Activin A antibody preparation and method of use thereof

Abstract

To provide an activin A antibody preparation and a method for using the same. [Solution] The present invention provides a novel pharmaceutical formulation comprising an antibody that specifically binds to human activin A. In addition to the anti-activin A antibody, the formulation may contain a histidine buffer, an organic cosolvent, and a heat stabilizer. The pharmaceutical formulation of the present invention exhibits a remarkable degree of antibody stability after storage for several months and after exposure to thermal and other physical stresses. Fibrodysplasia ossificans progressive (FOP), also known as Muncmeyer's disease, is an autosomal dominant disorder characterized by early onset and transient and progressive ossification of skeletal muscle and associated connective tissue.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 040,589, filed on 18 June 2020, the entirety of which is expressly incorporated herein by reference.

[0002] This invention relates to the field of therapeutic antibody preparations. More specifically, this invention relates to the field of pharmaceutical preparations comprising antibodies that specifically bind to human activin A. [Background technology]

[0003] Fibrodysplasia ossificans progressive (FOP), also known as Muncmeyer's disease, is an autosomal dominant disorder characterized by early onset and transient and progressive ossification of skeletal muscle and associated connective tissue. In FOP subjects, bone forms in the soft tissues outside the normal skeleton, a process known as heterotopic ossification (HO), which leads to the development of a secondary skeleton and can gradually restrict the patient's mobility. Removal of new bone formation has been shown to be ineffective and leads to the progression of further new bone growth.

[0004] FOP is driven by mutations in the intracellular domain of ACVR1 (ALK2), most of which change arginine 206 to histidine (R206H) (Pignolo, RJet al. 2011, Orphanet J. Rare Dis. 6:80). ACVR1 is a type I receptor for bone morphogenetic proteins (BMPs). The R206H mutation is thought to increase the receptor's sensitivity to activation and make it more resistant to silencing. Certain types of drugs have been used to alleviate pain and swelling associated with FOP during flare-up, but no effective medical treatment for FOP is currently known. Antibodies against activin A are an example of therapeutic polymers that require appropriate formulation. While several anti-activin A antibodies are known, there remains a need in the art for novel pharmaceutical formulations containing anti-activin A antibodies that are sufficiently stable and suitable for patient administration. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pignolo,RJet al.2011,Orphanet J.Rare Dis.6:80 [Overview of the project] [Means for solving the problem]

[0006] Methods for producing antibodies useful as human therapeutic agents include the generation of chimeric antibodies and humanized antibodies (see, for example, US6,949,245). For example, see WO94 / 02602 (Abgenix) and US6,596,541 (Regeneron Pharmaceuticals), which are specifically incorporated herein by reference, and describe methods for generating non-human transgenic mice capable of producing human antibodies. U.S. Patent No. 9,718,881 discloses an antibody against human activin A, which is incorporated herein by reference in its entirety.

[0007] Therapeutic antibodies must be formulated in a manner that not only produces antibodies suitable for administration to patients, but also maintains their stability during storage and subsequent use. For example, therapeutic antibodies in solution are prone to fragmentation, precipitation, aggregation, and undesirable chemical modifications unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation, but also on the amounts and ratios of the excipients to each other. Furthermore, other considerations besides stability must be taken into account when preparing liquid antibody formulations. Examples of such additional considerations include the viscosity of the solution that can be contained by a given formulation, the concentration of the antibody, and the visual quality and appeal of the formulation. Therefore, when formulating therapeutic antibodies, great care must be taken to arrive at a formulation that maintains stability, contains the appropriate concentration of antibody, has a suitable viscosity, and possesses other properties that allow the formulation to be conveniently administered to patients.

[0008] The present invention satisfies the above-mentioned need by providing a pharmaceutical formulation containing a human antibody that specifically binds to human activin A.

[0009] In one embodiment, a liquid pharmaceutical formulation is provided, comprising (i) a human antibody that specifically binds to activin A, (ii) a buffer, (iii) an organic cosolvent, and (iv) a heat stabilizer.

[0010] In another embodiment, the present invention provides a pharmaceutical formulation comprising (i) an anti-human activin A antibody or its antigen-binding moiety, (ii) a buffer solution with a pH of 6.3 ± 0.3, (iii) an organic co-solvent, and (iv) one or more heat stabilizers.

[0011] In some embodiments, the antibody or its antigen-binding portion comprises the following six CDR sequences: (a) HCDR1 having GGSFSSHF (SEQ ID NO: 1), (b) HCDR2 having the sequence ILYTGGT (SEQ ID NO: 2), (c) HCDR3 having the sequence ARARSGITFTGIIVPGSFDI (SEQ ID NO: 3), (d) LCDR1 having the sequence QSVSSSY (SEQ ID NO: 4), (e) LCDR2 having the sequence GAS (SEQ ID NO: 5), and (f) LCDR3 having the sequence QQYGSSPWT (SEQ ID NO: 6).

[0012] In some embodiments, the antibody has a molecular weight of about 145,235.3 Da.

[0013] In some embodiments, the antibody concentration is 60 mg / mL ± 6 mg / mL.

[0014] In some embodiments, the buffer is a histidine buffer. In some embodiments, the histidine concentration is 10 mM ± 2 mM.

[0015] In some embodiments, the organic co-solvent is polysorbate 20. In some embodiments, the polysorbate 20 concentration is 0.05% w / v ± 0.025%.

[0016] In some embodiments, the one or more heat stabilizers include sucrose and arginine. In some embodiments, the sucrose concentration is 5% ± 1% (w / v) and the arginine concentration is 70 mM ± 14 mM.

[0017] H In some embodiments, the pharmaceutical formulation comprises 60 mg / mL ± 6 mg / mL of antibody, 10 mM ± 2 mM of histidine, pH 6.3 ± 0.3, 0.05% w / v ± 0.025% of polysorbate 20, 5% w / v ± 1% of sucrose, and 70 mM ± 14 mM of arginine.

[0018] In some embodiments, after 56 days of storage at 40°C and 75% relative humidity (RH), at least 90% of the antibody or its antigen-binding portion has a native conformation, or at least 30% of the antibody or its antigen-binding portion is in the major charge form.

[0019] In some embodiments, after 56 days of storage at 40°C and 75% RH, at least 93% of the antibody or its antigen-binding portion has a native conformation, or at least 34.5% of the antibody or its antigen-binding portion is in the major charge form.

[0020] In some embodiments, after 56 days of storage at 40°C and 75% RH, at least 97% of the antibody or its antigen-binding portion has a native conformation, or at least 45% of the antibody or its antigen-binding portion is in the major charge form.

[0021] In some embodiments, after 6 months of storage at 25°C and 60% RH, at least 90% of the antibody or its antigen-binding portion has a native conformation, or at least 40% of the antibody or its antigen-binding portion is in the major charge form.

[0022] In some embodiments, after 6 months of storage at 25°C and 60% RH, at least 95% of the antibody or its antigen-binding portion has a native conformation, or at least 45% of the antibody or its antigen-binding portion is in the major charge form.

[0023] In some embodiments, after 6 months of storage at 25°C and 60% RH, at least 98% of the antibody or its antigen-binding portion has a native conformation, or at least 50% of the antibody or its antigen-binding portion is in the major charge form.

[0024] In some embodiments, after 12 months of storage at 2–8°C, at least 94% of the antibody or its antigen-binding moiety has the native conformation, or at least 45% of the antibody or its antigen-binding moiety is the main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding moiety prior to storage.

[0025] In some embodiments, after 12 months of storage at 2–8°C, at least 96% of the antibody or its antigen-binding moiety has the native conformation, or at least 50% of the antibody or its antigen-binding moiety is the main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding moiety prior to storage.

[0026] In some embodiments, after 12 months of storage at 2–8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0027] In some embodiments, after 18 months of storage at 2–8°C, at least 94% of the antibody or its antigen-binding portion has the native conformation, or at least 45% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0028] In some embodiments, after 18 months of storage at 2–8°C, at least 96% of the antibody or its antigen-binding portion has the native conformation, or at least 50% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0029] In some embodiments, after 18 months of storage at 2–8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0030] In some embodiments, after 24 months of storage at 2–8°C, at least 94% of the antibody or its antigen-binding portion has the native conformation, or at least 45% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0031] In some embodiments, after 24 months of storage at 2–8°C, at least 96% of the antibody or its antigen-binding portion has the native conformation, or at least 50% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0032] In some embodiments, after 24 months of storage at 2–8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is the main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage.

[0033] In another aspect, the present invention provides a pharmaceutical formulation comprising (a) 60 mg / mL ± 10 mg / mL of anti-human activin A antibody or its antigen-binding moiety, (b) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3, (c) 0.05% ± 0.025% polysorbate 20, (d) 70 mM ± 14 mM arginine, and (e) 5% ± 1% sucrose, wherein the antibody or its antigen-binding moiety comprises at least 90%, at least 91%, at least 92%, at least 93%, and less than 90% of SEQ ID NO: 7. In one embodiment, the heavy chain includes a heavy chain variable region having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with SEQ ID NO: 7, and includes or consists of SEQ ID NO: 8. In one embodiment, the heavy chain includes a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with SEQ ID NO: 9, and includes or consists of SEQ ID NO: 9. In one embodiment, the heavy chain includes a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with SEQ ID NO: 10, and includes or consists of SEQ ID NO: 10.

[0034] In some embodiments, the formulation is contained in a container. In some embodiments, the container is a vial. In some embodiments, the vial is made of glass. In some embodiments, the glass is Type 1 borosilicate glass with a FluroTec® coated 4432 / 50 butyl rubber stopper.

[0035] In some embodiments, the formulation is suitable for intravenous administration to human subjects requiring it. In some embodiments, the formulation is suitable for subcutaneous administration to human subjects requiring it.

[0036] In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is a lyophilized formulation.

[0037] In another embodiment, the present invention provides a kit comprising a pharmaceutical formulation disclosed herein, a container, and instructions for use thereof.

[0038] In some embodiments, the container is a glass vial fitted with a chlorobutyl stopper coated with FluroTec®.

[0039] In another aspect, the present invention provides a method for treating a disease or disorder related to activin A activity, comprising administering a therapeutically effective amount of one or more of the pharmaceutical compositions disclosed herein to a subject in need thereof.

[0040] In some embodiments, the disease or disorder associated with activin A activity is fibrous ossification of the progression of ostosis (FOP).

[0041] Other embodiments of the present invention will become apparent in the detailed review described below. In certain embodiments, for example, the following items are provided: (Item 1) (i) an anti-human activin A antibody or its antigen-binding moiety, (ii) a buffer solution with a pH of 6.3 ± 0.3, (iii) an organic cosolvent, and (iv) one or more heat stabilizers, comprising a pharmaceutical product Agent. (Item 2) The antibody or its antigen-binding portion is one of the following six CDR sequences: (a) HCDR1 having sequence GGSFSSHF (sequence number 1), (b) HCDR2 having sequence ILYTGGT (sequence number 2), (c) HCDR3 having sequence ARARSGITFTGIIVPGSFDI (sequence number 3), (d) LCDR1 having sequence QSVSSSY (sequence number 4), (e) LCDR2 having sequence GAS (sequence number 5), (f) A pharmaceutical preparation according to item 1, comprising LCDR3 having sequence QQYGSSPWT (sequence number 6). (Item 3) The pharmaceutical preparation according to item 1 or 2, wherein the antibody has a molecular weight of approximately 145,235.3 Da. (Item 4) A pharmaceutical preparation according to any one of items 1 to 3, wherein the antibody or the antigen-binding portion thereof is 60 mg / mL ± 6 mg / mL. (Item 5) A pharmaceutical preparation according to any one of items 1 to 4, wherein the buffer is a histidine buffer. (Item 6) The pharmaceutical preparation described in item 5, wherein the histidine concentration is 10 mM ± 2 mM. (Item 7) The pharmaceutical preparation according to any one of the preceding items, wherein the organic cosolvent is polysorbate 20. (Item 8) The pharmaceutical preparation described in item 7, wherein the concentration of polysorbate 20 is 0.05% w / v ± 0.025%. (Item 9) A pharmaceutical preparation according to any one of the preceding items, wherein the one or more heat stabilizers include sucrose and arginine. (Item 10) The pharmaceutical preparation according to item 9, wherein the sucrose concentration is 5% ± 1% (w / v) and the arginine concentration is 70 mM ± 14 mM. (Item 11) A pharmaceutical preparation according to any one of the preceding items, comprising 60 mg / mL ± 6 mg / mL of antibody, 10 mM ± 2 mM histidine, pH 6.3 ± 0.3, 0.05% w / v ± 0.025% polysorbate 20, 5% w / v ± 1% sucrose, and 70 mM ± 14 mM arginine. (Item 12) A pharmaceutical preparation according to any one of the preceding items, wherein, after 56 days of storage at 40°C and 75% relative humidity (RH), at least 90% of the antibody or its antigen-binding portion has a native conformation, or at least 30% of the antibody or its antigen-binding portion has a main charge form. (Item 13) A pharmaceutical preparation according to any one of the preceding items, wherein, after 56 days of storage at 40°C and 75% RH, at least 93% of the antibody or its antigen-binding portion has a native conformation, or at least 34.5% of the antibody or its antigen-binding portion is in a main charge form. (Item 14) A pharmaceutical preparation according to any one of the preceding items, wherein, after 56 days of storage at 40°C and 75% RH, at least 97% of the antibody or its antigen-binding portion has a native conformation, or at least 45% of the antibody or its antigen-binding portion is in a main charge form. (Item 15) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after storage for 6 months at 25°C and 60% RH, at least 90% of the antibody or its antigen-binding portion has a native conformation, or at least 40% of the antibody or its antigen-binding portion is in a primary charge form. (Item 16) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after storage for 6 months at 25°C and 60% RH, at least 95% of the antibody or its antigen-binding portion has a native conformation, or at least 45% of the antibody or its antigen-binding portion is in a primary charge form. (Item 17) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after storage for 6 months at 25°C and 60% RH, at least 98% of the antibody or its antigen-binding portion has a native conformation, or at least 50% of the antibody or its antigen-binding portion is in a primary charge form. (Item 18) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 12 months of storage at 2 to 8°C, at least 94% of the antibody or its antigen-binding portion has the native conformation, or at least 45% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 19) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 12 months of storage at 2 to 8°C, at least 96% of the antibody or its antigen-binding portion has the native conformation, or at least 50% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 20) A pharmaceutical formulation according to any one of items 1 to 11, wherein, after 12 months of storage at 2 to 8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 100% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 21) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 18 months of storage at 2 to 8°C, at least 94% of the antibody or its antigen-binding portion has the native conformation, or at least 45% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 22) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 18 months of storage at 2 to 8°C, at least 96% of the antibody or its antigen-binding portion has the native conformation, or at least 50% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 23) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 18 months of storage at 2 to 8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 95% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 24) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 24 months of storage at 2 to 8°C, at least 94% of the antibody or its antigen-binding portion has the native conformation, or at least 45% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 99% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 25) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 24 months of storage at 2 to 8°C, at least 96% of the antibody or its antigen-binding portion has the native conformation, or at least 50% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 99% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 26) A pharmaceutical preparation according to any one of items 1 to 11, wherein, after 24 months of storage at 2 to 8°C, at least 98% of the antibody or its antigen-binding portion has the native conformation, or at least 55% of the antibody or its antigen-binding portion is a main charge variant, and / or the antibody retains at least 99% of the efficacy of the antibody or its antigen-binding portion prior to storage. (Item 27) A pharmaceutical preparation comprising (a) 60 mg / mL ± 10 mg / mL of anti-human activin A antibody or its antigen-binding moiety, (b) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3, (c) 0.05% ± 0.025% polysorbate 20, (d) 70 mM ± 14 mM arginine, and (e) 5% ± 1% sucrose, wherein the antibody or its antigen-binding moiety comprises a heavy chain variable region containing SEQ ID NO: 7 and a light chain variable region containing SEQ ID NO: 8. (Item 28) The aforementioned preparation is a pharmaceutical preparation according to any one of the preceding items, contained in a container. (Item 29) The pharmaceutical preparation described in item 28, wherein the container is a vial. (Item 30) The pharmaceutical preparation according to item 29, wherein the vial is made of glass. (Item 31) The pharmaceutical formulation according to item 30, wherein the glass is a type 1 borosilicate glass having a 4432 / 50 butyl rubber stopper coated with FluroTec®. (Item 32) The pharmaceutical preparation according to any one of the preceding items, wherein the preparation is suitable for intravenous administration to a human subject requiring it. (Item 33) The pharmaceutical preparation described in any one of items 1 to 31, wherein the preparation is suitable for subcutaneous administration to human subjects requiring it. (Item 34) The pharmaceutical preparation described in any one of the preceding items, wherein the preparation is a liquid preparation. (Item 35) A pharmaceutical preparation according to any one of items 1 to 33, wherein the preparation is a lyophilized preparation. (Item 36) A kit comprising a pharmaceutical preparation described in any of the preceding items, a container, and instructions for use thereof. (Item 37) The kit according to item 36, wherein the container is a glass vial fitted with a chlorobutyl stopper coated with FluroTec®. (Item 38) A method for treating a disease or disorder related to activin A activity, comprising administering a therapeutically effective amount of any one of items 1 to 35 to a subject in need thereof. (Item 39) The method according to item 38, wherein the disease or disorder associated with the activin A activity is fibrous ossification of progression (FOP). [Brief explanation of the drawing]

[0042] [Figure 1] The heavy and light chain amino acid sequences of exemplary anti-activin A monoclonal antibodies (SEQ ID NO: 9 and SEQ ID NO: 10, respectively) are shown. [Figure 2] This study demonstrates the effect of altering the formulation on the degradation rate of anti-activin A antibodies when the formulation is incubated at 40°C / 75% RH for two months. For particle levels measured by HIAC and MFI, the results shown represent the final particle levels after two months of incubation (for HIAC results, see USP). <788> (Restrictions are indicated). Formulation 15 is a control formulation with all factors nominal. This line is a baseline showing the rate of change in the response of the control formulation. [Figure 3]This study demonstrates the effect of altering the formulation on the degradation rate of anti-activin A antibodies when the formulation is incubated at 25°C / 60% RH for 6 months. For particle levels measured by HIAC and MFI, the results shown represent the final particle levels after 6 months of incubation (for HIAC results, see USP). <788> (Restrictions are indicated). Formulation 15 is a control formulation with all factors nominal. This line is a baseline showing the rate of change in the response of the control formulation. [Figure 4] According to the DOE design, the effect of changing the formulation component on the stability of the anti-activin A antibody compared to the control formulation, designated as "Delta Control," is shown (8 cycles of freeze and thaw stress, freezing at -30°C, thawing at room temperature; the control formulation is F15). Particle level results by HIAC are shown as the final particle level after 8 cycles of freeze and thaw, and are based on USP <788> It is compared to the limit specified. [Figure 5] According to the DoE design, the effect of changing the formulation component on the stability of the anti-activin A antibody compared to the control formulation, indicated as "delta control" (120 minutes of vortex stirring; the control formulation is F15). Particle level results by HIAC are shown as the final particle level after 120 minutes of stirring, and are USP <788> It is compared to the limit specified. [Figure 6] According to the DOE design, the effect of altering the formulation components on the long-term stability of the anti-activin A antibody when the formulation is stored at 2-8°C for 12 months is shown (see Table 24 for a description of the formulation). USP <788> The specified limits are shown in the plot indicating particle levels by HIAC. The results are shown as a function of storage time at 2–8°C. [Figure 7] According to the DOE design, the effect of altering the formulation components on the long-term stability of the anti-activin A antibody when the formulation is stored at 2-8°C for 24 months is shown (see Table 24 for a description of the formulation). USP <788> The specified limits are shown in the plot indicating particle levels by HIAC. The results are shown as a function of storage time at 2–8°C. [Modes for carrying out the invention]

[0043] Before describing the present invention, please understand that the present invention is not limited to such methods and conditions, as the specific methods and experimental conditions described may vary. Also, please understand that the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention, as it is limited only by the appended claims.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention belongs. As used herein, the term “about” means that, when used in relation to a particular enumerated number, the value may vary by no more than 5% from the enumerated value. For example, as used herein, the expression “about 100” includes 95 and 105, as well as all values ​​in between (e.g., 95.00, 95.01, 95.02, 95.03, 95.04, ..., 104.96, 104.97, 104.98, 104.99, 105.00).

[0045] Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials will be described below. All publications referenced herein are incorporated herein by reference in their entirety.

[0046] Pharmaceutical preparations As used herein, the term “pharmaceutical formulation” means a combination of at least one active ingredient (e.g., a small molecule, polymer, compound, etc., capable of exerting a biological effect in humans or non-human animals) and at least one inactive ingredient that, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to humans or non-human animals. As used herein, the term “formulation” means “pharmaceutical formulation” unless otherwise specifically indicated. The present invention provides a pharmaceutical formulation comprising at least one therapeutic polypeptide. According to certain embodiments of the present invention, the therapeutic polypeptide is an antibody or an antibody antigen fragment thereof that specifically binds to human activin A protein. More specifically, the present invention includes a pharmaceutical formulation comprising (i) a human antibody that specifically binds to human activin A, (ii) a buffer which is histidine, (iii) an organic cosolvent which is a nonionic surfactant, and (iv) a stabilizer which is a carbohydrate or an inorganic salt, or a combination of a carbohydrate and an inorganic salt. Specific exemplary components and formulations included in the present invention are described in detail below.

[0047] Anti-activin A antibody The pharmaceutical formulations of the present invention may include a human antibody or an antigen-binding fragment thereof that specifically binds to human activin A. As used herein, the term "activin A" means human activin A, which is a homodimeric or heterodimeric protein. The homodimeric protein contains a homodimeric beta-A subunit pair. The heterodimeric protein contains a beta subunit and beta-B, beta-C, or beta-E subunits (i.e., beta-A-beta-B, beta-A-beta-C, or beta-A-beta-E). Each of these subunits is expressed as a precursor polypeptide containing a signal peptide, a propeptide, and a mature polypeptide. An exemplary form of the human beta-A subunit precursor polypeptide is a 426-amino acid polypeptide called Swiss Prot P08476, where residues 1-20 are the signal peptide, residues 21-310 are the propeptide, and residues 311-426 are the mature polypeptide. An exemplary form of the beta-B subunit precursor polypeptide is called Swiss Prot P09529, where residues 1-28 are the signal peptide, residues 29-292 are the propeptide, and residues 293-407 are the mature polypeptide. An exemplary form of the beta-C subunit is Swiss The sequence is called Prot P55103, in which residues 1-18 are a signal peptide, residues 19-236 are a propeptide, and residues 237-352 are a mature polypeptide. An exemplary form of the beta-E subunit precursor is called Swiss Prot P58166, in which residues 1-19 are a signal peptide, residues 20-236 are a propeptide, and residues 237-350 are a mature polypeptide. Several variants of these sequences are known, as described in the Swiss Prot Database. References to activin A include any of the beta-A homodimer, beta-A beta-B, beta-A beta-C, and beta-A beta-E heterodimer forms, and their subunits, as well as their precursors that attach to propeptides and / or signal peptides as defined by exemplary Swiss Prot sequences or other naturally occurring human forms of these sequences that provide the subunits. Activin A signals via binding to ACVR2A or ACVR2B, but it is not known to be a ligand for ACVR1. Activin A abnormally signals via mutant ACVR, transmitting osteogenic signals and inducing ectopic osteogenesis.

[0048] As used herein, the term “antibody” is generally intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their polymers (e.g., IgM), but immunoglobulin molecules consisting only of heavy chains (i.e., lacking light chains) are also included in the definition of the term “antibody.” Each heavy chain comprises a heavy chain variable region (HCVR or V in this specification). H It includes a heavy chain constant region (abbreviated as CH1, CH2, and CH3). Each light chain has a light chain variable region (LCVR or V as specified herein). L It includes the (abbreviated as) and the light chain constant region. The light chain constant region contains one domain (CL1). V H and V LThe region can be further subdivided into a hypervariable region called the complementary decision region (CDR), which is dotted with more conserved regions called the framework region (FR). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0049] Unless otherwise specifically indicated, the term “antibody” as used herein should be understood to include the complete antibody molecule and its antigen-binding fragment. As used herein, the terms “antigen-binding portion” or “antibody-binding fragment” (or simply “antibody portion” or “antibody fragment”) of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to human activin A or its epitope.

[0050] As used herein, “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities, as an obvious exception to bispecific (or multispecific) antibodies that specifically bind to activin A on the one hand and to another epitope on the other (for example, an isolated antibody that specifically binds to human activin A substantially does not contain antibodies that specifically bind to antigens other than human activin A). Furthermore, an isolated antibody may substantially not contain other cellular material or chemical substances.

[0051] The term "specifically binds" or similar means that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 × 10⁻⁶. -6It can be characterized by a dissociation constant of M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. However, isolated antibodies that specifically bind to human activin A may have cross-reactivity to other antigens, such as activin A molecules from other species (orthologs). In the context of the present invention, multispecific (e.g., bispecific) antibodies that bind to human activin A, as well as one or more additional antigens, are considered to "specifically bind" to human activin A.

[0052] An exemplary anti-human activin A antibody that may be included in the pharmaceutical formulation of the present invention is defined in U.S. Patent No. 9,718,881, the disclosure of which is incorporated herein by reference in its entirety.

[0053] Antibodies may also include antibodies that specifically bind to activin A. Such antibodies can specifically bind to any or all of the beta-A-beta-A, beta-A-beta-B, beta-A-beta-C, and beta-A-beta-E forms of activin A. Some antibodies specifically bind to only one of these forms (i.e., beta-A-beta-A, beta-A-beta-B, beta-A-beta-C, or beta-A-beta-E). Specificity for the beta-A-beta-B, beta-A-beta-C, and beta-A-beta-E forms can be conferred by an epitope within the beta-B, beta-C, or beta-E subunit, respectively, or to an epitope contributed by both components of the heterodimer. Specificity for beta-A-beta can be conferred by an epitope contributed by both molecules within the homodimer (e.g., at the subunit interface). Some antibodies specifically bind to all of these forms of activin A, in which case the epitope is typically located on the beta-A subunit. Antibodies typically have epitopes within the mature polypeptide components of the precursor protein. Some antibodies exist in the form of alpha(Swiss Prot P05111)beta A or alphabeta B heterodimers and specifically bind to any or all forms of activin A without binding to human inhibin. Some antibodies specifically bind to any or all forms of activin A and also bind to any or both forms of human inhibin. Such antibodies are thought to inhibit activin A signaling via one or more of their counterreceptors, ACVR2A and / or ACVR2B and / or BMPR2, but understanding the mechanism is not necessary for the use of such antibodies in methods of treating FOP.

[0054] A number of antibodies against activin A have been described. For example, US20150037339 discloses human antibodies designated as H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10447P2, H4H10448P2, H4H10452P2.

[0055] Preferred antibodies have an affinity for activin A of at least 10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、10 12 M -1 、or 10 13 M -1 (measured at 25 °C in Example 3 of US Patent No. 9,718,881). Some antibodies have an affinity in the range of 10 9 ~10 12 M -1 . Preferred antibodies inhibit activin A signaling with an IC50 of less than 4 nM, preferably less than 400 pM or less than 40 pM. Some antibodies inhibit signaling with an IC50 in the range of 4 nM to 10 pM or 3.5 nM to 35 pM.

[0056] Signal transduction inhibition can be measured in Example 6 of U.S. Patent No. 9,718,881, which is summarized below. Human A204 rhabdomyosarcoma cell line was transfected with the Smad 2 / 3-luciferase reporter plasmid to produce the A204 / CAGAx12-Luc cell line. A204 / CAGAx12-Luc cells were maintained in McCoy's 5A supplemented with 10% fetal bovine serum, penicillin / streptomycin / glutamine, and 250 μg / mL G418. For bioassay, A204 / CAGAx12-Luc cells were seeded at 10,000 cells / well on 96-well assay plates in low-serum medium, 0.5% FBS, and OPTIMEM, and incubated overnight at 37°C and 5% CO2. Activin A is sequentially diluted 1:3 to 100–0.002 nM and added to cells, starting with a control that does not contain activin. Antibodies are sequentially diluted 1:3 to 100–0.002 nM, 1000–0.02 nM, or 300–0.005 nM, and added to cells having a constant concentration of 100 pM activin A, including a control sample containing either an appropriate isotype control antibody or no antibody.

[0057] Some antibodies inhibit the binding of activin A to ACVR2A and / or ACVR2B and / or BMPR2 by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 99% so that the measurement is taken when the receptor is expressed from cells, or when the extracellular domain is fused with the Fc domain as a fusion protein and the fusion protein is immobilized on a support (e.g., a Biacore sensor chip). In such measurements, the antibody and activin A should be present in equimolar amounts, and the receptor or extracellular domain should be in excess.

[0058] The exemplary antibody used in this embodiment is designated as H4H10446P in U.S. Patent No. 9,718,881. It is a heavy chain variable region and heavy chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 162, 164, 166, and 168, respectively, in U.S. Patent No. 9,718,881. It is a light chain variable region and light chain CDR, CDRL1, CDRL2, and CDRL3 having the amino acid sequences of SEQ ID NOs. 146, 148, 150, and 152, respectively, in US2015 / 0037339. H4H10446P inhibits activin A-mediated signaling via ACVR2A and / or ACVRIIB, but does not strongly inhibit the binding of activin A to ACRIIA or ACVR2B. This also includes other antibodies that compete with H4H10446P for binding to human activin A, or for binding to the same epitopes on human activin A as H4H10446P, and that share the inhibition of signal transduction.

[0059] Another exemplary antibody for use in the method of the present invention is H4H10430P, designated in U.S. Patent No. 9,718,881. It is a heavy chain variable region and heavy chain CDRs CDRH1, CDRH2, and CDRH3 having the amino acid sequences of SEQ ID NOs. 66, 68, 70, and 72, respectively, in U.S. Patent No. 9,718,881. It is a light chain variable region and light chain CDRs CDRL1, CDRL2, and CDRL3 having the amino acid sequences of SEQ ID NOs. 74, 76, 78, and 80, respectively, in U.S. Patent No. 9,718,881. This antibody inhibits the binding of activin A to ACRV2A and / or ACVR2B, thereby inhibiting signal transduction mediated by one or both of these receptors. This also includes other antibodies that compete with H4H10430P for binding to activin A or to the same epitope on activin A as H4H10430P, and that share the property of inhibiting activin A binding and signal transduction via ACVR2A and ACVR2B.

[0060] An exemplary antibody for use in the method of the present invention is galetosumab. The recombinant monoclonal antibody galetosumab is a covalent heterotetramer consisting of two disulfide-bonded human heavy chains (IgG4 isotypes), each covalently bonded to a human kappa light chain via a disulfide bond. Based on the primary sequence, the glycan-free antibody has a predicted molecular weight of 145,235.3 Da, assuming the formation of 16 canonical disulfide bonds and the removal of Lys453 from the C-terminus of each heavy chain. Each heavy chain contains a serine-to-proline mutation at amino acid Pro234 within the hinge region of the Fc domain to reduce the tendency of the IgG4 isotype antibody to form half-antibodies in solution. One N-linked glycosylation site (Asn303) is present on each heavy chain and is located within the constant region of the Fc domain of the molecule. The complementarity-determining regions (CDRs) within the galetosumab heavy and light chain variable domains jointly form the binding sites for their targets: activin A, activin AB, and activin AC. Figure 1 shows the heavy and light chain amino acid sequences, the location of the CDRs within each polypeptide chain, the location of the heavy chain N-linked glycosylation site, and the predicted disulfide bond structure of the galetosumab monoclonal antibody.

[0061] In some embodiments, anti-human activin A antibodies contain one or more amino acid substitutions within one or more framework regions compared to a standard heavy chain variable region, which is reasonably expected to result in a change in the charge distribution across the exposed surface of the antibody and thus affect its interaction with the surrounding solvent and excipients.

[0062] The amount of antibody or antigen-binding fragment contained in the pharmaceutical formulation of the present invention may vary depending on the desired specific characteristics of the formulation, as well as the specific circumstances and purposes for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation is a liquid formulation that may contain antibody or antigen-binding fragment that specifically binds to human activin A in amounts of 20 mg / mL ± 2 mg / mL to 200 mg / mL ± 20 mg / mL, 30 ± 3 mg / mL to 150 ± 15 mg / mL, 40 ± 4 mg / mL to 100 ± 1 mg / mL, 45 ± 4.5 mg / mL to 80 ± 8 mg / mL, 50 ± 5 mg / mL to 60 ± 6 mg / mL, 55 ± 5.5 mg / mL to 60 ± 6 mg / mL, about 50 mg / mL, 50 mg / mL, 60 mg / mL ± 6 mg / mL, about 60 mg / mL, 60 mg / mL, about 70 mg / mL, or 70 mg / mL.

[0063] Excipients and pH The pharmaceutical formulation of the present invention comprises one or more excipients. As used herein, the term “excipient” means any non-therapeutic agent added to the formulation to provide a desired viscosity, viscosity, or stabilizing effect.

[0064] In certain embodiments, the pharmaceutical formulation of the present invention also comprises at least one organic co-solvent in a type and amount that stabilizes the human activin A antibody under rough handling or stress conditions such as agitation, including vortexing, or freeze-thawing. In some embodiments, “stabilize” means that at least 95% (on a molar basis) of the activin A antibody is maintained in its native state, i.e., not fragmented or aggregated, by rough handling, for example, by vortexing the antibody-organic co-solvent solution for about 30, 60, or about 120 minutes, or by freezing and thawing the antibody-organic co-solvent solution for 4 or 8 cycles.

[0065] In certain embodiments, the organic cosolvent is a nonionic surfactant such as alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181, poloxamer 188, and poloxamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN® 20, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate. Poloxamer 188 is also known as PLURONIC® F68.

[0066] The amount of nonionic surfactant contained in the pharmaceutical formulation of the present invention may vary depending on the desired specific properties of the formulation, as well as the specific circumstances and purposes for which the formulation is intended. In certain embodiments, the formulation may contain 0.01% ± 0.0015% to 1% ± 0.15% of surfactant. For example, formulations of the present invention may contain about 0.0085%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, and about 0.17%. It may contain approximately 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.15%, or 1.2% of polysorbate 20.

[0067] The pharmaceutical formulations of the present invention may also include one or more stabilizers of a type and amount that stabilize the human activin A antibody under thermal stress conditions. In some embodiments, “stabilized” means that a solution containing the antibody and the heat stabilizer retains more than 90% of the antibody in its native conformation when maintained at about 2–8°C for up to about 18 months, at about 25°C for up to about 6 months, or at about 40°C for up to about 56 days. In some embodiments, “stabilized” means that a solution containing the antibody and its antigen-binding fragment, as well as the heat stabilizer, retains more than 90% of the antibody in its native conformation when maintained at about 2–8°C for up to about 24 months. As used herein, “native” means the primary form of the antibody by size exclusion, which is generally the intact monomer of the antibody.

[0068] In certain embodiments, the heat stabilizer is a sugar or sugar alcohol selected from sucrose, sorbitol, glycerol, trehalose, and mannitol, or any combination thereof, and the amount contained in the formulation may vary depending on the specific circumstances and intended purpose in which the formulation is used. In certain embodiments, the formulation may contain about 5% to about 40% sugar or sugar alcohol, about 1% to about 20% sugar or sugar alcohol, about 5% to about 15% sugar or sugar alcohol, about 7.5% to about 12.5% ​​sugar or sugar alcohol, about 10% sugar or sugar alcohol, 10% ± 1.5% sugar or sugar alcohol, or about 10% sugar or sugar alcohol. For example, the pharmaceutical formulations of the present invention may contain 1%±0.2%, 2%±0.4%, 3%±0.6%, 4%±0.8%, 5%±1%, 6%±1.2%, 7%±1.4%, 8%±1.6%, 9%±1.8%, 10%±2%, 11%±2.2%, 12%±2.4%, 13%±2.6%, 14%±2.8%, or about 15%±3% (w / v) of sugar or sugar alcohol (e.g., sucrose).

[0069] In certain embodiments, the heat stabilizer is an amino acid (e.g., arginine), and the amount of amino acid (e.g., arginine) contained in the formulation may vary depending on the specific circumstances and intended purpose in which the formulation is used. In certain embodiments, the formulation may contain about 0 mM to about 150 mM arginine, about 10 mM to about 140 mM arginine, about 20 mM to about 130 mM arginine, about 30 mM to about 120 mM arginine, about 40 mM to about 110 mM arginine, about 50 mM to about 100 mM arginine, about 60 mM to about 90 mM arginine, about 70 mM to about 80 mM arginine, or about 70 mM arginine. For example, the pharmaceutical formulation of the present invention may contain 0 mM, 10 mM ± 2 mM arginine, 20 mM ± 4 mM arginine, 30 mM ± 6 mM arginine, 40 mM ± 8 mM arginine, 50 mM ± 10 mM arginine, 60 mM ± 12 mM arginine, 70 mM ± 14 mM arginine, approximately 70 mM arginine, or 70 mM arginine.

[0070] The pharmaceutical formulations of the present invention may also include a buffer or buffer system that helps maintain a stable pH and assist in stabilizing the human activin A antibody. In some embodiments, “stabilized” means that at least 90% of the antibody is in its native conformation, as determined by size exclusion chromatography, when the solution containing the antibody and buffer is maintained at about 2–8°C for up to about 18 months, at about 25°C for up to about 6 months, or at about 40°C for up to about 56 days. In some embodiments, “stabilized” means that at least 90% of the antibody is in its native conformation, as determined by size exclusion chromatography, when the solution containing the antibody or its antigen-binding fragment and buffer is maintained at about 2–8°C for up to about 24 months. “Native” or “native conformation” means an antibody fraction that has not agglutinated or degraded. This is generally determined by an assay that measures the relative size of the antibody entity, such as a size exclusion chromatography assay. Non-agglutinated and non-fragmented antibodies elute in fractions equal to the native antibody and are generally the main elution fraction. Agglutinated antibodies elute in fractions that are larger in size than native antibodies. Fragmented antibodies elute in fractions that are smaller in size than native antibodies.

[0071] In some embodiments, “stabilized” means that when the solution contains the antibody and the buffer is maintained at approximately 2–8°C for up to approximately 18 months, at approximately 25°C for up to approximately 6 months, or at approximately 40°C for up to approximately 56 days, at which point

[0072] The pharmaceutical formulations of the present invention may have a pH of about 4.5 to about 7.0. For example, the formulations of the present invention may have a pH of about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In some embodiments, the pH is 6.3±0.5, 6.3±0.4, 6.3±0.3, 6.3±0.2, 6.3±0.1, about 6.3, or 6.3.

[0073] In some embodiments, the buffer or buffer system includes at least one buffer having a buffer range that completely or partially overlaps in the pH range of 5.5 to 7.4, for example, a buffer having a useful buffer range of pH 4.8 to 8.8. In one embodiment, the buffer has a pH of about 6.3. In a particular embodiment, the buffer includes a histidine buffer. In a particular embodiment, the histidine is present at concentrations of 5 mM ± 1 mM to 25 mM ± 5 mM, 6 mM ± 1.2 mM to 20 mM ± 4 mM, 7 mM ± 1.4 mM to 15 mM ± 3 mM, 8 mM ± 1.6 mM to 12 mM ± 2.4 mM, 9 mM ± 1.8 mM to 11 mM ± 2.2 mM, 10 mM ± 2 mM, about 10 mM, or 10 mM. In a particular embodiment, the buffer system contains 10 mM ± 2 mM of phosphate at a pH of 6.3 ± 0.3.

[0074] Example formulation According to one aspect of the present invention, a liquid pharmaceutical formulation comprises (i) a human antibody (e.g., galetosumab) that specifically binds to human activin A at a concentration of 60 mg / mL ± 6 mg / mL, (ii) a buffer system that buffers at a pH of approximately 6.3 ± 0.3, (iii) a heat stabilizer containing sugar and salt, and (iv) an organic cosolvent.

[0075] According to one embodiment, the pharmaceutical preparation comprises (i) a human antibody that specifically binds to human activin A in a concentration of 20±2 mg / mL to 200±20 mg / mL, (ii) a histidine buffer buffered at pH 6.3±0.3, (iii) sucrose and arginine, and (iv) a nonionic detergent such as polysorbate.

[0076] According to one embodiment, the pharmaceutical formulation comprises (i) 50 mg / ml ± 5 mg / mL of human IgG1 antibody that specifically binds to human activin A and includes HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (ii) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3; (iii) 5% ± 1% sucrose; (iv) 70 mM ± 14 mM arginine; and (v) 0.05% ± 0.025% polysorbate 20.

[0077] According to one embodiment, the pharmaceutical formulation comprises (i) 60 mg / ml ± 6 mg / mL of human antibodies that specifically bind to human activin A and include HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (ii) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3; (iii) 5% ± 1% sucrose; (iv) 70 mM ± 14 mM arginine; and (v) 0.05% ± 0.025% polysorbate 20.

[0078] According to one embodiment, the pharmaceutical formulation comprises (i) 70 mg / ml ± 7 mg / mL of human IgG1 antibody that specifically binds to human activin A and includes HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and (ii) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3, (iii) 5% ± 1% sucrose, (iv) 70 mM ± 14 mM arginine, and (iv) 0.05% ± 0.025% polysorbate 20, at the following concentrations.

[0079] According to one embodiment, the pharmaceutical formulation comprises (i) a human antibody in a concentration of 60 mg / ml ± 6 mg / mL that specifically binds to human activin A and includes the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 5; (ii) 10 mM ± 2 mM histidine, pH 6.3 ± 0.3; (iii) 5% ± 1% sucrose; (iv) 70 mM ± 14 mM arginine; and (v) 0.05% ± 0.025% polysorbate 20.

[0080] The following table lists exemplary formulations and primary container closures used during preclinical and clinical development of galetosumab. [Table 1]

[0081] Additional non-limiting examples of pharmaceutical formulations encompassed by the present invention are set out elsewhere in this specification, including the examples presented below.

[0082] Stability and robustness of pharmaceutical formulations The pharmaceutical formulations of the present invention typically exhibit a high level of stability. As used herein in reference to pharmaceutical formulations, the term “stable” means that the antibodies within the pharmaceutical formulation retain an acceptable degree of physical and / or chemical structure or biological function after storage under defined conditions. A formulation may be stable even if the antibodies contained therein do not retain 100% of their physical and / or chemical structure or biological function after storage for a defined time period. Under certain circumstances, the retention of at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the antibody after storage for a defined time period may be considered “stable.”

[0083] Stability can be measured, in particular, by determining the percentage of native antibodies remaining in the formulation after storage for a defined amount of time at a defined temperature and / or relative humidity (RH). The percentage of native antibodies can be determined, in particular, by size exclusion chromatography (e.g., size exclusion ultrahigh performance liquid chromatography [SE-UPLC]), reversed-phase chromatography (e.g., reversed-phase ultrahigh performance liquid chromatography [RP-UPLC]), and / or microchip capillary electrophoresis (e.g., reduced or unreduced MCE), where native means non-aggregated and non-fragmented. “Acceptable stability,” where the phrase is used herein, means that at least 90% of the native form of the antibody can be detected in the formulation after storage for a defined amount of time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native form of the antibody can be detected in the formulation after storage for a defined amount of time at a defined temperature. The defined time period after stability has been measured may be at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 56 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The defined temperature at which the pharmaceutical formulation may be stored when assessing stability may be any temperature between approximately -80°C and approximately 45°C, for example, storage at approximately -80°C, approximately -30°C, approximately -20°C, approximately 0°C, approximately 2°C to 8°C, approximately 5°C, approximately 25°C, approximately 40°C, or approximately 45°C. When evaluating stability, the defined relative humidity (RH) at which a pharmaceutical formulation can be stored may be approximately 20–90% RH, approximately 20% RH, approximately 25% RH, approximately 30% RH, approximately 35% RH, approximately 40% RH, approximately 45% RH, approximately 50% RH, approximately 55% RH, approximately 60% RH, approximately 65% ​​RH, approximately 70% RH, approximately 75% RH, approximately 80% RH, approximately 85% RH, or approximately 90% RH.Stress conditions may be applied to pharmaceutical formulations to evaluate stability, such as vortexing or freeze-thaw cycles. Stress conditions include, for example, vortexing an antibody-organic cosolvent solution for about 30 minutes, 60 minutes, or about 120 minutes, or freezing and thawing an antibody-organic cosolvent solution for 4 or 8 cycles. Thermal stress may be applied to pharmaceutical formulations to evaluate stability. Thermal stress includes, for example, holding the formulation at about 25°C, about 35°C, about 37°C, about 40°C, or about 45°C for about 14 days, about 28 days, or about 56 days. In certain embodiments, stress may be applied to a pharmaceutical formulation at about 40°C and about 75% RH for about 56 days.

[0084] For example, a pharmaceutical product may be considered stable if, after 24 months of storage at 2–8°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibody or its antigen-binding fragment detected by SE-UPLC, RP-UPLC, or MCE is native (i.e., in the native peak fraction). A pharmaceutical product may be considered stable if, after 18 months of storage at 2–8°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibody detected by SE-UPLC, RP-UPLC, or MCE is native (i.e., in the native peak fraction). A pharmaceutical formulation may be considered stable if, after 6 months of storage at 25°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native (i.e., in the native peak fraction). A pharmaceutical formulation may be considered stable if, after 6 months of storage at 25°C and 60% RH, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native (i.e., in the native peak fraction). A pharmaceutical formulation may also be considered stable if, after 6 months of storage at 25°C, at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native.A pharmaceutical formulation may be considered stable if, after 3 months of storage at 25°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native (i.e., in the native peak fraction). A pharmaceutical formulation may be considered stable if, after 1 month of storage at 25°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native (i.e., in the native peak fraction). A pharmaceutical formulation may also be considered stable if, after 56 days of storage at 40°C, at least 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native. A pharmaceutical formulation may also be considered stable if, after 56 days of storage at 40°C, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native. A pharmaceutical formulation may also be considered stable if, after 56 days of storage at 40°C and 75% RH, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native. A pharmaceutical formulation may also be considered stable if, after 6 months of storage at -20°C, at least 98.5%, 99%, or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native.A pharmaceutical formulation may also be considered stable if, after 18 months of storage at -30°C, at least 99% or 99.5% of the antibodies detected by SE-UPLC, RP-UPLC, or MCE are native. A pharmaceutical formulation may also be considered stable if, after 6 months of storage at -80°C, at least 99% or 99.5% of the antibodies detected by SE-UPLC are native.

[0085] Stability can be measured, in particular, by determining the proportion of antibody that moves in the primary fraction of antibody during ion exchange ("primary charge form") compared to all combined peak regions, and stability is proportional to the fraction of antibody in primary charge form. Charge form can be determined, in particular, by cation exchange chromatography (e.g., cation exchange ultrahigh performance liquid chromatography [CEX-UPLC]) or imaged capillary isoelectric focusing (iCIEF). While we do not wish to be bound by theory, deamidation of an antibody can make the antibody more negatively charged and therefore more acidic than an undeamidated antibody (see, e.g., Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). "Acceptable stability," where the phrase is used herein, means that at least 30% of the antibody is in the primary charge form detected in the formulation after storage for a defined amount of time at a defined temperature. In certain embodiments, an acceptable degree of stability is such that at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the antibody can be detected in its principal charge form after storage for a defined amount of time at a given temperature, or under heat, freeze / thaw, or agitation stress. The defined time period after stability has been measured may be at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.When evaluating stability, the temperatures at which the pharmaceutical formulation may be stored are any temperature between approximately -80°C and approximately 45°C, for example, approximately -80°C, approximately -30°C, approximately -20°C, approximately 0°C, approximately 2°C to 8°C, approximately 5°C, approximately 25°C, approximately 37°C, approximately 40°C, or approximately 45°C. When evaluating stability, the relative humidity (RH) at which the pharmaceutical formulation may be stored is approximately 20-90% RH, approximately 20% RH, approximately 25% RH, approximately 30% RH, approximately 35% RH, approximately 40% RH, approximately 45% RH, approximately 50% RH, approximately 55% RH, approximately 60% RH, approximately 65% ​​RH, approximately 70% RH, approximately 75% RH, approximately 80% RH, approximately 85% RH, or approximately 90% RH. For example, a pharmaceutical product may be considered stable if, after 24 months of storage at 2-8°C, approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or 61% or more of the antibody or its antigen-binding fragment is in the main charge form. A pharmaceutical product may be considered stable if, after 18 months of storage at 2-8°C, approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or 61% or more of the antibody is in the main charge form. A pharmaceutical formulation may also be considered stable if, after 6 months of storage at 25°C and 60% RH, approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or 61% or more of the antibody is in the primary charge form. A pharmaceutical formulation may also be considered stable if, after 6 months of storage at 25°C, approximately 45% or more of the antibody is in the primary charge form. A pharmaceutical formulation may also be considered stable if, after 56 days of storage at 40°C and 75% RH, approximately 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or 61% or more of the antibody is in the primary charge form. A pharmaceutical formulation may also be considered stable if, after 56 days of storage at 40°C, approximately 34% or more of the antibody can be detected in its primary charge form.

[0086] Measuring the binding affinity of an antibody to a target may also be used to assess stability or efficacy. The term "efficacy" refers to any factor contributing to the usefulness or biological activity of an antibody that recognizes an antigen. Efficacy may be assayed by ligand binding assays or functional assays, such as ELISA, flow cytometry, and / or other in vitro cell-based assays. For example, the formulation of the present invention may be considered stable if, after storage for a defined amount of time (e.g., 7 days to 24 months) at temperatures such as -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 40°C, and 45°C, the anti-activin A antibody contained in the formulation maintains at least 50% and up to approximately 150% of the efficacy measured as the binding affinity of the antibody before storage. Binding affinity may be determined, for example, by ELISA or plasmon resonance. Biological activity may be determined by activin A activity assays, such as by contacting cells expressing activin A with the formulation containing the anti-activin A antibody. The binding of antibodies to such cells can be measured directly, for example, by FACS analysis. Alternatively, the downstream activity of the activin A signaling pathway can be measured in the presence of the antibody and compared to the activity of the activin A signaling pathway in the absence of the antibody. In some embodiments, activin A may be endogenous to the cell. In other embodiments, activin A may be ectopically (heterogeneously) expressed within the cell.

[0087] Additional methods for evaluating the stability of antibodies in formulations are demonstrated in the examples presented below.

[0088] Container and method of administration The pharmaceutical formulations of the present invention may be contained in any container suitable for storing or administering drugs and other therapeutic compositions. For example, the pharmaceutical formulations may be contained in sealed and sterile plastic or glass containers having a defined volume, such as vials, ampoules, syringes, cartridges, bottles, or IV bags. Different types of vials, including clear and opaque (e.g., amber) glass or plastic vials, may be used to contain the formulations of the present invention. Similarly, any type of syringe may be used to contain or administer the pharmaceutical formulations of the present invention.

[0089] The pharmaceutical formulations of the present invention may be contained in a “normal tungsten” syringe or a “low tungsten” syringe. As will be understood by those skilled in the art, the process of making a glass syringe generally involves the use of a high-temperature tungsten rod that functions to puncture the glass, thereby creating a hole from which liquid can be drawn out and discharged from the syringe. This process results in the deposition of trace amounts of tungsten on the inner surface of the syringe. Subsequent washing and other processing steps may be used to reduce the amount of tungsten in the syringe. As used herein, the term “normal tungsten” means that the syringe contains tungsten at a concentration of 500 parts per billion (ppb) or more. The term “low tungsten” means that the syringe contains tungsten at a concentration of less than 500 ppb. For example, the low-tungsten syringe according to the present invention may contain tungsten in amounts of approximately 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, less than 10 ppb or less.

[0090] Rubber plungers used in syringes and rubber stoppers used to close the openings of vials may be coated to prevent contamination of the pharmaceutical contents of the syringe or vial, or to maintain their stability. Therefore, according to certain embodiments, the pharmaceutical formulations of the present invention may be contained in a syringe containing a coated plunger, or in a vial sealed with a coated rubber stopper. For example, the plunger or stopper may be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for use with vials and syringes containing the pharmaceutical formulations of this disclosure are referenced, for example, in U.S. Patents 4,997,423, 5,908,686, 6,286,699, 6,645,635, and 7,226,554, the contents of which are incorporated herein by whole reference. Certain exemplary coated rubber stoppers and plungers that may be used in the context of the present invention are commercially available under the trade name "FluroTec®" from West Pharmaceutical Services, Inc. (Lionville, PA). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent pharmaceuticals from adhering to rubber surfaces.

[0091] According to a particular embodiment of the present invention, a pharmaceutical formulation may be contained within a low-tungsten syringe comprising a fluorocarbon-coated plunger.

[0092] Pharmaceutical formulations may be administered to patients by injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), parenteral routes such as transdermal, mucous membrane, nasal, or pulmonary, or by oral administration. Numerous reusable pens or auto-injector delivery devices may be used to subcutaneously deliver the pharmaceutical formulations of the present invention. Examples include AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II, and III (Novo Examples include, but are not limited to, Nordisk (Copenhagen, Denmark), NOVOPEN JUNIOR (trademark) (Novo Nordisk, Copenhagen, Denmark), BD (trademark) pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (trademark), OPTIPEN PRO (trademark), OPTIPEN STARLET (trademark), and OPTICLIK (trademark) (sanofi-aventis, Frankfurt, Germany). Examples of disposable pens or auto-injector delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR® pen (sanofi-aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (Eli Lilly), SURECLICK® auto-injector (Amgen, Thousand Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® pen (Abbott Labs, Abbott Park, IL).

[0093] The use of microinfusers for delivering the pharmaceutical formulations of the present invention is also intended herein. As used herein, the term "microinfuser" means a subcutaneous delivery device designed to slowly administer a large volume (e.g., up to approximately 2.5 mL or more) of a therapeutic formulation over a long period of time (e.g., about 10, 15, 20, 25, 30 minutes or more). See, for example, US6,629,949, US6,659,982, and Meehan et al., J. Controlled Release. See 46:107-116 (1996). Microinfusers are particularly useful for delivering large volumes of therapeutic proteins contained in high-concentration solutions (e.g., approximately 100, 125, 150, 175, 200 mg / mL or higher) or viscous solutions.

[0094] In one embodiment, the pharmaceutical formulation is administered via intravenous infusion so that the formulation is diluted in an IV bag containing a physiologically acceptable solution. In one embodiment, the pharmaceutical composition is a sterile preparation compounded in an intravenous infusion bag so that a single dose of the pharmaceutical is diluted in 100 mL, 250 mL (or other similar amounts suitable for intravenous infusion delivery) of physiological buffer (e.g., 0.9% saline). In some embodiments, the infusion bag is made of polyvinyl chloride (e.g., VIAFLEX, Baxter, Deerfield, Illinois). In some embodiments, the infusion bag is made of polyolefin (EXCEL IV Bags, Braun Medical Inc., Bethlehem, Pennsylvania).

[0095] Therapeutic use of pharmaceutical preparations The pharmaceutical formulations of the present invention are useful in the treatment, prevention, and / or improvement of any disease or disorder associated with activin A activity, including, in particular, diseases or disorders mediated by activin A. Exemplary, non-limiting diseases and disorders that may be treated or prevented by administration of the pharmaceutical formulations of the present invention include fibrodysplasia ossificans progressive (FOP). FOP is a rare genetic disorder in which heterotopic ossification forms histologically and biomechanically "normal" bone in external skeletal locations (e.g., connective tissue). This disorder is accidental but cumulative, causing permanent disability that increases in severity. FOP is a severe, progressive, very rare genetic disorder in which muscles, tendons, and ligaments are gradually replaced by bone (a process known as heterotopic ossification (HO)). HO of the jaw, spine, and rib cage can make it difficult to speak, eat, or breathe, cause weight loss, and worsen loss of mobility and skeletal deformities. People with FOP also experience accidental localized inflammation known as "flare-ups," but HO can be asymptomatic or symptomatic. Most people with FOP are wheelchair-bound by age 30, and the median survival age is approximately 40. Deaths often result from complications such as pneumonia, heart failure, and aspiration arising from HO, as well as loss of movement in the chest, neck, and jaw. [Examples]

[0096] The following examples are provided to give a complete disclosure and explanation of how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be the present invention. Although efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are molar parts, molecular weight is average molecular weight, concentration percentage (%) means the mass of solute in grams divided by the volume of solution in milliliters and multiplied by 100% (e.g., 10% sucrose means 0.1 grams of sucrose per milliliter of solution), temperature is in degrees Celsius, and pressure is atmospheric pressure or near-atmospheric pressure. Early formulation development activities included screening organic cosolvents, thermal stabilizers, and buffers in liquid and lyophilized formulations of anti-activin A antibody to identify excipients that are compatible with the protein, enhance its stability, and maintain near physiological osmotic pressure and low viscosity for intravenous and subcutaneous injection. We also investigated buffering conditions to determine the optimal pH for maximum protein stability.

[0097] Example 1: Exemplary anti-activin A antibody preparation In the First-in-Human (FIH) trial, a lyophilized drug (DP) intended for both intravenous (IV) and subcutaneous (SC) administration was developed. The FIH formulation was developed by evaluating the effects of different components of the formulation on the stability of anti-activin A antibodies, such as galetosumab. These components included pH, buffer, heat stabilizers, and surfactants. The main degradation pathways for galetosumab are the formation of high and low molecular weight species and acidic charge variants. Analytical methods were developed to monitor these degradation products. Furthermore, general quality characteristics were monitored, including protein concentration, pH, solubility, and bioefficacy. Based on the formulation development studies conducted, a lyophilized formulation containing histidine, sucrose, and polysorbate 80 at pH 6.3 was developed. The lyophilized formulation developed for FIH was stable for at least 36 months.

[0098] Galetosumab was delivered via IV and SC administration in a Phase I clinical study. A single, dual-use lyophilized formulation was developed to reconstitute lyophilized galetosumab DP with sterile water for injection (WFI) to either 50 mg / mL galetosumab for IV infusion or 150 mg / mL galetosumab for SC infusion. Formulation development activities included evaluation of buffers, pH, organic cosolvents, surfactants, and sucrose (as a heat stabilizer) to identify excipients that would optimize protein stability. Galetosumab DP was produced by lyophilizing an optimized aqueous buffer formulation containing 10 mM histidine, pH 6.3, 0.1% (w / v) polysorbate 80, 5% (w / v) sucrose, and 50 mg / mL galetosumab. The table lists the compositions of galetosumab DP after reconstitution using different volumes of WFI for IV or SC administration. [Table 2]

[0099] The effect of buffer and pH on the thermal stability of galetosumab was initially investigated in liquid formulations by incubating 5 mg / mL of galetosumab at 45°C for 28 days in a series of buffer systems within a fluctuating pH range. Maximum protein stability was observed when galetosumab was formulated in 10 mM histidine buffer at pH 5.5–6.5 (Table). The effect of pH on the thermal stability of galetosumab was further investigated in liquid formulations in histidine buffer in the pH range of 6.0–6.6. Since the formation of high molecular weight (HMW) species was minimized at this pH, pH 6.3 was selected for the DP formulation (Table). This target pH also allows for an appropriate pH range for maximum stability that can be achieved in a large-scale manufacturing environment. Based on these results, 10 mM histidine buffer at pH 6.3 was selected for the galetosumab DP formulation.

[0100] The effects of surfactants and organic co-solvents on the stirring stress stability, freeze / thaw stability, and thermal stability of 10 mg / mL galetosumab were investigated in liquid formulations. The results of these studies are summarized in the table. Galetosumab was unstable when stirred in the absence of organic co-solvents or surfactants. All organic co-solvents and surfactants tested protected galetosumab from stirring-induced instability (Table). 10 mg / mL galetosumab was unstable when exposed to multiple freeze / thaw cycles in the absence of organic co-solvents or surfactants (Table). All organic co-solvents and surfactants tested protected galetosumab from freeze / thaw-induced instability (Table). In a subsequent study, 70 mg / mL galetosumab was exposed to eight freeze and thaw cycles, but no visual impairment or increase of HMW species was observed, even in solutions without surfactants or co-solvents (Table). 70 mg / mL represents the concentration of galetosumab frozen and thawed in the manufacturing environment during clinical trials of galetosumab. Polysorbate 80 was selected as the surfactant for the initial galetosumab DP formulation because it has a history of safe use in monoclonal antibody formulations, stabilizes the protein against instability induced by stirring stress and freeze / thaw, and minimizes adverse effects on thermal stability (Table to Table).

[0101] The stabilizing effect of sucrose on galetosumab was evaluated. 10 mg / mL galetosumab in liquid formulation showed improved stability when formulated with 5% sucrose and incubated under accelerated conditions compared to the absence of sucrose (Table). Galetosumab was sufficiently stable for initial clinical use when formulated in the presence of histidine, polysorbate 80, and sucrose at pH 6.3.

[0102] Based on initial formulation development studies, the following formulation was selected for clinical use: 50 mg / mL galetosumab, 10 mM histidine, pH 6.3, 5% (w / v) sucrose, and 0.1% (w / v) polysorbate 80. DP was prepared by filling 5.74 mL of this formulation into 20 mL glass vials and lyophilizing them. Study stability studies demonstrated that this formulation was stable for 36 months (Table 9). [Table 3] [Table 4] [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 7] [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2]

[0103] Example 2: Exemplary anti-activin A antibody preparation The objective of the drug product (DP) commercial development activity was to develop an anti-activin A antibody (e.g., galetosumab) DP having the following attributes: a liquid formulation in a glass vial with a concentration of galetosumab sufficient to deliver a dose of 10 mg / kg of galetosumab by intravenous (IV) infusion; the galetosumab vial should contain 300 mg in 5 mL; a nearly equimolar formulation that can be diluted for IV infusion with commonly available diluents; a stable formulation compatible with Type 1 clear glass vials and standard serum stoppers as the primary packaging system; a sterile DP that supports long-term stability with a storage period of 24 months or more at 2-8°C; a robust formulation that minimizes the formation of high molecular weight (HMW) species of galetosumab, minimizes changes in the relative distribution of galetosumab charge variant species, minimizes the presence of visible and invisible particles, and maintains biological activity when subjected to handling and thermal stress; and a formulation that stabilizes high galetosumab concentrations and is suitable for use in subcutaneous (SC) administration.

[0104] This study describes the optimization of the late-stage formulation, confirmation of the market formulation of galetosumab, and the robustness of galetosumab. The market formulation of galetosumab is a liquid containing 60 mg / mL of galetosumab in an aqueous solution containing 10 mM L-histidine, pH 6.3, 5% (w / v) sucrose, 70 mM L-arginine HCl, and 0.05% (w / v) polysorbate 20. This formulation stabilizes galetosumab during long-term storage at 2–8°C and when subjected to accelerated and stressed conditions, including stirring, freezing and thawing, and thermal stress. For Phase 2 clinical research and commercialization, a liquid DP formulation suitable for IV administration was developed. This formulation was built upon knowledge gained during initial development, as well as additional formulation development studies conducted to refine and optimize the formulation for specific clinical indications and optimize product stability. The galetosumab formulation consists of 60 mg / mL of galetosumab; 10 mM histidine, pH 6.3, 5% sucrose, 70 mM arginine HCl, and 0.05% polysorbate 20. DP is manufactured by filling a 10 mL glass vial with 5.61 mL of galetosumab and sealing it with an elastomer stopper. Long-term stability studies have been initiated and have shown the formulation to be stable for at least 12 months. Stability studies will continue for 60 months. Robustness studies have been initiated. Previous long-term, accelerated, and stress stability studies have shown the formulation to be robust with respect to pH, protein concentration, and excipient concentration. Minor variations that may occur during the manufacturing process do not significantly affect the quality of galetosumab DP.

[0105] The drug development studies conducted during the galetosumab development process resulted in a stable formulation of galetosumab that met the objectives set to address clinical and commercial needs. The goal of late-stage clinical development was to produce a formulation that delivers 300 mg of galetosumab in 5 mL of solution. A secondary objective was to have a formulation that could be easily converted to a subcutaneous formulation for future use. Formulation development studies were conducted with the aim of developing a liquid formulation containing 60 mg / mL of galetosumab for use in intravenous administration. Formulation development activities for the liquid formulation included evaluation of histidine buffer concentration, surfactant concentration, and other stabilizers.

[0106] Selection of buffer and pH for galetosumab liquid formulations Early FIH development activities determined that the optimal pH for galetosumab was 6.3. pH remained constant throughout development. Histidine buffer was used for the lyophilized formulation in the early Phase 1 study of galetosumab, based on buffer screening studies. The same buffer was selected for later formulation development. The stability of 50 mg / mL galetosumab was tested when incubated at 45°C for 28 days at pH 6.3 with histidine concentrations of 5 mM, 10 mM, or 25 mM to assess the optimal histidine concentration. The results of this stress test indicate that 10 mM histidine at pH 6.3 falls within the stable region of the design space, with sufficient space at both ends to accommodate slight variations in buffer concentration (Table) or pH (Table). Based on these results, the same buffer composition and pH used for the lyophilized histidine formulation at 10 mM concentration and pH 6.3 were selected as the buffer, buffer concentration, and pH for the later liquid formulation.

[0107] Selection of heat stabilizers for galetosumab liquid formulations Initial development studies of galetosumab demonstrated that 5% (w / v) sucrose is necessary to stabilize galetosumab against freeze-thaw stress. Therefore, 5% (w / v) sucrose was added to the bulk active pharmaceutical ingredient (DS) to adequately stabilize bulk DS against HMW species formation when exposed to freeze-thaw cycles that DS may be exposed to during manufacturing (the freeze-thaw stability of DS is discussed in Module S.7.1). Studies were conducted to evaluate the stability of 50 mg / mL liquid galetosumab when incubated at 45°C for 28 days or subjected to 8 freeze-thaw cycles, with sucrose concentrations in the formulation varying between 0 and 10%. The results of these stress tests show that 5% sucrose falls within the stable region of the design space, which is at both ends of a space sufficient to accommodate the small variations in sucrose concentration that may be experienced as a result of normal manufacturing variations (Tables). To adequately stabilize galetosumab against thermal stress, as well as freeze-thaw stress, and to provide an acceptable osmotic pressure in the final formulation, 5% sucrose was selected as a stabilizer.

[0108] Arginine was evaluated as an additional stabilizer for the galetosumab liquid formulation. During evaluation, the range of arginine concentrations from 0 to 100 mM was assessed (Table). Stress test results showed that 70 mM arginine falls within the stable region of the design space, with sufficient space at both ends to accommodate the slight variations in arginine concentration that may be experienced as a result of normal manufacturing variations (Table). 70 mM arginine was selected as the stabilizer concentration in the galetosumab liquid formulation because it reduces the formation of HMW species and acidic charge variants observed under stress stability conditions and provides an acceptable osmotic pressure in the final formulation.

[0109] Selection of surfactants for galetosumab liquid formulations 0.1% (w / v) polysorbate 80 was selected for the galetosumab Phase 1 formulation. Further tests comparing the stability of 50 mg / mL galetosumab in the presence of polysorbate 20 or polysorbate 80 showed that both surfactants provided comparable stability to 50 mg / mL galetosumab when incubated at 45°C for 28 days or at 25°C for 6 months (Table). For further development of the galetosumab liquid formulation, polysorbate 20 was selected. The stability of 50 mg / mL galetosumab was evaluated by stirring for 30 minutes or by incubation at 45°C for 28 days at various polysorbate 20 concentrations. Stress test results showed that 0.05% polysorbate 20 fell within the stable region of the design space, with sufficient space at both ends to accommodate slight variations in polysorbate 20 concentration that may be experienced as a result of normal manufacturing variations (Tables). A 0.05% (w / v) polysorbate 20 concentration was selected for the galetosumab liquid formulation.

[0110] conclusion The late formulation was selected using the results of studies testing the stability of galetosumab under stress conditions, incubation at 45°C, vortex stirring, and freeze / thaw cycles. Based on the data collected from these studies, a galetosumab formulation containing 10 mM histidine, pH 6.3, 5% sucrose, 70 mM arginine-HCl, and 0.05% polysorbate 20 was selected and subsequently demonstrated to be suitable for clinical or commercial use. The excipient concentrations were selected so that minor variations that may occur during normal manufacturing do not have an observable effect on the stability or quality of galetosumab DP. The selected formulation contains 60 mg / mL of galetosumab, providing a dose of 300 mg in a 5 mL drawable volume. The suitability of this formulation has been confirmed based on long-term development stability data of 18 months at 5°C, accelerated stability data of 6 months at 25°C / 60% RH, and stress stability testing including 56-day stability data at 40°C / 75% RH, 8 cycles of freeze and thaw, and 120 minutes of agitation (vortexing). [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0111] Example 3: Stability Study The suitability of the galetosumab liquid formulation, developed and intended for late-stage clinical and commercial use, was confirmed in long-term, accelerated, and stress stability studies. In addition, the robustness of the commercial formulation was confirmed using a Design of Experiments (DOE) approach.

[0112] The study was initiated to evaluate the storage, acceleration, and stress stability of study lots of galetosumab DP. The FDS lots used in these studies represent FDS manufactured for clinical use. The DP composition used in these studies is identical to that used in the Phase 2 clinical study and was produced by filling 5.5 mL of FDS into 10 mL Type 1 glass vials.

[0113] Research, storage, acceleration, and stress stability of galetosumab drugs Galetosumab DP was physically and chemically stable when stored at 2–8°C for at least 12, 18, or 24 months.

[0114] (Table). No recognizable changes were detected in any of the monitored attributes. The analysis results of galetosumab DP after incubation under stress and accelerated temperature conditions are shown in the table. After incubation for 56 days at 40°C / 75% RH, some formation of HMW species, LMW species, and charge variants (an increase in the relative proportion of acidic species (region 1)) was detected. After incubation for 6 months at 25°C / 60% RH, no increase in HMW species was observed by SE-UPLC, but some increase in acidic species (region 1) was observed by CEX-UPLC and icIEF. Furthermore, after incubation for 6 months at 25°C / 60% RH, slight increases in LMW species were observed by unreduced and reduced MCE, respectively. No significant changes were observed in any of the other monitored attributes. Liquid DP is exposed to room temperature only for a short time. Temperatures above room temperature should be avoided.

[0115] The results of the analysis of galetosumab DP after multiple cycles of agitation or freeze-thaw are shown in the table. No significant changes were observed in any of the monitored attributes when galetosumab DP was subjected to 120 minutes of agitation or 8 cycles of freeze-thaw. The results of this stability study confirm that the galetosumab formulation developed for commercial use is appropriate and provides a stable drug product. [Table 17-1] [Table 17-2] [Table 18-1] [Table 18-2] [Table 19-1] [Table 19-2]

[0116] conclusion Results from studies on the long-term storage, accelerated, and stress stability of galetosumab DP support the clinical application of this product. Galetosumab DP can withstand short-term exposure to room temperature without compromising its physical or chemical stability. Galetosumab DP is stored at 2°C to 8°C, and exposure to temperatures above 2°C to 8°C is limited.

[0117] Example 4: Robustness Study Normal variations in the composition of galetosumab DP can occur during manufacturing, including variations in the concentration of galetosumab, the concentration of excipients (histidine, arginine, sucrose, or polysorbate 20), and / or the pH of the formulation. Since variations in any of these formulation factors may potentially affect the stability or quality of DP, formulation robustness studies were conducted to evaluate the effects of such variations within a defined range.

[0118] Two DOE studies were used to evaluate the effects of each individual formulation factor, as well as the impact of inter-factor interactions on formulation stability.

[0119] Preliminary robustness study – A DOE study involving accelerated and stress stability assessments to identify key formulation factors that may affect the stability of galetosumab DP.

[0120] Final Robustness Study – A comprehensive DOE study evaluating key formulation factors identified from preliminary robustness studies to assess the robustness of galetosumab formulations, including stress, acceleration, and long-term storage stability.

[0121] The nominal chemical formula of the commercially available galetosumab formulation is 60 mg / mL galetosumab, 10 mM histidine, 5% (w / v) sucrose, 70 mM arginine-HCl, and 0.05% (w / v) polysorbate 20 at pH 6.3.

[0122] The objective of this preliminary robustness study was to examine the primary and combined effects of several factors (interactions and secondary effects) on the stability of galetosumab and to identify factors that should be included in a comprehensive robustness study (primary effects are formulation factors, interactions describe interactions between two different factors, and secondary effects describe interactions between factors themselves). This study examined all primary effects. Subsets of interactions and secondary effects were selected to be examined based on a risk assessment given by prior experience with galetosumab. Based on the risk assessment, a six-factor D-optimal DOE study was developed to characterize and explore the excipient design space. A 24-run study was developed considering primary effects and interactions and secondary effects as defined in the risk assessment. This design was prepared, evaluated, and confirmed in JMP12.1 to be able to cover the design space with adequate power supply and good estimation capacity. The preliminary robustness study design is described in Tables 1, 2, and 3. [Table 20] [Table 21] [Table 22]

[0123] result In preliminary galetosumab robustness studies, the stability of galetosumab as a function of various formulations was evaluated at both 25°C and 45°C. However, the factors selected for the final robustness study were based on degradation rates determined only at 45°C. If the rate of change of response in a particular factor (including major effects, interactions, and quadratic equation theory) was p ≤ 0.05, that factor was considered statistically significant and could be considered for the final robustness study. The table shows a summary of the results of the DOE analysis, indicating statistically significant responses and related matters. The responses examined in the DOE analysis were the daily rate of change of HMW, LMW, and monomers by SE-UPLC, as well as the daily rate of change of acidic species, major peaks, and basic species by CEX-UPLC and icIEF. Other responses, including visual appearance, turbidity by OD405nm, pH, protein concentration (recovery), and particles invisible to the naked eye by MFI, were measured but were considered pass / fail or for informational purposes and were not used in the DOE analysis. [Table 23-1]

[0124] Visual inspection revealed that all samples passed the visual inspection for turbidity (OD405nm), pH, protein recovery, and invisible particles (MFI). No significant increase in OD405nm was observed in any sample, and no change in protein concentration was observed. All concentration measurements were within 5% of the t=0 value. Some fluctuations and some increasing trends were observed in particle concentration (MFI). Some trends were statistically significant, but the significant trends were not substantially significant.

[0125] conclusion The results of the DOE analysis showed that the following non-major effect factors had a statistically significant effect on the stability of galetosumab under thermal stress conditions (28-day incubation at 45°C). ●[Histidine]*pH ●[Galetosumab]*pH ●[histidine]*[histidine] ●[Arginine]*[Arginine] These interactions and quadratic equations, along with all main effects, were included in the final robustness study. A detailed summary of the preliminary robustness study, including data from incubations at 45°C and 25°C, is discussed in report EXP-09 Jul 2019-0096 garetosmab Pre-PAR study.

[0126] Final robustness study design for galetosumab The range of formulation factors tested in this study was defined as being wider than or equal to the range expected to occur when considering contributions from both manufacturing and assay variations. Formulation parameters in this robustness study included galetosumab concentration (±10%, the specification limit), buffer and stabilizer concentrations (±20%), surfactant concentration (±50%), and pH (±0.3 units, the specification limit) (Table).

[0127] To evaluate the impact of key formulation factors identified from preliminary robustness studies on the storage and stress stability of galetosumab formulations, a six-factor D-optimal DOE design was performed to characterize and explore the excipient design space. A 14-run study (Table) was designed considering all major effects, as well as secondary interactions and secondary effects identified as statistically significant in the preliminary robustness studies. This design was prepared, evaluated, and confirmed in JMP12.1 to have sufficient power supply and good estimation capability to cover the design space. In addition to the test formulations, one formulation with all factors at nominal levels was included as a control but excluded from the DOE analysis. A 5.5 mL formulation representative of the commercially available DP presentation was filled into a 10 mL Type 1 glass vial. Each formulation was evaluated for its long-term storage stability at 2°C to 8°C, accelerated stability at 25°C / 60%RH and under stress conditions, 40°C / 75%RH, and under stirring, freeze-and-thaw stress (storage and stress conditions are summarized in Table 26). [Table 23-2]

[0128] ).

[0129] As summarized in Table 26, all formulations were characterized and evaluated for physical and chemical properties and stability, including visual inspection, pH, turbidity, protein concentration and recovery, purity in terms of molecular weight and morphology (assessed by SE-UPLC and microchip capillary electrophoresis (MCE)), charge variants (assessed by CEX-UPLC and imaged capillary isoelectric focusing (icIEF)), and analysis of particles invisible to the naked eye (assessed by light shielding method (HIAC) and MFI). [Table 24] [Table 25] [Table 26]

[0130] Results (stress temperature 40°C / 75%RH) Galetosumab samples from the formulations listed in Table 24 were incubated at 40°C / 75%RH for 2 months. The responses considered most important in DOE analysis were the formation of molecular weight variants by SE-UPLC and MCE, and the formation of charge variants by CEX-UPLC and icIEF. Additional factors, including visual inspection, pH, turbidity, protein concentration and recovery, and particle level, were measured but were deemed pass / fail or informational and were not used in DOE analysis. Factors that resulted in statistically significant changes in the measured responses are listed in the table. Several factors had a statistically significant effect on the response, but the effect was small, resulting in relatively small changes depending on the variation of the factor under these stress conditions. The factor with the greatest effect was pH on the rate of charge variant formation by CEX-UPLC. The effect was statistically significant but small. The rate of acidic species formation changed by 3.8% per month when the pH changed from 6.0 to 6.6. Overall, under the tested stress conditions, formulation variability had little effect on the stability of galetosumab. The variability is shown in Figure 2. In all cases, diffusion relative to the control was not significant with respect to the overall stability of the formulation. [Table 27]

[0131] Results (accelerated temperature 25℃ / 60%RH) Galetosumab samples from the formulations listed in the table were incubated at 25°C / 60%RH for 6 months. The responses considered most important in DOE analysis were the formation of molecular weight variants by SE-UPLC and MCE, and the formation of charge variants by CEX-UPLC and icIEF. Additional factors, including visual inspection, pH, turbidity, protein concentration and recovery, and particle level, were measured but were deemed pass / fail or informational and were not used in DOE analysis. Factors that resulted in significant changes in the measured responses are listed in the table. Several factors had a statistically significant effect on the response, but the effect was small, and the variation in factors under these accelerated conditions resulted in a variation rate of less than 1% per month. Under the accelerated conditions tested, the variation in formulations had little effect on the stability of galetosumab. The variation rates are shown in the figure. In all cases, diffusion relative to the control was not significant with respect to the overall stability of the formulation. [Table 28-1] [Table 28-2]

[0132] Results (stress conditions: stirring, as well as freezing and thawing) The stability of the galetosumab formulations listed in Table 24 was evaluated after 4 or 8 cycles of freeze-thawing, or after 30, 60, or 120 minutes of vortex stirring. The quality attributes evaluated were molecular weight variant formation by SE-UPLC and MCE, charge variant formation by CEX-UPLC and icIEF, visual inspection, pH, turbidity, protein concentration and recovery, and particle level. Figures and diagrams summarize the effects of variations in formulation composition compared to a control formulation where all components of the formulation were nominal, after 8 cycles of freeze-thawing or 120 minutes of vortex stirring (shown as "delta control" on the graph), according to the DOE design. These results indicate that altering the formulation composition does not have a significant effect on the stability of galetosumab compared to the control formulation under the stress conditions in which it is used.

[0133] Results (long-term storage temperature 2~8℃) Galetosumab samples prepared according to the DOE design from the formulations listed in Table 24 were stored at 2–8°C. Long-term stability data for 24 months are now available for galetosumab robustness studies. The effects of varying galetosumab concentration, pH, histidine concentration, arginine concentration, sucrose concentration, and polysorbate 20 concentration on the long-term stability of galetosumab were investigated. Quality attributes evaluated included molecular weight variant formation by SE-UPLC and MCE, charge variant formation by CEX-UPLC and icIEF, visual inspection, pH, turbidity, protein concentration and recovery, and particle level. Figure 7 summarizes the effects of varying formulation components compared to a control formulation where all components were nominal according to the DOE design. These results indicate that varying the formulations within the tested range did not significantly affect the stability or quality of galetosumab after 12 or 24 months of storage at 5°C. No precipitate or visible particles were detected by either visual inspection or turbidity measurement (OD at 405 nm). No significant changes were observed in protein recovery (RP-UPLC). The pH of the formulation was stable. No significant increase in invisible particles was observed by HIAC, and no significant difference in the number of invisible particles was observed compared to the control formulation: all values ​​for invisible particles measured by HIAC are USP <788> The particle count was below the acceptable limit set by the method, and particles invisible to the naked eye were also measured by MFI. Although the particle count was variable, no significant changes in invisible particles were observed for 10 μm or 25 μm particles compared to the control or between formulations. A tendency to increase the number of 2-10 μm particles was observed over time, but the difference was not considered large.

[0134] Statistical analysis of the results showed that the differences between formulations were mainly due to random variability and not to changes in factors (Figures 6-7). No significant differences were observed in the levels of HMW species, LMW species, or native galetosumab compared to the control formulation, as determined by SE-UPLC or MCE. No significant differences were observed in the levels of acidic or basic charge variants compared to the control formulation, as determined by CEX-UPLC or icIEF.

[0135] The results of this study demonstrate that variations in formulation factors (galetosumab concentration, pH, histidine concentration, arginine concentration, sucrose concentration, and polysorbate 20 concentration) within the study range do not significantly affect the long-term stability of galetosumab. The 60 mg / mL galetosumab formulation is robust with respect to all quality attributes within the range of the tested formulation compositions.

[0136] Summary of the robustness of galetosumab formulations Formulation robustness studies were conducted to evaluate the effect of variations in formulation parameters on the stability of the galetosumab formulation. DOE studies evaluating long-term storage, accelerated stability, and stress stability demonstrated that variations in formulation parameters within the study range did not significantly affect the quality and stability of galetosumab.

[0137] Specifically, the proposed 60 mg / mL commercially available galetosumab formulation is robust to ±10% variation in protein concentration, ±20% variation in sucrose, arginine, and / or histidine concentrations, ±50% variation in polysorbate 20 concentration, and / or variation in pH units of ±0.3. Overall, the results from the 60 mg / mL galetosumab robustness study support the idea that variations in the composition of the galetosumab formulation within the study range do not adversely affect the stability or quality of galetosumab DP under recommended storage conditions (2–8°C).

[0138] conclusion Based on the results from the presented formulation development studies and clinical experience, a commercially available galetosumab formulation for intravenous infusion has been developed, containing the following components: 60 mg / mL galetosumab, 10 mM L-histidine, 5% (w / v) sucrose, 70 mM L-arginine HCl, 0.05% (w / v) polysorbate 20, and pH 6.3. This commercially available IV formulation meets the defined objectives for formulation development. The developed formulation is a liquid formulation in a glass vial with a concentration of galetosumab sufficient to deliver a dose of 10 mg / kg galetosumab via intravenous (IV) infusion. Galetosumab DP is 60 mg / mL and is manufactured in a 5 mL drawable volume providing 300 mg per vial. The formulation is a nearly isotonic formulation compatible with 0.9% sodium chloride infusion or 5% dextrose infusion for IV infusion. The formulation is stable and compatible with Type 1 clear glass vials and standard serum stoppers as the primary packaging system. The formulation is sterile DP, supporting long-term stability with a storage period of 24 months or more at 2–8°C. No significant changes in any galetosumab quality attributes were observed when stored for up to 12, 18, or 24 months at 2–8°C. The formulation is robust, minimizing the formation of high molecular weight (HMW) species of galetosumab, minimizing changes in the relative distribution of charge variant species of galetosumab, maintaining particles invisible to the naked eye at a safe level, and maintaining biological activity when exposed to handling and thermal stress. The formulation is readily convertible for use in subcutaneous (SC) administration. The active pharmaceutical ingredient containing galetosumab is 200 mg / mL.

[0139] Example 5: Excess The formulation did not contain any excess, but a slight overfill was included to compensate for normal variations in filling volume encountered during the automated filling termination process and to ensure that the correct volume could be drawn from the vial. Clinical DP was prepared with a minimum filling volume of 5.44 mL. Each vial contained 0.44 mL of overfill, which was sufficient to accurately draw 5.0 mL (300 mg) of DP from the vial.

[0140] The excess was not designed to compensate for losses during manufacturing, degradation during manufacturing, degradation during storage (shelf life), or extension of the expiration date. These excess amounts were intended to allow for precise withdrawal and administration of 5.0 mL of DP (e.g., 300 mg of galetosumab).

[0141] Example 6: Method used to evaluate stability The study stability of galetosumab DP was evaluated using different assays. Color and appearance were assessed by visual inspection. Turbidity was measured by the increase in optical density (OD) at 405 nm. Particulate matter analysis was determined by light shielding (HIAC). Particulate matter analysis was evaluated by Micro-Flow Imaging® (MFI). Protein concentration was evaluated by reverse-phase ultrahigh-performance liquid chromatography (RP-UPLC). The purity of each individual DP was evaluated using assays: size exclusion ultrahigh-performance liquid chromatography (SE-UPLC); reduced and unreduced microchip capillary electrophoresis (MCE).

[0142] Charge variant analysis was determined using assays: cation exchange ULC (CEX-UPLC) and imaged capillary isoelectric focusing (iCIEF). Charge variants are reported as percentages in regions 1, 2, and 3. Region 1 corresponds to acidic species eluting before the major peak, region 2 corresponds to the major peak, and region 3 corresponds to basic species eluting after the major peak.

[0143] Efficacy was evaluated using bioassays. The relative efficacy of each sample was determined by bioassay and defined as follows: (IC 50 Reference sample / IC 50 Test sample × 100%. The measured effectiveness of the storage stability sample must be within 50-150% of the measured effectiveness of the reference standard substance.

[0144] Unofficial Sequence List Sequence ID 1 GGSFSSHF Sequence ID 2 ILYTGGT Sequence ID 3 ARARSGITFTGIIVPGSFDI Sequence ID 4 QSVSSSY Sequence ID 5 GAS Sequence ID 6 QQYGSSPWT Sequence ID 7 [ka] Sequence ID 8 [ka]

Claims

[Claim 1] The invention described in the specification.

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