Method for purifying an immunoglobulin g degrading enzyme (IDES) from streptococcus pyogenes

Abstract

The present disclosure relates to a method for purifying an immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, preferably for removing IdeS charge variants and IdeS obtainable from said method.

Description

[0001]METHOD FOR PURIFYING AN IMMUNOGLOBULIN G DEGRADING ENZYME (IDES) FROM STREPTOCOCCUS PYOGENES TECHNICAL FIELD The present disclosure relates to a method for purifying an immunoglobulin degrading enzyme (IdeS) from Streptococcus pyogenes, preferably for removing IdeS charge variants and IdeS obtainable from said method. BACKGROUND IdeS (immunoglobulin G (IgG)-degrading protease) is an enzyme originating from Streptococcus pyogenes hydrolyzing IgG and can represent a great interest for therapeutic applications where IgG such neutralizing antibodies (NAbs) clearance is required. IdeS is a cysteine protease of the papain superfamily cleaving human IgG in positions 236 and 237 of the lower hinge region of IgG heavy chains in a two-step mechanism. It first cleaves one of the heavy chains generating a single-cleaved IgG molecule (scIgG) and then cleaves the remaining heavy chain, leading to the release of one F(ab’)2 fragment and one non-covalently linked homodimeric Fc fragment. The lower hinge region of IgG is critical for interaction with Fc receptors and complement binding, hence this enzymatic activity can prevent IgG mediated immune response. IdeS was first identified as a secreted immunogenic protein form by Lei B., et al. (2000). Infection and immunity, 68(12), 6807–6818. The function as an IgG-degrading protease was first identified by von Pawel-Rammingen et al. EMBO J.2002 Apr 2;21(7):1607-15. In this publication, IdeS was produced as a GST tagged protein in E. coli. IdeS is expressed as a 38 kDa protein containing a single cysteine residue (Genbank reference, QJC39417.1). Amino acids 1-29 constitute the signal sequence and is cleaved off upon secretion, leaving a 35 kDa mature protein of PI of 6.13. IdeS protein sequence contains also only one cysteine (no disulfide bonds). The first 3D structure of IdeS C94S variant was solved by Wenig, K., et al. (2004). Proceedings of the National Academy of Sciences of the United States of America, 101(50), 17371–17376. E. coli is a frequently used expression system for recombinant pharmaceutical proteins (Walsh G. (2018). Nature biotechnology, 36(12), 1136–1145). High-level expression of recombinant protein expression usually involves the formation of misfolded forms of the protein, commonly referred to as inclusion bodies that necessitates its refolding under enabling conditions to generate an active protein (Buscajoni, L., et al. (2022). Biotechnology advances, 61, 108050). Soluble production of recombinant proteins in E. coli cytoplasm can be hampered by the presence of modifying of degrading host cell proteins (HCPs) that are predominant and require the design of a specific downstream process for their removal. In solution, IdeS is reported as a monomer protein, but presence of high molecular particles can be also retrieved (Vindebro, R., et al. (2013). FEBS letters, 587(12):1818–1822). However, IdeS presents in its sequence L-asparagine (Asn), L-glutamine (Gln) residues that can be subjected to deamidation, a post-translational modification influenced by factors such as protein structure (primary, secondary and higher structure), temperature and pH and Methionine (Met) that can be subjected to oxidation. These modifications introduce negative charges (deamidation) or modify IdeS polarity (Met oxidation). These modifications can influence the structure and the function of the protein. The presence of inactive aggregates and / or charge variants represents a challenge for the design of the manufacturing process since these forms are difficult to separate from the main active component. SUMMARY The inventors have designed and developed a specific purifying method of recombinant IdeS obtained in E. coli allowing the enrichment of IdeS protein and separation of host cell protein, host nucleic acids and IdeS charge variants. The inventors unexpected showed that a three-step chromatography including anion exchange chromatography, hydrophobic interaction chromatography and hydrophobic cation exchange chromatography allow to obtain an enrichment to more than 50% of IdeS with a high clearance of contaminants (host cell DNA and proteins) with no more than 50 ng / mg of host cell protein and less than 3 pg / mg of residual DNA that represents an acceptable level to address safety for human use. The present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a chromatography anion ligand, preferably quaternary ammonium ligand, optionally washing said anion exchange chromatography with a wash buffer, preferably comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with an elution buffer, preferably a gradient salt buffer comprising a neutral salt, more preferably NaCl at a concentration comprised between 55 to 100 mM. ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a chromatography hydrophobic ligand, preferably butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, preferably wherein said feed solution and wash buffer comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer, preferably comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M. iii) contacting a feed solution comprising the eluted fraction of step ii) with a mixed mode chromatography, preferably a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a chromatography mixed mode ligand, preferably 4-aminobenzamidacetic acid ligand, preferably wherein said feed solution comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer, preferably which is a gradient salt buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M or 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. In a preferred embodiment, the initial solution is contacted with the anion exchange chromatography in step i) with a load of initial solution comprised between 20 and 35 g / L, more preferably 25 and 30 g / L of total protein per L of resin, again more preferably no more than 28 g / L and / or with a load conductivity comprised between 3 and 4 mS / cm, preferably 3 and 3.5 mS / cm, more preferably between 3.1 and 3.3 mS / cm to remove the IdeS charge variants, in particular IdeS acidic charge variants. In a particular embodiment, said initial solution is a lysate of a cell expressing a recombinant IdeS or any variant thereof, preferably expressing a codon-optimized sequence comprising or consisting of SEQ ID NO: 3, in a bacterial cell, preferably E. coli. In a preferred embodiment, IdeS or any variants thereof comprised in the initial solution of step i) is not precipitated. In a preferred embodiment, the initial solution is contacted with the anion exchange chromatography in step i) at a load flow rate comprised between 100 and 300 cm / h, preferably 150 and 200 cm / h, more preferably at 180 cm / h and / or with a load of initial solution comprised between 25 and 95 g / L, preferably 30 and 45 g / L, more preferably 32 and 42 g / L of total protein per L of resin. In a particular embodiment, the anion exchange chromatography is pre-equilibrated with an equilibration buffer comprising a neutral salt, preferably NaCl at a concentration comprised between 1 to 10 mM, preferably 2 to 6 mM, more preferably 4 mM. In another particular embodiment, the hydrophobic interaction chromatography is pre-equilibrated with an equilibration buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M. In a preferred embodiment, the feed solution is contacted with the hydrophobic interaction chromatography in step ii) with a load of feed solution comprised between 20 and 40 g of total protein per L of resin. In another preferred embodiment, the feed solution is contacted with the mixed mode chromatography in step iii) with a load of sample comprised between 12 and 20 g of total protein per L of resin. FIGURE LEGENDS Figure 1: Separation profile using Capto Q anion exchange chromatography resin. A. Standard separation profile including a 7.5mM NaCl wash step and a 90 mM NaCl elution step, B. Optimized separation profile. including 4mM NaCl condition during binding step, 25mM NaCl during wash step and 90 mM NaCl elution step Figure 2: Anion exchange chromatography (AEX) separation performances. Figure 3: SDS-PAGE analysis of AEX elution pools. A. Standard conditions, B. Optimized conditions. Figure 4: Separation profile using Capto Butyl anion exchange chromatography resin. Figure 5: HCP clearance factor of Capto-Butyl step Figure 6: Overall DSP process performance for IdeS purification. Figure 7: Example of charge variants distribution profile in AEX-HPLC for IdeS GMP DS batch #8010122001 Figure 8: Evolution of the distribution of charge variants in IdeS non GMP DP batch DP22-072 after 0 (T0M, 5°C), 1 month (T1M,5°C) and 6 months (T6M, 5°C) of storage at 5 ± 3°C. Figure 9: Evolution of the distribution of charge variants in IdeS non GMP DP batch DP22-072 and correlation with potency (a) or subvisible particles content (b) after 0 (T0M, 5°C), 1 month (T1M,5°C) and 6 months (T6M, 5°C) of storage at 5 ± 3°C Figure 10: Basic and acidic 3 charge variant profile obtained during development and comparison with IdeS GMP DS. Figure 11: SDS-PAGE analysis of AEX flow-through fractions using different loading capacity (42 (A&B) or 28 (C) g of total protein per Liter of resin (g / Lr)) and different clarified load conductivities: 4.6 mS / cm (A); 3.1 mS / cm (B) and 3.2 mS / cm (C). Narrow indicates protein mass corresponding to IdeS. Figure 12: Acidic 3 variant content (%) present in IdeS eluted pool following AEX chromatogrphy performed using different loading capacity (42 or 28 g of total protein per Liter of resin (g / Lr)) and different clarified load conductivities: from 2.7 to 4.6 mS / cm. DETAILED DESCRIPTION The predominant presence of inactive aggregates and charge variants represents a challenge for the design of the manufacturing process since these forms are difficult to separate from the main active component. To solve this issue, the inventors have developed a process of manufacturing combining anion exchange chromatography, hydrophobic interaction chromatography and mixed mode chromatography that allow to enrich the solution highly efficiently with IdeS without host cell protein and charge variants. The present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution comprising IdeS with an anion exchange chromatography under conditions thar allow the IdeS to bind to a chromatography anion ligand, optionally wash said anion exchange chromatography with a wash buffer and elution the fraction comprising IdeS with an eluting buffer, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions thar allow the IdeS to bind to a chromatography hydrophobic ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, and eluting the fraction comprising IdeS with an elution buffer, iii) contacting a feed solution comprising an eluted fraction of step ii) with a mixed mode chromatography, preferably a hydrophobic cation exchange chromatography under conditions thar allow the IdeS to bind to a chromatography mixed mode ligand, optionally washing said hydrophobic cation exchange chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer. IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes) (GenBank: QJC39417, updated on April 20, 2020; SEQ ID NO: 1 (IdeS protein without peptide signal)) is an extracellular cysteine protease produced by the human pathogen S. pyogenes. IdeS digests IgG at a specific site below the hinge region. IdeS cleaves some subclasses of IgG in various animals and efficiently converts IgG into Fc and (Fab)2fragments. The N-terminal methionine and the signal sequence of IdeS are typically removed to form the mature IdeS protein. Amino acids 1-29 constitute the signal sequence and is cleaved off upon secretion, leaving a 35 kDa mature protein of PI of 6.13. IdeS is encoded by IdeS gene (GenBank: JN035367, updated on July 06, 2011, SEQ ID NO: 2). In another embodiment, the cysteine protease can be an IdeS functional variant, which retains the cysteine protease activity. IdeS activity may be assessed by any suitable method, for example, by incubating an IdeS variant with a sample containing IgG and determining the presence of IgG cleavage products (i.e., Fc and F(ab’)2 fragments) by using for example SDS-PAGE. According to the present disclosure, the term “IdeS” refers to IdeS protein as described above and any functional variants thereof. Said IdeS functional variant comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with SEQ ID NO: 1. As used herein, the term “sequence identity” or “identity” refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimally along the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 30 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. The terms apply to amino acid polymers containing naturally occurring amino acids as well as amino acid polymers in which one or more amino acid residues are a non-naturally occurring amino acid or a chemical analogue of a naturally occurring amino acid. An amino acid polymer may contain one or more amino acid residues that has been modified by one or more natural processes, such as post-translational processing, and / or one or more amino acid residues that has been modified by one or more chemical modification techniques known in the art. The IdeS functional variant of the invention refers to an amino acid sequence having an amino acid sequence that differs from a native sequence by less than 30, 25, 20, 15, 10 or 5 substitutions, insertions and / or deletions. In a preferred embodiment, the variant differs from the native sequence by one or more conservative substitutions, preferably by less than 15, 10 or 5 conservative substitutions. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Production of IdeS The initial solution comprising the IdeS to be separated can be obtained from a lysate of a biological sample or from a lysate of a cell culture capable of synthesising the recombinant protein. The various methods for synthesising a protein by recombinant methods are well known to those skilled in the art and in particular consist of introducing into the cell an expression cassette comprising a sequence encoding the IdeS or a functional variant thereof under the control of regulatory elements necessary for its expression. In a preferred embodiment, the IdeS or a functional variant thereof may be produced by transforming a cell, typically a bacterial cell (e.g., E.coli), with an expression cassette, preferably a plasmid encoding the IdeS or a functional variant thereof. The coding sequence of IdeS or functional variant thereof may be easily determined by the skilled person based on the polypeptide sequence. In a preferred embodiment said nucleic acid molecule may comprise a codon-optimized sequence encoding IdeS or a functional variant thereof, more preferably said codon-optimized sequence encoding IdeS comprises or consists of SEQ ID NO: 3. In a particular embodiment, said plasmid comprises an antibiotic resistance gene such as kanamycin resistance gene to allow the selection of transformed E.coli. In another particular embodiment, said plasmid does not comprise an antibiotic resistance gene. In a more preferred embodiment, IdeS coding sequence can be induced for four hours, for example by cloning IdeS coding sequence under the lac operator and inducing expression by isopropyl -β-D-thiogalactopyranoside (IPTG). Preferably, the cells are separated for instance by centrifugation then lysed. Cell lysis is a technique well known to those skilled in the art and can be carried out chemically, mechanically or enzymatically. The cell debris can then be separated from the solution (i.e., initial solution) comprising recombinant product (i.e., IdeS), for example by centrifugation. The initial solution, for example comprises a recombinant cell lysate which comprises IdeS and impurities. The term “impurity” or “contaminant”, as used herein refers to any foreign or objectionable molecule, including host-cell proteins, host-cell nucleic acids such as a DNA, an RNA, or protein variants, such as aggregates, high molecular weight species, low molecular weight species and fragments, deamidated species and charge variants. The term “host cell protein” (HCP), as used herein, is intended to refer to non-IdeS protein that can be derived from host cells (e.g., E.coli) used to produce the IdeS protein. The term “host cell nucleic acids”, “host cell DNA” or “host cell RNA” as used herein, is intended to refer to nucleic acids derived from host cells (e.g., E.coli) used to produce IdeS protein. As used herein, the term “protein-variant” refers to variants of the protein of interest (i.e., IdeS protein) formed during manufacturing and / or storage of the protein of interest. Specific examples of protein variant include high molecular weight species, low molecular weight species, fragments of the protein, post-translational or chemical modified protein, including deamidated, isomerized, mismatched S-S linked, oxidized or altered conjugate forms (e.g., glycosylation, phosphorylation) resulting in charge variants or aggregates including dimers and higher multiples of the protein of interest. The term “charge variant”, as used herein, refers to an alteration of a protein (e.g., through post- translational modification or chemical degradation) which modifies the isoelectric pH (pI) values, leading to charge heterogeneity and can include acidic and basic variants. IdeS protein comprises L-asparagine (Asn), L-glutamine (Gln) residues that can be subjected to deamidation and methionine (Met) that can be subjected to oxidation. These modifications introduce negative charges (deamidation) or modify IdeS polarity (Met oxidation) and can influence the structure and the function of the protein. In particular, the inventors showed that 7 out of the 22 Asn can be partially deamidated, 3 out of the 22 Met residues can be oxidized. The inventors showed that IdeS is prone to aggregation or to generate particles. The presence of particles of different sizes, visible and subvisible was also reported in the formulated active substance during storage or after freeze-thaw cycles. The presence of inactive aggregates and charge variants that are predominant represents a challenge for the design of the manufacturing process since these forms are difficult to separate from the main active component. To solve this issue, a process of manufacturing has been specifically developed to find a unique combination of solutions allowing to stabilize IdeS and enrich the solution with the main component. The method for purifying IdeS or a functional variant thereof according to the present disclosure allows to generate a preparation comprising an IdeS protein and having a reduced level of impurity, in particular having a reduced level of host-cell protein, host-cell DNA and protein variants such as aggregates and charge variants. According to the present disclosure, the initial solution comprising IdeS protein comprises one or more contaminants selected from host-cell proteins, host-cell nucleic acids, protein-variants such as aggregates or protein charge variants. In a preferred embodiment, said initial solution is a lysate of a cell, preferably a bacterial cell (e.g., E. coli) expressing a recombinant nucleic acid encoding an IdeS protein or a functional variant thereof, more preferably comprising or consisting of SEQ ID NO: 3. In a particular embodiment, the initial solution is a lysate of a cell expressing an IdeS protein or a variant thereof, preferably comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with SEQ ID NO: 1, more preferably lysate of a bacterial cell (e.g., E. coli) expressing a recombinant nucleic acid encoding an IdeS protein or a variant thereof, more preferably a recombinant nucleic acid comprising or consisting of SEQ ID NO: 3. In a preferred embodiment, the IdeS in the initial solution is not precipitated. Protein precipitation is the process that lowers the solubility of the protein by addition of a precipitating agent such as ethanol or methanol. Anion exchange chromatography The initial solution comprising the IdeS protein and one or more contaminants is separated in a first step by anion-exchange chromatography, preferably strong anion-exchange chromatography. Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange matrix containing positively charged groups. Examples of functional groups of such anion exchanger that are attached to a matrix support are e.g., primary, secondary, and particularly tertiary or quaternary animo groups such as aminoethyl, diethylaminoethyl, dimethylaminoethyl, trimethylaminoethyl, trimethylaminomethyl, diethyl-(2- hydroxypropyl)-aminoethyl, polyethyleneimine (PEI), quaternary aminoalkyl, quaternary aminoethane (QAE), and quaternary ammonium (Q), preferably quaternary ammonium (Q). Suitable chromatographic support matrixes for the anion exchange chromatography are known in the art. Examples are agarose-based resins and beads, dextran beads, polystyrene beads and polystyrene / divinyl-benzene resins, preferably agarose-based resins. In a preferred embodiment, the anion exchange chromatography is a quaternary amine-based anion exchange chromatography mounted on an agarose matrix. Prior to loading the initial solution comprising IdeS, the anion-exchange chromatography may be pre-equilibrated with an equilibration buffer. In a preferred embodiment, said equilibration buffer comprises NaCl or other neutral salts such as potassium chloride, ammonium chloride or sodium sulfate at a concentration comprised between 0 to 6 mM, preferably 2 to 6 mM, more preferably 3 to 5 mM (e.g., NaCl 4mM). The initial solution comprising the IdeS, preferably a cell lysate, preferably a bacterial cell (e.g., E. coli) lysate expressing a recombinant nucleic acid encoding an IdeS protein or a functional variant thereof, more preferably comprising or consisting of SEQ ID NO: 3, is contacted with the anion exchange chromatography and the IdeS binds to the ligand attached to the stationary phase of the anion exchange chromatography (e.g. quaternary ammonium). According to the present disclosure, the initial solution comprising IdeS or any functional variant thereof is prepared prior to be contacted with anion-exchange chromatography, preferably by adjusting the pH in the range of 6 and 10, preferably 7 and 9, more preferably 7.5-8.5, again more preferably pH 8. The control of acidic forms content is key to ensuring the efficacy (potency) and the safety (subvisible particles) of IdeS as active pharmaceutical ingredient. In the present application, the inventors have determined the parameters that make it possible to purify IdeS efficiently while eliminating acidic charge variants. In a preferred embodiment, in order to eliminate the acidic charge variant, the conductivity of the initial solution is in a range comprised between 2 and 3.5 mS / cm, preferably in a range comprised between 2.5 and 3.5 mS / cm, preferably between 2.5 and 3 mS / cm, again more preferably at 2.8 mS / cm. According to the present disclosure, the initial solution is then contacted with the anion-exchange chromatography as described above under conditions that allow for the IdeS to bind to the anion exchange chromatography support. In some embodiments, the initial solution can be contacted to the column at a linear flow rate in the range of 100-300 cm / h, more preferably 150-200 cm / h (e.g., 180 cm / h). The interaction strength of the ligand can also be influenced by the ligand loading on the anion- exchange chromatography support. According to a preferred embodiment of the present disclosure, in particular to eliminate the acidic charge variant, the loading capacity of the initial solution is comprised between 20 to 95 g / L, preferably 20 to 60 g / L, more preferably 20 to 35 g / L, again more preferably 20 to 30 g / L, again more preferably 25 to 30 g / L (e.g., 28 g / L) of total protein per liter of resin. After binding of the IdeS to the anion-exchange chromatography materials, a washing step can be conducted in order to remove contaminants from the anion exchange chromatography material. In a preferred embodiment said wash buffer comprises a neutral salt, preferably NaCl at a concentration comprised between 5 and 40 mM, preferably between 15 and 35 mM, more preferably 20 and 30 mM (e.g., 25mM). After the optional washing step(s), the anion exchange chromatography material is eluted with a salt gradient and IdeS is collected as an eluate. In a preferred embodiment, the elution step is performed with salt gradient of a neutral salt, preferably NaCl at a concentration from 55 to 100mM. In a preferred embodiment of the present disclosure, the initial solution, equilibration buffer, wash buffer and elution buffer has preferably a pH in the range of 6 and 10, preferably 7 and 9, more preferably 7.5-8.5, again more preferably pH 8. In a more preferred embodiment, said initial solution, equilibration buffer, wash buffer and elution buffer comprises a buffering agent, typically a buffering agent comprising at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid, more preferably TRIS, again more preferably TRIS at a concentration comprised between 1 mM to about 50 mM, preferably between 10 to 30 mM (e.g., 20 mM). Hydrophobic interaction chromatography (HIC) In a second step, feed solution comprising the eluted solution of the anion exchange chromatography step (i.e., first step) is contacted with a hydrophobic interaction chromatography under conditions which facilitate the binding of said IdeS to said hydrophobic interaction chromatography ligand. Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobic interactions between immobilized hydrophobic ligands and non-polar regions of the surface of the protein. The adsorption increases with high salt concentration in the mobile phase and the elution is achieved by decreasing the salt concentration of the eluent. Suitable chromatographic support matrixes for the hydrophobic interaction chromatography are known in the art. HIC support matrix can comprise as non-limiting examples agarose, methacrylate, polystyrene, dextran, polystyrene or polystyrene / divinyl-benzene, preferably agarose. In a more preferred embodiment, the HIC support matrix includes porous beads, preferably agarose porous beads. According to the present disclosure, the HIC support is a matrix substituted with hydrophobic ligands such as ethyl, butyl, phenyl, or hexyl, preferably with butyl and / or phenyl ligands, more preferably butyl ligands. In a preferred embodiment, the HIC according to the present disclosure comprises agarose porous beads substituted with butyl ligands. According to the present disclosure, a feed solution comprising the anion exchange chromatography eluted solution is prepared prior to be contacted with hydrophobic interaction chromatography by adding a salt that promotes adsorption of IdeS in the hydrophobic interaction chromatography support. The expression “salt” used herein is intended to refer to one or more salts capable of making the IdeS so relatively hydrophobic that it will bind to the hydrophobic interaction chromatography material. Said salts can be ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride and potassium sulfate, preferably said salt is ammonium sulfate ((NH₄)₂SO₄). In a preferred embodiment, said feed solution comprises a salt at a concentration comprised between 0.5 and 3 M, preferably between 0.5 and 2 M, more preferably 0.9 and 1.5 M. In a more preferred embodiment, said feed solution comprises ammonium sulfate at a concentration comprised between 0.5 and 3 M, preferably between 0.5 and 2 M, more preferably 0.9 and 1.5 M. In a preferred embodiment, the conductivity of the feed solution is adjusted at least to 100 mS / cm, more preferably at least to 150 mS / cm, more preferably to a range comprised between 150 and 200 mS / cm, again more preferably to a range comprised between 170 and 190 mS / cm (e.g., 180 mS / cm). The hydrophobic interaction strength of the ligand can also be influenced by the ligand loading on the HIC support. According to a preferred embodiment of the present disclosure, the loading capacity of the feed solution is comprised between 20 to 40 g / L of total protein per liter of resin. Prior to loading the feed solution comprising the eluted solution of the anion exchange chromatography step (i.e., first step), the HIC may be pre-equilibrated with an equilibration buffer with a salt concentration and pH similar to the salt concentration and pH of the feed solution to ensure that the IdeS protein is tightly bound to the hydrophobic ligand. In a preferred embodiment, said HIC is pre-equilibrated with an equilibration buffer comprising a salt, preferably ammonium sulfate at a concentration comprised between 0.5 and 3 M, preferably between 0.5 and 2 M, more preferably between 0.9 and 1.5 M. According to the present disclosure, the feed solution is then contacted with the HIC as described above. After binding of the IdeS to the hydrophobic interaction chromatography materials, a washing step can be conducted in order to remove contaminants from the hydrophobic interaction chromatography material. In a preferred embodiment said wash buffer comprises a salt, preferably ammonium sulfate at a concentration comprised between 0.5 and 3 M, preferably between 0.5 and 2 M, more preferably 0. 9 and 1.5 M. After the optional washing step(s), the hydrophobic interaction chromatography material is eluted with an elution buffer, and IdeS is collected as an eluate. In a preferred embodiment, the elution buffer comprises a salt, preferably ammonium sulfate at a concentration below 0.9 M, preferably within a range from 0.8 to 0.9 M. In a preferred embodiment of the present disclosure, the feed solution, equilibration buffer, wash buffer and elution buffer has preferably a pH in the range of 6 and 10, preferably 7 and 9, more preferably 7.5 and 8.5, again more preferably pH 8. In a more preferred embodiment, said feed solution, equilibration buffer, wash buffer and elution buffer comprises a buffering agent, typically a buffering agent comprising at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid, more preferably Tris, again more preferably Tris at a concentration comprised between 1 mM to about 50 mM, preferably between 10 to 30 mM (e.g., 20 mM). Mixed mode chromatography (Hydrophobic cation exchange chromatography) In a third step, a feed solution comprising the eluted solution of the HIC (i.e., second step) is contacted with a mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) under conditions which facilitate the binding of said IdeS to said mixed mode chromatography ligand. Mixed mode chromatography (MMC) combines the multimodal interactions including electrostatic, hydrophobic and hydrogen bond interactions. According to the present disclosure, said mixed mode chromatography is preferably a hydrophobic cation exchange chromatography that comprises onto a solid phase a mixed-mode ligands comprising a cationic exchange part and a hydrophobic part. In a preferred embodiment, the cationic exchange part is a carboxylic acid, and the hydrophobic group is a phenyl group or an aliphatic hydrocarbon chain, preferably a phenyl group. The cationic exchange part and hydrophobic part can be joined by a chain. Examples of such chains are peptide- containing chains, such as —R1—C(O)—NH—R2— where R1 and R2 are alkyl groups and one or both of R1 and R2 can be absent. A specific example is —C(O)—NH—CH2—. In a preferred embodiment, the mixed mode ligand is 4-aminobenzamidoacetic acid, also known as para-aminohippuric acid, whose formula is as follows: A commercial example of the p-Aminohippuric acid ligand on a hydrophobic cation exchange support is NuviaTMcPrimeTM, which is commercially available from Bio-Rad Laboratories, Inc. (Hercules, Calif.). Any solid support can be used for mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography). The solid support can be, for example, porous or non-porous and can be in the form, for example, of a matrix, bead, or particle, preferably beads with pores of a median diameter of 0.5 micron or greater. In a preferred embodiment, the matrix is a macroporous highly crosslinked polymer. Examples of monomers that can be polymerized to achieve useful matrices are vinyl acetate, vinyl propylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, vinyl pyrrolidone (vinyl pyrrolidinone), with functional groups in some cases. According to the present disclosure, a feed solution comprising the HIC eluted solution is prepared prior to be contacted with a mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) by adding a salt that promotes adsorption of IdeS in the hydrophobic cation exchange chromatography support. The expression “salt” used herein is intended to refer to one or more salts capable of making the IdeS so relatively hydrophobic that it will bind to the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) material. Said salts can be ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride and potassium sulfate, preferably said salt is ammonium sulfate ((NH₄)₂SO₄). In a preferred embodiment, said feed solution comprises a salt at a concentration comprised between 0.5 and 5 M, preferably between 1 and 4 M, more preferably between 2.5 and 3.5 M. In a more preferred embodiment, said feed solution comprises ammonium sulfate at a concentration comprised between 0.5 and 5 M, preferably between 1 and 4 M, more preferably between 2.5 and 3.5 M, again more preferably 3M. In a preferred embodiment, the conductivity of the feed solution is adjusted at least to 100 mS / cm, more preferably at least to 150 mS / cm, more preferably to a range comprised between 150 and 200 mS / cm, again more preferably to a range comprised between 170 and 190 mS / cm, again more preferably to 180 mS / cm. The interaction strength of the ligand can also be influenced by the ligand loading on the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) support. According to a preferred embodiment of the present disclosure, the loading capacity of the feed solution is comprised between 5 to 40 g / L, preferably 10 to 30 g / L, more preferably 12 to 20 g / L of total protein per liter of resin. Prior to loading the feed solution comprising IdeS, the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) may be pre-equilibrated with an equilibration buffer. In a preferred embodiment, said mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) is pre-equilibrated with an equilibration buffer comprising a salt, preferably ammonium sulfate at a concentration comprised between 0.5 and 5 M, preferably between 0.5 and 3 M, more preferably between 1 and 2 M, again more preferably at 1.5 M. According to the present disclosure, the feed solution is then contacted with the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) as described above. The feed solution can be contacted to the mixed mode chromatography support (e.g., hydrophobic cation exchange chromatography) under conditions that allow for the IdeS to bind to the mixed mode Support. In some embodiments, the feed solution can be contacted to the column at a linear flow rate in the range of 100 to 600 cm / h, preferably 150 to 450 cm / h, more preferably 300 to 400 cm / h (e.g., 360 cm / h). After binding of the IdeS to the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) materials, a washing step can be conducted in order to remove contaminants from the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) material. In a preferred embodiment said wash buffer comprises a salt, preferably ammonium sulfate at a concentration comprised between 0.5 and 5 M, preferably between 0.5 and 3 M, more preferably between 1 and 2 M, again more preferably at 1.5 M. In a preferred embodiment of the present disclosure, the feed solution, equilibration buffer and wash buffer has preferably a pH in the range of 6 and 10, preferably 7 and 9, more preferably 7.5-8.5. In a more preferred embodiment, said feed solution, equilibration buffer, and wash buffer comprises a buffering agent, typically a buffering agent comprising at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, Tris, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid, more preferably Tris, again more preferably Tris at a concentration comprised between 1 mM to about 50 mM, preferably between 10 to 30 mM (e.g., 20 mM). After the optional washing step(s), the mixed mode chromatography (e.g., hydrophobic cation exchange chromatography) material is eluted with an elution buffer, and IdeS is collected as an eluate. In a preferred embodiment, the elution step is performed with salt gradient buffer, preferably sulfate ammonium gradient buffer, more preferably at a concentration comprised between 1.5 to 0 M, preferably 1.2 M to 0.375 M, preferably 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. In a preferred embodiment of the present disclosure, the elution buffer has preferably a pH in the range of 2 and 6, preferably 3 and 5, more preferably 3.5-4.5. In a more preferred embodiment, said elution buffer comprises a buffering agent, typically a buffering agent comprising at least one component selected from the groups consisting of acids and salts of MES, PIPES, ACES, BES, TES, HEPES, Tris, histidine, imidazole, glycine, glycylglycine, glycinamide, phosphoric acid, acetic acid (e.g. sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid, more preferably phosphate sodium, again more preferably at a concentration comprised between 1 mM to about 50 mM, preferably between 10 to 30 mM (e.g., 20 mM). In a preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind an anion exchange chromatography ligand which is a quaternary ammonium ligand, optionally washing said anion exchange chromatography with a wash buffer, preferably comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with an elution buffer, preferably a gradient salt buffer comprising a neutral salt, more preferably NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a hydrophobic interaction chromatography ligand which is a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, preferably wherein said feed solution and wash buffer comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer, preferably comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a hydrophobic cation exchange chromatography which is a 4-aminobenzamidacetic acid ligand, preferably wherein said feed solution comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer, preferably a gradient salt buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M, more preferably 1.1 to 0.75 M. In a more preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a quaternary ammonium ligand, optionally washing said anion exchange chromatography with a wash buffer, comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with a gradient salt buffer comprising a neutral salt, more preferably NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, wherein said feed solution and wash buffer comprise ammonium sulfate, preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer comprising ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a 4- aminobenzamidacetic acid ligand, wherein said feed solution comprises ammonium sulfate, preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer which is a gradient ammonium sulfate buffer at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. In a more preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a quaternary ammonium ligand, optionally washing said anion exchange chromatography with a wash buffer, comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with a gradient salt buffer comprising NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, wherein said feed solution and wash buffer comprise ammonium sulfate at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer comprising ammonium sulfate at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a 4- aminobenzamidacetic acid ligand, wherein said feed solution comprises ammonium sulfate at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer which is a gradient ammonium sulfate buffer at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. The control of acidic forms content is key to ensuring the efficacy (potency) and the safety (subvisible particles) of IdeS as active pharmaceutical ingredient. In the present application, the inventors have determined the parameters that make it possible to purify IdeS efficiently while eliminating acidic charge variants. In a more preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) and removing IdeS acidic charge variant comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind an anion exchange chromatography ligand which is a quaternary ammonium ligand, wherein said initial solution is contacted with the anion exchange chromatography with a load of initial solution comprised between 20 and 35 g / L, more preferably 25 and 30 g / L of total protein per L of resin, again more preferably no more than 28 g / L and / or with a load conductivity comprised between 3 and 4 mS / cm, preferably 3 and 3.5 mS / cm, more preferably between 3.1 and 3.3 mS / cm. optionally washing said anion exchange chromatography with a wash buffer, preferably comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with an elution buffer, preferably a gradient salt buffer comprising a neutral salt, more preferably NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a hydrophobic interaction chromatography ligand which is a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, preferably wherein said feed solution and wash buffer comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer, preferably comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a hydrophobic cation exchange chromatography which is a 4-aminobenzamidacetic acid ligand, preferably wherein said feed solution comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer, preferably a gradient salt buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M, more preferably 1.1 to 0.75 M. In a more preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) and removing IdeS acidic charge variant comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a quaternary ammonium ligand, wherein said initial solution is contacted with the anion exchange chromatography with a load of initial solution comprised between 20 and 35 g / L, more preferably 25 and 30 g / L of total protein per L of resin, again more preferably no more than 28 g / L and / or with a load conductivity comprised between 3 and 4 mS / cm, preferably 3 and 3.5 mS / cm, more preferably between 3.1 and 3.3 mS / cm. optionally washing said anion exchange chromatography with a wash buffer, comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with a gradient salt buffer comprising a neutral salt, more preferably NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, wherein said feed solution and wash buffer comprise ammonium sulfate, preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer comprising ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a 4- aminobenzamidacetic acid ligand, wherein said feed solution comprises ammonium sulfate, preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer which is a gradient ammonium sulfate buffer at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. In a more preferred embodiment, the present disclosure relates to a method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) and removing IdeS acidic charge variant comprising the steps of: i) contacting an initial solution (e.g., cell lysate) comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a quaternary ammonium ligand, wherein said initial solution is contacted with the anion exchange chromatography with a load of initial solution comprised between 20 and 35 g / L, more preferably 25 and 30 g / L of total protein per L of resin, again more preferably no more than 28 g / L and / or with a load conductivity comprised between 3 and 4 mS / cm, preferably 3 and 3.5 mS / cm, more preferably between 3.1 and 3.3 mS / cm. optionally washing said anion exchange chromatography with a wash buffer, comprising a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM, and eluting the fraction comprising the IdeS with a gradient salt buffer comprising NaCl at a concentration comprised between 55 to 100 mM, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, wherein said feed solution and wash buffer comprise ammonium sulfate at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M, and eluting the fraction comprising IdeS with an elution buffer comprising ammonium sulfate at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M, iii) contacting a feed solution comprising the eluted fraction of step ii) with a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a 4- aminobenzamidacetic acid ligand, wherein said feed solution comprises ammonium sulfate at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer which is a gradient ammonium sulfate buffer at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M more preferably 1.1 to 0.75 M. In another aspect, the present disclosure also relates to the IdeS protein solution obtainable from the three steps chromatography method as disclosed above. The IdeS protein solution that is eluted from the three-step chromatography as disclosed above is highly concentrated for IdeS as compared to the starting sample preparation. An enrichment to more than 50% of the main component is achieved with the method according to the present disclosure. Clearance of impurities (host cell DNA and protein) is achieved to acceptable level to address safety concern for human use with no more than 50 ng / mg of host cell protein and / or less than 3 pg / mg of residual DNA. In some embodiments, the IdeS protein solution obtainable from the three steps chromatography method comprises no more than 50 ng / mg of host cell protein and / or less than 3 pg / mg of host cell DNA. In some embodiments, the IdeS protein solution obtainable from the three steps chromatography method comprises no more than 25% of the acidic charge variant on total eluted components. Charge variant distribution between the components is quantified using AEX-HPLC with detection at 215 nm. The relative percentage of the main charge variant (corresponding to an acidic variant) is calculated by dividing the peak area for the corresponding component by the total peak area. In some embodiments, the IdeS protein solution obtainable from the three steps chromatography method is enriched to more than 50%, 60%, 70%, 80% or more in quantity in comparison to initial solution. Various methods of assessing the purity of IdeS are known in the art. For example, in Some embodiments, the purity of the IdeS that is eluted can be analyzed by gel electrophoresis (e.g., SDS- PAGE) and bands corresponding to IdeS protein may be visualized by staining (e.g., Coomassie staining). The purity of the IdeS that is purified can also be assessed by measuring the amount of host cell protein, Charge variants and / or host cell DNA contaminants, if any, that are present in the final purified product. The amount of host cell protein can be measured, for example, by immunoassay (e.g., ELISA) and the amount of host cell DNA can be measured, for example, by PCR (e.g., quantitative PCR against a housekeeping gene or ribosomal RNA gene). In another aspect, the present disclosure provides a kit for use in the methods described herein. A kit can optionally include written instructions as well as packaging material. In some embodiments, the kits comprise (1) and anion exchange chromatography support as described above, (2) a hydrophobic interaction chromatography as described above, (3) a mixed-mode chromatography support comprising hydrophobic and cation exchange moieties as described above. Other reagents described herein in the context of the methods (e.g., equilibration, wash, and / or elution solutions) can also optionally be included in the kits. EXAMPLES IdeS Drug Substance is produced using a genetically engineered E. coli strain followed by multiple chromatography steps to reach high product purity and formulated by ultrafiltration / diafiltration (UF / DF). The IdeS gene [JN035367.1] nucleotide sequence was used as reference sequence to chemically synthesize a codon optimized coding sequence (SEQ ID NO: 3). The synthesized sequence was cloned into pET28b+ expression plasmid providing the key element towards recombinant IdeS production to obtain pET28b+ / IDES plasmid. The inventors established a master cell bank (MCB) of E.coli BL21 ΔDE3 (XT7) from Xpress biologics SA transformed with the pET28b+ / IDES plasmid for the process development and manufacturing of the recombinant IdeS. The number and modes of chromatography steps have been specifically designed to remove process and product-related impurities to acceptable levels for an active pharmaceutical ingredient and to stabilize IdeS in solution. 1. IdeS production in E. coli IdeS was produced in E.coli BL21 ΔDE3 (XT7) cells transformed with pET28b+ / IdeS plasmid in a bioreactor with a fed batch process where expression of IdeS is induced by the addition of IPTG. The fermentation step ends 4 hours after induction of IdeS expression by IPTG. The culture is collected, and the cells are separated. The supernatant is discarded, and whole bacterial pellet is recovered and distributed in bags which are stored at ≤-60°C until further processing. All the bacteria pellets are thawed, pooled and resuspended in 20 mM Tris pH 8.0 buffer. Cell membranes are disrupted through 3 high pressure homogenization cycles (to generate a cell lysate containing the recombinant product. The cell lysate is clarified by centrifugation and then depth filtration followed by filtration steps to generate one clarified lysate. 2. Development of IdeS purification process for the removal of process and product impurities: A three-chromatography step process was designed for the purification of IdeS. To streamline the process, the developed process excludes an initial step of protein precipitation. The purpose of the first purification step is to capture soluble IdeS and to remove process related impurities. According to the pH and conductivity of the cell extract and the PI of 6.13, anion exchange chromatography (AEX) was selected as the most appropriate approach to use a neutral / mildly basic buffer system. To keep a unique ammonium sulfate system buffer during the remaining purification process, hydrophobic interaction chromatography (HIC) step following by multimodal mode chromatography (MMC) were used to achieve the purification of IdeS. 2.1 AEX DEVELOPMENT AEX step was developed using strong anion exchange chromatography based on quaternary ammonium. Several resins were screened (Q Sepharose FF and Capto Q). The standard operating conditions are the following: ^ Capto-Q is the chromatography resin equilibrated with 20 mM Tris pH 8.0 buffer. ^ pH and conductivity of the clarified lysate is adjusted to a target pH of 8.0 and a target conductivity of 3.2 mS. ^ The column is washed with 20 mM Tris pH 8.0, and then eluted with 20 mM Tris pH 8.0, 100 mM NaCl. Figure 1 shows a typical profile of elution of HCP and IdeS charge variants. Since there is only a slight separation between HCPs and IdeS, their removal can be only controlled by the pooling strategy. The main component of charge variant is of 40% and HCP is at least of 30 µg / mg in the elution pool (Figure 2). Residual host cell protein is quantified by ELISA. The absorbance in UV- A and the ratio of absorbance at 260 nm and 280 nm is used to measure the DNA contaminants. Under these conditions, eluting protein remains residually contaminated with DNA as shown by A 260 / 280 ratio of above 1 (e.g., the ratio higher than 0.6-0.7 usually indicates nucleic acid contamination). Key process parameters were investigated to define operating conditions allowing to prevent co- elution with HCP and charge variants. Chromatography flowrate. The chromatography column was operated up to 360 cm / h.180 cm / h was determined as an optimum flow rate to minimize risk of too high back pressure for column and to increase column separation capability. Loading capacity. Protein loading capacity was tested within the range of 22-190 g of total protein per liter of resin.32- 42 g of total of total protein per liter of resin is the optimal range. Overloading (95 g / L) conducted to negatively impact the separation’s efficiency and to oversaturate the resin. Underloading (22 g / L) impacted both IdeS recovery and removal of impurities, in particular a protein contaminant of 50 kDa. System buffer design. Different system buffers have been tested as detailed below: ^ Equilibration with up to 4 mM NaCl in 20 mM Tris pH 8.0 buffer ^ Wash steps with up to 25 mM NaCl in 20 mM Tris pH 8.0 buffer ^ Elution steps within a range of 55 to 100 mM NaCl in 20 mM Tris pH 8.0 buffer Combination of these changes for the different process parameters listed above conducted to modify the profile of HCP and IdeS charge variants (Figure 1b) and greatly improves the separation between HCP and IdeS. Depending on the combination selected, the main component of charge variant (IdeS charge variant of interest) can reach up to 60% and HCP reduces to just above 10 µg / mg in the elution pool (Figure 2). These changes allow the removal of a main 50 kDa protein contaminant (Figure 3). 2.2. HIC DEVELOPMENT HIC step was developed using different degrees of hydrophobicity of medium and several resins were screened (Phenyl HP resin Capto Butyl ImpRes). The standard operating conditions are the following: ^Capto Butyl ImpRes resin is the chromatography resin equilibrated with 20mM Tris pH 8.0, 1.5M (NH₄)₂SO₄ and operated using a flow rate of 180 cm / hrs. ^The conductivity of the AEX elution pool is adjusted to 180 ± 3 mS with 20 mM Tris pH 8.0, 3 M (NH₄)₂SO₄ buffer to allow the binding of IdeS to a CaptoButyl ImpRes resin. ^An elution gradient is applied from 20 mM Tris pH 8.0, 1.5 M (NH₄)₂SO₄ to 20 mM Tris pH 8.0, 0.1M (NH₄)₂SO₄ is applied. Figure 4 shows a typical profile of elution of HCP and IdeS charge variants. Since there is only a slight separation between HCPs and IdeS, their removal can only be controlled by the pooling strategy. Under these conditions, removing of contaminated DNA is achieved. A 260 / 280 ratio of the eluted fractions are close of 0.5, ratio corresponding to “pure” proteins. Key process parameters were investigated to define operating conditions allowing to prevent co- elution with HCP and product related impurities such as charge variants. ^Adjustment of AEX elution / Equilibration of the column within a range from 1.5 to 0,9M (NH₄)₂SO₄ in in 20 mM Tris pH 8.0 buffer ^Wash step within a range from 1,5 to 0,9M (NH₄)₂SO₄ in 20 mM Tris pH 8.0 buffer ^Elution steps within a range from 0,8 to 0,9M (NH₄)₂SO₄ in in 20 mM Tris pH 8.0 buffer ^Loading capacity within the range from 20 to 40 g / L. Combination of these changes for the different process parameters listed above conducted to modify the profile of HCP and IdeS charge variants (Figure 4) and greatly improves the separation between HCP and IdeS. Depending on the combination selected, the clearance factor of the chromatography step, defined as the ratio between host cell proteins introduced in the feed solution and the residual host cell proteins in the elution solution, can vary from no clearance (standard condition) up to near to 10-fold (Figure 5). 2.3. MIXED MODE CHROMATOGRAPHY (MMC) DEVELOPMENT Different resins were screened during the development of the MMC step: ^Ceramic Hydroxyapatite CHT type, ^Capto Adhere ImpRes usually used to remove aggregate, ^Eshmuno HCX is multi-mode cation exchanger media designed for direct capture of recombinant proteins at higher salt concentration with ligands, ^Nuvia cPrime is hydrophobic cation exchange resin with ligands –phenyl, amide and carboxy groups. Among the different resins tested, only the hydrophobic cation exchange resin (e.g., nuvia cPrime) leads to an efficient purification of IdeS without high loss of material. Thus, Nuvia cPrime has been selected. The standard operating conditions are the following: ^Nuvia cPrime resin is the chromatography resin equilibrated with 20mM Tris pH 8.0, 1.5M (NH₄)₂SO₄ and operated using a flow rate of 360 cm / h. ^The conductivity of the HIC elution pool is adjusted to 180 ± 3 mS with 20 mM Tris pH 8.0, 3 M (NH₄)₂SO₄ buffer to allow the binding of IdeS to a Nuvia cPrime resin. ^An elution gradient from 20 mM Tris pH 8.0, 1.5 M (NH₄)₂SO₄ to 20 mM Tris pH 8.0, 0.375M (NH₄)₂SO₄ is applied Key process parameters were also investigated. ^ Elution steps using (NH₄)₂SO₄ gradient from 1.5 M to 0 M in 20mM Sodium phosphate buffer, pH 4.0 ^ Loading capacity within the range from 12 to 20 g / L. Under these conditions, above 1.5 log of removal of host cell protein could be achieved. 2.4 CONCLUSION A 3-steps process was developed for the purification of IdeS which displays these specific features allowing to streamline its execution. ^ direct capture of IdeS from cellular extract, ^ Simple buffer system only based on two buffers: Tris and ammonium sulfate, ^ combination of set-up of process parameters allowing to recover IdeS containing more than 50% of charge variant main component and no more than about 50 ng / mg of host cell protein (Figure 6) and less than 3 pg / mg of residual DNA. The stabilization of IdeS in solution during DSP was achieved by minimizing duration of holding time between chromatography steps and applying appropriate dilution in ammonium sulfate buffer. IdeS is then formulated and concentrated by ultrafiltration / diafiltration. A unique process of IdeS manufacturing based on a specific combination of process steps and controlled parameters has been developed. With this process, active IdeS can be manufactured: ^ Stability issue is solved by controlling dilution of process intermediate samples, ^ Clearance of impurities (host cell DNA and protein) is achieved to acceptable level to address safety concern for human use. ^ Enrichment to more than 50% of the main component is achieved. 3. Improvement of IdeS purification process for the removal of acidic variants: 3.1 Charge variant distribution profile in IdeS DSP process was further developed to improve the efficacy and potency profile of IdeS as an active pharmaceutical ingredient, in particular, to correct the level of charge variants. Charge variant distribution between the components is quantified using AEX-HPLC with detection at 215 nm. The relative percentage of the main component is calculated by dividing the peak area for the corresponding component by the total peak area. The relative percentage of the basic variants, corresponding to the components eluting before the main component is calculated by dividing the peak area for the corresponding components by the total peak area. The relative percentage of the acidic variants, corresponding to the components eluting after the main component is calculated by dividing the peak area for the corresponding components (three main components: acidic 1, acidic 2, acidic 3) by the total peak area. The relative percentage of the main charge variant (corresponding to an acidic variant) is calculated by dividing the peak area for the corresponding component by the total peak area. Exemplary AEX-HPLC chromatograms and integration strategy for estimated amounts of potential impurities are provided in Figure 7 and Table 1 respectively. Time retention (min) RRT %Area Integration approach Remarks 10.875 0.725 23.3% Basic variants 14.995 1 59.0% Main component 15.393 1.027 5.8% 3 1.083 6.6% Ac (Acidic 1) 16.24 idic variants 33 1.216 5.2% (grou (Acidic 2) 18.2 ping) (Acidic 3) Table 1: Integration approach strategy for IdeS 3.2 Evolution of Charge variants & correlation with other attributes When stored frozen at ≤ -60°C, IdeS is stable for all tested attributes for at least up to 14 months. However, IdeS is not stable when stored in liquid form. On the contrary, when stored in a liquid form, for instance, stability data at 5 ± 3°C shows a decrease of the main protein component with regards to ionic charge variants (Figure 8). The decrease in the main protein component is primarily at the expense of the acidic forms, notably acidic 1 and acidic 2 forms, while the basic forms remain overall stable. Stability data showed the increase of acidic variants is associated with a loss of potency (Figure 9a) and an increase in subvisible particles (Figure 9b). The key finding is then that the control of acidic forms content is key to ensuring the efficacy (potency) and the safety (subvisible particles) of IdeS as active pharmaceutical ingredient. 3.3 Uncontrolled increase of acidic 3 content during downstream processing of IdeS A first version of the downstream process was implemented, leading to the extraction of IdeS from E. coli cells and the manufacturing of clinical material such as IdeS GMP DS batch #8010122001. The downstream process was further developed to achieve better clearance of impurities (e.g., clearance of host cell DNA and protein). Higher clearance performance was achieved by combining different control parameters such as: • Increasing AEX loading capacity from 35.5 to 42 g of total protein per liter of resin (g / Lr). The protein loading capacity was tested within the range of 22-190 g / Lr.32-42 g / Lr is the optimal range with a set-point at 42 g / Lr to maximize DSP process productivity. Overloading (95 g / Lr) negatively impacted the separation’s efficiency and oversaturated the resin. Underloading (22 g / Lr) impacted both IdeS recovery and removal of impurities, in particular a protein contaminant of 50 kDa. • Controlling HIC loading capacity at 25 g / L of total protein per liter of resin for optimum clearance of process-related impurities. Unexpectedly, during DSP development, although changes increased clearance of product-related impurities including basic variants, they were unexpectedly associated with an uncontrolled increase in acidic charge variants, particularly the acidic 3 variants (Figure 10). Compared to the acidic 3 variant content of 5.2% in IdeS GMP DS batch #8010122001, development DS batches showed levels as high as 30% (Figure 10). This increase was noticeable during the AEX step elution fraction. AEX conditions were further investigated to identify potential ways to mitigate this issue. 3.4 AEX chromatography development Method Different AEX chromatography assays were performed to test the impact of loading capacity and conductivity of the Load solution on both productivity and charge variant profile of IdeS. The different Load solutions was prepared as follows. Frozen E. coli cell pellets expressing IdeS are thawed, resuspended, and subjected to high-pressure homogenization. The resulting cell lysate was then clarified through a series of steps: centrifugation, depth filtration, and membrane filtration. Then, the conductivity of the clarified harvest containing IdeS was adjusted by dilution with purified water or NaCl to reach values ranging from 2.7 mS / cm to 4.6 mS / cm. The AEX separation was performed using pre-equilibrated Capto Q anion exchange chromatography resin. To evaluate the influence of the loading capacity, loading volume of the pH / conductivity-adjusted lysate was set to either 28 g / Lr (with a clarified lysate conductivity of 3.2 mS / cm) or 42 g / Lr (with clarified lysate conductivities of 2.7, 3.1, and 4.6 mS / cm). Other parameters, such as wash volume, buffer composition, and elution volume and composition, remained constant Results & discussion In order to eliminate acidic charge variants to ensure the efficacy and safety of IdeS as a pharmaceutical agent while allowing a good purification productivity of IdeS, the inventors determined the key parameters influencing the presence of acidic charge variants. The results can be summarized as follows ^ As expected, lowering the loading capacity reduce the productivity. For a given eluate pooling strategy and a similar load conductivity (3.1 mS / cm), the productivity decreased from 6 to 4 g of protein eluted per liter of resin for a capacity of 42 g / Lr to 28 g / Lr at similar conductivity. ^ At a given capacity (42 g / Lr), the magnitude of the conductivity ranging from 3.1 to 4.6 mS / cm has a more unexpected impact since the productivity decrease 2-fold, from 6 to 3 g / Lr. SDS-analysis of the elution fractions from the different AEX experiments (Figure 11) showed an earliest and unexpected elution of IdeS forms, which is impacted by the conductivity. ^ Charge variants analysis of the elution fractions allowed also to discover that both conductivity and loading capacity influence the content of acidic 3 (Figure 12): o Increase of loading capacity conducted to increase acidic 3 forms o Increase of conductivity conducted to increase acidic 3 forms These results could be explained by a molecular displacement phenomenon in AEX, where acidic 3 forms might exhibit a higher interaction with the AEX stationary phase compared to the main component, leading to their elution before the main forms. When the overall binding capacity of the AEX stationary phase is reduced either because of an increase the load quantity and / or the conductivity, the interaction of main forms with AEX stationary phase is not sufficient to prevent its elution in the flow-through. This was phenomenon was unexpected since apparently, no E-coli proteins having highest interaction with the AEX stationary phase (i.e. eluting at higher ionic strength) seem to act as a displacer. No visible molecular displacement of E. coli proteins can be detected in SDS-PAGE gels (Figure 12). Conclusion Recombinant IdeS present basic and acidic charge variants. Their control is key to produce a safe and active pharmaceutical ingredient. Control of acidic variants are essential since the low content is both associated to higher potency and lower presence of sub-visible particles. In that context, tight control of acidic 3 form requires to adapt loading capacity (e.g., maximum at around 28 g of total protein per liter of the resin) and the load conductivity (e.g. in a range around of 3.1-3.3 mS / cm). Sequences for use in practicing the disclosure: SEQ ID NO: 1 (IdeS protein without peptide signal) MDSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAG NMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVF PDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELT EGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKED NIGAQVLGLFTLSTGQDSWNQTN SEQ ID NO: 2 (nucleic acid sequence encoding IdeS) (GenBank: JN035367.1) ATGAGAAAAAGATGCTATTCAACTTCAGCTGTAGTATTGGCAGCAGTGACTTTATTTGCTCTATCGGTAGAT CGTGGTGTTATAGCAGATAGTTTTTCTGCTAATCAAGAGATTAGATATTCGGAAGTAACACCTTATCATGTT ACTTCCGTTTGGACCAAAGGAGTTACTCCTCCAGCAAAATTCACTCAAGGCGAAGATGTTTTTCACGCTCCT TATGTTGCTAACCAAGGATGGTATGATATTACCAAAACATTCAATGGAAAAGACGATCTTCTTTGCGGGGCT GCCACAGCAGGGAATATGCTTCACTGGTGGTTCGATCAAAACAAAGAAAAAATTGAAGCATATCTAAAAAAA CACCCAGATAAACAAAAAATCATGTTTGGTGATCAAGAATTATTGGATGTAAGAAAAGTTATTAATACCAAA GGTGACCAAACAAATAGCGAGCTTTTTAATTATTTCCGAGATAAAGCTTTCCCCGGTTTGTCAGCACGCCGA ATTGGAGTTATGCCTGATCTTGTTTTAGATATGTTTATCAATGGTTATTACTTAAATGTTTATAAGACACAG ACTACTGATGTCAATAGAACCTATCAAGAGAAAGATCGCCGAGGTGGTATTTTTGACGCCGTATTTACAAGA GGTGATCAAAGTAAGCTATTGACAAGTCGTCATGATTTTAAAGAAAAAAATCTCAAAGAAATCAGTGATCTC ATTAAGAAAGAGTTAACCGAAGGCAAGGCTCTAGGCCTATCACACACCTACGCTAACGTACGCATCAACCAT GTTATAAACCTGTGGGGAGCTGACTTTGATTCTAACGGGAACCTTAAAGCTATTTATGTAACAGACTCTGAT AGTAATGCATCTATTGGTATGAAGAAATACTTTGTTGGTGTTAATTCCGCTGGAAAAGTAGCTATTTCTGCT AAAGAAATAAAAGAAGATAATATTGGTGCTCAAGTACTAGGGTTATTTACACTTTCAACAGGGCAAGATAGT TGGAATCAGACCAATTAA SEQ ID NO: 3 (IdeS codon-optimized sequence) ATGGATAGCTTTAGCGCCAATCAAGAAATTCGTTATAGCGAAGTTACCCCGTATCATGTTACCAGCGTTTGG ACCAAAGGTGTTACCCCTCCGGCAAATTTTACCCAGGGTGAAGATGTTTTTCATGCACCGTATGTTGCAAAT CAAGGCTGGTATGATATCACCAAAACCTTCAATGGTAAAGATGATCTGCTGTGTGGTGCAGCAACCGCAGGT AATATGCTGCATTGGTGGTTTGATCAGAACAAAGATCAGATCAAACGCTACCTGGAAGAACATCCGGAAAAA CAGAAAATCAATTTTAACGGCGAGCAGATGTTCGATGTGAAAGAAGCAATTGACACCAAAAACCATCAGCTG GATAGCAAACTGTTCGAGTACTTTAAAGAAAAAGCGTTCCCGTATCTGAGCACCAAACATCTGGGTGTGTTT CCGGATCATGTTATCGATATGTTTATTAACGGTTATCGTCTGAGCCTGACCAATCATGGTCCGACACCGGTT AAAGAAGGTAGCAAAGATCCGCGTGGTGGTATTTTTGATGCAGTTTTTACCCGTGGTGATCAGAGCAAACTG CTGACCAGCCGTCATGATTTTAAAGAGAAAAACCTGAAAGAGATCAGCGACCTGATCAAAAAAGAACTGACC GAAGGTAAAGCACTGGGTCTGAGCCATACCTATGCAAATGTTCGTATTAACCATGTGATTAATCTGTGGGGT GCAGATTTTGATAGCAATGGTAATCTGAAAGCCATCTATGTTACCGATAGCGATAGTAATGCAAGCATTGGC ATGAAGAAATACTTTGTGGGTGTTAATAGCGCAGGTAAAGTTGCAATTAGCGCGAAAGAAATCAAAGAGGAT AATATTGGCGCACAGGTTCTGGGTCTGTTTACCCTGAGCACCGGTCAGGATAGCTGGAATCAGACCAACTAA TAA

Claims

CLAIMS 1. A method for purifying an immunoglobulin G degrading enzyme from Streptococcus pyogenes (IdeS) comprising the steps of: i) contacting an initial solution comprising IdeS with an anion exchange chromatography under conditions that allow the IdeS to bind to a chromatography anion ligand, preferably quaternary ammonium ligand, optionally washing said anion exchange chromatography with a wash buffer and eluting the fraction comprising the IdeS with an elution buffer, ii) contacting a feed solution comprising the eluted fraction of step i) with a hydrophobic interaction chromatography under conditions that allow the IdeS to bind to a chromatography hydrophobic ligand, preferably butyl ligand, optionally washing said hydrophobic interaction chromatography with a wash buffer, and eluting the fraction comprising IdeS with an elution buffer, iii) contacting a feed solution comprising the eluted fraction of step ii) with a mixed mode chromatography, preferably a hydrophobic cation exchange chromatography under conditions that allow the IdeS to bind to a chromatography mixed mode ligand, preferably 4-aminobenzamidacetic acid ligand, optionally washing said mixed mode chromatography with a wash buffer and eluting the fraction comprising IdeS with an elution buffer.

2. The method according to claim 1 for removing IdeS acidic charge variants, wherein the initial solution is contacted with the anion exchange chromatography in step i) with a load of initial solution comprised between 20 and 35 g / L, more preferably 25 and 30 g / L of total protein per L of resin, again more preferably no more than 28 g / L.

3. The method according to claim 1 or 2 wherein the initial solution is contacted with the anion exchange chromatography in step i) with a load conductivity comprised between 3 and 4 mS / cm, preferably 3 and 3.5 mS / cm, more preferably between 3.1 and 3.3 mS / cm.

4. The method according to any one of claims 1 to 3 wherein said initial solution is a lysate of a cell expressing a recombinant IdeS or any functional variants thereof, preferably expressing a codon-optimized sequence comprising or consisting of SEQ ID NO: 3.

5. The method according to claim 4 wherein said initial solution is a lysate of a bacterial cell, preferably lysate of E.coli expressing a recombinant IdeS or any functional variants thereof.

6. The method according to any one of claims 1 to 5 wherein IdeS comprised in the initial solution of step i) is not precipitated.

7. The method according to any one of claims 1 to 6 wherein the initial solution is contacted with the anion exchange chromatography in step i) at a load flow rate comprised between 100 and 300 cm / h, preferably 150 and 200 cm / h, more preferably at 180 cm / h..

8. The method according to any one of claims 1 to 7 wherein the anion exchange chromatography is pre-equilibrated with an equilibration buffer comprising a neutral salt, preferably NaCl at a concentration comprised between 1 to 10 mM, preferably 2 to 6 mM, more preferably 4 mM.

9. The method according to any one of claims 1 to 8 wherein the wash buffer in step i) comprises a neutral salt, preferably NaCl at a concentration comprised between 5 to 40 mM, preferably 15 to 35 mM, more preferably 20 to 30 mM.

10. The method according to any one of claims 1 to 9 wherein the elution buffer in step i) is a gradient salt buffer comprising a neutral salt, preferably NaCl at a concentration comprised between 55 to 100 mM.

11. The method according to any one of claims 1 to 10 wherein the hydrophobic interaction chromatography is pre-equilibrated with an equilibration buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M.

12. The method according to any one of claims 1 to 11 wherein the feed solution, and optionally wash buffer of step ii) comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 3 M, preferably 0.5 and 2 M, more preferably 0.9 and 1.5 M.

13. The method according to any one of claims 1 to 12 wherein the feed solution is contacted with the hydrophobic interaction chromatography in step ii) with a load of feed solution comprised between 20 and 40 g of total protein per L of resin.

14. The method according to any one of claims 1 to 13 wherein the elution buffer in step ii) comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration below 0.9, preferably within a range between 0.8 and 0.9 M.

15. The method according to any one of claims 1 to 14 wherein the feed solution of step iii) comprises a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 0.5 to 5 M, preferably 1 and 4 M, more preferably 2.5 and 3.5 M, again more preferably 3M.

16. The method according to any one of claims 1 to 15 wherein the feed solution is contacted with the mixed mode chromatography in step iii) with a load of feed solution comprised between 12 and 20 g of total protein per L of resin.

17. The method according to any one of claims 1 to 16 wherein the elution buffer in step iii) comprises a gradient salt buffer comprising a salt selected from the group consisting of: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration comprised between 1.5 M to 0M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M more preferably 1.1 to 0.75 M..

18. IdeS protein solution obtainable from the method according to any one of claims 1 to 17, preferably comprising no more than 50 ng / mg of host cell protein and less than 3 pg / mg of host cell DNA.

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