Pharmaceutical preparations containing heterodimeric relaxin fusion proteins and their use

Abstract

This disclosure relates to pharmaceutical formulations comprising heterodimeric relaxin fusion polypeptides, specifically heterodimeric relaxin 2 fusion polypeptides, and their uses. Accordingly, this disclosure provides pharmaceutical formulations comprising relaxin fusion polypeptides, kits comprising the same, and their uses, including therapeutic methods. The pharmaceutical formulations of the present invention may be useful specifically for the treatment of cardiovascular diseases, such as heart failure.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to European Patent Application Publication No. 23160008.1, filed on 3 March 2023, the contents of which are incorporated herein by reference.

[0002] (Sequence Listing) This application includes a sequence listing, which is incorporated herein by reference in its entirety. The sequence listing, created on 16 February 2024, is named 201258-WO-PCT Sequence listing.xml and has a size of 82,163 bytes.

[0003] (Field of Invention) This disclosure relates to the field of pharmaceutical formulations. Specifically, it provides stable pharmaceutical formulations for peptide-Fc fusion proteins. [Background technology]

[0004] Biologics are a type of drug whose active ingredient is derived from a biological origin. Therefore, biologics include macromolecular therapies such as proteins, antibodies, peptides, and nucleic acids, as well as cell-based therapies. Compared to small molecule therapies, biologics offer healthcare professionals effective and safe alternatives for treating a wide range of diseases and disorders, thanks to their high specificity to targets and superior safety profiles. Thus, biologics are rapidly increasing in proportion among the therapeutic tools available to physicians for the treatment of a wide range of diseases and disorders.

[0005] However, the structural complexity and size of biologics make them prone to instability in the formulation. Biologics are susceptible to physical and / or chemical degradation, which can lead not only to decreased efficacy and shortened shelf life but also to safety concerns. Chemical degradation can include deamination, isomerization, oxidation, hydrolysis, and glycation, while physical degradation can include aggregation, particle formation, precipitation, surface adsorption, and denaturation. Both forms of degradation can negatively impact both the efficacy and safety of biologics.

[0006] Well-designed manufacturing and purification processes can often produce highly pure products. However, many biological products can still degrade over time during storage, transport, and administration. In some cases, the stability of a biological product is inherent to its molecular sequence. On the other hand, in some other cases, exogenous factors, such as host cell proteins co-purified with the target biological product, or impurities from certain excipients, can act as "catalysts" to induce either chemical or physical degradation.

[0007] Relaxin is a peptide hormone belonging to the insulin superfamily. In humans, the relaxin peptide family includes seven peptides with high structural similarity but low sequence similarity: relaxin-1, -2, and -3, as well as insulin-like peptides INSL3, INSL4, INSL5, and INSL6. Naturally occurring relaxin consists of A and B polypeptide chains covalently linked by two interchain disulfide bonds. Chain A has an additional intrachain disulfide bond. In women, relaxin-2 expression peaks during pregnancy, and relaxin is thought to be involved in placental development and fetal implantation. However, relaxin has also been found to have anti-fibrotic properties. Heterodimeric fusions, such as HFUS1 (also known as RELAX0023), are recombinant fusion proteins consisting of the Fc portion of human IgG1 linked to human relaxin-2. They have been shown to retain the antifibrotic activity of human relaxin, and as a result, have demonstrated efficacy as a treatment for heart failure, as described in International Publication No. 2021 / 255127.

[0008] There remains a need to develop formulations of these heterodimer fusion proteins with relaxin activity, such as HFUS1, in a way that meets the requirements for drug shelf life. [Overview of the project]

[0009] During the formulation development of the heterodimer fusions described herein, several challenges related to shelf life and molecular stability were addressed. These included a tendency for self-association leading to aggregation, a tendency for amino acid cleavage within a specific pH range, and a tendency to form particles.

[0010] Therefore, the object of this disclosure is to provide a stable formulation of a heterodimer fusion having relaxin activity, such as HFUS1, that satisfies the requirements for the shelf life of the drug and addresses one or more of the above-mentioned problems.

[0011] Therefore, the present disclosure relates to pharmaceutical formulations of heterodimeric fusions having relaxin activity, such as HFUS1.

[0012] In one aspect, there is provided a pharmaceutical formulation comprising a heterodimeric fusion and a lipase-resistant surfactant, wherein the heterodimeric fusion (i) a first heterodimerization domain connected to at least one relaxin A-chain polypeptide or a variant thereof, and (ii) a second heterodimerization domain connected to at least one relaxin B-chain polypeptide or a variant thereof, and the first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity. The first heterodimerization domain heterodimerizes with the second heterodimerization domain, and the heterodimeric fusion has relaxin activity.

[0013] In some embodiments, the relaxin A-chain and the relaxin B-chain are covalently linked by one or more (e.g., two) inter-chain linkages, optionally one or more (e.g., two) inter-chain disulfide bonds. In some embodiments, the relaxin A-chain and the relaxin B-chain are not covalently linked to each other by an amino acid linker.

[0014] In some embodiments, the relaxin A-chain is a relaxin-2 A-chain and the relaxin B-chain is a relaxin-2 B-chain.

[0015] In certain embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fc region, such as an immunoglobulin G (IgG) Fc region (the "first Fc region" and the "second Fc region"). The first and second Fc regions may include the constant domains CH2 and / or CH3. In certain embodiments, the first and second Fc regions include CH2 and CH3.

[0016] In alternative embodiments, the first and second heterodimerization domains are derived from an immunoglobulin Fab region.

[0017] In further alternative embodiments, the first and second heterodimerized domains heterodimerize to form parallel coiled coils.

[0018] In some embodiments, relaxin A chain is connected to a first heterodimerization domain (e.g., a first Fc region) via a connector, and relaxin B chain is connected to a second heterodimerization domain (e.g., a second Fc region) via a connector. In certain embodiments, one or both connectors are polypeptides.

[0019] In some embodiments, at least one connector is a polypeptide having a length of 6 to 40 amino acids. In certain embodiments, both connectors are polypeptides having a length of 6 to 40 amino acids. In certain embodiments, at least one connector is a polypeptide having a length of 21 amino acids. In certain embodiments, both connectors are polypeptides having a length of 21 amino acids. In one embodiment, both connectors have the sequence GGGGSGGGGSGGGGSGGGGGS (Sequence ID 5).

[0020] In certain embodiments, the C-terminus of the first heterodimerization domain (e.g., the first Fc region) is connected to the N-terminus of the relaxin A chain, and the C-terminus of the second heterodimerization domain (e.g., the second Fc region) is connected to the N-terminus of the relaxin B chain. In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., the first Fc region) is connected to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., the second Fc region) is connected to the C-terminus of the relaxin B chain.

[0021] In some embodiments, the first and second heterodimerization domains (e.g., the first and second Fc regions) include heterodimerization-promoting amino acid mutations and / or modifications, which may be asymmetric heterodimerization-promoting amino acid mutations and / or modifications. In certain embodiments, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations. In certain embodiments, the "Fc knob" and "Fc hole" mutations are located in the CH3 domain. In some embodiments, the first and second Fc regions are derived from human IgG1 immunoglobulin, and optionally, C-terminal lysine (K447, by EU index as in Kabat) may not be present in the CH3 domain of the first and / or second Fc regions. In certain embodiments, the first Fc region includes an "Fc knob" mutation and the second Fc region includes an "Fc hole" mutation. Alternatively, the first Fc region has an "Fc hole" mutation and the second Fc region has an "Fc knob" mutation. In certain embodiments, the heterodimerization-promoting amino acid mutations include "Fc hole" mutations Y349C, T366S, L368A, and Y407V, or their conserved substitutions, in one CH3 domain, and "Fc knob" mutations S354C and T366W, or their conserved substitutions, in the other CH3 domain, with amino acid numbering following the EU index as found in Kabat. In certain embodiments, the first and / or second Fc regions include amino acid mutations L234F, L235E, and P331S, with amino acid numbering following the EU index as found in Kabat.

[0022] In any embodiment of the present disclosure, the relaxin-2 A chain polypeptide comprises the sequence shown in SEQ ID NO: 1 or a variant thereof, and the relaxin-2 B chain polypeptide comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. In some embodiments, the relaxin-2 A chain polypeptide comprises the amino acid mutations K9H, K17M, or K17I.

[0023] In some embodiments, both connectors have the array GGGGSGGGGSGGGGSGGGGGS (array number 5).

[0024] Furthermore, this disclosure provides a pharmaceutical formulation comprising a heterodimer fusion and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) FcX-con-A fusion polypeptide and (ii) comprising an FcY-con-B fusion polypeptide, At this time, A is the relaxin A chain or a variant thereof, for example, the relaxin-2 A chain or a variant thereof. B is the relaxin B chain or its variant, for example, the relaxin-2 B chain or its variant. FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V or its conservative substitution. FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation S354C:T366W or its conserved substitution. con is a connector, for example, a connector polypeptide such as the sequence GGGGSGGGGSGGGGSGGGGGS (array 5). The amino acid numbering follows the EU index as found in Kabat, and FcX heterodimerizes with FcY, with the heterodimer fusion possessing relaxin activity.

[0025] In certain embodiments, the heterodimer fusion comprises or consists of a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO: 20.

[0026] In some embodiments of any aspect of the present disclosure, the heterodimer fusion further comprises one or more Fabs, optionally comprising one Fab ligated to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab ligated to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

[0027] In some embodiments of any aspect of the present disclosure, the heterodimer fusion further comprises a second relaxin A chain polypeptide or a variant thereof connected to the N-terminus of a first heterodimerization domain (e.g., a first Fc region), and a second relaxin B chain polypeptide or a variant thereof connected to the second heterodimerization domain (e.g., a second Fc region), wherein optionally, the second relaxin A chain is connected to the first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide, and the second relaxin B chain is connected to the second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0028] This disclosure also provides a pharmaceutical formulation comprising a heterodimer fusion and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) FcX-BLA and FcY, optionally FcY-BLA, or (ii) FcY-BLA and FcX, optionally including FcX-BLA, At this time, FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof. FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof. B is the relaxin B chain or its variant, for example, the relaxin-2 B chain or its variant. A is the relaxin A chain or a variant thereof, for example, the relaxin-2 A chain or a variant thereof. L is a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60) at will. The amino acid numbering follows the EU index as found in Kabat, where FcX heterodimerizes with FcY, and the heterodimer fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerized domains as described herein. In some embodiments, the relaxin B chain is connected to FcX and / or FcY via a connector, and optionally, the connector polypeptide has a length of 6 to 40 amino acids, for example, 21 amino acids.

[0029] This disclosure further provides a pharmaceutical formulation comprising a heterodimer fusion and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) FcX-ALB and FcY, optionally FcY-ALB, or (ii) FcY-ALB and FcX, optionally including FcX-ALB, At this time, FcY is an immunoglobulin (e.g., IgG1) Fc region having an "Fc hole" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof. FcX is an immunoglobulin (e.g., IgG1) Fc region having an "Fc knob" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof. A is the relaxin A chain or a variant thereof, for example, the relaxin-2 A chain or a variant thereof. B is the relaxin B chain or its variant, for example, the relaxin-2 B chain or its variant. L is a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60) at will. The amino acid numbering follows the EU index as found in Kabat, where FcX heterodimerizes with FcY, and the heterodimer fusion has relaxin activity. Alternatively, FcX and FcY are non-Fc heterodimerized domains as described herein. In some embodiments, the relaxin A chain is connected to FcX and / or FcY via connectors, and optionally, the connector polypeptide has a length of 6 to 40 amino acids, for example, 21 amino acids.

[0030] In some embodiments of any aspect of this disclosure, the ratio of the relaxin activity of the heterodimer fusion to the relaxin activity of the reference relaxin protein is about 0.001 to about 10.

[0031] In some embodiments of any aspect of the present disclosure, the formulation contains less than approximately 10,000, approximately 6,000, approximately 5,000, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or approximately 50 particles per mL having a diameter of 2 μm, 5 μm, 10 μm, 15 μm, 250, approximately 100, or approximately 50.

[0032] In some embodiments of any aspect of this disclosure, the concentration of the lipase-resistant surfactant is 0.001%(w / v) to 1%(w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.005%(w / v) to 0.2%(w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.02%(w / v) to 0.06%(w / v). In certain embodiments, the concentration of the lipase-resistant surfactant is 0.04%(w / v).

[0033] In some embodiments of any aspect of this disclosure, the lipase-resistant surfactant is not enzymatically hydrolyzable by lipoprotein lipase (LPL), lipase 9, phospholipase 2, or phospholipase 2A. In certain embodiments, the lipase-resistant surfactant is not enzymatically hydrolyzable by LPL. In some embodiments, the lipase-resistant surfactant does not contain ester bonds that are enzymatically hydrolyzable by lipoprotein lipase, lipase 9, phospholipase 2, or phospholipase 2A. In some embodiments, the lipase-resistant surfactant does not contain ester bonds that are enzymatically hydrolyzable by lipoprotein lipase. In some embodiments, the lipase-resistant surfactant is a water-soluble nonionic triblock copolymer formed by polyethylene oxide (PEO) blocks and polypropylene oxide (PPO) blocks. In certain embodiments, the water-soluble nonionic triblock copolymer is poloxamer 188 (P188). Alternatively, the lipase-resistant surfactant is D-α-tocopherol polyethylene glycol succinate (TPGS). In further embodiments, the lipase-resistant surfactant is selected from P188, TPGS, Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG300, PEG400, Brij58, and Brij35.

[0034] In some embodiments of any aspect of this disclosure, the formulation further comprises a buffer with a pH of about 3 to about 10, optionally about 5.5 to about 7.5. In certain embodiments, the formulation has a pH in the range of 6 to 7. In certain embodiments, the formulation has a pH of 6.5.

[0035] In some embodiments of any aspect of this disclosure, the buffer concentration is 0.1 mM to 100 mM, for example, 5 mM, 10 mM, 15 nm, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In certain embodiments, the buffer concentration is 10 mM to 30 mM. In certain embodiments, the buffer concentration is 20 mM.

[0036] In some embodiments, the buffer is selected from acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycylglycine, and mixtures thereof. In certain embodiments, the buffer is selected from citrate buffer and histidine buffer. In certain embodiments, the buffer is histidine, histidine hydrochloride, or histidine / histidine hydrochloride buffer. In certain embodiments, the buffer is histidine / histidine hydrochloride buffer (i.e., a combination of histidine and histidine hydrochloride). In one embodiment, the buffer is L-histidine / L-histidine hydrochloride monohydrate.

[0037] In some embodiments of any aspect of this disclosure, the formulation further comprises an excipient, optionally, an ionic excipient. In some embodiments, the concentration of the excipient is 100 mM to 300 mM. In certain embodiments, the concentration of the excipient is 140 mM to 240 mM, for example, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, or 240 mM. In certain embodiments, the concentration of the excipient is 190 mM. Examples of ionic excipients for use in the formulations described herein include salts and charged amino acids. Ionic excipients may include combinations of salts and charged amino acids. Examples of charged amino acids include arginine and lysine. Examples of salts include chlorides, succinates, acetates and sulfates, as well as carbonates, glucons, lactates and malates. In certain embodiments, the ionic excipient is a hydrochloric acid (HCl) salt of a charged amino acid.

[0038] In some embodiments, the excipient is an ionic excipient selected from arginine salts or lysine salts. In some embodiments, the ionic excipient is selected from arginine HCl or lysine HCl. In certain embodiments, the ionic excipient is arginine HCl. It will also be understood that the buffer solution itself may be an ionic excipient as described herein. Thus, in some embodiments, the buffer solution is an ionic excipient.

[0039] In some embodiments of any aspect of the present disclosure, the formulation further comprises a sugar, optionally, sucrose. Other sugars that may be used include, but are not limited to, trehalose, lactose, mannitol, melibiose, mellitose, raffinose, mannotriose, stachyose, polyols, sugar alcohols with a molecular weight of trivalent or greater (e.g., glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol), glucose, maltose, maltulose, isomaltulose, lactulose, and cyclodextrin.

[0040] In some embodiments of any aspect of this disclosure, the concentration of the heterodimer fusion is 0.1 to 100 mg / mL, optionally 0.2 to 50 mg / mL, and optionally 1 to 30 mg / mL. In some embodiments, the formulation comprises 0.2 to 50 mg / mL of the heterodimer fusion, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5. In some embodiments, the formulation contains 50 mg / mL of the heterodimer fusion. In other embodiments, the formulation contains 30 mg / mL of the heterodimer fusion. In other embodiments, the formulation contains 33 mg / mL of the heterodimer fusion. In other embodiments, the formulation contains 5 mg / mL of the heterodimer fusion. In other embodiments, the formulation contains 1 mg / mL of the heterodimer fusion. In other embodiments, the formulation comprises a heterodimer fusion at a concentration of 1.1 mg / mL. In some embodiments, the heterodimer fusion comprises or consists of a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO: 20.

[0041] In one embodiment, the Disclosure further provides a pharmaceutical formulation described herein for use in treatment. The Disclosure also provides a method for treating a subject having a disease or disorder, the method comprising administering a pharmaceutical formulation described herein to the subject.

[0042] In one embodiment, the Disclosure further provides the pharmaceutical formulations described herein for use in the treatment of subjects having heart failure, such as heart failure with pulmonary hypertension (e.g., group II pulmonary hypertension). The Disclosure also provides a method for treating subjects having heart failure, such as heart failure with pulmonary hypertension (e.g., group II pulmonary hypertension), the method comprising administering the pharmaceutical formulations described herein to the subject. In some embodiments, the heart failure is heart failure with reduced ejection fraction, heart failure with moderate ejection fraction, or heart failure with preserved ejection fraction. In some embodiments, the subject has a mean pulmonary artery pressure of about 25 mmHg or greater, a pulmonary artery wedge pressure (PAWP) of greater than 15 mmHg, and / or a right ventricular systolic pressure of about 40 mmHg or greater. In some embodiments, the subject is fitted with a blood pressure monitoring device, which may be a pulmonary artery pressure monitoring device. In some embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. In some embodiments, the subject has a pulmonary vascular resistance of less than 3.0 Wood units. In other embodiments, the subject has a pulmonary vascular resistance of 3.0 Wood units or more.

[0043] In some embodiments, the fusion polypeptide or pharmaceutical formulation is suitable for subcutaneous injection and / or administered to the subject by subcutaneous injection. In some embodiments, the fusion polypeptide or pharmaceutical formulation is suitable for self-administration and / or administered by self-administration.

[0044] In some embodiments, administration of a pharmaceutical formulation results in one or more of the following compared to baseline levels before administration: a decrease in PVR, a decrease in mPAP, a decrease in ePAD, an increase in cardiac stroke volume (SV), a decrease in systemic vascular resistance (SVR), and / or an increase in estimated glomerular filtration rate (eGFR), an increase in ejection fraction, and / or an increase in cardiac output.

[0045] In one embodiment, the present disclosure further provides a kit comprising a pharmaceutical formulation described herein.

[0046] Aspects and embodiments of this disclosure are described in the appended claims. These and other aspects and embodiments of this disclosure are also described herein. [Brief explanation of the drawing]

[0047] [Figure 1A] The purity loss of HFUS1 at 5°C, 25°C, and 40°C for 3 months, 3 months, and 1 month, respectively, is shown for 50 mg / mL (A) and 10 mg / mL (B). [Figure 1B] The purity loss of HFUS1 at 5°C, 25°C, and 40°C for 3 months, 3 months, and 1 month, respectively, is shown for 50 mg / mL (A) and 10 mg / mL (B). [Figure 2] This shows the unfolding temperature profile of HFUS1 measured by differential scanning calorimetry thermogram. [Figure 3] The self-diffusion coefficient and hydrodynamic radius of HFUS1 at concentrations of 0.005 g / mL, 0.008 g / mL, 0.011 g / mL, 0.016 g / mL, and 0.020 g / mL are shown. [Figure 4A] This bar graph shows the amino acid cleavage at the C-terminus of relaxin chain B in HFUS1, as evaluated by mass spectrometry. F4, F5, F6, and F9 correspond to formulations 4, 5, 6, and 9, respectively, as shown in Table 4. [Figure 4B] This bar graph shows the change in the percentage of trisulfide bonds in HFUS1 as evaluated by mass spectrometry. F4, F5, F6, and F9 correspond to formulations 4, 5, 6, and 9, respectively, as shown in Table 4. [Figure 4C] This bar graph shows the methionine 271 (M271) oxidation of relaxin chain B in HFUS1, as evaluated by mass spectrometry. F4, F5, F6, and F9 correspond to formulations 4, 5, 6, and 9, respectively, as shown in Table 4. [Figure 5A] The purity profile of HFUS1 in histidine-arginine HCl at pH 5.5-7.0 is shown, along with the monthly percentage changes (%) in monomers, aggregation, and fragmentation during storage at 40°C (A), 25°C (B), and 5°C (C). [Figure 5B] The purity profile of HFUS1 in histidine-arginine HCl at pH 5.5-7.0 is shown, along with the monthly percentage changes (%) in monomers, aggregation, and fragmentation during storage at 40°C (A), 25°C (B), and 5°C (C). [Figure 5C] The purity profile of HFUS1 in histidine-arginine HCl at pH 5.5-7.0 is shown, along with the monthly percentage changes (%) in monomers, aggregation, and fragmentation during storage at 40°C (A), 25°C (B), and 5°C (C). [Figure 6] This bar graph shows the amino acid cleavage at the C-terminus of relaxin at pH levels ranging from 5.5 to 7.0, and at temperatures of 0°C, 5°C, 25°C, and 40°C. [Figure 7] This shows the purity loss of AZ3427 due to protease inhibitors (PIs) as evaluated by HPSEC. [Figure 8] This bar graph shows the amino acid cleavage of AZ3427 by protease inhibitors (PIs) as evaluated by mass spectrometry. [Figure 9A] The particle formation after storage at 5°C is shown. (A) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 6 months of storage, (B) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 12 months of storage, and (C) shows an optimized sample of the AZ3427 formulation after 12 months of storage (various buffers, excipients and pH), where formulations 1 to 8 represent formulations 1 to 8 listed in Table 4, respectively. [Figure 9B] The particle formation after storage at 5°C is shown. (A) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 6 months of storage, (B) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 12 months of storage, and (C) shows an optimized sample of the AZ3427 formulation after 12 months of storage (various buffers, excipients and pH), where formulations 1 to 8 represent formulations 1 to 8 listed in Table 4, respectively. [Figure 9C]The particle formation after storage at 5°C is shown. (A) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 6 months of storage, (B) shows a pH screening sample of the HFUS1 formulation in histidine-arginine HCl after 12 months of storage, and (C) shows an optimized sample of the AZ3427 formulation after 12 months of storage (various buffers, excipients and pH), where formulations 1 to 8 represent formulations 1 to 8 listed in Table 4, respectively. [Figure 10] The FTIR spectra of HFUS1 particles compared to the protein and PS80 reference are shown. The highlighted boxes indicate IR signals similar to the protein reference, as well as traces of the PS80 signal. [Figure 11A] (A) Visual inspection of HFUS1 formulations in 2R vials after storage at 5°C for 9 months and (B) 12 months. [Figure 11B] (A) Visual inspection of HFUS1 formulations in 2R vials after storage at 5°C for 9 months and (B) 12 months. [Figure 12A] (A) Visual inspection of HFUS1 formulations in 1 mL pre-filled syringes after storage at 5°C for 9 months and (B) 12 months. [Figure 12B] (A) Visual inspection of HFUS1 formulations in 1 mL pre-filled syringes after storage at 5°C for 9 months and (B) 12 months. [Figure 13A] The following shows the number of particles per 1 mL of the HFUS1 formulation as measured by microflow imaging. (A) represents particles with a diameter of 1 μm or more and less than 2 μm, (B) represents particles with a diameter of 2 μm or more, and (C) represents particles with a diameter of 10 μm or more. [Figure 13B] The following shows the number of particles per 1 mL of the HFUS1 formulation as measured by microflow imaging. (A) represents particles with a diameter of 1 μm or more and less than 2 μm, (B) represents particles with a diameter of 2 μm or more, and (C) represents particles with a diameter of 10 μm or more. [Figure 13C]The following shows the number of particles per 1 mL of the HFUS1 formulation as measured by microflow imaging. (A) represents particles with a diameter of 1 μm or more and less than 2 μm, (B) represents particles with a diameter of 2 μm or more, and (C) represents particles with a diameter of 10 μm or more. [Figure 14] These are LC-MS chromatograms of the total ions of PS-80 in stressed and unstressed samples (POE is polyoxymethylene). [Figure 15A] (A) HPSEC of HFUS1 formulations containing PS80 and P188 stored at 40°C for 3 months, and (B) at 5°C for 12 months (PS80 sample) or 18 months (P188 sample). MPP is the main product peak, which is the sum of monomers and shoulders. [Figure 15B] (A) HPSEC of HFUS1 formulations containing PS80 and P188 stored at 40°C for 3 months, and (B) at 5°C for 12 months (PS80 sample) or 18 months (P188 sample). MPP is the main product peak, which is the sum of monomers and shoulders. [Figure 16] This shows the change in isoelectric point, measured by capillary isoelectric focusing (cIEF), of HFUS1 formulations containing PS80 and P188, stored at 5°C for 12 months (PS80 sample) and 18 months (P188 sample). [Figure 17A] The HPSEC values ​​of HFUS1 formulations stored at (A) 40°C, (B) 40°C, (C) 25°C, and (D) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 17B] The HPSEC values ​​of HFUS1 formulations stored at (A) 40°C, (B) 40°C, (C) 25°C, and (D) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 17C]The HPSEC values ​​of HFUS1 formulations stored at (A) 40°C, (B) 40°C, (C) 25°C, and (D) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 17D] The HPSEC values ​​of HFUS1 formulations stored at (A) 40°C, (B) 40°C, (C) 25°C, and (D) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 18A] Capillary gel electrophoresis (CGE) of HFUS1 formulations at 0 months and 1 month after storage at (A) 40°C, (B) 25°C, and (C) 5°C is shown. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change of the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 18B] Capillary gel electrophoresis (CGE) of HFUS1 formulations at 0 months and 1 month after storage at (A) 40°C, (B) 25°C, and (C) 5°C is shown. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change of the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 18C] Capillary gel electrophoresis (CGE) of HFUS1 formulations at 0 months and 1 month after storage at (A) 40°C, (B) 25°C, and (C) 5°C is shown. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change of the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 19A]The capillary isoelectric focusing (CIEF) results of HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 19B] The capillary isoelectric focusing (CIEF) results of HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 19C] The capillary isoelectric focusing (CIEF) results of HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C are shown. The percentage change per month is indicated. "50L DEV LOT (P1) 0.04% P188 target" corresponds to formulation F1 (target) in Table 6. The x-axis shows the change in the test formulation relative to F1 (target) and correlates with the formulations listed in Table 6. [Figure 20A] (A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 20B] (A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 20C-01](A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 20C-02] (A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 20D-01] (A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 20D-02] (A) Microflow imaging (MFI) of HFUS1 formulations stored for 3 months at 40°C for particles with a diameter of 2 μm or larger, (B) 40°C for particles with a diameter of 10 μm or larger, (C) 5°C for particles with a diameter of 2 μm or larger, and (D) 5°C for particles with a diameter of 10 μm or larger. "F0" to "F8" correspond to the formulations listed in Table 6. [Figure 21A] Table 7 shows the HPSEC (percentage of monomers) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. "Target (0.25 MG / ML)", "Worst Case (0.25 MG / ML)", "Target (1 MG / ML)", and "Worst Case (1 MG / ML)" correspond to formulations P1 to P4, respectively. [Figure 21B]Table 7 shows the HPSEC (percentage of monomers) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. "Target (0.25 MG / ML)", "Worst Case (0.25 MG / ML)", "Target (1 MG / ML)", and "Worst Case (1 MG / ML)" correspond to formulations P1 to P4, respectively. [Figure 21C] Table 7 shows the HPSEC (percentage of monomers) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C. "Target (0.25 MG / ML)", "Worst Case (0.25 MG / ML)", "Target (1 MG / ML)", and "Worst Case (1 MG / ML)" correspond to formulations P1 to P4, respectively. [Figure 22A] (A) Capillary gel electrophoresis (CGE) of low-concentration HFUS1 formulations stored at 40°C, (B) 25°C, and (C) 5°C is shown. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 22B] (A) Capillary gel electrophoresis (CGE) of low-concentration HFUS1 formulations stored at 40°C, (B) 25°C, and (C) 5°C is shown. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 22C] (A) Capillary gel electrophoresis (CGE) of low-concentration HFUS1 formulations stored at 40°C, (B) 25°C, and (C) 5°C is shown. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 23A] The capillary isoelectric focusing (CIEF) (main peak %) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C is shown. The percentage change per month is indicated. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 23B] The capillary isoelectric focusing (CIEF) (main peak %) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C is shown. The percentage change per month is indicated. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 23C]The capillary isoelectric focusing (CIEF) (main peak %) of low-concentration HFUS1 formulations stored at (A) 40°C, (B) 25°C, and (C) 5°C is shown. The percentage change per month is indicated. P1 to P4 correspond to formulations P1 to P4 in Table 7, respectively. [Figure 24A] Microflow imaging (MFI) of low-concentration HFUS1 formulations stored at 5°C, 25°C, and 40°C is shown for (A) particles with a diameter of 25 μm or larger, and (B) particles with a diameter of 10 μm or larger. "Target (0.25 MG / ML)", "Worst Case (0.25 MG / ML)", "Target (1 MG / ML)", and "Worst Case (1 MG / ML)" correspond to formulations P1 to P4 in Table 7, respectively. [Figure 24B] Microflow imaging (MFI) of low-concentration HFUS1 formulations stored at 5°C, 25°C, and 40°C is shown for (A) particles with a diameter of 25 μm or larger, and (B) particles with a diameter of 10 μm or larger. "Target (0.25 MG / ML)", "Worst Case (0.25 MG / ML)", "Target (1 MG / ML)", and "Worst Case (1 MG / ML)" correspond to formulations P1 to P4 in Table 7, respectively. [Figure 25] Illustrative forms of heterodimer fusions according to some embodiments of the present disclosure are shown. The form of each fusion polypeptide of the heterodimer fusion is given with respect to FcX, FcY, A, B, con, and L, where FcX ("Fc knob") and FcY ("Fc hole") are two Fc regions containing heterodimerization-promoting amino acid mutations and / or modifications, A ("RlxA") and B ("RlxB") are relaxin A chain and relaxin B chain polypeptides, "con" is a connector polypeptide, L is a linker polypeptide, HC X and HC Y are the heavy chains of the antibody, LC is the light chain of the antibody, Hinge is the hinge region of the antibody, and Fab is the Fab fragment of the antibody. [Figure 26A]The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26B] The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26C]The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26D] The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26E]The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26F] The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26G]The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26H] The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 26I]The following shows the number of particles obtained by microflow imaging (MFI) of various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27A] The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27B]The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27C] The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27D]The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27E] The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27F]The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27G] The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27H]The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 27I] The following shows the number of particles obtained by the photo-shielding (HIAC) method for various HFUS1 formulations stored at the following temperatures: (A) 5°C for particles with a diameter of 2 μm or more, (B) 5°C for particles with a diameter of 10 μm or more, (C) 5°C for particles with a diameter of 25 μm or more, (D) 25°C for particles with a diameter of 2 μm or more, (E) 25°C for particles with a diameter of 10 μm or more, (F) 25°C for particles with a diameter of 25 μm or more, (G) 40°C for particles with a diameter of 2 μm or more, (H) 40°C for particles with a diameter of 10 μm or more, and (I) 40°C for particles with a diameter of 25 μm or more. The data corresponds to the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 28A]The HPSEC values ​​for HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C are shown. The monthly change in monomers is indicated. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 28B] The HPSEC values ​​for HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C are shown. The monthly change in monomers is indicated. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 28C] The HPSEC values ​​for HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C are shown. The monthly change in monomers is indicated. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 29A]The capillary isoelectric focusing (cIEF) of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 29B] The capillary isoelectric focusing (cIEF) of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 29C] The capillary isoelectric focusing (cIEF) of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 30A]The efficacy of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 30B] The efficacy of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 30C] The efficacy of HFUS1 formulations stored at (A) 5°C, (B) 25°C, and (C) 40°C is shown. The data correlate with the formulations listed in Table 8, with "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 31A]The data shows the cleavage of amino acids at the C-terminus of relaxin chain B in HFUS1 formulations, evaluated by RP-HPLC after storage at (A) 5°C, (B) 25°C, and (C) 40°C. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 31B] The data shows the cleavage of amino acids at the C-terminus of relaxin chain B in HFUS1 formulations, evaluated by RP-HPLC after storage at (A) 5°C, (B) 25°C, and (C) 40°C. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Figure 31C] The data shows the cleavage of amino acids at the C-terminus of relaxin chain B in HFUS1 formulations, evaluated by RP-HPLC after storage at (A) 5°C, (B) 25°C, and (C) 40°C. The data correlate with the formulations listed in Table 8, where "33 mg / mL" = PFS-control, "33 mg / mL pH6" = PFS-1, "33 mg / mL pH7" = PFS-2, "33 mg / mL low Arg HCl" = PFS-3, "33 mg / mL high Arg HCl" = PFS-4, "33 mg / mL 0.02% P188" = PFS-5, and "33 mg / mL 0.06% P188" = PFS-6. [Modes for carrying out the invention]

[0048] definition Unless otherwise defined, all technical terms, expressions, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those generally understood by those skilled in the art to which the claimed subject matter pertains. Where applicable, terms having a generally understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from the generally understood meaning in the art.

[0049] Concentrations, quantities, volumes, percentages, and other numerical values ​​may be presented in range form as specified herein. Such range forms are used solely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly stated as limits to the range, but also all individual numerical values ​​or subranges encompassed within that range, as if each numerical value and subrange were explicitly stated.

[0050] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for the same extent as each individual publication is incorporated by reference. If any definition contained herein contradicts or is inconsistent with any definition contained herein in a patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail.

[0051] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this disclosure. The following descriptions are illustrative of this disclosure and should not be construed as limiting the scope of the embodiments intended to be described herein.

[0052] References to subject matter disclosed or described "in this specification" relate to subject matter disclosed or described in any part of this application.

[0053] overview During the formulation development of heterodimer fusions, such as HFUS1, several challenges related to shelf life and molecular stability were addressed. These included HFUS1's tendency to self-associate, leading to aggregation, its tendency to cleave amino acids within a specific pH range, and its tendency to form particles.

[0054] Therefore, this disclosure describes several intentionally designed tests to identify the root cause of the instability of HFUS1, as well as formulation development and optimization work to identify a stable liquid formulation of the molecule to meet the drug's shelf-life requirements.

[0055] The inventors demonstrated that HFUS1 has a tendency to self-associate (see Example 1). High levels of molecular self-association can lead to the formation of soluble aggregates, which can then become precursors to large-sized insoluble aggregates, ultimately resulting in particles and significantly affecting the molecular stability profile. The inventors established that formulation optimization is necessary to reduce this tendency for self-association and aggregation. Surprisingly, it was determined that surfactants in the formulation play a crucial role in particle formation related to free fatty acids (FFAs), which can act as nuclei that induce aggregation of the HFUS1 protein. By optimizing the surfactant, particle formation was significantly reduced, and the tendency for aggregation was decreased (see Example 3). Further reduction of aggregation was also found by optimizing the pH, buffer, and excipients used in the formulation of HFUS1. Overall, the histidine-arginine HCl system (i.e., a histidine-based buffer system containing an arginine hydrochloride ionic excipient) was confirmed to provide the highest colloidal and stereostructural stability for HFUS1 (see Example 2).

[0056] The inventors also established that proteases are likely responsible for the fragmentation and cleavage of HFUS1. This enzymatic activity is likely to occur at lower pH levels, where proteases are most effective at cleaving molecules. Therefore, it is important to maintain the formulation's pH in a higher range, as demonstrated in pH optimization tests (see Example 2). The optimal pH range of 5.5–7.5, specifically 6–7, and more specifically 6.5, minimizes the effects of molecular fragmentation and chemical degradation such as amino acid (AA) cleavage.

[0057] As described above, the inventors identified the formation of visible particles in the HFUS1 formulation over time (see Example 3). Several underlying causes of particle formation were hypothesized, as described in Example 3. Surprisingly, the inventors established that the presence of the surfactant PS80 was the cause of particle formation. The inventors demonstrated that enzymatic hydrolysis of the ester bonds of PS80 by lipoprotein lipase (LPL) present in the formulation was likely the cause of PS80 degradation, which in turn led to the formation of impurities such as free fatty acids (FFA), which can act as nuclei that induce HFUS1 protein aggregation resulting in particles. Meanwhile, degradation reduced PS80 levels, and therefore, it lost its protective / surfactant effect to prevent particle formation during storage. The use of lipase-resistant surfactants such as poloxamer 188 (P188) and D-α-tocopherol polyethylene glycol succinate (TPGS) was able to mitigate particle formation in the HFUS1 formulation.

[0058] The effect of HFUS1 concentration on stability was also evaluated, and HFUS1 showed good stability in the formulations described herein over a wide range of HFUS1 concentrations. Data for HFUS1 concentrations ranged from 0.25 mg / mL to 50 mg / mL, and this was particularly confirmed for formulations with HFUS1 concentrations of 0.25 mg / mL, 1 mg / mL, 5 mg / mL, 33 mg / mL, and 50 mg / mL (see Examples 4, 5, and 6).

[0059] In summary, the histidine-arginine HCl system was selected for its effect in reducing the molecular self-association tendency, and an optimized pH range was identified that effectively prevents AA cleavage and fragmentation of the molecule (see Example 2). Furthermore, a detailed investigation was conducted to understand the cause of the particle formation problem, and PS80 was identified as the root cause. This led to the optimization of the surfactant in the formulation system, and alternative surfactants such as P188 and TPGS mitigated the particle formation problem (see Example 3). P188 was selected as the primary surfactant for the formulation. Finally, comprehensive formulation stability testing was performed to evaluate the robustness of the formulation system. The histidine / histidine hydrochloride-arginine HCl formulation showed excellent stability profile and robustness. The effect of HFUS1 concentration on stability was also evaluated, and HFUS1 also showed good stability over the wide range of concentrations tested (see Examples 4-6).

[0060] Therefore, the Specified herein provides a pharmaceutical formulation comprising a heterodimer fusion, for example, HFUS1, and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) a first heterodimerization domain attached to at least one relaxin A chain polypeptide or a variant thereof, (ii) comprising at least one relaxin B chain polypeptide or a second heterodimerization domain attached to a variant thereof, The first heterodimerizing domain heterodimerizes with the second heterodimerizing domain, and the heterodimer fusion has relaxin activity.

[0061] Furthermore, this specification also provides pharmaceutical formulations described herein for therapeutic use. Methods for treating subjects with a disease or disorder are also provided, the methods comprising administering the pharmaceutical formulations described herein to the subject.

[0062] Furthermore, this specification also provides pharmaceutical formulations described herein for use in the treatment of subjects having heart failure, or optionally heart failure with pulmonary hypertension (e.g., Class II pulmonary hypertension). Furthermore, this specification also provides a method for treating subjects having heart failure, or optionally heart failure with pulmonary hypertension (e.g., Class II pulmonary hypertension), the method comprising administering the pharmaceutical formulations described herein to the subject.

[0063] Furthermore, this specification also provides kits containing the pharmaceutical formulations described herein.

[0064] Heterodimer fusion with relaxin activity Relaxin The pharmaceutical formulations of this disclosure include a heterodimer fusion having relaxin activity, for example, HFUS1.

[0065] As described in International Publication No. 2021 / 255127, the heterodimer fusion described herein, e.g., HFUS1, may exhibit relaxin activity when the relaxin A and B chains are not covalently bonded to each other via an amino acid linker. Advantageously, heterodimerization of the heterodimerization domain induces precise folding and heterodimerization of the relaxin A and B chains (see Example 2 in International Publication No. 2021 / 255127). Furthermore, unlike wild-type relaxin proteins, the heterodimer fusion, e.g., HFUS1, does not require processing by intracellular proteolysis for biological activity.

[0066] As used herein, the term “heterodimer fusion” refers to a heterodimer of fusion polypeptides, where one fusion polypeptide comprises a first heterodimerization domain attached to a first subunit of the heterodimer protein (e.g., relaxin A chain), and the other fusion polypeptide comprises a second heterodimerization domain attached to a second subunit of the heterodimer protein (e.g., relaxin B chain). In some embodiments, the heterodimer fusion is HFUS1 (also known as RELAX0023). HFUS1 is a heterodimer fusion comprising a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO: 20.

[0067] The heterodimer fusion used in the formulations of this disclosure may comprise relaxin A and B chain polypeptides derived from relaxins selected from relaxin-1, relaxin-2, and relaxin-3. In certain embodiments, the relaxin A chain polypeptide is relaxin-2 A chain polypeptide or a variant thereof, and the relaxin B chain polypeptide is relaxin-2 B chain polypeptide or a variant thereof. In certain embodiments, the relaxin A chain polypeptide comprises human relaxin-2 A chain polypeptide or a variant thereof, and human relaxin-2 B chain polypeptide or a variant thereof.

[0068] The terms “chain,” “polypeptide,” and “peptide” are interchangeable herein and refer to a chain of two or more amino acids linked together by peptide bonds.

[0069] In some embodiments, the relaxin-2 A chain polypeptide has the sequence shown in SEQ ID NO: 1 or a variant thereof, and the relaxin-2 B chain polypeptide has the sequence shown in SEQ ID NO: 2 or a variant thereof. The variant may include one or more amino acid substitutions, deletions, and / or insertions. In some embodiments, the relaxin-2 A chain polypeptide contains one or more amino acid mutations selected from K9E, K9H, K9L, K9M, R18E, R18H, R22A, R22I, R22M, R22Q, R22S, R22Y, F23E, F23A, and F23I. In certain embodiments, the relaxin-2 A chain contains the amino acid mutation K9H.

[0070] Relaxin A and B chain variants are known in the art. Furthermore, guidelines for the design of relaxin A and B chain variants are available to those skilled in the art. For example, it is understood that variants may retain amino acids required for relaxin function. For example, relaxin-2 B chain variants may contain the conserved motif Arg-XXX-Arg-XX-lle (Claasz AA et al. (2002) Eur.J.Biochem. 269(24):6287-6293) or Arg-XXX-Arg-XX-Val (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480). Variants may contain one or more amino acid substitutions and / or insertions. For example, relaxin-2 B chain variants may have one or more additional amino acids compared to SEQ ID NO: 62, e.g., K30 and R31, as well as N-terminal V-2, A-1, and M-1. Alternatively, or in addition, the variant may contain one or more amino acid derivatives. For example, the first amino acid of the relaxin-2 B-chain variant may be pyroglutamate.

[0071] In certain embodiments, relaxin A and relaxin B chains are covalently bonded by two interchain disulfide bonds (see Example 2 in International Publication No. 2021 / 255127).

[0072] The relaxin family of peptides mediates biological effects, at least partially, through the activation of G protein-coupled receptors (GPCRs) and subsequent stimulation or inhibition of the cAMP signaling pathway by Gs or Gi protein subunits, respectively. Relaxin-2 is known to activate GPCR RXFP1 (also known as LGR7) and, to a lesser extent, GPCR RXFP2 (also known as LGR8), thereby stimulating the Gs-cAMP-dependent signaling pathway and resulting in an increase in the second messenger molecule, cAMP.

[0073] As used herein, the term “relaxin activity” refers to the ability of relaxin to bind to and / or activate the relaxin receptor and / or initiate a signaling cascade within the cell. In embodiments where relaxin activity is relaxin-2 activity, relaxin activity may refer to the ability to bind to and / or activate receptor RXFP1 and / or receptor RXFP2. The term “relaxin activity” may be used interchangeably with “biological activity.”

[0074] Relaxin activity can be determined by measuring the binding of relaxin molecules to relaxin receptors and / or by measuring downstream events from binding to relaxin receptors.

[0075] Relaxin activity can be determined in vitro and / or in vivo. In some embodiments, relaxin activity is determined in vitro.

[0076] Relaxin activity can be determined by measuring the amount and / or presence of molecules downstream from the relaxin activation of the receptor. For example, relaxin activity can be determined by measuring cAMP production after relaxin activation of the receptor. Methods for detecting relaxin-inducible cAMP production are known in the art. Such methods include cAMP ELISA, HTRF cAMP assay, and HitHunter® cAMP assay. In some embodiments, relaxin activity is determined by measuring relaxin-inducible cAMP production by HTRF cAMP assay, for example, as performed in Example 3 of International Publication No. 2021 / 255127. Relaxin activity can also be determined by measuring nitric oxide (NO) production after relaxin activation of the receptor. Relaxin activity can also be determined by measuring the activation of molecules downstream from the relaxin activation of the receptor. For example, relaxin activity can be determined by measuring the activation of p42 / 44 MAPK.

[0077] Alternatively, or in addition, relaxin activity can be determined by measuring the activation of known relaxin target genes. For example, relaxin activity can be determined by measuring the activation of transcription of a known relaxin target gene, VEGF, in THP-1 cells. Methods for determining the activation of gene transcription are known in the art and include quantitative PCR analysis of mRNA. Relative expression of VEGF mRNA can be measured by quantitative real-time PCR induction of VEGF transcripts after incubation with relaxin in THP-1 cells, as described in Xiao et al. (2013) Nat Commun. 4:1953.

[0078] Alternatively, or in addition, relaxin activity may be determined by measuring one or more downstream effects of relaxin. For example, reduction of cardiac hypertrophy can be measured by echocardiography, left ventricular weight relative to body weight, and / or tibial length according to standard methods. In another example, relaxin activity may be determined by measuring fibrosis reduction by Masson's trichrome staining. In yet another example, relaxin activity may be determined by measuring the regulation of connective tissue metabolism, e.g., inhibition of profibrotic factors (e.g., TGF-β), inhibition of fibroblast proliferation and differentiation, and / or activation of MMP-mediated extracellular matrix degradation (Bathgate RA et al. (2013) Physiol Rev. 93(1):405-480).

[0079] In some embodiments, relaxin activity is determined by measuring the recovery of isoproterenol-induced cardiac hypertrophy (measured as cardiac weight relative to tibial length) and fibrosis (measured as collagen content relative to cardiac weight), as demonstrated, for example, in Example 7 of International Publication No. 2021 / 255127.

[0080] The activity of the heterodimer fusions of this disclosure, for example, HFUS1, can be determined in relation to a reference relaxin protein. In some embodiments, the reference relaxin protein is a recombinant protein. In certain embodiments, the reference relaxin protein is a relaxin protein having a relaxin A-chain and relaxin B-chain array of a mature relaxin protein. Recombinant relaxins having a relaxin A-chain and relaxin B-chain array of a mature relaxin protein are commercially available. For example, recombinant human relaxin-2, mouse relaxin-1, and INSL3 are available from R&D systems (catalog numbers 6586-RN, 6637-RN, and 4544-NS, respectively).

[0081] In some embodiments, the reference relaxin protein has the same relaxin A and B chains as the heterodimer fusion of the Disclosure, or differs from the relaxin A and B chains of the heterodimer fusion of the Disclosure by up to 10 amino acids, for example, 1 or 2 amino acids. In some embodiments, the first amino acid of the B chain of reference relaxin-2 is D, which is deleted in the relaxin B chain of the heterodimer fusion of the Disclosure.

[0082] The reference relaxin protein may be selected from the following: (i) Recombinant human relaxin-2 (referred to herein as RELAX0013), and (ii) Recombinant mouse relaxin-1 (referred to herein as RELAX0014), and (iii) Recombinant Fc-fused relaxin-2 (referred to herein as RELAX0010, described in International Publication No. 2018 / 138170), in which relaxin A and relaxin B are fused in a single strand and Fc is a half-life extension Fc region, and (iv) Recombinant Fc-fused relaxin-2 (referred to herein as RELAX0009, described in International Publication No. 2018 / 138170), in which relaxin A and relaxin B are fused in a single strand and Fc is a half-life extension Fc region, and (v) Recombinant Fc-fusion relaxin-2 in which relaxin A and relaxin B are fused in a single strand (referred to herein as RELAX0126, described in International Publication No. 2013 / 004607), and (vi) Recombinant Fc-fusion relaxin-2 in which relaxin A and relaxin B are fused in a single strand (referred to herein as RELAX0127, described in International Publication No. 2013 / 004607), and (vii) Recombinant Fc-fusion relaxin in which relaxin A and relaxin B are fused in a single strand (referred to herein as RELAX0128, described in International Publication No. 2013 / 004607).

[0083] In certain embodiments, the reference relaxin protein is a relaxin-2 protein having relaxin-2 A and relaxin-2 B chain arrays of a mature relaxin-2 protein disclosed in UniProtKB / Swiss-Prot accession number P04090.1.

[0084] The heterodimer fusions of this disclosure, for example HFUS1, may be considered to have relaxin activity if they exhibit at least a certain percentage of the activity of a reference relaxin protein. For example, a fusion polypeptide may be considered to have relaxin activity if it has at least about half the activity of a reference relaxin protein. The heterodimer fusions of this disclosure have an activity ratio of the fusion polypeptide activity to the activity of a reference relaxin protein of about 10 -5 ~approximately 1, approximately 10 -4 ~approximately 1, approximately 10 -3 ~approximately 1, approximately 10 -2 Relaxin activity may be considered to exist if the ratio of the activity of the heterodimer fusion of the present disclosure to the activity of the reference relaxin protein is approximately 1 to 10. 5 , approximately 1 to approximately 10 4 , approximately 1 to approximately 10 3 If the values ​​are approximately 1 to 100, 1 to 50, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 2, it may be considered to have relaxin activity.

[0085] In some embodiments, the relaxin activity of the heterodimer fusion, for example HFUS1, is about 0.001 to about 10 relative to the relaxin activity of the reference relaxin protein.

[0086] Relaxin activity can be determined as the EC50 value. As used herein, the term “EC50” (maximum half dose effective concentration) refers to the effective concentration of a therapeutic compound that induces an intermediate response between baseline and maximum after a given exposure time.

[0087] Heterodimizing domain A heterodimer fusion used in the formulations of this disclosure, for example HFUS1, comprises a first heterodimer domain and a second heterodimer domain. In certain embodiments, the first and second heterodimer domains are derived from an immunoglobulin Fc region.

[0088] The term "Fc region" is defined as the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally contains two constant domains, the CH2 domain and the CH3 domain, and optionally the CH4 domain.

[0089] The first and second Fc regions may include immunoglobulin domains CH2 and / or CH3. In certain embodiments, the first and second Fc regions include immunoglobulin domains CH2 and CH3.

[0090] The Fc region may be derived from an immunoglobulin of any species (e.g., IgG), specifically from human immunoglobulin (e.g., human IgG). In embodiments where the Fc region is derived from IgG, the Fc region may be derived from any subclass of IgG (e.g., IgG1, IgG2, IgG3, IgG4), particularly IgG1. In certain embodiments, the first and second Fc regions are derived from human IgG1 immunoglobulin. In other embodiments, the first and second Fc regions are derived from human IgG4 immunoglobulin.

[0091] In certain embodiments, the first and second Fc regions include heterodimerization-promoting amino acid mutations and / or modifications. Such modifications may include the introduction of asymmetric complementary modifications to each of the first and second Fc regions, resulting in both strands being compatible with each other and therefore capable of forming a heterodimer, but each strand being unable to dimerize with itself. Such modifications may include insertions, deletions, conserved and non-conserved substitutions, and rearrangements. Incorporating such modifications provides a method for increasing the yield of heterodimers produced by recombinant cell cultures compared to other undesirable end products such as homodimers.

[0092] The first and second Fc regions may include any heterodimerization-promoting amino acid mutations and / or modifications known in the art. Combinations of modifications can be used to maximize the efficiency of association while minimizing the impact on antibody stability.

[0093] In the "knob-in-hole" method, heterodimerization can be facilitated by introducing steric hindrance between contact residues. A "protrusion" is generated by replacing one or more small amino acid side chains ("Fc knobs") at the interface of one Fc region with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "caves" of the same or similar size as the larger side chains are created on the interface of other Fc regions ("Fc holes") by replacing the amino acid with the larger side chain with an amino acid with the smaller side chain (e.g., alanine or valine). "Knob-in-hole" modifications are described in detail, for example, Ridgway JB et al. (1996) Protein Eng. 9(7) 617-621 and Merchant AM et al. (1998) Nat. Biotechnol. 16(7): 677-681.

[0094] Other modifications that may be used to generate heterodimers include, but are not limited to, those that produce favorable electrostatic interactions between the two Fc regions. For example, one or more positively charged amino acids may be introduced into one Fc region, and one or more negatively charged amino acids may be introduced into corresponding positions in the other Fc region. Alternatively, or in addition, the Fc region may be modified to include mutations that introduce cysteine ​​residues capable of forming disulfide bonds. Alternatively, or in addition, the Fc region may include one or more modifications to hydrophilic and hydrophobic residues at the interchain interface to make heterodimer formation more entropically and enthalpy than homodimer formation.

[0095] Therefore, in some embodiments, heterodimerization-promoting amino acid mutations and / or modifications cause steric hindrance between contact residues (e.g., by "knob-in-hole"), induce favorable electrostatic interactions between two Fc regions, introduce cysteine ​​residues capable of forming disulfide bonds, and / or modify hydrophilic and hydrophobic residues at the interface between two Fc regions.

[0096] In certain embodiments, the heterodimerization-promoting amino acid mutations are "Fc knob" and "Fc hole" mutations. In certain embodiments, the "Fc knob" and "Fc hole" mutations are located in the CH3 domain.

[0097] In some embodiments, the first and second Fc regions are derived from human IgG1 immunoglobulin and include "Fc X" and "Fc Y" with mutations in the CH3 domain, and the "Fc X" and "Fc Y" mutations are selected from the combinations (or their conservative substitutions) listed in Table 1.

[0098] [Table 1] * The amino acid numbering follows the EU index, as used in Kabat.

[0099] In certain embodiments, "Fc Y" is an "Fc hole" containing Y349C, T366S, L368A, and Y407V, or their conservative substitutions, and "Fc X" is an "Fc knob" containing S354C and T366W, or their conservative substitutions, and the amino acid numbering follows the EU index as found in Kabat.

[0100] The term "EU index as found in Kabat" refers to the numbering system for human IgG1 EU antibodies described in Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service. National Institutes of Health. Bethesda, MD. All amino acid positions referenced in this application refer to EU index positions.

[0101] In some embodiments, the first Fc region has an "Fc hole" mutation, and the second Fc region has an "Fc knob" mutation. In alternative embodiments, the first Fc region has an "Fc knob" mutation, and the second Fc region has an "Fc hole" mutation.

[0102] It will be understood that the Fc region may contain further amino acid modifications to the wild-type Fc region. The Fc region may be modified, for example, to increase the affinity of the IgG molecule for FcRn. International Publication 02 / 060919 discloses a modified immunoglobulin containing an Fc region having one or more amino acid modifications, which is incorporated herein by reference in its entirety. Methods for constructing an Fc region having one or more amino acid modifications are known in the art.

[0103] In some embodiments, the first and / or second Fc region includes one or more amino acid modifications that can reduce or eliminate the effector function of the Fc region. In some embodiments, the amino acid modifications reduce or avoid cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0104] In some embodiments, the first and / or second Fc region comprises one or more amino acid modifications, which can increase the half-life of the heterodimer fusion, e.g., HFUS1.

[0105] In some embodiments, the first and / or second Fc region comprises at least one of the following amino acid mutation combinations: (i) M252Y, S254T and T256E, or their conservative substitutions, (ii) L234F, L235Q and K322Q, or their conservative substitutions, (iii) L234F, L235E and P331S, or their conservative substitutes, (iv) M252Y, S254T, T256E, L234F, L235Q and K322Q, or their conservative substitutions, (v) M252Y, S254T, T256E, L234F, L235E and P331S, or their conservative substitutions, The numbering of amino acids follows the EU index, as in Kabat.

[0106] In some embodiments, the first and / or second Fc regions may further include amino acid mutations L234F, L235E, and P331S, or their conserved substitutions, and the amino acid numbering follows the EU index as found in Kabat.

[0107] In some embodiments, the Fc region containing the "Fc hole" mutation has the sequence or a variant thereof described in SEQ ID NO: 3, and the Fc region containing the "Fc knob" mutation has the sequence or a variant thereof described in SEQ ID NO: 4.

[0108] In some embodiments, the Fc region includes a variant of SEQ ID NO: 3 having the amino acid mutation Y349C, which is reverted to Y349, and a variant of SEQ ID NO: 4 having the amino acid mutation S354C, which is reverted to S354, such that the Fc region cannot form a stabilizing disulfide bond.

[0109] In some embodiments, the Fc region includes a variant of SEQ ID NO: 3 and / or a variant of SEQ ID NO: 4, with the first five residues DKTHTCPPC (SEQ ID NO: 69) being modified. In some embodiments, this region is replaced with the sequence DKTHTACPPC (SEQ ID NO: 70). In alternative embodiments, this region is replaced with the sequence GGAGGACPPC (SEQ ID NO: 71). In alternative embodiments, this region is replaced with the sequence ACPPC (SEQ ID NO: 72).

[0110] In alternative embodiments, the first and second heterodimerization domains are derived from the immunoglobulin Fab region. In some embodiments, the heterodimerization domain includes the CH1 and CL regions. The Fab region, including the L and Fd chains, has been found to mediate efficient heterodimerization (Schoonjans R et al. (2000) J.Immunol. 165 (12):7050-7057). Therefore, in alternative embodiments, the heterodimerization domain includes the L and Fd chains. In some embodiments, the L and Fd chains heterodimerize to form a disulfide-bridged stabilized heterodimer.

[0111] In further alternative embodiments, the first and second heterodimerizing domains heterodimerize to form parallel coiled coils. Heterodimerated coiled coils are described, for example, in Aronsson et al. (2015) Sci. Rep. 5:14063. In some embodiments, the heterodimerizing domains include amino acid mutations and / or modifications to prevent the formation of undesirable folded aggregates and / or to promote the formation of parallel coiled coils.

[0112] The first and second heterodimerization domains (e.g., the first and second Fc regions) can form half-life extension portions. Therefore, in some embodiments, the heterodimer fusion of the present disclosure, e.g., HFUS1, has an extended half-life compared to reference relaxin.

[0113] As used herein, the term “half-life” is used to refer to the time it takes for the concentration of a fusion protein in plasma to decrease to 50% of its original level. The “half-life” of a protein in plasma may depend on various factors such as the size of the protein, its stability, its clearance rate, metabolic turnover rate, in vivo proteolysis, and absorption rate by the body or specific tissues. Methods for determining the half-life of a protein are known in the art and are described in the following examples.

[0114] As shown in International Publication No. 2021 / 255127, the heterodimer fusions described herein, such as HFUS1, which have first and second heterodimerizing domains derived from immunoglobulin Fc, have a half-life of at least 5 hours in a mouse model (see Example 6 in International Publication No. 2021 / 255127). In comparison, the half-life of human relaxin-2 after IV administration is approximately 0.09 ± 0.04 hours, or 5.4 ± 2.4 minutes, in humans (Chen SA et al. (1993) Pharm. Res. 10(6):834-838).

[0115] An extended half-life will be recognized as advantageous because it allows for safer and more convenient dosing schedules, such as administering therapeutic proteins at lower doses that can be given less frequently. Furthermore, achieving lower doses may offer additional benefits, such as providing an improved safety profile and / or in vivo activation of multiple mechanisms of action.

[0116] connector Either or both of the relaxin A and B chains can be connected to their respective heterodimerization domains by connector polypeptides. In some embodiments, the relaxin A chain is connected to a first heterodimerization domain (e.g., a first Fc region) via a connector polypeptide, and the relaxin B chain is connected to a second heterodimerization domain (e.g., a second Fc region) via a connector polypeptide.

[0117] The connector polypeptide may be of any suitable length, for example, about 6 to 40 amino acids long, for example, about 6 to 21 amino acids long. In some embodiments, the connector polypeptide is at least 6 amino acid residues long, in particular at least 11 amino acids long, in particular at least 16 amino acids long. In some embodiments, the connector polypeptide is less than 40 amino acids long. Connector polypeptides of different or the same length can be used in each arm of the heterodimer fusion described herein, for example, HFUS1. In some embodiments, at least one connector polypeptide has a length of 21 amino acids. In certain embodiments, both connector polypeptides have a length of 21 amino acids. The connector polypeptide may have any amino acid sequence. Connector polypeptides of different or the same amino acid formulations can be used in each arm of the heterodimer fusion described herein, for example, HFUS1.

[0118] In some embodiments, one or both connector polypeptides contain a proline and alanine repeat (PA)x (SEQ ID NO: 73). In certain embodiments, x is 3 to 15, optionally the connector polypeptide is longer than 16 amino acids, and optionally the connector polypeptide consists of a 21-amino acid sequence PAPAPAPAPAPAPAPAPAPAG (SEQ ID NO: 6).

[0119] In some embodiments, one or both connector polypeptides include glycerin and serine repeats, such as those described in Chen X et al. (2013) Adv. Drug. Deliv. Rev. 65(10):1357-1369. In some embodiments, one or both connector polypeptides include the motif (GGGGS)n (SEQ ID NO: 74), where n can be 1 to 8, for example, n is 4. In some embodiments, one or more connector polypeptides consist of a 21-amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5). In some embodiments, both connector polypeptides consist of a 21-amino acid sequence GGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 5).

[0120] In some embodiments, one connector polypeptide comprises a proline and alanine repeat as described herein, and the other connector polypeptide comprises a glycine and serine repeat as described herein.

[0121] Alternatively, one or both of the relaxin A and B chains may be linked to their respective heterodimerized domains by synthetic connector polypeptides, such as polyethylene glycol (PEG) polymer chains. Thus, the relaxin A chain may be linked to a first heterodimerized domain (e.g., a first Fc region) via a synthetic connector such as a polyethylene glycol (PEG) polymer chain, and the relaxin B chain may be linked to a second heterodimerized domain (e.g., a second Fc region) via a synthetic connector such as a polyethylene glycol (PEG) polymer chain, and the synthetic connector may be covalently or noncovalently linked to the heterodimerized domain (e.g., the Fc region). The process of PEGylation, i.e., linking a PEG polymer chain to a molecule, can be carried out according to methods known in the art.

[0122] stability As shown in International Publication No. 2021 / 255127, the heterodimer fusions described herein, e.g., HFUS1, have unexpectedly excellent physical and chemical stability. Therefore, in some embodiments, the heterodimer fusions described herein, e.g., HFUS1, have superior physical and / or chemical stability compared to the reference relaxin protein.

[0123] The physical stability of relaxin can be determined, for example, by measuring its purity and aggregation by HP-SEC, as in Example 9 of International Publication No. 2021 / 255127. The chemical stability of relaxin can be determined, for example, by measuring molecular fragmentation and modification by LC-MS, as in Example 9 of International Publication No. 2021 / 255127.

[0124] Surprisingly, as shown in International Publication No. 2021 / 255127, the heterodimer fusions described herein, e.g., HFUS1, have superior physical and chemical stability compared to recombinant Fc-fusion relaxins in which relaxin A and relaxin B are fused in a single chain (as opposed to relaxin A and B in separate fusion polypeptides). International Publication No. 2013 / 004607 describes recombinant single-chain relaxin fusion polypeptides fused to an immunoglobulin Fc region, e.g., fusion polypeptides referred to herein as RELAX0127 and RELAX0128. Thus, in some embodiments, the heterodimer fusions described herein, e.g., HFUS1, have superior physical and / or chemical stability compared to RELAX0127 and RELAX0128.

[0125] A heterodimer fusion, such as HFUS1, may include a half-life extension portion in addition to the first and second heterodimerization domains. In some embodiments, the half-life extension portion is a proteinaceous half-life extension portion. The proteinaceous half-life extension portion may be selected from the group consisting of the Fc region of immunoglobulins, albumin-binding domains, and serum albumin. In further embodiments, the half-life extension portion is a chemical substance that is not a protein or peptide, such as a polyethylene glycol (PEG) polymer chain.

[0126] The half-life extension portion may be bound to the N-terminus or C-terminus of the first or second heterodimerization domain. In some embodiments, the half-life extension portion is bound to the N-terminus of the first or second heterodimerization domain. In other embodiments, the half-life extension portion is bound to the C-terminus of the first or second heterodimerization domain. Methods for attaching the half-life extension portion to a heterodimer fusion, e.g., HFUS1, are known in the art. For example, the half-life extension portion may be attached by chemical conjugation or recombinant techniques. The half-life extension portion may be attached to a heterodimer fusion, e.g., HFUS1, directly or via a connector (e.g., a connector polypeptide). The use of a connector polypeptide may be particularly appropriate when the fusion polypeptide contains a proteinaceous half-life extension portion, such as an Fc region.

[0127] Exemplary Embodiments The heterodimer fusion used in the formulations of this disclosure, for example, HFUS1, may have various forms and / or sequences.

[0128] The term "fusion polypeptide" may be used to refer to a first heterodimerized domain fused to the relaxin A chain and / or a second heterodimerized domain fused to the relaxin B chain. The fusion polypeptide used in the formulations of this disclosure may be a recombinant fusion polypeptide, i.e., one produced by recombinant DNA technology.

[0129] In certain embodiments, the C-terminus of a first heterodimerization domain (e.g., a first Fc region) is attached to the N-terminus of the relaxin A chain, and the C-terminus of a second heterodimerization domain (e.g., a second Fc region) is attached to the N-terminus of the relaxin B chain. In some embodiments, the relaxin A chain polypeptide and / or the relaxin B chain polypeptide have a free C-terminus.

[0130] In alternative embodiments, the N-terminus of the first heterodimerization domain (e.g., the first Fc region) is attached to the C-terminus of the relaxin A chain, and the N-terminus of the second heterodimerization domain (e.g., the second Fc region) is attached to the C-terminus of the relaxin B chain. In some embodiments, the relaxin A chain polypeptide and / or the relaxin B chain polypeptide have a free N-terminus.

[0131] A heterodimer fusion used in the formulations of the present disclosure, e.g., HFUS1, may further comprise one or more Fabs. In some embodiments, the heterodimer fusion comprises one Fab ligated to the N-terminus of a first heterodimerization domain (e.g., a first Fc region) and a second Fab ligated to the N-terminus of a second heterodimerization domain (e.g., a second Fc region).

[0132] A heterodimer fusion used in the formulations of the present disclosure, e.g., HFUS1, may further comprise a second relaxin A chain polypeptide or a variant thereof and a second relaxin B chain polypeptide or a variant thereof. In some embodiments, the second relaxin A chain polypeptide or a variant thereof is ligated to the N-terminus of a first heterodimerization domain (e.g., a first Fc region), the second relaxin B chain polypeptide or a variant thereof is ligated to a second heterodimerization domain (e.g., a second Fc region), and optionally, the second relaxin A chain is ligated to the first heterodimerization domain (e.g., a first Fc region) via a connector (e.g., a connector polypeptide), and the second relaxin B chain is ligated to the second heterodimerization domain (e.g., a second Fc region) via a connector (e.g., a connector polypeptide).

[0133] Therefore, in some embodiments, the form of the heterodimer fusion, e.g., HFUS1, is selected from the following: (i) FcX-con-A / FcY-con-B (see Figure 25, for example), (ii) FcX-con-B / FcY-con-A (see Figure 25, for example), (iii) A-con-FcX / B-con-FcY (see Figure 25, for example), (iv) B-con-FcX / A-con-FcY (see Figure 25, for example), (v) Fab-FcX-con-A / Fab-FcY-con-B (see Figure 25, for example), (vi) Fab-FcX-con-B / Fab-FcY-con-A, (vii) A-con-FcX-con-A / B-con-FcY-con-B (see Figure 25, for example), (viii) B-con-FcX-con-B / A-con-FcY-con-A, (ix) FcX-con-BLA and FcY, and optionally FcY-con-BLA (see Figure 25, for example), (x) FcY-con-BLA and FcX, and optionally FcX-con-BLA. (xi) FcX-con-ALB and FcY, optionally FcY-con-ALB, and (xii) FcY-con-ALB and FcX, optionally FcX-con-ALB, At this time, FcY is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V, or a conservative substitution thereof. FcX is an Fc region having an "Fc knob" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation S354C:T366W, or a conservative substitution thereof. "con" is a connector polypeptide, B is relaxin B chain or a variant thereof. A is relaxin A chain or a variant thereof. L is a linker polypeptide that optionally has the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60).

[0134] In another embodiment, the heterodimer fusion used in the formulations of the present disclosure is (i) XBLA and Y, YBLA (optional), (ii) YBLA and X, optionally XBLA, At this time, X and Y are heterodimerization domains as described herein, B is the relaxin B chain or its variant, for example, the relaxin-2 B chain or its variant. A is the relaxin A chain or a variant thereof, for example, the relaxin-2 A chain or a variant thereof. L is a linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60) at will. In this case, X heterodimerizes with Y, and the heterodimer fusion has relaxin activity.

[0135] In yet another embodiment, the heterodimer fusion used in the formulations of the present disclosure is (i) XALB and Y, optionally YALB or (ii) Including YALB and X, optionally XALB, At this time, X and Y are heterodimerization domains as described herein, A is the relaxin A chain or a variant thereof, for example, the relaxin-2 A chain or a variant thereof. B is the relaxin B chain or its variant, for example, the relaxin-2 B chain or its variant. L is an optionally linker polypeptide having the amino acid sequence GGGSGGGSGG (SEQ ID NO: 60), At this time, X heterodimerizes with Y, and the heterodimer fusion has relaxin activity.

[0136] In certain embodiments, the heterodimer fusion comprises the fusion polypeptide Rlx011DD described in SEQ ID NO: 11 and Rlx014DD described in SEQ ID NO: 20. In certain embodiments, the heterodimer fusion consists of the fusion polypeptide Rlx011DD described in SEQ ID NO: 11 and Rlx014DD described in SEQ ID NO: 20 (referred to as "HFUS1" or "RELAX0023"). In alternative embodiments, the heterodimer fusion comprises the fusion polypeptide Rlx013DD described in SEQ ID NO: 17 and Rlx012DD described in SEQ ID NO: 14.

[0137] In certain aspects of the present disclosure, the heterodimer fusions used in the formulations of the present disclosure, e.g., HFUS1, comprise combinations of fusion polypeptides selected from the combinations of FcX and FcY shown in Table 2.

[0138]

Table 2

[0139] In one aspect, a heterodimer fusion comprising the fusion polypeptides described in SEQ ID NO: 11 and SEQ ID NO: 20 is provided.

[0140] In an alternative aspect, a heterodimer fusion comprising the fusion polypeptides described in SEQ ID NO: 17 and SEQ ID NO: 14 is provided.

[0141] The fusion polypeptides used in the formulations of this disclosure may be produced by any method known in the art and as described in International Publication No. 2021 / 255127. In some embodiments, the fusion polypeptides used in the formulations of this disclosure are produced by recombinant expression of nucleic acid molecules encoding the fusion polypeptide in host cells.

[0142] Expression vectors containing nucleic acid molecules can be constructed using methods known to those skilled in the art. Suitable vectors include, for example, plasmids, phagemids, phages, or viral vectors.

[0143] Vectors containing nucleic acid molecules can be introduced into host cells by conventional techniques. Suitable host cells are known in the art. In some embodiments, the host cell is a mammalian cell such as HEK293 cells or CHO cells.

[0144] Transfected cells can be cultured by conventional techniques to produce fusion polypeptides used in the formulations of this disclosure.

[0145] Once the fusion polypeptides of this disclosure are produced, for example, by recombinant expression, they can be purified by any method known in the art. Exemplary protein purification techniques include chromatography (e.g., ion exchange, affinity, and / or sizing column chromatography), centrifugation, and differential solubility. International Publication No. 2021 / 255127 provides isolated fusion polypeptides separated from cell cultures by at least one optional purification step.

[0146] Stable pharmaceutical formulations This disclosure provides a pharmaceutical formulation comprising a heterodimer fusion, for example, HFUS1, and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) a first heterodimerization domain attached to at least one relaxin A chain polypeptide or a variant thereof, (ii) comprising at least one relaxin B chain polypeptide or a second heterodimerization domain attached to a variant thereof, The first heterodimerizing domain heterodimerizes with the second heterodimerizing domain, and the heterodimer fusion has relaxin activity.

[0147] In some embodiments, the pharmaceutical formulation further comprises a buffer. In some embodiments, the pharmaceutical formulation further comprises an excipient.

[0148] Lipase-resistant surfactants During the development of the pharmaceutical formulations of this disclosure, the inventors identified the formation of visible particles in the HFUS1 formulation over time (see Example 3). Surprisingly, the inventors established that the presence of the surfactant PS80 was the cause of particle formation. The inventors demonstrated that enzymatic hydrolysis of the ester bond of polysorbate 80 (PS80) by lipase (a host cell protein) present in the formulation (resulting from co-purification with HFUS1 during recombinant manufacturing) is likely the cause of PS80 degradation, which in turn led to the formation of impurities such as free fatty acids (FFA) that can function as nuclei to induce HFUS1 protein aggregation, resulting in particles. Furthermore, the degradation of PS80 over time can lead to an effective reduction in the amount of PS80 in the formulation, potentially adversely affecting the protective effect of the surfactant. The use of lipase-resistant surfactants such as poloxamer 188 (P188) and D-α-tocopherol polyethylene glycol succinate (TPGS) was able to mitigate particle formation in the HFUS1 formulation.

[0149] A "surfactant" refers to a surfactant that reduces the surface tension of the liquid in which it is dissolved. Surfactants can be included in pharmaceutical formulations for various reasons, such as to prevent or control aggregation, particle formation, or surface adsorption in liquid formulations, or to prevent or control similar phenomena during freeze-drying or redissolution of freeze-dried formulations. Examples of surfactants include amphiphilic organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. Common characteristics of surfactants include the ability to reduce the surface tension of water, the ability to reduce the interfacial tension between oil and water, and the ability to form micelles. Surfactants may be anionic, nonionic, cationic, amphoteric, bipolar, or combinations thereof.

[0150] Surfactants are typically amphiphilic molecules containing both hydrophilic and lipophilic groups. The hydrophilic-lipophilic balance (HLB) number can be used as a measure of the ratio between these groups and can range from 0 to 60, defining the affinity of the surfactant to water or oil. Molecules with an HLB number greater than 10 have an affinity for water (hydrophilic), while molecules with an HLB number less than 10 have an affinity for oil (lipophilic). Nonionic surfactants have an HLB number in the range of 0 to 20.

[0151] The critical micelle concentration (CMC) is the concentration of surfactant that is above the concentration at which micelles form. Below the CMC, the surface tension decreases with increasing surfactant concentration. Above the CMC, additional surfactant added to the system forms micelles.

[0152] Lipases are a group of enzymes that can hydrolyze triglycerides into their components, fatty acids and glycerol. Examples of lipases include lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, pharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, and lingual lipase.

[0153] In some embodiments, lipase-resistant surfactants cannot be enzymatically hydrolyzed by lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, pharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, or tongue lipase. In some embodiments, lipase-resistant surfactants cannot be enzymatically hydrolyzed by lipoprotein lipase.

[0154] In some embodiments, the lipase-resistant surfactant does not contain ester bonds that can be enzymatically hydrolyzed by lipoprotein lipase, lipase 9, phospholipase 2, phospholipase 2A, pharyngeal lipase, hepatic lipase, pancreatic lipase, endothelial lipase, bile salt-dependent lipase, lysosomal lipase, hormone-sensitive lipase, gastric lipase, or tongue lipase. In some embodiments, the lipase-resistant surfactant does not contain ester bonds that can be enzymatically hydrolyzed by lipoprotein lipase.

[0155] In some embodiments, the lipase-resistant surfactant is a water-soluble nonionic triblock copolymer formed by polyethylene oxide (PEO) blocks and polypropylene oxide (PPO) blocks. In certain embodiments, the water-soluble nonionic triblock copolymer is poloxamer 188 (P188).

[0156] Poloxamer 188 (P188, CAS number 9003-11-6) or Pluronic F68 is a nonionic triblock copolymer with a specified number of repeats of PEO and PPO, and a molecular weight of approximately 7680–9510 Da. P188 is amphiphilic due to the presence of two hydrophilic side chains (PPO) bound to a hydrophobic central core (PEO). Poloxamer has 29 HLB numbers. See Chen et al. (2022). Poloxamer 188 (P188), A Potential Polymeric Protective Agent for Central Nervous System Disorders: A Systematic Review. Curr Neuropharmacol. 20(4):799-808. P188 has a melting point of approximately 51°C–53°C. P188 has a CMC of approximately 24 mg / mL–32 mg / mL at 37°C. See Moghimi et al. (2004). Causative factors behind poloxamer 188 (Pluronic F68,Flocor(TM))-induced complement activation in human sera. A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels. Biochimica et Biophysica Acta 1689;103 - 113.

[0157] Alternatively, a lipase-resistant surfactant is D-α-tocopherol polyethylene glycol succinate (TPGS). Formulations containing TPGS are described in International Publication No. 2022 / 101826.

[0158] As described in International Publication No. 2022 / 101826, TPGS (also known as tocophersolane) is a water-soluble synthetic derivative of natural α-tocopherol (vitamin E) formed by covalently bonding tocopherol succinate, an ester formed by the esterification of tocopherol and succinic acid, to a polyethylene glycol (PEG) portion via an esterification reaction. The general structure of TPGS is as follows:

[0159] [ka]

[0160] TPGS is amphiphilic due to the presence of a polar hydrophilic head (polyethylene glycol) and a lipophilic tail (phytyl chain of d-α-tocopherol). TPGS surfactants can contain PEG moieties with varying molecular weights. In one embodiment, the PEG moiety of TPGS has a molecular weight of approximately 1000 Da, and the TPGS molecule is called D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000). As used herein, the term TPGS includes TPGS1000. TPGS1000 has an HLB number of 13.2. See Wu and Hopkins. (1999). "Characteristics of D-alpha-tocopheryl PEG1000 succinate for applications as an absorption enhancer in drug delivery systems." Pharm Tech. 23:52-60. TPGS has a melting point of approximately 37°C to 41°C, or approximately 38°C, is stable at a pH of approximately 4.5 to 7.5, and is approximately 20% water-soluble at 20°C. Vitamin E TPGS is a very stable form of vitamin E. See PMC Isochem. (2015). "Vitamin E TPGS: NF and Food Grade." Available at pmcisochem.fr / page / info-center. TPGS is generally considered safe (GRAS) and is approved by the Federal Drug Administration (FDA) as an inactive ingredient for oral and topical formulations. TPGS1000 has a CMC of 0.02% (w / w) at 37°C. See Wu and Hopkins. (1999). "Characteristics of D-alpha-tocopheryl PEG1000 succinate for applications as an absorption enhancer in drug delivery systems." Pharm Tech. 23:52-60.

[0161] TPGS formulations typically contain a mixture of monomers and dimers. The monomer contains a single vitamin E molecule covalently bonded to a water-soluble moiety such as polyethylene glycol (PEG) via a linker, where the water-soluble moiety, e.g., PEG, has free, unreacted terminal reactive groups, e.g., free terminal hydroxyl groups. The dimer contains two vitamin E molecules covalently bonded to a water-soluble moiety such as polyethylene glycol (PEG) via one or more linkers. The hydroxyl groups at both ends of the water-soluble moiety, e.g., both ends of the PEG moiety, react with the linker bonded to the vitamin E molecules, so that no free terminal reactive groups, e.g., hydroxyl groups, are present. The monomer and dimer are formed during the esterification reaction. In some embodiments, the TPGS formulation comprises at least about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% of TPGS monomers and about 30%, about 25%, about 20%, about 15%, about 14%, about 13%, about 12%, about 11%, or less than about 10% of TPGS dimers. In some embodiments, the TPGS formulation comprises at least about 85% of TPGS monomers and less than about 15% of TPGS dimers.

[0162] In further embodiments, the lipase-resistant surfactant is selected from P188, TPGS, Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG300, PEG400, Brij58, and Brij35.

[0163] In some embodiments of any aspect of this disclosure, the concentration of the lipase-resistant surfactant is 0.001%(w / v) to 1%(w / v), optionally 0.001%(w / v) to 0.5%(w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.005%(w / v) to 1%(w / v), optionally 0.005%(w / v) to 0.2%(w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.01%(w / v) to 0.15%(w / v). In some embodiments, the concentration of the lipase-resistant surfactant is 0.01%(w / v) to 0.1%(w / v). In certain embodiments, the concentration of the lipase-resistant surfactant is 0.02%(w / v) to 0.06%(w / v), optionally 0.02%(w / v), 0.03%(w / v), 0.04%(w / v), 0.05%(w / v), or 0.06%(w / v). In certain embodiments, the concentration of the lipase-resistant surfactant is 0.04%(w / v).

[0164] In some embodiments, the lipase-resistant surfactant is poloxamer 188 (P188). Therefore, in some embodiments, the formulations of the Disclosure contain about 0.01% (w / v) to about 0.1% (w / v) of poloxamer 188 (P188). In some embodiments, the formulations of the Disclosure contain about 0.02% (w / v) to about 0.06% (w / v) of poloxamer 188 (P188). In some embodiments, the formulations of the Disclosure contain about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), or about 0.06% (w / v) of poloxamer 188 (P188). In certain embodiments, the formulations of the Disclosure contain about 0.04% (w / v) of poloxamer 188 (P188).

[0165] Buffers and pH During the development and optimization of the pharmaceutical formulations of this disclosure, including the heterodimer fusion described herein, e.g., HFUS1, the inventors demonstrated that proteases are likely responsible for the fragmentation and cleavage of HFUS1. This enzymatic activity is likely to occur at lower pH levels, where proteases are most effective in cleaving molecules. Therefore, the inventors demonstrated that the pH of the formulation may be important to minimize the effects of chemical degradation, such as molecular fragmentation and amino acid (AA) cleavage, as demonstrated in pH optimization tests (see Example 2).

[0166] In some embodiments of any aspect of this disclosure, the formulation comprises a buffer with a pH of about 3 to about 10, optionally about 5 to about 8. In some embodiments, the formulation comprises a buffer with a pH of about 5.5 to about 7.5. At lower pH values, protease activity is higher, but the beneficial effects of ionic excipients (e.g., arginine-HCl) and lipase-resistant surfactants (e.g., P188) can still be obtained.

[0167] In some embodiments, the formulation has a pH in the range of about 6 to about 7. In some embodiments, the formulation has a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the formulation has a pH of about 6. In some embodiments, the formulation has a pH of about 7. In certain embodiments, the formulation has a pH of about 6.5.

[0168] As used herein, “buffer solution” refers to an acid-base conjugated component that is resistant to changes in pH, as is known in the art.

[0169] In some embodiments of any aspect of this disclosure, the buffer concentration is about 0.1 mM to about 100 mM, optionally about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the buffer concentration is about 1 mM to about 50 mM, about 10 to about 50 mM, or about 10 to about 30 mM. In certain embodiments, the buffer concentration is about 10 mM to about 30 mM.

[0170] In some embodiments, the buffer concentration is approximately 0.1 mM, approximately 0.5 mM, approximately 1 mM, approximately 5 mM, approximately 10 mM, approximately 11 mM, approximately 12 mM, approximately 13 mM, approximately 14 mM, approximately 15 mM, approximately 16 mM, approximately 17 mM, approximately 18 mM, approximately 19 mM, approximately 20 mM, approximately 21 mM, approximately 22 mM, approximately 23 mM, approximately 24 mM, approximately 25 mM, approximately 26 mM, approximately 27 mM, approximately 28 mM, approximately 29 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 75 mM, or approximately 100 mM. In some embodiments, the buffer concentration is approximately 10 mM. In some embodiments, the buffer concentration is approximately 15 mM. In some embodiments, the buffer concentration is approximately 25 mM. In some embodiments, the buffer concentration is about 30 mM. In certain embodiments, the buffer concentration is about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In certain embodiments, the buffer concentration is about 20 mM.

[0171] In some embodiments, the buffer is selected from acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycylglycine, and mixtures thereof. In certain embodiments, the buffer is selected from histidine, citrate, acetate, phosphate, tris, succinate, and mixtures thereof. In certain embodiments, the buffer is selected from citrate buffer and histidine buffer. In some embodiments, the buffer is citrate buffer. In some embodiments, the buffer is histidine buffer.

[0172] In certain embodiments, the buffer is histidine, histidine hydrochloride, or histidine / histidine hydrochloride buffer. In certain embodiments, the buffer is histidine / histidine hydrochloride buffer (i.e., a combination of histidine and histidine hydrochloride). In one embodiment, the buffer is L-histidine / L-histidine hydrochloride monohydrate.

[0173] In some embodiments, the formulations of the Disclosure contain about 10 mM to about 50 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 10 mM to about 30 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 15 mM to about 25 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 17 mM to about 23 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 10 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 15 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the Disclosure contain about 25 mM histidine / histidine hydrochloride buffer. In some embodiments, the formulations of the present disclosure contain about 30 mM histidine / histidine hydrochloride buffer. In certain embodiments, the formulations of the present disclosure contain about 20 mM histidine / histidine hydrochloride buffer.

[0174] Excipients During the development and optimization of the pharmaceutical formulations of this disclosure, including the heterodimer fusion described herein, e.g., HFUS1, the inventors demonstrated that the heterodimer fusion, e.g., HFUS1, has a tendency to self-associate (see Example 1). High levels of self-association of the molecule can lead to the formation of soluble aggregates, which can become precursors to large-sized insoluble aggregates, ultimately resulting in particles and significantly affecting the molecular stability profile. Optimization of the pH, buffer, and excipients used in the pharmaceutical formulations of this disclosure helped reduce aggregation. Overall, the histidine-arginine HCl system was found to provide the highest colloidal and stereostructural stability for the heterodimer fusion, e.g., HFUS1 (see Example 2).

[0175] Accordingly, in some embodiments of any aspect of this disclosure, the formulation further comprises an excipient for reducing protein aggregation.

[0176] The pharmaceutical formulations of this disclosure may comprise one or more excipients. Pharmaceutically acceptable excipients are known in the art; see, for example, "Remington's Pharmaceutical Sciences" (by Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, PA), which is incorporated herein in whole.

[0177] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia, the European Pharmacopeia, or any other generally accepted pharmacopoeia for use in animals, or more specifically, in humans.

[0178] In some embodiments, the excipient concentration is approximately 10 mM to approximately 500 mM, optionally 50 mM to 500 mM. In some embodiments, the excipient concentration is 100 mM to 300 mM. In some embodiments, the excipient concentration is 100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, or 250 mM to 300 mM. In certain embodiments, the excipient concentration is 140 mM to 240 mM. In some embodiments, the excipient concentration is 140 mM. In some embodiments, the excipient concentration is 150 mM. In some embodiments, the excipient concentration is 160 mM. In some embodiments, the excipient concentration is 170 mM. In some embodiments, the excipient concentration is 180 mM. In some embodiments, the excipient concentration is 190 mM. In some embodiments, the excipient concentration is 200 mM. In some embodiments, the excipient concentration is 210 mM. In some embodiments, the excipient concentration is 220 mM. In some embodiments, the excipient concentration is 230 mM. In some embodiments, the excipient concentration is 240 mM. In certain embodiments, the excipient concentration is 190 mM.

[0179] In one embodiment, the pharmaceutical preparation contains at least one amino acid as an excipient in an amount of approximately 1 mg / mL to approximately 50 mg / mL, approximately 5 mg / mL to approximately 25 mg / mL, approximately 10 mg / mL to approximately 20 mg / mL, or approximately 1 mg / mL to approximately 10 mg / mL. In another embodiment, the pharmaceutical preparation contains at least one amino acid as an excipient in an amount ranging from approximately 1 mg / mL, approximately 5 mg / mL, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, or approximately 25 mg / mL to approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, or approximately 50 mg / mL. In one embodiment, the pharmaceutical preparation contains at least one amino acid as an excipient in an amount of approximately 1 mg / mL, approximately 5 mg / mL, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 35 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, or approximately 50 mg / mL.

[0180] In some embodiments, the excipient is an ionic excipient. In some embodiments, the ionic excipient is an amino acid salt. "Amino acid salt" refers to a cationic or anionic form of an amino acid combined with a counterion having the opposite charge. In some embodiments, the amino acid salt is a pharmacokinetically acceptable salt. In some embodiments, the amino acid salt is an inorganic salt. In some embodiments, the amino acid salt is an organic salt. In some embodiments, the amino acid salt includes sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, hydrochlorides, sulfates, nitrates, or phosphates. In some embodiments, the amino acid salt includes organic acid salts such as acetates, citrates, maleates, malates, or oxalates. In some embodiments, the formulation includes an amino acid salt selected from salt forms of arginine, cysteine, glycine, lysine, ornithine, proline, alanine, glutamine, glutamic acid, histidine, valine, or combinations thereof. In some embodiments, the amino acid salt includes arginine, lysine, or histidine. In some embodiments, the amino acid salt is selected from arginine salts or lysine salts. In some embodiments, the ionic excipient is selected from arginine HCl or lysine HCl. In certain embodiments, the ionic excipient is arginine HCl.

[0181] In some embodiments, the formulations of the Disclosure contain about 100 mM to about 300 mM arginine HCl. In certain embodiments, the formulations of the Disclosure contain about 140 mM to about 240 mM arginine HCl. In some embodiments, the formulations of the Disclosure contain about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, or about 240 mM arginine HCl. In certain embodiments, the formulations of the Disclosure contain about 180 mM to about 200 mM arginine HCl. In certain embodiments, the formulations of the Disclosure contain about 190 mM arginine HCl.

[0182] Additional ingredients In some embodiments of any aspect of this disclosure, the formulation further comprises a sugar. The presence of the sugar can improve the osmotic pressure of the formulation. In certain embodiments, the concentration of the sugar is sufficient so that the formulation is isotonic or nearly isotonic. In some embodiments, the sugar is selected from sucrose, trehalose, fructose, glucose, mannose, melibiose, meletitose, raffinose, mannotriose, stachyose, sorbose, xylose, lactose, maltose, maltulose, isomaltulose, lactulose, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, polyol, sugar alcohols with a molecular weight of trivalent or greater (e.g., glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol), and mixtures thereof. In certain embodiments, the sugar is sucrose.

[0183] In some embodiments, the sugar concentration is 1 mg / mL to about 300 mg / mL, about 10 mg / mL to about 200 mg / mL, about 50 mg / mL to about 100 mg / mL, or about 80 mg / mL to about 90 mg / mL. In some embodiments, the sugar concentration is about 30 mg / mL to about 90 mg / mL.

[0184] Concentration of heterodimer fusion The inventors evaluated the effect of the concentration of a heterodimer fusion, such as HFUS1, on the stability of pharmaceutical formulations. The heterodimer fusion, such as HFUS1, showed good stability in formulations with various heterodimer fusion concentrations, such as HFUS1 (see Examples 4-6).

[0185] Accordingly, in some embodiments of any aspect of this disclosure, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 0.1 to 100 mg / mL, optionally 0.2 to 50 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 0.25 mg / mL to 50 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 25 mg / mL to 35 mg / mL. In some embodiments, the concentrations of the heterodimer fusion, e.g., HFUS1, in the formulation are 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL. The concentrations are mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, or 50 mg / mL. In certain embodiments, the concentration of the heterodimer fusion in the formulation, for example, HFUS1, is 0.25 mg / mL, 1 mg / mL, 5 mg / mL, or 50 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 0.25 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 1 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 1.1 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 5 mg / mL. In some embodiments, the concentration of the heterodimer fusion, e.g., HFUS1, in the formulation is 30 mg / mL.In some embodiments, the concentration of the heterodimeric fusion protein in the formulation, such as HFUS1, is 33 mg / mL. In some embodiments, the concentration of the heterodimeric fusion protein in the formulation, such as HFUS1, is 50 mg / mL.

[0186] In some exemplary embodiments, the formulation comprises a heterodimeric fusion protein (e.g., HFUS1) at a concentration of 0.2 mg / mL to 50 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0187] In some embodiments, the formulation comprises a heterodimeric fusion protein (e.g., HFUS1) at a concentration of 50 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0188] In some embodiments, the formulation comprises a heterodimeric fusion protein (e.g., HFUS1) at a concentration of 50 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.0.

[0189] In some embodiments, the formulation comprises a heterodimeric fusion protein (e.g., HFUS1) at a concentration of 50 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 7.0.

[0190] In some embodiments, the formulation comprises a heterodimeric fusion protein (e.g., HFUS1) at a concentration of 50 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0191] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion protein (e.g., HFUS1), 20 mM of histidine / histidine hydrochloride buffer, 240 mM of arginine HCl, and 0.04% (w / v) of poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0192] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion protein (e.g., HFUS1), 20 mM of histidine / histidine hydrochloride buffer, 190 mM of arginine HCl, and 0.02% (w / v) of poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0193] In some embodiments, the formulation comprises 50 mg / mL of a heterodimeric fusion protein (e.g., HFUS1), 20 mM of histidine / histidine hydrochloride buffer, 190 mM of arginine HCl, and 0.06% (w / v) of poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0194] In some embodiments, the formulation comprises 5 mg / mL of a heterodimeric fusion protein (e.g., HFUS1), 20 mM of histidine / histidine hydrochloride buffer, 190 mM of arginine HCl, and 0.04% (w / v) of poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0195] In some embodiments, the formulation comprises 1.1 mg / mL of a heterodimeric fusion protein (e.g., HFUS1), 20 mM of histidine / histidine hydrochloride buffer, 190 mM of arginine HCl, and 0.04% (w / v) of poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0196] In some embodiments, the formulation comprises 1 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0197] In some embodiments, the formulation comprises 1 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 7.0.

[0198] In some embodiments, the formulation comprises 0.25 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0199] In some embodiments, the formulation comprises 0.25 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 7.0.

[0200] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.0.

[0201] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0202] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 7.0.

[0203] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 150 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0204] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 230 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0205] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.02% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0206] In some embodiments, the formulation comprises 33 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.06% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0207] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.0.

[0208] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0209] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 7.0.

[0210] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 150 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0211] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 230 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0212] In some embodiments, the formulation comprises 30 mg / mL of a heterodimer fusion (e.g., HFUS1), 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.02% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0213] In some embodiments, the formulation comprises a heterodimer fusion protein (e.g., HFUS1) at 30 mg / mL, 20 mM histidine / histidine hydrochloride buffer, 190 mM arginine HCl, and 0.06% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.5.

[0214] Stability The inventors have conducted several deliberately designed tests (see Example 1) to identify the root causes of the instability of the heterodimer fusion proteins, such as HFUS1, used in the formulations of the present disclosure, and have also conducted formulation development and optimization efforts (see Examples 2-6) to identify a stable liquid formulation of the molecule that meets the expiration requirements of the pharmaceutical product.

[0215] A "stable" formulation refers to a formulation in which the heterodimer fusion protein, such as HFUS1, retains its physical stability, chemical stability, or biological activity during storage. "Chemical stability" can be evaluated by detecting and quantifying the chemically altered forms of the heterodimer fusion protein, such as HFUS1, which include, for example, deamidation, such as asparagine (Asn) deamidation, isomerization, such as aspartic acid (Asp) isomerization, oxidation, such as methionine (Met) oxidation, cleavage / hydrolysis / fragmentation, such as antibody hinge region fragmentation, succinimide formation, racemization, beta-elimination, glycation, adduct formation, disulfide scrambling, N-terminal extension, C-terminal processing, and differences in glycosylation. "Physical stability" can be evaluated by detecting and quantifying physically altered forms of the heterodimer fusion protein, such as HFUS1, such as, but not limited to, denaturation, aggregation, precipitation or particle formation, and physically altered forms due to surface adsorption.

[0216] Stability can be evaluated qualitatively or quantitatively by a variety of different methods, including, but not limited to, evaluation of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography or by visual inspection), evaluation of charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis, amino-terminated or carboxy-terminated sequence analysis, mass spectrometry, SDS-PAGE, peptide mapping analysis, or evaluation of biological activity by in vitro, in vivo, or in situ assays, for example, heterodimer fusions exhibiting HFUS1 activity.

[0217] A heterodimer fusion, e.g., HFUS1, is "stable" in a pharmaceutical formulation if the physical stability, chemical stability, or biological activity of the heterodimer fusion, e.g., HFUS1, at a given time point is within approximately 0.1%, approximately 0.5%, approximately 1%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, or approximately 25% (within standard error) of the physical stability, chemical stability, or biological activity of the heterodimer fusion, e.g., HFUS1, at an initial time point, e.g., when the pharmaceutical formulation is prepared.

[0218] Generally, stability is determined with respect to a selected temperature and a selected time. In some embodiments, the formulation is stable at temperatures of about 5°C ± 10°C and about 5°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months or about 36 months. In some embodiments, the formulation is stable at temperatures of about 25°C ± 10°C and about 25°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months or about 36 months. In some embodiments, the formulation is stable at temperatures of about 40°C ± 10°C and about 40°C ± 5°C for at least about 2 weeks, about 1 month, about 2 months or about 3 months, about 6 months, about 9 months, about 12 months, about 24 months or about 36 months.

[0219] In some embodiments, the formulation is stable at temperatures of approximately 2°C to approximately 8°C, or approximately 2°C, approximately 4°C, approximately 5°C, approximately 6°C, or approximately 8°C for at least approximately 2 weeks, approximately 1 month, approximately 3 months, or approximately 6 months, and up to approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months. In some embodiments, the formulation is stable at temperatures of approximately 22°C to approximately 28°C, or approximately 22°C, approximately 24°C, approximately 25°C, approximately 26°C, or approximately 28°C for at least approximately 2 weeks, approximately 1 month, approximately 3 months, or approximately 6 months, and up to approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months. In some embodiments, the formulation is stable at temperatures of approximately 37°C to approximately 43°C, or approximately 37°C, approximately 39°C, approximately 40°C, approximately 41°C, or approximately 43°C for at least approximately 2 weeks, approximately 1 month, approximately 3 months, or approximately 6 months, and up to approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months.

[0220] In some embodiments, the formulation is stable at 40°C for up to approximately 3 months. In some embodiments, the formulation is stable at 40°C for up to approximately 6 months. In some embodiments, the formulation is stable at 25°C for up to approximately 6 months. In some embodiments, the formulation is stable at 25°C for up to approximately 12 months. In some embodiments, the formulation is stable at 25°C for up to approximately 24 months. In some embodiments, the formulation is stable at 5°C for up to approximately 6 months. In some embodiments, the formulation is stable at 5°C for up to approximately 12 months. In some embodiments, the formulation is stable at 5°C for up to approximately 24 months. In some embodiments, the formulation is stable at 5°C for up to approximately 36 months.

[0221] In some embodiments, the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is chemically stable. In some embodiments, when stored at temperatures of about 40°C, about 25°C, about 2°C to about 8°C, or about 5°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months, the increase in the chemically altered form of the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is less than about 25%, about 20%, about 15%, about 10%, or about 5%, respectively. In some embodiments, the increase in heterodimer fusions, e.g., one or more chemically altered forms of HFUS1, is about 25%, about 20%, about 15%, about 10%, or less than about 5%, and these include chemically altered forms by, for example, deamidation, e.g., asparagine (Asn) deamidation; oxidation, e.g., oxidation of methionine, cysteine, histidine, tyrosine, tryptophan, or phenylalanine; intraresidial and interresidial cyclization (aspartic acid and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motif); cleavage / hydrolysis / fragmentation; β-elimination; glycation; and disulfide scrambling.

[0222] In some embodiments, the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is physically stable. In some embodiments, when stored at temperatures of about 40°C, about 25°C, about 2°C to about 8°C, or about 5°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, or about 6 months, and up to about 9 months, about 12 months, about 24 months, or about 36 months, the increase in the physically altered form of the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is less than about 25%, about 20%, about 15%, about 10%, or about 5%. In one embodiment, the increase in one or more physically altered forms of the heterodimer fusion, e.g., HFUS1, e.g., physically altered forms due to denaturation, aggregation, precipitation, or particle formation, is less than about 25%, about 20%, about 15%, about 10%, or about 5%.

[0223] In some embodiments, the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is biologically stable. In some embodiments, when stored at temperatures of about 40°C, about 25°C, about 2°C to about 8°C, or about 5°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, or about 6 months, and up to about 9 months, about 12 months, about 24 months, or about 36 months, the decrease in the biological activity of the heterodimer fusion in the pharmaceutical formulation, e.g., HFUS1, is less than about 25%, about 20%, about 15%, about 10%, or about 5%, respectively.

[0224] In some embodiments, the concentration of lipase-resistant surfactants in the pharmaceutical formulation remains stable during storage. In some embodiments, when stored at temperatures of about 40°C, about 25°C, about 2°C to about 8°C, or about 5°C for at least about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months, the decrease in the concentration of lipase-resistant surfactants in the pharmaceutical formulation is less than about 25%, about 20%, about 15%, about 10%, or about 5%, respectively.

[0225] In some embodiments, the stability of a pharmaceutical formulation is evaluated by measuring the amount of particulate impurities. Particulate impurities can include visible, invisible, and submicron impurities. Visible impurities have a diameter of about 100 μm or greater than about 150 μm and can be detected by visual inspection. Invisible particles generally range in size from about 1 μm to about 100 μm or about 150 μm. Submicron particles have a diameter of less than about 1 μm. Invisible particles generally pose the greatest risk when present in a pharmaceutical formulation, especially in formulations for parenteral administration, including subcutaneous, intravenous, or intramuscular administration, because they may, in some cases, induce a harmful immunogenic response. Invisible particles with a diameter of about 10 μm or greater, or about 5 μm or greater, may block pulmonary blood vessels after intravenous injection. Methods for detecting and quantifying particulate impurities are known and include dynamic light scattering (DLS) or static light scattering (SLS), nanoparticle tracking analysis (NTA), optical microscopy, electrical sensing zone (ESZ) method, flow imaging, resonant mass spectrometry, electron microscopy, Fourier transform infrared (FTIR) microscopy, and Raman microscopy. In some embodiments, invisible particles are detected using microflow imaging (MFI). In some embodiments, invisible particles are detected using light shielding methods, such as a High Accuracy Products (HIAC) system, for example, a HIAC system model 9703 with an HRLD150 sensor.

[0226] In some embodiments of any aspect of the present disclosure, the pharmaceutical formulation contains less than approximately 10,000, approximately 6,000, approximately 5,000, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or approximately 50 particles / mL with a diameter of approximately 2 μm, approximately 5 μm, approximately 10 μm, approximately 150, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or approximately 50 particles / mL. In some embodiments, the pharmaceutical formulation contains approximately 10,000, approximately 6,000, approximately 5,000, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or less than approximately 50 particles / mL with a diameter greater than 2 μm. In some embodiments, the pharmaceutical formulation contains approximately 10,000, approximately 6,000, approximately 5,000, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or less than approximately 50 particles / mL with a diameter greater than 10 μm.

[0227] A pharmaceutical formulation may contain a number of invisible particles within the limits of the USP (no more than 6,000 particles with diameters of 10 μm or larger and 25 μm or larger, respectively, as defined in Pharmacopeia US.2014.USP787 and 788). Therefore, in certain embodiments, the pharmaceutical formulation may contain less than approximately 6,000 particles / mL with a diameter greater than approximately 10 μm. In certain embodiments, the pharmaceutical formulation may contain less than approximately 600 particles / mL with a diameter greater than approximately 25 μm.

[0228] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months.In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0229] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 2 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0230] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months.In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0231] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 5 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0232] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months.In some embodiments, the pharmaceutical formulation contains less than approximately 6,000 or 5,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0233] In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C, approximately 30°C ± 10°C, approximately 25°C ± 10°C, approximately 20°C ± 10°C, or approximately 5°C ± 3°C for at least approximately 2 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 24 months, or approximately 36 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 3 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, when the pharmaceutical formulation is stored at a temperature of approximately 40°C ± 10°C for up to approximately 6 months, it contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 3 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 30°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 25°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 25°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 20°C ± 10°C for up to approximately 6 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 20°C ± 5°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 12 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 24 months. In some embodiments, the pharmaceutical formulation contains less than approximately 1,000 particles / mL with a diameter greater than approximately 10 μm when stored at a temperature of approximately 5°C ± 3°C for up to approximately 36 months.

[0234] Treatment method This disclosure includes therapies that involve administering the pharmaceutical formulations of this disclosure to animals, in particular mammals, such as humans, to prevent, treat, or improve symptoms associated with a disease, disorder, or infection.

[0235] Accordingly, the pharmaceutical formulations of this disclosure may be used, for example, in therapies for treating a disease or disorder. Also provided are methods for treating a disease or disorder, comprising administering a therapeutically effective amount of the fusion polypeptide described herein, e.g., HFUS1, to a subject or patient in need thereof. Use or methods may include administering a therapeutically effective schedule having the fusion polypeptide described herein, e.g., HFUS1, at a frequency less frequent than a therapeutically effective dosing schedule of wild-type relaxin molecule.

[0236] The heterodimer fusions described herein are described as useful for treating a variety of disorders, including heart failure and heart failure with pulmonary hypertension (see International Publication No. 2023 / 111112, incorporated herein by reference). Therefore, it will be understood that the pharmaceutical formulations of this disclosure may be used for the treatment of cardiovascular diseases, such as heart failure, and more specifically, heart failure with pulmonary hypertension.

[0237] The pharmaceutical formulations of the present invention may also be used in the treatment of kidney disease, lung disease and fibrotic disorders, such as fibrotic disorders of the kidneys, heart, lungs and liver, as well as in wound healing (Sherwood OD (2004) "Endocrine Reviews" 25(2):205-234). The fusion polypeptide described herein, for example HFUS1, may also be used to improve insulin resistance in diabetic patients (Bonner JS et al. (2013) "Diabetes" 62(9):3251-3260). The pharmaceutical formulations of the present disclosure may also be used in various forms of pulmonary hypertension. The pharmaceutical formulations of the present disclosure may also be used in disorders resulting from or caused by hardening of the arterial wall, decreased arterial wall elasticity, and decreased arterial compliance and extensibility, such as hypertension, kidney disease, peripheral artery disease, carotid and cerebrovascular disease (i.e., stroke and dementia), diabetes, microvascular disease resulting in end-organ damage, coronary artery disease, and heart failure.

[0238] This disclosure includes methods for treating subjects having heart failure, particularly heart failure with pulmonary hypertension, by administering the pharmaceutical formulations described herein, and the use of such pharmaceutical formulations for use in such methods. In particular, subjects may be animals, particularly mammals, and more specifically humans.

[0239] Use or method may include administering a therapeutically effective schedule having the heterodimer fusion / fusion polypeptide described herein, e.g., HFUS1, at a frequency less frequent than a therapeutically effective dosing schedule for the wild-type relaxin molecule.

[0240] As used herein, the term “heart failure” includes acute heart failure, chronic heart failure (CHF), and acute decompensated heart failure (ADHF). The term “heart failure” may also include more specific diagnoses such as heart failure with preserved ejection fraction (HFpEF), heart failure with moderate ejection fraction, or heart failure with reduced ejection fraction (HFrEF). This may also include heart failure due to hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0241] As used herein, the term “pulmonary hypertension” can typically be defined as a subject having a mean pulmonary artery pressure of approximately 20 mmHg or higher, and optionally 25 mmHg or higher, at rest. It can also typically be defined as a mean pulmonary artery pressure of approximately 30 mmHg or higher during or immediately after exercise. Therefore, a subject may have a mean pulmonary artery pressure in the range of approximately 20 mmHg to approximately 30 mmHg, optionally 25 mmHg to approximately 30 mmHg, or higher. Alternatively or additionally, a subject may have the following: a. Right ventricular systolic pressure of approximately 40 mmHg or higher, b. Pulmonary artery wedge pressure (PAWP) greater than 15 mmHg, and / or c. The following pulmonary vascular resistance, i. Wood units less than 3.0, ii. Wood units of 3.0 or more.

[0242] Therefore, in some cases, pulmonary hypertension may be classified as Group II pulmonary hypertension, as defined by the World Health Organization. This may also be referred to as "heart failure with pulmonary hypertension due to left heart disease." In other cases, pulmonary hypertension may be classified as Group I pulmonary artery hypertension, as defined by the World Health Organization (see Ryan et al., 2012, "Pulm.Circ". 2(1):107-121).

[0243] The parameters of pulmonary hypertension and heart failure can be measured or estimated using techniques known in the art. These include, for example, echocardiography, pulmonary artery catheterization, and implantable monitoring devices. In certain embodiments, the subject may wear a blood pressure monitoring device, and optionally a pulmonary artery pressure monitoring device, as known in the art. In certain embodiments, the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device. Typically, the device is implanted before treatment with the heterodimer fusion described herein, e.g., HFUS1. Alternatively, the subject wears the device during or after treatment.

[0244] As used herein, the term “heart failure with pulmonary hypertension” refers to a subset of heart failure subjects who also suffer from pulmonary hypertension (HF+PH subjects).

[0245] "Treatment" means improvement and / or elimination of one or more symptoms or causes of a target disease. In some embodiments, this may include modulating the level of one or more biological markers or functions within the non-affected range (compared to a healthy cohort). For example, the pharmaceutical formulations of the Disclosure may reduce pulmonary vascular resistance (PVR) in a subject. For example, PVR may be reduced by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more after treatment compared to baseline PVR (before administration of the heterodimer fusion described herein, e.g., HFUS1 to the subject). Thus, the pharmaceutical formulations of the Disclosure may reduce PVR in a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more compared to baseline PVR (before administration of the pharmaceutical formulation of the Disclosure to the subject). In addition, or alternatively, the pharmaceutical formulations of the present invention can reduce the mean pulmonary artery pressure (mPAP) in a subject. For example, mPAP can be reduced by at least 1 mmHg to 15 mmHg or more. Thus, the pharmaceutical formulations of the present disclosure can reduce the mean pulmonary artery pressure in a subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg or more. Similarly, the pharmaceutical formulations of the present disclosure can reduce the estimated diastolic pulmonary artery pressure (ePAD) in a subject. For example, ePAD can be reduced by at least 1 mmHg to 15 mmHg or more. Accordingly, the pharmaceutical formulations of this disclosure can reduce the estimated diastolic pulmonary artery pressure in the subject by at least 1 mmHg, at least 2 mmHg, at least 3 mmHg, at least 4 mmHg, at least 5 mmHg, at least 6 mmHg, at least 7 mmHg, at least 8 mmHg, at least 9 mmHg, at least 10 mmHg, at least 11 mmHg, at least 12 mmHg, at least 13 mmHg, at least 14 mmHg, or at least 15 mmHg or more.In addition, or alternatively, the pharmaceutical formulations of the Disclosure may increase the ejection fraction (EF%) in a subject as a measure of cardiac output. For example, the EF% may increase by at least 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, or 1% to 50% or more. Thus, the pharmaceutical formulations of the Disclosure may increase the ejection fraction (EF%) in a subject by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more. In addition, or alternatively, the pharmaceutical formulations of the Disclosure may increase the ejection fraction in a subject. (a) Increase in cardiac stroke volume (SV), (b) A decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR), (c) Increased ejection fraction, and / or (d) Increase in cardiac output, This can be achieved compared to baseline levels before administration. A combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion.

[0246] Therefore, the pharmaceutical formulations of this disclosure are subject to the following conditions: (a) Decrease in PVR, (b) Decrease in mPAP, (c) Decline in ePAD, (d) Increase in cardiac stroke volume (SV), (e) A decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR), (f) Increased ejection fraction, and / or (g) Increase in cardiac output, These changes may occur compared to baseline levels before administration. A combination of a decrease in SVR and an increase in eGFR indicates improved organ perfusion. Changes in one or more of these parameters may occur after 1 to 24 weeks of treatment. In some embodiments, changes in one or more of these parameters occur after 24 weeks of treatment.

[0247] In certain embodiments, the reduction in mPAP described herein may result in improvement of dyspnea, as described in Solomonica A, et al. (2013) "Circ Heart Fail" 6:53-60.

[0248] The pharmaceutical formulations of this disclosure are suitable for parenteral administration to subjects or patients. In some embodiments, the subjects or patients are mammals, specifically humans.

[0249] Wild-type human relaxin-2 has a half-life of several minutes in vivo. As a result, it must be administered to hospitalized patients by continuous intravenous infusion and exhibits serious side effects, including hypotension. In contrast, it will be understood that embodiments of the pharmaceutical formulations of this disclosure may be administered to subjects or patients by injection, for example, by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the pharmaceutical formulation is administered by subcutaneous injection. Administration by injection, such as subcutaneous injection, is more comfortable for the subject or patient and offers the advantage of having the opportunity to administer to the subject or patient in a non-hospital setting. In some embodiments, the pharmaceutical formulation is administered by self-administration.

[0250] In some embodiments, the fusion polypeptide (i.e., including heterodimer fusions), e.g., HFUS1, used in the formulations of the present disclosure has a longer half-life compared to wild-type relaxin, which allows for lower overall exposure based on molar concentration. For example, the fusion polypeptide (i.e., including heterodimer fusions), e.g., HFUS1, used in the formulations of the present disclosure can be administered at a lower frequency than wild-type relaxin, thus providing a more convenient dosing schedule.

[0251] manufactured goods In one embodiment, a product is provided. In some embodiments, the product includes a device or container containing a pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1, and a lipase-resistant surfactant as described herein. In some embodiments, the product includes a device or container containing a pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1, and a lipase-resistant surfactant selected from poloxamer 188 (P188) and D-α-tocopherol polyethylene glycol succinate (TPGS). In some embodiments, the product includes a device or container containing a pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1, and poloxamer 188 (P188). In some embodiments, the container or device is a syringe, e.g., a pre-filled syringe, an auto-injector, a bottle, a vial, or a test tube. In certain embodiments, the container or device is a syringe, optionally a pre-filled syringe. Thus, in certain embodiments, the product comprises a syringe, e.g., a pre-filled syringe, containing a pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1 and poloxamer 188 (P188). In some embodiments, the product comprises a device or container containing the pharmaceutical formulation and labels on or associated with the device or container providing instructions for use. In some embodiments, the product further comprises other materials desirable from a commercial or user perspective, such as buffers, diluents, filters, needles, syringes, or accompanying documentation containing instructions for use.

[0252] In one embodiment, a kit comprising the pharmaceutical formulation of the Disclosure is provided. The kit may include a package containing the pharmaceutical formulation and instructions. In some embodiments, the kit comprises at least one device or container containing the pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1, and a lipase-resistant surfactant as described herein. In some embodiments, the at least one device or container is selected from syringes, e.g., pre-filled syringes, auto-injectors, bottles, vials, or test tubes. In certain embodiments, the at least one device or container is a syringe, e.g., a pre-filled syringe.

[0253] This disclosure provides a kit containing the pharmaceutical formulation of this disclosure. The kit may include a package containing the pharmaceutical formulation of this disclosure and instructions for administering the formulation.

[0254] In some embodiments, the kit comprises at least one device or container containing the pharmaceutical formulation of the Disclosure, and an injection device. In some embodiments, the injection device is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the injection device is adapted for subcutaneous administration. In certain embodiments, the injection device is a syringe, e.g., a pre-filled syringe, containing the pharmaceutical formulation comprising a heterodimer fusion having relaxin activity as defined herein, e.g., HFUS1, and a lipase-resistant surfactant as defined herein, e.g., poloxamer 188 (P188).

[0255] In some embodiments, the pharmaceutical formulations of this disclosure are formulated in single-dose vials or in a container-closed system (e.g., pre-filled syringes). Such containers may optionally be accompanied by a warning in the form prescribed by the government agency regulating the manufacture, use, or sale of the pharmaceutical or biological product, which reflects the approval by the agency for manufacture, use, or sale for human administration. In some embodiments, the kit includes instructions.

[0256] The embodiments described above should be understood as illustrative examples. Further embodiments are conceivable. It should be understood that any feature described in relation to any one embodiment may be used alone or in combination with other features described, in combination with one or more features of any other embodiments, or in any combination of any other embodiments. Furthermore, equivalents and modifications not described above may be adopted without departing from the scope of this disclosure, as defined in the appended claims.

[0257] Other examples and variations are described in the appended claims and are within the scope of this disclosure. All documents referenced herein, including all data, tables, figures, and text presented therein, are incorporated herein by reference in their entirety.

[0258] array

[0259] [Table 3-1]

[0260] [Table 3-2]

[0261] [Table 3-3]

[0262] [Table 3-4]

[0263] [Table 3-5]

[0264] [Table 3-6]

[0265] [Table 3-7]

[0266] [Table 3-8]

[0267] [Table 3-9]

[0268] [Table 3-10]

[0269] [Table 3-11]

[0270] [Table 3-12]

[0271] [Table 3-13] [Examples]

[0272] Materials and methods material All materials used in the tests were USP or multi-standard grade. All solutions and buffers were prepared using USP or HPLC water and filtered through a 0.2 μm PVDF filter (Millipore, Millex GV, SLG033RB) before further use. HFUS1 samples for formulation screening and stability testing were prepared under sterile conditions in a biosafety cabinet (BSC). Bulk materials were stored at 5°C. All formulations were filtered through a 0.22 μm PVDF filter unit, filled into 2R glass vials or pre-filled syringes (PFS), and stored in incubators set to 5, 25, and 40°C for stability measurement time points.

[0273] method Determination of protein concentration The HFUS1 protein concentration was determined by measuring the absorbance at 280 nm using a Trinean HT-A280 with a procedure modified according to SOP DV-050465. The measured extinction coefficient of 1.47 (mg / mL) was used to calculate the protein concentration. -1 cm -1 I used it.

[0274] Purity determination by size exclusion chromatography Size exclusion chromatography was performed on an Agilent Technologies 1200 / 1100 HPLC system with an 8°C temperature-controlled autosampler, using a TSKgel G3000SWXL, 5 μm, 250 Å column (7.8 × 300 mm), a Guard Column for 7.8 mm TSKgel columns from Tosoh Bioscience, and Agilent ChemStation software. The system was primed with a mobile phase (0.1 M anhydrous disodium phosphate, 0.1 M sodium sulfate, pH 6.8) at 1.0 mL / min for at least 30 minutes, with the UV lamp switched on 30 minutes prior to analysis and the pressure monitored up to 130 bar. Samples were diluted to 10 mg / mL in PBS and filtered using a PVDF membrane and an Ultrafree-MC centrifugal filter (Merck) with a pore size of 0.45 μm. All samples were analyzed at 280 nm with a column flow rate of 1.0 mL / min, a pause time of 20 minutes, an injection volume of 25 μL, and a column temperature of 20°C.

[0275] Determination of the isoelectric point The isoelectric point of HFUS1 was determined using Maurice of ProteinSimple, according to the manufacturer's protocol (Bio-Techne®), via capillary isoelectric focusing (cIEF).

[0276] Appearance evaluation Before performing the visual inspection of the formulations, all samples and standards were equilibrated at room temperature. The outside of the glass vials was wiped with alcohol to remove any particles or residues from the glass surface. Visual inspection was performed manually in a white and black background light cabinet (Apollo II Liquid Viewer, Adelphi Manufacturing Co. Ltd.) with a 3000–3400 lux range. The cabinet lights were turned on 20 minutes before use to ensure appropriate light intensity.

[0277] The determination of visible particles was performed by comparing the sample to particle standards with increasing concentrations of barium sulfate. The particle standards were numbered from 0 to 7, with 7 being the maximum number of particles, and served as a guideline for determining the visible particles in the sample. The sample was rotated 10 to 20 times to ensure homogeneity and compared to the standard at the height of the middle cloth on the panel and directly below the top illumination of the panel, against a black background.

[0278] Particle analysis that is not visible to the naked eye Non-visually observable particles were counted using either MFI or HIAC, as described in the text.

[0279] For the MFI, the MFI5200 series model was used with a 100 μm, 1.6 mm silane-coated flow cell (Protein simple, United States). Before use, the system was washed with approximately 10 mL of non-ionized ultrapure water, followed by 1 mL of 10% Decon, and then rinsed with 10 mL of non-ionized ultrapure water using the System Flush Preview mode. The cell was dried to check that the silane coating was intact and that no particles were adhering to its surface. If the cell was contaminated, the washing procedure was repeated. To evaluate the cleanliness of the system, a water background run was performed by priming the cell with 250 μL of water at 1 mL / min and analyzing approximately 800 μL of water sample under optimized illumination. Live images were monitored to check for large particles or bubbles entering the system.

[0280] For HIAC analysis, a High Accuracy Products (HIAC) system model 9703 equipped with an HRLD150 sensor was used. Before analysis, the sample was diluted to 25 mg / mL and degassed for 30 minutes before measurement. For each sample, the number of particles in a representative buffer was obtained to evaluate the contribution of the diluted buffer to the total number of particles. Four measurements were performed for each sample.

[0281] Differential scanning calorimetry (DSC) of unfolding temperature DSC experiments were performed using an automated MicroCal VP-Capillary DSC system from Malvern Panalytical (United Kingdom). Samples were diluted to 5 mg / mL using the corresponding formulation buffer and filtered through a 0.22 μm PVD centrifugation filter unit. Lysozyme (3 mg / mL) was used as a standard at the start and end of every run to evaluate system suitability. Scans were performed from 25°C to 100°C, with a scan rate of 95°C / hour, a filtering cycle of 24 seconds, and a wash cycle between protein sample sets. Each sample was analyzed under the same conditions using the corresponding formulation buffer as a reference. Data analysis was performed using Origin v7.0552 software. Buffer blank scans were subtracted from the thermograms and normalized to appropriate protein concentrations. Thermodynamic parameters were determined by fitting the data using a Non-2-state model with cursor initialization for each transition.

[0282] Dynamic light scattering (DLS) measurement DLS measurements were performed using a Wyatt DynaPro PlateReader II (Wyatt, Santa Barbara, CA) with a laser at a wavelength of 820.17 nm. Three independent samples were prepared for each sample, and 30 μL of each sample was loaded into wells on a 384-well black untreated polystyrene plate (Thermo Scientific Nunc, UK) with protein concentrations ranging from 4 to 20 mg / mL. For each well, 10 DLS measurements of 5 seconds each were acquired at 20°C, and data were discarded if the polydispersity percentage was >15%. Cumulant analysis was performed using Wyatt Dynamics Software, and the protein self-diffusion coefficient (D) for the three samples under each condition was directly measured and then averaged. D is given by the formula: D = D0(1 + k D. c) (wherein D0 is the protein diffusion coefficient at infinite dilution) changes in proportion to the protein concentration (c), and therefore the protein-protein interaction parameter k changes accordingly. D This can be determined from the D vs. c plot.

[0283] Measurement of amino acid cleavage by reversed-phase high-performance liquid chromatography (RP-HPLC) Reverse-phase high-performance liquid chromatography was used to measure the main product peak and b-chain cleavage, thereby achieving separation by differential affinity with the hydrophobic chains of the column packing material. The test sample concentration was adjusted, mixed with denaturation buffer in the presence of a reducing agent, and incubated at a specified temperature for a specific time. The sample was then injected into a reverse-phase column and eluted with an increasing gradient of organic solvent. Peaks eluted according to their hydrophobicity.

[0284] More specifically, an Agilent 1260 Infinity series or equivalent instrument was used for reversed-phase high-performance liquid chromatography. The column used was a Phenomenex, Aeris WIDEPORE 3.6 μm XB-C8 HPLC column, 4.6 × 150 mm. 100 μg of each sample was denatured in the presence of a reducing agent (1 M DTT) (8 M guanidine, 130 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA pH 7.6), then mixed and incubated at 37°C for 45 minutes ± 5 minutes. The final concentration of each sample was 0.5 mg / mL. For each sample, 10 μL was injected into the column and eluted using a 40-minute gradient increasing the organic solvent (Mobile phase A: 95% water, 5% acetonitrile, 0.1% TFA and Mobile phase B: 80% acetonitrile, 20% water, 0.1% TFA). Peaks were eluted according to hydrophobicity and detected at 220 nm.

[0285] Efficacy assay to evaluate the binding of HFUS1 to the RXFP1 receptor THP1 cells endogenously expressing the RXFP1 receptor were engineered to express CRE-NanoLuc upon RXFP1 receptor activation. In this assay, HFUS1 binds to the RXFP1 receptor on the surface of THP1-CRE NanoLuc cells. This leads to the production of cyclic adenosine monophosphate (cAMP), which is then signaled via the cAMP response element (CRE) to promote the expression of the nanoluciferase enzyme. Luciferase enzyme expression is then measured by the addition of a chemiluminescent substrate. EC50 values, representing the HFUS1 concentration at which maximal luciferase expression is observed, are generated using a four-parameter semi-logistic curve fit for the HFUS1 reference standard (HFUS1 formulated in 20 mM histidine / histidine HCl, 190 mM arginine HCl, 0.02% (w / v) PS80, pH 6.5) and test samples. The relative potency of each HFUS1 sample was evaluated by dividing the EC50 value of the reference standard by the EC50 value of each sample and multiplying by 100%.

[0286] Example 1: Preliminary stability evaluation of HFUS1 The liquid stability of HFUS1 was first evaluated in histidine buffered formulations containing sucrose and polysorbate (PS) 80 at HFUS1 concentrations of 10 and 50 mg / mL. Molecular purity loss during storage at 5, 25, and 40°C was measured by HPSEC and is shown in Figure 1. The percentage decrease in major product peak (MMP)% at different temperatures was obtained from the slope of the linear regression of the data points: a) at 50 mg / mL, -0.2% at 5°C, -0.5% at 25°C, and -2.0% at 40°C; b) at 10 mg / mL, -0.1% at 5°C, -0.4% at 25°C, and -2.3% at 40°C.

[0287] The three-dimensional stability of a molecule is determined by the unfolding temperature (T) of the molecule in histidine / sucrose buffer. m The DSC trace was evaluated by measuring the following. The DSC trace is shown in Figure 2. HFUS1 has two unfolding events. m The starting temperature was approximately 57°C, the first unfolding occurred at 63°C, and the second unfolding occurred at 82°C.

[0288] HFUS1 protein-protein interaction parameters (k D ) was evaluated by DLS using the method described in the Methods section. As a rule of thumb, negative k D- The values ​​indicate a high attractive tendency between HFUS1 molecules, while positive values ​​indicate repulsive protein interactions. The self-diffusion coefficient and hydrodynamic radius of HFUS1 are shown in Figure 3. (The k of the molecule...) D This was calculated as -16 mL / g, which indicates the tendency for molecular self-association.

[0289] On the other hand, no phase separation or changes in appearance were observed in HFUS1 during storage. In general, HFUS1 showed an acceptable stability profile during evaluation. However, despite the low degradation rates at 25 and 40°C, from the limited point in time of data (up to 3 months), the molecule showed a relatively high aggregation rate by HPSEC at 5°C, i.e., -0.21% per month at 50 mg / mL. If this rate continues, it will lead to approximately 2.5% aggregation or monomer loss per year, making it difficult for the molecule to meet the shelf life requirements. This high aggregation rate at 5°C is negative k D As reflected by the results, this was likely due to the tendency of molecules in the formulation to self-associate. When molecules are prone to self-association, as the association continues, they may form soluble aggregates (usually detectable by HPSEC), which are precursors to insoluble large aggregates and ultimately particles, significantly affecting the molecular stability profile. This data indicates that formulation optimization is necessary to reduce this tendency for self-association and aggregation.

[0290] Example 2: Optimization and Development of Phase 1 Formulation The HFUS1 formulation was optimized. This focused on screening pH and buffer types, as well as the use of excipients to replace sucrose as a stabilizer. The pH range of 4.5–6.5 was evaluated and covered using citrate or histidine buffer systems. Meanwhile, arginine HCl or lysine HCl was used to replace sucrose in the formulation. PS80 was retained as a surfactant. HFUS1 was used at a concentration of 50 mg / mL. The interaction parameter k was determined using DLS. D DSC for the onset of unfolding temperature, HPSEC for purity, and visual evaluation for particle formation level were measured. A summary of the results is shown in Table 4. Results obtained from preliminary tests of the histidine-sucrose formulation were included for comparison. Furthermore, the histidine-sucrose formulation was repeated in this test, and was made to serve as a control, considering that a new HFUS1 material was used in this optimization test.

[0291] [Table 4] Abbreviations: mon-monomer, agg-aggregation, frag-fragmentation, and opal-opalescence a Within acceptable target thresholds, i Near the maximum allowable threshold, e Exceeding the tolerance threshold

[0292] In repeated studies (histidine-sucrose formulation, control), HFUS1 showed similar K levels to those measured in the feasibility study. D This showed that lower T at 40℃ 開始 It exhibits higher aggregation. This difference is likely due to variability in the materials used in this test. The feasibility study used transient expression-derived materials, while the optimization study used Chinese hamster ovary (CHO) cells. This indicates that process variability is likely to affect the quality of the molecular product, and that a robust formulation is needed to meet stability requirements.

[0293] As shown in Table 4, significant purity loss by HPSEC was observed at lower pH levels (pH 4.5 or pH 5.0) after storage under stress conditions (40°C) for one month. Formulation 2 showed high levels of aggregation and fragmentation, while Formulation 1 resulted in significant phase separation and precipitation. Lower unfolding onset temperatures were observed for Formulations 1, 2, 3, and 4, indicating that the molecular steric stability is also lower at lower pH levels. Buffer types and excipients appear to affect molecular stability. Several modifications were investigated at the same pH. At pH 5.5, formulations containing citrate or histidine buffer systems, as well as formulations containing arginine HCl, lysine HCl, and sucrose, were also present. Sucrose and lysine HCl showed slightly higher T 開始 This appears to provide better stereostructural stability, while the histidine system has a higher K D It provided better colloidal stability for molecules represented by the value, namely, formulation 4 (histidine) had a higher K than formulation 3 (citrate). D It has. Arginine HCl is K better than sucrose at pH 5.5 D Without showing, for example, formulation 3 (arginine HCl) has a K similar to that of formulation 7 (sucrose) at -12.6. D It has -11.5. However, at pH 6.0, arginine HCl (formulation 5) shows some improvement after storage at 40°C compared to sucrose (formulation 9), and at higher K D and had lower aggregation. Overall, formulation 6 had K Di It exhibits the best stability profile, with the highest pH and lowest purity loss. This is likely due to the combined effect of pH, buffering system, and the use of arginine HCl.

[0294] To evaluate the chemical stability of molecules in the formulations and assess the effect of pH on the molecules, the histidine-arginine HCl formulation system was further evaluated by mass spectrometry (MS). The histidine-sucrose formulation was included as a control for comparison. The attributes evaluated were: 1) cleavage of amino acids (AA) at the C-terminus of relaxin chain B, 2) change in trisulfide bond percentage, and 3) methionine 271 (M271) oxidation in relaxin chain B. The results are summarized in Figure 4.

[0295] No significant changes in trisulfide bond percentage were observed across the tested pH range. However, pH 5.5 showed the highest cleavage compared to pH 6.0 and pH 6.5, with the latter appearing to be the best. pH 5.5 also showed higher oxidation compared to higher pH levels, but the difference was not as pronounced compared to cleavage. To further evaluate the histidine-arginine HCl formulation system, pH optimization tests were performed. Histidine-arginine HCl formulations were prepared at pH 5.5, 6.0, 6.5, 6.8, and 7.0. Focusing on HPSEC for purity loss of HFUS1, the data are shown in Figure 5.

[0296] Fragmentation of HFUS1 appeared to be high at lower pH levels. At both 25°C and 40°C, pH 5.5 showed the highest molecular fragmentation, followed by pH 6.0. At both temperatures, fragmentation decreased from pH 6.5 to 0 and similarly thereafter. No fragmentation was observed at 5°C at any of the pH levels tested. Regarding aggregation, different profiles were observed at different temperatures. At 40°C, the highest aggregation was observed at pH 5.5. A V-shaped curve was observed as pH increased, showing the slowest aggregation rate at pH 6.5, while at 5°C, the aggregation rate also increased with increasing pH, but all were at low levels, showing a clear trend. The 5°C condition is most representative of real-time storage of pharmaceuticals, and in the histidine-arginine HCl system, all formulations offer improved stability compared to histidine-sucrose. Even at pH 7.0, which had the highest aggregation rate, a rate of 0.05% per month represents a decrease of approximately 0.6% per year. Molecular cleavage within this pH range was also evaluated by mass spectrometry, and the data are summarized in Figure 6. Similar to previous findings, pH 5.5 showed the highest cleavage compared to formulations at higher pH levels. pH 6.8 and pH 7.0 showed lower cleavage, a finding consistent with molecular fragmentation measured by HPSEC.

[0297] The high fragmentation or cleavage observed at low pH (5.5 and 6.0) is hypothesized to be due to protease activity. Molecules may be sensitive to certain enzymes co-eluted during purification, resulting in the chemical degradation of such molecules. To test this hypothesis, a protease inhibition test was performed. A formulation containing histidine-arginine HCl at pH 5.5 was selected due to the highest fragmentation and cleavage observed. The HFUS1 material was divided into four parts. The first was evaluated as is. A small amount of protease inhibitor (PI) cocktail was added to the second. The protease inhibitor cocktail was dissolved in DMSO, and therefore DMSO was added to the third as a control. EDTA was added to the last. The samples were stored at 40°C for 4 weeks, and then the material was evaluated for purity by HPSEC and for AA cleavage by MS. The data are summarized in Figures 7 and 8.

[0298] Samples treated with PI or EDTA showed a clear reduction in fragmentation and AA cleavage, while the control and DMSO-treated samples showed no significant change and both exhibited high levels of fragmentation and cleavage. This data supports the hypothesis that proteases are likely responsible for molecular fragmentation and cleavage. This enzymatic activity may be higher at lower pH levels, where proteases are most effective at cleaving molecules. Therefore, maintaining the formulation's pH in a higher range is crucial, as demonstrated in the pH optimization test. In this formulation optimization test, the histidine-arginine HCl system was confirmed to provide the highest colloidal and stereostructural stability for HFUS1. This formulation exhibited reduced molecular self-association tendencies and lower aggregation. An optimal pH range was also identified, minimizing the effects of chemical degradation such as molecular fragmentation and AA cleavage.

[0299] Example 3: Problems with particle formation of HFUS1 and mitigation through surfactant screening During long-term stability storage at 5°C, the optimized formulations began to exhibit visible particles. Particle formation varied depending on the conditions. Formulations at pH 6.5 and 6.8 began exhibiting particles after 6 months, and eventually, all other formulations exhibited particles after longer storage, as shown in Figure 9. Considering that these formulations had varying pH levels, buffer types, and excipients, it was unlikely that any of these were the root cause of particle formation. Therefore, it was considered more likely that this particle formation was related to the presence of PS80, a common factor in all formulations. Meanwhile, to identify the properties of the particles, they were investigated using Fourier transform infrared (FTIR). FTIR traces are shown in Figure 10. The particles were found in the CH region, similar to that of proteins, between 3000 and 2800 cm⁻¹. -1 , and the ester bond region is 1740 cm, similar to that of PS80. -1A signal was observed in the vicinity. While this data is not conclusive, it is clear that the particles are essentially proteinaceous. It is noteworthy that similar particle formation was observed for histidine-arginine HCl formulations in another stability test using a new batch of HFUS1 material (for GLP toxicity testing using lead clones). This indicates that the particles are not only specific to a particular batch of HFUS1 material, but that they have appeared in all HFUS1 materials tested to date.

[0300] The host cell protein (HCP) levels of the HFUS1 active pharmaceutical ingredient material were measured. HCPs are process-related protein impurities that are co-purified with HFUS1 molecules that could not be removed by the purification process. The HCP levels in the HFUS1 material were high, ranging from approximately 100 to 650 ng / mg. HCP species were analyzed by proteomics identification tests based on mass spectrometry. The identified HCPs included lipoprotein lipases (LPLs), known to degrade polysorbates, as well as proteases or peptidases that can degrade proteins or cleave terminal amino acids. While we do not wish to be bound by theory, this leads to several hypotheses regarding the cause of particle formation: 1) LPLs degrade polysorbate 80 in the formulation, resulting in impurities such as free fatty acids (FFAs). Since free fatty acids are insoluble in water, they precipitate as particles. 2) Free fatty acid impurities can act as nuclei to induce aggregation of the HFUS1 protein, resulting in particles, while degradation reduces PS80 levels, thus losing their protective / surfactant effect to prevent particle formation during storage. 3) Proteases can degrade HFUS1, and degraded HFUS1 has exposed patches that are susceptible to interactions that lead to aggregation, as well as 4) some proteases or HCPs can also interact with HFUS1 and cause aggregation.

[0301] Given that the particles identified by FTIR were essentially proteinaceous, it was unlikely that they were solely insoluble FFA. On the other hand, HFUS1 in the optimized histidine-arginine-HCl formulation (e.g., formulation 6 in Table 4) did not show abnormal fragmentation or AA cleavage, and therefore, it was unlikely that the particles were caused by HFUS1 being degraded by protease. In this case, hypotheses 2 and 4 were the most likely mechanisms of HFUS1 particle formation. To confirm whether the presence of PS80 plays a role in particle formation, new stability tests were set up by replacing PS80 in the histidine-arginine-HCl formulation with alternative surfactants such as poloxamer 188 (P188) and D-α-tocopherol polyethylene glycol succinate (TPGS). Two different HFUS1 concentrations, namely 50 and 5 mg / mL, were tested. The formulations were stored at 5°C in vials and pre-filled syringes (PFS), and particle formation was examined at different time points (Figures 11 and 12). After 6 months of storage, the 50 mg / mL formulation containing PS80 began to show visible particles with higher opalescence traces. Particle levels increased after 9 months, and high levels of visible particles were observed after 12 months. The 5 mg / mL PS80 formulation did not show particle formation until 9 months, but visible particles were observed at 12 months. In comparison, formulations containing TPGS and P188 did not show visible particles after 12 months of storage.

[0302] Furthermore, MFI was used to examine particles in the HFUS1 formulation that were not visible to the naked eye. As shown in Figure 13, the number of particles per mL in the 50 mg / mL formulation containing PS80 increased significantly after 6 months, but the formulations containing 50 mg / mL TPGS and P188 did not show a significant increase.

[0303] The levels of PS80 and P188 in the HFUS1 formulations after storage were also tested by HPLC equipped with an evaporative light scattering detector (ELSD). The data are summarized in Table 5. ELSD data for the PS80 formulation were obtained after 5 months, while P188 data were obtained after 11 months. PS80 levels decreased below the limit of quantification for the 50 mg / mL HFUS1 formulation, but the 5 mg / mL formulation also showed a decrease in PS80 levels. This indicates that PS80 was degraded in the formulation, and although we do not wish to be bound by theory, this was likely caused by the presence of LPL, which cleaves the ester bond of PS80 via enzymatic hydrolysis. This also explains why formulations with lower HFUS1 concentrations showed slower PS80 degradation over time. This was likely due to lower levels of co-purified LPL in the formulations. Conversely, formulations containing P188 instead of PS80 did not show any change in surfactant levels. P188 is a type of water-soluble nonionic triblock copolymer formed from polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks. The PEO and PPO blocks are linked together by ether bonds that are resistant to enzymatic hydrolysis by LPL.

[0304] [Table 5]

[0305] The effect of LPL on PS80 degradation was further investigated by LC-MS testing. A histidine-arginine HCl preparation containing 50 mg / mL of HFUS1 and PS80 (e.g., preparation 6 in Table 4) was divided into three parts. The first part served as the initial control sample, the next part was maintained at 40°C for two weeks, and the last part was treated with lipase and also maintained at 40°C for two weeks. The LC-MS chromatograms of the samples are shown in Figure 14. The HFUS1 sample subjected to heat stress showed degradation by hydrolysis, with accumulation of polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monooleate, and disubstituted and trisubstituted species. This profile was similar to that of the lipase-treated sample, which showed more pronounced hydrolysis.

[0306] These data and tests indicate that the polysorbate 80 degradation hypothesis was responsible for the particle formation observed with the HFUS1 formulation. On the other hand, the use of alternative lipase-resistant surfactants such as P188 or TPGS was able to mitigate the particle formation problem.

[0307] In addition to visual and non-visible particle analysis, HPSEC and cIEF were also used to evaluate the purity and chemical degradation profiles of HFUS1 formulations containing different surfactants. HPSEC data are shown in Figure 15, and cIEF data are shown in Figure 16. Formulations containing P188 and PS80 showed similar degradation profiles, and no significant differences were observed in samples stored in vials or pre-filled syringes (PFS). Therefore, HFUS1 is stable in histidine-arginine HCl formulations containing P188, and there are no significant changes in either physical or chemical stability after 18 months of storage.

[0308] Example 4: Robustness of HFUS1 formulation Following formulation optimization focusing on buffer species, pH, excipients, and surfactant screening, an optimized formulation was developed in histidine buffer (pH 6.5) containing arginine-HCl and poloxamer 188. This formulation provides long-term protection against HFUS1 and exhibits low chemical and physical degradation. The robustness of this formulation system was evaluated in this study, which involved varying the concentrations of arginine-HCl and surfactant, as well as the pH. Different formulations were stored at 5, 25, and 40°C, and their stability profiles were evaluated. A summary of the various conditions tested is shown in Table 6. The formulations were stored in either 2R glass vials or PFS.

[0309] [Table 6]

[0310] The formulations were tested using HPSEC, CGE, cIEF, and MFI, and the data are summarized in Figures 17, 18, 19, and 20. HPSEC was used to evaluate the purity and aggregation of HFUS1 during storage. CGE was used to detect molecular fragmentation, and cIEF was used to detect chemical instability. Particles not visible to the naked eye were evaluated using MFI, and the formulations were also visually inspected (data not shown).

[0311] After storage at 40°C for 3 months and at 5°C for over 12 months, no particle formation problems were observed by MFI and visual inspection. For comparison, a control formulation containing PS80 instead of P188 showed high levels of both invisible and visible particles. This data further confirms that the use of P188 mitigated the particle problems observed in the PS80 system. The formulation showed good physical stability, with no significant change in the main product peak (MPP%) detected by HPSEC after storage at 5°C for over 12 months and at 25°C for over 6 months. The rate of MPP% decrease at 40°C showed small differences between different formulation conditions, but this rate remained very low (<1% decrease per month). The concentration of HFUS1 affected molecular aggregation, with the 5 mg / mL sample showing a much lower rate of MPP% decrease per month (Figure 17). The chemical stability of the formulation was evaluated by CGE and cIEF. For CGE, minimal or negligible decrease in MPP% was observed in the stored formulation. This indicated a low level of molecular fragmentation. Similarly, cIEF data showed negligible changes in MPP% after 12 months of storage at 5°C, indicating low molecular chemical instability. While the decrease in MPP% at 25°C and 40°C was more pronounced, all remained at acceptable levels. In general, histidine-arginine HCl formulations demonstrated excellent stability and robustness. Formulation F1 / F1P was selected as the overall preferred formulation.

[0312] Example 5: Formulation stability at lower HFUS1 concentrations The stability of HFUS1 formulations at lower protein concentrations was also tested. The formulations were filled into 1 mL PFS containers, stored at 5, 25, and 40°C, and tested at multiple time points. A summary of the formulation conditions is shown in Table 7. The concentrations evaluated were 0.25 mg / mL and 1 mg / mL of HFUS1, which were formulated in target formulations (P1 and P3) and in formulations with lower arginine HCl concentrations and higher pH, which were likely to be less stable (P2 and P4). In the robustness test described in Example 4, higher aggregation was observed at lower arginine HCl levels, and the pH 7.0 sample showed a higher degradation rate by cIEF. This test in Example 5 enabled the generation of formulation data at lower HFUS1 concentrations and supported the suitability of the formulations across a wide range of HFUS1 concentrations. The formulations were tested by visual inspection (data not shown), MFI, HPSEC, cIEF, and CGE, and the data are summarized in Figures 21-24.

[0313] [Table 7]

[0314] HPSEC data showed that HFUS1 remained stable at all tested temperatures without monomer % loss after 6 months of storage at 5°C and 25°C, and 3 months at 40°C. No apparent aggregation was observed. CGE was used to monitor any molecular fragmentation during storage, and no loss of purity was observed at 5°C and 25°C, and only minimal loss was observed at 40°C. cIEF was used to detect any chemical degradation indicated by changes in the main peak. After storage at 40°C, a loss of approximately 18% of the main peak was observed per month. This was lower than the rate observed with higher concentration formulations (e.g., 50 mg / mL and 5 mg / mL). On the other hand, degradation at 25°C was slightly higher than that of the 50 mg / mL and 5 mg / mL formulations. These degradation rates at 25 and 40°C were at acceptable levels, and importantly, the degradation rate was minimal at 5°C. After 6 months of storage, almost no changes were observed. Furthermore, the tendency for particle formation was also evaluated by visual inspection (visible particles) and MFI (invisible particles). The particle level of HFUS1 in the formulation remained low during storage. Typically, formulations with lower protein concentrations have a lower risk of aggregation and particle formation, while the risk of chemical degradation is higher. This dataset at low concentrations indicates that HFUS1 does not have any chemical or physical stability issues at the concentrations tested.

[0315] Example 6: Formulation stability at an HFUS1 concentration of 33 mg / mL Following the development of an optimized formulation, univariate robustness stability testing was performed at 33 mg / mL in 1 mL PFS. PFS was selected as the intended long-term storage container for this molecule.

[0316] A concentration of 33 mg / mL corresponds to the highest concentration within ±10% of the 30 mg / mL formulation. The stability risk associated with the formation of invisible particles is considered to be highest at the highest protein concentration.

[0317] This test evaluated the robustness of the formulations at the lower and upper limits of the specified excipients, surfactants, and pH. This was done by varying the concentrations of arginine-HCl and surfactants, as well as the pH. Different formulations were stored at 5, 25, and 40°C and tested at multiple time points to evaluate their stability profiles. A summary of the various conditions tested is shown in Table 8.

[0318] [Table 8]

[0319] The formulations were tested using MFI, HIAC, HPSEC, cIEF, efficacy testing, and RP-HPLC, and the data are summarized in Figures 26 to 31.

[0320] Non-visible particles were evaluated by MFI and HIAC, and the formulations were also visually inspected (data not shown). HIAC is the official method for counting non-visible particles, and MFI is used as an orthogonal method. The purity of HFUS1 during storage was evaluated using HPSEC. Meanwhile, any chemical degradation indicated by changes in the main peak was detected using cIEF. The binding of HFUS1 to the RXFP1 receptor over time was evaluated using a potency assay. Amino acid (AA) cleavage at the C-terminus of relaxin chain B was monitored using RP-HPSEC.

[0321] After storage at 40°C for 3 months, 25°C for 6 months, or 5°C for 9 months, no tendency for particle formation was observed by MFI or visual inspection (Figure 26). HIAC data after the same storage periods showed no trend in the number of undetectable particles under any conditions, and the number of undetectable particles was well within the USP limits under all tested conditions (particle sizes ≥10 μm and ≥25 μm did not exceed 6000 and 600, respectively; Pharmacopeia US.2014, USP787 and 788) (Figure 27). This data further confirms that the use of P188 mitigated the particle problem observed in the PS80 system.

[0322] HPSEC data showed that all HFUS1 formulations remained stable with no monomer loss after 9 months of storage at 5°C. At pH 7, higher monomer loss was observed compared to pH 6 after 6 months of storage at 25°C (-0.11% / mo vs. -0.05% / mo) and 3 months at 40°C (-0.71% / mo vs. -0.36% / mo). These data demonstrate the good physical stability of the formulations, with no monomer loss at the intended long-term storage temperature of -5°C and a decrease of <1% per month at the other two temperatures (Figure 28).

[0323] The cIEF data also showed negligible changes in the main peak after 6 months of storage at 5°C, indicating low molecular chemical instability. The decrease in the main peak was more pronounced at 25°C and 40°C, with the highest rate of change observed at pH 7 compared to other formulation conditions (Figure 29). The changes in the cIEF profile were expected under harsh and accelerated conditions in which the molecule would not be exposed during its shelf life, and the degradation rate was minimal at 5°C for all formulations tested.

[0324] The efficacy results showed no change in molecular activity for all tested formulations after storage at 5°C for 6 months. Changes in efficacy at the other two temperatures, 25°C and 40°C, were similar for all formulations (Figure 30).

[0325] RP-HPLC results at 5°C showed no change in the main product peak for any formulation up to 9 months. The pH 6 formulation showed higher chain B cleavage at 25°C and 40°C compared to the higher pH 7 formulation (Figure 31).

[0326] conclusion Several challenges arose during the formulation development of HFUS1. In one formulation, the molecule tended to self-associate, posing a risk of aggregation. This molecule also exhibited a tendency for AA cleavage. This cleavage occurred on chain B of the relaxin peptide, which is involved in the binding of the molecule to the target. Furthermore, this molecule possessed high levels of process-related HCPs, and some species of HCPs present led to degradation to PS80, resulting in high levels of molecular particle formation. This particle formation was not significantly affected by pH, buffer type, and type of stabilizing excipient, but was primarily caused by PS80 degradation. Extensive formulation development and optimization tests were conducted. Arginine HCl was selected for its effect in reducing the molecule's self-association tendency, and an optimized pH range was identified that effectively prevented AA cleavage and fragmentation of the molecule. Detailed investigations were conducted to understand the cause of the particle formation problem, and PS80 was identified as the root cause. This led to the optimization of the surfactant in the formulation system, and alternative lipase-resistant surfactants such as P188 and TPGS mitigated the particle formation problem. P188 was selected as the primary surfactant for the formulation. Comprehensive formulation stability testing was conducted to evaluate the robustness of the formulation system. The histidine-arginine HCl formulation showed an excellent stability profile and robustness. The effect of HFUS1 concentration on stability was also evaluated, and HFUS1 showed good stability at low-concentration formulations (e.g., 0.25 mg / mL, 1 mg / mL, and 5 mg / mL) as well as at higher concentrations (e.g., 33 mg / mL and 50 mg / mL).

Claims

1. A pharmaceutical formulation comprising a heterodimer fusion and a lipase-resistant surfactant, wherein the heterodimer fusion is (i) a first heterodimerization domain attached to at least one relaxin A chain polypeptide or a variant thereof, (ii) comprising at least one relaxin B chain polypeptide or a variant thereof, The first heterodimerizing domain heterodimerizes with the second heterodimerizing domain, and the heterodimer fusion has relaxin activity, thereby providing a pharmaceutical formulation.

2. The pharmaceutical formulation according to claim 1, wherein the relaxin A chain polypeptide and the relaxin B chain polypeptide are covalently bonded by at least one interchain disulfide bond.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the relaxin A chain and the relaxin B chain are not covalently bonded to each other by an amino acid linker.

4. A pharmaceutical preparation according to any one of claims 1 to 3, wherein the relaxin A chain is a relaxin-2 A chain and the relaxin B chain is a relaxin-2 B chain.

5. The pharmaceutical formulation according to any one of claims 1 to 4, wherein the relaxin A chain is connected to the first heterodimerized domain via a connector, and the relaxin B chain is connected to the second heterodimerized domain via a connector, and optionally one or both connectors are polypeptides.

6. The pharmaceutical formulation according to claim 5, wherein one or both of the connectors have a length of 6 to 40 amino acids, for example, one or both connectors have a length of 21 amino acids.

7. The pharmaceutical formulation according to any one of claims 1 to 6, wherein the first heterodimerized domain and the second heterodimerized domain are derived from an immunoglobulin Fc region ("first Fc region" and "second Fc region," respectively), and optionally, the first Fc region and the second Fc region include constant domains CH2 and CH3.

8. The pharmaceutical formulation according to claim 7, wherein the C-terminus of the first Fc region is connected to the N-terminus of the relaxin A chain, and the C-terminus of the second Fc region is connected to the N-terminus of the relaxin B chain.

9. The pharmaceutical formulation according to claim 7 or 8, wherein the first Fc region and the second Fc region include heterodimerization-promoting amino acid mutations and / or modifications, and optionally, the heterodimerization-promoting amino acid mutations are "Fc knob" mutations and "Fc hole" mutations, for example, "Fc knob" mutations and "Fc hole" mutations located in the CH3 domain.

10. The pharmaceutical preparation according to any one of claims 7 to 9, wherein the first Fc region and the second Fc region are derived from human IgG1 immunoglobulin.

11. The aforementioned heterodimerization-promoting amino acid mutations a. Y349C, T366S, L368A, and Y407V, which are "Fc hole" mutations in a single CH3 domain, and b. Includes S354C and T366W, which are "Fc knob" mutations in other CH3 domains. The pharmaceutical formulation according to claim 10, wherein the amino acid numbering follows the EU index, such as in Kabat.

12. a. The first Fc region contains an "Fc knob" mutation, and the second Fc region contains an "Fc hole" mutation, or b. The pharmaceutical formulation according to claim 11, wherein the second Fc region includes an "Fc knob" mutation and the first Fc region includes an "Fc hole" mutation.

13. The pharmaceutical formulation according to any one of claims 10 to 12, wherein the first Fc region and / or the second Fc region comprises amino acid mutations L234F, L235E, and P331S, and the amino acid numbering follows the EU index as found in Kabat.

14. The pharmaceutical formulation according to any one of claims 4 to 13, wherein the relaxin-2 A chain polypeptide comprises the sequence shown in SEQ ID NO: 1 or a variant thereof, and the relaxin-2 B chain polypeptide comprises the sequence shown in SEQ ID NO: 2 or a variant thereof.

15. The pharmaceutical formulation according to claim 14, wherein the relaxin-2A chain polypeptide comprises amino acid mutations K9H, K17M, or K17I.

16. A pharmaceutical formulation according to any one of claims 5 to 15, wherein both connectors have the sequence GGGGGSGGGGGSGGGGGGGS (Sequence ID 5).

17. The aforementioned heterodimer fusion is (i) FcX-con-A fusion polypeptide and (ii) FcY-con-B fusion polypeptide, At this time, A is a relaxin A chain or a variant thereof, for example, a relaxin-2 A chain or a variant thereof. B is the relaxin B chain or a variant thereof, for example, the relaxin-2 B chain or a variant thereof. FcY is the Fc region containing the constant domains CH2 and CH3 of human IgG1 immunoglobulin, and includes "Fc hole" amino acid mutations and / or modifications, and optionally includes amino acid mutations such as Y349C:T366S:L368A:Y407V. FcX is an Fc region having an "Fc knob" amino acid mutation and / or modification, optionally comprising the constant domains CH2 and CH3 of human IgG1 immunoglobulin, and optionally comprising the amino acid mutation S354C:T366W, which contains an "Fc knob" amino acid mutation and / or modification. con is a connector polypeptide having the sequence GGGGGSGGGGGSGGGGGGGSGGGGGS (SEQ ID NO: 5) at will. The pharmaceutical formulation according to claim 1, wherein the amino acid numbering follows the EU index as in Kabat, and FcX heterodimerizes with FcY.

18. The pharmaceutical formulation according to any one of claims 1 to 17, wherein the heterodimer fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO:

20.

19. The pharmaceutical formulation according to any one of claims 8 to 18, wherein the heterodimer fusion further comprises one or more Fabs, and optionally the heterodimer fusion comprises one Fab attached to the N-terminus of the first Fc region and a second Fab attached to the N-terminus of the second Fc region.

20. The pharmaceutical formulation according to any one of claims 8 to 19, wherein the heterodimer further comprises a second relaxin A chain polypeptide or a variant thereof connected to the N-terminus of the first Fc region, and a second relaxin B chain polypeptide or a variant thereof connected to the N-terminus of the second Fc region, wherein optionally the second relaxin A chain is connected to the first Fc region via a connector polypeptide, and the second relaxin B chain is connected to the second Fc region via a connector polypeptide.

21. The aforementioned heterodimer fusion is (i) FcX-B-L-A and FcY, optionally FcY-B-L-A, or (ii) FcY-B-L-A and FcX, optionally including FcX-B-L-A, At this time, FcY is an immunoglobulin Fc region having an "Fc hole" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation Y349C:T366S:L368A:Y407V. FcX is an immunoglobulin Fc region having an "Fc knob" amino acid mutation and / or modification, and optionally includes a CH3 domain having the amino acid mutation S354C:T366W. B is the relaxin B chain or a variant thereof, for example, the relaxin 2 B chain or a variant thereof. A is a relaxin A chain or a variant thereof, for example, a relaxin 2A chain or a variant thereof. L is a linker polypeptide having the amino acid sequence GGGSGGGGGG (SEQ ID NO: 60) at will. The pharmaceutical formulation according to claim 1, wherein the amino acid numbering follows the EU index as in Kabat, and FcX heterodimerizes with FcY.

22. The pharmaceutical formulation according to claim 21, wherein the relaxin B chain is connected to FcX and / or FcY via a connector, and optionally the connector polypeptide has a length of 6 to 40 amino acids, for example, 21 amino acids.

23. The pharmaceutical formulation according to any one of claims 1 to 22, wherein the formulation contains approximately 10,000, approximately 6,000, approximately 5,000, approximately 1,000, approximately 750, approximately 600, approximately 500, approximately 250, approximately 150, approximately 100, or less than approximately 50 particles with a diameter of 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, or more than 25 μm per mL.

24. The pharmaceutical formulation according to any one of claims 1 to 23, wherein the concentration of the lipase-resistant surfactant is 0.001% (w / v) to 1% (w / v).

25. The pharmaceutical formulation according to any one of claims 1 to 24, wherein the concentration of the lipase-resistant surfactant is 0.02% (w / v) to 0.06% (w / v), optionally 0.04% (w / v).

26. The pharmaceutical formulation according to any one of claims 1 to 25, wherein the lipase-resistant surfactant cannot be enzymatically hydrolyzed by lipase, and optionally the lipase is selected from lipoprotein lipase, lipase 9, phospholipase 2, and phospholipase 2A.

27. The pharmaceutical formulation according to any one of claims 1 to 26, wherein the lipase-resistant surfactant does not contain an ester bond that can be enzymatically hydrolyzed by a lipase, and optionally the lipase is selected from lipoprotein lipase, lipase 9, phospholipase 2, and phospholipase 2A.

28. The pharmaceutical formulation according to any one of claims 1 to 27, wherein the lipase-resistant surfactant is a water-soluble nonionic triblock copolymer formed by polyethylene oxide (PEO) blocks and polypropylene oxide (PPO) blocks, and optionally the water-soluble nonionic triblock copolymer is poloxamer 188 (P188).

29. The pharmaceutical formulation according to any one of claims 1 to 27, wherein the lipase-resistant surfactant is selected from P188, D-α-tocopherol polyethylene glycol succinate (TPGS), Kolliphor HS15, Kolliphor EL, Kolliphor RH40, PEG300, PEG400, Brij58, and Brij35, and optionally the lipase-resistant surfactant is TPGS.

30. The pharmaceutical formulation according to any one of claims 1 to 29, wherein the formulation further comprises a buffer solution having a pH of approximately 3 to approximately 10, and optionally approximately 5.5 to approximately 7.

5.

31. The pharmaceutical formulation according to claim 30, wherein the formulation has a pH in the range of 6 to 7.

32. The pharmaceutical formulation according to claim 31, wherein the formulation has a pH of 6.

5.

33. The pharmaceutical preparation according to any one of claims 30 to 32, wherein the concentration of the buffer solution is 0.1 mM to 100 mM, and optionally 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

34. The pharmaceutical formulation according to claim 33, wherein the concentration of the buffer solution is 10 mM to 30 mM, and optionally 20 mM.

35. The pharmaceutical formulation according to any one of claims 30 to 34, wherein the buffer is selected from acetate, acetic acid, succinate, succinic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, lactate, lactic acid, tartaric acid, maleic acid, glycine, gluconate, citrate, histidine, imidazole, bicarbonate and carbonic acid, sodium benzoate, benzoic acid, edetate, malate, tris, glycylglycine, and mixtures thereof, and optionally, the buffer is selected from citrate buffer and histidine buffer.

36. The pharmaceutical preparation according to any one of claims 30 to 35, wherein the buffer is histidine, histidine hydrochloride, or histidine / histidine hydrochloride buffer, optionally histidine / histidine hydrochloride buffer.

37. The pharmaceutical preparation according to any one of claims 1 to 36, wherein the preparation further comprises an excipient, optionally wherein the excipient is an ionic excipient.

38. The pharmaceutical preparation according to claim 37, wherein the concentration of the excipient is 10 mM to 500 mM.

39. The pharmaceutical preparation according to claim 38, wherein the concentration of the excipient is 140 mM to 240 mM, and optionally 190 mM.

40. The pharmaceutical preparation according to any one of claims 37 to 39, wherein the excipient is an ionic excipient selected from arginine salts or lysine salts.

41. The pharmaceutical preparation according to claim 40, wherein the ionic excipient is selected from arginine HCl or lysine HCl, and optionally selected from arginine HCl.

42. The pharmaceutical preparation according to any one of claims 1 to 41, wherein the preparation further comprises a sugar, optionally wherein the sugar is sucrose.

43. A pharmaceutical preparation according to any one of claims 1 to 42, wherein the concentration of the heterodimer fusion is 0.1 to 100 mg / mL, and optionally 0.2 to 50 mg / mL.

44. The pharmaceutical formulation according to claim 43, wherein the formulation comprises a heterodimer fusion in a concentration of 0.2 to 50 mg / mL, a 20 mM histidine / histidine hydrochloride buffer, a 190 mM arginine HCl, and 0.04% (w / v) poloxamer 188 (P188), and the formulation has a pH of 6.

5.

45. The pharmaceutical formulation according to claim 44, wherein the heterodimer fusion comprises a fusion polypeptide having the amino acid sequence of SEQ ID NO: 11 and a fusion polypeptide having the amino acid sequence of SEQ ID NO:

20.

46. The pharmaceutical formulation according to any one of claims 1 to 45, wherein the formulation comprises a heterodimer fusion at a concentration of 50 mg / mL.

47. The pharmaceutical formulation according to any one of claims 1 to 45, wherein the formulation comprises a heterodimer fusion at a concentration of 30 mg / mL.

48. The pharmaceutical formulation according to any one of claims 1 to 45, wherein the formulation comprises a heterodimer fusion in a concentration of 5 mg / mL, 1.1 mg / mL, or 1 mg / mL.

49. A pharmaceutical preparation according to any one of claims 1 to 48, for use in treatment.

50. A pharmaceutical preparation according to any one of claims 1 to 48, for use in the treatment of a subject having heart failure accompanied by pulmonary hypertension.

51. The pharmaceutical preparation for use according to claim 49 or claim 50, wherein the pharmaceutical preparation is administered to the subject by subcutaneous injection.

52. A pharmaceutical formulation for use according to any one of claims 49 to 51, wherein the fusion polypeptide or pharmaceutical formulation is administered by self-administration.

53. A pharmaceutical formulation for use according to any one of claims 50 to 52, wherein the heart failure is heart failure having a reduced ejection fraction, heart failure having a moderate ejection fraction, or heart failure having a preserved ejection fraction.

54. A pharmaceutical preparation for use according to any one of claims 50 to 53, wherein the subject has a mean pulmonary artery pressure of approximately 25 mmHg or more, a pulmonary artery wedge pressure (PAWP) of more than 15 mmHg, and / or a right ventricular systolic pressure of approximately 40 mmHg or more.

55. A pharmaceutical formulation for use according to any one of claims 50 to 54, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units.

56. A pharmaceutical formulation for use according to any one of claims 50 to 54, wherein the subject has a pulmonary vascular resistance of 3.0 Wood units or more.

57. A pharmaceutical formulation for use according to any one of claims 50 to 56, wherein the subject is fitted with a blood pressure monitoring device, and optionally a pulmonary artery pressure monitoring device.

58. The pharmaceutical formulation for use according to claim 57, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

59. The administration of the aforementioned pharmaceutical preparation a) Decrease in PVR, (b) Decrease in mPAP, (c) Decline in ePAD, (d) Increase in cardiac stroke volume (SV), (e) A decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR), (f) Increased ejection fraction, and / or (g) Increased cardiac output, A pharmaceutical formulation for use according to any one of claims 49 to 58, which brings about one or more of the above compared to baseline levels before administration.

60. A kit comprising the pharmaceutical composition according to any one of claims 1 to 48.

61. A method for treating a subject having a disease or disorder, comprising administering a pharmaceutical preparation according to any one of claims 1 to 48 to the subject.

62. A method for treating a subject having heart failure accompanied by pulmonary hypertension, comprising administering a pharmaceutical preparation according to any one of claims 1 to 48 to the subject.

63. The method according to claim 61 or claim 62, wherein the heterodimer fusion or pharmaceutical preparation is administered to the subject by subcutaneous injection.

64. The method according to any one of claims 61 to 63, wherein the heterodimer fusion or pharmaceutical preparation is administered by self-administration.

65. The method according to any one of claims 62 to 64, wherein the heart failure is heart failure with a reduced ejection fraction, heart failure with a moderate ejection fraction, or heart failure with a preserved ejection fraction.

66. The method according to any one of claims 62 to 65, wherein the subject has a mean pulmonary artery pressure of approximately 25 mmHg or more, a pulmonary artery wedge pressure (PAWP) of more than 15 mmHg, and / or a right ventricular systolic pressure of approximately 40 mmHg or more.

67. The method according to any one of claims 62 to 66, wherein the subject has a pulmonary vascular resistance of less than 3.0 Wood units.

68. The method according to any one of claims 62 to 66, wherein the subject has a pulmonary vascular resistance of 3.0 Wood units or more.

69. The method according to any one of claims 62 to 68, wherein the subject is fitted with a blood pressure monitoring device, and optionally a pulmonary artery pressure monitoring device.

70. The method according to any one of claims 62 to 69, wherein the pulmonary artery pressure monitoring device is a CardioMEMS pressure monitoring device.

71. The administration of the aforementioned heterodimer fusion or pharmaceutical preparation is a) Decrease in PVR, (b) Decrease in mPAP, (c) Decline in ePAD, (d) Increase in cardiac stroke volume (SV), (e) A decrease in systemic vascular resistance (SVR) and / or an increase in estimated glomerular filtration rate (eGFR), (f) Increased ejection fraction, and / or (g) Increased cardiac output, The method according to any one of claims 61 to 70, which brings about one or more of the above compared to baseline levels before administration.

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