Purification of Soluble Complement Receptors and Their Variants

Abstract

The present invention relates to the purification of soluble complement receptor proteins and their variants by hydrophobic interaction chromatography (HIC) using a chromatography material having a particle size of less than 60 μm.

Description

Technical Field

[0001] Related Application Data This application claims priority based on European Patent Application No. 22179637.8, titled "Purification of soluble complement receptor and variants thereof", filed on June 17, 2022. The entire content thereof is incorporated herein by reference.

[0002] Sequence Listing This application is filed together with an electronic form of the sequence listing. The entire content of the sequence listing is incorporated herein by reference.

[0003] Field of the Invention The present invention relates to the purification of complement receptor proteins and variants thereof. The present invention provides a hydrophobic interaction chromatography (HIC) process that enables the high-yield purification of soluble complement receptors and variants thereof, particularly soluble complement receptor type 1 and variants thereof.

Background Art

[0004] The purification of proteins from a mixture may be based on differences in molecular properties such as size, charge, and solubility. The corresponding protocols are called size exclusion chromatography, ion exchange chromatography, differential precipitation, etc. The principle of chromatography is usually based on a system having at least two phases, a stationary phase and a mobile phase, and the mobile phase passes through the stationary phase. The mixture containing the target protein is dissolved in the mobile phase. The stationary phase interacts with the target protein in a non-covalent bond form. Due to this interaction, the target protein is retained and the remaining mobile phase can pass through the stationary phase. Thereafter, the target protein is eluted using an appropriate elution buffer.

[0005] HIC separates molecules based on their surface polarity. The chromatography material presents nonpolar groups (hydrophobic ligands) on its surface, which interact with the hydrophobic surface regions of a given molecule. The strength of the hydrophobic interaction depends particularly on the level of hydrophobicity of the chromatography material, i.e., the density of the hydrophobic ligand, pH, solvent, and its ionic strength.

[0006] The complement system is part of the innate immune system and contains a number of cell surface and soluble proteins that help eliminate foreign microorganisms while protecting the host from complement-related damage. The complement system includes soluble components C1 - C9, which are activated when their major components are fragmented, and the fragments activate further complement proteins, resulting in a proteolytic cascade. Activation of the complement system causes increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, direct killing of cells, and tissue damage.

[0007] Complement receptor type 1 (CR1) is a major regulator of complement activation. CR1 is present on the membranes of erythrocytes, monocytes / macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 binds to C3b and C4b and functions as a negative regulator of C3 activation, capable of inhibiting each of the classical pathway, the lectin pathway, and the alternative pathway, and is thus referred to as the C3b / C4b receptor.

[0008] The primary sequence of CR1 has been determined (Non-Patent Document 1, Non-Patent Document 2; Non-Patent Document 3). CR1 is composed of 30 short consensus repeat sequences containing 60 - 70 amino acids, 29 of which are conserved. The naturally occurring soluble form of CR1 (sCR1) has been detected in the plasma of healthy individuals and certain systemic lupus erythematosus (SLE) individuals (Non-Patent Document 4).

[0009] In recent years, a human sCR1 variant cleaved at amino acid 1392 has been found to retain the complement regulatory activity of the full-length protein and to be a potent inhibitor of complement activation. This fragment, called CSL040, exhibits affinity for C3b and C4b, as well as activity promoting their cleavage and decay, and was able to prevent organ damage in a glomerulonephritis model through a reduction in cell infiltration and urinary albumin. CSL040 has a modular structure that includes three long homologous repeat domains, LHR-A, LHR-B, and LHR-C, but does not include LHR-D. Therefore, CSL040 has been reported as a therapeutic candidate for complement-mediated disorders (Non-Patent Document 5). Aspects of CSL040 and additional sCR1 variants are described in Patent Document 1.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0012] In known purification protocols, the yield and / or purity are insufficient, thus requiring further purification steps. Therefore, an improved method for purifying complement receptor proteins and their variants is needed.

Means for Solving the Problems

[0013] The inventors have found that the yield of soluble complement receptors using hydrophobic interaction chromatography (HIC) can be improved by a particulate HIC material containing agarose and a hydrophobic ligand having a low particle size. Accordingly, the present invention is defined in the following items and relates to the methods described further herein.

[0014] Item 1: A method for purifying a soluble complement receptor protein (sCR) or a variant thereof, particularly CR1 or a variant thereof, comprising subjecting a liquid containing the complement receptor protein or a variant thereof to hydrophobic interaction chromatography (HIC), wherein the HIC comprises applying the liquid to a particulate HIC material containing agarose and a hydrophobic ligand, and the particle size of the particulate HIC material is less than 60 μm.

[0015] Item 2: The method according to Item 1, wherein the particle size is less than 55 μm, preferably less than 50 μm.

[0016] Item 3: The method according to item 1, wherein the particle size is 20 μm to 55 μm, particularly 30 μm to 55 μm, preferably 30 μm to 50 μm.

[0017] Item 4: The method according to any one of the preceding items, wherein the particle size is the median particle size of the cumulative volume distribution determined according to ASTM E 1772-95 (reapproved 2001) or ISO 13319-1:2021, particularly ASTM E 1772-95 (reapproved 2001).

[0018] Item 5: The method according to any one of the preceding items, wherein the agarose is cross-linked agarose.

[0019] Item 6: The method according to item 5, wherein the agarose is 6% cross-linked agarose.

[0020] Item 7: The method according to any one of the preceding items, wherein the HIC material has a ligand density of 5 μmol / mL to 60 μmol / mL, particularly 5 μmol / mL to 30 μmol / mL.

[0021] Item 8: The method according to any one of the preceding items, wherein the HIC material has a ligand density of 10 μmol / mL to 60 μmol / mL, particularly 10 μmol / mL to 30 μmol / mL.

[0022] Item 9: The method according to any one of the preceding items, wherein the hydrophobic ligand is selected from the group consisting of an aryl group, an alkyl group, and combinations thereof.

[0023] Item 10: The method according to any one of the preceding items, wherein the hydrophobic ligand is selected from the group consisting of a butyl group, a phenyl group, an octyl group, and combinations thereof.

[0024] Item 11: The method according to item 10, wherein the hydrophobic ligand is a butyl group.

[0025] Item 12: The method according to item 10, wherein the hydrophobic ligand is an octyl group.

[0026] Method according to item 10, wherein the hydrophobic ligand is a phenyl group.

[0027] Method according to any one of the preceding items, wherein the liquid applied to the HIC material contains at least one salt, in particular at a concentration of at least 0.4 M, preferably at least 0.5 M.

[0028] Method according to item 14, wherein the concentration of at least one salt, in particular sulfate, in the liquid is less than 1 M.

[0029] Method according to item 14 or 15, wherein at least one salt contains sulfate anions.

[0030] Method according to item 16, wherein at least one salt is selected from sodium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate and combinations thereof.

[0031] Method according to any one of the preceding items, wherein the liquid applied to the HIC material contains 0.4 M to 0.75 M of sodium sulfate.

[0032] Method according to any one of the preceding items, wherein the liquid applied to the HIC material has a pH of about 5 to about 8.

[0033] Method according to any one of the preceding items, further comprising: (1) washing the HIC material having the bound soluble complement receptor protein or a variant thereof, in particular wherein the washing buffer has a salt concentration of more than 0.1 M, preferably more than 0.3 M; and (2) applying an elution buffer to the HIC material to elute the soluble complement receptor protein or a variant thereof from the HIC material, wherein the elution buffer has a salt concentration of less than 0.3 M, preferably less than 0.1 M.

[0034] Method according to item 20, wherein said eluting comprises applying a gradient of decreasing salt concentration.

[0035] Item 22: The yield of the HIC process is more than 80%, the method according to any one of the preceding items.

[0036] Item 23: The host cell protein content in the HIC eluate is less than 1 μg / mg protein, preferably less than 0.5 μg / mg protein, more preferably less than 150 ng / mg protein, the method according to any one of the preceding items.

[0037] Item 24: The host cell protein depletion rate of HIC exceeds 50, the method according to any one of the preceding items.

[0038] Item 25: The soluble complement receptor protein or its variant is purified from the bioreactor harvest, especially the cell-free bioreactor harvest, the method according to any one of the preceding items.

[0039] Item 26: The soluble complement receptor protein or its variant contains an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2, especially a soluble complement receptor protein variant consisting of SEQ ID NO: 2, the method according to any one of the preceding items.

[0040] Item 27: The soluble complement receptor protein or its variant is an sCR1 variant lacking the long homology repeat region LHR-D and / or an sCR1 variant not containing the amino acid sequence corresponding to amino acids 1393-1971 of SEQ ID NO: 1, the method according to any one of the preceding items.

[0041] Item 28: Before the HIC, the following steps: a. Subjecting the mixture containing the complement receptor protein or its variant to cation exchange capture chromatography, especially using NaCl. b. Subjecting the composition to anion exchange chromatography, especially using NaCl. The method according to any one of the preceding items, further comprising.

[0042] Item 29: The method according to any one of the preceding items, wherein the soluble complement receptor protein or a variant thereof is a recombinant protein, particularly a recombinant soluble complement receptor type 1 protein or a variant thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0043]

Figure 1

Figure 2

[0044] The present invention relates to a method for purifying a soluble complement receptor protein or a variant thereof, comprising subjecting a liquid containing the complement receptor protein or a variant thereof to hydrophobic interaction chromatography (HIC), wherein the HIC comprises applying the liquid to a particulate HIC material comprising agarose and a hydrophobic ligand, wherein the particle size of the particulate HIC material is less than 60 μm.

[0045] General Throughout this specification, unless otherwise specified or required by the context, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, composition of matter, group of steps, or group of compositions of matter.

[0046] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds individually or collectively recited or shown herein, as well as any combinations or any two or more of the foregoing steps or features.

[0047] The present disclosure is not limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0048] Any example of the present disclosure is considered to be applicable to any other example of the present disclosure, with appropriate modifications, unless otherwise specifically stated. In other words, any specific example of the present disclosure can be combined with any other specific example of the present disclosure (except when mutually exclusive).

[0049] Any example of the present disclosure that discloses a particular feature or group of features, or a method or method step, is not to be construed as providing express support for excluding that particular feature or group of features, or that method or method step.

[0050] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0051] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y", and provides clear support for both meanings or either meaning.

[0052] Soluble complement receptor proteins and variants thereof Complement receptor type 1 (CR1), also known as C3b / C4b receptor or CD35, is a member of the complement activation control factor family. Naturally occurring CR1 is present on the membranes of erythrocytes, monocytes / macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes, and mediates cell binding to complement-activated particles and immune complexes. Human CR1 has a 41-amino acid signal peptide, a 1930-residue extracellular domain, a 25-residue transmembrane domain, and a 43-amino acid C-terminal cytoplasmic region.

[0053] Soluble complement receptor type 1 (sCR1) is naturally generated by cleavage of cell surface CR1 and plays a role in the control of complement activation at sites of inflammation. It should be understood that references to "sCR1" herein refer to soluble CR1, particularly the cleaved form of CR1 lacking the transmembrane and cytoplasmic domains. For purposes of naming only, and not by way of limitation, an exemplary sequence of human sCR1 including the signal peptide is shown in SEQ ID NO: 1. Amino acid positions are referred to herein with reference to the sCR1 protein consisting of 1971 amino acids (as shown, for example, in SEQ ID NO: 1). Full-length sCR1 contains four long homology repeat (LHR) regions, namely LHR-A, B, C, and D.

[0054] Sequences of sCR1 from other species can be determined using the sequences provided herein and / or sequences available in publicly accessible databases and / or using standard techniques such as those described in Ausubel el al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).

[0055] In some embodiments, the complement receptor protein or variant thereof used in the methods of the invention is sCR1 or a variant thereof, particularly an sCR1 variant such as CSL040. In some embodiments, the complement receptor protein or variant thereof used in the methods of the invention is human CR1 or a variant derived from human CR1, such as a variant comprising LHR-A, B and / or C of human CR1. In one embodiment, the soluble complement receptor protein or variant thereof is the soluble form of CR1. An exemplary sequence of human CR1 is shown in UniProt entry number P17927. In some embodiments, the complement receptor protein or variant thereof comprises SEQ ID NO: 2.

[0056] As used herein, the phrase "corresponding" when referring to the positions of the amino acids of SEQ ID NO: 1 is to be understood as referring to the amino acid residues or positions within the sCR1 sequence and not necessarily to a sequence that includes the entirety of SEQ ID NO: 1. For example, reference to the "positions corresponding to amino acids 42-939 of SEQ ID NO: 1" in an sCR1 sequence having a 41 amino acid N-terminal truncation (i.e., mature sCR1) refers to the amino acids at positions 1-898 of the N-terminal truncated sequence. In one embodiment, the soluble complement receptor protein or variant thereof comprises or consists of amino acids 42-1971 of SEQ ID NO: 1.

[0057] As used herein, the term "variant" refers to sCR1 that has undergone one or more amino acid substitutions, deletions, additions and / or truncations. Variants include naturally occurring isoforms of soluble CR1. Variants include sCR1 that has been truncated and comprises SEQ ID NO: 2. One example of such a variant is CSL040. Further variants are described herein and in the references cited below.

[0058] The term "recombinant" shall be understood to mean a product of artificial genetic recombination. Recombinant proteins include, for example, proteins expressed by artificial recombinant means when present within a cell, tissue, or subject in which they are expressed.

[0059] The term "protein" shall be construed to include a single polypeptide chain, i.e., a continuous series of amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other by covalent or non-covalent bonds (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using appropriate chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0060] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a continuous series of amino acids linked by peptide bonds.

[0061] Various sCR1 variants are disclosed in International Publication No. WO 2019 / 218009 A1 and corresponding U.S. Patent Application Publication No. US 2021 / 238238 (Application No. 17 / 053,981). These documents and the variants disclosed therein are hereby incorporated by reference in their entirety. In case of any conflict with the incorporated documents, the content of this specification shall prevail. Various sCR1 variants are also disclosed in Wymann et al. (2021) J Biol Chem. 296; see above for reference. In one embodiment, the sCR1 variant is amino acids 42 to 939 of SEQ ID NO: 1; amino acids 42 to 1392 of SEQ ID NO: 1; amino acids 42 to 1971 of SEQ ID NO: 1; amino acids 490 to 939 of SEQ ID NO: 1; amino acids 490 to 1392 of SEQ ID NO: 1; amino acids 490 to 1971 of SEQ ID NO: 1; or Comprising or consisting of their corresponding arrays.

[0062] In one embodiment, the sCR1 variant corresponds to SEQ ID NO: 1 that is cleaved at position 939 or 1392. In one embodiment, the sCR1 variant corresponds to SEQ ID NO: 1 that is cleaved at position 42 or 490.

[0063] In one embodiment, the sCR1 variant of the present disclosure does not include the amino acid sequence corresponding to amino acids 1-41 of SEQ ID NO: 1.

[0064] In one embodiment, the sCR1 variant of the present disclosure does not include the amino acid sequence corresponding to amino acids 940-1971 of SEQ ID NO: 1.

[0065] In one embodiment, the sCR1 variant of the present disclosure does not include the amino acid sequence corresponding to amino acids 1393-1971 of SEQ ID NO: 1.

[0066] In one embodiment, the sCR1 variant of the present disclosure does not include the amino acid sequence corresponding to amino acids 1-489 of SEQ ID NO: 1.

[0067] In one embodiment, the sCR1 variant corresponds to amino acids 42-1392 of SEQ ID NO: 1. In one embodiment, the sCR1 variant comprises an amino acid sequence selected from the group consisting of: (i) the amino acid sequence corresponding to amino acids 42-939 of SEQ ID NO: 1; and (ii) the amino acid sequence corresponding to amino acids 490-1392 of SEQ ID NO: 1. The sCR1 variant comprising residues 42-939 and / or residues 490-1392 of SEQ ID NO: 1 has increased complement inhibitory activity.

[0068] In one embodiment, the sCR1 variant of the present disclosure comprises a long homology repeat (LHR) region selected from the group consisting of: (i) LHR-A and LHR-B, but lacking LHR-C and LHR-D; (ii) LHR-A, LHR-B, and LHR-C, but lacking LHR-D; (iii) LHR-B and LHR-C, but lacking LHR-A and LHR-D; and (iv) LHR-B, LHR-C, and LHR-D, but lacking LHR-A. In such an embodiment, the LHR region LHR-A comprises an amino acid sequence corresponding to amino acids 42 to 489 of SEQ ID NO: 1; LHR-B comprises an amino acid sequence corresponding to amino acids 490 to 939 of SEQ ID NO: 1; LHR-C comprises an amino acid sequence corresponding to amino acids 940 to 1392 of SEQ ID NO: 1; and LHR-D comprises an amino acid sequence corresponding to amino acids 1393 to 1971 of SEQ ID NO: 1. In a convenient embodiment, the sCR1 variant of the present disclosure comprises the long homology repeat (LHR) regions LHR-A, LHR-B, and LHR-C, but does not comprise LHR-D.

[0069] In one embodiment, the sCR1 variant is a recombinant protein. In one embodiment, the sCR1 variant is a recombinant sCR1 variant comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% sequence identity to SEQ ID NO: 1, particularly an amino acid sequence having a length of at least 500, at least 600, at least 700, at least 800, or at least 900 amino acids.

[0070] In one embodiment, the sCR1 variant is a monomer (i.e., one copy of the sCR1 variant). In one embodiment, the sCR1 variant is a dimer or is dimerized (i.e., two copies of the sCR1 variant are linked in a fusion protein). In one embodiment, the sCR1 variant is a multimer or is multimerized (i.e., multiple copies of the sCR1 variant are linked in a fusion protein), see, for example, WO 2019 / 218009 A1.

[0071] In one embodiment, an sCR1 variant for use in the present disclosure comprises at least two sialylated sugar chains (e.g., di-, tri-, or tetra-sialylated sugar chains). For example, a composition for use in any of the methods described herein comprises a sialylated sCR1 variant glycoform. In one example, a sialylated sCR1 variant glycoform for use in any of the methods described herein comprises a di-, tri- or tetra-sialylated glycoform. Methods for producing variant sCR1 glycoforms that comprise at least two sialylated sugar chains (e.g., di-, tri-, or tetra-sialylated sugar chains) will be apparent to those skilled in the art and / or are described herein. In some embodiments, at least 30% of the sialylated sCR1 variant glycoform comprises mono-, di-, tri- and / or tetra-sialylated sugar chains.

[0072] Exemplary methods for determining the biological activity of the sCR1 variants of the present disclosure will be apparent to those skilled in the art. For example, methods for determining the inhibitory activity of the classical pathway, the lectin pathway and / or the alternative pathway are known in the art.

[0073] Exemplary compounds that can be conjugated to the sCR1 variants of the present disclosure and such conjugation methods are known in the art. In one example, an sCR1 variant of the present disclosure is conjugated to a half-life extending moiety. For example, the half-life extending moiety can be albumin, an antibody Fc region, or a functional fragment or variant thereof. sCR1 variants conjugated to a half-life extending moiety such as human serum albumin (HSA) or IgG4Fc are known in the art and, for example, some examples are described in the incorporated patent documents mentioned above. In one embodiment, the sCR1 variant comprises a half-life extending moiety selected from the group consisting of human serum albumin or a functional fragment thereof, an immunoglobulin Fc region or a functional fragment thereof, afamin, alpha-fetoprotein, vitamin D binding protein, an antibody fragment that binds to albumin, and a polymer.

[0074] HIC material The method of the present invention involves subjecting a particularly aqueous liquid containing said complement receptor protein or a variant thereof to hydrophobic interaction chromatography (HIC), where said HIC involves applying said liquid to a particulate (e.g., substantially spherical) HIC material comprising agarose and a hydrophobic ligand, where the particle size of said particulate HIC material is less than 60 μm.

[0075] As used herein, the term "particle size" refers to the median particle size of the cumulative volume distribution. This is determined in accordance with ASTM E 1772-95 (Reapproved 2001) "Standard Test Method for Particle Size Distribution of Chromatography Media by Electric Sensing Zone Technique" and / or ISO 13319-1:2021 "Determination of particle size distribution - Electrical sensing zone method - Part 1: Aperture / orifice tube method". In some embodiments, the particle size is determined in accordance with ASTM E 1772-95. In some embodiments, the particle size is determined in accordance with ISO 13319-1:2021.

[0076] The HIC material comprises or consists essentially of agarose having a covalently attached hydrophobic group. The hydrophobic group may be a hydrophobic hydrocarbon group. Preferably, the hydrophobic group is a hydrophobic alkyl group or a hydrophobic aryl group. More preferably, the hydrophobic group is a butyl group, a phenyl group, or an octyl group. Most preferably, the hydrophobic group is a phenyl group.

[0077] The HIC material typically consists of particles. In some embodiments, the particles are substantially spherical.

[0078] According to the present invention, the particle size of the particles is less than 60 μm. Preferably, the particle size is less than 55 μm, particularly less than 50 μm. In one embodiment, the particle size is 40 μm or less.

[0079] In some embodiments, the particle size is greater than 10 μm, particularly greater than 20 μm, preferably greater than 30 μm.

[0080] In other preferred embodiments, the particle size is from about 10 μm to about 55 μm, or from about 20 μm to about 50 μm, or from about 25 μm to about 45 μm. Also, a particle size of from about 30 μm to about 40 μm is also convenient. In some further embodiments, the particle size is from about 20 μm to about 55 μm, particularly from about 30 μm to about 55 μm, preferably from about 30 μm to about 50 μm.

[0081] In some embodiments, the HIC material used in the method of the present invention has a ligand density of from about 10 μmol / mL to about 60 μmol / mL, particularly from about 10 μmol / mL to about 50 μmol / mL, preferably from about 10 μmol / mL to about 30 μmol / mL. In some embodiments, the HIC material used in the method of the present invention has a ligand density of from 20 μmol / mL to 40 μmol / mL. In some embodiments, the HIC material used in the method of the present invention has a ligand density greater than 5 μmol / mL, particularly greater than 10 μmol / mL, preferably greater than 20 μmol / mL. In some embodiments, the HIC material used in the method of the present invention has a ligand density of less than 60 μmol / mL, particularly less than 50 μmol / mL. One skilled in the art can determine the ligand density using methods known in the art. In the case of an aromatic ligand, such as phenyl, the ligand density can be determined by measuring the absorbance of the hydrolyzed and dried sample of the HIC material. The ligand density is calculated from the molar extinction coefficient ε of the aromatic ligand using Lambert-Beer's law. In the case of an alkyl ligand, such as butyl or octyl, the ligand density can be determined by cleaving the ether bond with boron tribromide and quantifying the bromoalkane (e.g., bromobutane or bromooctane) produced by gas chromatography. When referring to the ligand density in units of "μmol / mL", this relates to the amount of ligand (μmol) relative to the resin (mL).

[0082] In some embodiments, the HIC material used in the method of the present invention has a ligand density of from about 0.2 μmol / mg to about 0.8 μmol / mg, particularly from about 0.25 μmol / mg to about 0.6 μmol / mg, preferably from about 0.3 μmol / mg to about 0.4 μmol / mg. When referring to the ligand density in units of "μmol / mg", this relates to the amount of ligand (μmol) relative to the resin (mg).

[0083] HIC materials suitable for use in the method of the present invention include, but are not limited to, the following, all of which are available from Cytiva®. · Capto® Phenyl ImpRes having the following characteristics. Matrix: high-flow agarose; hydrophobic ligand: phenyl group; particle size: about 40 μm; ligand density: about 9 μmol / mL medium. · Phenyl Sepharose® High Performance having the following characteristics. Matrix: 6% cross-linked agarose beads; hydrophobic ligand: phenyl group; particle size: about 34 μm; ligand density: about 25 μmol / mL medium. · Butyl Sepharose® High Performance having the following characteristics. Matrix: 6% cross-linked agarose beads; hydrophobic ligand: butyl group; particle size: about 34 μm; ligand density: about 50 μmol / mL medium.

[0084] In certain embodiments, the agarose is cross-linked agarose. Cross-linked agarose beads are typically available with agarose contents of 2%, 4%, and 6%, with 6% cross-linked agarose being preferred.

[0085] Salt solutions for protein binding to HIC columns Protein adsorption to HIC columns is preferred at high salt concentrations, but the actual concentration can vary significantly depending on the protein being selected and the nature of the specific HIC ligand. A liquid containing sCR1 or its variant can be loaded onto the HIC column, for example, by adding solid salt to the aforementioned eluent or by in-line dilution with a high-salt buffer. Details of the salts and concentrations suitable for protein binding to the column are described below.

[0086] Suitable salts that can be used include, but are not limited to, sodium chloride, magnesium chloride, sodium sulfate, ammonium sulfate, lithium sulfate, and potassium sulfate. In some embodiments, sulfates are used. Alkaline sulfates or ammonium sulfate, particularly ammonium sulfate, lithium sulfate, or sodium sulfate, have been shown to be advantageous. Sodium sulfate is particularly advantageous.

[0087] In some embodiments, sulfates are used at a concentration that avoids precipitation of the protein. In some embodiments, sulfates are used at a concentration of at least 0.4 M, preferably at least 0.5 M, where the concentration is low enough to avoid precipitation of the soluble complement receptor. Protein precipitation can be detected, for example, by turbidity measurement (see Examples). Examples of suitable concentration ranges are described herein below.

[0088] In some embodiments, in a liquid, salts, particularly sulfates, are used at a concentration of 500-900 mM, such as about 700 mM, to apply sCR1 or its variant onto an HIC material.

[0089] When ammonium sulfate is used, the concentration should be less than 2 M. In some embodiments, the appropriate concentration of ammonium sulfate in the loading buffer can be from about 0.5 M to about 1.5 M, preferably from about 0.5 M to about 1 M. Unfavorable ammonium sulfate concentrations have been found to have a negative impact on yield, for example, in terms of the possibility of protein precipitation.

[0090] When sodium sulfate is used, the concentration is preferably less than 1 M. In some embodiments, the appropriate concentration of sodium sulfate in the loading buffer can be from about 0.4 M to about 0.8 M, particularly from about 0.5 M to about 0.75 M, preferably from about 0.6 M to about 0.75 M, such as about 0.7 M. Unfavorable sodium sulfate concentrations have been found to have a negative impact on yield, for example, in terms of the possibility of protein precipitation.

[0091] When lithium sulfate is used, the concentration is preferably less than 1 M. In some embodiments, the appropriate concentration of sodium sulfate in the loading buffer can be from about 0.4 M to about 1 M, particularly from about 0.6 M to about 1 M, preferably about 1 M. An unfavorable lithium sulfate concentration has been found to have a negative impact on the yield, for example, in terms of the possibility of protein precipitation.

[0092] When potassium sulfate is used, the concentration is preferably less than 0.7 M. In some embodiments, the appropriate concentration of sodium sulfate in the loading buffer can be from about 0.3 M to about 0.8 M, particularly from about 0.4 to about 0.6 M, preferably about 0.5 M. An unfavorable potassium sulfate concentration has been found to have a negative impact on the yield, for example, in terms of the possibility of protein precipitation.

[0093] In some embodiments, the salt concentration of other salts can be in the range of about 0.5 M to about 2 M.

[0094] Preferably, at least one salt is an inorganic salt. In some convenient embodiments, only one inorganic salt is used, or at least 90% by weight, particularly at least 95% by weight, of the inorganic salts used is due to one inorganic salt. In other embodiments, a plurality of salts may be used, particularly a mixture of inorganic salts described herein.

[0095] Preferably, a salt concentration lower than the saturation limit of the protein, i.e., lower than the concentration at which the soluble complement receptor protein precipitates, particularly lower than the concentration that causes precipitation, but only up to 1 M lower than the concentration that causes precipitation, particularly only up to 0.5 M lower than the concentration that causes precipitation, is used. For example, if it is described that precipitation of the soluble complement receptor occurs at a salt concentration of 1.2 M as determined by turbidity, a suitable concentration may be 0.8 M, which is lower than 1.2 M but exactly 0.4 M lower than it (less than 1 M lower than it). At relatively high salt concentrations, but not high enough to cause precipitation of the soluble complement receptor, particularly in the case of sulfates, particularly high yields have been found. When the salt concentration is quite low, or when the salt concentration is high and protein precipitation occurs, the yield has been found to be low.

[0096] After loading sCR1 or its variant onto an HIC material, e.g., an HIC column, the HIC material may be washed using a high-salt concentration buffer as the loading buffer. Thereby, protein contaminants and other impurities are removed.

[0097] Elution of the protein Elution can be achieved in a stepwise or gradient form by various methods: (a) changing the salt concentration, (b) changing the polarity of the solvent, or (c) adding a surfactant. By decreasing the salt concentration, the adsorbed proteins are eluted in increasing order of hydrophobicity. In some embodiments, elution can be performed by applying a linear salt concentration gradient elution from the high salt concentration to a low concentration such as 0 mM. The change in polarity may be affected by the addition of a solvent such as ethylene glycol or (iso)propanol, thereby reducing the strength of the hydrophobic interaction. Surfactants function as protein substitutes and have mainly been used in the purification of membrane proteins.

[0098] Preferably, elution is performed by applying an aqueous elution buffer, particularly to an HIC material having a lower salt concentration than the loading buffer. Often, this is done by applying a gradient in which the salt concentration decreases. For example, sCR1 or a variant thereof may elute with a linear gradient to 0 mM salt.

[0099] Preferably, the yield of the HIC step is at least 80%, particularly at least 85%, preferably at least 90%.

[0100] The depletion rate of host cell proteins is preferably at least 50, more preferably at least 60 or more.

[0101] Further purification steps The method of the invention may include further purification steps (e.g., before and / or after HIC) including cation exchange chromatography (capture chromatography), anion exchange chromatography, and filtration.

[0102] The mixture containing sCR1 or its variant may be subjected to ion exchange chromatography as a first step. Various anion or cation substituents may be attached to the matrix to form an anion or cation support for chromatography. Anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Cation exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonic acid (S). Cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32, and CM-52 are available from Whatman Ltd., Maidstone, Kent, U.K. SEPHADEX®-type and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX®, as well as DEAE-, Q-, CM-, and S-SEPHAROSE® are available from Pharmacia AB. Furthermore, both DEAE and CM derivatized ethylene glycol-methacrylate copolymers, such as TOYOPEARL DEAE-650S and TOYOPEARL CM-650S, are available from Tosoh Bioscience, King of Prussia, PA.

[0103] The order of the purification steps, specifically in this order, is as follows: 1. Recovery and clarification of the cell culture supernatant 2. Capture cation exchange chromatography by applying a linear salt concentration gradient of NaCl 3. Inactivation of the virus 4. Depth filtration 5. Anion exchange chromatography in flow-through and / or bind-and-elute mode using NaCl 6. Hydrophobic interaction chromatography using Phenyl Sepharose HP in bind-and-elute mode 7. Filtration step may include.

[0104] Exemplary HIC Protocol An exemplary protocol for the HIC purification of CSL040 is described below: By using sodium sulfate as a chaotropic salt, CSL040 can be bound to the resin before eluting the product by reducing the high-salt conditions using a linear gradient. The Phenyl Sepharose HP column is packed to a bed height of 15 ± 1 cm. The column capacity is 15 - 20 g CSL040 / L resin. Solid sodium sulfate is added to the eluent of Toyopearl (trademark) NH2 750F up to a calculated concentration of 700 mM to achieve proper binding of CSL040 to the Phenyl Seoharose HP resin. The solid salt is added slowly while stirring constantly at room temperature until the conductivity level reaches about 80 mS / cm. pH adjustment is not performed after the addition of sodium sulfate. The raw material is applied to the column at a target linear flow rate of 115 cm / hour. After loading and washing after loading with HIC EQ-buffer (700 mM sodium sulfate, 148 mM sodium phosphate, pH 5.5 ± 0.1), the target protein is eluted by linear gradient elution from 700 mM sodium sulfate to 0 mM in 10 CV. Recovery is started when the UV signal exceeds 500 mAU / mm and stopped when the signal falls below 150 mAU / mm. The volume of the eluate approximates about 4 - 5 CV. Residual HMWC and CHO HCP in the Toyopearl NH2 750F eluate are further depleted (HMWC is 1% or less, and the CHO HCP level is less than 100 ppm). HIC EQ-buffer: 700 mM sodium sulfate, 148 mM sodium phosphate, pH 5.5. HIC elution buffer: 148 mM sodium phosphate, pH 5.5.

Example

[0105] Example 1: Generation of CSL040 and Its Derivatives CSL040 is a soluble variant of human CR1 that includes LHR domains A, B, and C. The amino acid sequence of mature CSL040 lacking the signal peptide is shown in SEQ ID NO: 2, which is the polypeptide used in this example. CSL040 derivatives were generated and expressed as described in Example 1 of U.S. Patent Application Publication No. 2021 / 238238 (corresponding to U.S. Patent Application No. 17 / 053,981 and International Publication No. 2019 / 218009 A1). CSL040 was generated accordingly. The disclosure of U.S. Patent Application Publication No. 2021 / 238238 is hereby incorporated by reference in its entirety. The generation of CSL040 derivatives with an 8x His tag is also described in Wyman et al., "A novel soluble complement receptor 1 fragment with enhanced therapeutic potential." Journal of Biological Chemistry 296 (2021).

[0106] For purification, the harvest from the cell-free bioreactor was adjusted for pH and conductivity before loading onto cation exchange chromatography to capture the target protein. CSL040 was eluted by applying a linear salt gradient of sodium chloride. Each eluate was virus inactivated and incubated at room temperature for at least 2 - 4 hours. After depth filtration, the filtrate was applied to an anion exchange chromatography resin. The target protein was eluted with an eluate containing sodium chloride.

[0107] The resulting partially purified CSL040 was used for further experiments.

[0108] Example 2: Effect of Salt The effects of the type and concentration of salts on the product quality were tested. To evaluate the effects of different chaotropic salts on product-related quality parameters (e.g., aggregation level), the quality of the CSL040 product was tested in the presence of different salt concentrations. Solid salts were added to the partially purified product to obtain different salt concentrations (see Table 1). The product-related quality attributes were tested by analytical size exclusion chromatography, showing the percentages of high molecular weight and low molecular weight components. Further, the turbidity (NTU - Nephelometric Turbidity Unit) of the samples was tested to evaluate the applicable salt concentrations at which the salting-out effect of the protein could be visualized. From Table 2, it is clearly seen that when sodium sulfate concentrations above 0.75 M and ammonium sulfate concentrations above 1 M are applied, the turbidity increases, indicating protein precipitation. The results are shown in Tables 1 and 2.

[0109]

Table 1

[0110]

Table 2

[0111] Example 3: High-Throughput Resin Screening In a high-throughput setting using TECAN-based and Cytiva PreDictor plates and Tosoh Seeker plates, the protein yield and host protein content (CHO HCP) were determined for different HIC resin materials. The following materials were compared.

[0112]

Table 3

[0113] The resin to be tested was transferred to a 96-well plate containing 20 μL to 50 μL of each test resin. Two loading densities (20 mg / mL and 40 mg / mL) were tested, and the binding capacity of each resin, the purity of the target protein, and the residual impurity level (e.g., CHO HCP) were evaluated to identify the appropriate HIC resin for CSL040 purification. Briefly, it is a high-throughput resin screening method. Each well was equilibrated with 5 equivalent volumes of resin using an equilibration buffer containing the same salt concentration as the loading material to be tested. After applying each salt-added loading material, the resin was washed with 5 equivalent volumes of resin with a high-salt concentration equilibration buffer. Elution of the target protein was achieved by reducing the salt concentration by applying a two-step elution. In the first step, the salt concentration was halved, and in the second step, the salt concentration was reduced to 0 mM. Each eluate was analyzed by UV measurement (yield) for product content and CHO HCP content. The yields are shown in Table 4 below.

[0114]

Table 4

[0115] Example 4: Column format To confirm the results of the high-throughput screening, this was repeated on a 1 mL column scale using sodium sulfate on the selected resin. Therefore, to the partially purified CSL040, each amount of solid sodium sulfate was added to a concentration of 0.7 M. After loading to a target concentration of 20 mg CSL040 / mL resin, the column was washed with an equilibration buffer containing an equal concentration of sodium sulfate, and then CSL040 was eluted from the resin using a linear gradient over 10 column volumes to a final sodium sulfate concentration of 0 M. Each yield was calculated based on the protein concentration by UV measurement in the eluate pool. Further experiments were conducted using different salts and / or salt concentrations. The results are summarized in Table 5.

[0116]

Table 5

[0117] An exemplary chromatogram of Phenyl Sepharose HP chromatography using sodium sulfate as a salt is shown in Figure 1. An exemplary chromatogram of Phenyl Sepharose HP chromatography using lithium sulfate as a salt is shown in Figure 2. Tests using potassium sulfate as a salt in Phenyl Sepharose HP chromatography were also successful.

Claims

1. A method for purifying a soluble complement receptor protein or a variant thereof, comprising subjecting a liquid containing the complement receptor protein or a variant thereof to hydrophobic interaction chromatography (HIC), wherein the HIC comprises applying the liquid to a particulate HIC material containing agarose and a hydrophobic ligand, and the particle size of the particulate HIC material is less than 60 μm.

2. The method according to claim 1, wherein the agarose is crosslinked agarose.

3. The method according to claim 1, wherein the particle size is 30 μm to 50 μm.

4. The method according to claim 1, wherein the HIC material has a ligand density of 5 μmol / mL to 60 μmol / mL, particularly 10 μmol / mL to 30 μmol / mL.

5. The method according to claim 1, wherein the hydrophobic ligand is selected from the group consisting of aryl groups, alkyl groups, and combinations thereof.

6. The method according to claim 1, wherein the liquid applied to the HIC material contains at least one salt at a concentration of at least 0.4 M.

7. The method according to claim 6, wherein the concentration of the at least one salt is lower than the concentration at which the soluble complement receptor protein precipitates.

8. The method according to claim 6, wherein the at least one salt contains a sulfate anion.

9. The method according to claim 8, wherein the at least one salt is selected from sodium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate and combinations thereof, and in particular from sodium sulfate, lithium sulfate, ammonium sulfate and combinations thereof.

10. The method according to claim 1, wherein the liquid applied to the HIC material has a pH of about 5 to about 8.

11. The method according to claim 1, further comprising (1) washing the HIC material having the bound soluble complement receptor protein or a variant thereof, and (2) applying an elution buffer to the HIC material to elute the soluble complement receptor protein or a variant thereof from the HIC material, wherein the elution buffer has a salt concentration of less than 0.3 M.

12. The method according to claim 1, wherein the host cell protein content in the HIC eluate is less than 1 μg / mg protein.

13. The method according to claim 1, wherein the host cell depletion rate of the HIC exceeds 50.

14. The method according to claim 1, wherein the soluble complement receptor protein or its variant is a recombinant protein, particularly recombinant soluble complement receptor protein type 1 or its variant.

15. Prior to the HIC mentioned above, the following steps are taken: a. A step of subjecting a mixture containing the complement receptor protein or a variant thereof to cation exchange capture chromatography. b. The process of subjecting the composition to anion exchange chromatography. The method according to claim 1, further comprising:

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