Purification of immunoglobulins from plasma

Abstract

The present invention relates to the purification of target molecules like immunoglobulins from plasma. The use of a certain type of ion exchanger based on a crosslinked polyvinylether results in especially high yields of the target molecule.

Claims

1. A method for purifying a target molecule from a plasma sample comprising: a) providing a plasma sample comprising the target molecule b) subjecting said plasma sample to an ion exchange chromatography on a polyvinylether matrix carrying between 600 and 1200 mol/g anion exchange groups whereby purified target molecule is eluted from the matrix wherein the polyvinylether matrix is a copolymer obtained by copolymerisation of at least one compound from the group a) and one compound from group b) with a) being at least one hydrophilically substituted alkyl vinyl ether of the formula I ##STR00007## where R1, R2, R3, independently of one another, are H or C1 to C6 alkyl, and R4 is a radical which carries at least one hydroxyl group and b) being at least one crosslinking agent of formula II, formula III or formula IV: ##STR00008## where X is a divalent alkyl radical having 2 to 5 C atoms in which one or more methylene groups which are not adjacent and are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H atoms of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)-alkyl, N(C1-C8)-alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, and ##STR00009## where Y1 and Y2 in formula III and IV are, independently of one another: C1 to C10 alkyl or cycloalkyl, where one or more non-adjacent methylene groups or methylene groups which are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH; or C6 to C18 aryl, where one or more H in the aryl system may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH; and A is a divalent alkyl radical having 2 to 5 C atoms, in which one or mom non-adjacent methylene groups or methylene groups which are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH.

2. Method according to claim 1, wherein the polyvinylether matrix is a copolymer obtained by copolymerisation of a hydrophilically substituted alkyl vinyl ether selected from: 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether; and divinylethyleneurea (1,3-divinylimidazolin-2-one) as crosslinking agent.

3. Method according to claim 1, wherein the anion exchange groups have been attached to the matrix by subjecting the polyvinylether matrix to cerium catalyzed graft polymerization.

4. Method according to claim 1, wherein the polyvinylether matrix carries 700 to 1100 mol/g of positively charged anion exchange groups which are graft polymerized to the matrix.

5. Method according to claim 1, wherein the anion exchange groups comprise trimethylammoniumalkyl groups.

6. Method according to claim 1, wherein the ion exchange chromatography is performed in a flow-through mode.

7. Method according to claim 1, wherein the target molecule is an immunoglobulin.

8. Method according to claim 1, wherein the target molecule is IgG and IgG is separated from IgA, IgM, albumin, and factor XIa in the plasma sample.

9. Method according to claim 1, wherein the matrix in the ion exchange chromatography step b) is eluted with a buffer having a pH between 4 and 7.4.

10. Method according to claim 1, wherein the plasma sample comprises protein and plasma sample is applied to the matrix in an amount such that there are 25 to 150 g of protein in the plasma sample per liter of the matrix.

11. Method according to claim 1, wherein loading and elution of the matrix in the ion exchange chromatography step b) is performed with an acetate buffer comprising between 0.005 and 1 M acetate.

12. Method according to claim 1, wherein the matrix is made of polyvinylether particles with an average particle size diameter of between 20 and 250 m.

13. Method according to claim 1, wherein, after elution of the target molecule from the matrix with a buffer, in a subsequent step c) the matrix is eluted again with a buffer having a pH below the pH of the buffer used in step b) whereby in step c) IgA, IgM and factor XIa are eluted from the matrix.

14. The method of claim 2, wherein: in formula II, X is a divalent alkyl radical having 2 to 3 C atoms, in which one or more methylene groups which are not adjacent and are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H atoms of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)-alkyl, N(C1-C8)-alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, and in formula III, A is a divalent alkyl radical having 2 to 3 C atoms, in which one or more non-adjacent methylene groups or methylene groups which are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The starting material of the present purification process can be any sample comprising the target molecule to be purified. Typically the sample is or has been obtained from blood or plasma. Advantageously it is an immunoglobulin-containing plasma protein fraction. The starting material for this can be normal human plasma or may originate from donors with high titers of specific antibodies, e. g. hyperimmune plasma.

(2) According to the method of the present invention such sample comprising the target molecule is subjected to at least one purification step in which the sample is loaded onto a chromatography matrix comprising anion exchange functionalities.

(3) It has been found that the use of a certain type of chromatography matrix according to the present invention results in especially higher yields of 5-10% combined with higher purity of the target molecule compared to for example methacrylate copolymer based strong anion exchangers like Macro-Prep High Q or sepharose based anion exchangers like Q Sepharose FF. E.g. compared to Q Sepharose FF the purity expressed as sum out of IgA and IgM in the IgG target fraction is typically 5 times higher, compared to Macro-Prep High Q the yield of the target protein IgG is typically 7.7% higher, see Table 1 for reference.

(4) This remarkable effect is achieved by using a hydrophilic polyvinylether matrix carrying between 600 and 1200 mol/g anionic groups.

(5) The matrix to be used is preferably based on a hydrophilic crosslinked polymer based on a copolymer at least comprising

(6) a) at least one hydrophilically substituted alkyl vinyl ether of the formula I

(7) ##STR00004##
where R1, R2, R3, independently of one another, can be H or C1 to C6 alkyl, preferably H or CH.sub.3,
and R4 is a radical which carries at least one hydroxyl group
and
b)
at least one crosslinking agent conforming to formula II and/or III and/or IV with

(8) ##STR00005##
where X is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more methylene groups which are not adjacent and are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H atoms of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)-alkyl, N(C1-C8)-alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, and

(9) ##STR00006##
where Y1 and Y2 in formula III and IV are, independently of one another, C1 to C10 alkyl or cycloalkyl, where one or more non-adjacent methylene groups or methylene groups which are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH,
or C6 to C18 aryl, where one or more H in the aryl system may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH and
A is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more non-adjacent methylene groups or methylene groups which are not located in the direct vicinity of N may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH or N and one or more H of the methylene groups may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH.sub.2, C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH.

(10) R4 in formula I is typically an alkyl radical, a cycloaliphatic radical or an aryl radical which carries at least one hydroxyl group.

(11) This means the polymer is formed by copolymerisation of at least one compound from the group of the hydrophilically substituted alkyl vinyl ethers of the formula I and at least one compound from the group of the crosslinking agents of the formula II and/or III and/or IV. Preferably, only one compound from the group of the hydrophilically substituted alkyl vinyl ethers of the formula I and one compound from the group of the crosslinking agents of the formula II, III or IV is employed.

(12) In a preferred embodiment, R4 in formula I is

(13) a straight-chain or branched C1 to C10 alkyl radical, in which one or more non-adjacent methylene groups may be replaced by O, CO, S, SO, SO.sub.2, NH, NOH, N and/or in which one or more H atoms may be substituted, independently of one another, by C1-C6-alkyl, C5-C10-aryl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH and in which at least one OH group is present either on the C1 to C10 alkyl radical or on a substituent,
or a cycloaliphatic radical, typically having 5 to 10 C atoms, in which one or more non-adjacent methylene groups may be replaced by O, CO, S, SO,

(14) SO.sub.2, NH, NOH, N and/or in which one or more H atoms of the cycloaliphatic radical may be substituted, independently of one another, by C1-C6-alkyl, C5-C10-aryl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the cycloaliphatic ring or on a side chain or substituent, or a

(15) C6 to C18 aryl radical, where one or more H atoms in the aryl radical may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, C5-C10-aryl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the aryl radical or on a side chain or substituent, or a
C5 to C18 heteroaryl radical, where one or more H atoms in the heteroaryl radical may be substituted, independently of one another, by hydroxyl

(16) groups, C1-C6-alkyl, C5-C10-aryl, halogen, NH.sub.2, NH(C1-C8)alkyl, N(C1-C8)alkyl.sub.2, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the heteroaryl radical or on a side chain or substituent.

(17) In a particularly preferred embodiment, R4 in formula I is

(18) a straight-chain or branched C1 to C10 alkyl radical, in which one or more non-adjacent methylene groups may be replaced by O, S, SO.sub.2 or NH and/or in which one or more H atoms may be substituted, independently of one another, by C1-C6-alkyl, C5-C10-aryl, C1-C6-alkoxy or C1-C6-alkyl-OH and in which at least one OH group is present either on the C1 to C10 alkyl radical or on a substituent,
or a cycloaliphatic radical, typically having 5 to 10 C atoms, in which one or more non-adjacent methylene groups may be replaced by O, S, SO.sub.2 or NH and/or in which one or more H atoms of the cycloaliphatic radical may be substituted, independently of one another, by C1-C6-alkyl, C5-C10-aryl, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the cycloaliphatic ring or on a side chain or substituent, or a
C6 to C14 aryl radical, where one or more H atoms in the aryl radical may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, C5-C10-aryl, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the aryl radical or on a side chain or substituent, or a
C6 to C14 heteroaryl radical, in which at least one N atom is present as heteroatom and where one or more H atoms in the heteroaryl radical may be substituted, independently of one another, by hydroxyl groups, C1-C6-alkyl, C5-C10-aryl, C1-C6-alkoxy or C1-C6-alkyl-OH, where at least one OH group is present either on the heteroaryl radical or on a side chain or substituent.

(19) In a preferred embodiment, the hydrophilically substituted alkyl vinyl ether employed is a compound of the formula I in which R4 is a radical which carries a hydroxyl group.

(20) In a preferred embodiment, the hydrophilically substituted alkyl vinyl ether employed is 1,2-ethanediol monovinyl ether, 1,3-propanediol monovinyl ether, 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, 1,6-hexanediol monovinyl ether or diethylene glycol monovinyl ether and the cycloaliphatic vinyl ether employed is cyclohexanedimethanol monovinyl ether, particularly preferably 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether.

(21) The crosslinking agents employed are preferably compounds of the formula II. Preference is given to the use of divinylpropyleneurea (1,3-divinyl-tetra-hydropyrimidin-2-one) or particularly preferably divinylethyleneurea (1,3-divinylimidazolin-2-one).

(22) The proportion of the hydrophilically substituted alkyl vinyl ethers with respect to the weight of the polymer is typically between 1% (by weight) and 90% (by weight) or a maximum proportion by weight of the alkyl vinyl ether which corresponds to a molar ratio of 2:1, based on a bifunctional crosslinking agent, if the alkyl vinyl ether does not homopolymerise. The proportion of the hydrophilically substituted alkyl vinyl ethers is preferably between 10 and 80% (% by weight), particularly preferably between 35 and 60%. Accordingly, the proportion of the crosslinking agent is between 10 and 99 (% by weight), preferably between 20 and 90%, particularly preferably between 40 and 65%.

(23) In another preferred embodiment, the polymer is porous having pore sizes between 2 and 200 nm, more preferred between 30 and 150 nm

(24) In another embodiment, the polymer is in the form of particles having average particle size diameters diameter between 25 and 250 m, most preferred between 30 to 90 m.

(25) The polymer carries ligands comprising an anion exchange group.

(26) In a preferred embodiment, the polymer has been derivatised by means of structures which have been attached to the polymer by graft polymerisation.

(27) In a preferred embodiment, the polymer has been derivatied by means of structures which have been attached to the polymer by graft polymerisation with cerium(IV) catalysis, preferrably according to U.S. Pat. No. 5,453,186 page 9 example 8, where preferably the charged group is the positively charged trimethylammoniumalkyl group.

(28) Further details about the material to be used in the method of the present invention and about its production can be found in WO 2007/014591.

(29) Ligands are known to the person skilled in the art in the area of chromatography. Ligands are substituents which can be introduced into the support material as early as during the synthesis of the base material or subsequently and influence the surface properties of the support material. In particular, targeted derivatisation of support materials by means of ligands produces support materials having certain chromatographic properties. In particular, ligands to be used in the present invention can have the following terminal groups:

(30) an ionic or ionisable group, for example

(31) NR.sup.7R.sup.8 or NR.sup.7R.sup.8R.sup.9,

(32) in which

(33) R.sup.7 and R.sup.8, independently of one another,

(34) H, alkyl having 1-5 C atoms
and
R.sup.9 alkyl having 1-5 C atoms
with the proviso that, if XNR.sup.7R.sup.8R.sup.9, R.sup.7 and R.sup.8 cannot be H, -guanidinium

(35) In a preferred embodiment the polymer to be used as a matrix in the method of the present invention is derivatised by graft polymerisation with tentacle-like structures, which can in turn carry the corresponding ligands or be functionalised by means of the latter. The grafting is preferably carried out in accordance with EP 0 337 144 page 12 example 8 or U.S. Pat. No. 5,453,186 page 9 example 8 using N-(2-Trimethylammoniumethyl)-acrylamide. The polymerisation catalyst employed is cerium(IV) ions, since this catalyst forms free-radical sites on the surface of the base material, from which the graft polymerisation of the monomers is initiated.

(36) The polymerisation is terminated by termination reactions involving the cerium salts. For this reason, the (average) chain length can be influenced by the concentration ratios of the base material, the initiator and the monomers. Furthermore, uniform monomers or also mixtures of different monomers can be employed; in the latter case, grafted copolymers are formed.

(37) Suitable monomers for the preparation of the graft polymers and further details about the grafting procedure are e.g. disclosed in WO 2007/014591, EP 0337 144, especially page 12, example 8 and U.S. Pat. No. 5,453,186 page 9, example 8.

(38) Preferably the matrix is derivatised with ionic groups by graft polymerisation whereby the resulting chains that are grafted onto the base polymer matrix have a length of between 2 and 100, preferably 5 and 60, in particular between 10 and 30 monomer units, each unit typically carrying one ionic group.

(39) Preferred ionic groups are positively charged Trimethylammoniumethyl groups.

(40) The matrix might carry additional other functional groups like hydrophobic or hydrophilic groups in addition to the anion exchange groups but in any case it has anion exchange groups.

(41) The ionic capacity of the anion exchange matrix to be used in the present invention is typically between 600 and 1200 mol/g, preferably between 700 and 1000 mol/g, most preferred between 800 and 1000 mol/g.

(42) Suitable materials to be used in the method of the invention are Eshmuno QPX and Eshmuno QPX Hicap from Merck KGaA, Germany. Those resins comprise polyvinylether beads synthesized according to the procedure disclosed in WO 2007/014591, to which polymer structures are grafted utilizing grafting techniques according to EP 0337 144 page 12 example 8 and U.S. Pat. No. 5,453,186 page 9 example 8 and yielding surface polymer structures carrying positively charged trimethylammoniumethyl groups, where the charge density in the case of Eshmuno QPX and Eshmuno QPX Hicap resins is adjusted to 600 to 1200 mol/g.

(43) For performing the method of the present invention, the sample is subjected to an anion exchange chromatography whereby the chromatography matrix is a hydrophilic polyvinylether functionalized with anionic groups as described above.

(44) The sample is preferably a pre-purified plasma sample comprising immunoglobulin G, preferably 75 to 99% by weight of IgG. The samples comprise preferably at least 80% by weight, more preferred at least 85% by weight, especially preferred more than 90% by weight most preferred between 92 and 98% by weight of immunoglobulin G. % by weight is in this case related to the mass of the dried plasma sample.

(45) Before applying the sample to the matrix, the matrix can be washed and/or equilibrated.

(46) Washing can be done with a moderately acidic pH 4.0-5.5 buffer like acetate buffer, optionally with salt, for example NaCl. The buffer typically has a concentration between 200 and 1000 mM/l.

(47) Equilibration is done with an equilibration buffer with a pH between 4 and 7.4. Preferably the pH of the equilibration buffer is the same as the pH of the sample. The concentration of the equilibration buffer is typically in the range of 0.005 to 2 Mol/l, preferably in the range of 0.005 to 0.05 Mol/l.

(48) Equilibration buffer is typically identical to the sample buffer.

(49) Prior to washing and/or equilibrating the matrix with the equilibration buffer it is possible to treat the matrix with a basic aqueous liquid having a pH of more than 10, preferably around 14. Such a treatment is known to a person skilled in the art. It is suitable to remove potential impurities from the matrix. Suitable liquids are aqueous sodium hydroxide or aqueous potassium hydroxide. The basic aqueous liquid can be removed from the matrix directly with the equilibration buffer or with a slightly acidic aqueous washing buffer like acetic acid buffer, preferably in a e.g. acetate concentration between 200 to 1000 mM/l.

(50) The sample is typically applied to the chromatography matrix in a buffer (also called loading buffer or sample buffer). The buffer preferably has a pH between 4.0 and 7.4. Suitable buffers are carbonic acid/silicate buffer, acetic acid buffers, citrate buffers, phosphate buffers, glycine buffers and/or 2-(N-morpholino)ethanesulfonic acid (MES) buffers. Most preferred is an acetate buffer.

(51) The buffers are typically used in concentrations between 5 and 500 mmol/l, preferably between 5 and 100 mmol/l, most preferred between 5 and 50 mmol/l.

(52) The IgG concentration in the sample feed is typically adjusted to between 1 to 50 g/l.

(53) The amount of sample to be loaded on the matrix is variable in a wide range.

(54) The matrix can be loaded with very small amounts of the sample, like e.g. 10 g sample per 1 liter of matrix volume. It is also possible to load up to 150 g sample per liter of matrix volume. Preferably more than 25 g/l matrix volume are loaded, most preferred between 25 and 100 g/l.

(55) The chromatographic purification of the sample can be performed in the bind-and-elute mode or preferably in the flow-through mode. In the flow-through mode, the target molecule is essentially not bound or adsorbed to the matrix. That means the target molecule moves through the matrix essentially with the solvent fronti.e. the front of the loading bufferand is recovered from the matrix essentially together with the solvent front. It has been found that when using the matrix according to the present invention in the flow-through mode, the target molecule can be typically eluted from the matrix with the 5-fold, preferably the three-fold, very preferred the two-fold volume of eluent with regard to the volume of the matrix. The eluent is in this case identical with the loading buffer. The impurities are retained on the matrix. With the flow-through mode at least 80%, preferably more than 90%, most preferred more than 95% of the target molecule can be recovered from the matrix.

(56) It has been further found that the binding of the impurities to the matrix is very stable as long as the matrix is eluted with the loading buffer. This offers the possibility to also enlarge the volume of the loading buffer used for elution of the target molecule to more than the 5-fold volume of the matrix, if necessary, so that the target molecule can be eluted nearly complete (about 97% yield) while its purity is still very high (typically >99.5%).

(57) The method of the present invention performed in the flow-through mode is especially suitable to separate immunoglobulin G as target molecule from impurities like IgA, IgM, albumin and Serine Proteases like factor XIa.

(58) But it is also suitable for the isolation of an IgM containing product or for the isolation of IgA, IgM and/or factor XIa. It has been found that when performing the chromatographic purification in the flow-through mode, immunoglobulin G is eluted essentially with the solvent front. After elution of IgG, the elution buffer (which for the elution of IgG is identical to the loading buffer) can be changed to support elution of further secondary target molecules like IgA, IgM and factor XIa. For example the secondary target molecules may be a mixture of IgA and IgM. For this, the acidity of the elution buffer is typically increased. Preferably the elution buffer for this application has a pH between 4 and 5.5 whereby the pH is in any case lower than the pH of the loading buffer. Typically the pH of the buffer used to elute IgA, IgM and/or factor XIa is between 0.5 and 2 pH units below the pH of the loading buffer.

(59) Consequently, the method of the present invention not only allows to purify one target molecule, like for example IgG, but also two or more target molecules, like IgG as well as IgA, IgM and/or factor XIa, for example IgG as well as a mixture of IgA and IgM.

(60) The method of the present invention can be used as a single, separate purification step but it can also preferably be combined with other purification steps that are performed prior or after the method of the invention.

(61) The preferred target molecule to be purified with the method of the invention is IgG. Known procedures for the purification of IgG typically comprise several steps including precipitation, filtration and chromatographic steps. Such methods are for example disclosed in Production of Plasma Proteins for Therapeutic Use, edited by J. Bertolini, N. Goss, J. Curling; John Wiley and sons Inc., 2013., see e.g. chapter 13.

(62) The method of the present invention can favorably be applied to substitute one or more of the purification steps of the known methods.

(63) Preferably, the crude plasma sample is first subjected to a precipitation step in which 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 can for example be achieved by ethanol precipitation yielding IgG enriched intermediates called fraction I+II+III or fraction II+III or fraction II. Following acid precipitation and filtration steps, the feed for the anion exchange chromatography can be obtained.

(64) It is also possible to perform additional chromatographic steps.

(65) Following anion chromatography the purified IgG is typically being nanofiltered and formulated.

(66) Unexpectedly, it has been found that the use of the certain type of chromatographic matrix not only offers the possibility to purify target molecules like IgG very effectively with yields >95% by simple flow-through ion exchange chromatography, it also ensures the separation of IgG from IgA below 25 mg/L, IgM below 25 mg/L, albumin (below detection limit) and factor XIa (below detection limit). The very good separation of IgG from these substances can further be used to additionally isolate one or more of those substances in also very good purity and yield.

(67) The purity and yield that can be achieved with the chromatographic matrix to be used according to the present invention is better compared to equivalent anion exchange steps on different matrices. While known procedures often combine an anion exchange purification step with a successive cation exchange polishing step, such a polishing step is typically not needed when the anion exchange purification is performed according to the present invention.

(68) The entire disclosures of all applications, patents, and publications cited above and below, as well as of corresponding EP 14002852.3, filed Aug. 15, 2014, are hereby incorporated by reference.

EXAMPLES

Example 1Purification of IgG

(69) The chromatographic matrix (Eshmuno QPX Hicap, Merck KGaA) is equilibrated with 500 mM acetate buffer (pH 6.5) and subsequently treated with 25 mM Acetate buffer (pH 6.5). The sample is a solution comprising 15 g/l IgG with the following impurities: IgA (1000 mg/l) and IgM (500 mg/l) and factor XIa (50 pM/l) in 25 mM acetate buffer (pH 6.5).

(70) The sample is applied to the matrix equivalent to a protein loading of 75 g/l of the matrix at 130 cm/h. The matrix is then eluted with 25 mM acetate buffer, pH 6.5 until IgG is completely eluted from the matrix. IgG is recovered in more than 95% yield, whereby the purified IgG comprises less than 25 mg/l IgM and IgA. The amount of factor XIa can be reduced to 0.0 pM. IgM and IgA can be further obtained from the matrix in a purity of more than 95% and with a yield of more than 95% by elution with an acidic buffer (300 mM Acetate buffer, pH 4.5). All steps run at 130 cm/h linear velocity.

Example 2Purification of IgG on Various Matrices

(71) General procedure:

(72) Feed composition: 20 g/l IgG und 1 g/l IgA und 1 g/l IgM in 25 mM acetat pH 6.5.

(73) Pre-Equilibration: 500 mM acetate buffer, pH 6.5

(74) Equilibration: 25 mM acetate buffer, pH 6.5

(75) Loading/Elution: 25 mM acetate buffer, pH 6.5

(76) Flow Rate: >130 cm/h

(77) Loading: 5 g/L (sum of IgM and IgA)

(78) Wash: 250 mM acetate buffer, pH 4.5

(79) Table 1 shows the yield, purity (concentration of impurities) and recovery data.

(80) TABLE-US-00001 TABLE 1 Fractogel Macro- Q Eshmuno EMD TMAE Prep Sepharose QPX Hicap (M) High Q FF Yield IgG 95.7 2.1 85-90 85-91 88 [%] Recovery 101 2 100 97 95 IgG [%] IgA@10% 9.1 2.2 14 4 >40 [mg/L] IgM@10% 34.0 2.3 30 26 >150 [mg/L] IgG 4 [%] >1.7 <1.3 <1.3 n.d. Factor XIa <0.001 n.d. 0.11 n.d. [ng/mL] Albumin [%] <0.1% n.d. <0.1% n.d. Experiments 5 2 2 1

(81) Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

(82) In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.

(83) The entire disclosures of all applications, patents and publications, cited herein and of corresponding European Application No. EP 14002852.3, filed Aug. 15, 2014 are incorporated by reference herein.

(84) The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

(85) From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.