High-concentration anti-Aβ protofibril antibody preparation and method of using same

Abstract

Provided herein 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.

Description

[Technical field]

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

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

[0003] The disease generally involves a generalized decline in cognitive function that progresses slowly, with end-stage subjects becoming bedridden. AD subjects typically survive only 3-10 years after onset, although extreme cases of 2-20 years have been known (Hebert, LE, et al., Alzheimer disease in the US population: prevalence estimates using the 2000 census. Arch Neurol. 2003;60:1119-1122). Despite the fact that AD is rarely listed as the cause of death on death certificates, and therefore mortality attributable to AD is greatly underestimated, 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 the age of 65 (2010 Alzheimer's disease facts and figures, supra).

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

[0005] Anti-Aβ antibodies and other proteins can be administered to subjects intravenously, subcutaneously, intramuscularly, and by other means. The dosage and / or dosage form of the antibody can present many challenges to the development of a suitable pharmaceutical formulation. For example, at high antibody concentrations, antibody stability can be problematic due to the formation of interprotein aggregates or fragmentation. Aggregation generally increases with increasing antibody concentration. Furthermore, high concentrations of stabilizers and other excipients are required to achieve long-term protein stability and shelf life for high-concentration antibody formulations. 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] Provided herein is 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. Also provided herein is a pharmaceutical formulation comprising 80-300 mg / ml of an isolated anti-Aβ protofibril antibody or fragment thereof that binds to Aβ protofibrils, where 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 antibodies (e.g., 100 mg / mL or 200 mg / mL), the protein-protein aggregation rate is unexpectedly low and comparable 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 with significantly lower concentrations of anti-Aβ protofibril antibodies (e.g., about 0.3% initial aggregate level for 100 mg / mL anti-Aβ protofibril antibody vs. about 0.8% initial aggregate level for 10 mg / mL anti-Aβ protofibril antibody). In some embodiments, the pharmaceutical formulations disclosed herein exhibit a lower sub-visible particle generation rate than formulations with significantly lower concentrations of anti-Aβ protofibril antibodies (e.g., 10.6 particles / mL for 100 mg / mL anti-Aβ protofibril antibody vs. 12.6 particles / mL for 10 mg / mL anti-Aβ protofibril antibody). In some embodiments, the pharmaceutical formulations disclosed herein exhibit a lower aggregation rate, lower initial aggregation level, lower protein fragmentation rate, and / or lower sub-visible particle formation when compared to formulations with significantly lower concentrations of anti-Aβ protofibril antibodies. Lower aggregation rates, initial aggregation levels, protein fragmentation rates, and / or subvisible particle occurrence may result in higher stability and / or longer product shelf life. Additionally, excipients in the pharmaceutical formulations disclosed herein may be present in lower amounts than currently 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 infusion 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 in SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (Kabat et al., 5th ed., USDepartment 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 may be used in the present disclosure. In some embodiments, the heavy chain constant region is selected from IgG1 and allelic variants thereof. The amino acid sequence of a 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 a κ-λ chain constant region and any allelic variants thereof as discussed 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 allelic variants thereof. 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, the 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 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 with high selectivity to soluble Aβ aggregate species. For example, BAN2401 has been reported to exhibit approximately 1000-fold and 5-fold to 10-fold higher selectivity for soluble Aβ protofibrils compared to Aβ monomers or insoluble Aβ 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 set forth in SEQ ID NOs: 11 and 12, and are described in WO 2007 / 108756 and Journal of Alzheimer's Disease 43:575-588 (2015).

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

[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 ranging from 80 mg / mL to 300 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration ranging from 85 mg / mL to 275 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration ranging from 90 mg / mL to 250 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration ranging from 95 mg / mL to 225 mg / mL. In some embodiments, the isolated anti-Aβ protofibril antibody is present at a concentration ranging from 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, the 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 further comprises at least one additional component. In some embodiments, the at least one additional component in the pharmaceutical formulation is selected from a buffering agent. In some embodiments, the buffering agent is a citrate buffering agent. In some embodiments, the buffering agent is a histidine buffering agent. In some embodiments, the at least one additional component in the pharmaceutical formulation is selected from an emulsifier. In some embodiments, the 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, the 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, the at least one additional ingredient 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 comprises arginine (and / or arginine hydrochloride). In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is in the range of about 100 mM to about 400 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is in the range of about 110 mM to about 380 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is in the range of about 120 mM to about 360 mM. In some embodiments, the concentration of arginine (and / or arginine hydrochloride) in the pharmaceutical formulation is in the range of 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 comprises 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 to 100 mM. In some embodiments, the concentration of citric acid monohydrate in the pharmaceutical formulation 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. 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 to 5.5. In some embodiments, the pH of the pharmaceutical formulation is in the range of 4.0 to 6.0, 4.2 to 5.8, 4.3 to 5.7, 4.4 to 5.6, or 4.5 to 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 deemed appropriate by a person skilled in the art. The route of administration of the compositions of the present disclosure may be intravenous or subcutaneous. In some embodiments, the pharmaceutical formulations are formulated as a sterile, nonpyrogenic liquid for intravenous administration. In some embodiments, the pharmaceutical formulations are formulated as a sterile, nonpyrogenic liquid for subcutaneous administration. In some embodiments, the pharmaceutical composition is a saline solution.

[0024] In some embodiments, the pharmaceutical formulation is a liquid dosage form comprising at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds Aβ protofibrils, and further comprising, for example, citric acid monohydrate, arginine, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 100 mg / mL of at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds Aβ protofibrils, 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 comprising at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds Aβ protofibrils, and further comprising, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 100 mg / mL or 200 mg / mL of at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds Aβ protofibrils, 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 is a liquid dosage form comprising at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds Aβ protofibrils, and further comprising, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 200 mg / mL of at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds 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 is a liquid dosage form comprising at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibrils, and further comprising, for example, histidine, histidine hydrochloride, arginine hydrochloride, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 200 mg / mL of at least one isolated anti-Aβ protofibril antibody, such as BAN2401, or a fragment thereof that binds to Aβ protofibrils, 50 mM citric acid (and / or citric acid 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] BAN2401 and methods involving 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 by reference in their entireties. [Brief description of the drawings]

[0029] [Figure 1] 4 shows percent aggregation for 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 5° C. [Diagram 2]4 shows the percent fragmentation of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 5° C. [Diagram 3] 4 shows the monomer percentage of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 5° C. [Figure 4] 1 shows percent aggregation for 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 25° C. [Diagram 5] 4 shows the percent fragmentation of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 25° C. [Figure 6] 1 shows the monomer percentage of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, 2, and 3 months at 25° C. [Figure 7] 1 shows the percent aggregation of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 5° C. and at 0, 1, 2, and 3 months. [Figure 8] 1 shows the percent fragmentation of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 5° C. and at 0, 1, 2, and 3 months. [Figure 9] 1 shows the monomer percentage of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 5° C. and at 0, 1, 2, and 3 months. [Figure 10] 1 shows the percent aggregation of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 0, 1, 2, and 3 months at 25° C. [Figure 11] 1 shows the percent fragmentation of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 0, 1, 2, and 3 months at 25° C. [Figure 12]1 shows the monomer percentage of formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 0, 1, 2, and 3 months at 25° C. [Figure 13] 1 shows percent aggregation for 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, and 2 months at 25° C. [Figure 14] 4 shows the percent fragmentation of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, and 2 months at 25° C. [Figure 15] 1 shows the monomer percentage of 10 mg / mL, 100 mg / mL, and 200 mg / mL formulations of BAN2401 at 0, 1, and 2 months at 25° C. [Figure 16] 1 shows the percent monomer aggregation in formulations of BAN2401 as a function of arginine concentration. [Figure 17] 1 shows a flowchart of the manufacturing process for BAN2401 10 mg / mL injection. [Figure 18] 1 shows a flowchart of the manufacturing process for BAN2401 100 mg / mL injection. [Figure 19] 1 shows the pH values ​​of formulations of BAN2401 at 5° C. and pH values ​​at 0 and 12 months. [Figure 20] 1 shows the pH values ​​of formulations of BAN2401 at 25° C. and pH values ​​at 0, 1, and 3 months. [Figure 21] 1 shows the pH value of formulations of BAN2401 at 5° C. and at 25° C. with stirring after 1 month. [Figure 22] 4 shows the absorbance at 405 nm of formulations of BAN2401 at 0, 1, 3, 6, 9, and 12 months at 5° C. [Figure 23] 1 shows the absorbance at 405 nm of formulations of BAN2401 at 0, 1, and 3 months at 25° C. [Figure 24] 1 shows the absorbance at 405 nm of formulations of BAN2401 at 5° C. and 25° C. with stirring at 1 month. [Diagram 25]4 shows percent aggregation of BAN2401 formulations at 0, 1, 3, 6, 9, and 12 months at 5° C. [Figure 26] 1 shows the percent fragmentation of BAN2401 formulations at 0, 1, 3, 6, 9, and 12 months at 5° C. [Figure 27] 4 shows the monomer percentage of BAN2401 formulations at 0, 1, 3, 6, 9, and 12 months at 5° C. [Figure 28] 1 shows percent aggregation of formulations of BAN2401 at 0, 1, and 3 months at 25° C. [Figure 29] 1 shows the percent fragmentation of BAN2401 formulations at 0, 1, and 3 months at 25° C. [Diagram 30] 1 shows the monomer percentage of BAN2401 formulations at 0, 1, and 3 months at 25° C. [Diagram 31] 1 shows the percent aggregation of BAN2401 formulations at 5° C. and 25° C. with stirring for 1 month. [Diagram 32] 1 shows the percentage of fragmentation of BAN2401 formulations at 5° C. and 25° C. with agitation for 1 month. [Diagram 33] 1 shows the percent monomer of BAN2401 formulations at 5° C. and 25° C. with stirring at 1 month. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition Below are definitions of terms used in this application.

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

[0032] The phrase "and / or," as used herein, refers to "either or both" of the elements so connected, i.e., elements that are present in some cases as a coordinate conjunction and in other cases as a disjunction. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended language such as "comprising," in some embodiments refers to only A (optionally including elements other than B); in other embodiments refers to only B (optionally including elements other than A); in yet other embodiments refers to both A and B (optionally including other elements), and so forth.

[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 listed in the list of elements, and does not exclude any combinations of the elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers can optionally be present, whether or not related to the elements specifically identified. 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 refer in one embodiment to at least one, optionally including more than one, A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements), and so forth.

[0034] When a number is recited alone or as part of a numerical range, it should be understood that the numerical value can vary above and below the stated value by reasonable variations in the recited value, as would be recognized by one of ordinary skill in the art.

[0035] As used herein, a "fragment" of an antibody includes a portion of an antibody, such as a portion containing the antigen-binding region or a 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, a "histidine buffer" may include histidine, histidine hydrochloride, or a combination thereof, where the histidine hydrochloride may be histidine hydrochloride monohydrate.

[0038] As used herein, a "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 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) 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 pharma- ceutically acceptable buffer, optionally a histidine buffer or a citrate buffer; and c. pH of 4.5 to 5.5 The present invention provides an aqueous pharmaceutical formulation comprising any one or more of the following:

[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 pharma- ceutically acceptable buffer, wherein the buffer is a histidine buffer, optionally wherein the histidine buffer is at a concentration of about 25 mM; and optionally including: a.0.01% w / v~0.1% w / v polysorbate 80; b. 100 mM to 400 mM arginine, arginine hydrochloride, or a combination thereof; and c. pH of 4.5 to 5.5 The present invention provides an aqueous pharmaceutical formulation comprising any one or more of the following:

[0042] Some embodiments include (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 pharma- ceutically acceptable buffer; The aqueous pharmaceutical formulation comprising: 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 may be present in the following concentrations:

[0044] 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 or more. 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.

[0045] 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 or more. 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.

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

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

[0049] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ protofibril antibody or fragment thereof is present in 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 include arginine as follows:

[0054] In some embodiments, the pharmaceutical formulation comprises arginine, hi 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 formulation, 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 formulation, 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 arginine, arginine hydrochloride, or combinations thereof.

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

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

[0059] In some embodiments of the pharmaceutical formulation, the concentration of arginine is in the range of 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 formulation, the concentration of arginine is 350 mM.

[0061] Polysorbate 80 (PS80) For any of the aqueous pharmaceutical formulations described herein, the formulation may include polysorbate 80 as follows:

[0062] In some embodiments of the pharmaceutical formulation, the concentration of polysorbate 80 is in the range of 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 in 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 in a concentration of 0.02% w / v.

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

[0066] Buffer For any of the aqueous pharmaceutical formulations described herein, the formulation may include a pharma- ceutically acceptable buffer as follows:

[0067] In some embodiments of the pharmaceutical formulation, the pharma- ceutically 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 pharma- ceutically 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 formulation, the concentration of 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.

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

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

[0076] In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate. In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, where 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 formulation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, where 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 formulation, the histidine buffer comprises histidine and histidine hydrochloride monohydrate, where the histidine comprises from 0.2 to 0.3 mg / mL of histidine and from 4.4 to 4.9 mg / mL of histidine hydrochloride, and optionally the histidine hydrochloride is a monohydrate.

[0077] pH For any of the aqueous pharmaceutical formulations described herein, the formulation may comprise a pH as follows:

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

[0079] In some embodiments of the pharmaceutical formulation, 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 comprises methionine.

[0085] In some embodiments, (a)80mg / mL~240mg / mL BAN2401, (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 Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0086] In some embodiments, (a) 80 mg / mL to 240 mg / mL of 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; (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 Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0087] In some embodiments, (a) 80 mg / mL to 240 mg / mL of an isolated anti-Aβ protofibril antibody or a fragment thereof, 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 Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0088] In some embodiments, the present disclosure provides: (a)80mg / mL~120mg / mL BAN2401, (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 Disclosed is 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.

[0089] In some embodiments, the present disclosure provides: (a) 80 mg / mL to 120 mg / mL of 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; (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 Disclosed is 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.

[0090] In some embodiments, the present disclosure provides: (a) 80 mg / mL to 120 mg / mL of an isolated anti-Aβ protofibril antibody or a fragment thereof, 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) 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 Disclosed is 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.

[0091] In some embodiments, the present disclosure provides: (a) 100 mg / mL BAN2401; (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0092] In some embodiments, the present disclosure provides: (a) 100 mg / mL of 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; (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0093] In some embodiments, the present disclosure provides: (a) 100 mg / mL of an isolated anti-Aβ protofibril antibody or fragment thereof 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) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0094] In some embodiments, the present disclosure provides: (a) 200 mg / mL BAN2401; (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0095] In some embodiments, the present disclosure provides: (a) 200 mg / mL of 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; (b) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0096] In some embodiments, the present disclosure provides: (a) 200 mg / mL of an isolated anti-Aβ protofibril antibody or fragment thereof 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) 200 mM arginine hydrochloride, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0097] In some embodiments, the present disclosure provides: (a) 100 mg / mL BAN2401; (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer Disclosed is 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.

[0098] In some embodiments, the present disclosure provides: (a) 100 mg / mL of 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; (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer Disclosed is 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.

[0099] In some embodiments, the present disclosure provides: (a) 100 mg / mL of an isolated anti-Aβ protofibril antibody or fragment thereof 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) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer Disclosed is 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.

[0100] In some embodiments, the present disclosure provides: (a)150mg / mL~250mg / 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 Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0101] In some embodiments, the present disclosure provides: (a) 150 mg / mL to 250 mg / mL of 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; (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 Disclosed is an aqueous pharmaceutical formulation comprising The pharmaceutical formulation has a pH in the range of 4.5 to 5.5.

[0102] In some embodiments, the present disclosure provides: (a) 150 mg / mL to 250 mg / mL of an isolated anti-Aβ protofibril antibody or a fragment thereof, 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) 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 Disclosed is an aqueous pharmaceutical formulation comprising 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] Methods for reducing aggregate formation In some embodiments, provided herein is a method of reducing aggregate formation 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 protofibrils 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 arginine, arginine hydrochloride, or a combination thereof, and optionally providing a pharma- ceutically acceptable buffer, the buffer optionally being a histidine buffer. Disclosed is a method comprising:

[0105] In some embodiments of the method of 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 of reducing aggregate formation, arginine, arginine hydrochloride, or a combination thereof is present at a concentration between 150 mM and 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 protofibrils 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. Disclosed is a method comprising:

[0108] In some embodiments of the method of 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 methods of reducing fragmentation, arginine, arginine hydrochloride, or a combination thereof is present at a concentration between 100 mM and 400 mM.

[0110] In some embodiments of the method of 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 of reducing fragmentation, the pharmaceutical formulation further comprises a pharma- ceutically acceptable buffer, where the buffer is a histidine buffer, optionally wherein 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.

[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, comprising: (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 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; (b) adding arginine, arginine hydrochloride, or a combination thereof; and (c) providing a pharma- ceutically acceptable histidine buffer; Disclosed is a method comprising:

[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, comprising: (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 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; (b) providing a histidine buffer; and (c) providing arginine, arginine hydrochloride, or a combination thereof; 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 a fragment thereof that binds to human Aβ protofibrils, at a concentration of 80 mg / mL to 300 mg / mL; b. 100mM to 400mM arginine, c. 0.01% w / v to 0.1% w / v polysorbate 80, and d. A pharma- ceutically acceptable buffer. An aqueous pharmaceutical formulation comprising: The pharmaceutical formulation has a pH in the range of 4.5 to 5.5; 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; and The pharmaceutical formulation, wherein the arginine is arginine, arginine hydrochloride, or a combination thereof.

[0116] [Embodiment 02] 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.

[0117] [Embodiment 03] 2. The pharmaceutical formulation of claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 100 mg / mL.

[0118] [Embodiment 04] 2. The pharmaceutical formulation of claim 1, wherein the isolated anti-Aβ protofibril antibody or fragment thereof is present at a concentration of 200 mg / mL.

[0119] [Embodiment 05] 5. The pharmaceutical formulation according to claim 1, further comprising methionine.

[0120] [Embodiment 06] 6. The pharmaceutical formulation according to claim 1, wherein the pharma- ceutically acceptable buffer is a citrate buffer or a histidine buffer.

[0121] [Embodiment 07] 7. The pharmaceutical formulation according to claim 1, comprising 10 to 100 mM citrate buffer or 10 to 100 mM histidine buffer.

[0122] [Embodiment 08] 8. The pharmaceutical formulation according to claim 1, comprising 125 to 350 mM arginine.

[0123] [Embodiment 09] 9. The pharmaceutical formulation of claim 1, comprising 200 mM arginine, the arginine being arginine hydrochloride.

[0124] [Embodiment 10] 10. The pharmaceutical formulation of claim 1, comprising 200 mM arginine, which is arginine hydrochloride, and 25 mM histidine buffer.

[0125] [Embodiment 11] a. 80 mg / mL to 240 mg / mL of 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; b. 140 mM to 260 mM arginine hydrochloride; c. 0.02% w / v to 0.08% w / v polysorbate 80, and d. 15mM to 35mM histidine buffer An aqueous pharmaceutical formulation comprising: A pharmaceutical formulation having a pH in the range of 4.5 to 5.5.

[0126] [Embodiment 12] a. 80 mg / mL to 120 mg / mL of 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; b. 240mM to 360mM arginine, c. 0.02% w / v to 0.08% w / v polysorbate 80, and d. 30mM to 50mM 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.

[0127] [Embodiment 13] 1. A method for reducing aggregate formation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) providing an aqueous pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof, the antibody 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 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.

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

[0129] [Embodiment 15] 15. The method of claim 14, wherein the 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 formulation further comprises a pharma- ceutically acceptable buffer.

[0131] [Embodiment 17] 17. The method of claim 16, wherein the pharma- ceutically acceptable buffer is a histidine buffer or a citrate buffer.

[0132] [Embodiment 18] 1. A method for reducing fragmentation of an isolated anti-Aβ protofibril antibody or a fragment thereof, comprising: (i) providing an aqueous pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof, the antibody 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 said aqueous pharmaceutical composition; wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5.

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

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

[0135] [Embodiment 21] a. 80 mg / mL to 240 mg / mL of 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; b. 140 mM to 260 mM arginine, arginine hydrochloride, or a combination thereof; c. 0.01% w / v to 0.1% w / v polysorbate 80; and d. 15mM to 35mM histidine buffer or 30mM to 50mM 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] 22. The pharmaceutical formulation of embodiment 21, comprising 100 mg / mL of the antibody.

[0137] [Embodiment 23] 22. The pharmaceutical formulation of embodiment 21, comprising 200 mg / mL of the antibody.

[0138] [Embodiment 24] 22. The pharmaceutical formulation of embodiment 21, comprising 90 mg / mL to 220 mg / mL, 100 mg / mL to 210 mg / mL, or 110 mg / mL to 200 mg / mL of the antibody.

[0139] [Embodiment 25] 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 histidine.

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

[0141] [Embodiment 27] 27. The pharmaceutical formulation of any one of embodiments 21-26, 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 arginine, arginine hydrochloride, or a combination thereof.

[0142] [Embodiment 28] 28. The pharmaceutical formulation 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 Polysorbate 80.

[0143] [Embodiment 29] 29. The pharmaceutical formulation of any one of embodiments 21 to 28, comprising a citrate buffer.

[0144] [Embodiment 30] 30. The pharmaceutical formulation of any one of embodiments 21 to 29, comprising a histidine buffer.

[0145] [Embodiment 31] 31. The pharmaceutical formulation of any one of embodiments 21 to 30, further comprising methionine.

[0146] [Embodiment 32] a. 100 mg / ml to 200 mg / ml of an antibody 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. 25mM histidine buffer An aqueous pharmaceutical formulation comprising: A pharmaceutical formulation having a pH in the range of 4.5 to 5.5.

[0147] [Embodiment 33] 33. The pharmaceutical formulation of embodiment 32, comprising 100 mg / mL of the antibody.

[0148] [Embodiment 34] 33. The pharmaceutical formulation of embodiment 32, comprising 200 mg / mL of the antibody.

[0149] [Embodiment 35] 1. A method for reducing antibody aggregate formation and / or fragmentation, comprising: a. Providing an aqueous pharmaceutical formulation comprising an isolated anti-Aβ protofibril antibody or a fragment thereof, the antibody 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 300 mg / ml; and b. adding to said aqueous pharmaceutical composition 1) arginine, arginine hydrochloride, or a combination thereof, or 2) a histidine buffer. The method includes:

[0150] [Embodiment 36] 36. The method according to embodiment 35, wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5.

[0151] [Embodiment 37] 36. The method of embodiment 35, wherein the arginine, arginine hydrochloride, or a combination thereof is present at a concentration of 150-250 mM.

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

[0153] [Embodiment 39] 39. The method of embodiment 38, comprising 15-25 mM histidine buffer.

[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 pharma- ceutically acceptable buffer.

[0156] [Embodiment 42] 42. The method of embodiment 41, wherein the pharma- ceutically acceptable buffer is a histidine buffer or a citrate buffer. EXAMPLES

[0157] Example 1: Protein concentration test Three protein concentrations (10, 100, and 200 mg / mL) were prepared and examined in the concentration study. The 10 mg / mL material was BAN2401 purified drug substance (PDS), and the 100 and 200 mg / mL materials were generated from 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 percentage of PS80 in the 10, 100, and 200 mg / mL samples was 0.02%, 0.16%, and 0.32%, respectively. All samples were filtered through a 0.2 gm filter and aliquoted into sterile polypropylene (PP) tubes for stability testing (Note: a PS80 removal step was not available at the time this concentration study was performed).

[0158] Sample stability was assessed over a 3 month period at two temperatures (5 and 25°C) using native HPLC-SEC, pH, and DLS. Additional characterization assays, such as PS80 testing, were performed on T=0 samples. Sample testing details are shown in Table 1.

[0159] [Table 1]

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

[0161] Native HPLC-SEC was performed on stability samples stored at 5 and 25° C. to determine aggregation and physical degradation of concentrated BAN2401.

[0162] HPLC-SEC (native) analysis was performed utilizing 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. Sample injection volumes were 15, 1.5, or 0.8 pg for protein concentrations of 10, 100, or 200 mg / mL, respectively. This ensured that approximately 150 pg of protein was injected onto the column throughout the study. The relative peak area (%) results are reported in Figures 1-6 for monomer, aggregates, and fragments.

[0163] At both temperatures (5 and 25°C), it was observed that the aggregate percentage and aggregation rate increased with increasing protein concentration. For the 100 and 200 mg / mL samples, the starting aggregate percentage was almost twice that of the 10 mg / mL sample, indicating that the concentration step alone caused protein aggregation. 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 only an aggregation rate of 0.07% per month under the same conditions. In summary, BAN2401 at 100 mg / mL and above did not appear to be physically stable in the formulation.

[0164] Protein concentration was assessed by UV by measuring absorbance at 280 and 320mu on a Beckman DU-800 spectrophotometer. Samples were diluted 500-fold and prepared in duplicate. Protein concentration was calculated using the following formula: Concentration = (A280-A320) / ε x dilution factor The extinction coefficient ε was calculated using 1.32.

[0165] Protein concentrations were measured using a UV-Vis spectrophotometer and the results are listed in Table 2. Overall, concentrations remained unchanged throughout the three months at both temperatures.

[0166] [Table 2]

[0167] Example 2: pH Screening Test The stability of 200 mg / mL BAN2401 was evaluated at five different pH values. To prepare the samples, BAN2401 PDS after removal of PS80 was concentrated and diafiltered with a buffer containing 50 mM sodium citrate, 100 mM sodium chloride at pH 4.5. Final concentration adjustment was performed by dilution to achieve a concentration of 200 mg / mL protein and 0.02% PS80. The resulting material was divided into five aliquots; four of these aliquots were titrated with 10 N sodium hydroxide to generate 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 subaliquotted into sterile polypropylene (PP) tubes for stability evaluation.

[0168] Sample stability was assessed over a 3 month period using native HPLC-SEC, pH, and DLS at two temperatures (5 and 25° C.). In addition, an agitation study was performed at 1 month where a subset of the 5° C. samples were agitated horizontally at 250 RPM in a 25° C. incubator for 3 days. Sample testing details are shown in Table 3.

[0169] [Table 3]

[0170] SEC-HPLC analysis was performed utilizing 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. Sample injection volumes were 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 onto the column throughout the study. Relative peak area (%) results were reported for monomer, aggregates, and fragments.

[0171] Aggregation and physical degradation of concentrated BAN2401 was determined by performing native HPLC-SEC on stability samples stored at 5 and 25°C. The results are shown in Figures 7-12. BAN2401 appeared to be more stable at lower pH at both temperatures (5 and 25°C). Formulations at pH 4.5 and 5.0 exhibited a slower increase in aggregate formation, starting with a low aggregate percentage, at a rate of about 0.07% per month at 5°C. In addition to aggregation, protein fragmentation was also analyzed. The results suggested that BAN2401 fragmentation was more pronounced at lower pH for BAN2401 formulations. Based on these considerations, the BAN2401 formulation 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 at different pH values).

[0172] Example 3: Excipient Screening Study In this study, 12 excipients were screened. To prepare a 200 mg / mL BAN2401 sample, BAN2401 after PS80 removal was concentrated in a TFF step followed by 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 fraction was spiked with a stock solution containing the particular excipient. For the majority of excipients, two concentrations were examined, with the exception of sodium chloride and ascorbic acid, which 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., sample in base buffer without excipients), were filtered through a 0.2 gm filter and then aseptically filled into BD syringes using a manual stoppering tool. Control samples were also filled into glass vials. Vials and BD syringes were placed at 5 and 25° C. Sample stability was assessed using native HPLC-SEC and pH over a two month period.

[0175] Samples for excipient screening studies were prepared in suboptimal buffer (50 mM citrate, 0.02% PS80, pH 6.0) conditions to amplify the positive effect from excipients in preventing aggregate formation. Native HPLC-SEC was performed to determine the stability profile (aggregation) of each formulation over a period of 2 months at 25°C.

[0176] As shown in Figures 13-15, the formulation containing 160 mM arginine (F14) produced the lowest aggregation rate among the formulations. In 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 basal buffer without excipients) showed 2.4% aggregates at the same time point under the same conditions.

[0177] In addition, the formulation containing arginine had the lowest starting aggregate percentage, indicating that arginine had 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 may have been within the range of assay variability.

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

[0179] Formulations containing sodium chloride (F16-F18) did not show any effect on stability when compared to the control sample. The same observation was made for formulations containing PS80 (F19 and F20): no stability effect was observed due to the different PS80 contents. The formulation containing ascorbic acid (F15) showed a dramatic effect with an increase in aggregate and fragment percentage from the beginning of the study.

[0180] The pH of the two arginine formulations (F13 and F14) was measured to ensure that there was no pH drift over time due to the presence of arginine in the formulation. No change in pH was observed, as shown in Table 4. The osmolality of the two formulations was also measured. 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 examined using Design Expert® 7.0. The factors selected for study, and the levels investigated for each factor, are shown in Table 6 below.

[0183] [Table 6]

[0184] Sample randomization was generated by D-optimal design in Design Expert® 7.0. The resulting sample set contained 25 data points with 4 replicates, as shown in Table 7. Each formulation was prepared and evaluated.

[0185] [Table 7]

[0186] Design Expert® 7.0 estimated the relationship between arginine concentration and aggregate levels in a 100 mg / mL BAN2401 formulation at pH 5.0 containing 0.02% polysorbate 80. The results of this estimation are shown in FIG.

[0187] Example 5: PS80 Test To evaluate the change in the quality of the drug product under stress conditions, agitation and freeze-thaw tests were performed using formulation buffer (pH 5.0, 350 mM arginine, 50 mM citric acid). In this experiment, formulations with various polysorbate 80 concentrations were examined to determine the effect of polysorbate 80 on subvisible particle generation under such stress conditions. The formulations examined and the applied stress conditions are shown in Table 8.

[0188] [Table 8]

[0189] Samples were prepared as described in Example 4. A 10% PS80 solution was added to achieve the target PS80 concentration in the formulation, and final protein concentration adjustments were made by dilution with formulation buffer. The formulation was passed through a 0.2 gm filter and filled into 2 mL vials with a fill volume of 1.3 mL. Samples were placed in a horizontal orientation (vials lying on their side) on an orbital shaker, which was then placed in the refrigerator or on the lab bench. Samples were shaken at 250 rpm for 3 days. Other samples were frozen by placing in a -20°C chamber for 2 hours, then thawed by removing and leaving at room temperature for 2 hours. This freeze-thaw cycle was repeated three times. Samples for agitation and freeze-thaw testing were evaluated for appearance, aggregates, and fragment levels by SEC-HPLC, and subvisible particles by Micro Flow Imaging (MFI).

[0190] The results of agitation and freeze-thaw studies on BAN2401 formulations containing various levels of PS80 are summarized below.

[0191] SEC-HPLC analysis SEC-HPLC analysis was performed utilizing 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. Sample injection volumes were 5.0 μL or 2.5 μL for protein concentrations of 50 or 100 mg / mL, respectively. This ensured that approximately 250 μg of protein was injected onto the column throughout the study. Relative peak area (%) results were reported for monomer, aggregates, and fragments.

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

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

[0194] After shaking at 250 rpm for 3 days at ambient temperature, the appearance of the formulations was such that the sample without PS80 was turbid and had 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 and free of precipitate. All samples subjected to freeze / thaw cycles were also clear and free of precipitate.

[0195] Aggregate and fragment levels for BAN2401 formulations subjected to agitation and freeze-thaw are shown in Table 9. The stress condition that caused the most instability was agitation under ambient conditions. Under these conditions, samples without PS80 showed the most formation of high molecular weight species and the greatest loss of monomer. Increasing the PS80 concentration abolished 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 results are reported for the 2.25-100 μm, 5-100 μm, 10-100 μm, and 25-100 μm ranges.

[0198] An increase in subvisible particles (2-10 pm) was observed in samples without PS80 subjected to shaking and freeze-thawing. Increasing the concentration of PS80 in the formulation progressively reduced the occurrence of subvisible particles. This effect was observed across the entire size range tested. There was no significant change in subvisible particles in formulations containing 0.05% and 0.1% polysorbate 80. Therefore, a PS80 concentration of 0.05% was selected as 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 agitation and freeze-thaw tests. A PS80 concentration of 0.05% was selected to prevent possible precipitation during shipping and to minimize the formation of subvisible particles.

[0200] Example 6: Preparation of an Intravenous (IV) Formulation of BAN2401 A. BAN2401 10mg / mL formulation BAN2401 10 mg / mL intravenous injection formulation ("10 mg / mL Injection") was manufactured by conventional cGMP aseptic processes for preparation of sterile aqueous formulations. BAN2401 10 mg / mL Injection was made from the corresponding BAN2401 bulk drug product below without the addition of any excipients or diluents. An exemplary IV formulation containing 10 mg / mL BAN2401 is shown in Table 10.

[0201] [Table 10]

[0202] The filtered BAN2401 drug substance solution was aseptically filled into vials as shown in Figure 17 (referred to as "Formulation A" in Figure 17). The pooled drug substance was passed through a 0.2 pm filter for a bioburden reduction filtration step. A final sterile filtration was performed through two 0.2 pm filters in succession, with pre- and post-filtration filter integrity tests. The sterile bulk drug substance product was aseptically filled into vials. Filling accuracy was confirmed by measuring the vial fill weight during the filling operation. The filled vials were stoppered and then sealed with an aluminum overseal. After seaming, the product was stored at 5±3°C.

[0203] The vials were analyzed using Method 1 (light obscuration) according to 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 100mg / mL formulation For preparation of the sterile aqueous formulation, the BAN2401 100 mg / mL formulation ("100 mg / mL Injection") was manufactured by conventional cGMP aseptic processes. BAN2401 100 mg / mL was made from the corresponding BAN2401 drug substance without the addition of any excipients or 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 pm filter for a bioburden reduction filtration step. A final sterile filtration was performed through two 0.2 pm filters in succession, with pre- and post-filtration filter integrity tests. The sterile bulk drug substance product was aseptically filled into vials. Filling accuracy was confirmed by measuring the vial fill weight during the filling operation. The filled vials were stoppered and then sealed with an aluminum overseal. After seaming, the product was stored at 5±3°C.

[0209] The vials were analyzed using Method 1 (light obscuration) according to 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 prepared. As shown in Table 16, for a second exemplary formulation containing 100 mg / mL of BAN2401, the following materials can be used:

[0213] [Table 16]

[0214] B. BAN2401 200mg / mL formulation The following materials can be used for an exemplary SC formulation containing 200 mg / mL BAN2401, as shown in Table 17. The stability of BAN2401 in these formulations (FSC1, FSC2, and FSC3) was evaluated in conjunction with an evaluation of material stability in three closed containers: i) Becton-Dickenson Hypak Biotech glass prefilled syringes (PFS) and stoppers ii) West Crystal Zenith Plastic PFS and Stoppers iii) West 5mL glass vials and stoppers

[0215] [Table 17]

[0216] BAN2401 was prepared by TFF at a target protein concentration of 200 mg / mL as summarized below. Separate TFF runs were performed to prepare BAN2401 material in each formulation buffer, except for FSC1a and FSC1b. For two of these formulations, one TFF run was performed and the resulting concentrated material was split into two half lots. A small amount of the sterile filtered material in each final formulation buffer was stored frozen at -20°C for filling into appropriate closed containers for syringe testing, rather than filling at time 0.

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

[0218] The concentration / diafiltration step was performed using a 0.02m 2 The TFF run was performed using a Pall Centramate LV system equipped with a membrane area of ​​1000 μg / mL. BAN2401 material (pulled from a GMP production lot prior to the addition of PS80) was loaded into the TFF system and concentrated 10-15x (stage 1). The material was then diafiltered with up to 5 diavolumes of formulation buffer, where diafiltration was monitored by pH and conductivity checks of the permeate (stage 2). After diafiltration, the material was further concentrated to a target protein concentration of 210-250 mg / mL (stage 3). The retentate was collected and sampled for protein concentration determination.

[0219] During 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 Millipore centrifugal filter units (30,000 MWCO). To perform this concentration step, the filter units were equilibrated with BAN2401 formulation buffer, and then the BAN2401 material was centrifuged at 3600 RPM (approximately 3000 x g) at 20 °C for 30 min intervals until the protein concentration of the retentate was expected to be higher than 200 mg / mL. The retentate was collected from the filter units and pooled. After thorough mixing, the pooled retentate was sampled for protein concentration measurement.

[0220] After protein concentration, samples were drawn 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. Final protein concentration adjustments were performed by dilution with the appropriate formulation buffer. Finally, 10% PS80 solution was added to BAN2401 to achieve 0.02% PS80 in the final solution, and the protein solution was mixed thoroughly by end-over-end rotation.

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

[0222] C. Stability Studies on Exemplary 200 mg / mL SC Formulations Containing BAN2401 Sample stability was evaluated using assays for pH (Figures 19-21), absorbance at 405 nm (Figures 22-24) (to detect increased yellowing), and size exclusion chromatography (SEC) (Figures 25-33) at 5°C (3 months, 6 months, 9 months, 12 months) and 25°C (1 month, 3 months) and then 5°C. See Table 17 for exemplary formulations FSC1-FSC4; note that in Figures 19-33, "F1a" refers to FSC1a, "F1b" refers to FSC1b, "F2" refers to FSC2, and "F3" refers to FSC3. Additional characterization assays were performed on the t=0 samples, including protein concentration, differential scanning calorimetry (DSC), PS80, and osmolality. Sub-visible particle testing was performed on the t=0 and t=6 month samples. A 3-day agitation test was performed on samples stored for 1 month at 5° C. Samples for this agitation test were transferred to 25° C. after 1 month of storage at 5° C. and agitated at 250 rpm for 3 days.

[0223] Test results showed that 200 mg / mL BAN2401 in all FSC1a-FSC3 formulations had similar aggregate and fragment levels after 12 months of storage at 2-8°C. The stability of BAN2401 at 200 mg / mL in the three FSC1a-FSC3 formulations was evaluated. Overall, the stability of BAN2401 in each formulation tested appeared similar, maintaining greater than 98.2% monomeric form after 12 months of storage at 5°C, regardless of the sealed container tested. In addition, the pH remained stable and there was no appreciable increase in A405 yellowing after 12 months of storage at 5°C.

[0224] (a) Protein concentration (T=0 sample) Protein concentration was measured using a UV-Vis spectrophotometer and the results are shown in Table 18.

[0225] [Table 18]

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

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

[0228] [Table 19]

[0229] This measurement was performed by quantifying oleic acid, a hydrolysis product of PS80. Using basic hydrolysis, PS80 releases oleic acid in a 1:1 molar ratio. Reverse-phase HPLC can then be used to separate oleic acid from other PS80 hydrolysis products and the matrix. Oleic acid was monitored using absorbance at 195 nm without derivatization [J. Chromatography B, 878 (2010) 1865-1870]. Experimentally, mixing the sample with sodium hydroxide released oleic acid, which was subsequently extracted with acetonitrile. The extract was diluted with potassium phosphate solution and a sample volume of 100 μL was injected onto a Waters Symmetry C18 column. Separation was performed using an isocratic elution containing 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 samples was determined using a freezing point osmometer and the results are shown in Table 20.

[0231] [Table 20]

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

[0233] (d) Subvisible particles Subvisible particle analysis was performed using a Fluid Technologies FlowCam instrument. The results for T=O are listed in Table 21. When the material was stored at 5°C, there appears to be an increase in total particle concentration and particle counts of particles larger than 10 μm over time. The increase in change, in decreasing order, is FSC1a. <FSC1b<FSC2<FSC3であった。

[0234] [Table 21]

[0235] Subvisible particle analysis was performed using a Fluid Imaging Technologies FlowCam equipped with a 20x objective and a 50um flow cell. Prior to running the BAN2401 samples, the flow cell was filled with dI H 2 Flush with HO and dI HO. 2 A measurement was performed on the flow cell to ensure it was clean. 0.2 mL of di H 2 The flow cell was considered ready for use when the total number of panicles counted per O was 2 or less. 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. The autoimage rate was 14, given an efficiency of 19.6% and a run time of 10 min. The resulting panicle concentrations were corrected for sample dilution.

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

[0237] (f) HPLC-SEC High performance liquid chromatography size exclusion chromatography (HPLC-SEC) analysis was performed utilizing a TSK 03000 SWXL column with a 0.2 M sodium phosphate, pH 7.0 mobile phase at a flow rate of 1.0 mL / min. 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 onto the column throughout the study. Relative peak area (%) results were reported for monomer, aggregates and fragments, as shown in Figures 25-33.

[0238] Physical degradation of BAN2401 was determined by performing HPLC-SEC on stability samples stored at 5 and 25°C. After 12 months of storage at 5°C, the aggregate percentage was similar for the three formulations at approximately 1.1%. The fragment percentages generated for all formulations tested ranged from 0.4 to 0.5%. After 12 months of storage at 5°C, the monomer content ranged from 98.4 to 98.6% for all formulations and sealed containers tested. With the current degradation rates, it is possible that BAN2401 in all formulations tested may have greater than 97% monomer after storage at 5°C for up to 24 months.

[0239] After storage for up to 3 months at 25° C., fragmentation after 3 months ranged from 0.4 to 0.8%. Monomer content was slightly higher than 98% for all formulations, with the exception of FSC3, which had slightly less than 98% monomer 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: Drug Substance (DS) process intermediate was concentrated and equilibrated with the corresponding formulation buffer (Table 22) by centrifugal filter unit. After concentration and equilibration, PS80 dissolved in formulation buffer was added to achieve the desired concentration and protein concentration was adjusted to the final concentration. Each candidate formulation was filled into vials with a fill 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 Tables 23, 24, and 25. After removal from storage, the vials were stored at 5° C. until the time of testing. Storage conditions were as follows: Long-term conditions: Stored upright at 5℃±3℃ Accelerated conditions: Stored upright at 25°C±2°C and 60%±5%RH Stress conditions (freezing and thawing): Frozen at -30°C in an upright position and thawed at room temperature. Stress loading conditions (agitation): 5°C, horizontal, 250 rpm with a vibration amplitude of 50 mm on a reciprocating shaker

[0243] [Table 23]

[0244] [Table 24]

[0245] [Table 25]

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

[0247] C. Results and Discussion (a) Selection of arginine concentration To select the target arginine concentration for the formulation, candidate formulations with various arginine concentrations (150-350 mmol / L, F1-F5) were evaluated for physical properties and subjected to freeze-thaw, long-term, and accelerated stability studies and compared to formulation F0 (see Table 22). The results of the physical properties and freeze-thaw studies are shown in Table 26. The results of the long-term and accelerated stability studies 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 values ​​increased. F1, F2 and F3 showed lower osmolality values ​​when compared to F4 and F5. Therefore, the arginine concentration was restricted to 250 mmol / L or less. Viscosity values ​​of F1-F5 were within a narrow range (7.3-8.1 cP) and did not correlate with arginine concentration within 150-350 mmol / L. The results of appearance, pH, and PS80 and protein concentrations were approximately on target.

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

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

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

[0255] (f) Arginine concentration As shown in Table 26, F4 and F5 (arginine concentrations of 300 and 350 mmol / L) were deemed not feasible when considering osmolality. 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, accelerated stability studies showed that formulations with higher arginine concentrations showed lower aggregate formation rates, but the difference in rates was not significant in the arginine concentration range evaluated. Taking into account both isotonicity and aggregate formation rate, 200 mmol / L based on the F2 formulation was selected as the target arginine concentration.

[0256] (g) Selection of protein concentration To select the target protein concentration for the formulation, candidate formulations with various protein concentrations (200-300 mg / mL, F2, F6, F7) and formulation F0 were evaluated for physical properties and subjected to freeze-thaw, long-term, and accelerated stability studies. The results of the physical properties and freeze-thaw tests 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 ​​compared to F7. The viscosity values ​​of F2, F6 and F7 were 7.8, 21 and 48 cP, respectively. A high viscosity above 20 cP can cause difficulties in the manufacture of DS due to increased pump back pressure and reduced membrane flux during ultrafiltration and diafiltration steps. In addition, a highly concentrated solution can cause difficulties in the DP filling process by clogging the filling needle, which leads to variability in the filling weight. The desired viscosity is 20 cP. 3 F2 was feasible as it was reported to be: Appearance, pH, and PS80 and protein concentration results were nearly on target.

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

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

[0261] (k) Accelerated stability testing 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 (0.7-0.8% increase) for all candidates (F2, F6 and F7) at 3 months. Therefore, all candidates were considered viable. The amount of fragments by SEC also increased for all candidates, but at a similar rate to F0. With regard to IEX, the amount of acidic peaks by IEX increased for all candidates, but at a similar rate to F0.

[0262] (l) Protein concentration Considering the osmolality and viscosity results 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, freeze-thaw and stirring tests were performed on candidate formulations with different PS80 concentrations [0-0.10 (w / v)%, F8-F12] and formulation F0. The results of the freeze-thaw and stirring 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 three cycles of freeze-thaw testing. The particle count by MFI was relatively high in F8 (without PS80).

[0267] (o) Stirring test As shown in Table 31, formulations (F9-F12) containing 0.02% (w / v)-0.10% (w / v) PS80 were stable after agitation. For F8 (without PS80), changes were observed after agitation in terms of appearance, protein concentration, and aggregate amount 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 aggregate amount 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 (agitation test), formulations containing 0.02 (w / v)% to 0.10 (w / v)% PS80 were stable after three cycles of freeze-thawing and agitation for up to three days. Therefore, a PS80 concentration of 0.05 (w / v)% was selected as the target value.

[0269] (q) Conclusion In conclusion, the following formulations were selected as BAN2401 formulations for further testing:

[0270] [Table 32]

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

[0272] A. Sample Preparation Each candidate formulation (Table 33) was prepared as follows: The formulation was equilibrated with the corresponding formulation buffer through a centrifugal filter unit. For the preparation of F20 only, a formulation buffer with a pH of 3.5 was used until the pH of the filtered solution reached 4.0 due to the repulsion of protons to the concentrated charged protein near the semipermeable membrane, known as the Donnan effect. After concentration and equilibration, PS80 dissolved in the formulation buffer was added to achieve the desired concentration, and then the protein concentration was adjusted to the final concentration. Each candidate formulation was filled into a vial with a fill 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 testing began. Storage conditions were as follows: Long-term conditions: Stored upright at 5℃±3℃ Accelerated conditions: Stored upright at 25°C±2°C and 60%±5%RH Stress conditions (freezing and thawing): Frozen at -30°C in an upright position and thawed at room temperature. 1000lx: 25℃±2℃, 60%RH±5%RH, lying down, 1000lx light irradiation Dark place: 25℃±2℃, 60%RH±5%RH, laid down, covered with aluminum foil to prevent exposure to light.

[0275] To confirm aggregate levels after storage for more than 3 months, size exclusion HPLC was further evaluated for extended storage samples at 9 months for the long-term and 3 months for the accelerated stability studies, the latter samples being tested after 6 months of storage in a refrigerator.

[0276] [Table 34]

[0277] [Table 35]

[0278] [Table 36]

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

[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-F22) for any of the test items except for size exclusion HPLC (SEC). Formulation F20 (i.e., formulation with a low pH of 4.0) showed a low amount of aggregates by SEC at the start. In addition, a slightly lower rate of aggregate formation and a higher rate of fragmentation were observed in the accelerated stability test. The results of the formulations with pH 4.5-5.5 were confirmed to be comparable.

[0281] [Table 37]

[0282] [Table 38]

[0283] [Table 39]

[0284] [Table 40]

[0285] [Table 41]

[0286] E. Methionine supplementation As shown in Tables 41A and 41B, and Tables 42 to 46, no significant differences were observed between formulations with or without methionine (F18 and F15). The formulation with methionine (F18) showed a slightly lower rate of aggregate formation in extended long-term stability studies, accelerated stability studies, and photostability studies. F18 also showed a slightly lower oxidation of methionine residues (e.g., position 259 of the heavy chain) with 1000 lux light exposure up to 7 days. Freeze-thaw studies did not show any differences. 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 attributes were comparable between formulations of BAN2401 with pH changes from pH 4.5 to pH 5.5. The formulation at pH 4.0 showed a lower aggregate formation rate and, conversely, a faster fragmentation rate during storage. We found no significant differences between formulations with and without methionine at a concentration of 10 mmol / L based on the evaluation of quality and stability tests, including long-term, accelerated, freeze-thaw, and light. A slightly slower aggregate formation rate and slight inhibition of oxidation of amino acid residues were observed, but the differences were not significant. In conclusion, F15 and F21 at 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 into the effect of arginine concentration on the stability of BAN2401 formulations The effect of arginine concentration (0, 50, 100, and 200 mmol / L) on the stability of 200 mg / mL BAN2401 was 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 concentrated BAN2401 drug substance process intermediates by ultrafiltration. After sterile filtration, 0.4 mL samples were filled into vials. Sample information is shown in Table 47.

[0297] [Table 49]

[0298] B. Results The results of this stability study for samples F1-F4 are shown in Tables 48-51. There were no differences in pH and protein concentration between the samples, and arginine concentration levels were consistent with the target values ​​for the starting samples.

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

[0300] [Table 50]

[0301] [Table 51]

[0302] [Table 52]

[0303] [Table 53]

[0304] C. Discussion and Conclusions The results of this study showed that arginine concentrations of 50-200mmol / L prevented the increase of protein aggregates in the BAN2401 200mg / mL formulation. Batch-to-batch variation in arginine concentration may result in batch-to-batch variation in aggregate %. In addition, arginine concentration may affect the tendency of increasing aggregate % in stability studies.

[0305] When comparing the fragmentation rate of sample F1 with that shown in FIG. 11 of Example 2, which used a sample containing 200 mg / mL BAN2401, 50 mM sodium citrate, and 100 mM sodium chloride, the formulation containing histidine buffer appeared to be effective in reducing the fragmentation of BAN2401.

[0306] [Table 54]

[0307] [Table 55]

[0308] [Table 56]

[0309] Heavy Chain (SEQ ID NO:11): [ka] Light Chain (SEQ ID NO: 12): [ka]

Claims

1. An aqueous pharmaceutical formulation comprising: (a) an isolated anti-Aβ protofibril antibody that binds to human Aβ protofibrils at a concentration of 80 mg / mL to 300 mg / mL; (b) 100 mM to 400 mM of arginine, arginine hydrochloride, or a combination thereof; (c) 0.01% w / v to 0.1% w / v of polysorbate 80; and (d) a pharmaceutically acceptable buffer, wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the isolated anti-Aβ protofibril antibody comprises (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.

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

4. The pharmaceutical formulation according to claim 1, wherein the isolated anti-Aβ protofibril antibody 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 10 to 100 mM of a citrate buffer or 10 to 100 mM of a histidine buffer.

8. The pharmaceutical formulation according to any one of claims 1 to 7, comprising 125 to 350 mM of arginine, arginine hydrochloride, or a combination thereof.

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

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

11. An aqueous pharmaceutical formulation comprising: (a) an isolated anti-Aβ protofibril antibody at a concentration of 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 of arginine hydrochloride; (c) 0.02% w / v to 0.08% w / v of polysorbate 80; and (d) 15 mM to 35 mM of a histidine buffer, wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.

5.

12. An aqueous pharmaceutical formulation comprising: (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 at a concentration of 80 mg / mL to 120 mg / mL, (b) 240 mM to 360 mM of arginine, arginine hydrochloride, or a combination thereof, (c) 0.02% w / v to 0.08% w / v of polysorbate 80, and (d) 30 mM to 50 mM of citrate buffer wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.

5.

13. An aqueous pharmaceutical formulation comprising: (a) an isolated anti-Aβ protofibril antibody that binds to human Aβ protofibrils at a concentration of 80 mg / mL to 300 mg / mL, (b) a histidine buffer, and (c) 0.01% w / v to 0.1% w / v of polysorbate 80 wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and wherein the isolated anti-Aβ protofibril antibody comprises: (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.

14. The pharmaceutical formulation according to claim 13, wherein the isolated anti-Aβ protofibril antibody is present at a concentration of 100 mg / mL to 200 mg / mL.

15. The pharmaceutical formulation according to claim 13 or 14, wherein the histidine buffer is present at a concentration of about 25 mM.

16. The pharmaceutical formulation according to any one of claims 13 to 15, wherein the histidine buffer comprises histidine and histidine hydrochloride monohydrate.

17. The pharmaceutical formulation according to claim 1, comprising: 100 mg / mL of the isolated anti-Aβ protofibril antibody, 190 mM to 210 mM of arginine hydrochloride, 0.04% w / v to 0.06% w / v of polysorbate 80, and 15 mM to 35 mM of histidine buffer

18. The pharmaceutical formulation according to claim 1, comprising: 200 mg / mL of the isolated anti-Aβ protofibril antibody, 190 mM to 210 mM of arginine hydrochloride, 0.04% w / v to 0.06% w / v of polysorbate 80, and 15 mM to 35 mM of histidine buffer

19. The pharmaceutical formulation according to claim 1, comprising: 100 mg / mL of the isolated anti-Aβ protofibril antibody, 200 mM of arginine hydrochloride, 0.05% w / v of polysorbate 80, and 25 mM of histidine buffer

20. ​ ​ ​ ​ ​ The isolated anti-Aβ protofibril antibody at 200 mg / mL, arginine hydrochloride at 200 mM, polysorbate 80 at 0.05% w / v, and a histidine buffer at 25 mM The pharmaceutical preparation according to claim 1, comprising the above.

21. The pharmaceutical preparation according to any one of claims 17 to 20, wherein the histidine buffer is a combination of histidine and histidine hydrochloride monohydrate.

22. The pharmaceutical preparation according to any one of claims 17 to 21, wherein the pH of the pharmaceutical preparation is 5.

0.

23. The pharmaceutical preparation according to any one of claims 1 to 22, wherein the isolated anti-Aβ protofibril antibody is lecanemab.

24. Lecanemab at 100 mg / mL, a histidine buffer at 25 mM, arginine hydrochloride at 200 mM, and polysorbate 80 at 0.05% w / v An aqueous pharmaceutical preparation comprising the above, wherein the pH of the pharmaceutical preparation is about 5.

0.

25. Lecanemab at 200 mg / mL, a histidine buffer at 25 mM, arginine hydrochloride at 200 mM, and polysorbate 80 at 0.05% w / v An aqueous pharmaceutical preparation comprising the above, wherein the pH of the pharmaceutical preparation is about 5.

0.

26. Lecanemab at 100 mg / mL, arginine hydrochloride at 42.13 mg / mL, polysorbate 80 at 0.50 mg / mL, and a histidine buffer at 25 mM An aqueous pharmaceutical preparation comprising the above, wherein the pH of the pharmaceutical preparation is about 5.

0.

27. Lecanemab at 200 mg / mL, arginine hydrochloride at 42.13 mg / mL, polysorbate 80 at 0.50 mg / mL, and a histidine buffer at 25 mM An aqueous pharmaceutical preparation comprising the above, wherein the pH of the pharmaceutical preparation is about 5.

0.

28. The pharmaceutical preparation according to any one of claims 24 to 27, wherein the histidine buffer is a combination of histidine and histidine hydrochloride.

29. The pharmaceutical preparation according to any one of claims 24 to 27, wherein the histidine buffer is a combination of histidine and histidine hydrochloride monohydrate.

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