 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 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 Purficiation of Immunoglobulin (with the exception of
 step 5, all steps are carried out at 5.+-.3.degree. C.)
 Step 1: Preparation of Cohn Fraction II+III Paste
 Cohn fraction II+III 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). 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.RTM. matrix.
 The exact conditions (pH, ethanol concentration, temperature, protein
 concentration) for obtaining the fraction II-III 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: Extraction of Immunoglobulins from Cohn Fraction II+Ill Paste
 From 140 kg of fraction II+III paste including 30 kg of filter aid (Schenk,
 Germany) (corresponding to a starting 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.
 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% by weight. After precipitation for 4 hours, the PEG
 suspension is centrifuged to clarity in a flow-through centrifuge
 (Westfalia BKA28, Germany) and is depth filtered (50LA and 90LA, Cuno,
 France) and subsequently sterile filtered through a 0.22 .mu.m filter
 (Durapore, Millipore, 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 l of DEAE Sepharose
 FF.RTM.(Pharmacia Biotech, Sweden) and 56 1 of CM Sepharose FF.RTM.
 (Pharmacia Biotech, Sweden), respectively. The columns are connected in
 series so that the liquid flow 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 solution 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 equilibration
 buffer, 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, then IgG is eluted with a gradient of NaCl from 125 mM to 350 mM
 NaCl, 15 mM sodium acetate, 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/diafiltration
 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 until the conductivity is less than 1.4 mS/cm. The IgG content
 of the solution is determined spectrophotometrically by measuring at 280
 nm (A.sub.280). The sorbitol concentration is adjusted to 10% by weight
 and the solution is filtered through a 0.45 .mu.m filter (Pall
 Corporation, UK). Tween 80 and TNBP are then added to a final
 concentration of 1% and 0.3% by weight, respectively, for subsequent S/D
 treatment. The S/D treatment proceeds for at least 6 hours at 25.degree.
 C.
 Step 6: Removal of S/D by Ion Exchange Chromatography (II)
 Two serially connected columns packed with 28 l of DEAE and 56 l of CM
 Sepharose FF, respectively, are equilibrated with 15 mM sodium acetate, pH
 5.4. The S/D-treated 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 applied to
 the two columns connected in series. The ion exchange chromatography and
 the 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 6 is subjected to ultrafiltration and
 desalting 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 employed membrane is a polysulfone membrane 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/litre, 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 aseptically.
 Example 2
 Results From An Analytical Study Of A Product Obtained By The Present
 Process, Compared To Other IVIG Products

Errorl Bookmark Gammonativ Octagam Gammagard IVIG, SSI
 not defined. lyophilized liquid lyophilized liquid
 Purity 45.4%.sup.1 99.1% 94.6%.sup.1 99.8%
 Albumin 50 mg/ml.sup.2 small 3 mg/ml.sup.2 not
 detectable
 amounts
 Content of mono- 98.3%.sup.3 96.8% 97.6%.sup.3 99.3%
 mers + dimers
 polymers + aggreg 0.8%.sup.3 1.6% &lt;0.1%
 ACA 26% 30% 34% 32%
 PKA &lt;8.5 IE/ml &lt;8.5 IE/ml &lt;8.5 IE/ml &lt;8.5 IE/ml
 Haemaglutinin, 3%
 solution
 anti-A &gt; 1:2 negative negative negative negative
 anti-B &gt; 1:2 negative negative negative negative
 Fc function 169% 121% 132% 178%
 Subclass
 distribution
 IgG1 60.0% 61.9% 67.7% 56.6%
 IgG2 35.8% 33.1% 27.2% 39.4%
 IgG3 3.5% 3.6% 4.4% 2.6%
 IgG4 0.7% 1.4% 0.6% 1.5%
 IgA 2.96 mg/l 54.7 mg/l 0.85 mg/l 1.36 mg/l
 IgM 0.28 mg/l 39.1 mg/l 0.99 mg/l 0.16 mg/l
 Tween 80 &lt;20 ppm &lt;20 ppm not deter- &lt;20 ppm
 mined
 TNBP 2.0 ppm 1.5 ppm 1.5 ppm 1.5 ppm
 PEG 0.01 mg/ml 0.01 mg/ml 1.6 mg/ml.sup.4 0.02
 mg/ml
 pH 6.7 5.7 6.7 5.6
 Total protein con- 97 g/l 45 g/l 50 g/l 51 g/l
 centration
 Maltose or glucose 20 mg/ml 92 mg/ml 15 mg/ml 88 mg/ml
 .sup.1 without correction for HSA; .sup.2 declared by producer; .sup.3
 corrected for HSA peak; .sup.4 used as a stabilizer
 Error! Bookmark not Defined.Purity (Protein Composition)
 Pharmacopoeia purity requirements for an IVIG-preparation is at least 95%
 IgG, that is not more than 5% non-IgG-contaminating proteins present.
 Purity is regarded as being of very high importance for several reasons.
 From a rational point of view, only the protein which 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, the gel is fixed, dried, and stained. Protein
 bands are finally monitored by scanning. It appears from the table above
 that the product of the invention is virtually pure (99.8%).
 Albumin
 The albumin content was analyzed by crossed immuno-electrophoresis
 essentially as described by C. B. Laurell (Anal Biochem (1965),. 10,
 358-361). 5 .mu.l 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.
 Error! Bookmark not Defined.Content of IgG Monomers and Dimers
 The content of IgG monomers and dimers can be analyzed by gel permeation
 chromatography, 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
 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 process of the invention is
 largely free of polymers and aggregates.
 Polymers can be analyzed by gel permeation chromatography, and any protein
 peaks with retention times shorter than the retention time for dimeric IgG
 are considered polymeric as described in Ph.Eur.
 According to Ph.Eur. and other guidelines, the content of protein
 aggregates should preferably be less than 3%. The product of the present
 process contains no measurable aggregates and is thus considered to
 contain less than 0.1% polymers and aggregates.
 Anti-complementary Activity (ACA) and Prekallikrein Activator Activity
 (PKA)
 ACA and PKA are measured as described in Ph.Eur.
 ACA should preferably be as low as possible. According to Ph.Eur. the
 complement consumption should be less than or equal to 50%. The complement
 consumption of the measured sample of the product of the invention is
 about 30%, i.e. comparable to that of the other products analyzed. It
 should be noted that 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&lt;35 lU/ml when measured as outlined in Ph.Eur. PKA of the product of
 invention as well as of the other products analyzed is less than the
 quantitation level of the method, i.e. below 8.5 lE/ml.
 Haemagglutinins Error! Bookmark not Defined
 The IgM fraction of plasma immunoglobulins comprises the haemagglutinins,
 that is antibodies against blood type A and B antigens. The presence of
 such antibodies may cause unwanted adverse effects due to a possible
 haemolytic 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-function
 Retained antigen binding activities are essential for the biological
 functions of the IVIG. This is also true for the immunomodulating
 activities. On the other hand, a retained Fc-function is essential for the
 effect of IVIG on various phagocytic cells and activation of the
 complement system. Fc-function can be demonstrated using various
 techniques, but an accepted methodology described in Ph.Eur. measures the
 complement-activating potential of antibodies in the preparation against
 rubella antigen. Activity is compared to that of a biological 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 preparation. It appears that the Fc-function of the product of
 the invention is very well preserved, 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 Suppl. 10). The concentrations 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 g IgG1/l serum, 1.1-5.9 g IgG2/l
 serum, 0.15-1.3 g IgG3/l serum, and 0.06-1.9 g IgG4/l serum (R. Djurup et
 al. Scand J. Clin Lab Invest 48, 77-83). Thus, the subclass distribution
 of all the products is acceptable.
 IgA-content
 The presence of IgA is known to potentially cause sensibilisation of
 IgA-deficient 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 immunoglobulin 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-IgA is used for the detection of bound IgA. 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 considerably lower IgA-content than that of the other
 analyzed liquid product. The physico-chemical similarities between IgG and
 IgA make it difficult to separate these immunoglobulins during a
 purification process. However, the two anion/cation exchange
 chromatography steps in the process reduce the IgA-content to a very low
 level.
 IgM-content
 IgM in an Ig-preparation can be monitored using an ELISA-technique, e.g.
 where a polyclonal anti-IgM is used to capture IgM, and a labelled
 anti-IgM is used for detection. Standards 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 IgM-content of the product of the
 invention is very low and markedly lower than that of the other liquid
 product.
 Tween 80. TNBP and PEG80
 Tween 80, TNBP and PEG are measured by standard procedures. In general, the
 content of these additives should be as low as possible.
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 pH of the analyzed liquid products is acidic, pH 5.6-5.7, whereas the
 analyzed lyophilized 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 products fulfil this requirement. The protein
 concentration is measured by the method of Kjeldahl.
 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 glucose is determined by use of a commercial kit
 (Boehringer Mannheim, Germany) with maltose as a reference.
 It appears that the two lyophilized products stabilized by albumin and
 albumin as well as PEG, respectively, also contain a saccharide stabilizer
 in concentrations from about 15 mg/ml 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. By 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 appear 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 patients), secondary immunodeficiency
 syndrome (6 patients), idiopathic thrombocytopenic purpura (15 patients)
 and patients with chronic inflammatory demyelinating polyneuropathia (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, serum-creatinine and virus markers
 have been determined in all 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
 T.sub.1/2, 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 pneumonias were registered retrospectively for a
 6-month period during which the patients had been treated with other
 registered IVIG medicaments. In the following 6 months during which the
 patients were treated with Immunoglobulin SSI, liquid, the same parameters
 were registered.
 The conclusion is that Immunoglobulin SSI, liquid is just as effective as
 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 raised from &lt;30.times.10.sup.9 /L before the treatment with
 Immunoglobulin SSI, liquid to &gt;50.times.10.sup.9 /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 infrequent, and thus not
 surprising. Details of the rise of platelets and the duration of the rise
 are under way.
 The conclusion is that Immunoglobulin SSI, liquid is just as effective as
 other IVIG medicaments in the treatment of low platelet count in patients
 with chronic idiopathic thrombogenic purpura.
 According to clinicians, and patients suffering from chronic inflammatory
 demyelinating polyneuropathia, the IVIG, SSI has shown identical efficacy
 to the IVIG administrered prior to the trial. IVIG, SSI was tolerated by
 the patients equally well as other IVIG products were tolerated by the
 patients.
 Safety
 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, SSI. 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 medicaments.
 Example 4
 Results From Stability Study For IVIG Liquid
 In order to test 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.degree. C.-8.degree. 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.

0 months of 6 months of 12 months of
 storage storage storage
 Appearance Slightly opales- Slightly opales- Slightly opalescent
 cent and colour- cent and colour- and colourless
 less less
 Content of mono- 100% 99.6% 99.5%
 mers + dimers
 polymers + aggreg &lt;0.1% &lt;0.1% &lt;0.1%
 ACA 39.7% 38.2% 37.3%
 PKA &lt;8.5 IE/ml &lt;8.5 IE/ml &lt;8.5 IE/ml
 Fc function 107% 113% 111%
 Subclass distribution
 IgG1 59.2% 57.7% 57.1%
 IgG2 36.4% 38.1% 38.6%
 IgG3 2.7% 2.6% 2.5%
 IgG4 1.8% 1.6% 1.7%
 pH 5.5 5.5 5.5
 Protein composition 99.8% 99.7% 99.1%
 (% IgG)
 Total protein 48.8 g/l 48.3 g/l 49.2 g/l
 concentration
 Osmolality 348 mOsm/kg 347 mOsm/kg 350 mOsm/kg
 All the above mentioned tests were carried out in accordance with Ph.Eur.
 and as described 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 known, 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-function indicates that the presence of intact functional
 IgG is maintained during storage. Thus, no proteases are present in the
 samples, as they would have degraded the proteins and thereby lowered the
 Fc-function. Denaturation 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
 over time, 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.degree. 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 log.sub.10 of Hepatitis A Virus (HAV)
 removal of 7.2 log.sub.10 of Polio Virus
 Virus validation studies have been performed employing two enveloped
 viruses, the following virus reductions were obtained:
 removal of 7.6 log.sub.10 of HIV
 removal of 7.5 log.sub.10 of BVDV
 Virus Inactivation By A S/D Treatment Step
 Treatment of the immunoglobulin solution with 1% Tween 80+0.3% TNBP, at
 25.degree. C. for&gt;6 hours.
 Virus validation studies have been performed employing four enveloped
 viruses, the following virus reductions were obtained:
 inactivation of 7.4 log.sub.10 of HIV
 inactivation of 5.3 log.sub.10 of Sindbis Virus
 inactivation of 4.1 log.sub.10 of BVDV
 inactivation of 5.1 log.sub.10 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 efficiently removed by PEG precipitation. The
 PEG precipitation step is therefore validated as an efficient virus
 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 virus 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 BVDV in the process are 15 and
 11.6, respectively. By this the product of the present process can be
 regarded as virus safe.