HK40017180A - Formulations of human anti-rankl antibodies, and methods of using the same - Google Patents

Description

Formulations of human anti-RANKL antibodies and methods of use thereof

Cross Reference to Related Applications

Us provisional patent application No. 62/492,056, filed 2017, 4, 28, c. § 119(e), claim 7, and the disclosure of which is hereby incorporated by reference herein.

Incorporation of electronically submitted material by reference

Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing filed concurrently herewith and identified as follows: a 49 kilobyte ASCII (text) file named "51689 a _ seqlistingtxt"; created on 2018, month 4 and day 20.

Background

Technical Field

The present invention relates to human anti-RANKL monoclonal antibodies, including high concentration aqueous formulations of denosumab and its biological analogs (biosimilar).

Prior Art

Dinosuzumab is commercially available in the form of a solution at 60mg/mL and 70mg/mL strength.

Increased concentrations of protein formulations can cause stability problems, for example, leading to the formation of aggregates of High Molecular Weight Species (HMWS). In some protein formulations, HMWS may be of particular interest, especially those that retain most of the native configuration of the monomeric counterpart. Aggregation may also affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

The filling and finishing operations and administration may involve the step of flowing the protein solution through a piston pump, peristaltic pump, or injection needle. This process may impart shear and mechanical stress, which may cause denaturation of the protein and aggregation. This phenomenon may be exacerbated when the protein solution is concentrated.

Disclosure of Invention

Provided according to the present invention is a disclosure demonstrating for the first time that the addition of an amino acid aggregation inhibitor to an aqueous solution comprising a high concentration of an anti-RANKL antibody may result in a reduction of the amount of antibody aggregates formed over time and a slowing of the rate of formation of such aggregates. The present disclosure also provides the effect of pH on aggregate formation in concentrated aqueous solutions of anti-RANKL antibodies, wherein a reduction in aggregate formation is observed when the pH of the aqueous solution is in the range of about 5.0 to less than 5.2. The disclosure provided herein further suggests that the anti-RANKL antibody is stabilized by the interaction between the amino acid aggregation inhibitor and the antibody. Without being bound by any particular theory, it is envisaged 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 the concentrated antibody solution. Accordingly, the present disclosure relates to stable aqueous pharmaceutical formulations comprising high concentrations of anti-RANKL antibody, which formulations comprise low amounts (e.g., less than about 2%) of aggregates.

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

Another aspect of the 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 antigen-binding portion thereof and an amino acid aggregation inhibitor. In exemplary aspects, the amino acid aggregation inhibitor comprises an amino acid containing a charged side chain, an aromatic amino acid, or a hydrophobic amino acid. In exemplary cases, the amino acid comprising a charged side chain is an amino acid comprising a positively charged side chain, such as arginine and lysine, for example. In exemplary aspects, the aromatic amino acid comprises phenyl orIndole. Optionally, the aromatic amino acid further comprises a C between the alpha carbon and the phenyl or indole₁-C₆An alkyl chain. 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 of greater than about 2.5 on the ktte-dolite hydrophobicity scale (Kyte and Doolittle hydropathicity scale). Optionally, the hydrophobic amino acid is valine, leucine, or isoleucine. Other amino acid aggregation inhibitors as described herein are contemplated.

In exemplary cases, the aqueous pharmaceutical formulation further comprises a tonicity modifier, a surfactant, a buffer, or any combination thereof.

Another aspect of the present disclosure is the presentation of the formulation for storage or use, for example, in a single use vial, a single use syringe, or a glass, glass-lined or glass-coated container. Exemplary aspects of the present disclosure are containers, optionally vials, pre-filled syringes (PFS), or glass containers, comprising any of the aqueous pharmaceutical formulations described herein. In exemplary cases, the container contains about 1mL or less (e.g., about 0.5mL) of the aqueous pharmaceutical formulation.

Another aspect of the present disclosure provides a method of making a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof, comprising combining the anti-RANKL monoclonal antibody or antigen-binding portion thereof at a concentration of greater than 70mg/mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. Aspects of the present disclosure include a stable aqueous pharmaceutical formulation made according to any of the methods of making a stable aqueous pharmaceutical formulation described herein.

Another aspect of the disclosure provides methods of preventing or treating diseases responsive to human anti-RANKL monoclonal antibodies or antigen-binding portions thereof using formulations as described herein. In exemplary aspects, the use encompasses therapeutic treatment of a subject, the therapeutic treatment encompassing treating or preventing a bone-related event (SRE), 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 encompasses (a) treating or preventing SRE in a subject having solid tumor bone metastases; (b) treating or preventing SRE in an adult or mature bone juvenile subject having unresectable or surgically resected giant cell tumors that are likely to cause severe morbidity; (c) treating a bisphosphonate refractory malignant hypercalcemia in a subject; (d) treating or preventing SRE in a subject having multiple myeloma or solid tumor bone metastasis; (e) treating osteoporosis in postmenopausal women at high risk of fracture; (f) a treatment that increases bone mass in women at high fracture risk receiving adjuvant aromatase inhibitor therapy for breast cancer; (g) a treatment that increases bone mass in men at high fracture risk receiving androgen deprivation therapy for non-metastatic prostate cancer; (h) a treatment that increases bone mass in men with osteoporosis at high risk of fracture; (i) therapy with calcium or vitamin D.

Other aspects of the disclosure include methods of preventing bone related events (SREs) in a patient in need thereof, methods of treating giant cell tumors of bone in a patient in need thereof, methods of treating malignant hypercalcemia in a patient in need thereof, methods of treating osteoporosis in a patient in need thereof, and methods of increasing bone mass in a patient in need thereof. The methods comprise administering to the patient an effective amount of any of the formulations described herein. In exemplary cases, the formulation is delivered subcutaneously to the patient.

Another aspect of the disclosure provides the use of dinotefuran or another human anti-RANKL monoclonal antibody or an antigen-binding portion thereof for the manufacture of a medicament as described herein to treat a patient in need of a human anti-RANKL monoclonal antibody.

Another aspect of the disclosure is a kit comprising a composition or article of manufacture described herein, and a package insert, package label, instructions, or other indicia directing or disclosing any of the methods or embodiments disclosed herein.

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

Another aspect of the present disclosure is a method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof, the method comprising the step of preparing an aqueous pharmaceutical formulation comprising a mixture of the human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof 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 the disclosure is a method of reducing the level of HMWS aggregates in a solution of dinoteumab or another human anti-RANKL monoclonal antibody.

Other aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. While these compositions, articles, and methods are susceptible of embodiment in various forms, the following description includes specific embodiments with the understanding that the present disclosure is illustrative, and is not intended to limit the invention to the specific embodiments described herein. With respect to the compositions, articles, and methods described herein, it is contemplated that optional features, including but not limited to components, compositional ranges thereof, substituents, conditions, and steps, are selected from the various aspects, embodiments, and examples provided herein.

Drawings

Fig. 1, fig. 2 and fig. 8 show the percentage of HMWS at 37 ℃ as a function of formulation and time for various high concentration formulations of dinotezumab as monitored by SE-UHPLC. The legend of figure 1 is consistent with the formulations having the abbreviations shown in table 1. The legend of fig. 8 corresponds to the letters shown in table 5.

Fig. 3 shows size exclusion chromatograms of various high concentration dinoteumab formulations after 1 month storage at 37 ℃. The legend of fig. 3 is consistent with the formulations having the abbreviations shown in table 2.

Fig. 4 is a graph of% HMWS as a function of time monitored by SE-UHPLC for each formulation with the corresponding F # shown in table 3A.

FIG. 5 is a pair of size exclusion chromatograms of the formulations listed in Table 3A. The legend of fig. 5 is consistent with the formulation names mentioned in table 3B.

Fig. 6 is a graph of% HMWS as a function of storage time at 37 ℃ as monitored by SE-UHPLC for each formulation having the corresponding F # shown in table 4A.

Fig. 7A shows a size exclusion chromatogram at pH 4.8 for formulations having the dinosumab concentrations listed in table 4A.

Fig. 7B shows a size exclusion chromatogram at pH 5.1 of a formulation having the concentration of dinosaumab listed in table 4A.

Fig. 9 is a graph of% HMWS as a function of storage time at 37 ℃ monitored by SE-UHPLC for each formulation having the corresponding F # shown in table 6B.

Figure 10 shows that the size exclusion chromatogram varies with formulation after storage of formulations with the names indicated in table 6B for 1 month at 37 ℃.

Fig. 11A is a graph of the percentage of HMWS as a function of time monitored by SE-UHPLC at 37 ℃ for formulations having the letters indicated in table 7B.

Fig. 11B is a graph of the percentage of HMWS as a function of time monitored by SE-UHPLC at 40 ℃ for formulations having the letters indicated in table 7C.

Figure 12A shows a size exclusion chromatogram of the formulation of table 7B.

Figure 12B shows a size exclusion chromatogram of the formulation of table 7C.

Fig. 13, 14 and 15 are graphs of the percentage of HMWS as a function of storage time at 37 ℃ as monitored by SE-UHPLC for each formulation with the corresponding formulation letter shown in table 8A. Fig. 16, 17, and 18 are chromatographic overlays of the formulations listed in table 8A after 1 month storage at 37 ℃. Fig. 13 and 16 relate to formulations comprising aromatic amino acids, fig. 14 and 17 relate to formulations comprising polar/charged amino acids, and fig. 15 and 18 relate to formulations comprising hydrophobic amino acids.

FIGS. 19-24 are graphs of% deuterium incorporation as a function of time (log (sec)) at 4 ℃ for light chain amino acids 28-33 (FIG. 19), light chain amino acids 108-116 (FIG. 20), light chain amino acids 125-132 (FIG. 21), heavy chain amino acids 47-59 (FIG. 22), heavy chain amino acids 243-253 (FIG. 23), and heavy chain amino acids 392-399 (FIG. 24) for each of formulations 35-38.

FIGS. 25-30 are graphs of% deuterium incorporation as a function of time (log (sec)) at 37 ℃ for light chain amino acids 28-33 (FIG. 25), light chain amino acids 108-117 (FIG. 26), light chain amino acids 124-131 (FIG. 27), heavy chain amino acids 47-59 (FIG. 28), heavy chain amino acids 242-253 (FIG. 29), and heavy chain amino acids 392-399 (FIG. 30) for each of formulations 35-38.

Fig. 31 is a graph of the percentage of HMWS as a function of formulation and time monitored by SE-UHPLC at 37 ℃ for formulations having the formulation names indicated in table 10.

Figure 32 is a graph of the percentage of LMWS as a function of formulation and time at 37 ℃ as monitored by SE-UHPLC for formulations having the formulation names indicated in table 11.

Fig. 33 is a graph of the percentage of HMWS monitored by SE-UHPLC at 37 ℃ as a function of formulation and time for formulations having the formulation designations indicated in table 12.

Figure 34 is a graph of the percentage LMWS as a function of formulation and time at 37 ℃ as monitored by SE-UHPLC for formulations having the formulation names indicated in table 13.

Fig. 35 is a graph of the percentage of HMWS as a function of formulation and time monitored by SE-UHPLC at 37 ℃ for formulations having the formulation names indicated in table 14.

Figure 36 is a graph of the percentage LMWS as a function of formulation and time at 37 ℃ as monitored by SE-UHPLC for formulations having the formulation names indicated in table 15.

Figure 37 is a size exclusion chromatography overlay for each formulation with the formulation name indicated in table 10.

Figure 38 is a size exclusion chromatography overlay for each formulation with the formulation name indicated in table 12.

Figure 39 is a size exclusion chromatography overlay for each formulation with the formulation name indicated in table 14.

Fig. 40A and 40B are graphs of isothermal chemical denaturation curves at pH 4.5, pH 4.8, and pH 5.0 with dinoteuzumab in the absence of arginine. Fig. 40A is a graph of denatured dinotezumab fraction as a function of denaturant concentration. FIG. 40B is a graph plotting dF/d [ denaturant ] as a function of denaturant concentration.

Fig. 41A and 41B are graphs of isothermal chemical denaturation curves at pH 4.5, pH 4.8, and pH5.2 containing dinoteuzumab in the presence of 75mM arginine hydrochloride. Fig. 41A is a graph of denatured dinotezumab fraction as a function of denaturant concentration. FIG. 41B is a graph plotting dF/d [ denaturant ] as a function of denaturant concentration.

Fig. 42 and 43 are graphs of the percentage of HMWS monitored by SE-UHPLC as a function of time at 25 ℃ over 3 months and at 37 ℃ over 2 months, respectively, for formulations having the formulation names in table 17.

Detailed Description

It is desirable to provide a more concentrated aqueous solution of dinoteumab and other human anti-RANKL antibodies and antigen-binding portions thereof that is as stable as or more stable than dilute solutions. A more concentrated solution may provide convenience to the patient, for example by allowing a smaller volume, such as 1mL of injection, to deliver 120mg of active agent (such as dinotefuran), rather than 1.7mL or 2mL of injection of a more dilute active formulation. Furthermore, an even smaller volume of injection solution would be allowed to deliver a lower dose of active agent, e.g., 0.5mL of 120mg/mL concentration dinotezumab to deliver a 60mg dose. It is also desirable to provide more stable aqueous solutions of dinotefuran and other human anti-RANKL antibodies and antigen-binding portions thereof than previously known solutions. The stable concentrated formulation will also have other benefits, such as allowing for handling and shipping of smaller volumes of product, and allowing for longer product shelf life.

Aggregates in biological products can vary in origin, size, and type. Of particular interest are aggregates that may affect the efficacy or safety of biological products, such as aggregates that may enhance immune responses and cause adverse clinical effects. Of particular interest may be high molecular weight aggregates, also known as High Molecular Weight Species (HMWS), especially those that retain most of the native configuration of the monomeric counterpart. Aggregation may also affect the subcutaneous bioavailability and pharmacokinetics of therapeutic proteins.

There may be various causes for aggregate formation. In general, protein aggregation leads to conformational instability (which is a result of changes in protein structure) and colloidal instability (which is controlled by intermolecular forces). Where a critical nucleation event is required to induce precipitation, protein aggregation kinetics are characterized by the inclusion of a time lag phase.

Aggregation due to configurational instability involves unfolding and association steps. Unfolding of the protein molecule exposes hydrophobic amino acid residues. Hydrophobic residues of unfolded molecules may then undergo association, thereby leading to aggregation (e.g., in the form of dimers, trimers, other polymers, and higher aggregates). This association is concentration dependent. An increase in protein concentration in the aqueous solvent generally increases the rate and extent of aggregation, including heat-induced aggregation. Thus, additives in solution that affect the free energy of protein unfolding may affect conformational stability.

Colloidal instability creates aggregates via protein-protein intramolecular association forces. Such forces may be affected by one or more factors including ionic strength, solution pH, and buffer type.

Dinosuzumab is commercially available in the form of a solution at 60mg/mL and 70mg/mL strength. Attempts to formulate higher concentration solutions of dinosaumab using the same excipients showed that this higher concentration affected product stability through the concomitant and proportional increase in HMWS. For example, the concentration of 120mg/mL dinotezumab has a concentration 70% higher than 70mg/mL dinotezumab and is double the concentration of 60 mg/mL.

Thus, a stable aqueous formulation according to the present disclosure will resist aggregate formation to a greater extent than previously known formulations. One aspect of the present disclosure is a stable aqueous formulation characterized by a pH of 5.0 to less than 5.2. Another non-exclusive aspect of the present disclosure is a stable aqueous formulation comprising an amino acid aggregation inhibitor. Related dosage presentation forms (e.g., in single use vials, syringes, and glass containers) and related methods of treatment are also provided. Methods of making stable aqueous pharmaceutical formulations are also provided.

As described below, pH and amino acid aggregation inhibitors (e.g., arginine-arginine dipeptide, arginine-phenylalanine dipeptide) are two pathways that have been shown to reduce HMWS levels and HMWS formation rates of deambuzumab at 120 mg/mL. HMWS can be described as irreversible (e.g., covalent) or reversible (e.g., non-covalent self-associating 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 basic amino acid highly charged at neutral to acidic pH) can interfere with charged protein intermolecular forces. Without intending to be bound by any particular theory, it is contemplated that HMWS formation of dinotezumab at 120mg/mL is based on protein charge, and that these formulation changes are destroying the charge forces involved in the HMWS formation mechanism. Furthermore, without intending to be bound by any particular theory, it is contemplated that hydrophobic protein self-association interactions may also be present in HMWS formation, as arginine contains short aliphatic chains in the side chains. This aliphatic chain may disrupt hydrophobic interactions between proteins. Inclusion of phenylalanine in the formulation to additionally reduce HMWS levels further supports this concept. Without being bound by any particular theory, arginine stabilizes the anti-RANKL antibody differently than phenylalanine, such that if arginine interacts with the antibody via hydrophobic interactions, arginine may interact with the antibody in one or more other ways.

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

Unless otherwise indicated, it is contemplated that the formulations, dosage-presentation forms, and methods include embodiments comprising any combination of one or more of the other optional elements, features, and steps (including those shown in the figures) described further below.

In jurisdictions where patenting methods practiced in humans is prohibited, the meaning of "administering" a composition to a human subject should be limited to specifying a controlled substance that the human subject will self-administer by any technique (e.g., oral, inhalation, topical administration, injection, insertion, etc.). The broadest reasonable interpretation is intended that is consistent with the law or law that defines the subject matter of the available patents. In the jurisdiction where patenting of methods for administration to a human is not prohibited, "administering" a composition includes both methods for administration to a human and the activities described above.

As used herein, the term "comprising" indicates that other agents, elements, steps or features may be included in addition to the stated items.

It should be understood that each maximum numerical limitation provided throughout this specification includes ranges formed with each corresponding lower numerical limitation, as if such ranges were expressly written. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such ranges were expressly written. Every numerical range provided throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if all such narrower numerical ranges were expressly written herein. Dimensions and values disclosed herein are to be understood to include the values recited by the disclosure and the corresponding exact numerical values, for example, a value described as "about 10 mM" is to be understood to include "10 mM" as an alternative disclosure.

The term "therapeutically effective amount" as used herein refers to an amount of a compound sufficient to treat, ameliorate or prevent an identified disease or condition, or to exhibit a detectable therapeutic, prophylactic or inhibitory effect. The effect can be detected, for example, by an improvement in clinical condition or a reduction in symptoms. The precise effective amount of a subject will depend upon the weight, size and health of the subject, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. Where a drug has been approved by the U.S. Food and Drug Administration (FDA), a "therapeutically effective amount" refers to a dose approved by the FDA or its foreign agency for the treatment of an identified disease or condition.

The present disclosure provides stable aqueous pharmaceutical formulations as shown by a reduction in the amount of aggregates and/or a reduction in the rate of aggregate formation upon storage. As described herein, the stability of such formulations is evidenced by a reduction in the amount of HMWS and/or a reduction in the rate of HMWS formation over various periods of time and upon storage at various temperatures. In general, higher stability formulations are associated with the presence of lower amounts of HMWS, lower rates of HMWS formation, and/or higher antibody main peaks at higher storage temperatures relative to lower temperatures. As used herein, the term "high molecular weight species" or "HMWS" refers to both high-order aggregates of antibody of a formulation as well as low-order aggregates of antibody of a formulation. Lower aggregates include, for example, dimer species. Aggregate amounts and rates of formation can be measured or monitored by a number of techniques, such as SE-UHPLC. In some cases, the SE-UHPLC chromatogram of the antibody shows a peak at about 5.8 minutes representing the amount of HMWS of the aqueous pharmaceutical formulation, a peak at about 6.7 minutes representing the dimer species, and a peak at about 8.0 minutes representing the amount of the intact unaggregated form of the antibody. Storage at 37 ℃ allows for accelerated stability analysis relative to storage at 4 ℃, so that the stability of a particular formulation can be determined in a shorter period of time relative to storage at 4 ℃. For example, storage at 37 ℃ for 1 month, 2 months, or 3 months may indicate or predict storage at 4 ℃ for 36 months.

In one type of embodiment, a stabilized formulation as described herein will exhibit a reduced degree and rate of HMWS formation after 3 months of storage at 37 ℃ as compared to an isoconcentrate control formulation consisting of 10mM acetate, 5% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 as excipients and having a solution pH of 5.2.

In another type of embodiment, a stabilized formulation as described herein and including an amino acid aggregation inhibitor will exhibit a reduced degree of HWMS formation upon storage at 37 ℃ for 1 month as compared to an equivalent control formulation that does not contain the amino acid aggregation inhibitor. For example, the degree of formation may be reduced compared to a control formulation after 1 month of storage at 37 ℃ to such an extent that the% amount of HMWS as measured by SE-UPHLC is reduced by 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, a stabilized formulation as described herein will have a lower amount of HMWS (as measured by SE-UHPLC) after 1 month of storage at 37 ℃. For example, the amount of HMWS may be in the range of no more than 2% or less than 2%, or no more than 1.9% or less than 1.9%, or no more than 1.8% or less than 1.8%, or no more than 1.7% or less than 1.7%, or no more than 1.6% or less than 1.6%, or no more than 1.5% or less than 1.5%, or no more than 1.4% or less than 1.4%, or no more than 1.3% or less than 1.3%, or no more than 1.2% or less than 1.2%, for example from about 0.01% to about 2%, or from about 0.01% to about 1.9%, or from about 0.01% to about 1.8%, or from about 0.01% to about 1.7%, or from about 0.01% to about 1.6%, or from about 0.01% to about 1.5%, or from about 0.01% to about 1.4%, or from about 0.01% to about 1.3%, or from about 0.01% to about 1.2%. In another type of embodiment, the amount of HMWS monitored by SE-UHPLC after 1 month of storage at 37 ℃ may exceed 2%, such as exceed 2% and up to 3%, while the reduced aggregation rate provided by the amino acid aggregation inhibitor will allow for a suitable shelf life of the product, such as up to three years or up to two years.

In another type of embodiment, a stabilized formulation as described herein will have a lower amount of HMWS (as measured by SE-UHPLC) after 3 months of storage at 37 ℃. For example, the amount of HMWS may be in the range of no more than 2% or less than 2%, or no more than 1.9% or less than 1.9%, or no more than 1.8% or less than 1.8%, or no more than 1.7% or less than 1.7%, or no more than 1.6% or less than 1.6%, or no more than 1.5% or less than 1.5%, or no more than 1.4% or less than 1.4%, or no more than 1.3% or less than 1.3%, or no more than 1.2% or less than 1.2%, for example from about 0.01% to about 2%, or from about 0.01% to about 1.9%, or from about 0.01% to about 1.8%, or from about 0.01% to about 1.7%, or from about 0.01% to about 1.6%, or from about 0.01% to about 1.5%, or from about 0.01% to about 1.4%, or from about 0.01% to about 1.3%, or from about 0.01% to about 1.2%.

In another type of embodiment, a stabilized formulation as described herein will have a lower amount of HMWS (as measured by SE-UHPLC) after 36 months of storage at 4 ℃. For example, the amount of HMWS may be in the range of no more than 2% or less than 2%, or no more than 1.9% or less than 1.9%, or no more than 1.8% or less than 1.8%, or no more than 1.7% or less than 1.7%, or no more than 1.6% or less than 1.6%, or no more than 1.5% or less than 1.5%, or no more than 1.4% or less than 1.4%, or no more than 1.3% or less than 1.3%, or no more than 1.2% or less than 1.2%, for example from about 0.01% to about 2%, or from about 0.01% to about 1.9%, or from about 0.01% to about 1.8%, or from about 0.01% to about 1.7%, or from about 0.01% to about 1.6%, or from about 0.01% to about 1.5%, or from about 0.01% to about 1.4%, or from about 0.01% to about 1.3%, or from about 0.01% to about 1.2%.

In another type of embodiment, a stabilized formulation as described herein will have a higher amount of the major peak of dinotezumab or other antibody (or antigen-binding portion thereof) after 1 month storage at 37 ℃ (as measured by SE-UHPLC). For example, the amount of the major peak may be at least 95% or more than 95%, or at least 96% or more than 96%, or at least 97% or more than 97%, or at least 97.5% or more than 97.5%, or at least 98% or more than 98%, or at least 98.1% or more than 98.1%, or at least 98.2% or more than 98.2%, or at least 98.3% or more than 98.3%, or at least 98.4% or more than 98.4%, or at least 98.5% or more than 98.5%, or at least 98.6% or more than 98.6%, for example, 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 98.1% to about 99.9%, or about 98%, or about 98.6%, such as about 95% to about 99.9%, or more than 98.9%.

In another type of embodiment, a stabilized formulation as described herein will have a higher amount of the major peak of dinotezumab or other antibody (or antigen-binding portion thereof) after 3 months of storage at 37 ℃ (as measured by SE-UHPLC). For example, the amount of the major peak may be at least 95% or more than 95%, or at least 96% or more than 96%, or at least 97% or more than 97%, or at least 97.5% or more than 97.5%, or at least 98% or more than 98%, or at least 98.1% or more than 98.1%, or at least 98.2% or more than 98.2%, or at least 98.3% or more than 98.3%, or at least 98.4% or more than 98.4%, or at least 98.5% or more than 98.5%, or at least 98.6% or more than 98.6%, for example, 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 98.1% to about 99.9%, or about 98%, or about 98.6%, such as about 95% to about 99.9%, or more than 98.9%.

In another type of embodiment, a stabilized formulation as described herein will have a higher amount of the major peak of dinotezumab or other antibody (or antigen-binding portion thereof) after 36 months of storage at 4 ℃ (as measured by SE-UHPLC). For example, the amount of the major peak may be at least 95% or more than 95%, or at least 96% or more than 96%, or at least 97% or more than 97%, or at least 97.5% or more than 97.5%, or at least 98% or more than 98%, or at least 98.1% or more than 98.1%, or at least 98.2% or more than 98.2%, or at least 98.3% or more than 98.3%, or at least 98.4% or more than 98.4%, or at least 98.5% or more than 98.5%, or at least 98.6% or more than 98.6%, for example, 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 98.1% to about 99.9%, or about 98%, or about 98.6%, such as about 95% to about 99.9%, or more than 98.9%.

In other embodiments, it is contemplated that a stabilized formulation will have a lower amount of HMWS and a higher amount of the main peak upon storage according to the above-described regulations.

In exemplary aspects, the aqueous pharmaceutical formulation comprises no more than about 4% High Molecular Weight Substance (HMWS) and/or comprises more than about 96% of the major peak of the antibody after storage, as measured by SE-UHPLC. In exemplary aspects, the aqueous pharmaceutical formulation comprises no more than about 3% High Molecular Weight Substance (HMWS) and/or comprises more than about 97% of the main peak of the antibody after storage, as measured by SE-UHPLC. In exemplary aspects, the aqueous pharmaceutical formulation comprises less than about 2% HMWS and/or more than about 98% of the major antibody peak after storage as measured by SE-UHPLC. In exemplary aspects, the storage is at a temperature of about 2 ℃ to about 8 ℃ (e.g., about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃) 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 exemplary aspects, the storage is stored at about 20 ℃ to about 30 ℃ (e.g., about 21 ℃ to about 30 ℃, about 22 ℃ to about 30 ℃, about 23 ℃ to about 30 ℃, about 24 ℃ to about 30 ℃, about 25 ℃ to about 30 ℃, about 26 ℃ to about 30 ℃, about 27 ℃ to about 30 ℃, about 28 ℃ to about 30 ℃, about 20 ℃ to about 29 ℃, about 20 ℃ to about 28 ℃, about 20 ℃ to about 27 ℃, about 20 ℃ to about 26 ℃, about 20 ℃ to about 25 ℃, about 20 ℃ to about 24 ℃, about 20 ℃ to about 23 ℃, about 20 ℃ to about 22 ℃) for about 1 month (e.g., about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days). In exemplary aspects, the storing comprises a first storing followed by a second storing, and the first storing is at about 2 ℃ to about 8 ℃ for at least 12 months, 24 months, or 36 months, and the second storing is at about 20 ℃ to about 30 ℃ for about 1 month. In exemplary cases, the aqueous pharmaceutical formulation comprises no more than 2% HMWS or less than 2% HMWS, or no more than 1.9% HMWS or less than 1.9% HMWS, or no more than 1.8% HMWS or less than 1.8% HMWS, or no more than 1.7% HMWS or less than 1.7% HMWS, or no more than 1.6% HMWS or less than 1.6% HMWS, or no more than 1.5% HMWS or less than 1.5% HMWS, or no more than 1.4% HMWS or less than 1.4% HMWS, or no more than 1.3% HMWS or less than 1.3% HMWS, or no 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 the uhse-UHPLC. In alternative or other aspects, the aqueous pharmaceutical formulation comprises more than 98% of the antibody major peak, or at least 95% or more than 95% of the antibody major peak, or at least 96% or more than 96% of the antibody major peak, or at least 97% or more than 97% of the antibody major peak, or at least 97.5% or more than 97.5% of the antibody major peak, or at least 98% or more than 98% of the antibody major peak, or at least 98.1% or more than 98.1% of the antibody major peak, or at least 98.2% or more than 98.2% of the antibody major peak, or at least 98.3% or more than 98.3% of the antibody major peak, or at least 98.4% or more than 98.4% of the antibody major peak, or at least 98.5% or more than 98.5% of the antibody major peak, or at least 98.6% or more than 98.6% of the antibody major peak, for example optionally at about 95.99% to about 9.99% of the antibody major peak, Or about 96% to about 99.9% antibody major peak, or about 97% to about 99.9% antibody major peak, or about 97.5% to about 99.9% antibody major peak, or about 98% to about 99.9% antibody major peak, or about 98.1% to about 99.9% antibody major peak, or about 98.2% to about 99.9% antibody major peak, or about 98.3% to about 99.9% antibody major peak, or about 98.4% to about 99.9% antibody major peak, or about 98.5% to about 99.9% antibody major peak, or about 98.6% to about 99.9% antibody major peak, as measured by SE-UHPLC.

As used herein, the term "antibody" refers to a protein having the conventional immunoglobulin form, comprising heavy and light chains, and comprising variable and constant regions. For example, the antibody may be an IgG, which is a "Y-shaped" structure of two pairs of identical polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region is typically about 100-110 or more amino acids, comprises three Complementarity Determining Regions (CDRs), is primarily responsible for antigen recognition, and is very different from other antibodies that bind different antigens. See, e.g., Janeway et al, "Structure of The Antibody molecules and immunoglobulin Genes" [ Structure of Antibody molecules and immunoglobulin Genes ], Immunobiology: The immunoglobulin System in Health and Disease [ Immunobiology: immune system of health and disease ], 4 th edition, einwei Science Ltd (Elsevier Science Ltd.)/garland press (garland publishing), (1999).

Briefly, in an antibody scaffold, CDRs are embedded within a framework in the heavy and light chain variable regions, where they constitute the regions primarily responsible for antigen binding and recognition. The variable region comprises at least three heavy chain CDRs or three light chain CDRs (Kabat et al, 1991, Sequences of proteins of immunological Interest [ immune-related protein Sequences ], Public Health Service [ Public Health agency ] N.I.H., Besserda, Md.; also see Chothia and Lesk,1987, J.mol.biol. [ J. Mobiol. ]196: 901-917; Chothia et al, 1989, Nature [ Nature ]342:877-883), located within the framework region (framework regions 1-4, FR1, FR2, FR3, and FR4 are designated by Kabat et al, 1991; also see Chothia and Lesk,1987, supra).

Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α or ε, and the 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 IgG 4. IgM has subclasses, including but not limited to IgM1 and IgM 2. Embodiments of the present disclosure include all such antibody classes or isotypes. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, such as a human kappa-type or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. Thus, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any of IgG1, IgG2, IgG3, or IgG 4. In exemplary aspects, the anti-RANKL antibody is an IgG1, IgG2, or IgG4 antibody.

In various aspects, the antibody can be a monoclonal antibody or a polyclonal antibody. In some aspects, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, e.g., a mouse, rat, rabbit, goat, horse, chicken, hamster, pig, human, and the like. In this regard, the antibody may be considered a mammalian antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a pig antibody, a human antibody, and the like. In certain aspects, the anti-RANKL antibody is a monoclonal human antibody. In certain aspects, the recombinant protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains constant domains from one species and variable domains from a second species, or more generally, amino acid sequence segments from at least two species. The term "humanized" when used with respect to an antibody refers to an antibody having at least CDR regions from non-human origin engineered to have a structure and immunological function more similar to that of a human antibody of origin than the antibody of origin. For example, humanization may involve grafting CDRs from a non-human antibody (e.g., a mouse antibody) into a human antibody. Humanization may also involve selecting amino acid substitutions to make non-human sequences appear more similar to human sequences.

In various aspects, the antibody is cleaved into fragments by enzymes, such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to produce F (ab')₂Fragment and pFc' fragment. In exemplary aspects, the aqueous pharmaceutical formulation comprises an antibody fragment that retains at least one antigen (RANKL) binding site, e.g., Fab, Fc, F (ab')₂Or pFC'. With respect to the aqueous pharmaceutical formulations and methods of the present disclosure, the antibody may lack certain portions of the antibody and may be an antibody fragment that binds to RANKL. In exemplary aspects, the antibody fragment is an antigen-binding portion of an anti-RANKL antibody.

The antibody protein product may be an antigen-binding form based on antibody fragments that retain full antigen-binding capacity, such as scFv, Fab and VHH/VH. The smallest antigen-binding fragment that retains its entire antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. The V region is either linked to an scFv fragment (variable single chain fragment) using a soluble flexible amino acid peptide linker to stabilize the molecule, or a constant (C) domain is added to the V region to produce a Fab fragment [ antigen binding fragment ]. Both scFv and Fab are widely used fragments, both of which can be readily produced in a host, e.g., a prokaryotic host. Other antibody protein products include disulfide stabilized scFv (ds-scFv), single chain fab (scfab), and dimeric and multimeric antibody formats, such as bifunctional, trifunctional, and tetrafunctional antibodies, or different formats of miniantibodies (miniAb) comprising scFv linked to an oligomerizing domain. The smallest fragments are the camelid heavy chain Ab and VHH/VH of the single domain Ab (sdab). The most commonly used building blocks for the novel antibody format are single chain variable (V) domain antibody fragments (scFv), which comprise V domains (VH and VL domains) from heavy and light chains connected by a peptide linker of about 15 amino acid residues. A peptide antibody or peptide-Fc fusion is another antibody protein product. The structure of the peptide antibody consists of a biologically active peptide grafted onto an Fc domain. Peptide antibodies are well described in the art. See, e.g., Shimamoto et al, mAbs 4(5): 586-.

Other antibody protein products include Single Chain Antibodies (SCAs), bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, bispecific or trispecific antibodies, and the like. Bispecific antibodies can be divided into five main classes: BsIgG, additional IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al, Molecular Immunology 67(2) part A: 97-106 (2015).

In exemplary aspects, the anti-RANKL antibody, or antigen-binding portion thereof, comprises, consists essentially of, or consists of these antibody protein products (e.g., scFv, Fab VHH/VH, Fv fragments, ds-scFv, scFab, diabody, polyiody (e.g., diabody, triabody, tetrabody), minibody, peptide antibody VHH/VH of camelid heavy chain antibody, sdAb, diabody, triabody, tetrabody, bispecific or trispecific antibody, bsig, episgg, BsAb fragment, bispecific fusion protein, and BsAb conjugate).

In exemplary aspects, the anti-RANKL antibody, or antigen-binding portion thereof, comprises, consists essentially of, or consists of an antibody protein product in monomeric form or in polymeric, oligomeric, or polymeric form. In certain embodiments where an 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 epitopes recognized and bound by the antibody.

The human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) antibody or antigen-binding portion thereof used in the formulation is an antibody or antigen-binding portion thereof that specifically binds to human RANKL protein or human Osteoprotegerin (OPGL) protein or a fragment 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 antigen-binding portion thereof. For example, the formulations 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 portions 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 the formation, function and survival of osteoclasts. For example, human anti-RANKL antibodies inhibit the interaction of RANKL with its receptor RANK.

An example of a human anti-RANKL monoclonal antibody is dinotefuran, which is used asAndsold in a commercially available form.A 120mg dose formulation of dinosaumab in 1.7mL solution (70mg/mL) in a single use vial containing 120mg of dinosaumab, acetate (18mM), sorbitol (4.6%), water for injection (USP), and sodium hydroxide (to pH 5.2).Available as a 60mg dose formulation of dinosaumab in 1mL solution (60 mg/mL).Each 1mL single use pre-filled syringe of (a) contains 60mg of dinotezumab (60mg/mL solution), 4.7% sorbitol, 17mM acetate, 0.01% polysorbate 20, water for injection (USP), and sodium hydroxide (to ph 5.2). Formulations as described herein and including dinotezumab or a portion thereof are specifically contemplated. Dinotezumab is a fully human IgG2 monoclonal antibody that binds to human RANKL. Dinosuzumab has an approximate molecular weight of 147kDa and is expressed in a Chinese Hamster Ovary (CHO) cell line. Dinosuzumab variable Light Chain (LC) and variable heavy chain (H)C) Are shown in SEQ ID NO 1 and 2, respectively, and full-length LC and HC are shown in SEQ ID NO 3 and 4, respectively. In some aspects, the nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 (Dinovobiocin variable LC) is the nucleic acid of SEQ ID NO: 19. In some aspects, the nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 (Dinosuzumab variable HC) is the nucleic acid of SEQ ID NO: 20. In some aspects, the nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 (full length Dinosuzumab LC) is the nucleic acid of SEQ ID NO: 21. In some aspects, the nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 (full length Dinosuzumab HC) is the nucleic acid of SEQ ID NO: 23. The mature form of amino acids 21-235, denoted full-length LC, is shown as SEQ ID NO 13, while the mature form of amino acids 20-467, denoted full-length HC, is shown as SEQ ID NO 14. In some aspects, the nucleic acid comprising a 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 comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 (mature form HC) is the nucleic acid of SEQ ID NO: 24. In addition, the Dinosuzumab LC CDR is shown as SEQ ID NO:5(LC CDR1), SEQ ID NO:6(LC CDR2) and SEQ ID NO:7(LC CDR 3). Dinosuzumab HC CDRs are shown as SEQ ID NO:8(HC CDR1), SEQ ID NO:9(HC CDR2), and SEQ ID NO:10(HC CDR 3). Deanusumab and claims thereto have been described in international patent application No. WO 03/002713 and U.S. patent No. 7,364,736, the disclosures of both of which are incorporated herein by reference in their entirety.

As used herein, the term "dinotefuran" includes biological analogs of dinotefuran. As used herein, "biosimilar" (of approved reference products/biopharmaceuticals, such as protein therapeutics, antibodies, etc.) refers to a biological product that is similar to a reference product based on data derived from the following studies: (a) analytical studies showing that biological products are highly similar to reference products but with minor differences in clinically inactive components; (b) animal studies (including assessment of toxicity); and/or (c) clinical studies (including assessment of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate use conditions for which the reference product is licensed and intended for use and for which the biological product seeks license. In one embodiment, the biologically similar biological product and the reference product utilize one or more of the same mechanism of action under one or more conditions of use specified, recommended, or suggested in the proposed mark, but only to the extent that one or more mechanism of action of the reference product is known. In one embodiment, one or more conditions of use specified, recommended, or suggested in the indicia proposed for the biological product have been previously approved for use in a reference product. In one embodiment, the biologic product is administered by the same route, dosage form, and/or intensity as the reference product. In one embodiment, the mechanism that manufactures, processes, packages, or contains the biologic meets design criteria that ensure that the biologic continues to be safe, pure, and effective. The reference product may be approved within at least one of the united states, europe, or japan. A biological analog can be, for example, an antibody that has the same major amino acid sequence as a marketed antibody, but may be made in a different cell type or by a different production, purification, or formulation method.

These formulations may comprise a human anti-RANKL antibody comprising at least one of the amino acid sequences of SEQ ID NOs 1-4, 13, 14 or a portion thereof. These formulations may comprise a polypeptide comprising at least one 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 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, or 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, 6, 7, 8, 9 or 10 or at least six of the CDR amino acid sequences shown in SEQ ID NO 5, 6, 7, 8, 9 or 10.

These formulations may comprise at least one human anti-RANKL antibody comprising at least one amino acid sequence which is at least 80% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or at least one amino acid sequence which is at least 85% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or at least one amino acid sequence which is at least 90% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or at least one human anti-RANKL antibody comprising at least one amino acid sequence which is at least 91% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence which is at least 92% identical to any of SEQ ID NOs 1-4, 13 and 14 and which inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence which is at least 93% identical to any of SEQ ID NOs 1-4, 13 and 14 and which inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence which is at least 94% identical to any of SEQ ID NOs 1-4, 13 and 14 and which inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence which is at least 95% identical to any of SEQ ID NOs 1-4, 13 and 14 and which inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence which is at least 92% identical to any of SEQ ID NOs 1-4, 13 and 14 and which inhibits the interaction between RAN, 13 and 14, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 97% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 98% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK, or a human anti-RANKL antibody comprising at least one amino acid sequence that is at least 99% identical to any of SEQ ID NOs 1-4, 13 and 14 and inhibits the interaction between RANKL and its receptor RANK.

In exemplary embodiments, the aqueous pharmaceutical formulation comprises an anti-RANKL antibody or antigen-binding portion thereof, including an antibody protein product as described herein. In exemplary aspects, the anti-RANKL antibody, or antigen-binding portion thereof, comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID No. 5. Alternatively or additionally, the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID No. 6. In an alternative or other aspect, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID No. 10. In some cases, the anti-RANKL antibody, or antigen-binding portion thereof, comprises SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 10. In exemplary aspects, the anti-RANKL antibody, or antigen-binding portion thereof, comprises (i) a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID No. 7; (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO 8, optionally SEQ ID NO 27; (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO. 9; or (iv) any combination thereof. In some aspects, the anti-RANKL antibody or antigen-binding portion thereof comprises (a) a light chain variable domain comprising the light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising the light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (B) a heavy chain variable domain comprising heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 8 (optionally SEQ ID N:27), a heavy chain variable domain comprising heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 9, and a heavy chain variable domain comprising heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 10. In exemplary aspects, the anti-RANKL antibody or a thereofThe antigen-binding portion comprises: (A) a light chain variable domain selected from the group consisting of: (i) a light chain variable domain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID No. 1; (ii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO 19; and (iii) a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO. 19; or (B) a heavy chain variable domain selected from the group consisting of: (i) a heavy chain variable domain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to seq id No. 2; (ii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID No. 20; and (iii) a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO: 20; or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B). In exemplary aspects, the anti-RANKL antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In exemplary cases, the antigen binding portion is a Fab, Fab ', F (ab') 2, or single chain Fv. In exemplary aspects, the anti-RANKL antibody is an IgG₁、IgG₂Or IgG₄An 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 exemplary aspects, the anti-RANKL antibody, or antigen-binding portion thereof, comprises: (A) a light chain selected from the group consisting of: (i) comprises 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%, up to SEQ ID No. 3 or SEQ ID No. 13)98% less, at least 99%) of the same amino acid sequence; (ii) a light chain comprising an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO 21 or 23; and (iii) a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO:21 or 23; or (B) a heavy chain selected from the group consisting of: (i) a heavy chain comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID No. 4 or SEQ ID No. 14; (ii) a heavy chain comprising an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO. 22 or 24; and (iii) a heavy chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO. 22 or 24; or (C) the light chain variable domain of (A) and the heavy chain variable domain of (B).

The concentration of dinotezumab or other human anti-RANKL antibody or antigen-binding portion thereof in the aqueous formulation can generally be in any useful range, for example, in the range of about 0.1 to about 200 mg/mL. As the concentration increases, the viscosity increases, which may prevent the formulation from being processed into sterile dose presentation forms for pharmaceutical use.

In one aspect, formulations having improved stability by an amino acid aggregation inhibitor may be present at any concentration of dinotefuran or other human anti-RANKL antibody or antigen-binding portion thereof, including from about 10mg/mL to about 200mg/mL, or from about 15mg/mL to about 150mg/mL, or from about 30mg/mL to about 200mg/mL, or from about 60mg/mL to about 180mg/mL, or from about 60mg/mL to about 160mg/mL, or from about 60mg/mL to about 150mg/mL, or from about 60mg/mL to about 140mg/mL, or from about 60mg/mL to about 130mg/mL, or from about 60mg/mL to about 120mg/mL, or from about 60mg/mL to about 110mg/mL, or from about 60mg/mL to about 100mg/mL, or, Or about 60mg/mL to about 90mg/mL, or about 60mg/mL to about 80mg/mL, or about 60mg/mL to about 70mg/mL, or about 70mg/mL to about 200mg/mL, or about 70mg/mL to about 180mg/mL, or about 70mg/mL to about 160mg/mL, or about 70mg/mL to about 150mg/mL, or about 70mg/mL to about 140mg/mL, or about 70mg/mL to about 130mg/mL, or about 70mg/mL to about 120mg/mL, or about 70mg/mL to about 110mg/mL, or about 70mg/mL to about 100mg/mL, or about 70mg/mL to about 90mg/mL, or about 70mg/mL to about 80mg/mL, for example 120 mg/mL.

In another aspect, for formulations having a pH value of about 5.0 to less than 5.2, it is contemplated that the concentration of dinotefuran or other human anti-RANKL antibody or antigen-binding portion thereof comprises a range of greater than 70mg/mL, or at least 71mg/mL, or at least about 75mg/mL, or at least about 80mg/mL, or at least about 85mg/mL, or at least about 90mg/mL, or at least about 95mg/mL, or at least about 100mg/mL, or at least about 105mg/mL, or at least about 110mg/mL, or at least about 115mg/mL, or at least about 120mg/mL and up to about 200 mg/mL. For example, contemplated ranges include 71mg/mL to about 200mg/mL, or about 75mg/mL to about 180mg/mL, or about 75mg/mL to about 160mg/mL, or about 75mg/mL to about 150mg/mL, or about 75mg/mL to about 140mg/mL, or about 75mg/mL to about 130mg/mL, or about 75mg/mL to about 120mg/mL, or about 75mg/mL to about 110mg/mL, or about 75mg/mL to about 100mg/mL, or about 75mg/mL to about 90mg/mL, or about 120mg/mL to about 200mg/mL, or about 120mg/mL to about 180mg/mL, or about 120mg/mL to about 160mg/mL, or about 120mg/mL to about 140mg/mL, for example 120 mg/mL.

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

Dinosaumab and other human anti-RANKL monoclonal antibodies and antigen-binding portions thereof can be prepared according to the description provided in international patent publication WO 2003002713 a 2.

Formulation studies described below on highly concentrated solutions of dinosaumab (e.g., 120mg/mL) showed that HMWS formation (rate and extent) was greatly increased below pH5 and especially at lower pH values (e.g., pH 4.5). It has been demonstrated that the formation of dimer species increases with increasing pH. Balancing the two effects, it is contemplated that the formulations described herein will have a pH in the range of about 5.0 to less than 5.2, or about 5.0 to about 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, such as about 5.0, about 5.05, about 5.1, or about 5.15.

The studies described herein also show that by including an amino acid aggregation inhibitor it is possible to obtain independent stability and aggregation reducing effects. Thus, it is contemplated that when an amino acid aggregation inhibitor is included, the formulation pH may be in the range of about 4.9 to about 5.4, or about 5.0 to about 5.2, or about 5.0 to less than 5.2, or about 5.0 to 5.19, or about 5.0 to about 5.15, or about 5.0 to about 5.10, such as about 5.0, about 5.05, about 5.1 or about 5.15, or about 5.2.

The aqueous formulation may be buffered. When used, the buffer may be an organic buffer. The buffer system may be centered, for example, at about pH 4 to 5.5, or 4.5 to 5, at 25 ℃. For example, the buffer system may have a pKa within one pH unit of pH 5.0-5.2 at 25 ℃. One such buffer system is an acetic acid/acetate salt having a pKa of about 4.75 at 25 ℃. Another such buffer system is glutamic acid/glutamate having a pKa of about 4.27 at 25 ℃. Other alternative buffer systems envisaged include ion-based systems including succinate (pKa 4.21 at 25 ℃), propionate (pKa 4.87 at 25 ℃), malate (pKa 5.13 at 25 ℃), pyridine (pKa 5.23 at 25 ℃), and piperazine (pKa 5.33 at 25 ℃). It is envisaged that the buffer may be provided as a sodium salt (or disodium salt, as the case may be) or in the alternative as a potassium, magnesium or ammonium salt. For example, buffers may be based on acetate, citrate, succinate, phosphate, and hydroxymethyl aminomethane (Tris). Especially buffers based on acetate, glutamate and succinate, e.g. acetate or glutamate, are envisaged.

Comparison of HMWS formation by size exclusion ultra high performance liquid chromatography (SE-UHPLC) in a 120mg/mL dinosaumab formulation with acetate or glutamate buffer but otherwise equivalent showed no difference between buffer types when evaluated at 37 ℃ storage for four weeks.

When used, the buffer will be included in an amount sufficient to maintain the selected pH of the formulation under storage conditions for the shelf life of the product (e.g., 3 years at 4 ℃, or 1 month at 25 ℃, or 2 weeks at 25 ℃, or 7 days at 25 ℃). The buffer concentration may be in the range of about 2mM to about 40mM, or about 5mM to about 20mM, or about 10mM to about 25mM, or about 15mM to about 25mM, for example 10mM, or 15mM, or 18mM, or 25 mM. For example, the acetate buffer used with an anti-RANKL monoclonal antibody (e.g., dinotezumab) and phenylalanine may be in the range of about 2mM to about 30mM, or about 16mM to about 41mM, or about 25mM to about 39mM, or about 30mM to about 34 mM. In other words, the diafiltration buffer used to concentrate the antibody to a concentration greater than 70mg/mL (e.g., 120mg/mL) may be in the range of 5mM to about 30mM or about 15mM to about 25mM or at about 20 mM. It is also contemplated to provide a self-buffering amino acid-stabilized formulation. In exemplary aspects, the buffer is included in an amount sufficient to maintain the selected pH of the formulation over the shelf life of the product (e.g., 36 months at about 2 ℃ to about 8 ℃, optionally followed by about 1 month at about 20 ℃ to about 30 ℃) under storage conditions.

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

The formulation may also include one or more stabilizers against protein aggregation and other formulation excipients. It is contemplated that such stabilizers and excipients include, but are not limited to, amino acid aggregation inhibitors, tonicity modifiers, surfactants, solubilizing agents (e.g., N-methyl-2-pyrrolidone), PEG conjugates, and cyclodextrins (e.g., N-methyl-2-pyrrolidone))。

The term "amino acid aggregation inhibitor" refers to an amino acid or combination of amino acids (e.g., a mixture, or a dipeptide, or an oligopeptide having 2 to 10 residues), 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 analog, and reduces HMWS or inhibits HMWS formation. Sodium, potassium and hydrochloride salts are contemplated. Additionally, arginine salts are contemplated with hydrochlorides, glutamates, butyrates and glycolates. When a combination of amino acids is used, these amino acids may all be present in their free base form, may all be present in their salt form, or may be present in part in their free base form and others in their salt form. In addition to or in place of the dipeptides and oligopeptides, mixtures of one or more amino acids may be used, for example mixtures of arginine and phenylalanine. In alternative embodiments, only one type of amino acid aggregation inhibitor is present in the aqueous pharmaceutical formulation. In exemplary aspects, only one amino acid is present in the formulation, e.g., only L-arginine or only L-phenylalanine.

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

In another aspect, it is contemplated to use one or more amino acids having an aromatic ring in its side chain, such as phenylalanine, tyrosine, tryptophan, or a combination thereof. Phenylalanine is especially envisaged.

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

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

Amino acid analogs that exhibit aggregation reducing or inhibiting effects may also be used in the methods or formulations of the 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 derivatives 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-acetyl arginine and n-acetyl lysine are not used alone, but may be used in combination with another amino acid aggregation inhibitor. Like the amino acids, amino acid analogs are used in the methods or formulations of the invention in their free base form or in their salt form.

The amino acid aggregation inhibitors used in the methods or formulations of the present invention protect therapeutically active proteins from various stresses, thereby increasing or/and maintaining the stability of the protein or protein-containing formulation during the lifetime of the protein (before storage and during storage, before use). As used herein, the term "stress" includes, but is not limited to, heat, freezing, pH, light, agitation, oxidation, dehydration, surface, shear, freeze/thaw, pressure, heavy metals, phenolic compounds, denaturants, and the like from any source, such as transportation. Thermal stresses are particularly envisaged. The term stress encompasses any factor that modulates (i.e., decreases, maintains, or increases) the stability of a protein or protein-containing formulation. Increasing and/or maintaining stability with the addition of an amino acid aggregation inhibitor occurs in a concentration-dependent manner. That is, increasing the concentration of an amino acid aggregation inhibitor increases and/or maintains the stability of the protein or protein-containing formulation of the invention when the protein or protein-containing formulation normally exhibits aggregate formation in the absence of the amino acid aggregation inhibitor. As shown in the examples below, inclusion of an amino acid aggregation inhibitor in the formulation may also reduce the amount of HMWS that has formed. Such amino acid aggregation inhibitors include, for example, arginine and arginine-phenylalanine dipeptides. In light of the disclosure herein, the amount of a particular amino acid aggregation inhibitor used in a method or formulation of the invention to reduce aggregate formation, thereby increasing the stability of the protein and thus the stability of the formulation over the lifetime of the protein, can be readily determined for dinoteumab or any particular human anti-RANKL monoclonal antibody of interest.

The presence of amino acid aggregation inhibitors in the formulation has been shown to reduce the amount of dimer species and their rate of dynamic formation. For example, the inclusion of arginine at a concentration of 75mM in a dinosaumab formulation having a pH of 5.2 reduced the amount of dimer species and its rate of dynamic formation by approximately 0.3% and 25%, respectively, when compared to a similar formulation at pH5.2 without arginine after 1 month at 37 ℃. In contrast, it was found that monoclonal antibodies other than human anti-RANKL monoclonal antibodies were not stabilized by including arginine, but instead caused an increase in HMWS. Thus, another method of the present disclosure is a method of reducing HMWS in a formulation of dinoteumab or another human anti-RANKL monoclonal antibody by adding an amino acid aggregation inhibitor, such as arginine or phenylalanine.

Thus, in exemplary embodiments, the aqueous pharmaceutical formulation comprises an amino acid aggregation inhibitor, the latter optionally being an amino acid. In exemplary aspects, the amino acid is an L-stereoisomer amino acid (L-amino acid), but a D-stereoisomer amino acid (D-amino acid) is contemplated. In some aspects, the inhibitor of amino acid aggregation comprises an amino acid comprising a charged side chain, also referred to herein as a "charged amino acid". The term "charged amino acid" refers to an amino acid that comprises a side chain that is either negatively charged (i.e., deprotonated) or positively charged (i.e., protonated) in aqueous solution at 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 those of the 20 encoded amino acids, as well as atypical or non-naturally occurring or non-encoded amino acids. Thus, in exemplary aspects, the inhibitor of amino acid aggregation is an amino acid comprising a positively charged side chain. In exemplary cases, the amino acid comprising a positively charged side chain comprises a side chain structure of formula I or formula II:

wherein n is 1 to 7, wherein R₁And R₂Each of which is independently selected from the group consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、NH、NH₂(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₇And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₇Is H or OH, wherein optionally R₁And R₂One of them being a free amino group (-NH)₃ ⁺)；

Wherein m is 1 to 7, wherein R₃And R₄Each of which is independently selected from group a consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₈And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₈Is H or OH, wherein R5 is optionally present and when present is selected from group A,optionally, wherein R is₃And R₄And R₅Each of which is H.

In exemplary aspects, 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, R₁Is NH or NH₂. In exemplary aspects, R₂Is NH₂Or NH₃ ⁺. In exemplary cases, the amino acid comprising a positively charged side chain is arginine. In exemplary aspects, 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, R₃And R₄Each is H. In some cases, R₅Is present and is 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 exemplary aspects, the aqueous pharmaceutical composition comprises L-arginine hydrochloride or L-lysine hydrochloride.

In exemplary aspects, the amino acid aggregation inhibitor is an aromatic amino acid. In some cases, the aromatic amino acid comprises a phenyl group or an indole. In exemplary aspects, the aromatic amino acid comprises a C between the alpha carbon and a phenyl or indole₁-C₆Alkyl chains (e.g. C)₁-C₃An alkyl chain). In exemplary cases, the aromatic amino acid is L-phenylalanine. In other cases, the aromatic amino acid is L-tryptophan.

In exemplary aspects, 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 greater the positive value of the score, the more hydrophobic the amino acid. In some cases, "A simple method for displaying the hydropathic character of a protein [ a simple method for demonstrating the hydrophilic character of a protein ] on the Kate-Durit hydrophobicity Scale (Kyte J, Doolittle RF (5 months 1982) ]]". J.mol.biol. [ journal of molecular biology]157(1) 105-32) the hydrophobicity was scored. In thatIn some aspects, the hydrophobic amino acid has a score of greater than about 2.5 on the katter-dolite hydrophobicity scale. In certain aspects, the hydrophobic amino acid comprises a branched or straight chain C₂-C₁₂Alkyl or C₄-C₈Cycloalkyl, nitrogen heteroatom containing C₄-C₈A side chain of a heterocycle, optionally wherein the heterocycle is imidazole, pyrrole, or indole. For purposes herein, the term "cycloalkyl" encompasses any carbocyclic ring, including carbobicyclic or tricyclic rings.

In exemplary aspects, the hydrophobic amino acid comprises C₃-C₈Alkyl, optionally the hydrophobic amino acid comprises a branched chain C₃Alkyl or branched C₄An alkyl group. In certain aspects, the hydrophobic amino acid is L-valine, L-leucine, or L-isoleucine.

The amino acid aggregation inhibitor is used in an amount effective to provide increased stability, and may be used at a concentration in the range of about 10mM to about 200mM, for example, in the range of about 30mM to about 120mM, or about 38mM to about 150mM, or about 38mM to about 113mM, or about 38mM to about 75mM, such as about 10mM, about 38mM, about 75mM, about 113mM, or about 150 mM. In exemplary aspects, the aqueous pharmaceutical formulation comprises about 5mM to about 300mM amino acid aggregation inhibitor, optionally about 25mM to about 90mM amino acid aggregation inhibitor. In some aspects, when the inhibitor of amino acid aggregation is an amino acid comprising a positively charged side chain, optionally L-arginine, the aqueous pharmaceutical formulation comprises about 5mM to about 150mM (e.g., about 10mM to about 150mM, about 15mM to about 150mM, about 20mM to about 150mM, about 25mM to about 150mM, about 5mM to about 140mM, about 5mM to about 130mM, about 5mM to about 120mM, about 5mM to about 110mM, about 5mM to about 100mM, about 5mM to about 90mM) inhibitor of amino acid aggregation. In some aspects, when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally L-arginine, the aqueous pharmaceutical formulation comprises about 30mM to about 80mM (e.g., about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM) amino acid aggregation inhibitor.

In some aspects, when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine, the aqueous pharmaceutical formulation comprises about 5mM to about 180mM (e.g., about 10mM to about 180mM, about 15mM to about 180mM, about 20mM to about 180mM, about 25mM to about 180mM, about 5mM to about 170mM, about 5mM to about 160mM, about 5mM to about 150mM, about 5mM to about 140mM, about 5mM to about 130mM, about 5mM to about 120mM, about 5mM to about 110mM) amino acid aggregation inhibitor. In exemplary cases, when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine, the aqueous pharmaceutical formulation comprises about 5mM to about 100mM (e.g., about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM) amino acid aggregation inhibitor, optionally about 20mM to about 50mM 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 comprises about 5mM to about 300mM 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 comprises about 5mM to about 200mM (e.g., about 10mM to about 200mM, about 20mM to about 200mM, about 30mM to about 200mM, about 40mM to about 200mM, about 50mM to about 200mM, about 60mM to about 200mM, about 70mM to about 200mM, about 80mM to about 200mM, about 90mM to about 200mM, about 100mM to about 200mM, about 5mM to about 290mM, about 5mM to about 280mM, about 5mM to about 270mM, about 5mM to about 260mM, about 5mM to about 250mM, about 5mM to about 240mM, about 5mM to about 230mM, about 5mM to about 220mM, about 5mM to about 210mM) amino acid aggregation inhibitor, optionally about 20mM to about 50mM amino acid inhibitor. In an exemplary aspect, the aqueous pharmaceutical composition comprises: about 30mM to about 80mM L-arginine hydrochloride; about 20mM to about 50mM L-phenylalanine; about 20mM to about 50mM L-tryptophan; about 30mM to about 80mM L-lysine hydrochloride; about 20mM to about 50mM L-leucine; about 20mM to about 50mM L-isoleucine; about 20mM to about 50mM L-valine; or any combination thereof.

In exemplary aspects, 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 exemplary aspects, when the amino acid aggregation inhibitor is an amino acid comprising 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 (having a polar head and a hydrophobic tail) surfactant. The surfactant preferentially accumulates at the interface, thereby reducing the interfacial tension. Surfactants may optionally be included in the formulation. The use of surfactants may also help to mitigate the formation of large protein particles.

In one type of embodiment, the surfactant can be a nonionic surfactant. Examples include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 80); alkylaryl polyethers, e.g. oxyethylated alkylphenols (e.g. Triton)^TMX-100); poloxamers (e.g. poloxamers)Such asF68) And combinations of any of the foregoing belonging to one surfactant class or to multiple surfactant classes. Especially polysorbate 20 and polysorbate 80 are envisaged.

Surfactant concentrations in the range of about 0.004% (w/v) to about 0.1% (w/v) (e.g., for polysorbate 20 or polysorbate 80) are suitable, e.g., about 0.004% to about 0.05%, or about 0.004% to about 0.02%, or about 0.01%. In exemplary aspects, the formulation comprises at least about 0.004 (w/v)% surfactant and optionally less than about 0.15 (w/v)%. In exemplary aspects, the surfactant is present in the formulation about 0.005 (w/v)% to about 0.015 (w/v)% 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)%.

The stabilized aqueous formulation may be suitable for administration by any acceptable route, including parenterally, and in particular subcutaneously. For example, subcutaneous administration may be to the upper arm, thigh, or abdomen. Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intranodal, and intrasplenic. The subcutaneous route is preferred.

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

In exemplary aspects, the aqueous pharmaceutical formulation has a conductivity in the range of about 500 μ S/cm to about 5500 μ S/cm, optionally wherein the conductivity is in the range of about 2500 μ S/cm to about 5500 μ S/cm when the formulation comprises an amino acid containing a positively charged side chain, or in the range of about 500 μ S/cm to about 2000 μ S/cm when the formulation comprises an aromatic amino acid or lacks an amino acid aggregation inhibitor. The aqueous pharmaceutical formulation of any one of the preceding claims, having a viscosity of no more than about 6cP at 5 ℃, optionally wherein the viscosity is about 4.5cP to about 5.5 cP. In certain aspects, the aqueous pharmaceutical formulation has a viscosity of less than about 13cP, optionally from about 2.0cP to about 10cP, optionally from about 2.5cP to about 4cP at 25 ℃.

Tonicity modifiers or tonicity modifiers are known in the art and include compounds such as salts (e.g., sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, calcium carbonate, sodium lactate), sugars (e.g., polydextrose, glucose, lactose, trehalose), and sugar alcohols (e.g., mannitol, sorbitol, xylitol, glycerol, propylene glycol). In certain aspects, the tonicity modifier is selected from the group consisting of: sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof. In an exemplary case, the tonicity modifier is sorbitol. Sorbitol can be used, for example, at a concentration in the range of 0.1% (w/v) to 5% (w/v), or 1.2% (w/v) to 5% (w/v), e.g., 3.6% (w/v), 4.6% (w/v), or 4.7% (w/v). Optionally, the formulation comprises about 1.0 (w/w)% to about 5.0 (w/w)% tonicity modifier. For example, the formulation comprises about 2.0 (w/w)% to about 5.0 (w/w)% sorbitol, or about 3.5 (w/w)% to about 5.0 (w/w)% sorbitol, or about 4.0% (w/w) to about 5.0 (w/w)% sorbitol. In some aspects, the formulation does not contain any sorbitol or does not contain sorbitol. In exemplary aspects, the formulation does not contain any tonicity modifier.

Other excipients known in the art may be used in the formulation as long as they do not negatively impact stability. Sugars and polyols can be used to protect proteins from aggregation, including providing freeze/thaw stability. Such compounds include sorbitol, mannitol, glycerol, erythritol, caprylate, tryptophan, sarcosinate and glycine. Stabilizers for preparing lyophilized formulations, e.g., stabilizing sugars, e.g., disaccharides, such as trehalose and sucrose, may also be used. The lyophilized formulation may also include a bulking agent, as is 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., N-methyl-2-pyrrolidone)). Pharmaceutically acceptable acids and bases may be used to adjust the pH of the solution, for example sodium hydroxide.

For parenteral administration, the formulation may be in the form of a pyrogen-free parenterally acceptable sterile aqueous solution comprising dinotezumab or another human anti-RANKL monoclonal antibody, with or without other therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the dinotezumab or another human anti-RANKL monoclonal antibody (with or without at least one additional therapeutic agent) is formulated as a sterile isotonic solution. The formulation will contain a pharmaceutically acceptable excipient, such as a USP (united states pharmacopeia) grade excipient.

"preservatives" are compounds that may be included in pharmaceutical formulations to reduce bacterial effects therein, for example, thereby facilitating the manufacture of multi-purpose formulations. Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, wherein the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols including phenol, butanol, and benzyl alcohol; alkyl parabens, including methyl paraben and propyl paraben; catechol, m-cresol, cyclohexanol, 3-pentanol, and m-cresol. In the alternative, the formulation may be preservative-free. For example, the formulation may be preservative-free when present in a single-use dosage form.

Although aqueous solution forms of the formulation have been described herein, the stabilized formulation can also be subsequently lyophilized to prepare a lyophilizate. Thus, unless the context dictates otherwise, it is contemplated that reference to a formulation and method of use thereof includes a lyophilizate obtained from the stabilized aqueous solution.

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

In certain embodiments, the invention is directed to kits for producing single dose administration units. In certain embodiments, the kits may each contain a first container having a dry formulation of dinoteumab or other human anti-RANKL monoclonal antibody made from the solution formulations described herein and a second container having sterile water or an aqueous solution. In certain embodiments of the invention, kits are included that contain single chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilizate syringes).

The stabilized formulations described herein may be used with one or more other therapeutic agents, such as calcium and vitamin D compounds. The stabilized formulations described herein can be administered to a patient receiving therapy with an additional therapeutic agent, or the stabilized formulations described herein can be co-administered with an additional therapeutic agent.

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

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

In another aspect, the formulation can be used to treat a patient with giant cell tumor of the bone comprising administering an effective amount of the stabilized formulation described herein. In one type of embodiment, the patient has giant cell tumor of bone that is recurrent, unresectable, or has the potential to cause severe morbidity from surgical resection. For example, the patient may be an adult or a bone-maturing adolescent.

In another aspect, the formulation is useful for treating a patient with malignant hypercalcemia of bone comprising administering an effective amount of the stabilized formulation described herein. In one aspect, the malignant disease is refractory to bisphosphonate therapy. The method or use may comprise administering the formulation in an amount effective to reduce or maintain serum calcium in the patient at a level of less than or equal to about 11.5 mg/dL.

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

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

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

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

In another aspect, the formulation can be used to treat Idiopathic Subthalangeal Stenosis (ISS).

In another aspect, the formulation is useful for breast and ovarian cancer prevention in BRCA-1 mutant healthy women.

Optionally, the formulation may be used in combination with an immune checkpoint inhibitor. Optionally, the immune checkpoint inhibitor is specific for a protein that functions in an immune checkpoint pathway, such as CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1, or TIM 3. Optionally, the immune checkpoint inhibitor is an antibody, antigen binding fragment thereof, or antibody protein product specific for CTLA4, LAG3, PD-1, PD-L1, PD-L2, B7-H3, B7H4, BTLA, SLAM, 2B4, CD160, KLRG-1, or TIM 3. Such immune checkpoint inhibitors include, but are not limited to: attuzumab (atezolizumab), Avelumab (avelumab), ipilimumab (ipilimumab), tremelimumab (tremelimumab), BMS-936558, MK3475, CT-011, AM-224, MDX-1105, IMP321, MGA 271. PD-1 inhibitors include, for example, pembrolizumab (pembrolizumab) and nivolumab (nivolumab). PD-L1 inhibitors include, for example, atilizumab, avizumab, and dolacizumab (durvalumab). CTLA4 includes, for example, ipilimumab. In another aspect, the formulation may optionally be used in combination with a PD-1 antibody (e.g., nivolumab, pentolizumab) for treating melanoma patients with bone metastases. In another aspect, the formulation may optionally be used in combination with a CTLA4 inhibitor, such as ipilimumab, for treating a breast cancer patient.

In another aspect, the formulation can be used to treat a giant cell rich tumor, such as parathyroid hyperactivity disorder or secondary aneurysmal bone cysts.

In another aspect, the formulation may be used to treat progressive metastatic castration resistant prostate cancer (mCRPC). In another aspect, the formulation is useful for treating castration sensitive prostate cancer. In another aspect, the formulation is useful for treating hormone resistant prostate cancer.

In another aspect, the formulation may be used to treat metastatic breast cancer (mBC). In another aspect, the formulation can be used to treat preoperative breast cancer. In another aspect, the formulation can be used to treat early breast cancer. In other aspects, the formulation can be used to treat hormone receptor negative RANK positive or RANK negative primary breast cancer. In another aspect, the formulation is useful for treating post-menopausal HER2 negative breast cancer.

In another aspect, the formulation is useful for treating myelodysplastic syndrome, for example, in elderly patients.

In another aspect, the formulation may be used to treat cancer therapy-induced bone loss (CTIBL).

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

In another aspect, the formulation can be used to induce immunomodulation in patients with or without immunotherapy.

In another aspect, the formulation is useful for preventing or treating bone loss associated with osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, and rheumatoid arthritis. In another aspect, the formulation may be used to prevent or treat an inflammatory condition accompanied by bone loss. In another aspect, the formulation may be used to prevent or treat autoimmune conditions with bone loss. In another aspect, the formulation is useful for preventing or treating bone loss associated with cancer (including breast, prostate, thyroid, kidney, lung, esophagus, rectum, bladder, cervix, ovary, liver, and gastrointestinal cancer), multiple myeloma, lymphoma, and Hodgkin's disease.

The formulation may be administered on any suitable schedule. In one embodiment, the administration is on a once every four week schedule. Optionally, the administering may include administering on days 8 and 15 of the first month of therapy. In another type of embodiment, the administration may be on a schedule of once every six months. For example, a schedule of once every six months is envisaged for osteoporosis and to increase bone mass. Other contemplated maintenance doses are every 3 weeks, every 3 months, and every 6 weeks.

In some aspects, the aqueous pharmaceutical formulation is used to treat a patient with multiple myeloma or solid tumor bone metastasis. In certain aspects, the formulation is administered to the upper arm, thigh, or abdomen in a subcutaneous injection at a dose of about 120mg every 4 weeks.

In some aspects, the aqueous pharmaceutical formulation is used to treat a patient having giant cell tumor of the bone. In certain aspects, the formulation is administered at a dose of about 120mg every 4 weeks, with an additional 120mg dose administered on days 8 and 15 of the first month of therapy. In some aspects, the formulation is administered subcutaneously to the upper arm, thigh, or abdomen of a patient. In some cases, calcium and vitamin D are administered to the patient to treat or prevent hypocalcemia.

In some aspects, the aqueous pharmaceutical formulation is used to treat a patient suffering from malignant hypercalcemia. In certain aspects, the formulation is administered at a dose of about 120mg every 4 weeks, with an additional 120mg dose administered on days 8 and 15 of the first month of therapy. In some aspects, the formulation is administered subcutaneously to the upper arm, thigh, or abdomen.

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

One type of formulation according to the present disclosure will contain dinotefuran, 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 a 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 the present disclosure would contain dinotezumab, acetate and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include a 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 dinotefuran at a concentration of about 108mg/mL to about 132mg/mL, about 28.8mM to about 35.2mM acetate, 33.3mM to about 40.7mM phenylalanine, 3.51% (w/v) to about 4.29% (w/v) sorbitol, and about 0.009% (w/v) to about 0.011% (w/v) polysorbate 20 at pH 5.1, and may optionally be included in a PFS optionally containing about 1mL or less than about 1mL (e.g., about 0.5mL) of the formulation. For example, the formulation may include dinotefuran at a concentration of 120mg/mL, 32mM acetate, 37mM phenylalanine, 3.9% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20 at pH 5.1, and may optionally be included in a PFS optionally containing about 1mL or less than about 1mL (e.g., about 0.5mL) of the formulation. The formulation can be made by concentrating dinotefuran using diafiltration buffer (pH 4.7) containing 20mM acetate, 4.2% (w/v) sorbitol, and 40mM phenylalanine.

Another type of formulation according to the present disclosure would contain dinotezumab, 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 a 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 the present disclosure would contain dinotezumab, acetate, arginine, and phenylalanine. The formulation may optionally include sorbitol. The formulation may optionally include a 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 the present disclosure would contain dinotezumab, 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 a 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.

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

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

The amino acid aggregation inhibitor may be introduced into the formulation by any suitable method. For example, the amino acid aggregation inhibitor may be introduced into the formulation via simple addition (spiking) into the formulation, e.g., as described in the examples below. In another approach, the amino acid aggregation inhibitor may be introduced into the formulation via diafiltration against a buffer solution containing the amino acid aggregation inhibitor, e.g., as described in the examples below. The amino acid aggregation inhibitor may be introduced into the formulation before or after concentrating the anti-RANKL monoclonal antibody above 70 mg/mL. As shown in the examples below, it is beneficial to add the amino acid aggregation inhibitor to the solution prior to concentration, as it can inhibit aggregation during concentration.

Accordingly, the present disclosure provides methods of making stable aqueous pharmaceutical formulations comprising human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibodies or antigen-binding portions thereof. In exemplary cases, the method comprises combining the anti-RANKL monoclonal antibody or antigen-binding portion thereof at a concentration of greater than 70mg/mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier. The antibody or antigen-binding portion may be any of those described herein, and the concentration of the antibody or antigen-binding portion thereof 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 can be in a molar ratio to an antibody as described herein. The amounts and selection of aggregation inhibitor, surfactant, tonicity modifier and buffer are as described above. The present disclosure also provides formulations made by the manufacturing methods described herein.

Formulations according to the disclosure herein may include pH adjustment of a high concentration solution of an anti-RANKL monoclonal antibody described herein (e.g., dinotezumab), e.g., a solution at a concentration above 70mg/mL or 120 mg/mL. In another aspect, the formulation may be prepared by pH adjustment of a low concentration solution of the anti-RANKL monoclonal antibody (e.g., dinotefuran), followed by concentration of the solution to the desired higher final concentration. Suitable pH adjusting agents are known in the art.

Examples

The following is a list of specific contemplated embodiments:

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

2. The formulation of embodiment 1, having a pH 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 of example 2, having a pH of about 5.1.

4. The formulation of any one of embodiments 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 antigen-binding portion thereof and an amino acid aggregation inhibitor.

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

7. The formulation of example 6, having a pH of about 5.1.

8. The formulation of any one of the preceding embodiments, further comprising a pH buffer.

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

10. The formulation of any one of the preceding embodiments, wherein the concentration of the antibody, or antigen-binding portion thereof, is in the range of greater than 70mg/mL to about 200 mg/mL.

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

12. The formulation of embodiment 11, wherein the concentration of the antibody, or antigen-binding portion thereof, is about 120 mg/mL.

13. The formulation of any one of the preceding embodiments, wherein the antibody is dinotezumab or a biological analog thereof.

14. The formulation of embodiment 13, wherein the antibody is dinotefuran.

15. The formulation of any one 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 of embodiment 15, wherein the amino acid aggregation inhibitor comprises a mixture of at least two amino acids.

17. The formulation of embodiment 16, wherein the amino acids comprise arginine and phenylalanine.

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

20. The formulation of any one of the preceding embodiments, wherein the amino acid aggregation inhibitor is selected from one or more basic amino acids, a dipeptide thereof, or an oligopeptide having 2 to 10 residues and containing one or more basic amino acids.

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

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

23. The formulation of any one of the preceding embodiments, wherein the amino acid aggregation inhibitor comprises an arginine residue, or the amino acid aggregation inhibitor comprises arginine.

24. The formulation of any one of the preceding embodiments, wherein the amino acid aggregation inhibitor comprises an arginine-phenylalanine dipeptide.

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

26. The formulation of any one of the preceding embodiments, further comprising a surfactant.

27. The formulation of embodiment 26, wherein the surfactant is selected from the group consisting of: a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 80), or one or more alkylaryl polyethers such as an oxyethylated alkylphenol (e.g., oxyethylated alkylphenol)X-100), or one or more poloxamers (e.g., one or more poloxamers)Such asF68) And combinations thereof.

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

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

30. The formulation of any one of the preceding embodiments, further comprising a buffering agent.

31. The formulation of embodiment 30, wherein the buffer is centered at a range of about pH 4 to about pH 5.5 at 25 ℃.

32. The formulation of embodiment 30 or embodiment 31, wherein the buffer has a pKa at 25 ℃ within a pH unit of pH 5.0-5.2.

33. The formulation of any one of embodiments 30 to 32, wherein the buffer comprises acetate.

34. The formulation of any one of embodiments 30 to 32, wherein the buffer comprises glutamate.

35. The formulation of any one of the preceding embodiments, further comprising a tonicity modifier.

36. The formulation of embodiment 35, wherein the tonicity modifier is selected from one or more of sorbitol, mannitol, sucrose, trehalose, glycerol and combinations thereof.

37. The formulation of embodiment 36, wherein the tonicity modifier comprises sorbitol.

38. The formulation of any one of the preceding embodiments, further comprising one or more additional excipients selected from the group consisting of sugars, polyalcohols, solubilizing agents (e.g., N-methyl-2-pyrrolidone), hydrophobic stabilizing agents (e.g., proline), polyethylene glycols, cyclodextrins, and combinations thereof.

39. The formulation of any of the preceding embodiments, which comprises less than 2% of the high molecular weight substance of the human anti-RANKL monoclonal antibody after three months storage at 37 ℃ according to SE-UHPLC.

40. The formulation of any of the preceding embodiments, which comprises less than 2% of the high molecular weight substance of the human anti-RANKL monoclonal antibody after 36 months storage at 4 ℃ according to SE-UHPLC.

41. The formulation of any one of the preceding embodiments, which comprises at least 98% of the main peak of antibody after three months storage at 37 ℃ according to SE-UHPLC.

42. The formulation of any one of the preceding embodiments, which comprises at least 98% of the main peak of antibody after 36 months storage at 4 ℃ according to SE-UHPLC.

43. The formulation of any one of the preceding embodiments, comprising dinotefuran; an amino acid aggregation inhibitor selected from one or more of arginine, a dipeptide thereof, or an oligomer having 2 to 10 residues and comprising arginine; an acetate buffer; sorbitol; and a surfactant, and the formulation has a pH value in the range of about 5.0 to less than 5.2.

44. The formulation of embodiment 43, wherein the amino acid aggregation inhibitor is selected from arginine, arginine-arginine, or arginine-phenylalanine.

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

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

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

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

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

50. The formulation of embodiment 49, wherein the pH is about 5.10.

51. The formulation of any one of the preceding embodiments, wherein the formulation is suitable for subcutaneous injection.

52. The formulation of any one of the preceding embodiments, wherein the formulation is sterile and preservative-free.

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

54. The formulation of any one of the preceding embodiments, wherein the human anti-RANKL monoclonal antibody or antigen-binding portion thereof is an antibody.

55. The formulation of any one of embodiments 1 to 53, wherein the human anti-RANKL monoclonal antibody or antigen-binding portion thereof is an antigen-binding portion.

56. A vial, pre-filled syringe or glass container containing the formulation of any one of examples 1 to 55.

57. The vial, pre-filled syringe, or glass container of embodiment 56, containing about 1mL or less of the formulation.

58. A method of preventing a bone related event (SRE) in a patient in need thereof comprising administering an effective amount of the formulation of any one of examples 1-55.

59. The method of embodiment 58, wherein the SRE is selected from the group consisting of: pathological fractures, radiation therapy for bones, surgery for bones, and spinal cord compression.

60. The method of embodiment 58 or embodiment 59, wherein the patient has solid tumor bone metastases.

61. The method of embodiment 60, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma.

62. The method of embodiment 58 or embodiment 59, wherein the patient has multiple myeloma.

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

64. A method of treating giant cell tumor of bone in a patient in need thereof comprising administering an effective amount of the formulation of any one of examples 1-55.

65. The method of embodiment 64, wherein the patient has recurrent, unresectable, or surgically removed giant cell tumor of bone that may cause severe morbidity.

66. A method of treating hypercalcemia of malignancy in a patient in need thereof comprising administering an effective amount of the formulation of any one of example 1 to example 55.

67. The method of embodiment 66, wherein the malignant disease is refractory to bisphosphonate therapy.

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

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

70. The method of any one of embodiment 58 to embodiment 69, comprising administering the formulation on a once every four week schedule.

71. The method of any one of embodiment 58 to embodiment 70, comprising administering the formulation on days 8 and 15 of the first month of therapy.

72. A method of treating osteoporosis in a patient in need thereof, comprising administering an effective amount of the formulation of any one of examples 1-55.

73. The method of embodiment 72, wherein the patient is a postmenopausal woman at risk of high fracture.

74. The method of embodiment 72, wherein the patient is a male at risk of high fracture.

75. A method of increasing bone mass in a patient in need thereof comprising administering an effective amount of the formulation of any one of example 1 to example 55.

76. The method of embodiment 75, wherein the patient has osteoporosis.

77. The method of embodiment 75, wherein the patient is a female at high risk of fracture who has received adjuvant aromatase inhibitor therapy for breast cancer.

78. The method of embodiment 75, wherein the patient is a male at risk of high fracture who is on androgen deprivation therapy for non-metastatic prostate cancer.

79. The method of any one of embodiments 75-78, comprising administering the formulation in an amount effective to reduce the incidence of new vertebral and/or non-vertebral fractures.

80. The method of any one of embodiment 75-embodiment 79, comprising administering the formulation in an amount effective to reduce bone resorption.

81. The method of any one of embodiments 75-80, comprising administering the formulation in an amount effective to increase bone density in at least one region of the patient selected from the lumbar spine, total hip, and femoral neck.

82. The method of any one of embodiments 75-81, comprising administering the formulation in an amount effective to increase bone mass in cortical bone and/or cancellous bone of the patient.

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

84. The method of any one of embodiment 75 to embodiment 83, comprising administering the formulation on a schedule of once every six months.

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

86. The method of any one of embodiment 58 to embodiment 85, comprising administering the formulation subcutaneously.

87. The method of embodiment 86, comprising administering the formulation subcutaneously to the upper arm, thigh, or abdomen.

88. The method of any one of embodiments 58-87, wherein the patient receives one or both of calcium and vitamin D.

89. A method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a concentration of more than 70mg/mL, comprising:

preparing the aqueous pharmaceutical formulation comprising the human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a pH in the range of about 5.0 to less than 5.2;

wherein the aqueous pharmaceutical formulation exhibits improved stability at a pH value in the range of about 5.0 to less than 5.2 compared to an equivalent aqueous pharmaceutical formulation not at a pH value in the range of about 5.0 to less than 5.2.

90. A method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof, comprising:

preparing the aqueous pharmaceutical formulation comprising a mixture of the human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof 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.

Examples of the invention

The following examples are provided for illustration and are not intended to limit the scope of the invention. Throughout the examples provided herein, the following abbreviations are used: DF, percolating; PS20, polysorbate 20; HCl, hydrochloride salt; UF/DF, ultrafiltration/diafiltration; f #, preparation number; HMWS, high molecular weight species; SE-UHPLC, size exclusion ultra high performance liquid chromatography. Additionally, throughout these examples, the composition of DF buffer or dialysis buffer used to make the final formulation comprising dinotezumab, and the estimated concentrations of the components of the final formulation are provided. The final concentration of certain components of the final formulation stored and subsequently analyzed for stability may differ from the concentration of DF or dialysis buffer, depending on the presence or absence of a counter ion (e.g., hydrochloride). In the absence of counter ions, the formulations have low ionic strength. In this case, the acetate is co-concentrated with dinotezumab such that the final formulation contains a higher concentration of acetate relative to the concentration of DF or dialysis buffer. For example, when neither DF buffer nor the final formulation contains a counter ion (e.g., hydrochloride salt) and thus has a low ionic strength, using DF buffer containing 10mM acetate results in about 23mM acetate present in the final dinotezumab (120mg/mL) formulation (pH 5.1). Similarly, DF buffer containing 20mM acetate in the absence of counter ion (e.g., hydrochloride salt) resulted in the presence of about 32mM acetate in the final dinotezumab (120mg/mL) formulation (pH 5.1). When a counter ion (e.g., arginine hydrochloride) is present, the acetate is not co-concentrated with dinotezumab, and thus the acetate concentration of DF buffer and the acetate concentration of the final composition are generally equal. Additionally, excipients may be subject to size exclusion, or may be subject to non-specific interactions. For example, in a 120mg/mL dinoteuzumab formulation, phenylalanine and sorbitol concentrations were approximately 7% to 10% lower and arginine concentrations were approximately 10% to 15% lower than those indicated in DF buffers. According to the above, throughout the following examples, the concentrations of the components of the final formulation are provided taking into account the excipient exclusion and acetate co-concentration effects described above.

Example 1

Twelve formulations were initially evaluated for the effect of minimizing the amount (%) of HMWS and its formation over time in a high concentration liquid formulation of dinotezumab (120 mg/mL). Formulation alternatives include variations in buffer type, stabilizers, and solution pH. The formulations a to L tested are described in table 1 below. All cited buffer values relate to the buffer concentration against which the diafiltration antibody is directed. After buffer exchange, various excipients and surfactants were added to the solution to the levels indicated in the table. Although the acetate concentration in the formulations of the invention was not measured, the 120mg/mL dinosaumab formulation diafiltered against 10mM acetate had an approximate final acetate value between 25mM and 35mM acetate with sorbitol.

Dinosuzumab (70mg/mL) in acetate (pH5.2) was UF/DF relative to 10mM acetate (pH5.2) and concentrated to 160 mg/mL. Stock solutions were prepared in 10mM acetate (pH5.2) consisting of:

35% sorbitol

1% polysorbate 20

1% polysorbate 80

30％F-68

3％Triton^TM X-100

250mM L-arginine hydrochloride

250mM N-acetyl arginine (NAR)

250mM N-acetyl lysine (NAK)

250mM proline

250mM polyethylene glycol (PEG)3350

250mMCyclodextrin

To obtain formulations a to J, 160mg/mL of material prepared with 10mM acetate (pH5.2) was diluted to 120mg/mL with 10mM acetate (pH5.2) followed by the addition of the corresponding sorbitol, excipients and/or surfactant stock solutions to the target final concentrations listed in table 1. To obtain formulations K and L, two separate aliquots from 160mg/mL of material, self-buffered formulations and glutamate formulations, respectively, were subjected to additional buffer exchange by centrifugation. Formulations K and L were then diluted to 120mg/mL with the respective buffers, followed by the addition of the corresponding sorbitol and polysorbate 20 stock solutions to the target final concentrations listed in the formulation tables in table 1.

TABLE 1

FIG. 1 shows the percentage of HMWS as a function of formulation and time, monitored by SE-UHPLC at 37 ℃. Formulation L, consisting of approximately 10mM glutamate buffer, 10mM L-arginine hydrochloride, 2.4% (w/v) sorbitol as tonicity modifier, 0.01% (w/v) polysorbate 20 as surfactant and at a pH of 5.0, showed a reduction in the initial amount of HMWS at 37 ℃ (indicating a certain reduction in the extent of aggregates that have formed) and a reduction in the dynamic formation of HMWS.

Example 2

Evaluation of 10mM acetate, 75mM L-arginine, 2.4% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 excipient formulation and 10mM acetate, 5% (w/v) sorbitol, 0.01% (w/v) polysorbate 20 excipient formulations each containing high concentrations (120mg/mL) of dinotezumab, at a temperature of 37 ℃ showed the effect of pH and amino acid aggregation inhibitors on the rate and extent of HMWS formation for up to 1 month. The formulations tested are described in table 2 below. All cited buffer and excipient values relate to the buffer and excipient concentrations to which the diafiltration antibody is directed.

To prepare test samples M to Q, a 3mL aliquot of dinosaumab (70mg/mL) in acetate (pH5.2) was dialyzed against 500mL of DF buffer described below, with a total of 3 buffer changes performed to achieve 100 ten thousand-fold dilutions of the previous formulation, to ensure complete buffer exchange. The material was then over-concentrated using a centrifuge-concentrator, then diluted to 120mg/mL, and polysorbate 20 was added to a final concentration of 0.01%.

TABLE 2

FIG. 2 shows the percentage of HMWS as a function of formulation and time, monitored by SE-UHPLC at 37 ℃. Figure 3 shows the size exclusion chromatogram as a function of formulation after 1 month of storage at 37 ℃.

As the pH of the solution decreases, the formation of large aggregates increases. At pH values below 4.8 and especially 4.5, the large aggregates were predominantly HWMS, with a significant increase in large aggregates at pH 4.5 for the formulations tested. As shown in fig. 3, formulations P and Q had the lowest amount of advanced HWMS (residence time about 6 minutes), followed by comparative formulations O, N and M with reduced pH.

However, as the pH increases, an increase in dimer species is generally caused. As shown in figure 3, formulation N had the lowest amount of dimer material (residence time about 6.8 minutes), followed by formulations M, O, P and Q.

The presence of arginine at a concentration of 75mM in formulation O caused a decrease in the amount of dimer species and its dynamic rate of formation by approximately 0.3% and 25%, respectively, when compared to formulation P having the same pH but without arginine, after 1 month at 37 ℃.

Example 3

This example shows the effect of pH on high concentration dinotefuran formulations.

Dinosuzumab (at 120mg/mL) was formulated with acetate, sorbitol and polysorbate 20(PS20) with or without an amino acid aggregation inhibitor at three different pH values: 4.8, 5.1 and 5.4. In this study, the amino acid aggregation inhibitor was L-arginine hydrochloride. All formulations were made by exchanging the buffer of the initial solution containing the lower concentration of dinotezumab, followed by over-concentration of the dinotezumab material, followed by dilution of the dinotezumab material with the desired amounts of buffer, excipients, and surfactant. Briefly, an aliquot of dinosaumab (70mg/mL) (starting material) in acetate (pH5.2) was dialyzed against DF buffer as described in table 3A, with a total of 3 buffer changes performed to achieve 100 ten thousand dilutions of the starting material to ensure complete buffer exchange. The buffer-exchanged dinotefuran material was then concentrated to a dinotefuran concentration above 120mg/mL using a centrifuge-concentrator, and the concentrated material was subsequently diluted to reach a concentration of 120mg/mL dinotefuran. PS20 was added to a final concentration of 0.01%.

It is believed that the high concentration of protein contributes to the solution pH based on its charge state. The acetate concentration of formulation 1 was increased to reach the target final pH and formulations 2 and 3 were matched to the acetate concentration of formulation 1. The acetate concentrations of formulations 4 to 6 required even higher amounts of acetate in order to match the final acetate concentrations of formulations 1 to 3, since the acetate failed to co-concentrate in the presence of the hydrochloride salt. Formulation 7 served as a control to ensure that increased acetate concentrations in formulations 4-6 did not interfere with protein stability in arginine hydrochloride formulations.

The different formulations of dinotefuran made and tested in this study are described in table 3A.

TABLE 3A

Final formulation comprised 120mg/mL dinosaumab and PS20 at a final concentration of 0.01% (w/v) and had the indicated pH values. The sorbitol concentration was estimated to be 8.5% lower than that of DF buffer. The arginine concentration was estimated to be 12.5% lower than that of DF buffer.

A sample of each formulation was filled into a container in a 1mL fill volume and stored at a temperature of 37 ℃ for 4 weeks. SE-UHPLC was used to assess aggregation inhibition and stability against aggregation inhibition over time (e.g. based on formation of HMWS and dimeric species). The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

The percentage of HMWS as a function of formulation and time was monitored by SE-UHPLC at 37 ℃. Fig. 4 represents a graph of the HMWS percentages of formulations 1-7 as a function of time, and table 3B provides data points for the graph.

TABLE 3B

The F # s shown in the left column correspond to the F # s of Table 3A.

Figure 5 shows a size exclusion chromatogram of each formulation after 1 month of storage at 37 ℃. The arginine-free formulation is shown in the left panel, and the arginine-containing formulation is shown in the right panel.

As shown in figure 4, the potency of the formulation containing arginine was superior to the control formulation without arginine hydrochloride, and the potency of the pH 5.1 formulation was superior to the comparable formulations of pH 4.8 and pH 5.4. For formulations 1 to 3 without arginine hydrochloride, the dimer species increased as the solution pH increased to 5.4 (fig. 5A). For formulations 4 to 6 containing arginine hydrochloride, the formation of larger aggregates increased when the solution pH was decreased to 4.8, and the dimer species increased when the solution pH was increased to 5.4 (fig. 5B). Formulation 6 with arginine hydrochloride present had the lowest amount of total HMWS at a solution pH of 5.1 compared to formulation 2 without arginine hydrochloride present. Additionally, formulation 7 performance shows that increasing acetate buffer concentration from 10mM to 40mM has a relatively small effect on HMWS formation.

Example 4

This example shows the relationship between pH and HMWS formation for different formulations of dinosaumab containing different concentrations of dinosaumab.

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

A total of 8 formulations (formulations 8 to 15; described in Table 4A) were evaluated in this study. To prepare these formulations, two aliquots of dinosaumab (70mg/mL) in acetate (pH5.2) were dialyzed against the corresponding DF buffers described in table 4A. Dialysis protocol nos. 1 and 2 were subjected to a total of 3 buffer changes to achieve 100 ten thousand dilutions of the previous formulation to ensure complete buffer exchange. For formulations 8, 9, 12 and 13, after dialysis, aliquots of each of dialysis protocols No. 1 and No. 2 described in table 4A were removed to prepare dilution steps. The remaining material was then over-concentrated using a centrifuge-concentrator, followed by dilution to the corresponding dinoteumab concentration listed in table 4A and addition of PS20 to reach a final concentration of 0.01%.

TABLE 4A

Final formulation comprised PS20 at a final concentration of 0.01% (w/v) and had the indicated pH value. The estimated concentration of acetate was 10mM and the sorbitol concentration was estimated to be about 8.5% lower than the DF buffer sorbitol concentration. Arginine concentration was estimated to be 65 mM. The sorbitol concentration was estimated to be 2.2% (w/v).

The formulations were filled into containers in 1mL fill volumes and stored at a temperature of 37 ℃ for 1 month. SE-UHPLC was used to assess aggregation inhibition and stability against aggregation inhibition over time (e.g. based on formation of HMWS and dimeric species). The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Fig. 6 shows a plot of the percentage HMWS as a function of storage time at 37 ℃ for each formulation as monitored by SE-UHPLC, and table 4B provides data points for the plot.

TABLE 4B

Figures 7A and 7B show that the size exclusion chromatogram varies with formulation after 1 month of storage at 37 ℃. As shown in fig. 6,% HMWS increased with increasing protein concentration. Formulations 8 to 11 at pH 4.8 consistently had higher levels of HMWS compared to the corresponding formulations at pH 5.1 (formulations 12 to 15). The% HMWS increase at pH 4.8 was due to the large aggregate peak at about 5.75 minutes shown in fig. 7A (above). Although the% HMWS at solution pH 5.1 had increased dimer species with increasing protein concentration, the total HMWS was lower than the corresponding protein concentration at solution pH 4.8 (fig. 7B (bottom)).

The difference in HMWS levels at pH 5.1 relative to pH 4.8 becomes greater with increasing concentration of dinotezumab, with the greater the higher the concentration of dinotezumab, the greater the difference.

Example 5

Formulations containing various concentrations of arginine, NAR, and two dipeptides consisting of arginine-arginine (Arg-Arg) and arginine-phenylalanine (Arg-Phe) were evaluated for stabilization of solutions with a concentration of 120mg/mL dinosaubi.

The formulations tested are described in table 5 below. All cited acetate and excipient (except dipeptide) values are in terms of the buffer and excipient concentrations against which the diafiltration antibody is directed. After buffer exchange, each dipeptide was added to the solution to the levels 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 co-UF/DF in a single pool against DF buffer (pH 4.0) containing 10mM acetate, 3.6% sorbitol. After UF/DF, the library of formulations Y and Z was divided into 2 and subsequently spiked with Arg-Arg or Arg-Phe dipeptides from a 1M 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 such that the final acetate concentration in formulations S-X was about 25 mM. Sorbitol is preferentially excluded during concentration, resulting in a reduction of about 7% to 8% (w/v) from the initial concentration.

The formulations were filled into containers at a fill volume of 1.0 mL. The formulations were stored at a temperature of 2 ℃ to 8 ℃ for 12 months and at 25 ℃, 30 ℃ and 37 ℃ for 3 months. Stability (based on HMWS formation) was assessed using SE-UHPLC. The stability of the dipeptide formulations after one month storage at 37 ℃ was compared to the arginine hydrochloride formulation at 37 ℃, as shown in figure 8.

TABLE 5

FIG. 8 shows the percentage of HMWS as a function of formulation and time as monitored by SE-UHPLC at 37 ℃. The results show that the amino acid aggregation inhibitor inhibits the formation of HMWS. For example, arginine-phenylalanine dipeptide showed significant improvement such that HMWS was reduced by about 0.3% compared to other formulations. The rank order of the lowest to highest HMWS is Z < < V < Y ≈ T ≈ W ≈ X ≈ U < S < R. As can be seen in the figure, the formulation containing arginine-arginine (Arg-Arg) dipeptide (formulation Y) and the formulation containing arginine-phenylalanine (Arg-Phe) dipeptide (formulation Z) reduced HMWS formation compared to the control formulation lacking arginine and lacking the arginine-containing dipeptide (formulation R). Formulation Z contained a minimal amount of HMWS, superior to formulation Y.

Example 6

This example shows that aggregation inhibition and stability to dinotezumab varies with different concentrations of arginine and phenylalanine and 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 dinotezumab. In this study, formulations containing various concentrations of arginine hydrochloride, various concentrations of phenylalanine, and combinations of arginine hydrochloride and phenylalanine were evaluated for stabilization of solutions containing dinotezumab (120 mg/mL).

The test formulations (formulations 16 to 20) are described in table 6A below. To prepare these formulations, aliquots of dinosaumab (70mg/mL) in acetate (pH5.2) were dialyzed against DF buffer as described in table 6A, with a total of 3 buffer changes to achieve 100 ten thousand-fold dilutions of the previous formulation, ensuring complete buffer exchange. The material was then over-concentrated using a centrifuge-concentrator, then diluted to 120mg/mL, and polysorbate 20 was added to a final concentration of 0.01%. Formulation 16 was considered a control formulation.

TABLE 6A

Final formulation comprised 120mg/mL dinosaumab and PS20 at a final concentration of 0.01% (w/v) and had the indicated pH values. The sorbitol and phenylalanine concentrations were estimated to be about 8.5% lower than the concentration of DF buffer. Arginine concentration was estimated to be about 12.5% lower than the concentration of DF buffer.

The formulations were filled into containers at a fill volume of 1.0 mL. These formulations were stored at 37 ℃ for 1 month. SE-UHPLC was used to assess aggregation inhibition and stability against aggregation inhibition over time (e.g. based on formation of HMWS and dimeric species). The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

FIG. 9 shows the percentage of HMWS as a function of formulation and time as monitored by SE-UHPLC at 37 ℃. Figure 10 shows the size exclusion chromatogram as a function of formulation after 1 month of storage at 37 ℃. Table 6B below shows the percentage of HMWS monitored by SE-UHPLC at 37 ℃ as a function of formulation and time.

TABLE 6B

All formulations containing an amino acid aggregation inhibitor, arginine or phenylalanine (formulations 17 to 20) outperformed the sorbitol control formulation lacking any amino acid aggregation inhibitor (formulation 16). All phenylalanine-containing formulations (formulations 18, 19 and 20) similarly contained low levels of HMWS when compared to the control formulation and arginine hydrochloride formulation (formulations 16 and 17, respectively) (figure 9). As shown in fig. 9, the HMWS formation rates of the formulations containing arginine hydrochloride and phenylalanine (formulations 17-19) were similar. The combined formulation comprising 38mM arginine and 38mM phenylalanine (76 nM total, formulation 20) showed better stability (figure 9) than the 75mM arginine formulation (formulation 17), but not better stability (figure 9) than the 75mM phenylalanine formulation (formulation 19).

Example 7

This example shows aggregation inhibition and stability to dinotefuran as a function of different concentrations of phenylalanine.

In previous studies, arginine hydrochloride and arginine hydrochloride-phenylalanine dipeptide were identified to minimize initial initiation levels of dinotezumab and the rate of HMWS formation. Formulations containing arginine hydrochloride, various concentrations of phenylalanine, and a combination of arginine hydrochloride and phenylalanine were evaluated for stabilization of solutions containing dinotezumab (120 mg/mL).

The formulations tested are described in table 7A below. To prepare test samples a-E, aliquots of dinosaumab (70mg/mL) in acetate (pH5.2) were dialyzed against DF buffer described below, with a total of 3 buffer changes performed to achieve 100 ten thousand-fold dilutions of the previous formulation, ensuring complete buffer exchange. The material was then over-concentrated to approximately 130mg/mL to 150mg/mL using a centrifuge-concentrator, followed by dilution to 120mg/mL and addition of polysorbate 20 to a final concentration of 0.01%. Formulation a was considered a control formulation.

The formulations were filled into containers at a fill volume of 1.0 mL. These formulations were stored at 37 ℃ for 1 month. Stability (based on HMWS formation) was assessed using SE-UHPLC. The stability profiles of these formulations after one month storage at 37 ℃ were compared to sorbitol and arginine hydrochloride/sorbitol formulations at 37 ℃, as shown in figure 11A.

To prepare test samples F to K, aliquots of dinosaumab (70mg/mL) in acetate (pH5.2) were subjected to ultrafiltration/diafiltration (UF/DF) against DF buffer described below for a total of 12 diafiltration volumes to ensure complete buffer exchange. The material was then over-concentrated to approximately 200mg/mL using ultrafiltration, followed by dilution to 120mg/mL and addition of polysorbate 20 to a final concentration of 0.01%. The acetate concentration in these formulations was 20 mM. Formulation F was considered a control formulation. All cited acetate and excipient values relate to the acetate and excipient concentrations to which the dialysis antibody is directed.

The formulations were filled into containers at a fill volume of 1.0 mL. These formulations were stored at a temperature of 40 ℃ for 1 month. Stability (based on HMWS formation) was assessed using SE-UHPLC. The stability profiles of these formulations after one month storage at 40 ℃ were compared to sorbitol and arginine hydrochloride/sorbitol formulations at 40 ℃, as shown in figure 11B.

TABLE 7A

Final formulation comprised 120mg/mL dinosaumab and PS20 at a final concentration of 0.01% (w/v) and had the indicated pH values. The sorbitol and phenylalanine concentrations were estimated to be about 8.5% lower than the concentration of DF buffer. Arginine concentration was estimated to be about 12.5% lower than the concentration of DF buffer.

Fig. 11A and table 7B show the percentage of HMWS monitored by SE-UHPLC at 37 ℃ as a function of formulation and time. Fig. 11B and table 7C show the percentage of HMWS monitored by SE-UHPLC at 40 ℃ as a function of formulation and time. Figure 12A and figure 12B show size exclusion chromatograms as a function of formulation after 1 month of storage at 37 ℃ and 40 ℃, respectively.

TABLE 7B

TABLE 7C

All phenylalanine formulations (formulations C, D, E, G-K) contained lower levels of HMWS when compared to sorbitol and arginine hydrochloride/sorbitol formulations (formulations a and B, respectively). The combined formulation of arginine hydrochloride and phenylalanine had similar stability when compared to the arginine/sorbitol formulation (formulation B). All formulations outperformed the sorbitol control formulations (formulations a and F).

Example 8

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

Different amino acid aggregation inhibitors were evaluated by preparing eight hydrophobic, aromatic or polar/charged amino acid containing formulations to determine their effect on minimizing the amount (%) of HMWS and the formation of HMWS over time in a high concentration liquid formulation of dinotezumab (120 mg/mL). The formulation comprised one of eight L-amino acids and a reduced amount of sorbitol relative to a control formulation (formulation 26) that did not contain any amino acid aggregation inhibitor and a higher amount of sorbitol present due to isotonicity.

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

I. aromatic amino acids:

(a)38mM phenylalanine (formulation 27);

(b)38mM tryptophan (formulation 28);

polar/charged amino acids:

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

(b)75mM lysine (formulation 30);

(c)75mM histidine (formulation 31);

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 dinosaumab (70mg/mL) in acetate (pH5.2) were dialyzed against DF buffer as described in table 8A, with a total of 3 buffer changes to achieve 100 ten thousand-fold dilutions of the previous formulation, 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 change to the target pH of 5.1 after protein concentration due to the south of the road effect (Donnan effect) and acetate co-concentration. However, after the protein was concentrated to 120mg/mL, the pH did not change to the target pH of 5.1, but was maintained at pH 4.0. To reach pH 5.1 for the histidine formulation, titration with dilute (0.1N) NaOH was required. The remaining formulations were over-concentrated using a centrifuge-concentrator unit, then diluted to 124 and 128mg/mL and polysorbate 20 was added to a final concentration of 0.01% (w/v).

TABLE 8A

Final formulation comprised 120mg/mL dinosaumab and PS20 at a final concentration of 0.01% (w/v) and had the indicated pH values. The sorbitol and phenylalanine concentrations were estimated to be about 8.5% lower than the DF buffer sorbitol concentration. The arginine concentration was estimated to be about 12.5% lower than that of DF buffer. The letters in () appearing after F # correspond to fig. 13 to fig. 18.

The formulations were filled into containers at a fill volume of 1.0 mL. These formulations were stored at a temperature of 37 ℃ for 4 weeks. SE-UHPLC was used to assess aggregation inhibition and stability against aggregation inhibition over time (e.g. based on formation of HMWS and dimeric species). The aggregation inhibition profiles of these formulations were compared under initial conditions and during and after the storage period.

Fig. 13-15 show plots of the percentage of HMWS as a function of storage time at 37 ℃ for each formulation as monitored by SE-UHPLC, and table 8B provides data points for each plot. Fig. 16-18 show chromatographic overlays of the formulations listed in table 8A after 1 month storage at 37 ℃. Fig. 13 and 16 relate to formulations comprising aromatic amino acids, fig. 14 and 17 relate to formulations comprising polar/charged amino acids, and fig. 15 and 18 relate to formulations comprising hydrophobic amino acids.

TABLE 8B

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

As shown in fig. 13-15, all of the formulations containing the amino acid aggregation inhibitor (formulations 27-34) showed some improvement in stability relative to the acetate/sorbitol formulation (formulation 26). The formulations containing aromatic amino acids (formulations 27 and 28) showed the greatest reduction in% HMWS. The phenylalanine containing formulation (formulation 27) also showed a substantial reduction in HMWS and the tryptophan containing formulation showed the greatest reduction relative to the control (formulation 26). The dinosaumab formulations containing polar/charged amino acids (formulations 29 to 31) generally showed a greater amount of higher order aggregates (fig. 17) compared to other formulations with amino acid stabilizers (fig. 16 and 18), and this particular histidine formulation generally showed a greater amount of HWMS (fig. 14) when compared to the acetate/sorbitol formulation (formulation 26). The results for the histidine formulation may be biased by the dialysis process, the longer duration spent at pH 4.0, and the titration of the formulation with dilute NaOH. The formulations containing hydrophobic amino acids (formulations 32 to 34) all showed consistent improvements in HMWS formation.

Example 9

This example shows the possible mechanism of action of arginine and phenylalanine in stabilizing dinoteumab. Deuterium-hydrogen exchange mass spectrometry (HDX-MS) is a sensitive and stable technique for characterizing protein-protein/ligand/excipient interactions. This method detects changes in backbone amide hydrogen bonds due to interaction with excipients.

Deuterium-deuterium exchange mass spectrometry (HDX-MS) was performed using dinosaumab (3mg/mL concentration) in 10mM acetate buffer (pH5.2) ("a 52") in the presence of L-arginine (formulation 35), L-phenylalanine (formulation 36), or L-glycine (formulation 37) and compared to dinosaumab formulation lacking any amino acid aggregation inhibitor (formulation 38). The experiments were carried out at 4 deg.C (using 75mM concentration of L-arginine, L-phenylalanine or L-glycine) and 37 deg.C (using 150mM concentration of L-arginine, L-phenylalanine or L-glycine). After analysis of more than 530 peptides, a small number of regions with significant conformational changes were identified. Several representative peptides from these regions are captured in fig. 19-30.

FIGS. 19-24 are graphs of% deuterium incorporation as a function of time (log (sec)) at 4 ℃ for light chain amino acids 28-33 (FIG. 19), light chain amino acids 108-116 (FIG. 20), light chain amino acids 125-132 (FIG. 21), heavy chain amino acids 47-59 (FIG. 22), heavy chain amino acids 243-253 (FIG. 23), and heavy chain amino acids 392-399 (FIG. 24) for each of formulations 35-38.

FIGS. 25-30 are graphs of% deuterium incorporation as a function of time (log (sec)) at 37 ℃ for light chain amino acids 28-33 (FIG. 25), light chain amino acids 108-117 (FIG. 26), light chain amino acids 124-131 (FIG. 27), heavy chain amino acids 47-59 (FIG. 28), heavy chain amino acids 242-253 (FIG. 29), and heavy chain amino acids 392-399 (FIG. 30) for each of formulations 35-38.

Although these data support that Arg and Gly have similar interaction effects on dinoteumab, Arg has a slightly stronger HDX footprint (conformational change) on dinoteumab: strong stabilization in the Fab LC 28-33 region; has weak stabilizing effect in Fab LC108-132, HC 47-59, Fc CH3 HC 392-399 regions; and weak destabilization in the Fc CH2243-253 region. Without intending to be bound by any particular theory, it is contemplated that the arginine hydrochloride effect is due to a combination of preferential exclusion from the interactions of the dinotezumab surface and weak surface, while glycine acts by preferential exclusion.

However, phenylalanine did not show significant structural perturbation to dinotezumab. Without intending to be bound by any particular theory, it is contemplated that the phenylalanine stabilizing effect may be accomplished by one or more of the following mechanisms: side chain interactions, as there was no effect on the peptide backbone (no HDX footprint); and/or cation-pi interactions with arginine/lysine side chains that do not affect the backbone hydrogen bonding network.

Example 10

This example shows the possible mechanism of action of phenylalanine stabilized dinotezumab.

To investigate the specific effect of Phe on dinotefuran, molecular dynamics simulations were performed. Specifically, the Fab domain of dinotezumab was solvated with excess Phe in the simulation cassette and simulated twice for 10 ns. In summary, Phe residues that bind to Fab for more than 90% of the time were selected for further analysis. Nine such cases were identified. In 5 of 9 long retention observations, Phe residues bound to the VH/VL interface (variable heavy chain/variable light chain) region as well as the CH/CL (constant heavy chain/constant light chain) region. In one example, it is believed that the Phe side chain interacts with the side chain of hydrophobic residues (e.g., V93, Y95, and W112 for heavy chains, and a44 and P45 for light chains) at the VH/VL interface. In another example, it is believed that the side chain loop of Phe interacts 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 interface of CH1 and CL. Without intending to be bound by any particular theory, this observation leads to the following notion: the specific role of Phe in mitigating dinosaumab aggregation is due to the interaction of the phenyl group with hydrophobic residues forming the interface of the heavy constant 1(Hc) chain and the light constant (Lc) chain (e.g., R30, G31, R32, and Y33 of CDR1 of the light chain, a52 of CDR2 of the light chain, and M106 of CDR3 of the heavy chain). This interaction is hypothesized to replace the previously hydrophobic surface with the relatively more charged (and therefore hydrophilic) surface of the NH3(+) and COO (-) groups from the Phe excipient.

Example 11

Stability assessments were performed on various anti-RANKL antibody constructs (isotypes IgG1, IgG2 and IgG 4). As described above, arginine hydrochloride and phenylalanine both minimized the starting HMWS and HWMS levels over time when compared to the acetate/sorbitol control formulation of dinosaumab, which is an IgG2 immunoglobulin. This assessment was done to compare the possibility 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 Regions (CDRs) when compared to dinotezumab, but contained different constant domain scaffolds. The different IgG2 constructs tested in this study had different CDRs relative to dinotezumab, but contained the same constant domain scaffold.

Each test antibody construct was purified and concentrated from 8mg/mL to 70mg/mL using a centrifuge. Each concentrated volume was divided into three aliquots and subsequently dialyzed against acetate buffer formulated with sorbitol, sorbitol/phenylalanine and sorbitol/arginine hydrochloride to prepare formulations 39 to 47 as described in table 9. Concentration using a centrifuge the post-dialysis samples were over concentrated to above 120 mg/mL. The antibody protein was diluted to 120mg/mL with the corresponding buffer.

TABLE 9

Final formulation comprised PS20 at a final concentration of 0.01% (w/v) and had the indicated pH value. The sorbitol and phenylalanine concentrations were estimated to be about 8.5% lower than the concentration of DF buffer. Arginine concentration was estimated to be about 12.5% lower than the concentration of DF buffer.

These formulations were filled into glass vial containers at a fill volume of 1.0 mL. These formulations were stored at 37 ℃ for 1 month. SE-UHPLC was used to assess aggregation inhibition and stability against aggregation inhibition over time (e.g. based on HMWS formation). Aggregation inhibition profiles of these formulations were compared under initial conditions and after a period of storage. The stability of the formulations within the immunoglobulin class was compared after storage.

Fig. 31, fig. 33 and fig. 35 (and the related tables 10, 12 and 14 below) show the percentage of HMWS monitored by SE-UHPLC at 37 ℃ in the case of immunoglobulin G (IgG 1, IgG2 and IgG4, respectively) as a function of formulation and time. Fig. 32, fig. 34 and fig. 36 (and related table 11, table 13 and table 15 below) show the percentage of low molecular weight substances (LMWS, e.g. protein fragmentation) monitored by SE-UHPLC at 37 ℃ in the case of immunoglobulin G (IgG 1, IgG2 and IgG4, respectively) as a function of formulation and time. Figure 37, figure 38 and figure 39 show the size exclusion chromatography overlay as a function of formulation after storage at 37 ℃ of t-4 w.

Table 10: comparison of% HMW, IgG1(A, B, C) at 37 ℃ for 4 weeks

Table 11: comparison of% LMWS, IgG1(A, B, C) at 37 ℃ for 4 weeks

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

Table 13: comparison of% LMWS, IgG2(D, E, F) at 37 ℃ for 4 weeks

Table 14: comparison of% HMW, IgG4(G, H, I) at 37 ℃ for 4 weeks

Table 15: comparison of% LMWS, IgG4(G, H, I) at 37 ℃ for 4 weeks

As shown in fig. 31 and 32, IgG1 molecules with similar CDR regions as the previous dinotezumab sample showed an approximate 0.2% reduction in HMWS with the addition of phenylalanine when compared to the acetate/sorbitol control formulation. IgG2 samples with different CDRs and as depicted in fig. 33 and fig. 34 showed an increase in HMWS in the acetate/phenylalanine/sorbitol formulation when compared to the control acetate/sorbitol formulation. As shown in fig. 35 and fig. 36, the acetate/sorbitol and acetate/phenylalanine/sorbitol formulations have similar stability for the IgG4 sample type, with acetate/sorbitol/arginine having higher HMWS formation. In all cases using the IgG1, IgG2, and IgG4 sample types, the acetate/sorbitol/arginine-containing formulations showed increased HMWS degradation when compared to the acetate/sorbitol (control) and acetate/phenylalanine/sorbitol formulations.

Since protein fragmentation was greatly increased in the acetate/arginine/sorbitol formulations as depicted in fig. 37 and fig. 38, the relationship between fragments and antibody isotypes is shown in fig. 32, fig. 34, and fig. 36. It has been shown in the literature that monoclonal antibody fragmentation-mediated aggregation may result from antibodies stored at 37 ℃ [ Perico n. et al, j.pharm.sci. [ journal of pharmaceutical science ] (2009)98, pages 3031 to 3042 ]. This mechanism was possible in this evaluation because fragmentation was greatest in the acetate/arginine/sorbitol formulation. Fragmentation in the acetate/phenylalanine/sorbitol formulation is minimized, potentially resulting in less HMWS material. The IgG4 sample types were not accelerated to fragment or aggregate.

Based on the data collected in this study, as well as previous data, i.e., molecular modeling data collected using dinotezumab, a strong correlation between the amino acid sequence of the CDR and the relative effect of phenylalanine on reducing HMWS can be established. Reduction of HMW species was observed in dinosaumab (IgG2) and IgG1 variants with the same CDR amino acids, but no reduction of HMWs was observed in IgG2 variants with different CDR domains. It will be found that the amino acid sequences contained within the CDR domains are susceptible to interaction with phenylalanine and subsequent inhibition of aggregation. The IgG4 molecule also had identical CDR regions when compared to dinotezumab, but minimal changes in aggregation were detected during the study. The IgG4 molecule differs from the IgG1 and IgG2 versions primarily in its hinge amino acid length and its functionally active structure. Because IgG1 and IgG2 antibody isotypes have extended structures that are typically described as "Y," the IgG4 Fab CH1 domain interacts with the CH2 domain to form a more compact structure [ aalbese r.c. et al, Immunology ] (2002),105, pages 9 to 19 ]. This compact structure inhibits fragmentation and aggregation reactions commonly seen in the IgG1 and IgG2 formats.

Example 12

A study was conducted to monitor the stability of dinotezumab formulated as described below and in combination with table 16 (formulations 51 to 55). The various diafiltration buffers used to generate the final formulation of Ph 5.1 at a concentration of 120mg/Ml dinoteuzumab differed in acetate concentration and initial Ph values. In addition, sorbitol levels were adjusted to maintain isotonicity (about 300mOsm/Kg) of the final product. 70mg/mL dinosaumab was diafiltered for each buffer no more than 7 diafiltration volumes, followed by ultrafiltration to about 180gm/mL, and diluted with diafiltration buffer and polysorbate to 120mg/mL dinosaumab concentration and 0.01% polysorbate 20. Stability was assessed using SE-UHPLC after storage at 37 ℃ and showed that the stability of dinotezumab in these formulations was highly similar. As the initial acetate concentration increases, the initial HMW species decreases slightly. In contrast, the aggregation rate was slightly increased in formulations containing lower levels of acetate.

TABLE 16

Final formulation comprised 120mg/mL dinotezumab and PS20 at a final concentration of 0.01% (w/v), and pH 5.1.

Example 13

The following examples report the results of studies of the effect of arginine on the chemical denaturation stability of dinotefuran at the following three different pH values: 4.5, 4.8 and 5 (or 5.2).

All chemical denaturation experiments were performed using a Uncariained Labs instrument HUNK with a fluorescence detector. The excitation wavelength was 280nm and emission scans between 300 and 500nm were recorded. For each denaturation experiment, proteins, buffer and denaturant (guanidine hydrochloride) were dispensed into 36 wells with a linear increase in denaturant concentration to obtain a 36-point curve for each condition. The data points were fitted using curve fitting software supplied by the instrument manufacturer (Uncariamed Labs, Inc.). The two-state model is used because only a single transition (native) has been demonstrated to existdenated). Experiments were performed with 0-6M urea in 10mM acetate 5.0% (w/v) sorbitol and titrated to the desired pH 4.5, 4.8 or 5 (5.2). In all experiments, the concentration of the dinotefuran protein was 7 mg/mL.

Fig. 40 shows the isothermal chemical denaturation curves for dinotefuran in the absence of arginine at pH 4.5, 4.8, and 5.0. C of chemical denaturants required for 50% unfolding at pH tested in the absence of arginine_1/2Similarly.

Fig. 41 shows isothermal chemical denaturation curves for dinotefuran in the presence of 75mM arginine hydrochloride at pH 4.5, 4.8 and 5.2. Chemical denaturation stability was significantly increased at pH5.2 when compared to pH 4.8 and 4.5. Denaturant guanidine hydrochloride C at pH5.2 relative to lower pH values_1/2Increase by 1M. Thus, the protective properties of arginine are surprising and highly dependent on pH.

Example 14

The following example provides the results of a study of the effect of arginine and phenylalanine on the stability of high concentration dinotezumab formulations in syringes over time.

In previous studies, arginine hydrochloride and phenylalanine were identified to reduce the initial initiation level and HMWS formation rate of dinotezumab. In this study, formulations containing arginine hydrochloride, phenylalanine, and a combination of arginine hydrochloride and phenylalanine were evaluated for stabilization of solutions containing dinotezumab (120mg/mL) and stored in syringes at two different temperatures for three months.

The formulations tested are described in table 17 below. To prepare formulations 56 to 59, dinosaumab (70mg/mL) in acetate (ph5.2) was diafiltered against Diafiltration (DF) buffer described below to 8 diafiltration volumes to ensure complete buffer exchange. The material was then ultrafiltered to above 180mg/mL, followed by dilution to 120mg/mL and addition of polysorbate 20 to a final concentration of 0.01%. Formulation 56 was considered a control formulation. The acetate, arginine hydrochloride, and phenylalanine values listed are for DF buffer and, when no other counter ions are present, provide estimated levels in the final composition of 120mg/mL dinotezumab, taking into account excipient exclusion and acetate co-concentration. Viscosities at 5 ℃ and 25 ℃ were measured using a Paar modular compact rheometer at shear rates up to 1000s-1 (reciprocal of second). These formulations were filled into glass pre-filled syringes (PFS) at a fill volume of 1.0 mL. The syringe parallel groups were stored at a temperature of 25 ℃ for 3 months and 37 ℃ for 2 months, respectively. Stability (e.g., based on HMWS formation) was assessed using SE-UHPLC.

TABLE 17

Each final formulation contained 120mg/mL dinosaumab and 0.01% PS20 and the pH values indicated in the abbreviated formulation names

Fig. 42 and 43 show the percentage of HMWS monitored by SE-UHPLC as a function of formulation and time at 25 ℃ for 3 months and 37 ℃ for 2 months, respectively.

Tables 18-21 show the same data in tabular form as well as the increase in HMWS relative to the initial HMWS level.

Table 18: the% HMWS levels over 12 weeks at 25 ℃

Table 19: HMWS increase over 12 weeks at 25 ℃

Table 20: the% HMWS levels over 8 weeks at 37 ℃

Table 21: HMWS increase at 37 ℃ over 8 weeks

This example shows that the addition of arginine, phenylalanine, and combinations thereof each reduces the level of initial HMWS (t ═ 0) in high concentration dinoteumab formulations. At 25 ℃, there was a reduction in the increase in HMWS in phenylalanine formulation 59 compared to control formulation 56. At 37 ℃, formulations 57 and 59 had reduced HMWS formation compared to control sorbitol formulation 56. The formulation containing arginine hydrochloride and phenylalanine formed HMWS at higher rates at 37 ℃ relative to other formulations, indicating that the combination of excipients destabilizes the dinotezumab in this formulation at this higher temperature.

The foregoing description is provided for clarity of understanding only, and no unnecessary limitations are to be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification, where compositions are described as comprising components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless otherwise described. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

Practice of the methods disclosed herein and the individual steps thereof can be performed manually and/or with the assistance of or automation provided by electronic equipment. Although the methods have been described with reference to particular embodiments, those of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may 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. In addition, some individual steps may be combined, omitted, or further subdivided into other steps.

All patents, publications, and references cited herein are incorporated by reference in their entirety. In case of conflict between the present disclosure and the incorporated patents, publications and references, the present disclosure should be taken as a priority.

Claims (208)

1. An aqueous pharmaceutical formulation comprising (i) a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof and (ii) an amino acid aggregation inhibitor.

2. An aqueous pharmaceutical formulation comprising (i) a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a concentration of greater than 70mg/mL, wherein the aqueous pharmaceutical formulation has a pH value in the range of about 5.0 to less than 5.2.

3. The aqueous pharmaceutical formulation of claim 1 or 2, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID No. 5.

4. The aqueous pharmaceutical formulation of claim 3, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO 6.

5. The aqueous pharmaceutical formulation of claim 4, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO 10.

6. The aqueous pharmaceutical formulation of any one of claims 3 to 5, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises (i) a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO 7; (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO. 8; (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO. 9; or (iv) any combination thereof.

7. The aqueous pharmaceutical formulation of any one of claims 3 to 6, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises (A) a light chain variable domain comprising the light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5, a light chain variable domain comprising the light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain variable domain comprising the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (B) a heavy chain variable domain comprising the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:8, a heavy chain variable domain comprising the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

8. The aqueous pharmaceutical formulation of any one of claims 1 to 7, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises:

(A) a light chain variable domain selected from the group consisting of:

i. a light chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID No. 1;

a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO 19;

a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO. 19; or

(B) A heavy chain variable domain selected from the group consisting of:

i. a heavy chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID No. 2;

a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID No. 20;

a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO. 20; or

(C) A light chain variable domain of (A) and a heavy chain variable domain of (B).

9. The aqueous pharmaceutical formulation of any one of claims 1 to 8, wherein the anti-RANKL antibody is a fully human antibody, a humanized antibody or a chimeric antibody.

10. The aqueous pharmaceutical formulation of any one of claims 1 to 8, wherein the antigen-binding moiety is Fab, Fab ', F (ab') 2, or single chain Fv.

11. The aqueous pharmaceutical formulation of any one of claims 1 to 8, wherein the anti-RANKL antibody is an IgG₁、IgG₂Or IgG₄An antibody, optionally comprising a kappa light chain.

12. The aqueous pharmaceutical formulation of claim 11, wherein the anti-RANKL antibody comprises the sequence of SEQ ID NO 15.

13. The aqueous pharmaceutical formulation of claim 12, wherein the anti-RANKL antibody comprises the sequence of SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18.

14. The aqueous pharmaceutical formulation of any one of claims 1 to 13, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises:

(A) a light chain selected from the group consisting of:

i. a light chain comprising an amino acid sequence at least 80% identical to SEQ ID NO 3 or SEQ ID NO 13;

a light chain comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO 21 or 23;

a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO. 21 or 23; or

(B) A heavy chain selected from the group consisting of:

i. a heavy chain comprising an amino acid sequence at least 80% identical to SEQ ID NO 4 or SEQ ID NO 14;

a heavy chain comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO 22 or 24;

a heavy chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO. 22 or 24; or

(C) A light chain variable domain of (A) and a heavy chain variable domain of (B).

15. The aqueous pharmaceutical formulation of any one of claims 1 and 3 to 14, wherein the concentration of the antibody or antigen-binding portion thereof is greater than 70mg/mL, optionally in the range of about 70mg/mL to about 300 mg/mL.

16. The aqueous pharmaceutical formulation of any one of the preceding claims, wherein the concentration of the antibody or antigen-binding portion thereof is in the range of more than 70mg/mL to about 300mg/mL, optionally in the range of more than about 70mg/mL to about 200 mg/mL.

17. The aqueous pharmaceutical formulation of claim 15 or 16, wherein the concentration of the antibody, or antigen-binding portion thereof, is in the range of about 100 to about 140 mg/mL.

18. The aqueous pharmaceutical formulation of any one of the preceding claims, wherein the concentration of the antibody or antigen-binding portion thereof is about 120mg/mL ± 12 mg/mL.

19. The aqueous pharmaceutical formulation of claims 2-18, further comprising an amino acid aggregation inhibitor.

20. The aqueous pharmaceutical formulation of any one of claims 1 and 3-19, wherein the amino acid aggregation inhibitor comprises an amino acid comprising a charged side chain, an aromatic amino acid, or a hydrophobic amino acid.

21. The aqueous pharmaceutical formulation of claim 20, wherein the amino acid comprising a charged side chain is an amino acid comprising a positively charged side chain.

22. The aqueous pharmaceutical formulation of claim 21, wherein the amino acid comprising a positively charged side chain comprises a side chain structure of formula I or formula II:

wherein n is 1 to 7, wherein R₁And R₂Each of which is independently selected from the group consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、NH、NH₂(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₇And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₇Is H or OH, wherein optionally R₁And R₂One of them being a free amino group (-NH)₃ ⁺)；

Wherein m is 1 to 7, wherein R₃And R₄Each of which is independently selected from group a consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₈And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₈Is H or OH, wherein R₅Optionally present and when present selected from group A, optionally wherein R₃And R₄And R₅Each of which is H.

23. The aqueous pharmaceutical formulation of claim 22, wherein n is in the range of 2 to 4.

24. The aqueous pharmaceutical formulation of claim 23, wherein R₁Is NH or NH₂。

25. The aqueous pharmaceutical formulation of claim 24, wherein R₂Is NH₂Or NH₃ ⁺。

26. The aqueous pharmaceutical formulation of claim 25, wherein the amino acid comprising a positively charged side chain is arginine.

27. The aqueous pharmaceutical formulation of claim 22, wherein m is in the range of 3 to 5.

28. The aqueous pharmaceutical formulation of claim 27, wherein R₃And R₄And R₅Each of which is optionally present, wherein when present, R₅Is H.

29. The aqueous pharmaceutical formulation of claim 28, wherein the amino acid comprising a positively charged side chain is lysine.

30. The aqueous pharmaceutical formulation of any one of claims 21 to 29, wherein the amino acid comprising a positively charged side chain is present in the formulation in the form of a salt.

31. The aqueous pharmaceutical formulation of claim 30, wherein the salt is a hydrochloric acid (HCl) salt.

32. The aqueous pharmaceutical formulation of claim 31, comprising L-arginine hydrochloride or L-lysine hydrochloride.

33. The aqueous pharmaceutical formulation of claim 20, wherein the aromatic amino acid comprises phenyl or indole.

34. The aqueous pharmaceutical formulation of claim 33, wherein the aromatic amino acid comprises a C between the alpha carbon and the phenyl or indole₁-C₆An alkyl chain.

35. The aqueous pharmaceutical formulation of claim 34, wherein the alkyl chain is C₁-C₃An alkyl chain.

36. The aqueous pharmaceutical formulation of claim 35, wherein the aromatic amino acid is L-phenylalanine.

37. The aqueous pharmaceutical formulation of claim 35, wherein the aromatic amino acid is L-tryptophan.

38. The aqueous pharmaceutical formulation of claim 20, wherein the hydrophobic amino acid has a score of more than about 2.5 on the katter-dolite hydrophobicity scale.

39. The aqueous pharmaceutical formulation of claim 20 or 38, wherein the hydrophobic amino acid comprises a branched or straight chain C-containing₂-C₁₂Alkyl or C₄-C₈Cycloalkyl, nitrogen heteroatom containing C₄-C₈A side chain of a heterocycle, optionally wherein the heterocycle is imidazole, pyrrole, or indole.

40. The aqueous pharmaceutical formulation of claim 39, wherein the hydrophobic amino acid comprises C₃-C₈An alkyl group.

41. The aqueous pharmaceutical formulation of claim 40, wherein the hydrophobic amino acid comprises a branched chain C₃Alkyl or branched C₄An alkyl group.

42. The aqueous pharmaceutical formulation of claim 41, wherein the hydrophobic amino acid is L-valine, L-leucine, or L-isoleucine.

43. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising about 5mM to about 300mM amino acid aggregation inhibitor, optionally comprising about 25mM to about 90mM amino acid aggregation inhibitor.

44. The aqueous pharmaceutical formulation of claim 43, comprising about 5mM to about 150mM amino acid aggregation inhibitor when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally L-arginine.

45. The aqueous pharmaceutical formulation of claim 44, comprising about 30mM to about 80mM amino acid aggregation inhibitor.

46. The aqueous pharmaceutical formulation of claim 43, comprising about 5mM to about 180mM amino acid aggregation inhibitor when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine.

47. The aqueous pharmaceutical formulation of claim 46, when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine, it comprises about 5mM to about 100mM amino acid aggregation inhibitor, optionally about 20mM to about 50mM amino acid aggregation inhibitor.

48. The aqueous pharmaceutical formulation of claim 43, comprising about 5mM to about 300mM amino acid aggregation inhibitor when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine.

49. The aqueous pharmaceutical formulation of claim 48, comprising about 5mM to about 200mM amino acid aggregation inhibitor, optionally about 20mM to about 50mM amino acid aggregation inhibitor, when the amino acid aggregation inhibitor is a hydrophobic amino acid, optionally L-valine, L-isoleucine, or L-leucine.

50. The aqueous pharmaceutical formulation of any one of claims 43 to 49, comprising:

a. about 30mM to about 80mM L-arginine hydrochloride;

b. about 20mM to about 50mM L-phenylalanine;

c. about 20mM to about 50mM L-tryptophan;

d. about 30mM to about 80mM L-lysine hydrochloride;

e. about 20mM to about 50mM L-leucine;

f. about 20mM to about 50mM L-isoleucine;

g. about 20mM to about 50mM L-valine; or

h. Any combination thereof.

51. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising only one amino acid aggregation inhibitor.

52. The aqueous pharmaceutical formulation of any one of the preceding claims, wherein 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: 200.

53. The aqueous pharmaceutical formulation of claim 52, wherein the molar ratio is about 20: about 90.

54. The aqueous pharmaceutical formulation of any one of the preceding claims, wherein when the amino acid aggregation inhibitor is an amino acid comprising 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.

55. The aqueous pharmaceutical formulation of claim 54, wherein the molar ratio is about 45: 180.

56. The aqueous pharmaceutical formulation of any one of the preceding claims, further comprising a tonicity modifier, optionally selected from the group consisting of: sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof.

57. The aqueous pharmaceutical formulation of claim 56, wherein the tonicity modifier comprises sorbitol.

58. The aqueous pharmaceutical formulation of claim 56 or 57, comprising about 1.0 (w/w)% to about 5.0 (w/w)% tonicity modifier.

59. The aqueous pharmaceutical formulation of claim 58, comprising about 2.0 (w/w)% to about 5.0 (w/w)% sorbitol or about 3.5 (w/w)% to about 5.0 (w/w)% sorbitol or about 4.0% (w/w) to about 5.0 (w/w)% sorbitol.

60. The aqueous pharmaceutical formulation of any one of claims 1 to 59, which is free of sorbitol and optionally free of any tonicity modifier.

61. The aqueous pharmaceutical formulation of any one of the preceding claims, further comprising a surfactant.

62. The aqueous pharmaceutical formulation of claim 61, wherein the surfactant is selected from the group consisting of: a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 80), or one or more alkylaryl polyethers such as an oxyethylated alkylphenol (e.g., oxyethylated alkylphenol)X-100), or one or more poloxamers (e.g., one or more poloxamers)Such asF68) And combinations thereof.

63. The aqueous pharmaceutical formulation of claim 62, wherein the surfactant is polysorbate 20.

64. The aqueous pharmaceutical formulation of any one of claims 61 to 63, comprising at least about 0.004 (w/v)% surfactant and optionally comprising less than 0.15 (w/v)% surfactant.

65. The aqueous pharmaceutical formulation of claim 64, comprising about 0.005 (w/v)% to about 0.015 (w/v)% surfactant.

66. The aqueous pharmaceutical formulation of any one of the preceding claims, further comprising a buffer, optionally wherein the buffer is centered at a range of about pH 4.0 to about pH 5.5 at 25 ℃.

67. The aqueous pharmaceutical formulation of claim 66, wherein the buffer has a pKa within a pH unit of pH 5.0-5.2 at 25 ℃.

68. The aqueous pharmaceutical formulation of claim 66 or 67, comprising about 5mM to about 60mM buffer.

69. The aqueous pharmaceutical formulation of claim 68, comprising about 5mM to about 50mM buffer.

70. The aqueous pharmaceutical formulation of claim 69, comprising about 9mM to about 45mM buffer.

71. The aqueous pharmaceutical formulation of any one of claims 66-70, wherein the buffer is acetate or glutamate.

72. The aqueous pharmaceutical formulation of any one of the preceding claims, having a pH value in the range of about 5.0 to 5.19.

73. The aqueous pharmaceutical formulation of claim 72, having a pH value in the range of about 5.0 to about 5.15.

74. The aqueous pharmaceutical formulation of claim 73, having a pH value in the range of about 5.0 to about 5.1.

75. The aqueous pharmaceutical formulation of any one of claims 1 and 3 to 74, 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 less than 5.2 or about 5.0 to 5.19 or about 5.0 to about 5.15 or about 5.0 to about 5.1.

76. The aqueous pharmaceutical formulation of claim 75, having a pH of about 5.1.

77. The aqueous pharmaceutical formulation of any one of the preceding claims, having a viscosity of no more than about 6cP at 5 ℃, optionally wherein the viscosity is about 4.5cP to about 5.5 cP.

78. The aqueous pharmaceutical formulation of any one of the preceding claims, having a viscosity of less than about 13cP at 25 ℃.

79. The aqueous pharmaceutical formulation of claim 78, having a viscosity in the range of about 2.0cP to about 10cP, optionally about 2.5cP to about 4 cP.

80. The aqueous pharmaceutical formulation of any one of the preceding claims, having a conductivity in the range of about 500 μ S/cm to about 5500 μ S/cm, optionally wherein the conductivity is in the range of about 2500 μ S/cm to about 5500 μ S/cm when the formulation comprises an amino acid containing a positively charged side chain, or in the range of about 500 μ S/cm to about 2000 μ S/cm when the formulation comprises an aromatic amino acid or lacks an amino acid aggregation inhibitor.

81. The aqueous pharmaceutical formulation of any one of the preceding claims, having an osmolality in the range of about 200 to about 500mOsm/kg or about 225 to about 400mOsm/kg or about 250 to about 350 mOsm/kg.

82. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of the antibody as measured by SE-UHPLC after storage at about 2 ℃ to about 8 ℃ for at least 12 months, 24 months or 36 months.

83. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the main peak of antibody after storage for about 1 month at about 20 ℃ to about 30 ℃, as measured by SE-UHPLC.

84. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of antibodies after a first storage at about 2 ℃ to about 8 ℃ for at least 12 months, 24 months or 36 months and a second storage at about 20 ℃ to about 30 ℃ for about 1 month, as measured by SE-UHPLC.

85. The aqueous pharmaceutical formulation of any one of the preceding claims, comprising less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the main peak of the antibody after storage for about one month at 37 ℃ or 3 months at 30 ℃, as measured by SE-UHPLC.

86. A container, optionally a vial, a pre-filled syringe (PFS), or a glass container, containing the aqueous pharmaceutical formulation of any one of claims 1 to 85.

87. The container of claim 86, containing about 1mL or less of the formulation, optionally about 0.5 mL.

88. A method of manufacturing a stable aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof, comprising combining the anti-RANKL monoclonal antibody or antigen-binding portion thereof at a concentration above 70mg/mL with an amino acid aggregation inhibitor, a buffer, a surfactant, and optionally a tonicity modifier.

89. The method of claim 88, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID No. 5.

90. The method of claim 89, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable domain comprising a light chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO 6.

91. The method of claim 90, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain variable domain comprising a heavy chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO 10.

92. The method of any one of claims 89 to 91, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises (i) a light chain variable domain comprising a light chain CDR3 sequence comprising the amino acid sequence set forth in SEQ ID No. 7; (ii) a heavy chain variable domain comprising a heavy chain CDR1 sequence comprising the amino acid sequence set forth in SEQ ID NO. 8; (iii) a heavy chain variable domain comprising a heavy chain CDR2 sequence comprising the amino acid sequence set forth in SEQ ID NO. 9; or (iv) combinations thereof.

93. The method of any one of claims 88 to 92, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises (A) a light chain variable domain comprising the light chain CDR1 comprising the amino acid sequence of SEQ ID NO 5, a light chain variable domain comprising the light chain CDR2 comprising the amino acid sequence of SEQ ID NO 6, and a light chain variable domain comprising the light chain CDR3 comprising the amino acid sequence of SEQ ID NO 7; and (B) a heavy chain variable domain comprising the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:8, a heavy chain variable domain comprising the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a heavy chain variable domain comprising the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

94. The method of any one of claims 88 to 93, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises:

(A) a light chain variable domain selected from the group consisting of:

i. a light chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID No. 1;

a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO 19;

a light chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO. 19; or

(B) A heavy chain variable domain selected from the group consisting of:

i. a heavy chain variable domain comprising an amino acid sequence at least 80% identical to SEQ ID No. 2;

a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID No. 20;

a heavy chain variable domain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of the polynucleotide consisting of SEQ ID NO. 20; or

(C) A light chain variable domain of (A) and a heavy chain variable domain of (B).

95. The method of any one of claims 88 to 94, wherein the anti-RANKL antibody is a fully human antibody, a humanized antibody or a chimeric antibody.

96. The method of any one of claims 88 to 95, wherein the antigen binding portion is a Fab, Fab ', F (ab') 2, or single chain Fv.

97. The method of any of claims 88 to 96, wherein the anti-RANKL antibody is an IgG₁、IgG₂Or IgG₄An antibody, optionally wherein the antibody comprises a kappa light chain.

98. The method of claim 97, wherein the anti-RANKL antibody comprises the sequence of SEQ ID NO 15.

99. The method of claim 98, wherein the anti-RANKL antibody comprises the sequence of SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18.

100. The method of any one of claims 88 to 99, wherein the anti-RANKL antibody or antigen-binding portion thereof comprises:

(A) a light chain selected from the group consisting of:

i. a light chain comprising an amino acid sequence at least 80% identical to SEQ ID NO 3 or SEQ ID NO 13;

a light chain comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO 21 or 23;

a light chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO. 21 or 23; or

(B) A heavy chain selected from the group consisting of:

i. a heavy chain comprising an amino acid sequence at least 80% identical to SEQ ID NO 4 or SEQ ID NO 14;

a heavy chain comprising an amino acid sequence encoded by a polynucleotide sequence at least 80% identical to SEQ ID NO 22 or 24;

a heavy chain comprising an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide consisting of SEQ ID NO. 22 or 24; or

(C) A light chain variable domain of (A) and a heavy chain variable domain of (B).

101. The method of any one of claims 88 to 100, comprising combining about 10mg/mL to about 300mg/mL of antibody, or antigen-binding portion thereof, with the amino acid aggregation inhibitor, buffer, surfactant, and optionally the tonicity modifier.

102. The method of any one of claims 88 to 101, wherein the stable aqueous pharmaceutical formulation comprises more than 70mg/mL to about 200mg/mL of an antibody or antigen-binding portion thereof.

103. The method of claim 101 or 102, wherein the stable aqueous pharmaceutical formulation comprises about 100 to about 140mg/mL of antibody or antigen-binding portion thereof.

104. The method of any one of claims 88 to 103, wherein the stable aqueous pharmaceutical formulation comprises about 120mg/mL ± 12mg/mL antibody or antigen-binding portion.

105. The method of claim 104, wherein the stable aqueous pharmaceutical formulation comprises about 120mg/mL ± 5mg/mL antibody or antigen-binding portion.

106. The method of any one of claims 88 to 105, wherein the inhibitor of amino acid aggregation comprises an amino acid comprising a charged side chain, an aromatic amino acid, or a hydrophobic amino acid.

107. The method of claim 106, wherein the amino acid comprising a charged side chain is an amino acid comprising a positively charged side chain.

108. The method of claim 107, wherein the amino acid comprising a positively charged side chain comprises a side chain structure of formula I or formula II:

wherein n is 1 to 7, wherein R₁And R₂Each of which is independently selected from the group consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、NH、NH₂(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₇And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₇Is H or OH, wherein optionally R₁And R₂One of them being a free amino group (-NH)₃ ⁺)；

Wherein m is 1 to 7, wherein R₃And R₄Each of which is independently selected from group a consisting of: H. c₁-C₁₈Alkyl, (C)₁-C₁₈Alkyl) OH, (C)₁-C₁₈Alkyl) NH₂、(C₁-C₁₈Alkyl) SH, (C)₀-C₄Alkyl) (C₃-C₆) Cycloalkyl group, (C)₀-C₄Alkyl) (C₂-C₅Heterocycle), (C)₀-C₄Alkyl) (C₆-C₁₀Aryl) R₈And (C)₁-C₄Alkyl) (C₃-C₉Heteroaryl) in which R is₈Is H or OH, wherein R₅Optionally present and when present selected from group A, optionally wherein R₃And R₄And R₅Each of which is H.

109. The method of claim 108, wherein n is in the range of 2 to 4.

110. The method as recited in claim 109, wherein R₁Is NH or NH₂。

111. The method as recited in claim 110, wherein R₂Is NH₂Or NH₃ ⁺。

112. The method of claim 111, wherein the amino acid comprising a positively charged side chain is arginine.

113. The method of claim 108, wherein m is in the range of 3 to 5.

114. The method as recited in claim 113, wherein R₃And R₄And R₅Each of which is optionally present, wherein when present, R₅Is H.

115. The method of claim 114, wherein the amino acid comprising a positively charged side chain is lysine.

116. The method of any one of claims 107 to 115, wherein the amino acid comprising a positively charged side chain is present in the formulation in the form of a salt.

117. The method of claim 116, wherein the salt is a hydrochloric acid (HCl) salt.

118. The method of claim 117, comprising L-arginine hydrochloride or L-lysine hydrochloride.

119. The method of claim 106, wherein the aromatic amino acid comprises phenyl or indole.

120. The method of claim 119, wherein the aromatic amino acid comprises a C between the alpha carbon and the phenyl or indole₁-C₆An alkyl chain.

121. The method of claim 120, wherein the alkyl chain is C₁-C₃An alkyl chain.

122. The method of claim 121, wherein the aromatic amino acid is L-phenylalanine.

123. The method of claim 121, wherein the aromatic amino acid is L-tryptophan.

124. The method of claim 106, wherein the hydrophobic amino acid has a score of more than about 2.5 on the katter-dolite hydrophobicity scale.

125. The method of claim 106 or 124, wherein the hydrophobic amino acid comprises a C comprising a branched or straight chain₂-C₁₂Alkyl or C₄-C₈Cycloalkyl, nitrogen heteroatom containing C₄-C₈A side chain of a heterocycle, optionally wherein the heterocycle is imidazole, pyrrole, or indole.

126. The method of claim 125, wherein the hydrophobic amino acid comprises C₃-C₈An alkyl group.

127. The method of claim 126, wherein the hydrophobic amino acid comprises a branched chain C₃Alkyl or branched C₄An alkyl group.

128. The method of claim 127, wherein the hydrophobic amino acid is L-valine, L-leucine, or L-isoleucine.

129. The method of any one of claims 88 to 128, comprising combining about 5mM to about 300mM amino acid aggregation inhibitor, optionally about 15mM to about 200mM or about 25mM to about 90mM amino acid aggregation inhibitor, with the antibody or antigen-binding portion.

130. The method of claim 129, comprising combining about 5mM to about 150mM of an inhibitor of amino acid aggregation with the antibody or antigen-binding portion, wherein the inhibitor of amino acid aggregation is an amino acid comprising a positively charged side chain, optionally L-arginine.

131. The method of claim 130, comprising combining about 30mM to about 80mM of an inhibitor of amino acid aggregation with the antibody or antigen-binding portion.

132. The method of claim 129, comprising combining about 5mM to about 180mM of an inhibitor of amino acid aggregation with the antibody or antigen-binding portion, wherein the inhibitor of amino acid aggregation is an aromatic amino acid, optionally L-phenylalanine.

133. The method of claim 132, comprising combining about 5mM to about 100mM amino acid aggregation inhibitor, optionally about 20mM to about 50mM amino acid aggregation inhibitor, with the antibody or antigen-binding portion.

134. The method of claim 129, comprising combining about 5mM to about 300mM of an inhibitor of amino acid aggregation with the antibody or antigen-binding portion, wherein the inhibitor of amino acid aggregation is a hydrophobic amino acid, optionally L-valine, L-isoleucine or L-leucine.

135. The method of claim 134, comprising combining about 5mM to about 200mM amino acid aggregation inhibitor, optionally about 20mM to about 50mM amino acid aggregation inhibitor, with the antibody or antigen-binding portion.

136. The method of any one of claims 88 to 135, comprising combining the antibody or antigen binding portion with:

a. about 30mM to about 80mM L-arginine hydrochloride;

b. about 20mM to about 50mM L-phenylalanine;

c. about 20mM to about 50mM L-tryptophan;

d. about 30mM to about 80mM L-lysine hydrochloride;

e. about 20mM to about 50mM L-leucine;

f. about 20mM to about 50mM L-isoleucine;

g. about 20mM to about 50mM L-valine; or

h. Any combination thereof.

137. The method of any one of claims 88 to 136, comprising combining the antibody or antigen binding portion with only one amino acid aggregation inhibitor.

138. The method of any one of claims 88 to 137, wherein when the amino acid aggregation inhibitor is an aromatic amino acid, optionally L-phenylalanine, the aqueous pharmaceutical formulation comprises the antibody and amino acid aggregation inhibitor in a molar ratio of the amino acid aggregation inhibitor to the antibody of about 10: 200.

139. The process of claim 138, wherein the molar ratio is about 20: about 90.

140. The method of any one of claims 88 to 139, wherein when the amino acid aggregation inhibitor is an amino acid comprising a positively charged side chain, optionally L-arginine, the aqueous pharmaceutical formulation comprises the antibody and amino acid aggregation inhibitor in a molar ratio of the amino acid aggregation inhibitor to the anti-RANKL antibody of about 20: 300.

141. The process of claim 140, wherein the molar ratio is about 45: 180.

142. The method of any one of claims 88 to 141 wherein the antibody or antigen binding portion is combined with a tonicity modifier selected from the group consisting of: sorbitol, mannitol, sucrose, trehalose, glycerol, and combinations thereof.

143. The method of claim 142, wherein the tonicity modifier comprises sorbitol.

144. The method of claim 142 or 143, comprising about 1.0 (w/w)% to about 5.0 (w/w)% tonicity modifier.

145. The method of claim 144, comprising about 2.0 (w/w)% to about 5.0 (w/w)% sorbitol or about 3.5 (w/w)% to about 5.0 (w/w)% sorbitol or about 4.0% (w/w) to about 5.0 (w/w)% sorbitol.

146. The method of any one of claims 88 to 145, which is free of sorbitol and optionally free of any tonicity modifier.

147. The method of any one of claims 88 to 146, wherein the surfactant is selected from the group consisting of: a polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 80), or one or more alkylaryl polyethers such as an oxyethylated alkylphenol (e.g., oxyethylated alkylphenol)X-100), or one or more poloxamers (e.g., one or more poloxamers)Such asF68) And combinations thereof.

148. The method of claim 147, wherein the surfactant is a polyoxyethylene sorbitan fatty acid ester.

149. The method of claim 148, wherein the surfactant is polysorbate 20.

150. The method of any one of claims 147 to 149 comprising at least about 0.004 (w/v)% surfactant and optionally less than 0.15 (w/v)% surfactant.

151. The method of claim 150, comprising about 0.005 (w/v)% to about 0.015 (w/v)% surfactant.

152. The method of any one of claims 88 to 151, comprising combining the antibody or antigen-binding portion with a buffer, wherein the buffer is centered at a range of about pH 4.0 to about pH 5.5 at 25 ℃.

153. The method of claim 152, wherein the buffer has a pKa at 25 ℃ within a pH unit of pH 5.0-5.2.

154. The method of claim 152 or 153, wherein the aqueous pharmaceutical formulation comprises about 5mM to about 60mM buffer.

155. The method of claim 154, wherein the aqueous pharmaceutical formulation comprises about 5mM to about 50mM buffer.

156. The method of claim 155, the aqueous pharmaceutical formulation comprising about 9mM to about 45mM buffer.

157. The method of any one of claims 152-156, wherein the buffer is acetate or glutamate.

158. The method of any one of claims 88 to 157, wherein the aqueous pharmaceutical formulation has a pH value within the range of about 5.0 to 5.19.

159. The method of claim 158, wherein the aqueous pharmaceutical formulation has a pH value within the range of about 5.0 to about 5.15.

160. The method of claim 159, wherein the aqueous pharmaceutical formulation has a pH value within the range of about 5.0 to about 5.1.

161. The method of any one of claims 88 to 160, wherein the aqueous pharmaceutical formulation has 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 less than 5.2 or about 5.0 to 5.19 or about 5.0 to about 5.15 or about 5.0 to about 5.1.

162. The method of claim 161, wherein the aqueous pharmaceutical formulation has a pH of about 5.1.

163. The method of any one of claims 88 to 162, wherein the aqueous pharmaceutical formulation has a viscosity of not more than about 6cP at 5 ℃, optionally wherein the viscosity is about 4.5cP to about 5.5 cP.

164. The method of any one of claims 88 to 163, wherein the aqueous pharmaceutical formulation has a viscosity of less than about 13cP at 25 ℃.

165. The method of claim 164, wherein the aqueous pharmaceutical formulation has a viscosity within the range of about 2.0cP to about 10cP, optionally about 2.5cP to about 4 cP.

166. The method of any one of claims 88 to 165, wherein the aqueous pharmaceutical formulation has a conductivity in the range of about 500 μ S/cm to about 5500 μ S/cm, optionally wherein the conductivity is in the range of about 2500 μ S/cm to about 5500 μ S/cm when the formulation comprises an amino acid containing a positively charged side chain, or in the range of about 500 μ S/cm to about 2000 μ S/cm when the formulation comprises an aromatic amino acid or lacks an amino acid aggregation inhibitor.

167. The method of any one of claims 88 to 166, wherein the aqueous pharmaceutical formulation has an osmolality within the range of about 200 to about 500mOsm/kg or about 225 to about 400mOsm/kg or about 250 to about 350 mOsm/kg.

168. The method of any one of claims 88 to 167, wherein the aqueous pharmaceutical formulation has less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of antibody as measured by SE-UHPLC after storage at about 2 ℃ to about 8 ℃ for at least 12 months, 24 months, or 36 months.

169. The method of any one of claims 88 to 168, wherein the aqueous pharmaceutical formulation has less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of antibody after storage for about 1 month at about 20 ℃ to about 30 ℃, as measured by SE-UHPLC.

170. The method of any one of claims 88 to 169, wherein the aqueous pharmaceutical formulation has less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of antibody after a first storage of at least 12 months, 24 months, or 36 months at about 2 ℃ to about 8 ℃ and a second storage of about 1 month at about 20 ℃ to about 30 ℃, as measured by SE-UHPLC.

171. The method of any one of claims 88 to 170, wherein the aqueous pharmaceutical formulation has less than 2% High Molecular Weight Substance (HMWS) and/or more than 98% of the major peak of antibody after storage for about one month at 37 ℃ or 3 months at 30 ℃, as measured by SE-UHPLC.

172. A stable aqueous pharmaceutical formulation manufactured according to any one of the preceding claims.

173. Use of the aqueous pharmaceutical formulation of any one of claims 1-85 and 172 for therapeutic treatment of a subject.

174. The use of claim 173, wherein the therapeutic treatment encompasses treating or preventing a skeletal-related event (SRE), treating or preventing giant cell tumor of bone, treating or preventing malignant hypercalcemia, treating or preventing osteoporosis, or increasing bone mass in a subject.

175. The use of claim 174, wherein the therapeutic treatment encompasses (a) treating or preventing SREs in a subject having solid tumor bone metastases; (b) treating or preventing SRE in an adult or mature bone juvenile subject having unresectable or surgically resected giant cell tumors that are likely to cause severe morbidity; (c) treating a bisphosphonate refractory malignant hypercalcemia in a subject; (d) treating or preventing SRE in a subject having multiple myeloma or solid tumor bone metastasis; (e) treating osteoporosis in postmenopausal women at high risk of fracture; (f) a treatment that increases bone mass in women at high fracture risk receiving adjuvant aromatase inhibitor therapy for breast cancer; (g) a treatment that increases bone mass in men at high fracture risk receiving androgen deprivation therapy for non-metastatic prostate cancer; (h) a treatment that increases bone mass in men with osteoporosis at high risk of fracture; (i) therapy with calcium or vitamin D.

176. A method of preventing a bone related event (SRE) in a patient in need thereof, comprising administering an effective amount of the formulation of any one of claims 1-85 and 172.

177. The method of claim 176, wherein the SRE is selected from the group consisting of: pathological fractures, radiation therapy directed to the bone, surgery directed to the bone, and spinal cord compression.

178. The method of claim 176 or 177, wherein the patient has solid tumor bone metastases.

179. The method of claim 178, wherein the solid tumor is selected from breast cancer, prostate cancer, lung cancer, non-small cell lung cancer, and renal cell carcinoma.

180. The method of claim 178 or 179, wherein the patient has multiple myeloma.

181. The method of any one of claims 176 to 180, comprising administering the formulation in an amount effective to reduce the bone turnover marker urinary creatinine-modified N-terminal telopeptide (uNTx/Cr), optionally by at least 80%.

182. A method of treating giant cell tumor of bone in a patient in need thereof, comprising administering an effective amount of the formulation of any one of claims 1-85 and 172.

183. The method of claim 182, wherein the patient has giant cell tumor of bone that is recurrent, unresectable, or has the potential to cause severe morbidity from surgical resection.

184. A method of treating malignant hypercalcemia in a patient in need thereof comprising administering an effective amount of the formulation of any one of claims 1-85 and 172.

185. The method of claim 184, wherein the malignant disease is refractory to bisphosphonate therapy.

186. The method of claim 184 or 185, comprising administering the formulation in an amount effective to reduce or maintain serum calcium in the patient at a level less than or equal to about 11.5 mg/dL.

187. The method of any one of claims 184 to 186, wherein the formulation comprises the human anti-RANKL antibody at a concentration of about 120 mg/mL.

188. The method of any one of claims 176 to 187, comprising administering the formulation on a once every four week schedule.

189. The method of any one of claims 176 to 188, comprising administering the formulation on days 8 and 15 of the first month of therapy.

190. A method of treating osteoporosis in a patient in need thereof, comprising administering an effective amount of the formulation of any one of claims 1-85 and 172.

191. The method of claim 190, wherein the patient is a postmenopausal female at risk of high fracture.

192. The method of claim 191, wherein the patient is a male at risk of high fracture.

193. A method of increasing bone mass in a patient in need thereof, comprising administering an effective amount of the formulation of any one of claims 1-85 and 172.

194. The method of claim 193, wherein the patient has osteoporosis, optionally wherein the patient is a male with osteoporosis at risk of high fracture.

195. The method of claim 194, wherein the patient is a female at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer.

196. The method of claim 194, wherein the patient is a male at risk of high fracture receiving androgen deprivation therapy for non-metastatic prostate cancer.

197. The method of any one of claims 193-196, comprising administering the formulation in an amount effective to reduce the incidence of a new vertebral fracture and/or a non-vertebral fracture.

198. The method of any one of claims 193 to 197, comprising administering the formulation in an amount effective to reduce bone resorption.

199. The method of any one of claims 193 to 198, comprising administering the formulation in an amount effective to increase bone density in at least one region of the patient selected from the lumbar spine, total hip, and femoral neck.

200. The method of any one of claims 193-199, comprising administering the formulation in an amount effective to increase bone mass in cortical bone and/or cancellous bone of the patient.

201. The method of any one of claims 193-200, comprising administering the formulation in an amount effective to reduce the bone resorption marker serum type 1C-terminal peptide (CTX).

202. The method of any one of claims 193-201, comprising administering the formulation on a schedule of once every six months.

203. The method of any one of claims 193 to 202, comprising administering the formulation in a volume of 1mL or less.

204. The method of any one of claims 176 to 203, comprising administering the formulation subcutaneously.

205. The method of claim 204, comprising administering the formulation subcutaneously to the upper arm, thigh, or abdomen.

206. The method of any one of claims 176-205, wherein the patient receives one or both of calcium and vitamin D.

207. A method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a concentration of more than 70mg/mL, comprising:

preparing said aqueous pharmaceutical formulation comprising said human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof at a pH in the range of about 5.0 to less than 5.2;

wherein the aqueous pharmaceutical formulation exhibits improved stability at a pH value in the range of about 5.0 to less than 5.2 compared to an equivalent aqueous pharmaceutical formulation not at a pH value in the range of about 5.0 to less than 5.2.

208. A method of improving the stability of an aqueous pharmaceutical formulation comprising a human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof, comprising:

preparing said aqueous pharmaceutical formulation comprising a mixture of said human anti-human nuclear factor kappa-B receptor activator ligand (anti-RANKL) monoclonal antibody or antigen-binding portion thereof 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.