Preparations of human anti-RANKL antibodies and methods for using them

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 with related applications The interests asserted herein under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 492,056, filed on 28 April 2017, are incorporated herein by reference.

[0002] Reference to electronically submitted data The computer-readable nucleotide / amino acid sequence list submitted concurrently with this specification is incorporated herein by reference in its entirety and is identified as: "51689A_Seqlisting.txt", a 49-kilobyte ASCII (text) file created on April 20, 2018.

[0003] background Areas of disclosure This invention relates to human anti-RANKL monoclonal antibodies, such as denosumab and high-concentration aqueous formulations of its biosimilars. [Background technology]

[0004] A brief explanation of related technologies Denosumab is commercially available in solution form at concentrations of 60 mg / mL and 70 mg / mL.

[0005] As the concentration of protein formulations increases, stability issues such as aggregation, which can lead to the formation of high molecular weight species (HMWS), may occur. HMWS, particularly those that preserve a large portion 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] Filling and finishing operations, as well as administration, may involve steps of flowing the protein solution through a piston pump, peristaltic pump, or injection needle. Such processes can impart shear and mechanical stresses that can cause protein denaturation and aggregation. This phenomenon may worsen as the protein solution becomes more concentrated. [Overview of the Initiative]

[0007] The disclosure provided in accordance with the present invention demonstrates for the first time that the amount of antibody aggregates formed over time decreases, and the rate of such aggregate formation slows down, by adding an amino acid aggregation inhibitor to an aqueous solution containing a high concentration of anti-RANKL antibody. The disclosure also provides the effect of pH on aggregate formation in concentrated aqueous solutions of anti-RANKL antibody, namely, the observation that aggregate formation is reduced 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 occurs due to the interaction between the amino acid aggregation inhibitor and the antibody. Although not bound by any particular theory, it is intended that hydrophobic interactions and other types of intermolecular interactions between the amino acid aggregation inhibitor and the anti-RANKL antibody result in 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 anti-RANKL antibody that contains only low concentrations (e.g., less than about 2%) of aggregates.

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

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

[0010] In exemplary cases, an aqueous pharmaceutical formulation further includes a tonicity modifier, a surfactant, a buffer, or any combination thereof.

[0011] Another aspect of this disclosure is the presentation of formulations for storage or use, for example, in disposable vials, disposable syringes, or glass, glass-covered, or glass-covered primary containers. Exemplary aspects of this disclosure are containers, optionally vials, pre-filled syringes (PFS), or glass containers, containing any of the aqueous pharmaceutical formulations described herein. In the exemplary examples, the container contains an aqueous pharmaceutical formulation of about 1 mL or less (e.g., about 0.5 mL).

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

[0013] Another aspect of this disclosure provides a method of using the formulations described herein to prevent or treat a disease in response to a human anti-RANKL monoclonal antibody or its antigen-binding moiety. In a specific embodiment, this use encompasses therapeutic treatments in subjects including the treatment or prevention of bone-related events (SREs), the treatment or prevention of giant cell tumors of bone, the treatment or prevention of hypercalcemia in malignant tumors, the treatment or prevention of osteoporosis, or bone mass increase. For example, therapeutic interventions include: (a) treatment or prevention of SRE in subjects with bone metastases from solid tumors; (b) treatment or prevention of SRE in subjects who are adults or skeletal mature adolescents with giant cell tumors of bone that are unresectable or for which surgical resection may result in a high incidence; (c) treatment of hypercalcemia of malignant tumors that are refractory to bisphosphonate therapy in subjects; (d) treatment or prevention of SRE in subjects with multiple myeloma or bone metastases originating 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 receiving androgen deprivation therapy for non-metastatic prostate cancer; (h) treatment to increase bone mass in men with osteoporosis at high risk of fracture; and (i) treatment with calcium or vitamin D.

[0014] Further aspects of this disclosure include methods for preventing bone-related events (SREs) in patients requiring prevention of SREs, methods for treating giant cell tumors of bone in patients requiring treatment of giant cell tumors of bone, methods for treating hypercalcemia of malignant tumors in patients requiring treatment of hypercalcemia of malignant tumors, methods for treating osteoporosis in patients requiring treatment of osteoporosis, and methods for increasing bone mass in patients requiring bone mass increase. The methods include administering an effective amount of any one of the formulations described herein to a patient. In exemplary cases, the formulation is delivered to the patient subcutaneously.

[0015] Another aspect of this disclosure provides the use of denosumab, or another human anti-RANKL monoclonal antibody or its antigen-binding moiety, in the manufacture of a medicament described herein for the treatment of a patient requiring a human anti-RANKL monoclonal antibody.

[0016] Another aspect of this disclosure is a kit comprising the compositions or articles disclosed herein, along with accompanying documentation, package labels, instructions, or other indications that indicate or disclose any of the methods or embodiments disclosed herein.

[0017] Another aspect of the present disclosure is a method for improving the stability of an aqueous pharmaceutical formulation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety at a concentration greater than 70 mg / mL, comprising the step of preparing an aqueous pharmaceutical formulation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety at a pH in the range of about 5.0 to less than 5.2, wherein the aqueous pharmaceutical formulation exhibits improved stability at a pH in the range of about 5.0 to less than 5.2 compared to an equivalent aqueous pharmaceutical formulation that does not have a pH in 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 on the understanding that the present disclosure is illustrative and 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] [Figure 1] The percent of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C is shown for various high-concentration denosumab formulations. The legend in Figure 1 corresponds to the formulations having the abbreviations shown in Table 1. [Figure 2] The percent of HMWS monitored by SE-UHPLC as a function of formulation and time at 37 °C is shown for various high-concentration denosumab formulations. [Figure 3]The size exclusion chromatograms of various high-concentration denosumab formulations after storage at 37°C for one month are shown. The legend in Figure 3 corresponds to the formulations with abbreviations shown in Table 2. [Figure 4] Table 3A shows a graph of HMWS% monitored by SE-UHPLC as a function of time for each formulation with the corresponding F# shown. [Figure 5] The size exclusion chromatograms of the formulations listed in Table 3A are shown. The legend in Figure 5 corresponds to the formulation names listed in Table 3B. [Figure 6] Table 4A shows a graph of HMWS% monitored by SE-UHPLC as a function of storage time at 37°C for each formulation with the corresponding F#. [Figure 7] A shows the size exclusion chromatogram of the formulation at pH 4.8 with the denosumab concentrations listed in Table 4A. B shows the size exclusion chromatogram of the formulation at pH 5.1 with the denosumab concentrations listed in Table 4A. [Figure 8] For various high-concentration denosumab formulations, the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C is shown. The legend in Figure 8 corresponds to the letters shown in Table 5. [Figure 9] Table 6B shows a graph of HMWS% monitored by SE-UHPLC as a function of storage time at 37°C for each formulation with the corresponding F#. [Figure 10] Table 6B shows the size exclusion chromatogram as a function of the formulations after storage at 37°C for one month for the formulations with the names listed. [Figure 11] A is a graph of HMWS percentage monitored by SE-UHPLC as a function of time at 37°C for formulations with the letters shown in Table 7B. B is a graph of HMWS percentage monitored by SE-UHPLC as a function of time at 40°C for formulations with the letters shown in Table 7C. [Figure 12]A shows the size exclusion chromatogram of the formulation in Table 7B. B shows the size exclusion chromatogram of the formulation in Table 7C. [Figure 13] The graphs in Table 8A show the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37°C for each formulation with the corresponding formulation letter. Figure 13 is for formulations containing aromatic amino acids. [Figure 14] The graphs in Table 8A show the HMWS percentage monitored by SE-UHPLC as a function of storage time at 37°C for each formulation with the corresponding formulation letter. Figure 14 is for formulations containing polar / charged amino acids. [Figure 15] The graphs in Table 8A show the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37°C for each formulation with the corresponding formulation letter. Figure 15 is for formulations containing hydrophobic amino acids. [Figure 16] The images show chromatographic overlays of the formulations listed in Table 8A after storage at 37°C for one month. Figure 16 relates to formulations containing aromatic amino acids. [Figure 17] The chromatographic overlays of the formulations listed in Table 8A after storage at 37°C for one month are shown. Figure 17 relates to formulations containing polar / charged amino acids. [Figure 18] The chromatographic overlays of the formulations listed in Table 8A after storage at 37°C for one month are shown. Figure 18 relates to formulations containing hydrophobic amino acids. [Figure 19] The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for the light chain amino acid formulations 28-33. [Figure 20] The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for light chain amino acids 108-116. [Figure 21]The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for light chain amino acids 125-132. [Figure 22] The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for the heavy chain amino acid 47-59. [Figure 23] The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for heavy chain amino acids 243-253. [Figure 24] The graphs show the percentage of deuterium uptake at 4°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for heavy chain amino acids 392-399. [Figure 25] The graphs show the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for light chain amino acids 28-33. [Figure 26] The graphs show the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the light chain amino acids 108-117 for formulations 35-38. [Figure 27] The graphs show the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for light chain amino acids 124-131. [Figure 28] The graphs show the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for the heavy chain amino acid formulations 47-59. [Figure 29] The graphs show the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for heavy chain amino acids 242-253. [Figure 30] This graph shows the percentage of deuterium uptake at 37°C as a function of time (log(seconds)) for each of the formulations 35-38, specifically for heavy chain amino acids 392-399. [Figure 31]Table 10 shows a graph of HMWS percentage monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 32] Table 11 shows graphs of LMWS percentages monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 33] Table 12 shows a graph of HMWS percentage monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 34] Table 13 shows a graph of LMWS percentage monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 35] Table 14 shows a graph of HMWS percentage monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 36] Table 15 shows a graph of LMWS percentage monitored by SE-UHPLC as a function of formulation and time at 37°C for the formulations listed. [Figure 37] Table 10 shows the size exclusion chromatogram overlays for each formulation with the formulation name shown. [Figure 38] Table 12 shows the size exclusion chromatogram overlays for each formulation with the formulation name shown. [Figure 39] Table 14 shows the size exclusion chromatogram overlays for each formulation with the formulation name shown. [Figure 40] This graph includes isothermal chemical denaturation curves for denosumab in the absence of arginine at pH 4.5, 4.8, and 5.0. Figure 40A is a graph of the fraction of denatured denosumab as a function of denaturant concentration. Figure 40B is a graph plotting dF / d[denaturant] as a function of denaturant concentration. [Figure 41]Figures 41A and 41B are graphs containing isothermal chemical denaturation curves of denosumab in the presence of 75 mM arginine HCl at pH 4.5, 4.8, and 5.2. Figure 41A is a graph of the fraction of denatured denosumab as a function of denaturant concentration. Figure 41B is a graph plotting dF / d[denaturant] as a function of denaturant concentration. [Figure 42] This graph shows the percentage of HMWS monitored by SE-UHPLC as a function of time over 3 months at 25°C for the formulations whose names are listed in Table 17. [Figure 43] This graph shows the percentage of HMWS monitored by SE-UHPLC as a function of time over 2 months at 37°C for the formulations whose names are listed in Table 17. [Modes for carrying out the invention]

[0022] It would be desirable to provide concentrated aqueous solutions of denosumab and other human anti-RANKL antibodies and their antigen-binding moieties that are as stable as or more stable than diluted solutions. More concentrated solutions can provide patient convenience by enabling smaller doses, such as a 1 mL injection, rather than a 1.7 mL or 2 mL injection of a more diluted active formulation, to deliver, for example, 120 mg of the active ingredient, such as denosumab. Furthermore, even smaller injectable solutions can deliver lower doses of the active substance; for example, 0.5 mL of 120 mg / mL denosumab can deliver a 60 mg dose. It would also be desirable to provide aqueous solutions of denosumab and other human anti-RANKL antibodies and their antigen-binding moieties that are more stable than conventionally known solutions. Stable concentrated formulations also offer other advantages, such as enabling handling and shipping of smaller quantities of product and allowing for longer product storage.

[0023] Aggregates in biological products may vary in origin, size, and type. Aggregates that may affect the efficacy or safety of biological products, such as those that 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), are of particular concern, especially those that preserve a large portion of the native conformation of the monomeric counterpart. Aggregates may also potentially affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

[0024] The formation of aggregates can have various causes. Generally, protein aggregation is due to structural instability resulting from structural changes in proteins, and colloidal instability governed by intermolecular forces. When a significant nucleation event is required to induce precipitation, the dynamics of protein aggregation can be characterized by the inclusion of a delayed-time phase.

[0025] Aggregation due to structural instability involves the steps of unfolding and association. Unfolding of a protein molecule exposes hydrophobic amino acid residues. These hydrophobic residues of the unfolded molecule can then association, leading to aggregates (e.g., dimers, trimers, other polymers, and higher-order aggregates). Such associations are concentration-dependent. Increasing the protein concentration in an aqueous solvent generally increases the rate and degree of aggregation, such as thermally induced aggregation. Therefore, additives that affect the free energy of protein unfolding in solution can affect structural stability.

[0026] Colloidal instability leads to aggregates due to intermolecular association forces between proteins. Such forces can be influenced by one or more factors, including ionic strength, solution pH, and the type of buffer.

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

[0028] Therefore, the stabilized aqueous formulations according to this disclosure will resist aggregate formation to a greater extent than conventionally known formulations. One aspect of this disclosure is a stabilized aqueous formulation characterized by a pH of 5.0 to less than 5.2. Another non-exclusive aspect of this disclosure is a stabilized aqueous formulation comprising an amino acid aggregation inhibitor. Forms of provision of the relevant formulations, such as disposable vials, syringes and glass containers, and related therapeutic methods can also be provided. Methods for producing stable aqueous pharmaceutical formulations are 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 HMWS levels and HMWS formation rates at 120 mg / mL denosumab. HMWS can be described as intermolecular protein interactions that are irreversible (e.g., covalent) or reversible (e.g., non-covalent self-association interactions). There are four generally accepted causes of protein self-association reactions that can lead to increased viscosity and HMWS: hydrophobicity, charge, polarity, and dipole interactions. Both formulation pH and arginine (a highly charged basic amino acid at neutral to acidic pH values) can interfere with the intermolecular forces of charged proteins. While not intended to be bound by any particular theory, HMWS formation at 120 mg / mL denosumab is thought to be based on the charge of the protein, and these changes in formulations are thought to disrupt the charge forces involved in the mechanism of HMWS formation. Furthermore, without intending to be bound by any particular theory, since arginine contains a short aliphatic hydrocarbon chain in its side chain, it is conceivable that hydrophobic protein self-association interactions are present 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, then arginine stabilizes the anti-RANKL antibody in a different way than phenylalanine, so 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 HMWS levels and formation rates may have similar positively charged groups at neutral to acidic pH values ​​compared to arginine, and / or be hydrophobic with properties similar to phenylalanine. Examples of these additives include lysine, N-acetylarginine, N-acetyllysine, tyrosine, tryptophan, and leucine.

[0031] Unless otherwise stated, the formulations, dispensing methods, and methods of delivery 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) described below.

[0032] In jurisdictions where patents for methods performed on the human body are prohibited, “administering” a composition to a human subject shall be limited to prescribing a controlled substance for self-administration by a human subject using any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest and most reasonable interpretation is intended to be consistent with the laws or regulations defining patentable subject matter. In jurisdictions where patents for methods performed on the human body are not prohibited, “administering” a composition includes both methods performed on the human body and the aforementioned activities.

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

[0034] All maximum numerical limits given throughout this specification should be understood to include, as an alternative, the range formed by all corresponding smaller numerical limits, as if such numerical ranges were explicitly stated. All minimum numerical limits given throughout this specification should include, as an alternative, the range formed by all higher numerical limits, as if such ranges were explicitly stated. All numerical ranges given throughout this specification should include all narrower numerical ranges contained within such wider numerical ranges, as if all such narrower numerical ranges were explicitly stated herein. Dimensions and values ​​disclosed herein should be understood to include disclosures of both the stated value and the corresponding exact numerical value; for example, a value stated as "about 10mM" should be understood to include "10mM" as an alternative disclosure.

[0035] As used herein, the term “therapeutic effective dose” refers to a sufficient amount of a compound to treat, improve or prevent a specified disease or condition, or to exhibit a detectable therapeutic, preventive, or inhibitory effect. This effect may be detected, for example, by improvement of the clinical condition or reduction of symptoms. The exact effective dose for a subject depends on the subject's weight, size, and health status, the nature and severity of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. If the drug is approved by the U.S. Food and Drug Administration (FDA), “therapeutic effective dose” refers to the dose approved by the FDA or its corresponding foreign agency for the treatment of a specified disease or condition.

[0036] This disclosure provides stabilized (or stable) aqueous pharmaceutical formulations, indicated by a decrease in the amount of aggregates and / or the rate of aggregate formation after storage. As described herein, the stability of such formulations is indicated by a decrease in the amount of HMWS and / or the rate of HMWS formation after storage for various times and at various temperatures. Generally, formulations with higher stability are associated with lower HMWS amounts, lower HMWS formation rates and / or higher antibody principal peaks at higher storage temperatures compared to lower temperatures. As used herein, the terms “high molecular weight species” or “HMWS” refer to higher-order aggregates of the antibody in the formulation, as well as lower-order aggregates of the antibody in the formulation. Lower-order aggregates include, for example, dimeric species. The amount and rate of aggregation can be measured or monitored by techniques such as SE-UHPLC. SE-UHPLC chromatograms of antibodies, in some examples, show a peak around 5.8 min representing the amount of HMWS in the aqueous pharmaceutical formulation, a peak around 6.7 min representing the dimer species, and a peak around 8.0 min reflecting the amount of intact, non-aggregated antibody. Storage at 37°C compared to 4°C can accelerate stability assays, allowing the stability of a particular formulation to be measured in a shorter time than at 4°C. For example, storage at 37°C for one, two, or three months may suggest or predict storage at 36 months at 4°C.

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

[0038] In another type of embodiment, a stabilized formulation containing an amino acid aggregation inhibitor described herein will result in a reduced degree of HWMS formation after storage at 37°C for one month compared to an equivalent control formulation without an amino acid aggregation inhibitor. The degree of formation may be reduced, for example, by a range of about 0.1% to 2%, or about 0.1% to 1%, compared to a control formulation stored at 37°C for one month, such as the HMWS% amount by SE-UPHLC being 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%, or about 0.7%, or about 0.1% to 2%, or about 0.1% to 1%.

[0039] In another type of embodiment, the stabilized formulation described herein will have a small amount of HMWS after storage at 37°C for one month, as measured 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, or less than 1.3%, or 1. The amount may be less than 0.2%, or less than 1.2%, for example, in the range of approximately 0.01% to 2%, or approximately 0.01% to 1.9%, or approximately 0.01% to 1.8%, or approximately 0.01% to 1.7%, or approximately 0.01% to 1.6%, or approximately 0.01% to 1.5%, or approximately 0.01% to 1.4%, or approximately 0.01% to 1.3%, or approximately 0.01% to 1.2%. In another type of embodiment, the amount of HMWS after 1 month of storage at 37°C by SE-UHPLC may be greater than 2%, for example, greater than 2% up to 3%, while the reduction in aggregation rate brought about by amino acid aggregation inhibitors can enable a suitable product storage period, up to 3 years, or up to 2 years.

[0040] In another type of embodiment, the stabilized formulation described herein would have a small amount of HMWS after storage at 37°C for 3 months, as measured by SE-UHPLC. For example, the amount of HMWS could 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, or less than 1.3%, or 1. The percentage may be less than 0.2%, or less than 1.2%, for example, in the range of approximately 0.01% to 2%, or approximately 0.01% to 1.9%, or approximately 0.01% to 1.8%, or approximately 0.01% to 1.7%, or approximately 0.01% to 1.6%, or approximately 0.01% to 1.5%, or approximately 0.01% to 1.4%, or approximately 0.01% to 1.3%, or approximately 0.01% to 1.2%.

[0041] In another type of embodiment, the stabilized formulation described herein will have a small amount of HMWS after storage at 4°C for 36 months, as measured 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, or less than 1.3%, or 1. The percentage may be less than 0.2%, or less than 1.2%, for example, in the range of approximately 0.01% to 2%, or approximately 0.01% to 1.9%, or approximately 0.01% to 1.8%, or approximately 0.01% to 1.7%, or approximately 0.01% to 1.6%, or approximately 0.01% to 1.5%, or approximately 0.01% to 1.4%, or approximately 0.01% to 1.3%, or approximately 0.01% to 1.2%.

[0042] In another type of embodiment, the stabilized formulation described herein would have a high amount of denosumab or other antibody (or its antigen-binding moiety) main peak after storage at 37°C for one month, as measured by SE-UHPLC. For example, the amount of the main peak would be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or less It could also be in the range of 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example, approximately 95-99.9%, or approximately 96-99.9%, or approximately 97-99.9%, or approximately 97.5-99.9%, or approximately 98-99.9%, or approximately 98.1-99.9%, or approximately 98.2-99.9%, or approximately 98.3-99.9%, or approximately 98.4-99.9%, or approximately 98.5-99.9%, or approximately 98.6-99.9%.

[0043] In another type of embodiment, the stabilized formulation described herein would have a high amount of the primary peak of denosumab or other antibody (or its antigen-binding moiety) after storage at 37°C for 3 months, as measured by SE-UHPLC. For example, the amount of the primary peak would be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or less It could also be in the range of 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example, approximately 95-99.9%, or approximately 96-99.9%, or approximately 97-99.9%, or approximately 97.5-99.9%, or approximately 98-99.9%, or approximately 98.1-99.9%, or approximately 98.2-99.9%, or approximately 98.3-99.9%, or approximately 98.4-99.9%, or approximately 98.5-99.9%, or approximately 98.6-99.9%.

[0044] In another type of embodiment, the stabilized formulation described herein will have a high amount of denosumab or other antibody (or its antigen-binding moiety) main peak after storage at 4°C for 36 months by SE-UHPLC. For example, the amount of the main peak will be at least 95%, or greater than 95%, or at least 96%, or greater than 96%, or at least 97%, or greater than 97%, or at least 97.5%, or greater than 97.5%, or at least 98%, or greater than 98%, or at least 98.1%, or greater than 98.1%, or at least 98.2%, or greater than 98.2%, or at least 98.3%, or greater than 98.3%, or at least 98.4%, or greater than 98.4%, or less It could also be in the range of 98.5%, or greater than 98.5%, or at least 98.6%, or greater than 98.6%, for example, approximately 95-99.9%, or approximately 96-99.9%, or approximately 97-99.9%, or approximately 97.5-99.9%, or approximately 98-99.9%, or approximately 98.1-99.9%, or approximately 98.2-99.9%, or approximately 98.3-99.9%, or approximately 98.4-99.9%, or approximately 98.5-99.9%, or approximately 98.6-99.9%.

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

[0046] In exemplary embodiments, the aqueous pharmaceutical formulation contains approximately 4% or less of high molecular weight species (HMWS) and / or more than 96% of the antibody main peak, as measured by SE-UHPLC after storage. In exemplary embodiments, the aqueous pharmaceutical formulation contains approximately 3% or less of high molecular weight species (HMWS) and / or more than 97% of the antibody main peak, as measured by SE-UHPLC after storage. In exemplary embodiments, the aqueous pharmaceutical formulation contains approximately 2% or less of HMWS and / or more than 98% of the antibody main peak, as measured by SE-UHPLC after storage. In exemplary embodiments, 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 a period of 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, or optionally longer). Exemplary properties indicate that storage is appropriate at approximately 20°C to 30°C (for example, approximately 21°C to 30°C, approximately 22°C to 30°C, approximately 23°C to 30°C, approximately 24°C to 30°C, approximately 25°C to 30°C, approximately 26°C to 30°C, approximately 27°C to 30°C, approximately 28°C to 30°C, approximately 28°C to 30°C, approximately 20°C to 29°C, approximately 20°C to 2 The storage period is approximately one month (for example, approximately 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 days) at temperatures of approximately 20°C to 8°C. In exemplary embodiments, storage includes a first storage period followed by a second storage period, the first storage period being approximately 12, 24, or 36 months at approximately 2°C to 8°C, and the second storage period being approximately one month at approximately 20°C to 30°C.In a typical example, an aqueous pharmaceutical formulation contains 2% or less HMWS, or less than 2% HMWS, or 1.9% or less HMWS, or less than 1.9% HMWS, or 1.8% or less HMWS, or less than 1.8% HMWS, or 1.7% or less HMWS, or less than 1.7% HMWS, or 1.6% or less HMWS, or less than 1.6% HMWS, or 1.5% or less HMWS, or less than 1.5% HMWS, or 1.4% or less HMWS, or less than 1.4% HMWS, or 1.3% or less HMWS, or less than 1.3% HMWS, Alternatively, it may include HMWS of 1.2% or less, or HMWS of less than 1.2%, for example, HMWS of approximately 0.01% to 2%, or approximately 0.01% to 1.9%, or approximately 0.01% to 1.8%, or approximately 0.01% to 1.7%, or approximately 0.01% to 1.6%, or approximately 0.01% to 1.5%, or approximately 0.01% to 1.4%, or approximately 0.01% to 1.3%, or approximately 0.01% to 1.2%, and this HMWS may optionally be measured by SE-UHPLC.In an alternative or additional embodiment, the aqueous pharmaceutical formulation has an antibody main peak of more than 98%, or 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%, 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% A peak, or a primary antibody peak with over 98.5% antibody concentration, or a primary antibody peak with at least 98.6% antibody concentration, or a primary antibody peak with over 98.6% antibody concentration, for example, a primary antibody peak in the range of approximately 95% to approximately 99.9%, or a primary antibody peak in the range of approximately 96% to approximately 99.9%, or a primary antibody peak in the range of approximately 97% to approximately 99.9%, or a primary antibody peak in the range of approximately 97.5% to approximately 99.9%, or a primary antibody peak in the range of approximately 98% to approximately 99.9%, and The antibody main peaks include those in the range of approximately 98.1% to 99.9%, or 98.2% to 99.9%, or 98.3% to 99.9%, or 98.4% to 99.9%, or 98.5% to 99.9%, or 98.6% to 99.9%, and these peaks are optionally measured by SE-UHPLC.

[0047] As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, a variable region and a constant region. For example, an antibody may be an IgG antibody, which is a “Y-type” 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 a variable region and a constant region. In the IgG format, the variable region generally consists of about 100–110 or more amino acids and includes three complementarity-determining regions (CDRs), which are primarily involved in antigen recognition and substantially vary between other antibodies that bind to different antigens. For example, Janeway et.al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th See ed. Elsevier Science Ltd. / Garland Publishing, (1999).

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

[0049] Human light chains are classified into kappa light chains and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, defining antibody isotypes 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 this disclosure encompass all such classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, e.g., a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. Therefore, in exemplary embodiments, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody containing one of IgG1, IgG2, IgG3, or IgG4. In exemplary embodiments, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody.

[0050] In various embodiments, antibodies can be monoclonal or polyclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to natural antibodies produced by mammals, such as mice, rats, rabbits, goats, horses, chickens, hamsters, pigs, and humans. In this regard, antibodies can be considered mammalian antibodies, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, pig antibodies, and human antibodies. In certain embodiments, anti-RANKL antibodies are monoclonal human antibodies. In certain embodiments, recombinant proteins are chimeric antibodies or humanized antibodies. The term “chimeric antibody” is used herein to refer to an antibody that contains a constant domain from one species and a variable domain from a second species, or more generally, a sequence of amino acids from at least two species. The term “humanized,” when used in relation to antibodies, refers to an antibody derived from a non-human source that has been engineered to have a structure and immune function more similar to a true human antibody than to the original source antibody, and has at least a CDR region. For example, humanization may involve transplanting a CDR derived from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization may also involve selected amino acid substitutions to make the non-human sequence appear like a human sequence.

[0051] In various embodiments, antibodies are 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 exemplary embodiments, an aqueous pharmaceutical formulation comprises an antibody fragment, e.g., Fab, Fc, F(ab')2, or pFc', which holds at least one antigen (RANKL) binding site. With respect to aqueous pharmaceutical formulations and methods of this disclosure, the antibody may lack a particular portion of the antibody and may be an antibody fragment that binds to RANKL. In exemplary embodiments, 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 full antigen-binding sites is the Fv fragment, which consists of a fully variable (V) region. A soluble and flexible amino acid peptide linker is used to stabilize the molecule by conjugating the V region to an scFv (single-stranded fragment variable) fragment, or by adding 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 readily produced in a host (e.g., a prokaryotic host). Other antibody protein products include disulfide-bonded scFv (ds-scFv), single-stranded Fab (scFab), and minibodies (miniAb) containing different formats consisting of dimeric and multimeric antibody formats such as dia, tria, and tetramomers, or scFv linked to an oligomeric domain. The smallest fragments are the VHH / VH of the camelid heavy chain Ab, as well as single-domain Ab (sdAb). The most frequently used building blocks for creating novel antibody formats are single-stranded variable (V)-domain antibody fragments (scFv) containing V domains (VH and VL domains) derived from the heavy and light chains, linked by a peptide linker of approximately 15 amino acid residues. Peptibodies, or peptide-Fc fusions, are yet another antibody protein product. The structure of a peptide body consists of a biologically active peptide transplanted onto an Fc domain. Peptibodies are well described in this field; see, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0053] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or trispecific antibodies. Bispecific antibodies can be classified into five main classes: BsIgG, IgG with ligation, 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 exemplary embodiments, an anti-RANKL antibody or its antigen-binding moiety comprises, substantially consists of, or comprises one of these antibody protein products (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camel heavy chain antibody, sdAb, diabody, triabody, tetrabody, bispecific or triplicate antibodies, BsIgG, added IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate).

[0055] In exemplary embodiments, an anti-RANKL antibody or its antigen-binding moiety comprises, substantially comprises, or comprises an antibody protein product in monomeric or polymeric form, oligomeric or multimeric form. In certain embodiments in which the antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered to be bispecific, triplicate, or polyspecific, or divalent, trivalent, or polyvalent, 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 its antigen-binding moiety used in the formulation is an antibody or its antigen-binding moiety that specifically binds to the human osteoproteglin (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 referred to herein as a human anti-RANKL monoclonal antibody or its antigen-binding moiety. For example, the formulation described herein may include a human anti-RANKL monoclonal antibody that specifically binds to the amino acid sequence of human RANKL (SEQ ID NO: 12) or a portion thereof. The human RANKL protein is a transmembrane protein or soluble protein encoded by the polynucleotide sequence of SEQ ID NO: 11, which is known to be essential for the formation, function, and survival of osteoclasts. 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 marketed as Xgeva® and Prolia®. Xgeva® is a 120 mg dose formulation of denosumab in a 1.7 mL solution (70 mg / mL) in a single-use vial, containing 120 mg of denosumab, 18 mM sorbitol, 4.6% sorbitol, water for injection (USP), and sodium hydroxide to adjust the pH to 5.2. Prolia® is available as a 60 mg dose formulation of denosumab in a 1 mL solution (60 mg / mL). Each 1 mL disposable syringe of Prolia® contains 60 mg of denosumab (60 mg / mL solution), 4.7% sorbitol, 17 mM sorbitol, 0.01% polysorbate 20, water for injection (USP), and sodium hydroxide to adjust the pH to 5.2. Formulations comprising denosumab or a portion thereof, as described herein, are particularly intended. 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 the 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 NOs: 1 and 2, respectively, and the full-length LC and HC are shown as SEQ ID NOs: 3 and 4, respectively. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 1 (denosumab variable LC) is, in some embodiments, the nucleic acid of SEQ ID NOs: 19. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 2 (denosumab variant HC) is, in some embodiments, the nucleic acid of SEQ ID NOs: 20. The nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 3 (full-length denosumab LC) is, in some embodiments, the nucleic acid of SEQ ID NOs: 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 LC, represented as amino acids 21-235 of full-length LC, is shown as SEQ ID NO: 13, and the mature form of HC, represented as amino acids 20-467 of full-length HC, is shown as SEQ ID NO: 14.A nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13 (the mature form of LC) is, in some embodiments, the nucleic acid of SEQ ID NO: 22. A nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (the mature form of HC) is, in some embodiments, the nucleic acid of SEQ ID NO: 24. Furthermore, denosumab LC CDR is indicated as SEQ ID NO: 5 (LC CDR1), SEQ ID NO: 6 (LC CDR2), and SEQ ID NO: 7 (LC CDR3). Denosumab HC CDR is indicated 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 incorporated herein by reference in their entirety.

[0058] As used herein, the term “denosumab” includes biosimilars of denosumab. As used herein, “biosimilar” (of an authorized reference product / biological agent, e.g., protein therapies, antibodies, etc.) refers to a biological agent that is similar to a reference product based on data derived from (a) analytical studies demonstrating that the biological agent is very similar to the reference product despite minor differences in clinically inactive components, (b) animal studies (including toxicity assessments), and / or (c) one or more clinical studies (including immunogenicity and pharmacokinetic or pharmacodynamic assessments) sufficient to demonstrate safety, purity and efficacy under one or more appropriate conditions of use in which the reference product is authorized, intended for use, and for which a license is sought. In one embodiment, the biosimilar biological agent and the reference product utilize the same one or more mechanisms of action under the one or more conditions of use specified, recommended, or suggested in the proposed representation, to the extent that one or more mechanisms of action are known for the reference product. In one embodiment, one or more conditions of use specified, recommended or suggested in the proposed labeling for the biologic product are previously approved for the reference product. In one embodiment, the route of administration, dosage form, and / or potency of the biologic product are the same as those of the reference product. In one embodiment, the facility in which the biologic product is manufactured, processed, packaged or stored meets standards designed to ensure that the biologic product remains safe, pure, and potent. The reference product may be approved in at least one of the United States, Europe, or Japan. A biosimilar may be, for example, an antibody having the same primary amino acid sequence as a commercially available antibody, but may be produced in a different cell type or by different manufacturing, purification, or formulation methods.

[0059] The formulation may contain a human anti-RANKL antibody comprising at least one of the amino acid sequences of SEQ ID NOs: 1-4, 13, 14 or a portion thereof. The formulation may contain a human anti-RANKL antibody comprising at least one of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10, or at least two of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10, or at least three of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10, or at least four of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10, or at least five of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10, or at least six of the CDR amino acid sequences shown as SEQ ID NOs: 5, 6, 7, 8, 9, or 10.

[0060] The formulation is a human anti-RANKL antibody that is at least 80% identical to any one of SEQ ID NOs: 1-4, 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: 1-4, 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: 1-4, 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: 1-4, 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: 1-4, 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 sequence numbers 1-4, 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 sequence numbers 1-4, 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 sequence numbers 1-4, 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 sequence numbers 1-4, 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 sequence numbers 1-4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK, or sequence numbers 1-4,This may include a human anti-RANKL antibody that is at least 98% identical to one of sequences 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 99% identical to one of sequences 1-4, 13, and 14 and contains at least one amino acid sequence that inhibits the interaction between RANKL and its receptor, RANK.

[0061] In exemplary embodiments, the aqueous pharmaceutical formulation comprises an anti-RANKL antibody or its antigen-binding moiety (such as an antibody protein product) as described herein. In exemplary embodiments, the anti-RANKL antibody or its antigen-binding moiety comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence described in SEQ ID NO: 5. In alternative or additional embodiments, the anti-RANKL antibody or its antigen-binding moiety comprises a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence described in SEQ ID NO: 6. In alternative or additional embodiments, the anti-RANKL antibody or its antigen-binding moiety comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence described in SEQ ID NO: 10. In some examples, the anti-RANKL antibody or its antigen-binding moiety comprises SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 10. In exemplary embodiments, an anti-RANKL antibody or its antigen-binding moiety includes (i) a light chain variable domain including a light chain CDR3 sequence containing the amino acid sequence described in SEQ ID NO: 7, (ii) a heavy chain variable domain including a heavy chain CDR1 sequence containing the amino acid sequence described in SEQ ID NO: 8, or optionally SEQ ID NO: 27, (iii) a heavy chain variable domain including a heavy chain CDR2 sequence containing the amino acid sequence described in SEQ ID NO: 9, or (iv) any combination thereof. In some embodiments, an anti-RANKL antibody or its antigen-binding moiety includes (A) a light chain variable domain including a light chain CDR1 sequence containing the amino acid sequence of SEQ ID NO: 5, a light chain variable domain including a light chain CDR2 sequence containing the amino acid sequence of SEQ ID NO: 6, and a light chain variable domain including a light chain CDR3 sequence containing the amino acid sequence of SEQ ID NO: 7, and (B) a heavy chain variable domain including a heavy chain CDR1 sequence containing the amino acid sequence of SEQ ID NO: 8 (or optionally SEQ ID NO: 27), a heavy chain variable domain including a heavy chain CDR2 sequence containing the amino acid sequence of SEQ ID NO: 9, and a heavy chain variable domain including a heavy chain CDR3 sequence containing the amino acid sequence of SEQ ID NO: 10.In exemplary embodiments, the anti-RANKL antibody or its antigen-binding moiety is a light chain variable domain selected from the group consisting of (A)(i) a light chain variable domain comprising 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%) of an amino acid sequence 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, or (iii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide complement comprising SEQ ID NO: 19, or (B)( i) a heavy chain variable domain selected from the group consisting of (i) a heavy chain variable domain containing an amino acid sequence identical to 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%) of SEQ ID NO: 2, (ii) a heavy chain variable domain containing an amino acid sequence encoded by a polynucleotide sequence containing SEQ ID NO: 20, and (iii) a heavy chain variable domain containing an amino acid sequence encoded by a polynucleotide that hybridizes to a complement of the polynucleotide comprising SEQ ID NO: 20 under stringent conditions, or (C) a light chain variable domain of (A) and a heavy chain variable domain of (B). In exemplary embodiments, the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In exemplary embodiments, the antigen-binding moiety is Fab, Fab', F(ab')2, or single-stranded Fv. In exemplary embodiments, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody, and optionally, the anti-RANKL antibody contains the sequence of SEQ ID NO: 15. In some embodiments, the anti-RANKL antibody contains the sequence of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.In exemplary embodiments, the anti-RANKL antibody or its antigen-binding moiety is a light chain selected from the group consisting of (A)(i) a light chain containing 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%) of an amino acid sequence identical to SEQ ID NO: 3 or SEQ ID NO: 13, (ii) a light chain containing an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 21 or 23, or (iii) a light chain containing an amino acid sequence encoded by a polynucleotide that hybridizes to a complement of the polynucleotide consisting of SEQ ID NO: 21 or 23 under stringent conditions, or (B) A heavy chain selected from the group consisting of (i) a heavy chain comprising 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%) of an amino acid sequence identical to SEQ ID NO: 4 or SEQ ID NO: 14, (ii) a heavy chain comprising an amino acid sequence encoded by a 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 to a complement of the polynucleotide comprising SEQ ID NO: 22 or 24 under stringent conditions, or (C) comprising 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 antibodies or their antigen-binding moieties in aqueous formulations can generally be within any useful range, e.g., about 0.1 to about 200 mg / mL. As the concentration increases, there is an increase in viscosity, which may hinder the processing of the formulation into sterile compounding forms for pharmaceutical use.

[0063] In one embodiment, the improved stability of the formulation by an amino acid aggregation inhibitor is that of denosumab or other human anti-RANKL antibody or its antigen-binding moiety, in the ranges of approximately 10 mg / mL to approximately 200 mg / mL, or approximately 15 mg / mL to approximately 150 mg / mL, or approximately 30 mg / mL to approximately 200 mg / mL, or approximately 60 mg / mL to approximately 200 mg / mL, or approximately 60 mg / mL to approximately 180 mg / mL, or approximately 60 mg / mL to approximately 160 mg / mL, or approximately 60 mg / mL to approximately 150 mg / mL, or approximately 60 mg / mL to approximately 140 mg / mL, or approximately 60 mg / mL to approximately 130 mg / mL, or approximately 60 mg / mL to approximately 120 mg / mL, or approximately 60 mg / mL to approximately 110 mg / mL, or approximately 60 mg / mL to approximately 100 mg / mL, and It can be present at any concentration, such as approximately 60 mg / mL to approximately 90 mg / mL, or approximately 60 mg / mL to approximately 80 mg / mL, or approximately 60 mg / mL to approximately 70 mg / mL, or approximately 70 mg / mL to approximately 200 mg / mL, or approximately 70 mg / mL to approximately 180 mg / mL, or approximately 70 mg / mL to approximately 160 mg / mL, or approximately 70 mg / mL to approximately 150 mg / mL, or approximately 70 mg / mL to approximately 140 mg / mL, or approximately 70 mg / mL to approximately 130 mg / mL, or approximately 70 mg / mL to approximately 120 mg / mL, or approximately 70 mg / mL to approximately 110 mg / mL, or approximately 70 mg / mL to approximately 100 mg / mL, or approximately 70 mg / mL to approximately 90 mg / mL, or approximately 70 mg / mL to approximately 80 mg / mL, for example, 120 mg / mL.

[0064] In another embodiment, the concentration of denosumab or other human anti-RANKL antibody or its antigen-binding moiety in a formulation having a pH of about 5.0 to less than 5.2 is intended to include a range of 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, the intended ranges are 71 mg / mL to approximately 200 mg / mL, or approximately 75 mg / mL to approximately 200 mg / mL, or approximately 75 mg / mL to approximately 180 mg / mL, or approximately 75 mg / mL to approximately 160 mg / mL, or approximately 75 mg / mL to approximately 150 mg / mL, or approximately 75 mg / mL to approximately 140 mg / mL, or approximately 75 mg / mL to approximately 130 mg / mL, or approximately 75 mg / mL to approximately 120 mg / mL Examples include mg / mL, or approximately 75 mg / mL to approximately 110 mg / mL, or approximately 75 mg / mL to approximately 100 mg / mL, or approximately 75 mg / mL to approximately 90 mg / mL, or approximately 120 mg / mL to approximately 200 mg / mL, or approximately 120 mg / mL to approximately 180 mg / mL, or approximately 120 mg / mL to approximately 160 mg / mL, or approximately 120 mg / mL to approximately 140 mg / mL, for example, approximately 120 mg / mL.

[0065] In exemplary embodiments, the aqueous pharmaceutical preparation contains the antibody or its antigen-binding portion at a concentration greater than 70 mg / mL, for example, 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, or greater than 275 mg / mL. In exemplary embodiments, the aqueous pharmaceutical preparation contains the antibody or its antigen-binding portion at a concentration of less than approximately 300 mg / mL, for example, less than approximately 275 mg / mL, less than approximately 250 mg / mL, less than approximately 225 mg / mL, less than approximately 200 mg / mL, less than approximately 175 mg / mL, or less than approximately 150 mg / mL. In exemplary embodiments, the concentration of the antibody or antigen-binding portion in the formulation is approximately 10 mg / mL to approximately 300 mg / mL, for example, approximately 25 mg / mL to approximately 300 mg / mL, approximately 50 mg / mL to approximately 300 mg / mL, approximately 75 mg / mL to approximately 300 mg / mL, approximately 125 mg / mL to approximately 300 mg / mL, approximately 150 mg / mL to approximately 300 mg / mL, approximately 175 mg / mL to approximately 300 mg / mL, approximately 200 mg / mL to approximately 300 mg / mL, approximately 225 mg / mL to approximately 300 mg / mL, approximately 250 mg / mL to approximately 300 mg / mL, and approximately 2 The ranges are approximately 75 mg / mL to 300 mg / mL, approximately 10 mg / mL to 275 mg / mL, approximately 10 mg / mL to 250 mg / mL, approximately 10 mg / mL to 225 mg / mL, approximately 10 mg / mL to 200 mg / mL, approximately 10 mg / mL to 175 mg / mL, approximately 10 mg / mL to 150 mg / mL, approximately 10 mg / mL to 125 mg / mL, approximately 10 mg / mL to 100 mg / mL, approximately 10 mg / mL to 75 mg / mL, approximately 10 mg / mL to 50 mg / mL, or approximately 10 mg / mL to 25 mg / mL.In exemplary embodiments, the aqueous pharmaceutical preparation contains an antibody or its antigen-binding portion in a range exceeding 70 mg / mL up to about 300 mg / mL, for example, in a range exceeding 80 mg / mL up to about 300 mg / mL, in a range exceeding 90 mg / mL up to about 300 mg / mL, in a range exceeding 100 mg / mL up to about 300 mg / mL, in a range exceeding 125 mg / mL up to about 300 mg / mL, in a range exceeding 150 mg / mL up to about 300 mg / mL, in a range exceeding 175 mg / mL up to about 300 mg / mL, and in a range exceeding 200 mg / mL up to about 3 The formulation contains the antibody or its antigen-binding moiety in concentrations ranging from approximately 100 mg / mL to approximately 275 mg / mL, from approximately 70 mg / mL to approximately 250 mg / mL, from approximately 70 mg / mL to approximately 225 mg / mL, from approximately 70 mg / mL to approximately 200 mg / mL, from approximately 70 mg / mL to approximately 175 mg / mL, from approximately 70 mg / mL to approximately 150 mg / mL, from approximately 70 mg / mL to approximately 125 mg / mL, and from approximately 70 mg / mL to approximately 100 mg / mL. In an exemplary embodiment, the aqueous pharmaceutical formulation contains the antibody or its antigen-binding moiety in concentrations ranging from approximately 100 mg / mL to approximately 140 mg / mL, for example, approximately 110 mg / mL, approximately 120 mg / mL, and approximately 130 mg / mL. In some embodiments, the aqueous pharmaceutical preparation contains an antibody or its antigen-binding moiety at concentrations of approximately 120 mg / mL ± 12 mg / mL, for example, approximately 108 mg / mL to approximately 132 mg / mL, approximately 115 mg / mL to approximately 125 mg / mL, approximately 116 mg / mL, approximately 117 mg / mL, approximately 118 mg / mL, approximately 119 mg / mL, approximately 120 mg / mL, approximately 121 mg / mL, approximately 122 mg / mL, approximately 123 mg / mL, and approximately 124 mg / mL.

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

[0067] Formulation studies of the high-concentration denosumab solutions described below (e.g., 120 mg / mL) showed a significant increase in HMWS formation (rate and degree) at pH less than 5, particularly at lower pH levels (e.g., pH 4.5). It was shown that the formation of dimer species increased with increasing pH. To balance these two effects, the formulations described herein are intended to have a pH in the range of approximately 5.0 to less than 5.2, or approximately 5.0 to approximately 5.19, or approximately 5.0 to approximately 5.15, or approximately 5.0 to approximately 5.10, e.g., approximately 5.0, approximately 5.05, approximately 5.1, or approximately 5.15.

[0068] The studies described herein have also shown independently stabilizing and aggregation-reducing effects made possible by the inclusion of amino acid aggregation inhibitors. Therefore, when amino acid aggregation inhibitors are included, the pH of the formulation is intended to 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 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, for example, about 5.0, about 5.05, about 5.1, or about 5.15, or about 5.2.

[0069] Aqueous formulations can be buffered. When used, the buffer may 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 acetate / acetate, which has a pKa of approximately 4.75 at 25°C. Another such buffer system is glutamate / glutamate, which has a pKa of approximately 4.27 at 25°C. Other alternative buffer systems that may be considered 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). The buffer is intended to be provided as a sodium salt (or disodium salt as needed), or alternatively as a potassium, magnesium, or ammonium salt. The buffer can be based on, for example, acetate, citrate, succinate, phosphate, and hydroxymethylaminomethane (Tris). Buffers based on acetate, glutamate, and succinate are particularly intended, for example, acetate or glutamate.

[0070] A comparison of HMWS formation in 120 mg / mL denosumab formulations with acetate or glutamate buffer (otherwise identical) by size exclusion ultra-high performance liquid chromatography (SE-UHPLC) showed no difference based on 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 may range from approximately 2 mM to approximately 40 mM, or approximately 5 mM to approximately 20 mM, or approximately 10 mM to approximately 25 mM, or approximately 15 mM to approximately 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 may range from approximately 2 mM to approximately 30 mM, or approximately 16 mM to approximately 41 mM, or approximately 25 mM to approximately 39 mM, or approximately 30 mM to approximately 34 mM. In other words, the diafiltration buffer used to concentrate antibodies to concentrations exceeding 70 mg / mL (e.g., 120 mg / mL) may be in the range of 5 mM to about 30 mM, or about 15 mM to about 25 mM, or about 20 mM. It is also intended to provide self-buffered amino acid-stabilized formulations. In exemplary embodiments, the buffer is contained in an amount sufficient to maintain the formulation at a selected pH under storage conditions for the product's shelf life, for example, 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] In some embodiments, the aqueous pharmaceutical formulation includes a buffer, which may be centered on a pH range of approximately 4.0 to approximately 5.5 at 25°C. In some embodiments, the buffer has a pKa within 1 pH unit of pH 5.0 to 5.2 at 25°C. In certain embodiments, the aqueous pharmaceutical formulation includes a buffer of approximately 5 mM to approximately 60 mM, approximately 5 mM to approximately 50 mM, or approximately 9 mM to approximately 45 mM (e.g., approximately 15 mM to approximately 30 mM, e.g., approximately 20 mM, approximately 25 mM buffer). In exemplary embodiments, the buffer is an acetate or glutamate.

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

[0074] The term "amino acid aggregation inhibitor" refers to any given amino acid or combination of amino acids (e.g., mixtures or dipeptides or oligopeptides having 2-10 residues), or amino acid analogs, in which any given amino acid exists in the form of its free base or a salt thereof (e.g., arginine HCl), which reduces or inhibits HMWS formation. Salts may include sodium salts, potassium salts, and hydrochloride salts. Furthermore, arginine salts, glutamates, butyrates, and glycolates with hydrochloride are also considered. When amino acid combinations are used, all amino acids may exist in the form of their free bases, all may exist in the form of their salts, or some may exist in the form of their free bases 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, may be used. In another embodiment, only one type of amino acid aggregation inhibitor is present in the aqueous pharmaceutical formulation. In exemplary embodiments, only one amino acid is present in the formulation, such as only L-arginine or only L-phenylalanine.

[0075] The invention intends to use one or more amino acids having a charged side chain, such as one or more of arginine, lysine, histidine, aspartic acid, and glutamic acid. The amino acids can be selected from basic amino acids, such as arginine, lysine, histidine, or combinations thereof. Arginine is particularly intended. Any stereoisomer of a particular amino acid (i.e., L, D, or DL ​​isomers), or combinations of these stereoisomers, may be used in the methods or formulations of the invention, insofar as the particular amino acid exists in the form of its free base or its base. The L-stereoisomer, such as L-arginine, is particularly intended. Optionally, the amino acid may have a positively charged side chain, such as arginine.

[0076] In another embodiment, it is intended to use one or more amino acids having aromatic rings in their side chains, such as phenylalanine, tyrosine, tryptophan, or a combination thereof. Phenylalanine is particularly intended.

[0077] In another embodiment, one or more hydrophobic amino acids, such as alanine, isoleucine, leucine, phenylalanine, valine, proline, or glycine, are intended to be used.

[0078] In another embodiment, one or more aliphatic hydrophobic amino acids, such as alanine, isoleucine, leucine, or valine, are intended to be used. Leucine is particularly intended.

[0079] Amino acid analogs exhibiting aggregation reduction or aggregation inhibition effects may 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. Analogs intended include, for example, amino, N-monoethyl, and n-acetyl derivatives. Other analogs intended include dipeptides or oligopeptides having 2 to 10 residues, such as arginine-arginine and phenylalanine-arginine. In one type of embodiment, it is considered that n-acetylarginine and n-acetyllysine may not be used alone but may be used in combination with other amino acid aggregation inhibitors. Similar to amino acids, amino acid analogs may be used in the methods or formulations of the present invention in either the form of their free base or a salt thereof.

[0080] The amino acid aggregation inhibitors (plural) used in the methods or formulations of the present invention protect therapeutically active proteins from various stresses, thereby increasing and / or maintaining the stability of the protein or protein-containing formulations throughout the lifespan 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 transport. Thermal stress is particularly intended. The term stress encompasses any factor that modulates (i.e., reduces, maintains, or increases) the stability of a protein or protein-containing formulation. The increase and / or maintenance of stability by the addition of an amino acid aggregation inhibitor occurs in a concentration-dependent manner. That is, increasing the concentration of the amino acid aggregation inhibitor leads to increased and / or maintenance of the stability of the protein or formulation of the present invention, where the protein or protein-containing formulation would normally exhibit aggregate formation in the absence of the amino acid aggregation inhibitor. The amount of HMWS already formed can also be reduced by including an amino acid aggregation inhibitor in the formulation, as shown in the following examples. For example, such amino acid aggregation inhibitors include arginine and arginine-phenylalanine dipeptides. Protein stability can be increased by determining the amount of a specific amino acid aggregation inhibitor used in the method or formulation to reduce aggregate formation, and therefore the improved stability of the formulation throughout the protein's entire lifespan can be easily determined for denosumab or any specific human anti-RANKL monoclonal antibody of interest, taking into account the disclosures herein.

[0081] The presence of amino acid aggregation inhibitors in formulations has been shown to reduce the amount and rate of dimer formation. For example, when arginine was added at a concentration of 75 mM to a denosumab formulation at pH 5.2, the amount and rate of dimer formation after 1 month at 37°C were reduced by approximately 0.3% and 25%, respectively, compared to a similar formulation at pH 5.2 without arginine. In contrast, monoclonal antibodies other than human anti-RANKL monoclonal antibodies were not stabilized by the addition of arginine, and instead increased HMWS. Therefore, another method of this disclosure is to reduce HMWS in a formulation of denosumab or another human anti-RANKL monoclonal antibody by adding an amino acid aggregation inhibitor, such as arginine or phenylalanine.

[0082] Therefore, in exemplary embodiments, the aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor, which is optionally an amino acid. In exemplary embodiments, the amino acid is intended to be a D-stereoisomerized amino acid (D-amino acid), but is also an L-stereoisomerized amino acid (L-amino acid). In some embodiments, the amino acid aggregation inhibitor comprises an amino acid containing a charged side chain, which is also referred herein as a “charged amino acid.” The term “charged amino acid” refers to an amino acid containing a side chain that is negatively charged (i.e., deprotonated) or positively charged (i.e., protonated) in aqueous solution at physiological pH. For example, negatively charged amino acids include aspartic acid and glutamic acid, while positively charged amino acids include arginine, lysine, and histidine. Charged amino acids include charged amino acids from the 20 coded amino acids, as well as atypical, unnatural, or non-coding amino acids. Therefore, in exemplary embodiments, the amino acid aggregation inhibitor is an amino acid containing a positively charged side chain. In illustrative examples, amino acids containing positively charged side chains include the side chain structure of formula I or formula II.

[0083] [ka] Where n is from 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] Where m is from 1 to 7, and each of R3 and R4 is independently H, C1-C 18 alkyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)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)R8 and (C1-C4 alkyl)(C3-C9 heteroaryl), selected from group A, where R8 is H or OH, R5 may optionally be present, and if present, is selected from group A, and optionally each of R3, R4, and R5 is H.

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

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

[0087] In exemplary embodiments, amino acid aggregation inhibitors are hydrophobic amino acids. Hydrophobicity can be measured or scored according to any one of the hydrophobic 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 hydrophobic 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, hydrophobic amino acids have a score greater than approximately 2.5 on the Kyte and Doolittle hydrophobic scale. In certain embodiments, hydrophobic amino acids are branched or linear C2-C2 12Alkyl, or C4-C8 cycloalkyl, a C4-C8 heterocycle containing a nitrogen heteroatom, optionally including a heterocycle side chain which is imidazole, pyrrole, or indole. For the purposes of this specification, the term "cycloalkyl" may encompass any carbon ring, such as a bicyclic or tricyclic carbon ring.

[0088] In exemplary embodiments, the hydrophobic amino acid comprises a C3-C8 alkyl group, and optionally the hydrophobic amino acid comprises a branched C3 alkyl group or a branched C4 alkyl group. In certain embodiments, the hydrophobic amino acid is 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 in concentrations ranging from about 10 mM to about 200 mM, for example, from about 30 mM to about 120 mM, or from about 38 mM to about 150 mM, or from about 38 mM to about 113 mM, or from about 38 mM to about 75 mM, for example, from 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, and optionally about 25 mM to about 90 mM of the amino acid aggregation inhibitor. In some embodiments, the aqueous pharmaceutical formulation contains an amino acid aggregation inhibitor in a concentration of approximately 5 mM to approximately 150 mM (e.g., approximately 10 mM to approximately 150 mM, approximately 15 mM to approximately 150 mM, approximately 20 mM to approximately 150 mM, approximately 25 mM to approximately 150 mM, approximately 5 mM to approximately 140 mM, approximately 5 mM to approximately 130 mM, approximately 5 mM to approximately 120 mM, approximately 5 mM to approximately 110 mM, approximately 5 mM to approximately 100 mM, approximately 5 mM to approximately 90 mM) when the amino acid aggregation inhibitor is an amino acid having a positively charged side chain, optionally L-arginine. In some embodiments, the aqueous pharmaceutical formulation contains an amino acid aggregation inhibitor in an amount of approximately 30 mM to approximately 80 mM (e.g., approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM) when the amino acid aggregation inhibitor is an amino acid having a positively charged side chain, optionally L-arginine.

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

[0091] Optionally, the aqueous flocculation formulation contains approximately 5 mM to approximately 300 mM of an amino acid flocculation inhibitor, where the amino acid flocculation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine. Optionally, the aqueous pharmaceutical formulation contains approximately 5 mM to approximately 200 mM (for example, approximately 10 mM to approximately 200 mM, approximately 20 mM to approximately 200 mM, approximately 30 mM to approximately 200 mM, approximately 40 mM to approximately 200 mM, approximately 50 mM to approximately 200 mM, approximately 60 mM to approximately 200 mM, approximately 70 mM to approximately 200 mM, approximately 80 mM to approximately 200 mM, approximately 90 mM to approximately 200 mM, approximately 100 mM to approximately 20 The product contains an amino acid aggregation inhibitor in the following concentrations: 0 mM, approximately 5 mM to approximately 290 mM, approximately 5 mM to approximately 280 mM, approximately 5 mM to approximately 270 mM, approximately 5 mM to approximately 260 mM, approximately 5 mM to approximately 250 mM, approximately 5 mM to approximately 240 mM, approximately 5 mM to approximately 230 mM, approximately 5 mM to approximately 220 mM, and approximately 5 mM to approximately 210 mM. Optionally, if the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine, the product contains an amino acid aggregation inhibitor in the following concentrations: approximately 20 mM to approximately 50 mM. In exemplary embodiments, the aqueous pharmaceutical composition comprises about 30 mM to about 80 mM L-arginine hydrochloride, about 20 mM to about 50 mM L-phenylalanine, about 20 mM to about 50 mM L-tryptophan, about 30 mM to about 80 mM L-lysine hydrochloride, about 20 mM to about 50 mM L-leucine, about 20 mM to about 50 mM L-isoleucine, about 20 mM to about 50 mM L-valine, or any combination thereof.

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

[0093] Surfactants are those that are amphiphilic (having a polar head and a hydrophobic tail). Surfactants preferentially accumulate at interfaces, resulting in a decrease in surface tension. Surfactants can optionally be included in formulations. The use of surfactants can also help reduce the formation of large protein particles.

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

[0095] The surfactant concentration in the range of approximately 0.004% (w / v) to approximately 0.1% (w / v) (for example, in the case of polysorbate 20 or polysorbate 80) is preferably, for example, approximately 0.004% to approximately 0.05%, or approximately 0.004% to approximately 0.02%, or approximately 0.01%. In exemplary embodiments, the formulation contains at least approximately 0.004% (w / v) of surfactant, optionally less than approximately 0.15% (w / v). In exemplary embodiments, approximately 0.005(w / v)% to approximately 0.015(w / v)% of surfactant is present in the formulation, and arbitrarily present in amounts of approximately 0.005(w / v)%, approximately 0.006(w / v)%, approximately 0.007(w / v)%, approximately 0.008(w / v)%, approximately 0.009(w / v)%, approximately 0.010(w / v)%, approximately 0.011(w / v)%, approximately 0.012(w / v)%, approximately 0.013(w / v)%, or approximately 0.014(w / v)%.

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

[0097] If the solution is in a form intended for administration to a target, it can be prepared to be isotonic with respect to the intended administration site. For example, the osmolality may be in the range of about 270 to about 350 mOsm / kG, or about 285 to about 345 mOsm / kG, or about 300 to about 315 mOsm / kG. For example, if the solution is in a form for parenteral administration, it may be isotonic with blood (osmolality of about 300 mOsm / kG). In exemplary embodiments, aqueous pharmaceutical formulations have osmolality in the range of about 200 mOsm / kg to about 500 mOsm / kg, or about 225 mOsm / kg to about 400 mOsm / kg, or about 250 mOsm / kg to about 350 mOsm / kg.

[0098] In exemplary embodiments, the aqueous pharmaceutical formulation has an conductivity in the range of about 500 μS / cm to about 5500 μS / cm, optionally, if the formulation contains an amino acid having 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. The aqueous pharmaceutical formulation according to any one of the prior claims, having a viscosity of about 6 cP or less at 5°C, optionally having a viscosity of about 4.5 cP to about 5.5 cP. In a particular embodiment, the aqueous pharmaceutical formulation has a viscosity of less than about 13 cP at 25°C, optionally about 2.0 cP to about 10 cP, optionally about 2.5 cP to about 4 cP.

[0099] Tonic modifiers, i.e., tonic modifiers, 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 tonic modifier is selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. In exemplary examples, the tonic modifier is sorbitol. Sorbitol can be used in concentrations ranging from, for example, 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 about 1.0% (w / w) to about 5.0% (w / w) of a tonic modifier. For example, the formulation contains about 2.0% (w / w) to about 5.0% (w / w) of sorbitol, or about 3.5% (w / w) to about 5.0% (w / w) of sorbitol, or about 4.0% (w / w) to about 5.0% (w / w) of sorbitol. In some embodiments, the formulation contains no sorbitol, i.e., no sorbitol. In exemplary embodiments, the formulation contains no tonic modifier at all.

[0100] Other additives known in the art may be used in the formulation, provided they do not adversely affect stability. Sugars and polyols may be used to protect proteins from aggregation, including providing freeze / thaw stability. Examples of such compounds include sorbitol, mannitol, glycerol, erythritol, caprylates, tryptophanates, sarcosides, and glycine. Stabilizers for preparing lyophilized formulations include stabilizing sugars, such as disaccharides like trehalose and sucrose. Lyophilized formulations may also contain bulking agents, as known in the art. Other additives known in the art for protein stabilization include solubilizers (e.g., N-methyl-2-pyrrolidone), polyethylene glycol (PEG), and cyclodextrins (e.g., Captisol®). The pH of the solution can be adjusted using pharmaceutically acceptable acids and bases, such as sodium hydroxide.

[0101] For parenteral administration, the formulation may be in the form of a pyrogenically 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 vehicle for parenteral injection is sterile distilled water formulated as a sterile isotonic solution containing denosumab or another human anti-RANKL monoclonal antibody, with or without at least one further therapeutic agent. The formulation contains pharmaceutically acceptable excipients, e.g., USP (United States Pharmacopeia) grade excipients.

[0102] A "preservative" is a compound that may be included in a pharmaceutical formulation to reduce the activity of bacteria within it, 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 in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include phenol, butyl and benzyl alcohol, alkylparabens such as methyl and propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Alternatively, a formulation may not contain a preservative. For example, a formulation provided in a single-use dosage form may not contain a preservative.

[0103] Although the formulations are described herein in their aqueous form, the stabilized formulations may also be lyophilized to prepare lyophilized products. Therefore, unless otherwise indicated in the context, references to the formulations and their uses are intended to include lyophilized products resulting from stabilized aqueous solutions.

[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, parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags, or vials or pre-filled syringes with a stopper that can be punctured with a subcutaneous needle. In certain embodiments, formulations may be stored either in a ready-to-use form or in a form that is reconstituted or diluted before administration (e.g., in a lyophilized form).

[0105] In certain embodiments, the present invention relates to a kit for preparing single-dose dosing units. In certain embodiments, the kit may include both a first container having a dried formulation of denosumab or other human anti-RANKL monoclonal antibody prepared from the solution formulation described herein, and a second container having sterile water or an aqueous solution. In certain embodiments of the present invention, the kit includes a pre-filled syringe (e.g., liquid syringes and rio-syringes) having one or more chambers.

[0106] The stabilized formulations described herein may be used in conjunction with one or more additional therapeutic agents, such as calcium and vitamin D compounds. The stabilized formulations described herein may be administered to patients receiving treatment with additional therapeutic agents, or may be administered concurrently with additional therapeutic agents.

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

[0108] In one embodiment, the formulation may be used to prevent bone-related events (SREs) in patients requiring prevention of SREs, the use of which involves administering an effective dose of the stabilized formulation described herein. SREs may be selected from, for example, pathological fractures, radiotherapy to the bone, surgery to the bone, and spinal cord compression. Patients may be patients with bone metastases from solid tumors. Solid tumors may be one or more of, for example, breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma. The amount of the formulation may be effective in optionally reducing the creatinine-corrected urinary N-terminal telopeptide (uNTx / Cr), a bone metabolism marker, by at least 80%. Patients may be patients with multiple myeloma.

[0109] In another embodiment, the formulation can be used to treat a patient with giant cell tumor of bone, the use of which involves administering an effective dose of the stabilized formulation described herein. In one type of embodiment, the patient has a giant cell tumor of bone that is recurrent, unresectable, or for which surgical excision would likely result in severe morbidity. The patient may be, for example, an adult or a young adult with a mature skeleton.

[0110] In another embodiment, the formulation may be used to treat patients with hypercalcemia due to bone malignancies, the use of which involves administering an effective dose of the stabilized formulation described herein. In one embodiment, the malignancy may be refractory to bisphosphonate treatment. This method or use may involve administering an effective dose of the formulation to reduce or maintain the patient's serum calcium level to approximately 11.5 mg / dL or less.

[0111] In another embodiment, the formulation may be used to treat osteoporosis in a patient requiring treatment for osteoporosis, the use of which involves 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 man at high risk of fracture.

[0112] In another embodiment, the formulation is used to increase bone mass in patients who require increased bone mass, and its use involves administering an effective amount of the stabilized formulation described herein. For example, the amount of the formulation administered may be an effective amount to reduce the incidence of new vertebral fractures and / or non-vertebral fractures. In another type of embodiment, the amount of the formulation administered may be an effective amount to reduce bone resorption. In another type of embodiment, the amount of the formulation may be an effective amount to increase the patient's bone density in at least one area selected from the lumbar spine, the entire hip joint, and the femoral neck. In another type of embodiment, the amount of the formulation may be an effective amount to increase bone mass in the patient's cortical bone and / or trabecular bone. In another type of embodiment, the amount of the formulation may be an effective amount to reduce the bone resorption marker serum type 1 C-telopetide (CTX). Patients requiring it may, in some cases, have osteoporosis. In another type of embodiment, patients requiring it may be high-risk women undergoing adjuvant aromatase inhibitor therapy for breast cancer. In another type of embodiment, patients requiring it may be high-risk men undergoing androgen deprivation therapy for non-metastatic prostate cancer. In another type of embodiment, the patient requiring it may be a man with osteoporosis, which puts him at high risk of fracture.

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

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

[0115] In another embodiment, the formulation can be used to treat idiopathic subglottic stenosis (ISS).

[0116] In another embodiment, the formulation can be used to prevent breast and ovarian cancer in healthy women with BRCA-1 mutations.

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

[0118] In another embodiment, the formulation may be used, for example, to treat giant cell-rich tumors in hyperparathyroidism or in cases associated with secondary aneurysmal bone cysts.

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

[0120] In another embodiment, the formulation can be used to treat metastatic breast cancer (mBC). In yet another embodiment, the formulation can be used to treat preoperative breast cancer. In yet another embodiment, the formulation can be used to treat early-stage breast cancer. In yet another embodiment, the formulation can be used to treat hormone receptor-negative, RANK-positive, or RANK-negative primary breast cancer. In yet another embodiment, the formulation can be used to treat postmenopausal HER2-negative breast cancer.

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

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

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

[0124] In another embodiment, the formulation can be used to induce immunomodulatory effects in patients with or without immunotherapy.

[0125] In another embodiment, the formulation may be used to prevent or treat bone loss associated with osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, and rheumatoid arthritis. In another embodiment, the formulation may be used to prevent or treat inflammatory conditions with bone loss. In another embodiment, the formulation may be used to prevent or treat autoimmune conditions with bone loss. In another embodiment, the formulation may be used to prevent or treat bone loss associated with cancers 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 appropriate schedule. In one embodiment, the administration schedule is once every four weeks. Optionally, administration may include doses on the 8th and 15th days of the first month of treatment. In another type of embodiment, administration may be on a schedule of once every six months. The once-every-six-months schedule is intended, for example, for use in osteoporosis and bone mass enhancement. Other intended maintenance doses are every three weeks, every three months, and every six weeks.

[0127] In some embodiments, aqueous pharmaceutical formulations are used to treat patients with bone metastases from multiple myeloma or solid tumors. In certain embodiments, the formulation is administered as a subcutaneous injection into the upper arm, upper thigh, or abdomen at a dose of approximately 120 mg every four weeks.

[0128] In some embodiments, aqueous pharmaceutical formulations are used to treat patients with giant cell tumors of bone. In certain embodiments, the formulation is administered in doses of approximately 120 mg every four weeks, with additional doses of 120 mg administered on the 8th and 15th days of the first month of treatment. In some embodiments, the formulation is administered subcutaneously to 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 embodiments, aqueous pharmaceutical formulations are used to treat patients with hypercalcemia due to malignant tumors. In certain embodiments, the formulation is administered in doses of approximately 120 mg every four weeks, with additional doses of 120 mg administered on the 8th and 15th days of the first month of treatment. In some embodiments, the formulation is administered subcutaneously to 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 to increase bone mass in men at high risk of fracture undergoing androgen deprivation therapy for non-metastatic prostate cancer, or in women at high risk of fracture undergoing adjuvant aromatase inhibitor therapy for breast cancer. In some embodiments, the aqueous pharmaceutical formulation is administered by a healthcare professional in doses of 60 mg every six months by subcutaneous injection into the upper arm, upper thigh, or abdomen. In some embodiments, the patient is also instructed to take 1000 mg of calcium and at least 400 IU of vitamin D daily.

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

[0132] Another type of formulation according to this disclosure contains denosumab, acetate, and phenylalanine. The formulation may optionally contain sorbitol. The formulation may optionally contain polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2. For example, the formulation may contain denosumab at a pH of 5.1 at a concentration of approximately 108 mg / mL to approximately 132 mg / mL, acetate at approximately 28.8 mM to approximately 35.2 mM, phenylalanine at 33.3 mM to approximately 40.7 mM, sorbitol at 3.51% (w / v) to approximately 4.29% (w / v), and polysorbate 20 at approximately 0.009% (w / v) to approximately 0.011% (w / v), and may optionally be contained in the PFS, which may optionally contain approximately 1 mL or less than approximately 1 mL (e.g., approximately 0.5 mL) of the formulation. For example, the formulation may contain denosumab at a concentration of 120 mg / mL at pH 5.1, 32 mM acetate, 37 mM phenylalanine, 3.9% (w / v) sorbitol, and 0.01% (w / v) polysorbate 20, and may optionally be contained in a PFS, which may optionally contain about 1 mL or less than about 1 mL (e.g., about 0.5 mL) of the formulation. This formulation can be prepared by concentrating denosumab in a dialysis filtration 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 is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally contain sorbitol. The formulation may optionally contain polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be 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 may optionally contain sorbitol. The formulation may optionally contain polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be 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 is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally contain sorbitol. The formulation may optionally contain polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be about 5.0 to about 5.2, or less than 5.2.

[0136] The formulations of this 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 the solution can then be concentrated to an amount greater than 70 mg / mL as described herein, for example, 120 mg / mL. Optionally, the solution can be initially over-concentrated, i.e., to a concentration of anti-RANKL monoclonal antibody (e.g., denosumab) higher than the final target concentration, and the over-concentrated solution can then be diluted, for example, with a pH-adjusted buffer solution, to the final target concentration and pH. For example, the over-concentration may 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 may 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 within a range enclosed by such concentrations, for example, about 1 mg / mL to about 70 mg / mL, or about 1 mg / mL to about 10 mg / mL.

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

[0138] The introduction of amino acid agglutination inhibitors into formulations can be carried out by any suitable method. For example, amino acid agglutination inhibitors can be introduced into formulations by simple addition (spiking), as described in the following examples. Alternatively, amino acid agglutination inhibitors can be introduced into formulations by dialysfiltration of a buffer containing the amino acid agglutination inhibitor, as described in the following examples. Amino acid agglutination inhibitors can be introduced into formulations before or after concentrating the anti-RANKL monoclonal antibody to more than 70 mg / mL. Adding amino acid agglutination inhibitors to the solution before concentration is beneficial because it suppresses agglutination during the concentration process, as shown in the following examples.

[0139] Accordingly, this 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 its antigen-binding moiety. In an exemplary example, this method involves mixing an anti-RANKL monoclonal antibody or its antigen-binding moiety at a concentration greater than 70 mg / mL with an amino acid agglutination inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. The antibody or antigen-binding moiety may be any of those described herein, and the concentration of the antibody or its antigen-binding moiety may be consistent with the teachings herein. The amino acid agglutination inhibitor may be any of those described herein. For example, the amino acid agglutination inhibitor may be a positively charged amino acid, an aromatic amino acid, or a hydrophobic amino acid. The amino acid agglutination inhibitor may be present in a molar ratio with the antibody described herein. The amounts and selections of the agglutination inhibitor, surfactant, tonicity modifier, and buffer are as described above. This disclosure also provides formulations prepared by the production methods described herein.

[0140] The formulations disclosed herein may include pH adjustment of a high-concentration solution of the anti-RANKL monoclonal antibody described herein (e.g., denosumab), for example, having a concentration greater than 70 mg / mL, i.e., 120 mg / mL. In another embodiment, the formulation may be prepared by pH adjustment of a low-concentration solution of the 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 intended embodiments: An aqueous pharmaceutical preparation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety having a concentration exceeding 1.70 mg / mL and a pH in the range of approximately 5.0 to less than 5.2.

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

[0144] 3. The formulation according to Embodiment 2, having a pH of approximately 5.1.

[0145] 4. A formulation according to any one of Embodiments 1 to 3, further comprising an amino acid aggregation inhibitor.

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

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

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

[0149] 8. A formulation according to any one of the prior 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 approximately 10 mg / mL to approximately 200 mg / mL.

[0151] 10. The formulation according to any one of the prior embodiments, wherein the concentration of the antibody or its antigen-binding portion is in the range of more than 70 mg / mL 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 approximately 100 mg / mL to approximately 140 mg / mL.

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

[0154] 13. The formulation according to any one of the prior 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 prior 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 include arginine and phenylalanine.

[0159] 18. The formulation according to any one of the prior 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 formulation according to any one of the prior embodiments, wherein the amino acid aggregation inhibitor is selected from one or more amino acids having a charged side chain, a dipeptide thereof, or an oligopeptide having 2 to 10 residues and containing one or more amino acids having a charged side chain.

[0161] 20. The formulation according to any one of the prior 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 formulation according to any one of the prior embodiments, wherein the amino acid aggregation inhibitor is selected from one or more dipeptides.

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

[0164] 23. The formulation according to any one of the prior embodiments, wherein the amino acid aggregation inhibitor comprises an arginine residue, or the amino acid aggregation inhibitor comprises arginine.

[0165] 24. The formulation according to any one of the prior embodiments, wherein the amino acid aggregation inhibitor comprises an arginine-phenylalanine dipeptide.

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

[0167] 26. A formulation according to any one of the prior embodiments, further comprising a surfactant.

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

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

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

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

[0172] 31. The buffering agent is the formulation according to Embodiment 30, wherein the pH range is mainly in the range of approximately pH 4 to approximately pH 5.5 at 25°C.

[0173] 32. The formulation according to Embodiment 30 or 31, wherein the buffer has a pKa within 1 pH unit of pH 5.0 to 5.2 at 25°C.

[0174] 33. The formulation according to any one of Embodiments 30 to 32, wherein the buffering agent contains an acetate.

[0175] 34. The formulation according to any one of embodiments 30 to 32, wherein the buffering agent comprises a glutamate salt.

[0176] 35. A formulation according to any one of the prior embodiments, further comprising a tonicity modifier.

[0177] 36. The formulation according to Embodiment 35, wherein the tonicity modifier 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 modifier comprises sorbitol.

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

[0180] A formulation according to any one of the prior embodiments, comprising less than 2% of a high molecular weight species of the human anti-RANKL monoclonal antibody obtained by SE-UHPLC after storage at 39.37°C for 3 months.

[0181] A formulation according to any one of the prior embodiments, comprising less than 2% of high molecular weight species of the human anti-RANKL monoclonal antibody obtained by SE-UHPLC after storage at 40.4°C for 36 months.

[0182] A formulation according to any one of the prior embodiments, comprising at least 98% of the antibody principal peaks obtained by SE-UHPLC after storage at 41.37°C for 3 months.

[0183] A formulation according to any one of the prior embodiments, comprising at least 98% of the antibody principal peaks obtained by SE-UHPLC after storage at 42.4°C for 36 months.

[0184] 43. A formulation according to any one of the prior embodiments, comprising denosumab, an amino acid aggregation inhibitor selected from one or more oligomers having 2 to 10 residues and containing arginine, an acetate buffer, sorbitol, and a surfactant, wherein the pH is in the range of approximately 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 a concentration of approximately 5 mM to approximately 25 mM.

[0188] 47. The formulation according to any one of Embodiments 43 to 46, wherein the sorbitol is present in a 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 polysorbate 20 and polysorbate 80.

[0190] 49. A formulation according to any one of Embodiments 43 to 48, wherein the pH is in the range of approximately 5.0 to approximately 5.15.

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

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

[0193] 52. The formulation according to any one of the prior embodiments, wherein the formulation is sterilized and free of preservatives.

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

[0195] 54. The formulation according to any one of the prior embodiments, wherein the human anti-RANKL monoclonal antibody or its antigen-binding portion is an antibody.

[0196] 55. A formulation according to any one of Embodiments 1 to 53, wherein the human anti-RANKL monoclonal antibody or its antigen-binding portion is the antigen-binding portion.

[0197] 56. A vial, pre-filled syringe, or glass container containing the formulation described in any one of Embodiments 1 to 55.

[0198] 57. A vial, pre-filled syringe, or glass container according to Embodiment 56, containing approximately 1 mL or less of the formulation.

[0199] 58. A method for preventing bone-related events (SREs) in a patient requiring prevention of SREs, comprising administering an effective amount of a formulation described in 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 fracture, radiotherapy to the bone, surgery to the bone, and spinal cord compression.

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

[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. The method according to any one of Embodiments 58 to 62, comprising administering an amount of the formulation effective to reduce, optionally at least 80%, the creatinine-corrected urinary N-terminal telopeptide (uNTx / Cr) bone metabolism marker.

[0205] 64. A method for treating a giant cell tumor of bone in a patient requiring treatment, comprising administering an effective dose of the formulation described in 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 prone to severe morbidity if surgically removed.

[0207] 66. A method for treating hypercalcemia of malignant tumors in a patient requiring treatment of hypercalcemia of malignant tumors, comprising administering an effective dose of the formulation described in any one of Embodiments 1 to 55.

[0208] 67. The method according to Embodiment 66, wherein the malignant tumor is refractory to bisphosphonate treatment.

[0209] 68. The method according to Embodiment 66 or 67, comprising administering an amount of the formulation effective in reducing or maintaining the patient's serum calcium level to a level of approximately 11.5 mg / dL or less.

[0210] 69. Any method according to Embodiments 58 to 68, wherein the formulation contains the human anti-RANKL antibody at a concentration of approximately 120 mg / mL.

[0211] 70. The method according to any one of Embodiments 58 to 69, comprising administering the formulation on a schedule of once every four weeks.

[0212] 71. The method according to any one of Embodiments 58 to 70, comprising administering the formulation on the 8th and 15th days of the first month of treatment.

[0213] 72. A method for treating osteoporosis in a patient requiring treatment for osteoporosis, comprising administering an effective amount of the preparation described in 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 male at high risk of fracture.

[0216] 75. A method for increasing bone mass in a patient requiring bone mass increase, comprising administering an effective amount of the formulation described in 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 undergoing adjuvant aromatase inhibitor therapy for breast cancer.

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

[0220] 79. The method according to any one of embodiments 75 to 78, comprising administering an amount of the formulation effective in reducing 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 preparation for reducing bone resorption.

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

[0223] 82. The method according to any one of Embodiments 75 to 81, comprising administering an amount of the preparation effective in increasing the bone mass of the cortical bone and / or trabeculae of the patient.

[0224] 83. The method according to any one of Embodiments 75 to 82, comprising administering an amount of the preparation effective in reducing the bone resorption marker serum type 1 C-telopetide (CTX).

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

[0226] The method according to any one of embodiments 58 to 84, comprising administering the formulation in a volume of 85.1 mL or less.

[0227] 86. The method according to any one of Embodiments 58 to 85, comprising administering the preparation subcutaneously.

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

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

[0230] 89. A method for improving the stability of an aqueous pharmaceutical preparation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety at a concentration exceeding 70 mg / mL, The preparation of the aqueous pharmaceutical formulation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety at a pH in the range of approximately 5.0 to less than 5.2, The method wherein the aqueous pharmaceutical formulation exhibits improved stability in the pH range of approximately 5.0 to less than 5.2 compared to equivalent aqueous pharmaceutical formulations that are not in the pH range of approximately 5.0 to less than 5.2.

[0231] 90. A method for improving the stability of an aqueous pharmaceutical preparation containing a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, The preparation of the aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, mixed with an amino acid aggregation inhibitor, The method wherein the aqueous pharmaceutical formulation exhibits improved stability due to the amino acid aggregation inhibitor compared to an equivalent aqueous pharmaceutical formulation that does not contain the amino acid aggregation inhibitor. [Examples]

[0232] The following examples are provided for illustrative purposes of the present invention and are not intended to limit the scope of the invention. Throughout 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 ultrahigh performance liquid chromatography. Furthermore, 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 certain components of the final formulation, when stored and subsequently analyzed for stability, may differ from the concentrations in the DF or dialysis buffer, depending on the presence or absence of a counterion (e.g., HCl). In the absence of a counterion, the formulation has lower ionic strength. In such examples, the acetate co-concentrates with denosumab so that the final formulation contains a higher concentration of acetate relative to the concentration in the DF or dialysis buffer. For example, using a DF buffer containing 10 mM acetate, if neither the DF buffer nor the final formulation contains counterions (e.g., HCl), and therefore the ionic strength is low, approximately 23 mM acetate is obtained in the final denosumab (120 mg / mL) formulation (pH 5.1). Similarly, a DF buffer containing 20 mM acetate yields approximately 32 mM acetate in the final denosumab (120 mg / mL) formulation at pH 5.1, without the presence of counterions (e.g., HCl). If counterions (e.g., HCl from arginine HCl) are present, the acetate does not co-concentrate with denosumab, so the acetate concentration in the DF buffer and the acetate concentration in the final composition are generally the same. Furthermore, additives can be eliminated by volumetric analysis or may be affected by nonspecific interactions. For example, in a 120 mg / mL denosumab formulation, the concentrations of phenylalanine and sorbitol are approximately 7-10% lower than those shown in the DF buffer, and the arginine concentration is approximately 10-15% lower. Taking the above into consideration, the concentrations of the components of the final formulation are provided through the following examples, taking into account the exclusion of the above-mentioned additives and the effect of acetate co-concentration.

[0233] Example 1 Initial evaluations of 12 formulations were conducted to minimize the amount (%) of HMWS in high-concentration liquid denosumab formulations (120 mg / mL) and their time-dependent formation. Substitutions of formulations included changes in buffer type, stabilizers, and solution pH. The formulations tested, A-L, are listed in Table 1 below. All buffer values ​​cited relate to the buffer concentration at which the antibody is dialyzed. Each additive and surfactant was added to the solution after buffer exchange to the levels shown in the table. Acetate concentration in this formulation was not measured, but 120 mg / mL denosumab formulations with dialyzed sorbitol relative to 10 mM acetate had approximate final acetate values ​​of 25 mM to 35 mM acetate.

[0234] 70 mg / mL denosumab in acetate at pH 5.2 was concentrated to 160 mg / mL by UF / DF in 10 mM acetate at pH 5.2. 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 (registered trademark) F-68

[0239] 3%Triton(TM)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 preparation using 10 mM acetate at pH 5.2 was diluted to 120 mg / mL using 10 mM acetate at pH 5.2, and then diluted to the target final concentrations listed in Table 1 by adding the corresponding sorbitol, additives, and / or surfactant stock solutions. To obtain formulations K and L, two separate aliquots from the 160 mg / mL preparations for the self-buffered formulation and the glutamate formulation underwent additional buffer exchange by centrifugation. Subsequently, the materials for formulations K and L were diluted to 120 mg / mL using buffer solutions, and then the corresponding sorbitol and polysorbate 20 stock solutions were added to the target final concentrations listed in Table 1.

[0247] [Table 1]

[0248] Figure 1 shows the percentage 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 tonic modifier, 0.01% (w / v) polysorbate 20 as a surfactant, and a pH of 5.0, showed both a decrease in the initial amount of HMWS, suggesting a reduction in some of the 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 to clarify the effect of pH and amino acid aggregation inhibitors on the rate and extent of HMWS formation. The tested formulations are described 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, an increase in the number of dimer species was generally observed. As shown in Figure 3, formulation N had the lowest amount of dimer species (retention time approximately 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 reduction of approximately 0.3% and 25% in the amount and formation rate of dimer species, respectively, after 1 month at 37°C, compared to formulation P, which has the same pH but does not contain 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 with acetate, sorbitol, and polysorbate 20 (PS20) at three different pH values: 4.8, 5.1, and 5.4, 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 an initial solution containing a lower concentration of denosumab, followed by overconcentration of the denosumab material, and then dilution of the denosumab material with the desired amount of buffer, additives, and surfactant. Briefly, aliquots of 70 mg / mL of denosumab in acetate (initial material) at pH 5.2 were dialyzed against the DF buffer listed in Table 3A, with a total of three buffer exchanges to achieve a 1,000,000-fold dilution of the initial material and ensure complete buffer exchange. Next, the denosumab material, after buffer exchange, was concentrated to a denosumab concentration exceeding 120 mg / mL using a centrifugal concentrator, and then the concentrated substance was diluted to a concentration of 120 mg / mL denosumab. PS20 was added to obtain 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 in formulation 1 was increased to achieve the target final pH, and the acetate concentrations in formulations 2 and 3 were matched to that of formulation 1. The acetate concentrations in formulations 4-6 required higher amounts of acetate to match the final acetate concentrations of formulations 1-3, as the acetate was not co-concentrated in the presence of the HCl salt. Formulation 7 served as a control to ensure that the increased acetate concentrations in formulations 4-6 did not impair the protein stability of the arginine hydrochloride formulations.

[0259] Table 3A lists the various denosumab formulations prepared and tested in this study.

[0260] [Table 3] * The final formulation contained denosumab 120 mg / mL and PS20 at a final concentration of 0.01% (w / v), and had the indicated pH. The sorbitol concentration was estimated to be 8.5% lower than that of the DF buffer. The arginine concentration was estimated to be 12.5% ​​lower than that of the DF buffer.

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

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

[0263] [Table 4] The F# shown in the left column corresponds to the F# in Table 3A.

[0264] Figure 5 shows the size exclusion chromatograms of each formulation after storage at 37°C for one 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 performed better than control formulations without arginine hydrochloride, and formulations at pH 5.1 performed better than comparative formulations at pH 4.8 and pH 5.4. In formulations 1-3 without arginine hydrochloride, the number of 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 number of 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 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, including various denosumab concentrations.

[0267] The pH sensitivity of HMWS formation at denosumab protein concentrations ranging from 15 mg / mL to 150 mg / mL was evaluated at various protein concentrations and a 75 mM arginine hydrochloride concentration. Two pH values, namely pH 4.8 and 5.1, were evaluated for each tested protein concentration: 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 acetate buffer at pH 5.2 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 of 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 concentrated in excess using a centrifugal concentrator, 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 against aggregation 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, during, and after the storage period.

[0271] Figure 6 represents a graph of the percentage 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 formulations after storage at 37°C for one month. As shown in Figure 6, HMWS% increased with increasing protein concentration. 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 attributed to a large aggregation peak at approximately 5.75 min, shown in Figure 7A (top). HMWS% at solution pH 5.1 has dimer species that increase with increasing protein concentration, but total HMWS was lower than at 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 with increasing denosumab concentration, and the difference was greater at higher denosumab concentrations.

[0275] Example 5 The stabilizing effect of various concentrations of formulations of arginine, NAR, and two dipeptides consisting of arginine-arginine (Arg-Arg) and arginine-phenylalanine (Arg-Phe) was evaluated against a denosumab solution at a concentration of 120 mg / mL.

[0276] The tested formulations are listed in Table 5 below. All cited values ​​for acetates and additives (except dipeptides) represent the concentrations of the buffer and additives in which the antibody was dialyzed. 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 in the DF buffer listed below. Formulations Y and Z were achieved together in a single pool by UF / DF in a DF buffer containing 10 mM acetate, 3.6% sorbitol, and pH 4.0. After UF / DF, the pools of formulations Y and Z were divided into two, and then Arg-Arg or Arg-Phe dipeptides were 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%. The acetates were co-concentrated without arginine to obtain a final acetate concentration of approximately 25 mM in formulations S-X. Sorbitol is preferentially removed during the concentration process, resulting in a reduction of approximately 7-8% (w / v) from the initial concentration.

[0277] The formulations were filled into containers with a filling volume of 1.0 mL. The formulations were stored at temperatures of 2°C to 8°C for up to 12 months, and at 25°C, 30°C, and 37°C for 3 months. Stability based on HMWS formation was evaluated using SE-UHPLC. The stability of these dipeptide formulations after 1 month at 37°C was compared with that of 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 generated 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 tested formulations (formulations 16 - 20) are described in Table 6A below. To prepare these formulations, an aliquot of 70 mg / mL denosumab in acetate buffer at pH 5.2 was 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] * The final formulation contained 120 mg / mL of denosumab and 0.01% (w / v) PS20 at a final concentration, and had the indicated pH. The concentrations of sorbitol and phenylalanine were estimated to be approximately 8.5% lower than those of the DF buffer. The arginine concentration was estimated to be approximately 12.5% ​​lower than that of the DF buffer.

[0284] The formulations were filled into containers with a filling volume of 1.0 mL. The formulations were stored at 37°C for up to one month. The stability against aggregation inhibition and aggregation inhibition over time, based on HMWS and dimer formation, was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions, during storage, and thereafter.

[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 the size exclusion chromatogram as a function of formulation after storage at 37°C for one month. Table 6B below 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 amino acid aggregation inhibitors, arginine, or phenylalanine (formulations 17-20) were superior to the sorbitol control formulation (formulation 16) which contained no amino acid aggregation inhibitor. All phenylalanine-containing formulations (formulations 18, 19, and 20) contained similarly low levels of HMWS compared to the control and the arginine HCl formulations (formulations 16 and 17, respectively) (Figure 9). The HMWS formation rate was similar among 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 not 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] Previous studies identified arginine hydrochloride and arginine hydrochloride-phenylalanine dipeptide as minimizing the initial onset level and rate of denosumab HMWS formation. The stabilizing effects of formulations containing arginine hydrochloride, formulations containing various concentrations of phenylalanine, and formulations containing combinations of arginine hydrochloride and phenylalanine were evaluated for a solution containing 120 mg / mL of denosumab.

[0290] The tested formulations are listed in Table 7A below. To prepare test samples A to E, aliquots of denosumab 70 mg / mL in acetate (pH 5.2) were dialyzed against the DF buffer described below, and a total of three buffer exchanges were performed to achieve a 1,000,000-fold dilution of the previous formulation and ensure complete buffer exchange. This material was then overconcentrated to approximately 130 mg / mL to 150 mg / mL using a centrifuge, 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 formulations were filled into containers with a filling volume of 1.0 mL. The formulations were stored at 37°C for up to one month. Stability based on HMWS formation was evaluated using SE UHPLC. The stability profiles of these formulations were compared with those of sorbitol and arginine hydrochloride / sorbitol formulations at 37°C after one month, as shown in Figure 11A.

[0292] To prepare test samples F-K, aliquots of 70 mg / mL denosumab in acetate (pH 5.2) were subjected to ultrafiltration / diafiltration (UF / DF) of a total of 12 dialysis volumes in the DF buffer listed below, ensuring complete buffer exchange. This material was then overconcentrated to approximately 200 mg / mL by ultrafiltration, followed by dilution to 120 mg / mL, and polysorbate 20 was added to a final concentration of 0.01%. In these preparations, the acetate concentration was 20 mM. Preparation F was considered the control preparation. All cited acetate and additive values ​​relate to the buffer concentration and additive concentration at which the antibody is dialyzed.

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

[0294] [Table 10] * The final formulation contained denosumab at a final concentration of 120 mg / mL and PS20 at a final concentration of 0.01% (w / v), and had the indicated pH. The concentrations of sorbitol and phenylalanine were estimated to be approximately 8.5% lower than those of the DF buffer. The arginine concentration was estimated to be approximately 12.5% ​​lower than that of the DF buffer.

[0295] Figure 11A and Table 7B show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 37°C. Figure 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 for one month at 37°C and 40°C, respectively.

[0296] [Table 11]

[0297] [Table 12]

[0298] Compared to both sorbitol preparations and arginine hydrochloride / sorbitol preparations (preparations A and B, respectively), all phenylalanine preparations (preparations C, D, E, G-K) contained lower levels of HMWS. The arginine hydrochloride and phenylalanine combination preparations exhibited similar stability to the arginine / sorbitol preparation (preparation B). All preparations performed better than the sorbitol control preparations (preparations 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 carried out by preparing eight formulations containing hydrophobic amino acids, aromatic amino acids, or polar / charged amino acids, and determining their effect on minimizing the amount (%) of HMWS in a high-concentration liquid denosumab formulation (120 mg / mL) and HMWS formation over time. This formulation contained one of the eight L-amino acids and a smaller amount of sorbitol compared to a control formulation (formulation 26) that contained no amino acid aggregation inhibitor and a larger amount of tonic sorbitol.

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

[0302] To prepare formulations 26-34, aliquots of denosumab at 70 mg / mL in acetate at pH 5.2 were dialyzed against the DF buffer described in Table 8A, with a total of three buffer exchanges to achieve a 1,000,000-fold dilution of the previous formulation and ensure complete buffer exchange. For the dialyzing of 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 5.1 during 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 but remained at pH 4.0. Titration with dilute (0.1N) NaOH was necessary to bring the pH of the histidine formulation to pH 5.1. The remaining formulations were overconcentrated using a centrifuge, subsequently diluted to 124-128 mg / mL, and polysorbate 20 was added to a final concentration of 0.01% (w / v).

[0303] [Table 13] *The final formulation contained denosumab 120 mg / mL and PS20 at a final concentration of 0.01% (w / v), and had the indicated pH. The concentrations of sorbitol and phenylalanine are estimated to be approximately 8.5% lower than the sorbitol concentration in DF buffer. The arginine concentration is estimated to be approximately 12.5% ​​lower than the arginine concentration in DF buffer. The letters in parentheses following F# correspond to Figures 13-18.

[0304] The formulations were filled into containers with a filling volume of 1.0 mL. The formulations were stored at 37°C for up to 4 weeks. The stability against aggregation inhibition and aggregation inhibition over time, based on HMWS and dimer formation, was evaluated using SE UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions, during storage, and thereafter.

[0305] Figures 13–15 show graphs of the percentage of HMWS monitored by SE-UHPLC as a function of storage time at 37°C for each formulation, with Table 8B providing the data points for the graphs. Figures 16–18 show chromatographic overlays of the formulations listed in Table 8A after storage at 37°C for one 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 13-15, all formulations containing amino acid aggregation inhibitors (Formulations 27-34) showed some improvement in stability compared to the acetate / sorbitol formulation (Formulation 26). Formulations containing aromatic amino acids (Formulations 27 and 28) showed the greatest decrease in HMWS%. Formulations containing phenylalanine (Formulation 27) also showed a significant decrease in HMWS, and formulations containing tryptophan showed the greatest decrease compared to the control (Formulation 26). Denosumab formulations containing polar / charged amino acids (Formulations 29-31) generally showed larger orders of magnitude of aggregates (Figure 17) compared to other formulations with amino acid stabilizers (Figures 16 and 18), and this particular histidine formulation showed a greater amount of HWMS overall compared to the acetate / sorbitol formulation (Formulation 26) (Figure 14). The results for histidine formulations may be biased due to the dialysis process, longer duration at pH 4.0, and titration of formulations with diluted NaOH. All formulations containing hydrophobic amino acids (formulations 32-34) showed consistent improvement in HMWS formation.

[0308] Example 9 This example demonstrates the possible mechanisms of action of arginine and phenylalanine in the stabilization of denosumab. Deuterium exchange mass spectrometry (HDX-MS) is a highly sensitive and robust technique for characterizing protein-protein / ligand / additive interactions. This method detects changes in skeletal 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 with a denosumab formulation without an amino acid aggregation inhibitor (formulation 38). Experiments were conducted at 4°C (75 mM concentration of L-arginine, L-phenylalanine, or L-glycine) and 37°C (150 mM concentration of L-arginine, L-phenylalanine, or L-glycine). After analyzing more than 530 peptides, a small number of 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 the deuterium uptake percentage as a function of time (log(seconds)) at 4°C for each of the formulations 35-38: 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).

[0311] Figures 25-30 are graphs of the deuterium uptake percentage as a function of time (log(seconds)) at 37°C for each of the formulations 35-38: 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).

[0312] These data support the idea that Arg and Gly have similar interaction effects on denosumab, although Arg has a slightly stronger HDX footprint (conformational change) 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 region, and slight destabilization in the Fc CH2 243-253 region. While not intended 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 action of glycine is thought to be solely due to preferential exclusion.

[0313] However, phenylalanine did not show a significant structural perturbation to denosumab. While we do not intend to be bound by any particular theory, the phenylalanine stabilizing effect may occur through one or more of the following mechanisms: side-chain interactions (without HDX footprint) that do not affect the peptide backbone, and / or cation-pi interactions with the arginine / lysine side chain without affecting the skeletal hydrogen bonding network.

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

[0315] Molecular dynamics simulations were performed to study the specific effects of Phe on denosumab. Specifically, the Fab domain of denosumab was solvated with excess Phe in a simulation box, and two 10-ns simulations were performed. Collectively, Phe residues that bound to Fab for more than 90% of the time were selected for further analysis. Nine such cases were identified. In five of the nine long-retention observations, the Phe residues bound to the boundaries of the VH / VL (variable heavy / variable light) and CH / CL (steady heavy / steady light) regions. In one case, the Phe side chain was thought to be interacting with the side chains of hydrophobic residues (e.g., V93, Y95, and W112 in the heavy chain and A44 and P45 in the light chain) at the VH / VL interface. In another example, the side ring of Phe was thought to interact with the NH3+ and COO(-) groups of residues (e.g., T165 in the light chain and G171, V172, and T174 in the heavy chain) at the CH1 and CL interface. While not intended to be bound by any particular theory, this observation leads to the idea that the specific effect of Phe in easing denosumab aggregation is due to the phenyl group and hydrophobic residues (e.g., R30, G31, R32, and Y33 in CDR1 of the light chain, A52 in CDR2 of the light chain, and M106 in CDR3 of the heavy chain) forming an interface between the heavy steady 1(Hc) chain and the light steady (Lc) chain. This interaction is hypothesized to replace the previously hydrophobic surface with a surface having a relatively larger charge (and consequently becoming hydrophilic) from the NH3+ and COO(-) groups of the Phe additive.

[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 minimized the onset of HMWS and the time course of HWMS compared to a denosumab (IgG2 immunoglobulin) acetate / sorbitol control formulation. This evaluation was performed 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 contained different constant-domain scaffolds. The different IgG2 constructs tested in this study had different CDRs than denosumab, but contained the same constant-domain scaffolds.

[0317] Each tested antibody construct was purified and concentrated to 8 mg / mL to 70 mg / mL using centrifugation. Each concentrated volume was divided into three aliquots and dialyzed against acetate buffers prepared with sorbitol, sorbitol / phenylalanine, and sorbitol / arginine hydrochloride, as shown in Table 9, to prepare formulations 39-47. The dialyzed samples were overconcentrated to over 120 mg / mL by centrifugation. The antibody proteins were diluted to 120 mg / mL in their respective buffers.

[0318] [Table 15] * The final formulation contained PS20 at a final concentration of 0.01% (w / v) and had the indicated pH. The concentrations of sorbitol and phenylalanine were estimated to be approximately 8.5% lower than those of the DF buffer. The arginine concentration was estimated to be approximately 12.5% ​​lower than that of the DF buffer.

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

[0320] Figures 31, 33, and 35 (and 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 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=4w.

[0321] [Table 16]

[0322] [Table 17]

[0323] [Table 18]

[0324] [Table 19]

[0325] [Table 20]

[0326] [Table 21]

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

[0328] A significant increase in protein fragmentation was observed in the acetate / arginine / sorbitol formulations shown in Figures 37 and 38; therefore, the relationship between fragmentation and antibody isoforms is shown in Figures 32, 34, and 36. The literature has shown that monoclonal antibody fragmentation-mediated agglutination 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 may be considered to be most pronounced in the acetate / arginine / sorbitol formulation, where fragmentation is greatest. This fragmentation is minimized in the acetate / phenylalanine / sorbitol formulation, potentially resulting in fewer HMWS species. The IgG4 sample type did not accelerate fragmentation or agglutination.

[0329] Data collected in this study, along with previous molecular modeling data collected for denosumab, establish a strong correlation between the amino acid sequence of the CDR and the relative effect of phenylalanine on reducing HMWS. A reduction in HMW species was observed in denosumab (IgG2) and IgG1 mutants with the same CDR amino acids, but no reduction in HMWS was observed in IgG2 mutants with different CDR domains. The amino acid sequence within the CDR domain appears to be susceptible to interaction with phenylalanine and subsequent inhibition of aggregation. The IgG4 molecule, while possessing the same CDR region as denosumab, showed only minimal aggregation changes during the study. The IgG4 molecule differs from the IgG1 and IgG2 versions primarily due to the length of its hinge amino acids and its functionally active structure. IgG1 and IgG2 antibody isoforms have an elongated structure specifically described as a "Y" shape, so the IgG4 Fab CH1 domain interacts with the CH2 domain to form a more compact structure [Aalberse RC et al., Immunology (2002), 105.pgs.9-19]. This compact structure was able to inhibit the fragmentation and aggregation reactions typically observed in IgG1 and IgG2 modalities.

[0330] Example 12 As described below, studies are conducted to monitor the stability of the denosumab formulations (formulations 51-55) related to Table 16. The diafiltration buffers differ in acetate concentration and starting pH, producing the final formulation with a pH of 5.1 at a denosumab concentration of 120 mg / ml. Furthermore, the sorbitol level is adjusted to maintain the isotonicity of the final product (approximately 300 mOsm / Kg). 70 mg / mL of denosumab is dialyzed into each buffer at a dialysis volume of more than 7 times, then ultrafiltered to approximately 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, showing that the 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 is slightly improved in formulations with lower levels of acetate.

[0331] [Table 22] * The final formulation contained 120 mg mL of denosumab and PS20 at a final concentration of 0.01% (w / v), with a pH of 5.1.

[0332] Example 13 The following examples report 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 emission scans were recorded between 300 and 500 nm. For each denaturation experiment, the protein, buffer, and denaturant (guanidinium HCl) were dispensed into 36 wells with a linear increase in denaturant concentration, and 36 curves were obtained for each condition. Curve fitting software provided by the instrument manufacturer (Unchained Labs) was used to fit the data points. A two-state model was used because there was evidence for only a single transition (native ↔ denatured). Experiments were performed using 0–6 M urea in 5.0% w / v sorbitol with 10 mM acetate, 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 amount of chemical denaturant required for 50% unfolding is C 1 / 2 This was similar under 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. A significant increase in chemical denaturation stability was observed at pH 5.2 compared to pH 4.8 and 4.5. 1 / 2 Therefore, at a lower pH of 5.2, the denaturing agent guanidinium HCl increases by only 1 M. Thus, the protective properties of arginine are remarkable and highly pH-dependent.

[0336] Example 14 The following examples provide the results of a study on the effects of arginine and phenylalanine on the time-dependent stability of high-concentration denosumab formulations in syringes.

[0337] Previous studies have shown that arginine hydrochloride and phenylalanine reduce the initial onset level and rate of denosumab HMWS formation. This study evaluated the stabilizing effects of formulations containing arginine hydrochloride, phenylalanine, and combinations of arginine hydrochloride and phenylalanine on a solution containing 120 mg / mL of denosumab, stored in syringes at two different temperatures for a period of less than three months.

[0338] The tested formulations are listed in Table 17 below. To prepare formulations 56-59, denosumab at 70 mg / mL in acetate, pH 5.2, was dialyzed into the following dialysis filtration (DF) buffer at 8 dialysate volumes to ensure buffer exchange. The material was then ultrafiltered to over 180 mg / mL, followed by dilution 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 DF buffer, and the estimated levels of the final composition at 120 mg / mL denosumab are provided considering the exclusion of additives and acetate co-concentration in the absence of other counterions. Viscosity at 5°C and 25°C is given for 1000 seconds. -1 Shear rates up to (reverse seconds) were measured using a Paar modular compact rheometer. The formulation was filled into glass pre-filled syringes (PFS) with a filling volume of 1.0 mL. The parallel syringe sets were stored at 25°C for 3 months and at 37°C for 2 months. Stability based on HMWS formation was evaluated using SE UHPLC.

[0339] [Table 23] * Each final formulation contains 120 mg / mL of denosumab and 0.01% PS20, and the pH is indicated in the formulation's abbreviation.

[0340] Figures 42 and 43 show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time over 3 months at 25°C and 2 months at 37°C, respectively.

[0341] Tables 18-21 present the same data in tabular format 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 demonstrates that the addition of arginine, phenylalanine, and combinations thereof reduces the initial HMWS (t=0) level in high-concentration denosumab formulations, respectively. At 25°C, the increase in HMWS is reduced in the phenylalanine formulation 59 compared to the control formulation 56. At 37°C, formulations 57 and 59 show reduced HMWS formation compared to the control sorbitol formulation 56. Formulations containing both arginine HCl and phenylalanine form 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] Since modifications within the scope of the present invention may be obvious to those skilled in the art, the above description is provided solely for clarity of understanding and should not be considered to impose any unnecessary limitations.

[0348] Throughout this specification and the following claims, unless the context specifically requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to mean the inclusion of any integer or step, or group of integers or steps, described herein, and not the exclusion of any other integer or step, or group of integers or steps.

[0349] Throughout this specification, where a composition is described as comprising components or materials, unless otherwise stated, the composition is substantially composed of or intended to consist of any combination of the listed components or materials. Similarly, where a method is described as comprising specific steps, unless otherwise stated, these methods are substantially composed of or intended to consist of any combination of the listed steps. The inventions disclosed exemplary herein can be adequately carried out in the absence of any element or step not specifically disclosed herein.

[0350] The methods and their individual steps disclosed herein may be carried out manually and / or by means provided by electronic devices or by automation. While the process has been described with reference to specific embodiments, those skilled in the art will readily understand that other methods may be used to perform the work related to the Method. For example, unless otherwise stated, the order of the various steps may be changed, as long as it does not deviate from the scope or spirit of the Method. Furthermore, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0351] All patents, publications, and references cited herein are incorporated herein in their entirety by reference. 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 an aqueous pharmaceutical preparation of 1.0 mL or less, wherein the aqueous pharmaceutical preparation is (a) Light chain variable region and human kappa light chain constant region of Sequence ID No. 1, and heavy chain variable region and human IgG ２ Antihuman nuclear factor kappa-B receptor activator ligand (anti-RANKL) antibody containing a constant region, and (b) Aromatic amino acids Pre-filled syringes, including [the specified ingredient].

2. A pre-filled syringe according to claim 1, wherein the concentration of anti-RANKL antibody in the aqueous pharmaceutical preparation exceeds 70 mg / mL.

3. A pre-filled syringe according to claim 2, wherein the concentration of anti-RANKL antibody in the aqueous pharmaceutical preparation exceeds 100 mg / mL.

4. A pre-filled syringe according to claim 3, wherein the concentration of anti-RANKL antibody in the aqueous pharmaceutical preparation is 100 to 140 mg / mL.

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

6. A 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 phenyl or indole. １ ~C ６ A pre-filled syringe according to claim 6, comprising an alkyl chain.

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

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

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

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

12. A pre-filled syringe according to claim 11, comprising 5 mM to 100 mM aromatic amino acids, and optionally 30 mM to 80 mM or 25 mM to 90 mM aromatic amino acids.

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

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

4.

16. A pre-filled syringe according to any one of claims 1 to 15, wherein the anti-RANKL antibody comprises the light chain of SEQ ID NO: 13 and the 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 preparation comprises denosumab, acetate, phenylalanine, sorbitol, and polysorbate 20 at a concentration of 120 mg / mL.

18. In the subjects, to treat bone-related events (SREs), giant cell tumors of bone, hypercalcemia, or osteoporosis, or to increase bone mass in the subjects, (a) Treatment of SRE in patients with bone metastases from solid tumors, (b) Treatment of SRE in adult or skeletal mature adolescent subjects with giant cell tumors of bone that are unresectable or for which surgical resection may result in a high morbidity. (c) Treatment of hypercalcemia in malignant tumors that are refractory to bisphosphonate therapy in the subjects, (d) Treatment of SRE in patients with multiple myeloma or bone metastases originating 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 who are receiving adjuvant aromatase inhibitor therapy for breast cancer. (g) Treatment to increase bone mass in men with non-metastatic prostate cancer who are at high risk of fracture and undergoing androgen deprivation therapy. (h) Treatment to increase bone mass in men with osteoporosis at high risk of fracture, and / or (i) Therapy with calcium or vitamin D A pre-filled syringe according to any one of claims 1 to 17.

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