(12) United States Patent
`Us 6,281,336 B1
`(10) Patent N0.:
`Laursen et al.
`(45) Date of Patent:
`Aug. 28, 2001
`
`U8006281336B1
`
`(54) PROCESS FOR PRODUCING
`IMMUNOGLOBULINS FOR INTRAVENOUS
`ADMINISTRATION AND OTHER
`IMMUNOGLOBULIN PRODUCTS
`
`0530447
`WO8606727
`WO 98/05686
`
`10/1997 (EP).
`11/1986 (W0).
`2/1998 (W0).
`OTHER PUBLICATIONS
`
`(75)
`
`Inventors:
`
`Inga Laursen, Hellerup; Barge
`Teisner, Odense C, both of (DK)
`
`(73) Assignee: Statens Serum Institut, Copenhagen S.
`(DK)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/328,497
`
`(22)
`
`Filed:
`
`Jun. 9, 1999
`
`Related U.S. Application Data
`Provisional application No. 60/102,055, filed on Sep. 28,
`1998.
`
`J Food Sci 58(6) 1993, 1282—90, Fichtali, et al. “Purification
`of antibodies .
`.
`. ”.
`:9
`J Am Chem Soc 68, 1946, 459—75, Cohn, et al. “Preparation
`and properties .
`.
`.
`.
`JAm Chem Soc 71, 1949, 541—50, Oncley, et al “Separation
`of antibodies .
`.
`. ”.
`
`Vox Sang 7, 1962, 414—24, Kistler et al, “Large scale
`production .
`.
`. ”.
`Biochem Biophys Acta 82, 1964, 463—75 Polson et al,
`“Fractionation of protein.”
`Vox Sang 23, 1972, 107—18, Polson et al., “Fractionation of
`plasma .
`.
`. ”
`Blood Separation and Plasma Fractionation, 1991, WileyL-
`iss, New York, p 266, Harns, J. (Ed.), Figure 3.
`Anal Biochem 10, 1965, 358—61, Laurell, “Antigen—anti-
`body crossed .
`.
`. ”.
`
`(60)
`
`(30)
`
`Foreign Application Priority Data
`
`* cited by examiner
`
`Jun. 9, 1998
`
`(EP)
`
`................................................. 98201909
`
`Int. Cl.7 ......................... A61K 39/395; C07K 16/00
`(51)
`(52) U.S. Cl.
`..................................... 530/390.1; 424/176.1;
`424/177.1, 530/390.5, 530/414, 530/416;
`530/417; 530/420; 530/421
`(58) Field of Search .............................. 424/176.1, 177.1;
`530/3901, 390.5, 414, 416, 417, 420, 421
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`3/1975 Falksveden ....................... 530/390.5
`3,869,436 *
`6/1981 Zufli
`............
`530/390.5
`4,272,521 *
`
`2/1982 Shandrom
`.......... 514/2
`4,314,997
`2/1982 Shanbrom ................. 514/1
`4,315,919
`
`8/1988 Neurath et al.
`.
`424/176.1
`4,764,369
`
`4,876,088 * 10/1989 Hirao et al.
`.....
`424/177.1
`4,880,913 * 11/1989 Doleschel et al.
`530/390.5
`5,164,487 * 11/1992 Kothe etal.
`530/390.5
`
`5,177,194 *
`1/1993 Sarno et al.
`...... 530/412
`.................... 530/390.1
`5,886,154 * 11/1999 Lebing et al.
`FOREIGN PATENT DOCUMENTS
`
`Primary Examiner—David Saunders
`(74) Attorney, Agent, or Firm—Birch, Stewart, Kolasch &
`Birch, LLP
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a process for purifying
`immunoglobulin G from a crude immunoglobulin-
`containing plasma protein fraction. Said process includes a
`number of steps of Which the anion exchange chromatog-
`raphy and the cation exchange chromatography are prefer-
`ably 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.
`
`2364792
`
`7/1974 (DE) .
`
`14 Claims, N0 Drawings
`
`AMGEN INC.
`
`Exhibit 1037
`
`Ex. 1037 - Page 1 of 14
`
`Ex. 1037 - Page 1 of 14
`
`AMGEN INC.
`Exhibit 1037
`
`

`

`US 6,281,336 B1
`
`1
`PROCESS FOR PRODUCING
`IMMUNOGLOBULINS FOR INTRAVENOUS
`ADMINISTRATION AND OTHER
`IMMUNOGLOBULIN PRODUCTS
`
`This application claims priority on provisional Applica-
`tion No. 60/102,055 filed on Sep. 28, 1998,
`the entire
`contents of which are hereby incorporated by reference.
`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 inven-
`tion 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 adminis-
`tered subcutaneously or intramuscularly.
`Congenital or acquired total or partial lack of immuno-
`globulin (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 injec-
`tions 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 intra-
`venous 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 admin-
`istration 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 immunoglo-
`bulins. Especially serious adverse effects of an anaphylactic
`nature could be seen in patients who developed antibodies to
`IgA. Consequently, methods of avoiding aggregate forma-
`tion and/or eliminating these aggregates during the prepa-
`ration 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 infec-
`tious episodes in patients with a congenital or acquired total
`or partial lack of immunoglobulins and to eliminate discom-
`fort 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 infec-
`tions quickly.
`In recent years, IVIG has furthermore proved to be
`efficient in other serious diseases, the treatment of which can
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`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 immunomodu-
`lating properties of IgG, e.g. a blockage of Fcy-receptors on
`phagocytic cells, increased metabolism of IgG, downregu-
`lation 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 produc-
`tion 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 combi-
`nation with the modest volume injected and the intramus-
`cular route of administration, these protective immunoglo-
`bulins can neutralize and render common viruses in plasma
`non-infectious. Especially when large doses of immunoglo-
`bulin are given intravenously, virus infections may occur as
`demonstrated in the early 1990’s. Therefore, it was recog-
`nized 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 anti-
`complementary 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 imme-
`diately prior to use.
`Dissolution of freeze-dried IVIG is slow (up to 30 min-
`utes for one vial). Several portions often have to be dis-
`solved 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 applica-
`tion leads to a liquid immunoglobulin product for intrave-
`
`Ex. 1037 - Page 2 of 14
`
`Ex. 1037 - Page 2 of 14
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`

`

`US 6,281,336 B1
`
`3
`nous 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 condi-
`tions for fractionation: pepsin cleavage is avoided, aggre-
`gates 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 prod-
`uct 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 pro-
`portion of non-immunoglobulin G proteins, aggregated
`immunoglobulins and particles including potentially
`infectious particles such as virus particles, without
`causing substantial precipitation of monomeric immu-
`noglobulin 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 substan-
`tial elution of immunoglobulin G;
`(f) eluting immunoglobulin G from the cation exchange
`resin with a substantially non-denaturating buffer hav-
`ing 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
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`(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, ot-globulins and
`B-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 suffi-
`ciently high so that, during the following precipitation step,
`a major proportion of the non-IgG-proteins, especially those
`of higher molecular weight, the aggregated immunoglobu-
`lins and other aggregated proteins as well as potentially
`infectious particles precipitate without substantial precipi-
`tation of monomeric IgG. This is generally achieved if the
`concentration of the IgG in the buffered and filtered suspen-
`sion 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.
`
`Ex. 1037 - Page 3 of 14
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`Ex. 1037 - Page 3 of 14
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`

`

`US 6,281,336 B1
`
`5
`It is preferred that the plasma protein fraction is sus-
`pended 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 main-
`tained 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 substan-
`tial 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° C., preferably 0—8° C., most
`preferably 1—4° 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 par-
`ticles” in the present context comprises e.g. virus particles
`(such as hepatitis viruses, HIV1 and HIV2) and bacteria.
`Substantially non-denaturating, water-soluble protein pre-
`cipitants 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.
`Apreferred precipitant is the organic agent PEG, particu-
`larly 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
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`6
`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 precipi-
`tation 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, form-
`ing 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 precipi-
`tate. 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° 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 precipitant.
`After completion of the protein precipitation, a clarified
`supernatant containing IgG almost exclusively in a mono-
`meric 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 super-
`natant is depth filtered to remove larger particles and aggre-
`gates. This is optionally followed by sterile filtration per-
`formed by use of a conventional sterilization filter (such as
`a 0.22 pm filter from Millipore or Sartorius), which elimi-
`nates 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 super-
`natant is applied to an anion exchange resin and subse-
`quently 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 wash-
`ing of the cation exchange resin.
`
`Ex. 1037 - Page 4 of 14
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`US 6,281,336 B1
`
`7
`the
`In a preferred embodiment of the present process,
`anion exchange resin and the cation exchange resin are
`connected in series. In the present context, the term “con-
`nected 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 chromatog-
`raphy 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: aga-
`rose based (such as Sepharose CL-6B®, Sepharose Fast
`Flow® and Sepharose High Performance®), cellulose based
`(such as DEAE Sephacel®), dextran based (such as
`Sephadex®), 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 diethylami-
`noethyl (DEAE), quaternary aminoethyl (QAE), and/or qua-
`ternary 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 cation 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 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® and the cation exchange resin CM
`Sepharose FF® and the columns are connected in series,
`
`8
`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. Apreferred buffer for the 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 1.2
`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 quan-
`titatively 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 substan-
`tially 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
`low