Pharmaceutical compositions of fusion proteins and methods of use thereof

Abstract

The present disclosure provides pharmaceutical compositions formulated for high concentrations of fusion proteins for subcutaneous administration, and methods of using the same. Additionally, the present disclosure provides methods for developing high-concentration protein formulations. In a first aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein, a detergent, and a buffer, wherein the fusion protein is present at a concentration of at least 120 mg / mL. In some embodiments, the fusion protein comprises a complementarity-determining region (CDR) having an amino acid sequence of SEQ ID NO: 2-7.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on August 3, 2023, is named 51196-031WO2_Sequence_Listing_8_3_23.xml, and is 50,310 bytes in size. [Background technology]

[0002] Subcutaneous administration of protein therapeutics offers several advantages over other administration methods. For example, subcutaneous administration has been shown to improve patient compliance, increase convenience, and improve pharmacokinetic and pharmacodynamic profiles. However, one of the challenges in developing formulations for subcutaneous administration is that the injection volume is typically less than 2 mL. As a result, proteins in pharmaceutical compositions must be present at high concentrations. Therefore, there is still a need for highly concentrated formulations to achieve adequate dosing. Several challenges remain associated with the development of highly concentrated formulations, such as viscosity, stability, and delivery. Summary of the Invention [Means for solving the problem]

[0003] In a first aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein, a detergent, and a buffer, wherein the fusion protein is present at a concentration of at least 120 mg / mL. In some embodiments, the fusion protein comprises a complementarity-determining region (CDR) having an amino acid sequence of SEQ ID NO: 2-7. In some embodiments, the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 55 and a second portion comprising an amino acid sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 56. In some embodiments, the fusion protein consists of the amino acid sequence of SEQ ID NO: 1 or a modification thereof. In some embodiments, the modification comprises converting the N-terminal glutamine of SEQ ID NO: 1 to pyroglutamate.

[0004] In some embodiments, the concentration of the fusion protein is 120 mg / mL to 250 mg / mL (e.g., 120 mg / mL to 250 mg / mL, 150 mg / mL to 250 mg / mL, 175 mg / mL to 250 mg / mL, 200 mg / mL to 250 mg / mL, 225 mg / mL to 250 mg / mL, 120 mg / mL to 225 mg / mL, 120 mg / mL to 200 mg / mL, 120 mg / mL to 175 mg / mL, 120 mg / mL to 150 mg / mL, or 150 mg / mL to 180 mg / mL). In some embodiments, the fusion protein is present at a concentration of at least 150 mg. In some embodiments, the concentration of the fusion protein is 150 mg / mL to 200 mg / mL (e.g., 150 mg / mL to 200 mg / mL, 150 mg / mL to 190 mg / mL, 150 mg / mL to 180 mg / mL, 150 mg / mL to 170 mg / mL, 150 mg / mL to 160 mg / mL, 160 mg / mL to 200 mg / mL, 170 mg / mL to 200 mg / mL, 180 mg / mL to 200 mg / mL, or 190 mg to 200 mg / mL) or more, for example, 250 mg / mL. In some embodiments, the concentration of the fusion protein is 170 mg / mL to 200 mg / mL (e.g., 170 mg / mL to 195 mg / mL, 170 mg / mL to 190 mg / mL, 170 mg / mL to 185 mg / mL, 170 mg / mL to 180 mg / mL, 170 mg / mL to 175 mg / mL, 175 mg / mL to 200 mg / mL, 180 mg / mL to 200 mg / mL, 185 mg / mL to 200 mg / mL, 190 mg / mL to 200 mg / mL, or 195 mg to 200 mg / mL). In some embodiments, the concentration of the fusion protein is about 190 mg / mL. In some embodiments, the concentration of the fusion protein is about 150 mg / mL.

[0005] In some embodiments, the detergent is a polysorbate. In some embodiments, the polysorbate is polysorbate 80 (PS80). In some embodiments, the concentration of the polysorbate is 0.001% to 1% (w / v) (e.g., 0.001% (w / v) to 0.8% (w / v), 0.001% (w / v) to 0.6% (w / v), 0.001% (w / v) to 0.4% (w / v), 0.001% (w / v) to 0.2% (w / v), 0.001% (w / v) to 0.05% ( In some embodiments, the concentration of polysorbate is about 0.05% (w / v). In some embodiments, the concentration of polysorbate is 0.05% (w / v) to 0.5% (w / v) (e.g., 0.05% (w / v) to 0.3% (w / v), 0.05% (w / v) to 0.1% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.2% (w / v) to 0.5% (w / v), or 0.3% (w / v) to 0.5% (w / v)). In some embodiments, the concentration of polysorbate is 0.1% (w / v) to 0.2% (w / v) (e.g., 0.1% (w / v), 0.11% (w / v), 0.12% (w / v), 0.13% (w / v), 0.14% (w / v), 0.15% (w / v), 0.16% (w / v), 0.17% (w / v), 0.18% (w / v), 0.19% (w / v), or 0.2% (w / v)). In some embodiments, the concentration of polysorbate is about 0.15% (w / v).

[0006] In some embodiments, the detergent is present in a concentration of 0.01% (w / v) to 1.5% (w / v) (e.g., 0.01% (w / v) to 0.4% (w / v), 0.01% (w / v) to 0.3% (w / v), 0.01% (w / v) to 0.2% (w / v), 0.01% (w / v) to 0.1% (w / v), 0.01% (w / v) to 1% (w / v), 0.1 %(w / v) to 1.5%(w / v), 0.2%(w / v) to 1.5%(w / v), 0.3%(w / v) to 1.5%(w / v), 0.4%(w / v) to 1.5%(w / v), 0.5%(w / v) to 1.5%(w / v), 0.8%(w / v) to 1.5%(w / v), or 1%(w / v) to 1.5%(w / v). In some embodiments, the detergent is present at a concentration of 0.01% (w / v) to 1% (w / v) (e.g., 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v), or 1% (w / v)). In some embodiments, the detergent is present at a concentration of about 0.05% (w / v) to about 0.1% (w / v) (e.g., 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), or 0.1% (w / v)). In some embodiments, the detergent is present at a concentration of about 0.05% (w / v). In some embodiments, the detergent is present at a concentration of about 0.1% (w / v).

[0007] In some embodiments, the buffering agent is acetate, histidine, phosphate, or succinate, or a combination thereof. In some embodiments, the buffering agent is acetate. In some embodiments, the acetate is sodium acetate. In some embodiments, the concentration of sodium acetate is about 10 mM to 150 mM (e.g., 10 mM to 140 mM, 10 mM to 130 mM, 10 mM to 120 mM, 10 mM to 100 mM, 10 mM to 75 mM, 10 mM to 50 mM, 20 mM to 150 mM, 30 mM to 150 mM, 75 mM to 150 mM, 100 mM to 150 mM, or 40 mM to 150 mM). In some embodiments, the concentration of sodium acetate is about 15 mM to 100 mM (e.g., about 15 mM to 80 mM, 15 mM to 60 mM, 15 mM to 40 mM, 15 mM to 20 mM, 20 mM to 100 mM, 40 mM to 100 mM, 60 mM to 100 mM, or 80 mM to 100 mM). In some embodiments, the concentration of sodium acetate is about 50 mM. In some embodiments, the concentration of sodium acetate is about 20 mM.

[0008] In some embodiments, the pharmaceutical composition further comprises an amino acid. In some embodiments, the amino acid is proline. In some embodiments, the amino acid is arginine. In some embodiments, the amino acid is glycine. In some embodiments, the amino acid is present in the pharmaceutical composition at a concentration of 100 mM to 200 mM (e.g., 100 mM to 190 mM, 100 mM to 180 mM, 100 mM to 170 mM, 140 mM to 200 mM, 150 mM to 200 mM, 160 mM to 200 mM, 110 mM to 190 mM, 120 mM to 180 mM, 130 mM to 170 mM, 140 mM to 180 mM, 150 mM to 170 mM, or 160 mM to 170 mM). In certain embodiments, the concentration of the amino acid is about 165 mM.

[0009] In some embodiments, the amino acids have a concentration of about 10 mM to 200 mM (e.g., 10 mM to 150 mM, 10 mM to 100 mM, 10 mM to 50 mM, 10 mM to 20 mM, 20 mM to 200 mM, 50 mM to 200 mM, 100 mM to 200 mM, or 150 mM to 200 mM). In some embodiments, the amino acids have a concentration of about 100 mM to 200 mM (e.g., 100 mM to 180 mM, 100 mM to 160 mM, 100 mM to 140 mM, 100 mM to 120 mM, 120 mM to 200 mM, 140 mM to 200 mM, 160 mM to 200 mM, or 180 mM to 200 mM).

[0010] In some embodiments, the pharmaceutical composition further comprises a tonicity agent. In some embodiments, the tonicity agent is a sugar, an amino acid, or a salt. In one embodiment, the salt is NaCl. In some embodiments, the sugar is sucrose, glucose, glycerol, or trehalose. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose is present at a concentration of about 1% (w / v) to about 15% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), or 15% (w / v)). In some embodiments, sucrose is present at a concentration of 2% (w / v) to 10% (w / v) (e.g., 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, sucrose is present at a concentration of about 4% (w / v) to about 9% (w / v) (e.g., 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, sucrose is present at a concentration of about 4% (w / v). In some embodiments, sucrose is present at a concentration of 8% (w / v) to 9% (w / v) (e.g., 8.1% (w / v), 8.2% (w / v), 8.3% (w / v), 8.4% (w / v), 8.5% (w / v), 8.6% (w / v), 8.7% (w / v), 8.8% (w / v), 8.9% (w / v), or 9% (w / v)). In some embodiments, sucrose is present at a concentration of about 8.6% (w / v).

[0011] In some embodiments, the pharmaceutical composition has a pH of about pH 3 to pH 8 (e.g., pH 3 to pH 6, pH 3 to pH 4, pH 4 to pH 8, or pH 6 to pH 8). In some embodiments, the pharmaceutical composition has a pH of about pH 4 to pH 7 (e.g., pH 4 to pH 6, pH 4 to pH 5, pH 5 to pH 7, or pH 6 to pH 7). In some embodiments, the pharmaceutical composition has a pH of about pH 5.4.

[0012] In some embodiments, the pharmaceutical composition has a viscosity of <17 cP at 20° C. In some embodiments, the pharmaceutical composition has a viscosity of <12 cP at 20° C. In some embodiments, the pharmaceutical composition has a viscosity of 10 cP or 11 cP at 20° C. In some embodiments, the pharmaceutical composition has a viscosity of 6 cP to 35 cP (e.g., 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30 cP, 31 cP, 32 cP, 33 cP, 34 cP, or 35 cP) at 20° C.

[0013] In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compound per month at 25° C. of between 0.05% (w / v) and 0.5% (w / v) (e.g., between 0.05% (w / v) and 0.4% (w / v), between 0.05% (w / v) and 0.3% (w / v), between 0.05% (w / v) and 0.2% (w / v), between 0.05% (w / v) and 0.1% (w / v), between 0.1% (w / v) and 0.5% (w / v), between 0.2% (w / v) and 0.5% (w / v), between 0.3% (w / v) and 0.5% (w / v), or between 0.4% (w / v) and 0.5% (w / v)). In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compounds per month at 25° C. over one month of about 0.3% (w / v). In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compounds per month at 37° C. over one month of about 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compounds per month at 37° C. over one month of about 4.6% (w / v) or about 5% (w / v). In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compounds per month at 37°C of 4.2% (w / v) to 5.8% (w / v), or 3.7% (w / v) to 5.5% (w / v).

[0014] In some embodiments, the formulation has a turbidity of about 1 to 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) per month at 37°C, where the turbidity is measured as optical density at 400 nm. In some embodiments, the pharmaceutical composition has a turbidity of about 3 or about 4 per month at 37°C. In some embodiments, the pharmaceutical composition has a turbidity of 1.4 to 4.5 per month at 37°C, or 2.3 to 5.8 per month at 37°C.

[0015] In some embodiments, the pharmaceutical composition has an osmolality of 100 Osm / H2O kg to 500 Osm / H2O kg (e.g., 100 Osm / H2O kg to 400 Osm / H2O kg, 100 Osm / H2O kg to 300 Osm / H2O kg, 100 Osm / H2O kg to 200 Osm / H2O kg, 200 Osm / H2O kg to 500 Osm / H2O kg, 300 Osm / H2O kg to 500 Osm / H2O kg, or 400 Osm / H2O kg to 500 Osm / H2O kg). In some embodiments, the pharmaceutical composition has an osmolality of about 245 Osm / H2O kg.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein, the fusion protein comprising a CDR having an amino acid sequence of SEQ ID NO: 2 to 7, a detergent, sucrose, and sodium acetate, wherein the fusion protein is present at a concentration of 120 mg / mL to 200 mg / mL, the sodium acetate is present at a concentration of 25 mM to 75 mM, the sucrose is present at a concentration of 2% (w / v) to 15% (w / v), the detergent is present at a concentration of 0.01% (w / v) to 0.2% (w / v), and the pharmaceutical composition has a pH of 4 to 7.

[0017] In some embodiments, the concentration of the fusion protein is about 150 mg / mL, the sodium acetate concentration is about 50 mM, the sucrose concentration is about 8.6% (w / v), the detergent is PS-80 at a concentration of about 0.05% (w / v), and the pharmaceutical composition has a pH of about 5.4.

[0018] In some embodiments, the concentration of the fusion protein is about 150 mg / mL, the sodium acetate concentration is about 50 mM, the sucrose concentration is about 8.6% (w / v), the detergent is PS-80 at a concentration of about 0.15% (w / v), and the pharmaceutical composition has a pH of about 5.4.

[0019] In one aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein, the fusion protein comprising a CDR having an amino acid sequence of SEQ ID NO: 2 to 7, a detergent, and sodium acetate, and the fusion protein is at a concentration of 150 mg / mL to 200 mg / mL (e.g., 150 mg / mL to 190 mg / mL, 150 mg / mL to 180 mg / mL, 150 mg / mL to 170 mg / mL, 150 mg / mL to 180 mg / mL, 150 mg / mL to 190 mg / mL, 150 mg / mL to 20 ... The detergent may be present at a concentration of 0.01% (w / v) to 0.1% (w / v) (e.g., 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), or 160 mg / mL to 200 mg / mL, 170 mg / mL to 200 mg / mL, 180 mg / mL to 200 mg / mL, or 190 mg / mL to 200 mg / mL). (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), or 0.1% (w / v)), and sucrose is present at a concentration of 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or In some embodiments, the fusion protein is present at a concentration of about 190 mg / mL, the detergent is present at a concentration of about 0.05% (w / v), the sucrose is present at a concentration of 4% (w / v), the sodium acetate is present at a concentration of about 20 mM, and the pharmaceutical composition has a pH of about pH 5.4. In some embodiments, the detergent is PS80. In some embodiments, PS80 is present at a concentration of about 1.0% (w / v).

[0020] In some embodiments, the fusion protein comprises a CDR having an amino acid sequence of SEQ ID NO: 2-7, detergent, sodium acetate, and proline, and the fusion protein is at a concentration of 150 mg / mL to 200 mg / mL (e.g., 150 mg / mL to 190 mg / mL, 150 mg / mL to 180 mg / mL, 150 mg / mL to 170 mg / mL, 150 mg / mL to 160 mg / mL, 160 mg / mL to 18 ... The detergent may be present at a concentration of 0.01% (w / v) to 0.1% (w / v) (e.g., 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), or 170 mg / mL to 200 mg / mL, 170 mg / mL to 200 mg / mL, 180 mg / mL to 200 mg / mL, or 190 mg / mL to 200 mg / mL). The sodium acetate is present at a concentration of 10 mM to 50 mM (e.g., 10 mM to 40 mM, 10 mM to 30 mM, 10 mM to 20 mM, 20 mM to 50 mM, 30 mM to 50 mM, or 40 mM to 50 mM), and the proline is present at a concentration of 100 mM to 200 mM (e.g., 100 mM to 200 mM). For example, the fusion protein may be present at a concentration of 100 mM to 180 mM, 100 mM to 160 mM, 100 mM to 140 mM, 100 mM to 120 mM, 120 mM to 200 mM, 140 mM to 200 mM, 160 mM to 200 mM, or 180 mM to 200 mM), and the pharmaceutical composition has a pH of 4 to 7 (e.g., pH 4 to 6, pH 4 to 5, pH 5 to 7, or pH 6 to 7). In some embodiments, the fusion protein is present at a concentration of about 190 mg / mL, the detergent is present at a concentration of about 0.05% (w / v), sodium acetate is present at a concentration of about 20 mM, and proline is present at a concentration of about 165 mM, and the pharmaceutical composition has a pH of about pH 5.4. In some embodiments, the detergent is PS80. In some embodiments, PS80 is present at a concentration of about 1.0% (w / v).

[0021] In some embodiments, the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 55, and a second portion comprising an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 56. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1.

[0022] In some embodiments, the pharmaceutical composition is formulated as a drug product.

[0023] In another aspect, the present disclosure provides a method for treating or preventing a disease described herein, comprising administering any one of the pharmaceutical compositions described herein to a subject in need of such treatment or prevention. In some embodiments, the disease is sickle cell disease. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered via a pre-filled syringe, an auto-injector, or an on-body device. In some embodiments, the subject is a human.

[0024] In another aspect, the disclosure provides a method for developing a high concentration formulation of a fusion protein for subcutaneous administration, the method comprising: i) performing an excipient screen comprising measuring the stability and viscosity of a solution comprising the fusion protein and one or more excipients over a period of time; ii) following step (i), measuring the viscosity, turbidity, and rate of formation of the high molecular weight compound in the solution of step (i) as a result of varying the pH, acetate concentration, and sucrose concentration; iii) following step (ii), measuring the viscosity, turbidity, and rate of formation of the high molecular weight compound in the solution of step (ii) as a result of varying the pH, sucrose concentration, excipients, and fusion protein concentration; and iv) following step (iii), performing a viscosity evaluation, wherein the viscosity of the formulation is measured at a range of temperatures and a range of fusion protein concentrations.

[0025] In some embodiments, the fusion protein comprises six CDRs having the amino acid sequences of SEQ ID NOs: 2-7. In some embodiments, the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 55, and a second portion comprising an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 56. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1, or a modification thereof. In some embodiments, the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

[0026] In some embodiments, the high concentration formulation has a fusion protein concentration of at least 120 mg / mL. In some embodiments, the high concentration formulation has a fusion protein concentration of at least 150 mg / mL. In some embodiments, the high concentration formulation has a fusion protein concentration of 120 mg / mL to 250 mg / mL (e.g., 120 mg / mL to 250 mg / mL, 150 mg / mL to 250 mg / mL, 175 mg / mL to 250 mg / mL, 200 mg / mL to 250 mg / mL, 225 mg / mL to 250 mg / mL, 120 mg / mL to 225 mg / mL, 120 mg / mL to 200 mg / mL, 120 mg / mL to 175 mg / mL, 120 mg / mL to 150 mg / mL, or 150 mg / mL to 180 mg / mL). In some embodiments, the solution in step (i) has a fusion protein concentration of about 200 mg / mL.

[0027] In some embodiments, the measurements in steps ii and iii are performed weekly, hi some embodiments, the measurements are performed for at least 4 weeks.

[0028] In some embodiments, the one or more excipients are selected from succinate, arginine, acetate, histidine, phosphate, or a combination thereof. In some embodiments, the one or more excipients are arginine. In some embodiments, the arginine is arginine hydrochloride. In some embodiments, the arginine is arginine acetate. In some embodiments, the arginine is arginine succinate. In some embodiments, the one or more excipients are succinate and arginine hydrochloride. In some embodiments, the one or more excipients are acetate. In some embodiments, the one or more excipients are acetate and arginine hydrochloride. In some embodiments, the one or more excipients are acetate and arginine acetate.

[0029] In some embodiments, the solution of step (i) further comprises sucrose, in a concentration of 1% (w / v) to 15% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), or 15% (w / v)). In some embodiments, sucrose is present at a concentration of 2% (w / v) to 10% (w / v) (e.g., 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, sucrose is present at a concentration of about 4% (w / v) to about 9% (w / v) (e.g., 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, sucrose is present at a concentration of about 4% (w / v). In some embodiments, sucrose is present at a concentration of 8% (w / v) to 9% (w / v) (e.g., 8.1% (w / v), 8.2% (w / v), 8.3% (w / v), 8.4% (w / v), 8.5% (w / v), 8.6% (w / v), 8.7% (w / v), 8.8% (w / v), 8.9% (w / v), or 9% (w / v)). In some embodiments, sucrose is present at a concentration of about 8.6% (w / v). It refers to a compound having the formula:

[0030] In some embodiments, the solution in step (ii) has a pH of 4 to 7 (e.g., a pH of 4 to 6, 4 to 5, 5 to 7, or 6 to 7). In some embodiments, the solution in step (ii) has a pH of 4.5 to 6.0 (e.g., a pH of 4.5 to 5.5, 4.5 to 5, 5 to 6, or 5.5 to 6). In some embodiments, the solution in step (ii) has an acetate concentration of 10 mM to 200 mM (e.g., 10 mM to 150 mM, 10 mM to 100 mM, 10 mM to 50 mM, 50 mM to 200 mM, 100 mM to 200 mM, or 150 mM to 200 mM). In some embodiments, the solution in step (ii) has an acetate concentration of 15 mM to 150 mM (e.g., 15 mM to 80 mM, 15 mM to 60 mM, 15 mM to 40 mM, 15 mM to 20 mM, 20 mM to 100 mM, 40 mM to 100 mM, 60 mM to 100 mM, 80 mM to 100 mM, 80 mM to 150 mM, 100 mM to 150 mM, or 60 mM to 150 mM).

[0031] In some embodiments, the solution in step (ii) has a sucrose concentration of 1% (w / v) to 20% (w / v) (e.g., 1% (w / v) to 15% (w / v), 1% (w / v) to 10% (w / v), 1% (w / v) to 5% (w / v), 5% (w / v) to 20% (w / v), 10% (w / v) to 20% (w / v), or 15% (w / v) to 20% (w / v)). In some embodiments, the solution in step (ii) has a sucrose concentration of 2% (w / v) to 10% (w / v) (e.g., 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the solution in step (ii) has a fusion protein concentration of about 150 mg / mL. In some embodiments, the solution in step (iii) has a pH of 5 to 6. In some embodiments, the solution in step (iii) has a sucrose concentration of 0% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the solution in step (iii) has a sucrose concentration of 0% (w / v) to 5% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), or 5% (w / v)). In some embodiments, the solution in step (iii) has a fusion protein concentration of about 100 mg / mL to 300 mg / mL (e.g., 100 mg / mL to 250 mg / mL, 100 mg / mL to 200 mg / mL, 100 mg / mL to 150 mg / mL, 150 mg / mL to 300 mg / mL, 200 mg / mL to 300 mg / mL, or 250 mg / mL to 300 mg / mL). In some embodiments, the solution in step (ii) has a fusion protein concentration of about 150 mg / mL to 230 mg / mL (e.g., 150 mg / mL to 200 mg / mL, 150 mg / mL to 175 mg / mL, 175 mg / mL to 230 mg / mL, 200 mg / mL to 230 mg / mL, or 220 mg / mL to 230 mg / mL).

[0032] In some embodiments, the formulation has a viscosity of 5 cP to 15 cP (e.g., 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, or 15 cP) at 20° C. In some embodiments, the formulation has a viscosity of about 10 cP or 11 cP at 20° C. In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 25° C. over one month of between 0.05% (w / v) and 0.5% (w / v) (e.g., between 0.05% (w / v) and 0.4% (w / v), between 0.05% (w / v), between 0.3% (w / v), between 0.05% (w / v), between 0.2% (w / v), between 0.05% (w / v) and 0.1% (w / v), between 0.1% (w / v) and 0.5% (w / v), between 0.2% (w / v) and 0.5% (w / v), between 0.3% (w / v) and 0.5% (w / v), or between 0.4% (w / v) and 0.5% (w / v)). In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 25° C. over one month of about 0.2% (w / v). In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 37° C. over one month of about 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 37° C. over one month of about 5% (w / v). In some embodiments, the formulation has a turbidity of about 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) per month at 37° C., where the turbidity is measured as optical density at 400 nm. In some embodiments, the formulation has a turbidity of about 3 per month at 37°C.

[0033] In another aspect, the present disclosure provides a bispecific construct that binds to properdin and human serum albumin, the bispecific construct comprising the amino acid sequence of SEQ ID NO: 1 or a modification thereof. The bispecific construct of claim 117, wherein the modification comprises conversion of the N-terminal glutamine of the amino acid sequence of SEQ ID NO: 1 to pyroglutamate. In another embodiment, the bispecific construct consists of the amino acid sequence of SEQ ID NO: 1 in which the N-terminal glutamine has been converted to pyroglutamate. [Brief explanation of the drawings]

[0034] [Figure 1A] 1 is a graph showing the effect of various buffer excipients on solution viscosity of a solution with 200 mg / mL of fusion protein over a range of temperatures. [Figure 1B] 1 is a graph showing the effect of the presence of proline on the solution viscosity of a solution with 200 mg / mL of fusion protein over a range of temperatures. [Figure 1C] 1 is a graph showing the effect of sucrose concentration on solution viscosity of a solution with 200 mg / mL of fusion protein over a range of temperatures. [Figure 2A] 1 is a graph showing the percentage of higher molecular weight compounds in solutions with various buffer excipient concentrations over a 4 week period at 37° C. [Figure 2B] 1 is a graph showing the percentage of higher molecular weight compounds in solutions with various sucrose concentrations or the presence of proline over a 4 week period at 37° C. [Figure 3A-3B] 3A and 3B are photographs showing turbidity in solutions with various buffer excipients (FIG. 2A) or sucrose concentrations or the presence of proline (FIG. 2B) over a 4-week period at 37° C. [Figure 4] 1 is a graph showing measurements of turbidity of solutions over a four week period for various sucrose concentrations. [Figure 5A] 1 is a graph showing measurement of the percentage of higher molecular weight compounds over three months at 37° C. in solutions with various concentrations of acetate. [Figure 5B] 1 is a graph showing measurement of the percentage of higher molecular weight compounds over three months at 25° C. in solutions with various concentrations of acetate. [Figure 6A] 1 is a graph showing measurements of the percentage of acid present in solution at 37° C. over a period of three months in solutions having various concentrations of acetate. [Figure 6B] 1 is a graph showing measurements of the percentage of acid present in solution at 25° C. over a period of three months in solutions having various concentrations of acetate. [Figure 7A] 1 is a graph showing measurements of the percentage of base present in solution at 37° C. over a period of three months in solutions having various concentrations of acetate. [Figure 7B] 1 is a graph showing measurements of the percentage of base present in solution at 25° C. over a period of three months in solutions having various concentrations of acetate. [Figure 8A] 1 is a graph showing measurement of solution turbidity over a 3 month period at 37° C. in solutions with various concentrations of acetate. [Figure 8B] 1 is a graph showing the measurement of solution turbidity at 25° C. over a period of three months in solutions having various concentrations of acetate. [Figure 9] A series of graphs showing the predicted effects of acetate concentration, sucrose concentration, and pH on the osmolality, viscosity at 25°C, and viscosity at 20°C of a solution of a polypeptide having the amino acid sequence of SEQ ID NO: 1 at a concentration of 150 mg / mL. [Figures 10A-10B] Figures 10A and 10B show a series of graphs showing the predicted effects of acetate concentration, sucrose concentration, and pH at 37°C (Figure 10A) and 25°C (Figure 10B) on the rate of high molecular weight compound formation, turbidity, acid formation rate, and base formation rate of a solution containing a polypeptide having the amino acid sequence of SEQ ID NO: 1 at a concentration of 150 mg / mL. [Figure 11A] 1 is a graph showing measurement of the percentage of higher molecular weight compounds over one month at 37° C. in solutions with various concentrations of acetate. [Figure 11B]1 is a graph showing measurement of the percentage of higher molecular weight compounds over one month at 25° C. in solutions with various concentrations of acetate. [Figure 12A] 1 is a graph showing measurements of the percentage of acid present in solution at 37° C. over a period of one month in solutions having various concentrations of acetate. [Figure 12B] 1 is a graph showing measurements of the percentage of acid present in solution at 25° C. over a period of one month in solutions having various concentrations of acetate. [Figure 13A] 1 is a graph showing measurements of the percentage of base present in solution at 37° C. over a period of one month in solutions having various concentrations of acetate. [Figure 13B] 1 is a graph showing measurements of the percentage of base present in solution at 25° C. over a period of one month in solutions having various concentrations of acetate. [Figure 14A] 1 is a graph showing measurement of solution turbidity over one month at 37° C. in solutions with various concentrations of acetate. [Figure 14B] 1 is a graph showing the measurement of solution turbidity over one month at 25° C. in solutions having various concentrations of acetate. [Figure 15] 1 shows a series of graphs illustrating the predicted effects of polypeptide concentration, proline concentration, sucrose concentration, and pH on the rate of higher molecular weight compound formation, turbidity, rate of acid formation, and rate of base formation of a solution. [Figures 16A-16B] Figures 16A and 16B show a series of graphs illustrating the predicted effects of polypeptide concentration, proline concentration, sucrose concentration, and pH at 37°C (Figure 16A) and 25°C (Figure 16B) on the rate of higher molecular weight compound formation, turbidity, rate of acid formation, and rate of base formation of a solution. [Figure 17] 1 is a graph showing the effect of sucrose concentration on the viscosity of solutions having a polypeptide (fusion protein) concentration of at least 200 mg / mL. DETAILED DESCRIPTION OF THE INVENTION

[0035] definition To facilitate understanding of this disclosure, several terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to this disclosure. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include general classifications for which specific examples may be used for illustration. While terminology herein is used to describe specific embodiments, its use does not limit the disclosure except as outlined in the claims.

[0036] As used herein, the term "about" refers to a value that is 10% more or less than 10% of the stated value.

[0037] As used herein, any value provided in a range of values includes both the upper and lower limits, and any value subsumed within the limits.

[0038] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single-chain version thereof. "Antibody" refers, for example, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin ("Ig") polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. LThe light chain constant region is composed of one domain, CL, and a light chain constant region. The light chain constant region is composed of one domain, CL. Within full-length light and heavy chains, the variable and constant domains are typically connected by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. V H Area and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0039] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Such "fragments" are, for example, about 8 to about 1500 amino acids in length, suitably about 8 to about 745 amino acids in length, suitably about 8 to about 300, e.g., about 8 to about 200 amino acids in length, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragments (V L , V H (ii) a F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region); (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L Domain and V HFv fragment consisting of domains; (v) V H dAb fragments comprise a V domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Additionally, the two domains of an Fv fragment are V and V. L and V H Although encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods, and V L Area and V H The domains pair to form a monovalent molecule (known as a single-chain Fv (scFv); Bird et al., Science 242:423-6; Huston, J. et al., Proc. Natl. Acad. Sci. USA, 85:5879-83, 1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0040] An antibody, immunoglobulin, or immunologically functional immunoglobulin fragment, or engineered polypeptide or fusion protein disclosed herein is said to "specifically" bind to an antigen when the molecule preferentially recognizes its antigen target in a complex mixture of proteins and / or macromolecules. As used herein, the term "specifically binds" refers to the K D is at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10-12 It refers to the ability to bind to an antigen containing an epitope that is equal to or less than M and / or to bind to an epitope with an affinity that is at least two-fold higher than its affinity for a nonspecific antigen.

[0041] As used herein, "complement-mediated disorder" refers to a disorder caused directly or indirectly by misregulation of the complement pathway, e.g., activation or inhibition of the complement pathway, or a disorder mediated directly or indirectly by one or more components of the complement pathway or products produced by the complement pathway. The term also refers to a disorder exacerbated by one or more components of the complement pathway or products produced by the complement pathway.

[0042] As used herein, "drug product" or "DP" refers to a pharmaceutical composition in its final configuration (e.g., in its final vial configuration) ready for administration to a subject. The concentration of a polypeptide in a DP may be the concentration at which the polypeptide is administered to a subject.

[0043] As used herein, the term "effective amount" refers to an amount of a pharmaceutical composition sufficient to produce a beneficial or desired result, such as a clinical result, when administered to a subject, e.g., a human subject. The amount of a given composition described herein that corresponds to such a beneficial or desired result will depend on various factors, such as the given agent, pharmaceutical formulation, route of administration, and the identity (e.g., age, sex, weight) of the subject to be treated.

[0044] As used herein, the term "fused to" refers to a polypeptide that has been engineered by combining two or more sequences, typically by cloning one sequence, e.g., a coding sequence, into an expression vector in frame with one or more second coding sequences so that the two or more coding sequences are transcribed and translated into a single, contiguous polypeptide. In addition to being produced by recombinant techniques, parts of a polypeptide can be "fused to" each other by chemical reaction or other means known in the art to create custom polypeptides.

[0045] As used herein, the term "heavy chain antibody" refers to an antibody that lacks the light chains found in conventional antibodies.

[0046] As used herein, the term "human antibody" refers to an Ig used, for example, by the immune system to bind to and neutralize pathogens. The term includes antibodies having variable and constant regions that substantially correspond to human germline Ig sequences. In some embodiments, human antibodies are produced in non-human mammals, including, but not limited to, rodents such as mice and rats, and lagomorphs such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In still other embodiments, human antibodies are recombinantly produced. As used herein, human antibodies include all or portions of antibodies, including, for example, heavy and light chains, variable regions, constant regions, proteolytic fragments, complementarity-determining regions (CDRs), and other functional fragments.

[0047] The term "VHH domain" refers to the variable domain present in naturally occurring heavy chain antibodies (e.g., "VHH antibodies" or "VHH single chain antibodies"), to distinguish it from the heavy chain variable domain (herein referred to as "VH domain") present in conventional four chain antibodies and the light chain variable domain (herein referred to as "VL domain") present in conventional four chain antibodies. VHH antibodies are produced by certain camelid species, such as camels and llamas. Camelids immunized against a specific antigen produce single chain antibodies (one heavy chain) that bind to the antigen. A single domain, heavy chain variable domain sequence, derived from a heavy chain antibody is referred to as a VHH or VHH. H H antibody, VHH or V H H antibody fragment, or VHH or V H It may be referred to as the H domain.

[0048] The term "linker" as used herein refers to one or more amino acid residues inserted or included between polypeptides of a fusion protein. A peptide linker can be inserted or included, for example, at the sequence level at the transition between polypeptides of a fusion protein. The identity and sequence of amino acid residues within a linker can vary depending on the desired secondary structure. For example, glycine, serine, and alanine are useful for providing maximum flexibility to the linker. Any amino acid residue can be considered a linker, optionally combined with one or more other amino acid residues, which may be the same or different from the first amino acid residue, to construct a larger peptide linker depending on the desired properties.

[0049] The term "bispecific" refers to a fusion protein capable of binding to two antigens. The term "multivalent fusion protein" refers to a fusion protein containing two or more antigen-binding sites. A "multispecific fusion protein" is a fusion protein capable of binding to two or more related or unrelated targets. Traditional four-chain antibodies are multivalent but not bispecific because each arm binds to the same antigen. VHH antibodies are monovalent. Bispecific antibodies can be engineered, for example, by fusing two or more monovalent VHH antibodies (or VHH variable domain fragments or other functional fragments), with or without a linker, such that the fusion protein is multivalent. A fusion protein is bispecific if the fused variable domains specifically bind to different antigens.

[0050] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to an expression system (e.g., a host cell or an in vitro expression system). One type of vector is a "plasmid," which refers to a circular double-stranded DNA (dsDNA) molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of coding sequences to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0051] As used herein, the term "operably linked" refers to the arrangement of flanking sequences that are configured or constructed to perform a desired function. Thus, a flanking sequence operably linked to a coding sequence is capable of effecting the replication, transcription, and / or translation of the coding sequence. A coding sequence can be operably linked, for example, to a promoter, which is capable of directing the transcription of the coding sequence. A flanking sequence need not be contiguous with the coding sequence to be considered operably linked, so long as it functions correctly.

[0052] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. A host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the fusion proteins of the present disclosure, including bacterial, yeast, baculovirus, and mammalian expression systems (and phage display expression systems).

[0053] As used herein, the terms "patient" and "subject" include human and animal subjects.

[0054] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when administered to a patient.

[0055] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of the fusion proteins of the present disclosure.

[0056] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in human treatment without undue toxicity. Pharmaceutically acceptable salts are known in the art (Berge, S. et al., J. Pharm. Sci., 66:1-19, 1977; Pharmaceutical Salts: Properties, Selection, and Use, (Eds. PH Stahl and (C.G. Wermuth), Wiley-VCH, 2008). Salts can be prepared in situ, for example, during the final isolation and purification of the compounds described herein, or can be prepared separately by reacting the free base group with a suitable organic acid. Representative acid addition salts include, but are not limited to, acetate, adipate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycosaminoglycan, glycerol, hydroxybenzoate ... Examples of the salts include cerium phosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate.Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations (including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, L-α-phosphatidylethanolamine, bis(2-ethylhexyl)amine, and soy lecithin, and the like). Representative amino acid salts include lysine, arginine, glycine, and histidine, and the like. Those skilled in the art will recognize that any reference to a drug compound includes within its scope pharmaceutically acceptable salts of the specified drug compound.

[0057] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but may also include subsequent rearrangements and mutations that occur, for example, during antibody maturation. The variable regions contain antigen-binding domains encoded by different genes that rearrange to form antibodies specific for foreign antigens (Lonberg, N., Nat. Biotechnol., 23:1117-25, 2005). In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (called somatic mutation or hypermutation) to increase the affinity of the antibody for foreign antigens. The constant regions will further change in response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to antigen may not have sequence identity to the original nucleic acid molecules, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0058] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include subjects having the disease or condition as well as those at risk of having the disease or condition, or those in whom the disease or condition is to be prevented.

[0059] As used herein, a "therapeutically effective" amount, e.g., of a fusion protein described herein, refers to an amount or dosage sufficient to bring about a desired therapeutic result. A therapeutically effective amount is an amount that, when administered, results in a reduction in the severity of disease symptoms (e.g., an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction), or is an amount sufficient to inhibit over a period of time one or more clinically defined pathological processes associated with the condition being treated. A therapeutically effective amount may vary depending on various factors and symptoms related to the patient being treated, as well as the severity of the disorder.

[0060] Detailed Description The present disclosure is based in part on the optimization of pharmaceutical compositions, which are desirable for the development of compositions for subcutaneous administration that allow fusion proteins to be present at high concentrations. A particular need has arisen for high-concentration formulations of pharmaceutical compositions containing biomolecules, such as enzymes, antibodies, VHH antibodies, bispecific VHH antibodies, and bispecific VHH antibody fragments. The pharmaceutical compositions described herein comprise a fusion protein (e.g., a VHH bispecific antibody comprising variable domains from two different camelid antibodies linked by a flexible linker), polysorbate, and a buffer. The formulations are optimized in terms of viscosity and stability so that the fusion protein is present at a concentration of at least 150 mg / mL.

[0061] Pharmaceutical Composition The fusion proteins described herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the fusion protein and one or more pharmaceutically acceptable carriers or excipients.

[0062] The pharmaceutical compositions described herein comprise a fusion protein present at a concentration of at least 150 mg / mL. For example, the concentration of the fusion protein in the pharmaceutical composition can be 150 mg / mL to 200 mg / mL (e.g., 150 mg / mL to 190 mg / mL, 150 mg / mL to 180 mg / mL, 150 mg / mL to 170 mg / mL, 150 mg / mL to 160 mg / mL, 160 mg / mL to 200 mg / mL, 170 mg / mL to 200 mg / mL, 180 mg / mL to 200 mg / mL, or 190 mg / mL to 200 mg / mL). In some embodiments, the concentration of the fusion protein is 170 mg / mL to 200 mg / mL (e.g., 170 mg / mL, 171 mg / mL, 172 mg / mL, 173 mg / mL, 174 mg / mL, 175 mg / mL, 176 mg / mL, 177 mg / mL, 178 mg / mL, 179 mg / mL, 180 mg / mL, 181 mg / mL, 182 mg / mL, 183 mg / mL, 184 mg / mL, 185 mg / mL, 186 mg / mL, 187 mg / mL, 188 mg / mL, 189 mg / mL, 190 mg / mL, 191 mg / mL, 192 mg / mL, 193 mg / mL, 194 mg / mL, 195 mg / mL, 196 mg / mL, 197 mg / mL, 198 mg / mL, 199 mg / mL, or 200 mg / mL). In some embodiments, the concentration of the fusion protein is about 190 mg / mL. In other embodiments, the concentration of the fusion protein is about 150 mg / mL. The methods and compositions described herein may allow for even higher concentrations, for example, up to 250 mg / mL or more.

[0063] The pharmaceutical compositions described herein may include a detergent. The pharmaceutical composition may have a detergent concentration of 0.01% (w / v) to 1.5% (w / v) (e.g., 0.01% (w / v) to 0.4% (w / v), 0.01% (w / v) to 0.3% (w / v), 0.01% (w / v) to 0.2% (w / v), 0.01% (w / v) to 0.1 (w / v), 0.01% (w / v) to 0.05% (w / v), 0.05% (w / v) to 0.5% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.2% (w / v) to 0.5% (w / v), 0.3% (w / v) to 0.5% (w / v), or 0.4% (w / v) to 0.5% (w / v)). In some embodiments, the detergent has a concentration of 0.01% (w / v) to 1% (w / v) (e.g., 0.01% (w / v) to 0.9% (w / v), 0.01% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.1% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.05% (w / v) to 1% (w / v), 0.1 (w / v) to 0.1% (w / v), or 0.5% to 1% (w / v)). In some embodiments, the detergent is present at a concentration of 0.01% (w / v) to 0.1% (w / v) (e.g., 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), or 0.1% (w / v)). In certain embodiments, the detergent is present at a concentration of about 0.05% (w / v). In other embodiments, the detergent is present at a concentration of about 0.1% (w / v). The detergent can be any detergent known to those of skill in the art. For example, the pharmaceutical compositions described herein may include polysorbate, TRITON® X-100, digitonin, saponin, n-dodecyl-β-D-maltoside, or any combination thereof. In some embodiments, the polysorbate may be polysorbate 80 (PS80) or polysorbate 20.

[0064] The pharmaceutical compositions described herein include one or more buffering agents. The buffering agent can be any buffering agent known in the art. For example, the buffering agent can be acetate, succinate, histidine, phosphate, or a combination thereof. The buffering agent can be present at any concentration that allows for buffering of the pharmaceutical composition. In some embodiments, the buffering agent has a concentration of about 10 mM to 200 mM (e.g., 10 mM to 150 mM, 10 mM to 100 mM, 10 mM to 50 mM, 50 mM to 200 mM, 100 mM to 200 mM, or 150 mM to 200 mM). For example, the buffer has a concentration of about 100 mM to 200 mM (e.g., 100 mM to 180 mM, 100 mM to 160 mM, 100 mM to 140 mM, 100 mM to 120 mM, 120 mM to 200 mM, 140 mM to 200 mM, 160 mM to 200 mM, or 180 mM to 200 mM).

[0065] In some embodiments, the pharmaceutical composition comprises an acetate buffer. For example, the acetate is sodium acetate. In some embodiments, the sodium acetate is present in the pharmaceutical composition at a concentration of 10 mM to 150 mM (e.g., 10 mM to 140 mM, 10 mM to 130 mM, 10 mM to 120 mM, 10 mM to 100 mM, 10 mM to 75 mM, 10 mM to 50 mM, 20 mM to 150 mM, 30 mM to 150 mM, 75 mM to 150 mM, 100 mM to 150 mM, or 40 mM to 150 mM). In some embodiments, the concentration of sodium acetate is about 15 mM to 100 mM (e.g., about 15 mM to 80 mM, 15 mM to 60 mM, 15 mM to 40 mM, 15 mM to 20 mM, 20 mM to 100 mM, 40 mM to 100 mM, 60 mM to 100 mM, or 80 mM to 100 mM). In some embodiments, the concentration of sodium phosphate is about 50 mM.

[0066] In some embodiments, the pharmaceutical composition includes an amino acid stabilizer, such as proline or arginine. In some embodiments, the amino acid stabilizer (e.g., proline) is present in the pharmaceutical composition at a concentration of 100 mM to 200 mM (e.g., 100 mM to 190 mM, 100 mM to 180 mM, 100 mM to 170 mM, 140 mM to 200 mM, 150 mM to 200 mM, 160 mM to 200 mM, 110 mM to 190 mM, 120 mM to 180 mM, 130 mM to 170 mM, 140 mM to 180 mM, 150 mM to 170 mM, or 160 mM to 170 mM). In certain embodiments, the concentration of proline is about 165 mM. In some embodiments, the arginine is arginine acetate, arginine succinate, or arginine hydrochloride.

[0067] The pharmaceutical compositions described herein may contain an isotonicity agent. The isotonicity agent may be any isotonicity agent known in the art. For example, the isotonicity agent may be a sugar, an amino acid, or a salt. The sugar may be sucrose, glucose, glycerol, or trehalose. In some embodiments, the sugar is sucrose. The sucrose may be present at a concentration of 1% (w / v) to about 15% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), or 15% (w / v)). For example, sucrose can be present at a concentration of about 4% (w / v) to about 9% (w / v) (e.g., 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, sucrose is present at a concentration of about 4% (w / v). In some embodiments, sucrose is present at a concentration of 8% (w / v) to 9% (w / v) (e.g., 8.1% (w / v), 8.2% (w / v), 8.3% (w / v), 8.4% (w / v), 8.5% (w / v), 8.6% (w / v), 8.7% (w / v), 8.8% (w / v), 8.9% (w / v), or 9% (w / v)).

[0068] The pH of the pharmaceutical composition may be from pH 3 to pH 8 (e.g., pH 3, pH 4, pH 5, pH 6, pH 7, or pH 8). In some embodiments, the pH of the pharmaceutical composition is from pH 4 to pH 7 (e.g., pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5, or pH 7). For example, the pH of the pharmaceutical composition may be pH 5.4.

[0069] The pharmaceutical composition may be formulated to have a viscosity of <17 cP at 20° C. In some embodiments, the pharmaceutical composition may be formulated to have a viscosity of <12 cP at 20° C. For example, the viscosity may be 10 or 11 cP at 20° C. In some embodiments, the pharmaceutical composition has a viscosity of 6 cP to 35 cP (e.g., 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30 cP, 31 cP, 32 cP, 33 cP, 34 cP, or 35 cP) at 20° C.

[0070] In some embodiments, the pharmaceutical composition may be formulated such that the percentage of higher molecular weight compound per month at 25° C. over one month is between 0.05% (w / v) and 0.5% (w / v) (e.g., between 0.05% (w / v) and 0.4% (w / v), between 0.05% (w / v) and 0.3% (w / v), between 0.05% (w / v) and 0.2% (w / v), between 0.05% (w / v) and 0.1% (w / v), between 0.1% (w / v) and 0.5% (w / v), between 0.2% (w / v) and 0.5% (w / v), between 0.3% (w / v) and 0.5% (w / v), or between 0.4% (w / v) and 0.5% (w / v)). In some embodiments, the pharmaceutical composition has a percentage of higher molecular weight compounds per month at 25° C. over one month of about 0.2% (w / v), or in the range of 0.1 to 0.2 or 0.1 to 0.3. The pharmaceutical composition can have a percentage of higher molecular weight compounds per month at 37° C. over one month of about 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v), or 4.2% (w / v) to 5.8% (w / v), or about 3.7% (w / v) to about 5.5% (w / v)). For example, the pharmaceutical composition may have a percentage of higher molecular weight compound per month at 37° C. of about 5% (w / v) or about 4.6% (w / v).

[0071] The pharmaceutical composition may have a turbidity of about 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) relative to the optical density (OD) at 400 nm after one month when stored at 37° C. For example, the pharmaceutical composition may have a turbidity of about 3.0 or 4.0 per month at 37° C., as measured by OD 400, or the pharmaceutical composition may have a turbidity of 1.4 to 4.5 or 2.3 to 5.8 per month at 37° C., as measured by OD 400.

[0072] In some embodiments, the pharmaceutical composition has an osmolality of 100 Osm / H2O kg to 500 Osm / H2O kg (e.g., 100 Osm / H2O kg to 400 Osm / H2O kg, 100 Osm / H2O kg to 300 Osm / H2O kg, 100 Osm / H2O kg to 200 Osm / H2O kg, 200 Osm / H2O kg to 500 Osm / H2O kg, 300 Osm / H2O kg to 500 Osm / H2O kg, or 400 Osm / H2O kg to 500 Osm / H2O kg). In some embodiments, the pharmaceutical composition has an osmolality of about 245 Osm / H2O kg.

[0073] Engineered fusion proteins Described herein are fusion proteins and formulations thereof. In some embodiments, the fusion protein is a bispecific antibody in which two antigen-binding polypeptides are linked (e.g., by a linker such as the linker). Such bispecific constructs can include an anti-properdin-binding polypeptide (e.g., a monovalent VHH antibody or a VHH variable domain) linked by a linker to a second polypeptide (e.g., a second monovalent antibody or a VHH variable domain). The second polypeptide can, for example, enhance the in vivo stability of the bispecific construct, target a different therapeutic target, or position two antigens in close proximity (e.g., thereby directing the first bound antigen toward the second bound antigen). In some embodiments, the second polypeptide is an albumin-binding molecule, albumin-binding peptide, or anti-albumin antibody (e.g., a monovalent antibody), or a modified form thereof (e.g., a variable domain of a llama antibody that specifically binds to human serum albumin). In addition to the present disclosure, albumin-binding peptides are known in the art (WO 2007 / 106120 (see Tables 1 to 9); Dennis, M. et al., J. Biol. Chem., 277:35035-43, 2002; the disclosures of which are incorporated herein by reference).

[0074] The antibodies described herein can inhibit properdin binding to, for example, C3b, C3Bb, and C3bBb. Inhibition of properdin results in a reduction of alternative pathway complement activation, demonstrating therapeutic benefit for patients suffering from alternative pathway dysregulation diseases in which the alternative pathway is overactivated.

[0075] The anti-properdin antibodies described herein can be generated by using full-length properdin, properdin polypeptides, and / or by using antigenic properdin epitope-bearing peptides, such as fragments of properdin polypeptides. Properdin peptides and polypeptides can be isolated and used to generate antibodies as natural polypeptides, recombinant polypeptides, or synthetic recombinant polypeptides. Any antigen useful for generating anti-properdin antibodies can be used to generate monovalent antibodies. Suitable monovalent antibody formats and methods for producing them are known in the art (WO 2007 / 048037 and WO 2007 / 059782, the contents of which are incorporated herein by reference in their entireties).

[0076] The anti-properdin antibody may be a monoclonal antibody or may be derived from a monoclonal antibody. Suitable monoclonal antibodies against a selected antigen may be prepared by known techniques ("Monoclonal Antibodies; A Manual of Techniques," Zola (CRC Press, 1988); "Monoclonal Hybridoma Antibodies; Techniques and Applications," Hurrell (CRC Press, 1982), the entire contents of which are incorporated herein by reference).

[0077] In other embodiments, the antibody may be a single-domain antibody, such as a VHH. Such antibodies naturally occur in camelids and sharks (Saerens, D. et al., Curr. Opin. Pharmacol., 8:600-8, 2008). Camelid antibodies are described, for example, in U.S. Pat. Nos. 5,759,808; 5,800,988; 5,840,526; 5,874,541; 6,005,079; and 6,015,695 (the entire contents of each are incorporated herein by reference). The cloned and isolated VHH domain is a stable polypeptide characterized by the full antigen-binding capacity of the original heavy-chain antibody. VHH domains possess unique structural and functional properties, combining the advantages of traditional antibodies (high target specificity, high target affinity, and low inherent toxicity) with important features of small molecule drugs (the ability to inhibit enzymes and access receptor cleavage). Furthermore, they are stable, have the potential to be administered by means other than injection, are easier to manufacture, and can be humanized (U.S. Pat. Nos. 5,840,526; 5,874,541; 6,005,079; 6,765,087; EP 1 589 107; WO 97 / 34103; WO 97 / 49805; U.S. Pat. Nos. 5,800,988; 5,874,541; and 6,015,695, the entire contents of each of which are incorporated herein by reference).

[0078] The anti-properdin component of the bispecific antibodies described herein comprises CDR sequences: CDR-H1 having an amino acid sequence at least 90% identical to GRISSIIHMA (SEQ ID NO: 2); CDR-H2 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to RVGTTVYADSVKG (SEQ ID NO: 3); and CDR-H2 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to LQYEKHGGADY (SEQ ID NO: 4). The bispecific antibodies described herein may comprise CDR-H1 having the amino acid sequence of GRISSIIHMA (SEQ ID NO: 2); CDR-H2 having the amino acid sequence of RVGTTVYADSVKG (SEQ ID NO: 3); and CDR-H3 having the amino acid sequence of LQYEKHGGADY (SEQ ID NO: 4).

[0079] Furthermore, the engineered fusion proteins described herein can specifically bind to serum albumin such that, when the engineered protein is bound to or otherwise associated with a serum albumin molecule, the binding of the serum albumin molecule to FcRn is not significantly reduced or inhibited compared to the binding of the serum albumin molecule to FcRn when the polypeptide is not bound. In this embodiment, "not significantly reduced or inhibited" means that the binding affinity of serum albumin to FcRn (measured using an appropriate assay, e.g., SPR) is not reduced by more than 50%, more than 30%, more than 10%, or more than 5%, or is not reduced at all. In this embodiment, "not significantly reduced or inhibited" also means that the half-life of the serum albumin molecule is not significantly reduced. In particular, the engineered polypeptide may bind to amino acid residues on serum albumin that are not involved in the binding of serum albumin to FcRn. More particularly, an engineered polypeptide, for example an engineered polypeptide capable of binding to amino acid residues or sequences of serum albumin that form part of domain I and / or domain II, can bind to amino acid residues or sequences of serum albumin that do not form part of domain III of serum albumin.

[0080] The anti-albumin component of the bispecific antibodies described herein may comprise CDR sequences including: CDR-H1 having an amino acid sequence at least 87% identical to GRPVSNYA (SEQ ID NO:5); CDR-H2 having an amino acid sequence at least 87% identical to INWQKTAT (SEQ ID NO:6); and CDR-H2 having an amino acid sequence at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identical to AAVFRVVAPKTQYDYDY (SEQ ID NO:7). The bispecific antibodies described herein may comprise CDR-H1 having the amino acid sequence of GRPVSNYA (SEQ ID NO:5); CDR-H2 having the amino acid sequence of INWQKTAT (SEQ ID NO:6); and CDR-H3 having the amino acid sequence of AAVFRVAPKTQYDYDY (SEQ ID NO:7).

[0081] In some embodiments, the fusion protein comprises an anti-properdin binding moiety and an anti-albumin binding moiety. In some embodiments in which the anti-properdin binding domain has an exposed N-terminus, the N-terminal glutamine can be converted to a cyclized pyroglutamate. Such modifications are known in the art (see, e.g., Liu et al., The Journal of Biological Chemistry 286(13:11211-11217, 2011)). The portion encoding the anti-properdin binding moiety can have at least 90% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following amino acid sequence: [ka]

[0082] In some embodiments, the anti-properdin binding portion of the fusion protein comprises SEQ ID NO: 55. The portion encoding the anti-albumin binding portion can have at least 90% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following amino acid sequence: [ka]

[0083] In some embodiments, the anti-properdin binding portion of the fusion protein comprises SEQ ID NO:56.

[0084] In some embodiments, the fusion protein is encoded by a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following nucleic acid sequence: [ka]

[0085] In some embodiments, the fusion protein is encoded by a nucleic acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the fusion protein is encoded by the nucleic acid sequence of SEQ ID NO: 57.

[0086] In some embodiments, the fusion protein has an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following amino acid sequence: [ka]

[0087] In some embodiments, the fusion protein has an amino acid sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1 with an N-terminal glutamine conversion of the sequence of SEQ ID NO: 1 to pyroglutamate.

[0088] In some embodiments, the C-terminal residue of the properdin-binding domain of the fusion protein can be fused directly or via a linker to the N-terminal residue of the human serum albumin-binding domain. In other embodiments, the C-terminal residue of the complement component human serum albumin-binding domain of the fusion protein can be fused directly or via a peptide to the N-terminal residue of the properdin-binding domain. The fusion proteins described herein may contain one or more modified amino acid residues. For example, the amino acid sequence of SEQ ID NO: 1 may contain one or more amino acid modifications. The amino acid modifications described herein include all amino acid modifications known in the art (see, e.g., Liu et al., The Journal of Biological Chemistry 286(13:11211-11217, 2011) and Manning et al., Pharmaceutical Research 27(4):544-575, 2010). In all contexts, known conversions of specific amino acids, such as those occurring during processing or purification of the fusion polypeptide, should be included, e.g., the conversion of an exposed N-terminal glutamine to pyroglutamate.

[0089] Linker As used herein, a linker is used to describe a linkage or connection between polypeptide or protein domains and / or associated non-protein moieties. In some embodiments, a linker is a link between at least two polypeptide constructs, such as two polypeptide constructs linked to each other in tandem (e.g., a monovalent antibody linked to a second polypeptide or monovalent antibody). A linker can attach the N-terminus or C-terminus of one antibody construct to the N-terminus or C-terminus of a second polypeptide construct.

[0090] Described herein are fusion proteins comprising engineered proteins that specifically bind to albumin and properdin, where the engineered proteins are directly fused or linked via one or more suitable linkers or spacers. A peptide linker can be inserted or included at the sequence level, for example, at the transition between the engineered proteins of the fusion protein. The identity and sequence of amino acid residues within the linker can vary depending on the desired secondary structure.

[0091] The linker can be a simple covalent bond, such as a peptide bond, a synthetic polymer, such as a polyethylene glycol (PEG) polymer, or any type of bond created by a chemical reaction, such as chemical conjugation. When the linker is a peptide bond, the C-terminal carboxylic acid group of one protein domain can react with the N-terminal amino group of another protein domain in a condensation reaction to form a peptide bond. Specifically, the peptide bond can be formed by synthetic means via conventional organic chemical reactions well known in the art, or by natural generation from host cells, where a polynucleotide sequence encoding the DNA sequences of both proteins, for example, two antibody constructs, in tandem can be directly transcribed and translated into a continuous polypeptide encoding both proteins by the necessary molecular machinery, such as DNA polymerase and ribosomes, in the host cell.

[0092] When the linker is a synthetic polymer, such as a PEG polymer, the polymer can be functionalized at each end with reactive chemical groups to react with the terminal amino acids at the linking ends of the two proteins.

[0093] When a linker (excluding the peptide bond mentioned above) is produced by chemical reaction, a chemical functional group, such as an amine, a carboxylic acid, an ester, an azide, or other functional group commonly used in the art, can be synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups can then react via synthetic chemical means to form a chemical bond, thereby linking the two proteins together. Such chemical conjugation procedures are routine for those skilled in the art.

[0094] As described herein, the linker between two peptide constructs can be an amino acid sequence containing 1 to 200 (e.g., 1 to 4, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 2 to 10, 2 to 12, 2 to 16, 1, 2, 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, 40, 50, 60, 70, 80, 90, 100, 150, 200) amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine.

[0095] Glycine, serine, and alanine are useful for providing maximum flexibility in the linker. Any amino acid residue can be considered a linker, optionally combined with one or more other amino acid residues, which may be the same or different from the first amino acid residue, to construct a larger peptide linker depending on the desired properties. In another embodiment, the linker is GGGGEGGGGEGGGGE (SEQ ID NO: 10). In another embodiment, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 11). Additional peptide linkers suitable for use in creating the fusion proteins described herein include, for example, G4S (SEQ ID NO: 12), (G4S)2 (SEQ ID NO: 13), (G4S)3 (SEQ ID NO: 14), (G4S)4 (SEQ ID NO: 15), (G4S)5 (SEQ ID NO: 16), (G4S)6 (SEQ ID NO: 17), (EAAAK)3 (SEQ ID NO: 18), PAPAP (SEQ ID NO: 19), G4SPAPAP (SEQ ID NO: 20), PAPAPG 4S (SEQ ID NO: 21), (GGGDS)2 (SEQ ID NO: 22), (GGGES)2 (SEQ ID NO: 23), GGGDSGGGGS (SEQ ID NO: 24), GGGASGGGGS (SEQ ID NO: 25), GGGESGGGGS (SEQ ID NO: 26), ASTKGP (SEQ ID NO: 27), ASTKGPSVFPLAP (SEQ ID NO: 28), G3P (SEQ ID NO: 29), G7P (SEQ ID NO: 30), PAPNLLGGP (SEQ ID NO: 31), G6 (SEQ ID NO: 32), G 12(SEQ ID NO: 33), APELPGGP (SEQ ID NO: 34), SEPQPQPG (SEQ ID NO: 35), (G3S2)3 (SEQ ID NO: 36), GGGGGGGGGSGGGS (SEQ ID NO: 37), GGGGSGGGGGGGGGS (SEQ ID NO: 38), (GGSSS)3 (SEQ ID NO: 39), (GS4)3 (SEQ ID NO: 40), G4A(G4S)2 (SEQ ID NO: 41), G4SG4AG4S (SEQ ID NO: 42), G3AS(G4S)2 (SEQ ID NO: 43), G4SG3ASG4S (SEQ ID NO: 44), Examples of suitable linkers include G4SAG3SG4S (SEQ ID NO: 45), (G4S)2AG3S (SEQ ID NO: 46), G4SAG3SAG3S (SEQ ID NO: 47), G4D(G4S)2 (SEQ ID NO: 48), G4SG4DG4S (SEQ ID NO: 49), (G4D)2G4S (SEQ ID NO: 50), G4E(G4S)2 (SEQ ID NO: 51), G4SG4EG4S (SEQ ID NO: 52), (G4E)2G4S (SEQ ID NO: 53), and GGGGAGGGGAGGGGS (SEQ ID NO: 54). A person skilled in the art can select a linker to, for example, reduce or eliminate post-translational modifications, such as glycosylation, e.g., xylosylation. In certain embodiments, the fusion protein comprises at least two sdAbs, Dabs, VHH antibodies, VHH antibody fragments, or a combination thereof, wherein at least one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against albumin and one of the sdAbs, Dabs, VHH antibodies, or VHH antibody fragments is directed against properdin, such that the resulting fusion protein is multivalent or multispecific. The binding domains or moieties may, for example, be directed against HSA, cynomolgus serum albumin, human properdin, and / or cynomolgus properdin.

[0096] Methods for formulating highly concentrated compositions Described herein is a method for developing a high-concentration formulation of a fusion protein for subcutaneous administration. The development of a high-concentration formulation for subcutaneous administration has several challenges, including the need to optimize viscosity, stability, and delivery. A design of experiments (DOE) approach may be used to develop a high-concentration formulation for subcutaneous administration of a fusion protein to a subject. The method described herein is designed to select potential viscosity-reducing excipients while minimizing adverse effects on the stability of the pharmaceutical composition.

[0097] Formulating a high-concentration formulation of a fusion protein can include a first step of performing an excipient screening. The excipient screening can include measuring the stability and viscosity of a solution containing the fusion protein and one or more excipients over a period of time. In a second step, the method can include, following the excipient screening, measuring the viscosity, turbidity, and rate of formation of a high-molecular-weight compound in the solution as a result of varying pH, acetate concentration, and sucrose concentration. As a third step, the method can include measuring the viscosity, turbidity, and rate of formation of a high-molecular-weight compound in the solution as a result of varying pH, sucrose concentration, excipient, and fusion protein concentration. A solution viscosity evaluation can be performed as a fourth step. The viscosity of the formulation is measured over a range of temperatures and a range of fusion protein concentrations.

[0098] Measurements of viscosity, turbidity, and rate of formation of the high molecular weight compound in the second and third steps may be performed weekly and may be measured over a period of at least four weeks.

[0099] For excipient screening, one or more excipients may be selected from succinate, arginine, acetate, or a combination thereof. One or more excipients may be arginine. For example, the arginine may be arginine hydrochloride, arginine acetate, or arginine succinate. One or more excipients may be succinate. In some embodiments, one or more excipients are succinate and arginine hydrochloride. One or more excipients may be acetate. In some embodiments, one or more excipients are acetate and arginine hydrochloride. In some embodiments, one or more excipients are acetate and arginine acetate.

[0100] In some embodiments, the solution described in step 2 has an acetate concentration of 10 mM to 200 mM (e.g., 10 mM to 150 mM, 10 mM to 100 mM, 10 mM to 50 mM, 50 mM to 200 mM, 100 mM to 200 mM, or 150 mM to 200 mM). For example, the solution has an acetate concentration of 15 mM to 100 mM (e.g., 15 mM to 80 mM, 15 mM to 60 mM, 15 mM to 40 mM, 15 mM to 20 mM, 20 mM to 100 mM, 40 mM to 100 mM, 60 mM to 100 mM, or 80 mM to 100 mM).

[0101] Alternatively, the solution in the first step involving excipient screening may be sucrose. The sucrose concentration in the solution may be 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the sucrose concentration is about 4% (w / v). The solution in the second step may have a sucrose concentration of 1% (w / v) to 20% (w / v) (e.g., 1% (w / v) to 15% (w / v), 1% (w / v) to 10% (w / v), 1% (w / v) to 5% (w / v), 5% (w / v) to 20% (w / v), 10% (w / v) to 20% (w / v), or 15% (w / v) to 20% (w / v)). For example, the solution described in step 2 can have a sucrose concentration of 2% (w / v) to 10% (w / v) (e.g., 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). The third step can have a solution with a sucrose concentration of 0% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). For example, the solution in step 3 may have a sucrose concentration of 0% (w / v) to 5% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), or 5% (w / v)).

[0102] The pH of the solution in the second step may be 4 to 7 (e.g., a pH of 4 to 6, 4 to 5, 5 to 7, or 6 to 7). For example, the pH of the solution is 4.5 to 6.0 (e.g., a pH of 4.5 to 5.5, 4.5 to 5, 5 to 6, or 5.5 to 6). The pH of the solution in the third step may be 5 to 6.

[0103] The fusion protein present in the high concentration formulation may comprise six CDRs having the amino acid sequences of SEQ ID NOs: 2-7. The fusion protein may comprise a first portion comprising an amino acid sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 55. Additionally, the fusion protein may comprise a second portion comprising an amino acid sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 56. In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO: 1, or a modification thereof. In some embodiments, the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

[0104] The fusion protein may have a concentration of about 150 mg / mL in the solution described in the second step. The fusion protein may have a concentration of about 190 mg / mL in the solution described in the second step. The solution in the second step may have a fusion protein concentration of about 150 mg / mL to 230 mg / mL (e.g., 150 mg / mL to 200 mg / mL, 150 mg / mL to 175 mg / mL, 175 mg / mL to 230 mg / mL, 200 mg / mL to 230 mg / mL, or 220 mg / mL to 230 mg / mL). In some embodiments, the solution in the third step has a fusion protein concentration of about 100 mg / mL to 300 mg / mL (e.g., 100 mg / mL to 250 mg / mL, 100 mg / mL to 200 mg / mL, 100 mg / mL to 150 mg / mL, 150 mg / mL to 300 mg / mL, 200 mg / mL to 300 mg / mL, or 250 mg / mL to 300 mg / mL). The fusion protein in a high-concentration formulation may be present at a concentration of at least 150 mg / mL. For example, the fusion protein in an excipient screen may be present at a concentration of about 200 mg / mL.

[0105] A high concentration formulation can have a viscosity of 5 cP to 15 cP (e.g., 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, or 15 cP) at 20° C. For example, the formulation has a viscosity of about 10 cP or 11 cP at 20° C. The formulation can have a percentage of higher molecular weight compound per month at 25° C. of 0.05% (w / v) to 0.5% (w / v) (e.g., 0.05% (w / v) to 0.4% (w / v), 0.05% (w / v) to 0.3% (w / v), 0.05% (w / v) to 0.2% (w / v), 0.05% (w / v) to 0.1% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.2% (w / v) to 0.5% (w / v), 0.3% (w / v) to 0.5% (w / v), or 0.4% (w / v) to 0.5% (w / v)). For example, the formulation has a percentage of higher molecular weight compound per month at 25° C. over one month of about 0.2% (w / v). In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 37° C. over one month of about 1% (w / v) to 10% (w / v) (e.g., 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), or 10% (w / v)). In some embodiments, the formulation has a percentage of higher molecular weight compound per month at 37° C. over one month of about 5% (w / v). In some embodiments, the formulation has a turbidity of about 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) per month at 37° C., where the turbidity is measured as optical density at 400 nm. In some embodiments, the formulation has a turbidity of about 3 or 4 per month at 37° C.

[0106] Treatment method The compositions described herein can be used in methods of treating a disease or disorder mediated by alternative complement pathway dysfunction in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of a high concentration formulation comprising a fusion protein described herein, in treating a disease mediated by alternative complement pathway dysregulation in a mammal (e.g., a human) by inhibiting alternative complement pathway activation. In some embodiments, the pharmaceutical compositions described herein can be used in methods of treating or preventing a disease or condition in a subject, wherein the disease is sickle cell disease.

[0107] The pharmaceutical compositions described herein can be administered by various methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is intravenous injection or infusion. Polypeptides may also be administered by intramuscular or subcutaneous injection. In some embodiments, the pharmaceutical compositions described herein may be administered subcutaneously to a subject. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Many methods for preparing such formulations are known to those skilled in the art (e.g., Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978). Methods applicable to the controlled or sustained release of antibodies, such as the monovalent single domain antibodies disclosed herein, are known (U.S. Pat. Nos. 6,306,406 and 6,346,274; U.S. Patent Application Publication Nos. 20020182254 and 20020051808, the teachings of each of which are incorporated herein by reference in their entirety).

[0108] In some embodiments, the fusion protein described herein is administered using a pre-filled syringe.In other embodiments, the fusion protein is administered using an auto-injector device.For example, the auto-injector device, such as the pen-type injector device for solution delivery, can be equipped with a single vial system. Such devices are commercially available from manufacturers such as, for example, BD Pen, BD Autojector®, Humaject®, NovoPen®, BD® Pen, AutoPen®, and OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, DosePro®, Medi-Ject® (e.g., Becton Dickinson, Franklin Lakes, NJ; Ypsomed, Burgdorf, Switzerland; www.ypsomed.com; Bioject, Portland, OR; National Medical Products, Weston Medical, Peterborough, UK; Medi-Ject (manufactured or developed by YpsoMate 2.25 or YpsoMate 2.25 Pro (Ypsomed) disposable injection devices). Recognized devices with dual vial systems include pen-type injector systems for reconstituting lyophilized drugs in cartridges for delivery of reconstitution solution, such as HumatroPen®. In one embodiment, the autoinjector is a YpsoMate 2.25 or YpsoMate 2.25 Pro (Ypsomed) disposable injection device.

[0109] kit Compositions containing the fusion proteins described herein can be provided in kits for use in treating a disease or condition. The kits can further include a label or package insert instructing a user of the kit, such as a subject with a disease or condition or a physician, to perform the methods described herein. In some embodiments, the kits include a container with a label and a composition containing a fusion protein described herein, the label indicating that the composition should be administered to a patient in need of the composition. The kits may optionally include a syringe or other device (e.g., an autoinjector, a prefilled syringe, or a wearable device) for administering the composition. In other embodiments, the kits include single-chamber or multi-chamber prefilled syringes (e.g., liquid syringes and dispersing syringes (lyosyringes)). In some embodiments, the kits include a cartridge containing a composition described herein for use in a medical device (e.g., an autoinjector or a wearable device). [Example]

[0110] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of the present disclosure.

[0111] Example 1. Application of a Design Experiment Strategy to Develop a High-Concentration Bispecific Antibody Formulation for Subcutaneous Administration Subcutaneous (SC) administration of protein therapeutics offers several advantages, including patient compliance / convenience, palatability, and improved PK / PD profiles. Injection volumes for SC administration are typically small (<2 mL), which in turn requires highly concentrated formulations to achieve adequate doses. Several challenges remain associated with the development of highly concentrated formulations, such as viscosity, stability, and delivery.

[0112] A design of experiments (DOE) approach was used to develop a high-concentration formulation of a 27 kDa bispecific antibody for subcutaneous administration. Given that viscosity can be a limiting factor in the development of ultra-high concentration (e.g., 150 mg / mL) formulations, the objective of the experiment was to select potential viscosity-reducing excipients while minimizing adverse effects on the stability of the pharmaceutical composition.

[0113] A preliminary excipient screen with arginine, arginine and counterion, and proline was performed to evaluate the stability of a solution of a fusion protein having the amino acid sequence of SEQ ID NO: 1 at a concentration of 200 mg / mL. st A DOE was performed to evaluate the effect of pH, acetate concentration, and sucrose concentration on the stability of pharmaceutical compositions with a fusion protein concentration of 150 mg / mL. Following this evaluation, the effects of pH, protein concentration, the presence of proline, and sucrose concentration were studied on the stability of the pharmaceutical compositions. Finally, a viscosity evaluation of the pharmaceutical compositions was performed.

[0114] For preliminary excipient screening, the following formulations were investigated: F1: 20 mM acetate, 8.6% (w / v) sucrose, 0.05% (w / v) PS-80, pH 5.4 F2: 20 mM acetate, 4% (w / v) sucrose, 0.05% (w / v) PS-80, pH 5.4 F3: 20 mM acetate, 4% (w / v) sucrose, 150 mM Arg-HCl, 0.05% (w / v) PS-80, pH 5.4 F4: 20 mM succinate, 4% (w / v) sucrose, 150 mM Arg-HCl, 0.05% (w / v) PS-80, pH 5.4 F5: 20 mM acetate, 4% (w / v) sucrose, 150 mM Arg-acetate, 0.05% (w / v) PS-80, pH 5.4 F6: 4% (w / v) sucrose, 150 mM Arg-succinate, 0.05% (w / v) PS-80, pH 5.4 F7: 20 mM acetate, 4% (w / v) sucrose, 150 mM proline, 0.05% (w / v) PS-80, pH 5.4 (one time point after 1 week)

[0115] The viscosity (Figures 1A, 1B, and 1C), the percentage of higher molecular weight (HMW) species present in the solution (Figures 2A and 2B), and the turbidity of the solutions as determined by optical density at 400 nm (Figure 4) were assessed after 1, 2, and 4 weeks of storage at 37°C, and photographs of the solutions were taken over time (Figures 3A and 3B). This excipient screen showed that arginine and proline did not reduce viscosity, and increasing levels of sucrose slightly increased viscosity. In addition, Arg-HCl was shown to destabilize the molecule, increasing turbidity and HMW species. Arg-acetate destabilized the molecule, increasing turbidity. Arg-succinate destabilized the molecule, increasing turbidity but decreasing HMW species. The presence of proline reduced the formation of HMW species. Increasing the concentration of sucrose minimized the increase in turbidity and reduced HMW formation at 37°C. Formulations with Arg revealed a significant increase in turbidity after 1 week at 37°C.

[0116] After preliminary excipient screening, 1 st A DOE study was performed to evaluate the effect of pH, acetate concentration, and sucrose concentration on the stability of pharmaceutical compositions containing 150 mg / mL of fusion protein. The rates of HMW formation were evaluated over one month at 37°C (Figure 5A) and 25°C (Figure 5B) for a composition containing 100 mM acetate, 2% (w / v) sucrose, and 0.05% PS80 at pH 5.4 (F4) and a composition containing 60.9 mM acetate, 2% (w / v) sucrose, and 0.05% PS80 at pH 6.0 (F5). The rates of acid formation, base formation, and turbidity increase were all similarly measured over one month at both 37°C (Figures 6A, 7A, and 8A, respectively) and 25°C (Figures 6B, 7B, and 8B, respectively).

[0117] In addition, predictive profiles were generated for compositions having 150 mg / mL of fusion protein to study the effect of acetate concentration, sucrose concentration, and composition pH on osmolality and viscosity at 25°C and 20°C (Figure 9). These profiles indicated that increasing acetate concentration increases osmolality but has little effect on viscosity, increasing sucrose concentration increases osmolality and increases viscosity, and increasing pH has little effect on osmolality and viscosity. Predictive profiles were also generated for compositions to evaluate the effect of acetate concentration, sucrose concentration, and composition pH on HMW formation rate, turbidity increase, acid formation rate, and base formation rate at 37°C (Figure 10A) and 25°C (Figure 10B). These predictions indicated that increasing acetate concentration has little effect on HMW formation rate, results in an increase in turbidity rate, and has little effect on acid formation rate. Increasing sucrose concentration had little effect on basic and acidic rate formation. Increasing pH had little effect on basic and acidic rate formation, but resulted in increased rates of HMW formation and turbidity. The results of these studies are summarized in Table 1 below.

[0118] [Table 1]

[0119] The results of this study resulted in a Phase 1 formulation of 150 mg / mL bispecific antibody having the sequence of SEQ ID NO: 1 in 20 mM sodium acetate, 8.6% w / v sucrose, 0.05% w / v PS80, pH 5.4. In addition, it is noted that the predictions are consistent with the experimental data, demonstrating that the model is robust.

[0120] Then, 1 st DOE, 2 ndA DOE study was performed to evaluate the effects of pH, protein concentration, the presence of proline, and sucrose concentration on the stability of pharmaceutical compositions. The rates of HMW formation over one month at 37°C (Figure 11A) and 25°C (Figure 11B) were evaluated for a composition (F2) containing 230 mg / mL fusion protein, 40 mM acetate, and 0.05% PS80 at pH 5.2, and a composition (F7) containing 221 mg / mL fusion protein, 40 mM acetate, 5% (w / v) sucrose, 165 mM proline, and 0.05% PS80 at pH 5.2. The rates of acid formation, base formation, and turbidity increase were all similarly measured over one month at both 37°C (Figures 12A, 13A, and 14A), respectively, and 25°C (Figures 12B, 13B, and 14B).

[0121] In addition, predictive profiles were generated for the compositions to study the effect of protein concentration, proline concentration, sucrose concentration, and composition pH on osmolality and viscosity at 25°C and 20°C (Figure 15). These profiles showed that increasing protein concentration increases osmolality and viscosity, increasing proline concentration increases osmolality and has little effect on viscosity, increasing sucrose concentration increases osmolality and has little effect on viscosity, and increasing pH has little effect on osmolality and viscosity. Predictive profiles were also generated for the compositions to evaluate the effect of protein concentration, proline concentration, sucrose concentration, and composition pH on HMW formation rate, turbidity increase, acid formation rate, and base formation rate at 37°C (Figure 16A) and 25°C (Figure 16B). These predictions indicated that increasing protein concentration increased the HMW formation rate, turbidity rate, and basic rate formation, and had little effect on acidic rate formation. Increasing proline concentration resulted in a decrease in HMW species formation and a decrease in basic rate formation, and had little effect on the turbidity rate and acidic rate formation. Increasing sucrose concentration had little effect on the rate of HMW formation at 37°C, and resulted in a decrease in basic rate formation, acidic rate formation, and HMW formation at 25°C. Increasing pH resulted in a decrease in HMW formation rate, basic rate formation, and acidic rate formation at 37°C, and an increase in turbidity rate, HMW formation rate, and turbidity rate at 25°C.

[0122] The viscosity of the compositions summarized in Table 2 was evaluated over a range of concentrations of fusion protein at 20° C., as shown in FIG.

[0123] [Table 2]

[0124] Based on stability data, the primary degradation pathway was the formation of HMW species at accelerated (25°C) conditions, and the formation of HMW species and increased turbidity at stressed (37°C) conditions. No significant changes in charge variants were observed in these studies. A formulation concentration of 190 mg / mL was selected based on viscosity. Based on these studies, the preferred formulations were determined to be 190 mg / mL in 20 mM sodium acetate, 4% (w / v) sucrose, 0.05% (w / v) PS80, pH 5.4, and 190 mg / mL in 20 mM sodium acetate, 165 mM proline, 0.05% (w / v) PS80, pH 5.4. The properties of each of these formulations are summarized in Table 3.

[0125] [Table 3]

[0126] Additional Embodiments All references cited herein, including database entry information (e.g., in GENBANK, UNIPROT, PUBMED), are hereby incorporated by reference as if each reference were specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0127] It will also be understood that each of the above-described elements, or two or more together, may find useful application in other types of methods different from those described above. Without further analysis, the foregoing will make the gist of the present disclosure so clear that others, by applying their current knowledge, can easily adapt it to various applications without omitting features that, in view of the prior art, constitute essential features of the general or specific aspects of the present disclosure as set forth in the appended claims. The foregoing embodiments are presented by way of example only.

Claims

1. A pharmaceutical composition comprising a fusion protein, a detergent, and a buffer, wherein the fusion protein is present at a concentration of at least 120 mg / mL.

2. The pharmaceutical composition of claim 2, wherein the fusion protein comprises a complementarity determining region (CDR) having an amino acid sequence of SEQ ID NO: 2-7.

3. 3. The pharmaceutical composition of claim 2, wherein the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 55 and a second portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

56.

4. 4. The pharmaceutical composition of claim 2 or 3, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 1 or a modification of the amino acid sequence of SEQ ID NO:

1.

5. 5. The pharmaceutical composition of claim 4, wherein the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the concentration of the fusion protein is 120 mg / mL to 250 mg / mL.

7. The pharmaceutical composition of any one of claims 1 to 5, wherein the fusion protein is present in a concentration of at least 150 mg.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the concentration of the fusion protein is 150 mg / mL to 200 mg / mL.

9. 9. The pharmaceutical composition of claim 8, wherein the concentration of the fusion protein is 170 mg / mL to 200 mg / mL.

10. 10. The pharmaceutical composition of claim 9, wherein the concentration of the fusion protein is about 190 mg / mL.

11. 9. The pharmaceutical composition of claim 8, wherein the concentration of the fusion protein is about 150 mg / mL.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the detergent is a polysorbate.

13. 13. The pharmaceutical composition of claim 12, wherein the polysorbate is polysorbate 80 (PS80).

14. The pharmaceutical composition according to claim 12 or 13, wherein the concentration of the polysorbate is 0.001% to 1% (w / v).

15. The pharmaceutical composition of claim 14, wherein the concentration of the polysorbate is 0.05% (w / v) to 0.5% (w / v).

16. 16. The pharmaceutical composition of claim 15, wherein the concentration of the polysorbate is 0.1% (w / v) to 0.2% (w / v).

17. 17. The pharmaceutical composition of claim 16, wherein the concentration of the polysorbate is about 0.15% (w / v).

18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the buffering agent is acetate, succinate, histidine, phosphate, or a combination thereof.

19. 19. The pharmaceutical composition of claim 18, wherein the buffering agent is acetate.

20. 20. The pharmaceutical formulation of claim 19, wherein the acetate is sodium acetate.

21. 21. The pharmaceutical composition of claim 20, wherein the concentration of sodium acetate is about 10 mM to 150 mM.

22. 22. The pharmaceutical composition of claim 21, wherein the concentration of sodium acetate is about 15 mM to 100 mM.

23. 23. The pharmaceutical composition of claim 22, wherein the concentration of sodium acetate is about 50 mM.

24. 22. The pharmaceutical composition of claim 21, wherein the concentration of sodium acetate is about 20 mM.

25. The pharmaceutical composition according to any one of claims 1 to 24, further comprising an amino acid.

26. 26. The pharmaceutical composition of claim 25, wherein the amino acid is arginine, proline, or glycine.

27. 27. The pharmaceutical composition of claim 26, wherein the concentration of the amino acid is about 100 mM to 200 mM.

28. 28. The pharmaceutical composition of claim 27, wherein the concentration of the amino acid is about 165 mM.

29. The pharmaceutical composition according to any one of claims 1 to 28, further comprising an isotonicity agent.

30. 30. The pharmaceutical composition of claim 29, wherein the tonicity agent is a sugar, an amino acid, or a salt.

31. 31. The pharmaceutical composition of claim 30, wherein the salt is NaCl.

32. 31. The pharmaceutical composition of claim 30, wherein the sugar is sucrose, glucose, glycerol, or trehalose.

33. 33. The pharmaceutical composition of claim 32, wherein the sugar is sucrose.

34. 33. The pharmaceutical composition of claim 32, wherein the sucrose is present at a concentration of about 1% (w / v) to about 15% (w / v).

35. 35. The pharmaceutical composition of claim 34, wherein the sucrose is present in a concentration of 2% (w / v) to 10% (w / v).

36. 35. The pharmaceutical composition of claim 34, wherein the sucrose is present in a concentration of 4% (w / v) to 9% (w / v).

37. 37. The pharmaceutical composition of claim 36, wherein the sucrose is present at a concentration of 8% (w / v) to 9% (w / v).

38. 38. The pharmaceutical composition of claim 37, wherein the sucrose is present at a concentration of about 8.6% (w / v).

39. 35. The pharmaceutical composition of claim 34, wherein the sucrose is present at a concentration of about 4% (w / v).

40. 40. The pharmaceutical composition of any one of claims 1 to 39, wherein the pH is from about pH 3 to pH 8.

41. 41. The pharmaceutical composition of claim 40, wherein the pH is between pH 4 and pH 7.

42. 42. The pharmaceutical composition of claim 41, wherein the pH is about pH 5.

4.

43. 43. The pharmaceutical composition of any one of claims 1 to 42, having a viscosity of 6 cP to 35 cP at 20°C.

44. 44. The pharmaceutical composition of any one of claims 1 to 43, having a viscosity of <17 cP at 20°C.

45. 45. The pharmaceutical composition of claim 44, having a viscosity of <12 cP at 20°C.

46. 46. The pharmaceutical composition of any one of claims 1 to 45, wherein the percentage of higher molecular weight compounds per month at 25°C over one month is between 0.05% (w / v) and 0.5% (w / v).

47. 47. The pharmaceutical composition of claim 46, wherein the percentage of higher molecular weight compounds per month at 25°C over one month is about 0.2% (w / v).

48. 48. The pharmaceutical composition of any one of claims 1 to 47, wherein the percentage of higher molecular weight compound per month at 37°C over one month is about 1% (w / v) to 10% (w / v).

49. 49. The pharmaceutical composition of claim 48, wherein the percentage of higher molecular weight compounds per month at 37°C over one month is about 4.6% (w / v) or about 5% (w / v).

50. 50. The pharmaceutical composition of any one of claims 1 to 49, having a turbidity of about 1 to 10 per month at 37°C, wherein the turbidity is measured as optical density at 400 nm.

51. 51. The pharmaceutical composition of claim 50, having a turbidity of about 3 or about 4 per month at 37°C.

52. Osmolality is 100 Osm / H ２ Okg~500Osm / H ２ 52. The pharmaceutical composition according to any one of claims 1 to 51, wherein the amount is 0 kg.

53. Osmolality is approximately 245 Osm / H ２ 53. The pharmaceutical composition of claim 52, wherein the amount is 0 kg.

54. 1. A pharmaceutical composition comprising a fusion protein, the fusion protein comprising a CDR having an amino acid sequence of SEQ ID NO: 2-7, a detergent, sucrose, and sodium acetate, wherein the fusion protein is present at a concentration of 120 mg / mL to 200 mg / mL, the sodium acetate is present at a concentration of 25 mM to 75 mM, the sucrose is present at a concentration of 2% (w / v) to 15% (w / v), the detergent is present at a concentration of 0.01% (w / v) to 0.2% (w / v), and the pharmaceutical composition has a pH of 4 to 7.

55. 55. The pharmaceutical composition of claim 54, wherein the concentration of the fusion protein is about 150 mg / mL, the sodium acetate concentration is about 50 mM, the sucrose concentration is about 8.6% (w / v), the detergent is PS-80 at a concentration of about 0.05% (w / v), and the pharmaceutical composition has a pH of about 5.

4.

56. 55. The pharmaceutical composition of claim 54, wherein the concentration of the fusion protein is about 150 mg / mL, the sodium acetate concentration is about 50 mM, the sucrose concentration is about 8.6% (w / v), the detergent is PS-80 at a concentration of about 0.15% (w / v), and the pharmaceutical composition has a pH of about 5.

4.

57. A pharmaceutical composition comprising a fusion protein, the fusion protein comprising a CDR having an amino acid sequence of SEQ ID NO: 2-7, a detergent, and sodium acetate, wherein the PS80 is present at a concentration of 0.01% (w / v) to 0.1% (w / v), the sucrose is present at a concentration of 1% (w / v) to 10% (w / v), the sodium acetate is present at a concentration of 10 mM to 50 mM, the pharmaceutical composition has a pH of 4 to 7, and the fusion protein is present at a concentration of 150 mg / mL to 200 mg / mL.

58. 58. The pharmaceutical composition of claim 57, wherein the fusion protein is present at a concentration of about 190 mg / mL, the PS80 is present at a concentration of about 0.05% (w / v) or about 0.1% (w / v), the sucrose is present at a concentration of 4% (w / v), the sodium acetate is present at a concentration of about 20 mM, and the pharmaceutical composition has a pH of about pH 5.

4.

59. 1. A pharmaceutical composition comprising a fusion protein, the fusion protein comprising a CDR having an amino acid sequence of SEQ ID NO: 2-7, a detergent, sodium acetate, and proline, wherein the detergent is present at a concentration of 0.01% (w / v) to 0.1% (w / v), the sodium acetate is present at a concentration of 10 mM to 50 mM, and the proline is present at a concentration of 100 mM to 200 mM, the pharmaceutical composition has a pH of 4 to 7, and the fusion protein is present at a concentration of 150 mg / mL to 200 mg / mL.

60. 60. The pharmaceutical composition of claim 59, wherein the fusion protein is present at a concentration of about 190 mg / mL, the detergent is present at a concentration of about 0.05% (w / v) or about 0.1% (w / v), the sodium acetate is present at a concentration of about 20 mM, the proline is present at a concentration of about 165 mM, and the pharmaceutical composition has a pH of about pH 5.

4.

61. 61. The pharmaceutical composition of any one of claims 54 to 60, wherein the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 55, and a second portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

56.

62. 62. The pharmaceutical composition of any one of claims 54 to 61, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 1 or a modification of the amino acid sequence of SEQ ID NO:

1.

63. 63. The method of claim 62, wherein the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

64. 64. The pharmaceutical composition of any one of claims 1 to 63, formulated as a drug product.

65. 65. A method of treating or preventing a disease or condition in a subject, comprising administering to a subject in need of such treatment or prevention the pharmaceutical composition of claim 64.

66. 66. The method of claim 65, wherein the disease is sickle cell disease.

67. 67. The method of claim 65 or 66, wherein the pharmaceutical composition is administered subcutaneously.

68. 68. The method of claim 67, wherein the pharmaceutical composition is administered via a pre-filled syringe, auto-injector, or on-body device.

69. 69. The method of any one of claims 65 to 68, wherein the subject is a human.

70. 1. A method for developing a highly concentrated formulation of a fusion protein for subcutaneous administration, comprising: i) performing an excipient screen comprising measuring the stability and viscosity of a solution comprising said fusion protein and one or more excipients over a period of time; ii) following step (i), measuring the viscosity, turbidity, and rate of formation of high molecular weight compounds in the solution of step (i) as a result of varying pH, acetate concentration, and sucrose concentration; iii) following step (ii), measuring the viscosity, turbidity, and rate of formation of high molecular weight compounds in the solution of step (ii) as a result of varying pH, sucrose concentration, excipients, and concentration of the fusion protein; iv) following step (iii), performing a viscosity evaluation; wherein the viscosity of the formulation is measured at a range of temperatures and a range of fusion protein concentrations.

71. 71. The method of claim 70, wherein the fusion protein comprises six CDRs having the amino acid sequences of SEQ ID NOs: 2-7.

72. 72. The method of claim 70 or 71, wherein the fusion protein comprises a first portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 55, and a second portion comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

56.

73. 73. The method of any one of claims 70 to 72, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 1 or a modification of the amino acid sequence of SEQ ID NO:

1.

74. 74. The method of claim 73, wherein the modification comprises converting the N-terminal glutamine of the sequence of SEQ ID NO: 1 to pyroglutamate.

75. 75. The method of any one of claims 70 to 74, wherein the high concentration formulation has a fusion protein concentration of at least 120 mg / mL.

76. 76. The method of any one of claims 70 to 75, wherein the high concentration formulation has a fusion protein concentration of 120 mg / mL to 250 mg / mL.

77. 76. The method of any one of claims 70 to 75, wherein the high concentration formulation has a fusion protein concentration of at least 150 mg / mL.

78. 78. The method of any one of claims 70 to 77, wherein the solution in step (i) has a fusion protein concentration of about 200 mg / mL.

79. 79. The method of any one of claims 70 to 78, wherein the measurements of steps ii and iii are performed weekly.

80. 80. The method of claim 79, wherein the measurements are performed for at least four weeks.

81. 81. The method of any one of claims 70 to 80, wherein the one or more excipients are selected from succinate, arginine, acetate, histidine, phosphate, or combinations thereof.

82. 82. The method of claim 81, wherein said one or more excipients is arginine.

83. 83. The method of claim 82, wherein the arginine is arginine hydrochloride.

84. 83. The method of claim 82, wherein the arginine is arginine acetate.

85. 83. The method of claim 82, wherein the arginine is arginine succinate.

86. 82. The method of claim 81, wherein the one or more excipients is a succinate.

87. 82. The method of claim 81, wherein the one or more excipients are succinate and arginine hydrochloride.

88. 82. The method of claim 81, wherein the one or more excipients is acetate.

89. 82. The method of claim 81, wherein the one or more excipients are acetate and arginine hydrochloride.

90. 82. The method of claim 81, wherein the one or more excipients are acetate and arginine acetate.

91. 91. The method of any one of claims 70 to 90, wherein the solution of step (i) further comprises sucrose.

92. 92. The method of claim 91, wherein the sucrose is present at a concentration of 8% (w / v) to 9% (w / v).

93. 93. The method of claim 92, wherein the sucrose is present at a concentration of about 8.6% (w / v).

94. 92. The method of claim 91, wherein the sucrose is at a concentration of 1% (w / v) to 10% (w / v).

95. 95. The method of claim 94, wherein the sucrose is at a concentration of about 4% (w / v).

96. 96. The method of any one of claims 70 to 95, wherein the solution in step (ii) has a pH of 4 to 7.

97. 97. The method of claim 96, wherein the solution in step (ii) has a pH of 4.5 to 6.

0.

98. 98. The method of any one of claims 70 to 97, wherein the solution in step (ii) has an acetate concentration of 10 mM to 200 mM.

99. 82. The method of claim 81, wherein the solution in step (ii) has an acetate concentration of 15 mM to 150 mM.

100. 100. The method of any one of claims 70 to 99, wherein the solution in step (ii) has a sucrose concentration of 1% (w / v) to 20% (w / v).

101. 101. The method of claim 100, wherein the solution in step (ii) has a sucrose concentration of 2% (w / v) to 10% (w / v).

102. 102. The method of any one of claims 70 to 101, wherein the solution in step (ii) has a fusion protein concentration of about 150 mg / mL.

103. 103. The dendrimer of any one of claims 70 to 102, wherein the solution in step (iii) has a pH of 5 to 6.

104. 104. The method of any one of claims 70 to 103, wherein the solution in step (iii) has a sucrose concentration of 0% (w / v) to 10% (w / v).

105. 105. The method of claim 104, wherein the solution in step (iii) has a sucrose concentration of 8% (w / v) to 9% (w / v).

106. 106. The method of any one of claims 70 to 105, wherein the solution in step (iii) has a fusion protein concentration of about 100 mg / mL to 300 mg / mL.

107. 107. The method of claim 106, wherein the solution of step (ii) has a fusion protein concentration of about 150 mg / mL to 230 mg / mL.

108. 108. The method of any one of claims 70 to 107, wherein the formulation has a viscosity of between 6 cP and 35 cP at 20°C.

109. 109. The method of any one of claims 70 to 108, wherein the formulation has a viscosity of 5 cP to 15 cP at 20°C.

110. 110. The method of claim 109, wherein the formulation has a viscosity of about 10 cP or 11 cP at 20°C.

111. 111. The method of any one of claims 70 to 110, wherein the formulation has a percentage of higher molecular weight compound per month at 25°C of 0.05% (w / v) to 0.5% (w / v).

112. 112. The method of claim 111, wherein the formulation has a percentage of higher molecular weight compounds per month at 25°C over one month of about 0.2% (w / v).

113. 113. The method of any one of claims 70-112, wherein the formulation has a percentage of higher molecular weight compound per month at 37°C of about 1% (w / v) to 10% (w / v).

114. 114. The method of claim 113, wherein the formulation has a percentage of higher molecular weight compounds per month at 37°C of about 4% (w / v) or about 5% (w / v).

115. 115. The method of any one of claims 70 to 114, wherein the formulation has a turbidity of about 1 to 10 per month at 37°C, where the turbidity is measured as optical density at 400 nm.

116. 116. The method of claim 115, wherein the formulation has a turbidity of about 3 or 4 per month at 37°C.

117. A bispecific construct that binds to properdin and human serum albumin, said bispecific construct comprising the amino acid sequence of SEQ ID NO:1 or a modification of the amino acid sequence of SEQ ID NO:

1.

118. 118. The bispecific construct of claim 117, wherein the modification comprises converting the N-terminal glutamine of the amino acid sequence of SEQ ID NO: 1 to pyroglutamate.

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