High concentration Anti-aβ protofibril antibody formulations and methods of use thereof

Abstract

This article provides an aqueous pharmaceutical formulation comprising a high concentration of isolated anti-Aβ fibrillary antibody or fragment thereof that binds to human Aβ fibrils, such as BAN2401, arginine, polysorbate 80, and a pharmaceutically acceptable buffer.

Description

Technical Field

[0001] This invention relates to an aqueous pharmaceutical formulation comprising a high concentration of isolated anti-Aβ fibrillary antibody or fragment thereof that binds to human Aβ fibrils, such as BAN2401, arginine, polysorbate 80, and a pharmaceutically acceptable buffer. Prior Technology

[0002] Alzheimer's disease (AD) is a progressive, neurodegenerative disorder of unknown cause and the most common form of dementia in older adults. In 2006, there were 26.6 million cases of AD worldwide (range: 11.4-59.4 million) (Brookmeyer, R. et al., Forecasting the global burden of Alzheimer's Disease. Alzheimer Dement. 2007; 3: 186-91). It was also reported that over 5 million people in the United States had AD (2010 Alzheimer's disease facts and figures. Alzheimer Dement. 2010; 6: 158-94). By 2050, the global prevalence of Alzheimer's disease (AD) is projected to reach 106.8 million (range: 47.2 million–221.2 million), with an estimated 11 million to 16 million in the United States alone. (Brookmeyer, see above, and 2010 Alzheimer's disease facts and figures, see above).

[0003] The disease is typically characterized by an overall decline in cognitive function, which progresses slowly and leaves terminally ill patients bedridden. Individuals with Alzheimer's disease (AD) typically survive only 3 to 10 years after symptom onset, although known extremes include 2 to 20 years. (Hebert, LE et al., Alzheimer's disease in the US population: prevalence estimates using the 2000 census. Arch Neurol. [Archives of Neurology] 2003; 60: 1119-1122.) Although deaths due to AD are significantly underestimated because death certificates rarely attribute the cause of death to AD, AD remains the seventh leading cause of death in the United States and the fifth leading cause of death among Americans over 65 years of age. (2010 Alzheimer's disease facts and figures, see above.)

[0004] Histologically, the disease is characterized by neuroinflammatory plaques, primarily found in the associative cortex, limbic system, and basal ganglia. The main component of these plaques is amyloid-β peptide (Aβ). Aβ exists in various conformational states: monomers, oligomers, fibrils, and insoluble filaments. The detailed mechanistic relationship between Alzheimer's disease pathogenesis and Aβ production remains unclear. However, several anti-Aβ antibodies are currently undergoing clinical trials as potential therapeutic agents for Alzheimer's disease.

[0005] Anti-Aβ antibodies and other proteins can be administered to subjects via intravenous, subcutaneous, intramuscular, and other routes. The dosage and / or formulation of the antibody can present numerous challenges in developing suitable drug formulations. For example, at high antibody concentrations, antibody stability can be problematic due to the formation or fragmentation of protein-protein aggregates. Typically, aggregation increases with increasing antibody concentration. Furthermore, high-concentration antibody formulations require high concentrations of stabilizers and other excipients to achieve long-term protein stability and shelf life. High-concentration antibody formulations are also often viscous, which can complicate the manufacture and administration of the drug formulation. Summary of the Invention

[0006] This article provides pharmaceutical formulations comprising a therapeutically effective amount of at least one isolated anti-Aβ fibrillary antibody or fragment thereof that binds to Aβ fibrils. This article also provides pharmaceutical formulations comprising 80-300 mg / mL of an isolated anti-Aβ fibrillary antibody or fragment thereof that binds to Aβ fibrils, wherein the antibody system is BAN2401 (also known as lecanemab). The pharmaceutical formulations provided herein have been found to be advantageous. For example, despite high concentrations of the anti-Aβ fibrillary antibody (e.g., 100 mg / mL or 200 mg / mL), the protein-protein aggregation rate is unexpectedly low and similar to that typically observed at much lower antibody concentrations (e.g., 10 mg / mL). In some embodiments, the currently disclosed pharmaceutical formulations exhibit a lower initial aggregation rate (e.g., approximately 0.3% for 100 mg / mL of anti-Aβ fibrillary antibody compared to formulations with significantly lower concentrations of anti-Aβ fibrillary antibody, compared to approximately 0.8% for 10 mg / mL of anti-Aβ fibrillary antibody). In some embodiments, the currently disclosed pharmaceutical formulations exhibit a lower rate of subvisible particle generation (e.g., 10.6 particles / mL for 100 mg / mL of anti-Aβ fibrillary antibody compared to 12.6 particles / mL for 10 mg / mL of anti-Aβ fibrillary antibody). In some embodiments, the currently disclosed pharmaceutical formulations exhibit reduced aggregation rate, reduced initial aggregation level, reduced protein fragmentation rate, and / or reduced subvisible particle formation compared to formulations with significantly lower concentrations of anti-Aβ fibrillary antibody. Low aggregation rates, initial aggregation levels, protein fragmentation rates, and / or the formation of subvisible particles can allow for increased stability and / or extended product shelf life. Furthermore, the excipients in the currently disclosed pharmaceutical formulations can be present in lower amounts compared to currently commercially available intravenous products. In some embodiments, the pH and osmolality of the currently disclosed pharmaceutical formulations, after dilution in intravenous solution, are acceptable for intravenous administration systems.

[0007] In some embodiments, the at least one anti-Aβ protoplast 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, the at least one anti-Aβ fibrillary antibody comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of an amino acid sequence containing 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) of an amino acid sequence containing SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3).

[0009] The assignment of amino acids to each domain is usually based on the definition of the sequence of the protein of immunological interest (SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST) ​​(Kabat et al., 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242, 1991, hereinafter referred to as the "Kabat Report").

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

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

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

[0013] In some embodiments, the at least one anti-Aβ fibrillary antibody is BAN2401, also known as lecanemab. BAN2401 is a humanized IgG1 monoclonal form of mAb158, a murine monoclonal antibody produced to target fibrils and disclosed in WO 2007 / 108756 and Journal of Alzheimer's Disease 43: 575-588 (2015). BAN2401 is at least one anti-Aβ fibrillary antibody that exhibits low affinity for Aβ monomers while binding to soluble Aβ aggregates with high selectivity. For example, BAN2401 has been reported to exhibit selectivity for soluble Aβ fibrils that is approximately 1000 times higher than that for Aβ monomers and 5 to 10 times higher than that for insoluble Aβ fibrils.

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

[0015] Other non-limiting examples of antibodies suitable as at least one anti-Aβ fibrillary antibody disclosed herein include the antibodies 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 fibrillary antibody is present at a concentration of at least 80 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration of at least 100 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration of at least 200 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration of at least 250 mg / mL. In some embodiments, the isolated antibody or a fragment thereof is present at a concentration ranging from 80 mg / mL to 300 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration ranging from 85 mg / mL to 275 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration ranging from 90 mg / mL to 250 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration ranging from 95 mg / mL to 225 mg / mL. In some embodiments, the isolated anti-Aβ fibrillary antibody is present at a concentration ranging from 100 mg / mL to 200 mg / mL. In some embodiments, the isolated antibody or a fragment thereof is present at concentrations 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 a 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) containing 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) containing the amino acid sequences of SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3).

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

[0018] In some embodiments, the pharmaceutical formulation comprises arginine (and / or arginine salt). In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation ranges from about 100 mM to about 400 mM. In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation ranges from about 110 mM to about 380 mM. In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation ranges from about 120 mM to about 360 mM. In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation ranges from about 125 mM to about 350 mM. In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation is 125 mM. In some embodiments, the concentration of arginine (and / or arginine salt) in the pharmaceutical formulation is 200 mM. In some embodiments, the concentration of arginine (and / or arginine salt) 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% 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. In some embodiments, the polysorbate 80 is present in the pharmaceutical formulation at concentrations 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, the polysorbate 80 is present in the pharmaceutical formulation at a concentration of 0.02% w / v. In some embodiments, the polysorbate 80 is present in the pharmaceutical formulation at a concentration of 0.05% w / v.

[0021] In some embodiments, the pharmaceutical formulation comprises citrate monohydrate. In some embodiments, the concentration of citrate monohydrate in the pharmaceutical formulation ranges from about 10 mM to 100 mM. In some embodiments, the concentration of citrate 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 citrate monohydrate in the pharmaceutical formulation is 50 mM.

[0022] In some embodiments, this disclosure provides a pharmaceutical formulation having a pH range of 4.5 to 5.5. In some embodiments, the pH range of the pharmaceutical formulation is 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 formulation disclosed herein may be in the form of a solution and / or any other suitable liquid formulation that those skilled in the art deem appropriate. The administration route of the disclosed composition may be intravenous or subcutaneous. In some embodiments, the pharmaceutical formulation is formulated as a sterile, pyrogen-free fluid for intravenous administration. In some embodiments, the pharmaceutical formulation is formulated as a sterile, pyrogen-free fluid 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 fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, and further comprising, for example, citrate monohydrate, arginine, arginine salt, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 100 mg / mL of at least one isolated anti-Aβ fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, 50 mM citrate monohydrate, 110 mM arginine, 240 mM arginine salt, and 0.05% (w / v) polysorbate 80, and 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 fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, and further comprising, for example, histidine, histidine salt, arginine salt, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 100 mg / mL or 200 mg / mL of at least one isolated anti-Aβ fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, 25 mM of histidine and histidine salt, 200 mM of arginine salt, and 0.05% (w / v) of polysorbate 80, and 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 fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, and further comprising, for example, histidine, histidine salt, arginine salt, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 200 mg / mL of at least one isolated anti-Aβ fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, 50 mM of histidine and histidine salt, 125 mM of arginine salt, and 0.02% (w / v) of polysorbate 80, and 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 fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, and further comprising, for example, histidine, histidine salt, arginine salt, and polysorbate 80. In some embodiments, the pharmaceutical formulation comprises 200 mg / mL of at least one isolated anti-Aβ fibrillary antibody or fragment thereof (e.g., BAN2401) that binds to Aβ fibrils, 50 mM of citrate (and / or citrate monohydrate), 125 mM of arginine (and / or arginine salt), and 0.02% (w / v) of polysorbate 80, and a pH of 5.0 ± 0.4.

[0028] BAN2401 and the methods of using BAN2401 disclosed in U.S. Provisional Application No. 62 / 749,614 and PCT International Application No. PCT / US 2019 / 043067 are incorporated herein by reference in their entirety. Simple Explanation of the Diagram

[0029] [Figure 1] illustrates the percentage of aggregation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 5°C.

[0030] [Figure 2] illustrates the percentage of fragmentation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 5°C.

[0031] [Figure 3] illustrates the percentage of monomers in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 5°C.

[0032] [Figure 4] illustrates the percentage of aggregation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 25°C.

[0033] [Figure 5] illustrates the percentage of fragmentation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 25°C.

[0034] [Figure 6] illustrates the percentage of monomers in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, 2, and 3 months at 25°C.

[0035] [Figure 7] illustrates the percentage of aggregation in formulations of BAN2401 at 5°C for 0, 1, 2, and 3 months, with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0036] [Figure 8] illustrates the percentage of fragmentation in BAN2401 formulations at 5°C for 0, 1, 2, and 3 months, with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0037] [Figure 9] illustrates the percentage of monomers in 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 5°C.

[0038] [Figure 10] illustrates the percentage of aggregation in formulations of BAN2401 at 25°C for 0, 1, 2, and 3 months, with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0039] [Figure 11] illustrates the percentage of fragmentation in BAN2401 formulations at 25°C for 0, 1, 2, and 3 months, with pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5.

[0040] [Figure 12] illustrates the percentage of monomers in formulations of BAN2401 at pH values ​​of 4.5, 5.0, 5.5, 6.0, and 6.5 at 25°C for 0, 1, 2, and 3 months.

[0041] [Figure 13] illustrates the percentage of aggregation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, and 2 months at 25°C.

[0042] [Figure 14] illustrates the percentage of fragmentation in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, and 2 months at 25°C.

[0043] [Figure 15] illustrates the percentage of monomers in formulations of BAN2401 at 10 mg / mL, 100 mg / mL, and 200 mg / mL at 0, 1, and 2 months at 25°C.

[0044] [Figure 16] illustrates the percentage of monomer aggregation in the formulation of BAN2401 as a function of arginine concentration.

[0045] [Figure 17] illustrates the process of manufacturing a 10 mg / mL injection of BAN2401.

[0046] [Figure 18] illustrates the process of manufacturing a 100 mg / mL injection of BAN2401.

[0047] [Figure 19] illustrates the pH values ​​of the BAN2401 formulation at 5°C at 0 and 12 months.

[0048] [Figure 20] illustrates the pH values ​​of the BAN2401 formulation at 25°C for 0, 1, and 3 months.

[0049] [Figure 21] illustrates the pH values ​​of the BAN2401 formulation after one month at 5°C and with stirring at 25°C.

[0050] [Figure 22] illustrates the absorbance of the BAN2401 formulation at 405 nm at 5°C for 0, 1, 3, 6, 9, and 12 months.

[0051] [Figure 23] illustrates the absorbance of the BAN2401 formulation at 405 nm at 0, 1, and 3 months at 25°C.

[0052] [Figure 24] illustrates the absorbance of the BAN2401 formulation at 405 nm after one month at 5°C and after stirring at 25°C.

[0053] [Figure 25] illustrates the percentage of aggregation in formulations of BAN2401 at 0, 1, 3, 6, 9, and 12 months at 5°C.

[0054] [Figure 26] illustrates the percentage of fragmentation in the formulation of BAN2401 at 0, 1, 3, 6, 9, and 12 months at 5°C.

[0055] [Figure 27] illustrates the percentage of monomers in the formulation of BAN2401 at 0, 1, 3, 6, 9, and 12 months at 5°C.

[0056] [Figure 28] illustrates the percentage of aggregation in the formulation of BAN2401 at 0, 1, and 3 months at 25°C.

[0057] [Figure 29] illustrates the percentage of fragmentation in the formulation of BAN2401 at 0, 1, and 3 months at 25°C.

[0058] [Figure 30] illustrates the percentage of monomers in the formulation of BAN2401 at 0, 1, and 3 months at 25°C.

[0059] [Figure 31] illustrates the percentage of aggregation in the formulation of BAN2401 after one month at 5°C and after stirring at 25°C.

[0060] [Figure 32] illustrates the percentage of fragmentation in the formulation of BAN2401 after one month at 5°C and after stirring at 25°C.

[0061] [Figure 33] illustrates the percentage of monomers in the formulation of BAN2401 after one month at 5°C and after stirring at 25°C. Implementation

[0062] This application claims priority to U.S. Provisional Application No. 62 / 992,746, filed March 20, 2020, and U.S. Provisional Application No. 63 / 027,263, filed May 19, 2020; the entire contents of both are incorporated herein by reference. [, definition , ] [, , ]

[0063] The following are definitions of the terms used in this application.

[0064] Unless the context clearly indicates otherwise, as used herein, the singular terms "a / an" and "the" include plural references.

[0065] As used herein, the phrase "and / or" means "either one or both" of the elements that are combined, that is, elements that are combined in some cases and not combined in others. Therefore, as a non-limiting example, "A and / or B," when used in conjunction with open-ended terms such as "including," may in some embodiments refer only to A (including elements other than B, if necessary); in other embodiments, only to B (including elements other than A, if necessary); in still other embodiments, both A and B (including other elements, if necessary); and so on.

[0066] As used herein, "at least one" means one or more elements in the list of elements, but is not necessarily required to include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently "at least one of A and / or B") may in one embodiment mean at least one (or more than one, if needed) A without B (and, if needed, include elements other than B); in another embodiment, it means at least one (or more than one, if needed) B without A (and, if needed, include elements other than A); in yet another embodiment, it means at least one (or more than one, if needed) A and at least one (or more than one, if needed) B (and, if needed, include other elements); and so on.

[0067] When a number is described alone or as part of a numerical range, it should be understood that the value may vary reasonably above or below the value, as is known to those skilled in the art.

[0068] As used herein, an "antibody fragment" comprises a portion of the antibody, such as an antigen-binding region or its variable region. Non-limiting examples of fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, biantibodies, linear antibodies, and single-chain antibody molecules.

[0069] As used herein, "fragmentation" or "fragmentation" refers to the degradation of an antibody or a fragment thereof when it is present in or added to a formulation. Fragments resulting from fragmentation or fragmentation may or may not bind to antigens to which the antibody or its fragments are bound.

[0070] As used herein, "histidine buffer" may comprise histidine, histidine salt, or a combination thereof, wherein histidine salt may be histidine salt monohydrate.

[0071] As used herein, a "citrate buffer" may contain citric acid, its salts, or a combination thereof, wherein the citric acid may be citric acid monohydrate or anhydrous citric acid. [, Non-limiting embodiments disclosed herein: , ] [, , ]

[0072] Some embodiments disclosed herein relate to aqueous pharmaceutical formulations.

[0073] In some embodiments, an aqueous pharmaceutical formulation is provided, the aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof in concentrations of 80–300 mg / ml, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; and (b) 100 mM to 400 mM arginine, wherein the arginine is arginine, arginine salt, or a combination thereof; the aqueous pharmaceutical formulation may, if necessary, contain any one or more of the following: a. Polysorbate 80, 0.01% w / v to 0.1% w / v; b. A pharmaceutically acceptable buffer, as needed, wherein the buffer is a histidine buffer or a citrate buffer; and c. pH 4.5 to 5.5.

[0074] In some embodiments, an aqueous pharmaceutical formulation is provided, the aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof in concentrations of 80–300 mg / ml, comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; and (b) A pharmaceutically acceptable buffer, wherein the buffer is a histidine buffer, wherein the concentration of the histidine buffer is about 25 mM as needed; the aqueous pharmaceutical formulation may contain any one or more of the following as needed: a. Polysorbate 80, 0.01% w / v to 0.1% w / v; b. 100 mM to 400 mM arginine, arginine salt, or combinations thereof; and c. pH 4.5 to 5.5.

[0075] Some embodiments relate to aqueous pharmaceutical formulations comprising: (a) Isolated anti-Aβ fibrillary antibodies or fragments thereof that bind to human Aβ fibrils at concentrations ranging from 80 mg / mL to 300 mg / mL. (b) Arginine from 100 mM to 400 mM, (c) Polysorbate 80, from 0.01% w / v to 0.1% w / v, and (d) Pharmaceutically acceptable buffers, The drug formulation has a pH range of 4.5 to 5.5, and The antibody or its fragment 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. Furthermore, arginine is arginine, arginine salt salt, or a combination thereof.

[0076] [Antibody Concentration] [ ]

[0077] For any aqueous pharmaceutical formulation described herein, the antibody may be present at the following concentrations. [ ]

[0078] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 100 mg / mL or higher.

[0079] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 200 mg / mL or higher.

[0080] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a 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 fibrillary antibody or a fragment thereof is present at a concentration ranging from 80 mg / mL to 240 mg / mL.

[0081] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration range of 100 mg / mL to 200 mg / mL.

[0082] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or fragment thereof is present at concentrations ranging from 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, and 100 to 200 mg / mL. In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or fragment thereof is present at concentrations ranging from 90 mg / mL to 220 mg / mL, 100 mg / mL to 210 mg / mL, or 110 mg / mL to 200 mg / mL.

[0083] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at concentrations 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.

[0084] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 100 mg / mL.

[0085] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 200 mg / mL.

[0086] In some embodiments of the pharmaceutical formulation, the isolated anti-Aβ fibrillary antibody or a fragment thereof is BAN2401.

[0087] Arginine [ ]

[0088] For any aqueous pharmaceutical formulation described herein, the formulation may contain arginine as follows. [ ]

[0089] In some embodiments, the pharmaceutical formulation comprises arginine. In some embodiments, the arginine is arginine, arginine salt, or a combination thereof.

[0090] In some embodiments of the pharmaceutical formulation, the concentration of arginine ranges from about 100 mM to about 400 mM.

[0091] In some embodiments of the pharmaceutical formulation, the concentration range of arginine is 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 range of arginine is 150 mM to 250 mM, 160 mM to 240 mM, 170 mM to 230 mM, 180 mM to 220 mM, 190 mM to 210 mM of arginine, arginine salt salt, or combinations thereof.

[0092] In some embodiments of this pharmaceutical formulation, the concentration of arginine is 125 mM.

[0093] In some embodiments of this pharmaceutical formulation, the concentration of arginine is 200 mM.

[0094] In some embodiments of the pharmaceutical formulation, the concentration range of the arginine is 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.

[0095] In some embodiments of this pharmaceutical formulation, the concentration of arginine is 350 mM.

[0096] [Polysorbate]

[80] [(] [PS80] [)] [ ]

[0097] For any aqueous pharmaceutical formulation described herein, the formulation may contain polysorbate 80 as follows. [ ]

[0098] In some embodiments of the pharmaceutical formulation, the concentration range of the polysorbate 80 is 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.

[0099] In some embodiments of the pharmaceutical formulation, the polysorbate 80 is present at concentrations 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.

[0100] In some embodiments of the pharmaceutical formulation, the polysorbate 80 is present at a concentration of 0.02% w / v.

[0101] In some embodiments of the pharmaceutical formulation, the polysorbate 80 is present at a concentration of 0.05% w / v.

[0102] [Buffer] [ ]

[0103] For any aqueous pharmaceutical formulation described herein, the formulation may contain a pharmaceutically acceptable buffer. [ ]

[0104] In some embodiments of this pharmaceutical formulation, the pharmaceutically acceptable buffer is a citrate buffer.

[0105] In some embodiments of the pharmaceutical formulation, the citrate buffer is present at a concentration of about 10 mM to about 100 mM.

[0106] In some embodiments of the pharmaceutical formulation, the concentration range of the citrate buffer is 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.

[0107] In some embodiments of this pharmaceutical formulation, the citrate buffer is present at a concentration of 50 mM.

[0108] In some embodiments of this pharmaceutical formulation, the pharmaceutically acceptable buffer is a histidine buffer.

[0109] In some embodiments of the pharmaceutical formulation, the histidine buffer is present at a concentration of about 10 mM to about 100 mM.

[0110] In some embodiments of the pharmaceutical formulation, the concentration range of the histidine buffer is 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.

[0111] In some embodiments of this pharmaceutical formulation, the histidine buffer is present at a concentration of 25 mM.

[0112] In some embodiments of this pharmaceutical formulation, the histidine buffer is present at a concentration of 50 mM.

[0113] In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine salt monohydrate. In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine salt monohydrate, wherein the concentration of histidine is about 0.1 to 0.3 mg / mL, and the concentration of histidine salt monohydrate is about 4 to 6 mg / mL. In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine salt monohydrate, wherein the concentration of histidine is about 0.26 mg / mL, and the concentration of histidine salt monohydrate is about 4.89 mg / mL. In some embodiments of the pharmaceutical formulation, the histidine buffer comprises histidine and histidine salt monohydrate, wherein the histidine comprises 0.2-0.3 mg / mL of histidine and 4.4 to 4.9 mg / mL of histidine salt, wherein the histidine salt is a monohydrate, as needed.

[0114] [pH]

[0115] For any aqueous pharmaceutical formulation described herein, the formulation may have the following pH values. [ ]

[0116] In some embodiments of the pharmaceutical formulation, the pH ranges from 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.

[0117] In some embodiments of this 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.

[0118] In some embodiments of this pharmaceutical formulation, the pH is 4.5 to 5.5.

[0119] In some embodiments of this pharmaceutical formulation, the pH is 5.0.

[0120] In some embodiments of the pharmaceutical formulation, the pharmaceutical formulation is suitable for intravenous injection.

[0121] In some embodiments of the pharmaceutical formulation, the pharmaceutical formulation is suitable for subcutaneous injection.

[0122] In some embodiments of the pharmaceutical formulation, the pharmaceutical formulation contains methionine.

[0123] In some embodiments, an aqueous pharmaceutical formulation is disclosed, comprising: (a) BAN2401 at concentrations ranging from 80 mg / mL to 240 mg / mL, (b) 140 mM to 260 mM arginine salts, (c) Polysorbate 80, from 0.01% w / v to 0.1% w / v, and (d) 15 mM to 35 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0124] In some embodiments, an aqueous pharmaceutical formulation is disclosed, comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations ranging from 80 mg / mL to 240 mg / mL, the isolated anti-Aβ fibrillary 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) 140 mM to 260 mM arginine salts, (c) Polysorbate 80, from 0.01% w / v to 0.1% w / v, and (d) 15 mM to 35 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0125] In some embodiments, an aqueous pharmaceutical formulation is disclosed, comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations from 80 mg / mL to 240 mg / mL, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 12. (b) 140 mM to 260 mM arginine salts, (c) Polysorbate 80, from 0.01% w / v to 0.1% w / v, and (d) 15 mM to 35 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0126] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) BAN2401 at concentrations ranging from 80 mg / mL to 120 mg / mL, (b) Arginine at 240 mM to 360 mM, (c) Polysorbate 80, from 0.03% w / v to 0.08% w / v, and (d) 30 mM to 70 mM citrate buffer, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0127] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations of 80 mg / mL to 120 mg / mL, the isolated anti-Aβ fibrillary 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) Arginine at 240 mM to 360 mM, (c) Polysorbate 80, from 0.03% w / v to 0.08% w / v, and (d) 30 mM to 70 mM citrate buffer, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0128] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations of 80 mg / mL to 120 mg / mL, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 12. (b) Arginine at 240 mM to 360 mM, (c) Polysorbate 80, from 0.03% w / v to 0.08% w / v, and (d) 30 mM to 70 mM citrate buffer, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0129] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL BAN2401, (b) 200 mM arginine salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0130] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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 salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0131] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 12. (b) 200 mM arginine salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0132] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) BAN2401 at 200 mg / mL, (b) 200 mM arginine salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0133] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 200 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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 salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0134] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 200 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 12. (b) 200 mM arginine salt, (c) 0.05% w / v polysorbate 80, and (d) 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5.

[0135] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL BAN2401, (b) 350 mM arginine, (c) 0.05% w / v polysorbate 80, and (d) 50 mM citrate buffer, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0136] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0137] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) 100 mg / mL of isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing 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, The pharmaceutical formulation has a pH in the range of 4.5 to 5.5, and the arginine is arginine, arginine salt, or a combination thereof.

[0138] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) BAN2401 at concentrations of 150 mg / mL to 250 mg / mL, (b) 100 mM to 150 mM arginine salt, (c) Polysorbate 80, from 0.01% w / v to 0.05% w / v, and (d) Histidine buffer at 35 mM to 65 mM,

[0139] The pharmaceutical formulation has a pH range of 4.5 to 5.5. In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations of 150 mg / mL to 250 mg / mL, the isolated anti-Aβ fibrillary 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) 100 mM to 150 mM arginine salt, (c) Polysorbate 80, from 0.01% w / v to 0.05% w / v, and (d) Histidine buffer at 35 mM to 65 mM, The drug formulation has a pH range of 4.5 to 5.5.

[0140] In some embodiments, this document discloses an aqueous pharmaceutical formulation comprising: (a) An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations of 150 mg / mL to 250 mg / mL, the isolated anti-Aβ fibrillary antibody or fragment thereof comprising: (i) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 12. (b) 100 mM to 150 mM arginine salt, (c) Polysorbate 80, from 0.01% w / v to 0.05% w / v, and (d) Histidine buffer at 35 mM to 65 mM, The drug formulation has a pH range of 4.5 to 5.5.

[0141] In some embodiments, the pH of the aqueous pharmaceutical formulation is 5.0. Methods to reduce aggregate formation [ ]

[0142] In some embodiments, this document discloses a method for reducing the formation of aggregates of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: (a) Provide a pharmaceutical formulation comprising, whereby a concentration of 50 mg / mL or higher of an isolated anti-Aβ fibrillary antibody or fragment thereof that binds to human Aβ fibrils, wherein the concentration is, as needed, from 80 mg / mL to 300 mg / mL, wherein the concentration is, as needed, from 100 mg / mL to 200 mg / mL, and (b) Add arginine, arginine salt, or a combination thereof, and provide a pharmaceutically acceptable buffer as needed, wherein the buffer is a histidine buffer as needed, wherein the isolated anti-Aβ fibrillary 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.

[0143] In some embodiments of the method for reducing aggregate formation, the pH of the pharmaceutical formulation is in the range of 4.5 to 5.5. In some embodiments of the method, the pH of the pharmaceutical formulation is 5.0.

[0144] In some embodiments of the method for reducing aggregate formation, the arginine, arginine salt, or a combination thereof is present at a concentration of 150 mM to 250 mM.

[0145] In some embodiments, this document discloses a method for reducing the fragmentation of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: (a) Provide a pharmaceutical formulation comprising, whereby a concentration of 50 mg / mL or higher of an isolated anti-Aβ fibrillary antibody or fragment thereof that binds to human Aβ fibrils, wherein the concentration is, as needed, from 80 mg / mL to 300 mg / mL, wherein the concentration is, as needed, from 100 mg / mL to 200 mg / mL, and (b) Add histidine buffer and, if necessary, further provide arginine, arginine salt, or a combination thereof, wherein the isolated anti-Aβ fibrillary 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.

[0146] In some embodiments of the method for reducing fragmentation, the pH of the pharmaceutical formulation ranges from 4.5 to 5.5. In some embodiments of the method, the pH of the pharmaceutical formulation is 5.0.

[0147] In some embodiments of the method for reducing fragmentation, the arginine, arginine salt, or a combination thereof is present at a concentration of 100 mM to 400 mM.

[0148] In some embodiments of the method for reducing fragmentation, the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80.

[0149] In some embodiments of the method for reducing fragmentation, the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer, wherein the buffer is a histidine buffer, and, if necessary, 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.

[0150] In some embodiments, this document discloses a method for reducing the aggregation and / or fragmentation of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: (a) Provide a pharmaceutical formulation comprising, as needed, a concentration of 50 mg / mL or higher of isolated anti-Aβ fibrillary antibody or a fragment thereof that binds to human Aβ fibrils, wherein the concentration is from 80 mg / mL to 300 mg / mL, or, as needed, from 100 mg / mL to 200 mg / mL. (b) Addition of arginine, arginine salt, or a combination thereof, and (c) Provide a pharmaceutically acceptable histidine buffer, wherein the isolated anti-Aβ fibrillary antibody or a fragment thereof comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2.

[0151] In some embodiments, this document discloses a method for reducing the aggregation and / or fragmentation of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: (a) Provide a pharmaceutical formulation comprising, as needed, a concentration of 50 mg / mL or higher of isolated anti-Aβ fibrillary antibody or a fragment thereof that binds to human Aβ fibrils, wherein the concentration is from 80 mg / mL to 300 mg / mL, or, as needed, from 100 mg / mL to 200 mg / mL. (b) Provide histidine buffer, and (c) Providing arginine, arginine salt, or a combination thereof, wherein the pH of the formulation is 4.5-5.5, wherein the isolated anti-Aβ fibrillary 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.

[0152] This article also provides the following implementation methods: [Embodiment 01] An aqueous pharmaceutical formulation comprising: a. Isolated anti-Aβ fibrillary antibodies or fragments thereof that bind to human Aβ fibrils, at concentrations ranging from 80 mg / mL to 300 mg / mL. b. Arginine at 100 mM to 400 mM, c. Polysorbate 80 at 0.01% w / v to 0.1% w / v, and d. Pharmaceutically acceptable buffers, The drug formulation has a pH range of 4.5 to 5.5. The isolated anti-Aβ protoplasmic antibody or fragment thereof comprises: (i) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain containing the amino acid sequence of SEQ ID NO: 2, and The arginine in question is arginine, arginine salt salt, or a combination thereof. [Embodiment 02] The pharmaceutical formulation as claimed in claim 1, wherein the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 100 mg / mL to 200 mg / mL. [Embodiment 03] The pharmaceutical formulation as claimed in claim 1, wherein the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 100 mg / mL. [Embodiment 04] The pharmaceutical formulation as claimed in claim 1, wherein the isolated anti-Aβ fibrillary antibody or a fragment thereof is present at a concentration of 200 mg / mL. [Embodiment 05] The pharmaceutical formulation as described in any one of claims 1-4 further comprises methionine. [Embodiment 06] The pharmaceutical formulation as described in any one of claims 1-5, wherein the pharmaceutically acceptable buffer is a citrate buffer or a histidine buffer. [Embodiment 07] The pharmaceutical formulation described in any one of claims 1-6 comprises 10 to 100 mM of citrate buffer or 10 to 100 mM of histidine buffer. [Embodiment 08] A pharmaceutical formulation as described in any one of claims 1-7, the pharmaceutical formulation comprising 125 to 350 mM of arginine. [Embodiment 09] A pharmaceutical formulation as described in any one of claims 1-8, the pharmaceutical formulation comprising 200 mM arginine, wherein the arginine is arginine salt. [Embodiment 10] A pharmaceutical formulation as described in any one of claims 1-9, the pharmaceutical formulation comprising 200 mM arginine and 25 mM histidine buffer, wherein the arginine is arginine salt. [Embodiment 11] An aqueous pharmaceutical formulation comprising: a. An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations from 80 mg / mL to 240 mg / mL, the isolated anti-Aβ fibrillary 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. 140 mM to 260 mM arginine salt, c. Polysorbate 80, from 0.02% w / v to 0.08% w / v, and d. 15 mM to 35 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5. [Embodiment 12] An aqueous pharmaceutical formulation comprising: a. An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations from 80 mg / mL to 120 mg / mL, the isolated anti-Aβ fibrillary 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. Arginine at 240 mM to 360 mM, c. Polysorbate 80, from 0.02% w / v to 0.08% w / v, and d. 30 mM to 50 mM citrate buffer, The drug formulation has a pH range of 4.5 to 5.5. Furthermore, the arginine in question is arginine, arginine salt salt, or a combination thereof. [Embodiment 13] A method for reducing the formation of aggregates of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: Provided is an aqueous pharmaceutical formulation comprising an isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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, wherein the isolated anti-Aβ fibrillary antibody or fragment thereof is present at a concentration of 80 mg / ml to 300 mg / ml, and Arginine, arginine salt, or a combination thereof are added to the aqueous pharmaceutical formulation, wherein the pH range of the pharmaceutical formulation is from 4.5 to 5.5. [Effective Method 14] The method as described in claim 13, wherein the arginine, arginine salt, or combination thereof is present at a concentration of 150 to 250 mM. [Embodiment 15] The method as described in claim 14, wherein the arginine, arginine salt, or combination thereof is present at a concentration of 200 mM. [Effective Method 16] The method as described in any one of claims 13 to 15, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer. [Effective Method 17] The method as described in claim 16, wherein the pharmaceutically acceptable buffer is a histidine buffer or a citrate buffer. [Embodiment 18] A method for reducing the fragmentation of isolated anti-Aβ fibrillary antibodies or fragments thereof, the method comprising: Provided is an aqueous pharmaceutical formulation comprising an isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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, wherein the isolated anti-Aβ fibrillary antibody or fragment thereof is present at a concentration of 80 mg / ml to 300 mg / ml, and Histidine buffer is added to the aqueous pharmaceutical composition, wherein the pH range of the pharmaceutical formulation is from 4.5 to 5.5. [Effective Method 19] The method as described in claim 18, comprising 15 to 35 mM of histidine buffer. [Embodiment 20] The method as described in any of claims 13-19, wherein the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80. [Embodiment 21] An aqueous pharmaceutical formulation comprising: a. An isolated anti-Aβ fibrillary antibody or fragment thereof at concentrations from 80 mg / mL to 240 mg / mL, the isolated anti-Aβ fibrillary 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. Arginine, arginine salt, or combinations thereof, in amounts ranging from 140 mM to 260 mM; c. Polysorbate 80 at 0.01% w / v to 0.1% w / v; and d. 15 mM to 35 mM histidine buffer or 30 mM to 50 mM citrate buffer. The drug formulation has a pH range of 4.5 to 5.5. [Effective Method 22] The pharmaceutical formulation according to Embodiment 21 contains 100 mg / mL of the antibody. [Embodiment 23] The pharmaceutical formulation according to Embodiment 21 contains 200 mg / mL of the antibody. [Embodiment 24] According to the pharmaceutical formulation described in Embodiment 21, the pharmaceutical formulation contains the antibody at concentrations of 90 mg / mL to 220 mg / mL, 100 mg / mL to 210 mg / mL, or 110 mg / mL to 200 mg / mL. [Embodiment 25] The pharmaceutical formulation according to any one of Embodiments 21-24 comprises a histidine buffer, wherein the histidine buffer comprises 15 mM to 30 mM, 15 mM to 25 mM, or 20 mM to 30 mM of histidine. [Embodiment 26] The pharmaceutical formulation according to any one of Embodiments 21-25 comprises a histidine buffer, wherein the histidine buffer comprises 0.2-0.3 mg / mL of histidine and 4.4 to 4.9 mg / mL of histidine salt, which is a monohydrate as needed. [Embodiment 27] The pharmaceutical formulation according to any one of Embodiments 21-26 comprises 150 mM to 250 mM, 160 mM to 240 mM, 170 mM to 230 mM, 180 mM to 220 mM, 190 mM to 210 mM of arginine, arginine salt salt, or a combination thereof. [Embodiment 28] The pharmaceutical formulation according to any one of Embodiments 21-27 comprises 0.02% w / v to 0.05%, or 0.04% w / v to 0.04% w / v of polysorbate 80. [Embodiment 29] The pharmaceutical formulation according to any one of Embodiments 21-28 comprises a citrate buffer. [Embodiment 30] The pharmaceutical formulation according to any one of Embodiments 21-29 comprises a histidine buffer. [Embodiment 31] The pharmaceutical formulation according to any one of Embodiments 21-30 further comprises methionine. [Embodiment 32] An aqueous pharmaceutical formulation comprising: a. An antibody at concentrations of 100 mg / ml to 200 mg / ml, comprising: (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2; b. 200 mM arginine salt; c. 0.05% w / v polysorbate 80; and d. 25 mM histidine buffer, The drug formulation has a pH range of 4.5 to 5.5. [Effective Method 33] The pharmaceutical formulation according to Embodiment 32 contains 100 mg / mL of the antibody. [Effective Method 34] The pharmaceutical formulation according to Embodiment 32 contains 200 mg / mL of the antibody. [Implementation Method 35] A method for reducing antibody aggregate formation and / or fragmentation, the method comprising: a. Provides an aqueous pharmaceutical formulation comprising an isolated anti-Aβ fibrillary antibody or a fragment thereof, the isolated anti-Aβ fibrillary 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, wherein the isolated anti-Aβ fibrillary antibody or fragment thereof is present at a concentration of 80 mg / ml to 300 mg / ml, and b. Add 1) arginine, arginine salt, or a combination thereof, or 2) histidine buffer to the aqueous pharmaceutical composition. [Effective Method 36] According to the method described in Effect 35, the pH range of the pharmaceutical formulation is from 4.5 to 5.5. [Embodiment 37] The method according to Embodiment 35, wherein the arginine, arginine salt salt, or combination thereof is present at a concentration of 150-250 mM. [Embodiment 38] The method according to Embodiment 37, wherein the arginine, arginine salt salt, or combination thereof is present at a concentration of 200 mM. [Effective Method 39] The method according to Embodiment 38 includes 15 to 25 mM of histidine buffer. [Effective Method 40] The method according to any one of Embodiments 35-39, wherein the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80. [Effective Method 41] The method according to any one of Embodiments 35-40, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable buffer. [Effective Method 42] The method according to Embodiment 41, wherein the pharmaceutically acceptable buffer is a histidine buffer or a citrate buffer. [Example] [ ] [Example] [1] [Protein Concentration Study] [ ]

[0153] Samples with three protein concentrations (10, 100, and 200 mg / mL) were prepared and tested in concentration studies. The 10 mg / mL sample was derived from pure BAN2401 drug substance (PDS), while the 100 and 200 mg / mL samples were produced from BAN2401 PDS using an Amicon Ultra-15 rotary filter. All samples were prepared in the same formulation buffer (25 mM sodium citrate, 125 mM sodium chloride, pH 5.7), but each sample had a different PS80 level. The percentages of PS80 in the 10, 100, and 200 mg / mL samples were 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: The PS80 removal process was not available for concentration studies.)

[0154] Sample stability was assessed over 3 months using non-denaturing HPLC-SEC, pH, and DLS at two temperatures (5°C and 25°C). Additional characterization assays, such as the PS80 assay, were performed on samples at T = 0. Table 1 shows detailed information on the test samples. [ [surface] [1] [. ] [Testing Schedule (Concentration Study)] [ ] Time point T = 0 T = 1 month T = 2 months T = 3 months temperature N / A 5°C 5°C 5°C 25°C 25°C 25°C Sample size 3 6 6 6 test Non-transferable SEC pH DLS Non-transferable SEC pH DLS Non-transferable SEC pH DLS Non-transferable SEC pH DLS

[0155] pH was measured using a pH meter with a microprobe. Calibration was performed using pH 4.0 and 7.0 standard solutions prior to measurement.

[0156] Aggregation and physical degradation of concentrated BAN2401 were evaluated by non-denaturing HPLC-SEC of stable samples stored at 5°C and 25°C.

[0157] HPLC-SEC (non-denaturing) analysis was performed using a TSK G3000 SWXL column (mobile phase: 0.2 M sodium phosphate, pH 7.0, flow rate: 1.0 mL / min). For protein concentrations of 10, 100, or 200 mg / mL, sample injection volumes were 15, 1.5, or 0.8 pg, respectively. This ensured that approximately 150 pg of protein was injected onto the column throughout the study. The results for the relative peak areas (%) of monomers, aggregates, and fragments are reported in Figures 1-6.

[0158] At both temperatures (5°C and 25°C), both the percentage of aggregates and the aggregation rate increased with increasing protein concentration. For the 100 and 200 mg / mL samples, the initial aggregation percentage was almost twice that of the 10 mg / mL sample. This indicates that the concentration process alone leads to protein aggregation. Additional aggregation occurred during storage at 5°C and 25°C. For the 100 mg / mL sample, the average aggregation rate was 0.17% per month at 5°C for 3 months, and 0.20% per month for the 200 mg / mL sample. For comparison, the 10 mg / mL sample showed an aggregation rate of only 0.07% per month under the same conditions. In conclusion, BAN2401 at concentrations of 100 mg / mL or higher appears to be physically unstable in formulations.

[0159] Protein concentration was assessed by measuring absorbance at 280 and 320 μm using a Beckman DU-800 spectrophotometer with UV light. Samples were diluted 500-fold and prepared in duplicate. Protein concentration was calculated using the following formula with an extinction coefficient ε of 1.32: Concentration = (A280 - A320) / ε x Dilution factor

[0160] Protein concentrations were measured using a UV-Vis spectrophotometer. The results are listed in Table 2. Overall, the concentrations remained constant throughout the three months at both temperatures. [ [surface] [2] [. ] [Protein Concentration Results (Concentration Study)] [ ] Sample ID Protein concentration (mg / ml) T = 0 T = 1 month T = 2 months T = 3 months 5°C 25°C 5°C 25°C 5°C 25°C 10 mg / ml 10.6 9.93 9.93 9.9 10.0 10.11 10.15 100 mg / ml 110.3 110.1 114.1 103.4 104.2 106.6 106.4 200 mg / ml 210.8 197.2 224.5 207.4 205.4 205.3 205.8 [Example] [2] [:] [pH] [Screening Study] [ ]

[0161] The stability of 200 mg / mL BAN2401 was evaluated at five different pH values. To prepare samples, PS80-free BAN2401 PDS was concentrated and percolated into a buffer (pH 4.5) containing 50 mM sodium citrate and 100 mM sodium chloride. Final concentration adjustments were made via dilution to achieve a concentration of 200 mg / mL protein and 0.02% PS80. The resulting material was aliquoted into five aliquots; four of the aliquots were titrated with 10 N sodium hydroxide to produce samples at different pH values ​​(i.e., pH 5.0, 5.5, 6.0, and 6.5). The resulting samples were filtered through a 0.2 μm filter and then aliquoted into sterile polypropylene (PP) tubes for stability assessment.

[0162] Sample stability was assessed over a 3-month period at two temperatures (5°C and 25°C) using non-denaturing HPLC-SEC, pH, and DLS. Additionally, a stirring study was conducted at the 1-month timepoint, in which a subset of the 5°C samples was stirred at 250 RPM in a 25°C incubator for 3 days. Table 3 shows detailed information on the tested samples. [ [surface] [3] [. ] [Test Schedule()] [pH] [Research)] [ ] Time point T = 0 T = 1 month T = 2 months T = 3 months temperature n / a 5°C 5°C 5°C 25°C 25°C 25°C agitation n / a agitation unnecessary unnecessary Sample size 5 15 10 10 test Non-transferable SEC pH DLS DSC Non-transferable SEC pH DLS Non-transferable SEC pH DLS Non-transferable SEC pH DLS

[0163] SEC-HPLC analysis was performed using a TSK G3000 SWXL column (mobile phase: 0.2 M sodium phosphate, pH 7.0, flow rate: 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. The relative peak areas (%) for monomers, aggregates, and fragments are reported.

[0164] Aggregation and physical degradation of concentrated BAN2401 were assessed by non-denaturing HPLC-SEC of stability samples stored at 5°C and 25°C. The results are shown in Figures 7-12. BAN2401 appeared to be more stable at both temperatures (5°C and 25°C) at lower pH values. Formulations at pH 4.5 and 5.0 showed lower initial aggregate percentages and exhibited slower aggregate growth, with a rate of approximately 0.07% per month at 5°C. In addition to aggregation, protein fragmentation was also analyzed. The results indicated more pronounced fragmentation of BAN2401 in the formulations at lower pH values. Based on these considerations, the BAN2401 formulation at pH 5.0 was considered the most stable (i.e., exhibiting less aggregation and / or physical degradation of BAN2401 compared to other formulations at different pH values). [Example] [3] [Excipient Screening Study] [ ]

[0165] Twelve excipients were screened in this study. To prepare samples containing 200 mg / mL BAN2401, BAN2401 with PS80 removed was concentrated via a TFF step followed by a percolation step with a basal buffer (50 mM citrate, 0.02% PS80, pH 6.0). The concentrated material was divided into twenty-four fractions. Each fraction was infused with a stock solution containing the specific excipient. For most excipients, two concentrations were examined, except for sodium chloride and ascorbic acid. For sodium chloride, samples were prepared at three concentration levels; for ascorbic acid, samples were prepared at one concentration level. Table 4 shows a list of the excipients used and their concentrations. [ [surface] [4] [. ] [List of Excipients and Corresponding Concentrations] [ ] [excipient] [concentration] [(Preparation)] [#] [)] none The control sample (F0) contained the protein in a base buffer without any excipients. sucrose 1% (F1), 6% (F2) Trehalose 0.5% (F3), 3.5% (F4) Mannitol 0.5% (F5), 1.8% (F6) proline 50 mm (F9), 400 mm (F10) Glycine 50 Mm (F11), 400 Mm (F12) Arginine 25 mm (F13), 160 mM (F14) NaCl 50mM(F16),150mM(F17),500mM(F18) PS80 0.05% v / v (F19), 0.1% v / v (F20) PEG400 1% (F21), 6% (F22) Dextran, LMW 1% (F23), 5% (F24) ascorbic acid 50 Mm (F15) Sorbitol 1% (F7), 6% (F8)

[0166] All samples, including controls (i.e., samples in excipient-free base buffer), were filtered to 0.2 gm and then aseptically filled into BD syringes using a manual stopper. Control samples were also filled into glass vials. The vials and BD syringes were incubated at 5°C and 25°C. Sample stability was assessed for 2 months using non-denaturing HPLC-SEC and pH.

[0167] Samples for the excipient screening study were prepared under suboptimal buffer conditions (50 mM citrate, 0.02% PS80, pH 6.0) to expand the positive role of excipients in preventing aggregate formation. The stability characteristics (aggregation) of each formulation at 25°C for 2 months were evaluated by non-denaturing HPLC-SEC.

[0168] As shown in Figures 13-15, among these formulations, the formulation containing 160 mM arginine (F14) produced the lowest aggregation rate. In comparison, the 160 mM arginine formulation showed only 1.4% aggregates after 2 months at 25°C, while the control (i.e., the formulation in the base buffer without excipients—F0) showed 2.4% aggregates at the same time under the same conditions.

[0169] Furthermore, the formulation containing arginine exhibited the lowest initial aggregation percentage, indicating that arginine inhibits aggregate formation during the protein concentration step. The arginine formulation (F14) showed a slightly higher degree of fragmentation compared to the control (F0). However, this difference is likely within the scope of assay variability.

[0170] The second best excipient in this study was the formulation with 400 mM proline (F10), although it was not as effective as the arginine formulation (F14) in controlling aggregation.

[0171] Compared to the control samples, the formulations containing sodium chloride (F16-F18) had no effect on stability. The same observation was made for the formulations containing PS80 (F19 and F20): no stability effect was observed by changing the PS80 content. The formulation containing ascorbic acid (F15) showed a significant effect of increasing the percentage of aggregates and fragments from the start of the study.

[0172] The pH of two arginine formulations (F13 and F14) was measured to verify that the presence of arginine in the formulations did not cause pH changes over time. As shown in Table 4, no pH changes were observed. The osmolality of both formulations was also measured. These samples were stored at -20°C before being thawed simultaneously for measurement. [ [surface] [5] [. ] [Arginine preparation()] [F13] [and] [F14] [)of] [pH] [and osmotic pressure molar concentration] [ ] [Arginine formulation] [(] [F13] [and] [F14] [Time point] [pH] [mOsm / kg] [F13] [,] [T = 0] No sample No sample [F14] [,] [T = 0] 5.86 No sample [F13] [,] [T = 1] [months] 5.97 231 [F14] [,] [T = 1] [months] 5.88 477 [F13] [,] [T = 2] [months] 5.97 230 [F14] [,] [T = 2] [months] 5.89 459 [Example] [4] [Arginine Concentration Study] [ ]

[0173] The relationship between arginine concentration and aggregate size in the formulation was studied using Design Expert® 7.0. Table 6 below shows the factors selected for the study and the exploration level for each factor. [[] [surface] [6]. [Example] [4] [Factors in the Chinese study] [ ] [factor] [Factor Range] [Factor Level] BAN2401 30 to 120 mg / mL 30, 53, 75, 98, 120 mg / mL pH 4.5 to 6.0 4.5, 4.9, 5.2, 5.6, and 6.0 Arginine 0 to 450 mM 0, 225, 338, and 450 mM Polysorbate 80 0% to 0.1% 0%, 0.025%, 0.05%, 0.075%, and 0.1%

[0174] Sample randomization was induced in Design Expert® 7.0 using D-optimal design. The resulting sample set contained 25 data points, including 4 replicates, as shown in Table 7. Each formulation was prepared and evaluated. [ [surface] [7] [. ] [For example] [4] [Prepared formulation] [ ] [entry] [BAN2401] [(] [mg / mL] [)] [pH] Arginine [(] [mM] [)] [Polysorbate]

[80] [(] [%] [)] 1 30 5.2 0 0.000 2 30 6.0 0 0.050 3 30 4.5 0 0.100 4 30 4.5 0 0.100 5 30 6.0 225 0.000 6 30 4.5 450 0.000 7 30 4.5 450 0.000 8 30 6.0 450 0.100 9 30 6.0 450 0.100 10 53 5.6 225 0.025 11 53 4.9 338 0.075 12 75 4.5 0 0.000 13 75 4.5 0 0.000 14 75 6.0 0 0.100 15 75 5.2 225 0.000 16 75 6.0 450 0.000 17 75 6.0 450 0.000 18 98 4.5 450 0.100 19 98 5.6 225 0.025 20 120 4.9 338 0.075 twenty one 120 6.0 0 0.000 twenty two 120 4.5 0 0.100 twenty three 120 5.2 225 0.050 twenty four 120 4.5 450 0.000 25 120 6.0 450 0.100

[0175] The relationship between arginine concentration and aggregate level in a 100 mg / mL BAN2401 formulation (pH 5.0) containing 0.02% polysorbate 80 was evaluated using Design Expert® 7.0. The estimation results are shown in Figure 16. [Example] [5] [:] [PS80] [Research] [ ]

[0176] To assess changes in drug product quality under stress conditions, agitation and freeze-thaw studies were conducted using formulation buffers (pH 5.0, 350 mM arginine, 50 mM citric acid). In this experiment, formulations with varying polysorbate 80 concentrations were investigated to confirm the effect of polysorbate 80 on subvisible particle formation under such stress conditions. Table 8 shows the formulations studied and the applied stress conditions. [ [surface] [8] [. ] [Study on the formulation and freeze-thaw stress by stirring] [ ] [Preparation] [Polysorbate]

[80] [Concentration] [Stress Conditions] 100 mg / mL BAN2401 50 mM citrate buffer 350 mM arginine pH 5.0 · 0% · 0.02% 0.05% 0.1% • Vibrate at 250 rpm for 3 days at 5°C. • Vibrate at 250 rpm for 3 days at ambient temperature. • Three freeze-thaw cycles (from -20°C to ambient temperature)

[0177] Samples were prepared in the same manner as described in Example 4. 10% PS80 solution was added to achieve the target PS80 concentration in the formulation, and the final protein concentration was adjusted by dilution with formulation buffer. The formulation was passed through a 0.2 gm filter and filled into 2 mL vials at a filling volume of 1.3 mL. The samples were placed horizontally (via the vials sideways) on a track-mounted shaker and then placed in a refrigerator or on a lab bench. The samples were shaken at 250 rpm for 3 days. Other samples were frozen for 2 hours by placing them at -20°C, then removed and allowed to thaw at room temperature for 2 hours. This freeze-thaw cycle was repeated three times. The visual appearance, aggregate and fragment levels of the samples from the agitation and freeze-thaw studies were evaluated by SEC-HPLC, and sub-visible particles were evaluated by microflow imaging (MFI).

[0178] The following summarizes the results of agitation and freeze-thaw studies of BAN2401 formulations containing different levels of PS80. [, SEC-HPLC , ] [, analyze , ] [, , ]

[0179] SEC-HPLC analysis was performed using a TSK G3000 SWXL column (mobile phase: 0.2 M sodium phosphate, pH 7.0, flow rate: 1.0 mL / min). For protein concentrations of 50 or 100 mg / mL, sample injection volumes were 5.0 µL or 2.5 µL, respectively. This ensured that approximately 250 µg of protein was injected onto the column throughout the study. The relative peak areas (%) for monomers, aggregates, and fragments are reported.

[0180] Regarding aggregate levels in formulations with varying polysorbate 80 concentrations and at pH 5 after 3 months of storage at 25°C, there was no significant difference in aggregate levels as the PS80 concentration increased from 0% to 0.06% and the formulation pH reached 5.0. It can be concluded that PS80 does not affect aggregate formation (i.e., dimers and trimers as measured by SEC-HPLC) during storage of the BAN2401 formulation at 25°C.

[0181] Regarding the effect of polysorbate 80 concentration on fragment levels in formulations at pH 5, polysorbate 80 concentration did not affect fragment levels in BAN2401.

[0182] Regarding the appearance of the preparations after 3 days of agitation at 250 rpm at ambient temperature, the sample without PS80 was cloudy with precipitated protein, while the other samples (0.02%, 0.05%, and 0.1% polysorbate 80) were clearly clear, particle-free solutions. All samples agitated at 250 rpm at 5°C for 3 days were clear and free of precipitate. All samples subjected to freeze / thaw cycles were also clear and free of precipitate.

[0183] Table 9 shows the aggregate and fragment levels in BAN2401 formulations subjected to agitation and freeze-thaw cycles. The stress condition causing the greatest instability was agitation under ambient conditions. Under these conditions, the PS80-free sample exhibited the most high molecular weight formation and the greatest monomer loss. This effect diminished with increasing PS80 concentration. [ [surface] [9] [. ] [Results at the aggregate and fragment levels from agitation and freeze-thaw studies] [ ] [Stress Conditions] [Polysorbate]

[80] [(] [%] [)] [HPLC] [purity(] [%] [area)] [HMW1] [, ( , ] [, a , ] [, ) , ] [HMW2, (a) , ] [monomer] [LMW] [, ( , ] [,b , ] [, ) , ] 5°C (control) 0 - 0.4 99.4 0.2 0.02 - 0.5 99.3 0.2 0.05 - 0.5 99.4 0.2 0.1 - 0.5 99.4 0.2 Vibrate at 5°C 0 0.5 0.6 98.7 0.2 0.02 - 0.5 99.3 0.2 0.05 - 0.5 99.3 0.2 0.1 - 0.5 99.3 0.2 Vibration at ambient temperature 0(c) 1.5 1.2 97.1 0.2 0.02 4.0 0.8 95.1 0.2 0.05 0.1 0.5 99.2 0.2 0.1 - 0.5 99.3 0.2 Three freeze / thaw cycles 0 - 0.4 99.4 0.2 0.02 - 0.5 99.4 0.2 0.05 - 0.4 99.4 0.2 0.1 - 0.5 99.4 0.2 (a) High molecular weight substances; (b) Low molecular weight substances; and (c) Turbid sample systems. [ ] [, Subvisible particle analysis , ] [, , ]

[0184] Subvisible particle analysis was performed using microfluidic imaging (DPA4100 flow imaging 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.

[0185] An increase in subvisible particles (2 to 10 pm) was observed in PS80-free samples subjected to vibration and freeze-thaw cycles. The formation of subvisible particles was increasingly suppressed with increasing PS80 concentration in the formulation. This effect was observed across all size ranges studied. No significant changes in subvisible particles were observed in formulations containing 0.05% and 0.1% polysorbate 80. Therefore, a PS80 concentration of 0.05% was chosen as the optimal concentration for the formulation.

[0186] Data showed that, as measured by SEC-HPLC, PS80 had no effect on the formation of BAN2401 aggregates (dimers and trimers) and fragments. Therefore, the PS80 concentration was selected based on the results of agitation and freeze-thaw studies. A PS80 concentration of 0.05% was chosen to prevent possible precipitation during transport and to minimize the formation of subvisible particles. [Example] [6] [:] [BAN2401] [intravenous () [IV] Preparation of the formulation [ ] [A.] [BAN2401 10 mg / mL] [Preparation] [ ]

[0187] A 10 mg / mL formulation of BAN2401 for intravenous injection (“10 mg / mL injection”) was manufactured using standard cGMP aseptic methods for preparing sterile aqueous formulations. The BAN2401 10 mg / mL injection was produced from the corresponding BAN2401 active pharmaceutical ingredient formulations (without the addition of any excipients or diluents) listed below. Table 10 shows exemplary IV formulations containing 10 mg / mL BAN2401. [ [surface]

[10] ] [. ] [Include] [BAN2401] [The contrast] [10 mg / mL IV] [Preparation ingredients.] [ ] [Components] [composition] BAN2401 10 mg / mL Sodium citrate / citric acid buffer 25 mM Sodium chloride 125 mM Polysorbate 80 0.02% (w / v) Water for Injection QS pH 5.7

[0188] As shown in Figure 17, the filtered BAN2401 active pharmaceutical ingredient (API) solution was aseptically filled into vials (referred to as "Formulation A" in Figure 17). The combined API was then filtered through a 0.2-pm filter to reduce bioburden. Final aseptic filtration was performed using two 0.2-pm filters in series, and filter integrity tests were conducted before and after filtration. The sterile bulk drug product was aseptically filled into vials. During filling, the filling accuracy was confirmed by measuring the vial filling weight. The filled vials were plugged and then sealed with an aluminum top seal. After crimping the cap, the product was stored at 5°C ± 3°C.

[0189] The vials were analyzed according to USP 788 using Method 1 (light obscuration method). The results are shown in Tables 11 and 12. [ [surface]

[11] [10 mg / mL] [Particle size distribution (number of particles) of injection vials] [ / ] [container)] [ ] [batch number] [A1] [A2] [A3] [A4] [A5] [A6] [A7] [A8] [A9] [A10] [A11] [A12] 25 µm 1 1 1 0 1 1 1 1 1 1 5 5 2 1 1 10 µm 126 145 469 241 469 225 107 617 249 315 215 273 231 191 125 5 µm - - - 2922 - - - - - - - - - 2 µm - - - 16636 - - - - - - - - - [[] [surface]

[12] . 10 mg / mL] [Particle size distribution (number of particles) of injection vials] [ / mL] [)] [ ] [batch number] [A1] [A2] [A3] [A4] [A5] [A6] [A7] [A8] [A9] [A10] [A11] [A12] 25 µm 0.1 0.1 0.1 0 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.5 0.2 0.1 0.1 10 µm 12.6 14.5 46.9 24.1 46.9 22.5 10.7 61.7 24.9 31.5 21.5 27.3 23.1 19.1 12.5 5 µm - - - 292.2 - - - - - - - - - 2 µm - - - 1664 - - - - - - - - - [Example] [7] [:] [BAN2401] [In the vein ()] [IV] [) and subcutaneous (] [SC] Preparation of [ ] formulations [ ] [A.] [BAN2401 100 mg / mL] [Preparation] [ ]

[0190] BAN2401 100 mg / mL formulation (“100 mg / mL injection”) was manufactured using standard cGMP aseptic methods for preparing sterile aqueous formulations. BAN2401 100 mg / mL was produced from the corresponding BAN2401 active pharmaceutical ingredient (without any excipients or diluents) (Table 13). [ [surface]

[13] [. ] [Include] [BAN2401] [Example] [100 mg / mL IV] [Preparation ingredients.] [ ] [Components] [composition] BAN2401 100 mg / mL Citric acid buffer 50 mM Arginine 110 mM Arginine salt 240 mM Polysorbate 80 0.05% (w / v) Water for Injection QS pH 5.0

[0191] As shown in Figure 18, the filtered BAN2401 active pharmaceutical ingredient (API) solution (100 mg / mL for injection) was aseptically filled into vials (referred to as "Formulation B" in Figure 18). The API was then filtered through a 0.2-pm filter to reduce bioburden. Final aseptic filtration was performed through two 0.2-pm filters in series, and filter integrity tests were conducted before and after filtration. The sterile bulk drug product was aseptically filled into vials. Filling accuracy was confirmed by measuring the weight of the vials during filling. The filled vials were plugged and then sealed with an aluminum top seal. After crimping the cap, the product was stored at 5°C ± 3°C.

[0192] The vials were analyzed according to USP 788 using Method 1 (light obscuration method). The results are shown in Tables 14 and 15. [ [surface]

[14] [100 mg / mL] [Particle size distribution (number of particles) of injection vials] [ / ] [Small bottle] [ ] [batch number] [B1] [B2] [B3] [B4] [25 µm] 0 0 1 0 [10 µm] 53 8 9 9 [5 µm] 631 72 51 60 [2 µm] 1954 321 249 267 [ [surface]

[15] [100 mg / mL] [Particle size distribution (number of particles) of injection vials] [ / mL] [)] [ ] [batch number] [B1] [B2] [B3] [B4] [25 µm] 0 0 0.2 0 [10 µm] 10.6 1.6 1.8 1.8 [5 µm] 126.2 14.4 10.2 12 [2 µm] 390.8 64.2 49.8 53.4

[0193] To manufacture another BAN2401 100 mg / mL injection. As shown in Table 16, the following materials may be used in a second exemplary formulation containing 100 mg / mL BAN2401. [ [surface]

[16] [. ] [Include] [BAN2401] [Another example] [100 mg / mL IV] [Preparation ingredients.] [ ] [Components] [composition] BAN2401 100 mg / mL histidine Total 25 mM Histidine HCl Arginine HCl 200 mM Polysorbate 80 0.05% (w / v) Water for Injection QS pH 5.0 ± 0.4 [B.] [BAN2401 200 mg / mL] [Preparation] [ ]

[0194] As shown in Table 17, the following materials can be used in exemplary SC formulations containing 200 mg / mL BAN2401. The stability of BAN2401 in these formulations (FSC1, FSC2, and FSC3) was evaluated in conjunction with material stability assessments of the following three container seals: i) Becton-Dickinson Hypak Biotech glass prefilled syringe (PFS) and stopper ii) West Crystal Zenith plastic PFS and stoppers iii) West 5 mL glass vial and stopper [ [surface]

[17] [. ] [Include] [BAN2401] [Example] [200 mg / mL SC] [Preparation ingredients.] [ ] [Components] [composition] Sample ID FSC1a FSC1b FSC2 FSC3 FSC4 BAN2401 200 mg / mL 200 mg / mL 200 mg / mL 200 mg / mL 200 mg / mL histidine Total 50 mM1 Total 50 mM1 Total 50 mM1 -- Total 25 mM1 Histidine HCl Citric acid -- -- -- 50 mM -- Arginine HCl 125 mM 125 mM 125 mM 125 mM 200 mM Polysorbate 80 0.02% (w / v) 0.02% (w / v) 0.02% (w / v) 0.02% (w / v) 0.05% (w / v) Methionine -- 5 mM -- -- -- Water for Injection QS QS QS QS QS pH 5.0 ± 0.4 5.0 ± 0.4 5.5 ± 0.4 5.0 ± 0.4 5.0 ± 0.4 Total concentration of histidine

[0195] BAN2401 at a target protein concentration of 200 mg / mL was prepared via TFF as described below. Except for FSC1a and FSC1b, a separate TFF operation was performed on each formulation buffer to prepare the BAN2401 material. For both formulations, one TFF operation was performed, and the resulting concentrated material was aliquoted into two half-lots. A small amount of sterile filter material from each final formulation buffer was not filled at zero but stored frozen at -20°C for injection testing after filling into appropriate container seals. [(a)] [BAN2401] [Preparation] [ ]

[0196] The protein concentration / percolation process via TFF can be divided into three stages: [1.] The material was concentrated to 100-150 mg / mL [2.] Percolation of the formulation buffer (5X) [3.] Concentrate to > 200 mg / mL

[0197] The concentration / percolation steps were performed using a Pall Centramate LV system with a membrane area of ​​0.02 m². BAN2401 material (removed from GMP batch manufacturing prior to the addition of PS80) was loaded into the TFF system and concentrated 10–15 times (Stage 1). The material was then percolated against a formulation buffer up to 5 times its original volume (Stage 2), with percolation monitored by pH and conductivity checks of the percolate. Following percolation, the material was further concentrated to a target protein concentration of 210–250 mg / mL (Stage 3). The percolate was collected and sampled to determine the protein concentration.

[0198] During the preparation of this formulation, the target protein concentration of 210 to 250 mg / mL was not achieved due to the high pressure in the TFF system. Therefore, the target protein concentration was achieved by using a Millipore 1-gal filtration unit (30,000 MWCO). To perform this concentration step, the filtration unit was equilibrated using a BAN2401 formulation buffer, and then the BAN2401 material was centrifuged at 3600 RPM (approximately 3000 xg) at 20°C for 30 minutes until the expected protein concentration in the leucovorin reached greater than 200 mg / mL. The leucovorin was recovered from the filtration unit and combined. After thorough mixing, the combined leucovorin was sampled for protein concentration measurement.

[0199] After protein concentration, samples were removed from the pool and diluted 500-fold with an appropriate formulation buffer. The absorbance of the diluted samples at 280 nm and 320 nm was measured relative to a blank buffer. Final protein concentration adjustment was performed by dilution with an appropriate formulation buffer. Finally, 10% PS80 solution was added to the BAN2401 to achieve a final PS80 concentration of 0.02%, and the protein solution was thoroughly mixed by inverting the container.

[0200] The final BAN2401 preparation material was filtered using a 0.2 µm syringe filter and then filled into vials or PFS. This step was performed aseptically in a biosafety cabinet. The resulting vials or PFS were placed in a freezer at -20°C. Vials were stored upside down, and PFS were stored horizontally to simulate worst-case conditions. [C.] [Targeting inclusion] [BAN2401] [Example] [200 mg / mL SC] [Stability test of the preparation.] [ ]

[0201] Sample stability was assessed at 5°C (3M, 6M, 9M, 12M) and 25°C (1M, 3M) and beyond using pH (Figs. 19-21), absorbance at 405 nm (Figs. 22-24) (to detect increased yellowing), and size exclusion chromatography (SEC) (Figs. 25-33), as well as subsequent measurements at 5°C. Exemplary formulations FSC1-FSC4 are shown in Table 17; note that in Figs. 19-33, "F1a" refers to FSC1a, "F1b" refers to FSC1b, "F2" refers to FSC2, and "F3" refers to FSC3. Additional characterization assays, including protein concentration, differential scanning calorimetry (DSC), PS80, and osmoregole concentration, were performed on samples at t = 0. Sub-visible particle testing was performed on samples at t = 0 and t = 6 months. A 3-day agitation study was conducted on samples stored at 5°C for 1 month. Samples used for agitation studies were stored at 5°C for one month and then transferred to 25°C, accompanied by agitation at 250 rpm for three days.

[0202] Test results showed that all FSC1a-FSC3 formulations containing 200 mg / mL BAN2401 exhibited similar aggregate and fragment levels after 12 months of storage at 2°C–8°C. The stability of 200 mg / mL BAN2401 in the three FSC1a-FSC3 formulations was evaluated. Overall, BAN2401 showed similar stability in each tested formulation, and after 12 months of storage at 5°C, it retained more than 98.2% of its monomers regardless of the container seal. Furthermore, after 12 months of storage at 5°C, the pH remained stable, and there was no significant increase in yellowing as detected by A405. [ ] [(a)] [Protein concentration ()] [T = 0] [sample)] [ ]

[0203] Protein concentrations were measured using a UV-Vis spectrophotometer. The results are listed in Table 18. [ [surface]

[18] [. ] [Protein Concentration Results] [ ] [sample] [ID] [Protein concentration ()] [mg / ml] [)] FSC1a 206 FSC1b 204 FSC2 201 FSC3 189

[0204] Protein concentration was assessed by measuring absorbance at 280 and 320 nm using a Beckman DU-800 spectrophotometer with a 1 cm quartz cuvette. Samples were diluted 500-fold and prepared in triplicate. Protein concentration was calculated using the following formula with an extinction coefficient ε of 1.32: Concentration = (A280 - A320) / ε x Dilution factor [(b)] [Polysorbate]

[80] [(] [T = 0] [sample)] [ ]

[0205] The PS80 content of the samples was measured by oleic acid quantification. The results are shown in Table 19. [ [surface]

[19] [. ] [Polysorbate]

[80] [result] [ ] [sample] [ID] Polysorbate 80% [FSC1a] 0.016 [FSC1b] 0.022 [FSC2] 0.019 [FSC3] 0.019

[0206] Oleic acid (a hydrolysis product of PS80) was measured quantitatively. Using alkaline hydrolysis, PS80 released oleic acid at a 1:1 molar ratio. Oleic acid was then separated from other PS80 hydrolysis products and the matrix using reversed-phase HPLC. Underrivatized oleic acid was monitored using absorbance at 195 nm. [J. Chromatography B, 878 (2010) 1865-1870]. Experimentally, the sample was mixed with sodium hydroxide to release oleic acid, followed by extraction with acetonitrile. The extract was diluted with potassium phosphate solution, and 100 µL of sample was injected into a Waters Symmetry C18 column. Separation was performed using an isocratic elution buffer containing 80% organic phase A (acetonitrile) and 20% aqueous phase B (20 mM potassium dihydrogen phosphate, pH 2.8). The concentration of PS80 in the sample was calculated from the peak area using a standard curve. [(c)] [Osmotic pressure molar concentration ()] [T = 0] [sample)] [ ]

[0207] The osmolality of the samples was determined using a freezing point osmoremeter. The results are shown in Table 20. [ [surface]

[20] [. ] [Osmotic Mole Concentration Results] [ ] [sample] [ID] [Osmotic pressure molar concentration ()] [mOsmol / kg H, 2, O] [)] [FSC1a] 294 [FSC1b] 309 [FSC2] 297 [FSC3] 327

[0208] Osmotic molar concentration was measured using a Precision Systems Osmette III freezing point osmoremeter. The sample was diluted 3-fold with WFI, and then 10 µL of the diluted sample was transferred to the instrument using the osmoremeter pipette. The resulting osmotic molar concentration measurements were calibrated for further sample dilution. [(d)] [Subvisible particles] [ ]

[0209] Subvisible particle analysis was performed using a Fluid Technologies FlowCarn instrument. The results at T = O are listed in Table 21. When the material was stored at 5°C, the total particle concentration and the number of particles larger than 10 μm appeared to increase over time. The magnitude of the increase was ordered as follows: FSC1a < FSC1b < FSC2 < FSC3. [ [surface] [twenty one]] [. ] [Subvisible particle results] [ ] Sample ID Particle count / mL, Total Particle count / mL ≥ 10 µm Particle count / mL ≥ 25 µm FSC1a 1.2 x 10⁴ 9 0 FSC1b 2.6 x 10⁴ 0 0 FSC2 2.8 x 10⁴ 18 0 FSC3 3.9 x 10⁴ 63 0

[0210] Sub-visible particle analysis was performed using a Fluid Imaging Technologies FlowCam with a 20x objective and a 50 μm flow cell. Before running the BAN2401 sample, the flow cell was flushed with dI H2O, and the dI H2O was measured to ensure the flow cell system was clean. The flow cell was considered usable if the total number of counted particles per 0.2 mL of dI H2O was ≤ 2. The sample was equilibrated to room temperature and then diluted 20-fold with deionized water. Repeated diluted samples were analyzed using a sample volume of 0.2 mL and a sample flow rate of 0.02 mL / min. The auto-imaging rate was 14, the efficiency was 19.6%, and the execution time was 10 min. The resulting particle concentration was corrected for sample dilution. [(e)] [pH]

[0211] Throughout the stability testing process, the pH value of each formulation was monitored. As shown in Figures 19-21, no significant changes were observed in the samples under any of the storage conditions tested. [(f)] [HPLC-SEC]

[0212] High-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) was performed using a TSK 03000 SWXL column (mobile phase: 0.2 M sodium phosphate, pH 7.0, flow rate: 1.0 mL / min). For a protein concentration of 200 mg / mL, the sample injection volume was 0.8 µL. This ensured that approximately 150 µg of protein was injected onto the column throughout the study. The results for the relative peak areas (%) of monomers, aggregates, and fragments are reported, as shown in Figures 25-33.

[0213] The physical degradation of BAN2401 was assessed by HPLC-SEC of stability samples stored at 5°C and 25°C. After 12 months of storage at 5°C, the percentage of aggregates in the three formulations was similar, approximately 1.1%. The percentage of fragments generated in all tested formulations ranged from 0.4% to 0.5%. For all tested formulations and container seals, the monomer content after 12 months of storage at 5°C ranged from 98.4% to 98.6%. At the current degradation rate, after up to 24 months of storage at 5°C, BAN2401 in all tested formulations could potentially contain more than 97% monomers.

[0214] After storage at 25°C for up to 3 months, the fragment content after 3 months ranged from 0.4% to 0.8%. Except for FSC3, all formulations had a monomer content slightly higher than 98%, while FSC3 had a monomer content slightly lower than 98% after storage at 25°C for 3 months. [Example] [8] [Protein, arginine, and polysorbate]

[80] [Concentration Selection] [ ] [A.] [Sample Preparation] [ ]

[0215] Each candidate formulation (F1-F12) was prepared as follows. The drug substance (DS) processing intermediate was concentrated and equilibrated using a centrifugal filtration device with the corresponding formulation buffer (Table 22). After concentration and equilibration, PS80 dissolved in the formulation buffer was added to achieve the desired concentration, and the protein concentration was adjusted to the final concentration. Each candidate formulation was filled into vials with a filling volume of 0.5 mL. The candidate formulations (F1-F12) are shown in Table 22. Formulation F0 was evaluated as a control. [ [surface] [twenty two]] [] [Preparation(s)] [F0] [) and candidate formulations [F1-F12] Preparation number BAN2401 buffer Stabilizer (arginine) Polysorbate 80 pH Fill volume mg / mL mmol / L mg / mL mmol / L mg / mL (w / v)% mg / mL mL F0 100 Citrate 50 9.6 350 61.0 0.05 0.5 5.0 5 / 0.5b F1 200 Histidine 25 3.9 150 26.1 0.05 0.5 5.0 0.5 F2a 200 Histidine 25 3.9 200 34.8 0.05 0.5 5.0 0.5 F3 200 Histidine 25 3.9 250 43.6 0.05 0.5 5.0 0.5 F4 200 Histidine 25 3.9 300 52.3 0.05 0.5 5.0 0.5 F5 200 Histidine 25 3.9 350 61.0 0.05 0.5 5.0 0.5 F6 250 Histidine 25 3.9 200 34.8 0.05 0.5 5.0 0.5 F7 300 Histidine 25 3.9 200 34.8 0.05 0.5 5.0 0.5 F8 200 Histidine 25 3.9 200 34.8 0 0 5.0 0.5 F9 200 Histidine 25 3.9 200 34.8 0.02 0.2 5.0 0.5 F10a 200 Histidine 25 3.9 200 34.8 0.05 0.5 5.0 0.5 F11 200 Histidine 25 3.9 200 34.8 0.08 0.8 5.0 0.5 F12 200 Histidine 25 3.9 200 34.8 0.10 1.0 5.0 0.5 a: F2 and F10 are the same formulations, prepared for different studies. b: When selecting the concentration of polysorbate 80, remove the drug solution from the drug product and fill it with 0.5 mL to match the filling volume with the container sealing system. [B.] [Stability Solution] [ ]

[0216] The stability protocols are shown in Tables 23, 24, and 25. Once removed from storage, the vials were stored at 5°C until testing. Storage conditions were as follows: • Long-term conditions: Store at 5°C ± 3°C, upright. • Acceleration conditions: Store at 25°C ± 2°C, 60% RH ± 5% RH, upright. • Stress conditions (freeze-thaw): Freeze at -30°C and thaw at room temperature, then place upright. • Stress conditions (stirring): 5°C, horizontal placement, vibration at 250 rpm on a reciprocating shaker, vibration amplitude of 50 mm. [ [surface] [twenty three]] [] [Stability Scheme (Long-Term and Acceleration Conditions)] [ ] [ ] [Test Point] [Test Project] [initial] [5]° [C ± 3]° [C] [, placed upright]

[25] ° [C ± 2]° [C] [,] [60% RH ± 5% RH] [, placed upright] [ ] [3M] [1M] [3M] Appearance F0-F7 F0-F7 F0-F7 F0-F7 pH F0-F7 F0-F7 F0-F7 F0-F7 Size exclusion HPLC F0-F7 F0-F7 F0-F7 F0-F7 Ion exchange HPLC F0-F7 F0-F7 N / A F0-F7 Polysorbate 80 F0-F7 F0-F7 F0-F7 F0-F7 N / A: Not applicable. [ [surface] [twenty four]] [] [Stability Scheme (Stress Condition:)] [F0-F7] [)] [ ] [ ] [Test Point] [Test Project] [initial] [freeze-thaw()] [-30]° [C] [⇔ ] [Room temperature)] [ ] [3] [Number of loops] Appearance F0-F7 F6-F7 pH F0-F7 F0-F7 Size exclusion HPLC F0-F7 F0-F7 Ion exchange HPLC F0-F7 F0-F7 Polysorbate 80 F0-F7 N / A protein concentration F0-F7 N / A viscosity F0-F7 N / A osmolar concentration F0-F7 N / A N / A: Not applicable. [ ] [ [surface]

[25] [] [Stability Scheme (Stress Condition:)] [F0] [、] [F8-F12] [)] [ ] [ ] [Test Point] [Test Project] [initial] [freeze-thaw()] [-30]° [C] [⇔ ] [Room temperature)] [agitation(] [5]° [C] [,placed horizontally,] [250 rpm] [)] [ ] [3] [Number of loops] [3] [sky] Appearance F0, F8-F12 F0, F8-F12 F0, F8-F12 pH F0, F8-F12 F0, F8-F12 F0, F8-F12 protein concentration F0, F8-F12 F0, F8-F12 F0, F8-F12 Foreign insoluble matter (visible particles) F0, F8-F12 F0, F8-F12 F0, F8-F12 Size exclusion HPLC (SEC) F0, F8-F12 F0, F8-F12 F0, F8-F12 Polysorbate 80 (PS80) F0, F9-F12 F0, F9-F12 F0, F9-F12 Microfluidic imaging (MFI) F0, F8-F12 F0, F8-F12 F0, F8-F12 N / A: Not applicable.

[0217] Because the viscosity of solutions is known to increase exponentially in high-concentration protein solutions, the protein concentration of each candidate formulation (F1-F5) was adjusted to 200 ± 10 mg / mL using a formulation buffer containing 0.05 (w / v)% PS80 for analysis. Other formulations were not diluted prior to measurement. [C.] [Results and discussion] [ ] [(a)] [Selection of Arginine Concentration] [ ]

[0218] To select the target arginine concentration in the formulation, physical properties were evaluated, and freeze-thaw, long-term, and accelerated stability studies were conducted on candidate formulations with different arginine concentrations (150 to 350 mmol / L, F1 to F5) 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. [(b)] [Physical Properties] [ ]

[0219] As shown in Table 26, the osmolality values ​​increased with increasing arginine concentration. Compared to F4 and F5, F1, F2, and F3 showed lower osmolality values. Therefore, the arginine concentration was limited to 250 mmol / L or lower. The viscosity values ​​of F1–F5 were within a narrow range (7.3–8.1 cP) and were not correlated with arginine concentrations in the range of 150–350 mmol / L. The results for PS80, as well as the appearance, pH, and concentration of the protein, were almost entirely satisfactory. [(c)] [Freeze-thaw stability study] [ ]

[0220] As shown in Table 26, no significant changes were observed in any of the test items after three cycles of freeze-thaw testing. [(d)] [Long-term stability study] [ ]

[0221] As shown in Table 27, after three months of storage, no significant changes were observed in any of the test items except for size exclusion HPLC (SEC). In candidate formulations F1-F5, the amounts of aggregates and fragments measured by SEC all showed a slight increase. The rate of increase was similar to that of formulation F0. [(e)] [Accelerated Stability Study] [ ]

[0222] As shown in Table 28, no significant changes were observed in any of the tests except for SEC and ion-exclusion HPLC (IEX). Although the amount of aggregates measured by SEC increased in all candidates (F1-F5, BAN2401 200 mg / mL), and as expected, at a faster rate than in formulation F0, all candidates were considered feasible considering the results under long-term conditions and the stability of F0 over its long shelf life. The higher arginine concentration resulted in slightly slower aggregate formation, consistent with the results described in Example 4. The amount of fragments measured by SEC also increased in all candidates, but at a rate similar to that in formulation F0. As for IEX, the amount of acidic peaks increased in all candidates, but at a rate similar to that in F0. [(f)] [Arginine concentration] [ ]

[0223] As shown in Table 26, F4 and F5 (arginine concentrations of 300 and 350 mmol / L) were deemed infeasible in terms of osmolality. Among the feasible candidates F1, F2, and F3 (arginine concentrations of 150, 200, and 250 mmol / L, respectively), F1 was closest to isotonicity. As shown in Table 28, in accelerated stability studies, formulations with higher arginine concentrations showed lower aggregate formation rates, but this difference was not significant within the assessed arginine concentration range. Considering isotonicity and aggregate formation rate, 200 mmol / L was selected as the target arginine concentration based on formulation F2. [(g)] [Choice of Protein Concentration] [ ]

[0224] To select the target protein concentration in the formulations, physical properties were evaluated, and freeze-thaw, long-term, and accelerated stability studies were performed on candidate formulations with different protein concentrations (200 to 300 mg / mL, F2, F6, F7) and formulation F0. The results of the physical properties and freeze-thaw studies are shown in Table 29. The results of the long-term and accelerated stability studies are shown in Tables 27 and 28. [(h)] [Physical Properties] [ ]

[0225] As shown in Table 29, F2 and F6 exhibited lower osmolality molar concentrations compared to F7. The viscosity values ​​for F2, F6, and F7 were 7.8, 21, and 48 cP, respectively. High viscosities exceeding 20 cP would make DS fabrication difficult by increasing pump back pressure and reducing transmembrane flux during ultrafiltration and diafiltration steps. Furthermore, higher solution concentrations would further complicate the DP filling process by causing packing weight changes due to clogging of the packing needle. Since the desired viscosity was reported to be no more than 20 cP³, the F2 system was feasible. Results for appearance, pH, and the concentrations of PS80 and protein were nearly satisfactory. [(i)] [Freeze-thaw research] [ ]

[0226] As shown in Table 29, no significant changes were observed in any of the test items after three cycles of freeze-thaw testing. [(j)] [Long-term stability study] [ ]

[0227] As shown in Table 27, after three months of storage, no significant changes were observed except for size exclusion HPLC (SEC). In candidate formulations F2, F6, and F7, the amount of aggregates measured by SEC increased slightly, but the rate of increase was similar to that in F0. [(k)] [Accelerated Stability Study] [ ]

[0228] As shown in Table 28, no significant changes were observed except for those measured by SEC and ion-exclusion HPLC (IEX). At the three-month time point, the amount of aggregates measured by SEC increased at a similar rate (0.7%–0.8%) in all candidates (F2, F6, and F7). Therefore, all candidates were considered feasible. The amount of fragments measured by SEC also increased in all candidates, but at a rate similar to that in F0. As for IEX, the amount of acidic peaks obtained by IEX increased in all candidates, but at a rate similar to that in F0. [(l)] [Protein Concentration] [ ]

[0229] Considering the results of osmotic pressure mole concentration and viscosity shown in Table 29 and the overall stability results shown in Tables 27 and 28, a protein concentration of 200 mg / mL was selected. [(m)] [PS80] [Concentration Selection] [ ]

[0230] To select the target PS80 concentration in the formulation, freeze-thaw and agitation studies were conducted on candidate formulations and formulation F0 with different PS80 concentrations [0 to 0.10 (w / v)%, F8-F12]. The results of the freeze-thaw and agitation studies are shown in Tables 30 and 31, respectively. [(n)] [Freeze-thaw research] [ ]

[0231] As shown in Table 30, no significant changes were observed in any of the test items after three cycles of freeze-thaw testing. In F8 (without PS80), the particle count obtained by MFI was relatively high. [(o)] [Stirring Study] [ ]

[0232] As shown in Table 31, formulations (F9 to F12) containing 0.02 (w / v)% to 0.10 (w / v)% PS80 were stable after stirring. After stirring, changes in appearance, protein concentration, and aggregate amount were observed in F8 (without PS80) using SEC. The appearance changed from a clear pale yellow liquid to a milky white liquid. The protein concentration decreased slightly from 206 mg / mL to 195 mg / mL. The amount of aggregates increased from 1.1% to 3.8%. Therefore, F8 (without PS80) was considered unfeasible. [(p)] [PS80] [concentration] [ ]

[0233] Based on the results shown in Tables 30 (Freeze-Thaw Study) and 31 (Agitation Study), formulations containing 0.02 (w / v)% to 0.10 (w / v)% PS80 were stable after three freeze-thaw cycles and agitation for up to three days. Therefore, a PS80 concentration of 0.05 (w / v)% was selected as the target. [(q)] [in conclusion] [ ]

[0234] In summary, the following formulations of BAN2401 were selected for further research. [ [surface]

[32] [] [Formulation for subsequent research] [ ] Preparation number BAN2401 buffer Stabilizer (arginine) Polysorbate 80 pH mg / mL mmol / L mg / mL mmol / L mg / mL (w / v)% mg / mL F2 200 Histidine 25 3.9 200 34.8 0.05 0.5 5.0 [Example] [9] [:different] [pH] [Value] [200 mg / mL BAN2401] [Stability Study of the Formulation] [ ]

[0235] Stability studies were conducted to test the stability of the 200 mg / mL BAN2401 formulation at different pH values. In addition, the effect of adding methionine on the stability of the pharmaceutical product (DP) was investigated. [A.] [Sample Preparation] [ ]

[0236] Each candidate formulation was prepared as follows (Table 33). The formulations were equilibrated with the appropriate formulation buffer using a centrifugal filtration apparatus. For the preparation of F20 only, a formulation buffer with a pH of 3.5 was used until the pH of the filtrate reached 4.0, because protons are repelled by concentrated charged proteins near the semipermeable membrane; this is known as the Donnan effect. After concentration and equilibration, PS80 dissolved in the formulation buffer was added to reach the desired concentration, and then the protein concentration was adjusted to the final concentration. Each candidate formulation was filled into vials with a filling volume of 0.5 mL. The formulation F0 was evaluated as a control (see Table 33). [ [surface]

[33] [] [Active Pharmaceutical Ingredients and Drug Candidates] [SC] [Composition of the preparation] [ ] [serial number] [BAN2401] [Buffer] [Stabilizer] [Polysorbate]

[80] [pH, a , ] [Fill Volume] mg / mL mmol / L mg / mL mmol / L mg / mL (w / v)% mg / mL mL Small bottle F0 100 Citrate 50 9.6 Arg 350 61.0 0.05 0.5 5.0 5 F13 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 4.5 0.5 F15 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 5.0 0.5 F17 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 5.5 0.5 F18 200 Histidine 25 3.9 Arg 200 Met 10 34.8 1.5 0.05 0.5 5.0 0.5 F20 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 4.0 0.5 F21b 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 5.0 0.5 F22 200 Histidine 25 3.9 Arg 200 34.8 0.05 0.5 6.0 0.5 Arg: Arginine, Met: Methionine a: To adjust the pH, for F0, arginine and arginine salt salts are combined; for F13 to F22, hydrochloride salt is added. b: The F21 series uses the same formulation as the F15. [B.] [Storage Solution] [ ]

[0237] The storage plan is shown in Tables 34, 35, and 36. Once removed from storage, the vials should be stored at 5°C until the test begins. Storage conditions are as follows: • Long-term conditions: Store at 5°C ± 3°C, upright. • Acceleration conditions: Store at 25°C ± 2°C, 60% RH ± 5% RH, upright. • Stress conditions (freeze-thaw): Freeze at -30°C and thaw at room temperature, then place upright. 1000 lx: 25°C ± 2°C, 60% RH ± 5% RH, laid flat at an exposure of 1000 lx. Darkness: 25°C ± 2°C, 60% RH ± 5% RH, placed flat without exposure by covering with aluminum foil

[0238] To confirm the aggregate level after storage for three months or longer, nine-month extended storage samples were additionally evaluated for size exclusion HPLC for long-term stability studies, and three-month extended storage samples for size exclusion HPLC for accelerated stability studies. The latter samples were tested after being stored in a refrigerator for six months. [ ] [C.] [Results and Discussion] [ ]

[0239] Physical properties were evaluated, and long-term and accelerated studies were performed on candidate formulations including pH changes (pH 4.0 to pH 6.0) and methionine addition. Freeze-thaw and photostability studies were also conducted to evaluate the efficacy of methionine addition. [D.] [pH] [Changes] [ ]

[0240] As shown in Table 37, Table 38A and 38B, Table 39, and Table 40, no significant difference between the candidate formulations (F13, F15, F17 and F20 to F22) was observed for any test item except size exclusion HPLC (SEC). Formulation F20, which is a lower pH formulation with pH 4.0, showed a lower amount of aggregates as measured by SEC at the initial time point. In addition, a slightly lower aggregate formation rate and a higher fragment formation rate were observed in accelerated stability studies. The results for formulations with a pH value of 4.5 to 5.5 were confirmed to be similar. [E.] [Addition of Methionine] [ ]

[0241] As shown in Tables 41A and 41B and Tables 42 through 46, no significant differences were observed between formulations containing or without methionine (F18 and F15). In extended long-term stability studies, accelerated stability studies, and photostability studies, the formulation containing methionine (F18) showed a slightly lower aggregate formation rate. F18 also showed slightly lower oxidation of the methionine residues (e.g., at position 259 in the heavy chain) after exposure to 1000 lux for up to seven days. Freeze-thaw studies showed no differences. The role of 10 mmol / L methionine as a stabilizer is limited. [F.] [in conclusion] [ ]

[0242] Among the BAN2401 formulations with a pH range of 4.5 to 5.5, the quality properties were similar. The formulation at pH 4.0 showed a lower aggregate formation rate, but conversely, a faster fragment formation rate during storage. Based on quality assessments, including long-term, accelerated, freeze-thaw, and light stability studies, we found no significant differences between formulations at 10 mmol / L containing or without methionine. Although a slightly slower aggregate formation rate and less inhibition of amino acid residue oxidation were observed, the differences were not significant. In conclusion, F15 and F21, composed of 200 mg / mL BAN2401, 25 mmol / L L-histidine / histidine salt, 200 mmol / L L-arginine, and 0.05 (w / v)% polysorbate 80 at pH 5.0 ± 0.5, were identified as promising drug substance candidates for further development. [Example]

[10] [Arginine concentration versus [BAN2401] [Study on the Influence of the Formulation on Stability] [ ]

[0243] The effect of arginine concentrations (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. [A.] [Sample Preparation] [ ]

[0244] Samples (F1-F4) were prepared by buffer exchange of the concentrated BAN2401 active pharmaceutical ingredient processing intermediate using ultrafiltration. After aseptic filtration, each 0.4 mL sample was filled into a vial. Sample information is shown in Table 47. [ [surface]

[47] [Sample Information] [ ] Preparation number BAN2401 buffer Stabilizer (arginine) Polysorbate 80 pH F1 200 mg / mL Histidine 25 mmol / L 0 mmol / L 0.05(w / v)% 5.0 F2 200 mg / mL Histidine 25 mmol / L 50 mmol / L 0.05(w / v)% 5.0 F3 200 mg / mL Histidine 25 mmol / L 100 mmol / L 0.05(w / v)% 5.0 F4 200 mg / mL Histidine 25 mmol / L 200 mmol / L 0.05(w / v)% 5.0 [B.] [result] [ ]

[0245] Tables 48 to 51 show the results of the stability studies for samples F1 to F4. There were no differences in pH and protein concentration among the samples, and the arginine concentration levels were consistent with the target levels of the initial samples.

[0246] The aggregate percentage of samples containing arginine (F2 to F4) was lower than that of samples without arginine (F1). [ [surface]

[48] [] [sample] [F1] [Test results (for arginine)] [0 mmol / L] [)] [ ] Test Project initial 1M 2M pH 5.1 - - Protein concentration (mg / mL) 195 - - SEC Single unit area % 98.3% 98.0% 97.8% Aggregate area % 1.6% 1.7% 1.9% Fragment area % 0.1% 0.2% 0.3% Arginine concentration (mmol / L) 1 - - [ [surface]

[49] [] [sample] [F2] [Test results (for arginine)] [50 mmol / L] [)] [ ] Test Project initial 1M 2M pH 5.0 - - Protein concentration (mg / mL) 192 - - SEC Single unit area % 98.4% 98.1% 97.8% Aggregate area % 1.5% 1.7% 1.8% Fragment area % 0.1% 0.2% 0.4% Arginine concentration (mmol / L) 47 - - [ [surface]

[50] [] [sample] [F3] [Test results (for arginine)] [100 mmol / L] [)] [ ] Test Project initial 1M 2M pH 5.0 - - Protein concentration (mg / mL) 198 - - SEC Single unit area % 98.4% 98.1% 97.8% Aggregate area % 1.5% 1.7% 1.8% Fragment area % 0.1% 0.2% 0.4% Arginine concentration (mmol / L) 87 - - [ [surface]

[51] [] [sample] [F4] [Test results (for arginine)] [200 mmol / L] [)] [ ] Test Project initial 1M 2M pH 5.0 - - Protein concentration (mg / mL) 191 - - SEC Single unit area % 98.4% 98.1% 97.9% Aggregate area % 1.5% 1.6% 1.7% Fragment area % 0.1% 0.2% 0.4% Arginine concentration (mmol / L) 173 - - [C.] [Discussion and Conclusion] [ ]

[0247] The results of this study indicate that arginine concentrations ranging from 50 to 200 mmol / L can prevent the increase of protein aggregates in BAN2401 200 mg / mL formulations. Variations in arginine concentration between batches may lead to variations in the percentage of aggregates between batches. Furthermore, in stability studies, arginine concentration may influence the trend of increasing aggregate percentages.

[0248] By comparing the fragmentation percentage in sample F1 with the fragmentation percentage shown in Figure 11 of Example 2, the formulation containing histidine buffer appears to exhibit a reducing effect on BAN2401 fragmentation, in which samples containing 200 mg / mL BAN2401, 50 mM sodium citrate, and 100 mM sodium chloride were used. [Sequence List] [ ] [ [surface]

[52] [. mAb] [Amino acid sequence in the variable region] [ ] [mAb] [IgG] [chain] [SEQ ID NO] [Amino acid sequence] BAN2401 Heavy chain 1 EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYWGQGTTVTVSS BAN2401 Light chain 2 DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIK [ [surface]

[53] [. mAb] [Amino acid sequence in the constant region] [ ] [mAb] [IgG] [chain] [category] [SEQ ID NO] [Amino acid sequence] BAN2401 Heavy chain IgG1 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK BAN2401 Light chain κ 4 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Table]

[54] ] [. mAb CDR] Amino acid sequence of [ ] [mAb] [IgG] Chain [SEQ ID NO] Amino acid sequence BAN2401 HCDR1 5 SFGMH HCDR2 6 YISSGSSTIYYGDTVKG HCDR3 7 ​EGGYYYGRSYYTMDY BAN2401 LCDR1 8 RSSQSIVHSNGNTYLE LCDR2 9 KVSNRFS LCDR3 10 FQGSHVPPT [heavy chain()] [SEQ ID NO: 11] [):] [ ] [ ] [Light Chain() [SEQ ID NO: 12] [):] [ ]

[0249] <![CDATA[ <110> EISAI R&D Management Co., Ltd. <![CDATA[ <120> High-concentration anti-Aβ fibrillary antibody formulation and its usage method <![CDATA[ <140> ]]> <![CDATA[ <141> 2021-03-22 <![CDATA[ <150> US 62 / 992,746 <![CDATA[ <151> 2020-03-20 <![CDATA[ <150> US 63 / 027,263 <![CDATA[ <151> 2019-05-19 <![CDATA[ <160> 12 ]]> <![CDATA[ <170> PatentIn Version 3.5 <![CDATA[ <210> 1]]> <![CDATA[ <211> 124]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[ <400> 1]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <![CDATA[ <210> 2]]> <![CDATA[ <211> 112]]> <![CDATA[ <212> PRT]]> <![CDATA[<2]]> 13> Artificial Sequences]]> <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[ <400> 2]]> <![CDATA[Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[ <210> 3]]> <![CDATA[ <211> 330]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[ <400> 3]]> Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <![CDATA[ <210> 4]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[ <400> 4]]> Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <![CDATA[ <210> 5]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 5]]> Ser Phe Gly Met His 1 5 <![CDATA[ <210> 6]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 6]]> Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val Lys 1 5 10 15 Gly <![CDATA[ <210> 7]]> <![CDATA[ <211> 15]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 7]]> Glu Gly Gly Tyr Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp Tyr 1 5 10 15 <![CDATA[ <210> 8]]> <![CDATA[ <211> 16]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 8]]> Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <![CDATA[ <210> 9]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 9]]> Lys Val Ser Asn Arg Phe Ser 1 5 <![CDATA[ <210> 10]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic peptide"]]> <![CDATA[ <400> 10]]> Phe Gln Gly Ser His Val Pro Pro Thr 1 5 <![CDATA[ <210> 11]]> <![CDATA[ <211> 454]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[ <400> 11]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Lys 450 <![CDATA[ <210> 12]]> <![CDATA[ <211> 219]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <221> Source <![CDATA[ <223> / Note="Description of artificial sequence: synthetic polypeptide"]]> <![CDATA[<400> 12]]> Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. An aqueous pharmaceutical formulation comprising: (a) an anti-Aβ fibrillary antibody that binds to human Aβ fibrillary fibers at a concentration of 80 mg / mL to 300 mg / mL, and (b) a histidine buffer, wherein the pharmaceutical formulation has a pH in the range of 4.5 to 5.5, wherein the anti-Aβ fibrillary antibody is lecanemab.

2. The pharmaceutical formulation of claim 1, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL to 200 mg / mL.

3. The pharmaceutical formulation of claim 1, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL.

4. The pharmaceutical formulation of claim 1, wherein the anti-Aβ fibrillary antibody is present at a concentration of 200 mg / mL.

5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the histidine buffer is present at a concentration of 10 mM to 100 mM.

6. The pharmaceutical formulation of any one of claims 1 to 4, wherein the histidine buffer is present at a concentration of 15 mM to 35 mM.

7. The pharmaceutical formulation of any one of claims 1 to 4, wherein the histidine buffer is present at a concentration of 25 mM.

8. A pharmaceutical formulation as claimed in any of claims 1 to 4, wherein the histidine buffer comprises histidine and histidine salt, or histidine and histidine salt monohydrate.

9. The pharmaceutical formulation of any one of claims 1 to 4 further comprises arginine, arginine salt, or a combination thereof.

10. The pharmaceutical formulation of any one of claims 1 to 4 further comprises 0.01% w / v to 0.1% w / v of polysorbate 80.

11. A pharmaceutical formulation as claimed in any of claims 1 to 4, wherein the pharmaceutical formulation has a pH of 5.

0.

12. A method for reducing the fragmentation of anti-Aβ fibrillary antibodies, comprising: providing an aqueous pharmaceutical formulation containing an anti-Aβ fibrillary antibody at a concentration of 80 mg / mL to 300 mg / mL, and adding a histidine buffer to the aqueous pharmaceutical formulation, wherein the pH range of the pharmaceutical formulation is from 4.5 to 5.5, and wherein the anti-Aβ fibrillary antibody is lecanemab.

13. The method of claim 12, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL to 200 mg / mL.

14. The method of claim 12, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL.

15. The method of claim 12, wherein the anti-Aβ fibrillary antibody is present at a concentration of 200 mg / mL.

16. The method of claim 12, wherein the histidine buffer is present at a concentration of 15 mM to 35 mM.

17. The method of claim 12, wherein the histidine buffer is present at a concentration of 25 mM.

18. The method of any one of claims 12 to 17, further comprising adding arginine, arginine salt, or a combination thereof.

19. The method of any one of claims 12 to 17, wherein after storage at 25°C for 3 months, the fragmentation of the anti-Aβ fibrillary antibody is less than 0.8%.

20. The method of any one of claims 12 to 17, wherein after storage at 5°C for 3 months, the fragmentation of the anti-Aβ fibrillary antibody is less than 0.4%.

21. The method of any one of claims 12 to 17, wherein the histidine buffer is a combination of histidine and histidine salt, or a combination of histidine and histidine salt monohydrate.

22. A method for reducing the aggregation of anti-Aβ fibrillary antibodies, comprising: providing an aqueous pharmaceutical formulation containing an anti-Aβ fibrillary antibody at a concentration of 80 mg / mL to 300 mg / mL, and adding arginine, arginine salt, 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, and wherein the anti-Aβ fibrillary antibody is lecanemab.

23. The method of claim 22, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL to 200 mg / mL.

24. The method of claim 22, wherein the anti-Aβ fibrillary antibody is present at a concentration of 100 mg / mL.

25. The method of claim 22, wherein the anti-Aβ fibrillary antibody is present at a concentration of 200 mg / mL.

26. The method of claim 22, wherein the arginine, arginine salt salt, or combination thereof is present at a concentration of 150 mM to 250 mM.

27. The method of claim 22, wherein the arginine, arginine salt, or combination thereof is present at a concentration of 200 mM.

28. The method of any of claims 22 to 27, further comprising adding a histidine buffer or a citrate buffer.

29. The method of any one of claims 22 to 27, wherein after storage at 25°C for 3 months, the aggregation of the anti-Aβ fibrillary antibody is less than 3%.

30. The method of any one of claims 22 to 27, wherein after storage at 5°C for 3 months, the aggregation of the anti-Aβ fibrillary antibody is less than 2%.

31. The method of any one of claims 12 to 17 and 22 to 27, wherein the pharmaceutical formulation further comprises 0.01% w / v to 0.1% w / v of polysorbate 80.

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