HK40017180B - Formulations of human anti-rankl antibodies, and methods of using the same - Google Patents

Description

Preparations and methods of use of human anti-RANKL antibodies

Cross-references to related applications

This document claims the benefit of U.S. Provisional Patent Application No. 62/492,056, filed April 28, 2017, under 35 U.S.C. § 119(e), the disclosure of which is hereby incorporated by reference.

By referencing materials submitted electronically.

By reference, incorporated in its entirety, is a computer-readable nucleotide/amino acid sequence listing, which was submitted concurrently with this paper and identified as follows: a 49-kilobyte ASCII (this paper) file named “51689A_Seqlisting.txt”; created on April 20, 2018.

Background Technology

Technical Field

This invention relates to human anti-RANKL monoclonal antibodies, including high-concentration aqueous formulations of denosumab and their biosimilars.

Related technologies

Dinosumab is commercially available in solutions with strengths of 60 mg/mL and 70 mg/mL.

Increased concentrations of protein formulations can cause stability issues, such as the formation of high molecular weight substances (HMWS) aggregates. HMWS may be of particular concern in some protein formulations, especially those that retain most of the native conformation of the monomeric counterpart. Aggregation can also affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

Filling and finishing operations, as well as steps involving the flow of protein solutions through piston pumps, peristaltic pumps, or injection needles, can introduce shear and mechanical stresses, potentially leading to protein denaturation and aggregation. This phenomenon may be exacerbated by higher concentrations of protein solutions.

Summary of the Invention

The present invention provides the following disclosure, demonstrating for the first time that the addition of an amino acid aggregation inhibitor to an aqueous solution containing a high concentration of anti-RANKL antibody results in a reduction in the amount of antibody aggregates formed over a period of time and a slowing of the rate of such aggregate formation. This disclosure also provides the effect of pH on aggregate formation in concentrated aqueous solutions of anti-RANKL antibody, wherein a reduction in aggregate formation is observed when the pH of the aqueous solution is in the range of about 5.0 to below 5.2. The disclosure provided herein further suggests that the antibody is stabilized through the interaction between the amino acid aggregation inhibitor and the anti-RANKL antibody. Without being bound by any particular theory, it is conceivable that hydrophobic interactions between the amino acid aggregation inhibitor and the anti-RANKL antibody, as well as other types of intermolecular interactions, have a stabilizing effect on concentrated antibody solutions. Therefore, the disclosure of the present invention relates to stable aqueous pharmaceutical formulations containing high concentrations of anti-RANKL antibody, which contain low amounts (e.g., less than about 2%) of aggregates.

Therefore, one aspect of this disclosure is an aqueous pharmaceutical preparation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion at a concentration higher than 70 mg/mL and having a pH value in the range of about 5.0 to below 5.2.

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 with an amino acid aggregation inhibitor. In an exemplary aspect, the amino acid aggregation inhibitor comprises an amino acid containing a charged side chain, an aromatic amino acid, or a hydrophobic amino acid. In an exemplary case, the amino acid containing a charged side chain is an amino acid containing a positively charged side chain, such as arginine and lysine. In an exemplary aspect, the aromatic amino acid comprises a phenyl or indole. Optionally, the aromatic amino acid further comprises a _C1 - _C6 alkyl chain between the α-carbon and the phenyl or indole. Amino acids, including, for example, phenylalanine and tryptophan, are exemplary amino acid aggregation inhibitors. In an exemplary case, the amino acid aggregation inhibitor is a hydrophobic amino acid having a score greater than about 2.5 on the Kyte and Doolittle hydrophobicity scale. Optionally, the hydrophobic amino acid is valine, leucine, or isoleucine. Imagine other amino acid aggregation inhibitors as described in this article.

In an exemplary case, the aqueous pharmaceutical formulation further comprises a tension modifier, a surfactant, a buffer, or any combination thereof.

Another aspect of this disclosure is the presentation of the formulation for storage or use, such as in a single-use vial, a single-use syringe, or a glass, glass-lined, or glass-coated container. An exemplary aspect of this disclosure is a container containing any of the aqueous pharmaceutical formulations described herein, optionally a vial, a pre-filled syringe (PFS), or a glass container. In an exemplary case, the container contains about 1 mL or less (e.g., about 0.5 mL) of the aqueous pharmaceutical formulation.

Another aspect of this disclosure provides a method for manufacturing a stable aqueous pharmaceutical preparation comprising a human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, comprising combining the anti-RANKL monoclonal antibody or its antigen-binding moiety at a concentration higher than 70 mg/mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a ground tension modifier. This aspect of the disclosure includes stable aqueous pharmaceutical preparations manufactured according to any of the methods for manufacturing stable aqueous pharmaceutical preparations described herein.

Another aspect of this disclosure provides methods for using formulations as described herein to prevent or treat diseases in which a human anti-RANKL monoclonal antibody or its antigen-binding portion responds. In an exemplary aspect, this use covers therapeutic treatment of a subject, which includes treating or preventing skeletal-related events (SREs), treating or preventing giant cell tumor of bone, treating or preventing malignant hypercalcemia, treating or preventing osteoporosis, or increasing bone mass in the subject. For example, the therapeutic treatment covers (a) treatment or prevention of SRE in subjects with bone metastases from solid tumors; (b) treatment or prevention of SRE in adult or skeletally mature adolescent subjects with unresectable or surgically resectable giant cell tumors of bone that could cause serious morbidity; (c) treatment of bisphosphonate-refractory malignant hypercalcemia in subjects; (d) treatment or prevention of SRE in subjects with multiple myeloma or bone metastases from solid tumors; (e) treatment of osteoporosis in postmenopausal women at high risk of fracture; (f) treatment to increase bone mass in women at high risk of fracture who are receiving adjuvant aromatase inhibitor therapy for breast cancer; (g) treatment to increase bone mass in men at high risk of fracture who are 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) therapy using calcium or vitamin D.

Other aspects of this disclosure include methods for preventing skeletal-related events (SREs) in patients of need, methods for treating giant cell tumors of bone in patients of need, methods for treating malignant hypercalcemia in patients of need, methods for treating osteoporosis in patients of need, and methods for increasing bone mass in patients of need. These methods include administering an effective amount of any of the preparations described herein to the patient. In an exemplary case, the preparation is delivered subcutaneously to the patient.

Another aspect of this disclosure provides the use of denosumab or another human anti-RANKL monoclonal antibody or its antigen-binding portion for the manufacture of a medicament as described herein for the treatment of patients requiring a human anti-RANKL monoclonal antibody.

Another aspect of this disclosure is a kit that includes the compositions or articles described herein, as well as packaging inserts, packaging labels, instructions, or other markings that guide or disclose any methods or embodiments disclosed herein.

Another aspect of this disclosure is a method for improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion at a concentration higher than 70 mg/mL. The method includes the step of preparing an aqueous pharmaceutical formulation comprising the human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion at a pH in the range of about 5.0 to below 5.2, wherein the aqueous pharmaceutical formulation exhibits improved stability at a pH in the range of about 5.0 to below 5.2 compared to an equivalent aqueous pharmaceutical formulation at a pH not in the range of about 5.0 to below 5.2.

Another aspect of this disclosure is a method for improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion thereof, the method comprising the step of preparing an aqueous pharmaceutical formulation comprising a mixture of the human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion and an amino acid aggregation inhibitor, wherein the aqueous pharmaceutical formulation exhibits improved stability in the presence of the amino acid aggregation inhibitor compared to an equivalent aqueous pharmaceutical formulation without the amino acid aggregation inhibitor.

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

Other aspects and advantages will become apparent to those skilled in the art from the following detailed summary, taken in conjunction with the accompanying drawings. While these compositions, articles, and methods are readily applicable to a wide variety of embodiments, specific embodiments are included in the following description, provided that this disclosure is illustrative and not intended to limit the invention to the particular embodiments described herein. With respect to the compositions, articles, and methods described herein, optional features are contemplated, including but not limited to components, their compositional ranges, substituents, conditions, and steps, selected from the various aspects, examples, and instances provided herein.

Attached Figure Description

Figures 1, 2, and 8 show the percentage of HMWS (Highly Matured Water Species) of various high-concentration dinosuzumab formulations as monitored by SE-U HPLC at 37°C, varying with formulation and time. The legend in Figure 1 corresponds to the formulations with the abbreviations shown in Table 1. The legend in Figure 8 corresponds to the letters shown in Table 5.

Figure 3 shows the size exclusion chromatograms of various high-concentration denosumab formulations after storage at 37°C for one month. The legend in Figure 3 is consistent with the formulations with the abbreviations shown in Table 2.

Figure 4 is a graph showing the change of %HMWS over time for each formulation with the corresponding F# shown in Table 3A, as monitored by SE-UHPLC.

Figure 5 shows a pair of size exclusion chromatograms of the formulations listed in Table 3A. The legend for Figure 5 is consistent with the names of the formulations mentioned in Table 3B.

Figure 6 shows the change of %HMWS with storage time for each formulation with the corresponding F# shown in Table 4A, monitored by SE-UHPLC at 37°C.

Figure 7A shows the size exclusion chromatogram of the formulation with the concentrations of denosumab listed in Table 4A at pH 4.8.

Figure 7B shows the size exclusion chromatogram of the formulation with the concentrations of denosumab listed in Table 4A at pH 5.1.

Figure 9 shows the change of %HMWS with storage time for each formulation with the corresponding F# shown in Table 6B, monitored by SE-UHPLC at 37°C.

Figure 10 shows the size exclusion chromatogram of formulations with the names indicated in Table 6B after storage at 37°C for 1 month, varying with the formulation.

Figure 11A is a graph showing the change in HMWS percentage over time for formulations with the letters indicated in Table 7B, monitored by SE-UHPLC at 37°C.

Figure 11B is a graph showing the change in HMWS percentage over time for formulations with the letters indicated in Table 7C, monitored by SE-UHPLC at 40°C.

Figure 12A shows the size exclusion chromatogram of the formulations in Table 7B.

Figure 12B shows the size exclusion chromatogram of the formulation in Table 7C.

Figures 13, 14, and 15 are graphs showing the change in the percentage of HMWS (Highly Magnetic Mixtures) over storage time for each formulation with the corresponding formulation letter shown in Table 8A, monitored by SE-UHPLC at 37°C. Figures 16, 17, and 18 are 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.

Figures 19 to 24 show the changes in deuterium inclusion percentage (%) of each of the following formulations 35 to 38 as a function of time (log(sec)): light chain amino acids 28-33 (Figure 19), 108-116 (Figure 20), 125-132 (Figure 21), 47-59 (Figure 22), 243-253 (Figure 23), and 392-399 (Figure 24) at 4°C.

Figures 25 to 30 show the changes in the percentage of deuterium inclusion at 37°C over time (log(sec)) for each of the following formulations 35 to 38: light chain amino acids 28-33 (Figure 25), 108-117 (Figure 26), 124-131 (Figure 27), 47-59 (Figure 28), 242-253 (Figure 29), and 392-399 (Figure 30).

Figure 31 shows the percentage of HMWS monitored by SE-UHPLC at 37°C for formulations with the formulation names indicated in Table 10 as a function of formulation and time.

Figure 32 shows the percentage of LMWS of the formulations with the formulation names indicated in Table 11 at 37°C as monitored by SE-UHPLC, as a function of formulation and time.

Figure 33 shows the percentage of HMWS monitored by SE-UHPLC at 37°C for formulations with the formulation names indicated in Table 12 as a function of formulation and time.

Figure 34 shows the percentage of LMWS of the formulations with the formulation names indicated in Table 13 at 37°C as monitored by SE-UHPLC, as a function of formulation and time.

Figure 35 shows the percentage of HMWS monitored by SE-UHPLC at 37°C for formulations with the formulation names indicated in Table 14 as a function of formulation and time.

Figure 36 shows the percentage of LMWS of the formulations with the formulation names indicated in Table 15 at 37°C as monitored by SE-UHPLC, as a function of the formulation and time.

Figure 37 is a size exclusion chromatogram overlay of each formulation with the formulation name indicated in Table 10.

Figure 38 is a size exclusion chromatogram overlay of each formulation with the formulation name indicated in Table 12.

Figure 39 is a size exclusion chromatogram overlay of each formulation with the formulation name indicated in Table 14.

Figures 40A and 40B are isothermal chemical denaturation curves of denosumab in the absence of arginine at pH 4.5, pH 4.8, and pH 5.0. Figure 40A shows the denatured denosumab fraction as a function of denaturant concentration. Figure 40B shows dF/d[denaturant] as a function of denaturant concentration.

Figures 41A and 41B are isothermal chemical denaturation curves of denosumab in the presence of 75 mM arginine hydrochloride at pH 4.5, pH 4.8, and pH 5.2. Figure 41A shows the denaturing denosumab fraction as a function of denaturant concentration. Figure 41B shows the dF/d[denaturant] ratio as a function of denaturant concentration.

Figures 42 and 43 show the changes in the percentage of HMWS over time for formulations with the names of the formulations in Table 17, as monitored by SE-UHPLC, after 3 months at 25°C and 2 months at 37°C, respectively.

Detailed Implementation

It is desirable to provide concentrated aqueous solutions of dinosuzumab and other human anti-RANKL antibodies and their antigen-binding portions that are as stable as or more stable than dilute solutions. Concentrated solutions will provide convenience to patients, for example, by allowing the administration of smaller volumes, such as 1 mL of injection, to deliver 120 mg of active agent (e.g., dinosuzumab), instead of the 1.7 mL or 2 mL injections of diluted active formulations. Furthermore, it will allow even smaller volumes of injection solutions to deliver lower doses of active agent, such as 0.5 mL of 120 mg/mL dinosuzumab to deliver a 60 mg dose. It is also desirable to provide aqueous solutions of dinosuzumab and other human anti-RANKL antibodies and their antigen-binding portions that are more stable than previously known solutions. This stable concentrated formulation will also have other benefits, such as allowing for the handling and transportation of smaller volumes of product, and allowing for a longer product shelf life.

Aggregates in bioproducts can vary in origin, size, and type. Of particular concern are aggregates that may affect the efficacy or safety of bioproducts, such as those that enhance immune responses or cause adverse clinical effects. High molecular weight aggregates, also known as high molecular weight substances (HMWS), especially those that retain most of the native configuration of their monomer counterparts, may be of particular interest. Aggregates may also affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

Aggregate formation can have various causes. Generally, protein aggregation leads to both conformational instability (a result of changes in protein structure) and colloidal instability (controlled by intermolecular forces). In cases where a critical nucleation event is required to induce precipitation, protein aggregation kinetics are characterized by the inclusion of time-delay phases.

Aggregation due to conformational instability involves unfolding and association steps. Protein molecules unfold, exposing hydrophobic amino acid residues. These hydrophobic residues can then associate, leading to aggregation (e.g., in the form of dimers, trimers, other polymers, and higher-order aggregates). This association is concentration-dependent. Increased protein concentration in aqueous solvents generally increases aggregation, including the rate and extent of heat-induced aggregation. Therefore, additives in solution that affect the free energy of protein unfolding may influence conformational stability.

Colloidal instability results in the formation of aggregates through intramolecular association forces between proteins. These forces can be influenced by one or more factors, including ionic strength, solution pH, and buffer type.

Dinosumarab is commercially available in solutions with strengths of 60 mg/mL and 70 mg/mL. Attempts to formulate higher concentrations of dinosumamarab using the same excipients showed that the higher concentration affected product stability via a proportional increase in HMWS (Highly Variable Molecular Weight). For example, a concentration of 120 mg/mL dinosumamarab represents 70% higher than 70 mg/mL dinosumamarab and is twice the concentration of 60 mg/mL.

Therefore, the stable aqueous formulations according to this disclosure will resist aggregate formation to a greater extent than previously known formulations. One aspect of this disclosure is a stable aqueous formulation characterized by a pH value of 5.0 to less than 5.2. Another non-exclusive aspect of this disclosure is a stable aqueous formulation comprising amino acid aggregation inhibitors. Related dosage forms (e.g., single-use vials, syringes, and glass containers) and related treatment methods are also provided. Additionally, methods for manufacturing stable aqueous pharmaceutical formulations are provided.

As described below, pH and amino acid aggregation inhibitors (e.g., arginine, arginine-arginine dipeptide, arginine-phenylalanine dipeptide) are two proven pathways to reduce HMWS levels and HMWS formation rates at 120 mg/mL with denosumab. HMWS can be described as irreversible (e.g., covalent) or reversible (e.g., non-covalent self-association interactions) intermolecular protein interactions. There are four widely accepted reasons for protein self-association reactions that may lead to increased viscosity and HMWS: hydrophobic interactions, charged interactions, polar interactions, and dipole interactions. Formulation pH and arginine (a highly charged basic amino acid at neutral to acidic pH) can interfere with intermolecular forces of charged proteins. Without intending to be bound by any particular theory, it can be hypothesized that HMWS formation with denosumab at 120 mg/mL is based on protein charge, and that these formulation changes are disrupting the charge forces involved in the HMWS formation mechanism. Furthermore, without intending to be bound by any particular theory, it is conceivable that hydrophobic protein self-association interactions may also exist in HMWS formation, because arginine contains a short aliphatic chain in its side chain. This aliphatic chain may disrupt the hydrophobic interactions between proteins. The inclusion of phenylalanine in the formulation to further reduce HMWS levels further supports this idea. Without being bound by any particular theory, arginine stabilizes anti-RANKL antibodies in a way different from phenylalanine, such that if arginine interacts with the antibody via hydrophobic interactions, arginine can interact with the antibody in one or more other ways.

Other excipients that may have a potentially positive effect on reducing HMWS levels and formation rates, compared to arginine, may have similar positively charged groups at neutral to acidic pH values and/or be hydrophobic in nature, similar to phenylalanine. Examples of such excipients may include lysine, N-acetylarginine, N-acetyllysine, tyrosine, tryptophan, and leucine.

Unless otherwise stated, these formulations, dosage forms, and methods are envisioned to include embodiments comprising one or more of the following optional elements, features, and steps (including those shown in the figures) further described below.

Regarding jurisdiction that prohibits patenting methods performed on humans, the meaning of "giving" a composition to a human subject should be limited to controlled substances that a human subject would self-administer using any technique (e.g., oral, inhalation, topical administration, injection, insertion, etc.). This should be interpreted in the broadest reasonable sense, consistent with laws or regulations that limit the patentable subject matter. Regarding jurisdiction that does not prohibit patenting methods performed on humans, "giving" a composition includes both methods performed on humans and the aforementioned activities.

As used herein, the term "comprising" indicates that it may include other reagents, elements, steps, or features in addition to the specified substance.

It should be understood that each maximum numerical limit provided throughout this specification includes, alternatively, the range formed with each corresponding lower numerical limit, as if such a range were explicitly stated. Each minimum numerical limit provided throughout this specification will include, alternatively, the range formed with each higher numerical limit, as if such a range were explicitly stated. Each numerical range provided throughout this specification will include each narrower numerical range falling within such a wider numerical range, as if all such narrower numerical ranges were explicitly stated herein. Dimensions and values disclosed herein should be understood to include both the disclosed values and their corresponding exact values; for example, a value described as “approximately 10 mM” should be understood to include “10 mM” as an alternative disclosure.

As used herein, the term "therapeutic effective amount" refers to an amount of compound sufficient to treat, improve, or prevent the identified disease or condition, or to exhibit a detectable therapeutic, preventative, or inhibitory effect. This effect can be detected, for example, by improvement of clinical symptoms or reduction of symptoms. The precise effective amount for a subject will depend on that subject's weight, body size, and health status, the nature and severity of the condition, and the chosen therapeutic agent or combination of agents. Where the drug has been approved by the U.S. Food and Drug Administration (FDA), "therapeutic effective amount" means a dose approved by the FDA or its foreign agents for the treatment of the identified disease or condition.

This disclosure provides for stabilized (or stable) aqueous pharmaceutical formulations, as indicated by a reduction in the amount of aggregates and/or a decrease in the rate of aggregate formation after storage. As described herein, the stability of such formulations is demonstrated by a reduction in the amount of high molecular weight substances (HMWS) and/or a decrease in the rate of HMWS formation after storage over various time periods and at various temperatures. Generally, higher stability formulations are associated with lower amounts of HMWS, lower HMWS formation rates, and/or higher antibody peaks at higher storage temperatures relative to lower temperatures. As used herein, the terms “high molecular weight substances” or “HMWS” refer to both higher-order aggregates of the antibody in the formulation and lower-order aggregates of the antibody in the formulation. Lower-order aggregates include, for example, dimer substances. The amount and rate of aggregate formation can be measured or monitored using various techniques, such as SE-UHPLC. In some cases, the SE-UHPLC chromatogram of the antibody shows a peak at approximately 5.8 min representing the amount of HMWS in the aqueous pharmaceutical formulation, a peak at approximately 6.7 min representing the dimer substance, and a peak at approximately 8.0 min representing the amount of the intact, unaggregated form of the antibody. Compared to storage at 4°C, storage at 37°C allows for accelerated stability analysis, enabling the determination of the stability of a specific formulation over a shorter period compared to storage at 4°C. For example, storage at 37°C for 1 month, 2 months, or 3 months may indicate or predict 36 months of stability at 4°C.

In one type of embodiment, compared with a control formulation of equal concentration consisting of 10 mM acetate as an excipient, 5% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20 and having a solution pH of 5.2, the stabilized formulation as described herein will show a reduced degree and rate of HMWS formation after storage at 37°C for 3 months.

In another type of embodiment, compared to an equivalent control formulation without amino acid aggregation inhibitors, a stabilized formulation as described herein and including amino acid aggregation inhibitors will show a reduced degree of HMWS formation after storage at 37°C for one month. For example, compared to the control formulation, after storage at 37°C for one month, the degree of formation may be reduced to such a reduction in the percentage of HMWS as measured by SE-UPHLC as at least about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, or about 0.7%, for example, in the range of about 0.1% to about 2% or about 0.1% to about 1%.

In another type of embodiment, the stabilized formulation, as described herein, will have a lower amount of HMWS (measured by SE-UHPLC) after being stored at 37°C for 1 month. For example, the amount of HMWS may not exceed 2% or less, or not exceed 1.9% or less, or not exceed 1.8% or less, or not exceed 1.7% or less, or not exceed 1.6% or less, or not exceed 1.5% or less, or not exceed 1.4% or less, or not exceed 1.3% or less, or not exceed 1.2% or less, for example, within the range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%. In another type of embodiment, the amount of HMWS monitored by SE-UHPLC after storage at 37°C for 1 month can exceed 2%, for example, more than 2% and up to 3%, while the reduced aggregation rate provided by the amino acid aggregation inhibitor will allow for a suitable product shelf life, for example, up to three years or up to two years.

In another type of embodiment, the stabilized formulation, as described herein, will have a lower amount of HMWS (measured by SE-UHPLC) after being stored at 37°C for 3 months. For example, the amount of HMWS may not exceed 2% or less, or not exceed 1.9% or less, or not exceed 1.8% or less, or not exceed 1.7% or less, or not exceed 1.6% or less, or not exceed 1.5% or less, or not exceed 1.4% or less, or not exceed 1.3% or less, or not exceed 1.2% or less, for example, within the range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%.

In another type of embodiment, the stabilized formulation, as described herein, will have a lower amount of HMWS (measured by SE-UHPLC) after being stored at 4°C for 36 months. For example, the amount of HMWS may not exceed 2% or less, or not exceed 1.9% or less, or not exceed 1.8% or less, or not exceed 1.7% or less, or not exceed 1.6% or less, or not exceed 1.5% or less, or not exceed 1.4% or less, or not exceed 1.3% or less, or not exceed 1.2% or less, for example, within the range of about 0.01% to about 2%, or about 0.01% to about 1.9%, or about 0.01% to about 1.8%, or about 0.01% to about 1.7%, or about 0.01% to about 1.6%, or about 0.01% to about 1.5%, or about 0.01% to about 1.4%, or about 0.01% to about 1.3%, or about 0.01% to about 1.2%.

In another type of embodiment, the stabilized formulation described herein, after being stored at 37°C for one month, will exhibit a high amount of a dominant peak of denosumab or other antibodies (or their antigen-binding moiety) (measured by SE-U HPLC). For example, the amount of the dominant peak may be at least 95% or more, or at least 96% or more, or at least 97% or more, or at least 97.5% or more, or at least 98% or more, or at least 98.1% or more, or at least 98.2% or more, or at least 98.3% or more, or at least 98.4% or more, or at least 98.5 ... 8.6% or more than 98.6%, for example, in the range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about 99.9%, or about 97.5% to about 99.9%, or about 98% to about 99.9%, or about 98.1% to about 99.9%, or about 98.2% to about 99.9%, or about 98.3% to about 99.9%, or about 98.4% to about 99.9%, or about 98.5% to about 99.9%, or about 98.6% to about 99.9%.

In another type of embodiment, the stabilized formulation, as described herein, will exhibit a high amount of a dominant peak of dinosuzumab or other antibodies (or their antigen-binding moiety) after storage at 37°C for 3 months (measured by SE-U HPLC). For example, the amount of the dominant peak may be at least 95% or more, or at least 96% or more, or at least 97% or more, or at least 97.5% or more, or at least 98% or more, or at least 98.1% or more, or at least 98.2% or more, or at least 98.3% or more, or at least 98.4% or more, or at least 98.5 ... 8.6% or more than 98.6%, for example, in the range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about 99.9%, or about 97.5% to about 99.9%, or about 98% to about 99.9%, or about 98.1% to about 99.9%, or about 98.2% to about 99.9%, or about 98.3% to about 99.9%, or about 98.4% to about 99.9%, or about 98.5% to about 99.9%, or about 98.6% to about 99.9%.

In another type of embodiment, the stabilized formulation, as described herein, will exhibit a high amount of a dominant peak of denosumab or other antibody (or its antigen-binding moiety) after storage at 4°C for 36 months (measured by SE-U HPLC). For example, the amount of the dominant peak may be at least 95% or more, or at least 96% or more, or at least 97% or more, or at least 97.5% or more, or at least 98% or more, or at least 98.1% or more, or at least 98.2% or more, or at least 98.3% or more, or at least 98.4% or more, or at least 98.5 ... 8.6% or more than 98.6%, for example, in the range of about 95% to about 99.9%, or about 96% to about 99.9%, or about 97% to about 99.9%, or about 97.5% to about 99.9%, or about 98% to about 99.9%, or about 98.1% to about 99.9%, or about 98.2% to about 99.9%, or about 98.3% to about 99.9%, or about 98.4% to about 99.9%, or about 98.5% to about 99.9%, or about 98.6% to about 99.9%.

In other embodiments, it is envisioned that the stabilized formulation, after being stored in accordance with the provisions described above, will have a lower amount of HMWS and a higher amount of main peak.

In one exemplary aspect, the aqueous pharmaceutical preparation, after storage, contains no more than about 4% high molecular weight substances (HMWS) and/or contains more than about 96% of the antibody peak, as measured by SE-UHPLC. In another exemplary aspect, the aqueous pharmaceutical preparation, after storage, contains no more than about 3% high molecular weight substances (HMWS) and/or contains more than about 97% of the antibody peak, as measured by SE-UHPLC. In yet another exemplary aspect, the aqueous pharmaceutical preparation, after storage, contains less than about 2% HMWS and/or more than about 98% of the antibody peak, as measured by SE-UHPLC. In an exemplary aspect, the storage is carried out at a temperature of about 2°C to about 8°C (e.g., about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C) for at least 12 months, 24 months, or 36 months (e.g., at least or about 12 months, at least or about 16 months, at least or about 20 months, at least or about 24 months, at least or about 28 months, at least or about 32 months, at least or about 36 months, optionally longer). In an exemplary aspect, the storage is carried out at about 20°C to about 30°C (e.g., about 21°C to about 30°C, about 22°C to about 30°C, about 23°C to about 30°C, about 24°C to about 30°C, about 25°C to about 30°C, about 26°C to about 30°C, about 27°C to about 30°C, about 28°C to about 30°C, about 28°C to about 30°C, about 20°C to about 29°C, about 20°C to about 28°C, about 20°C to about 27°C, about 20°C to about 26°C, about 20°C to about 25°C, about 20°C to about 24°C, about 20°C to about 23°C, about 20°C to about 22°C) for about one month (e.g., about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days). In an exemplary aspect, the storage includes a first storage and a subsequent second storage, wherein the first storage is carried out at a temperature of about 2°C to about 8°C for at least 12 months, 24 months, or 36 months, and the second storage is carried out at a temperature of about 20°C to about 30°C for about 1 month. In an exemplary case, the aqueous pharmaceutical preparation contains no more than 2% HMWS or less, or no more than 1.9% HMWS or less, or no more than 1.8% HMWS or less, or no more than 1.7% HMWS or less, or no more than 1.6% HMWS or less, or no more than 1.5% HMWS or less, or no more than 1.4% HMWS or less, or no more than 1.3% HMWS or less. Or not more than 1.2% HMWS or less than 1.2% HMWS, for example, optionally in the range of about 0.01% to about 2% HMWS, or about 0.01% to about 1.9% HMWS, or about 0.01% to about 1.8% HMWS, or about 0.01% to about 1.7% HMWS, or about 0.01% to about 1.6% HMWS, or about 0.01% to about 1.5% HMWS, or about 0.01% to about 1.4% HMWS, or about 0.01% to about 1.3% HMWS, or about 0.01% to about 1.2% HMWS, as measured by SE-UHPLC. In alternative or other respects, the aqueous pharmaceutical preparation contains more than 98% antibody peak, or at least 95% or more of antibody peak, or at least 96% or more of antibody peak, or at least 97% or more of antibody peak, or at least 97.5% or more of antibody peak, or at least 98% or more of antibody peak, or at least 98.1% or more of antibody peak, or at least 98.2% or more of antibody peak, or at least 98.3% or more of antibody peak, or at least 98.4% or more of antibody peak, or at least 98.5% antibody peak. Or more than 98.5% of the antibody peak, or at least 98.6% of the antibody peak, or more than 98.6% of the antibody peak, for example, optionally in the range of about 95% to about 99.9% of the antibody peak, or about 96% to about 99.9% of the antibody peak, or about 97% to about 99.9% of the antibody peak, or about 97.5% to about 99.9% of the antibody peak, or about 98% to about 99.9% of the antibody peak, or about 98.1% to about 99.9% of the antibody peak, or about 98.2% to about 99.9% of the antibody peak, or about 98.3% to about 99.9% of the antibody peak, or about 98.4% to about 99.9% of the antibody peak, or about 98.5% to about 99.9% of the antibody peak, or about 98.6% to about 99.9% of the antibody peak, as measured by SE-UHPLC.

As used herein, the term "antibody" refers to a protein that has a conventional immunoglobulin form, comprising heavy and light chains, and containing variable and constant regions. For example, an antibody can be IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG form, the variable region is typically about 100-110 or more amino acids, contains three complementarity-determining regions (CDRs), is primarily responsible for antigen recognition, and differs significantly from other antibodies that bind to different antigens. See, for example, Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd./Garland Publishing, (1999).

In short, in antibody scaffolds, CDRs are embedded within a framework of heavy and light chain variable regions, which constitute the regions primarily responsible for antigen binding and recognition. The variable regions contain at least three heavy chain CDRs or three light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service, N.I.H., Bethesda, Maryland; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), located within the framework regions (defined by Kabat et al., 1991 as framework regions 1-4, FR1, FR2, FR3, and FR4; see also Chothia and Lesk, 1987, ibid.).

Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Examples of this disclosure include all such antibody classes or isotypes. Light chain constant regions may be, for example, κ or λ type light chain constant regions, such as human κ or λ type light chain constant regions. Heavy chain constant regions may be, for example, α, δ, ε, γ, or μ type heavy chain constant regions, such as human α, δ, ε, γ, or μ type heavy chain constant regions. Therefore, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In an exemplary aspect, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody.

In various respects, the antibody may be a monoclonal or polyclonal antibody. In some respects, the antibody contains a sequence substantially similar to naturally occurring antibodies produced by mammals, such as mice, rats, rabbits, goats, horses, chickens, hamsters, pigs, humans, etc. In this regard, the antibody may be considered a mammalian antibody, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, pig antibodies, human antibodies, etc. In some respects, anti-RANKL antibodies are monoclonal human antibodies. In some respects, recombinant proteins are chimeric antibodies or humanized antibodies. The term "chimeric antibody" is used herein to refer to an antibody containing a constant domain from one species and a variable domain from a second species, or more generally, an antibody containing amino acid sequence segments from at least two species. The term "humanized" in relation to the use of an antibody refers to an antibody having at least a non-human source CDR region that has been engineered to have a structure and immunological function more similar to a true human antibody than the original source antibody. For example, humanization can involve grafting a CDR from a non-human antibody (such as a mouse antibody) into a human antibody. Humanization can also involve selecting amino acid substitutions to make a non-human sequence look more like a human sequence.

In various aspects, 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 an exemplary aspect, an aqueous pharmaceutical formulation comprises an antibody fragment, such as Fab, Fc, F(ab') ₂ , or pFc', that retains at least one antigen (RANKL) binding site. Regarding the aqueous pharmaceutical formulations and methods disclosed herein, the antibody may lack certain portions of the antibody and may be an antibody fragment bound to RANKL. In an exemplary aspect, the antibody fragment is the antigen-binding portion of an anti-RANKL antibody.

Antibody protein products can be antigen-binding fragments based on retaining complete antigen-binding capability, such as scFv, Fab, and VHH/VH. The smallest antigen-binding fragment retaining its complete antigen-binding site is the Fv fragment, which consists entirely of a variable (V) region. The molecule is stabilized by linking the V region to the scFv fragment (variable single-chain fragment) using a soluble flexible amino acid peptide linker, or by adding a constant (C) domain to the V region to produce the Fab fragment [antigen-binding fragment]. Both scFv and Fab are widely used fragments and can be readily produced in hosts, such as prokaryotic hosts. Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimer and polymeric antibody forms, such as bifunctional, trifunctional, and tetrafunctional antibodies, or various forms of mini-antibodies (miniAbs) comprising scFvs linked to oligomeric domains. The smallest fragments are camel heavy chain Abs and single-domain Abs (sdAbs) in VHH/VH. The most commonly used building blocks for constructing novel antibody forms are single-chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from the heavy and light chains linked by peptide linkers with approximately 15 amino acid residues. Peptide antibodies or peptide-Fc fusions are another antibody protein product. The structure of a peptide antibody consists of a biologically active peptide grafted onto the Fc domain. Peptide antibodies have been well described in this technique. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

Other antibody protein products include single-chain antibodies (SCAs), bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and bispecific or trispecific antibodies. Bispecific antibodies can be divided into five main categories: BsIgG, attached IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015).

In an exemplary aspect, an anti-RANKL antibody or its antigen-binding portion comprises or is substantially composed of these antibody protein products (e.g., scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimer antibodies, polyantibodies (e.g., bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies), mini antibodies, peptide antibodies VHH/VH of camel heavy chain antibodies, sdAb, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, bispecific or trispecific antibodies, BsIgG, additional IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates).

In exemplary aspects, an anti-RANKL antibody or its antigen-binding portion comprises, is substantially composed of, or consists of, an antibody protein product in monomeric or polymeric, oligomeric or polymeric form. In some embodiments where the antibody comprises two or more unique antigen-binding region fragments, the antibody is considered bispecific, trispecific or multispecific, or bivalent, trivalent or multivalent, depending on the number of unique antigenic determinants that the antibody recognizes and binds.

The human anti-human nuclear factor kappa B receptor activator ligand (anti-RANKL) antibody or its antigen-binding moiety used in this formulation is an antibody or its antigen-binding moiety that specifically binds to human RANKL protein or human osteoprotegerin (OPGL) protein or fragments thereof and inhibits or neutralizes the activity of 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 comprise 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. Human RANKL protein is a transmembrane or soluble protein encoded by the polynucleotide sequence of SEQ ID NO: 11, which is known to be essential for osteoclast formation, function, and survival. For example, human anti-RANKL antibodies inhibit the interaction between RANKL and its receptor RANK.

An example of a human anti-RANKL monoclonal antibody is dinosuzumab, which is sold commercially available. A single-use vial containing a 1.7 mL solution (70 mg/mL) of dinosuzumab at a dose of 120 mg contains 120 mg dinosuzumab, acetate (18 mM), sorbitol (4.6%), water for injection (USP), and sodium hydroxide (to pH 5.2). A single-use pre-filled syringe containing a 1 mL solution (60 mg/mL) of dinosuzumab at a dose of 60 mg contains 60 mg dinosuzumab (60 mg/mL solution), 4.7% sorbitol, 17 mM acetate, 0.01% polysorbate 20, water for injection (USP), and sodium hydroxide (to pH 5.2). Specific formulations as described herein and including dinosuzumab or portions thereof are also available. Dinosumab is a fully human IgG2 monoclonal antibody that binds to human RANKL. Dinosumab has an approximate molecular weight of 147 kDa and is expressed in the Chinese hamster ovary (CHO) cell line. The amino acid sequences of the variable light chain (LC) and variable heavy chain (HC) of Dinosumab are shown in SEQ ID NO:1 and 2, respectively, and the full-length LC and HC are shown in SEQ ID NO:3 and 4, respectively. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 (dinosumab variable LC) is the nucleic acid of SEQ ID NO:19. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 (dinosumab variable HC) is the nucleic acid of SEQ ID NO:20. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 (full-length Dinosumab LC) is the nucleic acid of SEQ ID NO:21. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 (full-length Dinosumab HC) is the nucleic acid of SEQ ID NO:23. The mature form LC, representing amino acids 21-235 of the full-length LC, is shown as SEQ ID NO:13, while the mature form HC, representing amino acids 20-467 of the full-length HC, is shown as SEQ ID NO:14. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:13 (mature form LC) is the nucleic acid of SEQ ID NO:22. In some aspects, the nucleic acid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 (mature form HC) is the nucleic acid of SEQ ID NO:24. Additionally, the Dinosumab LC CDRs are shown as SEQ ID NO:5 (LC CDR1), SEQ ID NO:6 (LC CDR2), and SEQ ID NO:7 (LC CDR3). Dinosumab HC CDRs are shown as SEQ ID NO:8 (HC CDR1), SEQ ID NO:9 (HC CDR2), and SEQ ID NO:10 (HC CDR3). Dinosumab and claims to protection thereof have been described in International Patent Application No. WO 03/002713 and U.S. Patent No. 7,364,736, the disclosures of which are incorporated herein by reference in their entirety.

As used herein, the term "dinosumab" includes biosimilars of dinosumab. As used herein, a "biosimilar" (of an approved reference product/biologic, such as a protein therapeutic agent, antibody, etc.) means a biologic product similar to a reference product based on data derived from studies that: (a) analytical studies showing a high degree of similarity to the reference product but with minor differences in clinically inactive components; (b) animal studies (including assessments of toxicity); and/or (c) clinical studies (including assessments of immunogenicity and pharmacokinetic or pharmacodynamic properties) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate conditions of use for which the reference product is licensed and intended to be used and for which the biologic product seeks license. In one embodiment, the biosimilar biologic product and the reference product utilize one or more of the same mechanisms of action under one or more conditions of use specified, recommended, or suggested in the proposed labeling, but only to the extent that one or more mechanisms of action of the reference product are known. In one embodiment, one or more conditions of use specified, recommended, or suggested in the proposed labeling for the biologic product have previously been approved for use in the reference product. In one embodiment, the biologic product has the same route of administration, dosage form, and/or strength as the reference product. In one embodiment, the organization that manufactures, processes, packages, or contains the biological product meets design criteria that ensure the continued safety, purity, and efficacy of the biological product. 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 major amino acid sequence as a marketed antibody but possibly manufactured in a different cell type or through a different production, purification, or formulation method.

These preparations may contain a human anti-RANKL antibody containing at least one or a portion of the amino acid sequences of SEQ ID NO:1-4, 13, and 14. These preparations may contain at least one, or at least two, of the CDR amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or at least three of the CDR amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Human anti-RANKL antibodies, such as at least four of the CDR amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or at least five of the CDR amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or at least six of the CDR amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

These formulations may comprise a human anti-RANKL antibody containing at least 80% identical amino acid sequence to any one of SEQ ID NO:1-4, 13, and 14 that inhibits the interaction between RANKL and its receptor RANK; or a human anti-RANKL antibody containing at least 85% identical amino acid sequence to any one of SEQ ID NO:1-4, 13, and 14 that inhibits the interaction between RANKL and its receptor RANK; or a human anti-RANKL antibody containing at least 90% identical amino acid sequence to any one of SEQ ID NO:1-4, 13, and 14 that inhibits the interaction between RANKL and its receptor RANK. NKL antibody, or a human anti-RANKL antibody comprising at least 91% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK; or a human anti-RANKL antibody comprising at least 92% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK; or a human anti-RANKL antibody comprising at least 93% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK. L antibody, or a human anti-RANKL antibody containing at least 94% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 that inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody containing at least 95% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 that inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody containing at least 96% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 that inhibits the interaction between RANKL and its receptor RANK. An antibody, or a human anti-RANKL antibody comprising at least 97% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least 98% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least 99% identical amino acid sequence to any one of SEQ ID NO:1-4, 13 and 14 and inhibiting the interaction between RANKL and its receptor RANK.

In an exemplary embodiment, the aqueous pharmaceutical preparation comprises an anti-RANKL antibody or its antigen-binding portion, including an antibody protein product as described herein. In an exemplary aspect, the anti-RANKL antibody or its antigen-binding portion comprises a light chain variable domain comprising a light chain CDR1 sequence containing the amino acid sequence shown in SEQ ID NO:5. Alternatively or otherwise, the anti-RANKL antibody or its antigen-binding portion comprises a light chain variable domain comprising a light chain CDR2 sequence containing the amino acid sequence shown in SEQ ID NO:6. Alternatively or otherwise, the anti-RANKL antibody or its antigen-binding portion comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence containing the amino acid sequence shown in SEQ ID NO:10. In some cases, the anti-RANKL antibody or its antigen-binding portion comprises SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:10. In an exemplary aspect, the anti-RANKL antibody or its antigen-binding portion comprises (i) a light chain variable domain comprising a light chain CDR3 sequence containing the amino acid sequence shown in SEQ ID NO:7; (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence containing the amino acid sequence shown in SEQ ID NO:8, optionally SEQ ID NO:27; (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence containing the amino acid sequence shown in SEQ ID NO:9; or (iv) any combination thereof. In some aspects, the anti-RANKL antibody or its antigen-binding portion comprises (A) a light chain variable domain comprising a light chain CDR1 containing the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising a light chain CDR2 containing the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising a light chain CDR3 containing the amino acid sequence of SEQ ID NO:7; and (B) a heavy chain variable domain comprising a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:8 (optionally SEQ ID N:27), a heavy chain variable domain comprising a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:10. In an exemplary aspect, the anti-RANKL antibody or its antigen-binding portion comprises: (A) a light chain variable domain selected from the group consisting of: (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 the same amino acid sequence as SEQ ID NO:1; (ii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO:19; and (iii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a complementary sequence of the polynucleotide comprising SEQ ID NO:19; or (B) a heavy chain variable domain selected from the group consisting of: (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 the same amino acid sequence as 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 (B) a heavy chain variable domain selected from the group consisting of: (i) 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 sequence comprising SEQ ID NO:19; or (B) a heavy chain variable domain comprising: (i) 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 sequence comprising SEQ ID NO:19; or (iv) a heavy chain variable domain comprising an amino acid sequence encoded by (i) a heavy 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 the same amino acid sequence as NO:2; (ii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO:20; and (iii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence hybridized under stringent conditions to a complementary sequence of the polynucleotide comprising SEQ ID NO:20; or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B). In an exemplary aspect, the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In an exemplary case, the antigen-binding moiety is Fab, Fab', F(ab')2, or a single-chain Fv. In one exemplary aspect, the anti-RANKL antibody is an _IgG1 , _IgG2 , or _IgG4 antibody, optionally wherein the anti-RANKL antibody comprises the sequence of SEQ ID NO:15. In some aspects, the anti-RANKL antibody comprises the sequence of SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In an exemplary aspect, the anti-RANKL antibody or its antigen-binding portion comprises: (A) a light chain selected from the group consisting of: (i) a light 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 the same amino acid sequence as SEQ ID NO:3 or SEQ ID NO:13; (ii) a light chain comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO:21 or 23; and (iii) a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a complementary sequence of a polynucleotide comprising SEQ ID NO:21 or 23; or (B) a heavy chain selected from the group consisting of: (i) a light chain comprising an amino acid sequence of 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 the same amino acid sequence as SEQ ID NO:3 or SEQ ID NO:13; or (B) a heavy chain selected from the group consisting of: (i) a light chain comprising an amino acid sequence of 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 the same amino acid sequence as SEQ ID NO:21 or 23; or (B) a heavy chain selected from the group consisting of: (i) a light chain comprising an amino acid sequence of at least 80% (e.g., at least 80%, at (ii) a heavy chain comprising an amino acid sequence of 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%) the same as NO:14; and (iii) a heavy chain comprising an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO:22 or 24; or (c) the variable domain of the light chain of (A) and the variable domain of the heavy chain of (B).

The concentration of denosumab or other human anti-RANKL antibody or its antigen-binding moiety in this aqueous formulation can generally be within any useful range, for example, from about 0.1 to about 200 mg/mL. As the concentration increases, the viscosity increases, which may prevent the formulation from being processed into a sterile dosage form for pharmaceutical use.

In one aspect, formulations with improved stability via amino acid aggregation inhibitors may be present at any concentration of denosumab or other human anti-RANKL antibodies or their antigen-binding moiety, including about 10 mg/mL to about 200 mg/mL, or about 15 mg/mL to about 150 mg/mL, or about 30 mg/mL to about 200 mg/mL, or about 60 mg/mL to about 200 mg/mL, or about 60 mg/mL to about 180 mg/mL, or about 60 mg/mL to about 160 mg/mL, or about 60 mg/mL to about 150 mg/mL, or about 60 mg/mL to about 140 mg/mL, or about 60 mg/mL to about 130 mg/mL, or about 60 mg/mL to about 120 mg/mL, or about 60 mg/mL to about 110 mg/mL, or about 60 mg/mL The concentrations are approximately 100 mg/mL, or 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.

In another respect, for formulations having a pH of about 5.0 to below 5.2, it is envisioned that the concentration of denosumab or other human anti-RANKL antibody or its antigen-binding moiety includes a range of more 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 envisioned range includes 71 mg/mL to about 200 mg/mL, or about 75 mg/mL to about 200 mg/mL, or about 75 mg/mL to about 180 mg/mL, or about 75 mg/mL to about 160 mg/mL, or about 75 mg/mL to about 150 mg/mL, or about 75 mg/mL to about 140 mg/mL, or about 75 mg/mL to about 130 mg/mL, or about 75 mg/mL to about 120 mg/mL, or about 75 mg/mL to about 110 mg/mL, or about 75 mg/mL to about 100 mg/mL, or about 75 mg/mL to about 90 mg/mL, or about 120 mg/mL to about 200 mg/mL, or about 120 mg/mL to about 180 mg/mL, or about 120 mg/mL to about 160 mg/mL, or about 120 mg/mL to about 140 mg/mL, such as 120 mg/mL.

In an exemplary aspect, the aqueous pharmaceutical preparation comprises the antibody or its antigen-binding portion at a concentration higher than 70 mg/mL, such as higher than 80 mg/mL, higher than 90 mg/mL, higher than 100 mg/mL, higher than 125 mg/mL, higher than 150 mg/mL, higher than 175 mg/mL, higher than 200 mg/mL, higher than 225 mg/mL, higher than 250 mg/mL, or higher than 275 mg/mL. In an exemplary aspect, the aqueous pharmaceutical preparation comprises the antibody or its antigen-binding portion at a concentration lower than about 300 mg/mL, such as lower than about 275 mg/mL, lower than about 250 mg/mL, lower than about 225 mg/mL, lower than about 200 mg/mL, lower than about 175 mg/mL, or lower than about 150 mg/mL. In an exemplary aspect, the concentration of the antibody or its antigen-binding portion in the preparation is in the range of about 10 mg/mL to about 300 mg/mL, for example, about 25 mg/mL to about 300 mg/mL, about 50 mg/mL to about 300 mg/mL, about 75 mg/mL to about 300 mg/mL, about 125 mg/mL to about 300 mg/mL, about 150 mg/mL to about 300 mg/mL, about 175 mg/mL to about 300 mg/mL, about 200 mg/mL to about 300 mg/mL, about 225 mg/mL to about 300 mg/mL, and about 250 mg/mL to about 300 mg/mL. The range is approximately 275 mg/mL to approximately 300 mg/mL, approximately 10 mg/mL to approximately 275 mg/mL, approximately 10 mg/mL to approximately 250 mg/mL, approximately 10 mg/mL to approximately 225 mg/mL, approximately 10 mg/mL to approximately 200 mg/mL, approximately 10 mg/mL to approximately 175 mg/mL, approximately 10 mg/mL to approximately 150 mg/mL, approximately 10 mg/mL to approximately 125 mg/mL, approximately 10 mg/mL to approximately 100 mg/mL, approximately 10 mg/mL to approximately 75 mg/mL, approximately 10 mg/mL to approximately 50 mg/mL, or approximately 10 mg/mL to approximately 25 mg/mL. In an exemplary aspect, the aqueous pharmaceutical preparation comprises a concentration in the range of above 70 mg/mL to about 300 mg/mL, for example, above 80 mg/mL to about 300 mg/mL, above 90 mg/mL to about 300 mg/mL, above 100 mg/mL to about 300 mg/mL, above 125 mg/mL to about 300 mg/mL, above 150 mg/mL to about 300 mg/mL, above 175 mg/mL to about 300 mg/mL, above 200 mg/mL to about 300 mg/mL. The antibody or its antigen-binding portion is present in concentrations ranging from about 70 mg/mL to about 275 mg/mL, from about 70 mg/mL to about 250 mg/mL, from about 70 mg/mL to about 225 mg/mL, from about 70 mg/mL to about 200 mg/mL, from about 70 mg/mL to about 175 mg/mL, from about 70 mg/mL to about 150 mg/mL, from about 70 mg/mL to about 125 mg/mL, and from about 70 mg/mL to about 100 mg/mL. In an exemplary aspect, the aqueous pharmaceutical preparation comprises the antibody or its antigen-binding portion in concentrations ranging from about 100 mg/mL to about 140 mg/mL, for example, about 110 mg/mL, about 120 mg/mL, and about 130 mg/mL. In some aspects, the aqueous pharmaceutical preparation contains the antibody or its antigen-binding portion at a concentration of about 120 mg/mL ± 12 mg/mL, such as about 108 mg/mL to about 132 mg/mL, about 115 mg/mL to about 125 mg/mL, about 116 mg/mL, about 117 mg/mL, about 118 mg/mL, about 119 mg/mL, about 120 mg/mL, about 121 mg/mL, about 122 mg/mL, about 123 mg/mL, or about 124 mg/mL.

Dinosumab and other human anti-RANKL monoclonal antibodies and their antigen-binding moieties can be prepared according to the description provided in International Patent Publication WO 2003002713 A2.

Studies on the formulation of high-concentration dinosumab solutions (e.g., 120 mg/mL) described below showed a significant increase in the rate and extent of HMWS formation below pH 5, and especially at lower pH values (e.g., pH 4.5). It has been demonstrated that the formation of dimer substances increases with increasing pH. To balance these two effects, it is envisioned that the formulations described herein would have a pH in the range of about 5.0 to below 5.2, or about 5.0 to about 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, for example, about 5.0, about 5.05, about 5.1, or about 5.15.

The studies described in this article also demonstrate that including amino acid aggregation inhibitors makes it possible to obtain independent stability and aggregation reduction effects. Therefore, it is envisioned that when amino acid aggregation inhibitors are included, the pH of the formulation can be in the range of about 4.9 to about 5.4, or about 5.0 to about 5.4, or about 5.0 to about 5.2, or about 5.0 to below 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.

This aqueous formulation can be buffered. When used, the buffer can be an organic buffer. The buffering system can be centered at, for example, a pH of about 4 to 5.5, or 4.5 to 5.5, or 4.5 to 5 at 25°C. For example, the buffering system can have a pKa within a pH unit of 5.0-5.2 at 25°C. One such buffering system is an acetate/acetate with a pKa of about 4.75 at 25°C. Another such buffering system is a glutamate/glutamate with a pKa of about 4.27 at 25°C. Other envisioned alternative buffering systems include ion-based systems, including 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). Buffers are envisioned to be provided as sodium salts (or disodium salts, as appropriate) or, alternatively, as potassium, magnesium, or ammonium salts. For example, buffers may be based on acetates, citrates, succinates, phosphates, and hydroxymethylaminomethane (Tris). Buffers based on acetates, glutamates, and succinates, such as acetates or glutamates, are particularly envisioned.

HMWS formation was compared by size exclusion ultra-high performance liquid chromatography (SE-UHPLC) among 120 mg/mL dinosuzumab formulations with acetate or glutamate buffers but otherwise equivalent, and no difference was found between the buffer types when evaluated after four weeks of storage at 37°C.

When used, the buffer will be included in an amount sufficient to maintain the selected pH of the formulation over the product's shelf life under storage conditions (e.g., 3 years at 4°C, 1 month at 25°C, 2 weeks at 25°C, or 7 days at 25°C). The buffer concentration may be in the range of about 2 mM to about 40 mM, or about 5 mM to about 20 mM, or about 10 mM to about 25 mM, or about 15 mM to about 25 mM, for example, 10 mM, or 15 mM, or 18 mM, or 25 mM. For example, an acetate buffer used with an anti-RANKL monoclonal antibody (e.g., denosumab) and phenylalanine may be in the range of about 2 mM to about 30 mM, or about 16 mM to about 41 mM, or about 25 mM to about 39 mM, or about 30 mM to about 34 mM. In other words, the dialysis buffer used to concentrate the antibody to a concentration higher than 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. A self-buffered, amino acid-stabilized formulation is also envisioned. In an exemplary aspect, the buffer is included in an amount sufficient to maintain the selected pH of the formulation during its shelf life under storage conditions (e.g., 36 months at about 2°C to about 8°C, optionally followed by about 1 month at about 20°C to about 30°C).

In some aspects, the aqueous pharmaceutical formulation comprises a buffer, and optionally, the buffer is centered in the range of about pH 4.0 to about pH 5.5 at 25°C. In some aspects, the buffer has a pKa in one pH unit of pH 5.0-5.2 at 25°C. In some aspects, the aqueous pharmaceutical formulation comprises about 5 mM to about 60 mM of buffer, about 5 mM to about 50 mM of buffer, or about 9 mM to about 45 mM of buffer (e.g., about 15 mM to about 30 mM of buffer, e.g., about 20 mM, about 25 mM of buffer). In an exemplary aspect, the buffer is an acetate or a glutamate.

The formulation may also include one or more stabilizers and other formulation excipients that target protein aggregation. Such stabilizers and excipients are envisioned to include, but are not limited to, amino acid aggregation inhibitors, tension modifiers, surfactants, solubilizers (e.g., N-methyl-2-pyrrolidone), PEG conjugates, and cyclodextrins (e.g.).

The term "amino acid aggregation inhibitor" refers to an amino acid or combination of amino acids (e.g., a mixture, a dipeptide, or an oligopeptide having 2 to 10 residues), wherein any given amino acid is present in its free base form or in its salt form (e.g., arginine hydrochloride) or in an amino acid analogue, and reduces or inhibits HMWS formation. Salts including sodium salts, potassium salts, and hydrochloride salts are envisioned. Additionally, arginine salts with hydrochloride, glutamate, butyrate, and glycolate are envisioned. When using combinations of amino acids, these amino acids may be present entirely in their free base form, entirely in their salt form, or possibly partially in their free base form and others in their salt form. In addition to dipeptides and oligopeptides, or in alternatives to dipeptides and oligopeptides, mixtures of one or more amino acids, such as a mixture of arginine and phenylalanine, may be used. In alternative embodiments, only one type of amino acid aggregation inhibitor is present in the aqueous pharmaceutical formulation. In an exemplary aspect, only one amino acid, such as L-arginine or L-phenylalanine only, is present in the formulation.

It is envisioned to use one or more amino acids with charged side chains, such as arginine, lysine, histidine, aspartate, and glutamate. The amino acid may be selected from basic amino acids, such as arginine, lysine, histidine, or combinations thereof. Arginine is particularly envisioned. Any stereoisomer of the specific amino acid (i.e., L, D, or DL isomers) or combinations of these stereoisomers may be used in the methods or formulations of the present invention, provided that the specific amino acid is present in its free base form or its salt form. L-stereomers, such as L-arginine, are particularly envisioned. Optionally, the amino acid is an amino acid having a positively charged side chain, such as arginine.

In another embodiment, it is envisioned to use one or more amino acids having an aromatic ring in their side chain, such as phenylalanine, tyrosine, tryptophan, or combinations thereof. Phenylalanine is particularly envisioned.

In another scenario, it is envisioned to use one or more hydrophobic amino acids, such as alanine, isoleucine, leucine, phenylalanine, valine, proline, or glycine.

In another approach, it is envisioned to use one or more aliphatic hydrophobic amino acids, such as alanine, isoleucine, leucine, or valine. Leucine is particularly envisioned.

Amino acid analogs exhibiting aggregation-reducing or inhibitory effects may also be used in the methods or formulations of the present invention. The term "amino acid analog" refers to a derivative of a naturally occurring amino acid. Contemplated analogs include, for example, amino derivatives, as well as N-monoethyl and n-acetyl derivatives. Other contemplated analogs include dipeptides or oligopeptides having 2 to 10 residues, such as arginine-arginine and phenylalanine-arginine. In one type of embodiment, it is contemplated that n-acetylarginine and n-acetyllysine are not used alone, but may be used in combination with another amino acid aggregation inhibitor. Like amino acids, amino acid analogs are used in the methods or formulations of the present invention in their free base form or salt form.

The amino acid aggregation inhibitors used in the methods or formulations of this invention protect therapeutically active proteins from various stresses, thereby increasing and/or maintaining the stability of the protein or protein-containing formulations throughout the protein's lifespan (before and during storage, before use). In this document, the term "stress" includes, but is not limited to, stresses from any source, such as heat, freezing, pH, light, agitation, oxidation, dehydration, surface, shear, freezing/thawing, pressure, heavy metals, phenolic compounds, denaturants, etc. Thermal stress is particularly contemplated. The term stress encompasses any factor that modulates (i.e., reduces, maintains, or increases) the stability of the protein or protein-containing formulation. The use of amino acid aggregation inhibitors to increase and/or maintain stability occurs in a concentration-dependent manner. That is, when the protein or protein-containing formulation of this invention normally exhibits aggregate formation in the absence of an amino acid aggregation inhibitor, increasing the concentration of the amino acid aggregation inhibitor increases and/or maintains the stability of the protein or protein-containing formulation. As shown in the following examples, including an amino acid aggregation inhibitor in the formulation can also reduce the amount of HMWS already formed. For example, such amino acid aggregation inhibitors include arginine and arginine-phenylalanine dipeptides. Based on the disclosure herein, for denosumab or any particular human anti-RANKL monoclonal antibody of interest, the amount of a specific amino acid aggregation inhibitor used in the methods or formulations of the present invention to reduce aggregate formation, thereby increasing protein stability and thus the stability of the formulation throughout the lifespan of the protein can be readily determined.

The presence of amino acid aggregation inhibitors in formulations has been shown to reduce the amount of dimer and its dynamic formation rate. For example, after one month at 37°C, the inclusion of 75 mM arginine in a denosumab formulation at pH 5.2 reduced the amount of dimer and its dynamic formation rate by approximately 0.3% and 25%, respectively, compared to a similar formulation without arginine at pH 5.2. In contrast, the inclusion of arginine was found to fail to stabilize monoclonal antibodies that are not human anti-RANKL monoclonal antibodies, but instead resulted in an increase in HMWS. Therefore, another approach disclosed herein is to reduce HMWS in denosumab or another human anti-RANKL monoclonal antibody formulation by adding an amino acid aggregation inhibitor, such as arginine or phenylalanine.

Therefore, in an exemplary embodiment, the aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor, which is optionally an amino acid. In an exemplary aspect, the amino acid is an L-stereoisomer (L-amino acid), but a D-stereoisomer (D-amino acid) is contemplated. In some aspects, the amino acid aggregation inhibitor comprises an amino acid containing a charged side chain, also referred to herein as a “charged amino acid.” The term “charged amino acid” refers to an amino acid containing a negatively charged (i.e., deprotonated) or positively charged (i.e., protonated) side chain in an aqueous solution having a physiological pH. For example, negatively charged amino acids include, for example, aspartic acid and glutamic acid, while positively charged amino acids include, for example, arginine, lysine, and histidine. Charged amino acids include charged amino acids from the 20 coding amino acids, as well as atypical or non-naturally occurring or non-coding amino acids. Therefore, in an exemplary aspect, the amino acid aggregation inhibitor is an amino acid containing a positively charged side chain. In an exemplary case, the amino acid containing a positively charged side chain comprises a side chain structure of Formula I or Formula II:

Where n is 1 to 7, and each of _R1 and _R2 is independently selected from the group consisting of: H, _C1 - _C18 alkyl, ( _C1 - _C18 alkyl)OH, ( _C1 - _C18 alkyl) _NH2 , NH, _NH2 ( _C1 - _C18 alkyl)SH, ( _C0 - _C4 alkyl)( _C3 - _C6 )cycloalkyl, ( _C0 - _C4 alkyl)( _C2 - _C5 heterocyclic), ( _C0 - _C4 alkyl)( _C6 - _C10 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 ⁺ );

Where m is 1 to 7, and each of _R3 and _R4 is independently selected from group A consisting of: H, _C1 - _C18 alkyl, ( _C1 - _C18 alkyl)OH, ( _C1 - _C18 alkyl) _NH2 , ( _C1 - _C18 alkyl)SH, ( _C0 - _C4 alkyl)( _C3 - _C6 )cycloalkyl, ( _C0 - _C4 alkyl)( _C2 - _C5 heterocyclic), ( _C0 - _C4 alkyl)( _C6 - _C10 aryl) _R8 and ( _C1 - _C4 alkyl)( _C3 - _C9 heteroaryl), where _R8 is H or OH, and R5 is optionally present and, when present, selected from group A, wherein optionally, each of _R3 , _R4 and _R5 is H.

In an exemplary aspect, the amino acid comprising a positively charged side chain comprises a side chain structure of Formula I, and n is in the range of 2 to 4. In alternative or other aspects, _R1 is NH or _NH2 . In an exemplary aspect, _R2 is _NH2 or _NH3 ⁺ . In an exemplary case, the amino acid comprising a positively charged side chain is arginine. In an exemplary aspect, the amino acid comprising a positively charged side chain comprises a side chain structure of Formula II, and m is in the range of 3 to 5. In some aspects, _R3 and _R4 are each H. In some cases, _R5 is present and optionally H. In some cases, the amino acid comprising a positively charged side chain is lysine. In some aspects, the amino acid comprising a positively charged side chain is present in the formulation as a salt, optionally as a hydrochloric acid (HCl) salt. Thus, in an exemplary aspect, the aqueous pharmaceutical composition comprises L-arginine hydrochloride or L-lysine hydrochloride.

In an exemplary aspect, the amino acid aggregation inhibitor is an aromatic amino acid. In some cases, the aromatic amino acid comprises a phenyl or indole. In an exemplary aspect, the aromatic amino acid comprises a _C1 - _C6 alkyl chain (e.g., a _C1 - _C3 alkyl chain) between the α-carbon and the phenyl or indole. In an exemplary case, the aromatic amino acid is L-phenylalanine. In other cases, the aromatic amino acid is L-tryptophan.

In an exemplary aspect, the amino acid aggregation inhibitor is a hydrophobic amino acid. Hydrophobicity can be measured or scored according to any of the hydrophobicity scales known in the art. Generally, the larger the positive value of the score, the stronger the hydrophobicity of the amino acid. In some cases, hydrophobicity is scored on the Kate-Doolittle hydrophobicity scale (Kyte J, Doolittle RF (May 1982). "A simple method for displaying the hydropathic character of a protein." J. Mol. Biol. [Journal of Molecular Biology] 157(1):105-32). In some aspects, the hydrophobic amino acid has a score greater than about 2.5 on the Kate-Doolittle hydrophobicity scale. In some respects, the hydrophobic amino acid comprises a side chain containing a branched or straight-chain _C2 - _C12 alkyl or _C4 - _C8 cycloalkyl group, or a _C4 - _C8 heterocycle containing a nitrogen heteroatom, optionally wherein the heterocycle is an imidazole, pyrrole, or indole. For the purposes of this document, the term "cycloalkyl" covers any carbon ring, including bicyclic or tricyclic carbon rings.

In an exemplary aspect, 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 some aspects, the hydrophobic amino acid is L-valine, L-leucine, or L-isoleucine.

The amino acid aggregation inhibitor is used in an amount that effectively provides increased stability and can be used at 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, at concentrations of about 10 mM, about 38 mM, about 75 mM, about 113 mM, or about 150 mM. In an exemplary aspect, the aqueous pharmaceutical formulation comprises about 5 mM to about 300 mM of amino acid aggregation inhibitor, optionally about 25 mM to about 90 mM of amino acid aggregation inhibitor. In some aspects, when the amino acid aggregation inhibitor is an amino acid containing a positively charged side chain, optionally L-arginine, the aqueous pharmaceutical formulation contains about 5 mM to about 150 mM (e.g., about 10 mM to about 150 mM, about 15 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, about 5 mM to about 140 mM, about 5 mM to about 130 mM, about 5 mM to about 120 mM, about 5 mM to about 110 mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM) an amino acid aggregation inhibitor. In some aspects, when the amino acid aggregation inhibitor is an amino acid containing a positively charged side chain, optionally L-arginine, the aqueous pharmaceutical formulation contains about 30 mM to about 80 mM (e.g., about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM) of amino acid aggregation inhibitor.

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

Optionally, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine, the aqueous pharmaceutical formulation contains about 5 mM to about 300 mM of the amino acid aggregation inhibitor. Alternatively, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine, the aqueous pharmaceutical formulation contains about 5 mM to about 200 mM (e.g., about 10 mM to about 200 mM, about 20 mM to about 200 mM, about 30 mM to about 200 mM, about 40 mM to about 200 mM, about 50 mM to about 200 mM, about 60 mM to about 200 mM, about 70 mM to about 200 mM, about 80 mM to about 200 mM). Amino acid aggregation inhibitors of 0 mM, about 90 mM to about 200 mM, about 100 mM to about 200 mM, about 5 mM to about 290 mM, about 5 mM to about 280 mM, about 5 mM to about 270 mM, about 5 mM to about 260 mM, about 5 mM to about 250 mM, about 5 mM to about 240 mM, about 5 mM to about 230 mM, about 5 mM to about 220 mM, about 5 mM to about 210 mM, optionally about 20 mM to about 50 mM. In an exemplary aspect, 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.

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

The surfactant is an amphoteric surfactant (with a polar head and a hydrophobic tail). The surfactant preferentially accumulates at the interface, thereby reducing interfacial tension. The formulation may optionally include the surfactant. The use of surfactants may also help mitigate the formation of large protein particles.

In one type of embodiment, the surfactant may be a nonionic surfactant. Examples include polyoxyethylene dehydrated sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80); alkyl aryl polyethers, such as oxyethylated alkylphenols (e.g., Triton ^™ X-100); poloxamers (e.g., F68); and combinations of any of the above belonging to one or more surfactant classes. Polysorbate 20 and polysorbate 80 are particularly contemplated.

The surfactant concentration is preferably in the range of about 0.004% (w/v) to about 0.1% (w/v) (e.g., for polysorbate 20 or polysorbate 80), such as about 0.004% to about 0.05%, or about 0.004% to about 0.02%, or about 0.01%. In an exemplary aspect, the formulation contains at least about 0.004% (w/v)% surfactant and optionally less than about 0.15% (w/v)%. In an exemplary aspect, the formulation contains about 0.005 (w/v)% to about 0.015 (w/v)% of surfactant, optionally about 0.005 (w/v)%, about 0.006 (w/v)%, about 0.007 (w/v)%, about 0.008 (w/v)%, about 0.009 (w/v)%, about 0.010 (w/v)%, about 0.011 (w/v)%, about 0.012 (w/v)%, about 0.013 (w/v)%, or about 0.014 (w/v)%.

Stable aqueous formulations are suitable for administration via any acceptable route, including parenteral, and specifically subcutaneously. For example, subcutaneous administration may be given to the upper arm, thigh, or abdomen. Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intratumoral, and intrasplenic administration. The subcutaneous route is preferred.

If the solution is intended to be administered to a subject, it can be made isotonic with the intended site of administration. For example, the osmotic pressure can 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 intended to be administered parenterally, it can be made isotonic with blood (about 300 mOsm/kG osmotic pressure). In an exemplary aspect, the aqueous pharmaceutical preparation has an osmotic pressure 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.

In an exemplary aspect, the aqueous pharmaceutical formulation has a conductivity in the range of about 500 μS/cm to about 5500 μS/cm, optionally wherein the conductivity is in the range of about 2500 μS/cm to about 5500 μS/cm when the formulation contains an amino acid with a positively charged side chain, or in the range of about 500 μS/cm to about 2000 μS/cm when the formulation contains an aromatic amino acid or lacks an amino acid aggregation inhibitor. The aqueous pharmaceutical formulation as described in any of the preceding claims has a viscosity of no more than about 6 cP at 5°C, optionally wherein the viscosity is about 4.5 cP to about 5.5 cP. In some aspects, the aqueous pharmaceutical formulation has a viscosity of less than about 13 cP, optionally about 2.0 cP to about 10 cP, optionally about 2.5 cP to about 4 cP at 25°C.

Tension modifiers or tension adjusters 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., polydextrose, glucose, lactose, trehalose), and sugar alcohols (e.g., mannitol, sorbitol, xylitol, glycerol, propylene glycol). In some aspects, the tension modifier is selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. In an exemplary case, the tension modifier is sorbitol. Sorbitol may be used, for example, at concentrations ranging from 0.1% (w/v) to 5% (w/v), or from 1.2% (w/v) to 5% (w/v), such as 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 tension 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 aspects, the formulation does not contain any sorbitol or contains no sorbitol. In an exemplary aspect, the formulation does not contain any tension modifier.

Other excipients known in the art may be used in this formulation, provided they do not negatively affect stability. Sugars and polyols may be used to protect proteins from aggregation, including providing freeze/thaw stability. Such compounds include sorbitol, mannitol, glycerol, erythritol, caprylate, tryptophan salt, sarcosine salt, and glycine. Stabilizers for preparing lyophilized formulations, such as stabilizing sugars, such as disaccharides like trehalose and sucrose, may also be used. Lyophilized formulations may also include building blocks, as known in the art. Other excipients known in the art for protein stabilization include solubilizers (e.g., N-methyl-2-pyrrolidone), polyethylene glycol (PEG), and cyclodextrins (e.g.). Pharmaceutically acceptable acids and bases may be used to adjust the pH of the solution, such as sodium hydroxide.

For parenteral administration, the preparation may be in the form of a pyrogen-free, sterile aqueous solution containing denosumab or another human anti-RANKL monoclonal antibody, with or without other therapeutic agents, in a pharmaceutically acceptable medium. In some embodiments, the medium for parenteral injection is sterile distilled water, wherein the denosumab or another human anti-RANKL monoclonal antibody (with or without at least one additional therapeutic agent) is formulated as a sterile isotonic solution. The preparation will contain pharmaceutically acceptable excipients, such as USP (United States Pharmacopeia) grade excipients.

"Preservatives" are compounds that may be included in pharmaceutical formulations to reduce bacterial activity, such as those that contribute to the manufacture of multipurpose formulations. Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethylammonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chloride in which the alkyl group is a long-chain compound), and benzyl chloride. Other types of preservatives include aromatic alcohols, including phenol, butanol, and benzyl alcohol; alkyl esters of p-hydroxybenzoate, including methylparaben and propylparaben; catechol, m-cresol, cyclohexanol, 3-pentanol, and m-cresol. Alternatively, the formulation may be preservative-free. For example, the formulation may be preservative-free when it is in a single-use dosage form.

Although the aqueous solution form of the formulation has been described herein, the stabilized formulation may subsequently be lyophilized to prepare a lyophilized agent. Therefore, unless the context otherwise requires, references to the formulation and its methods of use are contemplated to include the lyophilized agent obtained from the stabilized aqueous solution.

Pharmaceutical formulations intended for internal administration are typically sterile. In some embodiments, this may be achieved by filtration through a sterile filter membrane. In some embodiments, the parenteral composition is generally placed in a container with a sterile dispensing port, such as an intravenous solution bag, or a vial with a stopper that can be punctured by a subcutaneous needle, or in a pre-filled syringe. In some embodiments, the formulation may be stored in ready-to-use form or in a form that is reconstituted or diluted prior to administration (e.g., lyophilized).

In some embodiments, the present invention relates to kits for producing single-dose administration units. In some embodiments, these kits may each contain a first container having a dry formulation of dinosuzumab or other human anti-RANKL monoclonal antibody made from a solution formulation described herein, and a second container having sterile water or an aqueous solution. In some embodiments of the invention, kits include those containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes).

The stabilized formulations described herein may be used in conjunction with one or more other therapeutic agents, such as calcium and vitamin D compounds. The stabilized formulations described herein may be given to patients receiving therapy utilizing additional therapeutic agents, or may be given co-administered with additional therapeutic agents.

The stabilized formulations described herein, in any of the aspects and examples thereof, may be used for the prevention or treatment of any disease in response to denosulamab or another human anti-RANKL monoclonal antibody or its antigen-binding moiety. Such uses and related methods include, but are not limited to, the aspects and examples described below.

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

In another aspect, the formulation can be used to treat patients with giant cell tumor of bone, including administering an effective amount of the stabilized formulation described herein. In one type of embodiment, the patient has a recurrent, unresectable, or surgically resectable giant cell tumor of bone that would likely cause serious morbidity. For example, the patient may be an adult or a skeletally mature adolescent.

In another aspect, the formulation can be used to treat patients with malignant hypercalcemia of bone, including administering an effective amount of the stabilized formulation described herein. In one aspect, the malignancy is refractory to bisphosphonate therapy. The method or use may include administering an amount of the formulation that can effectively reduce or maintain serum calcium in the patient at a level below or equal to about 11.5 mg/dL.

In another aspect, the formulation can be used to treat osteoporosis in patients in need, including by administering an effective amount of the stabilized formulation described herein. For example, the patient could be a postmenopausal woman at high risk of fracture. In another type of embodiment, the patient could be a man at high risk of fracture.

In another aspect, the formulation is used to increase bone mass in patients in need, including administering an effective amount of the stabilized formulation described herein. For example, the amount of formulation administered may be an amount that effectively reduces the incidence of new vertebral and/or non-vertebral fractures. In another type of embodiment, the amount of formulation administered may be an amount that effectively reduces bone resorption. In another type of embodiment, the amount of formulation may be an amount that effectively increases bone mineral density in at least one region selected from the lumbar spine, the entire hip, and the femoral neck of the patient. In another type of embodiment, the amount of formulation may be an amount that effectively increases bone mass in the cortical bone and/or cancellous bone of the patient. In another type of embodiment, the amount of formulation may be an amount that effectively reduces the amount of the bone resorption marker serum C-terminal 1 peptide (CTX). Patients in need may optionally have osteoporosis. In another type of embodiment, the patient in need may be a woman at high risk of fracture who is receiving adjuvant aromatase inhibitor therapy for breast cancer. In another type of embodiment, the patient in need may be a man at high risk of fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer. In another type of embodiment, the patient in need may be a man with osteoporosis at high risk of fracture.

On the other hand, this preparation can be used as adjuvant therapy for postmenopausal women with early-stage breast cancer who are at high risk of disease recurrence and are receiving adjuvant/neoadjuvant cancer therapy.

On the other hand, this formulation can be combined with platinum-based chemotherapy as a first-line treatment for patients with metastatic non-small cell lung cancer.

On the other hand, this preparation can be used to treat idiopathic inferior laryngeal stenosis (ISS).

On the other hand, this formulation can be used for the prevention of breast and ovarian cancer in healthy women with BRCA-1 mutations.

Optionally, this formulation may be used in combination with an immune checkpoint inhibitor. Optionally, the immune checkpoint inhibitor is specific to proteins that function 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 antibody protein product specific to CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1, or TIM3. 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. PD-1 inhibitors include, for example, pembrolizumab and nivolumab. PD-L1 inhibitors include, for example, atezolizumab, avelumab, and durvalumab. CTLA4 inhibitors include, for example, ipilimumab. In another aspect, this formulation may optionally be combined with a PD-1 antibody (e.g., nivolumab, pembrolizumab) for the treatment of patients with melanoma having bone metastases. In another aspect, this formulation may optionally be combined with a CTLA4 inhibitor, such as ipilimumab, for the treatment of patients with breast cancer.

In another aspect, this formulation can be used to treat giant cell tumors, such as hyperparathyroidism or secondary aneurysmal bone cysts.

In another aspect, this formulation can be used to treat progressive metastatic castration-resistant prostate cancer (mCRPC). In another aspect, this formulation can be used to treat castration-sensitive prostate cancer. In another aspect, this formulation can be used to treat hormone-resistant prostate cancer.

In another aspect, this formulation can be used to treat metastatic breast cancer (mBC). In another aspect, this formulation can be used to treat preoperative breast cancer. In another aspect, this formulation can be used to treat early-stage breast cancer. In still other aspects, this formulation can be used to treat hormone receptor-negative RANK-positive or RANK-negative primary breast cancer. In yet another aspect, this formulation can be used to treat postmenopausal HER2-negative breast cancer.

On the other hand, this preparation can be used to treat, for example, myelodyplastic syndrome in elderly patients.

In another aspect, this formulation can be used to treat cancer treatment-induced bone loss (CTIBL).

On the other hand, this preparation can be used to treat uterine tumors of the cervix.

On the other hand, this formulation can be used to induce immunomodulatory effects in patients who are receiving or not receiving immunotherapy.

In another aspect, this formulation can be used to prevent or treat bone loss associated with osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, and rheumatoid arthritis. In another aspect, this formulation can be used to prevent or treat inflammatory conditions associated with bone loss. In another aspect, this formulation can be used to prevent or treat autoimmune conditions associated with bone loss. In another aspect, this formulation can be used to prevent or treat bone loss associated with cancer (including 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.

The preparation can be administered at any suitable schedule. In one embodiment, it is administered on a four-week schedule. Optionally, it may be administered on days 8 and 15 of the first month of treatment. In another type of embodiment, it may be administered on a six-month schedule. For example, a six-month schedule is envisioned for osteoporosis and increasing bone mass. Other envisioned maintenance doses are every 3 weeks, every 3 months, and every 6 weeks.

In some respects, this aqueous pharmaceutical preparation is used to treat patients with multiple myeloma or bone metastases from solid tumors. In some respects, the preparation is administered subcutaneously to the upper arm, thigh, or abdomen at a dose of approximately 120 mg every 4 weeks.

In some respects, this aqueous pharmaceutical preparation is used to treat patients with giant cell tumors of bone. In some respects, the preparation is administered at a dose of approximately 120 mg every 4 weeks, with additional 120 mg doses given on days 8 and 15 of the first month of treatment. In some respects, the preparation is administered subcutaneously to the patient's upper arm, thigh, or abdomen. In some cases, calcium and vitamin D are given to the patient to treat or prevent hypocalcemia.

In some respects, this aqueous pharmaceutical preparation is used to treat patients with malignant hypercalcemia. In some respects, the preparation is administered at a dose of approximately 120 mg every 4 weeks, with an additional 120 mg dose given on days 8 and 15 of the first month of treatment. In some respects, the preparation is administered subcutaneously to the upper arm, thigh, or abdomen.

In some respects, this aqueous pharmaceutical preparation 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 due to androgen deprivation therapy for non-metastatic prostate cancer, or in women at high risk of fracture due to breast cancer receiving adjuvant aromatase inhibitor therapy. In some respects, the aqueous pharmaceutical preparation is administered subcutaneously to the upper arm, thigh, or abdomen by a healthcare professional at a dose of 60 mg every 6 months. In some respects, the patient is also instructed to take 1000 mg of calcium daily and at least 400 IU of vitamin D daily.

One type of formulation according to this disclosure will contain dinosuzumab, acetate, and arginine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH value may optionally be from about 5.0 to about 5.2, or below 5.2.

Another type of formulation according to this disclosure will contain denosumab, acetate, and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be from about 5.0 to about 5.2, or below 5.2. For example, the formulation may include denosumab at pH 5.1, at a concentration of from about 108 mg/mL to about 132 mg/mL, from about 28.8 mM to about 35.2 mM acetate, from 33.3 mM to about 40.7 mM phenylalanine, from 3.51% (w/v) to about 4.29% (w/v) sorbitol, and from about 0.009% (w/v) to about 0.011% (w/v) polysorbate 20, and may optionally be included in a PFS optionally containing about 1 mL or less (e.g., about 0.5 mL) of the formulation. For example, the formulation may include 120 mg/mL of denosumab 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 included in a PFS optionally containing about 1 mL or less (e.g., about 0.5 mL) of the formulation. The formulation can be manufactured by concentrating denosumab using a percolation buffer (pH 4.7) containing 20 mM acetate, 4.2% (w/v) sorbitol, and 40 mM phenylalanine.

Another type of formulation according to this disclosure will contain dinosuzumab, glutamate, and arginine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH value may optionally be from about 5.0 to about 5.2, or below 5.2.

Another type of formulation according to this disclosure will contain denosumab, acetate, arginine, and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH may optionally be from about 5.0 to about 5.2, or below 5.2.

Another type of formulation according to this disclosure will contain denosumab, glutamate, arginine, and phenylalanine. The arginine is optionally L-arginine. The arginine is optionally L-arginine hydrochloride. The formulation may optionally include sorbitol. The formulation may optionally include polysorbate. The polysorbate may optionally be polysorbate 20. The pH value may optionally be from about 5.0 to about 5.2, or below 5.2.

The formulations according to this disclosure can be manufactured by any suitable method. In one type of method, a solution containing an anti-RANKL monoclonal antibody (e.g., denosumab) at a concentration below 70 mg/mL can be prepared by adding an appropriate amount of the amino acid aggregation inhibitor described herein, and then concentrating the solution to an amount above 70 mg/mL, for example, 120 mg/mL. Optionally, the solution can be overconcentrated first, i.e., to achieve a concentration of anti-RANKL monoclonal antibody (e.g., denosumab) above the final target concentration, and then the overconcentrated solution can be diluted to the final target concentration and pH, for example, with a pH-adjusted buffer solution. For example, overconcentration can yield 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 specifically 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 of any such concentration, for example, about 1 mg/mL to about 70 mg/mL or about 1 mg/mL to about 10 mg/mL.

The preparation can be concentrated by any suitable method. In one aspect, the concentration method may include centrifugation. In another aspect, the concentration method may include ultrafiltration.

The amino acid aggregation inhibitor can be introduced into the preparation by any suitable method. For example, it can be introduced into the preparation by simple addition (co-addition), such as as described in the examples below. In another method, it can be introduced into the preparation by percolation relative to a buffer solution containing the amino acid aggregation inhibitor, such as as described in the examples below. The amino acid aggregation inhibitor can be introduced into the preparation before or after concentrating the anti-RANKL monoclonal antibody to above 70 mg/mL. As shown in the examples below, it is advantageous to add the amino acid aggregation inhibitor to the solution before concentration because it inhibits aggregation during the concentration process.

Therefore, this disclosure provides a method for manufacturing a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety. In an exemplary embodiment, the method comprises combining the anti-RANKL monoclonal antibody or its antigen-binding moiety at a concentration greater than 70 mg/mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a toning 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 aggregation inhibitor may be any of those described herein. For example, the amino acid aggregation inhibitor may be a positively charged amino acid, an aromatic amino acid, or a hydrophobic amino acid. The amino acid aggregation inhibitor may be in a molar ratio with the antibody as described herein. The amounts and selections of the aggregation inhibitor, surfactant, toning modifier, and buffer are as described above. This disclosure also provides formulations manufactured by the manufacturing methods described herein.

Formulations disclosed herein may include a high-concentration solution of the anti-RANKL monoclonal antibody (e.g., denosumab) described herein, such as a solution with a concentration greater than 70 mg/mL or 120 mg/mL, pH adjusted. Alternatively, the formulation may be prepared by pH adjustment of a low-concentration solution of the anti-RANKL monoclonal antibody (e.g., denosumab), followed by concentration of that solution to a desired higher final concentration. Suitable pH adjusters are known in the art.

Example

The following is a list of specific hypothetical embodiments:

1. An aqueous pharmaceutical formulation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding portion at a concentration higher than 70 mg/mL and having a pH value in the range of about 5.0 to below 5.2.

2. The formulation as described in Example 1 has a pH value in the range of about 5.0 to 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.1.

3. The formulation as described in Example 2 has a pH value of approximately 5.1.

4. The formulation as described in any one of Examples 1 to 3, further comprising an amino acid aggregation inhibitor.

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

6. The formulation as described in Example 5, having a pH value in the range of about 5.0 to about 5.4, or about 5.0 to about 5.2, or about 5.0 to below 5.2, or about 5.0 to 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.1.

7. The formulation as described in Example 6 has a pH value of about 5.1.

8. The formulation as described in any of the foregoing embodiments further comprises a pH buffer.

9. The preparation as described in any one of Examples 5 to 8, wherein the concentration of the antibody or its antigen-binding portion is in the range of about 10 mg/mL to about 200 mg/mL.

10. The preparation as described in any of the foregoing 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.

11. The preparation as described in Example 10, wherein the concentration of the antibody or its antigen-binding portion is in the range of about 100 to about 140 mg/mL.

12. The preparation as described in Example 11, wherein the concentration of the antibody or its antigen-binding portion is about 120 mg/mL.

13. The formulation as described in any of the foregoing embodiments, wherein the antibody is denosumab or a biosimilar thereof.

14. The formulation as described in Example 13, wherein the antibody is denosumab.

15. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more amino acids, dipeptides thereof, or oligopeptides having 2 to 10 residues.

16. The formulation as described in Example 15, wherein the amino acid aggregation inhibitor comprises a mixture of at least two amino acids.

17. The formulation as described in Example 16, wherein the amino acid comprises arginine and phenylalanine.

18. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more hydrophobic amino acids, dipeptides thereof, or oligopeptides having 2 to 10 residues and containing one or more hydrophobic amino acids.

19. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more amino acids having charged side chains, dipeptides thereof, or oligopeptides having 2 to 10 residues and containing one or more amino acids having charged side chains.

20. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more basic amino acids, their dipeptides, or oligopeptides having 2 to 10 residues and containing one or more basic amino acids.

21. The formulation as described in any of the foregoing embodiments, wherein the amino acid aggregation inhibitor is selected from one or more dipeptides.

22. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more oligopeptides having 2 to 10 amino acid residues.

23. The formulation as described in any of the foregoing embodiments, wherein the amino acid aggregation inhibitor comprises an arginine residue, or the amino acid aggregation inhibitor comprises arginine.

24. The formulation as described in any of the foregoing embodiments, wherein the amino acid aggregation inhibitor comprises an arginine-phenylalanine dipeptide.

25. The formulation as described in any of the preceding embodiments, wherein the amino acid aggregation inhibitor is present in the formulation at a concentration ranging from about 10 mM to about 200 mM.

26. The formulation as described in any of the foregoing embodiments further comprises a surfactant.

27. The formulation as described in Example 26, wherein the surfactant is selected from the group consisting of: polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80), or one or more alkyl aryl polyethers, such as oxyethylated alkylphenols (e.g., X-100), or one or more poloxamers (e.g., F68) and combinations thereof.

28. The formulation as described in Example 26 or Example 27, wherein the surfactant is present at a concentration ranging from about 0.004% (w/v) to about 0.1% (w/v).

29. The formulation as described in Example 28, wherein the surfactant is present at a concentration of about 0.01% (w/v).

30. The formulation as described in any of the foregoing embodiments further comprises a buffer.

31. The formulation as described in Example 30, wherein the buffer is centered at 25°C in the range of about pH 4 to about pH 5.5.

32. The formulation as described in Example 30 or Example 31, wherein the buffer has a pKa in a pH unit between pH 5.0 and 5.2 at 25°C.

33. The formulation as described in any one of Examples 30 to 32, wherein the buffer comprises an acetate.

34. The formulation as described in any one of Examples 30 to 32, wherein the buffer comprises a glutamate.

35. The formulation as described in any of the foregoing embodiments further comprises a tension modifier.

36. The formulation as described in Example 35, wherein the tension modifier is selected from one or more of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof.

37. The formulation as described in Example 36, wherein the tension modifier comprises sorbitol.

38. The formulation as described in any of the foregoing embodiments further comprises one or more excipients selected from sugars, polyols, solubilizers (e.g., N-methyl-2-pyrrolidone), hydrophobic stabilizers (e.g., proline), polyethylene glycol, cyclodextrin, and combinations thereof.

39. The formulation as described in any of the foregoing embodiments, according to SE-UHPLC, after being stored at 37°C for three months, contains less than 2% of the high molecular weight substance of the human anti-RANKL monoclonal antibody.

40. The formulation as described in any of the foregoing embodiments, according to SE-UHPLC, after being stored at 4°C for 36 months, contains less than 2% of the high molecular weight substance of the human anti-RANKL monoclonal antibody.

41. The formulation as described in any of the foregoing embodiments, according to SE-UHPLC, contains at least 98% of the antibody main peak after being stored at 37°C for three months.

42. The formulation as described in any of the foregoing embodiments, according to SE-UHPLC, contains at least 98% of the antibody main peak after being stored at 4°C for 36 months.

43. The formulation as described in any of the preceding embodiments, comprising: dinosuzumab; an amino acid aggregation inhibitor selected from one or more of arginine, its dipeptide, or oligomers having 2 to 10 residues and containing arginine; an acetate buffer; sorbitol; and a surfactant, and the formulation having a pH value in the range of about 5.0 to below 5.2.

44. The formulation as described in Example 43, wherein the amino acid aggregation inhibitor is selected from arginine, arginine-arginine, or arginine-phenylalanine.

45. The formulation as described in Example 43, wherein the amino acid aggregation inhibitor comprises a mixture of arginine and phenylalanine.

46. The formulation as described in any one of Examples 43 to 45, wherein the acetate buffer is present in the range of about 5 mM to about 25 mM.

47. The formulation as described in any one of Examples 43 to 46, wherein the sorbitol is present in the range of 0.1% (w/v) to 5% (w/v).

48. The formulation as described in any one of Examples 43 to 47, wherein the surfactant is selected from one or more of polysorbate 20 and polysorbate 80.

49. The formulation as described in any one of Examples 43 to 48, wherein the pH value is in the range of about 5.0 to about 5.15.

50. The formulation as described in Example 49, wherein the pH value is about 5.10.

51. The formulation as described in any of the foregoing embodiments, wherein the formulation is suitable for subcutaneous injection.

52. The preparation as described in any of the foregoing embodiments, wherein the preparation is sterile and free of preservatives.

53. The formulation as described in any of the preceding 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 NO:8, 9 and 10 respectively, and light chain CDR1, CDR2 and CDR3 regions containing SEQ ID NO:5, 6 and 7 respectively.

54. The preparation as described in any of the foregoing embodiments, wherein the human anti-RANKL monoclonal antibody or its antigen-binding portion is an antibody.

55. The formulation as described in any one of Examples 1 to 53, wherein the human anti-RANKL monoclonal antibody or its antigen-binding portion is an antigen-binding portion.

56. A vial, prefilled syringe, or glass container containing a formulation as described in any one of Examples 1 to 55.

57. The vial, prefilled syringe or glass container as described in Example 56, containing about 1 mL or less of the preparation.

58. A method for preventing skeletal-related events (SREs) in patients in need, comprising administering an effective amount of the preparation as described in any one of Examples 1 to 55.

59. The method as described in Example 58, wherein the SRE is selected from the group consisting of: pathological fractures, radiotherapy to the bone, surgery to the bone, and spinal cord compression.

60. The method as described in Example 58 or Example 59, wherein the patient has bone metastases from a solid tumor.

61. The method as described in Example 60, wherein the solid tumor is selected from breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma.

62. The method as described in Example 58 or Example 59, wherein the patient has multiple myeloma.

63. The method as described in any one of Examples 58 to 62, comprising administering the formulation in an amount that can effectively reduce the bone turnover marker urinary creatinine modified N-terminal telopeptide (uNTx/Cr), optionally by at least 80%.

64. A method of treating a patient with giant cell tumor of bone, comprising administering an effective amount of the preparation as described in any one of Examples 1 to 55.

65. The method as described in Example 64, wherein the patient has a recurrent, unresectable, or surgically resectable giant cell tumor of bone that could cause serious complications.

66. A method of treating malignant hypercalcemia in a patient in need, comprising administering an effective amount of the preparation as described in any one of Examples 1 to 55.

67. The method as described in Example 66, wherein the malignancy is refractory to bisphosphonate therapy.

68. The method as described in Example 66 or Example 67, comprising administering an amount of the preparation that can effectively reduce or maintain the patient’s serum calcium at a level of less than or equal to about 11.5 mg/dL.

69. The method of any one of Examples 58 to 68, wherein the preparation comprises the human anti-RANKL antibody at a concentration of about 120 mg/mL.

70. The method as described in any one of Examples 58 to 69, comprising administering the preparation at a schedule of once every four weeks.

71. The method as described in any one of Examples 58 to 70, wherein the preparation is administered on the 8th and 15th days of the first month of treatment.

72. A method of treating osteoporosis in a patient in need, comprising administering an effective amount of the preparation as described in any one of Examples 1 to 55.

73. The method as described in Example 72, wherein the patient is a postmenopausal woman at high risk of fracture.

74. The method as described in Example 72, wherein the patient is a male at high risk of fracture.

75. A method for increasing bone mass in patients in need, comprising administering an effective amount of the preparation as described in any one of Examples 1 to 55.

76. The method as described in Example 75, wherein the patient suffers from osteoporosis.

77. The method as described in Example 75, wherein the patient is a woman at high risk of fracture who is receiving adjuvant aromatase inhibitor therapy for breast cancer.

78. The method as described in Example 75, wherein the patient is a male at high risk of fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer.

79. The method as described in any one of Examples 75 to 78, comprising administering an amount of the preparation that can effectively reduce the incidence of new vertebral fractures and/or non-vertebral fractures.

80. The method as described in any one of Examples 75 to 79, comprising administering an amount of the preparation that can effectively reduce bone resorption.

81. The method of any one of Examples 75 to 80, comprising administering an amount of the preparation that can effectively increase bone mineral density in at least one region selected from the lumbar spine, the entire hip, and the femoral neck of the patient.

82. The method as described in any one of Examples 75 to 81, comprising administering an amount of the preparation that can effectively increase the bone mass in the cortical bone and/or cancellous bone of the patient.

83. The method of any one of Examples 75 to 82, comprising administering the formulation in an amount that can effectively reduce the bone resorption marker serum type 1 C-terminal peptide (CTX).

84. The method as described in any one of Examples 75 to 83, comprising administering the preparation at a schedule of once every six months.

85. The method of any one of Examples 58 to 84, comprising administering the preparation in a volume of 1 mL or less.

86. The method of any one of Examples 58 to 85, comprising administering the preparation subcutaneously.

87. The method as described in Example 86, comprising subcutaneously administering the preparation to the upper arm, thigh, or abdomen.

88. The method as described in any one of Examples 58 to 87, wherein the patient receives one or both of calcium and vitamin D.

89. A method for improving the stability of an aqueous pharmaceutical preparation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety at a concentration exceeding 70 mg/mL, comprising:

Prepare an aqueous pharmaceutical preparation containing the human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, with a pH range of about 5.0 to below 5.2;

Compared to equivalent aqueous pharmaceutical formulations with pH values not in the range of about 5.0 to below 5.2, this aqueous pharmaceutical formulation exhibits improved stability at pH values in the range of about 5.0 to below 5.2.

90. A method for improving the stability of an aqueous pharmaceutical preparation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, comprising:

Prepare an aqueous pharmaceutical formulation comprising a mixture of the human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety with an amino acid aggregation inhibitor;

Compared to an equivalent aqueous pharmaceutical formulation without the amino acid aggregation inhibitor, the aqueous pharmaceutical formulation exhibits improved stability in the presence of the amino acid aggregation inhibitor.

Example

The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention. Throughout the examples provided herein, the following abbreviations are used: DF, percolation; PS20, polysorbate 20; HCl, hydrochloride; UF/DF, ultrafiltration/percolation; F#, formulation number; HMWS, high molecular weight substance; SE-UHPLC, size exclusion ultra-high performance liquid chromatography. Additionally, throughout these examples, the composition of the DF buffer or dialysis buffer used to manufacture the final formulation containing denosumab and the estimated concentrations of the components of the final formulation are provided. The final concentration of certain components of the final formulation, after storage and subsequent stability analysis, may differ from the concentration of the DF or dialysis buffer, depending on the presence or absence of counterions (e.g., hydrochloride). In the absence of counterions, the formulation has low ionic strength. In this case, acetate is co-concentrated with denosumab such that the final formulation contains a higher acetate concentration relative to the concentration of the DF or dialysis buffer. For example, when neither the DF buffer nor the final formulation contains an antiion (e.g., hydrochloride) and thus has low ionic strength, using a DF buffer containing 10 mM acetate results in approximately 23 mM acetate in the final denosumab (120 mg/mL) formulation (pH 5.1). Similarly, in the absence of an antiion (e.g., hydrochloride), a DF buffer containing 20 mM acetate results in approximately 32 mM acetate in the final denosumab (120 mg/mL) formulation (pH 5.1). When an antiion (e.g., arginine hydrochloride) is present, the acetate is not co-concentrated with denosumab, and therefore the acetate concentration in the DF buffer and the acetate concentration in the final composition are generally equal. Additionally, excipients can be affected by size exclusion or nonspecific interactions. For example, in a 120 mg/mL denosumab formulation, the concentrations of phenylalanine and sorbitol are approximately 7% to 10% lower than those indicated in the DF buffer, and the concentration of arginine is approximately 10% to 15% lower. Based on the above, and throughout the following examples, the concentrations of the components in the final formulation are provided, taking into account the excipient exclusion and acetate co-concentration effects described above.

Example 1

A preliminary assessment was conducted to evaluate the effect of twelve formulations on minimizing the amount (%) of high-concentration liquid dinosuzumab formulation (120 mg/mL) and its formation over time. Formulation alternatives included variations in buffer type, stabilizer, and solution pH. Formulations A through L tested are described in Table 1 below. All referenced buffer values are relative to the buffer concentration targeted by the percolating antibody. Excipients and surfactants were added to the solution after buffer exchange to the levels indicated in the table. Although acetate concentrations in the formulations of this invention were not measured, the 120 mg/mL dinosuzumab formulation percolated against 10 mM acetate had an approximate final acetate value between 25 mM and 35 mM with sorbitol.

Dinosumab (70 mg/mL) in acetate (pH 5.2) was subjected to unfractionated precipitate (UF/DF) relative to 10 mM acetate (pH 5.2) and concentrated to 160 mg/mL. A stock solution of the following composition was prepared in 10 mM acetate (pH 5.2):

35% Sorbitol

1% Polysorbate 20

1% Polysorbate 80

30% F-68

3% Triton ^™ X-100

250mM L-arginine hydrochloride

250mM N-acetylarginine (NAR)

250mM N-acetyllysine (NAK)

250mM proline

250mM polyethylene glycol (PEG) 3350

250mM cyclodextrin

To obtain formulations A through J, a 160 mg/mL substance prepared with 10 mM acetate (pH 5.2) was diluted to 120 mg/mL with 10 mM acetate (pH 5.2), followed by the addition of the corresponding stock solutions of sorbitol, excipients, and/or surfactants to the target final concentrations listed in Table 1. To obtain formulations K and L, two independent aliquots from the 160 mg/mL substance, representing the self-buffered formulation and the glutamate formulation respectively, were subjected to additional buffer exchange by centrifugation. Formulations K and L were then diluted to 120 mg/mL using the corresponding buffers, followed by the addition of the corresponding stock solutions of sorbitol and polysorbate 20 to the target final concentrations listed in the formulation table of Table 1.

Table 1

Figure 1 shows the percentage of HMWS monitored by SE-U HPLC at 37 °C as a function of formulation and time. Formulation L, consisting of approximately 10 mM glutamate buffer, 10 mM L-arginine hydrochloride, 2.4% (w/v) sorbitol as a tension modifier, and 0.01% (w/v) polysorbate 20 as a surfactant, and at pH 5.0, showed a decrease in the initial amount of HMWS at 37 °C (indicating a reduction in the amount of aggregates already formed) and a decrease in the dynamic formation of HMWS.

Example 2

The effects of pH and amino acid aggregation inhibitors on the rate and extent of HMWS formation were evaluated at 37°C for formulations containing 10 mM acetate, 75 mM L-arginine, 2.4% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20 excipients, and formulations containing 10 mM acetate, 5% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20 excipients (each containing a high concentration (120 mg/mL) of dinosumab) for up to 1 month. Table 2 below describes the formulations tested. All referenced buffer and excipient values are relative to the buffer and excipient concentrations targeted by the filtration antibody.

To prepare test samples M to Q, 3 mL aliquots of dinosuzumab (70 mg/mL) in acetate (pH 5.2) were dialyzed against less than 500 mL of the described DF buffer, with a total of 3 buffer replacements to achieve a 1,000,000-fold dilution of the previously prepared formulation, thus ensuring complete buffer exchange. The material was then over-concentrated using a centrifuge-concentrator, followed by dilution to 120 mg/mL, and polysorbate was added from 20% to a final concentration of 0.01%.

Table 2

Figure 2 shows the percentage of HMWS monitored by SE-U HPLC at 37°C as a function of the formulation and time. Figure 3 shows the size exclusion chromatogram as a function of the formulation after storage at 37°C for one month.

As the solution pH decreases, the formation of macroaggregates increases. Below pH 4.8, and especially at 4.5, the macroaggregates are primarily HWMS, with a significant increase in macroaggregates at pH 4.5 for the test formulations. As shown in Figure 3, formulations P and Q have the lowest amounts of high-grade HWMS (retention time approximately 6 minutes), followed by comparative formulations O, N, and M with lower pH values.

However, an increase in pH generally leads to an increase in dimer content. As shown in Figure 3, formulation N has the lowest amount of dimer content (retention time of approximately 6.8 minutes), followed by formulations M, O, P, and Q.

After one month at 37°C, the presence of 75 mM arginine in formulation O caused a decrease in the amount of dimer and its dynamic formation rate of approximately 0.3% and 25%, respectively, compared to formulation P, which had the same pH but did not contain arginine.

Example 3

This example demonstrates the effect of pH on high-concentration denosumab formulations.

Dinosumab (120 mg/mL) was formulated with acetate, sorbitol, and polysorbate 20 (PS20) at three different pH values, with or without an amino acid aggregation inhibitor: 4.8, 5.1, and 5.4. In this study, the amino acid aggregation inhibitor was L-arginine hydrochloride. All formulations were prepared by exchanging the buffer of an initial solution containing a low concentration of dinosumab, followed by over-concentration of the dinosumab material, and then diluting the dinosumab material with the desired amounts of buffer, excipients, and surfactant. Briefly, aliquots of dinosumab (70 mg/mL) (initial material) in acetate (pH 5.2) were dialyzed against the DF buffer as described in Table 3A, with a total of three buffer changes to achieve a 1,000,000-fold dilution of the initial material, thus ensuring complete buffer exchange. The buffer-exchanged denosumab material was then concentrated to a concentration above 120 mg/mL using a centrifuge-concentrator, and subsequently diluted to achieve a denosumab concentration of 120 mg/mL. PS20 was added to achieve a final concentration of 0.01%.

The high concentration of protein is considered to contribute to the solution pH based on its charge state. The acetate concentration of formulation 1 was increased to achieve the target final pH, and the acetate concentrations of formulations 2 and 3 were matched with those of formulation 1. Formulations 4 through 6 required even higher amounts of acetate to match the final acetate concentrations of formulations 1 through 3, as the acetate failed to co-concentrate in the presence of hydrochloride. Formulation 7 served as a control to ensure that the increased acetate concentrations in formulations 4 through 6 did not impair the protein stability in the arginine hydrochloride formulations.

Table 3A describes the different denosumab formulations manufactured and tested in this study.

Table 3A

*The final formulation contains 120 mg/mL dinosuzumab and PS20 at a final concentration of 0.01% (w/v), with the indicated pH. The sorbitol concentration is estimated to be 8.5% lower than that of the DF buffer. The arginine concentration is estimated to be 12.5% lower than that of the DF buffer.

Samples of each formulation were filled into containers at a 1 mL filling volume and stored at 37°C for up to 4 weeks. Aggregation inhibition and stability against aggregation inhibition over time (e.g., based on HMWS and dimer formation) were assessed using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

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

Table 3B

The F# shown in the left column is consistent with the F# in Table 3A.

Figure 5 shows the size exclusion chromatograms of each formulation after storage at 37°C for one month. The formulation without arginine is shown in the left figure, while the formulation containing arginine is shown in the right figure.

As shown in Figure 4, the formulations containing arginine were more effective than the control formulations without arginine hydrochloride, and the formulation at pH 5.1 was more effective than the equivalent formulations at pH 4.8 and pH 5.4. For formulations 1 to 3 without arginine hydrochloride, dimerization increased as the solution pH increased to 5.4 (Figure 5A). For formulations 4 to 6 containing arginine hydrochloride, the formation of larger aggregates increased as the solution pH decreased to 4.8, and dimerization increased as the solution pH increased to 5.4 (Figure 5B). Compared to formulation 2 without arginine hydrochloride, formulation 6 containing arginine hydrochloride had the lowest total HMWS at a solution pH of 5.1. Furthermore, formulation 7 showed that increasing the acetate buffer concentration from 10 mM to 40 mM had a relatively small effect on HMWS formation.

Example 4

This example demonstrates the relationship between pH and HMWS formation in different denosumab formulations containing varying concentrations of denosumab.

The pH sensitivity of HMWS formation at different protein concentrations (15 mg/mL to 150 mg/mL) and at 75 mM arginine hydrochloride was assessed. Two pH values, pH 4.8 and 5.1, were evaluated at each test protein concentration (15, 60, 120, and 150 mg/mL).

This study evaluated a total of eight formulations (formulations 8 to 15; described in Table 4A). To prepare these formulations, two aliquots of dinosumab (70 mg/mL) in acetate (pH 5.2) were dialyzed relative to the corresponding DF buffers described in Table 4A. Dialysis protocols 1 and 2 involved a total of three buffer replacements to achieve a 1,000,000-fold dilution of the previous formulation, ensuring complete buffer exchange. For formulations 8, 9, 12, and 13, after dialysis, the respective aliquots of dialysis protocols 1 and 2 described in Table 4A were removed to prepare the dilution step. The remaining material was then over-concentrated using a centrifuge-concentrator, followed by dilution to the corresponding dinosumab concentrations listed in Table 4A, and PS20 was added to achieve a final concentration of 0.01%.

Table 4A

*The final formulation contains PS20 at a final concentration of 0.01% (w/v) and has the indicated pH value. The estimated concentration of acetate is 10 mM, and the estimated concentration of sorbitol is approximately 8.5% lower than that of the DF buffer. The estimated concentration of arginine is 65 mM. The estimated concentration of sorbitol is 2.2% (w/v).

These formulations were filled into containers at 1 mL filling volumes and stored at 37°C for up to 1 month. Aggregation inhibition and stability against aggregation inhibition over time (e.g., based on HMWS and dimer formation) were assessed using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Figure 6 shows the percentage of HMWS for each formulation monitored by SE-UHPLC at 37°C as a function of storage time, and Table 4B provides the data points for this figure.

Table 4B

Figures 7A and 7B show the changes in size exclusion chromatograms with different formulations after storage at 37°C for one month. As shown in Figure 6, %HMWS increases with increasing protein concentration. Formulations 8 to 11 at pH 4.8 consistently exhibit higher levels of HMWS compared to their corresponding formulations at pH 5.1 (formulations 12 to 15). The increase in %HMWS at pH 4.8 is due to the large aggregate peak at approximately 5.75 minutes, as shown in Figure 7A (top). Although the %HMWS at solution pH 5.1 shows an increase in dimer content with increasing protein concentration, the total HMWS is lower than that at the corresponding protein concentration at solution pH 4.8 (Figure 7B (bottom)).

The difference in HMWS levels between pH 5.1 and pH 4.8 increased with increasing dinosuzumab concentration, with the difference being greater at higher dinosuzumab concentrations.

Example 5

The effects of formulations containing various concentrations of arginine, NAR, and two dipeptides consisting of arginine-arginine (Arg-Arg) and arginine-phenylalanine (Arg-Phe) on the stability of solutions containing 120 mg/mL of denosumab were evaluated.

The formulations tested are described in Table 5 below. All referenced acetate and excipient (except dipeptides) values are relative to the concentrations of the buffer and excipient targeted by the filtration antibody. After buffer exchange, each dipeptide was added to the solution to the level indicated in the table. Formulations R to X were obtained by UF/DF against the DF buffers listed below. Formulations Y and Z were obtained by UF/DF against a DF buffer (pH 4.0) containing 10 mM acetate and 3.6% sorbitol in a single library. After UF/DF, the libraries of formulations Y and Z were split into two, and subsequently doped with Arg-Arg or Arg-Phe dipeptides from a 1 M stock solution (pH 5.1) containing 3.6% sorbitol. Polysorbate 20 was added to each formulation to a final target concentration of 0.01%. Acetate co-concentration was performed in the absence of arginine to achieve a final acetate concentration of approximately 25 mM in formulations S to X. Sorbitol is preferentially excluded during the concentration process, resulting in a reduction of approximately 7% to 8% (w/v) from the initial concentration.

These formulations were filled into containers at 1.0 mL filler volumes. The formulations were stored at temperatures ranging from 2°C to 8°C for 12 months and at 25°C, 30°C, and 37°C for 3 months. Stability (based on HMWS formation) was assessed using SE-UHPLC. The stability of these dipeptide formulations after one month of storage at 37°C was compared with that of the arginine hydrochloride formulation at 37°C, as shown in Figure 8.

Table 5

Figure 8 shows the percentage of HMWS monitored by SE-UHPLC at 37°C as a function of formulation and time. The results indicate that amino acid aggregation inhibitors suppress HMWS formation. For example, the arginine-phenylalanine dipeptide showed significant improvement, resulting in approximately 0.3% less HMWS compared to other formulations. The order of lowest to highest HMWS was Z << V < Y ≈ T ≈ W ≈ X ≈ U < S < R. As shown in the figure, formulations containing arginine-arginine (Arg-Arg) dipeptide (Formulation Y) and arginine-phenylalanine (Arg-Phe) dipeptide (Formulation Z) reduced HMWS formation compared to the control formulation (Formulation R) which lacked arginine and an arginine-containing dipeptide. Formulation Z contained the least amount of HMWS, superior to formulation Y.

Example 6

This example demonstrates that the aggregation inhibition and stability of denosumab vary with different concentrations of arginine and phenylalanine, as well as with a comparative mixture of arginine and phenylalanine.

As described above, the identification of arginine hydrochloride (HCl) and arginine hydrochloride-phenylalanine dipeptide reduced the initial initiation level and HMWS formation rate of denosumab. In this study, the stabilizing effects of formulations containing various concentrations of arginine hydrochloride, various concentrations of phenylalanine, and combinations of arginine hydrochloride and phenylalanine on solutions containing denosumab (120 mg/mL) were evaluated.

The test formulations (formulations 16 to 20) are described in Table 6A below. To prepare these formulations, aliquots of dinosumab (70 mg/mL) in acetate (pH 5.2) were dialyzed against the DF buffer as described in Table 6A, with a total of three buffer replacements to achieve a 1,000,000-fold dilution of the previous formulation, ensuring complete buffer exchange. The material was then over-concentrated using a centrifuge-concentrator, followed by dilution to 120 mg/mL, and polysorbate was added from 20% to 0.01% of the final concentration. Formulation 16 was considered the control formulation.

Table 6A

*The final formulation contains 120 mg/mL dinosuzumab and PS20 at a final concentration of 0.01% (w/v), with the indicated pH. The concentrations of sorbitol and phenylalanine are estimated to be approximately 8.5% lower than those of the DF buffer. The concentration of arginine is estimated to be approximately 12.5% lower than that of the DF buffer.

These formulations were filled into containers at 1.0 mL filling volumes. The formulations were stored at 37°C for up to 1 month. Aggregation inhibition and stability against aggregation inhibition over time (e.g., based on HMWS and dimer formation) were assessed using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Figure 9 shows the percentage of HMWS monitored by SE-U HPLC at 37°C as a function of the formulation and time. Figure 10 shows the size exclusion chromatogram as a function of the formulation after storage at 37°C for one month. Table 6B below shows the percentage of HMWS monitored by SE-U HPLC at 37°C as a function of the formulation and time.

Table 6B

All formulations containing an amino acid aggregation inhibitor, arginine, or phenylalanine (formulations 17 to 20) were superior to the sorbitol control formulation (formulation 16) which lacked any amino acid aggregation inhibitor. When compared to the control formulation and the arginine hydrochloride formulation (formulations 16 and 17, respectively), all phenylalanine-containing formulations (formulations 18, 19, and 20) similarly contained low levels of HMWS (Figure 9). As shown in Figure 9, the HMWS formation rates of the formulations containing arginine hydrochloride and phenylalanine (formulations 17 to 19) were similar. The combined formulation containing 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).

Example 7

This example demonstrates that the inhibition of aggregation and stability of denosumab varies with different concentrations of phenylalanine.

In previous studies, we identified arginine hydrochloride and arginine hydrochloride-phenylalanine dipeptide as minimizing the initial initiation level and HMWS formation rate of denosumab. We evaluated the stabilizing effect of formulations containing arginine hydrochloride, various concentrations of phenylalanine, and combinations of arginine hydrochloride and phenylalanine on solutions containing denosumab (120 mg/mL).

The formulations tested are described in Table 7A below. To prepare test samples A through E, aliquots of dinosuzumab (70 mg/mL) in acetate (pH 5.2) were dialyzed against the DF buffer described below, with a total of three buffer replacements to achieve a 1,000,000-fold dilution of the previous formulation, ensuring complete buffer exchange. The material was then over-concentrated using a centrifuge-concentrator to approximately 130 mg/mL to 150 mg/mL, followed by dilution to 120 mg/mL and the addition of 20% to 0.01% polysorbate to a final concentration. Formulation A was considered the control formulation.

These formulations were filled into containers at 1.0 mL filling volumes. The formulations were stored at 37°C for one month. Stability was assessed using SE-UHPLC (based on HMWS formation). The stability profile of these formulations after one month of storage at 37°C is compared with that of sorbitol and arginine hydrochloride/sorbitol formulations at 37°C, as shown in Figure 11A.

To prepare test samples F through K, aliquots of dinosuzumab (70 mg/mL) in acetate (pH 5.2) were ultrafiltered/percolated (UF/DF) against the DF buffer described below, for a total of 12 percolation volumes to ensure complete buffer exchange. The material was then overconcentrated to approximately 200 mg/mL using ultrafiltration, followed by dilution to 120 mg/mL and the addition of polysorbate from 20% to 0.01% to a final concentration. The acetate concentration in these formulations was 20 mM. Formulation F was considered the control formulation. All cited acetate and excipient values are relative to the acetate and excipient concentrations targeted by the dialysis antibody.

These formulations were filled into containers at 1.0 mL filling volumes. The formulations were stored at 40°C for one month. Stability was assessed using SE-UHPLC (based on HMWS formation). The stability profile of these formulations after one month of storage at 40°C is compared with that of sorbitol and arginine hydrochloride/sorbitol formulations at 40°C, as shown in Figure 11B.

Table 7A

*The final formulation contains 120 mg/mL dinosuzumab and PS20 at a final concentration of 0.01% (w/v), with the indicated pH. The concentrations of sorbitol and phenylalanine are estimated to be approximately 8.5% lower than those of the DF buffer. The concentration of arginine is estimated to be approximately 12.5% lower than that of the DF buffer.

Figure 11A and Table 7B show the percentage of HMWS monitored by SE-U HPLC at 37°C as a function of preparation and time. Figure 11B and Table 7C show the percentage of HMWS monitored by SE-U HPLC at 40°C as a function of preparation and time. Figures 12A and 12B show the changes in size exclusion chromatograms with preparation after one month of storage at 37°C and 40°C, respectively.

Table 7B

Table 7C

When compared with sorbitol and arginine hydrochloride/sorbitol formulations (formulations A and B, respectively), all phenylalanine formulations (formulations C, D, E, G to K) contained lower levels of HMWS. When compared with the arginine/sorbitol formulation (formulation B), the combination formulation of arginine hydrochloride and phenylalanine showed similar stability. All formulations were superior to the sorbitol control formulations (formulations A and F).

Example 8

This example demonstrates the evaluation of different amino acid aggregation inhibitors.

Different amino acid aggregation inhibitors were evaluated by preparing formulations containing eight hydrophobic, aromatic, or polar/charged amino acids to determine their effect on minimizing the amount (%) of HMWS in a high-concentration liquid denosumab formulation (120 mg/mL) and HMWS formation over a specific time period. This formulation, containing one of eight L-amino acids and a reduced amount of sorbitol, was compared to a control formulation (formulation 26) that contained no amino acid aggregation inhibitors and had a higher amount of sorbitol due to isotonicity.

The tested amino acid aggregation inhibitors were grouped into one of three groups (Group I through Group III), and each group contained the following amounts of amino acid aggregation inhibitor:

I. Aromatic amino acids:

(a) 38mM phenylalanine (Formulation 27);

(b) 38mM tryptophan (Formulation 28);

II. Polar/charged amino acids:

(a) 75mM arginine hydrochloride (Formulation 29);

(b) 75mM lysine (preparation 30);

(c) 75mM histidine (Formulation 31);

III. Hydrophobic amino acids:

(a) 38mM leucine (Formulation 32);

(b) 38mM isoleucine (Formulation 33);

(c) 38mM valine (Formulation 34).

To prepare formulations 26 to 34, aliquots of dinosuzumab (70 mg/mL) in acetate (pH 5.2) were dialyzed against the DF buffer as described in Table 8A, with a total of three buffer replacements to achieve a 1,000,000-fold dilution of the previous formulations, ensuring complete buffer exchange. Dialysis of histidine formulation F used a buffer with an initial pH of 4.0, and the pH was expected to reach the target pH of 5.1 after protein concentration due to the Donnan effect and acetate co-concentration. However, after protein concentration to 120 mg/mL, the pH did not reach the target pH of 5.1, but remained at pH 4.0. To bring the histidine formulation to pH 5.1, titration with dilute (0.1N) NaOH was required. The remaining formulation was over-concentrated using a centrifuge-concentrator unit, then diluted to 124-128 mg/mL and polysorbate was added to a final concentration of 20 to 0.01% (w/v).

Table 8A

*The final formulation contains 120 mg/mL dinosuzumab and PS20 at a final concentration of 0.01% (w/v), with the indicated pH. The sorbitol and phenylalanine concentrations are estimated to be approximately 8.5% lower than the sorbitol concentration in the DF buffer. The arginine concentration is estimated to be approximately 12.5% lower than the arginine concentration in the DF buffer. The letters in parentheses following F# correspond to Figures 13 through 18.

These formulations were filled into containers at 1.0 mL filling volumes. The formulations were stored at 37°C for up to 4 weeks. Aggregation inhibition and stability against aggregation inhibition over time (e.g., based on HMWS and dimer formation) were assessed using SE-UHPLC. The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Figures 13 through 15 show the percentage of HMWS (high molecular weight loss) of each formulation monitored by SE-UHPLC at 37°C as a function of storage time, and Table 8B provides the data points for each figure. Figures 16 through 18 show the 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.

Table 8B

F# is provided in the left column and corresponds to the F# in Table 8A.

As shown in Figures 13 to 15, all formulations containing amino acid aggregation inhibitors (Formulations 27 to 34) showed some degree of improvement in stability compared to the acetate/sorbitol formulation (Formulation 26). Formulations containing aromatic amino acids (Formulations 27 and 28) showed the greatest reduction in %HMWS. Formulations containing phenylalanine (Formulation 27) also showed a significant reduction in HMWS compared to the control (Formulation 26), with the formulation containing tryptophan showing the greatest reduction. Dinosumab formulations containing polar/charged amino acids (Formulations 29 to 31) generally showed a larger amount of higher aggregates (Figure 17) compared to other formulations with amino acid stabilizers (Figures 16 and 18), and this particular histidine formulation generally showed a larger amount of HWMS compared to the acetate/sorbitol formulation (Formulation 26) (Figure 14). Results for histidine formulations may be biased by the dialysis process, the longer duration of dialysis at pH 4.0, and the titration of the formulation with dilute NaOH. Formulations containing hydrophobic amino acids (formulations 32 to 34) all showed consistent improvements in HMWS formation.

Example 9

This example illustrates the possible mechanisms by which arginine and phenylalanine play a role in stabilizing denosumarumab. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a sensitive and stable technique for characterizing protein-protein/ligand/excipient interactions. This method detects changes in the skeletal amide hydrogen bonds resulting from interactions with excipients.

Dinosumab (3 mg/mL) in 10 mM acetate buffer (pH 5.2) (“A52”) was subjected to hydrogen-deuterium exchange mass spectrometry (HDX-MS) in the presence of L-arginine (Formulation 35), L-phenylalanine (Formulation 36), or L-glycine (Formulation 37), and compared with a dinosumab formulation (Formulation 38) lacking any amino acid aggregation inhibitors. Experiments were conducted at 4 °C (using 75 mM L-arginine, L-phenylalanine, or L-glycine) and 37 °C (using 150 mM L-arginine, L-phenylalanine, or L-glycine). After analyzing over 530 peptides, a small number of regions with significant conformational changes were identified. Several representative peptides from these regions were captured in Figures 19–30.

Figures 19 to 24 show the changes in deuterium inclusion percentage (%) of each of the following formulations 35 to 38 as a function of time (log(sec)): light chain amino acids 28-33 (Figure 19), 108-116 (Figure 20), 125-132 (Figure 21), 47-59 (Figure 22), 243-253 (Figure 23), and 392-399 (Figure 24) at 4°C.

Figures 25 to 30 show the changes in the percentage of deuterium inclusion at 37°C over time (log(sec)) for each of the following formulations 35 to 38: light chain amino acids 28-33 (Figure 25), 108-117 (Figure 26), 124-131 (Figure 27), 47-59 (Figure 28), 242-253 (Figure 29), and 392-399 (Figure 30).

While these data support similar interaction effects between Arg and Gly on denosumab, Arg exhibits a slightly stronger HDX footprint (configurational changes) with denosumab: strong stabilizing effect in the Fab LC 28-33 region; weak stabilizing effect in the Fab LC 108-132 and HC 47-59, Fc CH3 HC 392-399 regions; and weak destabilizing effect in the Fc CH2 243-253 region. Without intending to be bound by any particular theory, it is hypothesized that the arginine hydrochloride effect is due to preferential exclusion resulting from a combination of surface and weak surface interactions on denosumab, while glycine acts through preferential exclusion.

However, phenylalanine did not show significant structural perturbation to denosumab. Without intending to be bound by any particular theory, it is hypothesized that the stabilizing effect of phenylalanine may be accomplished through one or more of the following mechanisms: side-chain interactions, since they have no effect on the peptide backbone (no HDX footprint); and/or cation-π interactions with the arginine/lysine side chains that do not affect the hydrogen bond network structure of the backbone.

Example 10

This example illustrates the possible mechanism of action of phenylalanine-stabilized denosumab.

To investigate the specific role of Phe in denosumab, molecular dynamics simulations were performed. Specifically, the Fab domain of denosumab was made to form a solvate with excess Phe in a simulation cassette, and two 10 ns simulations were performed. In summary, Phe residues that remained 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-stay observations, Phe residues bound to both the VH/VL interface (variable heavy chain/variable light chain) and the CH/CL (constant heavy chain/constant light chain) region. In one instance, the Phe side chain is believed to interact with the side chains of hydrophobic residues (e.g., V93, Y95, and W112 of the heavy chain, and A44 and P45 of the light chain) at the VH/VL interface. In another instance, it is believed that the side-chain rings of Phe interact with the NH3+ and COO(-) groups of residues (e.g., T165 of the light chain and G171, V172, and T174 of the heavy chain) at the CH1/CL interface. Without intending to be bound by any particular theory, this observation leads to the idea that the specific role of Phe in mitigating the aggregation of dinosuzumab is due to the interaction of the phenyl groups with hydrophobic residues (e.g., R30, G31, R32, and Y33 of the light chain CDR1, A52 of the light chain CDR2, and M106 of the heavy chain CDR3) that form the interface between the heavy constant 1 (Hc) and light constant (Lc) chains. This interaction is hypothesized to replace the previously hydrophobic surface with a relatively more charged (and therefore hydrophilic) surface from the NH3(+) and COO(-) groups of the Phe excipient.

Example 11

Stability assessments were performed on various anti-RANKL antibody constructs (isotypes IgG1, IgG2, and IgG4). As described above, both arginine hydrochloride and phenylalanine minimized initial HMWS and HWMS levels over a period of time compared to the acetate/sorbitol control formulation of denosumab (an IgG2 immunoglobulin). This assessment was performed to compare the likelihood of Arg-HCl and Phe reducing HMWS in formulations containing different anti-RANKL antibody constructs. The IgG1 and IgG4 constructs tested in this study contained the same complementarity-determining region (CDR) compared to denosumab, but contained different constant domain scaffolds. The different IgG2 constructs tested in this study had different CDRs relative to denosumab, but contained the same constant domain scaffolds.

Each test antibody construct was purified and concentrated from 8 mg/mL to 70 mg/mL using a centrifuge. Each concentrated volume was aliquoted into three equal aliquots and then dialyzed against acetate buffers formulated with sorbitol, sorbitol/phenylalanine, and sorbitol/arginine hydrochloride to prepare formulations 39 to 47 as described in Table 9. The dialyzed samples were then overconcentrated using a centrifuge to a concentration above 120 mg/mL. The antibody proteins were then diluted to 120 mg/mL with the appropriate buffer.

Table 9

*The final formulation contains 0.01% (w/v) PS20 and has the indicated pH value. The concentrations of sorbitol and phenylalanine are estimated to be approximately 8.5% lower than the concentration of the DF buffer. The concentration of arginine is estimated to be approximately 12.5% lower than the concentration of the DF buffer.

These formulations were filled into glass vials at 1.0 mL filling volumes. The formulations were stored at 37°C for up to one month. Aggregation inhibition and stability against aggregation inhibition over a period of time (e.g., based on HMWS formation) were assessed using SE-U HPLC. The aggregation inhibition profile of these formulations was compared under initial conditions and after the storage period. The stability of these formulations within this immunoglobulin class was compared after storage.

Figures 31, 33, and 35 (and related Tables 10, 12, and 14 below) show the percentage of HMWS (high molecular weight substances) monitored by SE-U HPLC at 37°C for immunoglobulin G (IgG1, IgG2, and IgG4, respectively) as a function of preparation and time. Figures 32, 34, and 36 (and related Tables 11, 13, and 15 below) show the percentage of low molecular weight substances (LMWS, e.g., protein fragmentation) monitored by SE-U HPLC at 37°C for immunoglobulin G (IgG1, IgG2, and IgG4, respectively) as a function of preparation and time. Figures 37, 38, and 39 show the particle size exclusion chromatographic overlay plots after storage at 37°C for t=4 weeks as a function of preparation.

Table 10: Comparison of %HMW and IgG1 (A, B, C) at 37°C for 4 weeks

Table 11: Comparison of %LMWS and IgG1 (A, B, C) at 37°C for 4 weeks

Table 12: Comparison of %HMW and IgG2 (D, E, F) at 37℃ for 4 weeks

Table 13: Comparison of %LMWS and IgG2 (D, E, F) after 4 weeks at 37°C

Table 14: Comparison of %HMW and IgG4 (G, H, I) at 37°C for 4 weeks

Table 15: Comparison of %LMWS and IgG4 (G, H, I) after 4 weeks at 37°C

As shown in Figures 31 and 32, IgG1 molecules with a CDR region similar to the previous Dinosumab sample showed a reduction in HMWS of approximately 0.2% with the addition of phenylalanine compared to the acetate/sorbitol control formulation. IgG2 samples with different CDRs, as depicted in Figures 33 and 34, showed an increase in HMWS in the acetate/phenylalanine/sorbitol formulation compared to the control acetate/sorbitol formulation. As shown in Figures 35 and 36, for the IgG4 sample type, both acetate/sorbitol and acetate/phenylalanine/sorbitol formulations exhibited similar stability, with acetate/sorbitol/arginine showing higher HMWS formation. In all cases utilizing IgG1, IgG2, and IgG4 sample types, formulations containing acetate/sorbitol/arginine showed increased HMWS degradation compared to both acetate/sorbitol (control) and acetate/phenylalanine/sorbitol formulations.

Because protein fragmentation was significantly increased in the acetate/arginine/sorbitol formulation as depicted in Figures 37 and 38, the relationship between fragments and antibody isotypes is shown in Figures 32, 34, and 36. Literature has shown that fragmentation-mediated aggregation of monoclonal antibodies may occur from antibodies stored at 37°C [Perico N. et al., J. Pharm. Sci. (2009) 98, pp. 3031-3042]. This mechanism is plausible in this assessment because fragmentation was greatest in the acetate/arginine/sorbitol formulation. Fragmentation was minimized in the acetate/phenylalanine/sorbitol formulation, potentially resulting in less HMWS material. IgG4 sample type did not accelerate fragmentation or aggregation.

Based on data collected in this study, along with previous data—specifically, molecular modeling data gathered using denosumab—a strong correlation was established between the amino acid sequence of the CDR and the relative effect of phenylalanine in reducing HMWS. A reduction in HMWS was observed in denosumab (IgG2) and IgG1 variants with the same CDR amino acid, but not in the IgG2 variant with a different CDR domain. It was found that the amino acid sequence contained within the CDR domain is susceptible to interaction with phenylalanine and subsequent aggregation inhibition. The IgG4 molecule also has the same CDR region as denosumab, but minimal changes in aggregation were detected during the study. The main differences between the IgG4 molecule and the IgG1 and IgG2 versions lie in its hinge amino acid length and its functionally active structure. Because IgG1 and IgG2 antibody isotypes typically have an extended structure described as a "Y" shape, the IgG4 Fab CH1 domain interacts with the CH2 domain to form a more compact structure [Aalberse R.C. et al., Immunology (2002), 105, pp. 9-19]. This compact structure can inhibit the fragmentation and aggregation reactions commonly seen in the IgG1 and IgG2 modes.

Example 12

Studies were conducted to monitor the stability of denosumab formulated as described below and in accordance with Table 16 (formulations 51 to 55). Various percolation buffers used to produce the final formulation of 120 mg/mL denosumab at pH 5.1 differed in acetate concentration and initial pH. Additionally, sorbitol levels were adjusted to maintain the isotonicity of the final product (approximately 300 mOsm/kg). 70 mg/mL denosumab was percolated against each buffer for no more than 7 times the percolation volume, followed by ultrafiltration to approximately 180 g/mL, and diluted with percolation buffer and polysorbate to a concentration of 120 mg/mL denosumab and 0.01% polysorbate 20. Stability was assessed using SE-U HPLC after storage at 37°C, and the stability of denosumab in these formulations was highly similar. Initial HMW decreased slightly with increasing initial acetate concentration. In contrast, the aggregation rate was slightly increased in formulations containing lower acetate levels.

Table 16

*The final formulation contains 120 mg/mL dinosumab and PS20 at a final concentration of 0.01% (w/v), with pH 5.1.

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).

All chemical denaturation experiments were performed using the Unchained Labs HUNK instrument with a fluorescence detector. Excitation wavelength was 280 nm, and emission scans between 300 and 500 nm were recorded. For each denaturation experiment, protein, buffer, and denaturant (guanidine hydrochloride) were dispensed into 36 wells, with the denaturant concentration increasing linearly, resulting in a 36-point curve for each condition. The data points were fitted using curve fitting software provided by the instrument manufacturer (Unchained Labs). A two-state model was used because only a single native denatured transition was demonstrated. Experiments were performed using 0–6 M urea in 10 mM acetate 5.0% (w/v) sorbitol and titrated to the desired pH of 4.5, 4.8, or 5 (5.2). The concentration of denosumab protein was 7 mg/mL in all experiments.

Figure 40 shows the isothermal chemical denaturation curves of dinosumab at pH 4.5, 4.8, and 5.0 in the absence of arginine. The C1 _/2 of the chemical denaturant required for 50% unfolding was similar under the three test pH conditions in the absence of arginine.

Figure 41 shows the isothermal chemical denaturation curves of denosumab at pH 4.5, 4.8, and 5.2 in the presence of 75 mM arginine hydrochloride. The chemical denaturing stability is significantly increased at pH 5.2 compared to pH 4.8 and 4.5. The C1 _/2 of the denaturing agent guanidine hydrochloride increases by 1 M at pH 5.2 relative to lower pH values. Therefore, the protective properties of arginine are surprisingly pH-dependent.

Example 14

The following examples provide the results of a study on the effects of arginine and phenylalanine on the stability of high-concentration denosumab formulations in syringes over a period of time.

In previous studies, arginine hydrochloride and phenylalanine were identified as reducing the initial initiation level and HMWS formation rate of denosumab. In this study, the stabilizing effect of formulations containing arginine hydrochloride, phenylalanine, and combinations of arginine hydrochloride and phenylalanine on solutions containing denosumab (120 mg/mL) was evaluated after storage in syringes at two different temperatures for up to three months.

The formulations tested are described in Table 17 below. To prepare formulations 56 to 59, dinosumab (70 mg/mL) in acetate (pH 5.2) was percolated to 8 times the percolation volume against the filtration (DF) buffer described below to ensure complete buffer exchange. The material was then ultrafiltered to a concentration above 180 mg/mL, followed by dilution to 120 mg/mL and the addition of polysorbate from 20% to 0.01% to a final concentration. Formulation 56 was considered a control formulation. The listed acetate, arginine hydrochloride, and phenylalanine values are relative to the DF buffer and provide estimated levels in the final composition of 120 mg/mL dinosumab, taking into account excipient exclusion and acetate co-concentration, in the absence of other counterions. Viscosities at 5°C and 25°C were measured using a Paar modular compact rheometer at shear rates up to 1000 s⁻¹ (reciprocal of a second). These formulations were filled into glass prefilled syringes (PFS) at a filling volume of 1.0 mL. Parallel sets of syringes were stored at 25°C for 3 months and at 37°C for 2 months, respectively. Stability (e.g., based on HMWS formation) was assessed using SE-UHPLC.

Table 17

*Each final formulation contains 120 mg/mL dinosuzumab and 0.01% PS20, as well as the pH indicated in the abbreviated formulation name.

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

Tables 18 to 21 present the same data and the increase in HMWS relative to the initial HMWS level in tabular form.

Table 18: %HMWS levels over 12 weeks at 25°C

Table 19: Increase in HMWS over 12 weeks at 25°C

Table 20: %HMWS levels over 8 weeks at 37°C

Table 21: Increase in HMWS over 8 weeks at 37°C

This example demonstrates that the addition of arginine, phenylalanine, and combinations thereof each reduces the initial HMWS (t=0) level in high-concentration Dinosumab formulations. At 25°C, the increase in HMWS in phenylalanine formulation 59 was reduced compared to control formulation 56. At 37°C, formulations 57 and 59 showed reduced HMWS formation compared to control sorbitol formulation 56. The formulations containing arginine hydrochloride and phenylalanine formed HMWS at a higher rate at 37°C compared to other formulations, indicating that the combination of these excipients destabilizes the Dinosumab in this formulation at this higher temperature.

The above description is provided for clarity only and should not be construed as an unnecessary limitation, as modifications within the scope of this invention will be obvious to those skilled in the art.

Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” should be understood to implicitly include the stated integer or step or group of integers or steps, rather than excluding any other integer or step or group of integers or steps.

Throughout this specification, when a composition is described as including components or materials, it is contemplated that such compositions may also consist substantially of or from any combination of the described components or materials, unless otherwise described. Similarly, when a method is described as including specific steps, it is contemplated that such methods may also consist substantially of or from any combination of the described steps, unless otherwise described. The invention illustratively disclosed herein may be suitably practiced in the absence of any elements or steps not expressly disclosed herein.

The methods disclosed herein and their individual steps can be performed manually and/or with the assistance of electronic devices or automation provided by them. While numerous methods have been described with reference to specific embodiments, those skilled in the art will readily appreciate that other ways of performing the actions associated with these methods can be used. For example, unless otherwise described, the order of the steps may be changed without departing from the scope or spirit of the method. Furthermore, some individual steps may be combined, omitted, or further subdivided into other steps.

All patents, publications, and references cited in this document are incorporated herein by reference in their entirety. In the event of any conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall prevail.

Claims (142)

1. An aqueous pharmaceutical formulation comprising (i) a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety and (ii) an amino acid aggregation inhibitor comprising phenylalanine or tryptophan. The anti-RANKL monoclonal antibody or its antigen-binding portion comprises (A) a light chain variable domain comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:7; and (B) a heavy chain variable domain comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:8, a heavy chain variable domain comprising a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:10. 2. The aqueous pharmaceutical preparation of claim 1, wherein the concentration of the human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety is higher than 70 mg/mL. 3. The aqueous pharmaceutical preparation according to any one of claims 1 to 2, wherein the anti-RANKL antibody or its antigen-binding portion comprises: A light chain variable domain containing the amino acid sequence of SEQ ID NO:1; and Heavy chain variable domain containing the amino acid sequence of SEQ ID NO:2. 4. The aqueous pharmaceutical preparation according to any one of claims 1 to 2, wherein the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. 5. The aqueous pharmaceutical preparation according to any one of claims 1 to 2, wherein the antigen-binding portion is Fab, Fab’, F(ab’)2 or a single-chain Fv. 6. The aqueous pharmaceutical formulation of claim 4, wherein the anti-RANKL antibody comprises a human κ light chain constant region and/or a γ heavy chain constant region. 7. The aqueous pharmaceutical formulation according to any one of claims 1 to 4 and 6, wherein the anti-RANKL antibody comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO:1 and a human κ light chain constant region. 8. The aqueous pharmaceutical preparation according to any one of claims 1 to 4, 6 and 7, wherein the anti-RANKL antibody comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:2 and a human _IgG2 constant region. 9. The aqueous pharmaceutical preparation according to any one of claims 1 to 4 and 6 to 8, wherein the anti-RANKL antibody is _IgG2 . 10. The aqueous pharmaceutical preparation of any one of claims 1 to 4 and 6 to 9, wherein the anti-RANKL antibody or its antigen-binding portion comprises: i. A light chain containing the amino acid sequence of SEQ ID NO:13; and i. A heavy chain containing the amino acid sequence of SEQ ID NO:14. 11. The aqueous pharmaceutical preparation according to any one of claims 1 to 10, wherein the concentration of the antibody or its antigen-binding moiety exceeds 70 mg/mL, optionally in the range of 70 mg/mL to 300 mg/mL. 12. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, wherein the concentration of the antibody or its antigen-binding portion is in the range of more than 70 mg/mL to 300 mg/mL, optionally in the range of more than 70 mg/mL to 200 mg/mL. 13. The aqueous pharmaceutical preparation of claim 11 or 12, wherein the concentration of the antibody or its antigen-binding portion is in the range of 100 to 140 mg/mL. 14. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, wherein the concentration of the antibody or its antigen-binding portion is 120 mg/mL ± 12 mg/mL. 15. The aqueous pharmaceutical preparation of claim 1, wherein the phenylalanine is L-phenylalanine. 16. The aqueous pharmaceutical preparation of claim 1, wherein the tryptophan is L-tryptophan. 17. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, comprising 5 mM to 300 mM amino acid aggregation inhibitors. 18. The aqueous pharmaceutical formulation of claim 17, comprising 5 mM to 180 mM amino acid aggregation inhibitors. 19. The aqueous pharmaceutical formulation of claim 18, comprising 5 mM to 100 mM amino acid aggregation inhibitor. 20. The aqueous pharmaceutical formulation of claim 19, comprising 25 mM to 90 mM amino acid aggregation inhibitors. 21. The aqueous pharmaceutical formulation of claim 20, comprising 20 mM to 50 mM amino acid aggregation inhibitor. 22. The aqueous pharmaceutical preparation of any one of claims 17 to 21, comprising: a. 20mM to 50mM phenylalanine; b. 20 mM to 50 mM tryptophan; or c. Any combination thereof. 23. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, comprising only one amino acid aggregation inhibitor. 24. The aqueous pharmaceutical preparation of any of the preceding claims, wherein the molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody is 10 to 200. 25. The aqueous pharmaceutical preparation of claim 24, wherein the molar ratio is 20 to 90. 26. The aqueous pharmaceutical preparation of any one of the preceding claims further comprises a tension modifier, which is optionally selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. 27. The aqueous pharmaceutical formulation of claim 26, wherein the tension modifier comprises sorbitol. 28. The aqueous pharmaceutical formulation of claim 26 or 27, comprising 1.0 (w/w)% to 5.0 (w/w)% tension modifier. 29. The aqueous pharmaceutical formulation of claim 28, comprising 2.0 (w/w)% to 5.0 (w/w)% sorbitol or 3.5 (w/w)% to 5.0 (w/w)% sorbitol or 4.0 (w/w)% to 5.0 (w/w)% sorbitol. 30. The aqueous pharmaceutical formulation according to any one of claims 1 to 29, wherein it is free of sorbitol and optionally free of any tension modifier. 31. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, further comprising a surfactant. 32. The aqueous pharmaceutical formulation of claim 31, wherein the surfactant is selected from the group consisting of: polyoxyethylene sorbitan fatty acid esters, or one or more alkyl aryl polyethers, or one or more poloxamers and combinations thereof. 33. The aqueous pharmaceutical formulation of claim 32, wherein the surfactant is polysorbate 20. 34. The aqueous pharmaceutical preparation of any one of claims 31 to 33, comprising at least 0.004 (w/v)% surfactant and optionally less than 0.15 (w/v)% surfactant. 35. The aqueous pharmaceutical formulation of claim 34, comprising 0.005 (w/v)% to 0.015 (w/v)% surfactant. 36. The aqueous pharmaceutical preparation of any of the preceding claims further comprises a buffer, optionally wherein the buffer is centered at a pH range of 4.0 to 5.5 at 25°C. 37. The aqueous pharmaceutical formulation of claim 36, wherein the buffer has a pKa in a pH unit of pH 5.0-5.2 at 25°C. 38. The aqueous pharmaceutical preparation of claim 36 or 37, comprising a 5 mM to 60 mM buffer. 39. The aqueous pharmaceutical preparation of claim 38, comprising a 5 mM to 50 mM buffer. 40. The aqueous pharmaceutical preparation of claim 39, comprising 9 mM to 45 mM buffer. 41. The aqueous pharmaceutical preparation according to any one of claims 36 to 40, wherein the buffer is an acetate or a glutamate. 42. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, having a pH value in the range of 5.0 to 5.4, 5.0 to 5.2, 5.0 to less than 5.2, 5.0 to 5.19, 5.0 to 5.15, or 5.0 to 5.1. 43. The aqueous pharmaceutical preparation of claim 42, having a pH value of about 5.1. 44. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, having a viscosity of not more than 6 cP at 5°C, optionally wherein the viscosity is 4.5 cP to 5.5 cP. 45. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, having a viscosity of less than 13 cP at 25°C. 46. The aqueous pharmaceutical formulation of claim 45, having a viscosity in the range of 2.0 cP to 10 cP, optionally 2.5 cP to 4 cP. 47. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, having a conductivity in the range of 500 μS/cm to 5500 μS/cm. 48. The aqueous pharmaceutical preparation as claimed in any of the preceding claims, having an osmotic pressure in the range of 200 mOsm/kg to 500 mOsm/kg, or 225 mOsm/kg to 400 mOsm/kg, or 250 mOsm/kg to 350 mOsm/kg. 49. The aqueous pharmaceutical preparation as described in any of the preceding claims, as measured by SE-UHPLC, contains less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 2°C to 8°C for at least 12 months, 24 months or 36 months. 50. The aqueous pharmaceutical preparation as described in any of the preceding claims, as measured by SE-UHPLC, contains less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 20°C to 30°C for about 1 month. 51. The aqueous pharmaceutical preparation as described in any of the preceding claims, as measured by SE-UHPLC, contains less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after a first storage of at least 12 months, 24 months or 36 months at 2°C to 8°C and a second storage of about 1 month at 20°C to 30°C. 52. The aqueous pharmaceutical preparation as described in any of the preceding claims, as measured by SE-UHPLC, contains less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 37°C for about one month or at 30°C for 3 months. 53. A medicament comprising, in a container, an aqueous pharmaceutical preparation as described in any one of claims 1 to 52, said container optionally being a vial, a prefilled syringe (PFS), or a glass container. 54. The medicament of claim 53, wherein it contains 1 mL or less of the preparation, optionally about 0.5 mL. 55. A method for manufacturing a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, comprising combining the anti-RANKL monoclonal antibody or its antigen-binding moiety at a concentration higher than 70 mg/mL with an amino acid aggregation inhibitor comprising phenylalanine or tryptophan, a buffer, a surfactant, and optionally a ground tension modifier. The anti-RANKL monoclonal antibody or its antigen-binding portion comprises (A) a light chain variable domain comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:7; and (B) a heavy chain variable domain comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:8, a heavy chain variable domain comprising a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:10. 56. The method of claim 55, wherein the anti-RANKL antibody or its antigen-binding portion comprises: A light chain variable domain containing the amino acid sequence of SEQ ID NO:1; and Heavy chain variable domain containing the amino acid sequence of SEQ ID NO:2. 57. The method of any one of claims 55 to 56, wherein the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. 58. The method of any one of claims 55 to 56, wherein the antigen-binding moiety is Fab, Fab’, F(ab’)2 or a single-chain Fv. 59. The method of claim 57, wherein the anti-RANKL antibody comprises a constant region of the human κ light chain and/or γ heavy chain. 60. The method of any one of claims 55 to 57 and 59, wherein the anti-RANKL antibody comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO:1 and a human κ light chain constant region. 61. The method of any one of claims 55 to 57, 59 and 60, wherein the anti-RANKL antibody comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:2 and a human _IgG2 constant region. 62. The method according to any one of claims 55 to 57 and 59 to 61, wherein the anti-RANKL antibody is _IgG2 . 63. The method of any one of claims 55 to 57 and 59 to 62, wherein the anti-RANKL antibody or its antigen-binding portion comprises: i. A light chain containing the amino acid sequence of SEQ ID NO:13; and i. A heavy chain containing the amino acid sequence of SEQ ID NO:14. 64. The method according to any one of claims 55 to 63, comprising combining an antibody or its antigen-binding portion at a concentration of 10 mg/mL to 300 mg/mL with the amino acid aggregation inhibitor, a buffer, a surfactant, and optionally the tension modifier. 65. The method of any one of claims 55 to 64, wherein the stable aqueous pharmaceutical preparation comprises an antibody or its antigen-binding portion at a concentration of more than 70 mg/mL to 200 mg/mL. 66. The method of claim 65 or 65, wherein the stable aqueous pharmaceutical preparation comprises 100 to 140 mg/mL of antibody or its antigen-binding portion. 67. The method of any one of claims 55 to 66, wherein the stable aqueous pharmaceutical preparation comprises 120 mg/mL ± 12 mg/mL of antibody or antigen-binding moiety. 68. The method of claim 67, wherein the stable aqueous pharmaceutical preparation comprises 120 mg/mL ± 5 mg/mL of antibody or antigen-binding moiety. 69. The method of claim 55, wherein the phenylalanine is L-phenylalanine. 70. The method of claim 55, wherein the tryptophan is L-tryptophan. 71. The method of any one of claims 55 to 70, comprising combining a 5 mM to 300 mM amino acid aggregation inhibitor with the antibody or antigen binding moiety. 72. The method of claim 71, further comprising combining a 15 mM to 200 mM amino acid aggregation inhibitor with the antibody or antigen binding moiety. 73. The method of claim 71, further comprising combining a 5 mM to 180 mM amino acid aggregation inhibitor with the antibody or antigen binding moiety. 74. The method of claim 73, further comprising combining a 5 mM to 100 mM amino acid aggregation inhibitor with the antibody or antigen binding moiety. 75. The method of claim 74, further comprising combining a 25 mM to 90 mM amino acid aggregation inhibitor with the antibody or antigen binding moiety. 76. The method of any one of claims 55 to 75, further comprising combining the antibody or antigen-binding portion with the following: a. 20mM to 50mM phenylalanine; b. 20 mM to 50 mM tryptophan; or c. Any combination thereof. 77. The method of any one of claims 55 to 76, comprising combining the antibody or antigen-binding portion with only one amino acid aggregation inhibitor. 78. The method of any one of claims 55 to 77, wherein the aqueous pharmaceutical formulation comprises the antibody and the amino acid aggregation inhibitor in a molar ratio of 10 to 200 of the amino acid aggregation inhibitor to the antibody. 79. The method of claim 78, wherein the molar ratio is 20 to 90. 80. The method of any one of claims 55 to 79, wherein the antibody or antigen-binding portion is combined with a tension modifier selected from the group consisting of sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. 81. The method of claim 80, wherein the tension modifier comprises sorbitol. 82. The method of claim 80 or 81, comprising 1.0 (w/w)% to 5.0 (w/w)% tension modifier. 83. The method of claim 82, comprising 2.0 (w/w)% to 5.0 (w/w)% sorbitol or 3.5 (w/w)% to 5.0 (w/w)% sorbitol or 4.0 (w/w)% to 5.0 (w/w)% sorbitol. 84. The method according to any one of claims 55 to 83, wherein it is free of sorbitol and optionally free of any tension modifier. 85. The method of any one of claims 55 to 84, wherein the surfactant is selected from the group consisting of: polyoxyethylene sorbitan fatty acid esters, or one or more alkyl aryl polyethers, or one or more poloxamers and combinations thereof. 86. The method of claim 85, wherein the surfactant is a polyoxyethylene sorbitan fatty acid ester. 87. The method of claim 86, wherein the surfactant is polysorbate 20. 88. The method of any one of claims 85 to 87, comprising at least 0.004 (w/v)% surfactant and optionally less than 0.15 (w/v)% surfactant. 89. The method of claim 88, comprising 0.005 (w/v)% to 0.015 (w/v)% surfactant. 90. The method of any one of claims 55 to 89, comprising combining the antibody or antigen-binding portion with a buffer, wherein the buffer is centered at 25°C in the range of pH 4.0 to pH 5.5. 91. The method of claim 90, wherein the buffer has a pKa within a pH unit of pH 5.0-5.2 at 25°C. 92. The method of claim 90 or 91, wherein the aqueous pharmaceutical preparation comprises a 5 mM to 60 mM buffer. 93. The method of claim 92, wherein the aqueous pharmaceutical preparation comprises a 5 mM to 50 mM buffer. 94. The method of claim 93, wherein the aqueous pharmaceutical preparation comprises a buffer of 9 mM to 45 mM. 95. The method of any one of claims 90 to 94, wherein the buffer is an acetate or a glutamate. 96. The method of any one of claims 55 to 95, wherein the aqueous pharmaceutical preparation has a pH value in the range of 5.0 to 5.4, 5.0 to 5.2, 5.0 to less than 5.2, 5.0 to 5.19, 5.0 to 5.15, or 5.0 to 5.1. 97. The method of claim 96, wherein the aqueous pharmaceutical preparation has a pH of about 5.1. 98. The method of any one of claims 55 to 97, wherein the aqueous pharmaceutical preparation has a viscosity of not more than 6 cP at 5°C, optionally wherein the viscosity is 4.5 cP to 5.5 cP. 99. The method of any one of claims 55 to 98, wherein the aqueous pharmaceutical preparation has a viscosity of less than 13 cP at 25°C. 100. The method of claim 99, wherein the aqueous pharmaceutical formulation has a viscosity in the range of 2.0 cP to 10 cP, optionally 2.5 cP to 4 cP. 101. The method according to any one of claims 55 to 100, wherein the aqueous pharmaceutical formulation has a conductivity in the range of 500 μS/cm to 5500 μS/cm. 102. The method according to any one of claims 55 to 101, wherein the aqueous pharmaceutical preparation has an osmotic pressure in the range of 200 mOsm/kg to 500 mOsm/kg, or 225 mOsm/kg to 400 mOsm/kg, or 250 mOsm/kg to 350 mOsm/kg. 103. The method of any one of claims 55 to 102, wherein the aqueous pharmaceutical formulation, as measured by SE-UHPLC, has less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 2°C to 8°C for at least 12 months, 24 months, or 36 months. 104. The method of any one of claims 55 to 103, wherein the aqueous pharmaceutical formulation, as measured by SE-UHPLC, has less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 20°C to 30°C for about one month. 105. The method of any one of claims 55 to 104, wherein the aqueous pharmaceutical preparation, as measured by SE-U HPLC, has less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after a first storage of at least 12 months, 24 months, or 36 months at 2°C to 8°C and a second storage of about 1 month at 20°C to 30°C. 106. The method of any one of claims 55 to 105, wherein the aqueous pharmaceutical formulation, as measured by SE-UHPLC, has less than 2% high molecular weight substances (HMWS) and/or more than 98% antibody main peak after being stored at 37°C for about one month or at 30°C for 3 months. 107. A stable aqueous pharmaceutical preparation, manufactured according to any one of claims 55 to 106. 108. Use of the aqueous pharmaceutical preparation as described in any one of claims 1 to 52 and 107 in the preparation of a medicament for therapeutic treatment of a subject. 109. The use as described in claim 108, wherein the therapeutic treatment covers treating or preventing skeletal-related events (SREs), treating or preventing giant cell tumor of bone, treating or preventing malignant hypercalcemia, treating or preventing osteoporosis, or increasing bone mass in a subject. 110. The use as described in claim 109, wherein the therapeutic treatment encompasses (a) treatment or prevention of SRE in subjects with bone metastases from solid tumors; (b) treatment or prevention of SRE in adult or skeletally mature adolescent subjects with unresectable or surgically resectable giant cell tumors of bone that could cause serious morbidity; (c) treatment of bisphosphonate-refractory malignant hypercalcemia in subjects; (d) treatment or prevention of SRE in subjects with multiple myeloma or bone metastases from solid tumors; (e) treatment of osteoporosis in postmenopausal women at high risk of fracture; (f) treatment to increase bone mass in women at high risk of fracture who are receiving adjuvant aromatase inhibitor therapy for breast cancer; (g) treatment to increase bone mass in men at high risk of fracture who are 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) therapy utilizing calcium or vitamin D. 111. Use of the preparation of the formulation according to any one of claims 1 to 52 and 107 in the preparation of a medicament for the prevention of skeletal-related events (SREs) in patients in need. 112. The use as described in claim 111, wherein the SRE is selected from the group consisting of: pathological fractures, radiotherapy to bone, surgery to bone, and spinal cord compression. 113. The use as described in claim 111 or 112, wherein the patient has bone metastases from a solid tumor. 114. The use as described in claim 113, wherein the solid tumor is selected from breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma. 115. The use as described in claim 113 or 114, wherein the patient has multiple myeloma. 116. The use according to any one of claims 111 to 115, wherein the drug comprises the formulation which effectively reduces the bone turnover marker urinary creatinine modified N-terminal telopeptide (uNTx/Cr), optionally by at least 80%. 117. Use of the formulation according to any one of claims 1 to 52 and 107 in the preparation of a medicament for treating giant cell tumor of bone in patients in need. 118. The use as described in claim 117, wherein the patient has a recurrent, unresectable, or surgically resectable giant cell tumor of bone that could cause serious morbidity. 119. Use of the preparation of any one of claims 1 to 52 and 107 in the preparation of a medicament for treating malignant hypercalcemia in patients in need. 120. The use as described in claim 119, wherein the malignancy is refractory to bisphosphonate therapy. 121. The use as described in claim 119 or 120, wherein the drug comprises an amount of the formulation that effectively reduces or maintains the patient’s serum calcium at a level of 11.5 mg/dL or less. 122. The use as described in any one of claims 119 to 121, wherein the preparation comprises the human anti-RANKL antibody at a concentration of 120 mg/mL. 123. The use as described in any one of claims 111 to 122, wherein the drug is prepared for administration on a schedule of once every four weeks. 124. The use as described in any one of claims 111 to 123, wherein the drug is prepared for administration on days 8 and 15 of the first month of the therapy. 125. Use of the preparation of any one of claims 1 to 52 and 107 in the preparation of a medicament for treating osteoporosis in patients in need. 126. The use as described in claim 125, wherein the patient is a postmenopausal woman at high risk of fracture. 127. The use as described in claim 126, wherein the patient is a male at high risk of fracture. 128. Use of the preparation of the formulation according to any one of claims 1 to 52 and 107 in the preparation of a medicament for increasing bone mass in patients in need. 129. The use as described in claim 128, wherein the patient has osteoporosis, optionally wherein the patient is a male with osteoporosis at high risk of fracture. 130. The use as described in claim 129, wherein the patient is a woman at high risk of fracture who is receiving adjuvant aromatase inhibitor therapy for breast cancer. 131. The use as described in claim 129, wherein the patient is a male at high risk of fracture who is receiving androgen deprivation therapy for non-metastatic prostate cancer. 132. The use as described in any one of claims 128 to 131, wherein the drug comprises an amount of the formulation that effectively reduces the incidence of new vertebral fractures and/or non-vertebral fractures. 133. The use as described in any one of claims 128 to 132, wherein the medicament comprises an amount of the formulation that effectively reduces bone resorption. 134. The use as claimed in any one of claims 128 to 133, wherein the drug comprises the formulation which effectively increases the bone mineral density of the patient in at least one region selected from the lumbar spine, the entire hip, and the femoral neck. 135. The use as claimed in any one of claims 128 to 134, wherein the drug comprises the formulation which effectively increases the amount of bone mass in the cortical bone and/or cancellous bone of the patient. 136. The use according to any one of claims 128 to 135, wherein the medicament comprises the formulation which effectively reduces the amount of the bone resorption marker serum type 1 C-terminal peptide (CTX). 137. The use as described in any one of claims 128 to 136, wherein the drug is prepared for administration on a schedule of once every six months. 138. The use as claimed in any one of claims 128 to 137, wherein the drug comprises the preparation in a volume of 1 mL or less. 139. The use as described in any one of claims 111 to 138, wherein the drug is prepared for subcutaneous administration. 140. The use as described in claim 139, wherein the drug is prepared for subcutaneous administration to the upper arm, thigh, or abdomen. 141. The use as described in any one of claims 111 to 140, wherein the patient receives one or both of calcium and vitamin D. 142. A method for improving the stability of an aqueous pharmaceutical preparation comprising a human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety, comprising: Prepare the aqueous pharmaceutical formulation comprising the human anti-human nuclear factor κ-B receptor activator ligand (anti-RANKL) monoclonal antibody or its antigen-binding moiety and an amino acid aggregation inhibitor comprising phenylalanine or tryptophan. Compared to an equivalent aqueous pharmaceutical formulation without the amino acid aggregation inhibitor, this aqueous pharmaceutical formulation exhibits improved stability in the presence of the amino acid aggregation inhibitor. The anti-RANKL monoclonal antibody or its antigen-binding portion comprises (A) a light chain variable domain comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO:7; and (B) a heavy chain variable domain comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO:8, a heavy chain variable domain comprising a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:10.