Methods of removing impurities during recombinant production of nomacopan

Abstract

The invention is directed to methods for purifying fusion proteins. The invention further relates to fusion proteins produced by the methods disclosed herein.

Description

[0001] METHODS OF REMOVING IMPURITIES DURING RECOMBINANT PRODUCTION OF NOMACOPAN

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for purifying fusion proteins which involve hydrophobic interaction chromatography and / or ammonium sulphate precipitation. The invention further relates to fusion proteins produced by the methods disclosed herein, which may be suitable for ophthalmic delivery.

[0004] INCORPORATION BY REFERENCE

[0005] All documents cited in the text and listed at the end of this description are incorporated herein by reference.

[0006] BACKGROUND TO THE INVENTION

[0007] Purification of therapeutic proteins expressed in host cells is challenging. Although various purification methods are known in the art, which process(es) are suitable depends on the protein and the types of impurities to be removed. This is especially true for ophthalmological compositions, e.g., for intravitreal administration, which require high levels of purity.

[0008] Purification of therapeutic proteins

[0009] Impurities

[0010] When recombinant proteins are produced in a host cell system, there will inevitably be process- related impurities, for example host cell proteins (HCPs). HCPs arise from unspecific secretion and cell lysis during the fermentation process. As used herein, the term “host cell protein (HCP)” encompasses any protein produced by the host cell which is not the target recombinant protein. Other impurities in the crude cell culture medium after fermentation include host cells, host cell debris, host cell DNA, media components (e.g., antibiotics), pyrogens (e.g., endotoxins and non-endotoxin pyrogens), and viruses. These impurities must be removed, or at least significantly reduced, to produce pharmaceutically acceptable compositions suitable for administration to subjects. A variety of techniques for removing such impurities can be found in the art, and include centrifugation-, precipitation-, chromatography-, and membrane filtration-based purification steps. Which purification methods will be effective depends on the biochemical properties of the therapeutic protein and the impurities.

[0011] Pyrogens

[0012] As used herein, the term “pyrogen” encompasses any substance that causes a rise in body temperature (known as a fever, a fever reaction, or a febrile reaction) in an animal subject such as a human. Pyrogens typically exert their effect through activation of the innate immune system. This can cause side effects and even fatal reactions, and thus it is important to remove pyrogens from pharmaceutical compositions. Additional responses include a chilly sensation, cutaneous vasoconstriction, pupillary dilation, decrease in respiration, increase in arterial blood pressure, muscular pain and nausea and malaise [1], Pyrogens can be microbial or non-microbial substances, and can be classified into two groups: endotoxin and non-endotoxin pyrogens (NEPs).

[0013] Endotoxins are substances that are confined within a microorganism (e.g., Gram-negative bacteria) and are released when the microorganism breaks down or dies. The term endotoxin is commonly used synonymously with lipopolysaccharide (LPS). While LPS is the most common endotoxin, there are several other endotoxins, for example, tetanus toxin, Shiga toxin, and diphtheria toxin. In general, endotoxins comprise three parts: a lipid component containing fatty acids and disaccharide phosphates (Lipid A), O-specific polysaccharide side chains (O-antigen) and a core polysaccharide chain [2], Identification and removal of endotoxins is a key part of generating pharmaceutical compositions.

[0014] NEPs include non-endotoxin microbial substances, including those derived from Gram-positive bacteria, viruses, yeast and / or fungi. Examples of NEPs include, but are not limited to, lipoproteins, peptidoglycans, wall teichoic acids, and lipoteichoic acids.

[0015] Limits on pyrogen levels in pharmaceutical compositions

[0016] Due to their potentially harmful effects, even low levels of pyrogens (endotoxins and / or NEPs) must be removed from pharmaceutical compositions. This applies to pharmaceutical compositions that are applied in different ways, e.g., intravenously, subcutaneously, intraperitoneally or intraocularly.

[0017] For example, for intravenous compositions, the US Food & Drug Administration (FDA) has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one-hour period [3], EU is a standard unit of measure for endotoxin activity initially established relative to the activity contained in 0.2 ng of the U.S. Reference Standard Endotoxin Lot EC-2 (USP standard reference material). Endotoxin is expressed in International Units (IU) and one EU is equal to one IU [4], The recommended limit for intraocular devices is even lower, at 0.2 EU / mL [5],

[0018] Ophthalmic compositions for retinal diseases

[0019] Ophthalmic compositions are used to treat retinal diseases including dry AMD (e.g., GA), diabetic retinopathy, ROP, uveitis (e.g., autoimmune uveitis, infective uveitis), optic neuritis (e.g., glaucoma associated optic neuritis), wet AMD (also known as neovascular AMD), diabetic macular oedema, and retinal vein occlusion. Other retinal diseases include Stargardt disease, polypoidal choroidal vasculopathy, retinitis pigmentosa, hypertension retinopathy, and sickle cell retinopathy. Retinal diseases of particular interest intermediate and advanced include dry AMD, especially GA, an advanced form of dry AMD. WO2020 / 216513 provides experimental evidence that confirms the efficacy of nomacopan and functional equivalents thereof in the treatment of eye conditions via intravitreal administration.

[0020] Complement C5 and / or LTB4 inhibitors

[0021] W02004 / 106369 ([6]) relates to complement inhibitors. A particular subset of the disclosed complement inhibitors are directed at C5 and prevent C5 being cleaved into C5a and C5b by any of the complement activation pathways. A particular example of such an inhibitor of C5 cleavage is a protein produced by ticks of the species Ornithodoros moubata, which in mature form is a protein consisting of amino acids 19 to 168 of the amino acid sequence shown in Figure 4 of [6], In [6], this protein is known by the names “rVA576”, “EV576” and “OmCI protein” and has more recently been known as “Coversin” [7], This protein is referred to herein as “nomacopan” which is the INN for the protein.

[0022] In the tick, nomacopan is expressed as a pre-protein having a leader sequence comprising amino acids 1 to 18 of the amino acid sequence of SEQ ID NO: 2 at the N-terminal end of the mature nomacopan protein. The leader sequence is cleaved off after translation. The mature protein has the sequence consisting of amino acids 19 to 168 of the amino acid sequence [6] of SEQ ID NO: 2.

[0023] Nomacopan also has the ability to inhibit leukotriene B4 (LTB4) activity by sequestering it within the body of the protein. The ability to bind LTB4 may be demonstrated by standard in vitro assays known in the art, for example by means of a competitive ELISA between nomacopan and an anti-LTB4 antibody competing for binding to labelled LTB4, by isothermal titration calorimetry or by fluorescence titration.

[0024] There are a number of further patent applications, such as WO 2007 / 028968, WO 2008 / 029167, WO 2008 / 029169, WO 2011 / 083317, WO 2015 / 185760, WO 2016 / 198133, WO 2018 / 193120, WO 2018 / 193121 , WO 2018 / 193122, WO 2020 / 053206, WO 2020 / 216513, and WO 2021058117 which relate to the use of nomacopan and functional equivalents thereof in various applications.

[0025] PASylation®

[0026] PASylation® is a technology developed by XL-protein (http: / / xl-protein.com / ) which involves the genetic fusion of a PAS or PA sequence to a polypeptide of interest. W02008 / 155134 [8] describes PAS sequences consisting of proline, alanine, and serine residues. Subsequently, WO2011 / 144756 [9] described PA sequences consisting of proline and alanine (but not serine) residues. These documents also disclose vectors for stable expression of the proteins described therein.

[0027] Fusion of a 600 amino acid long PAS polypeptide (‘PAS600’) to nomacopan has been described as increasing the plasma half-life in mice by 52-fold from 0.2 hours to 10.4 hours

[0010] ,

[0028] PCT / EP2023 / 083992 provides additional fusion proteins comprising nomacopan and a PA(S) polypeptide (i.e., PAS or PA polypeptides). SUMMARY

[0029] In one aspect, the invention provides a method for removing pyrogens from a composition comprising a fusion protein, wherein the method comprises: a) providing an input composition comprising the fusion protein and a pyrogen; and b) performing hydrophobic interaction chromatography (HIC) using a stationary phase comprising hexyl ligands to produce an output composition comprising the fusion protein, and wherein the fusion protein comprises: i) a bioactive polypeptide, wherein the bioactive polypeptide comprises amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide.

[0030] In some embodiments, the HIC is performed on a chromatographic column. In some embodiments, the stationary phase: i) comprises a hydroxylated polymethacrylic polymer, optionally wherein the hydroxylated polymethacrylic polymer is coupled to the hexyl ligands; ii) has a mean pore size of about 100 nm (1000 A); iii) has a mean particle size of about 100 pm; and / or iv) has a mean ligand density of from about 30 g / L to about 50 g / L.

[0031] In some embodiments, step b) comprises: i) conditioning the input composition; ii) equilibrating the HIC stationary phase; iii) loading the conditioned composition onto the HIC stationary phase; iv) washing the HIC stationary phase; and v) eluting the fusion protein from the HIC stationary phase using a salt gradient to produce the output composition. In some embodiments, steps i), ii) and / or iv) are performed using a buffer, optionally wherein the buffer is HEPES / ammonium sulphate buffer, optionally wherein the HEPES buffer has a pH of between about pH 6 to about pH 9, further optionally about pH 7 to about pH 8, preferably about pH 7.6; and / or the salt gradient is generated using a HEPES / ammonium sulphate buffer and HEPES buffer.

[0032] In some embodiments, the pyrogen is selected from: a non-endotoxin pyrogen (NEP), optionally wherein the NEP is a lipoprotein, peptidoglycan, wall teichoic acid, and / or a lipoteichoic acid; and / or an endotoxin, optionally wherein the endotoxin is a lipopolysaccharide (LPS).

[0033] In some embodiments, the pyrogens in the input composition are detectable by measuring their ability to induce monocytes to secrete at least one cytokine, optionally as measured by a monocyte activation test (MAT) assay. In some embodiments, the at least one cytokine comprises IL-6, IL-1 p, TNF-a and / or IFN-y, preferably IL-6.

[0034] In some embodiments, at least 80% or 85% of the total protein in the input composition is fusion protein, as measured by any one of reversed-phase high-performance liquid chromatography (RP- HPLC), size-exclusion HPLC (SE-HPLC), ion exchange HPLC (IEX-HPLC) and / or SDS-PAGE.

[0035] In some embodiments, the removal of pyrogens is as assessed by measuring the ability of pyrogens in the output composition to induce monocytes to secrete at least one cytokine compared to the pyrogens in the input composition, optionally as measured by a MAT assay. In some embodiments, the at least one cytokine comprises IL-6, IL-1 p, TNF-a and / or IFN-y, preferably IL-6. In some embodiments, the level of pyrogens in the output composition is reduced by at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, compared to the level of pyrogens in the input composition, optionally as measured by the MAT assay.

[0036] In some embodiments, the at least 85% or 90% of the total protein in the output composition is fusion protein, as measured by any one of RP-HPLC, SE-HPLC, IEX-HPLC and / or SDS-PAGE.

[0037] In some embodiments, after step b), the output composition is: i) filtered, optionally i. by ultrafiltration and diafiltration, optionally into a storage buffer; ii. by tangential flow filtration (TFF); and / or Hi. 0.2 pm sterile filtered; ii) formulated with at least one pharmaceutical carrier or excipient; and / or iii) filled into any sterile container. In some embodiments, the filtered output composition is suitable for administration to a subject, preferably a human subject.

[0038] In another aspect, the invention provides a composition produced by any of the methods described above and herein.

[0039] In a further aspect, the invention provides a method for removing host cell proteins (HCPs) from a composition comprising a fusion protein, the method comprising: a) providing an input composition comprising at least 30 L of cell culture medium or cell culture supernatant comprising the fusion protein and a HCP; b) performing ammonium sulphate precipitation to precipitate the fusion protein from the cell culture medium or cell culture supernatant; and c) resolubilizing the fusion protein to provide an output composition; and wherein the fusion protein comprises: i) a bioactive polypeptide, wherein the bioactive polypeptide comprises amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide.

[0040] In some embodiments, the method comprises providing an input composition comprising: i) at least 50 L, at least 100 L, at least 200 L, at least 400 L, or at least 600 L of cell culture medium or cell culture supernatant; or ii) between about 400 L to about 1000 L, or about 600 L to about 800 L, preferably about 650 L to about 750 L of cell culture medium or cell culture supernatant.

[0041] In some embodiments, the fusion protein is precipitated by addition of ammonium sulphate to a final concentration of between about 0.7 M (17.5% saturation) and about 1.4 M (35% saturation), preferably about 1 M (25% saturated).

[0042] In some embodiments, the method removes at least one of the following: host cell debris, host cell DNA, media components, pyrogens, and viruses.

[0043] In some embodiments, the total amount of fusion protein in the output composition is at least 70%, at least 75%, or at least 80% of the total amount of fusion protein in the input composition.

[0044] In some embodiments, at least 45% or at least 50% of the total protein in the output composition is fusion protein as measured by any one of RP-HPLC, SE-HPLC, IEX-HPLC and / or SDS-PAGE.

[0045] In any aspects of the invention, the bioactive polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to the sequence of amino acids 19 to 168 of SEQ ID NO: 2, and said bioactive polypeptide binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and / or binds to LTB4.

[0046] In any aspects of the invention, the bioactive polypeptide comprises or consists of amino acids 19 to 168 of SEQ ID NO: 2

[0047] In any aspects of the invention, the PA(S) sequence is a PAS sequence, optionally wherein the PAS sequence comprises or consists of 30 copies of SEQ ID NO: 15, preferably wherein the PAS sequence comprises or consists of SEQ ID NO: 31 .

[0048] In any aspects of the invention, the bioactive polypeptide comprises or consists of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 and the PA(S) polypeptide of the fusion protein comprises or consists of SEQ ID NO: 31 , and wherein the bioactive polypeptide is fused to the N terminus of the PA(S) polypeptide, optionally wherein the fusion comprises or consists of SEQ ID NO: 35.

[0049] In any aspects of the invention, the bioactive polypeptide binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and / or binds LTB4.

[0050] DETAILED DESCRIPTION

[0051] The invention is based on the inventors’ surprising observation that the level of impurities in an input composition comprising a fusion protein (e.g., PAS600-nomacopan) can be significantly reduced by performing particular methods.

[0052] The inventors found that performing a step of hydrophobic interaction chromatography (HIC) using a stationary phase comprising hexyl ligands (e.g., Toyopearl Hexyl 650C) significantly reduces the level of pyrogens, in particular NEPs, without significantly affecting the yield or the protein integrity, thereby producing an output composition particularly suitable for ophthalmic delivery.

[0053] The inventors also found that performing ammonium sulphate precipitation on cell culture medium or cell culture supernatant results in surprisingly good purity (e.g., removal of host cell proteins) and yield in a single step, even at large / commercial scales.

[0054] Fusion proteins

[0055] The invention provides methods for removing pyrogens and / or host cell proteins from compositions comprising a fusion protein. In both methods, the fusion protein comprises or consists of i) a bioactive polypeptide, wherein the first bioactive polypeptide comprises or consists of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide, and optionally a linker, for example between the bioactive polypeptide and the PA(S) polypeptide. Said bioactive polypeptide and PA(S) polypeptide may be referred to as the ‘first’ bioactive polypeptide and the ‘first’ PA(S) polypeptide, respectively, in embodiments where the fusion protein may further comprise an additional bioactive polypeptide(s) and / or an additional PA(S) polypeptide(s). The bioactive polypeptide may be any bioactive polypeptide described herein. The PA(S) polypeptide may be any PA(S) polypeptide described herein. Preferably, the PA(S) polypeptide is a PAS polypeptide. As used herein, the term “fusion protein” refers to a recombinant protein comprising a bioactive polypeptide (e.g. nomacopan) and a PA(S) polypeptide.

[0056] In some embodiments, the PA(S) polypeptide comprises at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids. In some embodiments, the PA(S) polypeptide comprises at least 650 amino acids, at least 700 amino acids, at least 750 amino acids, at least 800 amino acids, at least 850 amino acids, at least 900 amino acids, at least 950 amino acids, at least 1000 amino acids, at least 1050 amino acids, at least 1110 amino acids, at least 1150 amino acids, or at least 1200 amino acids.

[0057] In some embodiments, the PA(S) polypeptide comprises up to 600 amino acids, up to 650 amino acids, up to 700 amino acids, up to 750 amino acids, up to 800 amino acids, up to 850 amino acids, up to 900 amino acids, up to 950 amino acids, up to 1000 amino acids, up to 1050 amino acids, up to 1110 amino acids, up to 1150 amino acids, up to 1200 amino acids, up to 1250 amino acids, up to 1300 amino acids, up to 1350 amino acids, 1400 amino acids, up to 1450 amino acids, up to 1500 amino acids, up to 1550 amino acids, or up to 1600 amino acids. In preferred embodiments, the PA(S) polypeptide comprises up to 600 amino acids.

[0058] In some embodiments, the PA(S) polypeptide comprises at least 600 amino acids. In some embodiments, the PA(S) polypeptide comprises from 600 amino acids to 800 amino acids. In some embodiments, the PA(S) polypeptide comprises from 600 amino acids to 1000 amino acids. In some embodiments, the PA(S) polypeptide comprises from 600 amino acids to 1200 amino acids. In some embodiments, the PA(S) polypeptide comprises from 600 amino acids to 1400 amino acids. In some embodiments, the PA(S) polypeptide comprises from 600 amino acids to 1600 amino acids. In preferred embodiments, the PA(S) polypeptide consists of 600 amino acids.

[0059] The bioactive polypeptide typically comprises at least 140 amino acids. The bioactive polypeptide preferably comprises at least 145 amino acids, more preferably at least 146 amino acids, more preferably at least 147 amino acids, even more preferably at least 148 amino acids, yet more preferably at least 149 amino acids, most preferably at least 150 amino acids.

[0060] The bioactive polypeptide typically comprises up to 160 amino acids. The bioactive polypeptide preferably comprises up to 155 amino acids, more preferably up to 154 amino acids, more preferably up to 153 amino acids, even more preferably up to 152 amino acids, yet more preferably up to 151 amino acids, most preferably up to 150 amino acids. Preferably the bioactive polypeptide consists of 150 amino acids.

[0061] In some embodiments, the fusion protein comprises a minimum number of amino acids which is i) the minimum number of amino acids in the PA(S) polypeptide (as described above) plus ii) the minimum number of amino acids in the bioactive polypeptide (as described above). For example, in preferred embodiments, the fusion protein comprises at least 750 amino acids.

[0062] The PA(S) polypeptide may be fused to the N-terminus or the C-terminus of the bioactive polypeptide. Preferably, the PA(S) polypeptide is fused to the N-terminus of the bioactive polypeptide. Fusion may be direct (i.e., not via a linker) or indirect (i.e., via a linker).

[0063] Preferably, the C-terminus of the bioactive polypeptide is not fused to a PA(S) polypeptide. More preferably the C-terminus of the bioactive polypeptide is not fused to anything (it is free). This may be preferable because the C-terminus of nomacopan binds to C5.

[0064] In preferred embodiments, the fusion protein comprises or consists of: i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, or SEQ ID NO: 22, 23, 24, or 25, and ii) a PAS polypeptide comprising or consisting of at least 30 repeats of SEQ ID NO: 15, 16, 17, 18, 19, 20, or 21 , wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide.

[0065] In certain preferred embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, and ii) a PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In a particularly preferred embodiment, the fusion protein comprises or consists of SEQ ID NO: 35 (‘PAS600-nomacopan’).

[0066] Other components of fusion proteins

[0067] In addition to bioactive polypeptides and PA(S) polypeptides, fusion proteins may comprise other heterologous sequences. The term “heterologous sequence”, when used herein, is intended to designate any polypeptide other than the bioactive polypeptides and the PA(S) polypeptides described herein.

[0068] Heterologous sequences that can be present in the fusion protein are preferably present: a) At the N terminus of the fusion protein (e.g., N-terminal of the most N-terminal bioactive polypeptide or PA(S) polypeptide) b) Between components of the fusion protein, e.g., between a bioactive polypeptide and a PA(S) polypeptide c) At the C terminus of the fusion protein.

[0069] If the heterologous sequence is at the C-terminus, e.g., C-terminal to the most C-terminal bioactive polypeptide or PA(S) polypeptide, a cleavage sequence that permits the heterologous sequence to be removed may be present. This leaves the standard C-terminus of the bioactive polypeptide free for interaction with its biological target, e.g., binding to C5. Examples of heterologous sequences, that can be comprised in the fusion proteins, are the following: multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography. Many of these heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them

[0011] ,

[0070] Examples of such heterologous sequences include: i) affinity tags such as a polyhistidine tag (e.g., a His6-tag), a polyarginine-tag, the Strep-tag® II (Trp- Ser-His-Pro-GIn-Phe-Glu-Lys), the Twin-Strep® tag (Trp-Ser-His-Pro-GIn-Phe-Glu-Lys-Gly-Gly-Gly- Ser-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Ser-Ala-Trp-Ser-His-Pro-GIn-Phe-Glu-Lys), a GST tag, a FLAG tag, avidin, or an HA tag; ii) prokaryotic secretory signal peptides such as the signal peptide of OmpA, CspA, MalE, CGTase, pelB, CspB, TorA, DsbA or derivatives thereof; iii) eukaryotic secretory signal peptides such as the signal peptide of mating factor a, IgE, insulin, IgG kappa, albumin, azurocidin preproprotein or derivatives thereof; iv) a protease sensitive cleavage site such as a tobacco etch virus (TEV) or a SUMO protease (Ubl- specific protease 1) cleavage site; v) a targeting moiety directed towards human organs, tissues, or cell types; vi) additional functional / effector domains such as a binding protein, an antibody of fragments thereof, an enzyme for target degradation or prodrug activation; and / or vii) domains to improve protein production yields such as thioredoxin, small ubiquitin-like modifier (SUMO), glutathione-S-Transferase, and CspB-fusion tag.

[0071] Fusion proteins of the invention do not require linkers. PA(S) polypeptides are themselves unstructured and flexible and thus effectively serve as a linker. In fact, PA(S) polypeptides are known for use as linkers for example see

[0012] and

[0013] ,

[0072] Nevertheless, fusion proteins may additionally comprise linker sequences. For example, the bioactive polypeptide(s) and the PA(S) polypeptide(s) of the fusion proteins of the invention may be fused directly (i.e, without a linker) or indirectly, via a linker.

[0073] Any unstructured and / or flexible linker could be included within fusion proteins of the invention. In embodiments, the linker may be a peptide linker or non-peptide linker. A peptide linker may be 1-50, 2-30, 3-20, 5-10, 2-4, or 3-5 amino acids in length. In embodiments, the linker may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids in length. In embodiments, the linker may be up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids in length. In some embodiments, a linker may comprise or consist of glycine and / or serine, for example a linker may have the formula (Gly4Ser)n, and n is an integer, e.g., 1-10, 2-9, 3-8, 4-7, 5-6. Preferably a linker comprises one or more alanine residues, or consists of alanine residues. More preferably the linker sequence consists of a single alanine residue. Preferably, linkers do not alter the function of the polypeptide(s) to which they are fused. Linkers may be useful for reducing unwanted interactions between the constituent polypeptides of the fusion protein.

[0074] In some of the fusion proteins exemplified herein, there is a single alanine at the N-terminus of the bioactive polypeptide, e.g., nomacopan. In some constructs this alanine separates the bioactive polypeptide from a PA(S) polypeptide and thus may be regarded as a linker. However, this alanine is not intended to function as a linker and these fusion proteins do not require this alanine to function. The alanine is present due to the cloning procedure of PA(S) gene cassettes. In some embodiments, fusion proteins of the invention comprise a single alanine residue at the N-terminus of each bioactive polypeptide. In the sequence listing of this application, X is A (alanine) or is deleted (i.e., absent).

[0075] Fusion proteins of the invention may further comprise a single proline residue at the N-terminus of the fusion protein. This may optimize translation initiation when the fusion proteins are intracellularly produced in the cytoplasm of E. coli. Alternatively, fusion proteins of the invention may further comprise a single alanine residue at the N-terminus of the fusion protein. This may facilitate signal peptide cleavage in secretary production systems, if the N-terminal residue is not already an alanine.

[0076] Immature fusion proteins may comprise a fusion protein of the invention and, at the N-terminus of the fusion protein, a single methionine residue (the methionine would thus be N-terminal of any N- terminal proline or alanine in the fusion protein). This initial methionine is typically intracellularly cleaved by methionine aminopeptidase leading to a mature fusion protein.

[0077] Bioactive polypeptides

[0078] The fusion proteins purified by the methods of invention comprise at least one bioactive polypeptide (a ‘first bioactive polypeptide’). In preferred embodiments, the fusion protein comprises a single bioactive polypeptide, i.e., precisely one bioactive polypeptide.

[0079] In other embodiments, the fusion protein comprises at least two bioactive polypeptides (a ‘first bioactive polypeptide’ and a ‘second bioactive polypeptide). In certain embodiments, the fusion protein comprises precisely two bioactive polypeptides.

[0080] In the methods of the invention, the bioactive polypeptide comprises or consists of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof. As used herein, ‘nomacopan’ refers to a bioactive polypeptide consisting of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2.

[0081] Nomacopan was isolated from the salivary glands of the tick O. moubata. Nomacopan is an outlying member of the lipocalin family and was the first lipocalin family member shown to inhibit complement activation. Nomacopan inhibits the classical, alternative and lectin complement pathways by binding to C5 and preventing its cleavage by C5 convertase into C5a and C5b, thus inhibiting both the production of C5a, which is an active (e.g., proinflammatory) peptide, and the formation of the MAC. Nomacopan has been demonstrated to bind to C5 and prevent its cleavage by C5 convertase in rat, mouse and human serum with an IC50 of approximately 0.02mg / ml.

[0082] A bioactive polypeptide purified by the methods of the invention may thus comprise or consist of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or amino acids 1 to 168 of the amino acid sequence of SEQ ID NO: 2. The first 18 amino acids of the protein sequence given in SEQ ID NO: 2 form a signal sequence which is not required for C5 binding or for LTB4 binding activity and so this may optionally be dispensed with, for example, for efficiency of recombinant protein production.

[0083] Bioactive polypeptides that bind both C5 and LTB4

[0084] In some embodiments, in fusion proteins purified by methods of the invention, the bioactive polypeptide may bind to both C5 and to LTB4 (e.g., to both wild-type C5 and C5 from subjects with C5 polymorphisms that render treatment by eculizumab ineffective, or reduce the efficacy of treatment with eculizumab, and to LTB4).

[0085] Bioactive polypeptides that bind to LTB4 but have reduced or absent C5 binding

[0086] Bioactive polypeptides which do not bind or which show reduced binding to C5, but which do retain LTB4-binding activity are disclosed, for instance, in WQ2018 / 193121 , the entire contents of which are incorporated herein by reference. Such bioactive polypeptides which have reduced or absent C5- binding activity but which retain LTB4-binding ability may be used in all aspects of the present invention.

[0087] Such bioactive polypeptides which have reduced or absent C5-binding activity but which retain LTB4- binding ability may comprise or consist of the following sequences:

[0088] SEQ ID NO: 22 (SEQ ID NO: 5 of WO2018 / 193121) is the amino acid sequence of a modified nomacopan in which SEQ ID NO: 4 has been modified to change Met114 to Gin, Met116 to Gin, Leu117 to Ser, Asp118 to Asn, Alai 19 to Gly, Gly120 to Ser, Gly121 to Ala, Leu122 to Asp, Glu123 to Asp and Val124 to Lys. (nomacopan variant 1)

[0089] SEQ ID NO: 23 (SEQ ID NO: 6 of WO2018 / 193121) is the amino acid sequence of a modified nomacopan in which SEQ ID NO: 4 has been modified to change Ala44 to Asn, Met116 to Gin, Leu117 to Ser, Gly121 to Ala, Leu122 to Asp, Glu123 to Ala and Asp149 to Gly. (nomacopan variant 2, also referred to as ‘L-nomacopan’)

[0090] SEQ ID NO: 24 (SEQ ID NO: 7 of WO2018 / 193121) is the amino acid sequence of a modified nomacopan in which SEQ ID NO: 4 has been modified to change Ala44 to Asn, Met116 to Gin, Leu 122 to Asp and Asp149 to Gly. (nomacopan variant 3)

[0091] SEQ ID NO: 25 (SEQ ID NO: 8 of WO2018 / 193121) is the amino acid sequence of a modified nomacopan in which SEQ ID NO: 4 has been modified to change Ala44 to Asn. (nomacopan variant 4). The modified bioactive polypeptides that exhibit a reduced ability to bind to C5 compared to the unmodified nomacopan polypeptide may in some preferred embodiments exhibit no detectable binding to C5.

[0092] C5 binding may, for example, be reduced by at least 2, 5, 10, 15, 20, 50, 100 fold, or eliminated relative to the binding exhibited by the unmodified nomacopan polypeptide in SEQ ID NO: 4.

[0093] In some embodiments C5 binding is reduced by at least 50%, 60%, 70%, 80%, 90% or 95% relative to the unmodified nomacopan polypeptide in SEQ ID NO: 4.

[0094] Such bioactive polypeptides may e.g., bind C5 with a KD greater than 1 micromolar as determined by SPR according to the method described in

[0014] , or as set out in Example 2 of WO2018193121 and / or may inhibit sheep red blood cell lysis by less than 10% when present at a concentration of 0.02mg / mL in whole pooled normal serum with the CH50 lytic assay performed according to or similarly to that performed in

[0015] , The ability of the bioactive polypeptides to bind to C5 may also be determined by measuring the ability of the bioactive polypeptide to inhibit complement activation in serum.

[0095] In certain preferred embodiments, the bioactive polypeptide comprises or consists of variant 2.

[0096] These bioactive polypeptides are examples of functional equivalents of nomacopan which share the molecule’s ability to bind LTB4, but which do not bind C5 or which have reduced binding to C5.

[0097] Bioactive polypeptides that bind to C5 but have reduced or lack LK / E binding

[0098] In some embodiments the bioactive polypeptide has reduced or lacks leukotriene / hydroxyeicosanoid (LK / E) binding activity. In preferred embodiments the bioactive polypeptide has reduced or lacks leukotriene B4 (LTB4) binding activity.

[0099] In some embodiments, one or more of the amino acid residues to be mutated is selected from Phe36, Arg54, Leu57, Gly59, Val72, Met74, Phe76, Trp87, Phe89, Gln105, Arg107, His119, Asp121 and Trp133, wherein the numbering of amino acids is with reference to SEQ ID NO: 2.

[0100] In some embodiments, at least one amino acid mutation is selected from Phe36Trp and Gly59Trp, wherein the numbering of amino acids is with reference to SEQ ID NO: 2. For example, the bioactive polypeptide may comprise SEQ ID NO: 56 or SEQ ID NO: 57.

[0101] Homologues and sequence identity

[0102] A bioactive polypeptide may be a homologue or fragment of nomacopan which (i) retains its ability to bind to C5 and to prevent the cleavage of C5 by C5 convertase into C5a and C5b and / or (ii) which retains its ability to bind LTB4. In certain embodiments the bioactive polypeptide has property (i) and (ii). In other embodiments the bioactive polypeptide has property (ii), but reduced or no binding to C5 (e.g., one of nomacopan variants 1 to 4).

[0103] In some embodiments, the bioactive polypeptide is derived from a haematophagous arthropod. The term “haematophagous arthropod” includes all arthropods that take a blood meal from a suitable host, such as insects, ticks, lice, fleas and mites. Preferably, the bioactive polypeptide is derived from a tick, preferably from the tick O. moubata.

[0104] Homologues include paralogues and orthologues of the nomacopan sequence that is explicitly identified in SEQ ID NO: 2, including, for example, the nomacopan protein sequence from other tick species, including Rhipicephalus appendiculatus, R. sanguineus, R. bursa, A. americanum, A. cajennense, A. hebraeum, Boophilus microplus, B. annulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D. marginatus, D. variabilis, Haemaphysalis inermis, Ha. Leachii, Ha. Punctata, Hyalomma anatolicum anatolicum, Hy. Dromedarii, Hy. Marginatum marginatum, Ixodes ricinus, I. persulcatus, I. scapularis, I. hexagonus, Argas persicus, A. reflexus, O. erraticus, O. moubata moubata, O. m. porcinus, and O. savignyi.

[0105] The term “homologue” is also meant to include the equivalent nomacopan protein sequence from mosquito species, including those of the Culex, Anopheles and Aedes genera, particularly Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae; flea species, such as Ctenocephalides fells (the cat flea); horseflies; sandflies; blackflies; tsetse flies; lice; mites; leeches; and flatworms. The native nomacopan protein is thought to exist in O. moubata also in another three forms of around 18kDa and the term “homologue” is meant to include these alternative forms of nomacopan.

[0106] Methods for the identification of homologues of the nomacopan sequence given in SEQ ID NO: 2 will be clear to those of skill in the art. For example, homologues may be identified by homology searching of sequence databases, both public and private. Conveniently, publicly available databases may be used, although private or commercially-available databases will be equally useful, particularly if they contain data not represented in the public databases. Primary databases are the sites of primary nucleotide or amino acid sequence data deposit and may be publicly or commercially available. Examples of publicly-available primary databases include the GenBank database (http: / / www.ncbi.nlm.nih.gov / ), the EMBL database (http: / / www.ebi.ac.uk / ), the DDBJ database (http: / / www.ddbi.niq.ac.jp / ), the SWISS-PROT protein database (http: / / expasy.hcuqe.ch / ), PIR (http: / / pir.qeorqetown.edu / ), TrEMBL (http: / / www.ebi.ac.uk / ), the TIGR databases (see http: / / www.tiqr.org / tdb / index.html), the NRL-3D database (http: / / www.nbrfa.qeorqetown.edu), the Protein Data Base (http: / / www.rcsb.org / pdb), the NRDB database (ftp: / / ncbi.nlm.nih.gov / pub / nrdb / README), the OWL database (http: / / www.biochem.ucl.ac.uk / bsm / dbbrowser / OWL / ) and the secondary databases PROSITE (http: / / expasy.hcuge.ch / sprot / prosite.html), PRINTS (http: / / iupab.leeds.ac.uk / bmb5dp / prints.html),

[0107] Profiles (http: / / ulrec3.unil.ch / software / PFSCAN_form.html),

[0108] Pfam (http: / / www.sanger.ac.uk / software / pfam), Identify (http: / / dna.stanford.edu / identify / ), Blocks (http: / / www.blocks.fhcrc.org), and UniProt (https: / / www.uniprot.org) databases. The AlphaFold Protein Structure Database by DeepMind (https: / / alphafold.ebi.ac.uk / ) could also be used to identify homologues that have lower amino acid identity but very closely related folds. Examples of commercially-available databases or private databases include PathoGenome (Genome Therapeutics Inc.) and PathoSeq (previously of Incyte Pharmaceuticals Inc.). Typically, greater than 30% identity between two polypeptides (preferably, over a specified region such as the active site) is considered to be an indication of functional equivalence and thus an indication that two proteins are homologous. Preferably, proteins that are homologues have a degree of sequence identity with the nomacopan protein sequence identified in SEQ ID NO: 2 of greater than 60%. More preferred homologues have degrees of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively with the nomacopan protein sequence given in SEQ ID NO: 2. Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1]. The % identity may be over the full length of the relevant reference sequence (e.g., amino acids 1-168 of SEQ ID NO: 2 or amino acids 19-168 of SEQ ID NO: 2).

[0109] Bioactive polypeptides thus can be described by reference to a certain % amino acid sequence identity to a reference sequence e.g., amino acids 19-168 of SEQ ID NO: 2 or amino acids 1-168 of SEQ ID NO: 2 e.g., as a protein comprising or consisting of a sequence having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to amino acids 19-168 of SEQ ID NO: 2 or amino acids 1-168 of SEQ ID NO: 2. Preferably, bioactive polypeptides comprise or consist of a sequence having at least 90% identity to amino acids 19-168 of SEQ ID NO: 2 or amino acids 1-168 of SEQ ID NO: 2.

[0110] In the various aspects and embodiments of this disclosure, the bioactive polypeptides (e.g., modified nomacopan polypeptides) may differ from the unmodified nomacopan polypeptides in SEQ ID NO: 2 and SEQ ID NO: 4 by from 1 to 50, 2-45, 3-40, 4-35, 5-30, 6-25, 7-20, 8-25, 9-20, 10-15 amino acids, up to 1 , 2, 3, 4, 5, 7, 8, 9, 10, 20, 30, 40, 50 amino acids. These may be substitutions, insertions or deletions but are preferably substitutions. Where deletions are made these are preferably deletions of up to 1 , 2, 3, 4, 5, 7 or 10 amino acids, (e.g., deletions from the N or C terminus). Mutants thus include bioactive polypeptides containing amino acid substitutions, e.g., conservative amino acid substitutions that do not affect the function or activity of the protein in an adverse manner. This term is also intended to include natural biological variants (e.g., allelic variants or geographical variations within the species from which the nomacopan proteins are derived). Mutants with improved ability to bind wild-type C5 and / or C5 from subjects with a C5 polymorphism that render treatment by eculizumab ineffective, or reduce the efficacy of treatment with eculizumab and / or LTB4 may also be designed through the systematic or directed mutation of specific residues in the protein sequence.

[0111] These modifications may be made to the nomacopan polypeptide as set out in SEQ ID NO: 2 and SEQ ID NO: 4 and the molecule will remain useful and will be considered to be a functional equivalent of nomacopan provided that the resulting bioactive polypeptide retains (i) LTB4 binding activity and / or also (ii) C5 binding comparable with the nomacopan polypeptide as set out in SEQ ID NO: 2 and SEQ ID NO: 4, which can be determined e.g., using the tests referred to elsewhere herein (e.g., the binding to one or both of these is at least 80, 85, 90, 95% of the binding compared to the unmodified nomacopan polypeptide). As discussed elsewhere herein, both nomacopan and L-nomacopan have been shown to be effective in the treatment of a mouse model of autoimmune uveitis. L-nomacopan binds LTB4 but does not bind C5. Bioactive polypeptides may be defined by reference to their ability to bind to C5 and / or their ability to bind to LTB4. Those that bind LTB4 are of particular use in the invention. Those that bind LTB4 and C5 are also of particular use in the invention.

[0112] Given the requirement for functional variants to bind LTB4 and optionally also C5, when modifications are made, certain residues should be excluded from modification. These include conserved cysteine residues.

[0113] Other residues should be excluded from modification or, if substituted, should only be subject to conservative modification. These are the LTB4 binding residues. In embodiments where the bioactive polypeptide binds LTB4 and C5 then the C5 binding residues as defined below should preferably also be excluded from modification or, if substituted, should preferably only be subject to conservative modification. Given that the binding of LTB4 and C5 is relatively well understood it is possible to design a bioactive polypeptide that may have a percentage identity of around 65% to nomacopan but in which the changes are confined to residues which are not involved in LTB4 binding and optionally also C5 binding.

[0114] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of the mature nomacopan molecule (e.g., as set out in SEQ ID NO: 4 which corresponds to residues 19 to 168 of the full length protein including the signal sequence) is retained and at least five, ten or fifteen or each of the LTB4 binding residues set out below is retained or is subject to a conservative modification.

[0115] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and at least five, ten or fifteen or each of the LTB4 binding residues are retained or are subject to a conservative modification, wherein up to 2, 3, 4, 5, 10, 15, 20 of the LTB4 binding residues set out below are subject to a conservative modification.

[0116] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and at least five, ten or fifteen or each of the LTB4 binding residues set out below is retained.

[0117] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues set out below is retained or is subject to a conservative modification.

[0118] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues set out below is retained or is subject to a conservative modification, wherein up to 2, 3, 4, 5, 10, 15, 20 of the LTB4 binding residues are subject to a conservative modification.

[0119] For bioactive polypeptides that bind LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues set out below is retained. For bioactive polypeptides that bind C5 and LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of the mature nomacopan molecule (e.g., as set out in SEQ ID NO: 4 which corresponds to residues 19 to 168 of the full length protein including the signal sequence) is retained and at least five, ten or fifteen or each of the LTB4 binding residues are retained or are subject to a conservative modification and at least five, ten or fifteen or twenty or each of C5 binding residues set out below is retained or is subject to a conservative modification.

[0120] For bioactive polypeptides that bind C5 and LTB4, each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and at least five, ten or fifteen or each of the LTB4 binding residues and at least five, ten or fifteen or twenty or each of C5 binding residues set out below is retained or is subject to a conservative modification, wherein up to 2, 3, 4, 5, 10, 15, 20 of the LTB4 and C5 binding residues are subject to a conservative modification.

[0121] For bioactive polypeptides that bind C5 and LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and at least five, ten or fifteen or each of the LTB4 binding residues and at least five, ten or fifteen or twenty or each of C5 binding residues set out below is retained.

[0122] For bioactive polypeptides that bind C5 and LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues and each of C5 binding residues set out below is retained or is subject to a conservative modification.

[0123] For bioactive polypeptides that bind C5 and LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues and each of C5 binding residues set out below is retained or is subject to a conservative modification, wherein up to 2, 3, 4, 5, 10, 15, 20 of the C5 and / or LTB4 binding residues are subject to a conservative modification.

[0124] For bioactive polypeptides that bind C5 and LTB4, in some embodiments each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 is retained and each of the LTB4 binding residues and each of C5 binding residues set out below is retained.

[0125] Modifications made outside of these regions may be conservative or non-conservative.

[0126] In each of these embodiments the spacing between these six cysteine amino acid residues is preferably retained to preserve the overall structure of the molecule (e.g., the molecule comprises six cysteine residues that are spaced relative to each other at a distance of 32 amino acids apart, 62 amino acids apart, 28 amino acids apart, 1 amino acid apart and 21 amino acids apart as arranged from the amino terminus to the carboxyl terminus of the sequence according to amino acids 1 to 168 of the amino acid sequence in SEQ ID NO: 2). LTB4 binding residues

[0127] Resides that are thought to be involved in binding to LTB4 and are preferably retained in unmodified form or are subject to conservative changes only in the sequence of any molecule that is modified relative to SEQ ID NO: 2 or SEQ ID NO: 4 are Phe18, Tyr25, Arg36, Leu39, Gly41 , Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71 , Gln87, Arg89, His99, His101 , Asp103, and Trp115 (numbering according to SEQ ID NO: 4).

[0128] C5 binding residues

[0129] Resides that are thought to be involved in binding to C5 may be retained in unmodified form in the sequence of any molecule that is modified relative to SEQ ID NO: 2 or SEQ ID NO: 4 are Val26, Val28, Arg29, Ala44, Gly45, Gly61 , Thr62, Ser97, His99, His101 , Met 114, Met 116, Leu117, Asp118, Alai 19, Gly120, Gly121 , Leu122, Glu123, Val124, Glu125, Glu127, His146, Leu147 and Asp 149 (numbering according to SEQ ID NO: 4). These residues are among those that are modified in bioactive polypeptides that bind to LTB4 but which have been modified to reduce binding to C5.

[0130] LTB4 and / or C5 binding residues

[0131] There are two histidine residues involved in both LTB4 and C5 binding, His99 and H is 101 . The list of residues involved in LTB4 and / or C5 binding is therefore Phe18, Tyr25, Val26, Val28, Arg29, Arg36, Leu39, Gly41 , Pro43, Ala44, Gly45, Leu52, Val54, Met56, Phe58, Gly61 , Thr62, Thr67, Trp69, Phe71 , Gln87, Arg89, Ser97, His99, His101 , Asp103, Met 114, Trp115, Met 116, Leu117, Asp118, Alai 19, Gly120, Gly121 , Leu122, Glu123, Val124, Glu125, Glu127, His146, Leu147 and Asp 149 (numbering according to SEQ ID NO: 4).

[0132] Further examples of bioactive polypeptides that bind LTB4 but have reduced or absent C5 binding

[0133] As discussed above, bioactive polypeptides which do not bind or which show reduced binding to C5, but which do retain LTB4-binding activity are disclosed, for instance, in WO2018 / 193121 , the entire contents of which are incorporated herein by reference. Such bioactive polypeptides which have reduced or absent C5-binding activity but which retain LTB4-binding ability may be used in all aspects of the present invention.

[0134] Four exemplary bioactive polypeptides which have reduced or absent C5-binding activity but which retain LTB4-binding ability are disclosed in WO2018 / 193121 , specifically proteins having the amino acid sequences as set out in SEQ ID NO: 22 (SEQ ID NO: 5 of WO2018 / 193121 , variant 1), SEQ ID NO: 23 (SEQ ID NO: 6 of WO2018 / 193121 , variant 2), SEQ ID NO: 24 (SEQ ID NO: 7 of WO2018 / 193121 , variant 3) and SEQ ID NO: 25 (SEQ ID NO: 8 of WO2018 / 193121 , variant 4).

[0135] Such bioactive polypeptides are considered to be functional equivalents of nomacopan, however they share only the LTB4 binding properties thereof and have reduced or no binding to C5. Such bioactive polypeptides as defined in WO2018 / 193121 are described in more detail below and may be used in the present invention.

[0136] Further examples of bioactive polypeptides which have reduced or absent C5-binding activity but which retain LTB4-binding ability may comprise or consist of the following sequences:

[0137] SEQ ID NO: 26 (SEQ ID NO: 9 of WO2018 / 193121) is the amino acid sequence of the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132-142 of SEQ ID NO: 2).

[0138] SEQ ID NO: 27 (SEQ ID NO: 10 of WO2018 / 193121) is the amino acid sequence of the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 in nomacopan variant 1 (SEQ ID NO: 22).

[0139] SEQ ID NO: 28 (SEQ ID NO: 11 of WO2018 / 193121) is the amino acid sequence of the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 in nomacopan variant 2 (SEQ ID NO: 23).

[0140] SEQ ID NO: 29 (SEQ ID NO: 12 of WO2018 / 193121) is the amino acid sequence of the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 in nomacopan variant 3 (SEQ ID NO: 24).

[0141] The bioactive polypeptides which have reduced or absent C5-binding activity but which retain LTB4- binding ability may be described as “modified nomacopan polypeptides” (e.g., which exhibit leukotriene or hydroxyeicosanoid binding activity and reduced or absent C5 binding). References to a “modified nomacopan polypeptide” are to be understood as a reference to a modified version of either SEQ ID NO: 2 or SEQ ID NO: 4 i.e. the nomacopan polypeptide with or without the 18 amino acid signal sequence seen at the N-terminus of SEQ ID NO: 2.

[0142] In embodiments, bioactive polypeptides may exhibit leukotriene or hydroxyeicosanoid (typically LTB4) binding activity and reduced or absent C5 binding and can comprise SEQ ID NO: 4 in which from 1 to 30 amino acid substitutions are made, wherein

[0143] (i) in the positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)-(j) is made: a. Met114 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; b. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; c. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro; d. Asp118 is replaced with Asn, Gin, Arg, Lys, Gly, Ala, Leu, Ser, He, Phe, Tyr, Met Pro, His, or

[0144] Thr; e. Alai 19 is replaced with Gly, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His; f. Gly120 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His; g. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His; h. Leu122 is replaced with Asp, Glu, Asn, Ala, Gin, Arg, Lys, Pro, or His; i. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, He, Phe, Tyr, Pro, His, or

[0145] Thr; j. Val124 is replaced with Lys, Gin, Asn, Arg, Lys, Gly, Ala, Pro, His, or Thr; or / and wherein

[0146] (ii) Ala44 in SEQ ID NO: 4 is replaced with Asn, Asp, Gin, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met,

[0147] Pro, or His; or a fragment thereof in which up to five amino acids are deleted from the N terminus of the modified nomacopan polypeptide.

[0148] Leukotriene / eicosanoid (LK / E) binding activity as used herein refers to the ability to bind to leukotrienes and hydroxyeicosanoids including but not limited to LTB4, B4 isoleukotrienes and any hydroxylated derivative thereof, HETEs, HPETEs and EETs. LTB4 binding is of particular interest.

[0149] The modified nomacopan polypeptides which have reduced or absent C5-binding activity but which retain LTB4-binding ability may consist of SEQ ID NO: 2 or 4, modified in accordance with the description below, or may comprise SEQ ID NO: 2 or 4, modified in accordance with the description below.

[0150] The nomacopan polypeptide in SEQ ID NO: 2 and SEQ ID NO: 4 features a loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132-142 of SEQ ID NO: 2). This loop has the sequence shown below:

[0151] -Met-Trp-Met-Leu-Asp-Ala-Gly-Gly-Leu-Glu-Val- (SEQ ID NO: 26)

[0152] The first Met is at position 114 of SEQ ID NO: 4 and at position 132 of SEQ ID NO: 2.

[0153] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability, the nomacopan polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 is modified such that at positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)-(j) is made: a. Met114 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin or Ala; b. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin or Ala; c. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro, preferably Ser or Ala; d. Asp118 is replaced with Asn, Gin, Arg, Lys, Gly, Ala, Leu, Ser, lie, Phe, Tyr, Met Pro, His, or Thr, preferably Asn; e. Alai 19 is replaced with Gly, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His, preferably Gly or Asn; f. Gly120 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His, preferably Ser or Asn; g. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His, preferably Ala or Asn; h. Leu122 is replaced with Asp, Glu, Asn, Ala, Gin, Arg, Lys, Pro, or His, preferably Asp or Ala; i. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, lie, Phe, Tyr, Pro, His, or Thr, preferably Asp, Ala, Gin or Asn; j. Val124 is replaced with Lys, Gin, Asn, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Lys or Ala.

[0154] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability the nomacopan polypeptide in SEQ ID NO: 2 or SEQ ID NO: 4 can be modified such that at positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)-(j) is made: a. Met114 is replaced with Gin; b. Met116 is replaced with Gin; c. Leu117 is replaced with Ser; d. Asp118 is replaced with Asn; e. Alai 19 is replaced with Gly; f. Gly120 is replaced with Ser; g. Gly121 is replaced with Ala; h. Leu 122 is replaced with Asp; i. Glu123 is replaced with Asp, or Ala; j. Val124 is replaced with Lys.

[0155] In the modified nomacopan polypeptide two, three, four, five, six, seven, eight, nine, or ten of the substitutions (a)-(j) may be present. Preferably two or more, five or more, or eight or more of the substitutions (a)-(j) may be present.

[0156] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability the nomacopan polypeptide in SEQ ID NO: 2 or SEQ ID NO: 4 can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met114 is replaced with Gin; b. Met116 is replaced with Gin; c. Leu117 is replaced with Ser; d. Asp118 is replaced with Asn; e. Alai 19 is replaced with Gly; f. Gly120 is replaced with Ser; g. Gly121 is replaced with Ala; h. Leu 122 is replaced with Asp; i. Glu123 is replaced with Asp; j. Val124 is replaced with Lys.

[0157] Optionally in the modified nomacopan polypeptide referred to above Trp115 is not substituted. A preferred modified nomacopan polypeptide has a loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 that has the sequence Gln-Trp-Gln-Ser-Asn-Gly-Ser-Ala-Asp- Asp-Lys (SEQ ID NO: 27).

[0158] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability, the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin; b. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro, preferably Ser; c. Gly121 is replaced with Ala, Asp, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His, preferably

[0159] Ala; d. Leu122 is replaced with Asp, Glu, Asn, Gin, Arg, Lys, Pro, or His, preferably Asp; e. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, He, Phe, Tyr, Pro, His, or

[0160] Thr, preferably Asp.

[0161] In more particular embodiments; a. Met116 is replaced with Gin; b. Leu117 is replaced with Ser; c. Gly121 is replaced with Ala; d. Leu 122 is replaced with Asp; e. Glu123 is replaced with Ala.

[0162] Optionally in this modified nomacopan polypeptide referred to above Trp115 is not substituted. Optionally in this embodiment Met114, Trp115, Asp118, Alai 19, Gly120 and Val124 are not substituted, or are substituted with conservative substitutions as referred to elsewhere herein. A preferred modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability has a loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 that has the sequence Met-Trp-GIn-Ser-Asp-Ala-Gly-Ala-Asp-Ala-Val (SEQ ID NO: 28).

[0163] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability, the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin; b. Leu122 is replaced with Asp, Glu, Asn, Gin, Arg, Lys, Pro, or His, preferably Asp;

[0164] In more particular embodiments; a. Met116 is replaced with Gin; b. Leu 122 is replaced with Asp.

[0165] Optionally in this modified nomacopan polypeptide referred to above Trp115 is not substituted. Optionally in this embodiment Met114, Trp115, Leu117, Asp118, Alai 19, Gly120, Gly121 , Glu123 and Val124 are not substituted. A preferred modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability has a loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 that has the sequence Met-Trp-GIn-Leu- Asp-Ala-Gly-Gly-Asp-Glu-Val (SEQ ID NO: 29).

[0166] In the modified nomacopan polypeptide which has reduced or absent C5-binding activity, but which retains LTB4-binding ability the nomacopan polypeptide can be modified such that Ala44 in SEQ ID NO: 4 (Ala62 in SEQ ID NO: 2) is replaced with Asn, Asp, Gin, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His.

[0167] In preferred embodiments Ala44 in SEQ ID NO: 4 is replaced with Asn.

[0168] This substitution at position 44 of SEQ ID NO: 4 (or position 62 of SEQ ID NO: 2) may be made in combination with any of the other substitutions referred to herein.

[0169] In another modified nomacopan polypeptide which has reduced or absent C5-binding activity but which retains LTB4-binding ability the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)-(j) is present: a. Met114 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin or Ala, e.g., Gin; b. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin or Ala e.g., Gin; c. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro, preferably Ser or Ala, e.g., Ser; d. Asp118 is replaced with Asn, Gin, Arg, Lys, Gly, Ala, Leu, Ser, He, Phe, Tyr, Met Pro, His, or Thr, preferably Asn; e. Alai 19 is replaced with Gly, Asp, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His, preferably Gly or Asn, e.g., Gly; f. Gly120 is replaced with Ser, Asp, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His, preferably Ser or Asn, e.g., Ser; g. Gly121 is replaced with Ala, Asp, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His preferably Ala or Asn, e.g., Ala; h. Leu122 is replaced with Asp, Glu, Asn, Gin, Arg, Lys, Pro, or His, preferably Asp or Ala, e.g., Asp; i. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, lie, Phe, Tyr, Pro, His, or Thr, preferably Asp, Ala, Gin or Asn, e.g., Asp or Ala; j. Val124 is replaced with Lys, Gin, Asn, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Lys or Ala, e.g., Lys; and additionally Ala44 in SEQ ID NO: 4 (Ala62 in SEQ ID NO: 2) is replaced with Asn, Asp, Gin, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His, preferably Asn.

[0170] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability, the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin; b. Leu117 is replaced with Ser; c. Gly121 is replaced with Ala; d. Leu 122 is replaced with Asp; e. Glu123 is replaced with Ala; and Ala44 in SEQ ID NO: 4 is replaced with Asn. In preferred aspects of this embodiment the amino acid residues corresponding to positions 114 to

[0171] 124 of SEQ ID NO: 4 are as set out in SEQ ID NO: 28.

[0172] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability, the nomacopan polypeptide is modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin; b. Leu 122 is replaced with Asp; and Ala44 in SEQ ID NO: 4 is replaced with Asn

[0173] In preferred aspects of this embodiment the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO: 4 are as set out in SEQ ID NO: 29.

[0174] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability the nomacopan polypeptide can be modified such that Asp149 in SEQ ID NO: 4 is replaced with Gly, Gin, Asn, Ala, Met, Arg, Lys, Leu, Ser, He, Phe, Tyr, Pro, His, or Thr. In some embodiments the nomacopan polypeptide is modified such that Asp149 of SEQ ID NO: 4 is replaced with Gly. This substitution at position 149 of SEQ ID NO: 4 (position 167 of SEQ ID NO: 2) may be made in combination with any of the other substitutions referred to herein.

[0175] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin; b. Leu117 is replaced with Ser; c. Gly121 is replaced with Ala; d. Leu 122 is replaced with Asp; e. Glu123 is replaced with Ala; and Ala44 in SEQ ID NO: 4 is replaced with Asn and Asp149 of SEQ ID NO: 4 is replaced with Gly149.

[0176] In preferred aspects of this embodiment the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO: 4 are as set out in SEQ ID NO: 28.

[0177] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability, the nomacopan polypeptide can be modified such that at positions 114 to 124 of SEQ ID NO: 4 the following substitutions are present: a. Met116 is replaced with Gin; b. Leu 122 is replaced with Asp; and Ala44 in SEQ ID NO: 4 is replaced with Asn and Asp149 of SEQ ID NO: 4 is replaced with Gly149.

[0178] In preferred aspects of this embodiment the amino acid residues corresponding to positions 114 to 124 of SEQ ID NO: 4 are as set out in SEQ ID NO: 29.

[0179] In the various aspects and embodiments of this disclosure, the modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability differ from the unmodified nomacopan polypeptides in SEQ ID NO: 2 and SEQ ID NO: 4 by from 1 to 30 amino acids. Any modifications may be made to the nomacopan polypeptide in SEQ ID NO: 2 and SEQ ID NO: 4 provided that the resulting modified nomacopan polypeptide exhibits LK / E binding activity and reduced or absent C5 binding, compared to the unmodified nomacopan polypeptide.

[0180] In some embodiments the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 are retained in the modified nomacopan polypeptides of the invention.

[0181] In some modified nomacopan polypeptides, Asn60 and Asn84 according to the numbering of SEQ ID NO: 4 (Asn78 and Asn102 according to the numbering of SEQ ID NO: 2) are each replaced with Gin. This modification can be carried out by site directed mutagenesis to prevent N-linked glycosylation when the polypeptide is expressed in yeast or mammalian cell culture.

[0182] In some modified nomacopan polypeptides one or more of the following amino acids in SEQ ID NO: 4 are thought to be involved in binding to LTB4 and may therefore be retained in unmodified form: Phe18, Tyr25, Arg36, Leu39, Gly41 , Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71 , Gln87, Arg89, His99, His101 , Asp103, and Trp115. In some modified nomacopan polypeptides, at least five, ten or fifteen, or all of these amino acids are retained in unmodified form in the modified nomacopan polypeptides of the invention. In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability one or more of these amino acids may be conservatively substituted. In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability up to five, ten or fifteen, or all of these amino acids are conservatively substituted in the modified nomacopan polypeptides of the invention.

[0183] Amino acids at the following positions in SEQ ID NO: 4 are highly conserved between nomacopan and TSGP2 and TSGP3: 5, 6, 11 , 13-15, 20-21 , 24-27, 29-32, 35-41 , 45, 47-48, 50, 52-60, 64, 66, 69- 81 , 83, 84, 86, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150

[0016] ,

[0184] Amino acids at the following positions in SEQ ID NO: 4 are thought to be involved in binding to LTB4 and / or are highly conserved between nomacopan and TSGP2 and TSGP3: 5, 6, 11 , 13-15, 18, 20-21 , 24-27, 29-32, 35-41 , 43, 45, 47-48, 50, 52-60, 64, 66, 67, 69-81 , 83, 84, 86, 87, 89, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150. Amino acids at the following positions in SEQ ID NO: 4 are thought to be involved in binding to LTB4 and / or are highly conserved between nomacopan and TSGP2 and TSGP3 : 5, 6, 11 , 13-15, 18, 20- 21 , 24-25, 27, 30-32, 35-41 , 43, 47-48, 50, 52-60, 64, 66, 67, 69-81 , 83, 84, 86, 87, 89, 90-94, 98, 100, 102-104, 112-113, 115, 126, 128-129, 132-139, 145, 148, and 150.

[0185] In some modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability therefore the above amino acids are retained in unmodified form. In some embodiments, at least five, ten, or fifteen, or all of these amino acids are retained in unmodified form in the modified nomacopan polypeptides of the invention. In some embodiments one or more of these amino acids may be conservatively substituted. In some embodiments up to five, ten, fifteen, twenty, twenty-five, 30, 40, 50 or all of these amino acids are conservatively substituted in the modified nomacopan polypeptides of the invention.

[0186] The modified nomacopan polypeptides referred to herein typically differ from SEQ ID NO: 2 or SEQ ID NO: 4 by from 1 to 30, preferably from 2 to 25, more preferably from 3 to 20, even more preferably from 4 to 15 amino acids. Typically, the difference will be 5 to 12, or 6 to 10 amino acid changes. For example, from 1 to 30, or 2 to 25, 3 to 30, 4 to 15, 5 to 12, or 6 to 10 amino acid substitutions may be made in SEQ ID NO: 2 or SEQ ID NO: 4.

[0187] Modified nomacopan polypeptides which have the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132-142 of SEQ ID NO: 2) as set out in SEQ ID NO: 27 have 10 amino acid substitutions compared to SEQ ID NO: 4 as a result of the presence of this modified loop. In some embodiments, the modified nomacopan polypeptides referred to herein preferably therefore have 1-15, 2-10, 3-5, or up to 2, 3, 4 or 5 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 22 (e.g., in the loop of SEQ ID NO: 27).

[0188] Modified nomacopan polypeptides which have the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132-142 of SEQ ID NO: 2) as set out in SEQ ID NO: 28 have 5 amino acid substitutions compared to SEQ ID NO: 4 as a result of the presence of this loop. In some embodiments, the modified nomacopan polypeptides referred to herein preferably therefore have 1-20, 2-15, 3-10, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 23 (e.g., in the loop of SEQ ID NO: 28). The additional substitutions preferably include substitutions at position 44 and 149, as set out elsewhere herein.

[0189] Modified nomacopan polypeptides which have the loop between beta H and alpha2 at amino acid positions 114 to 124 of SEQ ID NO: 4 (amino acid positions 132-142 of SEQ ID NO: 2) as set out in SEQ ID NO: 29 have 2 amino acid substitutions compared to SEQ ID NO: 4 as a result of the presence of this loop. In some embodiments, the modified nomacopan polypeptides preferably therefore have 1-25, 2-12, 3-15, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 24 (e.g., substitutions in the loop of SEQ ID NO: 29). The additional substitutions preferably include substitutions at position 44 and 149, as set out elsewhere herein.

[0190] Modified nomacopan polypeptides which have the substitution at position 44 of SEQ ID NO: 4 as set out elsewhere herein preferably have 1-25, 2-12, 3-15, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 additional substitutions compared to SEQ ID NO: 4.

[0191] Substitutions other than those explicitly referred to above are preferably conservative substitutions as described herein.

[0192] Preferred modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability may comprise or consist of the amino acid sequences set out in one of SEQ ID NOs: 22, 23, 24, 25.

[0193] Examples of modified nomacopan polypeptides which have reduced or absent C5-binding activity, but which retain LTB4-binding ability include

[0194] 1 . A modified nomacopan polypeptide which exhibits leukotriene or hydroxyeicosanoid binding activity and reduced or absent C5 binding, said modified nomacopan polypeptide comprising SEQ ID NO: 4 in which from 1 to 30 amino acid substitutions are made, wherein

[0195] (i) in positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)- (j) is made: a. Met114 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; b. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; c. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro; d. Asp118 is replaced with Asn, Gin, Arg, Lys, Gly, Ala, Leu, Ser, He, Phe, Tyr, Met Pro, His, or Thr; e. Alai 19 is replaced with Gly, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His; f. Gly120 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His; g. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His; h. Leu122 is replaced with Asp, Glu, Asn, Ala, Gin, Arg, Lys, Pro, or His; i. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, lie, Phe, Tyr, Pro, His, or Thr; j. Val124 is replaced with Lys, Gin, Asn, Arg, Lys, Gly, Ala, Pro, His, or Thr; or / and wherein

[0196] (ii) Ala44 in SEQ ID NO: 3 is replaced with Asn, Asp, Gin, Glu, Arg, Lys, Leu, lie, Phe, Tyr, Met, Pro, or His; or a fragment thereof in which up to five amino acids are deleted from the N terminus of the modified nomacopan polypeptide. 2. A modified nomacopan polypeptide according to clause 1 wherein

[0197] (i) in positions 114 to 124 of SEQ ID NO: 4 one or more of the following substitutions (a)- (j) is made: a. Met114 is replaced with Gin; b. Met116 is replaced with Gin; c. Leu117 is replaced with Ser; d. Asp118 is replaced with Asn; e. Alai 19 is replaced with Gly; f. Gly120 is replaced with Ser; g. Gly121 is replaced with Ala; h. Leu 122 is replaced with Asp; i. Glu123 is replaced with Asp, or Ala; j. Val124 is replaced with Lys; or / and wherein

[0198] (ii) Ala44 in SEQ ID NO: 3 is replaced with Asn44; or a fragment thereof in which up to five amino acids are deleted from the N terminus of the modified nomacopan polypeptide.

[0199] 3. A modified nomacopan polypeptide according to clause 1 or clause 2 or fragment thereof, wherein in positions 114 to 124 of SEQ ID NO: 4 one or more of the substitutions (a)-(j) is present.

[0200] 4. A modified nomacopan polypeptide according to clause 3 or a fragment thereof, wherein two or more of the substitutions (a) - (j) are present.

[0201] 5. A modified nomacopan polypeptide according to clause 4 or a fragment thereof, wherein five or more of the substitutions (a) - (j) are present.

[0202] 6. A modified nomacopan polypeptide according to clause 5 or a fragment thereof, wherein each of the substitutions (a) - (j) is present, optionally wherein Trp 115 is not substituted.

[0203] 7. A modified nomacopan polypeptide according to clause 5 or a fragment thereof, wherein each of the substitutions (a) - (j) as defined in clause 2 is present, optionally wherein Trp 115 is not substituted.

[0204] 8. The modified polypeptide according to clause 7 or a fragment thereof, wherein Glu123 is replaced with Asp. A modified nomacopan polypeptide according to any one of clauses 1 to 8, or a fragment thereof which has a loop sequence between amino acid positions 114 to 124 of SEQ ID NO: 4 as set out in SEQ ID NO: 27 and which has 1-15 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 22. The modified nomacopan polypeptide according to clause 9, or a fragment thereof which has 2-10 additional substitutions compared to SEQ ID NO: 27 beyond those that are set out in SEQ ID NO: 22. The modified nomacopan polypeptide according to clause 9 or 10, or a fragment thereof which has 3-5 additional substitutions compared to SEQ ID NO: 27 beyond those that are set out in SEQ ID NO: 22. The modified nomacopan polypeptide according to any one of clauses 1 to 8 which consists of or comprises SEQ ID NO: 22. A modified nomacopan polypeptide according to any one of clauses 1 to 5, or a fragment thereof wherein: a. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin; b. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro, preferably Ser; c. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His, preferably Ala; d. Leu122 is replaced with Asp, Glu, Asn, Ala, Gin, Arg, Lys, Pro, or His, preferably Asp; and e. Glu123 is replaced with Asp, Ala, Gin, Asn, Arg, Lys, Gly, Leu, Ser, He, Phe, Tyr, Pro, His, or Thr, preferably Ala or Asp. A modified nomacopan polypeptide according to clause 13, or a fragment thereof, wherein in positions 114 to 124 of SEQ ID NO: 4: a. Met116 is replaced with Gin; b. Leu117 is replaced with Ser; c. Gly121 is replaced with Ala; d. Leu 122 is replaced with Asp; and e. Glu123 is replaced with Ala. A modified nomacopan polypeptide according to clause 13 or clause 14, or a fragment thereof, wherein Trp 115 is not substituted. 16. A modified nomacopan polypeptide according to clause 13, 14 or 15, or a fragment thereof, wherein Met114, Trp 115, Asp118, Alai 19, Gly120 and Val124 are not substituted.

[0205] 17. A modified nomacopan polypeptide according to any one of clauses 1 to 5 or 13 to 16, or a fragment thereof which has a loop sequence between amino acid positions 114 to 124 of SEQ ID NO: 4 as set out in SEQ ID NO: 28 and which has 1-20 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 23.

[0206] 18. The modified nomacopan polypeptide according to clause 17, or a fragment thereof which has 2- 15 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 23.

[0207] 19. The modified nomacopan polypeptide according to clause 17 or 18, or a fragment thereof which has 3-10 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 23.

[0208] 20. The modified nomacopan polypeptide according to any one of clauses 1 to 5 or 13 to 16 which consists of or comprises SEQ ID NO: 23.

[0209] 21 . A modified nomacopan polypeptide according to any one of clauses 1 to 4, or a fragment thereof, wherein: a. Met116 is replaced with Gin, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr, preferably Gin; b. Leu122 is replaced with Asp, Glu, Asn, Ala, Gin, Arg, Lys, Pro, or His, preferably Asp.

[0210] 22. A modified nomacopan polypeptide according to clause 21 or a fragment thereof, wherein a. Met116 is replaced with Gin; and b. Leu 122 is replaced with Asp.

[0211] 23. A modified nomacopan polypeptide according to clause 21 or clause 22, or a fragment thereof, wherein Trp 115 is not substituted.

[0212] 24. A modified nomacopan polypeptide according to clause 21 , 22 or 23, or a fragment thereof, wherein Met114, Trp 115, Leu117, Asp118, Alai 19, Gly120, Gly121 , Glu123 and Val124 are not substituted.

[0213] 25. A modified nomacopan polypeptide according to any one of clauses 1 to 4 or 21 to 24, or a fragment thereof which has a loop sequence between amino acid positions 114 to 124 of SEQ ID NO: 4 as set out in SEQ ID NO: 29 and which has 1-25 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 24.

[0214] 26. The modified nomacopan polypeptide according to clause 25, or a fragment thereof which has 2- 12 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 24. The modified nomacopan polypeptide according to clause 25 or 26, or a fragment thereof which has 3-15 additional substitutions compared to SEQ ID NO: 4 beyond those that are set out in SEQ ID NO: 24. The modified nomacopan polypeptide according to any one of clauses 1 to 4 or 21 to 24, which consists of or comprises SEQ ID NO: 24. A modified nomacopan polypeptide according to any one of clauses 1 to 11 or 13 to 28, or a fragment thereof, wherein Ala44 in SEQ ID NO: 4 is replaced with Asn, Asp, Gin, Glu, Arg, Lys, Leu, He, Phe, Tyr, Met, Pro, or His. A modified nomacopan polypeptide according to clause 29, or a fragment thereof, wherein Ala44 in SEQ ID NO: 4 is replaced with Asn. A modified nomacopan polypeptide according to any one of clauses 1 to 11 or 13 to 30, or a fragment thereof, wherein Asp149 in SEQ ID NO: 4 is replaced with Gly, Gin, Asn, Ala, Met, Arg, Lys, Leu, Ser, He, Phe, Tyr, Pro, His, or Thr. A modified nomacopan polypeptide according to clause 30 or 31 , wherein Ala44 in SEQ ID NO: 4 is replaced with Asn and Asp149 in SEQ ID NO: 4 is replaced with Gly. A modified nomacopan polypeptide according to any one of the preceding clauses, or a fragment thereof, wherein the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 4 are retained in unmodified form. A modified nomacopan polypeptide according to any one of clauses 1 to 11 , 13 to 19 or 21 to 27, wherein Asn60 and Asn84 are each replaced with Gin. A modified nomacopan polypeptide according to any one of the preceding clauses, or a fragment thereof, wherein one or more of the following amino acids is not substituted: Phe18, Tyr25, Arg36, Leu39, Gly41 , Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71 , Gln87, Arg89, His99, His101 , Asp103, and Trp115. A modified nomacopan polypeptide according to clause 35, or a fragment thereof, wherein all of the following amino acids are not substituted: Phe18, Tyr25, Arg36, Leu39, Gly41 , Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71 , Gln87, Arg89, His99, His101 , Asp103, and Trp115. A modified nomacopan polypeptide according to any one of clauses 1 to 36, or a fragment thereof wherein: a. none of amino acids 5, 6, 11 , 13-15, 20-21 , 24-27, 29-32, 35-41 , 45, 47-48, 50, 52-60, 64, 66, 69-81 , 83, 84, 86, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150 in SEQ ID NO: 4 are substituted; or b. none of amino acids 5, 6, 11 , 13-15, 18, 20-21 , 24-27, 29-32, 35-41 , 43, 45, 47-48, 50, 52-60, 64, 66, 67, 69-81 , 83, 84, 86, 87, 89, 90-94, 97-104, 112-113, 115, 125-129, 132- 139, 145, 148, and 150 in SEQ ID NO: 4 are substituted; or c. none of amino acids 5, 6, 11 , 13-15, 18, 20-21 , 24-25, 27, 30-32, 35-41 , 43, 47-48, 50, 52-60, 64, 66, 67, 69-81 , 83, 84, 86, 87, 89, 90-94, 98, 100, 102-104, 112-113, 115, 126, 128-129, 132-139, 145, 148, and 150 in SEQ ID NO: 4 are substituted.

[0215] 38. A modified nomacopan polypeptide according to clause 1 or clause 2 which comprises or consists of the sequence SEQ ID NO: 25.

[0216] 39. A modified nomacopan polypeptide according to any one of the preceding clauses or a fragment thereof which binds to LTB4.

[0217] Fragments

[0218] Bioactive polypeptides purified by the methods of the invention include fragments of nomacopan and fragments of functional equivalents of nomacopan, provided that the fragments retain the ability to (i) bind LTB4 and / or (ii) C5 (e.g., wild-type C5 and / or C5 from subjects with a C5 polymorphism that renders treatment by eculizumab ineffective, or reduce the efficacy of treatment with eculizumab). Preferably the functional fragment has property (i) and (ii). In other preferred embodiments the functional fragment has property (i) but reduced or absent C5 binding.

[0219] Fragments may include, for example, polypeptides derived from the nomacopan protein sequence (or homologue) which are less than 150 amino acids, less than 145 amino acids, provided that these fragments retain the ability to bind to LTB4 and optionally also C5.

[0220] Fragments may include, for example, polypeptides derived from the nomacopan protein sequence (or homologue) which are at least 140 amino acids, preferably at least 145 amino acids, more preferably at least 146 amino acids, more preferably at least 147 amino acids, even more preferably at least 148 amino acids, yet more preferably at least 149 amino acids, or most preferably at least 150 amino acids, provided that these fragments retain the ability to bind to LTB4 and optionally also C5.

[0221] Any functional equivalent or fragment thereof preferably retains the pattern of cysteine residues that is found in nomacopan. For example, said functional equivalent comprises six cysteine residues that are spaced relative to each other at a distance of 32 amino acids apart, 62 amino acids apart, 28 amino acids apart, 1 amino acid apart and 21 amino acids apart as arranged from the amino terminus to the carboxyl terminus of the sequence according to amino acids 1 to 168 of the amino acid sequence in SEQ ID NO: 2. Exemplary fragments of nomacopan protein are disclosed in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14. The DNA encoding the corresponding fragments are disclosed in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13. Included as such fragments are not only fragments of the O. moubata nomacopan protein that is explicitly identified herein in SEQ ID NO: 2, but also fragments of homologues (e.g., variants) of this protein, as described above. Such fragments of homologues will typically possess greater than 60% identity with fragments of the nomacopan protein sequence in SEQ ID NO: 2, although more preferred fragments of homologues will display degrees of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively with fragments of the nomacopan protein sequence in SEQ ID NO: 2. Preferably such fragments will retain the cysteine spacing referred to above. Fragments with improved properties may, of course, be rationally designed by the systematic mutation or fragmentation of the wild type sequence followed by appropriate activity assays. Fragments may exhibit similar or greater affinity for LTB4 as nomacopan and optionally also similar or greater affinity for C5 as nomacopan. These fragments may be of a size described above for fragments of the nomacopan protein.

[0222] As discussed above, in fusion proteins of the invention, bioactive polypeptides preferably bind to LTB4 and optionally also C5.

[0223] Conservative substitutions

[0224] Any substitutions are preferably conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0225] PA(S) polypeptides

[0226] As used herein, the term ‘PA(S) polypeptides’ refers to PAS polypeptides and PA polypeptides.

[0227] The fusion proteins purified by methods of the invention comprise at least one PA(S) polypeptide (a ‘first PA(S) polypeptide’). In preferred embodiments, the fusion protein comprises a single PA(S) polypeptide, i.e., precisely one PA(S) polypeptide. In other embodiments, the fusion protein comprises at least two PA(S) polypeptides (a ‘first PA(S) polypeptide’ and a ‘second PA(S) polypeptide). In certain embodiments, the fusion protein comprises precisely two PA(S) polypeptides. In embodiments comprising a single PA(S) polypeptide, the PA(S) polypeptide may be a PAS polypeptide or a PA polypeptide. Preferably, the PA(S) polypeptide is a PAS polypeptide. In embodiments comprising two PA(S) polypeptides, each PA(S) polypeptide may independently be a PAS polypeptide or a PA polypeptide. Preferably, both PA(S) polypeptides are PAS polypeptides. However, a fusion protein may comprise two PA polypeptides, or one PAS polypeptide and one PA polypeptide.

[0228] References herein to ‘a PA(S) polypeptide’, ‘the PA(S) polypeptide’, or ‘PA(S) polypeptides’ should be interpreted as references to ‘the first PA(S) polypeptide and / or the second PA(S) polypeptide’ unless explicitly stated otherwise.

[0229] Typically, the PA(S) polypeptide forms / adopts a random coil conformation. As used herein, the ‘random coil’ means any conformation of a polymeric molecule, including amino acid polymers (e.g., a PA(S) polypeptide), in which the individual monomeric elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomeric elements while still being chemically bound to said adjacent monomeric elements. In particular, a polypeptide or amino acid polymer forming a ‘random coil conformation’ substantially lacks a defined secondary and tertiary structure. The nature of polypeptide random coils and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature

[0017] ,

[0018] ,

[0019] , Methods for determining whether a PA(S) polypeptide forms / adopts a random coil conformation are described in [8] and [9], for example circular dichroism (CD) spectroscopy, size exclusion chromatography (SEC), and dynamic light scattering (DLS).

[0230] Typically, the PA(S) polypeptide forms / adopts a random coil confirmation under physiological conditions. As used herein, ‘physiological conditions’ means conditions (e.g., biochemical and biophysical parameters) in which proteins usually adopt their native conformation, for example as they are normally found in the body (e.g., in particular in body fluids such as the vitreous) of mammals and preferably in humans. With respect to ‘physiological conditions’ at which proteins adopt their native conformation / state, the most important parameters are temperature (37°C for the human body), pH (7.35-7.45 for human blood), osmolality (280-320 mOsm), and, if necessary, total protein content (66- 85 g / L serum).

[0231] In fusion proteins purified by methods of the invention, PA(S) polypeptide(s) typically mediate increased in vivo and / or in vitro stability. In other words, in fusion proteins of the present invention, the PA(S) polypeptide(s) mediate increased in vivo and / or in vitro stability of the fusion protein compared to the in vivo and / or in vitro stability of the bioactive polypeptide(s) alone. In preferred embodiments, the PA(S) polypeptide mediates increased in vivo stability, for example in the human body. In more preferred embodiments, the PA(S) polypeptide mediates increased intravitreal stability. As used herein, ‘in vivo stability’ means the capacity of a specific substance that is administered to the living body to remain biologically available and biologically active. Methods for measuring the in vivo stability of biologically active proteins are described in [8] and [9], As a result of forming / adopting a random coil conformation, the PA(S) polypeptide typically has a large hydrodynamic radius (Rh). A larger hydrodynamic radius may advantageously confer a longer half-life.

[0232] PAS polypeptides

[0233] As used herein a ‘PAS polypeptide’ is a polypeptide comprising, consisting essentially of, or consisting of proline, alanine, and serine residues. In some embodiments the fusion protein purified by methods of the invention comprises at least one PAS polypeptide (a ‘first PAS polypeptide’). In preferred embodiments, the fusion protein comprises a single PAS polypeptide, i.e., precisely one PAS polypeptide. In other embodiments, the fusion protein comprises at least two PAS polypeptides (a ‘first PAS polypeptide’ and a ‘second PAS polypeptide). In certain embodiments, the fusion protein comprises precisely two PAS polypeptides.

[0234] In embodiments comprising two PAS polypeptides, each PAS polypeptide may be the same (identical) or different. Each of the PAS polypeptides may be independently selected from the PAS polypeptides described herein. Thus, references herein to ‘a PAS polypeptide’, ‘the PAS polypeptide’, or ‘PAS polypeptides’ should be interpreted as references to ‘the first PAS polypeptide and / or the second PAS polypeptide’ unless explicitly stated otherwise.

[0235] In some embodiments, the PAS polypeptide consists essentially of proline, alanine, and serine residues. A PAS polypeptide consisting essentially of proline, alanine, and serine may comprise at least 90%, preferably at least 95%, more preferably 96%, yet more preferably 97%, yet more preferably 98%, even more preferably 99% proline, alanine, and serine (i.e., at least 90%, 95%, 96%, 97%, 98%, or 99% of the residues of the PAS polypeptide are proline, alanine, or serine). A PAS polypeptide consisting essentially of proline, alanine, and serine may comprise a sufficiently high proportion of proline, alanine, and serine to form a random coil conformation. Amino acids different from alanine, serine and proline may be selected from the group consisting of Arg, Asn, Asp, Cys, Gin, Giu, Giy, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai. Amino acids that do not have hydrophobic side chains, like Vai, He, Leu, Met, Phe, Tyr or Trp, and / or that do not have charged side chains, like Lys, Arg, Asp or Glu, are preferred. In preferred embodiments, the PAS polypeptide consists of proline, alanine, and serine residues. Typically, the PAS polypeptide comprises or consists of a plurality of PAS repeats, wherein typically each repeat consists of proline, alanine, and serine residues and wherein no more than 6 consecutive amino acid residues are identical. Typically, proline residues constitute more than 4% and less than 40% of the amino acids of each PAS repeat and / or each PAS polypeptide (typically alanine and serine residues comprise the remaining at least 60% to 96%). For example, each PAS repeat and / or PAS polypeptide may comprise more than about 4%, preferably more than about 5%, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% proline residues (i.e., more than about 4%, 5%, 6%, 8%, 10%, 15%, and 20% of the residues of the PAS repeat and / or PAS polypeptide are proline). PAS repeats preferably comprises less than about 40 % or less than about 35% proline residues. Each PAS repeat may comprise at least or may consist of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat comprises or consists of (an) alanine, serine, and proline residue(s).

[0236] In some embodiments, the PAS polypeptide comprises repeats of the formula: Serx[AlayProz] wherein x is an integer selected from 0 to 6, y is an integer selected from 1 to 6, and z is an integer selected from 1 to 6.

[0237] In some embodiments, each PAS repeat and / or the PAS polypeptide comprises or consists of: a) proline residues, wherein said proline residues constitute more than about 4 %, preferably more than about 5 %, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% of the amino acids constituting the random coil forming domain. Such an amino acid polymer purified by methods of the invention which forms random coil conformation preferably comprises less than about 40%, or less than about 35% of the amino acids constituting the random coil forming domain; b) alanine residues, wherein more than about 4% but less than about 50%, preferably more than about 10% but less than about 50% and most preferably more than about 20% but less than about 50% alanine residues; and / or c) serine residues, wherein more than about 4% and less than about 50%, preferably more than about 10% but less than about 50% and most preferably more than about 20% but less than about 50 % serine residues.

[0238] In some embodiments, each PAS repeat and / or PAS polypeptide comprises about 35% proline residues, about 50% alanine residues and about 15% serine residues of the amino acids constituting the random coil forming domain. Alternatively, each PAS repeat and / or PAS polypeptide may comprise about 35% proline residues, about 15% alanine residues and about 50% serine residues of the amino acids constituting the random coil forming domain. As used herein, ‘about’ means + / - 10%, preferably + / - 5%.

[0239] In preferred embodiments, the PAS polypeptide comprises or consists of PAS repeats having a sequence selected from the group consisting of: ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 15), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 16); APSSPSPSAPSSPSPASPSS (SEQ ID NO: 17); SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 18); SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 19); AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 20); and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 21). In even more preferred embodiments, the PAS polypeptide comprises or consists of repeats of SEQ ID NO: 15. In some embodiments, the PAS polypeptide comprises 400, 600, 800, 1000, or 1200 amino acids. In some embodiments, said 400, 600, 800, 1000, or 1200 amino acids consist of repeats of SEQ ID NO:

[0240] 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 , preferably of SEQ ID NO: 15.

[0241] In some embodiments, the PAS polypeptide comprises or consists of 20 repeats of SEQ ID NO: 15,

[0242] 16, 17, 19, 20, or 21 , preferably 20 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 30). In some embodiments, the PAS polypeptide comprises or consists of 30 repeats of SEQ ID NO: 15, 16, 17, 19, 20, or 21 , preferably 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31). In some embodiments, the PAS polypeptide comprises or consists of 40 repeats of SEQ ID NO: 15, 16, 17, 19, 20, or 21 , preferably 40 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 32). In some embodiments, the PAS polypeptide comprises or consists of 50 repeats of SEQ ID NO: 15, 16, 17, 19, 20, or 21 , preferably 50 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 33). In some embodiments, the PAS polypeptide comprises or consists of 60 repeats of SEQ ID NO: 15, 16, 17, 19, 20, or 21 , preferably 60 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 34).

[0243] Further exemplary PAS polypeptides are described in [8],

[0244] PA polypeptides

[0245] As used herein a ‘PA polypeptide’ is a polypeptide comprising, consisting essentially of, or consisting of proline and / or alanine residues. In some embodiments the fusion protein purified by methods of the invention comprises at least one PA polypeptide (a ‘first PA polypeptide’). In certain embodiments, the fusion protein comprises a single PA polypeptide, i.e., precisely one PA polypeptide. In other embodiments, the fusion protein comprises at least two PA polypeptides (a ‘first PA polypeptide’ and a ‘second PA polypeptide). In certain embodiments, the fusion protein comprises precisely two PA polypeptides.

[0246] In embodiments comprising two PA polypeptides, each PA polypeptide may be the same (identical) or different. Each of the PA polypeptides may be independently selected from the PA polypeptides described herein. Thus, references herein to ‘a PA polypeptide’, ‘the PA polypeptide’, or ‘PA polypeptides’ should be interpreted as references to ‘the first PA polypeptide and / or the second PA polypeptide’ unless explicitly stated otherwise.

[0247] In some embodiments, the PA polypeptide consists essentially of proline and alanine residues. A PA polypeptide consisting essentially of proline and alanine may comprise at least 90%, preferably at least 95%, more preferably 96%, yet more preferably 97%, yet more preferably 98%, even more preferably 99% proline and alanine. A PA polypeptide consisting essentially of proline and alanine may comprise a sufficiently high proportion of proline and alanine to form a random coil conformation. Amino acids different from proline and alanine may be selected from the group consisting of Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai. Amino acids that do not have hydrophobic side chains, like Vai, He, Leu, Met, Phe, Tyr or Trp, and / or that do not have charged side chains, like Lys, Arg, Asp or Glu, are preferred. PA polypeptides may comprise Ser but typically do not comprise Ser. In preferred embodiments, the PA polypeptide consists of proline and alanine residues. Typically, the PA polypeptide comprises or consists of a plurality of PA repeats, wherein typically each repeat consists of proline and alanine residues and wherein no more than 6 consecutive amino acid residues are identical. Typically, proline residues constitute more than 10% and less than 75% of the amino acids of each PA repeat and / or each PA polypeptide (typically alanine residues comprise the remaining at least 25% to 90%). For example, each PA repeat and / or PA polypeptide may comprise more than about 10%, preferably more than about 12%, even more preferably more than about 14%, particularly preferably more than about 18%, more particularly preferably more than about 20%, even more particularly preferably more than about 22%, 23% or 24% and most preferably more than about 25% proline residues. Each PA repeat and / or PA polypeptide preferably comprises less than about 75%, more preferably less than 70%, 65%, 60%, 55% or 50% proline residues, wherein the lower values are preferred. Even more preferably, each PA repeat and / or PA polypeptide comprises less than about 48%, 46%, 44%, 42% proline residues. Particularly preferred are PA repeats and PA polypeptides comprising less than about 41%, 40%, 39%, 38%, 37% or 36% proline residues, whereby lower values are preferred. Most preferably, each PA repeat and / or PA polypeptide comprises less than about 35% proline residues. Accordingly, each PA repeat and / or PA polypeptide may comprise about 25% proline residues and about 75% alanine residues. Alternatively, each PA repeat and / or PA polypeptide may comprise about 35% proline residues and about 65% alanine residues.

[0248] Each PA repeat may comprise at least or may consist of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat comprises or consists of (an) alanine and proline residue(s).

[0249] In some embodiments, the PA polypeptide comprises repeats of the formula: [ProxAlay] wherein x is an integer selected from 1 to 5 and y is an integer selected from 1 to 5.

[0250] In preferred embodiments, the PA polypeptide comprises or consists of PA repeats having a sequence selected from the group consisting of: AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 49), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 50), AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 51), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO: 52), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 53), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 54), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 55).

[0251] In some embodiments, the PA polypeptide comprises 400, 600, 800, 1000, or 1200 amino acids. In some embodiments, said 400, 600, 800, 1000, or 1200 amino acids consist of repeats of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, or SEQ ID NO: 55. Further exemplary PA polypeptides are described in [9], Exemplary fusion proteins

[0252] Examples of fusion proteins (i.e., comprises a which have reduced or absent C5-binding activity but which retain LTB4-binding ability may comprise or consist of the following sequences

[0253] Monovalent PAS-nomacopan fusion proteins

[0254] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, and ii) a PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 35 (‘PAS600-nomacopan’).

[0255] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, and ii) a PAS polypeptide comprising or consisting of 40 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 32), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 37 (‘PAS800-nomacopan’).

[0256] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, and ii) a PAS polypeptide comprising or consisting of 50 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 33), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In a particularly preferred embodiment, the fusion protein comprises or consists of SEQ ID NO: 39 (‘PASI OOO-nomacopan’).

[0257] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, and ii) a PAS polypeptide comprising or consisting of 60 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 34), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 41 (‘PAS1200-nomacopan’).

[0258] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), and ii) a PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 36 (‘PAS600-L-nomacopan’).

[0259] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), and ii) a PAS polypeptide comprising or consisting of 40 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 32), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 38 (‘PAS800-L-nomacopan’). In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), and ii) a PAS polypeptide comprising or consisting of 50 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 33), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 40 (‘PAS1000-L-nomacopan’).

[0260] In some embodiments, the fusion protein comprises or consists of i) a bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), and ii) a PAS polypeptide comprising or consisting of 60 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 34), wherein the PAS polypeptide is fused to the N-terminus of the bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 42 (‘PAS1200-L-nomacopan’).

[0261] Bivalent PAS-nomacopan fusion proteins (i.e., nomacopan-PAS-nomacopan and PAS-nomacopan- PAS-nomacopan fusion proteins)

[0262] In some embodiments, the fusion protein comprises or consists of: i) a first bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, ii) a PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), and iii) a second bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, wherein the first bioactive polypeptide is fused to the N-terminus of the PAS polypeptide and the second bioactive polypeptide is fused to the C-terminus of the PAS polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 43 (‘nomacopan-PAS600-nomacopan’).

[0263] In some embodiments, the fusion protein comprises or consists of: i) a first bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), ii) a PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), and iii) a second bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), wherein the first bioactive polypeptide is fused to the N-terminus of the PAS polypeptide and the second bioactive polypeptide is fused to the C-terminus of the PAS polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 44 (‘L-nomacopan- PAS600-L-nomacopan’).

[0264] In some embodiments, the fusion protein comprises or consists of: i) a first PAS polypeptide comprising or consisting of at least 20 repeats of SEQ ID NO: 15, 16, 17, 18, 19, 20, or 21 , ii) a first bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, or SEQ ID NO: 22, 23, 24, or 25, iii) a second PAS polypeptide comprising or consisting of at least 20 repeats of SEQ ID NO: 15, 16, 17, 18, 19, 20, or 21 , and iv) a second bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, or SEQ ID NO: 22, 23, 24, or 25, wherein the first PAS polypeptide is fused to the N-terminus of the first bioactive polypeptide and the second PAS polypeptide is fused to the C-terminus of the first bioactive polypeptide and to the N-terminus of the second bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of: i) a first PAS polypeptide comprising or consisting of 20 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 30), ii) a first bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, iii) a second PAS polypeptide comprising or consisting of at least 20 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 30), and iv) a second bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, wherein the first PAS polypeptide is fused to the N-terminus of the first bioactive polypeptide and the second PAS polypeptide is fused to the C-terminus of the first bioactive polypeptide and to the N- terminus of the second bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 45 (‘PAS400-nomacopan-PAS400-nomacopan’).

[0265] In some embodiments, the fusion protein comprises or consists of: i) a first PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), ii) a first bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, iii) a second PAS polypeptide comprising or consisting of at least 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), and iv) a second bioactive polypeptide comprising or consisting of amino acids 19-168 of SEQ ID NO: 2, wherein the first PAS polypeptide is fused to the N-terminus of the first bioactive polypeptide and the second PAS polypeptide is fused to the C-terminus of the first bioactive polypeptide and to the N- terminus of the second bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 47 (‘PAS600-nomacopan-PAS600-nomacopan’).

[0266] In some embodiments, the fusion protein comprises or consists of: i) a first PAS polypeptide comprising or consisting of 20 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 30), ii) a first bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), iii) a second PAS polypeptide comprising or consisting of at least 20 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 30), and iv) a second bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), wherein the first PAS polypeptide is fused to the N-terminus of the first bioactive polypeptide and the second PAS polypeptide is fused to the C-terminus of the first bioactive polypeptide and to the N-terminus of the second bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 46 (‘PAS400-L-nomacopan- PAS400-L-nomacopan’).

[0267] In some embodiments, the fusion protein comprises or consists of: i) a first PAS polypeptide comprising or consisting of 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), ii) a first bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), iii) a second PAS polypeptide comprising or consisting of at least 30 repeats of SEQ ID NO: 15 (i.e., SEQ ID NO: 31), and iv) a second bioactive polypeptide comprising or consisting of SEQ ID NO: 22, 23, 24, or 25 (preferably SEQ ID NO: 22), wherein the first PAS polypeptide is fused to the N-terminus of the first bioactive polypeptide and the second PAS polypeptide is fused to the C-terminus of the first bioactive polypeptide and to the N-terminus of the second bioactive polypeptide. In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 48 (‘PAS600-L-nomacopan- PAS600-L-nomacopan’). Hydrophobic interaction chromatography

[0268] The fusion proteins described herein are useful for treating diseases and conditions of the eye, especially of the retina, for example dry AMD and GA. The fusion proteins are typically administered via intraocular administration, preferably intravitreal administration. Therefore, the inventors sought to develop purification methods which result in compositions suitable for intraocular administration. Such compositions need to have low levels of pyrogens, which can cause inflammation in the eye. As explained in Example 1 , one challenge the inventors encountered was identifying a purification technique that could remove pyrogens without affecting the native structure of the protein. For example, although lipid ligands can be removed by a procedure known as delipidation, this must be performed at 37°C at a very hydrophobic surface, which often results in partly denaturing the protein.

[0269] The inventors therefore developed a HIC technique involving hexyl ligands which removed pyrogens under native conditions at room temperature with surprisingly good efficiency, without significantly impacting yield of the fusion protein.

[0270] The invention provides a method for removing pyrogens from a composition comprising a fusion protein, wherein the method comprises: a) providing an input composition comprising the fusion protein and a pyrogen, and b) performing at least one step of hydrophobic interaction chromatography (HIC) on using a stationary phase comprising hexyl ligands to produce an output composition comprising the fusion protein, and wherein the fusion protein comprises: i) a bioactive polypeptide, wherein the bioactive polypeptide comprises amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide.

[0271] As used in the context of HIC, the input composition is the composition that comprises a fusion protein as described herein and a pyrogen. In some embodiments, the input composition is conditioned with a buffer prior to loading onto the HIC stationary phase,

[0272] As used herein, the term “remove” refers to the reduction or depletion of a substance, such as an impurity, for example pyrogens or host cell proteins. Removal may refer to any amount of such substances from a composition (e.g. there is a reduction in the amount of the substance in an output composition compared to an input composition), and does not require the absence of the substance after the relevant method step or step(s). Although a method step referred to herein may also remove some fusion protein as well as removing the impurity (e.g. pyrogen), in general after a substance has been removed from a composition comprising a fusion protein and an impurity (e.g. pyrogen), the ratio of fusion protein to substance will be higher than before the relevant step is carried out. As used herein, the term “impurity” encompasses any substance other than the fusion protein (e.g. a pyrogen).

[0273] Input composition for HIC

[0274] In the invention, the purpose of the HIC step is to remove pyrogens, including endotoxins and NEPs, from compositions comprising the fusion proteins described herein. Removing pyrogens is particularly important when generating compositions that are suitable for ophthalmic administration

[0020] , Performing HIC is particularly useful on an input composition that has already been substantially purified, e.g. from cell culture medium into which the fusion protein has been secreted. Typically, HIC is performed as a final chromatography step to remove any residual pyrogens. For example, an input composition may have been subjected to at least one, at least two or at least three purification steps (e.g. centrifugation, chromatography, membrane filtration).

[0275] In some embodiments, the input composition comprises a fusion protein described herein and a pyrogen. In some embodiments, the pyrogen is an endotoxin, for example LPS. In some embodiments, the at pyrogen is a NEP, for example a lipoprotein, peptidoglycan, wall teichoic acid, and / or a lipoteichoic acid.

[0276] Various in vitro and in vivo methods for detecting pyrogens are known in the art, e.g., the rabbit pyrogen test (RPT), the Limulus amoebocyte lysate test (LAL), the monocyte activation test (MAT), and the recombinant factor C (rFC) test. Endotoxins are typically measured in Endotoxin Units (EU)

[0021] .

[0277] The RPT

[0022] involves intravenous injection of a composition into a rabbit and measuring the subsequent rise in temperature. This method has been the gold standard for assessing the level of pyrogens in pharmaceutical compositions for ~100 years. However, it only produces qualitative results, and the sensitivity is low. The robustness of the test is also limited, due to development of pyrogen tolerance in rabbits after repeated injections, or stress from the rabbits when performing the assay. Thus, regulatory bodies are moving towards mandating the use of in vitro alternatives, where possible.

[0278] The LAL assay

[0023] is the most common test for detecting endotoxins [1], LAL is an aqueous extract obtained after lysis of blood cells (amoebocytes), and this assay is based on the LAL obtained from horseshoe crab blood. The LAL naturally reacts with bacterial endotoxins in a coagulation reaction. Specifically, the presence of pyrogens initiates a series of enzymatic reactions that result in the activation of a pathway to the production of serine protease zymogens (e.g., factor C, factor B, and pro-clotting enzyme). The simplest LAL assay is the gel clot method, which is a qualitative test in which the formation of a clot or gel at the bottom of the sample tube is examined by eye and indicates the presence of endotoxin in a sample. The chromogenic LAL assay on the other hand is a quantitative test that produces a colour (chromogenic) as the result of the enzymatic reaction. The colour intensity is directly proportional to the quantity of endotoxins in the sample. The chromogenic LAL reaction can be quantified by absorbance detection using a spectrophotometer or an absorbance microplate reader

[0024] , While this method offers advantages over RPT testing, e.g., it has high sensitivity and allows quantification of endotoxins, it cannot detect NEPs. rFC is a genetically engineered equivalent of the Factor C protein which is part of the LAL cascade. In the rFC test, endotoxins in the test composition activate rFC, which couples with a marker to produce a quantifiable, fluorescent end product. The MAT assay is an alternative to using animal-based methods for the detection of both endotoxin and NEPs

[0025] , The MAT assay involves incubating monocytes with a test composition. If present, pyrogens will activate the monocytes, causing the release of inflammatory molecules e.g., a cytokine. The cytokine can then be detected using an immunological assay, typically an enzyme-linked immunosorbent assay (ELISA). This test therefore offers several advantages over the RPT and LAL tests., e.g., high sensitivity and quantification of both endotoxins and NEPs. Thus, the MAT assay is a better indicator of the total level of pyrogens in pharmaceutical compositions. There are different variants of the MAT assay available depending on (1) the source of monocytes, including whole blood, isolated primary monocytes (e.g., PBMCs) or monocytic cell lines; and (2) which cytokines are measured as a read-out, e.g., IL-6, IL-lp, TNF-a and / or IFN-y. The principle of these variants remains the same - a test composition is applied to monocytes and the inflammatory reaction is measured. Any endogenous mediator of the inflammatory response secreted by monocytes that is detectable may be used as the basis of a MAT assay. An exemplary MAT assay kit is the PyroMAT™ System (Merck Millipore), which uses the MM6 monocytic cell line. The test composition is incubated with the cell line and, after a 20-to-24-hour incubation, the concentration of IL-6 released is quantified using an ELISA. Other kits are known and available in the art.

[0279] In some embodiments, the pyrogen in the input composition comprises at least one (type of) endotoxin. In some embodiments, the pyrogen in the input composition comprises at least one (type of) NEP. In preferred embodiments, the pyrogen in the input composition comprises at least one endotoxin and at least one NEP.

[0280] Typically, pyrogens in the input composition induce monocytes to secrete a cytokine, e.g., in a MAT assay. In some embodiments, the pyrogens in the input composition are detectable by measuring their ability to induce monocytes to secrete at least one cytokine (i.e., at least one type of cytokine), for example as measured by a MAT assay. In some embodiments, the at least one cytokine is selected from: a pro-inflammatory cytokine (e.g., TNFa, IL-1 , IL-6), an anti-inflammatory cytokine (e.g., IL-4, 11-10, IL-13, IL-13, IL-1 ra, TGFp), a Th1 cytokine (e.g., IL-2, IFNy, IL-12), a Th2 cytokine (e.g., IL-4, IL-5, IL-6, IL-10, IL-13), IL-1 p, PGE2, and a combination thereof. In preferred embodiments, the at least one cytokine comprises IL-6, IL-lp, TNF-a and / or IFN-y. In more preferred embodiments, the at least one cytokine comprises IL-6.

[0281] A MAT assay may be performed on any composition. In some embodiments, the MAT assay comprises the following steps: a) incubating monocytes with a composition, optionally for 20 to 24 hours; and b) detecting and quantifying the amount of at least one cytokine released from the monocytes, optionally using an ELISA, further optionally wherein the cytokine is IL-6.

[0282] In some embodiments, the source of the monocytes is isolated primary monocytes (e.g., PBMCs) or monocytic cell lines, optionally a MM6 monocytic cell line. In some embodiments, the composition that is incubated with the monocytes is the input composition or is the input composition that has been processed before performing a MAT assay, for example by filtration, concentration and / or buffer exchange, preferably buffer exchange into a PBS buffer.

[0283] Typically, the input composition is reactive in a LAL assay, i.e., pyrogens in the input composition induce a coagulation reaction. In some embodiments, the pyrogens in the input composition are detectable by measuring their ability to induce a coagulation reaction (measured by a chromogenic reaction) in a LAL assay.

[0284] A LAL assay may be performed on any composition. In some embodiments, the LAL assay comprises the following steps: a) incubating a LAL reagent with a composition; and b) detecting and quantifying a chromogenic reaction, optionally using a spectrophotometer or an absorbance microplate reader.

[0285] In some embodiments, a series of endotoxins dilutions with known EU are also incubated with the LAL reagent to generate a standard curve for quantifying the EU in the input composition. In some embodiments, the composition that is incubated with the LAL reagent is the input composition or is the input composition that has been processed before performing a LAL assay, for example by filtration, concentration and / or buffer exchange, preferably buffer exchange into a PBS buffer.

[0286] In some embodiments, the input composition comprises more than 0.04 EU / mg, more than 0.05 EU / mg, more than 0.06 EU / mg, more than 0.08 EU / mg, more than 0.1 EU / mg, or greater than 0.1 EU / mg. Preferably, the input composition comprises more than 0.04 EU / mg of endotoxins.

[0287] As used herein, the term “purity” of the fusion protein means the proportion of total solute (e.g., protein) which is the fusion protein. For example, if a composition is described as having 90% purity of a fusion protein, this means that 90% of the total solute (e.g., protein) in the sample is the fusion protein.

[0288] Typically, the input composition has already been subjected to one or more purification steps that increase the purity of the fusion protein (e.g. starting from a cell culture medium containing the fusion protein), such as one or more steps of precipitation, centrifugation, and chromatography, although at least some pyrogen remains. Overall purity of the input composition (taking into account any / all impurities, not just pyrogens) can be measured using reverse-phase high-performance liquid chromatography (RP-HPLC), also known as high-pressure liquid chromatography. RP-HPLC is one of the most common techniques used to separate, quantify and identify individual compounds within a mixture

[0026] , In some embodiments of the invention, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the total protein in the input composition is the fusion protein, as measured RP-HPLC. In preferred embodiments, at least 85% of the total protein in the input composition is the fusion protein, as measured by RP-HPLC.

[0289] Purity of the input composition can alternatively be measured using size-exclusion chromatography high-performance liquid chromatography (SE-HPLC). SE-HPLC is particularly useful for determining purity of proteins from a mixture comprising fusion and impurities, such as degraded protein products

[0027] , In some embodiments, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of the total protein in the input composition is the fusion protein, as measured by SE-HPLC. In preferred embodiments, at least 90% of the total protein in the input composition is the fusion protein as measured by SE-HPLC.

[0290] In preferred embodiments, at least 85% of the total protein in the input composition is the fusion protein, as measured by RP-HPLC and at least 90% of the total protein in the input composition is the fusion protein, as measured by SE-HPLC.

[0291] Purity of the input composition can alternatively be measured using ion exchange high-performance liquid chromatography (IEX-HPLC)

[0028] , In some embodiments, at least 70%, least 75%, at least 80%, at least 85% or at least 90% of the total protein in the input composition is the fusion protein, as measured by IEX-HPLC. In preferred embodiments, at least 90% of the total protein in the input composition is the fusion protein, as measured by IEX-HPLC.

[0292] Purity of the input composition can alternatively be measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

[0029] , SDS-PAGE separates proteins based one size and charge and provides a simple method of analysing the purity of protein samples. In some embodiments, at least 70%, least 75%, at least 80%, at least 85% or at least 90% of the total protein in the input composition is the fusion protein, as measured by SDS-PAGE. In preferred embodiments, at least 90% of the total protein in the input composition is the fusion protein, as measured by SDS- PAGE.

[0293] Details of the HIC method

[0294] As used herein, hydrophobic interaction chromatography (HIC) is a type of liquid chromatography which separates compounds such as proteins according to differences in their surface hydrophobicity. Specifically, HIC uses reversible hydrophobic interactions between a target protein and a hydrophobic ligand of the HIC stationary phase. Purification of proteins by HIC is usually performed using high salt concentrations, for example by using by buffers of high ionic strength as a mobile phase, to enhance the hydrophobic interactions between the target protein and the HIC stationary phase (e.g. resin). The protein of interest is collected by applying a salt gradient, i.e. elution buffers of decreasing salt concentration.

[0295] Typically, HIC comprises the following steps: conditioning the input composition with a conditioning buffer; equilibrating the HIC stationary phase with an equilibrium buffer; loading the conditioned composition onto the HIC stationary phase; washing the HIC resin with a wash buffer; and eluting the target protein (i.e., the fusion protein) from the HIC stationary phase using a salt gradient to produce the output composition.

[0296] The conditioning buffer may be selected so that at least a proportion of the fusion protein binds to the HIC stationary phase (e.g., resin). In preferred embodiments, the buffer is a HEPES / ammonium sulphate buffer ((e.g. 50 mM HEPES, 1100 mM ammonium sulphate, pH 7.6). In preferred embodiments, the equilibrium buffer is a HEPES / ammonium sulphate buffer (e.g. 25 mM HEPES, 550 mM ammonium sulphate, pH 7.6). The wash buffer may be selected so that at least a proportion of the pyrogen is separated from the fusion protein. Typically, the wash buffer is the same as the equilibrium buffer. In preferred embodiments, the wash buffer is a HEPES / ammonium sulphate buffer (e.g. 25 mM HEPES, 550 mM ammonium sulphate, pH 7.6). The salt gradient may be continuous or step-wise. In preferred embodiments, elution is performed using a HEPES / ammonium sulphate buffer (e.g. 25 mM HEPES, 550 mM ammonium sulphate, pH 7.6) and a HEPES buffer (e.g. 25 mM HEPES, pH 7.6). Typically, at least one of the collected fractions (e.g., an aliquot of collected eluate) will have a reduced level of the pyrogen. Fractions may be stored e.g. at 5 ± 4 °C e.g., until further processing. Appropriate buffers for use in HIC may be selected by the skilled person. Examples of other appropriate buffers include a Tris / ammonium sulphate buffer (pH 7.8), a MES / ammonium sulphate buffer (pH 6.6), a Tris / ammonium sulphate buffer (pH 7.6) or a MOPS / ammonium sulphate buffer (pH 7.6). Thus, in some embodiments the conditioning buffer, the equilibrium buffer and / or the wash buffer is a Tris / ammonium sulphate buffer (pH 7.6), a MES / ammonium sulphate buffer (pH 6.6), a Tris / ammonium sulphate buffer (pH 7.6) and / or a MOPS / ammonium sulphate buffer (pH 7.6).

[0297] Thus, in preferred embodiments, HIC comprises the following steps: i) conditioning the input composition; ii) equilibrating the HIC stationary phase; iii) loading the conditioned composition onto the HIC stationary phase iv) washing the HIC stationary phase; and v) eluting the fusion protein from the HIC stationary phase using a salt gradient to produce the output composition.

[0298] In some embodiments, the method comprises performing HIC on multiple separate batches of input compositions, for example where the input composition is of a large volume.

[0299] Typically, HIC is performed using a stationary phase comprising hexyl ligands. As used herein, the term “hexyl ligand” refers to the presence of a hexyl group (i.e., CeHn). In some embodiments, the stationary phase further comprises a hydroxylated polymethacrylic polymer. In preferred embodiments, the stationary phase comprises a hydroxylated polymethacrylic polymer coupled to hexyl ligands, for example through a hydroxyl group (e.g., -OCH2CH2CH2CH2CH2CH3). In some embodiments, the stationary phase has a mean pore size of about 80 nm (800 A) to about 120 nm (1200 A), preferably about 100 nm (1000 A). In some embodiments, the stationary phase has a mean particle size of about 80 pm to about 120 pm, preferably 100 pm. In some embodiments, the stationary phase has a mean ligand density of about 30 g / L to about 50 g / L. For example, the stationary phase may be Toyopearl® Hexyl-650C (Tosoh Bioscience), which has all of the abovementioned characteristics. This resin has the highest hydrophobicity of the Toyopearl HIC resins. Thus, an advantage of using said resin is that it can be used for very hydrophilic proteins or where a low salt elution environment is required. Alternatively, HIC may be performed using a stationary phase comprising octyl ligands. As used herein the term “octyl ligand” refers to the presence of an octyl group (i.e., CaHis). In some embodiments, the stationary phase further comprises agarose. In some embodiments, the stationary phase comprises agarose coupled to octyl ligands. For example, the stationary phase may be Octyl Sepharose (Cytiva).

[0300] In some embodiments, the stationary phase is a particle or resin, preferably packed into a column. Thus, in some embodiments, the HIC is performed as column chromatography.

[0301] Alternative buffers suitable for use in the one or more HIC steps of the method are well known in the art. Appropriate buffers have a pH of from about pH 6 to about pH 9, preferably about pH 7 to about pH 8. In some embodiments, the buffer is a HEPES / ammonium sulphate buffer with a pH of about pH 7 to about pH 8, for example about pH 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, 7.8-8.0, preferably about pH 7.6.

[0302] Output composition of HIC

[0303] As used in the context of HIC, the output composition is the composition that is eluted from the HIC stationary phase. The output composition comprises the fusion protein and has a reduced level of the pyrogen, typically a pyrogen, compared to the input composition.

[0304] The HIC step generally reduces pyrogens. Particular examples of pyrogens are NEPs. The method may result in a reduction in the absolute amount of NEPs or a reduction in the NEPs as a proportion of any pyrogens that remain after the HIC step. Alternatively the method can be described as a method for removing NEPs.

[0305] In some embodiments, the reduced level of pyrogens in the output composition means that monocytes exposed to the output composition secrete a reduced level of at least one cytokine (i.e., at least one type of cytokine), for example as measured by a MAT assay. In some embodiments the at least one cytokine is selected from: a pro-inflammatory cytokine (e.g., TNFa, IL-1 , IL-6), an antiinflammatory cytokine (e.g., IL-4, 11-10, IL-13, IL-13, IL-1 ra, TGFp), a Th1 cytokine (e.g., IL-2, IFNy, IL- 12), a Th2 cytokine (e.g., IL-4, IL-5, IL-6, IL-10, IL-13), IL-1 p, PGE2, and a combination thereof. In preferred embodiments, the at least one cytokine comprises IL-6, IL-lp, TNF-a and / or IFN-y. In more preferred embodiments, the at least one cytokine comprises IL-6.

[0306] In some embodiments, the removal of pyrogens is as assessed by measuring the ability of pyrogens in the output composition to induce monocytes to secrete at least one cytokine compared to the pyrogens in the input composition, for example in a MAT assay. In some embodiments, the MAT assay comprises the following steps: a) incubating monocytes with a composition, optionally for 20 to 24 hours; and b) detecting and quantifying the amount of at least one cytokine released from the monocytes, optionally using an ELISA, further optionally wherein the cytokine is IL-6. In some embodiments, the source of the monocytes is isolated primary monocytes (e.g., PBMCs) or monocytic cell lines, optionally a MM6 monocytic cell line. In some embodiments, the composition that is incubated with the monocytes is the output composition or is the output composition that has been processed before performing a MAT assay, for example by filtration, concentration and / or buffer exchange, preferably buffer exchange into a PBS buffer.

[0307] In some embodiments, the pyrogen in the output composition is reduced by at least 5-fold, at least 10- fold, or at least 20-fold, compared to the input composition, optionally as measured by a MAT assay.

[0308] In some embodiments, the removal of pyrogens is as assessed by measuring the ability of pyrogens in the output composition to induce a coagulation reaction (as measured by a chromogenic reaction) in a LAL assay. In some embodiments, the LAL assay comprises the following steps: a) incubating the LAL reagent with a composition; and b) detecting and quantifying a chromogenic reaction, optionally using a spectrophotometer or an absorbance microplate reader; and / or

[0309] In some embodiments, a series of endotoxins dilutions with known EU are also incubated with the LAL reagent to generate a standard curve for quantifying the EU in the output and / or input composition. In some embodiments, the ability of pyrogens in the output composition to induce a coagulation reaction (as measured by the chromogenic reaction) is compared to that of pyrogens in the input composition. In some embodiments, the composition that is incubated with the monocytes is the output composition or is the output composition that has been processed before performing a LAL assay, for example by filtration, concentration and / or buffer exchange, preferably buffer exchange into a PBS buffer.

[0310] In some embodiments, the output composition comprises less than 0.1 EU / mg, less than 0.08 EU / mg, less than 0.06 EU / mg, less than 0.05 EU / mg or less than 0.04 EU / mg. Preferably, the output composition comprises less than 0.05 EU / mg of endotoxins, further preferably 0.04 EU / mg, even more preferably less than 0.033 EU / mg of endotoxins.

[0311] An exemplary output composition (or final drug product, which has for example been subjected to further steps downstream of the HIC method as described herein) has a maximum endotoxin level of 0.04 EU / mg. Where the fusion protein is present in this output composition or drug product at, e.g., a concentration of 50 mg / mL, the drug product will have no more than 2 EU / mL. Typically, the maximum volume injected into the eye is 0.1 mL. Thus, in this example, the maximum endotoxin value is 0.2 EU / eye.

[0312] In some embodiments, the yield of the HIC step is at least 70% or at least 75% (i.e., the total amount of fusion protein in the output composition comprises at least 70% or at least 75% of the total amount of fusion protein in the input composition).

[0313] In some embodiments, at least 85% or at least 90% of the total protein in the output composition is the fusion protein, as measured by any one of RP-HPLC, SE-HPLC, IEX-HPLC and SDS-PAGE. Post-HIC steps

[0314] After the HIC step, the output composition may be further processed to produce a pharmaceutical formulation. In some embodiments, the output composition is filtered. In preferred embodiments, the output composition is filtered by ultrafiltration and / or diafiltration. In preferred embodiments, the output composition is filtered by ultrafiltration and / or diafiltration are performed by tangential flow filtration (TFF). In some embodiments, the output composition is 0.2 pm sterile filtered. In some embodiments, the output composition is formulated, preferably into a pharmaceutical composition, for example by adding one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition is filled into sterile containers (e.g., vials, bottles, syringes). In some embodiments, the pharmaceutical composition is apportioned into a single dose, i.e., a unit dose. The pharmaceutical composition may be a final drug product.

[0315] Compositions produced by the methods and uses thereof

[0316] The invention provides compositions, including pharmaceutical compositions, produced by any of the methods described herein. In some embodiments, the compositions are suitable for direct administration into a subject, in particular, a human subject. The compositions may be suitable for administration by any route, for example intravenous, subcutaneous, or intraocular administration, preferably intravitreal administration. The compositions may be suitable for treating a disease or disorder, for example paroxysmal nocturnal hemoglobinuria (PNH), atypical haemolytic uremic syndrome (aHUS), neuromyelitis optica (NMOSD), myasthenia gravis (MG). The compositions may be particularly suitable for treating certain conditions of the eye, such as retinal diseases, in particular dry age-related macular degeneration (dry-AMD) and geographic atrophy (GA).

[0317] Pharmaceutical compositions

[0318] The invention provides compositions comprising fusion proteins of the invention. In some embodiments, the compositions are pharmaceutical compositions, for example comprising a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier”, in general will be a liquid but may include other agents provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Pharmaceutically acceptable carriers may e.g., contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. The pharmaceutical carrier employed will thus vary depending on the route of administration. A thorough discussion of pharmaceutically acceptable carriers is available in

[0030] , In a preferred embodiment the fusion protein is administered in an optically acceptable composition which may be a liquid, e.g., in a solution in water or PBS. In particular embodiments, formulation of pharmaceutically acceptable carrier solutions is well-known to those of skill in the art. It would be understood by the skilled artisan that particular embodiments contemplated herein may comprise other formulations, such as those that are well known in the pharmaceutical art.

[0319] In preferred embodiments, the pharmaceutical composition is for direct administration to an eye. The pharmaceutical composition may be for intravitreal, suprachoroidal, and / or subretinal administration, preferably intravitreal administration. In alternative embodiments, the pharmaceutical composition is for systemic delivery, such as by subcutaneous administration. The pharmaceutical composition may be for injection, e.g., via a hypodermic needle or microneedle. Preferably, the pharmaceutical compositions are for administration to the human eye.

[0320] Typically, pharmaceutical compositions are isotonic and / or sterile. In some embodiments, the fusion protein is administered in an optically acceptable composition which is an isotonic, sterile composition in PBS. In other embodiments, the fusion protein is administered as a pharmaceutical composition comprising suitable excipients. Common excipients include diluents (e.g., saline), counter ions (e.g., sodium sulfate), organic polymers (e.g., polyethylene glycol 400 (PEG400), PEG 3350 (Carbowax™400 or 3350)), surfactants (e.g., macrogol (e.g., Solutol® HS 15 (macrogol (15)-hydroxystearate, polyethylene glycol (15)-hydroxystrearate, polyoxyethylated 12-hydroxystearic acid, Kolliphor® HS15), polysorbate 20, polysorbate 21 , polysorbate 80, Pluronic® F108 (Poloaxmer 338)), suspending agents (e.g., ethyl cellulose polymers, 48-49.5% ethoxy content), cyclodextrins (e.g., sulfobutyl ether 7 betacyclodextrin (SBE-p-CD), ionic strength modifier and stabilizers (e.g., D-mannitol), buffers (e.g. sodium citrate, sodium phosphate), viscosity adjustment (e.g., polyvinyl alcohol) and microsphere technology (e.g., Medisorb® (poly lactic-co-glycolic acid (PLGA))

[0031] , In some embodiments, pharmaceutical compositions may include any one or more of these excipients. Typically, the buffer strength used is as low as possible while sill achieving a suitable window for long-term storage and maintaining the desired pH

[0032] ,

[0321] In some embodiments, the pharmaceutical composition is in a unit dose. As used herein a ‘unit dose’ (also referred to as a ‘dosage form’) means a pharmaceutical composition apportioned into a single dose. Preferably, the unit dose has a volume suitable for direct administration to the eye. Typically, a unit dose for intravitreal administration comprises up to 0.1 mL, for example from 0.05 mL to 0.1 mL. A unit dose may comprise a therapeutically or prophylactically effective amount as described herein. Alternatively, it may be necessary to administer a therapeutically or prophylactically effective amount as described herein as multiple (e.g., two) separate injections. Therefore, a unit dose may comprise a portion, e.g., half, of a therapeutically or prophylactically effective amount.

[0322] The invention provides pharmaceutical compositions produced by any of methods described herein for use in a method of treatment. The invention provides methods of treatment comprising administering to a subject a pharmaceutical composition produced by any of methods described herein. The invention provides pharmaceutical compositions produced by any of methods described herein for use in the manufacture of a medicament. The invention provides pharmaceutical compositions produced by any of methods described herein for use in a method of treating a retinal disease, for example dry-AMD, preferably GA. The invention provides methods of treating a retinal disease, for example dry-AMD, preferably GA, the method comprising administering to a subject a pharmaceutical composition produced by any of methods described herein. The invention provides pharmaceutical compositions produced by any of methods described herein for use in the manufacture of a medicament for the treatment of a retinal disease, for example dry-AMD, preferably GA.

[0323] The pharmaceutical compositions may be delivered (i.e., administered) by any known route of administration, for example locally or systemically. For example, the pharmaceutical compositions may be administered directly to the eye (e.g., intravitreally), topically to the surface of the eye, or systemically (e.g., subcutaneously). They are preferably administered directly into the eye (e.g., direct administration within the boundary of the eye as defined by the sclera), more preferably intravitreally. Intravitreal administration is well known in the art, e.g.,

[0033] ,

[0324] Ammonium sulphate precipitation

[0325] Salt-induced precipitation (also known as salting out) is a useful technique as an initial step in protein purification to concentrate the target protein and to remove impurities from a crude mixture comprising a protein, for example host cell proteins (HCPs) and other cell culture components. Such salts that are known in the art include ammonium sulphate, sodium chloride and sodium citrate. After precipitation, the protein is re-solubilised in buffer prior to performing further purification steps.

[0326] Ammonium sulphate precipitation is routinely used in the art for small-scale purification of proteins. However, it is not typically used for large-scale (e.g., commercial scale) purification, for several reasons. For example a major drawback of is that purity after ammonium sulphate precipitation is often poor because many HCPs precipitate out along with the protein of interest. There are also limits on the disposal of nitrogen containing compositions, such as ammonium sulphate, into sewage due to potential impact on the environment. Thus, when used at large scale, procedures for correctly handling the waste need to be setup. In addition, ammonium sulphate is expensive, due to the costly nature of the raw materials (i.e., ammonia and sulphur).

[0327] As shown in Example 2, the inventors found that ammonium sulphate precipitation is particularly suitable for purification of the fusion proteins described herein, and results in surprisingly high yields and levels of purity. Without being bound by theory, it is hypothesised that this is due to the distinctive biophysical properties of the fusion proteins described herein. In particular, PA(S) polypeptides have a strongly hydrophilic nature, despite having a lack of charged side chains which makes them especially susceptible to precipitation.

[0328] Additionally, PA(S) fusion proteins can be precipitated at low concentrations of ammonium sulphate, compared to HCPs and other impurities, e.g., endotoxins and viral particles. Thus, the use of a low concentration of ammonium sulphate ensures that the PA(S) fusion protein is precipitated more readily than other proteins / impurities, resulting in a high yield and high purity in a single purification step. This is advantageous because fewer downstream purification steps are needed to achieve the required purity.

[0329] Furthermore, ammonium sulphate precipitation can be applied to large volumes of cell culture medium or cell culture supernatant suitable for commercial production of the fusion protein (e.g., 750 L). This provides an advantage over other methods of purification, e.g., chromatography columns, which have a limited capacity. Additionally, the resulting precipitate can be re-solubilised in a significantly smaller volume than the starting volume, thus concentrating the fusion protein and avoiding the need for additional concentration steps. Provision of more concentrated compositions is beneficial for downstream purification steps as well as the final drug product.

[0330] Details of the ammonium sulphate precipitation method

[0331] The invention provides a method for removing host cell proteins (HCPs) from a composition comprising a fusion protein. The method comprises: providing an input composition (for the ammonium sulphate precipitation step) comprising at least 30 L of cell culture supernatant medium or cell culture supernatant comprising the fusion protein and a HCP; performing ammonium sulphate precipitation to precipitate the fusion protein from the cell culture medium or cell culture supernatant; and resolubilizing the fusion protein to provide an output composition. In some embodiments, only one ammonium sulphate precipitation step is performed. In other embodiments two or more ammonium sulphate precipitation steps are performed, optionally sequentially. When the method comprises two or more ammonium sulphate precipitation steps, said steps may be performed using the same concentration of ammonium sulphate or different concentrations of ammonium sulphate.

[0332] The method for removing host cell proteins (HCPs) from a composition comprising a fusion protein may additionally remove least one of the following: host cell debris, host cell DNA, media components, pyrogens, and viruses.

[0333] As used herein, the term “cell culture medium” encompasses the liquid portion collected from a cell culture vessel in which host cells expressing the fusion protein have been cultured (e.g. for at least 2, 4, 8, 12, 18, 24 hours or at least 2, 3, 4, 5, 6, 7 days) and comprises the fusion protein and impurities, such as host cell proteins, host cell debris, host cell DNA, media components, pyrogens, and viruses. The cell culture medium may or may not comprise the host cells. As used herein, the term “cell-culture supernatant” refers to the cell culture medium in which the host cells have been substantially removed (e.g., by centrifugation).

[0334] As used in the context of ammonium sulphate precipitation, the input composition is a composition that comprises a fusion protein as described herein and a host cell protein. The ammonium sulphate may be added directly to the input composition (e.g., the cell culture medium or cell culture supernatant). In some embodiments, the cell culture medium may be processed (e.g., centrifuged, diluted and / or filtered) before the ammonium sulphate is added. Therefore, in preferred embodiments, ammonium sulphate precipitation is performed on a cell culture supernatant. As used in the context of ammonium sulphate precipitation, the output composition is the re-solubilised composition that results from performing ammonium sulphate precipitation on the input composition.

[0335] In some embodiments, the fusion protein is precipitated by addition of ammonium sulphate to a final concentration of from about 0.7 M to about 1.3 M, from about 0.8 M to about 1.2 M, or between 0.9 M and 1.1 M. In some embodiments, the fusion protein is precipitated by addition of ammonium sulphate to a final concentration of from about 0.7 M to about 1 .3 M, from about 0.7 M to about 1 .2 M, from about 0.7 M to about 1.1 M, or from about 0.7 M to about 1 .0 M. In more preferred embodiments, the fusion protein is precipitated by addition of ammonium sulphate to final concentration of at about 1.0 M (25% saturated).

[0336] In some embodiments, a step of ammonium sulphate precipitation is performed using a stock concentration of ammonium sulphate that is between about 1 M and about 4 M, for example between about 3 M and about 4 M. In some embodiments, the stock concentration of ammonium sulphate is at least 2 M, at least 3 M, or about 4 M. In preferred embodiments, a step of ammonium sulphate precipitation is performed using an about 4 M stock concentration of ammonium sulphate.

[0337] In some embodiments, the fusion protein is precipitated by addition of ammonium sulphate to between 17.5% to 32.5% saturation, between 20% and 30% saturation, or between 22.5% and 27.5% saturation. In some embodiments, the fusion protein is precipitated by addition of ammonium sulphate to a final concentration of from about 17.5% to about 32.5% saturation, from about 17.5% to about 30% saturation, from about 17.5% to about 27.5% saturation, or from about 17.5% to about 25% saturation. In preferred embodiments, the fusion protein is precipitated by addition of ammonium sulphate to final concentration of at least 17.5% saturation. In more preferred embodiments, the fusion protein is precipitated by addition of ammonium sulphate to final concentration of at least 25% saturation.

[0338] In some embodiments, a step of ammonium sulphate precipitation is performed using a stock concentration of ammonium sulphate that is between about 25% and about 100% saturated, for example between about 75% and about 100% saturated. In some embodiments, the stock concentration of ammonium sulphate is at least 50% saturated, at least 75% saturated, or about 100% saturated. In preferred embodiments, a step of ammonium sulphate precipitation is performed using an about 100% saturated concentration of ammonium sulphate.

[0339] The concentration of an ammonium sulphate achieving 100% saturation is temperature dependent. Lower temperatures will decrease the concentration at which the solution is saturated. A saturated solution of ammonium sulphate is 4.1 M at 25 °C, but is 3.8 M at 0 °C. In some embodiments, ammonium precipitation is performed at a temperature of from about 15°C to about 25°C. In some embodiments, ammonium sulphate precipitate is performed at a temperature of about 20°C. In some embodiments, the input composition (for the ammonium sulphate precipitation step) comprises at least about 30 L, at least 50 L, at least about 100 L, at least about 200 L, at least about 400 L, or at least about 600 L of cell culture medium or cell culture supernatant. In some embodiments, the input composition comprises from about 400 L to about 1000 L, from about 600 L to about 800 L, or from about 650 L to about 750 L of cell culture medium or cell culture supernatant. In some embodiments, the input composition comprises at least about 1000 L, at least about 1200 L or at least about 1400 L of cell culture medium or cell culture supernatant. In some embodiments, the input composition comprises at least 1500 L, at least 2000 L, at least 3000 L, at least 4000 L, or at least 5000 L of cell culture medium or cell culture supernatant. In some embodiments, the input composition comprises at least 10,000 L, at least 20,000 L, at least 30,000 L, at least 40,000 L, or at least 50,000 L of cell culture medium or cell culture supernatant. In preferred embodiments, the input composition comprises from about 650 L to about 750 L of cell culture medium or cell culture supernatant.

[0340] Performing the ammonium sulphate precipitation provides a precipitate comprising the fusion protein. In some embodiments, one or more disc-stack centrifugation (DSC) steps are used to separate the precipitate from the solution. DSC uses the force of gravity to separate liquid with a specific density from other liquids and solids. Disc-stack centrifuges (also known as disc-stack separators) contain discs that are stacked on top of each other with clearances as small as 0.3 mm. the discs rotate with the bowl, separating the liquid into thin layers. DSC can be performed, e.g., on a cell culture medium and results in separation of the target protein from other soluble impurities. In some embodiments, only one DSC step is performed during ammonium sulphate precipitation. In other embodiments two or more subsequent DSC steps are performed during ammonium sulphate precipitation. When the method comprises two or more DSC steps, said steps may be performed using the same type of discstack centrifuge or different disc-stack centrifuges. In preferred embodiments, one or more DSC steps are performed during ammonium sulphate precipitation.

[0341] After ammonium sulphate precipitation, the output composition typically still comprises at least one impurity, in particular pyrogens. However, as explained above, in the invention the purity is surprisingly good. In some embodiments, the yield of the ammonium sulphate step is at least 70%, at least 75% or at least 80% (i.e., the total amount of fusion protein in the output composition comprises at least 70%, at least 75% or at least 80% of the total amount of fusion protein in the input composition).

[0342] In some embodiments, at least 45% or at least 50% of the total protein in the output composition is the fusion protein, as measured by any one of RP-HPLC, SE-HPLC, IEX-HPLC and SDS-PAGE. Other methods

[0343] In one aspect, the invention provides methods for removing pyrogens using HIC. In another aspect, the invention provides methods for removing HCPs and other impurities using ammonium sulphate precipitation. In a further aspect, both of the methods are performed, i.e., ammonium sulphate precipitation is performed before HIC. In a further aspect, HIC and ammonium sulphate precipitation are performed along with addition method steps to aid the removal of impurities (e.g., pyrogens and HCPs) from a composition comprising a fusion protein described herein. Accordingly references herein to “the method” or “the methods” should be interpreted as reference to methods comprising HIC, methods comprising ammonium sulphate precipitation, methods comprising HIC and ammonium sulphate precipitation or methods comprising HIC, ammonium sulphate precipitation and additional methods which are described below.

[0344] Methods of the invention may optionally comprise one or more additional steps before and / or after the ammonium sulphate precipitation and / or HIC steps detailed above. In some embodiments, the method comprises one or more, or all, of the following steps, optionally in the following order: a) Expression of the fusion protein in host cells, b) Harvesting the cell culture medium, c) Ammonium sulphate precipitation, d) A first anion exchange chromatography, e) Membrane absorption, f) A second anion exchange chromatography, and g) HIC.

[0345] Additional processing steps, such as dilution, concentration, filtration and / or buffer exchange can be performed at various stages of the method, details of which are provided below.

[0346] Details of steps c) and g) are described above. Details of all other steps are provided below. a) Expression of fusion proteins

[0347] Suitable host cells for recombinant expression include commonly used prokaryotic species or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities. Mammalian cell lines grown in vitro are also suitable, particularly when using virus-driven expression systems. Another suitable expression system is the baculovirus expression system that involves the use of insect cells as hosts. An expression system may also constitute host cells that have the DNA incorporated into their genome. Proteins, or protein fragments may also be expressed in vivo, for example in insect larvae or in mammalian tissues.

[0348] In preferred embodiments, the host cells secrete the fusion protein, for example into cell culture medium. Where the host cells are prokaryotic, they are typically E. coli cells, preferably E. coli cells having mutations in the thioredoxin reductase (trxB) and / or glutathione reductase (gor) genes such that cytoplasmic disulfide bond formation is reduced or prevented (e.g., E. coli Origami B cells). A further example of suitable E. coli cells is ESETEC® or ESETEC® 2.0 (modified E. coli K12 strains produced by WACKER)

[0034] , ESETEC® enable the controlled secretion of correctly folded recombinant proteins into the fermentation broth. Other preferred cell types are C. glutamicum cells.

[0349] A variety of techniques may be used to introduce the vectors according to the present invention into prokaryotic or eukaryotic cells. Suitable transformation or transfection techniques are well known in the art. In eukaryotic cells, expression systems may either be transient (e. g. episomal) or permanent (chromosomal integration) according to the needs of the system. Thus, the invention provides transformed or transfected host cells comprising a polynucleotide (e.g., a vector) as defined herein. The host cells expressing the fusion protein may have been cultured (e.g., for at least 2, 4, 8, 12, 18, 24, or at least 48 hours or at least 2, 3, 4, 5, 6, 7 days). The host cells may be cultured in cell culture media, optionally with a cell culture feed (e.g., glucose) and / or a cell culture serum. The host cells may be cultured in a bioreactor or fermenter, for example a 1500 L fermenter. b) Harvesting target protein

[0350] Typically, fusion proteins described herein are secreted from the host cells into the cell culture medium. The cell culture medium can then be harvested (i.e., collected) and the fusion proteins can be extracted from the other components, e.g., cell culture media and HCPs.

[0351] In some embodiments, the method comprises harvesting a cell culture medium from host cells.

[0352] In preferred embodiments, the method comprises harvesting a cell culture medium from the host cells described in step a). Optionally, the solution collected from the cell culture (e.g., the solution from a cell culture vessel) may be processed (e.g., centrifuged, diluted and / or filtered) before or after harvesting the cell culture medium. Thus, in some embodiments, a cell culture supernatant is harvested.

[0353] In some embodiments, the method comprises harvesting a cell culture medium or cell culture supernatant comprising a fusion protein described herein. In some embodiments, the method comprise harvesting said medium or cell culture supernatant using centrifugation, for example disc stack centrifugation, using the methods described below.

[0354] In some embodiments, the method comprises harvesting a composition comprising a fusion protein described herein and an impurity (e.g., a pyrogen and / or a HCP) using one or more centrifugation steps, for example a DSC. In some embodiments, the method comprises performing only one centrifugation step, for example a DSC harvesting said composition. In other embodiments, the method comprises performing two or more subsequent centrifugation steps, for example two or more DSC steps, when harvesting said composition. When the method comprises two or more DSC steps, said steps may be performed using the same type of disc-stack centrifuge or different disc-stack centrifuges. In preferred embodiments, a composition comprising a fusion protein described herein and a host cell protein is harvested using DSC. c) Ammonium sulphate precipitation

[0355] Ammonium sulphate precipitation can be performed according to the methods described in the above section (see Ammonium sulphate precipitation). In some embodiments, the method comprises performing an ammonium sulphate precipitation after harvesting a cell culture medium. In preferred embodiments, the method comprises performing ammonium sulphate precipitation after harvesting a cell culture medium as described in step b). Optionally, the harvested cell culture medium may be processed (e.g., centrifuged, diluted, filtered and / or buffer exchange) before ammonium sulphate precipitation is performed. t d) First anion exchange chromatography

[0356] Anion exchange chromatography is a form of ion exchange chromatography which is used to separate molecules based on their sent surface charge. Specifically, anion exchange chromatography uses a positively charged resin with affinity for molecules having net negative surface charges.

[0357] In some embodiments of any aspects of the invention, the method comprises only one anion exchange chromatography step. In other embodiments, the method comprises two or more anion- exchange chromatography steps, for example two, three, four or more anion-chromatography steps. When the method comprises more than one anion-exchange chromatography steps, said steps may be performed using the same type of chromatography resin or on different chromatography resins.

[0358] In preferred embodiments, a first anion exchange chromatography step is performed using a Nuvia-Q column. In some embodiments a first anion exchange chromatography step is performed using a Nuvia-Q column and a second anion exchange chromatography step is performed using a Q Sepharose column. Other suitable anion exchange columns are known in the art and include, but are not limited to, UNOsphere Q (Bio-Rad), POROS XQ, PORS HQ, POROS IC50 and POROS D50 (Thermo Fisher), Q-HP, Capto Q ImpRes (Cytiva), Toyopearl NH2-750F (Tosoh Bioscience).

[0359] Typically, anion exchange chromatography comprising the following steps: equilibrating the anion exchange column with a buffer ; loading the composition onto the anion exchange stationary phase; washing the anion exchange resin with the buffer; and eluting the target protein (i.e., the fusion protein) from the anion exchange stationary phase using a salt gradient.

[0360] Typically, the buffer used for equilibrating the anion exchange column is a standard buffer, defined by the manufacturer’s specifications. Typically, the composition comprising the target protein (i.e., fusion protein) is loaded onto the column in a loading buffer, the loading buffer is selected so that at least a proportion of the target fusion protein is retained on the anion exchange column. The wash buffer may be selected so that at least a proportion of an impurity (e.g. a HCP) is separated from the target fusion protein. The wash buffer is typically the same as the equilibrium buffer. In some embodiments, the column is washed in two steps. In other embodiments, the column is washed in three steps. The salt gradient may be continuous or step-wise. The collected fractions may have a reduced level of the impurity. Fractions may be stored e.g. at 5 ± 4 °C until further processing.

[0361] Suitable buffers for use in the anion exchange chromatography steps of the method are well known in the art and include, but are not limited to, Tris, Tris / sodium chloride, histidine, Bis-tris, Bis-tris propane, diethanolamine, ethanolamine, imidazole, L-Histidine, morpholine, N-ethylmorpholine, N- Methyl-diethanolamine, 2-amino-2-methyl-1 ,3-propanediol (AMPD), TEA, piperazine, piperidine, pyridine, 1 ,3-Diaminopropane, 2-amino-2-methyl-1 -propanol (AMP). The pH of the buffer used for loading and washing is selected so that the target protein is retained on the anion exchange chromatography resin. Appropriate buffers have a pH of from about pH 4 to about pH 12.

[0362] In some embodiments, the buffer is a histidine buffer with a pH of about 5.0-5.2, 5.2-5.4, 5.4-5.6, 5.6- 5.8, 5.8-6.0, preferably about pH 5.5. In some embodiments, the buffer is a Tris and / or Tris / sodium chloride buffer with a pH of about pH 8 to about pH 9, for example about pH 8.0-8.2, 8.2-8.4, 8.4-8.6, 8.6-8.8, 8.8-9.0, preferably about pH 8.0. In some embodiments, the buffer is a Tris and / or Tris / sodium chloride buffer with a pH of about pH 8 to about pH 9, for example about pH 8.0-8.2, 8.2- 8.4, 8.4-8.6, 8.6-8.8, 8.8-9.0, preferably about pH 8.8.

[0363] A first step of anion exchange chromatography can be performed to remove residual impurities, e.g., host cell proteins. Typically, before a first anion exchange chromatography step is performed, the composition to be purified is ultrafiltered in an appropriate buffer to facilitate the first chromatography step.

[0364] In some embodiments, the method comprises performing a first anion exchange chromatography after performing ammonium sulphate precipitation. In preferred embodiments, the method comprises performing a first anion exchange chromatography after ammonium sulphate precipitation as described in step c). Optionally, the composition produced by ammonium sulphate precipitation may be processed (e.g., diluted, filtered and / or buffer exchange) before performing a first anion exchange chromatography. In preferred embodiments, the first anion chromatography step comprises: a) equilibrating the anion exchange column; b) loading the composition onto the anion exchange stationary phase; c) washing the anion exchange resin; and d) eluting the fusion protein from the anion exchange stationary phase using a salt gradient.

[0365] In preferred embodiments, the composition is ultrafiltered into an appropriate loading buffer before being loaded onto the column. In preferred embodiments, the method comprises performing a first anion exchange chromatography step using a Nuvia-Q resin. In preferred embodiments, the method comprises equilibrating the Nuvia-Q column with a sodium chloride buffer (e.g. 2M sodium chloride, pH 8.8) and / or a Tris buffer (20 mM Tris, pH 8.8). In preferred embodiments, the method comprises washing the Nuvia-Q column with a wash buffer, for example in two or more steps, preferably with a Nuvia Q Buffer A (e.g. 20 mM Tris pH 8.8) and a Nuvia Q Buffer B (e.g. 20 mM Tris, 200 mM sodium chloride, pH 8.8). In preferred embodiments, the method comprises eluting the fusion protein from the stationary using a salt gradient.

[0366] After a first step of anion exchange chromatography, there is a reduction in residual impurities, e.g., host cell proteins. However, the composition may still contain an unacceptable level of residual impurities for ophthalmic use. In some embodiments, after a first anion exchange chromatography step, only fractions in which at least 50%, at least 55%, at least 60% or at least 65% of the total protein in the fraction is the fusion protein, as measured by RP-HPLC, are pooled. In preferred embodiments, after a first anion exchange chromatography step, only fractions in which at least 65% of the total protein in the fraction is the fusion protein, as measured by RP-HPLC, are pooled. e) Membrane adsorption

[0367] Membrane adsorbers for ion exchange chromatography are ready-to-use units in a syringe filter format. They comprise anion or cation exchange ligands covalently bound to the entire internal surface of the pores. This results in a chromatography matrix with superior flow rates and high binding capacity. An exemplary membrane absorber is the Sartobind® Q capsule (Satorius).

[0368] An intermediate step of membrane adsorption can be performed to remove further impurities, e.g., endotoxins and host cell DNA. Typically, a step of membrane adsorption is performed before or after anion exchange chromatography, for example as an intermediate step after a first step of anion exchange and before a second step of anion exchange chromatography.

[0369] In some embodiments, the method comprises only one membrane absorption step. In other embodiments, the method comprises two or more membrane absorption steps, for example two, three, four or more membrane absorption steps. When the method comprises more than one membrane absorption step, said steps may be performed using the same type of membrane absorber or different types of membrane absorbers.

[0370] In some embodiments, the method comprises performing a step of membrane adsorption after performing a first anion exchange chromatography step and / or before performing a second anion exchange chromatography step. In preferred embodiments, the method comprises performing a step of membrane adsorption after performing a first anion exchange chromatography step as described in step d) and / or before performing a second anion exchange chromatography step as described in step f). Optionally, the composition may be processed (e.g., diluted, filtered and / or buffer exchange) before and / or after performing membrane absorption.

[0371] In preferred embodiments, one or more steps of membrane absorption are performed using a Sartobind® Q capsule (Sartorius). f) Second anion exchange chromatography

[0372] A second step of anion exchange chromatography can be performed to remove further impurities, e.g., nomacopan truncation products and endotoxins. Typically, before a second anion exchange chromatography step is performed, the composition to be purified is ultrafiltered in an appropriate buffer to facilitate the first chromatography step.

[0373] In some embodiments, the method comprises performing a second anion exchange chromatography after performing a step of membrane adsorption. In preferred embodiments, the method comprises performing a second anion exchange chromatography after a step of membrane absorption as described in step e). Optionally, the composition may be processed (e.g., diluted, filtered and / or buffer exchange) before and / or after performing the second anion exchange chromatography. In preferred embodiment, the second anion chromatography step comprises: a) equilibrating the anion exchange column; b) loading the composition onto the anion exchange stationary phase; c) washing the anion exchange resin; and d) eluting the fusion protein from the anion exchange stationary phase using a salt gradient.

[0374] In preferred embodiments, the composition is ultrafiltered into an appropriate loading buffer before being loaded onto the column. In preferred embodiments, the method comprises performing a second anion exchange chromatography step using a Q Sepharose HP column. In preferred embodiments, the method comprises equilibrating the Q Sepharose HP column with a sodium chloride buffer (e.g. 2M sodium chloride, pH 8.0) and / or a Tris buffer (e.g. 20 mM Tris, pH 8.0). In preferred embodiments, the method comprises washing the Q Sepharose HP column with a wash buffer, for example in two or more steps, preferably with a Q Sepharose HP Buffer A (e.g. 20 mM Tris pH 8.0) and a Q Sepharose HP Buffer B (e.g. 20 mM Tris, 400 mM sodium chloride, pH 8.0). In preferred embodiments, the method comprises eluting the fusion protein from the stationary using a salt gradient.

[0375] In some embodiments, after a second anion exchange chromatography step, only fractions in which at least 70%, at least 75%, at least 80% or at least 85% of the total protein in the fraction is the fusion protein, as measured by RP-HPLC, and fractions in which at least 75%, at least 80%, at least 85%, at least 90% or least 90% of the total protein in the fraction is the fusion protein, as measured by SE- HPLC, are pooled. In preferred embodiments, after a second anion exchange chromatography step, only fractions in which at least 85% of the total protein in the fraction is the fusion protein, as measured by RP-HPLC, and fractions in which at least 90% of the total protein in the fraction is the fusion protein, as measured by SE-HPLC, are pooled. In preferred embodiments, pooled composition is split into two fractions before loading onto a HIC column. g) Hie

[0376] HIC can be performed according to the methods described in the above section (see Hydrophobic interaction chromatography). In some embodiments, the method comprises performing HIC after a second anion exchange chromatography. In preferred embodiments, the method comprises performing HIC after a second anion exchange chromatography as described in step f). Optionally, the composition may be processed (e.g., diluted, filtered and / or buffer exchange) before and / or after performing the HIC.

[0377] Additional filtration steps

[0378] Dead end / depth filtration and 0.2 urn filtration

[0379] Dead end filtration is used to remove particles and insoluble components by forcing a liquid composition through a membrane under pressure. Dead end filtration can be performed using depth filter capsules. Depth filter capsules can comprise a single- or a double-layer filter. A number of depth filter grades are available from 0.05 pm up to 10 pm. To minimize bioburden (e.g., cells, cell debris, insoluble HCPs, aggregates, or other genetic material), 0.2 pm filtration is typically performed at various stages of the method.

[0380] Depth filtration can be performed before or after one or more further purification steps, for example before or after ammonium sulphate precipitation. In particular, depth filtration is performed before one or more chromatography steps, for example anion exchange chromatography, HIC or tangential flow filtration (TFF). Loading unclarified samples onto a chromatography column may cause blockage and / or reduced performance. Thus, performing depth filtration before one or more chromatography steps improves performance of the chromatography.

[0381] Accordingly, in some embodiments, dead end filtration is performed using depth filter capsules. In some embodiments, the depth filter has a pore size of 0.2 pm to 6 pm. In some embodiments, the depth filter has a pore size of 0.2 pm. In some embodiments, the depth filter has a pore size of 3 pm. In some embodiments, the depth filter has a pore size of 4 pm. In some embodiments, the depth filter has a pore size of 6 pm. In some embodiments, a single-layer depth filter is used. In other embodiments, a double-layer depth filter is used.

[0382] In some embodiments, one or more depth filtration steps are performed after DSC. In some embodiments, only one depth filtration step is performed before or after DSC. In other embodiments two or more subsequent depth filtration steps are performed before or after DSC.

[0383] In some embodiments, one or more depth filtration steps are performed before ammonium sulphate precipitation. In some embodiments, one or more depth filtration steps are performed after ammonium sulphate precipitation. In some embodiments, only one depth filtration step is performed before or after ammonium sulphate precipitation. In other embodiments two or more subsequent depth filtration steps are performed before or after ammonium sulphate precipitation. In preferred embodiments, one or more depth filtration steps are performed after ammonium sulphate precipitation. In preferred embodiments, an additional step of 0.2 pm filtration is performed after depth filtration (and after ammonium sulphate precipitation).

[0384] In some embodiments, one or more depth filtration steps are performed before one or more anion exchange chromatography steps. In some embodiments, one or more depth filtration steps are performed after one or more anion exchange chromatography steps. In some embodiments, only one depth filtration step is performed before or after one or more anion exchange chromatography steps.

[0385] In other embodiments two or more subsequent depth filtration steps are performed before or after one or more anion exchange chromatography steps.

[0386] In some embodiments, one or more depth filtration steps are performed before one or more HIC steps. In some embodiments, one or more depth filtration steps are performed before or after one or more HIC steps. In some embodiments, only one depth filtration step is performed before or after one or more HIC steps. In other embodiments two or more subsequent depth filtration steps are performed before or after one or more HIC steps. In preferred embodiments, 0.2 pm filtration is performed after HIC.

[0387] When the method comprises two or more depth filtration steps, said steps may be performed using the same type of depth filter or different depth filters.

[0388] Ultrafiltration and diafiltration

[0389] Ultrafiltration and diafiltration can be performed at any stage of the method, for example where a buffer exchange is required. In preferred embodiments, ultrafiltration and diafiltration is performed: iii) after ammonium sulphate precipitation, and / or before a first step of anion exchange chromatography, ii) after a step of membrane absorption, and / or before a first second of anion exchange chromatography, and / or iii) after a step of HIC.

[0390] Typically, ultrafiltration and diafiltration is controlled using the transmembrane pressure (TMP) and the shear rate. Typically, tangential flow filtration (TFF, also known as “cross-filtration”) is commonly used in concentration and diafiltration processes, such as ultrafiltration and diafiltration._TFF is a polishing step which can be used to remove small-sized particle-related impurities through cycles of concentration and diafiltration though the pores of the filter. In contrast to other forms of filtration (where liquid is passed vertically through a filter membrane), liquid is passed parallel to the filter which allows for a consistent flow. The permeate is collected, while the retentive is gently cycled through. Typically, TFF is achieved using, as a filter, such as a hollow fibre filter. BRIEF DESCRIPTION OF FIGURES

[0391] Figure 1 A is a process flow chart for an exemplary HIC method performed according to the invention. In this example, the input composition had previously been subject to the steps shown in figure 3 up to and including the Q-HP Chromatography step. Thus, the input composition was the Q-HP chromatography pool. The hexyl conditioning buffer contained 0.0234 M HEPES, 0.0266 M HEPES- Na, and 1.1 M ammonium sulphate. The HIC solid phase was Toyopearl Hexyl 650C. Hexyl buffer A (0.00612 M HEPES, 0.01888 M HEPES-Na, and 0.55 M ammonium sulphate) and hexyl buffer B 0.0121 M HEPES, 0.0129 M HEPES-Na) were combined in different proportions (from 95% buffer A and 5% buffer B to 0% buffer A and 100% buffer B) thus creating a linear salt gradient, such that the fusion protein was eluted. The method was performed at 22°C.

[0392] Figure 1 B shows pyrogen detection in different PAS600-nomacopan samples using the Monocyte Activation Test (MAT). The samples were incubated with monocytes (human Mono-Mac 6 cell line) and, after 20 hours, the concentration of proinflammatory cytokines (IL-6) released was quantified using an enzyme-linked immunosorbent assay (ELISA). Sample 1 , Ph. Eur. Endotoxin reference standard at 2.5 EU / ml in endotoxin-free water; Sample 2, final protein preparation from the bioprocess without HIC at 48 mg / ml in PBS; Sample 3, final protein preparation from the bioprocess including the HIC step at 37 mg / ml in chromatography HEPES buffer; Sample 4, final protein preparation from the bioprocess including the HIC step at 36 mg / ml in PBS; Sample 5, PBS buffer as a control. Measurements were made in triplicate, error bars show the standard deviation. Bacterial endotoxin was determined with a chromogenic LAL testing platform using the Endosafe® nexgen-PTS test cartridges (Charles River, Wilmington, USA).

[0393] Figure 2 is a process flow chart for an exemplary ammonium sulphate precipitation method performed according to the invention. In this example, the input composition had previously been subject to the steps shown in figure 3 up to and including the harvest by centrifugation step. Thus, in this example, the input composition was undiluted cell culture supernatant which had been separated from cells by DSC. The stock buffer used was 4M ammonium sulphate and the target concentration was 1 M. A first precipitation was performed and the precipitate (solid phase) was separated from the liquid phase (including soluble impurities such as HCPs) by DSC. A second precipitation was performed by resolubilising the precipitate in 1 M ammonium sulphate. Again, the precipitate was separated from the liquid phase by DSC. Finally, the precipitate was resolubilized in 0.05M Tris buffer.

[0394] Figure 3 is a process flow chart for an exemplary method comprising an ammonium sulphate precipitation step according to the invention (“Primary Recovery”) and a HIC step according to the invention (“Hexyl-650 Chromatography”). Additional steps are also performed, resulting in the production of a final drug product suitable for administration to patients. EXAMPLES

[0395] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0396] Example 1

[0397] PA(S)-nomacopan fusion proteins are especially useful for treating diseases of the retina, in particular dry AMD and GA. The fusion proteins are typically delivered via intraocular administration, such as intravitreal administration, meaning that pharmaceutical compositions comprising the fusion proteins need to have low levels of pyrogens (endotoxins and NEPs) to avoid causing inflammation in the eye. When developing purification methods for these fusion proteins, one challenge the inventors encountered was identifying a purification technique that could efficiently reduce the level of pyrogens, without significantly impacting yield.

[0398] It is well-known that there is a ligand pocket at the centre of nomacopan (and thus of PA(S)- nomacopan fusion proteins) that is capable of binding to highly hydrophobic ligands, such as Cis fatty acids, e.g., the methyl ester of ricinoleic acid ((R)-12-hydroxy-cis-9-octadecenoic acid), tetraethyleneglycol diethylether, palmitic acid methyl ester, and / or stearic acid methyl ester

[0016] , The inventors hypothesised that the binding properties of this pocket could be exploited to purify PA(S)-nomacopan fusion proteins using HIC. The inventors searched for a HIC stationary phase comprising a ligand that structurally resembles the fatty acids naturally bound by nomacopan. The inventors identified a HIC stationary phase comprising hexyl (CeHn) ligands (Toyopearl Hexyl 650C, Tosoh Bioscience) as a candidate. Without wishing to be bound by any theory, the inventors hypothesized that the highly hydrophobic ligands of the HIC stationary phase may be able to compete with impurities, such as pyrogens, for binding to this pocket of nomacopan.

[0399] To test whether said HIC resin could successfully remove NEPs, the inventors generated compositions comprising PA(S)-nomacopan that were substantially free of impurities (e.g., 85% of the total protein in the input composition was the fusion protein, as measured by RP-HPLC, 90% of the total protein in the input composition was the fusion protein, as measured by SE-HPLC, 90% of the total protein in the input composition was the fusion protein, as measured by IEX-HPLC, and / or 90% of the total protein in the input composition was the fusion protein, as measured by SDS-PAGE). The compositions produced from earlier processing steps were split into two fractions. These compositions were used as the input composition for loading on the HIC resin.

[0400] The first fraction was conditioned in Hexyl Conditioning Buffer (HEPES / ammonium sulphate buffer, pH 7.6). A Toyopearl Hexyl 650C column (Tosoh Bioscience) is equilibrated with the Hexyl Equilibrium Buffer / Hexyl Buffer A (HEPES / ammonium sulphate buffer, pH 7.6). The conditioned input composition is then loaded onto the column. After loading, the column is washed with Hexyl Buffer A (HEPES / ammonium sulphate buffer, pH 7.6). Fusion proteins are then eluted using a salt gradient with Hexyl Buffer A (HEPES / ammonium sulphate buffer, pH 7.6) and Hexyl Buffer B (HEPES buffer, pH 7.6). The target protein elutes in the main peak and is collected in one main fraction. This fraction was collected as the output composition. The process was then repeated on the second fraction and the two (outcome composition) fractions were pooled. A flow-diagram of this process is shown in Figure 1A.

[0401] The inventors compared the levels of pyrogens in the input composition, i.e., the composition before HIC was applied, against the output composition, i.e., the composition after HIC was performed, using a LAL assay and a MAT assay.

[0402] The chromogenic LAL assay (Endosafe® nexgen-PTS test, Charles River) was performed on samples after each upstream processing step. These steps included a first anion exchange chromatography (Nuvia Q), a membrane absorption (Sartobind Q) and a second anion exchange chromatography (Q Sepharose). As shown in Table 1 , the output composition (bottom row of Table 1) had significantly lower levels of endotoxins compared to the input composition (penultimate row of Table 1).

[0403] Table 1 : LAL endotoxin testing

[0404] The MAT assay was performed using the PyroMAT® system (Merck Millipore), which measures IL-6 secretion from monocytes in response to a test composition. As shown in Fig 1 B, the HIC output composition induced significantly reduced IL-6 levels compared to the input composition, indicating a significant reduction in pyrogens.

[0405] In addition, high yields of fusion protein and high purity were achieved. For example, yields of more than 70% fusion protein were obtained after performing HIC, while the purity of the output composition was more than 85%, as measured by any one of RP-HPLC and SE-HPLC (i.e., more than 85% of the total protein in the output composition was the fusion protein).

[0406] Example 2

[0407] The inventors developed a method for large-scale purification of PA(S)-nomacopan fusion proteins from a cell culture supernatant using ammonium sulphate precipitation. Fusion proteins were first produced using E. coli ESETEC® 2.0 (Wacker). The cell culture medium was harvested using DSC and the fusion protein was precipitated by addition of ammonium sulphate to a final concentration of 0.7 M - 1 M. The resulting precipitate was separated from remaining soluble impurities by continuous centrifugation on the separator. The precipitated product was then re-solubilized by dilution in buffer. The particles and insoluble components contained in the resolubilized product were removed by dead end filtration using depth filter capsules. To minimize bioburden the dead-end filtrate was 0.2 pm filtered. A flow-diagram of this process is shown in Figure 2A.

[0408] The ammonium sulphate precipitation was found to achieve high yield, i.e., the total amount of fusion protein in the output composition was greater than 80% of the total amount of fusion protein in the input composition (see Table 2). This yield was reproduced across multiple batches.

[0409] Table 2: Yield of output composition from ammonium sulphate precipitation

[0410] ‘presumably due to analytical error lower yield by calculation

[0411] In addition, purity was assessed using SDS-PAGE. Ammonium sulphate precipitation was found to achieve remarkable purity of the fusion protein from the starting material (i.e., the proportion of total protein in the output composition after ammonium sulphate precipitation was significantly higher than in the input composition before ammonium sulphate precipitation was performed).

[0412] Example 3

[0413] The inventors developed an overall method for PAS-nomacopan drug substance (DS) manufacturing, which includes the HIC and ammonium sulphate methods described in Examples 1 & 2 respectively. This method is described below and depicted in Figure 3.

[0414] The DS manufacturing process starts with a pre-culture. A single vial from the PAS-nomacopan MCB is used to inoculate 2 x 100 mL of pre-culture media in 2 x 1000 mL shake flasks. The pre-culture is incubated until an optical density at 600 nm (OD600) of > 1.5 AU is reached. The pre-culture is then transferred into the production fermenter.

[0415] The production fermenter is filled with 600 L of batch media and inoculated with a calculated amount of pre-culture to reach the initial optical density of 0.0001 AU. The first phase of the process increases biomass only (growth phase) with pmax under non-limiting conditions. This growth phase continues until the glucose is consumed when a glucose feed is initiated. Four (4) h after feed start a phosphate buffer is added and a temperature shift is performed (30°C to 25°C). Four and three-quarter (4.75) h after feed start, the production phase is initiated by adding 0.125 mM IPTG, which induces the expression of the target protein. The production phase continues for 45 h after IPTG induction. At the end of fermentation, the culture broth is cooled (set point 12°C).

[0416] The supernatant is harvested using disc stack centrifugation and the soluble product is then precipitated from the culture supernatant by ammonium sulphate precipitation and the resulting precipitate is separated from remaining soluble impurities by continuous centrifugation on the separator. The precipitated product is then re-solubilized by dilution in buffer. The particles and insoluble components contained in the re-solubilized product are removed by dead end filtration using depth filter capsules. To minimize bioburden the dead-end filtrate is 0.2 pm filtered.

[0417] The filtrate is ultrafiltered to formulate the intermediate in an appropriate buffer system to facilitate the first chromatography step. This step is controlled using the transmembrane pressure (TMP) and the shear rate. The filtrate is initially concentrated by factor 4, then the retentate is diafiltrated by factor 3 using tris buffer (pH 8.8).

[0418] The diafiltrate is purified by anion exchange chromatography (AEC). The Nuvia Q column is equilibrated with sodium chloride and tris buffer (pH 8.8), then the diafiltrate is loaded onto the column. After loading, the column is washed in two steps with Tris and Tris / sodium chloride buffer (pH 8.8). Protein is eluted using a salt gradient with tris / sodium chloride and Tris buffer (pH 8.8). The target protein elutes in the main peak. Several fractions are collected. The fractions are stored at 5 ± 4 °C until further processing (< 20 h). Only fractions with a reverse phase high pressure liquid chromatography (rpHPLC) purity > 65 % are pooled (i.e., > 65 % of the total protein in the fraction composition is the fusion protein).

[0419] The Nuvia Q pool is further processed by the removal of remaining endotoxins and host cell DNA via a membrane absorber (Sartobind Q). The resulting filtrate is transferred to a low-salt buffer using tangential flow filtration (TFF) with 30 kDa membranes. The filtrate is initially concentrated by factor 3, then the retentate is diafiltrated by factor 3 using Tris buffer (pH 8.0). Afterwards the diafiltrate is 0.2 pm filtered and stored at 5 ± 4 °C until further processing (< 20 h).

[0420] The diafiltrate is loaded on an equilibrated Q Sepharose HP anion exchange column for the second chromatographic purification step. Equilibration is performed with sodium chloride and Tris buffer (pH 8.0). After loading, the column is washed in three steps with histidine buffer (pH 5.5), Tris and Tris / sodium chloride buffer (pH 8.0). Protein is eluted using a salt gradient with Tris buffer (pH 8.0) and Tris / sodium chloride buffer (pH 8.0). The target protein elutes in the main peak. Several fractions are collected. The fractions are stored at 5 ± 4 °C until further processing (< 69 h). Only fractions with a RP-HPLC purity > 85 % and a SE-HPLC purity > 90 % are used to generate the main pool (i.e., > 85 % or 90% of the total protein in the fraction composition is the fusion protein, as measured by RPHPLC or SE-HPLC, respectively). The pool is stored at 5 ± 4 °C until further processing (< 44 h). For further processing, the Q Sepharose HP pool is split into 2 fractions. Fraction 2 is stored at 5 ± 4 °C until further processing (~24 h). Fraction 1 is conditioned by diluting with HEPES / ammonium sulfate buffer (pH 7.6) in preparation for the third chromatographic hydrophobic interaction purification step (Toyopearl Hexyl 650C). The column is equilibrated with HEPES / ammonium sulfate buffer (pH 7.6), then the conditioned pool is loaded onto the column. After loading, the column is washed with HEPES / ammonium sulfate buffer (pH 7.6). Protein is eluted using a salt gradient with HEPES / ammonium sulfate buffer (pH 7.6) and HEPES buffer (pH 7.6). The target protein elutes in the main peak and is collected in one main fraction. The fraction is stored at 5 ± 4 °C until further processing (< 44 h).

[0421] Fraction 2 of the Q Sepharose HP pool is purified by the same process. Both main fractions of the Hexyl 650C are united and conditioned by ultrafiltration using 30 kDa membranes. This step is controlled by the TMP and the feed flowrate. The product solution is initially concentrated by factor 20, then the retentate is diafiltrated by factor 6 using formulation buffer. Finally, the retentate is concentrated to 60 mg / mL. The diafiltrate is stored at 5 ± 4 °C until further processing (< 24 h). The diafiltrate is 0.2 pm sterile filtered and filled into sterile 5 L bottles. The PAS-nomacopan DS is frozen and stored at -60 to -90 °C.

[0422] [1] Sandle T, Chapter 11 in Pharmaceutical Microbiology, Woodhead Publishing, 2016, Pages 131- 145,

[0423] [2] Pusterla, 2022, What are endotoxins?, BMG LABTECH blog, accessible at https: / / www.bmglabtech.com / en / blog / what-are-endotoxins /

[0424] [3] The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)).

[0425] [4] American National Standards Institute, Instrumentation Association for the Advancement of Medical Instrumentation. Bacterial endotoxins-Test methodologies, routine monitoring, and alternatives to batch testing. ST72: 2011 .

[0426] [5] Endotoxin Testing Recommendations for Single-Use Intraocular Ophthalmic Devices, Guidance for Industry and Food and Drug Administration Staff, CDRH, August 2015

[0427] [6] W02004 / 106369

[0428] [7] Jore, M. M. et al, Nature Structural & Molecular Biology 2016 volume 23, pages 378-386

[0429] [8] WO2008 / 155134

[0430] [9] WO2011 / 144756

[0431]

[0010] Kuhn et al, Bioconjugate Chem., 2016, 27 (10), pp 2359-2371

[0432]

[0011] Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003

[0433]

[0012] Lerchner et al. (2016) Protein Eng Des Sei. 29, 557-562

[0434]

[0013] Eggenstein et al. (2019) Protein Eng Des Sei. 32, 289-296

[0435]

[0014] Roversi et al. (2013) J Biol Chem. 288, 18789-18802

[0436]

[0015] Giclas 1994 (Giclas, P. C. (1994). Classical and alternative pathway evaluation (sections 13.1 and 13.2). In Current Protocols in Immunology, Vol. 3

[0437]

[0016] Roversi et al. (2007) J Mol Biol, 369:784-793

[0438]

[0017] Cantor (1980) Biophysical Chemistry, 2nd ed., W. H. Freeman and Company, New York

[0439]

[0018] Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York

[0440]

[0019] Smith (1996) Fold Des I :R95-R106

[0441]

[0020] Bantseev V, et al., Invest Ophthalmol Vis Sci. 2017 Mar 1 ;58(3):1545-1552.

[0442]

[0021] Malyala, P, & Singh, M. Journal of pharmaceutical sciences 97.6 (2008): 2041-2044.

[0443]

[0022] Hasiwa N, et al., ALTEX. 2013;30(2):169-208.

[0444]

[0023] Iwanaga, S., 1995. The limulus clotting reaction. Curr. Opin. Immunol. 5, 74.

[0024] Pusterla, 2022, The LAL assay: a living fossil exploited to detect bacterial contamination, BMG LABTECH blog, accessible at https: / / www.bmglabtech.com / en / blog / the-lal-assay-a-living-fossil- exploited-to-detect-bacterial-contamination /

[0445]

[0025] WO 2007 / 076411

[0446]

[0026] Josie D, Kovac S. Curr Protoc Protein Sci. 2010 Aug; Chapter 8:8.7.1-8.7.22.

[0447]

[0027] Kondaveeti, S et al. Application Note, Agilent, 2020

[0448]

[0028] Fekete S, et al., J Pharm Biomed Anal. 2015 Sep 10; 113:43-55

[0449]

[0029] Fling SP & Gregerson DS, Anal Biochem. 1986 May 15;155(1):83-8.

[0450]

[0030] Remington's Pharmaceutical Sciences; Mack Pub. Co., N.J. 1991

[0451]

[0031] Aguire et al. (2018) Pham Res 35:173

[0452]

[0032] Marra et al. (2011) AAPS PharmSciTech, Vol. 12, No. 1

[0453]

[0033] Fagan and Al-Qureshi (2013) Intravitreal injections: a review of the evidence for best practice, Clin Exp Opthalmol, 41 (5):500-507

[0454]

[0034] Thon and Koebsch Genetic Engineering & Biotechnology News BIOPROCESS Tutorial

Claims

CLAIMS1 . A method for removing pyrogens from a composition comprising a fusion protein, wherein the method comprises: a) providing an input composition comprising the fusion protein and a pyrogen, and b) performing hydrophobic interaction chromatography (HIC) using a stationary phase comprising hexyl ligands to produce an output composition comprising the fusion protein, and wherein the fusion protein comprises: i) a bioactive polypeptide, wherein the bioactive polypeptide comprises amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide.

2. The method of claim 1 , wherein the HIC is performed on a chromatographic column.

3. The method of claim 1 or 2, wherein the stationary phase: i) comprises a hydroxylated polymethacrylic polymer, optionally wherein the hydroxylated polymethacrylic polymer is coupled to the hexyl ligands; ii) has a mean pore size of about 100 nm (1000 A); iii) has a mean particle size of about 100 pm; and / or iv) has a mean ligand density of from about 30 g / L to about 50 g / L.

4. The method of any one of claims 1 to 3, wherein step b) comprises: i) conditioning the input composition; ii) equilibrating the HIC stationary phase; iii) loading the conditioned composition onto the HIC stationary phase; iv) washing the HIC stationary phase; and v) eluting the fusion protein from the HIC stationary phase using a salt gradient to produce the output composition.

5. The method of claim 4, whereinA. steps i), ii) and / or iv) are performed using a buffer, optionally wherein the buffer is HEPES / ammonium sulphate buffer, optionally wherein the HEPES buffer has a pH of between about pH 6 to about pH 9, further optionally about pH 7 to about pH 8, preferably about pH 7.6; and / orB. the salt gradient is generated using a HEPES / ammonium sulphate buffer and HEPES buffer.

6. The method of any one of claims 1 to 5, wherein the pyrogen is selected from: i) a non-endotoxin pyrogen (NEP), optionally wherein the NEP is a lipoprotein, peptidoglycan, wall teichoic acid, and / or a lipoteichoic acid; and / or ii) an endotoxin, optionally wherein the endotoxin is a lipopolysaccharide (LPS).

7. The method of any one of claims 1 to 6, wherein the pyrogens in the input composition are detectable by measuring their ability to induce monocytes to secrete at least one cytokine, optionally as measured by a monocyte activation test (MAT) assay.

8. The method of claim 7, wherein the at least one cytokine comprises IL-6, IL-1 p, TNF-a and / or IFN-y, preferably IL-6.

9. The method of any one of claims 1 to 8, wherein at least 80% or 85% of the total protein in the input composition is fusion protein, as measured by any one of reversed-phase high- performance liquid chromatography (RP-HPLC), size-exclusion HPLC (SE-HPLC), ion exchange HPLC (IEX-HPLC) and / or SDS-PAGE.

10. The method of any one of claims 1 to 9, wherein the removal of pyrogens is as assessed by measuring the ability of pyrogens in the output composition to induce monocytes to secrete at least one cytokine compared to the pyrogens in the input composition, optionally as measured by a MAT assay.

11. The method of claim 10, wherein the at least one cytokine comprises IL-6, IL-1 p, TNF-a and / or IFN-y, preferably IL-6.

12. The method of any one of claims 1 to 11 , wherein the level of pyrogens in the output composition is reduced by at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, compared to the level of pyrogens in the input composition, optionally as measured by the MAT assay.

13. The method of any one of claims 1 to 12, wherein the total amount of fusion protein in the output composition is at least 70% or at least 75% of the total amount of fusion protein in the input composition.

14. The method of any one of claims 1 to 13, wherein at least 85% or 90% of the total protein in the output composition is fusion protein, as measured by any one of RP-HPLC, SE-HPLC, IEX-HPLC and / or SDS-PAGE.

15. The method of any one of claims 1 to 14, wherein after step b), the output composition is: i) filtered, optionally i. by ultrafiltration and diafiltration, optionally into a storage buffer; ii. by tangential flow filtration (TFF); and / or Hi. 0.2 pm sterile filtered; ii) formulated with at least one pharmaceutical carrier or excipient; and / oriii) filled into any sterile container.

16. The method of claim 15, wherein the filtered output composition is suitable for administration to a subject, preferably a human subject.

17. A composition produced by the method of any preceding claim.

18. A method for removing host cell proteins (HCPs) from a composition comprising a fusion protein, the method comprising: a) providing an input composition comprising at least 30 L of cell culture medium or cell culture supernatant comprising the fusion protein and a HCP; b) performing ammonium sulphate precipitation to precipitate the fusion protein from the cell culture medium or the cell culture supernatant; and c) resolubilizing the fusion protein to provide an output composition; and wherein the fusion protein comprises: i) a bioactive polypeptide, wherein the bioactive polypeptide comprises amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof, and ii) a PA(S) polypeptide.

19. The method of claim 18, wherein the method comprises providing an input composition comprising: i) at least 50 L, at least 100 L, at least 200 L, at least 400 L, or at least 600 L of cell culture medium or cell culture supernatant; or ii) between about 400 L to about 1000 L, or about 600 L to about 800 L, preferably about 650 L to about 750 L of cell culture medium or cell culture supernatant.

20. The method of claim 18 or 19, wherein the fusion protein is precipitated by addition of ammonium sulphate to a final concentration of between about 0.7 M (17.5% saturation) and about 1.4 M (35% saturation), preferably about 1 M (25% saturated).21 . The method of any one of claims 18 to 20, wherein the method removes at least one of the following: host cell debris, host cell DNA, media components, pyrogens, and viruses.

22. The method of any one of claims 18 to 21 , wherein the total amount of fusion protein in the output composition is at least 70%, at least 75%, or at least 80% of the total amount of fusion protein in the input composition.

23. The method of any one of claims 18 to 22, wherein at least 45% or at least 50% of the total protein in the output composition is fusion protein as measured by any one of RP-HPLC, SE- HPLC, IEX-HPLC and / or SDS-PAGE.

24. The method of any preceding claim wherein the bioactive polypeptide comprises a sequence having at least 90% or at least 95% sequence identity to the sequence of amino acids 19 to 168 of SEQ ID NO: 2, and said bioactive polypeptide binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and / or binds to LTB4.

25. The method of any proceeding claim, wherein the bioactive polypeptide comprises or consists of amino acids 19 to 168 of SEQ ID NO: 2.

26. The method of any proceeding claim, wherein the PA(S) sequence is a PAS sequence, optionally wherein the PAS sequence comprises or consists of 30 copies of SEQ ID NO: 15, preferably wherein the PAS sequence comprises or consists of SEQ ID NO: 31 .

27. The method of any proceeding claim wherein the bioactive polypeptide comprises or consists of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 and the PA(S) polypeptide of the fusion protein comprises or consists of SEQ ID NO: 31 , and wherein the bioactive polypeptide is fused to the N terminus of the PA(S) polypeptide, optionally wherein the fusion protein comprises or consists of SEQ ID NO: 35.

28. The method of any proceeding claim, wherein the bioactive polypeptide binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and / or binds LTB4.

Images