Anti-sclerostin antibody preparations

Abstract

To avoid aggregation and other phenomena corresponding to the main physical instability of proteins, the present invention provides an optimized pharmaceutical composition that enhances the stabilization of therapeutic proteins and reduces aggregation, denaturation, or degradation during formulation, filling, transport, storage, and administration, thereby preventing loss of function and harmful immunogenic reactions. [Solution] (a) Anti-sclerostin antibody; (b) Buffering agents containing glutamic acid, histidine, or succinic acid; and (c) Polyol A pharmaceutical composition is provided, wherein the pharmaceutical composition has a pH of pH 4 to pH 7.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 885,672, filed on 12 August 2019, which is incorporated herein by reference in its entirety.

[0002] This application concerns pharmaceutical preparations containing anti-sclerostin antibodies.

[0003] Integration by referencing electronically submitted documents The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification is incorporated in its entirety by reference and identified as follows: a 17,909-byte ASCII (text) file named "53956_Seqlisting.txt" created on August 7, 2020.

[0004] Built-in by reference The following applications are incorporated herein by reference in their entirety: International Patent Application PCT / U.S. Patent Application Publication No. 2012 / 049331, filed 2 August 2012 (claiming priority to U.S. Provisional Patent Application No. 61 / 515,191, filed 4 August 2011), U.S. Patent Application No. 11 / 410,540, filed 25 April 2006 (U.S. Provisional Patent Application No. 60 / 792,645, filed 17 April 2006, U.S. Provisional Patent Application No. 60 / 782,244, filed 13 March 2006, U.S. Provisional Patent Application No. 60 / 776,847, filed 24 February 2006, and 20 Priority is claimed to U.S. Provisional Patent Application No. 60 / 677,583 filed on 3 May 2005; and U.S. Patent Application No. 11 / 411,003 filed on 25 April 2006 (registered as U.S. Patent No. 7,592,429) (priority is claimed to U.S. Provisional Patent Application No. 60 / 792,645 filed on 17 April 2006, U.S. Provisional Patent Application No. 60 / 782,244 filed on 13 March 2006, U.S. Provisional Patent Application No. 60 / 776,847 filed on 24 February 2006, and U.S. Provisional Patent Application No. 60 / 677,583 filed on 3 May 2005). The following applications are also incorporated herein by reference: U.S. Patent Application No. 12 / 212,327 filed September 17, 2008 (claiming priority to U.S. Provisional Patent Application No. 60 / 973,024 filed September 17, 2007), and U.S. Patent Application No. 12 / 811,171 filed June 29, 2010 (a U.S. national phase entry application under Section 371 of the U.S. Patent Act for International Patent Application No. PCT / US08 / 86864 filed December 15, 2008, claiming priority to U.S. Provisional Patent Application No. 61 / 013,917 filed December 14, 2007). [Background technology]

[0005] Protein-based drugs are among the fastest-growing therapeutics in (pre)clinical development and as marketed products. Compared to small molecule chemical agents, protein-based drugs exhibit high specificity and activity at relatively low concentrations and typically provide treatment for various cancers, autoimmune diseases, and metabolic disorders with significant impact (Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80, Wang, Int J Pharm. 1999 Aug 20;185(2):129-88).

[0006] Advances in commercial-scale purification processes now allow for the acquisition of high-purity recombinant proteins and other protein-based pharmaceuticals during initial production. However, proteins are only slightly stable and are highly susceptible to both chemical and physical degradation. Chemical degradation refers to covalent modifications such as deamide, oxidation, cleavage or formation of new disulfide crosslinks, hydrolysis, isomerization, or deglycosylation. Physical degradation includes protein unfolding, undesirable surface adsorption, and aggregation. Addressing these physical and chemical instabilities is one of the most challenging aspects of protein-based pharmaceutical development (Chi et al., Pharm Res, Vol.20, No.9, Sept 2003, pp.1325-1336; Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80).

[0007] Protein aggregation corresponds to a major phenomenon of protein physical instability and results from an inherent tendency to minimize the thermodynamically unfavorable interactions between the solvent and hydrophobic protein residues. Protein aggregation can be particularly problematic because it occurs during the processes of refolding, purification, sterilization, transportation, and storage. Aggregation can occur even under thermodynamically highly favorable (e.g., neutral pH and 37 °C) solution conditions and in the absence of stress (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325-1336; Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80; Wang, Int J Pharm. 1999 Aug 20;185(2):129-88; Mahler J Pharm Sci. 2009 Sep;98(9):2909-34.).

[0008] Maintaining the stability and activity of proteins in biological and biotechnology applications poses a serious challenge. There is a need in the art for optimized pharmaceutical compositions that enhance the stabilization of therapeutic proteins and prevent loss of function and harmful immunogenic reactions by reducing aggregation and denaturation or degradation during formulation, filling, transportation, storage, and administration. SUMMARY OF THE INVENTION

[0009] In one aspect, described herein is a pharmaceutical composition comprising an anti-sclerostin antibody; a buffer comprising glutamic acid, histidine, or succinic acid; and a polyol, wherein the pharmaceutical composition has a pH of pH 4 to pH 7.

[0010] In some embodiments, the buffer is present at a concentration of about 10 mM to about 50 mM. In some embodiments, the polyol is present in an amount of about 1% to about 10% w / v. In some embodiments, the polyol is sorbitol and is present in an amount of about 5% to about 10% w / v. In some embodiments, sorbitol is present in an amount of about 5% w / v.

[0011] In some embodiments, the pharmaceutical composition further comprises glycerol (for example, in an amount of about 1% to about 5% w / v).

[0012] In some embodiments, the pharmaceutical composition further comprises sucrose (for example, in an amount of about 1% to about 10% w / v).

[0013] In some embodiments, the pharmaceutical composition further comprises an amino acid other than histidine. In some embodiments, this amino acid is arginine. In some embodiments, arginine is present in an amount ranging from 10 mM to about 250 mM. In some embodiments, this amino acid is methionine. In some embodiments, methionine is present in an amount ranging from about 10 mM to about 100 mM.

[0014] In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, this surfactant is polysorbate 20, polysorbate 80, F16, or Triton.

[0015] In some embodiments, the pharmaceutical composition contains anti-sclerostin antibody at a concentration of at least 70 mg / mL. In some embodiments, the pharmaceutical composition contains anti-sclerostin antibody at a concentration of approximately 70 mg / mL to approximately 210 mg / mL.

[0016] In some embodiments, the anti-sclerostin antibody is romosozumab.

[0017] In some embodiments, the pharmaceutical composition contains 10 mM glutamic acid and 5% sorbitol at pH 4.5. In some embodiments, the pharmaceutical composition contains 10 mM glutamic acid and 5% sorbitol at pH 5.2. In some embodiments, the pharmaceutical composition contains 10 mM succinic acid and 5% sorbitol at pH 5.2. In some embodiments, the pharmaceutical composition contains 10 mM histidine and 5% sorbitol at pH 6.

[0018] While various embodiments herein are presented using the word “including” in various contexts, it should be understood that relevant embodiments may also be described using the words “consisting of” or “essentially consisting of.” Note that the terms “a” or “an” refer to one or more; for example, “immunoglobulin molecule” is understood to refer to one or more immunoglobulin molecules. Thus, the terms “one (a)” (or “one (an)”), “one or more” and “at least one” may be used interchangeably herein.

[0019] When describing a range of values, it should be understood that the described characteristic may be any individual value found within that range. For example, "pH approximately 4 to approximately 6" could be, but not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc., and any value between such values. In addition, "pH approximately 4 to approximately 6" should not be interpreted as meaning that the pH of the formulation of interest fluctuates in 2 pH increments within the range of pH 4 to pH 6 during storage, but rather as meaning that a value within that range can be selected for the pH of the solution, and that the pH remains buffered around that pH. In some embodiments, where the term "approximately" is used, this term means adding or subtracting 5%, 10%, 15%, or more to the enumerated number. The actual intended variation can be determined from the context.

[0020] In any of the scopes described herein, the endpoints of the scope are included within that scope. However, this description also intends the same scope in which the smaller endpoint and / or larger endpoint is excluded. Additional features and variations of the present invention will be apparent to those skilled in the art from the whole application, including the drawings and the detailed description, and all such features are intended as embodiments of the present invention. Similarly, the features described herein can be rearranged to form additional embodiments that are also intended as embodiments of the present invention, whether or not the combination of features is specifically described above as an embodiment or aspect of the present invention. Furthermore, only such limitations described herein as essential to the present invention should be considered as such; variations of the present invention lacking limitations not described herein as essential are also intended as embodiments of the present invention. [Brief explanation of the drawing]

[0021] [Figure 1] This graph shows the percentage of romosozumab high molecular weight (HMW) peak area in various formulations stored at 4°C for up to 24 months. [Figure 2] This graph shows the percentage of romosozumab high molecular weight (HMW) peak area in various formulations stored at 37°C for up to four weeks. [Figure 3] This graph shows the peak area percentage of romosozumab high molecular weight (HMW) in various formulations stored at 45°C for up to four weeks. [Figure 4] This graph shows the major peak area (%) of romosozumab in various formulations stored at 4°C for up to 24 months, as evaluated by cation exchange HPLC. [Figure 5] This graph shows the major peak area (%) of romosozumab in various formulations stored at -70°C for up to 24 months, as evaluated by cation exchange HPLC. [Figure 6]This graph shows the major peak area (%) of romosozumab in various formulations stored at 4°C, -30°C, and -70°C for up to 24 months, as evaluated by cation exchange HPLC. [Figure 7] This graph shows the major peak area (%) of romosozumab in various formulations stored at 4°C, 25°C, 37°C, 45°C, -30°C, and -70°C for up to 4 weeks, as evaluated by cation exchange HPLC. [Figure 8] This graph shows the acidic peak area (%) of romosozumab in various formulations stored at 4°C, 25°C, 37°C, 45°C, -30°C, and -70°C for up to 4 weeks, as evaluated by cation exchange HPLC. [Figure 9] This graph shows the acidic peak area (%) of romosozumab in various formulations stored at 4°C, -30°C, and -70°C for up to 24 months, as evaluated by cation exchange HPLC. [Figure 10] This graph shows the acidic peak area (%) of romosozumab in various formulations stored at 4°C, -30°C, and -70°C for up to 24 months, as evaluated by cation exchange HPLC. [Figure 11] These are chromatograms of romosozumab in formulation 4 after 3 months of storage at 4°C, 25°C, and 37°C, as evaluated by cation exchange HPLC. [Figure 12] This graph shows the major peak area (%) of romosozumab in various formulations stored at 4°C, -30°C, and -70°C over a two-year period. [Figure 13] This graph shows the acidic peak area (%) of romosozumab in various formulations stored at 4°C, -30°C, and -70°C for two years, as evaluated by cation exchange HPLC. [Figure 14] This graph shows the stability of the basic peak area of ​​romosozumab in various formulations stored at 4°C, -30°C, and -70°C for two years, as evaluated by cation exchange HPLC. [Figure 15]This graph shows the percentage of high molecular weight species of romosozumab in various formulations stored at 4°C over different time points (4 weeks, 3 months, 6 months, 1 year, 1.5 years, and 2 years), as evaluated by capillary electrophoresis SDS. [Figure 16] This graph shows the results of a high-concentration syringe test at time 0 (70 mg / mL romosozumab in various formulations) as evaluated by HIAC. [Figure 17] This graph shows the results of high-concentration syringe studies (70 mg / mL romosozumab in various formulations) at the 2-year mark, as evaluated by HIAC. [Figure 18] This graph shows the results of a high-concentration syringe test at time 0 (120 mg / mL romosozumab in various formulations) as evaluated by HIAC. [Figure 19] This graph shows the results of high-concentration syringe studies (120 mg / mL romosozumab in various formulations) at the 2-year mark, as evaluated by HIAC. [Modes for carrying out the invention]

[0022] This disclosure describes formulations comprising anti-sclerostin antibodies. Various embodiments of these formulations are described below. Section headings are used solely for reading convenience and are not intended to limit them. The entire specification is intended to be considered a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated.

[0023] In one embodiment, the pharmaceutical formulation described herein comprises (a) an anti-sclerostin antibody; (b) a buffer comprising glutamic acid, histidine, or succinic acid; and (c) a polyol, wherein the pharmaceutical composition is a pharmaceutical formulation having a pH of pH 4 to pH 7. As demonstrated in the examples, formulations comprising the combination of components described herein are stable under various conditions for long periods (up to 2 years) at various temperatures (e.g., -30°C, -70°C, and 4°C).

[0024] stability When used herein in relation to a composition comprising an antibody (or its antigen-binding fragment), the terms “stability” and “stable” refer to the resistance of the antibody (or its antigen-binding fragment) in the composition to aggregation, degradation, or fragmentation under given manufacturing, preparation, transport, and / or storage conditions. Antibody formulations with high stability exhibit high reliability and safety and are therefore advantageous for clinical use.

[0025] The stability of the antibody in the composition is optionally evaluated by testing the desired parameters of the antibody in the composition (e.g., aggregation, heavy and / or light chain degradation, chemical modification, etc.) over time. In this regard, the parameters are typically tested and compared at an initial time point (T0) and an evaluation time point (T1) while optionally exposing the antibody to one of several environmental conditions. The initial time point may be, for example, the time when the antibody is first incorporated into the composition or first tested for quality (i.e., tested to determine whether the antibody composition meets regulations or manufacturing specifications with respect to aggregation or degradation). The initial time point may also be the time when the antibody is reincorporated into the composition (e.g., reincorporated at a higher or lower concentration compared to the initial preparation). The evaluation time point is, in various embodiments, about one week (or about two weeks, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about ten weeks, or about three months, or about six months, or about one year) after the initial time point. Desired parameters (e.g., aggregation or degradation) of the antibody or its fragments in the composition can be evaluated under various storage conditions (e.g., temperatures of -30°C, 4°C, 20°C, or 40°C, shaking, pH, storage in various container materials (e.g., glass vials, pre-filled syringes, etc.), and the like).

[0026] Exemplary methods for determining the degree, and / or type, and / or size of aggregates present in antibody-containing compositions include, but are not limited to, size exclusion chromatography (SEC), high-speed size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques. Size exclusion chromatography (SEC) may be performed to separate molecules based on their size by passing them through a column packed with a suitable resin, with larger molecules (e.g., aggregates) eluting before smaller molecules (e.g., monomers). Molecules are generally detected by UV absorbance at 280 nm and can be collected for further characterization. High-pressure liquid chromatography columns are commonly used for SEC analysis (HP-SEC). Alternatively, analytical ultracentrifuges (AUC) may be used. AUC is an orthogonal technique for determining the sedimentation coefficient of macromolecules in liquid samples. Similar to SEC, AUC can separate and detect antibody fragments / aggregates from monomers and may even provide information on molecular weight. Antibody aggregation in a composition can also be characterized by particle counter analysis using a Coulter counter or by turbidity measurement using a turbidimeter. Turbidity is a measure of the amount of light scattered by particles in a solution and can therefore be used as a general indicator of protein aggregation. In addition, the state of aggregation and / or fragmentation of antibodies or antibody fragments in a composition can be characterized using non-reducing polyacrylamide gel electrophoresis (PAGE) or capillary gel electrophoresis (CGE).

[0027] Exemplary methods for determining antibody degradation include, but are not limited to, size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis using SDS (CE-SDS), and reversed-phase HPLC with in-line MS detection.

[0028] In various embodiments, less than 5% of the antibodies described herein in the composition are aggregated under the conditions of interest. For example, after storage at -30°C, 4°C, 20°C, or 40°C for a period of about one week (or about two weeks, or about three weeks, or about four weeks, or about five weeks, or about six weeks, or about seven weeks, or about eight weeks, or about ten weeks, or about three months, or about six months, or about one year), less than 4%, or less than 3%, or less than 2%, or less than 1% of the antibodies in the composition are aggregated. In some embodiments, after storage at about 4°C for two weeks, less than 5% (or less than 4%, or less than 3%, or less than 2%, or less than 1%) of the antibodies described herein in the composition are aggregated.

[0029] For example, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) of the antibody in the composition is optionally present in a non-aggregated (i.e., monomer) form after storage at -30°C, 4°C, 20°C, or 40°C for a period of about 1 week (or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 7 weeks, or about 8 weeks, or about 10 weeks, or about 3 months, or about 6 months, or about 1 year). In some embodiments, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or more) of the antibody is present in the composition in a non-aggregated form after two weeks of storage at approximately 4°C. In some embodiments, at least 99% of the antibody is present in the composition in a non-aggregated form after two weeks of storage at approximately 4°C, and / or at least 95% of the antibody present in the composition is in a non-aggregated form after two weeks of storage at 40°C.

[0030] In various embodiments, less than 5% of the antibodies described herein in the composition are degraded. For example, less than 4%, less than 3%, less than 2%, or 1%, or less, of the antibodies in the composition are degraded under the conditions of interest. For example, optionally, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) of the antibodies in the composition stored at about -30°C, about 4°C, about 20°C, or about 40°C for a period of about 1 week (or about 2 weeks, or about 3 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or about 6 months, or about 1 year) remain intact (i.e., undegraded). In some embodiments, at least 85% (or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or more) of the antibody remains intact (i.e., undegraded) after storage in the composition at about 4°C for a period of two weeks. In some embodiments, at least 99% of the antibody remains intact when stored in the composition at about 4°C for two weeks, and / or at least 95% remains intact when stored in the composition at about 40°C for two weeks.

[0031] The functional or activity stability of antibodies in a composition is also a concern herein. For example, assays for detecting and / or quantifying antibody binding to a target or sclerostin neutralization are known in the art. Optionally, an antibody exhibits about 50–100% activity under the conditions of interest compared to its activity at the initial point. For example, an antibody retains about 60–90% or 70–80% of its activity level compared to its activity at the initial point. Thus, the functional stability of an antibody includes retaining at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of its activity, and may include activity measurements exceeding 100% (e.g., 105%, 110%, 115%, 120%, 125%, or 150% or higher) compared to its activity at the initial point.

[0032] cushioning agent The pharmaceutical compositions described herein include a buffer, which may be optionally selected from the group consisting of histidine, glutamic acid, succinic acid, and combinations thereof. In some embodiments, the pharmaceutical composition includes at least one buffer selected from the group consisting of histidine, glutamic acid, succinic acid, and combinations thereof.

[0033] Buffers are often used to control the pH of a formulation. In some embodiments, buffers are added at concentrations that maintain the pH of the formulation at approximately 4–7, 4.5–6, or 5.2. The effect of pH on the formulation can be characterized using one or more of several methods, such as accelerated stability testing and calorimetry screening tests (Remmele RLJr., et al., Biochemistry, 38(16):5241-7(1999)).

[0034] Organic acids, phosphates, and Tris are suitable buffers in protein formulations (Table 1). The buffering capacity of a buffer species is maximum at pH equal to its pKa and decreases as pH increases or decreases from this value. 90 percent of the buffering capacity is located within 1 pH unit of its pKa. Buffering capacity also increases proportionally with increasing buffer concentration.

[0035] When selecting a buffer, several factors are typically considered. For example, the type of buffer and its concentration should be defined based on its pKa and the desired formulation pH. Similarly, buffers are preferably compatible with protein drugs and other formulation excipients and do not catalyze any degradation reactions. Polyanionic carboxylate buffers, such as citrates and succinates, may form covalent adducts with protein side-chain residues. A third aspect to consider is the stinging and irritating sensations that buffers may induce. For example, citrates are known to cause stinging upon injection (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2):218-21 (2006)). Drugs administered via the SC or IM pathway, where the drug solution remains at the site for a relatively long period, are more likely to cause stinging and irritation than drugs administered via the IV pathway, where the formulation is rapidly diluted in the blood at the time of administration. In the case of formulations administered by direct IV infusion, the total amount of buffer (and any other formulation components) needs to be monitored. For example, potassium ions administered in the form of potassium phosphate buffers have been reported to potentially induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am.Fam.Physician., 73(2):283-90 (2006)).

[0036] [Table 1]

[0037] The buffer system present in the formulation is selected to be physiologically compatible and to maintain the desired pH.

[0038] The buffer may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. The buffer may be present at concentrations of about 0.1 mM to about 1000 mM (1 M), or about 5 mM to about 200 mM, or about 5 mM to about 100 mM, or about 10 mM to about 50 mM. Suitable buffer concentrations include concentrations of about 200 mM or less. In some embodiments, the buffer in the formulation may be present at concentrations of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM, or about 5 mM. In some embodiments, the concentration of the buffer is at least about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffer is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM and 100 mM. In some embodiments, the concentration of the buffer is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM and 50 mM. In some embodiments, the concentration of the buffer is approximately 10 mM.

[0039] surfactant The pharmaceutical compositions described herein include at least one surfactant. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to overcome proteins in terms of interfacial location. The hydrophobic portion of the surfactant molecule occupies the interfacial location (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically near the critical micelle concentration of detergents), the surface layer of the surfactant molecule prevents protein molecules from adsorbing at the interface. This minimizes surface-induced degradation. Examples of surfactants include fatty acid esters of sorbitan polyethoxylate, i.e., polysorbate 20 and polysorbate 80 (see, e.g., Avonex®, Neupogen®, and Neulasta®). These two differ only in the length of the aliphatic chains that confer hydrophobic properties to molecules C-12 and C-18, respectively. Therefore, polysorbate-80 has higher surfactant activity and a lower critical micelle concentration compared to polysorbate-20. The surfactant poloxamer 188 has also been used in several commercially available liquid products such as Gonal-F®, Norditropin®, and Ovidrel®.

[0040] Detergents can also affect the thermodynamic conformational stability of proteins. Here again, the effect of a given excipient may be protein-specific. For example, polysorbates may decrease the stability of some proteins and increase the stability of others. The protein destabilization by detergents makes sense in terms of the hydrophobic tails of detergent molecules that can be involved in specific binding to partially or completely unfolded protein states. These types of interactions may lead to a shift in conformational equilibrium to a more extended protein state (i.e., increasing the exposure of the hydrophobic portion of the protein molecule by complementing the binding polysorbate). Alternatively, if the native state of a protein exhibits several hydrophobic surfaces, a detergent binding to the native state may stabilize its conformation.

[0041] Another aspect of polysorbates is their inherent susceptibility to oxidative degradation. Polysorbates often contain sufficient amounts of peroxides as raw materials to cause oxidation of protein residue side chains (particularly methionine). It is emphasized that excipients should be used in formulations at the lowest effective concentration, as oxidative damage can occur from the addition of stabilizers. In the case of surfactants, the effective concentration of a given protein is determined by the stabilization mechanism. If the stabilization mechanism of the surfactant is related to preventing surface denaturation, the effective concentration is assumed to be near the critical micelle concentration of the detergent. Conversely, if the stabilization mechanism is related to a specific protein-detergent interaction, the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (Randolph TW, et al., Pharm Biotechnol., 13:159-75 (2002)).

[0042] Furthermore, an appropriate amount of surfactant may be added to prevent surface-related aggregation during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Exemplary surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, zwitterionic surfactants, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholate. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Examples of zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Examples of nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN®-20, and TWEEN®-80. In another embodiment, examples of surfactants include: lauromacrogol 400; polyoxyl stearate 40; polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60; glycerol monostearate; polysorbate 40, 60, 65, and 80; soy lecithin and other phospholipids, e.g., DOPC, DMPG, DMPG, and DOPG; sucrose fatty acid esters; methylcellulose, and carboxymethylcellulose.

[0043] The pharmaceutical compositions described herein include at least one surfactant, either individually or in mixtures of various proportions. In some embodiments, the composition contains the surfactant at a concentration of about 0.001% to about 5% w / v (or about 0.004 to about 0.5% w / v, or about 0.001 to about 0.01% w / v, or about 0.004 to about 0.01% w / v). In some embodiments, the composition contains a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5% w / v. In some embodiments, the composition contains a surfactant at a concentration of about 0.004% to about 0.5% w / v. In some embodiments, the composition contains a surfactant at a concentration of about 0.004% to about 0.5% w / v. In some embodiments, the composition contains a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some embodiments, the composition contains a surfactant at a concentration of about 0.004 to about 0.01% w / v. In some embodiments, the composition contains a surfactant at concentrations of about 0.004, about 0.005, about 0.007, about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4% w / v to about 0.5% w / v. In some embodiments, the composition contains a surfactant incorporated at a concentration of about 0.001% to about 0.01% w / v.

[0044] Sugars The pharmaceutical compositions described herein contain at least one sugar. Sugars may be added as stabilizers or fillers. The term “stabilizer” as used herein refers to an excipient that can prevent aggregation or other physical degradation, and chemical degradation (e.g., autodegradation, deamidation, oxidation, etc.) in aqueous and solid states. Stabilizers used in pharmaceutical compositions include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, genthiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCl, polyhydroxy compounds, such as polysaccharides, such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid, and sodium chloride (Carpenter et al., Develop. Biol. Standard 74:225, (1991)).

[0045] In some embodiments, at least one sugar is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextrans, and linear unbranched dextrans, or combinations thereof. In some embodiments, at least one sugar is a disaccharide selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol, or combinations thereof.

[0046] In some embodiments, the pharmaceutical composition contains at least one sugar at a concentration of about 0.01% to about 40% w / v, or about 0.1% to about 20% w / v, or about 1% to about 15% w / v. In some embodiments, the pharmaceutical composition contains at least one sugar at a concentration of at least 0.5%, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, or at least 40% w / v. In some embodiments, the pharmaceutical composition contains at least one sugar at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, and about 14% to about 15% w / v. In some embodiments, the pharmaceutical composition contains at least one sugar at a concentration of about 1% to about 15% w / v. In further embodiments, the pharmaceutical composition contains at least one sugar at a concentration of about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12% w / v. In some embodiments, the pharmaceutical composition contains at least one sugar at a concentration of about 9% to about 12% w / v. In some embodiments, at least one sugar is present in the composition at a concentration of about 9% w / v. In some embodiments, at least one sugar is sorbitol, sucrose, trehalose, or mannitol, or a combination thereof.

[0047] In some embodiments, the formulation contains sorbitol in amounts of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12%. In some embodiments, the formulation contains sorbitol in amounts of approximately 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, the formulation contains sorbitol in amounts of approximately 5%.

[0048] In some embodiments, the formulation further contains sucrose, which is present in the composition in the range of 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% to about 15% w / v. In some embodiments, the formulation further contains sucrose in an amount of about 9%.

[0049] In some embodiments, the formulation further comprises glycerol. In some embodiments, the formulation further comprises glycerol in an amount of about 1%, about 2%, about 3%, about 4%, or about 5%. The formulation optionally further comprises glycerol in an amount of about 1% or about 2.5%.

[0050] If necessary, the formulation also includes appropriate amounts of fillers and molar osmotic pressure modifiers (e.g., sugars) suitable for forming a lyophilized "cake".

[0051] In some embodiments, the formulation further contains glycerol. In some embodiments, the formulation further contains glycerol in an amount of about 1%, about 2%, about 3%, about 4%, or about 5%. The formulation further contains glycerol in an amount of about 1% or about 2.5%.

[0052] In some embodiments, the formulation contains 10 mM glutamic acid and 5% sorbitol at pH 4.5.

[0053] In some embodiments, the formulation contains 10 mM glutamic acid and 5% sorbitol at pH 5.2.

[0054] In some embodiments, the formulation contains 10 mM succinic acid and 5% sorbitol at pH 5.2.

[0055] In some embodiments, the formulation contains 10 mM histidine and 5% sorbitol at pH 6.

[0056] Other considerations As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a subject requiring it. The terms “subject,” “individual,” “animal,” or “patient” are interchangeable herein and refer to any subject (in particular mammalian subjects) to which the administration of the pharmaceutical composition of the present invention is desirable. Examples of mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, and the like, with humans being preferred. The pharmaceutical compositions of this disclosure are stable and pharmaceutically acceptable, i.e., capable of exerting the desired therapeutic effect without causing any significant undesirable local or systemic effects in the subject to which the pharmaceutical composition is administered. The pharmaceutically acceptable compositions of this disclosure may be sterile and / or pharmaceutically inactive. Specifically, the term “pharmaceutically acceptable” may mean approved by a regulatory authority or other generally recognized pharmacopoeia for use in animals (more specifically humans).

[0057] The formulations provided by this disclosure include the antibodies described herein. In some embodiments, the antibodies are provided in a therapeutically effective amount. "Therapeutally effective amount" means the amount of the antibody that elicits the desired therapeutic effect. Therapeutic effect and toxicity can be determined by standard pharmaceutically procedures in cell culture or experimental animals (e.g., ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population)). The dose ratio between therapeutic effect and toxic effect is the therapeutic index and can be expressed as the ratio ED50 / LD50. Formulations exhibiting a large therapeutic index are generally preferred.

[0058] Protein preparations are generally administered parenterally. When administered parenterally, protein preparations must be sterile. Examples of sterile diluents include liquids that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid preparations such as preparations that can be reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. Examples of diluents include aqueous solutions of salts and / or buffers.

[0059] Excipients are additives included in drug formulations to provide or enhance the stability, delivery, and manufacturability of the drug product. Regardless of the reason for including excipients, they are essential components of the drug product and therefore must be safe and well-tolerated by patients. For protein drugs, the selection of excipients is particularly important, as these excipients can affect both the efficacy and immunogenicity of the drug. Therefore, protein formulations need to be developed with the appropriate selection of excipients that provide suitable stability, safety, and marketability.

[0060] The excipients described herein are organized either by their chemical species or their functional role in the formulation. When discussing each type of excipient, a brief description of its mode of stabilization is provided. With the teachings and guidance provided herein, those skilled in the art will be able to easily vary the amount or range of excipients without increasing viscosity to undesirable levels. Excipients may be selected to achieve a desired gravimetric osmolality (i.e., isotonic, hypotonic, or hypertonic), pH, desired stability, resistance to aggregation, decomposition, or precipitation, protection under freezing, lyophilization, or high-temperature conditions, or other properties of the final solution. Various types of excipients are known in the art. Exemplary excipients include: salts, amino acids, other isotonic agents, surfactants, stabilizers, fillers, cryoprotectants, lyophilization protectants, antioxidants, metal ions, chelating agents, and / or preservatives.

[0061] Furthermore, if a particular excipient is reported in a formulation, for example, in percent (%) w / v, a person skilled in the art will recognize that the equivalent molar concentration of this excipient is also intended.

[0062] Other stabilizers and fillers Examples of stabilizers include certain compounds that can act as cryoprotectants, freeze-drying protectants, and glass-forming agents. Cryoprotectants act to stabilize proteins during or in a frozen state at low temperatures. Freeze-drying protectants stabilize proteins in a freeze-dried solid form by preserving the protein's natural conformational properties during the dehydration step of freeze-drying. Glass properties are classified as "robust" or "fragile" depending on their relaxation properties with respect to temperature. For stability to be provided, it is important that cryoprotectants, freeze-drying protectants, and glass-forming agents remain in the same phase as the protein. Sugars, polymers, and polyols fall into this category and can sometimes perform all three roles.

[0063] Polyols encompass a class of excipients containing sugars (e.g., mannitol, sucrose, or sorbitol) and other polyhydric alcohols (e.g., glycerol and propylene glycol). Polyethylene glycol (PEG) polymers fall into this category. Polyols are commonly used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized parenteral protein formulations. Polyols can protect proteins from both physical and chemical degradation pathways.

[0064] Examples of C3-C6 polyols include: propylene glycol, glycerin (glycerol), threose, threitol, erythritol, ribose, arabinose, arabitol, lyxose, maltitol, sorbitol, glucose, mannose, mannitol, levrose, dextrose, maltose, trehalose, fructose, xylitol, inositol, galactose, xylose, fructose, sucrose, 1,2,6-hexanetriol, and similar. Higher-order sugars include dextran, propylene glycol, or polyethylene glycol. Reducing sugars such as fructose, maltose, or galactose are more readily oxidized than non-reducing sugars. Further examples of sugar alcohols include glucitol, maltitol, lactitol, or isomaltulose. Further exemplary freeze-drying protective agents include glycerin and gelatin, as well as sugars such as melibiose, meletitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyalcohols. Examples of monoglycosides include compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose, and maltulose.

[0065] amino acid In some embodiments, the pharmaceutical compositions described herein further comprise one or more amino acids as buffers, fillers, stabilizers, and / or antioxidants. Histidine and glutamic acid may be used to buffer protein formulations in pH ranges of 5.5–6.5 and 4.0–5.5, respectively. The amino acids glycine, proline, serine, and alanine stabilize proteins.

[0066] In some embodiments, the formulation further contains amino acids other than histidine.

[0067] In some embodiments, the formulation further optionally contains arginine in an amount ranging from about 10 mM to about 250 mM (for example, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM). In some embodiments, the formulation further contains arginine in an amount of about 100 mM.

[0068] In some embodiments, the formulation further optionally contains methionine in an amount ranging from about 10 mM to about 100 mM (for example, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM). In some embodiments, the formulation further contains methionine in an amount of about 20 mM.

[0069] Antioxidants In some embodiments, the pharmaceutical compositions described herein further comprise one or more antioxidants. Oxidation of protein residues arises from several different causes. In addition to the addition of specific antioxidants, careful control of several factors during the manufacturing process and storage of the product, such as atmospheric oxygen, temperature, exposure to light, and chemical contamination, is necessary to prevent oxidative protein damage. The most commonly used pharmaceutical antioxidants are reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations must be water-soluble and maintain their activity throughout the product's entire shelf life. Reducing agents and oxygen / free radical scavengers function by cleaving reactive oxygen species in solution. Chelating agents, such as EDTA, can exert their effect by binding to trace metal contaminants that promote free radical formation.

[0070] However, antioxidants themselves can induce other covalent or physical changes in proteins. The selection of the appropriate antioxidant depends on the specific stress and sensitivity of the protein.

[0071] metal ions In some embodiments, the pharmaceutical composition further comprises one or more metal ions. Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, certain metal ions are included in formulations when they are cofactors for proteins and are present in protein suspension formulations that form coordination complexes (e.g., zinc suspensions of insulin).

[0072] Preservatives In some embodiments, the pharmaceutical composition further comprises one or more preservatives. Preservatives may be necessary when developing parenteral formulations for multiple uses involving more than one dispensing from the same container. Possible preservatives include phenol, benzyl alcohol, meta-cresol, alkylparabens, e.g., methylparaben or propylparaben, benzalkonium chloride, and benzethonium chloride. Other examples of compounds having antimicrobial preservative activity include octadecyldimethylbenzylammonium chloride and hexamethonium chloride. Other types of preservatives include aromatic alcohols, e.g., butyl alcohol, phenol, benzyl alcohol; atechol, resorcinol, cyclohexanol, and 3-pentanol.

[0073] Some preservatives can cause injection site reactions, which is another factor to consider when selecting a preservative. However, this disclosure also intends to include pharmaceutical compositions that do not contain any preservatives.

[0074] Antibodies in the preparation The term "anti-sclerostin antibody" or "antibody that binds to sclerostin" refers to an antibody that binds to sclerostin of SEQ ID NO: 1 or a part thereof. Recombinant human sclerostin / SOST is commercially available, for example, from R&D Systems (Minneapolis, Minn., USA; Catalog No. 1406-ST-025 in 2006). U.S. Patent Nos. 6,395,511 and 6,803,453, and U.S. Patent Application Publication Nos. 2004 / 0009535 and 2005 / 0106683 generally refer to anti-sclerostin antibodies. Examples of anti-sclerostin antibodies suitable for use in the context of the present invention are also described in U.S. Patent Application Publication Nos. 2007 / 0110747 and 2007 / 0072797, which are hereby incorporated by reference in their entirety. Further information regarding materials and methods for generating anti-sclerostin antibodies can be found in U.S. Patent Application Publication No. 2004 / 0158045 (incorporated herein by reference).

[0075] The term "antibody" refers to a complete immunoglobulin molecule (including polyclonal versions, monoclonal versions, chimeric versions, humanized versions, and / or human versions having full-length heavy and / or light chains).

[0076] "Specifically binds" as used herein means that an antibody binds to an antigen preferentially over other proteins. In some embodiments, "specifically binds" means that an antibody has a high affinity for an antigen compared to other proteins. An antibody that specifically binds to an antigen has a dissociation constant (Kd) of 1×10 -7 M or less, 2×10 -7 M or less, 3×10 -7 M or less, 4×10 -7 M or less, 5×10 -7 M or less, 6×10 -7 M or less, 7×10 -7 M or less, 8×10 -7 M or less, 9×10 -7 M or less, 1×10 -8 M or less, 2×10-8 Below M, 3×10 -8 Below M, 4×10 -8 Below M, 5×10 -8 Below M, 6×10 -8 Below M, 7×10 -8 Below M, 8×10 -8 Below M, 9×10 -8 Below M, 1×10 -9 Below M, 2×10 -9 Below M, 3×10 -9 Below M, 4×10 -9 Below M, 5×10 -9 Below M, 6×10 -9 Below M, 7×10 -9 Below M, 8×10 -9 Below M, 9×10 -9 Below M, 1×10 -10 Below M, 2×10 -10 Below M, 3×10 -10 Below M, 4×10 -10 Below M, 5×10 -10 Below M, 6×10 -10 Below M, 7×10 -10 Below M, 8×10 -10 Below M, 9×10 -10 Below M, 1×10 -11 Below M, 2×10 -11 Below M, 3×10 -11 Below M, 4×10 -11 Below M, 5×10 -11 Below M, 6×10 -11 Below M, 7×10 -11 Below M, 8×10 -11 Below M, 9×10 -11 Below M, 1×10 -12 Below M, 2×10 -12 Below M, 3×10 -12 Below M, 4×10 -12 Below M, 5×10 -12 Below M, 6×10 -12 Below M, 7×10 -12 Below M, 8×10 -12 Below M, also 9×10 -12 Below M, the antigen has no binding affinity and the binding affinity has not been determined.

[0077] One part and one part of any form, antibody, 1×10-7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 × 10 -12 The antibody binds to sclerostin of SEQ ID NO: 1 or its naturally occurring variants with an affinity (Kd) of M or less. Affinity is determined using various techniques, one example being the affinity ELISA assay. In various embodiments, affinity is determined by the BIAcore assay. In various embodiments, affinity is determined by kinetic methods. In various embodiments, affinity is determined by equilibrium / solution methods. U.S. Patent Application Publication No. 2007 / 0110747 (this disclosure is incorporated herein by reference) contains a further description of affinity assays suitable for determining the affinity (Kd) of antibodies against sclerostin.

[0078] In some or any embodiments, the anti-sclerostin antibodies described herein preferably modulate sclerostin function in a cell-based assay and / or an in vivo assay as described in U.S. Patent Application Publication No. 2007 / 0110747, and / or bind to one or more of the epitopes described in U.S. Patent Application Publication No. 2007 / 0110747, and / or crossblock the binding of one of the antibodies described in U.S. Patent Application Publication No. 2007 / 0110747, and / or have their binding to sclerostin crossblocked by one of the antibodies described in U.S. Patent Application Publication No. 2007 / 0110747 (these documents are incorporated by reference in their entirety and for the purpose of describing the assays characterizing the anti-sclerostin antibodies).

[0079] "CDR" refers to the complementarity-determining region within the antibody variable sequence. Each of the heavy and light chain variable regions has three CDRs, which are called CDR1, CDR2, and CDR3, respectively. The term "set of six CDRs," as used herein, refers to the group of three CDRs present in the light chain variable region and heavy chain variable region that can bind to the antigen. The precise boundaries of CDRs are defined differently by different systems. The system described by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and (1991)), not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs may be called Kabat CDRs. Chothia and collaborators (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) described Kabat We found that certain sub-regions within a CDR adopt nearly identical peptide backbone structures despite exhibiting significant diversity at the amino acid sequence level. These sub-regions were named L1, L2, and L3, or H1, H2, and H3 (where "L" and "H" represent the light chain and heavy chain regions, respectively). These regions can be called Chothia CDRs, and these Chothia CDRs have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):73245 (1996)). Further definitions of CDR boundaries may not strictly follow one of the systems described above, but still... These overlap with CDRs. However, given predictions or experimental results suggesting that specific residues, groups of residues, or even the entire CDR may not significantly affect antigen binding, they may be shortened or lengthened.The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined by Kabat or Chothia.

[0080] CDRs can be obtained, for example, by constructing a polynucleotide encoding the desired CDR. Such polynucleotides can be prepared, for example, by using a polymerase chain reaction to synthesize the variable region using mRNA from an antibody-producing cell as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley-Liss, Inc. (1995)).

[0081] In various embodiments, the antibody contains at least one CDR sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. Here, CDR-H1 has the sequence shown in SEQ ID NO: 2, CDR-H2 has the sequence shown in SEQ ID NO: 3, CDR-H3 has the sequence shown in SEQ ID NO: 4, CDR-L1 has the sequence shown in SEQ ID NO: 5, CDR-L2 has the sequence shown in SEQ ID NO: 6, and CDR-L3 has the sequence shown in SEQ ID NO: 7. The anti-sclerostin antibody contains two or six CDRs in various embodiments.

[0082] In a preferred embodiment, the anti-sclerostin antibody comprises a set of six CDRs as follows: CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 5, CDR-L2 of SEQ ID NO: 6, and CDR-L3 of SEQ ID NO: 7.

[0083] In some or any embodiment, the antibody includes a light chain variable region containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 8 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) and a heavy chain variable region containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 9 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity). In various embodiments, the sequence difference compared to SEQ ID NO: 8 or 9 lies outside the CDR region in the corresponding sequence. In some or any embodiment, the antibody includes a light chain variable region containing the amino acid sequence described in SEQ ID NO: 8 and a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 9.

[0084] In some or any embodiment, the anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 11 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity), and all or part of a light chain (e.g., two light chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 10 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity).

[0085] In some or any embodiment, the anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 13 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity), and all or part of a light chain (e.g., two light chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 12 (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity).

[0086] Other examples of anti-sclerostin antibodies include, but are not limited to, those disclosed in International Patent Application Publications International Publication No. 2008 / 092894, International Publication No. 2008 / 115732, International Publication No. 2009 / 056634, International Publication No. 2009 / 047356, International Publication No. 2010 / 100200, International Publication No. 2010 / 100179, International Publication No. 2010 / 115932, and International Publication No. 2010 / 130830 (each of which is incorporated herein by reference in its entirety).

[0087] Those skilled in the art will understand that some proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the protein and the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperidine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a basic residue at the carboxyl terminus (e.g., lysine or arginine) by the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

[0088] Other modifications include: hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of α-amino groups of lysine side chains, arginine side chains, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86

[1983] (entirely incorporated by reference)); acetylation of N-terminal amines; and amidation of any C-terminal carboxyl group.

[0089] In some embodiments, the anti-sclerostin antibody in this formulation is at least about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml, about 74 mg / ml, about 75 mg / ml, about 76 mg / ml, about 77 mg / ml, about 78 mg / ml, about 79 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, About 94mg / ml, about 95mg / ml, about 96mg / ml, about 97mg / ml, about 98mg / ml, about 99mg / ml, about 100mg / ml, about 101mg / ml, about 102mg / ml, about 103mg / ml, about 104mg / ml, about 105mg / ml, about 106mg / ml, About 107mg / ml, about 108mg / ml, about 109mg / ml, about 110mg / ml, about 111mg / ml, about 112mg / ml, about 113mg / ml, about 114mg / ml, about 115mg / ml, about 116mg / ml, about 117mg / ml, about 118mg / ml, about 119m g / ml, about 120mg / ml, about 121mg / ml, about 122mg / ml, about 123mg / ml, about 124mg / ml, about 125mg / ml, about 126mg / ml, about 127mg / ml, about 128mg / ml, about 129mg / ml, about 130mg / ml, about 131mg / ml , about 132mg / ml, about 132mg / ml, about 133mg / ml, about 134mg / ml, about 135mg / ml, about 136mg / ml, about 137mg / ml, about 138mg / ml, about 139mg / ml, about 140mg / ml, about 141mg / ml, about 142mg / ml, about 143 mg / ml, approximately 144 mg / ml, approximately 145 mg / ml, approximately 146 mg / ml, approximately 147 mg / ml, approximately 148 mg / ml, approximately 149 mg / ml, approximately 150 mg / ml, approximately 151 mg / ml, approximately 152 mg / ml, approximately 153 mg / ml, approximately 154 mg / ml, approximately 155 mg / ml, approximately 156 mg / ml, approximately 157 mg / ml, approximately 158 mg / ml, approximately 159 mg / ml, or approximately 160 mg / ml, and for example, up to approximately 300 mg / ml, approximately 290 mg / ml, approximately 280 mg / ml, approximately 270 mg / ml, approximately 260 mg / ml, approximately 250 mg / ml,The concentration may be approximately 240 mg / ml, 230 mg / ml, 220 mg / ml, 210 mg / ml, 200 mg / ml, 190 mg / ml, 180 mg / ml, or 170 mg / ml. Any range characterized by the aforementioned combinations of endpoints is intended, and this range includes, but is not limited to, the following: approximately 70 mg / ml to 250 mg / ml, approximately 70 mg / ml to 200 mg / ml, approximately 70 mg / ml to 160 mg / ml, approximately 100 mg / ml to 250 mg / ml, approximately 100 mg / ml to 200 mg / ml, or approximately 100 mg / ml to 180 mg / ml.

[0090] viscosity In some embodiments, the viscosity of a composition containing one or more of the antibodies described herein is determined. The term “viscosity” as used herein refers to “absolute viscosity.” Absolute viscosity, sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity × density). The dimension of kinematic viscosity is L 2 / T, where L is length and T is time. In general, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s is equal to 1 cSt. Absolute viscosity is expressed in centipoise (cP) units. The SI unit of absolute viscosity is millipascal-second (mPa-s), where 1 cP = 1 mPa-s.

[0091] The viscosity of the composition may be measured several hours (e.g., 1 to 23 hours), several days (e.g., 1 to 10 days), several weeks (e.g., 1 to 5 weeks), several months (e.g., 1 to 12 months), or several years (e.g., 1 to 2 years, 1 to 3 years) after the addition of the antibody to the composition. Viscosity measurements may be performed at the storage temperature or administration temperature, for example, at 2 to 8°C or 25°C (room temperature). In some embodiments, the absolute viscosity of the liquid composition or reconstituted liquid composition at the storage temperature and / or administration temperature is 15 cP or less, or 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 cP or less. In some embodiments, the absolute viscosity of the liquid composition or reconstituted liquid composition is 6 cP or less.

[0092] In some embodiments, the viscosity of the antibody composition is measured before and after the addition of the antibody. Methods for measuring viscosity are known in the art, and include, for example, the use of a capillary viscometer or a cone-plate rheometer. Any method may be used, as long as the same method is used to compare the test formulation with the reference formulation.

[0093] Treatment method The antibodies and pharmaceutical compositions described herein are useful for the treatment or prevention of bone-related disorders, such as bone-related disorders associated with abnormal activity of osteoblasts or osteoclasts. In some embodiments, the antibody is administered to subjects suffering from bone-related disorders selected from the group consisting of: achondroplasia, occlavicular dysostosis, enchondromatosis, fibrous dysplasia, Gaucher disease, hypophosphatemic rickets, Marfan syndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteomycosis, sclerosing lesions, pseudoarthrosis, suppurative osteomyelitis, periodontal disease, and anti-epileptic drug-induced bone loss.Loss of bone, primary and secondary hyperparathyroidism, familial hyperparathyroidism, weight-loss-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, bone infiltration disorders, oral bone loss, mandibular osteonecrosis, juvenile Paget's disease, meroleostosis, metabolic bone disease, mastocytosis, sickle cell anemia / disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, tetracholastic anemia, mucopolysaccharide Diseases such as Fabry's disease, Turner syndrome, Down syndrome, Klinefelter syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, infantile-onset multi-system inflammatory disease, Winchester syndrome, Menkes' disease, Wilson's disease, ischemic bone disease (e.g., Legg-Calvé-Perthes disease and focal migratory osteoporosis), anemia, steroid-induced conditions, glucocorticoid-induced osteopenia, heparin-induced osteopenia, bone marrow disorders, scurvy, malnutrition, calcium deficiency, and osteoporosis. Osteopenia, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory state, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory bowel disease, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy-related bone loss, diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, fixation or disuse, reflex sympathetic dystrophy syndrome, focal osteoporosis, osteomalacia, bone loss associated with joint replacement, HIV-related bone loss, bone loss associated with growth hormone loss Decreased bone mass, cystic fibrosis-related bone loss, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone-free bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease-related facial bone loss, disease-related cranial bone loss, disease-related mandibular bone loss, disease-related cranial bone loss, aging-related bone loss, aging-related facial bone loss, aging-related cranial bone loss, aging-related mandibular bone loss, aging-related cranial bone loss, and space travel-related bone loss.

[0094] In some embodiments, the antibodies described herein are useful for improving outcomes in orthopedic procedures, dental procedures, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, and bone repair, such as fracture healing, nonunion, delayed healing, and facial reconstruction. Compositions comprising one or more antibodies may be administered before, during, and / or after a procedure, replacement, grafting, surgery, or repair.

[0095] In some embodiments, the antibodies described herein are useful for treating any fracture involving a gap between two segments of bone (e.g., a gap of at least about 1 mm between two segments of bone). In some or any embodiments, the gap is at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, or at least about 1 cm, or greater. In some or any embodiments, the gap is about 5 mm to 1 cm, or up to 1 cm. The terms “bone gap defect” and “partial skeletal defect” are used synonymously herein and refer to a gap between two segments of bone (e.g., a gap of at least 1 mm).

[0096] Exemplary bone gap defects include, but are not limited to, comminuted fractures, ununion fractures, partial skeletal defects, surgically created bone defects, surgically treated bone defects, and bone defects resulting from trauma or disease of the bone (e.g., arthritis, tumor removal (excision), or infection removal). In some or any embodiment, bone gap defects result from the removal of an infected portion of the bone or from the removal of cancer from the bone due to bone cancer (e.g., osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma, and chordoma). In some or any embodiment, bone gap defects are, for example, developmental deformities due to gene defects.

[0097] In some or any embodiment, a bone gap defect results from the removal of a bone portion containing a benign tumor. Exemplary benign bone tumors include, but are not limited to, osteomas, osteoid osteomas, osteoblastomas, osteochondromas, enchondromas, chondromyxofibromas, aneurysmal bone cysts, unilocular bone cysts, fibrous dysplasia of bone, and giant cell tumors of bone.

[0098] Antibodies do not need to heal the target of the disorder or provide complete protection from the development of bone-related disorders in order to achieve a beneficial biological response. Antibodies can be used prophylactically, meaning to provide overall or partial protection against bone-related disorders or their symptoms. Antibodies can be used therapeutically to improve bone-related disorders or their symptoms overall or partially, or to provide overall or partial protection against further progression of bone-related disorders or their symptoms. In fact, the materials and methods of the present invention are particularly useful for increasing bone mineral density and, optionally, for maintaining increased bone mineral density over a period of time.

[0099] In some embodiments, one or more doses of the antibody described herein are administered over a treatment period of, for example, about 1 week to about 18 months (e.g., about 1 month to about 12 months, about 1 month to about 9 months, or about 1 month to about 6 months, or about 1 month to about 3 months). In some embodiments, the subject is administered one or more doses of the antibody described herein over a treatment period of, for example, about 1 month to about 12 months (52 weeks) (e.g., about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months).

[0100] In addition, depending on the treatment plan selected for a particular target, it may be advantageous to administer the antibody in multiple doses or to space out the doses. In some embodiments, the antibody or fragment thereof is administered regularly over a period of one year (12 months, 52 weeks) or less (e.g., nine months or less, six months or less, or three months or less). In this regard, the antibody or fragment thereof is administered to a human once every three days, or every seven days, or every two weeks, or every three weeks, or every four weeks, or every five weeks, or every six weeks, or every seven weeks, or every eight weeks, or every nine weeks, or every ten weeks, or every eleven weeks, or every twelve weeks, or every thirteen weeks, or every fourteen weeks, or every fifteen weeks, or every sixteen weeks, or every seventeen weeks, or every eighteen weeks, or every nineteen weeks, or every twenty weeks, or every twenty-one weeks, or every twenty-two weeks, or every twenty-three weeks, or every six months, or every twelve months.

[0101] In some embodiments, one or more doses of the antibody are administered in amounts and for durations effective in increasing bone mineral density or treating bone disorders associated with decreased bone mineral density. In various embodiments, a subject (e.g., a human subject) is administered one or more doses containing approximately 50 milligrams to approximately 1,000 milligrams of antibody per week. For example, a single dose of antibody may contain at least approximately 5 mg, 15 mg, 25 mg, 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 120 mg, approximately 150 mg, approximately 200 mg, approximately 210 mg, approximately 240 mg, approximately 250 mg, approximately 280 mg, approximately 300 mg, approximately 350 mg, approximately 400 mg, approximately 420 mg, approximately 450 mg, approximately 500 mg, approximately 550 mg, approximately 600 mg, approximately 650 mg, approximately 700 mg, approximately 750 mg, approximately 800 mg, approximately 850 mg, 900 mg, approximately 950 mg, or up to approximately 1,000 mg of antibody. A range between any and all of these endpoints (e.g., approximately 50 mg to approximately 80 mg, approximately 70 mg to approximately 140 mg, approximately 70 mg to approximately 270 mg, approximately 75 mg to approximately 100 mg, approximately 100 mg to approximately 150 mg, approximately 140 mg to approximately 210 mg, or approximately 150 mg to approximately 200 mg, or approximately 180 mg to approximately 270 mg, or approximately 280 to approximately 410 mg) is also intended. This dose is administered at any interval, such as multiple times per week (e.g., twice or three times per week), once per week, once every two weeks, once every three weeks, or once every four weeks. In some or any embodiment, a dose of antibody in the range of approximately 120 mg to approximately 210 mg is administered twice a month. In some or any embodiment, a dose of approximately 140 mg of antibody is administered twice a week. In various configurations, a dose of approximately 210 mg of the antibody is administered once a month.

[0102] In some embodiments, a single or multiple dose of antibody may contain about 0.1 to about 50 milligrams per kg of body weight (e.g., about 5 to about 50 milligrams) (mg / kg), or about 1 to about 100 milligrams per kg of body weight (mg / kg). For example, antibody doses may be at least about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg g may contain approximately 37 mg / kg, approximately 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately 47 mg / kg, approximately 48 mg / kg, or approximately 49 mg / kg, or approximately 50 mg / kg, approximately 55 mg / kg, approximately 60 mg / kg, approximately 65 mg / kg, approximately 70 mg / kg, approximately 75 mg / kg, approximately 80 mg / kg, approximately 85 mg / kg, approximately 90 mg / kg, approximately 95 mg / kg, or up to approximately 100 mg / kg. The range between any and all of these endpoints (for example, approximately 1 mg / kg to 3 mg / kg, approximately 1 mg / kg to 5 mg / kg, approximately 1 mg / kg to 8 mg / kg, approximately 3 mg / kg to 8 mg / kg, approximately 1 mg / kg to 10 mg / kg, approximately 1 mg / kg to 20 mg / kg, approximately 1 mg / kg to 40 mg / kg, approximately 5 mg / kg to 30 mg / kg, or approximately 5 mg / kg to 20 mg / kg) is also intended.

[0103] Treatment monitoring Antibody-mediated increases in bone mineral density or bone mineral content can be measured using single and dual-energy X-ray absorptiometry, ultrasound, computed tomography, radiography, and magnetic resonance imaging. Bone mass can also be calculated from body weight or by other methods (see Guinness-Hey, Metab. Bone Dis. Relat. Res., 5:177-181 (1984)). In the art, for example, animal models that mimic the pathophysiology of human diseases such as osteoporosis and osteopenia are used to test the effects of pharmaceutical compositions and methods on parameters such as bone loss, bone resorption, bone formation, bone strength, or bone mineralization. Examples of such models include the ovariectomized rat model (Kalu, Bone and Mineral, 15:175-192 (1991); Frost and Jee, Bone and Mineral, 18:227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact., 1:193-207 (2001)). The efficacy of other sclerostin inhibitors can also be determined using the antibody activity measurement methods described herein.

[0104] In humans, bone mineral density can be clinically determined, for example, using dual-energy absorptiometry (DXA) of the hip and spine. Other techniques include quantitative computed tomography (QCT), ultrasound, single-energy absorptiometry (SXA), and X-ray absorptiometry. Common central skeletal sites for measurement include the spine and hip, while peripheral sites include the forearm, fingers, wrist, and heel. Except for ultrasound, the American Medical Association notes that BMD techniques generally involve the use of X-rays and are based on the principle that radiation attenuation depends on the thickness and composition of the tissue in the radiation path. All techniques involve comparing the results to a standard database.

[0105] Alternatively, the physiological response to one or more anti-sclerostin antibodies can be measured by monitoring bone marker levels. Bone markers are products of the bone remodeling process and are released by bone, osteoblasts, and / or osteoclasts. Fluctuations in bone resorption and / or bone formation "marker" levels suggest changes in bone remodeling / modeling. The International Osteoporosis Foundation (IOF) recommends the use of bone markers to monitor bone density therapy (see, for example, Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000) (incorporated herein by reference)). Markers indicating bone resorption (or osteoclast activity) include, for example, C-telopeptides (e.g., C-terminal telopeptide of type I collagen (CTX), or serum cross-linked C-telopeptides), N-telopeptides (N-terminal telopeptide of type I collagen (NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline, galactosylhydroxylysine, and tartrate-resistant acid phosphatases (e.g., serum tartrate-resistant acid phosphatase isoform 5b). Markers of bone formation / calcification include, but are not limited to, bone-specific alkaline phosphatase (BSAP), peptides released from the N-terminus and C-terminus elongation of type I procollagen (P1NP, PICP), and osteocalcin (OstCa). Several kits are commercially available for detecting and quantifying these markers in clinical samples such as urine and blood.

[0106] Combination therapy Treating a medical condition with two or more drugs targeting the same pathogen, biochemical pathway, or biological process may result in greater efficacy and reduced side effects compared to using the therapeutic doses of each drug alone. In some cases, the combined effect of drugs is additive (the combined effect is approximately equal to the sum of the effects of each drug alone), while in other cases, the effect is synergistic (the combined effect is greater than the sum of the effects of each drug given individually). As used herein, the term “combination therapy” means that two or more drugs are delivered simultaneously (for example, at the same time, or by administering one drug first followed by the second drug, for example, in sequence).

[0107] In some embodiments, the antibody is administered in conjunction with standard care treatment for addressing bone mineral density reduction (i.e., the antibody and standard care treatment are part of the same treatment plan). As used herein, the term “standard care” refers to treatment generally accepted by clinicians for certain patients diagnosed with certain diseases. In some embodiments, the antibody is administered in conjunction with a second bone strengthening agent useful for addressing bone mineral density reduction or bone defects. In some embodiments, the bone strengthening agent is selected from the group consisting of antiabsorbent agents, osteogenic agents (i.e., anabolic agents), estrogen receptor modulators (e.g., raloxifene, bazedoxifene, and rasofoxifene), and drugs having an inhibitory effect on osteoclasts. In some embodiments, the second bone strengthening agent is selected from the group consisting of bisphosphonates (e.g., but not limited to, alendronate sodium (FOSAMAX®), risedronate, ibandronate sodium (BONIVA®), and zoledronic acid (RECLAST®)); estrogen or estrogen analogues; anti-RANK ligand (RANKL) inhibitors, e.g., anti-RANKL antibodies (e.g., denosumab, PROLIA®); vitamin D or its derivatives or mimics; calcium sources, cathepsin-K (cat-K) inhibitors (e.g., odanacatib), tiborone, calcitonin, or calcitriol; and hormone replacement therapy. In some embodiments, the second bone strengthening agent may include, but is not limited to, parathyroid hormone (PTH) or its peptide fragments, PTH-related proteins (PTHrp), osteomorphonocytes, osteogenin, NaF, PGE2 agonists, statins, strontium ranerate, and sclerostin inhibitors (e.g., anti-sclerostin antibodies as described in U.S. Patent No. 7,592,429 or No. 7,872,106). In some embodiments, the second bone strengthening agent may be Tymlos® (avaloparatide), Forteo® (teriparatide), Preotact®, or Protelos®.In some embodiments, the second bone strengthening agent comprises bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, and / or BMP-15).

[0108] In some embodiments, the antibody-based combination therapy described herein may precede or follow the administration of an additional therapeutic agent (e.g., a second bone strengthening agent) at intervals of several minutes, several weeks, or several months. For example, the separate modalities may be administered within approximately 24 hours of each other, for example, within approximately 6 to 12 hours, or within approximately 1 to 2 hours, or within approximately 10 to 30 minutes of each other. In some situations, it may be desirable to significantly extend the treatment period, such as several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration of the various modalities. Repeated treatment with one or both of the combination therapies is particularly intended.

[0109] kit A pharmaceutical composition comprising one or more antibodies as described herein may be placed in a container (e.g., a vial or syringe) together with packaging material that provides instructions for the use of such a pharmaceutical composition. Generally, such instructions include specific expressions describing the antibody concentration and relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be essential for reconstituting the pharmaceutical composition in a particular embodiment. [Examples]

[0110] Example 1 - Evaluation of Stability For both protein and placebo, samples were packed into 3cc vials at a concentration of 1 mL. Romosozumab (70 mg / ml) was dialyzed to the formulation buffer identified in Table 2 below, aseptically filtered, and then packed aseptically. Storage temperatures were -70°C, -30°C, 4°C, 25°C, 37°C, and 45°C. Samples were stored for up to 24 months and removed for analysis at specified time points. Samples stored at accelerated temperatures of 25°C, 37°C, and 45°C were stored for 4 weeks.

[0111] [Table 2]

[0112] After 24 months of storage at 4°C, formulation 5 exhibited the lowest performance among the panel when measured by SE-HPLC analysis of high molecular weight species (Figure 1). The results revealed that formulations 1, 2, and 6 produced the lowest amount of HMW (dimer) form after 2 years of storage at 4°C. Similar stability profiles were observed for samples stored at -30°C and -70°C for 24 months (data not shown).

[0113] All tested formulations fell within 0.5% of the major peak range when measured by integration of peak area (data not shown). When stored at temperatures below 0°C, the pH data over two years showed a similar trend to that of the 4°C data, but less significantly (data not shown).

[0114] Similar stability profiles were observed over four weeks of storage from accelerated temperature storage at 37°C, however, the proportion of HMW species was higher compared to lower storage temperatures. The percentage of romosozumab HMW increased most significantly in formulation 1 when samples were stored at 45°C (Figures 2 and 3).

[0115] Light-obscuration sub-visible particle detection (HIAC) determined that the trend of particles invisible to the naked eye was similar for both romosozumab-containing and placebo samples (data not shown). All particle counts were below the compendial assay limit; however, it should be noted that while romosozumab in formulations 5, 7, 8, and 9 showed detectable levels throughout the entire stability period, the levels of particles invisible to the naked eye were not identical at either 10 μM or 25 μM at the final measurement point of 2 years (24 months). In the placebo sample, formulation 1 showed the highest level of particles invisible to the naked eye at 2 years, but was still within the USP limits for size and container (data not shown).

[0116] Cation exchange HPLC Long-term major peak stability data for romosozumab at 4°C and -70°C are shown in Figures 4–10. Overall, both temperatures show similar stability based on cation exchange HPLC. Regardless of temperature and time, the performance of the histidine formulation H6S was the most consistent (Figure 6 for comparison of major peak data, and Figure 9 for acidic peak data). Comparison of formulations stored at individual temperatures up to 2 years provides more detail, including additional time points, and is shown in Figures 4 and 5. Romosozumab stored at accelerated temperatures, including 25°C, 37°C, and 45°C, showed several different trends, and the major peak data at 25°C, 37°C, and 45°C were compared with that of the acetate-containing formulation stored at 4°C (Figures 4 and 7). The trends for the major peaks are reversed. While the trends for short-term stability are consistent, they differ when the results at relatively high temperatures are compared with the long-term stability figures.

[0117] Example 2 - pH and stability testing Nine formulations were acetate-based, and eight were glutamate-buffered, histidine-buffered, or succinate-buffered formulations. The following isotonic excipients were used alone or in combination: glycerol, sucrose, arginine, and methionine. All formulations were prepared by dialyzing romosozumab into each formulation listed in Table 3.

[0118] Each formulation tested contained 70 mg / mL of romosozumab. The volume filled into a 3cc vial was 0.5 mL. The samples in the vials were stored at -70°C, -30°C, 4°C, 25°C, 37°C, and 45°C. The samples were analyzed at appropriate time points as set by SEC-HPLC, GEX-HPLC, reduced CE-SDS, HIAC, and both reduced and unreduced SDS-PAGE.

[0119] Samples stored at accelerated temperatures were analyzed at 2 weeks, 4 weeks, 8 weeks, and 3 months. Samples stored at all other temperatures were analyzed up to 2 years.

[0120] [Table 3]

[0121] HIAC analysis conducted after two years of storage measured particle counts within USP guidelines for 10 and 25 micrometer size particles (data not shown).

[0122] Arginine preparation 26 appeared turbid after both 5 and 10 cycles of freeze-thaw at both -30°C and -70°C (data not shown). Due to this turbidity, particle analysis was not performed for these samples. All other preparations and placebo stored below 0°C had particle counts below the USP guidelines for 10 and 25 micrometer particles (data not shown). At 2 years, all samples stored at 4°C were well below the USP guideline limits (data not shown). Consistently, all tested preparations showed an increase in particle count at 3 months, but this trend was not observed at subsequent time points.

[0123] Size exclusion η High molecular weight species generally increased with increasing pH. Based on SE-HPLC data, formulations 17, 25, 26, and 28 performed similarly in suppressing HMW species formation at 4°C. Arginine-containing formulations suppressed high molecular weight morphology at accelerated temperatures. Tables 4 and 5 below show the results for romosozumab in various formulations stored at -30°C and -70°C at various time points (t0, 4 weeks, 3 months, 6 months, 1 year, 1.5 years, and 2 years), respectively.

[0124] [Table 4]

[0125] [Table 5]

[0126] Cation exchange HPLC Romosozumab in A52Su was analyzed by CEX-HPLC after 3 months of storage at 4°C, 25°C, and 37°C (Figure 11). The 2-year stability data, based on acidic peak data, shows that the performance of the arginine-containing formulations (formulations 25 and 26) was particularly good at 4°C (Figure 13). For comparison, basic peak stability data (Figure 14) and major peak stability data (Figure 12) are also shown.

[0127] Capillary electrophoresis - SDS After two years of storage at 4°C, succinate and arginine formulations, as well as acetate formulations at pH 4.8, exhibited the highest levels of high molecular weight species as measured by CE-SDS, as shown in Figure 15. All samples stored for two years showed similar profiles in terms of nonglycosylated heavy chain (NGHC) peak area between 0.3% and 0.4% (data not shown).

[0128] In summary, the data obtained in this example demonstrates that the arginine-containing formulation suppressed the high molecular weight species of romosozumab at accelerated temperatures compared to the other formulations tested.

[0129] Example 3 - Transport and concentration test of polysorbate 20 Romosozumab in formulation 4 was concentrated to 100 mg / mL using a Millipore agitated cell (Model 8400, 400 mL capacity) equipped with a PES membrane (10 kD cutoff). The concentrated romosozumab was dialyzed into each formulation, the concentration was adjusted to 70 mg / mL with a formulation buffer, and polysorbate 20 was added to the specified concentration.

[0130] The samples were transported under conditions that mimicked realistic transport conditions. Upon arrival, all samples were visually inspected together with static samples before storage at the specified temperature and before any freeze / thaw cycles.

[0131] [Table 6]

[0132] Size exclusion η Size exclusion HPLC analysis of romosozumab showed very small differences between samples stored at 4°C and samples subjected to real-time transport stress before stability storage (data not shown). The performance of various levels of polysorbate 20 was similar.

[0133] Counting of particles invisible to the naked eye by light shielding (HIAC) Formulated samples and placebos (both static and transported samples) were analyzed for particles invisible to the naked eye (HIAC). In particular, the counting results for both 10 μM and 25 μM showed that higher concentrations of polysorbate 20 tended to suppress particle formation over time (Tables 8-11).

[0134] [Table 7]

[0135] [Table 8]

[0136] [Table 9]

[0137] [Table 10]

[0138] Visual Analysis Both size exclusion HPLC and HIAC analysis did not show improvement in the performance of the formulations over time, and visual analysis indicated that certain formulations should be excluded from further testing. All samples at time zero were clear and scored 0 in visual analysis, meaning no particles were present (data not shown). However, both formulations 35 and 36, which are glutamate formulations, were opaque after 2 years of storage at all temperatures except for the sample at -20°C. Formulation 29, which contains both glycerol and arginine, was opaque after 2 years, except for two frozen samples (one at -20°C and one at -30°C). All samples also scored 0 (virtually no particles) at 2 years with respect to visible particles (data not shown).

[0139] In summary, the data obtained in this example demonstrates that the romosozumab formulation containing arginine was more stable under the various conditions tested compared to the other formulations tested.

[0140] Example 4 - High-Concentration Syringe Test Six syringe formulations and three vial formulations were tested under both static (non-shipment) and transport (shipment) modalities. Romosozumab concentrations were 70 mg / mL and 120 mg / mL. 1.0 mL was filled into a 1 cc syringe, and 2.0 mL into a 5 cc vial. The vial and syringe samples were transported by a domestic commercial freight carrier simulating realistic transport conditions, and then stored at either 4°C or 29°C for up to two years.

[0141] [Table 11]

[0142] Subvisible particle analysis by light shielding (HIAC) Results from the HIAC assay (detection of light-shielded subvisible particles) showed that all protein-containing preparations (presented in either vials or syringes) were below the USP guidelines for 10 μM and 25 μM particles. See Figures 16-19. In the case of succinate preparations, 0.010% (w / v) polysorbate 20 suppressed the formation of particles invisible to the naked eye at 70 mg / mL, but the effect was less pronounced at 120 mg / mL romosozumab. Regardless of the level of polysorbate 20, samples in vials contained fewer particles invisible to the naked eye compared to those in syringes. Overall, more particles invisible to the naked eye were detected at 120 mg / mL compared to 70 mg / mL.

[0143] Visual assay After two years of storage at 4°C or 29°C, the samples were visually evaluated. All placebo samples in vials and syringes were clear and particle-free after two years. All romosozumab samples in vials and syringes were also particle-free; however, many samples appeared "cloudy" or "turbid" (data not shown).

[0144] The turbid formulations were succinate compositions (one in a vial and two in syringes), and based on these results, these formulations were excluded from further investigation. The presence of polysorbate 20 did not appear to prevent the "turbid" results. Samples with lower romosozumab concentrations were "turbid," while the 120 mg / mL sample was only "cloudy." Only 32 vials of the formulation remained consistently "clear" after two years of storage.

[0145] Size exclusion HPLC (SE-HPLC) Size exclusion HPLC data showed an increase in high molecular weight (HMW) species during a two-year storage period at 4°C (data not shown). Romosozumab formulated at 120 mg / mL showed higher HMW generation over time at 4°C compared to the 70 mg / mL formulation, and this was observed in all formulations tested. HMW levels were also slightly higher in samples subjected to transport stress (data not shown). Although the difference was small, formulation 23 performed worst in both vials and syringes, likely due to the absence of polysorbate 20.

[0146] Data from samples stored at 29°C for two years showed significantly higher levels of HMW species when quantified by size exclusion HPLC compared to stability data at 4°C (data not shown). Samples subjected to transport stress also showed higher levels of HMW species compared to static samples. The result at 1.5 years for 120 mg / mL HMW% was low and deviated from this trend, and this observation applies to both temperature and static vs. transport samples, therefore this effect may be an assay artifact.

[0147] Cation exchange HPLC Both 70 mg / mL and 120 mg / mL romosozumab protein concentrations, stored at 4°C or subjected to transport stress, showed good stability when measured using cation exchange HPLC (data not shown).

[0148] Sequence List SEQUENCE LISTING <110> Amgen Inc. <120> ANTI-SCLEROSTIN ANTIBODY FORMULATIONS <130> PA25-567 <140> JP2025-284117 <141> 2020-08-07 <150> US 62 / 885,672 <151> 2019-08-12 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 190 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Human Sclerostin <400> 1 Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu 35 40 45 Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg 50 55 60 Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu 65 70 75 80 Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile 85 90 95 Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile 100 105 110 Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly 115 120 125 Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys 130 135 140 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly 145 150 155 160 Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala 165 170 175 Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr 180 185 190 <210> 2 <211> 5 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR1 <400> 2 Asp Tyr Asn Met His 1 5 <210> 3 <211> 17 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR2 <400> 3 Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 4 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo HCDR3 <400> 4 Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val 1 5 10 <210> 5 <211> 11 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romoLCDR1 <400> 5 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 6 <211> 7 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo LCDR2 <400> 6 Tyr Thr Ser Arg Leu Leu Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo LCD3 <400> 7 Gln Gln Gly Asp Thr Leu Pro Tyr Thr 1 5 <210> 8 <211> 123 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo light chain variable region <400> 8 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 107 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <223> romo heavy chain variable region <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody sequence <220> <221> MISC_FEATURE <223> romo light chain <400> 10 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Asp Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 11 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Humanized Antibody sequence <220> <221> MISC_FEATURE <223> romo heavy chain wild type <400> 11 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr 115 120 125 Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val 225 230 235 240 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 245 250 255 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 12 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> romo light chain wild type without signal sequence <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 13 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> romo heavy chain wild type without signal sequence <400> 13 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 210 215 220 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 290 295 300 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys

Claims

1. (a) Anti-sclerostin antibody; (b) Buffering agents containing glutamic acid, histidine, or succinic acid; and (c) Polyol A pharmaceutical composition containing, The aforementioned pharmaceutical composition has a pH of 4 to 7. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the buffering agent is present in an amount of about 10 mM to about 50 mM.

3. The pharmaceutical composition according to claim 1, wherein the polyol is present in an amount with an approximate concentration of about 1% to about 10% w / v.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polyol is sorbitol.

5. The pharmaceutical composition according to claim 4, wherein sorbitol is present in an amount of about 5% to about 10% w / v.

6. The pharmaceutical composition according to claim 4, wherein the sorbitol is present in an amount of about 5% w / v.

7. A pharmaceutical composition according to any one of claims 1 to 6, further comprising glycerol.

8. The pharmaceutical composition according to claim 7, wherein the glycerol is present at a concentration of about 1% to about 5% w / v.

9. The pharmaceutical composition according to claim 8, wherein the glycerol is present at a concentration of about 1% w / v.

10. The pharmaceutical composition according to claim 8, wherein the glycerol is present at a concentration of about 2.5% w / v.

11. A pharmaceutical composition according to any one of claims 1 to 10, further comprising sucrose.

12. The pharmaceutical composition according to claim 11, wherein the sucrose is present at a concentration of about 1% to about 10% w / v.

13. The pharmaceutical composition according to claim 12, wherein the sucrose is present at a concentration of about 9%.

14. A pharmaceutical composition according to any one of claims 1 to 13, further comprising an amino acid other than histidine.

15. The pharmaceutical composition according to claim 14, wherein the amino acid is arginine.

16. The pharmaceutical composition according to claim 15, wherein arginine is present in an amount of about 10 mM to about 250 mM.

17. The pharmaceutical composition according to claim 16, wherein arginine is present in an amount of approximately 100 mM.

18. A pharmaceutical composition according to any one of claims 1 to 6, 11, and 12, further comprising methionine.

19. The pharmaceutical composition according to claim 18, wherein methionine is present in an amount of about 10 mM to about 100 mM.

20. The pharmaceutical composition according to claim 19, wherein the methionine is present in an amount of about 20 mM.

21. A pharmaceutical composition according to any one of claims 1 to 20, further comprising a surfactant.

22. The pharmaceutical composition according to claim 21, wherein the surfactant is polysorbate 20, polysorbate 80, F16, or Triton.

23. The pharmaceutical composition according to any one of claims 1 to 22, comprising the anti-sclerostin antibody at a concentration of at least 70 mg / mL.

24. The pharmaceutical composition according to claim 23, comprising the anti-sclerostin antibody at a concentration of approximately 70 mg / mL to approximately 210 mg / mL.

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the anti-sclerostin antibody is romosozumab.

26. A pharmaceutical composition according to any one of claims 1 to 25, comprising 10 mM glutamic acid and 5% sorbitol at pH 4.

5.

27. A pharmaceutical composition according to any one of claims 1 to 25, comprising 10 mM glutamic acid and 5% sorbitol at pH 5.

2.

28. A pharmaceutical composition according to any one of claims 1 to 25, comprising 10 mM succinic acid and 5% sorbitol at pH 5.

2.

29. A pharmaceutical composition according to any one of claims 1 to 25, comprising 10 mM histidine and 5% sorbitol at pH 6.

30. A method for treating osteoporosis in a target subject, comprising administering to the target subject a pharmaceutical composition according to any one of claims 1 to 29.

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