Method for purifying 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 to a method for removing IdeS charge variants and IdeS obtainable from said method.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method for purifying immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes, preferably to a method for removing IdeS charge variants and IdeS obtainable from said method. BACKGROUND

[0002] IdeS (immunoglobulin G (IgG)-degrading protease) is an enzyme derived from Streptococcus pyogenes (S. pyogenes) that hydrolyzes IgG and can show important implications in therapeutic applications where neutralizing antibodies (NAbs) such as IgG need to be cleared. Streptococcus pyogenes IdeS is a cysteine protease of the papain superfamily that cleaves human IgG at positions 236 and 237 in the lower hinge region of the heavy chain in a two-step mechanism. It first cleaves one heavy chain, generating a single-cleaved IgG molecule (scIgG), and then cleaves the remaining heavy chain, resulting in 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, and thus this enzymatic activity can prevent IgG-mediated immune responses.

[0003] IdeS is a cysteine protease of the papain superfamily that cleaves human IgG at positions 236 and 237 in the lower hinge region of the heavy chain in a two-step mechanism. It first cleaves one heavy chain, generating a single-cleaved IgG molecule (scIgG), and then cleaves the remaining heavy chain, resulting in 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, and thus this enzymatic activity can prevent IgG-mediated immune responses.

[0004] Lei B., et al. (2000). Infection and immunity, 68(12), 6807-6818 first identified IdeS as a secreted immunogenic protein form. von Pawel-Rammingen et al. EMBO J. 2002 Apr 2;21(7):1607-15 first identified the function as an IgG-degrading protease. In this publication, IdeS was produced as a GST-tagged protein in E. coli.

[0005] 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 are cleaved off upon secretion, leaving a 35 kDa mature protein with a PI of 6.13. The IdeS protein sequence also contains only one cysteine (no disulfide bonds). Wenig, K., et al. (2004). Proceedings of the National Academy of Sciences of the United States of America, 101(50), 17371-17376 solved the first 3D structure of the IdeS C94S variant.

[0006] Escherichia coli is a commonly used expression system for recombinant drug proteins (Walsh G. (2018). Nature biotechnology, 36(12), 1136–1145). High-level expression of recombinant proteins often involves the formation of misfolded protein forms, commonly known as inclusion bodies, which require refolding under conditions that enable the production of active proteins (Buscajoni, L., et al. (2022). Biotechnology advances, 61, 108050).

[0007] The production of soluble recombinant proteins in the cytoplasm of E. coli may be hindered by the presence of modifications that degrade host cell proteins (HCPs), which are dominant and require the design of specific downstream processes to remove them.

[0008] In solution, IdeS has been reported as a monomeric protein, but it can also be re-exposed as a macroparticle (Vindebro, R., et al. (2013). FEBS letters, 587(12):1818–1822).

[0009] However, IdeS contains L-asparagine (Asn) and L-glutamine (Gln) residues in its sequence that can undergo deamidation, post-translational modifications influenced by factors such as protein structure (primary, secondary, and higher-order structures), temperature, and pH, and methionine (Met) residues that can undergo oxidation. These modifications introduce a negative charge (deamidation) or modify the polarity of IdeS (Met oxidation). These modifications can affect the structure and function of the protein.

[0010] The presence of inactive aggregates and / or charge variants presents a challenge for fabrication process design, as these forms are difficult to separate from the main active component. SUMMARY The inventors have designed and developed a specific purification method for recombinant IdeS obtained from *E. coli*, which allows for the enrichment of IdeS protein and the separation of host cell proteins, host nucleic acids, and IdeS charge variants. The inventors have unexpectedly demonstrated that a three-step chromatography process, comprising anion exchange chromatography, hydrophobic interaction chromatography, and hydrophobic cation exchange chromatography, allows for the acquisition of IdeS enriched to over 50%, with high clearance of contaminants (host cell DNA and proteins), and no more than 50 ng / mg of host cell protein and less than 3 pg / mg of residual DNA, representing an acceptable level of safety for human use.

[0012] This disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes, comprising the following steps: i) Contact the initial solution containing IdeS with anion exchange chromatography under conditions that allow IdeS to bind to the chromatographic anion ligand, preferably a quaternary ammonium ligand. Optionally, the anion exchange chromatography is washed with a washing buffer, preferably containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS is eluted with an elution buffer, preferably a gradient salt buffer containing a neutral salt, more preferably a NaCl with a concentration of 55 to 100 mM.

[0013] ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the hydrophobic ligands of the chromatography, preferably butyl ligands. Optionally, the hydrophobic interaction chromatography is washed with washing buffer. Preferably, the feed solution and washing buffer contain a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 3 M, more preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M. The fraction containing IdeS is eluted with an elution buffer, which preferably contains a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, with ammonium sulfate being preferred, more preferably at a concentration of less than 0.9 M, and more preferably from 0.8 to 0.9 M.

[0014] iii) Contacting the feed solution containing the elution fraction from step ii) with mixed-mode chromatography, preferably hydrophobic cation exchange chromatography, under conditions that allow IdeS to bind to the mixed-mode ligand, preferably 4-aminobenzamide acetate ligand, wherein the feed solution preferably contains a salt selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, preferably a gradient salt buffer containing salts selected from the following: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably 1.5M to 0M, preferably 1.2M to 0.375M or 1.1M to 0.6M, more preferably 1.1M to 0.75M.

[0015] In a preferred embodiment, the initial solution is contacted with the anion exchange chromatography in step i), wherein the initial solution is loaded with 20 to 35 g / L, more preferably 25 to 30 g / L total protein / L resin, even more preferably not exceeding 28 g / L, and / or wherein the loaded conductivity is 3 to 4 mS / cm, preferably 3 to 3.5 mS / cm, more preferably 3.1 to 3.3 mS / cm, to remove IdeS charge variants, particularly acidic IdeS charge variants.

[0016] In one specific embodiment, the starting solution is a cell lysate expressing recombinant IdeS or any variant thereof in bacterial cells, preferably Escherichia coli, and preferably expressing a codon-optimized sequence comprising SEQ ID NO: 3 or consisting of SEQ ID NO: 3. In a preferred embodiment, IdeS or any variant thereof contained in the initial solution of step i) does not precipitate.

[0017] In a preferred embodiment, in step i), the initial solution is contacted with anion exchange chromatography at a concentration of 100 cm⁻¹. 3 / h to 300cm 3 / h, preferably 150cm 3 / h to 200cm³ / h, more preferably 180cm³ / h 3 The loading flow rate is / h, and / or the initial solution loading is 25 to 95 g / L, preferably 30 to 45 g / L, more preferably 32 to 42 g / L total protein / L resin.

[0018] In one specific embodiment, anion exchange chromatography is pre-equilibrated with an equilibration buffer containing a neutral salt, preferably with a NaCl concentration of 1 to 10 mM, more preferably 2 to 6 mM, and even more preferably 4 mM.

[0019] In another specific embodiment, hydrophobic interaction chromatography is pre-equilibrated with an equilibration buffer comprising salts selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M.

[0020] In a preferred embodiment, in step ii), the feed solution is brought into contact with a hydrophobic interaction chromatography, wherein the feed solution is loaded with 20 to 40 g total protein / L resin.

[0021] In another preferred embodiment, in step iii), the feed solution is contacted with mixed-mode chromatography, wherein the sample loading is 12 to 20 g total protein / L resin. Attached Figure Description

[0022] Figure 1: Separation spectra using Capto Q anion exchange chromatography resin. A. Standard separation spectrum, including a 7.5 mM NaCl washing step and a 90 mM NaCl elution step; B. Optimized separation spectrum, including 4 mM NaCl conditions during the binding step, and 25 mM NaCl and 90 mM NaCl elution steps during the washing step. Figure 2 Anion exchange chromatography (AEX) separation performance.

[0023] Figure 3 SDS-PAGE analysis of AEX elution cells. A. Standard conditions, B. Optimized conditions.

[0024] Figure 4: Separation spectrum using Capto butyl anion exchange chromatography resin.

[0025] Figure 5 HCP scavenging factor of the Capto butyl step Figure 6 Overall DSP process performance for IdeS purification.

[0026] Figure 7 Example of charge variant distribution in AEX-HPLC for IdeS GMP DS batch #8010122001 Figure 8 Evolution of charge variant distribution in non-GMPDP batch DP22-072 after storage at 5±3℃ for 0 (T0M, 5℃), 1 month (T1M, 5℃), and 6 months (T6M, 5℃).

[0027] Figure 9 The evolution of charge variant distribution in non-GMP DP batch DP22-072 and its correlation with potency (a) or subvisible particle content (b) after storage at 5±3℃ for 0 (T0M, 5℃), 1 month (T1M, 5℃), and 6 months (T6M, 5℃). Figure 10 Three charge variant spectra of basic and acidic materials were obtained during the development process and compared with IdeS GMP DS.

[0028] Figure 11 SDS-PAGE analysis of AEX flow-through fractions using different loadings (42 (A&B) or 28 (C) g total protein / L resin (g / Lr)) and different clarifying loading conductivity (4.6 mS / cm (A); 3.1 mS / cm (B) and 3.2 mS / cm (C)). Narrow lines indicate the amount of protein corresponding to IdeS.

[0029] Figure 12Content (%) of three acidic variants in the IdeS elution cell after AEX chromatography using different loading amounts (42 or 28 g total protein / L resin (g / Lr)) and different clarifying loading conductivity (2.7 to 4.6 mS / cm). Detailed Implementation

[0030] The dominant presence of inactive aggregates and charge variants presents a challenge for fabrication process design, as these forms are difficult to separate from the main active component.

[0031] To address this problem, the inventors have developed a preparation method that combines anion exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography. This method allows for the efficient enrichment of IdeS in solutions without the presence of host cell proteins and charge variants.

[0032] This disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes, comprising the following steps: i) Contact the initial solution containing IdeS with anion exchange chromatography under conditions that allow IdeS to bind to the chromatographic anion ligands, optionally wash the anion exchange chromatography with washing buffer, and elute the fraction containing IdeS with elution buffer. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the hydrophobic ligands in the chromatography, optionally wash the hydrophobic interaction chromatography with washing buffer, and elute the fraction containing IdeS with elution buffer. iii) Contact the feed solution containing the elution fraction from step ii) with mixed-mode chromatography, preferably hydrophobic cation exchange chromatography, under conditions that allow IdeS to bind to the ligands in the mixed-mode chromatography, optionally wash the hydrophobic cation exchange chromatography with a washing buffer, and elute the fraction containing IdeS with an elution buffer.

[0033] IdeS (Immunoglobulin G degrading enzyme of Streptococcus pyogenes) (GenBank: QJC39417, updated April 20, 2020; SEQ ID NO: 1 (IdeS protein without peptide signal)) is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes. IdeS digests IgG at a specific site below the hinge region. IdeS cleaves several subclasses of IgG in various animals and efficiently converts IgG into Fc and (Fab)2 fragments. The N-terminal methionine and the signal sequence of IdeS are usually removed to form the mature IdeS protein. Amino acids 1-29 constitute the signal sequence and are excised upon secretion, leaving a 35 kDa mature protein with a PI of 6.13. IdeS is encoded by the IdeS gene (GenBank: JN035367, updated July 06, 2011, SEQ ID NO: 2).

[0034] In another embodiment, the cysteine ​​protease can be a functional variant of IdeS that retains cysteine ​​protease activity. IdeS activity can be assessed by any suitable method, for example, by incubating the 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.

[0035] According to this disclosure, the term "IdeS" refers to the IdeS protein as described above and any of its functional variants.

[0036] The functional variant of IdeS contains an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to SEQ ID NO: 1.

[0037] As used herein, the term “sequence identity” or “identity” refers to the percentage (%) of positional matches (identical amino acid residues) between two polypeptide sequences. Sequence identity is determined by comparing sequences during sequence alignment to maximize overlap and identity while minimizing sequence gaps. Specifically, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms, depending on the length of the two sequences. Global alignment algorithms (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) are preferred for aligning sequences of similar length, as they optimally align along their entire length. For sequences of substantially different lengths, local alignment algorithms (e.g., the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 30 1997; Altschul et al., 2005) are preferred. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways known to those skilled in the art, for example, using publicly available computer software available on internet websites 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 the alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For the purposes of this document, the % amino acid sequence identity value refers to the value generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to generate the best global alignment of two sequences, with all search parameters set to default values: score matrix = BLOSUM62, gap open = 10, gap extension = 0.5, terminal gap penalty = false, terminal gap open = 10, and terminal gap extension = 0.5.

[0038] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., a peptide isosteric group. This term applies to amino acid polymers containing naturally occurring amino acids, as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids or chemical analogs of naturally occurring amino acids. Amino acid polymers may contain one or more amino acid residues that have been modified by one or more natural processes such as post-translational processing, and / or one or more amino acid residues that have been modified by one or more chemical modification techniques known in the art.

[0039] The functional variants of the IdeS of this invention refer to amino acid sequences having fewer than 30, 25, 20, 15, 10, or 5 substituted, inserted, and / or deleted amino acid sequences that differ from the native sequence. In a preferred embodiment, the variant differs from the native sequence by one or more conserved substitutions, preferably fewer than 15, 10, or 5 conserved substitutions. Examples of conserved substitutions include 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).

[0040] IdeS production The initial solution containing the IdeS to be isolated can be obtained from lysates of biological samples or from cell cultures capable of synthesizing recombinant proteins. Various methods for synthesizing proteins via recombinant approaches are well known to those skilled in the art and particularly consist of introducing an expression cassette into cells containing a sequence encoding the IdeS or a functional variant thereof, under the control of regulatory elements necessary for its expression.

[0041] In a preferred embodiment, IdeS or a functional variant thereof can be generated by transforming cells, typically bacterial cells (e.g., Escherichia coli), with an expression cassette, preferably a plasmid encoding IdeS or a functional variant thereof.

[0042] Those skilled in the art can readily determine the coding sequence of IdeS or its functional variants based on the polypeptide sequence. In a preferred embodiment, the nucleic acid molecule may contain a codon-optimized sequence encoding IdeS or its functional variants; more preferably, the codon-optimized sequence encoding IdeS contains or consists of SEQ ID NO: 3.

[0043] In one specific embodiment, the plasmid contains an antibiotic resistance gene, such as a kanamycin resistance gene, to allow selection of transformed *E. coli*. In another specific embodiment, the plasmid does not contain an antibiotic resistance gene. In a more preferred embodiment, the IdeS coding sequence can be induced for 4 hours, for example, by cloning the IdeS coding sequence under the lac operon and inducing expression via isopropyl-β-D-thiogalactoside (IPTG).

[0044] Preferably, cells are separated, for example, by centrifugation followed by lysis. Cell lysis is a technique well known to those skilled in the art and can be performed chemically, mechanically, or enzymatically. The cell fragments can then be separated from the solution containing the recombinant product (i.e., the initial solution) by centrifugation, for example.

[0045] The initial solution may contain, for example, recombinant cell lysate, which contains IdeS and impurities.

[0046] As used herein, the term "impurity" or "contaminant" refers to any foreign or harmful molecule, including host cell proteins, host cell nucleic acids such as DNA and RNA, or protein variants such as aggregates, high molecular weight, low molecular weight and fragments, deamidated and charged variants.

[0047] As used herein, the term “host cell protein” (HCP) is intended to refer to non-IdeS proteins that can be derived from host cells (e.g., E. coli) used to produce IdeS proteins.

[0048] As used herein, the terms “host cell nucleic acid,” “host cell DNA,” or “host cell RNA” are intended to refer to nucleic acids derived from host cells (e.g., E. coli) used to produce the IdeS protein.

[0049] As used herein, the term "protein variant" refers to a variant of the target protein (i.e., an IdeS protein) that forms during the preparation and / or storage of the target protein. Specific examples of protein variants include high molecular weight classes, low molecular weight classes, fragments of proteins, post-translational or chemically modified proteins, including deamidated, isomerized, mismatched SS-linked, oxidized or altered conjugate forms (e.g., glycosylation, phosphorylation), resulting in charge variants or aggregates, including dimers of the target protein and at higher folds.

[0050] As used herein, the term “charge variant” refers to a protein alteration (e.g., by post-translational modification or chemical degradation) that changes the isoelectric point pH (pI) value, resulting in charge heterogeneity, and may include acidic and basic variants.

[0051] The IdeS protein contains L-asparagine (Asn) and L-glutamine (Gln) residues that can undergo deamidation, and methionine (Met) residues that can undergo oxidation. These modifications introduce a negative charge (deamidation) or modify the polarity of IdeS (Met oxidation) and can affect the protein's structure and function. In particular, the inventors have shown that 7 of the 22 Asn residues can be partially deamidated, and 3 of the 22 Met residues can be oxidized.

[0052] The inventors have demonstrated that IdeS readily aggregates or forms particles. The presence of visible and sub-visible particles of varying sizes in formulated active substances has also been reported during storage or after freeze-thaw cycles. The dominant presence of inactive aggregates and charge variants represents a challenge for process design, as these forms are difficult to separate from the primary active component.

[0053] To address this problem, a special preparation process has been developed to find a unique combination of schemes that allow for the stabilization of IdeS and enrichment of the solution with the main components.

[0054] The method for purifying IdeS or its functional variants according to this disclosure allows for the production of formulations containing IdeS protein and having reduced levels of impurities, particularly reduced levels of host cell proteins, host cell DNA, and protein variants such as aggregates and charge variants.

[0055] According to this disclosure, the initial solution containing the IdeS protein contains 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, the initial solution is a lysate of cells, preferably bacterial cells (e.g., Escherichia coli), which expresses and encodes the IdeS protein or a functional variant thereof, more preferably containing SEQ ID NO: 3 or a recombinant nucleic acid consisting of SEQ ID NO: 3.

[0056] In one specific embodiment, the initial solution is a cell lysate expressing the IdeS protein or a variant thereof, preferably containing 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 a bacterial cell (e.g., Escherichia coli) lysate expressing a recombinant nucleic acid encoding the IdeS protein or a variant thereof, more preferably containing SEQ ID NO: 3 or a recombinant nucleic acid consisting of SEQ ID NO: 3.

[0057] In a preferred embodiment, IdeS in the initial solution does not precipitate. Protein precipitation is achieved by adding a precipitating agent such as ethanol or methanol to reduce protein solubility.

[0058] Anion exchange chromatography An initial solution containing the IdeS protein and one or more contaminants is separated in the first step by anion exchange chromatography, preferably strong anion exchange chromatography.

[0059] Anion exchange chromatography is a process that uses an ion exchange matrix containing positively charged groups to separate substances based on their charges.

[0060] Examples of functional groups of such anion exchangers attached to the matrix support are, for example, primary, secondary, especially tertiary or quaternary aminoalkyl 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).

[0061] Suitable chromatography support matrices for anion exchange chromatography are known in the art. Examples are agarose-based resins and beads, dextran beads, polystyrene beads, and polystyrene / divinylbenzene resins, with agarose-based resins being preferred.

[0062] In a preferred embodiment, the anion exchange chromatography is a quaternary ammonium-based anion exchange chromatography immobilized on an agarose matrix.

[0063] Anion exchange chromatography can be pre-equilibrated with an equilibration buffer before loading an initial solution containing IdeS. In a preferred embodiment, the equilibration buffer contains 0 to 6 mM, preferably 2 to 6 mM, more preferably 3 to 5 mM NaCl (e.g., 4 mM NaCl) or other neutral salts such as potassium chloride, ammonium chloride, or sodium sulfate.

[0064] An initial solution of bacterial cell (e.g., Escherichia coli) lysate containing IdeS, preferably cell lysate, preferably expressing recombinant nucleic acid encoding IdeS protein or a functional variant thereof (more preferably containing SEQ ID NO: 3 or consisting of SEQ ID NO: 3) is contacted with anion exchange chromatography, and IdeS binds to a ligand (e.g., quaternary ammonium) attached to the stationary phase of the anion exchange chromatography.

[0065] According to this disclosure, prior to contact with anion exchange chromatography, it is preferable to prepare an initial solution containing IdeS or any functional variant thereof by adjusting the pH to a range of 6 to 10, preferably 7 to 9, more preferably 7.5 to 8.5, and even more preferably pH 8.

[0066] Controlling the content of acidic forms is crucial to ensuring the efficacy and safety (sub-visible particles) of IdeS as an active pharmaceutical ingredient. In this application, the inventors have identified parameters that enable the efficient purification of IdeS while eliminating acidic charge variants.

[0067] In a preferred embodiment, in order to eliminate acidic charge variants, the conductivity of the initial solution is 2 to 3.5 mS / cm, preferably 2.5 to 3.5 mS / cm, more preferably 2.5 to 3 mS / cm, and even more preferably 2.8 mS / cm.

[0068] According to this disclosure, the initial solution is then contacted with the anion exchange chromatography as described above, under conditions that allow IdeS to bind with the anion exchange chromatography support.

[0069] In some embodiments, the initial solution may be contacted with the column at a linear flow rate of 100 to 300 cm / h, more preferably 150 to 200 cm / h (e.g., 180 cm / h).

[0070] The interaction strength of ligands can also be affected by the ligands loaded on the anion exchange chromatography support. According to a preferred embodiment of this disclosure, particularly to eliminate acidic charge variants, the initial solution loading is 20 to 95 g / L, preferably 20 to 60 g / L, more preferably 20 to 35 g / L, even more preferably 20 to 30 g / L, and even more preferably 25 to 30 g / L (e.g., 28 g / L) total protein / L resin.

[0071] After IdeS is combined with anion exchange chromatography material, a washing step can be performed to remove contaminants from the anion exchange chromatography material.

[0072] In a preferred embodiment, the washing buffer contains a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and even more preferably 20 to 30 mM (e.g., 25 mM) of NaCl.

[0073] Following an optional washing step, the anion exchange chromatography material is eluted with a salt gradient, and IdeS is collected as the eluent.

[0074] In a preferred embodiment, the elution step is performed using a salt gradient of neutral salt, preferably NaCl with a concentration of 55 to 100 mM.

[0075] In a preferred embodiment of this disclosure, the initial solution, equilibration buffer, washing buffer, and elution buffer preferably have a pH in the range of 6 to 10, more preferably 7 to 9, more preferably 7.5 to 8.5, and even more preferably pH 8. In a more preferred embodiment, the initial solution, equilibration buffer, washing buffer, and elution buffer contain a buffer, typically a buffer containing at least one component selected from the following: MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycamide, 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, and even more preferably TRIS at a concentration of 1 mM to about 50 mM, preferably 10 to 30 mM (e.g., 20 mM).

[0076] Hydrophobic interaction chromatography (HIC) In the second step, the feed solution containing the elution solution from the anion exchange chromatography step (i.e., the first step) is contacted with the hydrophobic interaction chromatography under conditions that promote the binding of the IdeS to the hydrophobic interaction chromatography ligand.

[0077] Hydrophobic interaction chromatography (HIC) separates molecules based on hydrophobic interactions between immobilized hydrophobic ligands and nonpolar regions on the protein surface. Adsorption increases with higher salt concentrations in the mobile phase, and elution is achieved by decreasing the salt concentration of the eluent.

[0078] Suitable chromatographic support matrices for hydrophobic interaction chromatography are known in the art. HIC support matrices may comprise, as non-limiting examples, agarose, methacrylates, polystyrene, dextran, polystyrene, or polystyrene / divinylbenzene, preferably agarose. In a more preferred embodiment, the HIC support matrix comprises porous beads, preferably agarose porous beads.

[0079] According to this disclosure, the HIC support is a matrix substituted with hydrophobic ligands such as ethyl, butyl, phenyl or hexyl, preferably butyl and / or phenyl ligands, more preferably butyl ligands.

[0080] In a preferred embodiment, the HIC according to this disclosure comprises agarose porous beads substituted with butyl ligands.

[0081] According to this disclosure, a feed solution containing anion exchange chromatography eluent is prepared by adding a salt that promotes the adsorption of IdeS in the hydrophobic interaction chromatography support before contact with hydrophobic interaction chromatography.

[0082] As used herein, "salt" refers to one or more salts that make IdeS relatively hydrophobic, allowing it to bind to hydrophobically interacting chromatographic materials. The salt may be ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride, and potassium sulfate, preferably ammonium sulfate ((NH4)2SO4).

[0083] In a preferred embodiment, the feed solution contains a salt with a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M.

[0084] In a more preferred embodiment, the feed solution contains ammonium sulfate at a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M.

[0085] In a preferred embodiment, the conductivity of the feed solution is adjusted to at least 100 mS / cm, more preferably at least 150 mS / cm, more preferably 150-200 mS / cm, and even more preferably 170-190 mS / cm (e.g., 180 mS / cm).

[0086] The strength of the hydrophobic interaction of the ligands can also be affected by the ligands loaded on the HIC support. According to a preferred embodiment of this disclosure, the loading of the feed solution is 20 to 40 g / L total protein / L resin.

[0087] Before loading the feed solution containing the elution solution from the anion exchange chromatography step (i.e., the first step), HIC can be pre-equilibrated with an equilibration buffer with a salt concentration and pH similar to that of the feed solution to ensure that the IdeS protein binds tightly to the hydrophobic ligand.

[0088] In a preferred embodiment, the HIC is pre-equilibrated with an equilibration buffer containing a salt, preferably ammonium sulfate, at a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M.

[0089] According to this disclosure, the feed solution is then contacted with HIC as described above.

[0090] After IdeS is combined with hydrophobic interaction chromatography material, a washing step can be performed to remove contaminants from the hydrophobic interaction chromatography material.

[0091] In a preferred embodiment, the washing buffer contains a salt with a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M, preferably ammonium sulfate.

[0092] Following an optional washing step, the hydrophobic interaction chromatographic material is eluted with elution buffer, and IdeS is collected as the eluent.

[0093] In a preferred embodiment, the elution buffer contains a salt with a concentration of less than 0.9 M, preferably in the range of 0.8 to 0.9 M, preferably ammonium sulfate.

[0094] In a preferred embodiment of this disclosure, the feed solution, equilibration buffer, wash buffer, and elution buffer preferably have a pH in the range of 6 to 10, more preferably 7 to 9, more preferably 7.5 to 8.5, and even more preferably pH 8. In a more preferred embodiment, the feed solution, equilibration buffer, wash buffer, and elution buffer contain a buffer, typically a buffer containing at least one component selected from the following: MES, PIPES, ACES, BES, TES, HEPES, TRIS, histidine, imidazole, glycine, glycylglycine, glycamide, 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, and even more preferably Tris at a concentration of 1 mM to about 50 mM, preferably 10 to 30 mM (e.g., 20 mM).

[0095] Mixed-mode chromatography (hydrophobic cation exchange chromatography) In the third step, the feed solution containing the elution solution of HIC (i.e., the second step) is contacted with mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) under conditions that promote the binding of the IdeS to the mixed-mode chromatography ligand.

[0096] Mixed-mode chromatography (MMC) combines multi-mode interactions, including electrostatic, hydrophobic, and hydrogen bonding interactions.

[0097] According to this disclosure, the mixed-mode chromatography is preferably hydrophobic cation exchange chromatography, which contains a mixed-mode ligand comprising a cation exchange portion and a hydrophobic portion on a solid phase.

[0098] In a preferred embodiment, the cation exchange moiety is a carboxylic acid, and the hydrophobic group is a phenyl or aliphatic hydrocarbon chain, preferably phenyl. The cation exchange moiety and the hydrophobic moiety can be linked by a chain. Examples of such chains are peptide-containing chains, such as... —R1—C(O)—NH—R2— R1 and R2 are alkyl groups, and one or both of R1 and R2 may be absent.

[0099] A specific example is —C(O)—NH—CH2—.

[0100] In a preferred embodiment, the mixed-mode ligand is 4-aminobenzoic acid, also known as para-aminohippuric acid, with the following formula: A commercial example of a para-aminohippuric acid ligand on a hydrophobic cation exchange support is Nuvia. TM cPrime TM It is commercially available from Bio-Rad Laboratories, Inc. (Hercules, Calif.).

[0101] 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 of, for example, a matrix, beads, or particles, preferably beads with a median pore diameter of 0.5 micrometers or greater. In a preferred embodiment, the matrix is ​​a macroporous, highly cross-linked polymer.

[0102] Examples of monomers that can be polymerized to obtain a useful matrix are vinyl acetate, vinylpropylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, and vinylpyrrolidone (vinylpyrrolidone), which in some cases have functional groups.

[0103] According to this disclosure, a feed solution containing HIC elution solution is prepared by adding a salt that promotes the adsorption of IdeS in the hydrophobic cation exchange chromatography support before contacting mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography).

[0104] As used herein, the term "salt" is intended to refer to one or more salts that enable IdeS to be relatively hydrophobic, thus allowing it to bind with mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) materials. The salt may be ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride, and potassium sulfate, with ammonium sulfate ((NH4)2SO4) being preferred.

[0105] In a preferred embodiment, the feed solution contains a salt with a concentration of 0.5 to 5 M, preferably 1 to 4 M, and more preferably 2.5 to 3.5 M.

[0106] In a more preferred embodiment, the feed solution contains ammonium sulfate at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M.

[0107] In a preferred embodiment, the conductivity of the feed solution is adjusted to at least 100 mS / cm, more preferably at least 150 mS / cm, more preferably 150-200 mS / cm, even more preferably 170-190 mS / cm, and even more preferably 180 mS / cm.

[0108] The strength of ligand interactions can also be affected by the ligand loading on the support of mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography). According to a preferred embodiment of this disclosure, the loading of the feed solution is 5 to 40 g / L, preferably 10 to 30 g / L, and more preferably 12 to 20 g / L total protein / L resin.

[0109] Mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) can be pre-equilibrated with equilibration buffer before loading the feed solution containing IdeS.

[0110] In a preferred embodiment, the mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) is pre-equilibrated with an equilibration buffer containing a salt, preferably ammonium sulfate, at a concentration of 0.5 to 5 M, preferably 0.5 to 3 M, more preferably 1 to 2 M, and even more preferably 1.5 M.

[0111] According to this disclosure, the feed solution is then contacted with a mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) as described above.

[0112] The feed solution can be contacted with a mixed-mode chromatography support (e.g., hydrophobic cation exchange chromatography) under conditions that allow IdeS to bind to the mixed-mode support.

[0113] In some embodiments, the feed solution may be brought into contact with the tower at a linear flow rate of 100 to 600 cm / h, preferably 150 to 450 cm / h, more preferably 300 to 400 cm / h (e.g., 360 cm / h).

[0114] After IdeS is incorporated into a mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) material, a washing step can be performed to remove contaminants from the mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) material.

[0115] In a preferred embodiment, the washing buffer contains a salt, preferably ammonium sulfate, at a concentration of 0.5 to 5 M, preferably 0.5 to 3 M, more preferably 1 to 2 M, and even more preferably 1.5 M.

[0116] In a preferred embodiment of this disclosure, the feed solution, equilibration buffer, and wash buffer preferably have a pH in the range of 6 to 10, more preferably 7 to 9, and even more preferably 7.5 to 8.5. In a more preferred embodiment, the feed solution, equilibration buffer, and wash buffer comprise a buffer, typically a buffer containing at least one component selected from the following: MES, PIPES, ACES, BES, TES, HEPES, Tris, histidine, imidazole, glycine, glycylglycine, glycamide, 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, and even more preferably Tris at a concentration of 1 mM to about 50 mM, preferably 10 to 30 mM (e.g., 20 mM).

[0117] Following an optional washing step, the mixed-mode chromatography (e.g., hydrophobic cation exchange chromatography) material is eluted with elution buffer, and IdeS is collected as the eluent.

[0118] In a preferred embodiment, the elution step is performed using a salt gradient buffer, preferably an ammonium sulfate gradient buffer, more preferably at a concentration of 1.5 to 0 M, more preferably 1.2 M to 0.375 M, more preferably 1.1 M to 0.6 M, and more preferably 1.1 to 0.75 M.

[0119] In a preferred embodiment of this disclosure, the elution buffer preferably has a pH in the range of 2 to 6, more preferably 3 to 5, and even more preferably 3.5 to 4.5. In a more preferred embodiment, the elution buffer comprises a buffer, typically a buffer containing at least one component selected from the following: MES, PIPES, ACES, BES, TES, HEPES, Tris, histidine, imidazole, glycine, glycylglycine, glycamide, phosphate, acetic acid (e.g., sodium acetate), lactic acid, glutaric acid, citric acid, tartaric acid, malic acid, maleic acid, and succinic acid, more preferably sodium phosphate, and even more preferably a concentration of 1 mM to about 50 mM, preferably 10 to 30 mM (e.g., 20 mM).

[0120] In a preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes, comprising the following steps: i) Contact the initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to the anion exchange chromatography ligand (which is a quaternary ammonium ligand). Optionally, the anion exchange chromatography is washed with a washing buffer, preferably containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS is eluted with an elution buffer, preferably a gradient salt buffer containing neutral salts, more preferably NaCl at a concentration of 55 to 100 mM. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the hydrophobic interaction chromatography ligand (which is a butyl ligand). Optionally, the hydrophobic interaction chromatography is washed with washing buffer. Preferably, the feed solution and washing buffer contain a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 3 M, more preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M. The fraction containing IdeS is eluted with an elution buffer, which preferably contains a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, with ammonium sulfate being preferred, and more preferably at a concentration below 0.9 M, preferably in the range of 0.8 to 0.9 M. iii) Contact the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography under conditions that allow IdeS to bind with the hydrophobic cation exchange chromatography (which is a 4-aminobenzamide acetic acid ligand), preferably wherein the feed solution contains a salt selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, which preferably contains a gradient salt buffer selected from the following salts: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, more preferably 1.1M to 0.75M.

[0121] In a more preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes, comprising the following steps: i) Contact the initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to quaternary ammonium ligands. Optionally, the anion exchange chromatography is washed with a washing buffer containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS was eluted with a gradient salt buffer containing neutral salt, more preferably 55 to 100 mM NaCl. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the butyl ligand. Optionally, the hydrophobic interaction chromatography is washed with washing buffer. The feed solution and washing buffer contain ammonium sulfate, preferably at a concentration of 0.5 to 3 M, more preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M. The fraction containing IdeS was eluted with an elution buffer containing ammonium sulfate, more preferably at a concentration of less than 0.9, and most preferably 0.8 to 0.9 M. iii) Contact the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography under conditions that allow IdeS to bind to the 4-aminobenzamide acetic acid ligand, wherein the feed solution contains ammonium sulfate, preferably at a concentration of 0.5 to 5 M, more preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, wherein the elution buffer is a gradient ammonium sulfate buffer with a concentration of 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, and more preferably 1.1M to 0.75M.

[0122] In a more preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes, comprising the following steps: i) Contact the initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to quaternary ammonium ligands. Optionally, the anion exchange chromatography is washed with a washing buffer containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS was eluted with a gradient salt buffer containing 55 to 100 mM NaCl. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the butyl ligand. Optionally, the hydrophobic interaction chromatography is washed with washing buffer. The feed solution and wash buffer contain ammonium sulfate at a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M, and The fraction containing IdeS was eluted with an elution buffer containing ammonium sulfate at a concentration of less than 0.9, preferably 0.8 to 0.9 M. iii) Contacting the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography under conditions that allow IdeS to bind to the 4-aminobenzamide acetic acid ligand, wherein the feed solution contains ammonium sulfate at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, wherein the elution buffer is a gradient ammonium sulfate buffer with a concentration of 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, and more preferably 1.1M to 0.75M.

[0123] Controlling the content of acidic forms is crucial to ensuring the efficacy and safety (sub-visible particles) of IdeS as an active pharmaceutical ingredient. In this application, the inventors have identified parameters that enable the efficient purification of IdeS while eliminating acidic charge variants.

[0124] In a more preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes and removing the acidic variant of IdeS, comprising the following steps: i) Contacting an initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to anion exchange chromatography ligands (which are quaternary ammonium ligands), wherein the initial solution is contacted with the anion exchange chromatography, wherein the initial solution has a loading of 20 to 35 g / L, more preferably 25 to 30 g / L total protein / L resin, even more preferably not exceeding 28 g / L, and / or wherein the loading conductivity is 3 to 4 mS / cm, preferably 3 to 3.5 mS / cm, more preferably 3.1 to 3.3 mS / cm.

[0125] Optionally, the anion exchange chromatography is washed with a washing buffer, preferably containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS is eluted with an elution buffer, preferably a gradient salt buffer containing neutral salts, more preferably NaCl at a concentration of 55 to 100 mM. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the hydrophobic interaction chromatography ligand (which is a butyl ligand). Optionally, the hydrophobic interaction chromatography is washed with washing buffer. Preferably, the feed solution and washing buffer contain a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 3 M, more preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M. The fraction containing IdeS is eluted with an elution buffer, which preferably contains a salt selected from the group consisting of ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, and sodium sulfate, with ammonium sulfate being preferred, and more preferably at a concentration below 0.9 M, preferably in the range of 0.8 to 0.9 M. iii) Contact the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography under conditions that allow IdeS to bind with the hydrophobic cation exchange chromatography (which is a 4-aminobenzamide acetic acid ligand), preferably wherein the feed solution contains a salt selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, which preferably contains a gradient salt buffer selected from the following salts: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, preferably ammonium sulfate, more preferably 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, more preferably 1.1M to 0.75M.

[0126] In a more preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes and removing the acidic variant of IdeS, comprising the following steps: i) Contacting an initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to quaternary ammonium ligands, wherein the initial solution is contacted with the anion exchange chromatography, wherein the initial solution is loaded with 20 to 35 g / L, more preferably 25 to 30 g / L total protein / L resin, even more preferably not exceeding 28 g / L, and / or wherein the loaded conductivity is 3 to 4 mS / cm, preferably 3 to 3.5 mS / cm, more preferably 3.1 to 3.3 mS / cm.

[0127] Optionally, the anion exchange chromatography is washed with a washing buffer containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS was eluted with a gradient salt buffer containing neutral salt, preferably 55-100 mM NaCl. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the butyl ligand. Optionally, the hydrophobic interaction chromatography is washed with washing buffer. The feed solution and washing buffer contain ammonium sulfate, preferably at a concentration of 0.5 to 3 M, more preferably 0.5 to 2 M, and more preferably 0.9 to 1.5 M. The fraction containing IdeS was eluted with an elution buffer containing ammonium sulfate, more preferably at a concentration of less than 0.9, and most preferably 0.8 to 0.9 M. iii) Contacting the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography process under conditions that allow IdeS to bind to the 4-aminobenzamide acetic acid ligand, wherein the feed solution contains ammonium sulfate, preferably at a concentration of 0.5 to 5 M, more preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, wherein the elution buffer is a gradient ammonium sulfate buffer with a concentration of 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, and more preferably 1.1M to 0.75M.

[0128] In a more preferred embodiment, this disclosure relates to a method for purifying immunoglobulin G degrading enzyme (IdeS) from Streptococcus pyogenes and removing the acidic variant of IdeS, comprising the following steps: i) Contacting an initial solution containing IdeS (e.g., cell lysate) with anion exchange chromatography under conditions that allow IdeS to bind to quaternary ammonium ligands, wherein the initial solution is contacted with the anion exchange chromatography, wherein the initial solution is loaded with 20 to 35 g / L, more preferably 25 to 30 g / L total protein / L resin, even more preferably not exceeding 28 g / L, and / or wherein the loaded conductivity is 3 to 4 mS / cm, preferably 3 to 3.5 mS / cm, more preferably 3.1 to 3.3 mS / cm.

[0129] Optionally, the anion exchange chromatography is washed with a washing buffer containing a neutral salt, preferably at a concentration of 5 to 40 mM, more preferably 15 to 35 mM, and more preferably 20 to 30 mM NaCl. The fraction containing IdeS was eluted with a gradient salt buffer containing 55 to 100 mM NaCl. ii) Contact the feed solution containing the elution fraction from step i) with the hydrophobic interaction chromatography under conditions that allow IdeS to bind to the butyl ligand. Optionally, the hydrophobic interaction chromatography is washed with washing buffer. The feed solution and wash buffer contain ammonium sulfate at a concentration of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M, and The fraction containing IdeS was eluted with an elution buffer containing ammonium sulfate at a concentration of less than 0.9, preferably 0.8 to 0.9 M. iii) Contact the feed solution containing the elution fraction from step ii) with a hydrophobic cation exchange chromatography under conditions that allow IdeS to bind to the 4-aminobenzamide acetic acid ligand, wherein the feed solution contains ammonium sulfate at a concentration of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, and even more preferably 3 M. Optionally, the mixed-mode chromatography was washed with washing buffer, and The fraction containing IdeS is eluted with an elution buffer, wherein the elution buffer is a gradient ammonium sulfate buffer with a concentration of 1.5M to 0M, preferably 1.2M to 0.375M, 1.1M to 0.6M, and more preferably 1.1M to 0.75M.

[0130] In another respect, this disclosure also relates to IdeS protein solutions that can be obtained from the three-step chromatography method disclosed above.

[0131] Compared to the starting sample formulation, the IdeS protein solution eluted from the three-step chromatography disclosed above is highly concentrated for IdeS. Enrichment of the major component by more than 50% was achieved using the method according to this disclosure. Removal of impurities (host cell DNA and protein) reached acceptable levels to address safety concerns regarding human use of no more than 50 ng / mg host cell protein and / or less than 3 pg / mg residual DNA.

[0132] In some implementations, the IdeS protein solution obtainable from the three-step chromatography method contains no more than 50 ng / mg of host cell protein and / or less than 3 pg / mg of host cell DNA.

[0133] In some implementations, the IdeS protein solution obtainable from the three-step chromatography method contains no more than 25% of the acidic charge variant of the total eluted fraction.

[0134] The charge variant distribution among components was quantified using AEX-HPLC detection at 215 nm. The relative percentage of the major charge variant (corresponding to the acid variant) was calculated by dividing the peak area of ​​the corresponding component by the total peak area.

[0135] In some implementations, the amount of IdeS protein solution obtainable from the three-step chromatography method is enriched to more than 50%, 60%, 70%, 80% or more compared to the initial solution.

[0136] Various methods for assessing the purity of IdeS are known in the art. For example, in some embodiments, the purity of eluted IdeS can be analyzed by gel electrophoresis (e.g., SDS-PAGE), and bands corresponding to the IdeS protein can be visualized by staining (e.g., Coomassie staining). The purity of purified IdeS can also be assessed by measuring the amount of host cell proteins, charge variants, and / or host cell DNA contaminants (if any) present in the final purified product.

[0137] 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 for housekeeping genes or ribosomal RNA genes).

[0138] In another aspect, this disclosure provides kits for use with the methods described herein. The kits may optionally include written instructions and packaging materials. In some embodiments, the kit comprises (1) anion exchange chromatography support as described above, (2) hydrophobic interaction chromatography as described above, and (3) a mixed-mode chromatography support comprising the hydrophobic and cation exchange moieties as described above. Other reagents (e.g., equilibration, washing, and / or elution solutions) described herein in the context of the methods may also optionally be included in the kit.

[0139] Example IdeS drug substance is produced using genetically engineered E. coli strains, followed by multiple chromatographic steps to achieve high product purity and formulation via ultrafiltration / percolation (UF / DF).

[0140] The nucleotide sequence of the IdeS gene [JN 035367.1] was used as a reference sequence to chemically synthesize a codon-optimized coding sequence (SEQ ID NO: 3). The synthesized sequence was cloned into the pET28b+ expression plasmid, providing a key element for the production of recombinant IdeS, to obtain the pET28b+ / IDES plasmid.

[0141] The inventors have established a master cell bank (MCB) of Escherichia coli BL21ΔDE3 (XT7) from Xpress biologics SA transformed with pET28b+ / IDES plasmid for the process development and preparation of recombinant IdeS.

[0142] The number and pattern of chromatography steps have been specifically designed to remove process- and product-related impurities to levels acceptable to the active pharmaceutical ingredient and to stabilize IdeS in solution.

[0143] 1. IdeS production in Escherichia coli In a bioreactor employing a batch feed process, IdeS was generated in E. coli BL21ΔDE3(XT7) cells transformed with the pET28b+ / IdeS plasmid, wherein IdeS expression was induced by the addition of IPTG.

[0144] The fermentation step was terminated 4 hours after IPTG induction of IdeS expression. The culture was collected and cells were isolated. The supernatant was discarded, the whole bacterial pellet was recovered, and aliquoted into bags stored at ≤-60℃ until further processing.

[0145] All bacterial pellets were thawed, combined, and resuspended in 20 mM Tris pH 8.0 buffer. Cell membranes were disrupted by three autoclaving cycles to produce cell lysates containing recombinant products. The cell lysates were clarified by centrifugation, followed by deep filtration and a subsequent filtration step to produce a clear lysate.

[0146] 2. Development of an IdeS purification method for removing process and product impurities: A three-step chromatography method was designed for the purification of IdeS. To simplify the method, the initial step of protein precipitation was omitted.

[0147] The purpose of the first purification step is to capture soluble IdeS and remove process-related impurities. Based on the pH and conductivity of the cell extract and a PI of 6.13, anion exchange chromatography (AEX) was selected as the most suitable method using a neutral / weakly basic buffer system.

[0148] To maintain the unique ammonium sulfate system buffer during the remaining purification process, a hydrophobic interaction chromatography (HIC) step was used, followed by multimode chromatography (MMC) to purify IdeS.

[0149] 2.1 AEX Development AEX steps were developed using quaternary ammonium-based strong anion exchange chromatography. Several resins (Q Sepharose FF and CaptoQ) were screened.

[0150] The standard operating conditions are as follows: • Capto-Q is a chromatography resin equilibrated with 20 mM Tris pH 8.0 buffer.

[0151] • Adjust the pH and conductivity of the clarified pyrolyte to a target pH of 8.0 and a target conductivity of 3.2 mS.

[0152] • Wash the column with 20 mM Tris pH 8.0, then elute with 20 mM Tris pH 8.0 and 100 mM NaCl.

[0153] Figure 1 shows the typical elution spectra of HCP and IdeS charge variants. Due to the slight separation between HCP and IdeS, their removal could only be controlled through a co-occurrence strategy. The major component of the charge variants in the elution cell was 40%, and HCP was at least 30 μg / mg. Figure 2 Residual host cell proteins were quantified by ELISA. DNA contamination was measured using the ratio of absorbance in UV-A and absorbance at 260 nm and 280 nm. Under these conditions, eluted proteins remained residually contaminated with DNA, as indicated by an A260 / 280 ratio greater than 1 (e.g., a ratio greater than 0.6–0.7 typically indicates nucleic acid contamination).

[0154] The study investigated key process parameters to define operating conditions that would allow for the prevention of co-elution with HCP and charge variants.

[0155] Chromatography flow rate. The chromatography column was run at 360 cm / h. 180 cm / h was determined to be the optimal flow rate to minimize the risk of excessive back pressure on the column and to increase the column's separation capacity.

[0156] Protein loading. Test protein loading within the range of 22-190 g total protein per liter of resin. 32-42 g total protein per liter of resin is the optimal range. Overloading (95 g / L) negatively impacts separation efficiency and causes resin supersaturation. Underloading (22 g / L) affects the recovery and removal of impurities, particularly 50 kDa protein contaminants.

[0157] System buffer design. The following details the testing of different system buffers: • Equilibrate with up to 4 mM NaCl in 20 mM Tris pH 8.0 buffer. • Wash with up to 25 mM NaCl in 20 mM Tris pH 8.0 buffer. • Elution steps in 20 mM Tris pH 8.0 buffer with 55 to 100 mM sodium chloride. Combinations of these changes to the different process parameters listed above alter the distribution of HCP and IdeS charge variants (Figure 1b) and significantly improve the separation between HCP and IdeS. Depending on the chosen combination, the dominant component of the charge variant (target IdeS charge variant) can be as high as 60%, and HCP is reduced to just above 10 μg / mg in the elution cell. Figure 2 These changes allow for the removal of the main 50kDa protein contaminant ( ). Figure 3 ).

[0158] 2.2. HIC Development The HIC step was performed using media with varying degrees of hydrophobicity, and several resins (Phenyl HP resin, CaptoButyl ImpRes) were screened.

[0159] The standard operating conditions are as follows: • Capto Butyl ImpRes resin is a chromatography resin equilibrated with 20 mM Tris pH 8.0 and 1.5 M (NH4)2SO4 and operated at a flow rate of 180 cm / hr.

[0160] • Adjust the conductivity of the AEX elution cell to 180 ± 3 mS using 20 mM Tris pH 8.0 and 3 M (NH4)2SO4 buffer to allow IdeS to bind to Capto Butyl ImpRes resin.

[0161] • Apply elution gradients from 20 mM Tris pH 8.0, 1.5 M (NH4)2SO4 to 20 mM Tris pH 8.0, 0.1 M (NH4)2SO4.

[0162] Figure 4 shows typical elution profiles for the HCP and IdeS charge variants. Since there is only slight separation between HCP and IdeS, their removal can only be controlled through a merging strategy. Under these conditions, removal of contaminating DNA was achieved. The 260 / 280 ratio of the elution fractions is close to 0.5, which corresponds to “pure” protein.

[0163] The study investigated key process parameters to define operating conditions that allow for the prevention of co-elution with HCP and product-related impurities such as charge variants.

[0164] • Adjust the column’s AEX elution / equilibration to the range of 1.5–0.9 M (NH4)2SO4 in 20 mM Tris pH 8.0 buffer.

[0165] • Washing steps in 20 mM Tris pH 8.0 buffer with 1.5–0.9 M (NH4)2SO4. • Elution steps in 20 mM Tris pH 8.0 buffer with 0.8–0.9 M (NH4)2SO4. • Loading capacity is in the range of 20 to 40 g / L.

[0166] Combinations of these changes to the different process parameters listed above alter the distribution of HCP and IdeS charge variants (Figure 4) and significantly improve the separation between HCP and IdeS. Depending on the chosen combination, the clearance factor of the chromatography step (defined as the ratio between host cell proteins introduced into the feed solution and residual host cell proteins in the elution solution) can vary from no clearance (standard conditions) up to nearly 10-fold. Figure 5 ).

[0167] 2.3. Mixed Mode Tomography (MMC) Development Different resins were screened during the development of the MMC step: • Ceramic hydroxyapatite CHT type • Capto Adhere ImpRes are typically used to remove aggregates. • Eshmuno HCX is a multimode cation exchange medium designed for the direct capture of recombinant proteins with ligands at high salt concentrations.

[0168] Nuvia cPrime is a hydrophobic cation exchange resin with ligands - phenyl, amide and carboxyl groups.

[0169] Of the different resins tested, only hydrophobic cation exchange resins (e.g., Nuvia cPrime) resulted in efficient purification of IdeS without significant material loss. Therefore, Nuvia cPrime was selected.

[0170] The standard operating conditions are as follows: • Nuvia cPrime resin is a chromatography resin equilibrated with 20 mM Tris pH 8.0 and 1.5 M (NH4)2SO4 and operated at a flow rate of 360 cm / h.

[0171] • Adjust the conductivity of the HIC elution cell to 180 ± 3 mS using 20 mM Tris pH 8.0 and 3 M (NH4)2SO4 buffer to allow IdeS to bind to Nuvia cPrime resin.

[0172] • Apply elution gradients from 20 mM Tris pH 8.0, 1.5 M (NH4)2SO4 to 20 mM Tris pH 8.0, 0.375 M (NH4)2SO4.

[0173] Key process parameters were also studied.

[0174] • The elution step uses 20 mM sodium phosphate buffer, with a gradient of 1.5 M to 0 M of (NH4)2SO4 in pH 4.0. • Loading capacity is in the range of 12 to 20 g / L.

[0175] Under these conditions, more than 1.5 log of host cell protein removal can be achieved.

[0176] 2.4 Conclusion A three-step method for purifying IdeS was developed, which demonstrates these specific features that allow for simplification of its execution.

[0177] • IdeS can be captured directly from cell extracts. • Simple buffer systems are based on only two buffers: TRIS and ammonium sulfate. • Combinations of process parameter settings allow for the recovery of major components containing more than 50% charge variants and no more than approximately 50 ng / mg of host cell protein. Figure 6 IdeS with less than 3 pg / mg of residual DNA.

[0178] IdeS stability in solution during DSP was achieved by minimizing the duration of the holding time between chromatography steps and by applying appropriate dilution in ammonium sulfate buffer.

[0179] Then IdeS is formulated and concentrated by ultrafiltration / percolation.

[0180] A unique method for preparing IdeS based on a specific combination of process steps and controlled parameters has been developed. This method allows for the preparation of active IdeS: • Stability issues can be addressed by controlling the dilution of intermediate samples during the process.

[0181] • Impurities (host cell DNA and proteins) are removed to acceptable levels to address safety concerns for human use.

[0182] • More than 50% of the major components were enriched.

[0183] 3. Improvements to the IdeS purification method for removing acidic variants: 3.1 Charge Variant Distribution Spectrum in IdeS DSP methods were also developed to improve the efficacy and potency spectrum of IdeS as an active pharmaceutical ingredient, particularly by correcting the level of charge variants.

[0184] The charge variant distribution between components was quantified using AEX-HPLC detection at 215 nm.

[0185] The relative percentage of the major component is calculated by dividing the peak area of ​​the corresponding component by the total peak area. The relative percentage of the basic variants corresponding to the components eluted before the major components is calculated by dividing the peak area of ​​the corresponding component by the total peak area. The relative percentage of the acidic variants corresponding to the components eluted after the major components is calculated by dividing the peak area of ​​the corresponding component (the three major components: acid 1, acid 2, and acid 3) by the total peak area. The relative percentage of the major charge variants (corresponding to the acidic variants) is calculated by dividing the peak area of ​​the corresponding component by the total peak area. Figure 7 Table 1 provides exemplary AEX-HPLC chromatograms and integration strategies for estimating the amount of potential impurities.

[0186] Table 1: Integration Methods and Strategies for IdeS 3.2 Evolution of charge variants and their correlation with other properties When frozen at ≤-60°C, IdeS remains stable for at least 14 months across all tested properties.

[0187] However, IdeS is unstable when stored in liquid form. Conversely, stability data when stored in liquid form, for example at 5 ± 3 °C, show a reduction in the major protein component relative to the ionic charge variant. Figure 8 ).

[0188] The reduction of major protein components comes primarily at the expense of acidic forms, particularly acid 1 and acid 2, while basic forms remain generally stable.

[0189] Stability data showed that an increase in acidic variants was associated with a loss of potency. Figure 9 a) and the increase in subvisible particles ( Figure 9 b) Related.

[0190] The key finding is that controlling the content of the acidic form is crucial to ensuring the efficacy and safety (subvisible particles) of IdeS as an active pharmaceutical ingredient.

[0191] 3.3 Uncontrolled increase in acid 3 content during downstream processes of IdeS The implementation of the first version of the downstream process led to the preparation of IdeS extracted from E. coli cells and clinical materials such as IdeS GMPDS batch #8010122001.

[0192] Further development of downstream processes to achieve better impurity removal (e.g., removal of host cell DNA and proteins). Higher removal performance can be achieved by combining different control parameters, for example: • Increase the AEX loading from 35.5 to 42 g total protein / L resin (g / Lr). Test protein loading within the range of 22–190 g / Lr. 32–42 g / Lr is the optimal range, with 42 g / Lr as the set point to maximize DSP process yield. Overloading (95 g / Lr) adversely affects separation efficiency and causes resin supersaturation. Underloading (22 g / Lr) affects the recovery and removal of impurities, particularly 50 kDa protein contaminants.

[0193] • Control the HIC loading at 25 g / L total protein / L resin for optimal removal of process-related impurities.

[0194] Unexpectedly, during DSP development, although the changes increased the removal of product-related impurities, including basic variants, they were unexpectedly associated with an uncontrolled increase in acidic charge variants, particularly acidic 3 variants. Figure 10 ).

[0195] Compared to the 5.2% acid 3 variant content in IdeS GMP DS batch #8010122001, the development DS batch showed a level as high as 30%. Figure 10 ).

[0196] This increase was evident during the elution fraction of the AEX step. The AEX conditions were further investigated to identify potential methods to mitigate this problem.

[0197] 3.4 AEX Layered Development method Different AEX chromatography measurements were performed to test the effects of the loading amount and conductivity of the loaded solution on the yield and charge change spectrum of IdeS.

[0198] Different loading solutions were prepared as follows. Frozen E. coli cell pellets expressing IdeS were thawed, resuspended, and homogenized under high pressure. The resulting cell lysates were then clarified through a series of steps: centrifugation, deep filtration, and membrane filtration. The conductivity of the clarified harvest containing IdeS was then adjusted to a value from 2.7 mS / cm to 4.6 mS / cm by dilution with pure water or NaCl.

[0199] AEX separation was performed using pre-equilibrated Capto Q anion exchange chromatography resin.

[0200] To evaluate the effect of loading, the loading volume of pH / conductivity-adjusted pyrolytes was set at 28 g / Lr (clarified pyrolyte conductivity of 3.2 mS / cm) or 42 g / Lr (clarified pyrolyte conductivity of 2.7, 3.1 and 4.6 mS / cm).

[0201] Other parameters, such as wash volume, buffer composition, and elution volume and composition, are kept constant.

[0202] Results and Discussion In order to eliminate acidic charge variants to ensure the efficacy and safety of IdeS as a pharmaceutical agent, while allowing for good purification yield of IdeS, the inventors identified key parameters affecting the presence of acidic charge variants.

[0203] The results are summarized as follows: • As expected, reducing the loading reduced the yield. For a given eluent pooling strategy and similar loading conductivity (3.1 mS / cm), for amounts ranging from 42 g / Lr to 28 g / Lr, at similar conductivity, the yield decreased from 6 g of protein eluted per liter of resin to 4 g of protein.

[0204] • At a given concentration (42 g / Lr), the magnitude of conductivity in the range of 3.1 mS / cm to 4.6 mS / cm had a more unexpected effect, as the yield decreased by a factor of 2 from 6 g / Lr to 3 g / Lr. SDS-PAGE analysis of elution fractions from different AEX experiments ( Figure 11 This shows the earliest and unexpected elution of the IdeS form, which is affected by conductivity.

[0205] • Charge variant analysis of the elution fractions also allows for the discovery that both conductivity and loading affect the content of acid 3 ( Figure 12 ): o Increase the load to increase the acidity of form 3 o Increase conductivity to increase acidity in form 3 These results can be explained by molecular substitution in AEX, where the acidic 3 form may exhibit a higher interaction with the AEX stationary phase compared to the major component, causing them to elute before the major form.

[0206] When the total binding amount of the AEX stationary phase decreases due to an increase in loading and / or conductivity, the interaction between the dominant form and the AEX stationary phase is insufficient to prevent its elution during flow-through.

[0207] This phenomenon was unexpected because it is clear that no *E. coli* protein, which obviously has the highest interaction with the AEX stationary phase (i.e., elutes at higher ionic strengths), appears to act as a displacement agent. No visible molecular substitution of *E. coli* protein was detected in the SDS-PAGE gel. Figure 12 ).

[0208] in conclusion Recombinant IdeS exists in both basic and acidic charge variants. Controlling these variants is crucial for the production of safe and active pharmaceutical ingredients. Controlling the acidic variant is necessary because lower concentrations are associated with higher potency and lower levels of subvisible particles.

[0209] In this case, strict control of acidic form 3 requires adjustment of the loading (e.g., at a maximum of about 28 g of total protein per liter of resin) and the loading conductivity (e.g., in the range of about 3.1 to 3.3 mS / cm).

[0210] Sequences for practicing the disclosure: SEQ ID NO: 1 (IdeS protein without peptide signal) MDSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFING YRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFLTLSTGQDSWNQTN SEQ ID NO: 2 (Nucleic acid sequence encoding IdeS) (GenBank: JN035367.1) SEQ ID NO: 3 (IdeS codon-optimized sequence) ATGGATAGCTTTAGCGCCAATCAAGAAATTCGTTATAGCGAAGTTACCCCGTATCATGTTACCAGCGTTTGGACCAAAGGTGTTACCCCTCCGGCAAATTTTACCCAGGGTGAAGATGTTTTTCATGCACCGTATGTTGCAAATCAAGGCTGGTATGATATCACCAAAACCTTCAATGGTAAAGATGATCTGCTGTGTGGTGCAGCAACCGCAGGTAATATGCTGCATTGGTGGTTTGATCAGAACAAAGATCAGATCAAACGCTACCTGGAAGAACATCCGGAAAAACAGAAAATCAATTTTAACGGCGAGCAGATGTTCGATGTGAAAGAAGCAATTGACACCAAAAACCATCAGCTGGATAGCAAACTGTTCGAGTACTTTAAAGAAAAAGCGTTCCCGTATCTGAGCACCAAACATCTGGGTGTGTTTCCGGATCATGTTATCGATATGTTTATTAACGGTTATCGTCTGAGCCTGACCAATCATGGTCCGACACCGGTTAAAGAAGGTAGCAAAGATCCGCGTGGTGGTATTTTTGATGCAGTTTTTACCCGTGGTGATCAGAGCAAACTGCTGACCAGCCGTCATGATTTTAAAGAGAAAAACCTGAAAGAGATCAGCGACCTGATCAAAAAAGAACTGACCGAAGGTAAAGCACTGGGTCTGAGCCATACCTATGCAAATGTTCGTATTAACCATGTGATTAATCTGTGGGGTGCAGATTTTGATAGCAATGGTAATCTGAAAGCCATCTATGTTACCGATAGCGATAGTAATGCAAGCATTGGCATGAAGAAATACTTTGTGGGTGTTAATAGCGCAGGTAAAGTTGCAATTAGCGCGAAAGAAATCAAAGAGGATAATATTGGCGCACAGGTTCTGGGTCTGTTTACCCTGAGCACCGGTCAGGATAGCTGGAATCAGACCAACTAATAA

Claims

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

2. The method according to claim 1 for removing acidic charge variants, wherein in step i) the initial solution is contacted with an anion exchange chromatography, wherein the loading of the initial solution is between 20 and 35 g / L, more preferably between 25 and 30 g / L total protein per L resin, even more preferably not more than 28 g / L.

3. The method according to claim 1 or 2, wherein in step i) the initial solution is contacted with an anion exchange chromatography, wherein the loading conductivity is between 3 and 4 mS / cm, preferably between 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 the initial solution is a cell lysate expressing a recombinant IdeS or any functional variant thereof, preferably expressing a codon-optimized sequence comprising or consisting of SEQ ID NO:

3.

5. The method according to claim 4, wherein the initial solution is a bacterial cell lysate, preferably an E. coli lysate expressing a recombinant IdeS or any functional variant 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 in step i) the initial solution is contacted with the anion exchange chromatography at a loading flow rate of 100 cm / h to 300 cm / h, preferably 150 cm / h to 200 cm / h, more preferably 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 of 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 washing buffer in step i) comprises a neutral salt, preferably NaCI at a concentration of 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 NaCI at a concentration of 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 of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M.

12. The method according to any one of claims 1 to 11, wherein the feed solution and the optional washing buffer of step ii) comprise 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 of 0.5 to 3 M, preferably 0.5 to 2 M, more preferably 0.9 to 1.5 M.

13. The method according to any one of claims 1 to 12, wherein in step ii) the feed solution is contacted with the hydrophobic interaction chromatography, wherein the loading of the feed solution is 20 to 40 g 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 of below 0.9, preferably 0.8 to 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 of 0.5 to 5 M, preferably 1 to 4 M, more preferably 2.5 to 3.5 M, again more preferably 3 M.

16. The method according to any one of claims 1 to 15, wherein in step iii) the feed solution is contacted with the mixed mode chromatography, wherein the loading of the feed solution is 12 to 20 g 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, which 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 of 1.5 M to 0 M, preferably 1.2 M to 0.375 M, 1.1 M to 0.6 M, more preferably 1.1 to 0.75 M.

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

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