Formulations of human Anti-rankl antibodies, and methods of using the same

Abstract

To provide stable aqueous pharmaceutical formulations comprising a high concentration of an anti-RANKL antibody, the formulations comprising only low concentrations (e.g., less than about 2%) of aggregates.SOLUTION: The present invention provides an aqueous pharmaceutical formulation comprising (i) a human anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) monoclonal antibody, or an antigen-binding portion thereof and (ii) an amino acid aggregation inhibitor.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications The benefit of 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 62 / 492,056, filed on April 28, 2017, is hereby claimed, and its disclosure is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Data The computer - readable nucleotide / amino acid sequence listing submitted simultaneously with this specification is hereby incorporated by reference in its entirety and is identified as follows: a 49 - kilobyte ASCII (text) file, "51689A_Seqlisting.txt", created on April 20, 2018.

[0003] Background Field of the Disclosure The present invention relates to human anti - RANKL monoclonal antibodies, such as high - concentration aqueous formulations of denosumab and its biosimilars.

Background Art

[0004] Brief Description of the Related Art Denosumab is commercially available in solution form at concentrations of 60 mg / mL and 70 mg / mL.

[0005] As the concentration of a protein formulation increases, it can cause problems with stability, such as aggregation leading to the formation of high - molecular - weight species (HMWS). HMWS, especially those that preserve most of the native conformation of the monomeric counterpart, are of particular concern in some protein formulations. Aggregation can also potentially affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

[0006] The filling and finishing operations, as well as administration, may include flowing the protein solution through a piston pump, a peristaltic pump, or an injection needle. Such processes can impart shear stress and mechanical stress that can cause protein denaturation and aggregation. This phenomenon may worsen as the protein solution becomes more concentrated.

Summary of the Invention

[0007] The disclosure provided according to the present invention is the first to demonstrate that by adding an amino acid aggregation inhibitor to an aqueous solution containing a high concentration of an anti-RANKL antibody, the amount of antibody aggregates formed over time decreases, and the rate of formation of such aggregates slows down. The disclosure also provides the pH effect on aggregate formation in a concentrated aqueous solution of an anti-RANKL antibody, namely the effect that the formation of aggregates is observed to decrease when the pH of the aqueous solution is in the range of about 5.0 to less than 5.2. Further suggested by the disclosure presented herein is that the stabilization of the anti-RANKL antibody results from the interaction between the amino acid aggregation inhibitor and the antibody. Without being bound by any particular theory, it is contemplated that hydrophobic interactions and other types of intermolecular interactions between the amino acid aggregation inhibitor and the anti-RANKL antibody provide a stabilizing effect on the concentrated antibody solution. Accordingly, the disclosure of the present invention relates to a stable aqueous pharmaceutical formulation containing a high concentration of an anti-RANKL antibody that contains only low concentrations (e.g., less than about 2%) of aggregates.

[0008] Accordingly, one aspect of the disclosure is an aqueous pharmaceutical formulation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof at a concentration exceeding 70 mg / mL and having a pH in the range of about 5.0 to less than 5.2.

[0009] Another aspect of the disclosure is an aqueous pharmaceutical formulation comprising a human anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof and an amino acid aggregation inhibitor. In an exemplary embodiment, the amino acid aggregation inhibitor comprises an amino acid having a charged side chain, an aromatic amino acid, or a hydrophobic amino acid. In an exemplary example, the amino acid having a charged side chain is an amino acid having a positively charged side chain such as, for example, arginine and lysine. In an exemplary embodiment, the aromatic amino acid comprises phenyl or indole. Optionally, the aromatic amino acid further comprises a C1-C6 alkyl chain between the alpha carbon and the phenyl or indole. For example, amino acids such as phenylalanine and tryptophan are exemplary amino acid aggregation inhibitors. In an exemplary example, the amino acid aggregation inhibitor is a hydrophobic amino acid having a score greater than about 2.5 on the Kyte and Doolittle hydrophobicity scale. Optionally, the hydrophobic amino acid is valine, leucine or isoleucine. Further amino acid aggregation inhibitors are contemplated as described herein.

[0010] In an exemplary example, the aqueous pharmaceutical formulation further comprises an isotonicity regulator, a surfactant, a buffer, or any combination thereof.

[0011] Another aspect of the disclosure is the presentation of a formulation for storage or use, for example, storage or use in a disposable vial, a disposable syringe, or a primary container that is glass, glass-lined, or glass-coated. Exemplary embodiments of the disclosure are containers, optionally vials, prefilled syringes (PFS), or glass containers, comprising any of the aqueous pharmaceutical formulations described herein. In an exemplary example, the container comprises an aqueous pharmaceutical formulation of about 1 mL or less (e.g., about 0.5 mL).

[0012] Another aspect of the present disclosure is a method for manufacturing a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof, the method comprising mixing an anti-RANKL monoclonal antibody or an antigen-binding portion thereof at a concentration exceeding 70 mg / mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. Aspects of the present disclosure include stable aqueous pharmaceutical formulations manufactured according to any one of the methods for manufacturing stable aqueous pharmaceutical formulations described herein.

[0013] Another aspect of the present disclosure provides a method of using a formulation described herein for preventing or treating a disease responsive to a human anti-RANKL monoclonal antibody or an antigen-binding portion thereof. In a specific aspect, such use includes treating a subject for the treatment or prevention of skeletal-related events (SREs) in a subject, the treatment or prevention of giant cell tumors of bone, the treatment or prevention of hypercalcemia of malignancy, the treatment or prevention of osteoporosis, or an increase in bone mass. For example, the therapeutic treatment includes (a) treatment or prevention of SREs in a subject having bone metastases from a solid tumor, (b) treatment or prevention of SREs in an adult or skeletally mature adolescent subject having a giant cell tumor of bone that is inoperable or for which surgical resection may result in a high morbidity rate, (c) treatment of hypercalcemia of malignancy refractory to bisphosphonate treatment in a subject, (d) treatment or prevention of SREs in a subject having multiple myeloma or bone metastases from a solid tumor, (e) treatment of osteoporosis in postmenopausal women at high risk of fracture, (f) treatment to increase bone mass in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer, (g) treatment to increase bone mass in men at high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, (h) treatment to increase bone mass in men with osteoporosis at high risk of fracture, (i) treatment with calcium or vitamin D.

[0014] A further aspect of the present disclosure is a method of preventing skeletal-related events (SREs) in patients in need thereof, a method of treating giant cell tumor of bone in patients in need thereof, a method of treating hypercalcemia of malignancy in patients in need thereof, a method of treating osteoporosis in patients in need thereof, and a method of increasing bone mass in patients in need thereof. The method includes administering to the patient an effective amount of any one of the formulations described herein. In an exemplary example, the formulation is delivered subcutaneously to the patient.

[0015] Another aspect of the present disclosure provides the use of denosumab, or another human anti-RANKL monoclonal antibody or antigen-binding portion thereof, in the manufacture of a medicament described herein for treating patients in need of a human anti-RANKL monoclonal antibody.

[0016] Another aspect of the present disclosure is a kit comprising a composition or article disclosed herein together with a package insert, package label, instruction sheet, or other display that indicates or discloses any of the methods or embodiments disclosed herein.

[0017] Another aspect of the present disclosure is a method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a concentration greater than 70 mg / mL, the method comprising preparing an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a pH in the range of about 5.0 to less than 5.2, and the aqueous pharmaceutical formulation exhibits improved stability at a pH in the range of about 5.0 to less than 5.2 as compared to an equivalent aqueous pharmaceutical formulation that is not at a pH within the range of about 5.0 to less than 5.2.

[0018] Another aspect of the present disclosure is a method for improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof, the method comprising preparing an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof, mixed with an amino acid aggregation inhibitor, and the aqueous pharmaceutical formulation demonstrating improved stability by the amino acid aggregation inhibitor as compared to an equivalent aqueous pharmaceutical formulation without the amino acid aggregation inhibitor.

[0019] Another aspect of the present disclosure is a method for reducing the level of HMWS aggregates in a solution of denosumab or another human anti-RANKL monoclonal antibody.

[0020] Further aspects and advantages will be apparent to those skilled in the art upon consideration of the following detailed description in conjunction with the drawings. Compositions, articles, and methods are susceptible to various forms of embodiments, but the following description includes specific embodiments with the understanding that the present disclosure is exemplary and is not intended to limit the invention to the specific embodiments described herein. For the compositions, articles, and methods described herein, any features including, but not limited to, components, their composition ranges, substituents, conditions, and steps are contemplated to be selected from the various aspects, embodiments, and examples provided herein.

Brief Description of the Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] It would be desirable to provide a more concentrated aqueous solution of denosumab and other human anti-RANKL antibodies and antigen-binding portions thereof that is as stable as or more stable than a dilution solution. The more concentrated solution can provide patient convenience, for example, by enabling administration in a smaller volume, such as a 1 mL injection, rather than a 1.7 mL or 2 mL injection of a more diluted active formulation, to deliver 120 mg of an active ingredient such as denosumab. Furthermore, an even smaller amount of injection solution can deliver an even lower dose of the active substance, for example, 0.5 mL of denosumab at a concentration of 120 mg / mL to deliver a 60 mg dose. It would also be desirable to provide an aqueous solution of denosumab and other human anti-RANKL antibodies and antigen-binding portions thereof that is more stable than conventionally known solutions. A stable concentrated formulation also has other advantages such as enabling handling and shipping of a smaller amount of product and allowing for a longer shelf life of the product.

[0023] Aggregates in biological preparations can vary in origin, size, and type. Aggregates that can affect the efficacy or safety of biological preparations, such as those that can enhance the immune response and cause adverse clinical effects, are of particular concern. High molecular weight aggregates, also known as high molecular weight species (HMWS), especially those that preserve most of the native conformation of the monomeric counterpart, are of particular concern. Aggregation can also potentially affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

[0024] There can be various causes for aggregate formation. Generally, protein aggregation results from structural instability, which is a consequence of protein structural changes, and colloidal instability, which is governed by intermolecular forces. When important nucleation events are required to induce precipitation, the kinetics of protein aggregation can be characterized by including a lag time phase.

[0025] Aggregation due to structural instability involves steps of unfolding and association. Unfolding of protein molecules exposes hydrophobic amino acid residues. The hydrophobic residues of unfolded molecules can then lead to aggregates (such as dimers, trimers, other multimers, and higher-order aggregates, etc.) through subsequent association. Such association is concentration-dependent. An increase in the protein concentration in an aqueous solvent generally increases the rate and extent of aggregation, such as heat-induced aggregation. Therefore, additives that affect the free energy of protein unfolding in solution can affect structural stability.

[0026] Colloidal instability results in aggregates due to protein-protein intermolecular forces. Such forces can be affected by one or more factors including ionic strength, solution pH, and the type of buffer.

[0027] Denosumab is commercially available in solution form at concentrations of 60 mg / mL and 70 mg / mL. Attempts to formulate higher concentration solutions of denosumab using the same additives have shown that higher concentrations affect the stability of the product by an attendant and proportional increase in HMWS. For example, a concentration of denosumab 120 mg / mL has a concentration more than 70% higher than denosumab 70 mg / mL and is twice the 60 mg / mL concentration.

[0028] Accordingly, the stabilized aqueous formulations according to the present disclosure will be more resistant to aggregate formation to a greater extent than conventionally known formulations. One aspect of the present disclosure is a stabilized aqueous formulation characterized by a pH of from 5.0 to less than 5.2. Another non-exclusive aspect of the present disclosure is a stabilized aqueous formulation comprising an amino acid aggregation inhibitor. Associated dosage forms, such as disposable vials, syringes and glass containers, and associated treatment methods can also be provided. A method for producing a stable aqueous pharmaceutical formulation is further provided.

[0029] As described below, pH and amino acid aggregation inhibitors (e.g., arginine, arginine-arginine dipeptide, arginine-phenylalanine dipeptide) are two means that have been shown to reduce the HMWS level and HMWS formation rate of 120 mg / mL denosumab. HMWS can be described as an intermolecular protein interaction that is either irreversible (e.g., covalent) or reversible (e.g., non-covalent self-association interaction). There are four generally recognized causes of protein self-association reactions that can lead to an increase in viscosity and HMWS: hydrophobicity, charge, polar and dipole interactions. Both formulation pH and arginine (a highly charged basic amino acid at neutral to acidic pH values) can potentially interfere with the intermolecular forces of charged proteins. Without intending to be bound by a particular theory, the HMWS formation at 120 mg / mL of denosumab is thought to be based on the charge of the protein, and the changes in these formulations are thought to disrupt the charge forces involved in the HMWS formation mechanism. Furthermore, without intending to be bound by a particular theory, since arginine contains a short aliphatic hydrocarbon chain in its side chain, it is also conceivable that hydrophobic protein self-association interactions exist in the formation of HMWS. This aliphatic chain can disrupt the hydrophobic interactions between proteins. This idea is further supported by including phenylalanine in the formulation to further reduce the level of HMWS. Without being bound by any particular theory, if arginine interacts with the antibody via hydrophobic interactions, arginine stabilizes the anti-RANKL antibody in a different way than phenylalanine, such that arginine may interact with the antibody in one or more other ways.

[0030] Other additives that may potentially have a positive effect on reducing the level and formation rate of HMWS can have a similar positive charge at neutral to acidic pH values when compared to arginine and / or can be hydrophobic in nature similar to phenylalanine. Examples of these additives include lysine, N-acetylarginine, N-acetyllysine, tyrosine, tryptophan, and leucine.

[0031] The formulations, dispensing forms, and methods are intended to include embodiments that include any combination of one or more additional optional elements, features, and steps (including those shown in the drawings), as further described below, unless otherwise stated.

[0032] In jurisdictions that prohibit patents on methods practiced on the human body, the meaning of "administering" a composition to a human subject shall be limited to prescribing a controlled substance for self-administration by the human subject by any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods practiced on the human body, "administration" of a composition includes both methods practiced on the human body and the aforementioned activities.

[0033] As used herein, the term "comprising" indicates the potential inclusion of other agents, elements, steps, or features in addition to those specified.

[0034] All maximum numerical limitations given throughout this specification are to be understood to include, as alternative embodiments, ranges formed by all corresponding smaller numerical limitations, as if such numerical ranges were expressly written herein. The minimum numerical limitations given throughout this specification include, as alternative embodiments, ranges formed by all higher numerical limitations, as if such ranges were expressly written herein. All numerical ranges given throughout this specification include all narrower numerical ranges included within such broader numerical ranges, as if all such narrower numerical ranges were expressly written herein. The dimensions and values disclosed herein are to be understood to include the disclosure of both the recited values and the corresponding exact numerical values. For example, a value recited as "about 10 mM" is to be understood to include "10 mM" as an alternative disclosure.

[0035] As used herein, the term "therapeutically effective amount" refers to an amount of a compound sufficient to treat, ameliorate or prevent a specified disease or condition, or to exhibit a detectable therapeutic, prophylactic or inhibitory effect. The effect can be detected, for example, by improvement of a clinical condition or alleviation of symptoms. The exact effective amount for a subject will depend upon the subject's weight, size, and health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. Where the agent is approved by the U.S. Food and Drug Administration (FDA), a "therapeutically effective amount" refers to the dosage approved by the FDA or its corresponding foreign agency for the treatment of the specified disease or condition.

[0036] The present disclosure provides a stabilized (or stable) aqueous pharmaceutical formulation indicated by a reduced amount of aggregates and / or a decreased rate of aggregate formation after storage. As described herein, the stability of such a formulation is indicated by a reduced amount of HMWS and / or a decreased rate of HMWS formation after storage at various times and various temperatures. Generally, formulations having higher stability are associated with a lower amount of HMWS, a lower rate of HMWS formation and / or a higher antibody main peak at higher storage temperatures compared to lower temperatures. As used herein, the term "high molecular weight species" or "HMWS" refers to higher order aggregates of the antibody of the formulation, as well as lower order aggregates of the antibody of the formulation. Lower order aggregates include, for example, dimer species. The amount of aggregation and the rate of formation can be measured or monitored by techniques such as, for example, SE-UHPLC. The SE-UHPLC chromatogram of an antibody shows, in some examples, a peak around 5.8 minutes representing the amount of HMWS of the aqueous pharmaceutical formulation, a peak around 6.7 minutes representing dimer species, and a peak around 8.0 minutes reflecting the amount of intact non-aggregated form of the antibody. Storage at 37°C can facilitate stability assays such that the stability of a particular formulation is measured in a shorter time compared to the storage period at 4°C. For example, storage at 37°C for 1 month, 2 months, or 3 months can be indicative of or predicted to be equivalent to storage at 4°C for 36 months.

[0037] In one type of embodiment, the stabilized formulation described herein, as an additive, contains 10 mM acetate, 5% (w / v) sorbitol, 0.01% (w / v) polysorbate 20, and the degree and rate of HMWS formation after storage at 37 °C for 3 months will be reduced compared to an isoconcentration control formulation having a solution pH of 5.2.

[0038] In another type of embodiment, the stabilized formulation containing an amino acid aggregation inhibitor described herein will have a reduced degree of HWMS formation after storage at 37 °C for 1 month compared to an equivalent control formulation that does not contain the amino acid aggregation inhibitor. The degree of formation can be reduced such that, for example, the amount of HMWS% by SE-UPHLC is at least about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5% or about 0.6%, or about 0.7%, for example in the range of about 0.1% to about 2%, or about 0.1% to about 1% lower than that of the control formulation stored at 37 °C for 1 month.

[0039] In another type of embodiment, the stabilized formulations described herein will have a low amount of HMWS after storage at 37°C for 1 month by SE-UHPLC. For example, the amount of HMWS may be 2% or less, or less than 2%, or 1.9% or less, or less than 1.9%, or 1.8% or less, or less than 1.8%, or 1.7% or less, or less than 1.7%, or 1.6% or less, or less than 1.6%, or 1.5% or less, or less than 1.5%, or 1.4% or less, or less than 1.4%, or 1.3% or less, less than 1.3%, or 1.2% or less, or less than 1.2%, for example, in the range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%. In another type of embodiment, the amount of HMWS after storage at 37°C for 1 month by SE-UHPLC may be greater than 2%, for example, greater than 2% up to 3%, while the decrease in the aggregation rate brought about by the amino acid aggregation inhibitor can enable an appropriate product shelf life, up to a maximum of 3 years, or a maximum of 2 years.

[0040] In another type of embodiment, the stabilized formulations described herein will have a low amount of HMWS after storage at 37°C for 3 months by SE-UHPLC. For example, the amount of HMWS may be 2% or less, or less than 2%, or 1.9% or less, or less than 1.9%, or 1.8% or less, or less than 1.8%, or 1.7% or less, or less than 1.7%, or 1.6% or less, or less than 1.6%, or 1.5% or less, or less than 1.5%, or 1.4% or less, or less than 1.4%, or 1.3% or less, less than 1.3%, or 1.2% or less, or less than 1.2%, for example, in the range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%.

[0041] In another type of embodiment, the stabilized formulations described herein would have a low amount of HMWS after storage at 4 °C for 36 months by SE-UHPLC. For example, the amount of HMWS may be 2% or less, or less than 2%, or 1.9% or less, or less than 1.9%, or 1.8% or less, or less than 1.8%, or 1.7% or less, or less than 1.7%, or 1.6% or less, or less than 1.6%, or 1.5% or less, or less than 1.5%, or 1.4% or less, or less than 1.4%, or 1.3% or less, less than 1.3%, or 1.2% or less, or less than 1.2%, for example, from about 0.01% to about 2%, or from about 0.01% to about 1.9%, or from about 0.01% to about 1.8%, or from about 0.01% to about 1.7%, or from about 0.01% to about 1.6%, or from about 0.01% to about 1.5%, or from about 0.01% to about 1.4%, or from about 0.01% to about 1.3%, or from about 0.01% to about 1.2%.

[0042] In another type of embodiment, the stabilized formulations described herein would have a high amount of the main peak of denosumab or another antibody (or antigen-binding portion thereof) after storage at 37 °C for 1 month by SE-UHPLC. For example, the amount of the main peak may be at least 95%, or more than 95%, or at least 96%, or more than 96%, or at least 97%, or more than 97%, or at least 97.5%, or more than 97.5%, or at least 98%, or more than 98%, or at least 98.1%, or more than 98.1%, or at least 98.2%, or more than 98.2%, or at least 98.3%, or more than 98.3%, or at least 98.4%, or more than 98.4%, or at least 98.5%, or more than 98.5%, or at least 98.6%, or more than 98.6%, for example, from about 95 to about 99.9%, or from about 96 to about 99.9%, or from about 97 to about 99.9%, or from about 97.5 to about 99.9%, or from about 98 to about 99.9%, or from about 98.1 to about 99.9%, or from about 98.2 to about 99.9%, or from about 98.3 to about 99.9%, or from about 98.4 to about 99.9%, or from about 98.5 to about 99.9%, or from about 98.6 to about 99.9%.

[0043] In another type of embodiment, the stabilization formulations described herein would have a high amount of the main peak of denosumab or other antibody (or antigen-binding portion thereof) after storage at 37°C for 3 months by SE-UHPLC. For example, the amount of the main peak is at least 95%, or more than 95%, or at least 96%, or more than 96%, or at least 97%, or more than 97%, or at least 97.5%, or more than 97.5%, or at least 98%, or more than 98%, or at least 98.1%, or more than 98.1%, or at least 98.2%, or more than 98.2%, or at least 98.3%, or more than 98.3%, or at least 98.4%, or more than 98.4%, or at least 98.5%, or more than 98.5%, or at least 98.6%, or more than 98.6%, for example, in the range of about 95 to about 99.9%, or about 96 to about 99.9%, or about 97 to about 99.9%, or about 97.5 to about 99.9%, or about 98 to about 99.9%, or about 98.1 to about 99.9%, or about 98.2 to about 99.9%, or about 98.3 to about 99.9%, or about 98.4 to about 99.9%, or about 98.5 to about 99.9%, or about 98.6 to about 99.9%.

[0044] In another type of embodiment, the stabilized formulations described herein will have a high amount of the main peak of denosumab or other antibody (or antigen-binding portion thereof) after storage at 4 °C for 36 months by SE-UHPLC. For example, the amount of the main peak is at least 95%, or more than 95%, or at least 96%, or more than 96%, or at least 97%, or more than 97%, or at least 97.5%, or more than 97.5%, or at least 98%, or more than 98%, or at least 98.1%, or more than 98.1%, or at least 98.2%, or more than 98.2%, or at least 98.3%, or more than 98.3%, or at least 98.4%, or more than 98.4%, or at least 98.5%, or more than 98.5%, or at least 98.6%, or more than 98.6%, for example, in the range of about 95 to about 99.9%, or about 96 to about 99.9%, or about 97 to about 99.9%, or about 97.5 to about 99.9%, or about 98 to about 99.9%, or about 98.1 to about 99.9%, or about 98.2 to about 99.9%, or about 98.3 to about 99.9%, or about 98.4 to about 99.9%, or about 98.5 to about 99.9%, or about 98.6 to about 99.9%.

[0045] In a further embodiment, the stabilized formulation is intended to have a small amount of HMWS and a large amount of the main peak after storage, according to the above specification.

[0046] In an exemplary embodiment, the aqueous pharmaceutical formulation contains about 4% or less of high molecular weight species (HMWS) and / or more than about 96% of the antibody main peak, as measured by SE-UHPLC after storage. In an exemplary embodiment, the aqueous pharmaceutical formulation contains about 3% or less of high molecular weight species (HMWS) and / or more than about 97% of the antibody main peak, as measured by SE-UHPLC after storage. In an exemplary embodiment, the aqueous pharmaceutical formulation contains about 2% or less of HMWS and / or more than about 98% of the antibody main peak, as measured by SE-UHPLC after storage. In an exemplary embodiment, the storage is at a temperature of about 2°C to about 8°C (e.g., about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C) for at least 12 months, 24 months or 36 months (e.g., at least or about 12 months, at least or about 16 months, at least or about 20 months, at least or about 24 months, at least or about 28 months, at least or about 32 months, at least or about 36 months, optionally longer). In an exemplary embodiment, the storage is at about 20°C to about 30°C (e.g., about 21°C to about 30°C, about 22°C to about 30°C, about 23°C to about 30°C, about 24°C to about 30°C, about 25°C to about 30°C, about 26°C to about 30°C, about 27°C to about 30°C, about 28°C to about 30°C, about 28°C to about 30°C, about 20°C to about 29°C, about 20°C to about 28°C, about 20°C to about 27°C, about 20°C to about 26°C, about 20°C to about 25°C, about 20°C to about 24°C, about 20°C to about 23°C, about 20°C to about 22°C) for about 1 month (e.g., about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days). In an exemplary embodiment, the storage includes a first storage followed by a second storage, the first storage is at about 2°C to about 8°C for at least 12 months, 24 months, or 36 months, and the second storage is at about 20°C to about 30°C for about 1 month.In an exemplary example, the aqueous pharmaceutical formulation contains HMWS of 2% or less, or less than 2% of HMWS, or 1.9% or less of HMWS, or less than 1.9% of HMWS, or 1.8% or less of HMWS, or less than 1.8% of HMWS, or 1.7% or less of HMWS, or less than 1.7% of HMWS, or 1.6% or less of HMWS, or less than 1.6% of HMWS, or 1.5% or less of HMWS, or less than 1.5% of HMWS, or 1.4% or less of HMWS, or less than 1.4% of HMWS, or 1.3% or less of HMWS or less than 1.3% of HMWS, or 1.2% or less of HMWS or less than 1.2% of HMWS, for example, about 0.01% to about 2% of HMWS, or about 0.01% to about 1.9% of HMWS, or about 0.01% to about 1.8% of HMWS, or about 0.01% to about 1.7% of HMWS, or about 0.01% to about 1.6% of HMWS, or about 0.01% to about 1.5% of HMWS, or about 0.01% to about 1.4% of HMWS, or about 0.01% to about 1.3% of HMWS or about 0.01% to about 1.2% of HMWS, and this HMWS is optionally measured by SE-UHPLC.In an alternative or additional embodiment, the aqueous pharmaceutical formulation comprises an antibody main peak of greater than 98%, or at least 95% antibody main peak, or greater than 95% antibody main peak, or at least 96% antibody main peak, or greater than 96% antibody main peak, or at least 97% antibody main peak, or greater than 97% antibody main peak, or at least 97.5% antibody main peak, or greater than 97.5% antibody main peak, or at least 98% antibody main peak, or greater than 98% antibody main peak, or at least 98.1% antibody main peak, or greater than 98.1% antibody main peak, or at least 98.2% antibody main peak, or greater than 98.2% antibody main peak, or at least 98.3% antibody main peak, or greater than 98.3% antibody main peak, or at least 98.4% antibody main peak, or greater than 98.4% antibody main peak, or at least 98.5% antibody main peak, or greater than 98.5% antibody main peak, or at least 98.6% antibody main peak, or greater than 98.6% antibody main peak, for example, an antibody main peak in the range of about 95% to about 99.9%, or in the range of about 96% to about 99.9%, or in the range of about 97% to about 99.9%, or in the range of about 97.5% to about 99.9%, or in the range of about 98% to about 99.9%, or in the range of about 98.1% to about 99.9% antibody main peak, or in the range of about 98.2% to about 99.9%, or in the range of about 98.3% to about 99.9%, or in the range of about 98.4% to about 99.9%, or in the range of about 98.5% to about 99.9%, or in the range of about 98.6% to about 99.9%, and this peak is optionally measured by SE-UHPLC.

[0047] As used herein, the term “antibody” refers to a protein having the conventional immunoglobulin format that includes heavy and light chains and includes variable and constant regions. For example, the antibody may be an IgG antibody having a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region is generally about 100-110 or more amino acids, includes three complementarity determining regions (CDRs), is mainly involved in antigen recognition, and varies substantially among other antibodies that bind different antigens. See, for example, Janeway et.al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999).

[0048] Briefly, in the antibody backbone, the CDRs are embedded within the frameworks of the heavy and light chain variable regions that mainly constitute the regions involved in antigen binding and recognition. The variable region includes at least three heavy chain CDRs or three light chain CDRs (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md., and Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917, Chothia et al., 1989, Nature 342:877-883) within the framework regions (designated framework regions 1-4, FR1, FR2, FR3, and FR4 as designated by Kabat et al., 1991, and Chothia and Lesk, 1987, supra).

[0049] Human light chains are classified into kappa and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. Thus, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In an exemplary aspect, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody.

[0050] In various embodiments, the antibody can be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody comprises a sequence that is substantially similar to a natural antibody produced by a mammal, such as a mouse, rat, rabbit, goat, horse, chicken, hamster, pig, human, etc. In this regard, the antibody can be considered a mammalian antibody, such as a mouse antibody, rat antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, pig antibody, human antibody, etc. In certain embodiments, the anti-RANKL antibody is a monoclonal human antibody. In certain embodiments, the recombinant protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that comprises a constant domain from one species and a variable domain from a second species, or more generally, an antibody that comprises a contiguous sequence of amino acids from at least two species. The term "humanized," when used in reference to an antibody, refers to an antibody having at least the CDR regions derived from a non-human source that has been engineered to have a structure and immune function more similar to that of a true human antibody than the original source antibody. For example, humanization can include transplanting the CDRs from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization can also include amino acid substitutions selected to make the non-human sequences appear more like human sequences.

[0051] In various embodiments, the antibody is cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab’)2 fragment and a pFc’ fragment. In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an antibody fragment that retains at least one antigen (RANKL) binding site, such as Fab, Fc, F(ab’)2, or pFc’. With regard to the aqueous pharmaceutical formulations and methods of the present disclosure, the antibody may lack a particular portion of the antibody and may be an antibody fragment that binds RANKL. In an exemplary embodiment, the antibody fragment is the antigen-binding portion of an anti-RANKL antibody.

[0052] Antibody protein products can be antigen-binding formats based on antibody fragments that retain full antigen-binding ability, such as scFv, Fab, and VHH / VH. The smallest antigen-binding fragment that retains the complete antigen-binding site is the Fv fragment consisting entirely of the variable (V) region. A soluble and flexible amino acid peptide linker is used to join the V regions into an scFv (single-chain fragment variable) fragment to stabilize the molecule or to add a constant (C) domain to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab are widely used fragments that can be easily produced in a host, such as a prokaryotic host. Other antibody protein products include disulfide bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), and different formats of dimeric and multimeric antibody formats such as diabodies, triabodies, and tetrabodies, or miniAbs consisting of scFv linked to an oligomerization domain. The smallest fragments are the VHH / VH of camelid heavy-chain Abs and single-domain Abs (sdAbs). The most frequently used building block for creating novel antibody formats is the single-chain variable (V) domain antibody fragment (scFv), which contains V domains (VH and VL domains) derived from the heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another type of antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto the Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0053] Other antibody protein products include single-chain antibodies (SCA), diabodies, triabodies, tetra-bodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be classified into five main classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0054] In an exemplary embodiment, the anti-RANKL antibody or antigen-binding portion thereof comprises, consists essentially of, or consists of any one of these antibody protein products (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy-chain antibody, sdAb, diabody, triabody, tetrabody, bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate).

[0055] In an exemplary embodiment, the anti-RANKL antibody or antigen-binding portion thereof comprises, consists essentially of, or consists of an antibody protein product in monomeric form or polymeric form, oligomeric form or multimeric form. In certain embodiments where the antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent or multivalent, depending on the number of distinct epitopes recognized and bound by the antibody.

[0056] The human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof used in the formulation specifically binds to the human osteoprotegerin (OPGL) protein of the human RANKL protein or a fragment thereof, and inhibits or neutralizes the activity of the RANKL or OPGL protein, and / or inhibits the RANK / RANKL signaling pathway, and is an antibody or antigen-binding portion thereof, which is referred to herein as a human anti-RANKL monoclonal antibody or antigen-binding portion thereof. For example, the formulation described herein can include a human anti-RANKL monoclonal antibody that specifically binds to human RANKL (SEQ ID NO: 12) or a partial amino acid sequence thereof. The human RANKL protein is a transmembrane protein or a soluble protein encoded by the polynucleotide sequence of SEQ ID NO: 11, which is known to be essential for osteoclast formation, function, and survival. For example, the human anti-RANKL antibody inhibits the interaction between RANKL and its receptor RANK.

[0057] An example of a human anti-RANKL monoclonal antibody is denosumab, which is commercially available as Xgeva® and Prolia®. Xgeva® is a 120 mg dosage formulation of denosumab in a 1.7 mL solution (70 mg / mL) in a single-use vial containing 120 mg of denosumab, acetate (18 mM), sorbitol (4.6%), water for injection (USP), and sodium hydroxide for pH adjustment to 5.2. Prolia® is available as a 60 mg dosage formulation of denosumab in a 1 mL solution (60 mg / mL). The 1 mL disposable syringe of Prolia® contains 60 mg of denosumab (60 mg / mL solution), 4.7% sorbitol, 17 mM acetate, 0.01% polysorbate 20, water for injection (USP), and sodium hydroxide for pH adjustment to 5.2. Formulations containing denosumab or a portion thereof as described herein are particularly contemplated. Denosumab is a fully human IgG2 monoclonal antibody that binds to human RANKL. Denosumab has a molecular weight of approximately 147 kDa and is expressed in a Chinese hamster ovary (CHO) cell line. The amino acid sequences of the denosumab variable light chain (LC) and variable heavy chain (HC) are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the full-length LC and HC are shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 (denosumab variable LC) is, in some embodiments, the nucleic acid of SEQ ID NO: 19. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 (denosumab variant HC) is, in some embodiments, the nucleic acid of SEQ ID NO: 20. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 (full-length denosumab LC) is, in some embodiments, the nucleic acid of SEQ ID NO: 21. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 (full-length denosumab HC) is, in some embodiments, the nucleic acid of SEQ ID NO: 23. The mature form of the LC represented as amino acids 21-235 of the full-length LC is shown as SEQ ID NO: 13, and the mature form of the HC represented as amino acids 20-467 of the full-length HC is shown as SEQ ID NO: 14.Nucleic acids comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13 (mature form of LC) are, in some embodiments, the nucleic acid of SEQ ID NO: 22. Nucleic acids comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (mature form of HC) are, in some embodiments, the nucleic acid of SEQ ID NO: 24. Further, the denosumab LC CDRs are shown as SEQ ID NO: 5 (LC CDR1), SEQ ID NO: 6 (LC CDR2), and SEQ ID NO: 7 (LC CDR3). The denosumab HC CDRs are shown as SEQ ID NO: 8 (HC CDR1), SEQ ID NO: 9 (HC CDR2), and SEQ ID NO: 10 (HC CDR3). Denosumab is described and claimed in International Patent Application No. WO03 / 002713 and U.S. Patent No. 7,364,736, the disclosures of which are hereby incorporated by reference in their entirety.

[0058] As used herein, the term "denosumab" includes biosimilars of denosumab. As used herein, a "biosimilar" (of an approved reference product / biological agent, e.g., a protein therapeutic, an antibody, etc.) is a biological product that is similar to a reference product based on data derived from (a) analytical studies demonstrating that the biological product is highly similar to the reference product despite minor differences in clinically inactive components, (b) animal studies (including assessment of toxicity), and / or (c) one or more appropriate clinical studies (including assessment of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and potency in one or more of the conditions of use for which the reference product is approved, intended to be used, and for which a license is sought for the biological product. In one embodiment, the biosimilar biological product and the reference product utilize the same one or more mechanisms of action in one or more of the conditions of use defined, recommended, or suggested in the proposed labeling to the extent that one or more mechanisms of action are known for the reference product. In one embodiment, one or more of the conditions of use defined, recommended, or suggested in the proposed labeling for the biological product have been previously approved for the reference product. In one embodiment, the route of administration, dosage form, and / or strength of the biological product are the same as those of the reference product. In one embodiment, the facility in which the biological product is manufactured, processed, packaged, or held meets the criteria designed to ensure that the biological product continues to be safe, pure, and potent. The reference product may be approved in at least one of the United States, Europe, or Japan. A biosimilar can be, for example, an antibody having the same primary amino acid sequence as a commercially available antibody, but can be made in a different cell type or by a different method of manufacture, purification, or formulation.

[0059] The formulation can comprise a human anti-RANKL antibody comprising at least one of the amino acid sequences of SEQ ID NOs: 1 to 4, 13, 14 or a part thereof. The formulation can comprise at least one of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or can comprise at least two of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or can comprise at least three of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or can comprise at least four of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or can comprise at least five of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or can comprise at least six of the CDR amino acid sequences shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, and can comprise a human anti-RANKL antibody.

[0060] The preparation is a human anti-RANKL antibody that is at least 80% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 85% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 90% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 91% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 92% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 93% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 94% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 95% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 96% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK; or a human anti-RANKL antibody that is at least 97% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKA human anti-RANKL antibody comprising at least one amino acid sequence that is at least 98% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 99% identical to any one of SEQ ID NOs: 1 to 4, 13, and 14 and inhibits the interaction between RANKL and its receptor, RANK, can be included.

[0061] In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an anti-RANKL antibody or an antigen-binding portion thereof (such as an antibody protein product) as described herein. In an exemplary aspect, the anti-RANKL antibody or an antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 5. In an alternative or additional example, the anti-RANKL antibody or an antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 6. In an alternative or additional aspect, the anti-RANKL antibody or an antigen-binding portion thereof comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 10. In some examples, the anti-RANKL antibody or an antigen-binding portion thereof comprises SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 10. In an exemplary aspect, the anti-RANKL antibody or an antigen-binding portion thereof comprises (i) a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 7, (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO: 8, optionally SEQ ID NO: 27, (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO: 9, or (iv) any combination thereof. In some aspects, the anti-RANKL antibody or an antigen-binding portion thereof comprises (A) a light chain variable domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain variable domain comprising a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and (B) a heavy chain variable domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8 (optionally SEQ ID NO: 27), a heavy chain variable domain comprising a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain variable domain comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.In an exemplary embodiment, the anti-RANKL antibody or an antigen-binding portion thereof comprises a light chain variable domain selected from the group consisting of (A) (i) an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 1, (ii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO: 19, and (iii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of the polynucleotide consisting of SEQ ID NO: 19, or (B) (i) an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 2, (ii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO: 20, and (iii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of the polynucleotide consisting of SEQ ID NO: 20, or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B). In an exemplary embodiment, the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In an exemplary example, the antigen-binding portion is a Fab, Fab’, F(ab’)2, or single-chain Fv. In an exemplary embodiment, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody, and optionally, the anti-RANKL antibody comprises the sequence of SEQ ID NO: 15. In some embodiments, the anti-RANKL antibody comprises the sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.In an exemplary embodiment, the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain selected from the group consisting of (A) (i) a light chain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 3 or SEQ ID NO: 13, (ii) a light chain comprising an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 21 or 23, and (iii) a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of a polynucleotide consisting of SEQ ID NO: 21 or 23, or (B) (i) a heavy chain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 4 or SEQ ID NO: 14, (ii) a heavy chain comprising an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 22 or 24, and (iii) a heavy chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of a polynucleotide consisting of SEQ ID NO: 22 or 24, or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B).

[0062] The concentration of denosumab or other human anti-RANKL antibody or antigen-binding portion thereof in an aqueous formulation can generally be in any useful range, e.g., from about 0.1 to about 200 mg / mL. As the concentration increases, there is an increase in viscosity, which can impede processing into a sterile dosage form for pharmaceutical use of the formulation.

[0063] In one aspect, the improved stability of the formulation by the amino acid aggregation inhibitor is such that denosumab or other human anti-RANKL antibodies or antigen-binding portions thereof are present at any concentration from about 10 mg / mL to about 200 mg / mL, or from about 15 mg / mL to about 150 mg / mL, or from about 30 mg / mL to about 200 mg / mL, or from about 60 mg / mL to about 200 mg / mL, or from about 60 mg / mL to about 180 mg / mL, or from about 60 mg / mL to about 160 mg / mL, or from about 60 mg / mL to about 150 mg / mL, or from about 60 mg / mL to about 140 mg / mL, or from about 60 mg / mL to about 130 mg / mL, or from about 60 mg / mL to about 120 mg / mL, or from about 60 mg / mL to about 110 mg / mL, or from about 60 mg / mL to about 100 mg / mL, or from about 60 mg / mL to about 90 mg / mL, or from about 60 mg / mL to about 80 mg / mL, or from about 60 mg / mL to about 70 mg / mL, or from about 70 mg / mL to about 200 mg / mL, or from about 70 mg / mL to about 180 mg / mL, or from about 70 mg / mL to about 160 mg / mL, or from about 70 mg / mL to about 150 mg / mL, or from about 70 mg / mL to about 140 mg / mL, or from about 70 mg / mL to about 130 mg / mL, or from about 70 mg / mL to about 120 mg / mL, or from about 70 mg / mL to about 110 mg / mL, or from about 70 mg / mL to about 100 mg / mL, or from about 70 mg / mL to about 90 mg / mL, or from about 70 mg / mL to about 80 mg / mL, such as 120 mg / mL, etc.

[0064] In another aspect, the concentration of denosumab or other human anti-RANKL antibody or antigen-binding portion thereof in a formulation having a pH of from about 5.0 to less than 5.2 is contemplated to be greater than 70 mg / mL, or at least 71 mg / mL, or at least about 75 mg / mL, or at least about 80 mg / mL, or at least about 85 mg / mL, or at least about 90 mg / mL, or at least about 95 mg / mL, or at least about 100 mg / mL, or at least about 105 mg / mL, or at least about 110 mg / mL, or at least about 115 mg / mL, or at least about 120 mg / mL, and up to about 200 mg / mL. For example, contemplated ranges include 71 mg / mL to about 200 mg / mL, or about 75 mg / mL to about 200 mg / mL, or about 75 mg / mL to about 180 mg / mL, or about 75 mg / mL to about 160 mg / mL, or about 75 mg / mL to about 150 mg / mL, or about 75 mg / mL to about 140 mg / mL, or about 75 mg / mL to about 130 mg / mL, or about 75 mg / mL to about 120 mg / mL, or about 75 mg / mL to about 110 mg / mL, or about 75 mg / mL to about 100 mg / mL, or about 75 mg / mL to about 90 mg / mL, or about 120 mg / mL to about 200 mg / mL, or about 120 mg / mL to about 180 mg / mL, or about 120 mg / mL to about 160 mg / mL, or about 120 mg / mL to about 140 mg / mL, such as about 120 mg / mL.

[0065] In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an antibody or an antigen-binding portion thereof at a concentration greater than 70 mg / mL, such as greater than 80 mg / mL, greater than 90 mg / mL, greater than 100 mg / mL, greater than 125 mg / mL, greater than 150 mg / mL, greater than 175 mg / mL, greater than 200 mg / mL, greater than 225 mg / mL, greater than 250 mg / mL, greater than 275 mg / mL. In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an antibody or an antigen-binding portion thereof at a concentration of less than about 300 mg / mL, such as less than about 275 mg / mL, less than about 250 mg / mL, less than about 225 mg / mL, less than about 200 mg / mL, less than about 175 mg / mL, or less than about 150 mg / mL. In an exemplary embodiment, the concentration of the antibody or an antigen-binding portion thereof in the formulation ranges from about 10 mg / mL to about 300 mg / mL, such as from about 25 mg / mL to about 300 mg / mL, from about 50 mg / mL to about 300 mg / mL, from about 75 mg / mL to about 300 mg / mL, from about 125 mg / mL to about 300 mg / mL, from about 150 mg / mL to about 300 mg / mL, from about 175 mg / mL to about 300 mg / mL, from about 200 mg / mL to about 300 mg / mL, from about 225 mg / mL to about 300 mg / mL, from about 250 mg / mL to about 300 mg / mL, from about 275 mg / mL to about 300 mg / mL, from about 10 mg / mL to about 275 mg / mL, from about 10 mg / mL to about 250 mg / mL, from about 10 mg / mL to about 225 mg / mL, from about 10 mg / mL to about 200 mg / mL, from about 10 mg / mL to about 175 mg / mL, from about 10 mg / mL to about 150 mg / mL, from about 10 mg / mL to about 125 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 75 mg / mL, from about 10 mg / mL to about 50 mg / mL, or from about 10 mg / mL to about 25 mg / mL.In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an antibody or an antigen-binding portion thereof at a concentration in the range of greater than 70 mg / mL to about 300 mg / mL, such as in the range of greater than 80 mg / mL to about 300 mg / mL, greater than 90 mg / mL to about 300 mg / mL, greater than 100 mg / mL to about 300 mg / mL, greater than 125 mg / mL to about 300 mg / mL, greater than 150 mg / mL to about 300 mg / mL, greater than 175 mg / mL to about 300 mg / mL, greater than 200 mg / mL to about 300 mg / mL, greater than 70 mg / mL to about 275 mg / mL, greater than about 70 mg / mL to about 250 mg / mL, greater than about 70 mg / mL to about 225 mg / mL, greater than about 70 mg / mL to about 200 mg / mL, greater than about 70 mg / mL to about 175 mg / mL, greater than about 70 mg / mL to about 150 mg / mL, greater than about 70 mg / mL to about 125 mg / mL, greater than about 70 mg / mL to about 100 mg / mL. In an exemplary embodiment, the aqueous pharmaceutical formulation comprises an antibody or an antigen-binding portion thereof at a concentration in the range of about 100 mg / mL to about 140 mg / mL, such as at a concentration of about 110 mg / mL, about 120 mg / mL, about 130 mg / mL. In some embodiments, the aqueous pharmaceutical formulation comprises an antibody or an antigen-binding portion thereof at a concentration of about 120 mg / mL ± 12 mg / mL, such as in the range of about 108 mg / mL to about 132 mg / mL, about 115 mg / mL to about 125 mg / mL, at a concentration of about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL.

[0066] Denosumab and other human anti-RANKL monoclonal antibodies and antigen-binding portions thereof can be prepared according to the description provided in International Patent Publication WO2003002713A2.

[0067] The formulation studies on the high-concentration denosumab solution (e.g., 120 mg / mL) described below showed a significant increase in the formation (rate and extent) of HMWS at pH less than 5, particularly at a lower pH (e.g., pH 4.5). As the pH increased, an increase in the formation of dimer species was shown. To balance the two effects, the formulations described herein are contemplated to have a pH in the range of about 5.0 to less than 5.2, or about 5.0 to about 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, e.g., about 5.0, about 5.05, about 5.1, or about 5.15.

[0068] The studies described herein also showed an independently stabilized and reduced aggregation effect enabled by including an amino acid aggregation inhibitor. Thus, when an amino acid aggregation inhibitor is included, the pH of the formulation can be in the range of about 4.9 to about 5.4, or about 5.0 to about 5.4, or about 5.0 to about 5.2, or about 5.0 to less than 5.2, or about 5.0 to about 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, e.g., about 5.0, about 5.05, about 5.1 or about 5.15, or about 5.2.

[0069] The aqueous formulation can be buffered. When used, the buffer can be an organic buffer. The buffer system can be centered around pH 4 - 5.5, or 4.5 - 5.5, or around 4.5 - 5 at 25°C. For example, the buffer system can have a pKa within 1 pH unit of pH 5.0 - 5.2 at 25°C. One such buffer system is acetic acid / acetic acid salt, which has a pKa of about 4.75 at 25°C. Another such buffer system is glutamic acid / glutamic acid salt, which has a pKa of about 4.27 at 25°C. Other alternative buffer systems contemplated include ion-based systems such as succinate (pKa 4.21 at 25°C), propionate (pKa 4.87 at 25°C), malate (pKa 5.13 at 25°C), pyridine (pKa 5.23 at 25°C), and piperazine (pKa 5.33 at 25°C). It is contemplated that the buffer can be provided as a sodium salt (or disodium salt if necessary), or alternatively as a potassium, magnesium, or ammonium salt. The buffer can be based on, for example, acetate, citrate, succinate, phosphate, and hydroxymethylaminomethane (Tris). Buffer solutions based on acetate, glutamate, and succinate are particularly contemplated, for example acetate or glutamate.

[0070] Comparison of HMWS formation by size exclusion ultra-high performance liquid chromatography (SE-UHPLC) in a 120 mg / mL denosumab formulation having an acetate or glutamate buffer (otherwise the same) showed no difference due to buffer type when evaluated over 4 weeks of storage at 37°C.

[0071] When used, the buffer is included in an amount sufficient to maintain the selected pH of the formulation under storage conditions for the product's shelf life, e.g., 3 years at 4°C, or 1 month at 25°C, or 2 weeks at 25°C, or 7 days at 25°C. The buffer concentration can range from about 2 mM to about 40 mM, or from about 5 mM to about 20 mM, or from about 10 mM to about 25 mM, or from about 15 mM to about 25 mM, e.g., 10 mM, or 15 mM, or 18 mM, or 25 mM. For example, the acetate buffer used with an anti-RANKL monoclonal antibody (e.g., denosumab) and phenylalanine can range from about 2 mM to about 30 mM, or from about 16 mM to about 41 mM, or from about 25 mM to about 39 mM, or from about 30 mM to about 34 mM. In other words, the diafiltration buffer used to concentrate the antibody to a concentration above 70 mg / mL (e.g., 120 mg / mL) can range from 5 mM to about 30 mM, or from about 15 mM to about 25 mM, or about 20 mM. Also contemplated is providing a self-buffering amino acid-stabilized formulation. In an exemplary embodiment, the buffer is included in an amount sufficient to maintain the selected pH of the formulation under storage conditions for the product's shelf life, e.g., 36 months at about 2°C to about 8°C, optionally followed by about 1 month at about 20°C to about 30°C.

[0072] Aqueous pharmaceutical formulations in some embodiments include a buffer, and optionally, the buffer is centered around a range from about pH 4.0 to about pH 5.5 at 25°C. In some embodiments, the buffer has a pKa within 1 pH unit of pH 5.0 - 5.2 at 25°C. Aqueous pharmaceutical formulations in certain embodiments include a buffer from about 5 mM to about 60 mM, from about 5 mM to about 50 mM, or from about 9 mM to about 45 mM (e.g., from about 15 mM to about 30 mM, e.g., about 20 mM, about 25 mM buffer). In an exemplary embodiment, the buffer is acetate or glutamate.

[0073] The formulation may also contain one or more stabilizers against protein aggregation and other formulation additives. Such stabilizers and additives include, but are not intended to be limited to, amino acid aggregation inhibitors, tonicity modifiers, surfactants, solubilizers (e.g., N-methyl-2-pyrrolidone), PEG conjugation, and cyclodextrins (e.g., Captisol®).

[0074] The term "amino acid aggregation inhibitor" refers to an amino acid or combination of amino acids (e.g., a mixture or a dipeptide or an oligopeptide having 2 to 10 residues) or an amino acid analog in which any given amino acid is present in its free base form or in the form of its salt (e.g., arginine HCl), that reduces HMWS or inhibits the formation of HMWS. Salts contemplated include sodium salts, potassium salts, and hydrochloride salts. Further contemplated are arginine salts, glutamate salts, butyrate salts, and glycolate salts having hydrochloric acid. When a combination of amino acids is used, all of the amino acids may be present in their free base form, all may be present in the form of their salts, or some may be present in their free base form and others in the form of their salts. In addition to or instead of dipeptides and oligopeptides, a mixture of one or more amino acids, such as a mixture of arginine and phenylalanine, can be used. In another embodiment, only one type of amino acid aggregation inhibitor is present in the aqueous pharmaceutical formulation. In an exemplary aspect, only one amino acid is present, such as only L-arginine or only L-phenylalanine is present in the formulation.

[0075] It is contemplated to use one or more amino acids having a side chain with a charge, such as one or more of arginine, lysine, histidine, aspartic acid, and glutamic acid. The amino acid can be selected from basic amino acids, such as arginine, lysine, histidine, or combinations thereof. Arginine is particularly contemplated. Any stereoisomer of a particular amino acid (i.e., L, D, or DL isomer), or a combination of these stereoisomers, can be used in the methods or formulations of the present invention as long as the particular amino acid is present in its free base form or its salt form. In particular, the L-stereoisomer, such as L-arginine, is contemplated. Optionally, the amino acid has a side chain with a positive charge, such as arginine.

[0076] In another aspect, it is contemplated to use one or more amino acids having an aromatic ring in the side chain, such as phenylalanine, tyrosine, tryptophan, or combinations thereof. Phenylalanine is particularly contemplated.

[0077] In another aspect, it is contemplated to use one or more hydrophobic amino acids, such as alanine, isoleucine, leucine, phenylalanine, valine, proline, or glycine.

[0078] In another aspect, it is contemplated to use one or more aliphatic hydrophobic amino acids, such as alanine, isoleucine, leucine, or valine. Leucine is particularly contemplated.

[0079] Amino acid analogs that exhibit the effect of reducing or inhibiting aggregation can also be used in the methods or formulations of the present invention. The term "amino acid analog" refers to derivatives of naturally occurring amino acids. Envisioned analogs include, for example, amino and N-monoethyl and n-acetyl derivatives. Other envisioned analogs include dipeptides, or oligopeptides having 2 to 10 residues, such as arginine-arginine and phenylalanine-arginine. In one type of embodiment, it is contemplated that n-acetylarginine and n-acetyllysine are not used alone, but can be used in combination with another amino acid aggregation inhibitor. Similar to amino acids, amino acid analogs are used in the methods or formulations of the present invention either in the form of their free base or in the form of their salts.

[0080] The amino acid aggregation inhibitor(s) used in the method or formulation of the present invention protects the therapeutic active protein from various stresses, thereby increasing and / or maintaining the stability of the protein or the formulation containing the protein during the lifetime of the protein (before and during storage, before use). Here, "stress" includes, but is not limited to, heat, freezing, pH, light, agitation, oxidation, dehydration, surface, shear, freeze / thaw, pressure, heavy metals, phenolic compounds, denaturants, etc. from any source such as transportation. Heat stress is particularly contemplated. The term "stress" encompasses any factor that modulates (i.e., decreases, maintains, or increases) the stability of the protein or the formulation containing the protein. The increase and / or maintenance of stability by the addition of the amino acid aggregation inhibitor occurs in a concentration-dependent manner. That is, an increase in the concentration of the amino acid aggregation inhibitor leads the protein of the present invention or the formulation containing the protein to have increased and / or maintained stability of the protein or the formulation when the protein or the formulation containing the protein would normally show aggregate formation in the absence of the amino acid aggregation inhibitor. As shown in the following examples, the amount of pre-formed HMWS can also be reduced by including an amino acid aggregation inhibitor in the formulation. For example, such amino acid aggregation inhibitors include arginine and arginine-phenylalanine dipeptide. By determining the amount of a particular amino acid aggregation inhibitor used in the method or formulation to reduce the formation of aggregates, the stability of the protein can be increased. Thus, the improved stability of the formulation throughout the lifetime of the protein can be readily determined for denosumab, or any particular human anti-RANKL monoclonal antibody of interest, in view of the disclosure herein.

[0081] The presence of an amino acid aggregation inhibitor in a formulation has been shown to reduce the amount and the rate of formation of the dimer species. For example, when denosumab formulation at pH 5.2 contains arginine at a concentration of 75 mM, after 1 month at 37 °C, the amount and the rate of formation of the dimer species are reduced by about 0.3% and 25%, respectively, compared to a similar formulation without arginine at pH 5.2. In contrast, monoclonal antibodies that are not human anti-RANKL monoclonal antibodies have been found not to be stabilized by the inclusion of arginine and instead increase HMWS. Thus, another method of the present disclosure is a method of reducing HMWS in a formulation of denosumab or another human anti-RANKL monoclonal antibody by the addition of an amino acid aggregation inhibitor, such as arginine or phenylalanine.

[0082] Thus, in an exemplary embodiment, an aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor, which is optionally an amino acid. In an exemplary aspect, the amino acid is contemplated to be a D-stereoisomer amino acid (D-amino acid), but is an L-stereoisomer amino acid (L-amino acid). In some aspects, the amino acid aggregation inhibitor comprises an amino acid having a side chain with a charge, which is also referred to herein as an "amino acid with a charge". The term "amino acid with a charge" refers to an amino acid having a side chain that has a negative charge (i.e., is deprotonated) or a positive charge (i.e., is protonated) in an aqueous solution at physiological pH. For example, amino acids with a negative charge include, for example, aspartic acid and glutamic acid, while amino acids with a positive charge include, for example, arginine, lysine, and histidine. Amino acids with a charge include charged amino acids among the 20 coded amino acids, as well as non-canonical or non-natural or non-coded amino acids. Thus, in an exemplary aspect, the amino acid aggregation inhibitor is an amino acid having a side chain with a positive charge. In an exemplary example, an amino acid having a side chain with a positive charge comprises a side chain structure of formula I or formula II,

[0083] [Chemical formula] Wherein, n is 1 to 7, and each of R1 and R2 is independently H, C1-C 18 alkyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)NH2, NH, NH2(C1-C 18 alkyl)SH, (C0-C4 alkyl)(C3-C6) cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7 and (C1-C4 alkyl)(C3-C9 heteroaryl), where R7 is H or OH, and optionally one of R1 and R2 is a free amino group (-NH3 + ),

[0084]

Chemical formula

[0085] In an exemplary embodiment, the amino acid containing a side chain having a positive charge includes the side chain structure of formula I, and n is in the range of 2 to 4. In an alternative or additional embodiment, R1 is NH or NH2. In an exemplary embodiment, R2 is NH2 or NH3 +It is. In an exemplary example, the amino acid containing a side chain having a positive charge is arginine. In an exemplary embodiment, the amino acid containing a side chain having a positive charge includes the side chain structure of Formula II, and m ranges from 3 to 5. In some embodiments, each of R3 and R4 is H. In certain cases, R5 is present and is optionally H. In certain cases, the amino acid containing a side chain having a positive charge is lysine. The amino acid containing a side chain having a positive charge is present in the formulation as a salt, optionally as a hydrochloride (HCl) salt, in some embodiments. Thus, in an exemplary embodiment, the aqueous pharmaceutical composition contains L-arginine HCl or L-lysine HCl.

[0086] In an exemplary embodiment, the amino acid aggregation inhibitor is an aromatic amino acid. In some examples, the aromatic amino acid includes phenyl or indole. In an exemplary embodiment, the aromatic amino acid includes a C1-C6 alkyl chain (e.g., a C1-C3 alkyl chain) between the alpha carbon and phenyl or indole. In an exemplary example, the aromatic amino acid is L-phenylalanine. In other examples, the aromatic amino acid is L-tryptophan.

[0087] In an exemplary embodiment, the amino acid aggregation inhibitor is a hydrophobic amino acid. Hydrophobicity can be measured or scored according to any one of the hydrophobicity scales known in the art. Generally, the more positive the score, the more hydrophobic the amino acid. In some examples, hydrophobicity is scored using the Kyte and Doolittle hydrophobicity scale (Kyte J, Doolittle RF (May 1982). “A simple method for displaying the hydropathic character of a protein”. J. Mol. Biol. 157(1):105-32.). In some embodiments, the hydrophobic amino acid has a score greater than about 2.5 on the Kyte and Doolittle hydrophobicity scale. The hydrophobic amino acid in a particular embodiment is a branched or straight-chain C2-C 12It includes a side chain containing a heterocyclic ring which is alkyl, or C4-C8 cycloalkyl, or a C4-C8 heterocyclic ring containing a nitrogen heteroatom, optionally being imidazole, pyrrole, or indole. For the purposes of this specification, the term "cycloalkyl" may encompass any carbocyclic ring such as a bicyclic or tricyclic carbon ring.

[0088] In an exemplary embodiment, the hydrophobic amino acid contains C3-C8 alkyl, and optionally the hydrophobic amino acid contains branched C3 alkyl or branched C4 alkyl. The hydrophobic amino acid is, in certain embodiments, L-valine, L-leucine or L-isoleucine.

[0089] The amino acid aggregation inhibitor is used in an effective amount to provide increased stability, and can be used at a concentration in the range of about 10 mM to about 200 mM, such as in the range of about 30 mM to about 120 mM, or about 38 mM to about 150 mM, or about 38 mM to about 113 mM, or about 38 mM to about 75 mM, for example, about 10 mM, about 38 mM, about 75 mM, about 113 mM, or about 150 mM. In an exemplary embodiment, the aqueous pharmaceutical formulation contains about 5 mM to about 300 mM of the amino acid aggregation inhibitor, optionally about 25 mM to about 90 mM of the amino acid aggregation inhibitor. In some embodiments, when the amino acid aggregation inhibitor is an amino acid containing a side chain with a positive charge, optionally L-arginine, the aqueous pharmaceutical formulation contains about 5 mM to about 150 mM (for example, about 10 mM to about 150 mM, about 15 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, about 5 mM to about 140 mM, about 5 mM to about 130 mM, about 5 mM to about 120 mM, about 5 mM to about 110 mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM) of the amino acid aggregation inhibitor. In some embodiments, when the amino acid aggregation inhibitor is an amino acid containing a side chain with a positive charge, optionally L-arginine, the aqueous pharmaceutical formulation contains about 30 mM to about 80 mM (for example, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM) of the amino acid aggregation inhibitor.

[0090] In some embodiments, the aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor at about 5 mM to about 180 mM (e.g., about 10 mM to about 180 mM, about 15 mM to about 180 mM, about 20 mM to about 180 mM, about 25 mM to about 180 mM, about 5 mM to about 170 mM, about 5 mM to about 170 mM, about 5 mM to about 160 mM, about 5 mM to about 150 mM, about 5 mM to about 140 mM, about 5 mM to about 130 mM, about 5 mM to about 120 mM, about 5 mM to about 110 mM) when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine. In an exemplary example, the aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor at about 5 mM to about 100 mM (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM) when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine, and optionally an amino acid aggregation inhibitor at about 20 mM to about 50 mM.

[0091] Optionally, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine or L-leucine, the aqueous aggregation formulation contains from about 5 mM to about 300 mM of the amino acid aggregation inhibitor. Optionally, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine or L-leucine, the aqueous pharmaceutical formulation contains from about 5 mM to about 200 mM (e.g., from about 10 mM to about 200 mM, from about 20 mM to about 200 mM, from about 30 mM to about 200 mM, from about 40 mM to about 200 mM, from about 50 mM to about 200 mM, from about 60 mM to about 200 mM, from about 70 mM to about 200 mM, from about 80 mM to about 200 mM, from about 90 mM to about 200 mM, from about 100 mM to about 200 mM, from about 5 mM to about 290 mM, from about 5 mM to about 280 mM, from about 5 mM to about 270 mM, from about 5 mM to about 260 mM, from about 5 mM to about 250 mM, from about 5 mM to about 240 mM, from about 5 mM to about 230 mM, from about 5 mM to about 220 mM, from about 5 mM to about 210 mM) of the amino acid aggregation inhibitor, and optionally, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine or L-leucine, contains from about 20 mM to about 50 mM of the amino acid aggregation inhibitor. In an exemplary embodiment, the aqueous pharmaceutical composition contains from about 30 mM to about 80 mM of L-arginine hydrochloride, from about 20 mM to about 50 mM of L-phenylalanine, from about 20 mM to about 50 mM of L-tryptophan, from about 30 mM to about 80 mM of L-lysine hydrochloride, from about 20 mM to about 50 mM of L-leucine, from about 20 mM to about 50 mM of L-isoleucine, from about 20 mM to about 50 mM of L-valine, or any combination thereof.

[0092] In an exemplary embodiment, the concentration of the amino acid aggregation inhibitor is a molar ratio to the antibody. In some embodiments, the molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody is from about 10 to about 200 (e.g., from about 25 to about 150, from about 50 to about 100) when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine. Optionally, the molar ratio is from about 20 to about 90. In an exemplary embodiment, the molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody is from about 20 to 300 when the amino acid aggregation inhibitor is an amino acid containing a positively charged side chain, optionally L-arginine. Optionally, the molar ratio is from about 45 to about 180.

[0093] The surfactant is a surfactant that is amphiphilic (having a polar head and a hydrophobic tail). The surfactant preferentially accumulates at the interface and as a result reduces the interfacial tension. The surfactant can optionally be included in the formulation. The use of the surfactant can also help to reduce the formation of large proteinaceous particles.

[0094] In one type of embodiment, the surfactant may be a non-ionic surfactant. Examples include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), alkylaryl polyethers, such as oxyethylated alkylphenols (e.g., Triton™ X-100) and poloxamers (e.g., Pluronics®, e.g., Pluronic® F68), and any combination of the foregoing, within a class of surfactants or among multiple classes of surfactants. Polysorbate 20 and polysorbate 80 are particularly contemplated.

[0095] A surfactant concentration in the range of about 0.004% (w / v) to about 0.1% (w / v) (e.g., in the case of polysorbate 20 or polysorbate 80) is suitable, for example, about 0.004% to about 0.05%, or about 0.004% to about 0.02%, or about 0.01%. In an exemplary embodiment, the formulation contains at least about 0.004% (w / v) of the surfactant, optionally less than about 0.15 (w / v)%. In an exemplary embodiment, about 0.005 (w / v)% to about 0.015 (w / v)% of the surfactant is present in the formulation, optionally about 0.005 (w / v)%, about 0.006 (w / v)%, about 0.007 (w / v)%, about 0.008 (w / v)%, about 0.009 (w / v)%, about 0.010 (w / v)%, about 0.011 (w / v)%, about 0.012 (w / v)%, about 0.013 (w / v)%, or about 0.014 (w / v)% is present.

[0096] The stabilized aqueous formulation can be suitable for administration by any acceptable route, including parenteral, particularly subcutaneous. For example, subcutaneous administration can be performed on the upper arm, upper thigh or abdomen. Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intra-articular and intrasplenic. The subcutaneous route is preferred.

[0097] If the solution is in a form intended for administration to a subject, it can be formulated to be isotonic with the intended site of administration. For example, the osmolality can range from about 270 to about 350 mOsm / kg, or from about 285 to about 345 mOsm / kg, or from about 300 to about 315 mOsm / kg. For example, if the solution is in a form for parenteral administration, it can be isotonic with blood (osmolality of about 300 mOsm / kg). In an exemplary embodiment, the aqueous pharmaceutical formulation has an osmolality in the range of about 200 mOsm / kg to about 500 mOsm / kg, or from about 225 mOsm / kg to about 400 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg.

[0098] In an exemplary embodiment, the aqueous pharmaceutical formulation has a conductivity in the range of about 500 μS / cm to about 5500 μS / cm, and optionally, if the formulation contains an amino acid with a positively charged side chain, the conductivity is in the range of about 2500 μS / cm to about 5500 μS / cm, or if the formulation contains an aromatic amino acid or lacks an amino acid aggregation inhibitor, the conductivity is in the range of about 500 μS / cm to about 2000 μS / cm. An aqueous pharmaceutical formulation according to any one of the preceding claims, having a viscosity of about 6 cP or less at 5°C, and optionally a viscosity of about 4.5 cP to about 5.5 cP. The aqueous pharmaceutical formulation in a particular embodiment has a viscosity of less than about 13 cP at 25°C, optionally from about 2.0 cP to about 10 cP, optionally from about 2.5 cP to about 4 cP.

[0099] Tonicity regulators, i.e., tonicity adjusting agents, are known in the art and include compounds such as salts (e.g., sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, calcium carbonate, sodium lactate), sugars (e.g., dextran, dextrose, lactose, trehalose), and sugar alcohols (e.g., mannitol, sorbitol, xylitol, glycerol, propylene glycol). In certain embodiments, the tonicity regulator is selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. In an exemplary example, the tonicity regulator is sorbitol. Sorbitol can be used, for example, in the range of 0.1% (w / v) to 5% (w / v), or 1.2% (w / v) to 5% (w / v), for example 3.6% (w / v), 4.6% (w / v) or 4.7% (w / v). Optionally, the formulation contains from about 1.0% (w / w) to about 5.0% (w / w) tonicity regulator. For example, the formulation contains from about 2.0% (w / w) to about 5.0% (w / w) sorbitol, or from about 3.5% (w / w) to about 5.0% (w / w) sorbitol, or from about 4.0% (w / w) to about 5.0% (w / w) sorbitol. In some embodiments, the formulation does not contain any sorbitol, i.e., contains no sorbitol. In an exemplary embodiment, the formulation contains no tonicity regulator.

[0100] Unless it has an adverse effect on stability, other additives known in the art can be used in the formulation. Sugars and polyols can be used to protect proteins from aggregation, including providing freeze / thaw stability. Such compounds include sorbitol, mannitol, glycerol, erythritol, caprylate, tryptophanate, sarcoside, and glycine. Stabilizers for preparing lyophilized formulations include stabilizing sugars, such as disaccharides like trehalose and sucrose. Lyophilized formulations can also include bulking agents as known in the art. Other additives known in the art for protein stabilization include solubilizing agents (e.g., N-methyl-2-pyrrolidone), polyethylene glycol (PEG), and cyclodextrin (e.g., Captisol®). Pharmaceutically acceptable acids and bases, such as sodium hydroxide, can be used to adjust the pH of the solution.

[0101] For parenteral administration, the formulation can be in the form of a pyrogen-free, parenterally acceptable sterile aqueous solution containing denosumab or another human anti-RANKL monoclonal antibody, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the parenteral injection vehicle is sterile distilled water in which denosumab or another human anti-RANKL monoclonal antibody is formulated as a sterile isotonic solution, with or without at least one additional therapeutic agent. The formulation includes pharmaceutically acceptable additives, such as USP (United States Pharmacopeia) grade additives.

[0102] "Preservative" is a compound that can be included in a pharmaceutical formulation to reduce the action of bacteria therein, thereby facilitating, for example, the manufacture of multi-purpose formulations. Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include phenol, butyl and benzyl alcohol, alkyl parabens such as methyl and propyl paraben, aromatic alcohols such as catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Alternatively, the formulation may not contain a preservative. For example, a formulation provided in a single-use dosage form may not contain a preservative.

[0103] The formulation is described herein in its aqueous form, but the stabilized formulation can subsequently be lyophilized to prepare a lyophilizate. Thus, unless the context specifically indicates otherwise, references to the formulation and its method of use are intended to include the lyophilizate resulting from the stabilized aqueous solution.

[0104] Pharmaceutical formulations used for in vivo administration are typically sterile. In certain embodiments, this can be achieved by filtration through a sterile filtration membrane. In certain embodiments, the parenteral composition is generally placed in a container having a sterile access port, such as an intravenous solution bag, or a vial having a stopper pierceable with a hypodermic needle, or a prefilled syringe. In certain embodiments, the formulation can be stored either in a ready-to-use form or in a form that is reconstituted or diluted prior to administration (e.g., a lyophilized form).

[0105] In certain embodiments, the present invention relates to a kit for preparing a single-dose administration unit. In certain embodiments, the kit can include both a first container having a dry formulation of denosumab or another human anti-RANKL monoclonal antibody made from the solution formulations described herein, respectively, and a second container having sterile water or an aqueous solution. Certain embodiments of the present invention include kits that include prefilled syringes (e.g., liquid syringes and riosyringes) with single and multiple chambers.

[0106] The stabilized formulations described herein can be used with one or more additional therapeutic agents, such as calcium and vitamin D compounds. The stabilized formulations described herein can be administered to patients receiving treatment with additional therapeutic agents, or the stabilized formulations described herein can be co-administered with additional therapeutic agents.

[0107] The stabilized formulations can be used to prevent or treat any disease responsive to denosumab or another human anti-RANKL monoclonal antibody or antigen-binding portion thereof, in any of the aspects and embodiments described herein. Such uses and related methods include, but are not limited to, the aspects and embodiments described below.

[0108] In one aspect, the formulation can be used to prevent skeletal-related events (SREs) in patients in need of prevention of skeletal-related events (SREs), the use including administering the stabilized formulation described herein in an effective amount. SREs can be selected from the group consisting of, for example, pathologic fractures, radiation therapy to bone, surgery to bone, and spinal cord compression. The patient can be a patient having bone metastases from a solid tumor. The solid tumor can be, for example, one or more of breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma. The amount of the formulation can be effective to optionally reduce the bone metabolism marker urinary N-terminal telopeptide (uNTx / Cr) corrected for creatinine by at least 80%. The patient can be a patient with multiple myeloma.

[0109] In another aspect, the formulation can be used to treat a patient with giant cell tumor of bone, the use of which comprises administering an effective amount of the stabilized formulation described herein. In one type of embodiment, the patient has a giant cell tumor of bone that is recurrent, inoperable, or for which surgical resection is likely to result in a high morbidity. The patient may be, for example, an adult or a skeletally mature adolescent.

[0110] In another aspect, the formulation can be used to treat a patient with hypercalcemia associated with a bone malignancy, the use of which comprises administering an effective amount of the stabilized formulation described herein. In one aspect, the malignancy may be refractory to bisphosphonate treatment. This method or use can comprise administering an amount of the formulation effective to reduce or maintain the patient's serum calcium at a level of about 11.5 mg / dL or less.

[0111] In another aspect, the formulation can be used to treat osteoporosis in a patient in need thereof, the use of which comprises administering an effective amount of the stabilized formulation described herein. For example, the patient may be a postmenopausal woman at high risk of fracture. In another type of embodiment, the patient may be a male at high risk of fracture.

[0112] In another aspect, the formulation is used to increase bone mass in patients in need thereof, the use comprising administering an effective amount of the stabilized formulation described herein. For example, the amount of the formulation administered can be an amount effective to reduce the incidence of new vertebral and / or non-vertebral fractures. In another type of embodiment, the amount of the formulation administered can be an amount effective to reduce bone resorption. In another type of embodiment, the amount of the formulation can be an amount effective to increase the patient's bone density in at least one region selected from the lumbar spine, total hip, and femoral neck. In another type of embodiment, the amount of the formulation can be an amount effective to increase bone mass in the patient's cortical bone and / or trabecular bone. In another type of embodiment, the amount of the formulation can be an amount effective to reduce the bone resorption marker serum type 1 C-telopeptide (CTX). The patient in need thereof may optionally have osteoporosis. In another type of embodiment, the patient in need thereof can be a woman at high risk of fracture who is receiving adjuvant aromatase inhibitor therapy for breast cancer. In another type of embodiment, the patient in need thereof can be a man at high risk of fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer. In another type of embodiment, the patient in need thereof can be a man with osteoporosis at high risk of fracture.

[0113] In another aspect, the formulation can be used as adjuvant therapy for postmenopausal women with early breast cancer at high risk of recurrence of the disease who are receiving adjuvant / neoadjuvant therapy.

[0114] In another aspect, the formulation can be used as first-line treatment for patients with metastatic non-small cell lung cancer in combination with platinum-based chemotherapy.

[0115] In another aspect, the formulation can be used for the treatment of idiopathic subglottic stenosis (ISS).

[0116] In another aspect, the formulation can be used for the prevention of breast and ovarian cancer in healthy women with a BRCA-1 mutation.

[0117] Optionally, the formulation can be used in combination with an immune checkpoint inhibitor. Optionally, the immune checkpoint inhibitor is specific for a protein that functions in an immune checkpoint pathway, such as CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1 or TIM3. Optionally, the immune checkpoint inhibitor is an antibody specific for CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1 or TIM3, an antigen-binding fragment thereof, or an antibody protein product. Such immune checkpoint inhibitors include, but are not limited to, atezolizumab, avelumab, ipilimumab, tremelimumab, BMS-936558, MK3475, CT-011, AM-224, MDX-1105, IMP321, MGA271. Examples of PD-1 inhibitors include pembrolizumab and nivolumab. Examples of PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. Examples of CTLA4 inhibitors include ipilimumab. In another aspect, the formulation can be used in combination with a PD-1 antibody (e.g., nivolumab, pembrolizumab), optionally, to treat melanoma patients with bone metastases. In another aspect, the formulation can be used in combination with a CTLA4 inhibitor, such as ipilimumab, optionally, to treat breast cancer patients.

[0118] In another aspect, the formulation is used, for example, to treat tumors rich in giant cells in hyperparathyroidism or with secondary aneurysmal bone cysts.

[0119] In another aspect, the formulation can be used to treat metastatic castration-resistant prostate cancer (mCRPC). In another aspect, the formulation can be used to treat castration-sensitive prostate cancer. In another aspect, the formulation can be used to treat hormone-resistant prostate cancer.

[0120] In another aspect, the formulation can be used to treat metastatic breast cancer (mBC). In another aspect, the formulation can be used to treat pre - operative breast cancer. In another aspect, the formulation can be used to treat early - stage breast cancer. In other aspects, the formulation can be used to treat hormone receptor - negative, RANK - positive or RANK - negative primary breast cancer. In another aspect, the formulation can be used to treat post - menopausal HER2 - negative breast cancer.

[0121] In another aspect, the formulation can be used to treat myelodysplastic syndromes, for example, in elderly patients.

[0122] In another aspect, the formulation can be used to treat cancer - induced bone loss (CTIBL).

[0123] In another aspect, the formulation can be used to treat uterine tumors of the cervix.

[0124] In another aspect, the formulation can be used to induce an immunomodulatory effect in a patient, regardless of the presence or absence of immunotherapy.

[0125] In another aspect, the formulation can be used to prevent or treat bone loss associated with osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis and rheumatoid arthritis - related bone loss. In another aspect, the formulation can be used to prevent or treat inflammatory conditions associated with bone loss. In another aspect, the formulation can be used to prevent or treat autoimmune conditions associated with bone loss. In another aspect, the present formulation can be used to prevent or treat cancer - related bone loss such as breast cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer and gastrointestinal cancer, multiple myeloma, lymphoma and Hodgkin's disease.

[0126] The formulation can be administered on any suitable timing schedule. In one embodiment, the administration schedule is once every four weeks. Optionally, the administration can include administrations on the 8th and 15th days of the first month of treatment. In another type of embodiment, the administration can be performed on a schedule of once every six months. The once-every-six-months schedule is contemplated for use, for example, for osteoporosis and bone mass increase. Other maintenance dosages that are contemplated are every three weeks, every three months, and every six weeks.

[0127] In some aspects, the aqueous pharmaceutical formulation is used to treat patients having bone metastases from multiple myeloma or solid tumors. In certain aspects, the formulation is administered as a subcutaneous injection in the upper arm, upper thigh, or abdomen at a dosage of about 120 mg every four weeks.

[0128] In some aspects, the aqueous pharmaceutical formulation is used to treat patients with giant cell tumor of bone. In certain aspects, the formulation is administered at a dosage of about 120 mg every four weeks, with an additional 120 mg dosage administered on the 8th and 15th days of the first month of treatment. In some aspects, the formulation is administered subcutaneously in the patient's upper arm, upper thigh, or abdomen. In some cases, calcium and vitamin D are administered to the patient to treat or prevent hypocalcemia.

[0129] In some aspects, the aqueous pharmaceutical formulation is used to treat patients with hypercalcemia of malignancy. In certain aspects, the formulation is administered at a dosage of about 120 mg every four weeks, with an additional 120 mg dosage administered on the 8th and 15th days of the first month of treatment. In some aspects, the formulation is administered subcutaneously in the patient's upper arm, upper thigh, or abdomen.

[0130] In some embodiments, the aqueous pharmaceutical formulation is used to treat postmenopausal women with osteoporosis at high risk of fracture, or men at high risk of fracture who are undergoing androgen deprivation therapy for non-metastatic prostate cancer, or women at high risk of fracture who are undergoing adjuvant aromatase inhibitor therapy for breast cancer, to increase bone mass. In some embodiments, the aqueous pharmaceutical formulation is administered by subcutaneous injection in the upper arm, upper thigh or abdomen, by a healthcare professional, at a dose of 60 mg every 6 months. In some embodiments, patients are also instructed to take 1000 mg of calcium per day and at least 400 IU of vitamin D per day.

[0131] One type of the formulation of the present disclosure contains denosumab, acetate and arginine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation can optionally contain sorbitol. The formulation can optionally contain polysorbate. The polysorbate can optionally be polysorbate 20. The pH can optionally be about 5.0 to about 5.2, or less than 5.2.

[0132] Another type of formulation according to the present disclosure contains denosumab, acetate, and phenylalanine. The formulation can optionally contain sorbitol. The formulation can optionally contain polysorbate. The polysorbate can optionally be polysorbate 20. The pH can optionally be from about 5.0 to about 5.2, or less than 5.2. For example, the formulation can contain denosumab at a concentration of about 108 mg / mL to about 132 mg / mL, acetate at about 28.8 mM to about 35.2 mM, phenylalanine at 33.3 mM to about 40.7 mM, sorbitol at 3.51% (w / v) to about 4.29% (w / v), and polysorbate 20 at about 0.009% (w / v) to about 0.011% (w / v) at pH 5.1, and can optionally be contained in a PFS, which can optionally contain about 1 mL or less (e.g., about 0.5 mL) of the formulation. For example, the formulation can contain denosumab at a concentration of 120 mg / mL, acetate at 32 mM, phenylalanine at 37 mM, sorbitol at 3.9% (w / v), and polysorbate 20 at 0.01% (w / v) at pH 5.1, and can optionally be contained in a PFS, which can optionally contain about 1 mL or less (e.g., about 0.5 mL) of the formulation. This formulation can be prepared by concentrating denosumab using a diafiltration buffer containing 20 mM acetate, 4.2% (w / v) sorbitol, and 40 mM phenylalanine at pH 4.7.

[0133] Another type of formulation of the present disclosure contains denosumab, glutamate, and arginine. The arginine can optionally be L-arginine. The arginine can optionally be L-arginine hydrochloride. The formulation can optionally contain sorbitol. The formulation can optionally contain polysorbate. The polysorbate can optionally be polysorbate 20. The pH can optionally be from about 5.0 to about 5.2, or less than 5.2.

[0134] Another type of formulation of the present disclosure contains denosumab, acetate, arginine, and phenylalanine. The formulation can optionally contain sorbitol. The formulation can optionally contain polysorbate. The polysorbate can optionally be polysorbate 20. The pH can optionally be from about 5.0 to about 5.2, or less than 5.2.

[0135] Another type of formulation of the present disclosure contains denosumab, glutamate, arginine, and phenylalanine. The arginine can optionally be L-arginine. The arginine can optionally be L-arginine hydrochloride. The formulation can optionally contain sorbitol. The formulation can optionally contain polysorbate. The polysorbate can optionally be polysorbate 20. The pH can optionally be from about 5.0 to about 5.2, or less than 5.2.

[0136] The formulations of the present disclosure can be prepared by any suitable method. In one type of method, a solution containing an anti-RANKL monoclonal antibody (e.g., denosumab) can be prepared at a concentration of less than 70 mg / mL, an appropriate amount of the amino acid aggregation inhibitor described herein can be added to the solution, and then the solution can be concentrated to an amount greater than 70 mg / mL as described herein, e.g., 120 mg / mL. Optionally, the solution can first be concentrated in excess, i.e., to a concentration of anti-RANKL monoclonal antibody (e.g., denosumab) higher than the final target concentration, and then the over-concentrated solution can be diluted, e.g., using a pH-adjusted buffer solution, to the final target concentration and pH. For example, the excess concentration can result in an amount of anti-RANKL monoclonal antibody (e.g., denosumab) in the range of 130 mg / mL to 300 mg / mL or 180 mg / mL to 300 mg / mL. The initial concentration of denosumab before concentration is not particularly limited and can be, for example, about 1 mg / mL, or about 2 mg / mL, or about 5 mg / mL, or about 8 mg / mL, or about 10 mg / mL, or about 20 mg / mL, or about 30 mg / mL, or about 40 mg / mL, or about 50 mg / mL, or about 60 mg / mL or about 70 mg / mL, or a range bounded by such concentrations, e.g., about 1 mg / mL to about 70 mg / mL, or about 1 mg / mL to about 10 mg / mL.

[0137] Concentration of the formulation can be carried out by any suitable method. In one aspect, the concentration process can include centrifugation. In another aspect, the concentration process can include ultrafiltration.

[0138] The introduction of the amino acid aggregation inhibitor into the formulation can be carried out by any suitable method. For example, the amino acid aggregation inhibitor can be introduced into the formulation by simple addition (spiking) into the formulation, as described in the following examples. In another method, the amino acid aggregation inhibitor can be introduced into the formulation by diafiltration against a buffer containing the amino acid aggregation inhibitor, as described in the following examples, for example. The amino acid aggregation inhibitor can be introduced into the formulation before or after concentrating the anti-RANKL monoclonal antibody to a concentration exceeding 70 mg / mL. As shown in the following examples, adding the amino acid aggregation inhibitor to the solution before concentration is beneficial as it suppresses aggregation during the concentration process.

[0139] Accordingly, the present disclosure provides a method for preparing a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof. In an exemplary example, the method comprises mixing an anti-RANKL monoclonal antibody or an antigen-binding portion thereof at a concentration exceeding 70 mg / mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. The antibody or antigen-binding portion can be any of those described herein, and the concentration of the antibody or its antigen-binding portion can be consistent with the teachings herein. The amino acid aggregation inhibitor can be any of those described herein. For example, the amino acid aggregation inhibitor can be a positively charged amino acid, an aromatic amino acid, or a hydrophobic amino acid. The amino acid aggregation inhibitor can be present in a molar ratio with the antibody described herein. The amounts and selection of the aggregation inhibitor, surfactant, tonicity modifier, and buffer are as described above. The present disclosure also provides a formulation produced by the manufacturing method described herein.

[0140] Formulations according to the disclosure herein can include adjusting the pH of a high-concentration solution of an anti-RANKL monoclonal antibody (e.g., denosumab) as described herein, such as having a concentration greater than 70 mg / mL, i.e., a concentration of 120 mg / mL. In another aspect, the formulation can be prepared by adjusting the pH of a low-concentration solution of an anti-RANKL monoclonal antibody (e.g., denosumab) and then concentrating the solution to a desired higher final concentration. Suitable pH adjusters are known in the art.

[0141] Embodiment

[0142] The following is a list of specific contemplated embodiments: 1. An aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof having a concentration greater than 70 mg / mL and a pH in the range of about 5.0 to less than 5.2.

[0143] 2. The formulation according to embodiment 1 having a pH in the range of about 5.0 - 5.19, or about 5.0 - about 5.15, or about 5.0 - about 5.1.

[0144] 3. The formulation according to embodiment 2 having a pH of about 5.1.

[0145] 4. The formulation according to any one of embodiments 1 - 3 further comprising an amino acid aggregation inhibitor.

[0146] 5. An aqueous pharmaceutical formulation comprising a mixture of a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof and an amino acid aggregation inhibitor.

[0147] 6. The formulation according to embodiment 5 having a pH in the range of about 5.0 - about 5.4, or about 5.0 - about 5.2, or about 5.0 - less than 5.2, or about 5.0 - 5.19, or about 5.0 - about 5.15, or about 5.0 - about 5.1.

[0148] 7. The formulation according to embodiment 6, having a pH of about 5.1.

[0149] 8. The formulation according to any one of the preceding embodiments, further comprising a pH buffer.

[0150] 9. The formulation according to any one of embodiments 5 to 8, wherein the concentration of the antibody or its antigen-binding portion is in the range of about 10 mg / mL to about 200 mg / mL.

[0151] 10. The formulation according to any one of the preceding embodiments, wherein the concentration of the antibody or its antigen-binding portion is greater than 70 mg / mL and up to about 200 mg / mL.

[0152] 11. The formulation according to embodiment 10, wherein the concentration of the antibody or its antigen-binding portion is in the range of about 100 mg / mL to about 140 mg / mL.

[0153] 12. The formulation according to embodiment 11, wherein the concentration of the antibody or its antigen-binding portion is about 120 mg / mL.

[0154] 13. The formulation according to any one of the preceding embodiments, wherein the antibody is denosumab or a biosimilar thereof.

[0155] 14. The formulation according to embodiment 13, wherein the antibody is denosumab.

[0156] 15. The formulation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more amino acids, their dipeptides, or oligopeptides having 2 to 10 residues.

[0157] 16. The formulation according to embodiment 15, wherein the amino acid aggregation inhibitor comprises a mixture of at least two amino acids.

[0158] 17. The formulation according to embodiment 16, wherein the amino acids comprise arginine and phenylalanine.

[0159] 18. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more hydrophobic amino acids, their dipeptides, or oligopeptides having 2 to 10 residues and containing one or more hydrophobic amino acids.

[0160] 19. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more amino acids having a side chain with a charge, their dipeptides, or oligopeptides having 2 to 10 residues and containing one or more amino acids having a side chain with a charge.

[0161] 20. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more basic amino acids, their dipeptides, or oligopeptides having 2 to 10 residues and containing one or more basic amino acids.

[0162] 21. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more dipeptides.

[0163] 22. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more oligopeptides having 2 to 10 amino acid residues.

[0164] 23. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor contains an arginine residue, or the amino acid aggregation inhibitor contains arginine.

[0165] 24. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor contains arginine-phenylalanine dipeptide.

[0166] 25. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is present in the preparation at a concentration in the range of about 10 mM to about 200 mM.

[0167] 26. The pharmaceutical preparation according to any one of the preceding embodiments, further comprising a surfactant.

[0168] 27. The preparation according to embodiment 26, wherein the surfactant is selected from one or more polyoxyethylene sorbitan fatty acid esters (for example, polysorbate 20, polysorbate 80), or one or more alkylaryl polyethers, for example, oxyethylated alkylphenol (for example, Triton® X-100), or one or more poloxamers (for example Pluronics®, for example Pluronic® F68), and combinations thereof.

[0169] 28. The preparation according to embodiment 26 or 27, wherein the surfactant is present at a concentration in the range of about 0.004% (w / v) to about 0.1% (w / v).

[0170] 29. The preparation according to embodiment 28, wherein the surfactant is present at a concentration of about 0.01% (w / v).

[0171] 30. The preparation according to any one of the preceding embodiments, further comprising a buffering agent.

[0172] 31. The preparation according to embodiment 30, wherein the buffering agent centers around a range of about pH 4 to about pH 5.5 at 25°C.

[0173] 32. The preparation according to embodiment 30 or 31, wherein the buffering agent has a pKa within 1 pH unit of pH 5.0 - 5.2 at 25°C.

[0174] 33. The preparation according to any one of embodiments 30 - 32, wherein the buffering agent contains acetate.

[0175] 34. The preparation according to any one of embodiments 30 - 32, wherein the buffering agent contains glutamate.

[0176] 35. The preparation according to any one of the preceding embodiments, further comprising a tonicity regulator.

[0177] 36. The formulation according to embodiment 35, wherein the tonicity regulator is selected from one or more of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof.

[0178] 37. The formulation according to embodiment 36, wherein the tonicity regulator comprises sorbitol.

[0179] 38. The formulation according to any one of the preceding embodiments, further comprising one or more additional additives selected from sugars, polyols, solubilizers (such as N-methyl-2-pyrrolidone), hydrophobic stabilizers (such as proline), polyethylene glycol, cyclodextrin, and combinations thereof.

[0180] 39. The formulation according to any one of the preceding embodiments, comprising less than 2% of high molecular weight species of the human anti-RANKL monoclonal antibody obtained by SE-UHPLC after storage at 37 °C for 3 months.

[0181] 40. The formulation according to any one of the preceding embodiments, comprising less than 2% of high molecular weight species of the human anti-RANKL monoclonal antibody obtained by SE-UHPLC after storage at 4 °C for 36 months.

[0182] 41. The formulation according to any one of the preceding embodiments, comprising at least 98% of the main antibody peak obtained by SE-UHPLC after storage at 37 °C for 3 months.

[0183] 42. The formulation according to any one of the preceding embodiments, comprising at least 98% of the main antibody peak obtained by SE-UHPLC after storage at 4 °C for 36 months.

[0184] 43. The formulation according to any one of the preceding embodiments, comprising denosumab, an amino acid aggregation inhibitor selected from one or more of arginine, its dipeptides, or oligomers having 2 to 10 residues and containing arginine, an acetate buffer, sorbitol, and a surfactant, and having a pH in the range of about 5.0 to less than 5.2.

[0185] 44. The formulation according to embodiment 43, wherein the amino acid aggregation inhibitor is selected from arginine, arginine-arginine, or arginine-phenylalanine.

[0186] 45. The formulation according to embodiment 43, wherein the amino acid aggregation inhibitor comprises a mixture of arginine and phenylalanine.

[0187] 46. The formulation according to any one of embodiments 43 to 45, wherein the acetate buffer is present in the range of about 5 mM to about 25 mM.

[0188] 47. The formulation according to any one of embodiments 43 to 46, wherein the sorbitol is present in the range of 0.1% (w / v) to 5% (w / v).

[0189] 48. The formulation according to any one of embodiments 43 to 47, wherein the surfactant is selected from one or more of polysorbate 20 and polysorbate 80.

[0190] 49. The formulation according to any one of embodiments 43 to 48, wherein the pH is in the range of about 5.0 to about 5.15.

[0191] 50. The formulation according to embodiment 49, wherein the pH is about 5.10.

[0192] 51. The formulation according to any one of the preceding embodiments, wherein the formulation is suitable for subcutaneous injection.

[0193] 52. The formulation according to any one of the preceding embodiments, wherein the formulation is sterilized and does not contain a preservative.

[0194] 53. The formulation according to any one of the preceding embodiments, wherein the human anti-RANKL monoclonal antibody or antigen-binding portion thereof comprises (1) a heavy chain variable region comprising SEQ ID NO: 2 and a light chain variable region comprising SEQ ID NO: 1, or (2) heavy chain CDR1, CDR2, and CDR3 regions each comprising SEQ ID NO: 8, 9, and 10, and light chain CDR1, CDR2, and CDR3 regions each comprising SEQ ID NO: 5, 6, and 7.

[0195] 54. The pharmaceutical preparation according to any one of the preceding embodiments, wherein the human anti-RANKL monoclonal antibody or an antigen-binding portion thereof is an antibody.

[0196] 55. The pharmaceutical preparation according to any one of Embodiments 1 to 53, wherein the human anti-RANKL monoclonal antibody or an antigen-binding portion thereof is an antigen-binding portion.

[0197] 56. A vial, prefilled syringe, or glass container containing the pharmaceutical preparation according to any one of Embodiments 1 to 55.

[0198] 57. The vial, prefilled syringe, or glass container according to Embodiment 56, containing a pharmaceutical preparation of about 1 mL or less.

[0199] 58. A method for preventing skeletal-related events (SREs) in a patient in need of prevention of skeletal-related events (SREs), the method comprising administering an effective amount of the pharmaceutical preparation according to any one of Embodiments 1 to 55.

[0200] 59. The method according to Embodiment 58, wherein the SRE is selected from the group consisting of pathological fractures, radiotherapy to bone, surgery to bone, and spinal cord compression.

[0201] 60. The method according to Embodiment 58 or 59, wherein the patient has bone metastases from solid tumors.

[0202] 61. The method according to Embodiment 60, wherein the solid tumor is selected from breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma.

[0203] 62. The method according to Embodiment 58 or 59, wherein the patient has multiple myeloma.

[0204] 63. A method according to any one of embodiments 58 to 62, comprising administering an effective amount of said formulation to reduce the bone metabolism marker urinary N-terminal telopeptide (uNTx / Cr) corrected for creatinine, optionally by at least 80%.

[0205] 64. A method of treating giant cell tumor of bone in a patient in need of treatment for giant cell tumor of bone, comprising administering an effective amount of the formulation according to any one of embodiments 1 to 55.

[0206] 65. The method according to embodiment 64, wherein the patient has a giant cell tumor of bone that is recurrent, unresectable, or for which surgical resection is likely to result in a high morbidity.

[0207] 66. A method of treating hypercalcemia of malignancy in a patient in need of treatment for hypercalcemia of malignancy, comprising administering an effective amount of the formulation according to any one of embodiments 1 to 55.

[0208] 67. The method according to embodiment 66, wherein the malignancy is refractory to bisphosphonate treatment.

[0209] 68. The method according to embodiment 66 or 67, comprising administering an effective amount of said formulation to reduce or maintain the serum calcium of said patient at a level of about 11.5 mg / dL or less.

[0210] 69. Any method according to embodiments 58 to 68, wherein said formulation comprises said human anti-RANKL antibody at a concentration of about 120 mg / mL.

[0211] 70. Any method according to embodiments 58 to 69, comprising administering said formulation on a schedule of once every four weeks.

[0212] 71. Any method according to embodiments 58 to 70, comprising administering said formulation on the 8th and 15th days of the first month of treatment.

[0213] A method for treating osteoporosis in a patient in need of treatment for osteoporosis, comprising administering an effective amount of the pharmaceutical preparation according to any one of Embodiments 1 to 55.

[0214] 73. The method according to Embodiment 72, wherein the patient is a postmenopausal woman at high risk of fracture.

[0215] 74. The method according to Embodiment 72, wherein the patient is a man at high risk of fracture.

[0216] 75. A method for increasing bone mass in a patient in need of increasing bone mass, comprising administering an effective amount of the pharmaceutical preparation according to any one of Embodiments 1 to 55.

[0217] 76. The method according to Embodiment 75, wherein the patient has osteoporosis.

[0218] 77. The method according to Embodiment 75, wherein the patient is a woman at high risk of fracture who is receiving adjuvant aromatase inhibitor therapy for breast cancer.

[0219] 78. The method according to Embodiment 75, wherein the patient is a man at high risk of fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer.

[0220] 79. The method according to any one of Embodiments 75 to 78, comprising administering an effective amount of the pharmaceutical preparation to reduce the incidence of new vertebral fractures and / or non-vertebral fractures.

[0221] 80. The method according to any one of Embodiments 75 to 79, comprising administering an effective amount of the pharmaceutical preparation to reduce bone resorption.

[0222] 81. The method according to any one of Embodiments 75 to 80, comprising administering an effective amount of the pharmaceutical preparation to increase the bone density of the patient in at least one region selected from the lumbar spine, the total hip joint, and the femoral neck.

[0223] The method according to any one of embodiments 75 to 81, comprising administering an effective amount of said formulation to increase the bone mass of the cortical bone and / or trabecular bone of said patient.

[0224] 83. The method according to any one of embodiments 75 to 82, comprising administering an effective amount of said formulation to reduce the bone resorption marker serum type 1 C-telopeptide (CTX).

[0225] 84. The method according to any one of embodiments 75 to 83, comprising administering said formulation on a schedule of once every six months.

[0226] 85. The method according to any one of embodiments 58 to 84, comprising administering said formulation in a volume of 1 mL or less.

[0227] 86. The method according to any one of embodiments 58 to 85, comprising administering said formulation subcutaneously.

[0228] 87. The method according to embodiment 86, comprising administering said formulation subcutaneously to the upper arm, upper thigh or abdomen.

[0229] 88. The method according to any one of embodiments 58 to 87, wherein said patient is receiving administration of one or both of calcium and vitamin D.

[0230] 89. A method for improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof at a concentration exceeding 70 mg / mL, comprising: preparing said aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof at a pH in the range of about 5.0 to less than 5.2; said method, wherein said aqueous pharmaceutical formulation exhibits improved stability at a pH in the range of about 5.0 to less than 5.2 as compared to an equivalent aqueous pharmaceutical formulation that is not at a pH in the range of about 5.0 to less than 5.2.

[0231] 90. A method for improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof, comprising preparing the aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or an antigen-binding portion thereof, mixed with an amino acid aggregation inhibitor, wherein the aqueous pharmaceutical formulation exhibits improved stability by the amino acid aggregation inhibitor as compared to an equivalent aqueous pharmaceutical formulation not containing the amino acid aggregation inhibitor.

Example

[0232] The following examples are provided for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Through the examples presented herein, the following abbreviations are used: DF, diafiltration; PS20, polysorbate 20; HCl, hydrochloride; UF / DF, ultrafiltration / diafiltration; F#, formulation number; HMWS, high molecular weight species; SE-UHPLC, size exclusion ultra-high performance liquid chromatography. Further, through these examples, the composition of the DF buffer or dialysis buffer used to produce the final formulation containing denosumab, as well as the estimated concentrations of the components of the final formulation, are provided. The final concentrations of specific components of the final formulation that are stored and subsequently analyzed for stability may differ from the concentrations of the DF or dialysis buffer depending on the presence or absence of counterions (e.g., HCl). In the absence of counterions, the formulation has a low ionic strength. In such an example, acetate co-concentrates with denosumab such that the final formulation contains a higher concentration of acetate relative to the concentration of the DF or dialysis buffer. For example, when using a DF buffer containing 10 mM acetate, if neither the DF buffer nor the final formulation contains counterions (e.g., HCl) and thus has a low ionic strength, approximately 23 mM acetate is obtained in the final denosumab (120 mg / mL) formulation (pH 5.1). Similarly, with a DF buffer containing 20 mM acetate, approximately 32 mM acetate is provided in the final denosumab (120 mg / mL) formulation at pH 5.1 without containing counterions (e.g., HCl). When counterions (e.g., HCl of arginine HCl) are present, acetate does not co-concentrate with denosumab, so the acetate concentration of the DF buffer and the acetate concentration of the final composition are generally the same. Additionally, additives can be excluded by volumetric analysis or may be affected by non-specific interactions. For example, in a 120 mg / mL denosumab formulation, the phenylalanine and sorbitol concentrations are approximately 7-10% lower than the concentrations shown in the DF buffer, and the arginine concentration is approximately 10-15% lower. In view of the above, through the following examples, the concentrations of the components of the final formulation are provided taking into account the above additive exclusion and acetate co-concentration effects.

[0233] Example 1 The initial evaluation of the formulation of 12 was performed with respect to the amount (%) of HMWS in the high-concentration liquid denosumab formulation (120 mg / mL) and the effect of minimizing its formation over time. Alternatives to the formulation included changes in the type of buffer, stabilizer, and pH of the solution. The formulations tested, A - L, are described in Table 1 below. All buffer values cited are with respect to the buffer concentration in which the antibody is diafiltered. Each additive and surfactant was added to the solution after buffer exchange to the levels shown in the table. The acetate concentration in this formulation was not measured, but a 120 mg / mL denosumab formulation with sorbitol diafiltered against 10 mM acetate had an approximate final acetate value of 25 mM - 35 mM acetate.

[0234] 70 mg / mL denosumab in acetate at pH 5.2 was subjected to UF / DF against 10 mM acetate at pH 5.2 and concentrated to 160 mg / mL. The stock solution was prepared in 10 mM acetate at pH 5.2 consisting of the following.

[0235] 35% sorbitol

[0236] 1% polysorbate 20

[0237] 1% polysorbate 80

[0238] 30% Pluronic® F - 68

[0239] 3% Triton™ X - 100

[0240] 250 mM L - arginine HCl

[0241] 250 mM N - acetylarginine (NAR)

[0242] 250 mM N - acetyllysine (NAK)

[0243] 250 mM proline

[0244] 250 mM polyethylene glycol (PEG) 3350

[0245] 250 mM Captisol® cyclodextrin

[0246] To obtain Formulations A - J, a 160 mg / mL material prepared using 10 mM acetate at pH 5.2 was diluted to 120 mg / mL using 10 mM acetate at pH 5.2, and then the corresponding sorbitol, additive, and / or surfactant stock solution was added and diluted to the final concentration of interest listed in Table 1. To obtain Formulations K and L, two separate aliquots from 160 mg / mL materials of a self - buffered formulation and a glutamate formulation, respectively, were subjected to additional buffer exchange by centrifugation. Thereafter, the materials for Formulations K and L were diluted to 120 mg / mL using buffer, and then the corresponding sorbitol and polysorbate 20 stock solutions were added to bring them to the target final concentrations listed for the formulations in Table 1.

[0247]

Table 1

[0248] Figure 1 shows the percent of HMWS monitored by SE - UHPLC as a function of formulation and time at 37°C. Formulation L, consisting of approximately 10 mM glutamate buffer, 10 mM L - arginine HCl, 2.4% (w / v) sorbitol as a tonicity modifier, 0.01% (w / v) polysorbate 20 as a surfactant, and a pH value of 5.0, showed both a decrease in the starting amount of HMWS suggesting some reduction of already - formed aggregates, and a decrease in the kinetics of HMWS formation at 37°C.

[0249] Example 2 Formulations of additives of 10 mM acetate, 75 mM L-arginine, 2.4% (w / v) sorbitol, 0.01% (w / v) polysorbate 20, and formulations of additives of 10 mM acetate, 5% (w / v) sorbitol, 0.01% (w / v) polysorbate 20, each having a formulation with a high concentration (120 mg / mL) of denosumab, were evaluated at 37 °C for up to 1 month at temperature to clarify the effect of pH and amino acid aggregation inhibitors on the rate and extent of HMWS formation. The tested formulations are listed in Table 2 below. All buffer and additive values cited relate to the concentration of the buffer and additive in which the antibody is diafiltered.

[0250] To prepare test samples M - Q, 3 mL aliquots of denosumab in 70 mg / mL acetate at pH 5.2 were dialyzed against 500 mL of the following DF buffer for a total of 3 buffer exchanges to achieve a 1 million-fold dilution of the previous formulation and ensure complete buffer exchange. The material was then ultraconcentrated using a centrifugal concentrator and subsequently diluted to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%.

[0251] [Table 2]

[0252] Figure 2 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C. Figure 3 shows a size exclusion chromatogram as a function of the formulation after storage at 37 °C for 1 month.

[0253] As the pH of the solution decreased, the formation of large aggregates increased. Below pH 4.8, especially at 4.5, large aggregates were the main HWMS, showing a dramatic increase at pH 4.5 for the test formulations. As shown in Figure 3, formulations P and Q had the minimum amount of higher-order HWMS (retention time of about 6 minutes), followed by comparative formulations O, N, and M having decreasing pH values.

[0254] However, as the pH increased, generally an increase in the dimer species was brought about. As shown in Figure 3, formulation N had the least amount of dimer species (retention time of about 6.8 minutes), followed by formulations M, O, P, and Q.

[0255] The presence of arginine in formulation O at a concentration of 75 mM resulted in a decrease of about 0.3% and 25% in the amount of dimer species and its formation rate, respectively, after 1 month at 37°C, compared to formulation P having the same pH but no arginine.

[0256] Example 3 This example demonstrates the effect of pH on high-concentration denosumab formulations.

[0257] Denosumab (at a concentration of 120 mg / mL) was formulated at three different pH values: 4.8, 5.1, and 5.4, with acetate, sorbitol, and polysorbate 20 (PS20), and with or without an amino acid aggregation inhibitor. In this study, the amino acid aggregation inhibitor was L-arginine HCl. All formulations were prepared by exchanging the buffer of the initial solution containing a lower concentration of denosumab, followed by ultra-concentrating the denosumab material and then diluting the denosumab material with the desired amount of buffer, additives, and surfactant. Briefly, an aliquot of 70 mg / mL denosumab in acetate at pH 5.2 (initial substance) was dialyzed against the DF buffer described in Table 3A, with a total of three buffer exchanges to achieve a dilution of 1 million-fold of the initial material and to ensure complete buffer exchange. The buffer-exchanged denosumab material was then concentrated to a denosumab concentration of more than 120 mg / mL using a centrifugal concentrator and then diluted to a concentration of 120 mg / mL denosumab. PS20 was added to a final concentration of 0.01%.

[0258] High concentrations of protein were thought to affect the solution pH based on their charge state. The acetate concentration of Formulation 1 was increased to achieve the target final pH, and the acetate concentrations of Formulations 2 and 3 were made consistent with that of Formulation 1. For Formulations 4 - 6, since acetate was not co - concentrated in the presence of the HCl salt, a higher amount of acetate was required to match the final acetate concentration of Formulations 1 - 3. Formulation 7 served as a control to ensure that the increased acetate concentration in Formulations 4 - 6 did not interfere with the protein stability of the arginine hydrochloride formulation.

[0259] The various denosumab formulations prepared and tested in this study are listed in Table 3A.

[0260]

Table 3

[0261] Samples of each formulation were filled into containers with a fill volume of 1 mL and stored at a temperature of 37°C for up to 4 weeks. The stability against aggregation inhibition and aggregation over time based on HMWS and dimer species formation was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared at the initial conditions, during and after the storage period.

[0262] The percentage of HMWS was monitored by SE - UHPLC as a function of formulation and time at 37°C. Figure 4 represents a graph of the percentage of HMWS as a function of time for Formulations 1 - 7, and Table 3B provides the data points of the graph.

[0263]

Table 4

[0264] Figure 5 shows the size exclusion chromatograms of each formulation after storage at 37°C for 1 month. Formulations without arginine are shown in the left panel, and formulations containing arginine are shown in the right panel.

[0265] As shown in Figure 4, formulations containing arginine were performed better than the control formulation without arginine hydrochloride, and the formulation at pH 5.1 was performed better than the comparative formulations at pH 4.8 and pH 5.4. In formulations 1 - 3 without arginine hydrochloride, the dimer species increased as the solution pH increased to 5.4 (Figure 5A). For formulations 4 - 6 containing arginine hydrochloride, the formation of larger aggregates increased as the solution pH decreased to 4.8, and the dimer species increased as the solution pH increased to 5.4 (Figure 5B). At a solution pH of 5.1, formulation 6 in the presence of arginine hydrochloride showed the lowest amount of total HMWS compared to formulation 2 without the presence of arginine hydrochloride. Furthermore, the behavior of formulation 7 showed that increasing the acetate buffer concentration from 10 mM to 40 mM had a relatively small effect on HMWS formation.

[0266] Example 4 This example demonstrates the relationship between pH and HMWS formation for different denosumab formulations containing various denosumab concentrations.

[0267] The pH sensitivity of HMWS formation at denosumab protein concentrations of 15 mg / mL to 150 mg / mL was evaluated at various protein concentrations and an arginine hydrochloride concentration of 75 mM. Two pH values, namely pH 4.8 and 5.1, were evaluated at each protein concentration tested: 15, 60, 120, and 150 mg / mL.

[0268] A total of eight formulations (Formulations 8 - 15, described in Table 4A) were evaluated in this study. To prepare these formulations, two aliquots of 70 mg / mL denosumab in pH 5.2 acetate were dialyzed against each of the DF buffers described in Table 4A. Both dialysis setups #1 and #2 were each subjected to a total of three buffer exchanges to dilute the previous formulation by a factor of one million and ensure a complete buffer exchange. After dialysis, aliquots from each of dialysis setups #1 and #2 described in Table 4A were removed to prepare the dilution steps for Formulations 8, 9, 12, and 13. Subsequently, the remaining material was ultrafiltered using a centrifugal concentrator and diluted to the corresponding denosumab concentrations listed in Table 4A, and PS20 was added to a final concentration of 0.01%.

[0269]

Table 5

[0270] The formulations were filled into containers at a fill volume of 1 mL and stored at a temperature of 37°C for up to one month. The stability with respect to aggregation inhibition and aggregation over time based on the formation of HMWS and dimer species was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared at initial conditions and during and after the storage period.

[0271] Figure 6 represents a graph of the percent of HMWS monitored by SE - UHPLC as a function of storage time at 37°C for each formulation, and Table 4B provides the data points for the graph.

[0272]

Table 6

[0273] Figures 7A and 7B show size exclusion chromatograms as a function of the formulation after storage at 37 °C for 1 month. As shown in Figure 6, the HMWS% increased as the protein concentration increased. Formulations 8 - 11 at pH 4.8 consistently had higher levels of HMWS compared to the corresponding formulations at pH 5.1 (Formulations 12 - 15). The increase in HMWS% at pH 4.8 is due to a large aggregation peak at approximately 5.75 minutes shown in Figure 7A (top). The HMWS% at solution pH 5.1 has a dimer species that increases as the protein concentration increases, but the total HMWS was lower than the corresponding protein concentration at solution pH 4.8 (Figure 7b (bottom)).

[0274] The difference in HMWS levels between pH 5.1 and pH 4.8 increased as the denosumab concentration increased, and the higher the denosumab concentration, the greater the difference.

[0275] Example 5 Formulations with various concentrations of arginine, NAR, and two dipeptides consisting of arginine - arginine (Arg - Arg) and arginine - phenylalanine (Arg - Phe) were evaluated for their stabilizing effect on a solution with a denosumab concentration of 120 mg / mL.

[0276] The tested formulations are described in Table 5 below. The values for all acetates and additives (except dipeptides) cited are the concentrations of the buffer and additives through which the antibody is diafiltered. Each dipeptide was added to the solution to the levels shown in the table after buffer exchange. Formulations R - X were achieved by UF / DF against the DF buffer listed below. Formulations Y and Z were achieved together in a single pool by UF / DF against a DF buffer containing 10 mM acetate, 3.6% sorbitol, pH 4.0. After UF / DF, the pool of formulations Y and Z was split into two, and then the Arg - Arg or Arg - Phe dipeptide was added from a 1 M stock solution containing 3.6% sorbitol at pH 5.1. Polysorbate 20 was added to each formulation at a final formulation concentration of 0.01%. Acetate was co - concentrated without arginine to obtain a final acetate concentration of approximately 25 mM in formulations S - X. Sorbitol was preferentially excluded during the concentration process, resulting in a decrease of approximately 7 - 8% (w / v) from the initial concentration.

[0277] The formulations were filled into containers with a fill volume of 1.0 mL. The formulations are stored at temperatures of 2°C - 8°C for up to 12 months and at 25°C, 30°C, and 37°C for 3 months. The stability based on the formation of HMWS is evaluated using SE - UHPLC. The stability of these dipeptide formulations after 1 month at 37°C was compared to the arginine hydrochloride formulation at 37°C as shown in Figure 8.

[0278]

Table 7

[0279] Figure 8 shows the percent of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. The results indicate that the amino acid aggregation inhibitor inhibited the formation of HMWS. For example, the arginine-phenylalanine dipeptide showed a significant improvement, with about 0.3% less HMWS formed compared to other formulations. The rank order of HMWS from lowest to highest was Z << V < Y ≒ T ≒ W ≒ X ≒ U < S < R. As can be seen from the figure, both the arginine-arginine (Arg-Arg) (formulation Y) and arginine-phenylalanine (Arg-Phe) (formulation Z) dipeptide-containing formulations decreased HMWS formation compared to the control formulation (formulation R) lacking arginine and arginine-containing dipeptides. Formulation Z contained the lowest amount of HMWS and was superior to formulation Y.

[0280] Example 6 This example demonstrates the aggregation inhibition and stability of denosumab as a function of different concentrations of arginine and phenylalanine, and comparative mixtures of arginine and phenylalanine.

[0281] As described above, it was confirmed that arginine hydrochloride (HCl) and the arginine HCl-phenylalanine dipeptide decreased the initial onset level and rate of HMWS formation of denosumab. In this study, formulations containing the concentration of arginine HCl, the concentration of phenylalanine, and the combination of arginine HCl and phenylalanine were evaluated for their stabilizing effect on a solution containing denosumab at 120 mg / mL.

[0282] The formulations tested (formulations 16 - 20) are described in Table 6A below. To prepare these formulations, aliquots of 70 mg / mL denosumab in acetate buffer at pH 5.2 were dialyzed against the DF buffer described in Table 6A for a total of three buffer exchanges to achieve a dilution of one million-fold of the previous formulation and to ensure complete buffer exchange. The material was then concentrated in excess using a centrifugal concentrator and subsequently diluted to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%. Formulation 16 was considered the control formulation.

[0283]

Table 8

[0284] The formulation was filled into containers at a fill volume of 1.0 mL. The formulation was stored at a temperature of 37 °C for up to 1 month. The stability against aggregation inhibition and time-dependent aggregation inhibition based on the formation of HMWS and dimer species was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared at initial conditions and during and after the storage period.

[0285] Figure 9 shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C. Figure 10 shows a size exclusion chromatogram as a function of the formulation after storage at 37 °C for 1 month. The following Table 6B shows the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C.

[0286]

Table 9

[0287] All formulations containing an amino acid aggregation inhibitor, arginine or phenylalanine (Formulations 17 - 20) were superior to a sorbitol control formulation (Formulation 16) that contained no amino acid aggregation inhibitor. All phenylalanine - containing formulations (Formulations 18, 19 and 20) contained similarly low levels of HMWS when compared to the control and arginine HCl formulations (Formulations 16 and 17, respectively) (Figure 9). The rate of HMWS formation was similar between the arginine HCl and phenylalanine - containing formulations (Formulations 17 - 19), as shown in Figure 9. The combination formulation containing both 38 mM arginine and 38 mM phenylalanine (total 76 nM, Formulation 20) showed better stability than the 75 mM arginine formulation (Formulation 17) (Figure 9), but did not show better stability than the 75 mM phenylalanine formulation (Formulation 19) (Figure 9).

[0288] Example 7 This example demonstrates the aggregation inhibition and stability of denosumab as a function of different concentrations of phenylalanine.

[0289] In previous studies, arginine hydrochloride and arginine hydrochloride - phenylalanine dipeptide were identified as minimizing the initial onset level and rate of HMWS formation of denosumab. The stabilizing effect of formulations containing arginine hydrochloride, formulations containing various concentrations of phenylalanine, and formulations containing a combination of arginine hydrochloride and phenylalanine on a solution containing denosumab at 120 mg / mL was evaluated.

[0290] The formulations tested are described in Table 7A below. To prepare Test Samples A - E, an aliquot of 70 mg / mL denosumab in acetate (pH 5.2) was dialyzed against the DF buffer described below, with a total of three buffer exchanges to achieve a dilution of 1 million - fold of the previous formulation and to ensure complete buffer exchange. This material was then ultra - concentrated to approximately 130 mg / mL - 150 mg / mL using a centrifugal concentrator, followed by dilution to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%. Formulation A was considered the control formulation.

[0291] The formulation was filled into containers with a filling volume of 1.0 mL. The formulation was stored at a temperature of 37 °C for up to 1 month. The stability based on the formation of HMWS was evaluated using SE UHPLC. The stability profiles of these formulations were compared with those of the sorbitol and arginine hydrochloride / sorbitol formulations at 37 °C after 1 month at 37 °C, as shown in Figure 11A.

[0292] To prepare test samples F - K, an aliquot of denosumab at 70 mg / mL in acetate (pH 5.2) was subjected to a total of 12 dialysis volumes of ultrafiltration / diafiltration (UF / DF) against the following DF buffer to ensure complete buffer exchange. The material was then ultra - concentrated to approximately 200 mg / mL using ultrafiltration, followed by dilution to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%. In these formulations, the acetate concentration was 20 mM. Formulation F was considered the control formulation. All cited values for acetate and additives relate to the buffer and additive concentrations in which the antibody was dialyzed.

[0293] The formulation was filled into containers with a filling volume of 1.0 mL. The formulation was stored at a temperature of 40 °C for up to 1 month. The stability based on the formation of HMWS was evaluated using SE UHPLC. The stability profiles of these formulations were compared with those of the sorbitol and arginine hydrochloride / sorbitol formulations at 40 °C after 1 month at 40 °C, as shown in Figure 11B.

[0294]

Table 10

[0295] Figures 11A and Table 7B show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Figures 11B and Table 7C show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 40°C. Figures 12A and 12B show size exclusion chromatograms as a function of formulation after storage at 37°C and 40°C for 1 month, respectively.

[0296]

Table 11

[0297]

Table 12

[0298] When compared to both the sorbitol formulation and the arginine hydrochloride / sorbitol formulation (Formulations A and B, respectively), all phenylalanine formulations (Formulations C, D, E, G-K) contained lower levels of HMWS. The combined formulations of arginine hydrochloride and phenylalanine had similar stability compared to the arginine / sorbitol formulation (Formulation B). All formulations were superior to the sorbitol control formulations (Formulations A and F).

[0299] Example 8 This example demonstrates the evaluation of various amino acid aggregation inhibitors.

[0300] The evaluation of different amino acid aggregation inhibitors was performed by preparing eight formulations containing hydrophobic amino acids, aromatic amino acids, or amino acids with polarity / charge, and determining the amount (%) of HMWS in a high-concentration liquid denosumab formulation (120 mg / mL) and the effect on minimizing HMWS formation over time. This formulation contained one of eight L-amino acids and a lower amount of sorbitol compared to a control formulation (Formulation 26) that did not contain an amino acid aggregation inhibitor and had a greater amount of tonicity-providing sorbitol.

[0301] The amino acid aggregation inhibitors tested were grouped into one of three groups (Groups I - III) as follows and contained amino acid aggregation inhibitor amounts: I. Aromatic amino acids: (a) 38 mM phenylalanine (Formulation 27), (b) 38 mM tryptophan (Formulation 28), II. Polar / charged amino acids: (a) 75 mM arginine HCl (Formulation 29), (b) 75 mM lysine (Formulation 30), (c) 75 mM histidine (Formulation 31), III. Hydrophobic amino acids: (a) 38 mM leucine (Formulation 32), (b) 38 mM isoleucine (Formulation 33), (c) 38 mM valine (Formulation 34).

[0302] To prepare Formulations 26 - 34, an aliquot of 70 mg / mL denosumab in pH 5.2 acetate was dialyzed against the DF buffer described in Table 8A, with a total of three buffer exchanges to achieve a 1 million - fold dilution of the previous formulation and ensure complete buffer exchange. For the dialysis of the histidine formulation F, a buffer with an initial pH of 4.0 was used, and it was predicted that the pH would shift to the target pH of 5.1 upon protein concentration due to the Donnan effect and co - concentration of acetate. However, after concentrating the protein to 120 mg / mL, the pH did not shift to the target pH of 5.1 and remained at pH 4.0. Titration with dilute (0.1 N) NaOH was required to bring the pH of the histidine formulation to pH 5.1. The remaining formulations were concentrated in excess using a centrifugal concentrator, followed by dilution to 124 - 128 mg / mL, and polysorbate 20 was added to a final concentration of 0.01% (w / v).

[0303]

Table 13

[0304] The formulation was filled into containers at a fill volume of 1.0 mL. The formulation was stored at a temperature of 37°C for up to 4 weeks. The stability against aggregation and time - dependent aggregation based on the formation of HMWS and dimer species was evaluated using SEC - UHPLC. The aggregation - inhibition profiles of these formulations were compared at initial conditions and during and after the storage period.

[0305] Figures 13 - 15 represent graphs of the percentage of HMWS monitored by SEC - UHPLC as a function of storage time at 37°C for each formulation, and Table 8B provides the data points of the graphs. Figures 16 - 18 show chromatographic overlays of the formulations listed in Table 8A after storage at 37°C for 1 month. Figures 13 and 16 relate to formulations containing aromatic amino acids, Figures 14 and 17 relate to formulations containing polar / charged amino acids, and Figures 15 and 18 relate to formulations containing hydrophobic amino acids.

[0306] [Table 14] F# is provided in the left column and corresponds to F# in Table 8A.

[0307] As shown in Figures 13 to 15, all formulations (Formulations 27 to 34) containing amino acid aggregation inhibitors showed some improvement in stability compared to the acetate / sorbitol formulation (Formulation 26). Formulations containing aromatic amino acids (Formulations 27 and 28) showed the largest decrease in HMWS%. Also, the formulation containing phenylalanine (Formulation 27) showed a large decrease in HMWS, and the formulation containing tryptophan showed the largest decrease compared to the control (Formulation 26). Denosumab formulations containing polar / charged amino acids (Formulations 29 to 31) generally showed larger order aggregates (Figure 17) compared to other formulations having amino acid stabilizers (Figures 16 and 18), and this particular histidine formulation showed a greater amount of HWMS overall when compared to the acetate / sorbitol formulation (Formulation 26) (Figure 14). The results of the histidine formulation may be biased from the dialysis process, longer duration at pH 4.0, and titration of the formulation with dilute NaOH. All formulations containing hydrophobic amino acids (Formulations 32 to 34) showed consistent improvement in HMWS formation.

[0308] Example 9 This example demonstrates the possible mechanism of action of arginine and phenylalanine in the stabilization of denosumab. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a sensitive and robust technique for characterizing protein-protein / ligand / additive interactions. This method detects changes in backbone amide hydrogen bonds due to interactions with additives.

[0309] Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed using denosumab (concentration 3 mg / mL) in 10 mM acetate buffer (pH 5.2) (“A52”) in the presence of L-arginine (formulation 35), L-phenylalanine (formulation 36), or L-glycine (formulation 37), and compared to a denosumab formulation (formulation 38) that does not contain an amino acid aggregation inhibitor. The experiments were conducted at 4 °C (L-arginine, L-phenylalanine, or L-glycine at 75 mM concentration) and 37 °C (L-arginine, L-phenylalanine, or L-glycine at 150 mM concentration). After analyzing more than 530 peptides, a few regions with significant structural changes were identified. Some representative peptides derived from these regions are shown in Figures 19 - 30.

[0310] Figures 19 - 24 are graphs of % deuterium incorporation as a function of time (log(seconds)) at 4 °C for light chain amino acids 28 - 33 (Figure 19), light chain amino acids 108 - 116 (Figure 20), light chain amino acids 125 - 132 (Figure 21), heavy chain amino acids 47 - 59 (Figure 22), heavy chain amino acids 243 - 253 (Figure 23), and heavy chain amino acids 392 - 399 (Figure 24) for each of formulations 35 - 38.

[0311] Figures 25 - 30 are graphs of % deuterium incorporation as a function of time (log(seconds)) at 37 °C for light chain amino acids 28 - 33 (Figure 25), light chain amino acids 108 - 117 (Figure 26), light chain amino acids 124 - 131 (Figure 27), heavy chain amino acids 47 - 59 (Figure 28), heavy chain amino acids 242 - 253 (Figure 29), and heavy chain amino acids 392 - 399 (Figure 30) for each of formulations 35 - 38.

[0312] These data support that Arg has a slightly stronger HDX footprint (conformational change) on denosumab, while Arg and Gly have similar interaction effects on denosumab: strong stabilization in the Fab LC 28 - 33 region, slight stabilization in the Fab LC 108 - 132 and HC 47 - 59, Fc CH3 HC 392 - 399 regions, and slight destabilization in the Fc CH2 243 - 253 region. Without intending to be bound by any particular theory, the effect of arginine hydrochloride is thought to be due to a combination of preferential exclusion from the denosumab surface and weak surface interactions, while the role of glycine is thought to be due to preferential exclusion.

[0313] However, phenylalanine did not show significant structural perturbation to denosumab. Without intending to be constrained by any particular theory, the phenylalanine stabilization effect may occur via one or more of the following mechanisms: side-chain interactions without affecting the peptide backbone (no HDX footprint), and / or cation - pi interactions with arginine / lysine side-chains without affecting the backbone hydrogen bond network.

[0314] Example 10 This example demonstrates a possible mechanism for the action of phenylalanine - stabilized denosumab.

[0315] To study the specific effect of Phe on denosumab, molecular dynamics simulations were performed. Specifically, the Fab domain of denosumab was solvated with excess Phe in a simulation box, and two 10-ns simulations were conducted. Collectively, Phe residues that were bound to Fab over more than 90% of the time were selected for further analysis. Nine such cases were identified. In five of the nine observations of long residence times, the Phe residues bound at the boundaries of the VH / VL (variable heavy / variable light) and CH / CL (constant heavy / constant light) regions. In one example, the Phe side chain was thought to interact with the side chains of hydrophobic residues (e.g., V93, Y95, and W112 of the heavy chain and A44 and P45 of the light chain) at the VH / VL interface. In another example, the side chain ring of Phe was thought to interact with the NH3+ and COO(−) groups of residues (e.g., T165 of the light chain and G171, V172, and T174 of the heavy chain) at the CH1 and CL interface. Without intending to be bound by a particular theory, this observation leads to the idea that the specific effect of Phe in alleviating denosumab aggregation is due to the phenyl group and hydrophobic residues (e.g., R30, G31, R32, and Y33 of CDR1 of the light chain, A52 of CDR2 of the light chain, and M106 of CDR3 of the heavy chain) forming the interface between the heavy constant 1 (Hc) chain and the light constant (Lc) chain. This interaction is hypothesized to replace the previously hydrophobic surface with a surface having a relatively larger charge from the NH3+ and COO(−) groups from the Phe additive (resulting in hydrophilicity).

[0316] Example 11 The stability of multiple constructs of anti-RANKL antibodies (isotypes IgG1, IgG2, and IgG4) was evaluated. As described above, both arginine HCl and phenylalanine minimize the onset of HMWS and the levels of HWMS over time compared to the acetate / sorbitol control formulation of denosumab (an IgG2 immunoglobulin). This evaluation was conducted to compare the potential of Arg-HCl and Phe to reduce HMWS in formulations containing different anti-RANKL antibody constructs. The IgG1 and IgG4 constructs tested in this study contained the same complementarity-determining regions (CDRs) as denosumab but also included different constant domain scaffolds. The different IgG2 constructs tested in this study had CDRs different from denosumab but included the same constant domain scaffold.

[0317] Each of the tested antibody constructs was purified and concentrated from 8 mg / mL to 70 mg / mL using centrifugal concentration. Each concentrated volume was divided into three aliquots and dialyzed against acetate buffer prepared with sorbitol, sorbitol / phenylalanine, and sorbitol / arginine hydrochloride as described in Table 9 to prepare Formulations 39 - 47. The samples after dialysis were ultra-concentrated by centrifugation to over 120 mg / mL. The antibody proteins were diluted to 120 mg / mL with their respective buffers.

[0318]

Table 15

[0319] The formulation was filled into glass vial containers with a filling volume of 1.0 mL. The formulation was stored at a temperature of 37 °C for up to 1 month. The stability against aggregation inhibition based on the formation of HMWS and time-dependent aggregation inhibition was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared at the initial conditions and after the storage period. The stability of these formulations after storage was compared within the immunoglobulin class.

[0320] Figures 31, 33, and 35 (and the related Tables 10, 12, and 14 below) show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C using immunoglobulin G (IgG1, IgG2, and IgG4, respectively). Figures 32, 34, and 36 (and the related Tables 11, 13, and 15 below) show the percentage of low molecular weight species (LMWS, e.g., protein fragmentation) monitored by SE-UHPLC as a function of formulation and time at 37 °C using immunoglobulin G (IgG1, IgG2, and IgG4, respectively). Figures 37, 38, and 39 show size exclusion chromatogram overlays as a function of formulation after storage at 37 °C for t = 4 w.

[0321] [Table 16]

[0322] [Table 17]

[0323] [Table 18]

[0324] [Table 19]

[0325] [Table 20]

[0326]

Table 21

[0327] As shown in FIGS. 31 and 32, IgG1 molecules having a CDR region similar to the previous denosumab sample showed an approximately 0.2% decrease in HMWS upon addition of phenylalanine when compared to the acetate / sorbitol control formulation. IgG2 samples having different CDRs and shown in FIGS. 33 and 34 showed an increase in HMWS in the acetate / phenylalanine / sorbitol formulation when compared to the control acetate / sorbitol formulation. The acetate / sorbitol and acetate / phenylalanine / sorbitol formulations had the same stability as acetate / sorbitol / arginine, which has greater HMWS formation, also for the IgG4 sample type, as shown in FIGS. 35 and 36. In all cases of the IgG1, IgG2, and IgG4 sample types, the acetate / sorbitol / arginine-containing formulation showed an increase in HMWS degradation compared to the acetate / sorbitol (control) and acetate / phenylalanine / sorbitol formulations.

[0328] Due to a large increase in protein fragmentation in the acetate / arginine / sorbitol formulation shown in FIGS. 37 and 38, the relationship between fragmentation and antibody isotype was shown in FIGS. 32, 34, and 36. It has been shown in the literature that monoclonal antibody fragmentation-mediated aggregation can occur in antibodies stored at 37° C. [Perico N. et.al., J. Pharm. Sci. (2009) 98, pgs. 3031-3042]. In this evaluation, this mechanism can be considered to have the maximum fragmentation in the acetate / arginine / sorbitol formulation. This fragmentation is minimized in the acetate / phenylalanine / sorbitol formulation, and there may be fewer HMWS species. The IgG4 sample type did not accelerate fragmentation or aggregation.

[0329] From the data collected in this study and the previous molecular modeling data collected with denosumab, a strong correlation can be established between the amino acid sequence of the CDR and the relative effect of reducing HMWS with phenylalanine. A decrease in HMW species was observed with denosumab (IgG2) and an IgG1 variant having the same CDR amino acids, but no decrease in HMWS was observed with IgG2 variants having different CDR domains. The amino acid sequences contained within the CDR domain appear to be susceptible to interactions with phenylalanine and subsequent inhibition of aggregation. The IgG4 molecule also had the same CDR region when compared to denosumab, but only minimal aggregation changes were detected during the course of the study. The IgG4 molecule is different from the IgG1 and IgG2 versions, mainly due to the length of its hinge amino acids and its functionally active structure. Since the IgG1 and IgG2 antibody isotypes have an extended structure specifically described as "Y" shaped, the IgG4 Fab CH1 domain interacts with the CH2 domain to form a more compact structure [Aalberse R.C. et al., Immunology (2002), 105. pgs. 9-19]. This compact structure was able to inhibit the fragmentation and aggregation reactions typical of the IgG1 and IgG2 modalities.

[0330] Example 12 As described below, studies are conducted to monitor the stability of denosumab (Formulations 51-55) formulated in relation to Table 16. The diafiltration buffer has different acetate concentrations and starting pHs and produces a final formulation with a pH of 5.1 at a denosumab concentration of 120 mg / ml. Further, the sorbitol level is adjusted to maintain the isotonicity of the final product (about 300 mOsm / Kg). 70 mg / mL of denosumab is diafiltered against each buffer at a diafiltration volume of more than 7-fold, then ultrafiltered to about 180 gm / mL and diluted with diafiltration buffer and polysorbate to a denosumab concentration of 120 mg / mL and 0.01% polysorbate 20. Stability is evaluated using SE-UHPLC after storage at 37°C, indicating that denosumab stability in these formulations is very similar. The initial HMW species decreases slightly as the initial acetate concentration increases. In contrast, the aggregation rate improves slightly in formulations with lower levels of acetate.

[0331]

Table 22

[0332] Example 13 The following example reports the results of a study on the effect of arginine on the chemical denaturation stability of denosumab at three different pH values: 4.5, 4.8 and 5 (or 5.2).

[0333] All chemical denaturation experiments were performed using an Unchained Labs instrument - HUNK equipped with a fluorescence detector. The excitation wavelength was 280 nm, and the emission scan was recorded between 300 and 500 nm. For each denaturation experiment, protein, buffer, and denaturant (guanidinium HCl) were dispensed into 36 wells with a linear increase in denaturant concentration to obtain 36 - point curves for each condition. Curve - fitting software provided by the instrument manufacturer (Unchained Labs) was used to fit the data points. Since there was evidence of only a single transition (native ←→ denatured), a two - state model was used. The experiments were conducted using 0 - 6 M urea in 5.0% w / v sorbitol of 10 mM acetate and titrated to the required pH of 4.5, 4.8, or 5 (5.2). In all experiments, the concentration of denosumab protein was 7 mg / mL.

[0334] Figure 40 shows the isothermal chemical denaturation curves of denosumab at pH 4.5, 4.8, and 5.0 in the absence of arginine. In the absence of arginine, the concentration of chemical denaturant C required for 50% unfolding 1 / 2 is similar for the three pH conditions tested.

[0335] Figure 41 shows the isothermal chemical denaturation curves of denosumab in the presence of 75 mM arginine HCl at pH 4.5, 4.8, and 5.2. There was a significant increase in chemical denaturation stability at pH 5.2 compared to pH 4.8 and 4.5. C 1 / 2 at which, for lower pH compared to pH 5.2, the guanidinium HCl of the denaturant increases by only 1 M. Thus, the protective nature of arginine is remarkable and highly pH - dependent.

[0336] Example 14 The following examples provide the results of a study on the effect of arginine and phenylalanine on the stability over time of a high - concentration denosumab formulation in a syringe.

[0337] In previous studies, it was confirmed that arginine hydrochloride and phenylalanine decrease the initial onset level and rate of denosumab HMWS formation. In this study, the stabilizing effects of formulations containing arginine hydrochloride, phenylalanine, and a combination of arginine hydrochloride and phenylalanine on a solution containing 120 mg / mL denosumab stored in syringes at two different temperatures for up to three months were evaluated.

[0338] The tested formulations are described in Table 17 below. To prepare Formulations 56 - 59, 70 mg / mL denosumab in acetate at pH 5.2 was diafiltered against the following diafiltration (DF) buffer at 8 diavolumes to ensure buffer exchange. The material was then ultrafiltered to above 180 mg / mL and subsequently diluted to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%. Formulation 56 was considered the control formulation. The listed values for acetate, arginine HCl, and phenylalanine are for the DF buffer, and the estimated levels in the final composition at 120 mg / mL denosumab are provided considering additive exclusion and acetate co - concentration in the absence of other counterions. Viscosities at 5 °C and 25 °C were measured using a Paar modular compact rheometer at a shear rate up to 1000 s -1 (inverse seconds). The formulations were filled into glass pre - filled syringes (PFS) with a fill volume of 1.0 mL. Parallel syringe sets were stored at 25 °C for three months and at 37 °C for two months. Stability based on HMWS formation was evaluated using SE UHPLC.

[0339]

Table 23

[0340] Figures 42 and 43 show the percentage of HMWS monitored by SE - UHPLC as a function of formulation and time at 25 °C for three months and at 37 °C for two months, respectively.

[0341] Tables 18 to 21 show the same data in tabular form and also show the increase in HMWS relative to the initial level of HMWS.

[0342]

Table 24

[0343]

Table 25

[0344]

Table 26

[0345]

Table 27

[0346] This example shows that the addition of arginine, phenylalanine, and combinations thereof each reduces the level of initial HMWS (t = 0) in a high-concentration denosumab formulation. At 25°C, the increase in HMWS decreases in the phenylalanine formulation 59 compared to the control formulation 56. At 37°C, formulations 57 and 59 show a decrease in HMWS formation compared to the control sorbitol formulation 56. The formulation containing both arginine HCl and phenylalanine forms HMWS at a higher rate at 37°C compared to the other formulations, indicating that the combination of these additives destabilizes denosumab at such higher temperatures in this formulation.

[0347] Changes within the scope of the present invention may be apparent to those skilled in the art, so the above description is given for clarity of understanding only and should not be considered as imposing unnecessary limitations.

[0348] Throughout this specification and the following claims, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted as including the stated integer or step or group of integers or steps and not as excluding any other integer or step or group of integers or steps.

[0349] Throughout this specification, when a composition is described as including components or materials, unless otherwise specified, the composition is also contemplated to consist essentially of, or consist of, any combination of the recited components or materials. Similarly, when a method is described as including specific steps, unless otherwise stated, these methods are also contemplated to consist essentially of, or consist of, any combination of the recited steps. The invention disclosed herein by way of example can be practiced appropriately in the absence of any element or step not specifically disclosed herein.

[0350] The methods disclosed herein and the implementation of their individual steps can be performed manually and / or by means provided by electronic devices or by automation. Although the process has been described with reference to specific embodiments, those skilled in the art will readily understand that other methods for performing operations related to this method can be used. For example, unless otherwise specified, the order of the various steps may be changed without departing from the scope or gist of the method. Furthermore, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0351] All patents, publications and references cited herein are hereby incorporated by reference in their entirety into this specification. In the event of any conflict between this disclosure and the incorporated patents, publications and references, this disclosure shall prevail.

Claims

1. A pre-filled syringe containing 1.0 mL or less of an aqueous medical formulation, wherein the aqueous medical formulation comprises: (a) a light chain variable region of SEQ ID NO: 1 and a human kappa light chain constant region, and a heavy chain variable region of SEQ ID NO: 2 and a human IgG ２ Anti-human receptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibodies, including constant regions thereof; (b) aromatic amino acids A prefilled syringe comprising:

2. The pre-filled syringe according to claim 1, wherein the concentration of the anti-RANKL antibody in the liquid medical formulation exceeds 70 mg / mL.

3. The pre-filled syringe according to claim 2, wherein the concentration of the anti-RANKL antibody in the liquid medical formulation exceeds 100 mg / mL.

4. The pre-filled syringe according to claim 3, wherein the concentration of the anti-RANKL antibody in the liquid medical formulation is 100 to 140 mg / mL.

5. The pre-filled syringe according to claim 4, wherein the concentration of the anti-RANKL antibody in the liquid medical formulation is 120 mg / mL±12 mg / mL.

6. The pre-filled syringe according to any one of claims 1 to 5, wherein the aromatic amino acid comprises phenyl or indole.

7. Aromatic amino acids have a C between the alpha carbon and the phenyl or indole １ ~C ６ 7. The pre-filled syringe of claim 6, comprising an alkyl chain.

8. Alkyl chain is C １ ~C ３ The pre-filled syringe according to claim 7, wherein the alkyl chain is an alkyl chain.

9. 9. The pre-filled syringe according to claim 8, wherein the aromatic amino acid is phenylalanine.

10. 9. The pre-filled syringe according to claim 8, wherein the aromatic amino acid is tryptophan.

11. The pre-filled syringe according to any one of claims 1 to 10, comprising 5 mM to 180 mM of an aromatic amino acid.

12. 12. The pre-filled syringe of claim 11, comprising 5 mM to 100 mM of an aromatic amino acid, optionally 30 mM to 80 mM or 25 mM to 90 mM of an aromatic amino acid.

13. 13. The pre-filled syringe of any one of claims 1 to 12, further comprising a tonicity modifier, optionally selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof.

14. 14. The pre-filled syringe according to any one of claims 1 to 13, wherein the aqueous pharmaceutical formulation is self-buffered and / or does not contain sorbitol.

15. The pre-filled syringe according to any one of claims 1 to 14, wherein the aqueous pharmaceutical formulation has a pH in the range of 5.0 to 5.

4.

16. The pre-filled syringe according to any one of claims 1 to 15, wherein the anti-RANKL antibody comprises a light chain of SEQ ID NO: 13 and a heavy chain of SEQ ID NO:

14.

17. The pre-filled syringe according to any one of claims 1 to 16, wherein the aqueous pharmaceutical formulation comprises denosumab at a concentration of 120 mg / mL, acetate, phenylalanine, sorbitol, and polysorbate 20.

18. for treating a skeletal-related event (SRE), giant cell tumor of bone, hypercalcemia, or osteoporosis in a subject, or for increasing bone mass in a subject; or (a) treating SREs in subjects with bone metastases from solid tumors; (b) Treatment of SREs in adults or skeletally mature adolescents with giant cell tumors of bone that are unresectable or where surgical resection would result in significant morbidity; (c) treating hypercalcemia of malignancies that is refractory to bisphosphonate treatment in a subject; (d) treatment of SREs in subjects with multiple myeloma or bone metastases from solid tumors; (e) treatment of osteoporosis in postmenopausal women at high risk of fracture; (f) treatment to increase bone mass in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer; (g) Treatment to increase bone mass in men at high risk of fracture who are undergoing androgen deprivation therapy for non-metastatic prostate cancer; (h) treating men with osteoporosis who are at high risk of fracture to increase bone mass; and / or (i) Calcium or Vitamin D Therapy The pre-filled syringe according to any one of claims 1 to 17.

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