Purification of soluble complement receptors and variants thereof

Abstract

The present invention relates to the purification of soluble complement receptor proteins and variants thereof by hydrophobic interaction chromatography (HIC) using chromatography materials having a particle size of less than 60 [mu] m.

Description

[0001] Related application data

[0002] This application claims priority to European patent application No. 22179637.8, filed on June 17, 2022, entitled “Purification of soluble complement receptors and variants thereof”, the entire contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The entire contents of the sequence listing are hereby incorporated by reference in their entirety. Technical Field

[0005] The present invention relates to the purification of complement receptor proteins and variants thereof. The present invention provides a hydrophobic interaction chromatography (HIC) step which allows the purification of soluble complement receptors and variants thereof, in particular soluble complement receptor type 1 and variants thereof, in high yield. Background Art

[0006] Protein purification in a mixture can be based on differences in molecular properties, such as size, charge and solubility. The corresponding schemes are called size exclusion chromatography, ion exchange chromatography, differential precipitation, etc. The chromatography principle is usually based on a system with at least two phases (stationary phase and mobile phase), wherein 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 the form of non-covalent binding. This interaction allows the target protein to be detained, and the residual mobile phase flows through the stationary phase. The target protein can then be eluted using a suitable elution buffer.

[0007] HIC separates molecules based on their surface polarity. The chromatographic material displays non-polar groups (hydrophobic ligands) on its surface which interact with the hydrophobic surface area of ​​a given molecule. The strength of the hydrophobic interaction depends inter alia on the level of hydrophobicity of the chromatographic material (i.e. the density of the hydrophobic ligands), the pH, the solvent and its ionic strength.

[0008] The complement system is part of the innate immune system and involves many cell surface and soluble proteins that play a role in eliminating foreign microorganisms while protecting the host from complement-related damage. The complement system comprises soluble components C1-C9, and is activated when its major components are fragmented, and the fragments activate additional complement proteins leading to a proteolytic cascade. Activation of the complement system leads to increased vascular permeability, chemotaxis of phagocytes, activation of inflammatory cells, direct killing of cells, and tissue damage.

[0009] Complement receptor type 1 (CR1) is the main 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 is referred to as the C3b / C4b receptor because the C3b / C4b receptor acts as a negative regulator of C3 activation and can inhibit each of the classical, phosphatidic, and alternative pathways.

[0010] The primary sequence of CR1 has been determined (Klickstein et al., J. Exp. Med. 165: 1095-1112 (1987), Klickstein et al., J. Exp. Med. 168: 1699-1717 (1988); Hourcade et al., J. Exp Med. 168: 1255-1270 (1988)). The primary sequence of CR1 consists of 30 short common repeats containing 60-70 amino acids, 29 of which are conserved. Naturally occurring soluble CR1 (sCR1) has been detected in the plasma of normal individuals and some patients with systemic lupus erythematosus (SLE) (Yoon & Fearon J. Immunol. 134: 3332-3338 (1985)).

[0011] Recently, it was found that the human sCR1 variant truncated at amino acid 1392 retains the complement regulatory activity of the full-length protein, which is an effective inhibitor of complement activation. A fragment called CSL040 shows affinity for C3b and C4b and its cleavage and decay acceleration activity, and can prevent organ damage in glomerulonephritis models by reducing cell infiltration and urinary albumin. CSL040 has a modular structure comprising three long homologous repeat domains LHR-A, LHR-B and LHR-C, but does not include LHR-D. Therefore, the reported CSL040 is a candidate for the treatment of complement-mediated diseases (Wymann, S., Dai, Y., Nair, AG, Cao, H., Powers, GA, Schnell, A., ... & Hardy, MP (2021). A novel soluble complement receptor 1 fragment with enhanced therapeutic potential. Journal of Biological Chemistry, 296). Various aspects of CSL040 and other sCR1 variants are described in WO2019 / 218009.

[0012] The yield and / or purity of known purification schemes are insufficient and further purification steps are therefore required.Therefore, there is a need for improved methods for purifying complement receptor proteins and variants thereof. Summary of the invention

[0013] The inventors have found that the use of a particulate HIC material comprising agarose and a hydrophobic ligand having a low particle size can improve the yield of soluble complement receptors using HIC. Accordingly, the present invention relates to the methods defined in the following items and further described herein.

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

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

[0016] Item 3: The method of Item 1, wherein the particle size is 20 μm to 55 μm, in particular 30 μm to 55 μm, preferably 30 μm to 50 μm.

[0017] Item 4: The method of any 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 (2001 Reapproved) or ISO 13319-1:2021, in particular ASTM E 1772-95 (2001 Reapproved).

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

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

[0020] Item 7: The method of any of the preceding items, wherein the ligand density of the HIC material is 5 μmol / mL to 60 μmol / mL, in particular 5 μmol / mL to 30 μmol / mL.

[0021] Item 8: The method of any of the preceding items, wherein the ligand density of the HIC material is 10 μmol / mL to 60 μmol / mL, in particular 10 μmol / mL to 30 μmol / mL

[0022] Item 9: The method of any of the preceding items, wherein the hydrophobic ligand is selected from the group consisting of aromatic groups, alkyl groups, and combinations thereof.

[0023] Item 10: The method of any of the preceding items, wherein the hydrophobic ligand is selected from butyl, phenyl, octyl, and combinations thereof.

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

[0025] Item 12: The method of Item 10, wherein the hydrophobic ligand is octyl.

[0026] Item 13: The method of Item 10, wherein the hydrophobic ligand is phenyl.

[0027] Item 14: The method of any of the preceding items, wherein the liquid applied to the HIC material comprises at least one salt, in particular, the concentration of the salt is at least 0.4M, preferably at least 0.5M.

[0028] Item 15: The method of Item 14, wherein the concentration of at least one salt, especially sulfate, in the liquid is less than 1M.

[0029] Item 16: The method of Item 14 or 15, wherein at least one salt comprises a sulfate anion.

[0030] Item 17: The method of Item 16, wherein at least one salt is selected from sodium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, and combinations thereof.

[0031] Item 18: The method of any of the preceding items, wherein the liquid applied to the HIC material comprises 0.4M to 0.75M sodium sulfate.

[0032] Item 19: The method of any of the preceding items, wherein the pH of the liquid applied to the HIC material is from about 5 to about 8.

[0033] Item 20: The method of any of the preceding items, further comprising: (1) washing the HIC material to which the soluble complement receptor protein or its variant has been bound, in particular wherein the salt concentration of the washing buffer is greater than 0.1 M, preferably greater than 0.3 M, and (2) eluting the soluble complement receptor protein or its variant from the HIC material by applying an elution buffer to the HIC material, wherein the salt concentration of the elution buffer is less than 0.3 M, preferably less than 0.1 M.

[0034] Item 21: The method of Item 20, wherein the elution comprises applying a gradient of decreasing salt concentration.

[0035] Item 22: The method of any of the preceding items, wherein the yield of the HIC step is greater than 80%.

[0036] Item 23: The method of any of the preceding items, wherein 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.

[0037] Item 24: The method of any of the preceding items, wherein the host cell protein depletion factor of the HIC is greater than 50.

[0038] Item 25: The method of any of the preceding items, wherein the soluble complement receptor protein or variant thereof is purified from a bioreactor harvest, in particular a cell-free bioreactor harvest.

[0039] Item 26: The method of any of the preceding items, wherein the soluble complement receptor protein or a variant thereof comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2, in particular a soluble complement receptor protein variant consisting of SEQ ID NO: 2.

[0040] Item 27: The method of any of the preceding items, wherein the soluble complement receptor protein or variant thereof is a sCR1 variant lacking the long homologous repeat region LHR-D and / or a sCR1 variant that does not comprise the amino acid sequence corresponding to amino acids 1393 to 1971 of SEQ ID NO:1.

[0041] Item 28: The method of any of the preceding items, wherein the method further comprises the following steps before the HIC:

[0042] a. subjecting the mixture comprising the complement receptor protein or a variant thereof to cation exchange capture chromatography, in particular using NaCl,

[0043] b. subjecting the composition to anion exchange chromatography, in particular using NaCl.

[0044] Item 29: The method of any of the preceding items, wherein the soluble complement receptor protein or variant thereof is a recombinant protein, in particular a recombinant soluble complement receptor protein type 1 or a variant thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Depicting a chromatogram of Phenyl Sepharose HP chromatography using sodium sulfate as salt performed according to Example 4. The dashed line shows the conductivity curve and the solid line shows the UV signal.

[0046] Figure 2 Depicts the UV chromatogram of Phenyl Sepharose HP chromatography performed using lithium sulfate as salt according to Example 4. The dashed line shows the conductivity curve and the solid line shows the UV signal. DETAILED DESCRIPTION

[0047] The present invention relates to a method for purifying a soluble complement receptor protein or a variant thereof, the method 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 granular HIC material comprising agarose and a hydrophobic ligand, wherein the particle size of the granular HIC material is less than 60 μm.

[0048] General Description

[0049] In this specification, reference to an individual step, composition of matter, group of steps or group of compositions of matter shall be deemed to cover both one and more (i.e., one or more) of such steps, compositions of matter, groups of steps or groups of compositions of matter, unless specifically stated otherwise or the context requires otherwise.

[0050] Those skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of said steps or features or any two or more thereof.

[0051] The present invention is not to be limited in scope by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

[0052] Unless otherwise specifically stated, any embodiment of the present invention should be applicable to any other embodiment of the present invention after appropriate modification. In other words, any specific embodiment of the present invention can be combined with any other specific embodiment of the present invention (unless mutually exclusive).

[0053] Any example of the present invention disclosing a particular feature or group of features or method or method step is to be considered as providing explicit support for disclaimer of the particular feature or group of features or method or method step.

[0054] Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0055] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0056] Soluble complement receptor proteins and their variants

[0057] Complement receptor type 1 (CR1), also known as C3b / C4b receptor or CD35, is a member of the complement activation regulator 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 the binding of cells to particles and immune complexes with activated complement. 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.

[0058] Soluble complement receptor type 1 (sCR1) is naturally produced by cleavage of cell surface CR1 and plays a role in controlling complement activation at sites of inflammation. It should be understood that "sCR1" referred to herein refers to soluble CR1, particularly truncated CR1 lacking transmembrane and cytoplasmic domains. For purposes of nomenclature only and not limitation, an exemplary sequence of human sCR1 including a signal peptide is shown in SEQ ID NO:1. The positions of amino acids are referred to herein by reference to a sCR1 protein consisting of 1971 amino acids (e.g., as shown in SEQ ID NO:1). Full-length sCR1 comprises four long homologous repeat (LHR) regions, namely LHR-A, B, C and D.

[0059] The sequence of sCR1 from other species can be determined using the sequences provided herein and / or in publicly available databases and / or using standard techniques (e.g., as described in Ausubel et 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)).

[0060] In some embodiments, the complement receptor protein or variant thereof used in the methods of the present invention is sCR1 or a variant thereof, in particular a sCR1 variant, such as CSL040. In some embodiments, the complement receptor protein or variant thereof used in the methods of the present 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 a 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.

[0061] As used herein, the phrase "corresponding to" in reference to an amino acid position in SEQ ID NO: 1 is to be understood as referring to an amino acid residue or position within the sCR1 sequence and does not necessarily encompass the entire sequence of SEQ ID NO: 1. For example, in an sCR1 sequence having an N-terminal truncation of 41 amino acids (i.e., mature sCR1), reference to "positions corresponding to amino acids 42 to 939 of SEQ ID NO: 1" would refer to amino acids at positions 1 to 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 to 1971 of SEQ ID NO: 1.

[0062] 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 also include sCR1 that has undergone truncation and comprises SEQ ID NO:2. An example of such a variant is CSL040. Other variants are described herein and in the documents cited below.

[0063] The term "recombinant" is understood to mean the product of artificial genetic recombination. Recombinant protein also encompasses proteins expressed by artificial recombinant means when they are located in (eg, expressed in) a cell, tissue, or subject.

[0064] The term "protein" shall include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using a suitable chemical or disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0065] It will be understood from the preceding paragraph that the term "polypeptide" or "polypeptide chain" shall mean a series of consecutive amino acids linked by peptide bonds.

[0066] Various sCR1 variants are disclosed in WO 2019 / 218009 A1 and corresponding US2021 / 238238 A (application number 17 / 053,981). These documents and the variants disclosed therein are incorporated herein by reference in their entirety. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Wymann et al. (2021) J Biol Chem also discloses various sCR1 variants, see above. In one embodiment, the sCR1 variant comprises or consists of:

[0067] Amino acids 42 to 939 of SEQ ID NO: 1;

[0068] Amino acids 42 to 1392 of SEQ ID NO: 1;

[0069] Amino acids 42 to 1971 of SEQ ID NO: 1;

[0070] Amino acids 490 to 939 of SEQ ID NO: 1;

[0071] Amino acids 490 to 1392 of SEQ ID NO: 1;

[0072] Amino acids 490 to 1971 of SEQ ID NO: 1; or

[0073] Its corresponding sequence.

[0074] In one embodiment, the sCR1 variant corresponds to SEQ ID NO:1, said sCR1 variant is truncated at position 939 or 1392. In one embodiment, the sCR1 variant corresponds to SEQ ID NO:1, said sCR1 variant is truncated at position 42 or 490.

[0075] In one embodiment, the sCR1 variant of the invention does not comprise the amino acid sequence corresponding to amino acids 1 to 41 of SEQ ID NO:1.

[0076] In one embodiment, the sCR1 variant of the invention does not comprise the amino acid sequence corresponding to amino acids 940 to 1971 of SEQ ID NO:1.

[0077] In one embodiment, the sCR1 variant of the invention does not comprise the amino acid sequence corresponding to amino acids 1393 to 1971 of SEQ ID NO:1.

[0078] In one embodiment, the sCR1 variant of the invention does not comprise the amino acid sequence corresponding to amino acids 1 to 489 of SEQ ID NO:1.

[0079] In one embodiment, the sCR1 variant corresponds to amino acids 42 to 1392 of SEQ ID NO: 1. In one embodiment, the sCR1 variant comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence corresponding to amino acids 42 to 939 of SEQ ID NO: 1; and (ii) an amino acid sequence corresponding to amino acids 490 to 1392 of SEQ ID NO: 1. sCR1 variants comprising residues 42 to 939 and / or residues 490 to 1392 of SEQ ID NO: 1 have increased complement inhibitory activity.

[0080] In one embodiment, the sCR1 variant of the invention comprises a long homologous 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 embodiments, 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 an advantageous embodiment, the sCR1 variant of the present invention comprises the long homologous repeat (LHR) regions LHR-A, LHR-B, LHR-C, but not LHR-D.

[0081] 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, in particular 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.

[0082] 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 dimerized (i.e., two copies of the sCR1 variant are linked to form a fusion protein). In one embodiment, the sCR1 variant is a multimer or multimerized (i.e., multiple copies of the sCR1 variant are linked to form a fusion protein), see, e.g., WO 2019 / 218009A1.

[0083] In one embodiment, the sCR1 variants used in the present invention comprise at least two sialylated glycans (e.g., di-, tri-, or tetra-sialylated glycans). For example, the compositions used in any of the methods described herein comprise sialylated sCR1 variant glycoforms. In one example, the sialylated sCR1 variant glycoforms used in any of the methods described herein comprise di-, tri-, or tetra-sialylated glycoforms. Methods for producing variant sCR1 glycoforms comprising at least two sialylated glycans (e.g., di-, tri-, or tetra-sialylated glycans) will be apparent to the skilled artisan and / or as described herein. In some embodiments, at least 30% of the sialylated sCR1 variant glycoforms comprise mono-, di-, tri-, and / or tetra-sialylated glycans.

[0084] Exemplary methods for determining the biological activity of the sCR1 variants of the invention will be apparent to the skilled artisan.For example, methods for determining inhibitory activity of the classical, lectin and / or alternative pathways are known in the art.

[0085] Exemplary compounds that can be conjugated to the sCR1 variants of the present invention and methods for such conjugation are known in the art. In one example, the sCR1 variants of the present invention are 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 thereof or a variant. sCR1 variants conjugated to a half-life extending moiety such as human serum albumin (HSA) or IgG4 Fc are known in the art, for example, several examples are described in the patent documents incorporated above. In one embodiment, the sCR1 variant comprises a half-life extending moiety selected from 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 bound to albumin, and a polymer.

[0086] HIC Materials

[0087] The method of the present invention comprises subjecting a liquid (particularly aqueous) comprising the complement receptor protein or variant thereof to hydrophobic interaction chromatography (HIC), wherein the HIC comprises applying the liquid to a granular (e.g. substantially spherical) HIC material comprising agarose and a hydrophobic ligand, wherein the particle size of the granular HIC material is less than 60 μm.

[0088] As used herein, the term "particle size" refers to the median particle size of the cumulative volume distribution. It can be determined according to ASTM E1772-95 (2001 re-approved), "Standard Test Method for Particle Size Distribution of ChromatograpHy Media by Electric Sensing Zone Technique" and / or according to 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 according to ASTM E1772-95. In some embodiments, the particle size is determined according to ISO 13319-1:2021.

[0089] The HIC material comprises or consists essentially of agarose covalently linked to a hydrophobic group. The hydrophobic group may be a hydrophobic hydrocarbon group. Preferably, the hydrophobic group is a hydrophobic alkyl or a hydrophobic aryl. More preferably, the hydrophobic group is a butyl, phenyl or octyl group. Most preferably, the hydrophobic group is a phenyl group.

[0090] HIC materials are typically composed of particles. In some embodiments, the particles are substantially spherical.

[0091] 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, in particular less than 50 μm. In one embodiment, the particle size is 40 μm or less.

[0092] In some embodiments, the particle size is greater than 10 μm, particularly greater than 20 μm, preferably greater than 30 μm.

[0093] 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. Advantageously, the particle size is from about 30 μm to about 40 μm. 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.

[0094] In some embodiments, the ligand density of the HIC material used in the method of the present invention is from about 10 μmol / mL to about 60 μmol / mL, in particular 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 ligand density of the HIC material used in the method of the present invention is from 20 μmol / mL to 40 μmol / mL. In some embodiments, the ligand density of the HIC material used in the method of the present invention is greater than 5 μmol / mL, in particular greater than 10 μmol / mL, preferably greater than 20 μmol / mL. In some embodiments, the ligand density of the HIC material used in the method of the present invention is less than 60 μmol / mL, in particular less than 50 μmol / mL. Those skilled in the art are able to determine the ligand density using methods known in the art. For aromatic ligands, such as phenyl, the ligand density can be determined by measuring the absorbance of a hydrolyzed dry sample of the HIC material. The ligand density is then calculated from the molar absorbance ε of the aromatic ligand using Lambert-Beer's law. For alkyl ligands, 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., bromobutyl or bromooctane) formed by gas chromatography. When the unit "μmol / mL" is used to refer to the ligand density, this is related to the amount of ligand in μmol relative to the resin in mL.

[0095] In some embodiments, the ligand density of the HIC material used in the method of the present invention is about 0.2 μmol / mg to about 0.8 μmol / mg, in particular about 0.25 μmol / mg to about 0.6 μmol / mg, preferably about 0.3 μmol / mg to about 0.4 μmol / mg. When the unit "μmol / mg" is used to refer to the ligand density, this is related to the amount of ligand in μmol relative to mg of resin.

[0096] Suitable HIC materials for use in the methods of the present invention include, but are not limited to, the following materials, all of which can be obtained from The company obtained.

[0097] · Phenyl ImpRes, which has the following properties: Matrix: high flow agarose; Hydrophobic ligand: phenyl; Particle size: about 40 μm; Ligand density: about 9 μmol / mL culture medium.

[0098] ·Phenyl High Performance, it has the following characteristics: Matrix: 6% cross-linked agarose beads; Hydrophobic ligand: phenyl; Particle size: about 34 μm; Ligand density: about 25 μmol / mL culture medium.

[0099] Butyl High Performance, it has the following characteristics: Matrix: 6% cross-linked agarose beads; Hydrophobic ligand: butyl; Particle size: about 34 μm; Ligand density: about 50 μmol / mL culture medium.

[0100] In certain embodiments, the agarose is cross-linked agarose. Cross-linked agarose beads are typically used at 2%, 4%, and 6% agarose content, with 6% cross-linked agarose being preferred.

[0101] Salt solution for binding of proteins to HIC columns

[0102] High salt concentrations favor protein adsorption to the HIC column, but actual concentrations can vary widely, depending on the nature of the protein and the particular HIC ligand selected. For example, a liquid containing sCR1 or a variant thereof can be loaded onto a HIC column by spiking the eluate with a solid salt as described above or by in-line dilution with a high salt buffer. Details of suitable salts and concentrations for binding proteins to the column are provided below.

[0103] 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. Alkali sulfates or ammonium sulfate have proven to be advantageous, particularly ammonium sulfate, lithium sulfate, or sodium sulfate. Sodium sulfate is particularly advantageous.

[0104] In some embodiments, sulfate is used at a concentration that avoids protein precipitation, in some embodiments, sulfate is used at a concentration of at least 0.4 M, preferably at least 0.5 M, wherein the concentration is low enough to avoid precipitation of soluble complement receptors. For example, protein precipitation can be detected by turbidity measurement (see Examples). Examples of suitable concentration ranges are provided below.

[0105] In some embodiments, sCR1 or a variant thereof is applied to the HIC material using a salt, particularly a sulfate salt, at a concentration of 500 mM to 900 mM (eg, about 700 mM) in the liquid.

[0106] If ammonium sulfate is used, the concentration should be less than 2 M. In some embodiments, a suitable ammonium sulfate concentration in the loading buffer may be about 0.5 M to about 1.5 M, preferably about 0.5 M to about 1 M. Unfavorable ammonium sulfate concentrations have been found to negatively affect yield, for example due to possible protein precipitation.

[0107] If sodium sulfate is used, the concentration is preferably less than 1 M. In some embodiments, a suitable sodium sulfate concentration in the loading buffer may be about 0.4 M to about 0.8 M, particularly about 0.5 M to about 0.75 M, preferably about 0.6 M to about 0.75 M, for example about 0.7 M. It was found that unfavorable sodium sulfate concentrations can lead to a negative impact on the yield, for example in view of possible protein precipitation.

[0108] If lithium sulfate is used, the concentration is preferably less than 1 M. In some embodiments, a suitable sodium sulfate concentration in the loading buffer may be about 0.4 M to about 1 M, particularly about 0.6 M to about 1 M, preferably about 1 M. It has been found that unfavorable lithium sulfate concentrations may have a negative impact on the yield, for example in view of possible protein precipitation.

[0109] If potassium sulfate is used, the concentration is preferably less than 0.7 M. In some embodiments, a suitable sodium sulfate concentration in the loading buffer may be about 0.3 M to about 0,8 M, in particular about 0.4 to about 0.6 M, preferably about 0.5 M. It was found that unfavorable potassium sulfate concentrations can have a negative impact on the yield, for example in view of possible protein precipitation.

[0110] In some embodiments, the salt concentration of the other salts may range from about 0.5M to about 2M.

[0111] Preferably, at least one salt is an inorganic salt. In some advantageous embodiments, only one inorganic salt is used or at least 90%, in particular at least 95% (by weight) of the inorganic salts used belong to one inorganic salt. In other embodiments, more than one salt, in particular a mixture of the inorganic salts described herein, can be used.

[0112] Preferably, a salt concentration below the protein saturation limit is used, i.e., a salt concentration below the concentration at which the soluble complement receptor protein precipitates, particularly a salt concentration below the concentration that causes precipitation but not more than 1 M below the concentration that causes precipitation, particularly a salt concentration not more than 0.5 M below the concentration that causes precipitation. For example, if the soluble complement receptor precipitation occurs at a salt concentration of 1.2 M as determined by turbidity, a suitable concentration may be 0.8 M, which is less than 1.2 M but only 0.4 M below 1.2 M (0.4 M is less than 1 M below 1.2 M). It has been found that relatively high salt concentrations, but not sufficient to cause precipitation of the soluble complement receptor, result in particularly high yields, particularly with sulfate. It has been found that lower salt concentrations or higher salt concentrations cause protein precipitation, thereby providing lower yields.

[0113] After loading sCR1 or a variant thereof onto a HIC material (eg, a HIC column), the HIC material can be washed using a high salt buffer used as a loading buffer. This removes protein contaminants and other impurities.

[0114] Elution of proteins

[0115] Elution, whether in the form of stepwise elution or gradient elution, can be accomplished in a variety of ways: (a) by changing the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents. By reducing the salt concentration, the adsorbed proteins are eluted in the order of increasing hydrophobicity. In some embodiments, elution can be accomplished by applying a linear salt gradient elution from the high salt concentration to a lower concentration (such as 0 mM). The change in polarity may be affected by the addition of solvents (such as ethylene glycol or (iso)propanol), thereby reducing the strength of the hydrophobic interaction. Detergents act as protein displacers and are primarily used for the purification of membrane proteins.

[0116] Preferably, elution is performed by applying to the HIC material an aqueous elution buffer having a lower salt concentration than the loading buffer. Often, this is accomplished by applying a gradient of decreasing salt concentration. For example, sCR1 or a variant thereof may be eluted by a linear gradient to 0 mM salt.

[0117] Preferably, the yield of the HIC step is at least 80%, in particular at least 85%, preferably at least 90%.

[0118] The host cell protein depletion factor is preferably at least 50, more preferably at least 60 or higher.

[0119] Further purification steps

[0120] The methods of the invention may comprise additional purification steps (eg, before and / or after HIC) including cation exchange chromatography (capture chromatography), anion exchange chromatography, and filtration.

[0121] As a first step, a mixture comprising sCR1 or a variant thereof can be subjected to ion exchange chromatography. Various anionic or cationic substituents can be attached to a matrix to form an anionic or cationic matrix 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 sulfonate (S). Cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd., Maidstone, Kent, UK. Based on Crosslinked ion exchangers are also known. For example, DEAE-, QAE-, CM- and SP- and DEAE-, Q-, CM- and Available from PHarmacia AB. In addition, DEAE and CM derived ethylene glycol methacrylate copolymers such as TOYOPEARL DEAE-650S and TOYOPEARL CM-650S are available from Tosoh Bioscience (King of Prussia, PA).

[0122] A series of purification steps may comprise the following steps, in particular in the following order:

[0123] 1. Collection and clarification of cell culture supernatant,

[0124] 2. Capture cation exchange chromatography by applying a linear salt gradient of NaCl,

[0125] 3. Virus inactivation,

[0126] 4. Deep filtration,

[0127] 5. Anion exchange chromatography using NaCl in flow-through and / or bind elution mode,

[0128] 6. Hydrophobic interaction chromatography using phenyl sepharose HP in bind-elute mode,

[0129] 7. Filtration step.

[0130] Exemplary HIC Protocol

[0131] An exemplary scheme for HIC purification of CSL040 is described as follows: CSL040 can be bound to the resin using sodium sulfate as the chaotropic salt, and the product can then be eluted by reducing the high salt conditions using a linear gradient. A phenyl sepharose HP column was filled to a bed height of 15 cm ± 1 cm. The column capacity was 15 g-20 g CSL040 / L resin. TMThe NH2 750F eluate was doped with solid sodium sulfate at a calculated concentration of 700 mM to achieve the correct binding of CSL040 to phenyl agarose HP resin. Solid salt was slowly added to a conductivity level of about 80 mS / cm under continuous stirring at room temperature. No pH adjustment was performed after the addition of sodium sulfate. The feed was applied to the column at a target linear flow rate of 115 cm / h. After loading and post-loading washing with HIC-EQ buffer (700 mM sodium sulfate, 148 mM sodium phosphate, pH 5.5 ± 0.1), the target protein was eluted from 700 mM sodium sulfate to 0 mM by eluting with a linear gradient of 10 CV. Once the UV signal exceeded 500 mAU / mm, collection began, and collection stopped when the signal fell below 150 mAU / mm. The eluate volume was similar to about 4 CV-5 CV. The residual HMWC and CHO-HCP in the Toyopearl NH2 750F eluate were further depleted (HMWC ≤ 1%, CHO-HCP level < 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.

[0132] Example

[0133] Example 1: Production of CSL040 and its derivatives

[0134] CSL040 is a soluble variant of human CR1 comprising LHR domains A, B and C. The amino acid sequence of mature CSL040 lacking a signal peptide is shown in SEQ ID NO: 2, which is the polypeptide used in this example. CSL040 derivatives produced and expressed as described in Example 1 of US2021 / 238238A (U.S. Application No. 17 / 053,981, corresponding to WO 2019 / 218009A1). CSL040 was produced accordingly. The disclosure of US2021 / 238238 A is incorporated herein by reference in its entirety. The production of CSL040 derivatives with 8x His tags is also described in Wyman et al., "A novel soluble complement receptor 1 fragment with enhanced therapeutic potential." Journal of Biological Chemistry 296 (2021).

[0135] For purification, the cell-free bioreactor harvest was pH and conductivity adjusted before loading it onto a cation exchange chromatography to capture the target protein. CSL040 was eluted by applying a linear salt gradient of sodium chloride. The respective eluates were 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 eluent containing sodium chloride.

[0136] The resulting partially purified CSL040 was used for further experiments.

[0137] Example 2: Effect of Salt

[0138] The effect of test salt type and salt concentration on product quality. In order to evaluate the effect of different chaotropic salts on product-related quality parameters (such as aggregation level), the CSL040 product quality was tested at different salt concentrations. Solid salts were mixed into partially purified products to obtain different salt concentrations (see Table 1). The quality attributes associated with the product were tested by analyzing size exclusion chromatography, and the percentages of high and low molecular weight components were provided. In addition, the turbidity (NTU-scattering turbidity unit) of the test samples was used to evaluate the salt concentration applicable when the protein salting-out effect was visible. It can be clearly seen from Table 2 that applying a sodium sulfate concentration greater than 0.75M and an ammonium sulfate concentration greater than 1M resulted in higher turbidity, indicating protein precipitation. The results are shown in Tables 1 and 2.

[0139] Table 1. Sample composition and analysis.

[0140]

[0141]

[0142] Table 2. Turbidity tests for salt concentrations.

[0143]

[0144] Example 3: High-throughput resin screening

[0145] Protein yield and host protein content (CHO-HCP) of different HIC resin materials were determined in a high throughput setting using the TECAN system and Cytiva PreDictor and Tosoh Seeker plates. The following materials were compared.

[0146] Table 3. HIC resin materials.

[0147]

[0148] na – not obtained

[0149] Impres – Improved resolution

[0150] HS – High degree of substitution

[0151] LS-low degree of substitution

[0152] FF – Fast Flow

[0153] The resin to be tested is transferred to a 96-well plate, and each of the resins tested contains 20 μL to 50 μL. Two loading densities (20 mg / mL and 40 mg / mL) are tested to evaluate the binding capacity of each resin, the purity of the target protein, and the level of residual impurities (e.g., CHO HCP) to identify suitable HIC resins for CSL040 purification. The method of high-throughput resin screening is briefly introduced. Using an equilibrium buffer containing the same salt concentration as the loading material tested, each well is balanced with the equivalent of 5 resin volumes. After applying the loading material incorporating the corresponding salt, the resin is washed with a high-salt equilibrium buffer equivalent to 5 resin volumes. The elution of the target protein is achieved by applying a 2-step elution to reduce the salt concentration. The first step reduces the salt concentration by half, and the second step reduces the salt concentration to 0 mM. The product content of each eluate is analyzed by UV measurement (yield) and CHO-HCP content. The yield is shown in Table 4 below.

[0154] Table 4. High throughput resin screening of Cytiva and Tosoh resins using Cytiva PreDictor plates and Tosoh Seeker plates, respectively.

[0155]

[0156] Example 4: Column form

[0157] To confirm the high-throughput screening results, sodium sulfate was used to repeat these results on the selected resin at a 1 mL column scale. Therefore, the partially purified CSL040 was mixed with a corresponding amount of solid sodium sulfate to a concentration of 0.7 M. After loading to a target concentration of 20 mg CSL040 per mL resin, the column was washed with an equilibrium buffer containing an isotropic sodium sulfate, and then CSL040 was eluted from the resin using a linear gradient of 10 column volumes (to a final sodium sulfate concentration of 0 M). The corresponding yield was calculated based on the protein concentration measured by UV in the eluate pool. Further experiments were performed using different salts and / or salt concentrations. The results are summarized in Table 5.

[0158] Table 5. Column resins, conditions and yields.

[0159]

[0160] An exemplary chromatogram of phenyl sepharose HP chromatography using sodium sulfate as the salt is shown in Figure 1An exemplary chromatogram of phenyl sepharose HP chromatography using lithium sulfate as the salt is shown in Figure 2 Potassium sulfate was also successfully tested as a salt in phenyl sepharose HP chromatography.

Claims

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

2. The method of claim 1, wherein the agarose is cross-linked agarose.

3. The method according to claim 1 or 2, wherein the particle size is 30 μm to 50 μm.

4. The method according to any one of the preceding claims, wherein the ligand density of the HIC material is 5 to 60 μmol / mL, in particular 10 to 30 μmol / mL.

5. The method of any one of the preceding claims, wherein the hydrophobic ligand is selected from the group consisting of aryl, alkyl, and combinations thereof.

6. A method as claimed in any preceding claim, wherein the liquid applied to the HIC material comprises at least one salt in a concentration of at least 0.4M.

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

8. The method of claim 6 or 7, wherein the at least one salt comprises a sulfate anion.

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

10. The method of any one of the preceding claims, wherein the liquid applied to the HIC material has a pH of about 5 to about 8.

11. The method according to any one of the preceding claims, further comprising: (1) washing the HIC material to which the soluble complement receptor protein or variant thereof has been bound, and (2) eluting the soluble complement receptor protein or variant thereof from the HIC material by applying an elution buffer to the HIC material, wherein the salt concentration of the elution buffer is less than 0.3M.

12. A method as claimed in any preceding claim, wherein the host cell protein content in the HIC eluate is less than 1 μg / mg protein.

13. The method of any one of the preceding claims, wherein the host cell depletion factor of the HIC is greater than 50.

14. The method according to any one of the preceding claims, wherein the soluble complement receptor protein or variant thereof is a recombinant protein, in particular a recombinant soluble complement receptor protein type 1 or a variant thereof.

15. The method according to any one of the preceding claims, further comprising the following steps before the HIC: a. subjecting the mixture comprising the complement receptor protein or a variant thereof to cation exchange capture chromatography, b. Subjecting the composition to anion exchange chromatography.

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