High-concentration Anti-aβ protofibril antibody formulation and method of use thereof

Abstract

To provide a stable isolated anti-Aβ protofibril antibody formulation.SOLUTION: Provided is an aqueous pharmaceutical formulation, comprising: a high concentration of an isolated anti-Aβ protofibril antibody, or a fragment thereof that binds to human Aβ protofibrils, such as BAN2401; arginine; polysorbate 80; and a pharmaceutically acceptable buffer.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 992,746, filed Mar. 20, 2020, and U.S. Provisional Patent Application No. 63 / 027,263, filed May 19, 2020; the contents of both are hereby incorporated by reference in their entirety.

Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disorder of unknown etiology and is the most common form of dementia among the elderly. In 2006, there were 26.6 million cases of AD worldwide (range: 11.4 million - 59.4 million cases) (Brookmeyer, R., et al., Forecasting the global burden of Alzheimer’s Disease. Alzheimer Dement. 2007;3:186 - 91), while in the United States, it was reported that over 5 million people were living with AD (2010 Alzheimer’s disease facts and figures. Alzheimer Dement. 2010;6:158 - 94). By 2050, the number of AD patients worldwide is predicted to reach 106.8 million cases (range: 47.2 million - 221.2 million cases), while in the United States alone, the number of patients is estimated to be 11 million - 16 million cases. (Brookmeyer, supra, and 2010 Alzheimer’s disease facts and figures, supra).

[0003] This disease is generally associated with a general decline in cognitive function, which progresses slowly and results in a bedridden state in the terminal stages. AD patients typically survive only 3 to 10 years after onset, although extreme cases of 2 to 20 years are known (Hebert, L.E., et al., Alzheimer disease in the U.S. population: prevalence estimates using the 2000 census. Arch Neurol. 2003;60:1119-1122). Despite the fact that the mortality rate attributed to AD is grossly underestimated because it is rarely listed as the cause of death on death certificates, AD is the seventh most common cause of all deaths in the United States and the fifth most common cause of death among Americans over age 65 (2010 Alzheimer’s disease facts and figures, supra).

[0004] Histologically, this disease is characterized by senile plaques that are mainly found in the association cortex, limbic system, and basal ganglia. The main component of these senile plaques is amyloid-beta peptide (Aβ). Aβ exists in various conformational states - monomers, oligomers, protofibrils, and insoluble fibrils. The detailed mechanistic relationship between the onset of Alzheimer's disease and Aβ production is unknown. However, currently, several anti-Aβ antibodies are in clinical trials as potential therapeutic agents for Alzheimer's disease.

[0005] Anti-Aβ antibodies and other proteins can be administered to a subject by intravenous, subcutaneous, intramuscular, and other means. The dosage and / or dosage form of the antibody can present many challenges in the development of suitable pharmaceutical formulations. For example, at high antibody concentrations, the stability of the antibody can be a problem due to the formation of protein aggregates or fragmentation. Generally, aggregation increases with increasing antibody concentration. Furthermore, to achieve long-term protein stability and shelf life for high-concentration antibody formulations, high concentrations of stabilizers and other excipients are required. High-concentration antibody formulations are also often viscous, which can complicate the manufacture and administration of pharmaceutical formulations. SUMMARY OF THE INVENTION

Means for Solving the Problem

[0006] The present specification provides a pharmaceutical formulation comprising a therapeutically effective amount of at least one isolated anti-Aβ protofibril antibody or a fragment thereof that binds to Aβ protofibrils. The present specification also provides a pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to Aβ protofibrils at 80 - 300 mg / ml, wherein the antibody is BAN2401 (also known as lecanemab). The pharmaceutical formulations provided herein have been found to be advantageous. For example, despite high concentrations of anti-Aβ protofibril antibody (e.g., 100 mg / mL or 200 mg / mL), the protein - protein aggregation rate is unexpectedly low and is equivalent to the aggregation rate typically seen at much lower antibody concentrations (e.g., 10 mg / mL). In some embodiments, the pharmaceutical formulations disclosed herein exhibit a lower initial aggregate rate than formulations having a significantly lower concentration of anti-Aβ protofibril antibody (e.g., an initial aggregate level of about 0.3% for 100 mg / mL anti-Aβ protofibril antibody versus an initial aggregate level of about 0.8% for 10 mg / mL anti-Aβ protofibril antibody). In some embodiments, the pharmaceutical formulations disclosed herein exhibit a lower subvisible particle generation rate than formulations having a significantly lower concentration of anti-Aβ protofibril antibody (e.g., 10.6 particles / mL for 100 mg / mL anti-Aβ protofibril antibody versus 12.6 particles / mL for 10 mg / mL anti-Aβ protofibril antibody). In some embodiments, the pharmaceutical formulations disclosed herein exhibit a decrease in aggregation rate, a decrease in initial aggregation level, a decrease in protein fragmentation rate, and / or a decrease in subvisible particle formation when compared to formulations having a significantly lower concentration of anti-Aβ protofibril antibody. The low aggregation rate, initial aggregation level, protein fragmentation rate, and / or subvisible particle generation can result in higher stability and / or a longer product shelf life. Further, the excipients in the pharmaceutical formulations disclosed herein can be present in lower amounts than currently commercially available intravenous products. In some embodiments, the pH and osmolality of the pharmaceutical formulations disclosed herein are acceptable for intravenous administration after dilution in intravenous fluids.

[0007] In some embodiments, at least one anti-Aβ protofibril antibody comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0008] In some embodiments, at least one anti-Aβ protofibril antibody comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 5 (HCDR1), SEQ ID NO: 6 (HCDR2), and SEQ ID NO: 7 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3).

[0009] The amino acid assignments for each domain generally follow the definitions of SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (Kabat et al., 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, hereinafter referred to as the "Kabat report").

[0010] In some embodiments, at least one anti-Aβ protofibril antibody comprises a human constant region. In some embodiments, the human constant region of at least one anti-Aβ protofibril antibody comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE, and any allelic variants thereof as disclosed in the Kabat report. Any one or more of such sequences can be used in the present disclosure. In some embodiments, the heavy chain constant region is selected from IgG1 and its allelic variants. The amino acid sequence of the human IgG1 constant region is known in the art and is shown in SEQ ID NO: 3.

[0011] In some embodiments, the human constant region of at least one anti-Aβ antibody comprises a light chain constant region selected from the κ-λ chain constant region and any allelic variants as contemplated in the Kabat report. Any one or more of such sequences may be used in the present disclosure. In some embodiments, the light chain constant region is selected from κ and its allelic variants. The amino acid sequence of the human κ chain constant region is known in the art and is shown in SEQ ID NO: 4.

[0012] In some embodiments, at least one anti-Aβ protofibril antibody comprises human heavy and light chain variable region frameworks. In some embodiments, at least one anti-Aβ protofibril antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, at least one anti-Aβ protofibril antibody comprises a human IgG1 heavy chain constant region and a human Ig kappa light chain constant region. In some embodiments, at least one anti-Aβ protofibril antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 4.

[0013] In some embodiments, at least one anti-Aβ protofibril antibody is BAN2401, also known as lecanemab. BAN2401 is a humanized IgG1 monoclonal version of mAb158, a mouse monoclonal antibody produced to target protofibrils, and is disclosed in WO 2007 / 108756 pamphlet and Journal of Alzheimer’s Disease 43:575-588 (2015). BAN2401 is at least one anti-Aβ protofibril antibody that exhibits low affinity for Aβ monomers while binding to soluble Aβ aggregate species with high selectivity. For example, BAN2401 has been reported to exhibit approximately 1000-fold and 5- to 10-fold higher selectivity for soluble Aβ protofibrils compared to Aβ monomers or Aβ insoluble fibrils, respectively.

[0014] BAN2401 comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. The full-length sequences of the heavy and light chains of BAN2401 are shown in SEQ ID NOs: 11 and 12, and are described in WO 2007 / 108756 pamphlet and Journal of Alzheimer’s Disease 43:575-588 (2015).

[0015] Other non-limiting examples of antibodies suitable for use as at least one anti-Aβ protofibril antibody in the present disclosure include those disclosed in WO 2002 / 003911 pamphlet, WO 2005 / 123775 pamphlet, WO 2007 / 108756 pamphlet, WO 2011 / 001366 pamphlet, WO 2011 / 104696 pamphlet, and WO 2016 / 005466 pamphlet.

[0016] In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration of at least 80 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration of at least 100 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration of at least 200 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration of at least 250 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration in the range of 80 mg / mL to 300 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration in the range of 85 mg / mL to 275 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration in the range of 90 mg / mL to 250 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration in the range of 95 mg / mL to 225 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration in the range of 100 mg / mL to 200 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration of 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, or 300 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration of 100 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration of 200 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration of 250 mg / mL. In some embodiments, the isolated antibody or fragment thereof is present at a concentration of 300 mg / mL. In some embodiments, the isolated antibody or fragment thereof is BAN2401.In some embodiments, the isolated antibody or fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated antibody or fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 5 (HCDR1), SEQ ID NO: 6 (HCDR2), and SEQ ID NO: 7 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3).

[0017] In some embodiments, a pharmaceutical formulation comprising a therapeutically effective amount of at least one isolated anti-Aβ protofibril antibody or fragment thereof that binds to Aβ protofibrils further comprises at least one additional component. In some embodiments, at least one additional component in the pharmaceutical formulation is selected from buffering agents. In some embodiments, the buffering agent is a citrate buffer. In some embodiments, the buffering agent is a histidine buffer. In some embodiments, at least one additional component in the pharmaceutical formulation is selected from emulsifying agents. In some embodiments, at least one additional component in the pharmaceutical formulation is selected from citric acid (or citric acid monohydrate), sodium chloride, histidine (and / or histidine hydrochloride), arginine (and / or arginine hydrochloride), and polysorbate 80. In some embodiments, at least one additional component in the pharmaceutical formulation is selected from citric acid (and / or citric acid monohydrate), arginine (and / or arginine hydrochloride), and polysorbate 80. In some embodiments, at least one additional component in the pharmaceutical formulation is selected from histidine (and / or histidine hydrochloride), arginine (and / or arginine hydrochloride), and polysorbate 80.

[0018] In some embodiments, the pharmaceutical formulation contains arginine (and / or arginine hydrochloride). In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation ranges from about 100 mM to about 400 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation ranges from about 110 mM to about 380 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation ranges from about 120 mM to about 360 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation ranges from about 125 mM to about 350 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is 125 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is 200 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is 350 mM.

[0019] In some embodiments, the pharmaceutical formulation contains histidine. In some embodiments, the concentration of histidine in the pharmaceutical formulation ranges from about 10 mM to about 100 mM. In some embodiments, the concentration of histidine in the pharmaceutical formulation ranges from 10 mM to 100 mM, 12 mM to 80 mM, 14 mM to 60 mM, 15 mM to 55 mM, 15 mM to 35 mM, or 15 mM to 25 mM. In some embodiments, the concentration of histidine is 25 mM. In some embodiments, the concentration of histidine is 50 mM.

[0020] In some embodiments, the pharmaceutical formulation comprises polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation ranges from about 0.01 - 0.1% w / v, 0.01 - 0.08% w / v, 0.02 - 0.08% w / v, 0.03 - 0.07% w / v, or 0.04 - 0.06% w / v. In some embodiments, polysorbate 80 is present in the pharmaceutical formulation at a concentration of 0.01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, or 0.08% w / v. In some embodiments, polysorbate 80 is present in the pharmaceutical formulation at a concentration of 0.02% w / v. In some embodiments, polysorbate 80 is present in the pharmaceutical formulation at a concentration of 0.05% w / v.

[0021] In some embodiments, the pharmaceutical formulation comprises citric acid monohydrate. In some embodiments, the concentration of citric acid monohydrate in the pharmaceutical formulation ranges from about 10 mM - 100 mM. In some embodiments, the concentration of citric acid monohydrate in the pharmaceutical formulation ranges from 10 mM - 100 mM, 10 mM - 90 mM, 15 mM - 85 mM, 20 mM - 80 mM, 25 mM - 75 mM, 30 mM - 70 mM, 30 mM - 60 mM, or 30 mM - 50 mM. In some embodiments, the concentration of citric acid monohydrate in the pharmaceutical formulation is 50 mM.

[0022] In some embodiments, the present disclosure provides a pharmaceutical formulation having a pH in the range of 4.5 - 5.5. In some embodiments, the pH of the pharmaceutical formulation ranges from 4.0 - 6.0, 4.2 - 5.8, 4.3 - 5.7, 4.4 - 5.6, or 4.5 - 5.5. In some embodiments, the pH is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the pH is 5.0.

[0023] In some embodiments, the pharmaceutical formulations disclosed herein may be in the form of a solution and / or any other suitable liquid formulation that those skilled in the art would consider appropriate. The route of administration of the compositions of the present disclosure may be intravenous or subcutaneous. In some embodiments, the pharmaceutical formulation is formulated as a sterile and non-pyrogenic liquid for intravenous administration. In some embodiments, the pharmaceutical formulation is formulated as a sterile and non-pyrogenic liquid for subcutaneous administration. In some embodiments, the pharmaceutical composition is physiological saline.

[0024] In some embodiments, the pharmaceutical formulation is a liquid dosage form that contains at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibril, and further contains, for example, citric acid monohydrate, arginine, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation contains at least one isolated anti-Aβ protofibril antibody, such as BAN2401 at 100 mg / mL, or a fragment thereof that binds to Aβ protofibril, 50 mM citric acid monohydrate, 110 mM arginine, 240 mM arginine hydrochloride, and 0.05% (w / v) polysorbate 80, and has a pH of 5.0 ± 0.4.

[0025] In some embodiments, the pharmaceutical formulation is a liquid dosage form that contains at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibril, and further contains, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation contains at least one isolated anti-Aβ protofibril antibody, such as BAN2401 at 100 mg / mL or 200 mg / mL, or a fragment thereof that binds to Aβ protofibril, 25 mM histidine and histidine hydrochloride, 200 mM arginine hydrochloride, and 0.05% (w / v) polysorbate 80, and has a pH of 5.0 ± 0.4.

[0026] In some embodiments, the pharmaceutical formulation comprises at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibrils, and is in a liquid dosage form that further comprises, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises at least one isolated anti-Aβ protofibril antibody, such as BAN2401 at 200 mg / mL, or a fragment thereof that binds to Aβ protofibrils, 50 mM histidine and histidine hydrochloride, 125 mM arginine hydrochloride, and 0.02% (w / v) polysorbate 80, and has a pH of 5.0 ± 0.4.

[0027] In some embodiments, the pharmaceutical formulation comprises at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibrils, and is in a liquid dosage form that further comprises, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises at least one isolated anti-Aβ protofibril antibody, such as BAN2401 at 200 mg / mL, or a fragment thereof that binds to Aβ protofibrils, 50 mM citrate (and / or citrate monohydrate), 125 mM arginine (and / or arginine hydrochloride), and 0.02% (w / v) polysorbate 80, and has a pH of 5.0 ± 0.4.

[0028] Methods involving BAN2401 and the use of BAN2401 are disclosed in U.S. Provisional Patent Application No. 62 / 749,614 and PCT International Application No. PCT / US2019 / 043067, both of which are incorporated herein by reference in their entirety.

Brief Description of the Drawings

[0029]

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Mode for Carrying Out the Invention

[0030] Definition The following are the definitions of the terms used in this application.

[0031] As used herein, the singular terms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0032] As used herein, the phrase "and / or" means "either or both" of the elements so connected, i.e., elements that may be present in conjunctive or disjunctive connection in some cases. Thus, as a non-limiting example, when "A and / or B" is used in conjunction with an open-ended phrase such as "comprising", in some embodiments it refers to only A (optionally including elements other than B); in other embodiments it refers to only B (optionally including elements other than A); and in still other embodiments it refers to both A and B (optionally including other elements), etc.

[0033] As used herein, "at least one" means one or more of the elements in a list of elements, but does not necessarily include at least one of each and every element specifically recited in the list of elements, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically recited in the list of elements to which the phrase "at least one" refers, whether or not those other elements are related to the specifically recited elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") can, in one embodiment, refer to at least one A, optionally including two or more, with no B present (and optionally including elements other than B); in another embodiment, it can refer to at least one B, optionally including two or more, with no A present (and optionally including elements other than A); and in still another embodiment, it can refer to at least one A, optionally including two or more, and at least one B, optionally including two or more (and optionally including other elements), etc.

[0034] It should be understood that when a number is recited alone or as part of a numerical range, the number can vary up and down from the value explicitly stated by only a reasonable difference for the stated value, as would be recognized by one of ordinary skill in the art.

[0035] As used herein, an antibody "fragment" refers to a portion of an antibody, including, for example, the antigen-binding region or a portion containing the variable region thereof. Non-limiting examples of fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, linear antibodies, and single-chain antibody molecules.

[0036] As used herein, "fragmentation" or "fragmentation" refers to the degradation of an antibody or fragment thereof when it is in a formulation or when added to a formulation. The fragments produced by fragmentation or fragmentation may or may not have the ability to bind to the antigen to which the antibody or fragment thereof binds.

[0037] As used herein, "histidine buffer" may include histidine, histidine hydrochloride, or a combination thereof, where the histidine hydrochloride may be histidine hydrochloride monohydrate.

[0038] As used herein, "citrate buffer" may include citric acid, a salt thereof, or a combination thereof, where the citric acid may be citric acid monohydrate or anhydrous citric acid.

[0039] Non-limiting embodiments of the present disclosure: Certain embodiments of the present disclosure relate to aqueous pharmaceutical formulations.

[0040] In some embodiments, (a) an isolated anti-Aβ protofibril antibody or fragment thereof, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, at a concentration of 80 to 300 mg / ml; and (b) 100 mM to 400 mM arginine, which is arginine, arginine hydrochloride, or a combination thereof. and optionally including: a. 0.01% w / v~0.1% w / v polysorbate 80; b. A pharmaceutically acceptable buffer, optionally a histidine buffer or a citrate buffer; and c. A pH of 4.5 to 5.5 An aqueous pharmaceutical formulation comprising any one or more of the above is provided.

[0041] In some embodiments, (a) An isolated anti-Aβ protofibril antibody or a fragment thereof comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, at a concentration of 80 to 300 mg / ml; and (b) A pharmaceutically acceptable buffer, wherein the buffer is a histidine buffer, optionally at a concentration of about 25 mM comprising, optionally, the following: a. 0.01% w / v to 0.1% w / v polysorbate 80; b. 100 mM to 400 mM arginine, arginine hydrochloride, or a combination thereof; and c. A pH of 4.5 to 5.5 An aqueous pharmaceutical formulation comprising any one or more of the above is provided.

[0042] Some embodiments relate to (a) An isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibrils, at a concentration of 80 mg / mL to 300 mg / mL, (b) 100 mM to 400 mM arginine, (c) 0.01% w / v to 0.1% w / v polysorbate 80, and (d) A pharmaceutically acceptable buffer in an aqueous pharmaceutical formulation, the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the antibody or fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0043] Antibody concentration For any of the aqueous pharmaceutical formulations described herein, the antibody can be present at the following concentrations.

[0044] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration of 100 mg / mL or more. In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration of 100 mg / mL.

[0045] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration of 200 mg / mL or more. In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration of 200 mg / mL.

[0046] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration in the range of 80 mg / mL to 300 mg / mL. In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration in the range of 80 mg / mL to 240 mg / mL.

[0047] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration in the range of 100 mg / mL to 200 mg / mL.

[0048] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration in the ranges of 80 mg / mL to 300 mg / mL, 85 mg / mL to 275 mg / mL, 90 mg / mL to 250 mg / mL, 95 mg / mL to 225 mg / mL, 100 to 200 mg / mL. In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or a fragment thereof is present at a concentration in the ranges of 90 mg / mL to 220 mg / mL, 100 mg / mL to 210 mg / mL, or 110 mg / mL to 200 mg / mL.

[0049] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, or 300 mg / mL.

[0050] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL.

[0051] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 200 mg / mL.

[0052] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or fragment thereof is BAN2401.

[0053] Arginine For any of the aqueous pharmaceutical formulations described herein, the formulation may contain arginine as follows.

[0054] In some embodiments, the pharmaceutical formulation contains arginine. In some embodiments, the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0055] In some embodiments of the pharmaceutical formulation, the concentration of arginine ranges from about 100 mM to about 400 mM.

[0056] In some embodiments of the pharmaceutical preparation, the concentration of arginine ranges from 100 mM to 400 mM, 110 mM to 380 mM, 120 mM to 360 mM, 125 mM to 350 mM, 100 mM to 200 mM, 125 mM to 200 mM, or 150 mM to 200 mM. In some embodiments of the pharmaceutical preparation, the concentration of arginine ranges from 150 mM to 250 mM, 160 mM to 240 mM, 170 mM to 230 mM, 180 mM to 220 mM, 190 mM to 210 mM of arginine, arginine hydrochloride, or a combination thereof.

[0057] In some embodiments of the pharmaceutical preparation, the concentration of arginine is 125 mM.

[0058] In some embodiments of the pharmaceutical preparation, the concentration of arginine is 200 mM.

[0059] In some embodiments of the pharmaceutical preparation, the concentration of arginine ranges from 200 mM to 400 mM, 210 mM to 390 mM, 220 mM to 380 mM, 230 mM to 370 mM, 240 mM to 360 mM, 240 mM to 350 mM, or 250 mM to 350 mM.

[0060] In some embodiments of the pharmaceutical preparation, the concentration of arginine is 350 mM.

[0061] Polysorbate 80 (PS80) For any of the aqueous pharmaceutical preparations described herein, the preparation may contain polysorbate 80 as follows.

[0062] In some embodiments of the pharmaceutical preparation, the concentration of polysorbate 80 ranges from about 0.01% w / v to 0.1% w / v, 0.01% w / v to 0.08% w / v, 0.02% w / v to 0.08% w / v, 0.03% w / v to 0.07% w / v, or 0.04% w / v to 0.06% w / v.

[0063] In some embodiments of the pharmaceutical formulation, polysorbate 80 is present at a concentration of 0.01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, or 0.08% w / v.

[0064] In some embodiments of the pharmaceutical formulation, polysorbate 80 is present at a concentration of 0.02% w / v.

[0065] In some embodiments of the pharmaceutical formulation, polysorbate 80 is present at a concentration of 0.05% w / v.

[0066] Buffer For any of the aqueous pharmaceutical formulations described herein, the formulation may contain a pharmaceutically acceptable buffer as follows.

[0067] In some embodiments of the pharmaceutical formulation, the pharmaceutically acceptable buffer is a citrate buffer.

[0068] In some embodiments of the pharmaceutical formulation, the citrate buffer is present at a concentration of about 10 mM to about 100 mM.

[0069] In some embodiments of the pharmaceutical formulation, the concentration of the citrate buffer ranges from 10 mM to 100 mM, 10 mM to 90 mM, 15 mM to 85 mM, 20 mM to 80 mM, 25 mM to 75 mM, 30 mM to 70 mM, 30 mM to 60 mM, or 30 mM to 50 mM.

[0070] In some embodiments of the pharmaceutical formulation, the citrate buffer is present at a concentration of 50 mM.

[0071] In some embodiments of the pharmaceutical formulation, the pharmaceutically acceptable buffer is a histidine buffer.

[0072] In some embodiments of the pharmaceutical formulation, the histidine buffer is present at a concentration of about 10 mM to about 100 mM.

[0073] In some embodiments of the pharmaceutical preparation, the concentration of the histidine buffer is in the range of 10 mM to 100 mM, 12 mM to 80 mM, 14 mM to 60 mM, or 15 mM to 55 mM, 15 mM to 35 mM, or 15 mM to 25 mM.

[0074] In some embodiments of the pharmaceutical preparation, the histidine buffer is present at a concentration of 25 mM.

[0075] In some embodiments of the pharmaceutical preparation, the histidine buffer is present at a concentration of 50 mM.

[0076] In some embodiments of the pharmaceutical preparation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate. In some embodiments of the pharmaceutical preparation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, wherein the histidine is at a concentration of about 0.1 - 0.3 mg / mL and the histidine hydrochloride monohydrate is at a concentration of about 4 - 6 mg / mL. In some embodiments of the pharmaceutical preparation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, wherein the histidine is at a concentration of about 0.26 mg / mL and the histidine hydrochloride monohydrate is at a concentration of about 4.89 mg / mL. In some embodiments of the pharmaceutical preparation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, wherein the histidine comprises 0.2 - 0.3 mg / mL of histidine and 4.4 - 4.9 mg / mL of histidine hydrochloride, and optionally the histidine hydrochloride is the monohydrate.

[0077] pH For any of the aqueous pharmaceutical preparations described herein, the preparation may comprise a pH as follows.

[0078] In some embodiments of the pharmaceutical preparation, the pH is in the range of 4.0 - 6.0, 4.2 - 5.8, 4.3 - 5.7, 4.4 - 5.6 or 4.5 - 5.5.

[0079] In some embodiments of the pharmaceutical preparation, the pH is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

[0080] In some embodiments of the pharmaceutical formulation, the pH is 4.5 to 5.5.

[0081] In some embodiments of the pharmaceutical formulation, the pH is 5.0.

[0082] In some embodiments of the pharmaceutical formulation, the pharmaceutical formulation is suitable for intravenous injection.

[0083] In some embodiments of the pharmaceutical formulation, the pharmaceutical formulation is suitable for subcutaneous injection.

[0084] In some embodiments of the aqueous pharmaceutical formulation, the pharmaceutical formulation contains methionine.

[0085] In some embodiments, (a) 80 mg / mL to 240 mg / mL of BAN2401, (b) 140 mM to 260 mM of arginine hydrochloride, (c) 0.01% w / v to 0.1% w / v of polysorbate 80, and (d) 15 mM to 35 mM of histidine buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0086] In some embodiments, : (a) (i) An 80 mg / mL to 240 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, (b) 140 mM to 260 mM of arginine hydrochloride, (c) 0.01% w / v to 0.1% w / v of polysorbate 80, and (d) 15 mM to 35 mM of histidine buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0087] In some embodiments, (a) an isolated anti-Aβ protofibril antibody or fragment thereof at 80 mg / mL to 240 mg / mL, comprising (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 12; (b) 140 mM to 260 mM arginine hydrochloride, (c) 0.01% w / v to 0.1% w / v polysorbate 80, and (d) 15 mM to 35 mM histidine buffer is disclosed, and the pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0088] In some embodiments, herein, (a) BAN2401 at 80 mg / mL to 120 mg / mL, (b) 240 mM to 360 mM arginine, (c) 0.03% w / v to 0.08% w / v polysorbate 80, and (d) 30 mM to 70 mM citrate buffer is disclosed, and the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0089] In some embodiments, herein, (a) an isolated anti-Aβ protofibril antibody or fragment thereof at 80 mg / mL to 120 mg / mL, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; (b) 240 mM to 360 mM arginine, (c) 0.03% w / v to 0.08% w / v polysorbate 80, and (d) 30 mM to 70 mM citrate buffer is disclosed, This pharmaceutical preparation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0090] In some embodiments, this specification provides (a) (i) an isolated anti-Aβ protofibril antibody or a fragment thereof at 80 mg / mL to 120 mg / mL comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 12, (b) 240 mM to 360 mM arginine, (c) 0.03% w / v to 0.08% w / v polysorbate 80, and (d) 30 mM to 70 mM citrate buffer An aqueous pharmaceutical preparation comprising the above is disclosed. This pharmaceutical preparation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0091] In some embodiments, this specification provides (a) 100 mg / mL BAN2401, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer An aqueous pharmaceutical preparation comprising the above is disclosed. This pharmaceutical preparation has a pH in the range of 4.5 to 5.5.

[0092] In some embodiments, this specification provides (a) (i) an isolated anti-Aβ protofibril antibody or a fragment thereof at 100 mg / mL comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer An aqueous pharmaceutical preparation comprising the above is disclosed. This pharmaceutical preparation has a pH in the range of 4.5 to 5.5.

[0093] In some embodiments, this specification discloses (a) (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 12, of a 100 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer and an aqueous pharmaceutical preparation is disclosed, This pharmaceutical preparation has a pH in the range of 4.5 to 5.5.

[0094] In some embodiments, this specification discloses (a) 200 mg / mL BAN2401, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer and an aqueous pharmaceutical preparation is disclosed, This pharmaceutical preparation has a pH in the range of 4.5 to 5.5.

[0095] In some embodiments, this specification discloses (a) (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, of a 200 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer and an aqueous pharmaceutical preparation is disclosed, This pharmaceutical preparation has a pH in the range of 4.5 to 5.5.

[0096] In some embodiments, this specification discloses (a) (i) A 200 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 12, (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0097] In some embodiments, herein, (a) 100 mg / mL BAN2401, (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0098] In some embodiments, herein, (a) (i) A 100 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0099] In some embodiments, herein, (a) (i) A 100 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 12, (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer An aqueous pharmaceutical formulation comprising the same is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0100] In some embodiments, herein, (a) 150 mg / mL to 250 mg / mL BAN2401, (b) 100 mM to 150 mM arginine hydrochloride, (c) 0.01% w / v to 0.05% w / v polysorbate 80, and (d) 35 mM to 65 mM histidine buffer An aqueous pharmaceutical formulation comprising the same is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0101] In some embodiments, herein, (a) (i) A 150 mg / mL to 250 mg / mL isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, (b) 100 mM to 150 mM arginine hydrochloride, (c) 0.01% w / v to 0.05% w / v polysorbate 80, and (d) 35 mM to 65 mM histidine buffer An aqueous pharmaceutical formulation comprising the same is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0102] In some embodiments, herein, (a) (i) A heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and (ii) A light chain comprising the amino acid sequence of SEQ ID NO: 12, an isolated anti-Aβ protofibril antibody or a fragment thereof at a concentration of 150 mg / mL to 250 mg / mL, (b) 100 mM to 150 mM arginine hydrochloride, (c) 0.01% w / v to 0.05% w / v polysorbate 80, and (d) 35 mM to 65 mM histidine buffer An aqueous pharmaceutical formulation comprising is disclosed, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0103] In some embodiments, the aqueous pharmaceutical formulation has a pH of 5.0.

[0104] Method for reducing aggregate formation In some embodiments herein, a method for reducing aggregate formation of an isolated anti-Aβ protofibril antibody or a fragment thereof, (a) Providing a pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibril at a concentration of 50 mg / ml or higher, optionally at a concentration of 80 mg / mL to 300 mg / mL, and optionally at a concentration of 100 mg / mL to 200 mg / mL, and (b) Adding arginine, arginine hydrochloride, or a combination thereof, and optionally providing a pharmaceutically acceptable buffer, wherein the buffer is optionally a histidine buffer A method comprising is disclosed, wherein the isolated anti-Aβ protofibril antibody or a fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0105] In some embodiments of the method for reducing aggregate formation, the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5. In some embodiments of the method, the pH of the pharmaceutical formulation is 5.0.

[0106] In some embodiments of the method for reducing aggregate formation, arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 150 mM to 250 mM.

[0107] In some embodiments, provided herein is a method for reducing fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (a) providing a pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibril at a concentration of 50 mg / ml or more, optionally at a concentration of 80 mg / mL to 300 mg / mL, and optionally at a concentration of 100 mg / mL to 200 mg / mL, and (b) adding a histidine buffer, and optionally further providing arginine, arginine hydrochloride, or a combination thereof. The method is disclosed, wherein the isolated anti-Aβ protofibril antibody or a fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0108] In some embodiments of the method for reducing fragmentation, the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5. In some embodiments of the method, the pH of the pharmaceutical formulation is 5.0.

[0109] In some embodiments of the method for reducing fragmentation, arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 100 mM to 400 mM.

[0110] In some embodiments of the method for reducing fragmentation, the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v polysorbate 80.

[0111] In some embodiments of the method for reducing fragmentation, the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer, wherein the buffer is a histidine buffer, and optionally, the histidine buffer ranges from 10 mM to 100 mM, 12 mM to 80 mM, 14 mM to 60 mM, or 15 mM to 55 mM, 15 mM to 35 mM, or 15 mM to 25 mM.

[0112] In some embodiments, provided herein is a method for reducing aggregate formation and / or fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, (a) providing a pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibrils at a concentration of 50 mg / ml or greater, optionally at a concentration of 80 mg / mL to 300 mg / mL, and optionally at a concentration of 100 mg / mL to 200 mg / mL, (b) adding arginine, arginine hydrochloride, or a combination thereof, and (c) providing a pharmaceutically acceptable histidine buffer is disclosed, wherein the isolated anti-Aβ protofibril antibody or a fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0113] In some embodiments, provided herein is a method for reducing aggregate formation and / or fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, (a) providing a pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibrils at a concentration of 50 mg / ml or greater, optionally at a concentration of 80 mg / mL to 300 mg / mL, and optionally at a concentration of 100 mg / mL to 200 mg / mL, (b) providing a histidine buffer, and (c) providing arginine, arginine hydrochloride, or a combination thereof A method is disclosed, wherein the formulation has a pH of 4.5 to 5.5, and the isolated anti-Aβ protofibril antibody or fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0114] Also provided herein are the following embodiments:

[0115] [Embodiment 01] a. An isolated anti-Aβ protofibril antibody or fragment thereof that binds to human Aβ protofibril at a concentration of 80 mg / mL to 300 mg / mL, b. 100 mM to 400 mM arginine, c. 0.01% w / v to 0.1% w / v polysorbate 80, and d. A pharmaceutically acceptable buffer An aqueous pharmaceutical formulation comprising: The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, The isolated anti-Aβ protofibril antibody or fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and The arginine is arginine, arginine hydrochloride, or a combination thereof.

[0116] [Embodiment 02] The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL to 200 mg / mL.

[0117] [Embodiment 03] The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL.

[0118] [Embodiment 04] The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 200 mg / mL.

[0119] [Embodiment 05] The pharmaceutical preparation according to any one of claims 1 to 4, further comprising methionine.

[0120] [Embodiment 06] The pharmaceutical preparation according to any one of claims 1 to 5, wherein the pharmaceutically acceptable buffer is a citrate buffer or a histidine buffer.

[0121] [Embodiment 07] The pharmaceutical preparation according to any one of claims 1 to 6, comprising a 10 to 100 mM citrate buffer or a 10 to 100 mM histidine buffer.

[0122] [Embodiment 08] The pharmaceutical preparation according to any one of claims 1 to 7, comprising 125 to 350 mM arginine.

[0123] [Embodiment 09] The pharmaceutical preparation according to any one of claims 1 to 8, comprising 200 mM arginine which is arginine hydrochloride.

[0124] [Embodiment 10] The pharmaceutical preparation according to any one of claims 1 to 9, comprising 200 mM arginine which is arginine hydrochloride and a 25 mM histidine buffer.

[0125] [Embodiment 11] a. An isolated anti-Aβ protofibril antibody or a fragment thereof at 80 mg / mL to 240 mg / mL, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, b. A 140 mM to 260 mM arginine hydrochloride, c. A 0.02% w / v to 0.08% w / v polysorbate 80, and d. A 15 mM to 35 mM histidine buffer which is an aqueous pharmaceutical preparation comprising a pharmaceutical preparation having a pH in the range of 4.5 to 5.5.

[0126] [Embodiment 12] a. An isolated anti-Aβ protofibril antibody or a fragment thereof at a concentration of 80 mg / mL to 120 mg / mL, comprising (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2, b. 240 mM to 360 mM arginine, c. 0.02% w / v to 0.08% w / v polysorbate 80, and d. 30 mM to 50 mM citrate buffer An aqueous pharmaceutical formulation comprising: The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and arginine is arginine, arginine hydrochloride, or a combination thereof. A pharmaceutical formulation.

[0127] [Embodiment 13] A method for reducing aggregate formation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) Providing an aqueous pharmaceutical formulation containing an isolated anti-Aβ protofibril antibody or a fragment thereof containing a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2 at a concentration of 80 mg / ml to 300 mg / ml; and Adding arginine, arginine hydrochloride, or a combination thereof to the aqueous pharmaceutical formulation, wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5. A method.

[0128] [Embodiment 14] The method according to claim 13, wherein arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 150 to 250 mM.

[0129] [Embodiment 15] The method according to claim 14, wherein arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 200 mM.

[0130] [Embodiment 16] The method according to any one of claims 13 to 15, wherein the pharmaceutical preparation further comprises a pharmaceutically acceptable buffer.

[0131] [Embodiment 17] The method according to claim 16, wherein the pharmaceutically acceptable buffer is a histidine buffer or a citrate buffer.

[0132] [Embodiment 18] A method for reducing fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) providing an aqueous pharmaceutical preparation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 at a concentration of 80 mg / ml to 300 mg / ml; and adding a histidine buffer to the aqueous pharmaceutical composition The method, wherein the pH of the pharmaceutical preparation is in the range of 4.5 to 5.5.

[0133] [Embodiment 19] The method according to claim 18, comprising 15 to 35 mM of a histidine buffer.

[0134] [Embodiment 20] The method according to any one of claims 13 to 19, wherein the pharmaceutical preparation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80.

[0135] [Embodiment 21] a. An isolated anti-Aβ protofibril antibody or a fragment thereof at 80 mg / mL to 240 mg / mL, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; b. 140 mM to 260 mM of arginine, arginine hydrochloride, or a combination thereof; c. 0.01% w / v to 0.1% w / v of polysorbate 80; and d. 15 mM to 35 mM of a histidine buffer or 30 mM to 50 mM of a citrate buffer An aqueous pharmaceutical formulation comprising A pharmaceutical formulation having a pH in the range of 4.5 to 5.5.

[0136] [Embodiment 22] The pharmaceutical formulation according to Embodiment 21, comprising 100 mg / mL of an antibody.

[0137] [Embodiment 23] The pharmaceutical formulation according to Embodiment 21, comprising 200 mg / mL of an antibody.

[0138] [Embodiment 24] The pharmaceutical formulation according to Embodiment 21, comprising an antibody in the range of 90 mg / mL to 220 mg / mL, 100 mg / mL to 210 mg / mL, or 110 mg / mL to 200 mg / mL.

[0139] [Embodiment 25] The pharmaceutical formulation according to any one of Embodiments 21 to 24, comprising a histidine buffer, wherein the histidine buffer comprises 15 mM to 30 mM, 15 mM to 25 mM, or 20 mM to 30 mM of histidine.

[0140] [Embodiment 26] The pharmaceutical formulation according to any one of Embodiments 21 to 25, comprising a histidine buffer, wherein the histidine buffer comprises 0.2 to 0.3 mg / mL of histidine and 4.4 to 4.9 mg / mL of histidine hydrochloride, and optionally the histidine hydrochloride is monohydrate.

[0141] [Embodiment 27] The pharmaceutical formulation according to any one of Embodiments 21 to 26, comprising 150 mM to 250 mM, 160 mM to 240 mM, 170 mM to 230 mM, 180 mM to 220 mM, 190 mM to 210 mM of arginine, arginine hydrochloride, or a combination thereof.

[0142] [Embodiment 28] The pharmaceutical preparation according to any one of Embodiments 21 to 27, comprising 0.02% w / v to 0.05%, or 0.04% w / v to 0.04% w / v of polysorbate 80.

[0143] [Embodiment 29] The pharmaceutical preparation according to any one of Embodiments 21 to 28, comprising a citrate buffer.

[0144] [Embodiment 30] The pharmaceutical preparation according to any one of Embodiments 21 to 29, comprising a histidine buffer.

[0145] [Embodiment 31] The pharmaceutical preparation according to any one of Embodiments 21 to 30, further comprising methionine.

[0146] [Embodiment 32] a. An antibody at 100 mg / ml to 200 mg / ml, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; b. 200 mM arginine hydrochloride; c. 0.05% w / v polysorbate 80; and d. 25 mM histidine buffer An aqueous pharmaceutical preparation comprising: A pharmaceutical preparation having a pH in the range of 4.5 to 5.5.

[0147] [Embodiment 33] The pharmaceutical preparation according to Embodiment 32, comprising 100 mg / mL of an antibody.

[0148] [Embodiment 34] The pharmaceutical preparation according to Embodiment 32, comprising 200 mg / mL of an antibody.

[0149] [Embodiment 35] A method for reducing aggregate formation and / or fragmentation of an antibody, comprising: a. To provide an aqueous pharmaceutical formulation containing an isolated anti-Aβ protofibril antibody or a fragment thereof comprising (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2 at a concentration of 80 mg / ml to 300 mg / ml, and b. Adding to the aqueous pharmaceutical composition 1) arginine, arginine hydrochloride, or a combination thereof, or 2) a histidine buffer A method comprising.

[0150] [Embodiment 36] The method according to Embodiment 35, wherein the pH of the pharmaceutical formulation ranges from 4.5 to 5.5.

[0151] [Embodiment 37] The method according to Embodiment 35, wherein arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 150 to 250 mM.

[0152] [Embodiment 38] The method according to Embodiment 37, wherein arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 200 mM.

[0153] [Embodiment 39] The method according to Embodiment 38, comprising a histidine buffer of 15 to 25 mM.

[0154] [Embodiment 40] The method according to any one of Embodiments 35 to 39, wherein the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v polysorbate 80.

[0155] [Embodiment 41] The method according to any one of Embodiments 35 to 40, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer.

[0156] [Embodiment 42] The method according to Embodiment 41, wherein the pharmaceutically acceptable buffer is a histidine buffer or a citrate buffer. [Example]

[0157] Example 1: Protein Concentration Test Samples of three protein concentrations (10, 100, 200 mg / mL) were prepared and examined in a concentration test. The 10 mg / mL material was the BAN2401 purified drug substance (PDS), and the 100 and 200 mg / mL materials were prepared from the BAN2401 PDS using an Amicon Ultra-15 spin filter. All samples were in the same formulation buffer (25 mM sodium citrate, 125 mM sodium chloride, pH 5.7), except that each had a different PS80 level. The proportions of PS80 in the 10, 100, 200 mg / mL samples were 0.02%, 0.16%, and 0.32%, respectively. All samples were filtered through a 0.2 gm filter, aliquoted for stability testing, and placed in sterile polypropylene (PP) tubes (Note: The PS80 removal process was not available at the time this concentration test was conducted).

[0158] Sample stability was evaluated over 3 months at two temperatures (5 and 25 °C) using native HPLC-SEC, pH, and DLS. For the T = 0 samples, additional characterization assays such as PS80 inspection were performed. The details of the sample tests are shown in Table 1.

[0159] [Table 1]

[0160] pH was measured using a pH meter equipped with a microprobe. Calibration was performed using pH 4.0 and 7.0 standards prior to measurement.

[0161] Aggregation and physical degradation of concentrated BAN2401 were determined by performing native HPLC-SEC on stability samples stored at 5 and 25 °C.

[0162] HPLC-SEC (native) analysis was performed using a TSK G3000 SWXL column with a mobile phase of 0.2 M sodium phosphate, pH 7.0, at a flow rate of 1.0 mL / min. The sample injection volume was 15, 1.5, or 0.8 μL for protein concentrations of 10, 100, or 200 mg / mL, respectively. This ensured that approximately 150 pg of protein was injected onto the column through this test. Results of the relative peak areas (%) for the monomer, aggregate, and fragment are reported in FIGS. 1-6.

[0163] At both temperatures (5 and 25 °C), an increase in protein concentration was seen to result in higher aggregate ratios and aggregation rates. For the 100 and 200 mg / mL samples, the initial aggregate ratio was approximately twice that of the 10 mg / mL sample. This indicated that protein aggregation was caused by just the concentration step alone. Further aggregation occurred during storage at 5 and 25 °C. The average aggregation rate over 3 months at 5 °C was 0.17% per month for 100 mg / mL and 0.20% per month for 200 mg / mL. For comparison, the 10 mg / mL sample exhibited an aggregation rate of only 0.07% per month under the same conditions. In summary, BAN2401 at 100 mg / mL or higher appeared to have no physical stability in the formulation.

[0164] Protein concentration was evaluated by UV by measuring the absorbance at 280 and 320 nm with a Beckman DU-800 spectrophotometer. The samples were diluted 500-fold and prepared in duplicate. The protein concentration was calculated using the following formula: Concentration = (A280 - A320) / ε × dilution factor using an extinction coefficient ε of 1.32.

[0165] Protein concentration was measured using an ultraviolet-visible spectrophotometer. The results are presented in Table 2. Overall, the concentration remained unchanged over 3 months at both temperatures.

[0166]

Table 2

[0167] Example 2: pH Screening Test The stability of 200 mg / mL of BAN2401 was evaluated at five different pH values. To prepare the samples, the BAN2401 PDS after PS80 removal was concentrated and diafiltered with a pH 4.5 buffer containing 50 mM sodium citrate and 100 mM sodium chloride. The final concentration was adjusted by dilution to achieve a concentration of 200 mg / mL of protein and 0.02% of PS80. The resulting material was divided into five aliquots; four of these aliquots were titrated with 10 N sodium hydroxide to create samples of different pH (i.e., pH 5.0, 5.5, 6.0, and 6.5). The resulting samples were filtered through a 0.2 μm filter and divided into sub-aliquots for stability evaluation and placed in sterile polypropylene (PP) tubes.

[0168] The stability of the samples was evaluated over three months at two temperatures (5 and 25 °C) using native HPLC-SEC, pH, and DLS. In addition, a stirring test was performed at the one-month time point, where a subset of the 5 °C samples was stirred horizontally at 250 RPM for three days in a 25 °C incubator. The details of the sample tests are shown in Table 3.

[0169]

Table 3

[0170] SEC-HPLC analysis was performed using a TSK G3000 SWXL column with a 0.2 M sodium phosphate, pH 7.0 mobile phase at a flow rate of 1.0 mL / min. The sample injection volume was 15, 1.5, or 0.8 μL for protein concentrations of 10, 100, or 200 mg / mL, respectively. This ensured that approximately 150 μg of protein was injected into the column through this test. Results of the relative peak area (%) were reported for the monomer, aggregate, and fragment.

[0171] Native HPLC-SEC was performed on stability samples stored at 5 and 25 °C to determine aggregation and physical degradation of concentrated BAN2401. The results are shown in Figures 7 to 12. BAN2401 appeared to be more stable at lower pH values at both temperatures (5 and 25 °C). Formulations at pH 4.5 and 5.0 started with a low aggregate ratio and exhibited a more gradual increase in aggregate formation, at a rate of approximately 0.07% per month at 5 °C. In addition to aggregation, protein fragmentation was also analyzed. These results suggested that fragmentation of BAN2401 was more prominent at lower pH values in the formulations of BAN2401. Based on these considerations, the formulation of BAN2401 at pH 5.0 was considered to be the most stable (i.e., exhibited less aggregation and / or physical degradation of BAN2401 compared to other formulations with different pH values).

[0172] Example 3: Excipient Screening Test In this test, 12 excipients were screened. To prepare a 200 mg / mL sample of BAN2401, BAN2401 after PS80 removal was concentrated by a TFF step and subsequently subjected to a diafiltration step with a base buffer (50 mM citrate, 0.02% PS80, pH 6.0). The concentrated material was divided into 24 aliquots. Each aliquot was spiked with a stock solution containing a specific excipient. For most excipients, two concentrations were examined, with the exceptions of sodium chloride and ascorbic acid, for which samples were prepared at three and one concentration levels, respectively. A list of the excipients used and their concentrations is shown in Table 4.

[0173] [Table 4]

[0174] All samples, including the control (i.e., the sample in the base buffer without excipient), were filtered through a 0.2 gm filter and then aseptically filled into BD syringes using a manual stopper tool. The control samples were also filled into glass vials. The vials and BD syringes were placed at 5 and 25 °C. Sample stability was evaluated using native HPLC-SEC and pH over a period of 2 months.

[0175] Samples for the excipient screening test were prepared under sub-optimal buffer (50 mM citrate, 0.02% PS80, pH 6.0) conditions to amplify the positive effect from the excipient in preventing aggregate formation. The stability profile (aggregation) of each formulation over 2 months at 25 °C was determined by performing native HPLC-SEC.

[0176] As shown in Figures 13 - 15, the formulation containing 160 mM arginine (F14) produced the lowest aggregation rate among the formulations. As a comparison, the 160 mM arginine formulation exhibited only 1.4% aggregates after 2 months at 25 °C, while the control (i.e., formulation - F0 in the base buffer without excipient) showed 2.4% aggregates at the same time point under the same conditions.

[0177] In addition, since the starting aggregate ratio was the lowest for the formulations containing arginine, it is pointed out that arginine has the ability to suppress aggregate formation during the protein concentration step. Fragmentation was slightly higher in the arginine formulation (F14) when compared to the control (F0). However, this difference could have been within the range of assay variability.

[0178] The next good excipient in this test was the formulation containing 400 mM proline (F10), but its effect in controlling aggregation was not as effective as the arginine formulation (F14).

[0179] The formulations containing sodium chloride (F16 - F18) showed no effect on stability when compared to the control samples. The same observation was made for the formulations containing PS80 (F19 and F20): no stability effect due to different PS80 contents was observed. The formulation containing ascorbic acid (F15) showed a dramatic effect with an increasing proportion of aggregates and fragments from the beginning of the test.

[0180] By measuring the pH of two arginine formulations (F13 and F14), it was confirmed whether there was no pH drift over time due to the presence of arginine in the formulations. As shown in Table 4, no change in pH was observed. The osmolality of the two formulations was also measured. The samples were stored at -20 °C and then thawed simultaneously for measurement.

[0181]

Table 5

[0182] Example 4: Arginine Concentration Test The relationship between the concentration of arginine in the formulation and aggregates was tested by Design Expert® 7.0. The factors selected for the test and the levels investigated for each factor are shown in Table 6 below.

[0183]

Table 6

[0184] Using the D - optimal design method of Design Expert® 7.0, randomization of the samples was generated. As shown in Table 7, the obtained sample set included 25 data points with 4 replicates. Each formulation was prepared and evaluated.

[0185]

Table 7

[0186] The relationship between the arginine concentration and the aggregate level in a 100 mg / mL BAN2401 formulation with pH 5.0 containing 0.02% polysorbate 80 was estimated by Design Expert (registered trademark) 7.0. The results of this estimation are shown in Figure 16.

[0187] Example 5: PS80 Test To evaluate the quality change of drug products under stress conditions, stirring and freeze-thaw tests were carried out using a formulation buffer (pH 5.0, 350 mM arginine, 50 mM citric acid). In this experiment, by examining formulations with various polysorbate 80 concentrations, the effect of polysorbate 80 on the generation of subvisible particles under such stress conditions was confirmed. The examined formulations and the applied stress conditions are shown in Table 8.

[0188]

Table 8

[0189] Samples were prepared in the same manner as described in Example 4. A 10% PS80 solution was added to achieve the target PS80 concentration in the formulation, and the final protein concentration was adjusted by diluting with the formulation buffer. The formulation was passed through a 0.2 gm filter and filled into a 2 mL vial with a filling volume of 1.3 mL. The sample was placed horizontally on an orbital shaker (lying the vial on its side), and then placed in a refrigerator or on a laboratory bench. The sample was shaken at 250 rpm for 3 days. Other samples were frozen by placing them in a -20 °C chamber for 2 hours and then thawed by taking them out and leaving them at room temperature for 2 hours. This freeze-thaw cycle was repeated 3 times. For the samples used in the stirring and freeze-thaw tests, the appearance, aggregate, and fragment levels were evaluated by SEC-HPLC, and the subvisible particles were evaluated by Micro Flow Imaging (MFI).

[0190] The results of the stirring and freeze-thaw tests for BAN2401 formulations containing various levels of PS80 are summarized below.

[0191] SEC-HPLC analysis SEC-HPLC analysis was performed using a TSK G3000 SWXL column with a mobile phase of 0.2 M sodium phosphate, pH 7.0, at a flow rate of 1.0 mL / min. The sample injection volume was 5.0 μL or 2.5 μL for protein concentrations of 50 or 100 mg / mL, respectively, ensuring that approximately 250 μg of protein was injected into the column through this test. Results of relative peak areas (%) were reported for monomers, aggregates, and fragments.

[0192] At the aggregate levels of pH 5 formulations with various polysorbate 80 concentrations stored at 25°C for 3 months, when the PS80 concentration was increased from 0% to 0.06% at a formulation pH of 5.0, there was no significant difference in the aggregate level. It was concluded that PS80 does not affect the formation of aggregates, namely dimers and trimers, as measured by SEC-HPLC during the storage of the BAN2401 formulation at 25°C.

[0193] In the effect of polysorbate 80 concentration on the fragment level in the pH 5 formulation, the polysorbate 80 concentration does not affect the fragment level in BAN2401.

[0194] In the appearance of the formulation after shaking at 250 rpm for 3 days at ambient temperature, the sample without PS80 was turbid with protein precipitation, while the other samples (0.02%, 0.05%, and 0.1% polysorbate 80) were visibly clear, particle-free solutions. All samples shaken at 250 rpm for 3 days at 5°C were clear with no precipitate. All samples subjected to freeze / thaw cycles were also clear with no precipitate.

[0195] Table 9 shows the aggregate and fragment levels of the BAN2401 formulation subjected to agitation and freeze-thaw. The stress condition that caused the greatest instability was agitation under ambient conditions. Under these conditions, the sample without PS80 showed the most formation of high molecular weight species and the greatest monomer loss. Increasing the PS80 concentration nullified this effect.

[0196]

Table 9

[0197] Subvisible particle analysis Subvisible particle analysis was performed using microflow imaging (DPA4100 Flow microscope and BP-4100-FC-400-UN flow cell). The total sample volume was 0.9 mL, and the results were reported for the ranges 2.25 - 100 μm, 5 - 100 μm, 10 - 100 μm, and 25 - 100 μm.

[0198] An increase in subvisible particles (2 - 10 pm) was observed in samples without PS80 that were subjected to shaking and freeze - thaw cycles. As the PS80 concentration in the formulation was increased, the generation of subvisible particles was gradually suppressed. This effect was observed in all the size ranges tested. There was no significant change in the subvisible particles in formulations containing 0.05% and 0.1% polysorbate 80. Therefore, a PS80 concentration of 0.05% was selected as being optimal for this formulation.

[0199] This data may suggest that PS80 has no effect on the formation of BAN2401 aggregates (dimers and trimers) and fragments as measured by SEC - HPLC. Therefore, the PS80 concentration was selected based on the results of the agitation and freeze - thaw tests. A PS80 concentration of 0.05% was selected to prevent the possibility of precipitation during transportation and to minimize the formation of subvisible particles.

[0200] Example 6: Preparation of an intravenous (IV) formulation of BAN2401 A. BAN2401 10 mg / mL formulation For the preparation of the sterile aqueous formulation, the BAN2401 10 mg / mL intravenous formulation (the "10 mg / mL injection solution") was manufactured by conventional cGMP sterile methods. The BAN2401 10 mg / mL injection solution was prepared from the corresponding BAN2401 drug substance formulation without the addition of any excipients and diluents. An exemplary IV formulation containing 10 mg / mL BAN2401 is shown in Table 10.

[0201] [Table 10]

[0202] As shown in Figure 17, the filtered BAN2401 drug substance solution was aseptically filled into vials (referred to as "Formulation A" in Figure 17). The pooled drug substance was passed through a 0.2 µm filter for a bioburden reduction filtration step. Final sterilizing filtration was performed by passing it successively through two 0.2 µm filters, and filter integrity tests were conducted before and after filtration. The sterile bulk drug substance product was aseptically filled into vials. During the filling operation, the accuracy of filling was confirmed by measuring the vial filling weight. The filled vials were stoppered and then sealed with an aluminum overwrap. After crimping, the product was stored at 5 ± 3°C.

[0203] The vials were analyzed using Method 1 (light obscuration method) in accordance with USP 788. The results are shown in Tables 11 and 12.

[0204] [Table 11]

[0205] [Table 12]

[0206] Example 7: Preparation of Intravenous (IV) and Subcutaneous (SC) Formulations of BAN2401 A. BAN2401 100 mg / mL Formulation For the preparation of the sterile aqueous formulation, the BAN2401 100 mg / mL formulation (the "100 mg / mL injection") was manufactured by conventional cGMP aseptic methods. BAN2401 100 mg / mL was prepared from the corresponding BAN2401 drug substance without the addition of any excipients and diluents (Table 13).

[0207]

Table 13

[0208] As shown in Figure 18, the filtered BAN2401 drug substance (100 mg / mL injection) solution was aseptically filled into vials (referred to as "Formulation B" in Figure 18). The drug substance was passed through a 0.2 µm filter for a bioburden reduction filtration step. Final sterilizing filtration was performed by passing it successively through two 0.2 µm filters, and filter integrity tests were conducted before and after filtration. The sterile bulk drug substance product was aseptically filled into vials. During the filling operation, the accuracy of filling was confirmed by measuring the vial filling weight. The filled vials were stoppered and then sealed with an aluminum overwrap. After crimping, the product was stored at 5 ± 3°C.

[0209] The vials were analyzed using Method 1 (light obscuration method) in accordance with USP 788. The results are shown in Tables 14 and 15.

[0210]

Table 14

[0211]

Table 15

[0212] Another BAN2401 100 mg / mL injection was manufactured. As shown in Table 16, the following materials can be used for a second exemplary formulation containing 100 mg / mL of BAN2401.

[0213]

Table 16

[0214] B.BAN2401 200 mg / mL formulation As shown in Table 17, the following materials can be used for the exemplary SC formulations containing 200 mg / mL BAN2401. The stability of BAN2401 in these formulations (FSC1, FSC2, and FSC3) was evaluated in conjunction with the evaluation of the material stability in three types of sealed containers: i) Becton-Dickenson Hypak Biotech glass prefilled syringe (PFS) and stopper ii) West Crystal Zenith plastic PFS and stopper iii) West 5 mL glass vial and stopper

[0215]

Table 17

[0216] As summarized below, BAN2401 with a target protein concentration of 200 mg / mL was prepared by TFF. Separate TFF operations were performed to prepare the BAN2401 material in each formulation buffer, except for FSC1a and FSC1b. For two of these formulations, one performed the TFF operation and the resulting concentrated material was divided into two half lots. A small amount of the sterile filtered material in each final formulation buffer was not filled at time point 0 but was cryopreserved at -20 °C for filling into appropriate sealed containers for syringe testing.

[0217] (a) BAN2401 preparation The protein concentration / diafiltration process by TFF can be subdivided into three steps: 1. Concentration of the material to 100 - 150 mg / mL 2. Diafiltration (5×) with the formulation buffer 3. Concentration to >200 mg / mL

[0218] The concentration / diafiltration step was carried out using a Pall Centramate LV system equipped with a membrane area of 0.02 m 2 . The BAN2401 material (extracted from GMP lot production before adding PS80) was loaded into the TFF system and 10 - 15 fold concentration (first stage) was carried out. Next, diafiltration of the material was performed with up to 5 diavolumes of the formulation buffer, and here the pH and electrical conductivity of the permeate were checked to monitor the diafiltration (second stage). After diafiltration, the material was further concentrated to a target protein concentration of 210 - 250 mg / mL (third stage). The retentate was collected and samples were taken for protein concentration determination.

[0219] During the preparation of this formulation, the target protein concentration of 210 - 250 mg / mL was not reached due to the high pressure of the TFF system. Therefore, the target protein concentration was achieved by using a Millipore centrifugal filter unit (30,000 MWCO). To carry out this concentration step, the filter unit was equilibrated with the BAN2401 formulation buffer, and then the BAN2401 material was centrifuged at 3600 RPM (about 3000×g) at 20°C at 30 - minute intervals until the protein concentration of the retentate was expected to be higher than 200 mg / mL. The retentate was recovered from the filter unit and pooled. After thorough mixing, the pooled retentate was sampled for protein concentration measurement.

[0220] After concentrating the protein, samples were withdrawn from the pool and diluted 500 - fold with the appropriate formulation buffer. The absorbance of the diluted samples at 280 nm and 320 nm was measured against a buffer blank. By diluting with the appropriate formulation buffer, the final protein concentration was adjusted. Finally, a 10% PS80 solution was added to BAN2401 to achieve 0.02% PS80 in the final solution, and this protein solution was thoroughly mixed by inverting rotation.

[0221] The final BAN2401 formulation material was filtered using a 0.2 μm syringe filter and subsequently filled into vials or PFSs. This step was carried out aseptically in a biosafety cabinet. The resulting vials or PFSs were placed in a freezer at -20°C. To simulate worst-case conditions, the vials were stored upside down and the PFSs were stored horizontally.

[0222] Stability experiments on exemplary 200 mg / mL SC formulations containing C.BAN2401 At 5°C (3 months, 6 months, 9 months, 12 months) and 25°C (1 month, 3 months), sample stability was evaluated using assays for pH (Figs. 19 - 21), absorbance at 405 nm (Figs. 22 - 24) (to detect increased yellowing), and size exclusion chromatography (SEC) (Figs. 25 - 33). And then at 5°C. For exemplary formulations FSC1 - FSC4, see Table 17; note that in Figs. 19 - 33, "F1a" refers to FSC1a, "F1b" refers to FSC1b, "F2" refers to FSC2, and "F3" refers to FSC3. For t = 0 samples, additional characterization assays including protein concentration, differential scanning calorimetry (DSC), PS80, and osmolality were performed. Subvisible particle tests were performed on t = 0 and t = 6 month samples. A 3-day agitation test was performed on samples stored at 5°C for 1 month. The samples for this agitation test were transferred from storage at 5°C to 25°C 1 month later and agitated at 250 rpm for 3 days.

[0223] From the test results, it was shown that all 200 mg / ml BAN2401 of the FSC1a - FSC3 formulations had comparable levels of aggregates and fragments after storage at 2 - 8°C for 12 months. The stability of BAN2401 at 200 mg / mL in the three FSC1a - FSC3 formulations was evaluated. Overall, the stability of BAN2401 in each of the tested formulations appeared similar, maintaining more than 98.2% monomer after storage at 5°C for 12 months regardless of the sealed container tested. In addition, after storage at 5°C for 12 months, the pH remained stable and there was no appreciable increase in yellowing due to A405.

[0224] (a) Protein concentration (T = 0 sample) The protein concentration was measured using an ultraviolet-visible spectrophotometer. The results are shown in Table 18.

[0225]

Table 18

[0226] The protein concentration was evaluated by measuring the absorbance at 280 and 320 nm using a Beckman DU-800 spectrophotometer with a 1-cm quartz cuvette. The samples were diluted 500-fold and prepared in triplicate. The protein concentration was calculated using the following formula: Concentration = (A280 - A320) / ε × dilution factor using an extinction coefficient ε of 1.32.

[0227] (b) Polysorbate 80 (T = 0 sample) The PS80 content of the samples was measured by quantifying oleic acid. The results are shown in Table 19.

[0228]

Table 19

[0229] This measurement was carried out by quantifying oleic acid, which is a hydrolysis product of PS80. Using basic hydrolysis, PS80 releases oleic acid in a 1:1 molar ratio. Next, oleic acid can be separated from other PS80 hydrolysis products and the matrix using reverse-phase HPLC. Oleic acid was monitored using absorbance at 195 nm without derivatization [J. Chromatography B, 878 (2010) 1865 - 1870]. Experimentally, when the sample was mixed with sodium hydroxide, oleic acid was released and subsequently extracted with acetonitrile. The extract was diluted with potassium phosphate solution, and 100 μL of the sample volume was injected onto a Waters Symmetry C18 column. Separation was performed using isocratic elution with an 80% organic phase A (acetonitrile) and 20% aqueous phase B (20 mM potassium dihydrogen phosphate, pH 2.8). The PS80 concentration of the sample was calculated from the peak area using a standard curve.

[0230] (c) Osmolality (T = 0 sample) The osmolality of the sample was determined using a freezing point osmometer. The results are shown in Table 20.

[0231]

Table 20

[0232] The osmolality was measured using a Precision Systems Osmette III freezing point osmometer. The sample was diluted 3-fold with WFI, 10 μL of the diluted sample volume was withdrawn, and loaded into the instrument using an osmometer pipette. The obtained osmolality measurements were corrected for sample dilution.

[0233] (d) Subvisible particles Subvisible particle analysis was performed using a Fluid Technologies FlowCam instrument. The results at T = 0 are presented in Table 21. When the material was stored at 5 °C, there appeared to be an increase in the total particle concentration and the number of particles larger than 10 μm over time. The degree of increase in the change was, in ascending order, FSC1a < FSC1b < FSC2 < FSC3.

[0234]

Table 21

[0235] Subvisible particle analysis was performed using a Fluid Imaging Technologies FlowCam equipped with a 20x objective lens and a 50-μm flow cell. Prior to running the BAN2401 sample, the flow cell was flushed with dI H2O and measurements were performed with respect to dI H2O to ensure that the flow cell was clean. If the total number of particles counted per 0.2 mL of dI H2O was 2 or less, the flow cell was considered to be in a usable state. The samples were equilibrated to room temperature and then diluted 20-fold with deionized water. Duplicate diluted samples were analyzed using a sample volume of 0.2 mL and a sample flow rate of 0.02 mL / min. Given an efficiency of 19.6% and a run time of 10 minutes, the auto image rate was 14. The resulting particle concentration was corrected with respect to the sample dilution.

[0236] (e) pH The pH of each formulation was monitored throughout the stability test. As shown in Figures 19 - 21, no obvious changes were observed for the samples under any of the tested storage conditions.

[0237] (f) HPLC-SEC Using a TSK 03000 SWXL column, high performance liquid chromatography size exclusion chromatography (HPLC-SEC) analysis was performed at a flow rate of 1.0 mL / min with a 0.2 M sodium phosphate, pH 7.0 mobile phase. The sample injection volume was 0.8 μL for a protein concentration of 200 mg / mL. This ensured that approximately 150 μg of protein was injected into the column through this test. As shown in Figures 25 to 33, the results of the relative peak area (%) for the monomer, aggregate, and fragment were reported.

[0238] The physical degradation of BAN2401 was determined by performing HPLC-SEC on stability samples stored at 5 and 25 °C. After storage at 5 °C for 12 months, the aggregate rate was comparable at approximately 1.1% for the three formulations. The fragment rates that occurred for all the formulations tested were in the range of 0.4 - 0.5%. The monomer content after storage at 5 °C for 12 months was in the range of 98.4 - 98.6% for all the formulations and sealed containers tested. At the current degradation rate, it is possible that all the formulations of BAN2401 tested could have a monomer higher than 97% even after storage at 5 °C for up to 24 months.

[0239] After storage at 25 °C for up to 3 months, the fragment generation after 3 months was in the range of 0.4 - 0.8%. The monomer content was slightly higher than 98% for all the formulations, with the exception that FSC3 had a monomer slightly below 98% after 3 months at 25 °C.

[0240] Example 8: Selection of Protein, Arginine, and Polysorbate 80 Concentrations A. Sample Preparation Each candidate formulation (F1 - F12) was prepared as follows. The drug substance (DS) process intermediate was concentrated and equilibrated with the corresponding formulation buffer (Table 22) using a centrifugal filter unit. After concentration and equilibration, PS80 dissolved in the formulation buffer was added to achieve the predetermined concentration, and the protein concentration was adjusted to the final concentration. Each candidate formulation was filled into a vial with a filling volume of 0.5 mL. The candidate formulations (F1 - F12) are shown in Table 22. Formulation F0 was evaluated as a control.

[0241]

Table 22

[0242] B. Stability Protocol The stability protocols are shown in Table 23, Table 24, and Table 25. After removal from storage, the vials were stored at 5°C until testing. The storage conditions were as follows: · Long-term condition: stored upright at 5°C ± 3°C · Accelerated condition: stored upright at 25°C ± 2°C, 60% RH ± 5% RH · Stress load condition (freeze-thaw): frozen at -30°C upright and thawed at room temperature · Stress load condition (agitation): at 5°C, lying down, horizontal, with a reciprocating shaker at 250 rpm with a shaking amplitude of 50 mm

[0243]

Table 23

[0244]

Table 24

[0245]

Table 25

[0246] Since it is known that the viscosity of the solution increases exponentially in highly concentrated protein solutions, the protein concentration of each candidate formulation (F1 - F5) was adjusted to 200 ± 10 mg / mL with a formulated buffer containing 0.05 (w / v)% PS80 for analysis. Other formulations were not diluted before measurement.

[0247] C. Results and Discussion (a) Selection of Arginine Concentration To select the target arginine concentration of the formulation, the physical properties of candidate formulations (150 - 350 mmol / L, F1 - F5) with various arginine concentrations were evaluated, and freeze - thaw, long - term, and accelerated stability tests were conducted and compared with formulation F0 (see Table 22). The results of the physical properties and the freeze - thaw test are shown in Table 26. The results of the long - term and accelerated stability tests are shown in Tables 27 and 28.

[0248] [Table 26]

[0249] [Table 27]

[0250] [Table 28]

[0251] (b) Physical properties As shown in Table 26, as the arginine concentration increased, the osmolality value increased. F1, F2, and F3 showed lower osmolality values when compared with F4 and F5. Therefore, the arginine concentration was limited to 250 mmol / L or less. The viscosity values of F1 - F5 were in a narrow range (7.3 - 8.1 cP) and there was no correlation with the arginine concentration within 150 - 350 mmol / L. The results of appearance, pH, and the concentrations of PS80 and protein were almost as expected.

[0252] (c) Freeze - thaw stability test As shown in Table 26, after 3 cycles of the freeze - thaw test, no significant changes were observed in any of the test items.

[0253] (d) Long - term stability test As shown in Table 27, after 3 months of storage, no significant changes were observed in any test item except for size exclusion HPLC (SEC). In candidate formulations F1 - F5, the amount of aggregates and fragments by SEC slightly increased. The rate of increase for each was comparable to that of formulation F0.

[0254] (e) Accelerated stability test As shown in Table 28, no significant changes were observed in any test item except for SEC and ion exchange HPLC (IEX). As expected, the amount of aggregates by SEC increased in all candidates (F1 - F5, BAN2401 200 mg / mL), and the rate was faster than that of formulation F0. However, considering the results under long - term conditions and the stability of F0 with a long expiration date, all candidates were considered feasible. Consistent with the results described in Example 4, as the arginine concentration increased, aggregate formation became slightly slower. The amount of fragments by SEC also increased in all candidates, but the rate was comparable to that of formulation F0. Regarding IEX, the amount of acidic peaks increased in all candidates, and the rate was comparable to that of F0.

[0255] (f) Arginine concentration As shown in Table 26, considering the osmolality, F4 and F5 (arginine concentrations of 300 and 350 mmol / L) were considered infeasible. Among the feasible candidates F1, F2, and F3 (arginine concentrations of 150, 200, and 250 mmol / L), F1 was the closest to isotonicity. As shown in Table 28, in the accelerated stability test, formulations with higher arginine concentrations showed lower aggregate formation rates, but in the evaluated arginine concentration range, the difference in rates was not significant. Considering both isotonicity and aggregate formation rate, 200 mmol / L based on formulation F2 was selected as the target arginine concentration.

[0256] (g) Selection of protein concentration To select the target protein concentration of the formulation, the physical properties of candidate formulations (200 - 300 mg / mL, F2, F6, F7) and formulation F0 at various protein concentrations were evaluated, and freeze - thaw, long - term, and accelerated stability tests were conducted. The results of the physical properties and freeze - thaw test are shown in Table 29. The results of the long - term and accelerated stability tests are shown in Tables 27 and 28.

[0257]

Table 29

[0258] (h) Physical properties As shown in Table 29, F2 and F6 showed lower osmolality values when compared with F7. The viscosity values of F2, F6, and F7 were 7.8, 21, and 48 cP, respectively. When the viscosity exceeds 20 cP, difficulties may arise in the manufacture of DS due to an increase in the back pressure of the pump and a decrease in the membrane permeation flux during the ultrafiltration and diafiltration steps. In addition, when the solution has a high concentration, the filling needle becomes clogged, leading to variations in the filling weight, which makes the DP filling process difficult. The desirable viscosity is 20 cP 3 Since it is reported to be as follows, F2 was achievable. The results of the appearance, pH, and the concentrations of PS80 and protein were almost as expected.

[0259] (i) Freeze - thaw test As shown in Table 29, no significant changes were observed in any of the tested items after 3 cycles of the freeze - thaw test.

[0260] (j) Long - term stability test As shown in Table 27, after 3 months of storage, no significant changes were observed except for size - exclusion HPLC (SEC). The amount of aggregates by SEC increased slightly in candidate formulations F2, F6, and F7, but the rate of increase was similar to that of F0.

[0261] (k) Accelerated stability test As shown in Table 28, no significant changes were observed except for SEC and ion-exclusion HPLC (IEX). The amount of aggregates by SEC increased at a similar rate (an increase of 0.7 - 0.8%) at the 3-month time point for all candidates (F2, F6, and F7). Therefore, all candidates were considered feasible. The amount of fragments by SEC also increased for all candidates, but the rate was similar to that of F0. Regarding IEX, the amount of acidic peaks by IEX increased for all candidates, but the rate was similar to that of F0.

[0262] (l) Protein concentration Considering the results of osmolality and viscosity shown in Table 29, and the overall stability results shown in Tables 27 and 28, a protein concentration of 200 mg / mL was selected.

[0263] (m) Selection of PS80 concentration To select the target PS80 concentration of the formulation, candidate formulations with different PS80 concentrations [0 - 0.10 (w / v)%, F8 - F12] and formulation F0 were subjected to freeze-thaw and agitation tests. The results of the freeze-thaw and agitation tests are shown in Tables 30 and 31, respectively.

[0264]

Table 30

[0265]

Table 31

[0266] (n) Freeze-thaw test As shown in Table 30, no significant changes were observed in any of the tested items after 3 cycles of the freeze-thaw test. The particle count by MFI was relatively high for F8 (without PS80).

[0267] (o) Agitation test As shown in Table 31, the formulations (F9 - F12) containing 0.02 (w / v)% - 0.10 (w / v)% of PS80 were stable even after stirring. For F8 (without PS80), changes were observed after stirring with respect to appearance, protein concentration, and the amount of aggregates by SEC. The appearance changed from a clear yellowish liquid to a milky white liquid. The protein concentration slightly decreased from 206 to 195 mg / mL. The amount of aggregates increased from 1.1% to 3.8%. Therefore, F8 (without PS80) was considered not feasible.

[0268] (p) PS80 concentration Based on the results shown in Table 30 (freeze - thaw test) and Table 31 (stirring test), the formulations containing 0.02 (w / v)% - 0.10 (w / v)% of PS80 were stable even after 3 cycles of freeze - thaw and up to 3 days of stirring. Therefore, a PS80 concentration of 0.05 (w / v)% was selected as the target value.

[0269] (q) Conclusion As a conclusion, the following formulations were selected as the BAN2401 formulations for subsequent tests.

[0270]

Table 32

[0271] Example 9: Stability test for 200 mg / mL BAN2401 formulations at various pH values A stability test was conducted to test the stability of 200 mg / mL BAN2401 formulations having various pH values. In addition, the effect of methionine addition on the stability of the drug product (DP) was tested.

[0272] A. Sample preparation Each candidate formulation (Table 33) was prepared as follows. The formulation was equilibrated with the corresponding formulation buffer using a centrifugal filter unit. For the preparation of F20, as is known as the Donnan effect, since protons repel the concentrated charged proteins near the semipermeable membrane, a formulation buffer with a pH of 3.5 was used until the pH of the solution after filtration reached 4.0. After concentration and equilibration, PS80 dissolved in the formulation buffer was added to achieve a predetermined concentration, and then the protein concentration was adjusted to the final concentration. Each candidate formulation was filled into vials with a filling volume of 0.5 mL. Formulation F0 (see Table 33) was evaluated as a control.

[0273]

Table 33

[0274] B. Storage Protocol The storage protocols are shown in Tables 34, 35, and 36. After removal from storage, the vials were stored at 5°C until the start of the test. The storage conditions are as follows: · Long-term condition: Stored upright at 5°C ± 3°C · Accelerated condition: Stored upright at 25°C ± 2°C, 60% RH ± 5% RH · Stress load condition (freeze-thaw): Frozen at -30°C upright and thawed at room temperature 1000 lx: 25°C ± 2°C, 60% RH ± 5% RH, lying down, with 1000 lx light irradiation Dark place: 25°C ± 2°C, 60% RH ± 5% RH, lying down, without light irradiation by covering with aluminum foil.

[0275] To confirm the aggregate level after storage for more than 3 months, size exclusion HPLC was further evaluated as an extended storage sample at the 9-month time point for the long-term stability test and at the 3-month time point for the accelerated stability test. The latter samples were tested after storage in the refrigerator for 6 months.

[0276]

Table 34

[0277]

Table 35

[0278]

Table 36

[0279] C. Results and Discussion Regarding the candidate formulations, physical properties were evaluated, including pH change (pH 4.0 to pH 6.0) and addition of methionine, and long-term and accelerated tests were conducted. Freeze-thaw and photo-stability tests were also performed to evaluate the methionine addition efficiency.

[0280] D. pH Change As shown in Table 37, Table 38A and Table 38B, Table 39, and Table 40, no significant differences were observed among the candidate formulations (F13, F15, F17, and F20 to F22) for any of the test items, except for size exclusion HPLC (SEC). Formulation F20 (i.e., the formulation with a low pH of 4.0) showed aggregates by a low amount of SEC at the starting point. In addition, in the accelerated stability test, it was observed that the aggregate formation rate was slightly low and the fragment formation rate was high. It was confirmed that the results of the formulations at pH 4.5 to 5.5 were equivalent.

[0281]

Table 37

[0282]

Table 38

[0283]

Table 39

[0284]

Table 40

[0285]

Table 41

[0286] E. Methionine addition As shown in Table 41A, Table 41B, and Tables 42 to 46, no significant difference was observed between the formulations with or without methionine (F18 and F15). The formulation with methionine (F18) showed a slightly lower rate of aggregate formation in the extended long-term stability test, accelerated stability test, and photo-stability test. F18 also showed a slightly lower oxidation of methionine residues (e.g., position 259 of the heavy chain) upon irradiation with 1000 lux light for up to 7 days. No difference was shown in the freeze-thaw test. The effect of 10 mmol / L methionine as a stabilizer was limited.

[0287]

Table 42

[0288]

Table 43

[0289]

Table 44

[0290]

Table 45

[0291]

Table 46

[0292]

Table 47

[0293]

Table 48

[0294] F . Conclusion The quality characteristics were equivalent among the formulations of BAN2401 with a pH change from pH 4.5 to pH 5.5. At pH 4.0, s showed a lower aggregate formation rate and, conversely, a faster fragment formation rate during storage. Based on the evaluation of quality and stability tests, including long-term, accelerated, freeze-thaw, and light, the inventors found no significant difference between the formulations with and without 10 mmol / L methionine. A slightly slower aggregate formation rate and a slight suppression of the oxidation of amino acid residues were observed, but the difference was not significant. As a conclusion, F15 and F21 with a pH of 5.0 ± 0.5, composed of 200 mg / mL BAN2401, 25 mmol / L L-histidine / histidine hydrochloride, 200 mmol / L L-arginine, and 0.05 (w / v)% polysorbate 80, were determined to be good drug substance candidates for further development.

[0295] Example 10: Investigation on the Effect of Arginine Concentration on the Stability of BAN2401 Formulations The effects of arginine concentrations (0, 50, 100, and 200 mmol / L) on the stability of 200 mg / mL BAN2401 were evaluated. Samples at each arginine concentration level were stored under accelerated conditions (25 °C / 60% RH) and tested at 0, 1, and 2 months.

[0296] A. Sample Preparation Samples (F1 - F4) were prepared by buffer exchange of the concentrated BAN2401 drug substance intermediate by ultrafiltration. After sterile filtration, each 0.4 mL sample was filled into vials. The sample information is shown in Table 47.

[0297]

Table 49

[0298] B. Results The results of this stability test for Samples F1 - F4 are shown in Tables 48 - 51. There were no differences in pH and protein concentration among the samples, and the arginine concentration level was consistent with the target value of the starting sample.

[0299] The % aggregates for the samples containing arginine (F2 - F4) were lower than those for the sample without arginine (F1).

[0300] [Table 50]

[0301] [Table 51]

[0302] [Table 52]

[0303] [Table 53]

[0304] C. Discussion and Conclusion From the results of this test, it was shown that an arginine concentration of 50 - 200 mmol / L prevents an increase in protein aggregates in the 200 mg / mL formulation of BAN2401. Batch - to - batch variation in arginine concentration can result in batch - to - batch variation in % aggregates. Additionally, the arginine concentration can affect the increasing trend of % aggregates in the stability test.

[0305] When comparing the fragmentation ratio shown in Figure 11 of Example 2 using a sample containing 200 mg / mL BAN2401, 50 mM sodium citrate, and 100 mM sodium chloride with the fragmentation ratio of Sample F1, the formulation containing a histidine buffer appeared to show an effect of reducing the fragmentation of BAN2401.

[0306]

Table 54

[0307]

Table 55

[0308]

Table 56

[0309] Heavy chain (SEQ ID NO: 11):

Chemical Structure

Chemical Structure

Claims

1. An aqueous pharmaceutical formulation comprising: (a) an isolated anti-Aβ protofibril antibody or a fragment thereof that binds to human Aβ protofibrils, at a concentration of 80 mg / mL to 300 mg / mL; (b) 100 mM to 400 mM arginine; (c) 0.01% w / v to 0.1% w / v polysorbate 80; and (d) a pharmaceutically acceptable buffer, wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, the isolated anti-Aβ protofibril antibody or fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and the arginine is arginine, arginine hydrochloride, or a combination thereof.

2. The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL to 200 mg / mL.

3. The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL.

4. The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 200 mg / mL.

5. The pharmaceutical formulation according to any one of claims 1 to 4, further comprising methionine.

6. The pharmaceutical formulation according to any one of claims 1 to 5, wherein the pharmaceutically acceptable buffer is a citrate buffer or a histidine buffer.

7. The pharmaceutical formulation according to any one of claims 1 to 6, comprising a 10 to 100 mM citrate buffer or a 10 to 100 mM histidine buffer.

8. The pharmaceutical formulation according to any one of claims 1 to 7, comprising 125 to 350 mM arginine.

9. The pharmaceutical formulation according to any one of claims 1 to 8, comprising 200 mM arginine in the form of arginine hydrochloride.

10. The pharmaceutical formulation according to any one of claims 1 to 9, comprising 200 mM arginine in the form of arginine hydrochloride and 25 mM histidine buffer.

11. (a) an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, at a concentration of 80 mg / mL to 240 mg / mL; (b) 140 mM to 260 mM arginine hydrochloride; (c) 0.02% w / v to 0.08% w / v polysorbate 80, and (d) 15 mM to 35 mM histidine buffer An aqueous pharmaceutical formulation comprising: A pharmaceutical formulation having a pH in the range of 4.5 to 5.

5. **Claim 12** (a) (i) An isolated anti-Aβ protofibril antibody or a fragment thereof at 80 mg / mL to 120 mg / mL, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2, (b) 240 mM to 360 mM arginine, (c) 0.02% w / v to 0.08% w / v polysorbate 80, and (d) 30 mM to 50 mM citrate buffer An aqueous pharmaceutical formulation comprising: The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine hydrochloride, or a combination thereof. **Claim 13** A method for reducing aggregate formation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) Providing an aqueous pharmaceutical formulation containing an isolated anti-Aβ protofibril antibody or a fragment thereof at a concentration of 80 mg / ml to 300 mg / ml, said antibody or fragment comprising (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2, and Adding arginine, arginine hydrochloride, or a combination thereof to the aqueous pharmaceutical formulation wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 5.

5. **Claim 14** The method according to claim 13, wherein the arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 150 to 250 mM. **Claim 15** The method according to claim 14, wherein the arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 200 mM. **Claim 16** The method according to any one of claims 13 to 15, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer. **Claim 17** The method according to claim 16, wherein the pharmaceutically acceptable buffer is a histidine buffer or a citrate buffer. **Claim 18** A method for reducing fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) Providing an aqueous pharmaceutical formulation containing an isolated anti-Aβ protofibril antibody or a fragment thereof at a concentration of 80 mg / ml to 300 mg / ml, said antibody or fragment comprising (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2, and Adding a histidine buffer to the aqueous pharmaceutical composition A method, wherein the pH of the pharmaceutical preparation is in the range of 4.5 to 5.

5.

19. The method according to claim 18, comprising 15 to 35 mM of a histidine buffer.

20. The method according to any one of claims 13 to 19, wherein the pharmaceutical preparation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80.

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