Complement factor I preparations

Abstract

Provided herein are pharma- ceutically acceptable formulations comprising wild-type complement factor I (CFI) and variants thereof, in some embodiments, the wild-type CFI and variants thereof are part of a fusion construct, e.g., a fusion construct with human serum albumin. The formulations stabilize CFI against acute stress during storage in either liquid or lyophilized states. Also provided are methods of making the formulations and methods of using the formulations in the treatment of disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 293,013, filed December 22, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (VTEX_707_01WO_SeqList_ST26.xml; size: 31,757 bytes; and creation date: December 17, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Complement Factor I (CFI) is a soluble protein of the complement system that regulates complement activation by cleaving cell-bound or fluid-phase C3b and C4b. It is a soluble glycoprotein that circulates in human blood and acts to maintain the balance between the classical, lectin, and alternative pathways of the complement system. Dysregulated CFI, mutated and dysfunctional CFI, and CFI deficiency are associated with diseases involving the complement system. There is a need for pharma- ceutically acceptable CFI formulations useful for regulating the complement system. Provided herein are formulations that address this need. Summary of the Invention

[0004] The present disclosure provides formulations comprising wild-type Complement Factor I (CFI) or CFI mutants, and fusion proteins thereof. The provided formulations stabilize the active ingredients against acute stress and allow for storage in both liquid and lyophilized states. Also provided are methods of making the formulations and methods of using the formulations in the treatment of disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] The present disclosure provides formulations comprising wild-type CFI or CFI mutants, and fusion proteins thereof. The formulations provided stabilize the active ingredients against acute stress and allow for storage in both liquid and lyophilized states. Also provided are methods of making the formulations and methods of using the formulations in the treatment of disease.

[0006] I. Pharmaceutically Acceptable Formulations The pharma- ceutically acceptable formulations described herein provide stabilizing properties to the disclosed wild-type CFI or CFI variants, and fusion constructs thereof (interchangeably referred to herein as "active ingredients" or "active pharmaceutical ingredients") at a concentration range that allows for pharma- ceutically acceptable storage conditions. The stabilizing properties can be, for example, prevention of degradation, maintenance of concentration, prevention of aggregation, and / or maintenance of biological activity.

[0007] The formulations of the disclosure include wild-type CFI, a CFI variant, a fusion construct comprising wild-type CFI (e.g., CFI-HSA), or a fusion construct comprising a CFI variant (e.g., any CFI variant-HSA fusion in Table 2), a buffer, a tonicity modifying agent, a surfactant, and, optionally, a bulking agent, a cryoprotectant, a lyoprotectant, and / or a stabilizer.

[0008] The formulation may be designed to support storage of the active ingredient in a solid (dry) form (e.g., lyophilized cake or cryopreserved). In some embodiments, the formulation is a lyophilizate. In some embodiments, the formulation is a liquid formulation.

[0009] The formulation can be designed to support preservation of the active ingredient in solution (e.g., while maintaining stability, activity) as a liquid at a range of concentrations. Concentrations can be adjusted in the formulation for use in different types of administration (e.g., subcutaneous or intravenous). For example, the active ingredient is about 10 mg / mL to about 300 mg / mL, for example, about 10 mg / mL, about 15 mg / mL, 20 mg / mL, about 25 mg / mL, 30 mg / mL, about 35 mg / mL, 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL L, about 160 mg / mL, about 165 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL, about 205 mg / mL, about 210 mg / mL, about 215 mg / mL, about 220 mg / mL, about 225 mg / mL, about 230 mg / mL, about 235 mg / mL, about 245 mg / mL, about 250 mg / mL, about 255 mg / mL, about 260 mg / mL, about 265 mg / mL, about 270 mg / mL, about 275 mg / mL, about 280 mg / mL, about 285 mg / mL, about 290 mg / mL, about 295 mg / mL, or about 300 mg / mL. In some embodiments, the formulation comprises the active ingredient in a concentration of about 50 mg / mL. In some embodiments, the formulation comprises the active ingredient in a concentration of about 100 mg / mL. In some embodiments, the formulation comprises the active ingredient in a concentration of greater than 150 mg / mL. In some embodiments, the formulation comprises the active ingredient in a concentration of about 170 mg / mL. In some embodiments, the formulation comprises the active ingredient in a concentration of about 190 mg / mL.

[0010] The formulations of the present disclosure allow the active ingredient to remain stable (e.g., active) at any one or more of the following temperatures: -80°C+ / -2°C, -20°C+ / -2°C, 0°C+ / -2°C, 4°C+ / -2°C, 25°C+ / -2°C, 37°C+ / -2°C, 45°C+ / -2°C, or 60°C+ / -2°C.

[0011] The formulations of the present disclosure may allow the active ingredient to remain stable (e.g., active) for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years or more.

[0012] The pH of the formulations described herein is any pH that provides stabilizing properties to the CFI variants and fusion constructs described herein. In some embodiments, the formulation comprises a pH of about 5 to about 7.5. The pH can be, for example, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, about pH 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9, about pH 7, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, or about pH 7.5.

[0013] The formulation comprises a pharma- ceutically acceptable buffering agent. The buffering agent may be used to maintain the pH of the formulation in a desired range. Any suitable buffering agent may be used in the formulations of the present disclosure. Examples of pharma- ceutically acceptable buffering agents include histidine, acetate, citrate, succinate, tartrate, glutamate, glycine, bicarbonate, sulfate, nitrate, phosphate, and hydroxymethylaminomethane (Tris) buffers. In some embodiments, the buffering agent is present at about 1 mM to about 50 mM. In some embodiments, the buffering agent is present at about 20 nM. In some embodiments, the buffering agent comprises histidine, e.g., histidine hydrochloride. In some embodiments, the buffering agent comprises acetate, e.g., sodium acetate. In some embodiments, the buffering agent comprises succinate, e.g., sodium succinate.

[0014] The formulation includes a surfactant, sometimes referred to as a wetting agent and / or solubilizer. Without limitation, surfactant excipients can be used to adjust the solubility and bioavailability of biological molecules, increase the stability of biological molecules in the dosage form, and maintain a preferred polymorphic form. Examples of surfactants include polysorbate 20 (PS20, Tween 20), polysorbate 80 (PS80, Tween 80), PS80 / 20, poloxamers (Pluronic F68 and F127), Triton X-100, Brij 30, and Brij 35. In some embodiments, the formulation of the present disclosure includes a surfactant. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the formulation includes polysorbate 20. In some embodiments, the polysorbate 20 is about 0.01% to about 0.05%. In some embodiments, the polysorbate is about 0.02%. In some embodiments, the formulation includes polysorbate 80. In some embodiments, the polysorbate 80 is about 0.01% to about 0.05%. In some embodiments, the polysorbate 80 is about 0.02%.

[0015] The formulation includes a tonicity modifying agent, which may sometimes be referred to as a tonicity agent, tonicifier, or tonicifying agent. A tonicity modifying agent is used to adjust the osmolality of the formulation so that the osmolality of the formulation does not cause adverse effects such as undesired cell lysis when administered to a subject. Any pharma- ceutically acceptable tonicity modifying agent that is compatible with the active ingredients of the present disclosure may be suitable for use in the formulations described herein. Pharmaceutically acceptable tonicity modifying agents may include, for example, mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, potassium chloride, glycerol, glycerin, or arginine hydrochloride. In some embodiments, the formulation includes a tonicity modifying agent. In some embodiments, the tonicity modifying agent is arginine hydrochloride. In some embodiments, the formulation includes arginine hydrochloride. In some embodiments, the arginine hydrochloride is about 10 mM to about 200 mM. In some embodiments, the arginine hydrochloride is at about 70 mM. In some embodiments, the arginine hydrochloride is at about 135 mM. In some embodiments, sorbitol is the tonicity modifying agent, and the sorbitol is present at about 1% to about 10% v / v, e.g., about 5% v / v. In some embodiments, the tonicity modifying agent is trehalose, and the trehalose is present at about 1% to about 15% v / v, e.g., about 10% v / v. In some embodiments, the tonicity modifying agent is sodium chloride, and is present at about 1 mM to about 500 mM, e.g., about 150 mM. In some embodiments, the tonicity modifying agent is sorbitol or trehalose for non-ionic buffers, or sodium chloride for ionic buffers.

[0016] The formulation may include a bulking agent, such as trehalose or glycine. In some embodiments, the bulking agent is glycine and is present in the formulation at about 1% to about 5% v / v, such as about 2% v / v. In some embodiments, the bulking agent is glycine and is present in the formulation at about 1 mM to about 100 nM, such as about 60 mM.

[0017] The formulation may include a cryoprotectant and / or a lyoprotectant. Cryoprotectants and lyoprotectants are included in the formulation to protect biological molecules from denaturation during cycles of freezing and thawing. They can also aid in preserving biological integrity, e.g., maintaining biological activity during lyophilization (i.e., freeze drying) and reconstitution. In some examples, cryoprotectants and lyoprotectants may be referred to interchangeably. Any pharma- ceutically acceptable cryoprotectant and / or lyoprotectant that can provide stabilizing properties to the formulations containing the CFI variants and fusion constructs of the present disclosure may be used. Non-limiting examples of cryoprotectants and lyoprotectants include polyols, disaccharides, polysaccharides, glucose, glycine, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some embodiments, the formulation includes a cryoprotectant. In some embodiments, the cryoprotectant is at least one of sucrose, glycine, sorbitol, trehalose, and mannitol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the sucrose is about 2% to about 10%. In some embodiments, the sucrose is about 4% to about 5%. In some embodiments, the sucrose is about 8.5%. In some embodiments, the cryoprotectant is glycine. In some embodiments, the glycine is about 50 mM to about 150 mM, e.g., about 120 mM. In some embodiments, the glycine is about 120 mM. In some embodiments, the cryoprotectant is sorbitol. In some embodiments, glucose is present in the formulation at about 1% to about 10% v / v, e.g., about 4% v / v. In some embodiments, sorbitol is present at about 1% to about 5%. In some embodiments, sorbitol is about 2.5% v / v. In some embodiments, trehalose is present at about 1% to about 10% v / v, e.g., about 4% v / v. In some embodiments, sorbitol is about 2.5%. In some embodiments, the cryoprotectant is mannitol, hi some embodiments, the mannitol is at about 1 mM to about 100 mM.In some embodiments, the mannitol is about 60 mM.

[0018] The formulation may include a stabilizing agent, sometimes referred to as a stabilizer. A stabilizing agent is any excipient that provides stabilizing properties to the formulations containing the CFI variants and fusion constructs described herein. For example, the stabilizing agent may be calcium chloride (CaCl2), histamine, methionine, ascorbic acid, glutathione, vitamin E, poly(ethyleneimine), a chelating agent, an antimicrobial preservative, an antioxidant preservative, or any other pharma- ceutically acceptable stabilizing agent. In some embodiments, the formulation includes a stabilizing agent. In some embodiments, the stabilizing agent is calcium chloride. In some embodiments, the formulation includes calcium chloride. In some embodiments, the calcium chloride is about 20 mM to about 50 mM. In some embodiments, the calcium chloride is about 35 mM.

[0019] Exemplary formulations are provided in Tables 2.2 and 3.3.

[0020] In some embodiments, the formulation comprises 20 mM sodium acetate, 5% sorbitol, 0.02% PS80, and has a pH of about 5.

[0021] In some embodiments, the formulation comprises 20 mM sodium acetate, 5% sorbitol, 0.02% PS80, and has a pH of about 5.5.

[0022] In some embodiments, the formulation comprises 20 mM sodium acetate, 150 mM sodium chloride, 0.02% PS80, and has a pH of about 5.5.

[0023] In some embodiments, the formulation comprises 20 mM sodium acetate, 70 mM arginine hydrochloride, 2% sucrose, 60 mM glycine, 0.02% PS80, and has a pH of about 5.5.

[0024] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 70 mM arginine hydrochloride, 2% sucrose, 60 mM glycine, 0.02% PS80, and has a pH of about 6.

[0025] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 150 mM sodium chloride, 0.02% PS80, and has a pH of about 6.

[0026] In some embodiments, the formulation comprises 20 mM sodium succinate, 10% trehalose, 0.02% PS80, and has a pH of about 5.

[0027] In some embodiments, the formulation comprises 20 mM sodium succinate, 5% trehalose, 2% glycine, 0.02% PS80, and has a pH of about 5.

[0028] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 10% trehalose, 0.02% PS80, and has a pH of about 6.

[0029] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 5% trehalose, 2% glycine, 0.02% PS80, and has a pH of about 6.

[0030] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 10% trehalose, 0.02% PS80, and has a pH of about 7.

[0031] In some embodiments, the formulation comprises 20 mM histidine hydrochloride, 5% trehalose, 2% glycine, 0.02% PS80, and has a pH of about 7.

[0032] In some embodiments, the formulation comprises 20 mM histidine, 135 mM arginine hydrochloride, 0.02% polysorbate 20, and has a pH of about 5.8.

[0033] In some embodiments, the formulation comprises 20 mM histidine, 70 mM arginine hydrochloride, 4% sucrose, 0.02% polysorbate 20, and has a pH of about 5.8.

[0034] In some embodiments, the formulation comprises 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 20, and has a pH of about 5.8.

[0035] In some embodiments, the formulation comprises 20 mM histidine, 70 mM arginine hydrochloride, 120 mM glycine, 0.02% polysorbate 20, and has a pH of about 5.8.

[0036] In some embodiments, the formulation comprises 20 mM histidine, 70 mM arginine hydrochloride, 2.5% sorbitol, 0.02% polysorbate 20, and has a pH of about 5.8.

[0037] In some embodiments, the formulation comprises 20 mM histidine, 70 mM arginine hydrochloride, 4% trehalose, 0.02% polysorbate 20, and has a pH of about 5.8.

[0038] In some embodiments, the formulation comprises 20 mM histidine, 5% sucrose, 35 mM calcium chloride, 0.02% polysorbate 20, and has a pH of about 5.8.

[0039] II. Complement Factor I Variants and Fusion Constructs for Formulation A. Complement factor I mutants Provided herein are formulations that contain variants that contain one or more modifications with respect to wild-type CFI, referred to herein as "CFI variants." As used herein, a "modification" with respect to wild-type CFI includes a deletion of one or more amino acid residues, a deletion of one or more domains, a substitution of one or more amino acid residues, an insertion (i.e., addition) of one or more amino acid residues, an insertion (i.e., addition) of one or more domains, an inversion of one or more domains, and a substitution of one or more domains.

[0040] The CFI mutants of the disclosure do not act directly on C3, e.g., the mutants of the disclosure do not directly cleave C3, do not directly inhibit C3, do not directly inhibit the activation of C3, or do not directly reduce the activation of C3.

[0041] As used herein, "wild-type CFI" refers to any naturally occurring full-length CFI that is not a disease-causing CFI and can be of any species, with or without a signal sequence.

[0042] In some embodiments, the wild-type CFI is plasma-derived. In some embodiments, the wild-type CFI is human wild-type CFI. In some embodiments, the wild-type human CFI with a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 1 (shown in Table 1 below). In some embodiments, the wild-type CFI is human CFI. In some embodiments, the wild-type human CFI does not comprise a signal sequence. In some embodiments, the wild-type CFI without a signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 5 (shown in Table 1 below).

[0043] Wild-type CFI comprises a heavy chain and a light chain, which are also referred to as A chain and B chain, respectively. The heavy chain (A chain) has four domains: the FI membrane attack complex (FIMAC) domain (residues 36-90 of SEQ ID NO:5), the SRCR domain, which is further composed of multiple scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein 1 domain (LDLr1), and low density lipoprotein 2 domain (LDLr2). The light chain (B chain) consists of a serine protease domain (SPD). The interface between these chains is referred to as the A:B chain interface.

[0044] The CFI mutants of the present disclosure include one or more of the following: a deletion of one or more amino acid residues of wild-type CFI, a deletion of one or more CFI domains of wild-type CFI, a substitution of one or more amino acid residues of wild-type CFI, an insertion of one or more amino acid residues into wild-type CFI, an inversion of one or more CFI domains of wild-type CFI, and an insertion of one or more domains into wild-type CFI.

[0045] The CFI variants of the present disclosure can be generated by introducing one or more modifications into a CFI base molecule, where the domains of the CFI base molecule correspond to those domains found in wild-type CFI. Thus, the CFI base molecule can be the wild-type CFI of any species, or the CFI base molecule can include only a portion of wild-type CFI having only some of the domains of wild-type CFI of any species (e.g., already a CFI variant). In some embodiments, the CFI base molecule is wild-type mouse CFI. In some embodiments, the CFI base molecule is wild-type human CFI. In some embodiments, the CFI base molecule is wild-type non-human primate CFI. In some embodiments, the CFI base molecule includes only some of the domains of wild-type human CFI.

[0046] In some embodiments, the CFI variants provided herein modulate the activity of the complement system and have at least one improved characteristic compared to wild-type CFI. Such improved characteristics include, but are not limited to, an increase or decrease in any one or more of bioavailability, half-life, activity, potency, catalytic activity, cofactor affinity (e.g., affinity for factor H and / or CR1), substrate specificity, and substrate affinity (e.g., affinity for C3b and / or C4b). In some embodiments, the improved characteristic is an increase in half-life. In some embodiments, the improved characteristic is an increase in activity, which is discussed in more detail in the subsequent sections below. In other embodiments, the improved characteristic is an altered substrate specificity for C3b and / or C4b, allowing for synchrony of the CFI variants.

[0047] Provided in Table 1 are exemplary base molecules that can be used to generate CFI variants. The base molecules provided herein can be useful for regulating the complement system without further modification, or can be useful for regulating the complement system with further modification. For example, any one of the base molecules provided in Table 1 can be further modified to include one or more modifications, such as deletion of one or more amino acid residues, deletion of one or more CFI domains, substitution of one or more amino acid residues, or addition of one or more amino acid residues or CFI domains. The base molecules of Table 1 can be further moieties of fusion constructs, which are further described below.

[0048] [Table 1-1]

[0049] [Table 1-2]

[0050] In some embodiments, the base molecule itself can be a CFI variant, for example, in some embodiments, a CFI variant that contains only the serine protease domain (CFI-SPD) is itself a CFI variant. In some embodiments, a CFI variant is derived from any base molecule in Table 1 and contains modifications to loops that correspond to loops of unmodified CFI. In some embodiments, a CFI variant is derived from any base molecule in Table 1 and contains substitution mutations. In some embodiments, a CFI variant is derived from any base molecule in Table 1 and contains a deletion of one or more domains of CFI. In some embodiments, a CFI variant is derived from any base molecule in Table 1 and contains an inversion of the A and B strands of CFI.

[0051] In some embodiments, provided herein are CFI variants comprising at least one CFI domain, where the at least one CFI domain corresponds to a CFI domain of a wild-type CFI of any species. For example, the amino acid sequence of the at least one CFI domain can comprise an amino acid sequence derived from wild-type human CFI as set forth in SEQ ID NO:5. The CFI variants provided herein comprising an amino acid sequence derived from SEQ ID NO:5 can comprise one or more modifications with respect to the sequence as set forth in SEQ ID NO:5. For example, the one or more modifications can comprise a deletion of one or more amino acid residues, a substitution mutation of one or more amino acid residues, an addition of one or more amino acid residues, a deletion of one or more domains of CFI, a substitution of one or more domains of CFI, or an addition of one or more domains of CFI.

[0052] In some embodiments, provided herein is a CFI variant comprising at least one CFI domain of any species, wherein the at least one CFI domain comprises any one or more CFI domains selected from a serine protease domain (SPD), a factor I membrane attack complex (FIMAC) domain, a scavenger receptor cysteine ​​rich domain (SRCR), a low density lipoprotein receptor 1 (LDLr1), and a low density lipoprotein receptor 2 (LDLr2) domain. In some embodiments, the any one or more CFI domains are CFI domains of human CFI. In some embodiments, the any one or more CFI domains comprise an amino acid sequence derived from the sequence set forth in SEQ ID NO:5.

[0053] In some embodiments, the CFI mutant comprises all of the domains of wild-type CFI, i.e., one each of the SPD, FIMAC domain, SRCR domain, LDLr1 domain, and LDLr2 domain, and includes modifications in any one or more of these domains relative to wild-type CFI.

[0054] In some embodiments, the CFI mutant does not contain all of the domains corresponding to those of wild-type CFI. In some embodiments, the CFI mutant contains an SPD. In some embodiments, the CFI mutant contains only the SPD and lacks the A strand of CFI, referred to herein as "CFI-SPD." In some embodiments, the CFI-SPD contains the amino acid sequence set forth in SEQ ID NO: 12 (shown in Table 1), which is the SPD of human CFI. In some embodiments, the CFI-SPD does not contain any further modifications relative to the modifications of wild-type CFI SPD. In some embodiments, the CFI-SPD contains one or more modifications relative to the modifications of wild-type CFI SPD. In some embodiments, the CFI-SPD contains at least one modification relative to the amino acid sequence set forth in SEQ ID NO: 12.

[0055] Exemplary mutants of CFI are described in further detail below. Exemplary CFI mutants contain one or more substitutions of amino acid residues with respect to CFI having the amino acid sequence set forth in SEQ ID NO: 5. For example, a CFI mutant containing substitutions at positions S499 and I500 has substitutions at positions S499 and I500 in the amino acid sequence set forth in SEQ ID NO: 5.

[0056] Adjustment of activity and specificity The activity and specificity of the CFI variants provided herein can be tuned (adjusted) for a particular use and therapeutic indication. For example, activity and specificity can be adjusted by selection of a C3b degrading agent or a C4b degrading agent, or a degrading agent for both C3b and C4b. As referred to herein, protease activity against a substrate refers to the ability of a CFI variant of the present disclosure to cleave its substrates C4b and C3b. This can be expressed in several ways, for example, as an increase in C4b degrading agent activity, protease activity against C4b, C3b degrading agent activity, protease activity against C3b, yield of cleavage products, etc.

[0057] As used herein, a C3b degrading agent is a CFI mutant capable of cleaving C3b. Similarly, a C4b degrading agent is a CFI mutant capable of cleaving C4b. The use of a C3b degrading agent does not mean that it does not degrade C4b. A CFI mutant may be both a C3b degrading agent and a C4b degrading agent, and may, but does not necessarily, show specificity for one over the other.

[0058] The CFI variants provided herein have altered characteristics, including increased or decreased protease activity towards a substrate, as well as increased or decreased substrate specificity.

[0059] In some embodiments, the disclosed CFI variants that are specific C3b degrading agents are useful in the treatment of disease.

[0060] In some embodiments, the disclosed CFI variants that are specific C4b degrading agents are useful in the treatment of disease.

[0061] In some embodiments, CFI variants of the disclosure that are both C4b and C3b degraders and exhibit improved characteristics compared to wild-type CFI (e.g., increased activity against both C4b and C3b) are useful in the treatment of diseases.

[0062] Exemplary CFI Mutants Provided herein are CFI variants that contain or consist of at least one modification relative to wild-type CFI.

[0063] Without being limited thereto, the present disclosure contemplates exemplary CFI mutants set forth in Table 2. The mutants in Table 2 include modified CFIs described herein. For the avoidance of doubt, unless otherwise indicated, when a residue number is given, it refers to SEQ ID NO:5 (wild type human CFI) or a sequence corresponding thereto. For the avoidance of doubt, by way of example, a mutant whose description is P433A is a CFI mutant comprising a P433A substitution, e.g., a CFI mutant comprising a P433A substitution in SEQ ID NO:5 (or a sequence corresponding thereto), and the present disclosure also provides CFI mutants consisting of a P433A substitution, e.g., a CFI mutant in SEQ ID NO:5 having a P433A substitution.

[0064] The CFI variants of the present disclosure may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven or more modifications, such as substitutions, deletions, insertions, and fusions. The modifications, such as substitutions, for a given variant may be expressed in one of many ways recognized by those of skill in the art. For example, a hCFI variant having substitutions at D395A and E416A may be referred to as having the substitutions: "D395A and E416A", "D395A-E416A", "D395A+E416A", "D395A / E416A", or "D395A;E416A", which may be used interchangeably herein. In some examples, a CFI variant having substitutions at D395A and E416A may be referred to as "hCFI;D395A;E416A" or CFI variant (D395A;E416A)". As described herein, variants having other modifications, such as deletions, or combinations of modifications, such as deletions, fusions, and substitutions, may follow a similar nomenclature.

[0065] Table 2 provides exemplary CFI variants of the disclosure. This and other tables disclosing variants may include the following symbols and abbreviations and associated meanings: HSA = human serum albumin, CFI = complement factor I, Δ = deletion of the stated amino acid range, → or > = deletion of the stated sequence and substitution with the stated amino acid.

[0066] [Table 2-1]

[0067] [Table 2-2]

[0068] [Table 2-3]

[0069] [Table 2-4]

[0070] [Table 2-5]

[0071] [Table 2-6]

[0072] [Table 2-7]

[0073] [Table 2-8]

[0074] [Table 2-9]

[0075] B. Fusion constructs containing complement factor I Provided herein are formulations for fusion constructs comprising a first component (a CFI portion) comprising at least one domain of complement factor I and at least a second component, where the first component and the second and subsequent components are fused (e.g., adjacent or separated by an optional linker). These fusion constructs are referred to herein as "CFI fusion constructs" or simply "fusion constructs." In some embodiments, the fusion constructs comprise additional components, e.g., a third component, a fourth component, etc.

[0076] In some embodiments, the second and subsequent components of the fusion construct are proteins. In some embodiments, the second and / or subsequent components are not proteins.

[0077] The components of the fusion constructs of the present disclosure may be held together by optional linkers. They may be of any suitable length of at least one amino acid. The linkers may be flexible linkers and may be peptides of about 1 to about 20 amino acid residues in length, and the amino acid residues may include glycine residues. The linkers may also optionally include serine residues. Exemplary flexible linkers may include, but are not limited to, glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, or any other suitable flexible linkers known in the art. An exemplary linker is (GGSS) n where n is any number from about 1 to about 20 (SEQ ID NO: 35). An exemplary linker is (GGSS) n GG (SEQ ID NO: 36), where n is any number from about 1 to about 20. An exemplary linker is (GGSSGG) n(SEQ ID NO: 37), where n is any number from about 1 to about 20. In some embodiments, the linker is a protease-sensitive cleavable linker. Exemplary linkers linking fusion constructs can be 1-50, 5-50, 10-50, 15-50, 20-50, 25-50, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-10, 6-9, 6-8, or 6-7 amino acids in length.

[0078] CFI+ half-life extended fusion construct In some embodiments, the fusion construct comprises wild-type CFI or a CFI mutant (first component) and at least a second component, where the second component is a half-life extender. Since naturally occurring CFI has a relatively short half-life, in some embodiments, it may be advantageous to increase the half-life of CFI or its mutants. By using a second component that is a half-life extender, activity may be increased or another characteristic may be improved compared to wild-type CFI. For example, wild-type CFI or a CFI mutant may have their half-life extended by fusing CFI to a half-life extender.

[0079] Exemplary half-life extenders include, but are not limited to, albumin, such as human serum albumin, PEG, non-biodegradable polymers, biodegradable polymers, and Fc. In some embodiments, the second component is a protein and a half-life extender, such as albumin or Fc. In some embodiments, the second component is not a protein and is a half-life extender, such as PEG. In some embodiments, the half-life extender is a peptide repeat.

[0080] In some embodiments, the second component is a half-life extender and is albumin. Note that as used herein, albumin refers to any albumin, such as any serum albumin, or an albumin variant, or an albumin derivative. By way of example, an albumin variant includes any albumin that contains at least one modification corresponding to the amino acid sequence set forth in SEQ ID NO: 7 (wild-type human serum albumin (HSA)), or at least one modification corresponding to the amino acid sequence of any non-human species of albumin. In an exemplary embodiment, the albumin is human serum albumin (HSA), as provided in SEQ ID NO: 7.

[0081] An exemplary fusion construct comprising wild-type CFI and HSA is referred to herein as "CFI-HSA" and is discussed in more detail below. An exemplary fusion construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:21.

[0082] In some embodiments, a fusion construct of the present disclosure comprises albumin and a CFI variant of the present disclosure. Exemplary CFI variants are provided in Table 2 below.

[0083] Structural arrangement of fusion constructs In some embodiments, provided herein are fusion constructs comprising at least a first component, where the first component is either a wild-type CFI or a CFI variant (CFI moiety) provided herein, and a second component, where the first component and the second component are fused, and the second component is fused to the N-terminus of the CFI moiety. In some embodiments, the second component is fused to the C-terminus of the CFI moiety. In some embodiments, the second component is fused to the C-terminus of the CFI moiety, and a third component is further fused to the N-terminus of the CFI moiety. In some embodiments, the second component is fused to the N-terminus of the CFI moiety, and a third component is further fused to the C-terminus of the CFI moiety.

[0084] With reference to Table 3, SEQ ID NO:1 is the amino acid sequence of wild-type plasma-derived human CFI, designated "CFI-PD," and has a leader sequence. The wild-type CFI used for fusion with the second component may comprise the amino acid sequence of SEQ ID NO:5, without the leader sequence present in SEQ ID NO:1. Alternatively, the mouse Ig kappa chain V-III region MOPC63 leader sequence (SEQ ID NO:2) may be used in the recombinant production of any of the CFI fusion constructs provided herein. In some embodiments, provided herein are CFI fusion constructs comprising at least one CFI domain, wherein the at least one CFI domain comprises the amino acid sequence set forth in SEQ ID NO:5.

[0085] An exemplary fusion construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:21.

[0086] [Table 3-1]

[0087] [Table 3-2]

[0088] III. Use of CFI-containing preparations The formulations of the present disclosure can be used for treatment in subjects. As used herein, a subject includes any mammalian subject, including primates, rodents, livestock, zoo animals, and pets. In some embodiments, the mammalian subject is a human subject. In some embodiments, the mammalian subject is a non-human primate.

[0089] A. Treatment of Non-Ocular Conditions In some embodiments, the formulations provided herein are useful for treating a non-ocular condition in a subject. In some embodiments, provided herein is a method of treating an ocular condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of any one of the formulations provided herein.

[0090] In some embodiments, the non-ocular condition is characterized by a deficiency in CFI, hi some embodiments, the non-ocular condition is characterized by dysregulation of the complement system.

[0091] In some embodiments, the non-ocular condition is a systemic acute indication selected from the group consisting of acute glomerulonephritis, acute kidney injury, acute respiratory distress syndrome, bacterial meningitis, cerebral hemorrhage, burns, coronavirus infection, Epstein-Barr virus infection, hematopoietic stem cell transplantation, ischemia-reperfusion injury, Lyme disease, myocardial infarction, organ transplant, periodontitis, pneumonia, pre-eclampsia, schistosomiasis, sepsis, stroke, thromboembolism, and traumatic brain injury.

[0092] In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is selected from the group consisting of Alzheimer's disease, anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease (COPD), cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMg), Granulomatosis with Polyangiitis (GPA), Guillain-Barre syndrome (Guillain-Barre syndrome), and combination therapy with anti-inflammatory drugs. Syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy (IgAN), lupus nephritis (LN), membranous glomerulonephritis (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis (MS), non-insulin-dependent diabetes mellitus, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein-losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITH),and systemic chronic indications selected from the group consisting of ulcerative colitis, Lampert-Eaton myasthenic syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), thrombotic microangiography (TMA) and chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, and ischemia-reperfusion injury.

[0093] In some embodiments, diseases that may be treated by use of the formulations provided herein that include CFI mutants or fusion constructs that are C4b degraders include, but are not limited to, non-ocular conditions. In some embodiments, the non-ocular condition is a systemic chronic indication. In some embodiments, the non-ocular condition is, but is not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (AMSC), or other conditions that may be treated by use of the formulations provided herein that include CFI mutants or fusion constructs that are C4b degraders. sclerosis, ALS), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, antiphospholipid syndrome, asthma, atherosclerosis, atypical hemolytic uraemic syndrome (aHUS), autoimmune hemolytic anemia, bullous pemphigoid (BP), C3 glomerulopathy, chronic renal failure, chronic obstructive pulmonary disease (COPD), cold agglutinin disease (CAD), Crohn's disease, diabetic neuropathy, generalized myasthenia gravis (gMg), granulomatosis with polyangiitis (GPA), Guillain-Barré syndrome (GBS), hereditary angioedema (HAE), hidradenitis suppurativa (HS), IgA nephropathy, lupus nephritis (LN), membranous nephropathy (MN), microscopic polyangiitis (MPA), motor neuron disease, multifocal motor neuropathy (MMN), multiple sclerosis disease (MS), non-insulin-dependent diabetes mellitus, osteoarthritis, pancreatitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), post-transplant lymphoproliferative disease, protein-losing enteropathy, psoriasis, pyoderma gangrenosum, rheumatoid arthritis, schizophrenia (SZ), systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), warm autoimmune hemolytic anemia (WHA) and systemic chronic indications selected from the group consisting of Autoimmune hemolytic anemia (wAIHA), Immune-Complex Membranoproliferative Glomerulonephritis (IC-MPGN), and ulcerative colitis, Lampert-Eaton Myasthenic Syndrome (LEMS), CHAPLE syndrome (CD55 deficiency), Thrombotic Microangiography (TMA) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Huntington's Disease, and ischemia-reperfusion injury.

[0094] In some embodiments, the non-ocular condition is non-oncological.

[0095] In some embodiments, the non-ocular condition is oncological. In some embodiments, the non-ocular condition is oncological and is characterized by a solid tumor or a liquid tumor. In some embodiments, the non-ocular disease is characterized by a solid tumor and is selected from the group consisting of colorectal tumor, hormone refractory prostate cancer, melanoma, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic pancreatic cancer, metastatic gastric cancer, nasopharyngeal carcinoma, non-small cell lung cancer, pancreatic tumor, squamous cell carcinoma, and gastric tumor. In some embodiments, the non-ocular condition is characterized by a liquid tumor and is selected from the group consisting of acute myeloid leukemia, B-cell lymphoma, and Hodgkin's disease.

[0096] B. Treating Eye Conditions In some embodiments, the formulations provided herein are useful for treating an ocular condition in a subject. In some embodiments, provided herein is a method of treating an ocular condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of any of the formulations provided herein.

[0097] In some embodiments, the ocular condition is characterized by a deficiency in CFI. In some embodiments, the ocular condition is characterized by dysregulation of the complement system.

[0098] In some embodiments, the ocular condition is characterized by the presence of a dysfunctional CFI gene, hi some embodiments, the ocular condition is characterized by dysregulation of the complement system and low CFI levels.

[0099] In some embodiments, the ocular condition is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy, dry age-related macular degeneration (AMD), glaucoma, keratoconjunctivitis, neuromyelitis optica spectrum disorder (NMOSD), open angle glaucoma, polypoidal choroidal vasculopathy, Stargardt's disease, uveitis, and vitreous retinopathy.

[0100] In some embodiments, the ocular condition is non-oncological.

[0101] C. Administration In vivo administration of the formulations described herein can be performed intravenously or subcutaneously.

[0102] In an exemplary embodiment, administration of the formulations described herein is subcutaneous administration, hi some embodiments, subcutaneous administration is daily, every other day, twice weekly, or weekly.

[0103] In some embodiments, administration of the formulations described herein is intravenous administration.

[0104] As generally contemplated herein, the CFI mutants or fusion constructs described herein are delivered in an activated, two-chain form. In some instances, however, an inactive CFI mutant or fusion construct may be delivered in an inactive, single-chain form. In some embodiments, what is delivered includes both the single-chain inactive form and the two-chain active form.

[0105] D. Dosage In some embodiments, any of the formulations described herein may be administered to a subject in need thereof at a dosage of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, the dosage is about 1 mg / kg. In some embodiments, the administration of a therapeutic CFI, variant, or fusion construct in a formulation described herein is subcutaneous at a dosage of about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, administration of a therapeutic CFI, variant, or fusion construct in a formulation described herein is intravenous administration at a dosage of about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, administration of a therapeutic CFI variant or fusion construct described herein is daily administration, every other day administration, weekly administration, or twice weekly administration.

[0106] In some embodiments, the target level of a therapeutic CFI, variant, or fusion construct in a formulation provided herein in plasma is about 0.1 μg / ml, about 0.5 μg / ml, about 1 μg / ml, about 1.5 μg / ml, about 2 μg / ml, about 2.5 μg / ml, about 3 μg / ml, about 3.5 μg / ml, about 4 μg / ml, about 4.5 μg / ml, 5 μg / ml, about 5.5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg ml, about 6μg / ml, about 6.5μg / ml, about 7μg / ml, about 7.5μg / ml, about 8μg / ml, about 8.5μg / ml, about 9μg / ml, about 9.5μg / ml, about 10μg / ml ml, approximately 10.5μg / ml, approximately 11μg / ml, approximately 11.5μg / ml, approximately 12μg / ml, approximately 12.5μg / ml, approximately 13μg / ml, approximately 13.5μg / ml, approximately 14μg / m l, about 14.5μg / ml, 15μg / ml, about 15.5μg / ml, about 16μg / ml, about 16.5μg / ml, about 17μg / ml, about 17.5μg / ml, about 18μg / ml, Approximately 18.5μg / ml, approximately 19μg / ml, approximately 19.5μg / ml, approximately 20μg / ml, approximately 20.5μg / ml, approximately 21μg / ml, approximately 21.5μg / ml, approximately 22μg / ml, approximately The target level may be about 10 μg / ml, about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, about 200 μg / ml, about 250 μg / ml, or even about 300 μg / ml. EXAMPLES

[0107] Example 1: Preparation of CFI, CFI variants, and fusion proteins overview The methods provided below are applicable to the expression, purification, activation, and in vitro sialylation of wild-type CFI, CFI mutants, and fusion constructs containing wild-type CFI and CFI mutants.

[0108] In the case of Example 1, reference to CFI-HSA refers to human serum albumin (SEQ ID NO:21) fused to the N-terminus of human wild-type CFI.

[0109] Wild-type CFI-HSA protein was expressed in Chinese hamster ovary (CHO) cells, purified by anti-albumin affinity purification, activated with furin, and purified by size column. The activated CFI-HSA protein was subjected to in vitro sialylation to increase the total sialylation of CFI-HSA. Finally, the sialylated protein was purified using anti-albumin affinity purification and refined by size exclusion column chromatography.

[0110] Expression The CFI-HSA gene (SEQ ID NO:21) with human serum albumin at the amino terminus of the CFI protein was synthesized (ThermoFisher Scientific, Geneart, Regensburg, Germany). The protein was made with a signal sequence of SEQ ID NO:2 that was removed during expression. The amino-terminal albumin tag was connected to the CFI gene via a linker (SEQ ID NO:6). The gene for CFI-HSA was inserted into an expression vector (LakePharma, Hayward, CA) using standard molecular biology techniques. The resulting plasmid DNA was transformed into E. coli. The transfected E. coli was grown in 200 ml LB medium for expression of the plasmid DNA and harvested using standard techniques. The plasmid DNA was run on an agarose gel for quality assessment and sequence confirmation before proceeding to transfection.

[0111] 1.0 liter of suspension TunaCHO™ cells were seeded into shake flasks and grown using serum-free synthetic medium. On the day of transfection, the grown cells were seeded into new flasks containing fresh medium. Plasmid DNA was transiently transfected into CHO cells using Lipofectamine 2000 (ThermoFisher Scientific). Cells were maintained as batch-fed cultures until the end of the production run. Protein was expressed for 14 days at 37°C with 8% CO2 concentration and 125 RMP. Cells were centrifuged and the supernatant was collected for purification of secreted CFI-HSA at the end of the 14-day expression.

[0112] purification The supernatant containing the expressed CFI-HSA protein was passed through a 10 ml gravity-flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). The bound protein was washed with 10 column volumes of 20 mM sodium phosphate buffer. The bound CFI-HSA protein was eluted in two steps: first, with 3 column volumes of 20 mM Tris-HCl, pH 7.0 buffer containing 2 M MgCl2, and second, with 3 column volumes of 20 mM citric acid, pH 3.0. The elution from both steps 1 and 2 was collected in 5 ml fractions. Each fraction of the elution from step 2 was neutralized with 10% neutralization buffer (1.5 M Tris-HCl pH 7.4). All fractions were analyzed by reducing and non-reducing SDS-PAGE electrophoresis, and bands were visualized by SimplyBlue™ SafeStain (ThermoFisher Scientific). CFI-HSA migrates as a 130 kDa band on non-reducing gels and as 102 kDa and 28 kDa bands on reducing gels. Fractions with the greatest CFI-HSA concentration and purity were pooled for further processing.

[0113] Furin activation CFI-HSA is expressed as an inactive single-chain precursor protein and is activated by another serine protease, furin. Furin is an endoprotease that cleaves CFI at its conserved RRKR sequence (also called the furin recognition sequence), resulting in a heavy and light chain connected by a disulfide bond. The furin-processed mature two-chain protein is the activated form of the CFI protein.

[0114] Cleavage of CFI-HSA to produce the protein in its activated form was performed by incubating 4 μg of recombinant furin per mg of purified CFI-HSA in Tris-NaCl, 2.5 mM CaCl2, and 0.5% CHAPS for 18 h at 30°C. The CFI-HSA protein concentration was maintained at 1.4 mg / ml, which results in more than 90% activation of the protein. The activated protein was separated from the inactivated CFI-HSA and other proteins by size exclusion chromatography. Size exclusion chromatography (SEC) was performed using a HiLoad 16 / 600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) as the mobile phase. The collected fractions were analyzed by CE-SDS (LabChip GXII, PerkinElmer). The fractions containing the target protein were pooled and analyzed by SE-UPLC.

[0115] In Vitro Sialylation The activated CFI-HSA protein was subjected to in vitro sialylation. Briefly, sialylation was carried out in a two-step enzymatic reaction. First, the galactosylation reaction of CFI-HSA was carried out in a volume of 200 μl using a 1:200 molar ratio of galactosyltransferase (GalT1) enzyme and CFI-HSA in 10 mM UDP-galactose, 5 mM MnCl2, and 100 mM MES, pH 6.5 buffer. Galactosylated CFI-HSA was purified from the reaction mixture by CaptureSelect™ human albumin affinity chromatography as previously described. The sialylation reaction was then carried out in a volume of 250 μl at 37° C. for 1 h using a 1:50 molar ratio of the enzyme alpha 2,6-sialyltransferase and purified CFI-HSA in 80 μM alkaline phosphatase, 6.1 mM CMP-NANA, 10 mM ZnCl2, and 200 mM MES buffer, pH 6.5. Sialylated CFI-HSA protein was purified from the reaction mixture by CaptureSelect™ human albumin affinity chromatography. The extent and characteristics of sialic acid chains on CFI-HSA were determined by using Agilent / Prozyme analytical services, the GS-SAP method for total sialic acid quantification (Agilent GS48), and mass spectrophotometric (MS) analysis (Lake Pharma analytical services), as described in more detail below.

[0116] Briefly, total sialic acid quantification was performed by mixing 20 μl of each sample with 10 μl of release reagent in a 96-well plate. The reaction mixture was incubated at 80° C. for 2 h. The samples were cooled to room temperature and 10 μl of labeling reagent was added to each sample for further incubation at 50° C. for 3 h. The samples were cooled again to room temperature and 160 μl of de-ionized (dI) water was added to a total volume of 200 μl. 10 μl of sample was injected onto an Agilent UHPLC Poroshell C18 column and run at 30° C. in 4% methanol in water, 8% acetonitrile (line A1) and 100% ACN (line B1) at a flow rate of 0.4 ml / min. Peaks were recorded at 373 / 448 nm wavelength. A standard curve of total peak area versus picomoles (pmol) of sialic acid was generated by running 1-2000 pmol of NANA (Neu5Ac), supplied with the kit, on the same column. Total sialic acid in each sample was quantified by comparing the peak area of ​​the sample to the standard curve.

[0117] Mass spectrometry was performed by a standard trypsin Q-TOF mass spectrometer. Briefly, all samples were treated with DTT and iodoacetamide, reduced, alkylated, and then digested with trypsin. The digested samples were analyzed by a Waters ACQUITY UPLC coupled to a Xevo G2-XS-QTOF mass spectrometer using a protein BEH C18 column.

[0118] Sophistication The purified CFI-HSA protein was subjected to size-exclusion chromatography (SEC) using a HiLoad 16 / 600 Superdex 200 column (GE Healthcare Life Sciences) and phosphate buffer saline as the mobile phase. The collected fractions were analyzed by CE-SDS (LabChip GXII, PerkinElmer). The fractions containing the target protein were pooled, the concentration was brought to 5 mg / ml, and the samples were flash frozen for storage at -80°C.

[0119] Expression and purification of CFI-HSA mutants DNA for the CFI-HSA variants was generated either synthetically or by site-directed mutagenesis using standard techniques. Proteins were expressed in 250 ml suspension of TunaCHO™ cells as described herein for wild-type CFI-HSA protein, except that expression was for 7 days instead of 14 days. After 7 days, cells were centrifuged and conditioned medium was passed over a gravity-flow column of CaptureSelect™ human albumin affinity matrix (ThermoFisher Scientific). Proteins bound to the column were washed with 10 column volumes of 20 mM sodium phosphate buffer. Bound CFI-HSA protein was eluted in 5 ml fractions with 3 column volumes of 20 mM Tris-HCl, pH 7.0 buffer containing 2 M MgCl2. CFI-HSA or its variants were buffer exchanged (either by dialysis or spin concentrator) into 30 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, pH 7.4. Recombinant human furin was added to CFI-HSA at a molar ratio of 1:25 (furin:CFI-HSA) and the reaction mixture was incubated for 16 hours at 30° C. Two micrograms of the activation mixture was run on a 9% SDS-PAGE gel to assess activation efficiency. In general, over 80% activation was achieved.

[0120] Example 2: Formulation Development Study I For Example 2, references to CFI-HSA refer to human serum albumin (SEQ ID NO: 21) fused to the N-terminus of wild-type CFI. CFI-HSA was formulated at a concentration of approximately 150 mg / mL.

[0121] The study was designed to explore the physical and chemical properties of CFI-HSA in a range of liquid and lyophilized formulations to evaluate conditions that would provide optimal options for stability. For example, high concentration and stability of CFI-containing compositions is desired to enable subcutaneous administration.

[0122] The active pharmaceutical ingredient (API) investigated in this study was CFI-HAS (SEQ ID NO: 21). Materials used in this study included: (1) Drug substance: CFI-HSA, 153.4 mg / mL, F1 formulation (2) Chemicals and materials used to formulate and analyze CFI-HSA are as follows and are shown in Table 2.1:

[0123] [Table 4]

[0124] In this stability study, the stability of CFI-HSA was monitored in 12 formulations containing various tonicity modifiers / bulking agents across an optimal range of pH. Table 2.2 provides the 12 formulation matrices tested. (1) pH 5 to 7 (2) Buffer Solution (also referred to interchangeably herein as a buffering agent) (3) Tonicity modifying agents (sorbitol or trehalose for non-ionic buffers, sodium chloride for ionic buffers) (4) Bulking agent (trehalose or glycine) (5) Surfactants

[0125] [Table 5] * Form. = Formulation, API = Active Pharmaceutical Ingredient, CFI-HSA, Lyo = Lyophilized

[0126] Preparation of lyophilized formulations For each formulation, the CFI-HSADS solution was loaded into a dialysis cassette (Slide-A-Lyzer® Dialysis Cassette, 10,000 MWCO). The formulations were dialyzed into the respective formulation buffer.

[0127] The formulations were sterile filtered through a 0.2 μm PES membrane in a sterile BSC and filled to 0.2 mL into 2 cc sterile glass vials. The vials were then partially stoppered with a lyophilization vent stopper and lyophilized using the parameters outlined in Table 2. After lyophilization, the chamber was backfilled with nitrogen and the vials were stoppered. The vials were then removed, stoppered, crimped, and labeled. An additional vial was set aside and frozen pre-lyophilization for pre-lyophilization analysis in parallel with time zero.

[0128] Liquid formulation preparation For each formulation, the CFI-HSADS solution was loaded into a dialysis cassette (Slide-A-Lyzer® Dialysis Cassette, 10,000 MWCO). The formulations were dialyzed into the respective formulation buffer. The formulations were sterile filtered through a 0.2 μm PES membrane in a sterile BSC and filled to 0.2 mL into 2 cc sterile glass vials. The vials were then stoppered, crimped, and labeled.

[0129] Analysis method The following analytical methods were used: (1) Visual inspection: Visual inspection was performed under a white light source (13 W fluorescent tube) against a black or white background. Digital photographs of all formulations were taken at each time point. (2) Concentration measurement: The concentration of CFI-HSA was 1.041 mg / mL. -1 ×cm -1 The samples were analyzed by A280 via SoloVPE using an EC of 1000. (3) SE-HPLC: Size-exclusion HPLC chromatography was performed with the following parameters:

[0130] [Table 6] (4) FlowCAM: The FlowCAM particle imaging system combines optics, electronics, and fluidics for automated analysis of particles. The optical system is used to capture real-time images of particles in the fluid as they pass through a flow cell. The imaging software provides the ability to assess particle size and morphology. All samples analyzed were degassed at 75 torr for 30 minutes prior to analysis. (5) Sub-ambient temperature DSC: Approximately 10 μL of each sample was frozen at −60° C. using a Pyris Diamond DSC equipped with an intercooler II. The samples were warmed until thawed at a ramp rate of 10° C.min, and the heat flow during the warming process was recorded. (6) Fourier Transform Infrared Spectroscopy (FTIR): FTIR is a technique used to obtain infrared spectra of the absorption of gases, liquids, or solids. The IBI is the absorption spectrum of the sample material versus wavenumber [cm -1

[0036] An FTIR-660Plus spectrometer is used to collect the absorption of infrared radiation by . This data provides information about the secondary structure of the polypeptide.

[0131] Surfactant Screening CFI-HSA drug substance was diluted to 15 mg / mL with water. The solution was then sterile filtered through a 0.2 μm syringe filter and filled into 3 cc vials with a fill volume of 1.0 mL. Four vials were then directly spiked with stock solutions of either 1% PS20, 1% PS80, or 10% F-68, respectively, to reach final concentrations of 0.015% PS20, 0.015% PS80, or 0.15% F-68. Four of the filled vials were used as controls without surfactant. A total of four vials per condition were prepared. The vials per condition were placed on a shaker and agitated at 1000 rpm for 4 hours at ambient temperature. One vial per condition was incubated simultaneously for 4 hours at ambient temperature.

[0132] [Table 7]

[0133] The stability of CFI-HSA with and without surfactant was studied after 4 hours of agitation (1000 RPM on an orbital shaker, room temperature) and 4 hours statically at room temperature.

[0134] After stirring and settling, all samples appeared clear, colorless and free of visible particulates, regardless of surfactant.

[0135] After 4 hours of stirring or static incubation, similar chromatographic profiles were observed for all samples with and without PS80, PS20, and F68. All samples also showed similar purity and total peak area (Table 2.4).

[0136] [Table 8]

[0137] The FlowCam analysis is shown in Table 2.5. The surfactant-free sample shows a significant increase in particle counts. However, all surfactant-containing samples showed relatively low particle concentrations, regardless of surfactant.

[0138] [Table 9]

[0139] Based on these results, 0.02% polysorbate 80 was determined to be one optimal choice for the next round of formulation optimization studies.

[0140] Formulation optimization The objective of the accelerated stability study was to evaluate the stability of both liquid and lyophilized formulations containing 150 mg / mL CFI-HSA. All formulations were filled at a fill volume of 0.2 mL and F7-F12 were lyophilized. Pre-lyophilized samples were frozen at -70°C during the lyophilization cycle and thawed at time zero for analysis.

[0141] The study included incubation of all formulation candidates at refrigerated (5°C), stressed (25°C), and accelerated (40°C) storage temperatures. The temperature storage portion of the study was conducted over a period of four weeks. Table 2.6 summarizes the storage conditions used for formulation stability evaluation.

[0142] [Table 10]

[0143] At time zero, the vials were placed at the appropriate temperature according to Table 2.6. At each time point, a vial was withdrawn and analyzed.

[0144] At time zero, all pre-lyophilized formulations appeared clear, colorless, and free of visible particulates. The lyophilized formulations showed good cakes. All liquid and reconstituted lyophilized formulations appeared clear, slightly yellowish in color, and free of visible particulates. No significant changes were observed in the lyophilized cakes, regardless of storage temperature, throughout the study time points up to 4 weeks. Similarly, most liquid and reconstituted lyophilized formulations appeared clear and yellow with no visible particulates at all temperatures.

[0145] At time zero, all formulations showed concentrations close to the target, ranging from 130 mg / mL to 160 mg / mL. Throughout the study time points up to 4 weeks, most formulations at 5°C, 25°C, and 40°C showed concentrations within the experimental variability. Table 2.7 shows the stability concentration results.

[0146] [Table 11]

[0147] The FTIR spectrum of CFI-HSA is 1650-1660 cm -1The FTIR spectra of CFI-HSA appeared to be similar to those reported for albumin, which has a strong helical signal at 100 nm. No major changes in the FTIR spectra were observed when CFI-HSA was lyophilized in the candidate formulations.

[0148] At time zero, all formulations, including the reconstituted lyophilized formulation, showed comparable chromatographic profiles with minor HMW (high molecular weight) and LMW (low molecular weight) peaks (SE-HPLC results).

[0149] After 1 week of storage at 25-40°C, all liquid formulations showed an increase in HMWS and LMW. The increase in LMW was much faster at lower pH. However, all lyophilized formulations remained much more stable, especially with no signs of an increase in LMW. (SE-HPLC results)

[0150] After 2 weeks of storage at 5, 25, and 40° C., all liquid formulations showed an increase in HMWS and LMW. The increase in LMW was much faster at lower pH. However, all lyophilized formulations remained much more stable, especially with no signs of an increase in LMW. (SE-HPLC results)

[0151] The general degradation process remained consistent at 4 weeks at 5, 25, and 40° C.: all liquid formulations showed an increase in HMWS and LMW. The increase in LMW was much faster at lower pH. However, all lyophilized formulations remained more stable, especially with no signs of an increase in LMW. (SE-HPLC results)

[0152] The degradation rate of each tested formulation was compared over a 4 week period stored at 40° C. Of all the formulations tested, the lyophilized formulation containing both trehalose and glycine as bulking agents performed best in this study in the pH range of 6-7.

[0153] findings In this example, the stability of 150 mg / mL CFI-HSA in various formulation conditions was investigated. Conditions investigated included liquid and lyophilized formulations containing various buffers (20 mM sodium acetate, sodium succinate, or histidine.HCl), tonicity modifiers (sodium chloride, sorbitol, or trehalose), bulking agents (trehalose or glycine), and spanning pH values ​​ranging from 5.0 to 7.0. Formulations were investigated under static storage conditions at refrigerated (5°C), ambient (25°C), and accelerated (40°C) temperatures for up to 4 weeks. Analyses were performed over the course of the study by visual inspection, concentration, SE-HPLC, and FlowCAM. In addition, lyophilized formulations were also analyzed by DSC and FTIR.

[0154] Initially, a small surfactant screen was performed using commonly used surfactant stabilizers. This study evaluated the benefit of surfactants in stabilizing CFI-HSA against degradation after exposure to agitation-induced shear stress. 15 mg / mL CFI-HSA drug substance was spiked with the surfactants 0.015% PS20, 0.015% PS80, and 0.15% F68. Comparison of surfactant-containing vials after agitation stress by SE-HPLC showed comparable profiles for all samples regardless of agitation. However, MFI analysis demonstrated that samples without surfactant showed substantially increased subvisible particulate concentrations compared to those containing surfactant. This provided evidence that CFI-HSA is sensitive to shear stress and that improved stability could be achieved with the addition of surfactants in further studies. The current surfactant in the drug substance, 0.02% PS80, was selected for the accelerated stability study.

[0155] After preparation of the 12 candidate formulations, all samples appeared clear and free of visible particulates at time zero.

[0156] Throughout the entire study, no significant changes in visual appearance were observed in the lyophilized cakes, regardless of storage temperature. Similarly, most liquid and reconstituted lyophilized formulations appeared clear with a characteristic yellow hue at all temperatures. After 4 weeks, most formulations were near target concentration at all storage conditions.

[0157] FTIR results found no significant differences in the spectral profiles between pre-lyophilized and freeze-dried samples.

[0158] FlowCAM analysis showed relatively low subtracted subvisible particle concentrations for all samples, suggesting that precipitation or particles are not a significant factor for properly formulated CFI-HSA.

[0159] The accelerated studies were under extreme pressure conditions and may not be predictive. Nevertheless, analysis by SE-HPLC after 4 weeks at all temperatures found significantly higher LMW in the liquid formulations, especially in the low pH formulations such as F1, F2, F3, and F4. Even at pH 6, F5 and F6 showed an increase of about 1% in LMW during 4 weeks of storage at 5°C. Additives effective in inhibiting autodegradation of CFI-HSA may be useful to include. Autodegradation is stopped when CFI-HSA is lyophilized. No significant increase in LMW was observed when the lyophilized formulations were stored at 40°C for 4 weeks. Among those tested, formulations F8, F10, and F12, which contain glycine as a bulking agent, an ionic bulking agent, compared to nonionic trehalose, showed improved stability against aggregation. In addition, aggregation slowed as the pH increased from 5 to 7.

[0160] Key degradation products and stability indicative assays were identified as increased HMW and LMW by SE-HPLC. Based on all results generated in this study, one lead formulation for 150 mg / mL CFI-HSA could be lyophilized conditions of 20 mM histidine, 5.0% trehalose, 2.0% glycine, and 0.02% polysorbate 80 at pH 6.0-7.0.

[0161] Example 3: Formulation Development Study II Further formulations for the active ingredients described herein have been developed in indepenf studies. It is desirable to achieve high concentrations and stability of CFI-containing compositions to enable subcutaneous administration.

[0162] For Example 3, references to CFI-HSA refer to human serum albumin (SEQ ID NO: 21) fused to the N-terminus of wild-type CFI. CFI-HSA was formulated at concentrations ranging from 30 to 189 mg / mL.

[0163] Biophysical characterization and pH stability profile A stock solution of 5 mg / mL CFI-HSA was dialyzed against citrate phosphate buffers of pH 5, 5.5, 6, 6.5, 7, or 7.5 containing 135 mM sodium chloride. Following visual appearance to assess any potential precipitation, samples were 0.2 micron filtered, adjusted to 2 mg / mL in the corresponding buffer, and aseptically transferred to microfuge tubes. Sets of samples were stored at either -70°C, 25°C, or 40°C. After 7 days, sets were analyzed by appearance (opalescence and particles), protein concentration (OD280), SEC, DSF, DLS, icIEF, CE-SDS, and S2288 chromogenic activity assays.

[0164] After 7 days of storage at either 25° C. or 40° C., all samples were found to be visually clear and free of visually detectable particles at both temperatures and all pH levels tested. Protein concentrations were stable in all samples (Table 3.1).

[0165] [Table 12]

[0166] SEC-HPLC was used to assess aggregation under storage conditions. At 25°C storage, the main peak purity showed a steady increase as the pH decreased from 7.5 to 5.0. In contrast, at pH 5.0 and 40°C storage conditions, the main peak purity decreased sharply and low molecular weight species were spiked, indicating potential proteolysis had occurred. In summary, under these conditions, pH 5.5-6.0 appeared to be the optimal range for CFI-HSA stability.

[0167] To further evaluate aggregation under storage conditions, the hydrodynamic size of the aggregate collection of particles was measured by dynamic light scattering (DLS). Results showed that both the intensity weighted average (Zave) and polydispersity increased sharply at pH 5 but were comparable at pH 6-7.5. Meanwhile, the size of the major species (>99.8%) increased slightly with increasing pH. Taken together, the polydispersity and particle size data measured by DLS indicated an optimal storage pH of approximately 6, supporting the SEC-HPLC results.

[0168] CE-SDS was performed to assess purity changes using preformulations. Results showed a slight decrease in purity at pH 5 and 7.5 and comparable purity at pH 5.5-6.5 with both reduced and non-reduced CE-SDS. Similarly, an optimal pH level of 5.5-7.0 was suggested by differential scanning fluorimetry (DSF), while pH 5 was found to be the least stable by imaged capillary isoelectric focusing (icIEF).

[0169] Highly concentrated liquid formulation The feasibility of a high concentration formulation was evaluated. Purified CFI-HSA was dialyzed against 20 mM Histidine, 150 mM ArgHCl, pH 5.8 and concentrated to 189 mg / mL in a spin filter unit. A high concentration of arginine was used to increase solubility and prevent excess viscosity caused by concentrated CFI-HSA. To evaluate the effect of protein concentration and arginine concentration on viscosity, the 189 mg / mL concentrated sample was also diluted to 169 mg / mL in 20 mM His, pH 5.8 to reach 135 mM ArgHCl. The data presented in Table 3.2 showed that even at 189 mg / mL CFI-HSA the viscosity was only 10.6 centipoise (CP), indicating that the risk of excess viscosity was minimal using the formulation tested.

[0170] [Table 13]

[0171] Concentrated samples were 0.2 micron filtered and tested for aggregation using SEC. No change in aggregate levels (1.5%) was observed in a 188.7 mg / mL sample in 150 mM arginine that was diluted to 5 mg / mL and stored in an autosampler at 5° C. A 169.7 mg / mL sample that was diluted to 5 mg / ml and stored in an autosampler at 5° C. for one week showed only 1.72% aggregates. After one week at 5° C., a 169.7 mg / mL sample that was then diluted to 5 mg / ml showed 2.5% HMW species.

[0172] The bioactivity of the CFI-HSA sample concentrated to 169.7 mg / mL was assessed using the S2288 chromogenic assay. As shown in Figure 20, the activity of the 169.7 mg / mL sample is comparable to that of the control (Ctl) sample. The slight difference in the slope of the curves may be due to either imprecision in the final concentration in the assay after significant dilution of the viscous solution or some loss of bioactivity after approximately 1 month of storage at 5°C.

[0173] Taken together, these results suggest that a liquid formulation containing arginine hydrochloride and a histidine buffer can stabilize CFI-HSA at high concentrations (e.g., greater than 150 mg / mL).

[0174] Formulation Screening A panel of formulations (F1-F7) was screened to determine the effect of ionic strength, buffer type, cryoprotectant, and calcium chloride (CaCl2) on the stability of CFI-HSA. The formulations contained pharma- ceutically acceptable excipients and a range of concentrations of tonicity modifiers (also referred to interchangeably herein as isotonicity agents), cryoprotectants, lyoprotectants, stabilizers, and surfactants added to the histidine and arginine buffers previously tested. Table 3.3 shows formulations F1-F7 tested in this example and should not be confused with F1-F12 from Example 2.

[0175] [Table 14]

[0176] Stock CFI-HSA samples were concentrated to approximately 40 mg / mL and then dialyzed against the seven formulations listed in Table 3.3. The concentrated samples were 0.2 micron filtered and aseptically filled into glass vials and capped. One vial of each composition was subjected to three cycles of freezing and thawing and evaluated by appearance, OD280, and SEC. For short-term thermal stability studies, four vials of each composition were included in a five-week stability study, one each at -70°C, 5°C, 25°C, and 40°C. The five-week stability samples were analyzed by OD280, SEC, and CE-SDS. Additionally, peptide map MS / MS and bioactivity analysis were performed on selected samples to assess deamidation and oxidation.

[0177] After 5 weeks, visual inspection showed that all formulations were colloidally stable, except for F3, where samples had signs of precipitation when stored at 40° C. Protein concentration measurements at OD280 showed that CFI-HSA concentrations remained unchanged after three freeze / thaw cycles of storage for 1 or 5 weeks at all tested temperatures.

[0178] Purity assessment by SEC and CD-SDS showed that for all seven formulations, the -70°C samples were unchanged in purity, the 5°C samples were approximately 97% pure, the 25°C samples were approximately 96% pure, and the 40°C samples were all 90-92% pure of the major species. The significant decrease in purity at 40°C and the slight decrease in purity at 25°C over 5 weeks of storage in all seven formulations indicates that liquid high concentration formulations at room temperature are not feasible.

[0179] Bioactivity assays were performed to verify that CFI-HSA maintained activity after storage in the formulations and storage conditions tested (Table 3.4). The formulations are relatively comparable overall. Formulation F1 is slightly more stable than the other six formulations at 40°C. At both 25°C and 40°C, formulation F3 was the least stable, exhibiting 20-30% less activity compared to the -70°C control and a greater loss of activity than the F2, F4, or F1 samples.

[0180] [Table 15]

[0181] Peptide mapping MS / MS was performed on CFI-HSA formulations to determine the levels of deamidation and oxidation after the storage conditions tested. Samples of formulations F1, F2, F4, F5, and F6 were tested after 5 weeks of storage at 5° C. Frozen F4 (F / T) was run as a control. Results showed that the deamidation and oxidation levels at all sites were comparable between the different formulations.

[0182] Additional studies and formulations developed for clinical evaluation and drug manufacturing include the formulations in Table 3.5. CFI-HSA was buffer exchanged into the three new formulations in Table 3.5 and concentrated to 100 mg / mL or 150 mg / mL, but no precipitation, severe opalescence, or high viscosity was observed. To make the formulations more suitable for lyophilized products, 60 mM glycine or 60 mM mannitol was added to F2 and F3, respectively, to protect the structure of the lyophilized cake. Samples were then filter sterilized, filled, and tested for viscosity, SEC, and activity.

[0183] [Table 16]

Claims

1. A formulation comprising wild-type complement factor I (CFI) or a variant thereof (CFI variant), said formulation comprising a buffer, a surfactant, and a tonicity modifying agent.

2. 10. The formulation of claim 1, wherein the formulation comprises one or more of a bulking agent, a cryoprotectant, and a lyoprotectant.

3. 10. The formulation of claim 1, wherein the CFI or variant thereof is present at a concentration of about 10 mg / ml to about 300 mg / ml.

4. 10. The formulation of claim 1, wherein the buffering agent comprises acetate, histidine, or succinate.

5. 10. The formulation of claim 1, wherein the buffering agent is present at about 1 mM to about 50 mM.

6. 2. The formulation of claim 1, wherein the surfactant is polysorbate 80, polysorbate 20, polysorbate 80 / 20, or poloxamer F68.

7. 7. The formulation of claim 6, wherein the surfactant is present at about 0.001% to about 0.1% v / v.

8. 2. The formulation of claim 1, wherein the tonicity modifying agent is sorbitol, trehalose, sodium chloride, or arginine hydrochloride.

9. 3. The formulation of claim 2, wherein the bulking agent is trehalose or glycine.

10. The tonicity modifying agent, (i) sorbitol, present at about 1% to about 10% v / v; (ii) trehalose, present at about 1% to about 15% v / v; (iii) sodium chloride, present at about 1 mM to about 500 mM, and (iv) arginine hydrochloride, present at about 10 mM to about 200 mM The formulation of claim 1 , wherein the formulation is selected from:

11. 10. The formulation of claim 9, wherein the glycine is present at about 1% to about 5% v / v or about 1 mM to about 100 mM.

12. 3. The formulation of claim 2, wherein the cryoprotectant or lyoprotectant is selected from glucose, sucrose, glycine, sorbitol, trehalose, sucrose, or mannitol. (a) glucose is present at about 4% v / v; (b) sucrose is present at about 1% to about 10% v / v; (c) glycine is present at about 50 mM to about 150 mM; (d) trehalose is present at about 1% to about 10% v / v; (e) mannitol is present at about 1 mM to about 100 mM; or (f) sorbitol is present at about 1% to about 5% v / v; The formulation of claim 12.

14. The formulation is as shown in Table 1 or Table 2 below. Table 1 ＊ Form. = Formulation, API = Active Pharmaceutical Ingredient, CFI-HSA, Lyo = Lyophilized Table 2 2. The formulation of claim 1, selected from the formulations presented in

15. 10. The formulation of claim 1, wherein the formulation is in solid, lyophilized, or liquid form.

16. 10. The formulation of claim 1, wherein the formulation is stable at any one or more of -80°C + / - 2°C, -20°C + / - 2°C, 0°C + / - 2°C, 4°C + / - 2°C, 25°C + / - 2°C, 37°C + / - 2°C, 45°C + / - 2°C, or 60°C + / - 2°C.

17. 10. The formulation of claim 1, wherein the formulation is stable for at least one week, at least one month, or at least one year.

18. The formulation of claim 1 , wherein the formulation comprises wild-type CFI.

19. The formulation of claim 1, wherein the formulation comprises a CFI variant that includes at least one modification relative to wild-type CFI, the CFI variant being capable of regulating the complement system, and the CFI variant having at least one improved characteristic compared to the wild-type CFI.

20. The CFI mutants are as follows: Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 20. The formulation of claim 19, selected from those presented in

21. The formulation of claim 1, wherein the wild-type CFI or CFI variant is a first component of a fusion construct comprising a first component and at least a second component, and the wild-type CFI or CFI variant is fused to the second component.

22. A formulation comprising a fusion construct comprising wild-type CFI or a variant thereof (CFI variant), wherein the wild-type CFI or the CFI variant is fused to human serum albumin, and the formulation is selected from those presented in Table 1 or Table 2 of claim 14.

23. 23. The formulation of claim 22, wherein the fusion construct comprises the amino acid sequence of SEQ ID NO:

21.

24. A formulation according to any one of claims 1 to 23 for use in a method for treating a condition in a subject.

Images