HK1174044B - Chromatographic method for purifying fc-containing proteins - Google Patents

Description

Chromatographic methods for purifying FC-containing proteins

The present invention relates to chromatographic methods for purifying proteins and reagents for use in such methods.

Biomolecules (e.g., proteins, polynucleotides, polysaccharides, etc.) are of increasing commercial importance as pharmaceuticals, diagnostic reagents, food additives, detergents, research reagents, and many other applications. The requirements for such biomolecules (e.g. proteins) have not generally been met by isolating the molecules from natural sources, but rather require the use of biotechnological production methods.

Biotechnological production of proteins usually begins with isolation of DNA encoding the desired protein and its cloning into a suitable expression vector. After transfection of the expression vector into suitable prokaryotic or eukaryotic expression cells and subsequent selection of the transfected cells, the transfected cells are cultured in a fermenter so that the desired protein is expressed. The cells or culture supernatant are then collected, post-treated and the proteins contained therein purified.

In the case of eukaryotic expression systems, i.e. when cultured using mammalian cells (e.g. CHO or NSO cells), the concentration of the desired protein in the cell culture or cell culture supernatant obtainable in the expression step has increased 100-fold over the last 15 years, for example. Whereas at the same time the binding capacity of the chromatographic material used in the subsequent protein purification step was increased only 3-fold. For this reason, there is a need to improve, optimize the purification process of biomolecules (especially proteins) to enable it to be carried out on an industrial scale.

In biopharmaceutical applications, for example the use of proteins as pharmaceuticals (e.g. therapeutic antibodies), the isolation of impurities is critical in addition to the yield of the product. Impurities can be divided into process-dependent and product-dependent impurities. The process-dependent impurities contain components of the host cell, such as proteins (host cell proteins, HCPs) and nucleic acids, which result from cell culture (e.g. media components) or from post-processing (e.g. salts or dissolved chromatographic ligands). Product-dependent impurities are molecular variants of the product with different properties, including simplified forms (e.g. precursors and hydrolytic breakdown products) and modified forms (e.g. formed by deamination, mis-glycosylation or mis-ligation of disulfide bridges). The product-dependent molecular variants also include polymers and aggregates. Other impurities are contaminants, meaning all other chemical, biochemical or microbial sources of matter that are not directly part of the production process. Contaminants are for example viruses which are not desired in cell culture.

In biopharmaceuticals, impurities can cause safety concerns. This concern is even more acute if the therapeutic protein is introduced directly into the blood stream by injection or infusion, which is often the case in biopharmaceuticals. Thus, host cell components may cause allergic reactions or immunopathological effects. Furthermore, impurities may also lead to undesired immunogenicity of the administered protein, i.e. it may cause an undesired immune response to the therapeutic agent in the patient and may even cause life-threatening anaphylactic shock. Therefore, a suitable purification method is required in order to reduce the undesirable substances to an insignificant level by this method.

On the other hand, the economic aspect of biopharmaceuticals is not negligible. Thus, the production and purification methods used should not compromise the economic value of the biopharmaceutical products produced therefrom. Furthermore, it is also important to establish a time scale for the new purification process: in addition to the impact on cost, the development of methods needs to be consistent with preclinical and clinical studies of drugs. Thus, for example, some preclinical trials and all clinical trials can only be initiated if the biopharmaceutical is of sufficient purity and in sufficient quantity.

The following standard method consists of four basic steps, which may be the starting point for the development of antibody purification processes that can be carried out on an industrial scale: in the first step the target protein is isolated, concentrated and stabilized ("capture"). In the second step the virus is removed and in the third step purification is performed, removing the main impurities (e.g. nucleic acids, other proteins and endotoxins). The last step removes any trace contaminants ("polishing").

In addition to the filtration and precipitation steps, the (column) chromatographic method is also important. Thus, capture is often by a step involving affinity chromatography purification. Accordingly, many known column chromatography methods and chromatography materials may be used therein.

Affinity chromatography matrices, hereinafter also referred to as affinity matrices, are used as stationary phases in the industrial purification of various substances. By means of the immobilization of the ligands, it is possible to specifically enrich and purify substances having a certain affinity for the particular ligand used. For industrial purification of antibodies (immunoglobulins), in particular monoclonal antibodies, it has proven effective to use immobilized protein a as an initial purification step. Protein A is a protein of about 41kDA from Staphylococcus aureus, with high affinity (10 for human IgG)^-8M-10^-12M) CH that binds to the Fc region of an immunoglobulin₂/CH₃A domain. In protein A chromatography, immunoglobulins with protein A binding Fc region from mobile phaseThe fusion protein specifically binds to the protein a ligand, which is covalently bound to a carrier (e.g., agarose gel). Protein a from staphylococcus aureus (wild-type protein a) and genetically modified recombinant protein a (rec.a) interact with the constant region (Fc fragment) of the antibody by non-covalent interactions. This specific interaction can be used to efficiently separate impurities from antibodies. The interaction between the antibody and the protein a ligand can be deliberately stopped by changing the pH, and the antibody can be released or eluted from the stationary phase.

If the stationary phase is washed after the column is packed, the effectiveness of affinity chromatography can be increased. This washing refers to the use of a mobile phase that elutes impurities (rather than the target product) from the stationary phase. In the case of affinity chromatography of antibodies using protein a matrices, wash buffers containing arginine, isopropanol, NaCl or detergents have been used (WO2008031020, WO2007109163, WO2007081906, WO2003066662, Millipore Tech Brief TB1026EN00), but these components have not been combined into a single wash buffer. Combinations of two of these ingredients (salt and detergent), i.e. polymers such as polyethylene glycol, polypropylene glycol and copolymers composed thereof, are disclosed in us patent 6,870,034B 2.

Summary of The Invention

It has surprisingly been found that the use of a specific combination of these components in a single wash buffer in protein a chromatography results in a higher purity of the target protein to be purified than if the same components were used individually. In addition, the wash buffer of the invention is suitable for antibody purification on standard substrates and the elimination of optimization steps shortens the process development.

The present invention relates to a method for removing impurities from a composition containing a protein comprising an Fc domain of an immunoglobulin (target protein) by protein a chromatography, comprising the steps of:

a. loading a mobile phase comprising a target protein onto a stationary phase comprising protein a under conditions wherein the target protein binds to the stationary phase;

b. applying a wash buffer at a pH between 4 and 8 as mobile phase containing the following additives:

i. arginine with a concentration of 0.1-1mol/l,

sodium chloride in a concentration of 0.2 to 2mol/l,

an alcohol selected from the group consisting of isopropanol, n-propanol and ethanol at a concentration of 5-30% (w/v), and

polyvinylpyrrolidone and/or detergent in a concentration of 0.05-2% (w/v);

c. under conditions in which the target protein is eluted from the stationary phase, an elution buffer is used as the mobile phase.

In another aspect, the wash buffer pH is 4.5 to 8. In another aspect, the wash buffer pH is 5 to 8. In another aspect, the wash buffer pH is 6 to 8.

Preferably, the arginine concentration in the wash buffer is between 0.4 and 0.6mol/l, in particular 0.5 mol/l. The concentration of the sodium chloride in the washing buffer is preferably 0.9 to 1.1mol/l, in particular 1 mol/l. The alcohol used in the washing buffer is preferably isopropanol, at a concentration of 10-20% (v/v), in particular 15% (v/v).

Preference is given to using polyvinylpyrrolidone (PVP) in a concentration of 0.1-2% (w/v), in particular 0.25% (w/v). Additionally or alternatively, polyoxyethylene-sorbitan-monolaurate (Polysorbat 20, Polysorbat 80) may be used in a concentration of 0.05-2% (w/v).

In another aspect, the invention relates to a wash buffer for affinity chromatography having a pH of 4 to 8 comprising:

i. arginine with a concentration of 0.1-1mol/l,

sodium chloride in a concentration of 0.2 to 2mol/l,

an alcohol selected from the group consisting of isopropanol, n-propanol and ethanol at a concentration of 5-30% (v/v), and

polyvinylpyrrolidone or detergent at a concentration of 0.05-2% (w/v).

Drawings

FIG. 1 shows the yield (1a), turbidity (1b), monomer content (1c) and amount of HCP (1d) in the eluate of affinity chromatography after washing with different combinations and different compositions of wash buffer, exemplified by antibody BI-MAb 06 a. The values on the X-axis refer to the additives specifically added to the wash buffer in the examples.

FIG. 2 shows the yield (2a), turbidity (2b), monomer content (2c) and amount of HCP (2d) in the eluate of affinity chromatography after washing with different combinations and different compositions of wash buffer, exemplified by antibody BI-MAb 1003 a. The values on the X-axis refer to the additives specifically added to the wash buffer in the examples.

FIG. 3 compares the amount of HCP in the eluate of affinity chromatography, exemplified by antibody BI-MAb 07c, after washing with different combinations and different compositions of wash buffer. The values on the X-axis refer to the additives specifically added to the wash buffer in the examples.

FIG. 4 compares the amount of HCP in the eluate of affinity chromatography, exemplified by antibody BI-MAb 1001b, after washing with different combinations and different compositions of wash buffer. The values on the X-axis refer to the additives specifically added to the wash buffer in the examples.

Detailed Description

The present invention relates to methods for removing impurities, in particular host cell proteins (HOPs) and DNA, from protein compositions obtained in cell culture in which the proteins are expressed recombinantly or endogenously. In particular, the present invention relates to a method for purifying or concentrating a protein (target protein) which is reversibly immobilized on a stationary phase by means of a ligand and is therefore suitable for affinity chromatography.

The target protein may in particular be an immunoglobulin or a protein comprising the Fc domain of an immunoglobulin and capable of binding to protein a. In a preferred embodiment, it is an immunoglobulin consisting of two each of the heavy and light chains of an immunoglobulin. Antibodies consist of two identical heavy chains (H) and two identical light chains (L) joined together by covalent disulfide bridges to form a Y-shaped structure. The light chains each consist of one variable domain and one constant domain, denoted VL and CL, respectively. On the other hand, the heavy chains each have one variable domain and three to four constant domains, which are similarly referred to as VH and CH1, CH2, CH3, respectively, depending on the immunoglobulin. The variable domains of the light and heavy chains form the antigen binding site. The CH2 domain contains sugar chains that form the binding sites for the complement system. The CH3 domain contains an Fc receptor binding site.

Protein a binds to the Fc domain of immunoglobulins through interaction with the heavy chain. Binding affinity was highest for human IgG1, IgG2 and IgG2a and murine IgG2 b. It binds with moderate affinity to human IgM, IgA, IgE as well as murine IgG3 and IgG 1.

However, it does not react with human IgG3, IgD or murine immunoglobulins IgM, IgA and IgE.

Target proteins to which the method of the invention is applicable are all those proteins having an Fc domain, such as immunoglobulins. The immunoglobulin may be a polyclonal or monoclonal antibody expressed in a hybridoma cell or a recombinant host cell. The antibodies may be prepared originally by immunization of animals, particularly mammals, including transgenic animals, such as mice expressing human immunoglobulins. However, suitable target proteins also include fusion proteins in which any desired protein has been fused to the Fc domain of an immunoglobulin.

The protein a matrix for the purposes of the present invention is an affinity chromatography matrix which contains immobilized proteins as ligands. They comprise an affinity matrix containing, as ligand, a wild-type protein A, for example from Staphylococcus aureus (Staphylococcus aureus). The description of protein a is found in particular in: lofdahl, S. et al, 1983(Lindmark, R., Thoren-Tolling, K., Sjoquist J (1983); Binding of immunoglobulins to protein A and immunoglobulins in mammalian sera: J immunomethods 1983 Aug 12; 62 (1): 1-13.) and Lindmark et al, 1983(Lindmark, R., Thoren-Tolling, K., Sjoquist J (1983); Binding of immunoglobulins to protein A and immunoglobulins in mammalian sera: JImmunol Methods 1983 g 12; 62 (1): 1-13). In addition, the invention relates to a matrix using protein A produced by recombinant methods as a ligand. Recombinant protein A is disclosed, for example, in Duggleby C.J. and Jones, S.A.,1983(Duggleby, C.J.and Jones, S.A. (1983), Cloning and expression of Staphylococcus aureus protein A gene in Escherichia coli.Nucl.acid.Res.1983 May 25; 11 (10): 3065-76) or Li, R.et al, 1998(Li, R.Down, V.J., Stewart, D.J., Burton, S.J.Lowe, C.R., Design, Synthesis and application of aprotein A mimicry.Nat.Biotechnol.1998 Feb; 16 (2): 190-5) and are known in the art.

The protein a may be coupled to a variety of support materials, such as agarose, polysaccharides, dextran, silica gel, and glass beads. A non-limiting list of suitable carrier materials can be found in Harlow, e.and Lane, d.1999. A support material formed of agarose-based material is commonly used, namely "agarose gels (sepharoses)" produced by Amersham Pharmacia Biotech, Uppsala, Sweden, which is well known to those skilled in the art. Specific examples of protein A sepharoses can be found in the handbook written by the company in 2001, having the subject of "Affinity Chromatography". In addition, other protein A chromatography matrices are known to those skilled in the art, such as MabSelect (Amersham Pharmacia Biotech, Uppsala, Sweden), STREAMLINE^TMrProtein A (Amersham Pharmacia Biotech, Uppsala, Sweden), Poros A (Millipore, Durham, England). The process of the invention comprises treatment with a corresponding substrate, the list of which is given by way of illustration and not exhaustive.

Coupling of ligands is typically performed by activation of free amino, carboxyl or thio groups with cyanogen bromide, NHS or coupling thiols to the support matrix. For this subject, reference is made, for example, to the "affinity chromatography" handbook, Amersham Pharmacia Biotech, Uppsala, Sweden, 2001.

In a particularly preferred embodiment, polyvinylpyrrolidone (PVP, also known as povidone or povidone, CAS: 9003-39-8) is used in the wash buffer of the present invention. Polyvinylpyrrolidone is a water-soluble polymer consisting of N-vinylpyrrolidone monomer units. However, it can also be dissolved in other polar solvents. PVP is a hygroscopic amorphous powder that is white to light yellow in color. Standard commercially available polymers have a molar mass of about 2500 to 2,500,000 daltons.

Detergents may be used in addition to or in place of PVP in the wash buffer of the invention. Detergents are surfactants and amphiphilic molecules that can form micelles, i.e., aggregates of detergent molecules, with the hydrophilic head facing outward toward the aqueous solvent.

Detergents for the purposes of the present invention are both nonionic and ionic substances. However, nonionic detergents are preferred, such as polyethylene glycol ethers (type NP-40, Tergitol NP40, CAS: 127087-87-0), PEG-alkyl ether polyoxyethylene (23) lauryl ether (CAS: 9002-92-0), PEG-sorbitan fatty acid esters, such as polyoxyethylene (20) sorbitan-monolaurate (Polysorbat 20, CAS: 9005-64-5) or polyoxyethylene (20) sorbitan-monooleate (Polysorbat 80, CAS: 9005-65-6), alkylphenyl-PEG-ethers, such as tert-octylphenoxypolyethoxyethanol (Triton-X-100, CAS: 9002-93-1) or PEO-PPO block copolymers (Poloxamer derivatives), such as polyoxyethylene-polyoxypropylene block copolymer (Pluronic F68, lutrol F68, Poloxamer188, CAS: 9003-11-6 or Poloxamer 407, Pluronic F127, Lutrol F127, CAS: 9003-11-6).

The pH of the wash and elution buffers depends on the overall system and is therefore typically determined and optimized independently for each system.

In one aspect, the wash buffer pH is 4 to 8. It is well known to those skilled in the art that the pH at which a protein elutes from a column depends on a number of factors. These factors may include, inter alia, the buffer system used in binding, washing and elution, the presence of impurities, the geometry of the matrix particles, the nature of the coupling of the affinity ligand to the chromatography matrix. In particular the specificity of the protein is of decisive influence.

In some cases, there may be combinations that allow partial or complete elution of the protein from the affinity ligand even at a pH greater than 4. In this case, and if the loss of protein is unacceptable, the wash buffer must have a higher pH. It is clear to the person skilled in the art that in this case the pH of the wash buffer has to be adjusted accordingly. In more cases, however, the binding between protein and protein a affinity ligand will only be released at a pH below 4.

In another aspect, the wash buffer pH is 4.5 to 8. In another aspect, the wash buffer pH is 5 to 8. In another aspect, the wash buffer pH is 6 to 8.

In one exemplary embodiment, the invention may be carried out, for example, as follows:

protein a chromatography is typically the first purification step. The cell culture supernatant, free of cells, can be added directly to the column, or the concentrate can be prepared first by means of an ultrafiltration membrane (so-called UF/DF system).

The protein a column was first equilibrated with Phosphate Buffered Saline (PBS), which is roughly physico-chemically identical to the loading solution (charging pool). The column feed consists of the supernatant from a cell culture containing the product to be purified and other medium components necessary for the growth of the mammalian cells in question (e.g.CHO or NSO cells). Loading the cell-free supernatant from the cell culture or concentrate thereof onto the protein a column. The target protein is then bound to the protein a binding site on the column. The column is then washed with equilibration buffer until unbound media components and cell products are eluted. The disclosed wash buffer can be added after the equilibration buffer or directly after loading the column. The amount of wash buffer depends on the scale and is therefore given in an amount corresponding to the size of the column.

Typically, the volume of wash buffer is about 2 to 5 times the column volume (2-5 Bed Volumes (BV)). After addition of the wash buffer, the column can be treated again with the equilibration buffer or with another buffer so that no component of the wash buffer is eluted with the product. The buffer used herein must have a pH lower than the wash buffer but higher than the elution buffer, and similar to the pH of the buffer salts and elution buffers in the composition. A buffer at a pH below 4 is used for elution and may, for example, contain acetate or citrate as its main component. The elution buffer may contain acetate at a concentration of 10mM to 200mM, preferably 20mM to 100mM, most preferably 50mM to 100 mM; or may contain citrate at a concentration of 10 to 200mM, preferably 20 to 100 mM. The pH of both buffers should be in the above-mentioned pH range, i.e.below 4, preferably from 3 to 4, particularly preferably from 3.4 to 3.6. In addition, additives such as arginine or PVP may also be present. In addition, a glycine buffer may be used, the concentration of which is 10 to 200mM, preferably 25 to 100mM, and the pH of which is 2 to 3.5; or other buffers suitable for reducing binding of the Fc domain of the antibody to the protein a may be used. The eluate may then be subjected to further post-treatment, for example neutralization or incubation at low pH.

Examples

Experiments can be performed with a variety of proteins from different cell lines (CHO and NSO) which are fermented in different matrices and whose Fc parts belong to different IgG subtypes (IgG1, IgG2, IgG 4).

A test series starting from a standard was performed in which only the wash buffer was changed. Or by introducing an equilibration buffer without any additive, by using a wash buffer with only one additive, by adding several different buffers each with one additive in sequence, or by using a wash buffer with a combination of several additives.

The protein solution applied is always the same as the respective protein.

The impurities in the eluent were measured in each experiment. By comparison between these experiments, the wash buffer was determined which gave the smallest possible amount of each impurity in the eluent. Yield, haze and monomer content were also measured in some experiments.

Chromatography

Use automationThe chromatographic experiments were carried out on an FPLC Model 900 system (GE Healthcare). Four different products were used for this experiment. Of the products, cell-free culture supernatant or concentrated culture supernatant was used as a starting material, and the preparation was concentrated 10-fold with a 50kD Omega membrane (Pall) and then diafiltered 3 times with PBS.

The column used is in a volume of 1ml to 8ml and contains one of the chromatographic gels MabSelect or MabSelect Xtra (GEHealthcare).

Buffer solution

In all experiments, Phosphate Buffer Solution (PBS) at pH 7.4 containing 10mM phosphate, 5mM potassium chloride and 140mM sodium chloride was used as an equilibration buffer.

All the wash buffers were based on PBS buffer (pH 7.4) containing:

8mmol/L sodium monohydrogen phosphate (sodium monohydrogen phosphate),

1.5mmol/L potassium hydrogen phosphate (potassium hydrogen phosphate),

2.7mM potassium chloride, and

140mM sodium chloride.

The following substances are used as additives, alone or in various combinations (as indicated in the figure):

(1)860mmol/L sodium chloride (total content 1mol/L sodium chloride)

(2)0.25% (w/V) polyvinylpyrrolidone (PVP)

(3)15% (V/V) Isopropanol

(4)0.5mol/L of L-arginine

For elution, different buffers were used, with different acetate concentrations and pH, depending on the product. The following concentrations were used:

BI-Mab 06 a: 100mM acetate, pH 3.4

BI-Mab 1003 a: 50mM acetate, pH 3.4

BI-Mab 1001 b: 50mM acetate, pH 3.4

BI-Mab 07 c: 50mM acetate, pH 3.6

Analysis of

The eluates of these experiments were compared for impurity content and the yield was determined.

To determine the amount of cellular components, a common sandwich ELISA was used, and host cell proteins were measured as empirical parameters. For this analysis, polyclonal detection antibodies were used.

Monomer content was determined using an Agilent Series HPLC 1200(Waters) system and TSK 3000SW or TSK 3000SWXL columns (TosoH) were used depending on the protein. The gradient method was performed with Tris buffer pH 7.0 at a flow rate of 1 ml/min.

DNA was determined by the threshold method (Kung, V.T. et al, Picogram quantification of TotalDNA Using DNA-Binding Proteins in a Silicon Sensor-Based System, anal. biochem.1990,187, 220-227).

SDS-PAGE was performed using the Phast system (GE Healthcare). Samples were separated using Phast SDS gels (4% -15%, GE). Staining was carried out using the Heukeshoven silver stain method (Heukeshoven, Dernick 1988, Electrophoresis 9(1), pages 28-32).

To determine the amount of antibody on the column and in the eluate, PA 2-1001-00 protein A columns (applied BioSystems) and Agilent Series 1200HPLC systems (Waters) were used. Binding and elution were performed via a gradient from pH 7.4 to pH 2.8 in a PBS buffer system, and the measured antibodies were evaluated using an external calibration curve.

After calibration with the manufacturer's turbidity standards, the turbidity was measured using a 2100AN Turbimideter (Hach).

Results

Experiments were performed with the IgG1 subtype antibody BI-MAb 06a

The results show that the wash has a significant effect on yield, monomer, turbidity and Host Cell Protein (HCP).

The eluates showed the best values after washing with buffer containing all four additives in the quality standards for yield, monomer content and HCP removal. In addition, the combination of additives, either the combination of three components of 0.86mol/L sodium chloride, 0.25% (w/V) PVP and 15% (V/V) isopropanol or the combination of all four additives, 0.86mol/L sodium chloride, 0.25% (w/V) PVP, 15% (V/V) isopropanol and 0.5mol/L arginine, resulted in a significant reduction in turbidity.

Experiments were performed with the IgG1 subtype antibody BI-MAb 1003a

Optimal HCP removal was achieved with a wash buffer containing all four additional components. Similarly, a wash buffer containing a combination of sodium chloride, PVP and isopropanol also gave better values. Washing with buffer containing one of the four components alone resulted in more HCP.

The same is true of the turbidity results, the combination of the washing substances in the buffer gives better results.

Experiments with the IgG4 subtype antibody BI-MAb 07c

The eluate from the assay with a single wash buffer contains a higher concentration of HCP than the eluate from the assay with the combined wash buffers.

Experiments were performed with the IgG2 subtype antibody BI-MAb 1001b

The eluate from the assay with a single wash buffer contains a higher concentration of HCP than the eluate from the assay with the combined wash buffers.

These four experiments show that combining four additives (sodium chloride, PVP, isopropanol and arginine) in a single wash buffer has advantages over the use of the additive alone in the content of Host Cell Protein (HCP) in the eluate.

The use of the disclosed wash buffer also showed a significant decrease in turbidity of the eluate. The amount of monomer and yield are also increased (BI-MAb 06a), or at least kept at the same level (BI-MAb 1003 a).

The disclosed wash buffer is therefore suitable for significantly improving the protein production process. The introduction of a wash buffer combination removes impurities that may require additional purification steps to remove.

Claims (12)

1. A method for removing impurities from a composition containing a protein comprising an Fc domain of an immunoglobulin (target protein) by protein a chromatography, the method comprising the steps of:

a. loading a mobile phase comprising a target protein onto a protein a-containing stationary phase under conditions in which the target protein binds to the stationary phase;

b. applying a wash buffer as mobile phase having a pH between 6 and 8, comprising the following components:

i. arginine with a concentration of 0.1-1mol/l,

sodium chloride in a concentration of 0.2 to 2mol/l,

an alcohol selected from the group consisting of isopropanol, n-propanol and ethanol at a concentration of 5-30% (w/v), and

polyvinylpyrrolidone in a concentration of 0.05-2% (w/v);

c. under conditions in which the target protein is eluted from the stationary phase, an elution buffer is used as the mobile phase.

2. Method according to claim 1, characterized in that the arginine concentration in the washing buffer is between 0.4 and 0.6 mol/l.

3. The method according to claim 1, characterized in that the concentration of the sodium chloride in the washing buffer is 0.9-1.1 mol/l.

4. The method according to claim 2, characterized in that the concentration of the sodium chloride in the washing buffer is 0.9-1.1 mol/l.

5. The method according to claim 1, characterized in that the alcohol in the washing buffer is isopropanol and its concentration is 10-20% (w/v).

6. The method according to claim 2, characterized in that the alcohol in the washing buffer is isopropanol and its concentration is 10-20% (w/v).

7. The method according to claim 3, characterized in that the alcohol in the washing buffer is isopropanol and its concentration is 10-20% (w/v).

8. The method according to claim 4, characterized in that the alcohol in the washing buffer is isopropanol and its concentration is 10-20% (w/v).

9. A method according to any of the preceding claims, characterized in that the concentration of polyvinylpyrrolidone (PVP) is 0.1-2% (w/v).

10. The method according to any one of claims 1 to 8, characterized in that said impurity is a Host Cell Protein (HCP).

11. The method of claim 9, characterized in that said impurity is a Host Cell Protein (HCP).

12. A wash buffer for affinity chromatography having a pH of 6 to 8 comprising:

i. arginine with a concentration of 0.1-1mol/l,

sodium chloride in a concentration of 0.2 to 2mol/l,

an alcohol selected from the group consisting of isopropanol, n-propanol and ethanol at a concentration of 5-30% (v/v), and

polyvinylpyrrolidone in a concentration of 0.05-2% (w/v).