Stabilized formulation containing anti-ANGPTL3 antibody

Abstract

The present invention provides a stable pharmaceutical formulation comprising a human antibody that specifically binds to human angiopoietin-like protein 3 (ANGPTL3). In addition to an anti-ANGPTL3 antibody, the formulation may contain a buffer; an organic cosolvent; at least one viscosity modifier, and optionally at least one amino acid. The pharmaceutical formulation of the present invention can be administered intravenously or subcutaneously and exhibits a significant degree of antibody stability after storage for several months.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as PTC International Patent Application on May 24, 2020, and claims priority to U.S. Provisional Patent Application No. 62 / 852,643, filed on May 24, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations comprising human antibodies that specifically bind to human angiopoietin-like (protein) 3 (ANGPTL3). [Background technology]

[0003] Therapeutic macromolecules (e.g., antibodies) must be formulated not only to render the molecule suitable for administration to patients, but also to maintain its stability during storage and subsequent use. For example, therapeutic antibodies in liquid solution are prone to degradation, aggregation, or undesirable chemical modification if the solution is not properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation, but also on the amounts and ratios of the excipients relative to each other. Furthermore, when preparing liquid antibody formulations, other considerations aside from stability must be taken into account. Examples of such additional considerations include the solution viscosity and antibody concentration that a given formulation can accommodate, as well as the visual quality or attractiveness of the formulation. Therefore, when formulating therapeutic antibodies, great care must be taken to arrive at a formulation that remains stable, contains the appropriate concentration of antibody, has the appropriate viscosity, and other properties that allow the formulation to be conveniently administered to patients.

[0004] Antibodies against angiopoietin-like protein 3 (ANGPTL3) are an example of a therapeutically relevant macromolecule that requires appropriate formulation. Anti-ANGPTL3 antibodies are clinically useful for treating diseases or disorders related to lipid metabolism, cardiovascular diseases or disorders, and angiogenesis-related diseases or disorders.

[0005] The amino acid and nucleotide sequences of human ANGPTL3 are set forth in SEQ ID NOs: 161 and 162, respectively. Exemplary anti-ANGPTL3 antibodies are described, for example, in US Pat. Nos. 5,629,999, 5,729,949, 5,729,959, and 5,729,959.

[0006] Although anti-ANGPTL3 antibodies are known, there remains a need in the art for new pharmaceutical formulations comprising such antibodies that are sufficiently stable and suitable for administration to patients. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 9,018,356 B2 [Patent Document 2] International Publication No. 2008 / 073300 [Patent Document 3] U.S. Patent No. 7,935,796 Summary of the Invention [Problem to be solved by the invention]

[0008] For many commercialized monoclonal antibodies, the final product presentation is dictated by the method of administration. One such method is based on self-administration and patient preference for less frequent administration. Self-administration by subcutaneous injection is one preferred means of administering parenteral products developed for the long-term treatment of many diseases. Subcutaneous (SC) injections require administration in a total volume of ≤2 mL, preferably ≤1 mL. Less frequent administration requires a higher concentration of drug per dose and, consequently, a higher protein concentration formulation. Therefore, to enable less frequent administration, a high concentration of drug (>150 mg per dose) that can be delivered in 1 mL is desirable. Highly concentrated formulations also allow for smaller doses. For example, to deliver 15 mg / kg of drug to a 100 kg patient, i.e., 1500 mg of drug, 150 mL of a 10 mg / mL formulation is required, whereas only 10 mL of a 150 mg / mL formulation is required. Thus, highly concentrated formulations are preferred because they allow for smaller injection volumes.

[0009] It is important to consider both the stability and viscosity of this higher protein concentration formulation. Because the relationship between protein concentration and viscosity is exponential, small differences in protein concentration can significantly affect viscosity and thus the ability of the drug to be delivered to the patient. The steepness of the curve plotting viscosity (y-axis) versus protein concentration (x-axis) can be influenced by the addition of excipients, particularly those that increase (e.g., sugars) or decrease (e.g., salts) viscosity and temperature. Furthermore, viscosity is directly related to the ability to deliver the drug through a syringe. Endurance is the force required to continuously dispense the contents of a prefilled syringe. Endurance is measured using a syringe force tester (Instron). The relationship between endurance and viscosity is linear.

[0010] Self-administration using prefilled syringes or autoinjectors requires formulations with low viscosity (typically less than approximately 20 cPoise). Therefore, there is a need to identify viscosity-reducing excipients and evaluate their effects on the hydrodynamic properties and stability of antibody (especially anti-ANGPTL3 antibody) formulations. This data can be used to enable the development of high-concentration amino acid-based liquid formulations with acceptable viscosities that can be used in prefilled syringes and device development. [Means for solving the problem]

[0011] The present invention fulfills the aforementioned needs by providing a stable pharmaceutical formulation comprising a fully human monoclonal antibody that specifically binds to human angiopoietin-like protein 3 (ANGPTL3). H4H1276S is a fully human monoclonal antibody that targets ANGPTL3, a key protein that inhibits lipoprotein lipase (LPL) when active. Inhibition of ANGPTL3 by H4H1276S restores LPL activity and promotes triglyceride and vLDL disposal. Thus, H4H1276S is potentially required for several disease pathways, including severe hypertriglyceridemia and homozygous familial hypercholesterolemia.

[0012] In one aspect, a low-viscosity, stable, high-concentration liquid pharmaceutical formulation is provided, comprising: (i) a human antibody that specifically binds to human angiopoietin-like protein 3 (ANGPTL3); (ii) a buffer; (iii) an organic cosolvent; and (iv) at least one viscosity-modifying agent. In one embodiment, the stable, high-concentration liquid pharmaceutical formulation further comprises at least one amino acid. In another embodiment, the formulation comprises a stabilizer. In another aspect, a low-viscosity, stable, high-concentration liquid pharmaceutical formulation is provided, comprising: (i) a human antibody that specifically binds to human angiopoietin-like protein 3 (ANGPTL3); (ii) a buffer; (iii) an organic cosolvent; and (iv) at least two viscosity-modifying agents. In one embodiment, the stable, high-concentration liquid pharmaceutical formulation further comprises at least one amino acid. In another embodiment, the formulation comprises a stabilizer. The term "viscosity-modifying agent" includes a viscosity-lowering agent or an excipient.

[0013] In various embodiments, the antibody is provided at a concentration of about 5±0.75 mg / mL to about 250±37.5 mg / mL. In one embodiment, the antibody is provided at a concentration of 12.5 mg / mL±1.85 mg / mL, or about 12.5 mg / mL. In another embodiment, the antibody is provided at a concentration of 25 mg / mL±3.75 mg / mL, or about 25 mg / mL. In another embodiment, the antibody is provided at a concentration of 50 mg / mL±7.5 mg / mL, or about 50 mg / mL. In another embodiment, the antibody is provided at a concentration of 100 mg / mL±15 mg / mL, or about 100 mg / mL. In one embodiment, the antibody is provided at a concentration of 150 mg / mL±22.5 mg / mL, or about 150 mg / mL. In another embodiment, the antibody is provided at a concentration of 165 mg / mL±24.75 mg / mL, or about 165 mg / mL. In another embodiment, the antibody is provided at a concentration of 175 mg / mL ± 26.25 mg / mL, or about 175 mg / mL. In another embodiment, the antibody is provided at a concentration of 200 mg / mL ± 30 mg / mL, or about 200 mg / mL.

[0014] In certain embodiments, the formulation comprises any one of the anti-ANGPTL3 antibodies disclosed in U.S. Patent No. 9,018,356 B2, which is incorporated herein in its entirety. In certain embodiments, the anti-ANGPTL3 antibody comprises (a) a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions 1, 2, and 3 (HCDR1-HCDR2-HCDR3), each comprising the sequences of SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 72, respectively; and (b) a light chain variable region (LCVR) comprising light chain complementarity determining regions 1, 2, and 3 (LCDR1-LCDR2-LCDR3), each comprising the sequences of SEQ ID NO: 76, SEQ ID NO: 78, and SEQ ID NO: 80, respectively. In one embodiment, the antibody comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 66 and a LCVR comprising the amino acid sequence of SEQ ID NO: 74. In another embodiment, the antibody comprises a HCVR having at least about 90% sequence identity to SEQ ID NO: 66 and a LCVR having at least about 90% sequence identity to SEQ ID NO: 74. In yet another embodiment, the antibody comprises a HCVR having at least about 95% sequence identity to SEQ ID NO:66 and a LCVR having at least about 95% sequence identity to SEQ ID NO:74.

[0015] In one embodiment, the pH of the liquid formulation is pH 6.0±0.5, pH 6.0±0.4, pH 6.0±0.3, pH 6.0±0.2, pH 6.0±0.1, pH 6.0±0.05, pH 6.0±0.01, or pH 6.0. In one embodiment, the pH of the liquid formulation is about pH 6.0±0.3.

[0016] In one embodiment, the buffer is histidine. In certain embodiments, the histidine is at a concentration of 5 mM ± 1 mM to 50 mM ± 10 mM, preferably 5 mM ± 1 mM to 25 mM ± 5 mM. In one embodiment, the histidine is at a concentration of 10 mM ± 2 mM, or 10 mM ± 1 mM, or about 10 mM. In another embodiment, the histidine is at a concentration of 20 mM ± 4 mM, or 20 mM ± 2 mM, or about 20 mM. In yet another embodiment, the histidine is at a concentration of 40 nM ± 8 mM, or 40 nM ± 4 mM, or about 40 nM.

[0017] In certain embodiments, the organic cosolvent is a non-ionic polymer containing a polyoxyethylene moiety. In one embodiment, the organic cosolvent is a surfactant. In some embodiments, the organic cosolvent is any one or more of polysorbate, poloxamer 188, and polyethylene glycol 3350. In one embodiment, the organic cosolvent is polysorbate 80. In one embodiment, the organic cosolvent is polysorbate 20.

[0018] In one embodiment, the organic cosolvent is present at a concentration of about 0.01%±0.005% to about 1%±0.5% "weight to volume" or "w / v," e.g., 0.1 g / ml = 10% and 0.01 g / ml = 1%. In certain embodiments, the organic cosolvent is polysorbate at a concentration of 0.05%±0.025% to 0.5%±0.25% (w / v). In one embodiment, the organic cosolvent is polysorbate 80 at a concentration of 0.2%±0.1% w / v, or about 0.2% w / v. In another embodiment, the organic cosolvent is polysorbate 80 at a concentration of 0.1%±0.05% w / v, or about 0.1% w / v. In one embodiment, the organic cosolvent is polysorbate 20 at a concentration of 0.2%±0.1% w / v, or about 0.2% w / v. In another embodiment, the organic co-solvent is polysorbate 20, which is at a concentration of 0.1%±0.05% w / v or about 0.1% w / v.

[0019] In certain embodiments, a stabilizer is included in the formulation. In one embodiment, the stabilizer is a sugar. In another embodiment, the sugar is sucrose. In various embodiments, the stabilizer is at a concentration of 1%±0.2% w / v to 20%±4% w / v, 5%±1% w / v to 15%±3% w / v, or 1%±0.2% w / v to 10%±2% w / v. In one embodiment, the stabilizer is sucrose at a concentration of 5%±1% w / v or about 5% w / v. In another embodiment, the stabilizer is sucrose at a concentration of 9%±1.8% w / v or about 9% w / v. In another embodiment, the stabilizer is sucrose at a concentration of 10%±2% w / v or about 10% w / v.

[0020] In one embodiment, at least one amino acid is included in the formulation. In one embodiment, the amino acid is L-proline. In certain embodiments, the amino acid is at a concentration of 1%±0.2% to 5%±1% w / v. In one embodiment, the amino acid is proline at a concentration of 1.5%±0.3% w / v or about 1.5% w / v. In one embodiment, the amino acid is proline at a concentration of 3%±0.6% w / v or about 3% w / v.

[0021] In one embodiment, the at least one viscosity modifier is an excipient selected from the group consisting of arginine-HCl, sodium chloride, histidine-HCl, sodium acetate, calcium chloride, magnesium chloride, calcium acetate, and magnesium acetate. In one embodiment, the viscosity modifier is arginine-HCl. In certain embodiments, the viscosity modifier is at a concentration of 25 mM to about 75 mM. In one embodiment, the viscosity modifier is arginine-HCl at a concentration of 50 mM to about 75 mM.

[0022] In certain embodiments, the viscosity of the liquid pharmaceutical formulation at 25°C is less than or equal to about 20 cPoise ± 10%. In certain embodiments, the viscosity at 25°C is 1.0 cPoise ± 10% to 20 cPoise ± 10%. In certain embodiments, the viscosity of the liquid pharmaceutical formulation is ≦15 cPoise. In certain embodiments, the viscosity of the liquid pharmaceutical formulation is ≦20 cPoise. In certain embodiments, the viscosity of the liquid pharmaceutical formulation is ≦10 cPoise. In certain embodiments, the viscosity at 25°C is 5 cPoise ± 10%, 6.0 cPoise ± 10%, 7.0 cPoise ± 10%, 7.1 cPoise ± 10%, 7.2 cPoise ± 10%, 7.9 cPoise ± 10%, 8.3 cPoise ± 10%, 9.0 cPoise ± 10%, 9.6 cPoise ± 10%, 10.0 cPoise ± 10%, 10.6 cPoise ± 10%, 11.4 cPoise ± 10%, 11.6 cPoise ± 10%, 11.8 cPoise ± 10%, 12.0 cPoise ± 10%, 13.0 cPoise ± 10%, 14.0 cPoise ± 10%, 15.0 cPoise ± 10%, or 16 cPoise ± 10%.

[0023] In one embodiment, (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 0 mM to 40±8 mM; (iii) polysorbate 80 at 0% to 0.5%±0.25% (w / v); and (iv) arginine-HCl. and (v) 0 to 5% ± 1% proline, at a pH of about 5.3 to about 6.7. The anti-ANGPTL3 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) such that the HCVR / LCVR combination comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3), wherein these complementarity determining regions comprise the amino acid sequences of SEQ ID NOs: 68-70-72 / 76-78-80, respectively. In one embodiment, the anti-ANGPTL3 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) comprising the amino acid sequences of SEQ ID NOs: 66 and 74, respectively. In certain embodiments, the anti-PD1 antibody comprises an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 isotypes. In one embodiment, the antibody comprises a human IgG4 isotype.

[0024] In certain embodiments, a stable, low-viscosity liquid pharmaceutical formulation is provided comprising: (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 0 mM to 40±8 mM; (iii) polysorbate 80 at 0% to 0.5%±0.25% (w / v); (iv) arginine-HCl at 50±10 mM to 75±15 mM; and (v) proline at 0% to 5%±1%, at a pH of about 5.3 to about 6.7, wherein the anti-ANGPTL3 antibody comprises a HCVR and a LCVR, wherein the HCVR has at least about 90% sequence identity to SEQ ID NO: 66 and / or the LCVR has at least about 90% sequence identity to SEQ ID NO: 74.

[0025] A stable, low-viscosity liquid pharmaceutical formulation is provided, comprising: (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 0 mM to 40±8 mM; (iii) polysorbate 80 at 0% to 0.5%±0.25% (w / v); (iv) arginine-HCl at 50±10 mM to 75±15 mM; and (v) proline at 0% to 5%±1%, at a pH of about 5.3 to about 6.7; the anti-ANGPTL3 antibody comprises a HCVR and a LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with five or fewer amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with two or fewer amino acid substitutions.

[0026] In certain embodiments, the formulation of any of the preceding aspects has a characteristic selected from the group consisting of: (i) the formulation is stable to long-term storage at 25°C, 5°C, -20°C, -30°C, and -80°C, as described herein; (ii) the formulation is stable to agitation stress, as described herein; (iii) the formulation has a low viscosity (a viscosity of less than about 20 cPoise, preferably less than about 15 cPoise); (iv) the formulation is stable to up to ±50% variation in formulation excipient concentration, as described herein; (v) the formulation is isotonic with respect to physiological conditions; (vi) the formulation is stable and compatible with subcutaneous delivery devices and procedures; and (vii) the formulation is stable to long-term storage in a pre-filled syringe.

[0027] In certain embodiments of this aspect, a stable liquid formulation is provided comprising: (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 5 mM±1 mM to 20±4 mM; (iii) polysorbate 80 at 0.05%±0.025% to 0.3%±0.15% (w / v); (iv) arginine-HCl at 50±5 mM to 75±7.5 mM; and (v) proline at 1%±0.2% to 5%±1%, at a pH of about 6.0; wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74. In one embodiment, the stable liquid formulation of this aspect has a viscosity of less than about 20 cP. In another embodiment, the stable liquid formulation of this aspect has a viscosity of less than about 15 cP.

[0028] In one embodiment of this aspect, the stable liquid formulation comprises: (i) anti-ANGPTL3 antibody 50±7.5 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) proline 3%±0.6%; and (v) arginine-HCl 70±5 mM, at a pH of 6.0±0.3, wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74.

[0029] In another embodiment, the stable liquid formulation comprises (i) anti-ANGPTL3 antibody 100±15 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) proline 3%±0.6%; and (v) arginine-HCl 70±5 mM, at a pH of 6.0±0.3, wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74.

[0030] In another embodiment, the stable liquid formulation comprises: (i) anti-ANGPTL3 antibody 150±22.5 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM; and (v) proline 3%±0.6%, at a pH of 6.0±0.3, wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair SEQ ID NOs: 66 / 74. In one embodiment of the formulations disclosed herein, the viscosity is less than about 20 cPoise, and in another embodiment, less than about 15 cPoise.

[0031] In another embodiment of this aspect, the stable liquid formulation comprises: (i) anti-ANGPTL3 antibody 175±26.25 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM; and (v) proline 3%±0.6%, at a pH of 6.0±0.3, wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74.

[0032] In another embodiment of this aspect, the stable liquid formulation comprises: (i) anti-ANGPTL3 antibody 200±30.00 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM; and (v) proline 3%±0.6%, at a pH of 6.0±0.3, wherein the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74.

[0033] In one embodiment of the formulations disclosed herein, the formulation further comprises sucrose 5%±1% (w / v).

[0034] In one embodiment, after storage of the formulation at 45° C. for 21 days, > about 95% of the antibody is native and > about 45% of the antibody is in the main charge form. In one embodiment, after storage of the formulation at 5° C. for 36 months, > about 98% of the antibody is native and > about 55% of the antibody is in the main charge form. In one embodiment, after storage of the formulation at −20° C. for 9 months, > about 98% of the antibody is native and > about 61% of the antibody is in the main charge form. In one embodiment, after storage of the formulation at −30° C. for 36 months, > about 98% of the antibody is native and > about 56% of the antibody is in the main charge form.

[0035] The cation exchange chromatography elution profile of a monoclonal antibody generally contains three peaks: an early and a late eluting peak (the so-called acidic and basic variants, respectively) and the most abundant peak (in the middle) referred to as the main peak (or main charge form or variant).

[0036] In one aspect, the liquid pharmaceutical formulation of any of the preceding aspects is provided in a container. In one embodiment, the container is a polycarbonate vial. In another embodiment, the container is a glass vial. In one embodiment, the glass vial is a Type 1 borosilicate glass vial with a fluorocarbon-coated butyl rubber stopper. In another embodiment, the container is a microinjector. In another embodiment, the container is a syringe. In another embodiment, the container is a prefilled syringe. In one embodiment, the syringe has a fluorocarbon-coated plunger. In a specific embodiment, the syringe is a 1 mL or 2.25 mL long glass syringe containing less than about 500 parts per billion of tungsten with a 27G needle, a fluorocarbon-coated butyl rubber stopper, and a latex-free, non-cytotoxic rubber tip cap. In a specific embodiment, the syringe is a 1 mL long glass syringe with a 27G thin-walled needle, a FLUROTEC-coated 4023 / 50 rubber stopper, and an FM27 rubber tip cap. In another specific embodiment, the syringe is a 1 mL or 3 mL plastic syringe fitted with a 27G needle. In one embodiment, the plastic syringe is supplied by BECTON DICKINSON, Inc. In another embodiment, the container is Type 1 clear glass with a FluroTec® coated 4432 / 50 chlorobutyl stopper.

[0037] In one aspect, a kit is provided, comprising any one of the pharmaceutical compositions of the preceding aspects, a container, and instructions for use.In one embodiment, the container is a pre-filled syringe.In one embodiment, the syringe is a NUOVA OMPI 1mL or 2.25mL long glass syringe with a 27G thin-walled needle, a FLUROTEC coated 4023 / 50 rubber stopper, and an FM27 rubber tip cap.

[0038] In certain embodiments, the present invention provides a solution comprising: (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 5 mM±1 mM to 20±4 mM; (iii) polysorbate 80 at 0.05%±0.025% to 0.3%±0.15% (w / v); and (iv) arginine-HCl. and (v) proline 1%±0.2% to 5%±1%, at a pH of 6.0±0.3, wherein the antibody comprises an HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74, and wherein the formulation has attributes selected from the group consisting of: (i) after 36 months of storage at 5°C, ≧98% of the antibody is in its native form; (ii) after 36 months of storage at 5°C, ≧55% of the antibody is in its predominant charge variant; and (iii) the formulation is stable to agitation stress, and after 120 minutes of agitation stress in a clear glass vial, ≧98% of the antibody is in its native form.

[0039] In certain embodiments, the present invention provides a solution comprising: (i) a human antibody that specifically binds to human ANGPTL3 at 5±0.75 mg / ml to 250±37.5 mg / ml; (ii) histidine at 5 mM±1 mM to 20±4 mM; (iii) polysorbate 80 at 0.05%±0.025% to 0.3%±0.15% (w / v); and (iv) arginine-HCl. and (v) proline 1%±0.2% to 5%±1%, at a pH of 6.0±0.3, wherein the antibody comprises an HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NOs: 66 / 74, and wherein the formulation has attributes selected from the group consisting of: (i) the formulation is stable and compatible for use in subcutaneous and / or intravenous delivery devices; (ii) the formulation is chemically and physically stable to dilution with standard diluents known in the art (e.g., 0.9% sodium chloride or 5% dextrose); (iii) the formulation is stable for pre-filled syringe or autoinjector formats; and (iv) the formulation is compatible with standard infusion pumps (e.g., peristaltic pumps, fluid displacement pumps).

[0040] Other embodiments will become apparent from examination of the detailed description that follows. [Brief explanation of the drawings]

[0041] [Figure 1] 1 is a graph depicting the effect of H4H1276S concentration on viscosity. [Figure 2] This table summarizes the effect of pH on the stability of H4H1276S 150 mg / mL incubated at 45°C for 28 days. a. Samples pass color and appearance if they are clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. b. This is reported as the change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC in all five formulations. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 3] This table summarizes the effect of polysorbate 80 concentration on the stability of H4H1276S 150 mg / mL following agitation (vortexing for 120 minutes). a. Color and appearance pass if the sample is clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. b. This is reported as the change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC for all 12 formulations. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 4]This table summarizes the effect of polysorbate 80 concentration on the stability of H4H1276S 150 mg / mL following 28 days of incubation at 45°C. a. Samples pass color and appearance if they are clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. b. This is reported as the change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC in all five formulations. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 5] Table summarizing the effect of polysorbate 80 concentration on subvisible particle formation for H4H1276S 150 mg / mL following agitation (vortexing for 120 min) or incubation at 45°C for 28 days. a. Data were filtered using an ECD (μm) ≥ 5.00, aspect ratio < 0.85, and Ignore Edge particle filter. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 6]This table summarizes the effect of sucrose and proline on the stability of H4H1276S following 9 months of incubation at -20°C. a. Corresponds to a 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, 70 mM arginine-HCl formulation. b. Samples pass color and appearance if they are clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. c. Reported as a change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC in all five formulations. CEX-UPLC, cation exchange ultra-high performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-high performance liquid chromatography; SE-UPLC, size-exclusion ultra-high performance liquid chromatography. [Figure 7] This table summarizes the effect of sucrose and proline on the stability of H4H1276S following eight freeze / thaw cycles. a. Corresponding to a 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, 70 mM arginine-HCl formulation. b. Samples pass color and appearance if they are clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. c. Reported as the change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC in all five formulations. CEX-UPLC, cation exchange ultra-high performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-high performance liquid chromatography; SE-UPLC, size-exclusion ultra-high performance liquid chromatography. [Figure 8]This table summarizes the effect of sucrose and proline on the stability of H4H1276S following 21 days of incubation at 45°C. a. Corresponds to a 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, 70 mM arginine-HCl formulation. b. Samples pass color and appearance if they are clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. c. Reported as the change in purity compared to the starting material. The starting material (without incubation) contains ≥98.4% native peak by SE-UPLC and ≥62.7% main peak by CEX-UPLC in all five formulations. CEX-UPLC, cation exchange ultra-high performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-high performance liquid chromatography; SE-UPLC, size-exclusion ultra-high performance liquid chromatography. [Figure 9] Table summarizing the effect of sucrose and proline on the stability of H4H1276S following 36 months of incubation at -30°C. a. Color and appearance pass if the sample is clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. b. Reported as change in purity compared to the starting material. The starting material (without incubation) contains 98.8% native peak by SE-UPLC for both formulations, and 59.2% main peak for the sucrose formulation and 60.0% main peak for the proline formulation, as determined by CEX-UPLC. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 10]Table summarizing the effect of sucrose and proline on the stability of H4H1276S following 36 months of incubation at 5°C. a. Color and appearance pass if the sample is clear to slightly opalescent, essentially free of visible particulates, and colorless to pale yellow. b. Purity is reported as the change compared to the starting material. The starting material (without incubation) contains 98.8% native peak by SE-UPLC for both formulations, and 59.2% main peak for the sucrose formulation and 60.0% main peak for the proline formulation, as determined by CEX-UPLC. c. The average of three independent samples is reported. CEX-UPLC, cation exchange ultra-performance liquid chromatography; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SE-UPLC, size-exclusion ultra-performance liquid chromatography. [Figure 11] 1 is a graph depicting viscosity versus H4H1276S concentration for lead formulations. [Figure 12] Figures 12A and 12B are graphs depicting the relationship between H4H1276S concentration, temperature, and viscosity. The asterisk and circle at H4H1276S 150 mg / mL correspond to the recommended storage temperature of 5°C (circle) or the recommended administration temperature of 25°C (star). The asterisk and circle at H4H1276S 165 mg / mL correspond to formulations at +10% manufacturing specifications. The drug formulation in Figure 12A is 10 mM histidine, 70 mM arginine-HCl, 5% sucrose, and 0.1% polysorbate 80, pH 6. The drug formulation in Figure 12B is 10 mM histidine, 70 mM arginine-HCl, 3% sucrose, and 0.1% polysorbate 80, pH 6. [Figure 13A] 13A-13C are bar graphs showing the results of screening for viscosity-reducing excipients. In Figure 13A, viscosity was measured for various excipients added to the base formulation, and pH was adjusted for a few of the choices. [Figure 13B]13A and 13B are bar graphs showing the results of screening for viscosity-reducing excipients. In Figure 13B, viscosity was measured for various excipients added to different base formulations. [Figure 14] 1 is a bar graph providing the relative increase in HMW species (quantified as a percentage) for various excipients following incubation at 45° C. for 21 days. [Figure 15A] FIG. 15A is a bar graph depicting the stability of H4H1276S formulations containing viscosity-reducing excipients. In FIG. 15A, the relative increase in HMW and acidic species and viscosity is measured for formulations containing 70 mM Arg-HCl versus 25 mM Mg(OAc) for various concentrations of sucrose and / or L-proline. [Figure 15B] 15B is a line graph depicting the stability of H4H1276S formulations containing viscosity-reducing excipients. In FIG. 15B, frozen storage stability (-20°C) was measured in terms of % HMW species over time. [Figure 16A] 16A and 16B are contour plots showing temperature, H4H1276S concentration, and viscosity relative to one another for a formulation containing 10 mM histidine, pH 6, 70 mM Arg-HCl, and 3% (w / v) proline (FIG. 16A); and for a formulation containing 10 mM histidine, pH 6, 70 mM Arg-HCl, and 5% (w / v) sucrose (FIG. 16B). [Figure 16B] 16A and 16B are contour plots showing temperature, H4H1276S concentration, and viscosity relative to one another for a formulation containing 10 mM histidine, pH 6, 70 mM Arg-HCl, and 3% (w / v) proline (FIG. 16A); and for a formulation containing 10 mM histidine, pH 6, 70 mM Arg-HCl, and 5% (w / v) sucrose (FIG. 16B). [Figure 17]1 is a graph depicting viscosity versus H4H1276S concentration at 20° C. Viscosity was plotted as a function of protein concentration. The data was fit to an exponential curve using GraphPad Prism. The equation can be used to predict viscosity based on known concentrations, which is useful for defining manufacturing specifications. DETAILED DESCRIPTION OF THE INVENTION

[0042] Before describing the present formulations and methods, it is to be understood that this invention is not limited to the particular formulations and methods, and experimental conditions described, as such formulations and methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in connection with a specific recited numerical value or range of numerical values, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all intermediate values ​​(e.g., 99.1, 99.2, 99.3, 99.4, etc.). Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0044] definition As used herein, the expression "pharmaceutical formulation" refers to a combination of at least one active ingredient (e.g., a small molecule, macromolecule, compound, etc. capable of exerting a biological effect in humans or non-human animals) and at least one inactive ingredient that, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to humans or non-human animals. The term "formulation," as used herein, means "pharmaceutical preparation" unless otherwise indicated. The present invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to certain embodiments of the present invention, the therapeutic polypeptide is an antibody, or an antigen-binding fragment thereof, that specifically binds to human angiopoietin-like protein 3 (ANGPTL3).

[0045] The term "human angiopoietin-like protein 3" or "hANGPTL3," as used herein, refers to ANGPTL3 having the nucleic acid sequence set forth in SEQ ID NO: 162 and the amino acid sequence of SEQ ID NO: 161, or a biologically active fragment thereof.

[0046] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule made up of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR) and a heavy chain constant region (CVR). H ;Domain C H 1. C H 2 and C H Each light chain is composed of a light chain variable region (LCVR) and a light chain constant region (CVR). L ) HCVRs and LCVRs can be further subdivided into hypervariable regions termed complementarity-determining regions (CDRs), which are interspersed with more conserved regions termed framework regions (FRs). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0047] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can dispense with one or two CDRs for binding have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-quarter to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs have no amino acids in contact with the antigen (see also Vajdos et al., 2002 J Mol Biol 320:415-428).

[0048] CDR residues that do not contact antigen can be identified by molecular modeling and / or empirically from regions of the Kabat CDR outside the Chothia CDR. When a CDR or its residues are omitted, they are usually replaced with amino acids that occupy the corresponding positions in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0049] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present invention may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences.

[0050] The fully human anti-hANGPTL3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains when compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antibodies and antigen-binding fragments thereof described herein are derived from any of the amino acid sequences disclosed herein, with one or more amino acids in one or more framework and / or CDR regions mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations").

[0051] Starting with the heavy and light chain variable regions disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline back mutations or combinations thereof. H and / or V L All of the framework and / or CDR residues within a domain are backmutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline sequence from which the antibody was originally derived).

[0052] Furthermore, the antibodies described herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, and certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by such general methods are encompassed within the present invention.

[0053] The present invention also includes anti-ANGPTL3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes anti-ANGPTL3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or 1 conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In one embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with 10 or fewer conservative amino acid substitutions therein. In another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with 8 or fewer conservative amino acid substitutions therein. In another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with 6 or fewer conservative amino acid substitutions therein. In another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with 4 or fewer conservative amino acid substitutions therein. In yet another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 with two or one conservative amino acid substitution therein. In one embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with ten or fewer conservative amino acid substitutions therein. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with eight or fewer conservative amino acid substitutions therein. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with six or fewer conservative amino acid substitutions therein. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with four or fewer conservative amino acid substitutions therein. In yet another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 with two or one conservative amino acid substitution therein.

[0054] Unless otherwise indicated, the term "antibody," as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules") and antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any native, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies are derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage display antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0055] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide, or a constrained FR3-CDR3-FR4 peptide). Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the term "antigen-binding fragment" as used herein.

[0056] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V associated with domain H For antigen-binding fragments containing domains, V H and V L The domains can be positioned in any suitable arrangement relative to each other. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0057] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains may be directly linked to one another or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above, linked to one another and / or to one or more monomeric V H Or V L It may be non-covalently bound to the domain (eg, by a disulfide bond).

[0058] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0059] In certain embodiments, the antibodies or antibody fragments described herein may be conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic agent, an immunosuppressant, or a radioisotope (an "immunoconjugate").

[0060] The term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding occurs when the antibody binds to an antigen with a binding affinity of about 1 x 10 -6 M or lower equilibrium dissociation constant (K D ) (i.e., K D(A smaller value indicates tighter binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds to hANGPTL3 may exhibit cross-reactivity with other antigens, such as ANGPTL3 molecules from other species, e.g., cynomolgus monkey ANGPTL3, mouse ANGPTL3, rat ANGPTL3, and / or hANGPTL4. Furthermore, multispecific antibodies (e.g., bispecifics) that bind to hANGPTL3 and one or more additional antigens are nevertheless considered to be antibodies that "specifically bind" to hANGPTL3 as used herein.

[0061] The term “K D ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0062] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other mAbs with different antigen specificities (e.g., an isolated antibody that specifically binds to hANGPTL3 is substantially free of mAbs that specifically bind to antigens other than hANGPTL3). However, an isolated antibody that specifically binds to hANGPTL3 may have cross-reactivity with other antigens, such as ANGPTL3 molecules from other species, such as cynomolgus monkeys, mice, and rats, and / or other related proteins, such as human ANGPTL4.

[0063] As used herein, a "neutralizing," "blocking," or "inhibiting" antibody (or an antibody that "neutralizes," "blocks," or "inhibits" ANGPTL3 activity) is intended to refer to an antibody whose binding to ANGPTL3 directly inhibits at least one biological activity of ANGPTL3, as assessed by standard in vitro assays known in the art. The terms "neutralizing," "inhibiting," "blocking," and "inhibiting" may be used interchangeably herein. A "non-blocking" antibody refers to an antibody whose binding to ANGPTL3 does not directly block the targeted activity of ANGPTL3, as assessed by standard in vitro assays, but may still be an "interfering" antibody whose binding to ANGPTL3 indirectly inhibits, reduces, attenuates, or otherwise interferes with at least one biological activity of ANGPTL3 in vivo, for example, by enhancing the clearance of ANGPTL3 from the circulation. Clearance of ANGPTL3 from the circulation can be particularly enhanced by a combination of at least two non-blocking antibodies. Neutralization, inhibition, suppression, reduction, attenuation, or interference with the biological activity of ANGPTL3 can be assessed by measuring one or more indicators of the biological activity of ANGPTL3 by one or more of several standard in vitro or in vivo assays known in the art.

[0064] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time bispecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0065] The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0066] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST or GAP, as discussed below.

[0067] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0068] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters; GCG version 6.1. FASTA programs (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-402.

[0069] The phrase "therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The precise amount will depend on the purpose of treatment, the age and size of the subject being treated, the route of administration, and the like, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0070] Bioequivalent preparations The anti-hANGPTL3 antibodies and antibody fragments described herein include proteins having amino acid sequences that differ from those of the described mAbs but retain the ability to bind to human ANGPTL3. Such mutant mAbs and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequences, but exhibit biological activity essentially equivalent to that of the described mAbs. Similarly, the anti-hANGPTL3 antibody-encoding DNA sequences described herein include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode anti-hANGPTL3 antibodies or antibody fragments that are essentially biologically equivalent to the anti-hANGPTL3 antibodies or antibody fragments described herein. Examples of such mutant amino acid and DNA sequences are discussed below.

[0071] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, the two antigen-binding proteins or antibodies are pharmaceutical equivalents or pharmaceutical substitutes that exhibit no significant differences in their rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either single or multiple doses. Some antibodies may be considered equivalents or pharmaceutical substitutes if they exhibit a comparable extent of absorption but not a comparable rate of absorption, but still be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling, are not essential for achieving effective body drug concentrations for chronic use, and are not considered medically significant for the particular drug product being studied. In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0072] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without a clinically significant change in immunogenicity or an expected increase in the risk of adverse effects, including a decrease in efficacy, when compared to continuing treatment without such switching.

[0073] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism of action for the conditions of use, so long as such mechanism is known.

[0074] Bioequivalence may be demonstrated by in vivo and in vitro methods. Criteria for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which concentrations of the antibody or its metabolites are measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-conducted clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0075] Bioequivalent variants of the anti-hANGPTL3 antibodies of the present invention can be constructed, for example, by various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation.

[0076] Antibodies that specifically bind to ANGPTL3 The pharmaceutical formulations of the present invention may comprise a human antibody or antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 3 (ANGPTL3). Exemplary anti-human ANGPTL3 antibodies that can be included in the pharmaceutical formulations of the present invention are set forth in Patent Application Publication No. U.S. Pat. No. 9,018,356 B2, WO 2008 / 073300, and U.S. Pat. No. 7,935,796, the disclosures of which are incorporated by reference in their entireties.

[0077] In certain embodiments, an anti-ANGPTL3 antibody comprises an HCVR / LCVR amino acid sequence pair having a SEQ ID NO: selected from the group consisting of 2 / 10 ("H4H1248P"), 18 / 26 ("H4H1250P"), 34 / 42 ("H4H1263S"), 50 / 58 ("H4H1268S"), 66 / 74 ("H4H1276S"), 82 / 90 ("H4H1279P"), 98 / 106 ("H4H1282P"), 114 / 122 ("H4H1292P"), 130 / 138 ("H4H1295P"), 146 / 154 ("H4H1296P"), and 180 / 188 ("H1M896N").

[0078] According to certain embodiments of the invention, an anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 68, HCDR2 of SEQ ID NO: 70, and HCDR3 of SEQ ID NO: 72. In certain embodiments, an anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO: 66.

[0079] According to certain embodiments of the invention, an anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises a light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80. In certain embodiments, an anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises an LCVR of SEQ ID NO: 74.

[0080] According to certain embodiments of the invention, the anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises a HCVR having 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:66.

[0081] According to certain embodiments of the invention, the anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises an LCVR having 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:74.

[0082] According to certain embodiments of the invention, the anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 66 with five or fewer amino acid substitutions.

[0083] According to certain embodiments of the invention, the anti-human ANGPTL3 antibody or antigen-binding fragment thereof comprises an LCVR comprising the amino acid sequence of SEQ ID NO: 74 with no more than two amino acid substitutions.

[0084] Sequence identity may be measured by any method known in the art (eg, GAP, BESTFIT, and BLAST).

[0085] The present invention also includes formulations comprising anti-ANGPTL3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative amino acid substitutions. For example, the present invention includes formulations comprising anti-ANGPTL3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0086] In certain embodiments, the anti-ANGPTL3 antibody comprises an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 isotypes.

[0087] A non-limiting exemplary antibody used in the Examples herein is referred to as "H4H1276S" or "mAb1." This antibody is also referred to as H4H1276S in U.S. Patent No. 9,018,356 B2. mAb1 (H4H1276S) comprises the HCVR / LCVR amino acid sequence pair having SEQ ID NOs: 66 / 74 and the HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3 domains represented by SEQ ID NOs: 68-70-72 / SEQ ID NOs: 76-78-80.

[0088] The full-length sequence of H4H1276S is as follows: Heavy chain sequence (SEQ ID NO: 195) EVQLVESGGGVIQPGGSLRLSCAASGFTFDDYAMNWVRQGPGKGLEWVSAISGDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAFFYCAKDLRNTIFGVVIPDAF DIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain sequence (SEQ ID NO: 196) DIQMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0089] The amount of antibody, or antigen-binding fragment thereof, included within the pharmaceutical formulation of the present invention can vary depending on the particular properties desired for the formulation and the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation contains 5±0.75 mg / mL to 250±37.5 mg / mL of antibody; 10±1.5 mg / mL to 240±36 mg / mL of antibody; 20±3.0 mg / mL to 230±34.5 mg / mL of antibody; 25±3.75 mg / mL to 240±36 mg / mL of antibody; 50±7.5 mg / mL to 230±34.5 mg / mL of antibody; 60±9 mg / mL to 240±36 mg / mL of antibody; 70±10.5 mg / mL to 230±34.5 mg / mL of antibody; 80±12 mg / mL to 220±36 mg / mL of antibody. 0±33 mg / mL; antibody 90±13.5 mg / mL to 210±31.5 mg / mL; antibody 100±15 mg / mL to 200±30 mg / mL; antibody 110±16.5 mg / mL to 190±28.5 mg / mL; antibody 120±18 mg / mL to 180±27 mg / mL; antibody 130±19.5 mg / mL to 170±25.5 mg / mL; antibody 140±21 mg / mL to 160±24 mg / mL; antibody 150±22.5 mg / mL; or 175±26.25 mg / mL.For example, the formulations of the invention may contain an antibody or antigen-binding fragment thereof that specifically binds to human ANGPTL3, and the antibody or antigen-binding fragment thereof may be about 5 mg / mL; about 10 mg / mL; about 15 mg / mL; about 20 mg / mL; about 25 mg / mL; about 30 mg / mL; about 35 mg / mL; about 40 mg / mL; about 45 mg / mL; about 50 mg / mL; about 55 mg / mL; about 60 mg / mL; about 65 mg / mL; about 70 mg / mL; about 75 mg / mL; about 80 mg / mL; about 85 mg / mL; about 90 mg / mL; about 95 mg / mL; about 100 mg / mL; about 105 mg / mL; about 110 mg / mL; about 115 mg / mL; about 120 mg / mL; about 130 mg / mL; about 140 mg / mL; about 150 mg / mL; about 160 mg / mL; about 170 mg / mL; about 180 mg / mL; about 190 mg / mL; about 200 mg / mL; about 210 mg / mL; about 220 mg / mL; about 230 mg / mL; about 240 mg / mL; about 250 mg / mL; about 260 mg / mL; about 270 mg / mL; about 280 mg / mL; about 290 mg / mL; about 300 mg / mL; about 310 mg / mL; about 320 mg / mL; about 330 mg / mL; about 340 mg / mL; about 350 mg / mL; about 360 mg / mL; about 370 mg / mL; about 380 mg / mL; about 390 mg / mL; about 400 mg / mL; about 410 mg / mL; about 420 mg / mL; about 4 25 mg / mL; about 130 mg / mL; about 135 mg / mL; about 140 mg / mL; about 145 mg / mL; about 150 mg / mL; about 155 mg / mL; about 160 mg / mL; about 165 mg / mL; about 170 mg / mL; about 175 mg / mL; about 180 mg / mL; about 185 mg / mL; about 190 mg / mL; about 195 mg / mL; about 200 mg / mL; about 205 mg / mL; about 210 mg / mL; about 215 mg / mL; about 220 mg / mL; about 225 mg / mL; about 230 mg / mL; about 235 mg / mL; about 240 mg / mL; about 245 mg / mL; or about 250 mg / mL. In one embodiment, a formulation of the invention comprises about 150 mg / mL of an anti-human ANGPTL3 antibody or antigen-binding fragment thereof.

[0090] Excipients and pH The pharmaceutical formulations of the present invention include one or more excipients. The term "excipient," as used herein, means any non-therapeutic agent added to a formulation to impart a desired density, viscosity, or stabilizing effect.

[0091] In certain embodiments, the pharmaceutical formulation of the present invention comprises at least one organic co-solvent of a type and amount that stabilizes the anti-hANGPTL3 antibody under rough handling or agitation conditions, such as vortexing. In some embodiments, "stabilizing" refers to preventing the formation of aggregated antibodies (on a molar basis) that account for more than 3% of the total amount of antibody during rough handling. In some embodiments, rough handling refers to vortexing a solution containing the antibody and the organic co-solvent for about 60 minutes or about 120 minutes.

[0092] In certain embodiments, the organic cosolvent is a nonionic surfactant such as alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181, poloxamer 188, and poloxamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate. Poloxamer 188 is also known as PLURONIC F68. In certain embodiments, the organic cosolvent included in the formulations of the present invention is polysorbate 80.

[0093] The amount of nonionic surfactant included in the pharmaceutical formulations of the present invention can vary depending on the particular properties desired for the formulation and the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the formulations can contain 0.01%±0.005% to 0.5%±0.25% surfactant. For example, the formulations of the present invention can contain about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, or about 0.18% of polysorbate 20 or polysorbate 80. %; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; about 0.30%; about 0.35%; about 0.40%; about 0.45%; about 0.46%; about 0.47%; about 0.48%; about 0.49%; about 0.50%; about 0.55%; or about 0.575%. In certain embodiments, the formulations of the present invention comprise about 0.1% (w / v) polysorbate 80.

[0094] The pharmaceutical formulations of the present invention may also include one or more stabilizers of a type and amount that stabilize the anti-hANGPTL3 antibody under conditions of heat stress. In some embodiments, "stabilized" means that when a solution containing the antibody and the thermal stabilizer is maintained at about 45°C for up to about 28 days, a significant percentage of the antibody is in the native conformation. In some embodiments, "stabilized" means that when a solution containing the antibody and the thermal stabilizer is maintained at about 45°C for up to about 28 days, only a small percentage of the antibody is aggregated. As used herein, "native" refers to the predominant form of the antibody by size exclusion, which is generally an intact monomer of the antibody. The term "native" also refers to the non-aggregated and non-degraded form of the antibody.

[0095] In certain embodiments, the heat stabilizer is a sugar, such as sucrose, and the amount of sugar included in the formulation can vary depending on the particular context and intended purpose of the formulation. In certain embodiments, the formulation can contain about 1% to about 15% sugar; about 2% to about 14% sugar; about 3% to about 13% sugar; about 4% to about 12% sugar; about 5% to about 12% sugar; about 6% to about 11% sugar; about 7% to about 10% sugar; about 8% to about 11% sugar; or about 9% to about 11% sugar. For example, pharmaceutical formulations of the invention can contain 4%±0.8%, 5%±1%, 6%±1.2%, 7%±1.4%, 8%±1.6%, 9%±1.8%, 10%±2%, 11%±2.2%, 12%±2.4%, 13%±2.6%, or about 14%±2.8% sugar (e.g., sucrose). In certain embodiments, formulations of the invention are sugar-free.

[0096] The pharmaceutical formulations of the present invention may also include a buffer or buffer system, which helps maintain a stable pH and stabilize the anti-hANGPTL3 antibody. In some embodiments, "stabilizing" refers to minimizing the amount of aggregated antibody when a solution containing the antibody and buffer is maintained at about 45°C for up to about 28 days. In some embodiments, "stabilizing" refers to maximizing the amount of antibody in the native configuration, as determined by size exclusion chromatography, when a solution containing the antibody and buffer is maintained at about 45°C for up to about 28 days. "Native" or "native configuration" refers to the antibody fraction that is neither aggregated nor degraded. This is generally determined by an assay that measures the relative size of antibody entities, such as a size exclusion chromatography assay. Non-aggregated and non-degraded antibody elutes in a fraction equivalent to native antibody and is generally the major elution fraction. Aggregated antibody elutes in a fraction exhibiting a larger size than native antibody. Degraded antibody elutes in a fraction exhibiting a smaller size than native antibody.

[0097] In some embodiments, "stabilize" means that at least about 46% of the antibody is in its predominant charge form, as determined by cation exchange chromatography, when a solution containing the antibody and buffer is maintained at about 45° C. for up to about 28 days. "Predominant charge" or "predominant charge form" refers to the fraction of antibody that elutes from the ion exchange resin in a predominant peak, which is generally flanked on one side by a more "basic" peak and on the other by a more "acidic" peak.

[0098] The pharmaceutical formulations of the present invention may have a pH of about 5.2 to about 6.4. For example, the formulations of the present invention may have a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH is 6.0±0.4, 6.0±0.3, 6.0±0.2, 6.0±0.1, about 6.0, or 6.0.

[0099] In some embodiments, the buffer or buffer system comprises at least one buffer having a buffering range that completely or partially overlaps the pH range of 5.5 to 7.4. In certain embodiments, the buffer comprises a histidine buffer. In certain embodiments, histidine is present at a concentration of 5 mM ± 1 mM to 15 mM ± 3 mM; 6 mM ± 1.2 mM to 14 mM ± 2.8 mM; 7 mM ± 1.4 mM to 13 mM ± 2.6 mM; 8 mM ± 1.6 mM to 12 mM ± 2.4 mM; 9 mM ± 1.8 mM to 11 mM ± 2.2 mM; 10 mM ± 2 mM; or about 10 mM. In certain embodiments, the buffer system comprises histidine at 10 mM ± 2 mM at pH 6.0 ± 0.3.

[0100] The pharmaceutical formulations of the present invention may also contain one or more excipients that maintain a reduced viscosity or reduce the viscosity of formulations containing high concentrations of anti-ANGPTL3 antibody drug substance (e.g., typically about 150 mg / ml of antibody). In certain embodiments, at least one viscosity-adjusting agent is selected from the group consisting of arginine-HCl, sodium chloride, histidine-HCl, sodium acetate (pH 5), calcium chloride, magnesium chloride, calcium acetate, and magnesium acetate. In certain embodiments, the formulations of the present invention contain arginine-HCl.

[0101] In certain embodiments, the pharmaceutical formulation comprises at least one amino acid. In certain embodiments, the amino acid is proline, and the pharmaceutical formulations of the present invention contain proline, preferably as L-proline, at a concentration of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. In some embodiments, the formulation contains proline in an amount sufficient to maintain the viscosity of the liquid formulation at less than 20±3 cPoise, less than 15±2.25 cPoise, or less than 11±1.65 cPoise. In some embodiments, the formulation contains proline in an amount sufficient to maintain a viscosity of 15±2.25 cPoise or less. In certain embodiments, the formulation may contain about 1% to about 5% proline; about 2% to about 4% proline; or about 3% proline. For example, pharmaceutical formulations of the invention may contain 1%±0.2%; 1.5%±0.3%; 2%±0.4%; 2.5%±0.5%; 3%±0.6%; 3.5%±0.7%; 4%±0.8%; 4.5%±0.9%; or about 5%±1% proline.

[0102] During antibody purification processes, it may be desirable or necessary to exchange one buffer for another to achieve appropriate excipient concentrations, antibody concentrations, pH, etc. Buffer exchange can be achieved, for example, by ultrafiltration / diafiltration (UF / DF) using, for example, a semi-permeable tangential flow filtration membrane. However, the use of such techniques can result in the Gibbs-Donnan effect [Bolton et al., 2011, Biotechnol. Prog. 27(1):140-152]. The accumulation of positive charges on the product side of the membrane during protein concentration is electrically balanced by the preferential migration of positive ions to the opposite side of the membrane. A potential consequence of this phenomenon is that the final concentration of certain components (e.g., histidine, L-proline, etc.) may be lower than the intended target concentration of these components due to the electrostatic repulsion of positively charged diafiltration buffer excipients against the positively charged antibody protein during the UF / DF step. Thus, the present invention provides that, for example, the concentrations of histidine and / or L-proline vary from the amounts or ranges recited herein due to the Gibbs-Donnan effect.

[0103] Volume exclusion describes the behavior of highly concentrated samples in which a significant portion of the total volume of the solution is occupied by solutes, particularly large molecules such as proteins, which exclude solvent from this space. This, in turn, reduces the total volume of solvent available for other solutes to dissolve therein, which can result in unequal partitioning across the ultrafiltration membrane. Thus, the present invention includes formulations in which, for example, the concentrations of histidine and / or L-proline may vary from the amounts or ranges recited herein due to volume exclusion effects.

[0104] During the manufacture of the formulations of the present invention, variations in the composition of the formulation may occur. These variations may include the concentration of the active ingredient, the concentration of excipients, and / or the pH of the formulation. Because changes in any of these parameters can potentially affect the stability or efficacy of the pharmaceutical product, a validated tolerance range (PAR) study was conducted to evaluate whether variations in composition affect the stability or efficacy of the antibody within a limited range. Thus, the present invention includes formulations containing anti-ANGPTL3 antibodies that are stable and retain efficacy with up to a 50% variation in excipient concentration. For example, the present invention includes formulations in which the stability and efficacy of anti-ANGPTL3 antibody formulations are not affected by ±10%, ±20%, ±30%, ±40%, or ±50% variations in the concentrations of the antibody, histidine, arginine-HCl, and / or polysorbate.

[0105] Stability and viscosity of pharmaceutical formulations The pharmaceutical formulations of the present invention typically exhibit a high level of stability. The term "stable," as used herein in reference to a pharmaceutical formulation, means that the antibody within the pharmaceutical formulation retains an acceptable degree of chemical structure or biological function after storage under specified conditions. A formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after storage for a specified period of time. Under certain circumstances, retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody's structure or function after storage for a specified period of time may be considered "stable."

[0106] Stability can be measured, inter alia, by determining the percentage of native antibody remaining in the formulation after storage at a specified temperature for a specified time. The percentage of native antibody can be determined, inter alia, by size exclusion chromatography (e.g., size exclusion ultra-performance liquid chromatography [SE-UPLC]), where native means non-aggregated and non-degraded. "Acceptable stability," as the phrase is used herein, means that at least 90% of the native form of the antibody is detectable in the formulation after storage at a given temperature for a specified time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native form of the antibody is detectable in the formulation after storage at a specified temperature for a specified time. The specified time period for determining stability can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The specified temperature at which the pharmaceutical formulation can be stored when stability is assessed can be any temperature between about -80°C and about 45°C, such as about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if greater than about 95%, 96%, 97%, or 98% of the native antibody is detected by SE-UPLC after 6 months of storage at 5°C. A pharmaceutical formulation may also be considered stable if greater than about 95%, 96%, 97%, or 98% of the native antibody is detected by SE-UPLC after 6 months of storage at 25° C. A pharmaceutical formulation may also be considered stable if greater than about 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% of the native antibody is detected by SE-UPLC after 28 days of storage at 45° C.A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native antibody is detected by SE-UPLC after 12 months of storage at −20° C. A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native antibody is detected by SE-UPLC after 12 months of storage at −30° C. A pharmaceutical formulation may also be considered stable if greater than about 96%, 97%, or 98% of the native antibody is detected by SE-UPLC after 12 months of storage at −80° C.

[0107] Stability can be measured, inter alia, by determining the percentage of antibody formed in aggregates within a formulation after storage at a specified temperature for a specified time, with stability being inversely proportional to the percent aggregates formed. The percentage of aggregated antibody can be determined, inter alia, by size exclusion chromatography (e.g., size exclusion ultra-performance liquid chromatography [SE-UPLC]). "Acceptable stability," as the phrase is used herein, means that at most 5% of the antibody is detected in aggregate form (also referred to as high molecular weight - HMW-form) in the formulation after storage at a given temperature for a specified time. In certain embodiments, acceptable stability means that at most about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in aggregate form in the formulation after storage at a given temperature for a specified time. The specified time period for determining stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature between about −80° C. and about 45° C., e.g., about −80° C., about −30° C., about −20° C., about 0° C., about 4° C. to 8° C., about 5° C., about 25° C., about 35° C., about 37° C., or about 45° C. For example, a pharmaceutical formulation can be considered stable if, after 12 months of storage at 5° C., less than about 2%, 1%, 0.5%, or 0.1% of the antibody is detected in aggregated form. A pharmaceutical formulation may also be considered stable if less than about 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in aggregated form after 3 months of storage at 25° C. A pharmaceutical formulation may also be considered stable if less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the antibody is detected in aggregated form after 28 days of storage at 45° C.A pharmaceutical formulation may also be considered stable if less than about 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detected in aggregated form after 3 months of storage at -20°C, -30°C, or -80°C.

[0108] Stability can be measured, inter alia, by determining the percentage of antibody that migrates in a more acidic fraction ("acidic form") during ion exchange than in the main fraction ("mainly charged form") of the antibody, with stability being inversely proportional to the fraction of antibody in the acidic form. Without wishing to be bound by theory, deamidating an antibody can make it more negatively charged and therefore more acidic compared to non-deamidated antibodies (see, e.g., Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). The percentage of "acidified" antibody can be determined, inter alia, by ion exchange chromatography (e.g., cation exchange ultra-performance liquid chromatography [CEX-UPLC]). "Acceptably stable," as that phrase is used herein, means that at most 45% of the antibody in the more acidic form is detected in the formulation after storage at a specified temperature for a specified time. In certain embodiments, acceptable stability means that at most about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in the acidic form in the formulation after storage for a specified time at a given temperature. In one embodiment, acceptable stability means that less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in the acidic form in the formulation after storage for a specified time at a given temperature. The specified time period over which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, the pharmaceutical formulation can be stored at any temperature between about -80°C and about 45°C, for example, about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, or about 45°C.For example, a pharmaceutical formulation may be considered stable if less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in a more acidic form after 3 months of storage at -80°C, -30°C, or -20°C. A pharmaceutical formulation may also be considered stable if less than about 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in a more acidic form after storage at 5°C for 6 months. A pharmaceutical formulation may also be considered stable if less than about 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in a more acidic form after 6 months of storage at 25°C. A pharmaceutical formulation may also be considered stable if less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in the more acidic form after 28 days of storage at 45°C.

[0109] Other methods may be used to assess the stability of the formulations of the invention, such as, for example, differential scanning calorimetry (DSC) to determine thermal stability, controlled stirring to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidity. For example, the formulations of the invention may be stored at about 5° C. to about 25° C. for 6 months or longer, and the OD of the formulation may be measured at 100° C. or 150° C. 405 Change in OD of the formulation at time 0 405 to less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or even less).

[0110] Stability may be assessed using measurements of the biological activity or binding affinity of an antibody to its target. For example, a formulation of the present invention may be considered stable if, after storage for a specified period of time (e.g., 1-12 months) at, for example, 5°C, 25°C, 45°C, etc., the anti-ANGPTL3 antibody contained in the formulation binds to ANGPTL3 with at least 90%, 95%, or more of the binding affinity of the antibody prior to storage. Binding affinity may be determined, for example, by ELISA or surface plasmon resonance. Biological activity may be determined, for example, by an ANGPTL3 activity assay in which ANGPTL3-expressing cells are contacted with a formulation containing an anti-ANGPTL3 antibody. Binding of the antibody to such cells may be measured directly, for example, by FACS analysis. Alternatively, downstream activity of the ANGPTL3 system may be measured in the presence of the antibody and compared to the activity of the ANGPTL3 system in the absence of the antibody. In some embodiments, ANGPTL3 may be endogenous to the cells. In other embodiments, ANGPTL3 may be ectopically expressed in the cells.

[0111] Additional methods for assessing antibody stability in formulation are provided in the Examples presented below.

[0112] The liquid pharmaceutical formulations of the present invention may, in certain embodiments, exhibit low to moderate levels of viscosity. As used herein, "viscosity" may be "kinematic viscosity" or "absolute viscosity." "Kinematic viscosity" is a measure of the resistive flow of a liquid under the influence of gravity. When two liquids of equal volume are placed in the same capillary viscometer and allowed to flow by gravity, the liquid with higher viscosity will take longer to flow through the capillary than the liquid with lower viscosity. For example, if one liquid takes 200 seconds to complete its flow and another takes 400 seconds, then on the kinematic viscosity scale, the viscosity of the second liquid is twice that of the first liquid. "Absolute viscosity," sometimes referred to as dynamic viscosity or simple viscosity, is the product of kinematic viscosity and liquid density (absolute viscosity = kinematic viscosity x density). The dimensions of kinematic viscosity are L 2 / T, where L is length and T is time. Kinematic viscosity is generally expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is 1 cSt. Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa-s), with 1 cP = 1 mPa-s.

[0113] As used herein, a low level of viscosity, in relation to a liquid formulation of the present invention, refers to an absolute viscosity of less than about 20 centipoise (cP). For example, a liquid formulation of the present invention is considered to have a "low viscosity" if the formulation exhibits an absolute viscosity of about 20 cP, about 19 cP, about 18 cP, about 15 cP, about 12 cP, about 10 cP, about 9 cP, about 8 cP, or less, when measured using standard viscosity measurement techniques. As used herein, a medium level of viscosity, in relation to a liquid formulation of the present invention, refers to an absolute viscosity of about 35 cP to about 20 cP. For example, a liquid formulation of the present invention is considered to have a "medium viscosity" if the formulation exhibits an absolute viscosity of about 34 cP, about 33 cP, about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, about 20 cP, about 19 cP, 18 cP, about 17 cP, about 16 cP, or about 15 cP, when measured using standard viscosity measurement techniques.

[0114] As shown in the Examples below, the inventors have made the surprising discovery that low-viscosity liquid formulations containing high concentrations of anti-human ANGPTL3 antibodies (e.g., from about 50 mg / mL to at least 250 mg / mL) can be obtained by formulating the antibodies with about 1% to about 5% proline, without the need for stabilizers such as sucrose. Such formulations are stable to stresses during handling and to storage at temperatures ranging from 45°C to -80°C (as demonstrated herein), and exhibit viscosities of less than about 15 cP.

[0115] Example Formulations According to one embodiment of the present invention, the pharmaceutical formulation is a stable, low-viscosity, generally physiologically isotonic liquid formulation that includes: (i) a human antibody or antigen-binding fragment thereof (e.g., H4H1276S) that specifically binds to human ANGPTL3 at a concentration of about 25 to about 250 mg / mL; (ii) a buffer system that provides sufficient buffering at about pH 6.0±0.3; (iii) an organic cosolvent, which protects the structural integrity of the antibody; and (iv) a viscosity modifier that is a viscosity-lowering excipient. According to another aspect of the present invention, the pharmaceutical formulation is a stable, low-viscosity, generally physiologically isotonic liquid formulation comprising: (i) a human antibody or antigen-binding fragment thereof (e.g., H4H1276S) that specifically binds to human ANGPTL3, at a concentration of about 25 to about 250 mg / mL; (ii) a buffer system that provides sufficient buffering at about pH 6.0±0.3; (iii) an organic cosolvent, which protects the structural integrity of the antibody; (iv) a viscosity modifier that is a viscosity-lowering excipient; and (v) an amino acid, which serves to maintain a manageable viscosity for injection in volumes convenient for subcutaneous administration.

[0116] According to one embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 25 mg / ml ± 7.5 mg / mL; (ii) histidine at 10 mM ± 2 mM, buffered at pH 6.0 ± 0.3; (iii) polysorbate 80 at 0.1% w / v ± 0.05% w / v; (iv) arginine-HCl 70 ± 5 mM; and (v) L-proline at about 3% (w / v) ± 0.6%. In another embodiment, the antibody comprises an HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0117] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 50 mg / ml ± 7.5 mg / mL; (ii) histidine 10 ± 2 mM; (iii) polysorbate 80 0.1% ± 0.05% (w / v); (iv) proline 3% ± 0.6%; and (v) arginine-HCl 70 ± 5 mM at pH 6.0 ± 0.3. In another embodiment, the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0118] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 100±15 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) proline 3%±0.6%; and (v) arginine-HCl 70±5 mM, at pH 6.0±0.3. In another embodiment, the antibody comprises an HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0119] According to another embodiment, a stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 150±22.5 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM, and (v) proline 3%±0.6% at pH 6.0±0.3. In another embodiment, the antibody comprises an HCVR / LCVR comprising the amino acid sequence pair SEQ ID NO: 66 / 74. In certain embodiments of the formulations disclosed herein, the viscosity is less than about 20 cPoise; in further embodiments, the viscosity of the formulation is less than about 15 cPoise.

[0120] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 175±26.25 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM, and (v) proline 3%±0.6% at pH 6.0±0.3. In another embodiment, the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0121] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3 and comprises an HCDR1 of SEQ ID NO: 68, an HCDR2 of SEQ ID NO: 70, an HCDR3 of SEQ ID NO: 72, an LCDR1 of SEQ ID NO: 76, an LCDR2 of SEQ ID NO: 78, and an LCDR3 of SEQ ID NO: 80, at a concentration of 200±30.00 mg / mL; (ii) histidine 10±2 mM; (iii) polysorbate 80 0.1%±0.05% (w / v); (iv) arginine-HCl 70±5 mM, and (v) proline 3%±0.6% at pH 6.0±0.3. In another embodiment, the antibody comprises a HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0122] Additional non-limiting examples of pharmaceutical formulations encompassed by the present invention are described elsewhere herein, including in the working examples provided below.

[0123] Container and method of administration The pharmaceutical preparations of the present invention can be contained in any container suitable for storing pharmaceuticals and other therapeutic compositions.For example, the pharmaceutical preparations can be contained in a sealed and sterilized plastic or glass container with a specified volume, such as a vial, an ampoule, a syringe, a cartridge, or a bottle.Different types of vials can be used to contain the formulations of the present invention, including, for example, transparent and opaque (e.g., amber) glass or plastic vials.Similarly, any type of syringe can be used to contain or administer the pharmaceutical preparations of the present invention.

[0124] The pharmaceutical formulations of the present invention can be contained in "normal tungsten" syringes or "low tungsten" syringes. As those skilled in the art will recognize, the process of making glass syringes generally involves the use of a hot tungsten rod, which functions to puncture the glass, thereby creating a hole through which liquid can be drawn and expelled from the syringe. This process deposits trace amounts of tungsten on the interior surface of the syringe. Subsequent cleaning and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means that the syringe contains greater than or equal to 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe, in accordance with the present invention, can contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or less ppb of tungsten.

[0125] Rubber plungers used in syringes and rubber stoppers used to close openings in vials can be coated to prevent contamination or preserve the stability of the pharmaceutical contents of the syringe or vial. Thus, according to certain embodiments, the pharmaceutical formulations of the present invention can be placed in syringes containing coated plungers or in vials sealed with coated rubber stoppers. For example, the plungers or stoppers can be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for use in vials and syringes containing the pharmaceutical formulations of the present invention are referenced, for example, in U.S. Pat. Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the contents of which are incorporated herein by reference in their entireties. Specific exemplary coated rubber stoppers and plungers that can be used in the context of the present invention are commercially available under the trade name "FluroTec®" from West Pharmaceutical Services, Inc. (Lionville, PA). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent pharmaceuticals from adhering to rubber surfaces.

[0126] According to certain embodiments of the present invention, the pharmaceutical formulation may be contained in a low-tungsten syringe equipped with a fluorocarbon-coated plunger.

[0127] The pharmaceutical formulations can be administered to a patient by parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or by transdermal, mucosal, intranasal, intrapulmonary, or oral administration. Many reusable pen or autoinjector delivery devices can be used to deliver the pharmaceutical formulations of the invention subcutaneously. Examples are AUTOPEN(TM) (Owen Mumford, Woodstock, UK), DISETRONIC(TM) pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25(TM) pen, HUMALOG(TM) pen, HUMALIN70 / 30(TM) pen(Eli Lilly, Indianapolis, IN), NOVOPEN(TM) I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR(TM)(Novo Nordisk, Copenhagen, Denmark), BD(TM) pen(Becton Dickinson, Franklin Lakes, NJ), OPTIPEN(TM), OPTIPEN PRO(TM), OPTIPEN Examples of disposable pens or autoinjector delivery devices useful for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR pen (sanofi-aventis), the FLEXPEN (Novo Nordisk), and the KWIKPEN (Eli Lilly), the SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), the PENLET (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, LP), and the HUMIRA Pen (Abbott Labs, Abbott Park, IL).

[0128] The use of a microinjector to deliver the pharmaceutical formulations of the present invention is also contemplated herein. As used herein, the term "microinjector" refers to a subcutaneous delivery device designed to slowly administer large volumes (e.g., up to about 2.5 mL or more) of a therapeutic formulation over an extended period of time (e.g., about 10, 15, 20, 25, 30, or more minutes). See, e.g., U.S. Pat. No. 6,629,949; U.S. Pat. No. 6,659,982; and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinjectors are particularly useful for delivering large doses of therapeutic proteins at high concentrations (e.g., about 100, 125, 150, 175, 200, or more mg / mL) or contained within viscous solutions.

[0129] In certain embodiments, the stable liquid pharmaceutical formulation of any of the preceding aspects can be placed in a sterile glass vial and administered as an IV infusion.

[0130] In one embodiment, the container is a 20 mL type 1 clear borosilicate glass vial. In certain embodiments, the container is a 2 mL or 3 mL type 1 borosilicate glass vial with a FluroTec® coated 4432 / 50 butyl rubber stopper.

[0131] In one embodiment, a liquid pharmaceutical formulation of the invention containing about 25 mg / mL or 50 mg / mL of mAb1 is administered intravenously and can be contained in a glass vial.

[0132] In certain embodiments, the invention provides an autoinjector comprising any of the liquid formulations described herein. In some embodiments, the invention provides an autoinjector comprising a stable liquid formulation comprising about 50 mg / mL, about 100 mg / mL, about 150 mg / mL, or about 175 mg / mL of mAb1, about 10 mM histidine, about 70 mM arginine-HCl, about 3% proline, and about 0.1% polysorbate 80 at a pH of about 6.0.

[0133] In certain embodiments, the present invention provides a pre-filled syringe containing any of the liquid formulations described herein.In some embodiments, the present invention provides a pre-filled syringe containing a stable liquid formulation comprising mAb1 at about 50 mg / mL, about 100 mg / mL, about 150 mg / mL or about 175 mg / mL, histidine at about 10 mM, pH at about 6.0, arginine-HCl at about 70 mM, proline at about 3% and polysorbate 80 at about 0.1%.In certain embodiments, the syringe is a 1 mL or 2.25 mL glass syringe equipped with a 27 gauge thin-walled needle, a fluorocarbon-coated rubber plunger and a rubber needle shield.

[0134] In one embodiment, a liquid pharmaceutical formulation containing about 175 mg / mL±26.25 mg / mL of mAb1 is subcutaneously administered in a prefilled syringe with a volume of approximately up to 2 mL. In certain embodiments, the syringe is a 1 mL or 2.25 mL long glass syringe fitted with a 27 gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle shield. In one embodiment, the syringe is an OMPI 1 mL long glass syringe fitted with a 27 gauge needle, an FM27 rubber needle shield, and a FLUROTEC®-coated 4023 / 50 rubber plunger.

[0135] In one embodiment, a liquid pharmaceutical formulation containing about 150 mg / mL ± 22.5 mg / mL of mAb1 is administered subcutaneously in a prefilled syringe in a volume of about 1 to about 2 mL. In one embodiment, the syringe is a 1 mL or 2.25 mL long glass syringe fitted with a 27-gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle shield. In one embodiment, the syringe is an OMPI 1 mL long glass syringe fitted with a 27-gauge needle, an FM27 rubber needle shield, and a FLUROTEC®-coated 4023 / 50 rubber plunger.

[0136] Therapeutic uses of pharmaceutical preparations The pharmaceutical formulations of the present invention are particularly useful for treating, preventing, or ameliorating any disease or disorder associated with ANGPTL3 activity, including diseases or disorders mediated by ANGPTL3. The disease or disorder treatable using the formulations of the present invention is any disease or condition that is improved, ameliorated, inhibited, or prevented, or whose incidence is reduced, by removing, inhibiting, reducing, or otherwise interfering with ANGPTL3 activity, compared to the disease or condition (e.g., an ANGPTL3-mediated disease or disorder) without anti-hANGPTL3 antibody treatment.

[0137] Examples of diseases or disorders treatable using the formulations of the present invention include, but are not limited to, diseases or disorders involving lipid metabolism such as hyperlipidemia, hyperlipoproteinemia and atherogenic dyslipidemia, dyslipidemia including diabetic dyslipidemia, hypertriglyceridemia including severe hypertriglyceridemia with TG > 1000 mg / dL, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, lipoatrophy, and the like, where such diseases or disorders are caused by, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor (LDLR) activity and / or LDL receptor deficiency (e.g., LDLR - / - Hyperlipidemia can be caused by a variety of conditions, including homozygous familial hypercholesterolemia (homozygous familial hypercholesterolemia with α-amyloid), altered ApoC2, ApoE deficiency, increased ApoB, increased production and / or decreased clearance of very low-density lipoproteins (VLDL), certain drug treatments (e.g., glucocorticoid treatment-induced dyslipidemia), any genetic predisposition, diet, lifestyle, and the like. The formulations of the present invention can also prevent or treat diseases or disorders associated with or resulting from hyperlipidemia, hyperlipoproteinemia, and / or dyslipidemia, including, but not limited to, cardiovascular diseases or disorders such as atherosclerosis, aneurysm, hypertension, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, and the like; acute pancreatitis; nonalcoholic steatohepatitis (NASH); glycemic disorders such as diabetes; obesity, and the like.

[0138] Other examples of diseases or disorders treatable using the formulations of the present invention include cancers / tumors and non-tumor angiogenesis-related diseases or disorders, including ocular angiogenic diseases or disorders such as age-related macular degeneration, central retinal vein occlusion or branch retinal vein occlusion, diabetic retinopathy, retinopathy of prematurity, and the like, inflammatory diseases or disorders such as arthritis, rheumatoid arthritis (RA), psoriasis, and the like. [Example]

[0139] The following examples are presented so as to fully disclose and describe to those of ordinary skill in the art how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are molar parts, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0140] Development of anti-ANGPTL3 antibody formulations Late-stage clinical development planned for both IV and SC administration of relatively high doses. Therefore, formulation development studies were conducted with the goal of developing a 150 mg / mL liquid formulation that could be used for either IV or SC injection. A highly concentrated liquid formulation would be necessary to deliver doses of up to 450 mg of H4H1276S in one or two SC injections. For IV administration, a highly concentrated liquid formulation would also be advantageous because it would allow for the addition of a smaller amount of DP to the IV infusion bag. This would support the use of a dose of 15 mg of H4H1276S per kg of patient weight.

[0141] Initial formulation development activities for the lyophilized H4H1276S formulation were conducted at low protein concentrations (5-50 mg / mL H4H1276S) and included evaluation of buffers, pH, organic cosolvents, surfactants, and sucrose to identify excipients that enhance protein stability. Using knowledge gained from initial formulation development, formulation development activities for the 150 mg / mL liquid formulation included evaluation of viscosity-reducing excipients, pH, surfactants, and thermal stabilizers to identify excipients that enhance protein stability at the relatively high protein concentrations of 150-200 mg / mL H4H1276S while maintaining a solution with acceptable viscosity.

[0142] Throughout formulation development, antibody formulations were developed and optimized using three major protein stress conditions (representing extreme handling conditions to which antibody drugs are not expected to be exposed during handling, manufacturing, transport, storage, and labeling) to assess the effect of potential real-world stresses on drug stability. These stress conditions were: Room temperature agitation (vortexing) of protein solutions. Vortexing in glass vials exceeds the agitation that occurs during protein handling and manufacturing. Incubate the protein solution at elevated temperatures (37°C, 40°C, or 45°C) relative to the proposed DP storage conditions (2°C to 8°C). Exposing the protein to multiple freeze-thaw cycles: Because proteins will undergo at least one freeze-thaw cycle during the manufacture of DPs, the multiple freeze-thaw cycles simulate and exceed the actual stresses that proteins are expected to experience. It included. Anti-ANGPTL3 Antibodies: Anti-ANGPTL3 antibodies are described in U.S. Patent No. 9,018,356 B2, which is incorporated herein in its entirety. An exemplary antibody used in the Examples below is the fully human anti-ANGPTL3 antibody H4H1276S (disclosed in "356"), also referred to herein as "mAb1," which comprises a heavy chain variable region / light chain variable region HCVR / LCVR amino acid sequence pair comprising SEQ ID NOs: 66 / 74; and heavy and light chain complementarity determining region CDR sequences comprising SEQ ID NOs: 68 / 70 / 72 / 76 / 78 / 80. [Example]

[0143] Example prescriptions In certain embodiments, mAb1 is formulated as an aqueous buffered formulation containing 5 mg / ml ± 0.75 mg / ml to 250 mg / ml ± 45.0 mg / ml of mAb1, 10 mM ± 2 mM histidine, 0.1% ± 0.05% w / v polysorbate, 50 to 75 mM arginine-HCl, and 1% ± 0.02% to 5% ± 1% w / v proline at pH 6.0 ± 0.3. An exemplary formulation contains H4H1276S 150 mg / mL, 10 mM histidine, pH 6.0, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 3% (w / v) proline. [Example]

[0144] Methods used to assess formulation stability Physical stability of a formulation refers to properties such as color, appearance, pH, turbidity, and protein concentration. Chemical stability refers to the formation of high molecular weight (HMW) species, low molecular weight (LMW) species, charge variants, and other chemical modifications of the protein. The physical and chemical stability of antibody drugs (e.g., H4H1276S) was evaluated using the following assays: Visual inspection of color and appearance (able to detect the presence of visible particles in the solution) pH Turbidity measured by the increase in optical density (OD) at 405 nm Micro-Flow Imaging™ (MFI) for analysis of particles invisible to the naked eye Protein concentration by reversed-phase ultra-performance liquid chromatography (RP-UPLC), reported as percent protein recovery relative to starting material Purity as assayed for: - Size-exclusion ultra-performance liquid chromatography (SE-UPLC) - Reducing and non-reducing microchip capillary electrophoresis sodium dodecyl sulfate (MCE-SDS) Charge Variant Analysis: - Cation Exchange UPLC (CEX-UPLC) - Imaged capillary isoelectric focusing (iCIEF) Potency by biological assay: - The relative potency of each sample was determined by biological assay (IC 50 Reference sample / IC 50 The measured potency of the storage-stability sample should be within 50%-150% of the measured potency of the reference standard.

[0145] The chemical stability of the formulation assesses the formation of covalently modified forms (e.g., covalent aggregates, cleavage products, or charge variants) and non-covalently modified forms (e.g., non-covalent aggregates) of the protein. Larger and smaller molecular weight degradation products can be separated from the native antibody by SE-UPLC and MCE-SDS methods. [Example]

[0146] Viscosity reducer selection To understand how viscosity is affected by increasing concentrations of H4H1276S, formulations were prepared using different protein concentrations in 10 mM histidine, pH 6.0, 0.5% sucrose, and 0.1% polysorbate 80. This formulation is equivalent to the First-in-Human (FIH) formulation used for IV administration in the initial clinical trial. The viscosity of each sample was measured at 20°C, and the results are shown in Figure 1 (top curve). The measured viscosity at 150 mg / mL was greater than 40 centipoise (cP), significantly greater than the targeted acceptable viscosity of 20 cP. Therefore, it was inferred that the use of viscosity-reducing excipients would be required to achieve a formulation with a target protein concentration of 150 mg / mL and acceptable viscosity.

[0147] To identify suitable viscosity-reducing excipients, the effect of selected excipients on the viscosity of the H4H1276S formulation was examined. Excipients included arginine-HCl, sodium chloride, histidine-HCl, sodium acetate, calcium chloride, magnesium chloride, calcium acetate, and magnesium acetate. The addition of 70 mM arginine-HCl reduced viscosity at all protein concentrations tested (Figure 1, lower curve). Arginine-HCl was found to be the most effective at reducing viscosity compared to the other excipients. Furthermore, arginine-HCl had the least impact on stability. A summary of the excipients tested for viscosity reduction is provided in Table 1 below. Arginine-HCl sufficiently reduced viscosity and was therefore selected for additional development studies. Subsequent formulation development studies included 70-75 mM arginine-HCl to determine the optimal pH, surfactant concentration, and thermal stabilizer.

[0148] [Table 1] [Example]

[0149] pH Selection The effect of pH on the thermal stability of H4H1276S was investigated in liquid formulations by incubating 150 mg / mL of H4H1276S at varying pH ranges in 10 mM histidine containing 5% (w / v) sucrose or 2% (w / v) proline at 45°C for 28 days. Sucrose and proline were considered potential thermal stabilizers and were included so that the effects of buffer and pH could be studied using formulation compositions that more closely represent the final formulation. Based on the results from SE-UPLC and CEX-UPLC analysis (Table 2, shown in Figure 2), higher pH minimizes HMW species formation, while lower pH minimizes charge variant formation. Histidine buffer at pH 6.0 was chosen as the formulation buffer because it provided the best balance between HMW species formation and charge variant formation. [Example]

[0150] Surfactant concentration optimization Surfactants are often added to antibody formulations to protect proteins from agitation-induced aggregation. When developing the initial lyophilizable formulation, HMW species formation was observed when 50 mg / mL of H4H1276S was agitated. Addition of 0.1% (w / v) polysorbate 80 protected H4H1276S from agitation-induced instability. However, protein concentration, thermostabilizer content, and the presence of other excipients can affect the protein's sensitivity to agitation stress. Therefore, we evaluated the minimum amount of polysorbate 80 required to protect 150 mg / mL of H4H1276S from agitation stress. Polysorbate 80 concentrations (% w / v) of 0.0%, 0.02%, 0.05%, and 0.1% were tested in the presence of 5% sucrose, 2% sucrose and 1.3% proline, or 2% proline (% w / v). Samples were formulated in 10 mM histidine with 70 mM arginine to more closely represent the final formulation, pH 6. The results are summarized in Tables 3, 4, and 5 (shown in Figures 3, 4, and 5, respectively).

[0151] After 120 minutes of stirring (Table 3, shown in Figure 3), a significant increase in HMW species (4.8–6.3%) was observed for the surfactant-free formulation. Addition of 0.02% (w / v) polysorbate 80 was not sufficient to protect H4H1276S from stirring-induced instability, while 0.05% (w / v) polysorbate 80 and higher provided sufficient stabilization, regardless of the thermal stabilizer included in the formulation. The data demonstrate that at least 0.05% (w / v) polysorbate 80 is required to protect H4H1276S from stirring-induced instability, and that stirring-induced instability is not affected by the choice of thermal stabilizer when at least 0.05% (w / v) polysorbate 80 is used. Addition of polysorbate 80 does not affect the formation of HMW species for H4H1276S when incubated at 45°C, regardless of the thermal stabilizer included in the formulation (Table 4, shown in Figure 4). The relative change in HMW species from t = 0 for formulations without heat stabilizers was comparable to formulations with polysorbate. The increase in HMW species ranged from 3.1 to 3.2%, 3.5 to 3.8%, and 3.8 to 4.1% for formulations with sucrose only, sucrose and proline, or proline only, respectively.

[0152] Comparing the heat stabilizer groups, H4H1276S showed moderately improved stability when formulated with sucrose (vs. proline) and incubated under stress conditions. However, the difference was not considered significant. The difference in the total relative change in charge variant distribution from t = 0 for all formulations evaluated was considered within assay variability. The effect of agitation and 45°C incubation on microparticle formation was assessed using MFI, and the results are presented in Table 5 (shown in Figure 5). There was no discernible trend in microparticle formation, suggesting that microparticle formation was not affected by the concentration of polysorbate 80 or heat stabilizer included in the formulation. Although stabilization was achieved at a polysorbate concentration of 0.05% (w / v), 0.1% (w / v) polysorbate 80 was selected as the surfactant concentration. The higher concentration of polysorbate ensures robustness of the formulation, with stability equivalent to a 0.05% (w / v) polysorbate formulation, and is even more stable upon dilution into the IV bag. [Example]

[0153] Effect of heat stabilizers The addition of stabilizers to antibody formulations can enhance protein stability in liquid formulations and during frozen storage. Sucrose has been included as a heat stabilizer in previous formulations. However, sucrose can also increase the viscosity of the solution. Therefore, proline was evaluated as a heat stabilizer, with the expectation that it could improve the storage stability of monoclonal antibody product formulations by affecting the solubility and colloidal stability of the protein without increasing the viscosity of the final formulation (Table 6, bottom).

[0154] [Table 2]

[0155] To aid in the selection of ingredients for the bulk formulated drug substance composition and to evaluate the need for the presence of a thermal stabilizer in the formulation, the stability of H4H1276S 175 mg / mL in 10 mM histidine, arginine-HCl (no sucrose or proline), pH 6.0 and 70 mM was investigated by evaluating frozen storage stability at -20°C and freeze / thaw stability (freezing at -30°C and thawing at room temperature) (Tables 7 and 8, shown in Figures 6 and 7, respectively). While -30°C is the intended long-term storage condition for the formulated drug substance, frozen storage stability at -20°C was evaluated as an accelerated frozen storage condition for development. Following 9 months of incubation at -20°C, a 7.2% increase in HMW species was observed (Table 7, shown in Figure 6). Although a slight increase in HMW species was observed in this formulation following 8 freeze / thaw cycles (Table 8, shown in Figure 7), the frozen storage stability data at -20°C demonstrates the need for a heat stabilizer to support long-term bulk drug substance frozen storage.

[0156] To identify and optimize the concentration of thermal stabilizers in liquid formulations, H4H1276S 150 mg / mL was formulated in 10 mM histidine, pH 6, 70 mM arginine-HCl, and 0.1% (w / v) polysorbate 80 and incubated at 45°C with different concentrations of sucrose and proline to assess protein stability. Formulations were also incubated at -20°C and subjected to freeze / thaw cycles (freezing at -30°C and thawing at room temperature) to compare frozen storage stability, which is required to support storage of bulk formulations. Polysorbate 80 was included in these formulations to more closely represent the final product formulation. Viscosity at 20°C was measured for all samples at t=0 and is summarized in Table 6 above. Table 6 demonstrates that replacing sucrose with proline reduces viscosity in a concentration-dependent manner, suggesting that formulations containing proline may be advantageous for the final product if it does not negatively impact stability.

[0157] H4H1276S exhibited moderately improved stability when formulated with sucrose compared to proline and incubated at 45°C for 21 days (shown in Table 9 and Figure 8). For the 5% sucrose formulation, the total relative change from t=0 for HMW species formation was 2.7%, and for the 2% proline formulation, the total relative change from t=0 for HMW species was 3.3%. This difference is not considered significant. The difference in the total relative change from t=0 for charge variants is considered within the assay variability due to the different formulations evaluated.

[0158] No significant changes in any quality attributes were observed for all formulations containing sucrose and / or proline when incubated at -20°C or subjected to eight freeze / thaw cycles (freezing at -30°C and thawing at room temperature) (shown in Tables 7 and 8, Figures 6 and 7). In summary, proline-containing formulations exhibited lower viscosity and comparable stability compared to sucrose-containing formulations when stored under accelerated or stressed storage conditions. Two lead formulations were selected based on these results: 1) H4H1276S 150 mg / mL, histidine 10 mM, pH 6, arginine-HCl 70 mM, polysorbate 80 0.1% (w / v), and sucrose 5% (w / v); and 2) H4H1276S 150 mg / mL, histidine 10 mM, pH 6, arginine-HCl 70 mM, polysorbate 80 0.1% (w / v), and proline 3% (w / v). The proline concentration was slightly increased to ensure sufficient protein stability under typical long-term storage conditions. [Example]

[0159] Formulation Selection Long-term stability was investigated to compare the stability profiles of the two lead formulations (see Example 7 above). Stability data was collected at -30°C to evaluate long-term storage of the bulk formulation substance (Table 10, shown in Figure 9). Stability data was collected at 5°C to evaluate long-term storage of the drug product (Table 11, shown in Figure 10). The data show that comparable stability profiles were obtained with both lead formulations.

[0160] The relationship between viscosity, protein concentration, and temperature was used to facilitate the selection of formulations that could potentially be delivered over a range of protein concentrations and temperatures. The effect of protein concentration on the viscosity of the final formulation and how excipients affect that relationship were examined. Similarly, temperatures were considered with a view to a) drug withdrawal and / or administration (approximately room temperature); b) bulk manufacturing process steps (typically approximately 15-25°C); and c) storage.

[0161] To guide the selection of the final formulation and better characterize the effect of the thermal stabilizer on viscosity, formulations with different H4H1276S concentrations were prepared for two lead formulations (10 mM histidine, pH 6.0, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 5% (w / v) sucrose; and 10 mM histidine, pH 6, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 3% (w / v) proline). Viscosity was measured at temperatures ranging from 5°C to 35°C. Figure 11 shows the viscosity vs. H4H1276S concentration relationship at 20°C for both formulations. The proline-containing formulation (lower curve in Figure 11) consistently had lower viscosity across multiple H4H1276S concentrations when compared to the sucrose-containing formulation (upper curve in Figure 11).

[0162] This trend was much more pronounced at lower temperatures (Figures 12A and 12B). The data show that the formulation containing 3% (w / v) proline (Figure 12B) provides a wider operating range of viscosity values ​​that are considered acceptable. Because the stability of the two lead formulations was comparable and the proline-containing formulation had a favorable viscosity profile, the formulation containing 3% (w / v) proline was selected for the final drug product formulation.

[0163] The final H4H1276S liquid pharmaceutical formulation contains H4H1276S 150 mg / mL, histidine 10 mM, arginine-HCl 70 mM, proline 3% (w / v), and polysorbate 80 0.1% (w / v) at pH 6.0. The primary degradation pathways identified during development were high molecular weight species and charge variants. The final osmolality of the formulation is approximately 480 mOsm / kg, and the viscosity is approximately 15 cPoise (at 20°C), which is suitable for clinical use.

[0164] A summary of H4H1276S formulations developed for preclinical (GLP toxicology) and clinical use is outlined in Table 12 below.

[0165] [Table 3] [Example]

[0166] Summary of stability studies for H4H1276S drug Studies were performed to evaluate the storage and accelerated stability of H4H1276S 150 mg / mL drug product (DP) (data not shown). Stability was assessed in terms of color and appearance, turbidity (increase in OD at 405 nm), pH, particulate matter by MFI, % total protein recovered by RP-UPLC (reverse-phase ultra-performance liquid chromatography), % purity by non-reducing and reducing MCE-SDS (microchip capillary electrophoresis-sodium dodecyl sulfate), % purity by SE-UPLC (size-exclusion ultra-performance liquid chromatography), charge variant analysis by CEX-UPLC (cation-exchange ultra-performance liquid chromatography), charge variant analysis by iCIEF (imaging capillary isoelectric focusing), and % relative potency by bioassay.

[0167] The DP used in the storage and accelerated stability studies was manufactured by incubating 5.0 mL of formulated drug substance (FDS) in a 20 mL Type 1 clear glass vial. H4H1276S DP was physically and chemically stable when stored at 5°C for at least 12 months. No discernible changes in physical or chemical stability were detected in any of the attributes monitored.

[0168] Subsequent incubation at 25°C / 60% relative humidity (accelerated stability) or 45°C (stress stability) was performed. These accelerated and stress conditions were chosen to elucidate the degradation pathway for H4H1276S. For the accelerated stability study, after 3 months at 25°C / 60% RH, discernible formation of HMW species and charge variants was detected. After 1 month of incubation at 25°C / 60% RH, no discernible formation of HMW species or charge variants was observed, indicating that H4H1276S DP can be exposed to room temperature for short periods. As determined by bioassay analysis, H4H1276S maintained its potency after incubation under accelerated conditions.

[0169] Incubation at 45°C resulted in significant formation of HMW species and charge variants in just 7 days, demonstrating that the increase in HMW species and the formation of charge variants are the major degradation pathways for H4H1276S DP. H4H1276S DP was found to be physically and chemically stable when agitated for 120 minutes (vortexed at ambient temperature) or subjected to eight freeze / thaw cycles (frozen at -30°C and thawed at room temperature). No discernible changes in physical or chemical stability were detected in any of the monitored attributes.

[0170] Results from accelerated and stressed DP storage stability studies indicate that H4H1276S 150 mg / mL DP is stable during manufacturing and storage. Furthermore, H4H1276S formulations can withstand short-term exposure to room temperature without compromising physical or chemical stability. H4H1276S 150 mg / mL DP is preferably stored at 2°C to 8°C, with exposure to temperatures greater than 2°C and limited to 8°C. [Example]

[0171] Development of high-concentration liquid amino acid-based pharmaceuticals for H4H1276S, from excipient screening to characterization of hydrodynamic properties H4H1276S was lyophilized and then reconstituted to higher protein concentrations with a solution containing the desired excipients. The pre-lyophilized formulation contained 4 mL of H4H1276S 87.5 mg / mL with 10 mM histidine, pH 6, and 2.5% (w / v) sucrose. The lyophilized cake contained 350 mg of solid H4H1276S. The lyophilized cake was reconstituted with 2–2.4 mL of the liquid formulation to obtain a final H4H1276S concentration (nominal) of 160–175 mg / mL. The reconstitution solution was adjusted so that the final sample contained 20 mM histidine, pH 6, with 5% (w / v) sucrose and the test excipients. To adjust the pH to 6–5, acetate buffer was added to the reconstitution solution to achieve a final acetate concentration of 40 mM.

[0172] To determine the accelerated and frozen storage stability of H4H1276S formulations containing viscosity-reducing excipients, the viscosity of the test formulations was measured at 20° C. using a Rheosens viscometer. The test formulations were incubated in 2 mL glass vials under the following conditions: (i) 45° C. for 0, 7, 14, and 21 days; (ii) −20° C. for 0, 1, 2, 3, 6, and 9 months; and (iii) 5° C., −30° C., and −80° C. for 0 and 3 months. The resulting material was assayed for aggregate content by SEC and for charge variant formation by CEX.

[0173] To understand the relationship between protein concentration, temperature, and viscosity for H4H1276S formulations with reduced viscosity, 200 mg / mL H4H1276S formulations were prepared containing the following combinations of excipients, all containing 10 mM histidine at pH 6, along with 0.1% (w / v) polysorbate 80: Sucrose 5% (w / v) Sucrose 5% (w / v), L-Arg-HCl 70 mL Sucrose 3% (w / v), proline 1.3% (w / v), L-Arg-HCl 70mL Proline 3% (w / v) Proline 3% (w / v), L-Arg-HCl 70mL The 200 mg / mL formulations were diluted with the respective formulation buffer to protein concentrations ranging from 50 to 200 mg / mL. Viscosity measurements of each formulation were performed using a Rheosens viscometer at temperatures ranging from 5 to 35°C. Finally, data analysis was performed in GraphPad Prism and MiniTab.

[0174] The results of the screen for viscosity-reducing excipients are shown in Figures 13A and 13B. In Figure 13A, upon reconstitution, the base formulation contained 175 mg / mL H4H1276S, 20 mM histidine, pH 6, 0.1% (w / v) polysorbate 80, and 5% (w / v) sucrose. To achieve a pH solution, acetic acid was added to a final concentration of 40 mM. A comparable formulation containing 40 mM acetic acid, pH 6.0, had a viscosity of 64.1 cPoise, demonstrating that both the addition of acetic acid and the pH adjustment to 5.0 reduced the viscosity of the H4H1276S formulation. In Figure 13B, upon reconstitution, the base formulation contained 165 mg / mL H4H1276S, 20 mM histidine / 40 mM acetic acid, pH 5.0, 0.1% (w / v) polysorbate 80, and 5% (w / v) sucrose.

[0175] The addition of monovalent and divalent salts (L-Arg-HCl, NaCl, CaCl2, MgCl2, Ca(OAc)2, Mg(OAc)2) decreased the viscosity of the H4H1276S formulation. The viscosity of the H4H1276S formulation was also decreased by adjusting the pH from 6 to 5. Finally, sucrose increased the viscosity of the H4H1276S formulation, while L-proline had no effect on the viscosity of the H4H1276S formulation.

[0176] The formation of high molecular weight (HMW) species following 21 days of incubation at 45°C is shown in Figure 14. Selected H4H1276S formulations containing various VR (viscosity reducing) excipients were incubated at 45°C and analyzed by SEC for aggregate content. The relative increase in HMW species from the t=0 sample is plotted as a function of formulation. Under accelerated conditions (45°C for 21 days), the following: · L-Arg-HCl had little effect on the formation of HMW species; · Lowering the pH of H4H1276S formulations increased the formation of HMW species; · The relative increase in HMW species was similar in CaCl2- and MgCl2-containing formulations compared to L-Arg-HCl- and NaCl-containing formulations; L-proline acted as a thermostabilizer, reducing the formation of HMW species; and · Compared with the L-Arg-HCl formulation, the Ca(OAc)2- and Mg(OAc)2-containing formulations reduced the formation of HMW species; was observed. Therefore, L-Arg-HCl or Mg(OAc)2 (substantial viscosity reduction) in pH 6 buffer (better accelerated stability) were selected as lead viscosity-reducing agents for further development.

[0177] The stability of H4H1276S formulations containing viscosity-reducing excipients is summarized in Figures 15A and 15B. H4H1276S formulations containing 70 mM L-Arg-HCl or 25 mM Mg(OAc)2 were formulated at 150 mg / mL with various concentrations of sucrose and / or L-proline. Concentrations were adjusted to target an osmolality of approximately 300 mM to maintain isotonicity. Formulations containing only L-Arg-HCl or Mg(OAc)2 were prepared at 175 mg / mL H4H1276S. Figure 15A depicts the degradation of H4H1276S following 21 days of incubation at 45°C; samples were incubated at 45°C and analyzed by SEC for aggregate content and CEX for acidic charge variant formation. The relative increase in HMW or acidic species from the t=0 sample is plotted as a function of formulation. Figure 15B depicts the frozen storage stability of H4H1276S; samples were incubated at -20°C for 9 months and analyzed by SEC for aggregate content. The percentage of HMW species is plotted as a function of time. Test formulations were also incubated at -80°C, -30°C, and 5°C for 3 months (data not shown). No change in HMW species was observed in any of the formulations at -80°C. At 5°C or -30°C, formulations without heat stabilizers had increased HMW species content.

[0178] The Mg(OAc)2 formulation was slightly less stable than the formulation containing L-Arg-HCl under accelerated storage conditions, with increased formation of both HMW and acidic species. Replacing sucrose with L-proline reduced viscosity in a concentration-dependent manner. Addition of sucrose, L-proline, or a combination of both excipients fully protected H4H1276S from HMW species formation at -20°C. Therefore, 70 mM L-Arg-HCl was selected as the lead viscosity-reducing agent for further development. The presented data support the use of Mg(OAc)2 as a backup excipient.

[0179] The relationship between protein concentration, temperature, and viscosity is shown in Figures 16A and 16B. These figures show contour plots of viscosity versus protein concentration and temperature. Contour plots were generated in Minitab. The shapes of the points corresponding to H4H1276S 150 mg / mL and 165 mg / mL (or 150 mg / mL + 10%) are the recommended storage temperature of 5°C (circle) or the recommended administration temperature of 25°C (star).

[0180] The relationship between viscosity and protein concentration at 20°C is shown in Figure 17. Viscosity was plotted as a function of protein concentration, and the data was fit to an exponential curve using GraphPad Prism. The equation can be used to predict viscosity based on known concentrations, which is useful for defining manufacturing specifications for the final product and informing process development. For a formulation containing 3% L-proline and 70 mM L-Arg-HCl, the equation is: Viscosity = 0.444 e 0.023[H4H1276S] is.

[0181] The above contour plots and graphs show the following observations: Compared to the sucrose-containing formulations, the L-proline-containing formulations were consistently less viscous at multiple H4H1276S concentrations (and temperatures; data not shown) with or without 70 mM L-Arg-HCl.

[0182] The viscosity versus protein concentration curve (at 20°C) for the formulation containing a combination of 5% sucrose and 3% L-proline was very similar to the 3% proline formulation. The distinction between these two formulations was actually more apparent at lower temperatures and higher protein concentrations. Contour plots for these formulations had subtle but distinct differences (data not shown).

[0183] When considering both temperature and manufacturing specifications, the L-proline formulation offered a wider working space of viscosity values ​​that were deemed acceptable for SC administration (Figure 16A).

[0184] An amino acid-based formulation containing H4H1276S 150 mg / mL, histidine 10 mM, pH 6 together with L-Arg-HCl 70 mM and proline 3% was selected for H4H1276S DP development.

[0185] The formulation containing a combination of both sucrose and L-proline was discarded for further development because this formulation did not offer any advantage in terms of frozen storage stability (see above).

[0186] Therefore, a thorough understanding of the relationship between viscosity, protein concentration, and temperature is found herein to inform decision-making and facilitate the selection of a formulation that is stable on storage and deliverable in a prefilled syringe or autoinjector format. To describe the manufacturing specifications for a DP and process the relevant manufacturing steps (i.e., bulk drug substance), viscosity should ideally be considered as a range of protein concentration, with the selected target concentration located at the lower end of the viscosity versus protein concentration curve. [Example]

[0187] Compatible with intravenous delivery devices For delivery in a clinical setting, H4H1276S DP 150 mg / mL can be diluted to clinical doses of 5 mg / kg and 15 mg / kg in an intravenous (IV) bag containing saline for IV administration. The in-use stability of H4H1276S to support IV administration of clinical doses was evaluated. Two admixture concentrations, H4H1276S 0.5 mg / mL and H4H1276S 20 mg / mL, were investigated to bracket low and high admixture concentrations that can be administered in a clinical setting.

[0188] To evaluate delivery of admixtures from IV bags using IV pumps and infusion sets containing in-line filters, saline-containing IV bags made from polyvinyl chloride (PVC) containing di-(2-ethylhexyl) butaphthalate (DEHP) and two types of commonly used infusion pumps (peristaltic and fluid displacement) were tested. Several infusion sets containing substrates (PVC with DEHP, PVC and polyethylene with TOTM) and 0.2 μm polyethersulfone in-line filters were also evaluated.

[0189] Assay The compatibility of the H4H1276S admixture with the materials used in the IV administration device was assessed by the following assays: Visual inspection of color and appearance pH Turbidity measured by the increase in optical density (OD) at 405 nm Analysis of invisible particles on admixtures by light obscuration method (HIAC) Protein concentration by reversed-phase high-performance liquid chromatography (RP-UPLC) Purity by SE-UPLC Potency, by bioassay: The relative potency of each sample was determined using a bioassay (IC 50 Reference sample / IC 50 The measured potency of a storage-stable sample should be within 50-150% of the assumed potency of the reference standard. was evaluated using.

[0190] Study Procedures To evaluate whether H4H1276S is stable in admixtures and when delivered intravenously, 100 mL saline IV bags containing H4H1276S DP were subjected to various stress conditions. The IV bags containing the admixtures were first held at 5°C for 24 hours; then, the bags were incubated at 25°C for at least 8 hours. After these incubations were completed, each of the infusion sets being evaluated was connected to the IV bag, primed with the admixture, and held at ambient room temperature for 1 hour. Each admixture was then pumped through each infusion set at a rate of 25 mL / hour or 500 mL / hour. In the clinic, the dose could be administered using DP diluted in a 100 mL or 250 mL IV bag. In the compatibility study, each dose was tested using a 100 mL IV bag.

[0191] Research results H4H1276S 0.5 mg / mL and 20 mg / mL were diluted in saline and were physically and chemically stable under all conditions tested, including: i) 24 hours at 5°C in an IV bag, ii) 8 hours at 25°C in an IV bag, and iii) 1 hour at ambient temperature in all infusion sets tested.

[0192] Furthermore, the H4H1276S mixture was stable when pumped through each of the infusion sets evaluated using various infusion pumps at rates of 25 mL / h or 500 mL / h. No precipitate was detected by visual inspection or turbidity measurements. The pH of the solutions was stable, and no discernible decrease in protein concentration was observed. No discernible changes in the relative percentages of high or low molecular weight species were observed in this compatibility study, as determined by size-exclusion ultra-performance liquid chromatography (SE-UPLC). No meaningful changes in the level of subvisible particulates were observed after samples were pumped through the infusion sets at 500 mL / h compared to t=0, as determined by HIAC analysis. Finally, all samples tested maintained potency, as determined by bioassays.

[0193] Data support the following dose preparation and administration in the clinic: · Saline IV bags made from PVC containing DEHP are compatible with H4H1276S for IV administration. · H4H1276S can be diluted to a concentration of only 0.5 mg / mL in a PVC IV bag containing saline for IV administration; H4H1276S can be diluted up to 20.0 mg / mL in a PVC IV bag containing normal saline for IV administration; The H4H1276S admixture in saline was stable after incubation in a PVC IV bag for up to 24 hours at 5°C and 8 hours at 25°C. The diluted H4H1276S admixture was administered within 4 hours of preparation; · H4H1276S admixture in saline can be administered using a standard infusion pump; · The H4H1276S admixture may be administered using an infusion set constructed of PVC containing DEHP, PVC containing TOTM, or polyethylene; · H4H1276S blend is compatible with the use of in-line 0.2 μm polyethersulfone filters; · The H4H1276S mixture can be administered at flow rates ranging from 25 to 500 mL / hour. [Example]

[0194] Further stability studies for H4H1276S drug product The H4H1276S formulation was analyzed for stability upon extended storage at -20°C and 5°C for periods up to 36 months.

[0195] Initially, the formulation was H4H1276S 150 mg / mL, histidine 10 mM, pH 6.0, arginine-HCl 70 mM, proline 3% (w / v), and polysorbate 80 0.1% (w / v), with a fill volume of 2.0 mL in 5 mL Nalge-Nunc gamma-irradiated polycarbonate vials with silicone-lined closures.

[0196] [Table 4]

[0197] The formulation was then H4H1276S drug product 150 mg / mL, L-histidine 10 mM, pH 6.0, arginine-HCl 70 mM, L-proline 3% (w / v), and polysorbate 80 0.1% (w / v) in a 5.0 mL fill volume in 20 mL Type 1 clear glass vials with 20 mm FluroTec® coated West S2-451 4432 / 50 GRY B2-40 stoppers.

[0198] [Table 5]

[0199] The formulation bulk was stored for extended periods (up to 36 months) at -20° C. and the drug product was stored for extended periods (up to 24 months) at 5° C. As can be seen in Tables 13 and 14, formulation H4H1276S demonstrated stability, e.g., values ​​remained within acceptable ranges in all tests over the entire range of storage lengths. [Example]

[0200] container The primary container for antibody pharmaceuticals intended for clinical development and product commercialization is the prefilled syringe, which is presented as a standalone syringe for self-injection or incorporated into an autoinjector for self-administration. Antibody formulations can also be developed in glass vials (for delivery by intravenous infusion).

[0201] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A liquid pharmaceutical formulation comprising: (a) an antibody or antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 3 (ANGPTL3) at a concentration of 50 mg / mL ± 7.5 mg / mL to 250 mg / mL ± 37.5 mg / mL, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 66 and a light chain variable region (LCVR) of SEQ ID NO: 74, wherein the antibody comprises an Fc region selected from the group consisting of human IgG1, IgG2, and IgG4 isotypes; (b) a buffer, wherein the buffer is histidine; (c) an organic co-solvent selected from the group consisting of polysorbate, poloxamer 188, and polyethylene glycol 3350; and (d) at least one viscosity modifier selected from the group consisting of arginine-HCl, sodium chloride, histidine-HCl, sodium acetate, calcium chloride, magnesium chloride, calcium acetate, and magnesium acetate, and the liquid pharmaceutical formulation has a pH of 6.0±0.3 and further comprises at least one amino acid, wherein the amino acid is proline, and the proline concentration is >0-5% w / v±1%.

2. 2. The liquid pharmaceutical formulation of claim 1, wherein the concentration of the antibody or antigen-binding fragment thereof is 150 mg / mL ± 22.5 mg / mL.

3. 2. The liquid pharmaceutical formulation of claim 1, wherein the concentration of the antibody or antigen-binding fragment thereof is 175 mg / mL ± 26.25 mg / mL.

4. 2. The liquid pharmaceutical formulation according to claim 1, wherein the histidine concentration is 5 mM±1 mM to 20 mM±4 mM.

5. 10. The liquid pharmaceutical formulation of claim 1 or 4, wherein the histidine concentration is about 10 mM±2 mM.

6. The liquid pharmaceutical formulation according to any one of claims 1 to 5, wherein the organic co-solvent is a polysorbate.

7. 7. The liquid pharmaceutical formulation of claim 6, wherein the polysorbate concentration is 0.01% w / v ± 0.005% to 0.5% w / v ± 0.25%.

8. 8. The liquid pharmaceutical formulation according to claim 6 or 7, wherein the polysorbate concentration is 0.1% w / v ± 0.05%.

9. The liquid pharmaceutical formulation according to any one of claims 6 to 8, wherein the organic co-solvent is polysorbate 80.

10. The liquid pharmaceutical formulation according to any one of claims 1 to 9, wherein the at least one viscosity modifier is arginine HCl.

11. 11. The liquid pharmaceutical formulation of claim 10, wherein the arginine HCl concentration is from about 50 mM to about 75 mM.

12. 12. The liquid pharmaceutical formulation of claim 10 or 11, wherein the arginine HCl concentration is about 70 mM.

13. 2. The liquid pharmaceutical formulation of claim 1, wherein the proline concentration is 3% w / v ± 0.6%.

14. 1. A liquid pharmaceutical formulation comprising: i) an anti-ANGPTL3 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) of SEQ ID NO: 66 and a light chain variable region (LCVR) of SEQ ID NO: 74, wherein the antibody comprises an Fc region selected from the group consisting of human IgG1, IgG2, and IgG4 isotypes; ii) histidine 5 mM ± 1 mM to 20 mM ± 4 mM; iii) polysorbate 80 0.1% w / v ± 0.05% to 0.5% w / v ± 0.25%; iv) arginine-HCl 50 mM to 75 mM; and v) proline 1% w / v ± 0.2% to 5% w / v ± 1%; and a pH of 6.0 ± 0.

3.

15. 15. The liquid pharmaceutical formulation of claim 14, wherein the histidine concentration is 10 mM ± 2 mM, the polysorbate concentration is 0.1% w / v ± 0.05%, the arginine-HCl concentration is about 70 mM, and the proline concentration is 3% w / v ± 0.6%.

16. 16. The liquid pharmaceutical formulation of any one of claims 1 to 15, having a viscosity of less than about 20 cP.

17. 17. The liquid pharmaceutical formulation of any one of claims 1 to 16, wherein at least about 95% of the antibody or antigen-binding fragment thereof retains the native configuration after 21 days at 45°C.

18. 18. The liquid pharmaceutical formulation of any one of claims 1 to 17, wherein at least about 45% of the antibody or antigen-binding fragment thereof is the predominant charge variant of the antibody or antigen-binding fragment thereof after 21 days at 45°C.

19. 19. The liquid pharmaceutical formulation of any one of claims 1 to 18, wherein at least about 98% of the antibody or antigen-binding fragment thereof retains the native configuration after 36 months at 5°C.

20. 20. The liquid pharmaceutical formulation of any one of claims 1 to 19, wherein at least about 55% of the antibody or antigen-binding fragment thereof is the predominant charge variant of the antibody or antigen-binding fragment thereof after 36 months at 5°C.

21. 21. The liquid pharmaceutical formulation of any one of claims 1 to 20, wherein at least about 98% of the antibody or antigen-binding fragment thereof retains the native configuration after 36 months at -30°C.

22. 22. The liquid pharmaceutical formulation of any one of claims 1 to 21, wherein at least about 57% of the antibody or antigen-binding fragment thereof is the predominant charge variant of the antibody or antigen-binding fragment thereof after 36 months at -30°C.

23. The liquid pharmaceutical formulation according to any one of claims 1 to 22, in a pre-filled syringe or auto-injector.

24. The liquid pharmaceutical formulation according to any one of claims 1 to 22, which is in a glass vial.

25. A kit comprising the liquid pharmaceutical formulation of any one of claims 1 to 22, a container, and instructions.

26. 26. The kit of claim 25, wherein the container is a prefilled syringe or an autoinjector.

27. 25. The liquid pharmaceutical formulation of any one of claims 1 to 24, for use in a method for treating, preventing, or ameliorating any disease or disorder associated with ANGPTL3 activity or mediated by ANGPTL3 in a subject, the method comprising administering the liquid pharmaceutical formulation to the subject.

28. 28. The liquid pharmaceutical formulation of claim 27, which is administered subcutaneously to a subject.

Images