WO2000041721A1

WO2000041721A1 - Process for purifying immunoglobulins

Info

Publication number: WO2000041721A1
Application number: PCT/AU1999/001153
Authority: WO
Inventors: Joseph Bertolini; Gerard Seneviratne; Colin Richard Carbis
Original assignee: Csl Limited
Priority date: 1999-01-08
Filing date: 1999-12-23
Publication date: 2000-07-20
Also published as: AUPP806999A0

Abstract

The present invention relates to a method for increasing the ratio of IgA to at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM in a fluid, which method comprises exposing the fluid to a metal ion matrix.

Description

Process for purifying immunoglobulins

FIELD OF THE INVENTION

The present invention relates to a method for purifying immunoglobulins. In particular, the present invention relates to a method of purifying IgA, IgM and IgG.

BACKGROUND OF THE INVENTION

During the last century, immunoglobulin preparations were successfully used for the prophylaxis and treatment of various infectious diseases. Traditionally, immunoglobulin preparations were developed for systemic administration, and were largely comprised of IgG. However, the successful use of breast milk for the prophylaxis and treatment of infant diarrhoea highlighted the potential benefits of plasma (monomeric) and mucosal (secretory) IgA for immunotherapeutic use (Hammarstrom et al., 1994).

A clinical trial conducted by Eibl et al., (1988) indicates that oral- feeding with a plasma derived IgA rich immunoglobulin preparation (IgAbulin - 73% IgA and 26% IgG) may prevent the development of necrotizing enterocolitis (NEC). Oral-feeding with IgAbulin also displays a therapeutic effect in immunodeficient patients suffering from Clostridium difficile (Tjellstrom et al., 1993) or Campylobacter jejuni-induced diarrhoea (Hammarstrom et al., 1993).

Hemmingsson and Hammarstrom (1993) prophylactically administered IgA rich immunoglobulin preparations (IgAbulin) in a nasal spray, and reduced the incidence of respiratory tract infections in elite skiers, and elite rowers (personal communication Dr Martha Eibl). IgAbulin has also been administered in nose drops to reduce the incidence of Haemophilus influenzae in people identified as chronic nasopharyngeal carriers (Lindberg et al, 1993).

IgA can be fractionated from plasma or mucosal secretions using various combinations of precipitation and chromatographic techniques. Compounds used to remove selected proteins from solutions containing IgA include ZnS0₄, (NH₄)₂S0₄, ethanol, polyethylene glycol, λ-carrageenan and caprylic acid (Heremans, 1974; Schumacher, 1969; Pejaudier et al, 1972). Chromatography resins used to fractionate IgA include DEAE cellulose, Sephadex g-200, DEAE-Sephadex A-50, affinity chromatography on anti IgG and anti IgM immunosorbents, jacalin-Sepharose, Protein G-Sepharose, Fastflow-S Sepharose, Superose 6, thiophilic adsorption, Sephacryl S-300, anti-IgA antibody conjugated to cyanogen bromide activated Sepharose 4B, protein-A and protein-G affinity chromatography (Balint et al., 1982; Beetham et al., 1993; Collard et al., 1984; Cripps et al., 1983; Doellgast and Plaut, 1976; Gregory et al., 1987; Hutchens and Porath, 1986; Kobayashi et al., 1987; Leibl et al., 1995; Loomes et al., 1991; Oncley et al, 1949; Pejaudier et al, 1972; Rogue-Barreira and Campos-Neto 1985; Schumacher, 1969).

Although numerous IgA fractionation methods have been described in the literature, surprisingly few IgA immunotherapy products have been marketed by the pharmaceutical industry. It seems that the availability of IgA for immunotherapeutic use has been limited by technical difficulties. Methods for large scale fractionation of IgA fail to produce a highly purified preparation (i.e. greater than 90%), and small scale purification methods (e.g. anti-IgA affinity columns) cannot provide a commercially viable product. Accordingly, methods for large scale purification of IgA, which generate highly purified IgA preparations suitable for immunotherapeutic use, are desirable. In particular, IgA free of IgG is highly desirable due to the fact that IgG may adversely affect the anti-inflammatory properties of IgA.

SUMMARY OF THE INVENTION

The present inventors have now found unexpectedly that IgA may be separated from other proteins in solution such as ceruloplasmin, alpha anti- trypsin, IgG and IgM by metal chelate chromotography. Accordingly, in a first aspect, the present invention provides a method for increasing the ratio of IgA to at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM in a fluid which method comprises:

(a) exposing a fluid containing IgA and at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM to a metal ion matrix under conditions such that the IgA is adsorbed to the metal ion matrix; and

(b) selectively eluting the IgA from the metal ion matrix.

In a second aspect, the present invention provides a method for increasing the ratio of IgA to at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM in a fluid which method comprises

(a) exposing a fluid containing IgA and at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM to a metal ion matrix under conditions such that the at least one other protein is adsorbed to the metal ion matrix while the IgA is not substantially adsorbed to the matrix; and

(b) separating the fluid containing IgA from the metal ion matrix.

In a third aspect the present invention provides an improved method for purifying IgA from a plasma fraction which method comprises decreasing the concentration of at least one protein selected from the group consisting of IgG, IgM, complement (C3), haptoglobin, albumin, ceruloplasmin and alpha- antitrypsin in the plasma fraction to obtain a fraction enriched in IgA, the improvement comprising passing the fraction enriched in IgA through a metal ion matrix to further decrease the concentration of at least one protein selected from the group consisting of IgG, IgM, ceruloplasmin and alpha-antitrypsin .

By "metal ion matrix" we mean a matrix which has at least one metal ion attached thereto. The matrix may be any suitable solid support. For example, the matrix may be an agarose resin such as agarose, methylacrylate, dextran and silica. In a preferred embodiment, the metal ion is attached to the matrix by way of a chelate forming ligand. The chelate forming ligand may be any suitable ligand. Suitable chelate forming ligands will be known to those skilled in the art and include, for example, bis-carboxymethyl amino groups, 1,4,7-triazacyclononane (Jiang et al, 1998), iminodiacetate, tris (carboxymethyl) ethylenediamine (Porath and Olin, 1983), 2-hydroxy-3[N-(2- pyridylmethyl) glycine] propyl, α-alkyl nitrilotriacetic acid, and carboxymethylated aspartic acid (Yip and Hutchens, 1994). The metal ion may be any suitable ion, examples of which include zinc, copper, nickel, iron, manganese, chromium, cadmium, calcium, magnesium and the like. In a preferred embodiment, the metal ion is zinc. Within the parameters of the present invention, the fluid used as the starting material may be any proteinaceous material containing IgA, IgG or IgM and may be derived from a range of in vivo or in vitro sources. For example, the fluid may be derived from serum, plasma, plasma fractions, mucosal secretions (such as milk, colostrum, tears and saliva), or any mixture derived from recombinant sources or transgenic animals. In a preferred embodiment, the fluid is derived from a human.

The fluid used as starting material in a method of the present invention may be partially purified. For example, the fluid may be derived from a chroma tograp hie process. In a preferred embodiment, the starting material is a Cohn II + Cohn III fraction. The IgA solution obtained by a method of the present invention may be subjected to a further purification step. For example, the IgA solution may be subjected to a further chromatography process.

In a preferred embodiment, the starting material used in a method of the present invention, or an immunoglobulin solution obtained by a method of the present invention, is subjected to a process for the deactivation of viruses.

In a further preferred embodiment, the metal ion matrix is packed into a column. It will be appreciated by those skilled in the art that within the context of this preferred embodiment, the physical parameters may be adjusted in order to optimise the purification process. For example, the column flow rate, buffer, pH, column pressure, bed height, temperature and column diameter may all be optimised by routine trial and error.

In a fourth aspect, the present invention provides an aqueous solution including IgA in an amount of at least 90% (w/w), IgG in an amount of between 0-10% (w/w), IgM in an amount of between 0-10% [w/w], plasminogen in an amount less than 0.001 casein units, a conductivity lower than 60 mS/cm and a pH of between 5-9.

In a preferred embodiment of the fourth aspect of the present invention, the IgA, IgG and IgM are derived from a human. In a further preferred embodiment of the fourth aspect, the aqueous solution is obtained by a method according to the first or second aspects of the present invention.

When used herein the term "w/w" is intended to refer to the percentage of the specified protein in relation to the total protein in the sample.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As will be appreciated by those skilled in the art, the present invention provides a method which allows large scale purification of IgA and which generates highly purified preparations of IgA suitable for immunotherapeutic use.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are offered for illustration purposes, and are not intended to limit or define the invention in any manner.

Example 1: Immobilization of IgA on a Zn²⁺ affinity column

IgA was immobilized on a metal affinity column loaded with Zn²⁺. After washing away the unbound material, IgA was eluted by either changing the buffer conditions, or using a gradient or stepwise reduction in pH to 3 or 4. Alternatively, a gradient of increasing concentration of (e.g. ammonium chloride, glycine, histamine, histidine, or imidazole) could be used for competitive elution of IgA.

The purification process involved the following steps: (1) Ethanol (8%) was added to plasma, and the supernatant was removed by filtration.

(2) The supernatant was delipidated with aerosil.

(3) The delipidated solution was loaded onto a column packed with DEAE Sepharose Fast Flow, and equilibrated with lOmM sodium acetate at pH 5.2. IgG and transferrin were removed in the void volume, and albumin was eluted with 25mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM complement (C3) and small amounts of IgG and albumin co-eluted in 0.5M sodium chloride pH 5.2.

(4) A solution of 1.7M ammonium sulfate, 0.1M sodium acetate was added to an equal volume of the aqueous material eluted from the DEAE column with 0.5M sodium chloride at pH 5.2. The solution was adjusted to pH 6.0 to 7.5, and the final conductivity was between 118-124 mS/cm at 25°C.

(5) The solution described in step 4 was loaded onto a column packed with thiophilic resin, which had been equilibrated with 0.85M ammonium sulfate, 0.05M sodium acetate, pH 6.0-7.5, conductivity 118-124 mS/cm at 25°C.

(6) IgA rich material was eluted with 0.012M sodium acetate, 0.1M ammonium sulfate, pH 6.0-7.5, conductivity 19-22 mS/cm.

(7) Thiophilic resin was regenerated with three to four column volumes of 0.05M Tris acetate, pH 8.0, conductivity 2.3-2.5 mS/cm, followed by three to four column volumes of pyrogen free water (PFW).

(8) After three cycles, the column was regenerated with three to four column volumes of 20% isopropanol, 0.01M sodium hydroxide.

• (9) The IgA rich material eluted from the thiophilic column (step 6) was dialyzed against 0.05M Tris HC1, 0.15M sodium chloride, pH 7.5 - 8.0.

(10) A column was packed with chelate agarose, and regenerated with 0.05M EDTA, 0.5M sodium chloride, pH 7.0.

(11) Zn was immobilized on the resin by loading the column with a solution containing ZnCl₂ (3mg/ml) in PFW. Zn ⁺ loading was optimized by increasing the flow rate during the loading process. The resins were saturated with Zn ⁺ when the column pH returned to the pH of the metal ion solution. The pH of the loading solution should be neutral or weakly acidic to avoid hydroxy precipitates.

(12) The column was flushed with three column volumes of PFW to remove hydroxy precipitates that may have formed and to remove excess

Zn ions not immobilized on the agarose support.

(13) The column was equilibrated with five to seven column volumes of 0.05M Tris HCl, 0.15M sodium chloride, pH 7.5 - 8.0.

(14) The dialyzed solution described in step 9 was loaded onto a chelate agarose column after Zn²⁺ had been immobilized. (15) A solution that is more than 90% IgA (w/w) was eluted from the column with 0.1M sodium phosphate pH 6.5.

(16) The column was flushed with three column volumes of PFW.

(17) The column was regenerated with 0.1M sodium acetate, 0.8M sodium chloride, pH 4.5.

(18) After each cycle, the resins were regenerated by loading the column with three to four column volumes of 0.05M EDTA, 0.5M sodium chloride, pH 7.0. Zn²⁺ was re-immobilized on the resin by loading the column with ZnCl₂ (3mg/ml) in PFW.

Proteins detected in eluants from the last three chromatographic IgA fractionation processes (i.e. DEAE, thiophilic and metal chelate) are listed in Table 1. The protein fractions were analyzed by nephelometry.

Table 1 Percentage content of proteins in eluates collected from DEAE, thiophilic and metal chelate chromatography.

Example 2: Immobilization of IgA on a Cu 2 + affinity column

IgA was immobilized on a metal affinity column loaded with Cu²⁺. After washing away the unbound material, IgA was eluted by either changing the buffer conditions, or using a gradient or stepwise reduction in pH to 3 or 4. Alternatively, a gradient of increasing concentration of (e.g. ammonium chloride, glycine, histamine, histidine, or imidazole) could be used for competitive elution of IgA. The purification process involved the following steps:

(1) Ethanol (8%) was added to plasma, and the supernatant was removed by filtration.

(2) The supernatant was delipidated with aerosil. (3) The delipidated solution was loaded onto a column packed with

DEAE Sepharose Fast Flow, and equilibrated with lOmM sodium acetate at pH 5.2. IgG and transferrin were removed in the void volume, and albumin was eluted with 25mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM and small amounts of IgG and albumin co-eluted in 0.5M sodium chloride pH 5.2.

(4) A solution of 1.7M ammonium sulfate, 0.1M sodium acetate was added to an equal volume of the aqueous material eluted from the DEAE column with 0.5M sodium chloride at pH 5.2. The solution was adjusted to pH 6.0 - 7.5, and the final conductivity was between 118-124 mS/cm at 25°C. (5) The solution described in step 4 was loaded onto a column packed with thiophilic resin, which had been equilibrated with 0.85M ammonium sulfate, 0.05M sodium acetate, pH 6.0-7.5, conductivity 118-124 mS/cm at 25°C.

(6) IgA rich material was eluted with 0.012M sodium acetate, 0.1M ammonium sulfate, pH 6.0 - 7.5, conductivity 19-22 mS/cm.

(8) After every three cycles, the column was regenerated with three to four column volumes of 20% isopropanol, 0.01M sodium hydroxide.

(9) The IgA rich material eluted from the thiophilic column (step 6) was dialyzed against 0.05M Tris HC1, 0.15M sodium chloride, pH 7.5 - 8.0.

(10) A column was packed with chelate agarose, and regenerated with 0.05M EDTA, 0.5M sodium chloride, pH 7.0. (11) Cu²⁺ was immobilized on the resin by loading the column with a solution containing CuSO₄.5H₂0 (3mg/ml) in PFW. Cu²⁺ loading was optimized by increasing the flow rate during the loading process. The resin was saturated with Cu²⁺ when the column pH returned to the pH of the metal ion solution. The pH of the loading solution should be neutral or weakly acidic to avoid hydroxy precipitates. (12) The column was flushed with three column volumes of PFW to remove hydroxy precipitates that may have formed and to remove excess Cu²⁺ ions not immobilized on the agarose support.

(13) The column was equilibrated with five to seven column volumes of 0.05M Tris HC1, 0.15M sodium chloride, pH 7.5 - 8.0.

(14) The dialyzed solution described in step 9 was loaded onto the immobilized copper affinity resin.

(15) A solution that is more than 90% IgA (w/w) was eluted from the column with 0.01M histidine pH 6.0.

(16) The column was flushed with three column volumes of PFW.

After each cycle, the resin was regenerated by loading the column with three to four column volumes of 0.05M EDTA, 0.5M sodium chloride, pH 7.0. Cu²⁺ was re-immobilized on the resin by loading the column with CuSO₄.5H₂0 (3mg/ml) in PFW.

Proteins detected in eluants from the last three chromatographic IgA fractionation processes (i.e. DEAE, thiophilic and metal chelate) have been compiled in Table 2. The protein fractions were analyzed by nephelometry.

Table 2

Percentage content of proteins in eluates collected from DEAE, thiophilic and metal chelate chromatography.

Example 3: Immobilization of contaminating proteins on Mg²⁺, Mn²⁺ and Ca²⁺ affinity columns

Protein samples were loaded onto immobilized Mg ⁺, Mn ⁺ and Ca ⁺ affinity columns, and IgA was recovered from the non-bound fraction. The purification process involved the following steps:

DEAE Sepharose Fast Flow, and equilibrated with lOmM sodium acetate at pH 5.2. IgG and transferrin were removed in the void volume, and albumin was eluted with 25mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM and small amounts of IgG and albumin co-elute in 0.5M sodium chloride pH 5.2.

(4) A solution of 1.7M ammonium sulfate, O.lM sodium acetate was added to an equal volume of the aqueous material eluted from the DEAE column with 0.5M sodium chloride at pH 5.2. The solution was adjusted to pH 6.0 - 7.5, and the final conductivity was between 118-124 mS/cm at 25°C. (5) The solution described in step 4 was loaded onto a column packed with thiophilic resin, which has been equilibrated with 0.85M ammonium sulfate, 0.05M sodium acetate, pH 6.0 - 7.5, conductivity 118-124 mS/cm at 25°C.

(6) IgA rich material was eluted with 0.012M sodium acetate, O.lM ammonium sulfate, pH 6.0-7.5, conductivity 19-22 mS/cm.

(10) A column was packed with chelate agarose, and regenerated with 0.05M EDTA, 0.5M sodium chloride, pH 7.0. (11) Either Mg²⁺, Mn²⁺ or Ca²⁺ were immobilized on the resins by loading the column with a solution containing either MgCl₂.6H₂0 (5mg/ml), MnCl₂.4H₂0 (5mg/ml) or CaCl₂.2H0 (3mg/ml) in PFW. Mg²⁺, Mn²⁺ or Ca²⁺ loading was optimized by increasing the flow rate during the loading process. The resins were saturated with Mg ⁺, Mn ⁺ or Ca²⁺ when the column pH returned to the pH of the metal ion solution. The pH of the loading solution should be neutral or weakly acidic to avoid hydroxy precipitates.

(12) The column was flushed with three column volumes of PFW to remove hydroxy precipitates that may have formed and to remove excess Mg²⁺, Mn ⁺ or Ca²⁺ ions not immobilized on the agarose support.

(14) The dialyzed solution described in step 9 was loaded onto an immobilized Mg²⁺, Mn^z+ or Ca²⁺ affinity resin.

(15) The column was flushed with three column volumes of 0.05M Tris HC1, 0.15M sodium chloride, pH 7.5 - 8.0, and a solution that is more than 90% IgA (w/w) was collected from the void volume.

(16) The column was flushed with three column volumes of PFW.

(17) The column was regenerated with O.lM sodium acetate, 0.8M sodium chloride, pH 4.5.

(18) After each cycle, the resins were regenerated by loading the column with three to four column volumes of 0.05M EDTA, 0.5M sodium chloride, pH 7.0. Mg²⁺, Mn²⁺ or Ca²⁺ were re-immobilized on the resin by loading the column with either MgCl₂.6H₂0 (5mg/ml), MnCl₂.4H₂0 (5mg/ml) or CaCl₂.2H₂0 (3mg/ml) in PFW.

Proteins detected in eluants of the DEAE and thiophilic resins and the void of the metal chelate resins are listed in Table 3. The protein fractions were analyzed by nephelometry.

Table 3

Percentage of proteins detected in eluates collected from DEAE and thiophilic chromatography and the non-bound fraction after metal chelate chromatography.

Example 4: Immobilization of contaminating proteins on Ni²⁺, Zn²⁺, Mg ⁺, Mn²⁺ and Ca²⁺ affinity columns

In this example, protein binding to immobilized Ni , Zn 2 + , Mg 2 + , Mn 2 + and

Ca ,2 + affinity resins was reduced by loading and equilibrating the thiophilic eluant on a column with a phosphate buffer instead of a Tris buffer. The following example is offered for illustration purposes only, and is not intended to limit or define the use of buffers to increase or decrease the binding capacity of proteins to the immobilized metal ions.

(1) Ethanol (8%) is added to plasma, and the supernatant is removed by filtration.

(2) The supernatant is delipidated with aerosil.

(3) The delipidated solution is loaded onto a column packed with DEAE Sepharose Fast Flow, and equilibrated with lOmM sodium acetate at pH 5.2. IgG and transferrin are removed in the void volume, and albumin is eluted with 25mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM and small amounts of IgG and albumin co-elute in 0.5M sodium chloride pH 5.2.

(4) A solution of 1.7M ammonium sulfate, O.lM sodium acetate is added to an equal volume of the aqueous material eluted from the DEAE column with 0.5M sodium chloride at pH 5.2. The solution is adjusted to pH 6.0 - 7.5, and the final conductivity is between 118-124 mS/cm at 25°C.

(5) The solution described in step 4 is loaded onto a column packed with thiophilic resin, which has been equilibrated with 0.85M ammonium sulfate, 0.05M sodium acetate, pH 6.0 - 7.5, conductivity 118-124 mS/cm at 25°C. (6) IgA rich material is eluted with 0.012M sodium acetate, O.lM ammonium sulfate, pH 6.0 - 7.5, conductivity 19-22 mS/cm. (7) Thiophilic resin is regenerated with three to four column volumes of 0.05M Tris acetate, pH 8.0, conductivity 2.3-2.5 mS/cm, followed by three to four column volumes of pyrogen free water (PFW). (8) After three cycles, the column is regenerated with three to four column volumes of 20% isopropanol, 0.01M sodium hydroxide.

(9) The IgA rich material eluted from the thiophilic column (step 6) was dialyzed against 0.05M phosphate, 0.15M sodium chloride, pH 7.0 -7.5.

(11) Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺ and Ca²⁺ were immobilized on the resins by loading the columns with a solution containing either Ni(N03)2 (5mg/ml) ZnCl₂ (3mg/ml), MgCl₂.6H₂0 (5mg/ml), MnCl₂.4H₂0 (5mg/ml) or CaCl₂.2H₂0 (3mg/ml) in PFW. Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺ or Ca²⁺ loading was optimized by increasing the flow rate during the loading process. The resins were saturated with Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺ or Ca²⁺ when the column pH returned to the pH of the metal ion solution.

(12) The pH of the loading solution should be neutral or weakly acidic to avoid hydroxy precipitates. (13) The column was flushed with three column volumes of PFW to remove hydroxy precipitates that may have formed and to remove excess Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺or Ca²⁺ ions not immobilized on the agarose support. (14) The column was equilibrated with five to seven column volumes of 0.05M phosphate, 0.15M sodium chloride, pH 7.0 - 7.5. (15) The dialyzed solution described in step 9 was loaded onto the immobilized Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺ and Ca²⁺ affinity resins. (16) A solution that was more than 90% IgA [w/w] was collected from the non-bound fraction of immobilized Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺and Ca²⁺ affinity columns.

(17) The column was flushed with three column volumes of PFW.

(18) The column was regenerated with O.lM sodium acetate, 0.8M sodium chloride, pH 4.5.

(19) After each cycle, the resins were regenerated by loading the column with three to four column volumes of 0.05M EDTA, 0.5M sodium chloride, pH 7.0. Ni , Zn , Mg , Mn or Ca were immobilized on the resin by loading the column with either Ni(N0₃)₂ (5mg/ml) ZnCl₂ (3mg/ml), MgCl₂.6H₂0 (5mg/ml), MnCl₂.4H₂0 (5mg/ml) or CaCl₂.2H₂0 (3mg/ml) in PFW.

Proteins detected in eluants of the DEAE and thiophilic resins and the non-bound fraction of immobilized Ni , Zn , Mg , Mn and Ca affinity columns have been compiled in Table 4. The protein fractions were analyzed by nephelometry.

Table 4 Percentage of proteins detected in DEAE and thiophilic eluates and the non- bound fraction of immobilized Ni²⁺, Zn²⁺, Mg²⁺, Mn²⁺ and Ca²⁺ affinity columns.

All references cited above are incorporated herein in their entirity by reference.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims

Claims:

1. A method for increasing the ratio of IgA to at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM in a fluid, which method comprises exposing a fluid containing IgA and at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM to a metal ion matrix under conditions such that the IgA is adsorbed to the metal ion matrix; and selectively eluting the IgA from the metal ion matrix.

2. A method for increasing the ratio of IgA to at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM in a fluid which method comprises exposing a fluid containing IgA and at least one other protein selected from the group consisting of ceruloplasmin, alpha-antitrypsin, IgG and IgM to a metal ion matrix under conditions such that the at least one other protein is adsorbed to the metal ion matrix while the IgA is not substantially adsorbed to the matrix; and separating the fluid containing IgA from the metal ion matrix.

3. An improved method for purifying IgA from a plasma fraction which method comprises decreasing the concentration of at least one protein selected from the group consisting of IgG, IgM, complement (C3), haptoglobin, albumin, ceruloplasmin and alpha-antitrypsin in the plasma fraction to obtain a fraction enriched in IgA, the improvement comprising passing the fraction enriched in IgA through a metal ion matrix to further decrease the concentration of at least one protein selected from the group consisting of IgG, IgM, ceruloplasmin and alpha-antitrypsin .

4. A method as claimed in any one of claims 1 to 3 in which the matrix is selected from the group consisting of DEAE CL6B sepharose, DEAE fast flow sepharose, Q fast flow sepharose, Sepharose 4B, Sepharose 6B, Cibacron Blue 3GA-Sepharose CL-6B, epoxy-activated Sepharose 6B and Phenyl-Sepharose CL-4B.

5. A method as claimed in any one of claims 1 to 4 in which the metal ion is attached to the matrix by way of a chelate forming ligand.

6. A method as claimed in any one of claims 1 to 5 in which the chelate forming ligand is selected from the group consisting of bis-carboxymethyl amino groups, 1,4,7-triazacyclononane, iminodiacetate, tris (carboxymethyl) ethylenediamine, 2-hydroxy-3[N-(2-pyridylmethyl) glycine] propyl, α-alkyl nitrilotriacetic acid and carboxymethylated aspartic acid.

7. A method as claimed in any one of claims 1 to 6 in which the metal ion is selected from the group consisting of zinc, copper, nickel, iron, manganese, chromium, calcium, magnesium and cadmium.

8. A method as claimed in any one of claims 1 to 7 in which the fluid is derived from a human.

9. A method as claimed in any one of claims 1 to 8 in which the fluid is a Cohn II + Cohn III fraction.

10. A method as claimed in any one of claims 1 to 9 in which the obtained IgA solution is further purified by an additional chromatography process.

11. A method as claimed in any one of claims 1 to 10 in which the fluid starting material or the obtained IgA solution is subjected to a process for the deactivation of viruses.

12. An aqueous solution including IgA in an amount of at least 90% (w/w), IgG in an amount of between 0-10% (w/w), IgM in an amount of between 0- 10% (w/w), plasminogen in an amount less than 0.001 casein units, a conductivity lower than 60 mS/cm and a pH of between 5-9.

13. An aqueous solution as claimed in claim 12 in which the IgA, IgG and IgM are derived from a human.

14. An aqueous solution as claimed in claim 12 or claim 13 in which the aqueous solution is obtained by a method according to the first or second aspects of the present invention.