Stable formulations containing anti-angptl3 antibodies

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application was filed on May 24, 2020 as a PCT international patent application 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. Technical Field

[0003] This invention relates to the field of therapeutic antibody formulations. More specifically, this invention relates to the field of pharmaceutical formulations comprising human antibodies that specifically bind to human angiopoietin-like (protein) 3 (ANGPTL3). Background Technology

[0004] Therapeutic macromolecules (e.g., antibodies) must be formulated in a way that not only makes the molecules suitable for administration to patients but also maintains their stability during storage and subsequent use. For example, therapeutic antibodies in liquid solutions are prone to degradation, aggregation, or unwanted chemical modifications unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the type of excipients used in the formulation but also on the amount and proportion of the excipients relative to each other. Furthermore, in preparing liquid antibody formulations, other factors must be considered besides stability. Examples of these other considerations include the viscosity of the solution and the antibody concentration that a given formulation can accommodate, as well as the visible quality or attractiveness of the formulation. Therefore, great care must be taken when formulating therapeutic antibodies to obtain a formulation that remains stable, contains a sufficient concentration of antibody, has an appropriate viscosity, and possesses other properties that allow the formulation to be readily administered to patients.

[0005] Antibodies against angiopoietin-like protein 3 (ANGPTL3) are an example of therapeutically relevant macromolecules that require appropriate formulation. Anti-ANGPTL3 antibodies are clinically used to treat diseases or conditions related to lipid metabolism, cardiovascular disease or conditions, and angiogenesis.

[0006] The amino acid and nucleotide sequences of human ANGPTL3 are shown in SEQ ID NOS:161 and 162, respectively. Exemplary anti-ANGPTL3 antibodies are described, for example, in US 9,018,356B2, WO2008 / 073300 and US 7,935,796.

[0007] Although anti-ANGPTL3 antibodies are known, there is still a need in the field for novel pharmaceutical formulations containing such antibodies that are sufficiently stable and suitable for administration to patients. Invention Overview

[0009] For many commercially available monoclonal antibodies, the final product presentation depends on the method of administration. One such method is based on patient preferences for self-administration and lower dosing frequency. Subcutaneous self-administration is a preferred method of administration for parenteral products developed for the long-term treatment of many diseases. Subcutaneous (SC) injection requires administration in a total volume of ≤2 mL, preferably ≤1 mL. Lower dosing frequency requires higher drug concentrations per dose and correspondingly higher protein concentration formulations. Therefore, to enable lower dosing frequency, it is necessary to deliver high concentrations of drug (>150 mg per dose) in 1 mL. High-concentration formulations also allow for smaller dosing volumes. 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, while only 10 mL of a 150 mg / mL formulation is needed. Therefore, high-concentration formulations are preferred because they enable smaller injection volumes.

[0010] It is important to consider the stability and viscosity of such high protein concentration formulations. Because of the exponential relationship between protein concentration and viscosity, small differences in protein concentration can have a significant impact on viscosity and thus affect the patient's ability to deliver the drug. The steepness of the curve describing viscosity (y-axis) versus protein concentration (x-axis) can be affected by the addition of excipients, especially those that increase (e.g., sugar) or decrease (e.g., salt) viscosity, and temperature. Furthermore, viscosity is directly related to the ability to deliver the drug through a syringe. Sustaining force is the force required for continuous dispensing of the contents of a pre-filled syringe. It can be measured using a syringe thrust tester (Instron). The relationship between sustaining force and viscosity is linear.

[0011] Self-administering using pre-filled syringes or autoinjectors requires formulations with low viscosity (typically less than about 20 centipoise). Therefore, it is necessary to identify viscosity-reducing excipients and evaluate their impact on the rheological properties and stability of antibody formulations, particularly anti-ANGPTL3 antibodies. This data can be used to develop high-concentration, amino acid-based liquid formulations with acceptable viscosity for use in pre-filled syringes and devices.

[0012] This invention addresses the aforementioned need 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 targeting ANGPTL3, an important protein that inhibits lipoprotein lipase (LPL) in its active form. Inhibition of ANGPTL3 by H4H1276S restores LPL activity and promotes the processing of triglycerides and vLDL. Therefore, H4H1276S is potentially applicable to a variety of disease pathways, including severe hypertriglyceridemia and homozygous familial hypercholesterolemia.

[0013] On one hand, the present invention provides a stable, low-viscosity, high-concentration liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human angiopoietin-like protein 3 (ANGPTL3); (ii) a buffer; (iii) an organic co-solvent; and (iv) at least one viscosity modifier. 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. On the other hand, the present invention provides a stable, low-viscosity, high-concentration liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human angiopoietin-like protein 3 (ANGPTL3); (ii) a buffer; (iii) an organic co-solvent; and (iv) at least two viscosity modifiers. 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 modifier" includes substances or excipients that reduce viscosity.

[0014] In several 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.

[0015] In some embodiments, the formulation comprises any of the anti-ANGPTL3 antibodies disclosed in U.S. Patent No. 9,018,356B2, which is incorporated herein by reference in its entirety. In some 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) containing 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) containing the sequences of SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80, respectively. In one embodiment, the antibody comprises: an HCVR containing the amino acid sequence SEQ ID NO:66 and an LCVR containing the amino acid sequence SEQ ID NO:74. In another embodiment, the antibody comprises HCVR having at least about 90% sequence identity with SEQ ID NO:66 and LCVR having at least about 90% sequence identity with SEQ ID NO:74. In another embodiment, the antibody comprises HCVR having at least about 95% sequence identity with SEQ ID NO:66 and LCVR having at least about 95% sequence identity with SEQ ID NO:74.

[0016] In one embodiment, the liquid formulation has a pH value of 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 liquid formulation has a pH value of approximately pH 6.0±0.3.

[0017] In one embodiment, the buffer is histidine. In some embodiments, the concentration of histidine is from 5 mM ± 1 mM to 50 mM ± 10 mM, preferably from 5 mM ± 1 mM to 25 mM ± 5 mM. In one embodiment, the concentration of histidine is 10 mM ± 2 mM or 10 mM ± 1 mM or about 10 mM. In another embodiment, the concentration of histidine is 20 mM ± 4 mM or 20 mM ± 2 mM or about 20 mM. In yet another embodiment, the concentration of histidine is 40 nM ± 8 mM or 40 nM ± 4 mM or about 40 nM.

[0018] In some embodiments, the organic cosolvent is a nonionic polymer containing a polyoxyethylene moiety. In one embodiment, the organic solvent 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.

[0019] In one embodiment, the concentration of the organic co-solvent is from about 0.01% ± 0.005% to about 1% ± 0.5% "weight / volume" or "w / v", wherein, for example, 0.1 g / ml = 10% and 0.01 g / ml = 1%. In some embodiments, the organic solvent is polysorbate with a concentration of 0.05% ± 0.025% to 0.5% ± 0.25% (w / v). In one embodiment, the organic co-solvent is polysorbate 80 with a concentration of 0.2% ± 0.1% w / v, or about 0.2%. In another embodiment, the organic co-solvent is polysorbate 80 with a concentration of 0.1% ± 0.05% w / v or about 0.1% w / v. In one embodiment, the organic co-solvent is polysorbate 20 with a concentration of 0.2% ± 0.1% w / v, or about 0.2%. In another embodiment, the organic cosolvent is polysorbate 20 at a concentration of 0.1% ± 0.05% w / v or about 0.1% w / v.

[0020] In some embodiments, a stabilizer is included in the formulation. In one embodiment, the stabilizer is a sugar. In another embodiment, the sugar is sucrose. In several embodiments, the concentration of the stabilizer is from 1% ± 0.2% w / v to 20% ± 4% w / v, 5% ± 1% w / v to 15% ± 3% w / v, or 1% ± 0.2% 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 yet another embodiment, the stabilizer is sucrose at a concentration of 10% ± 2% w / v or about 10% w / v.

[0021] In one embodiment, at least one amino acid is included in the formulation. In one embodiment, the amino acid is L-proline. In some embodiments, the concentration of the amino acid is from 1% ± 0.2% to 5% ± 1% w / v. In one embodiment, the amino acid is proline at a concentration of 1.5% ± 0.3% or about 1.5%. In one embodiment, the amino acid is proline at a concentration of 3% ± 0.6% or about 3%.

[0022] In one embodiment, 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 some embodiments, the concentration of the viscosity modifier is from 25 mM to about 75 mM. In one embodiment, the viscosity modifier is arginine-HCl at a concentration of from 50 mM to about 75 mM.

[0023] In some embodiments, the viscosity of the liquid pharmaceutical formulation at 25°C is less than or equal to about 20 centipoise ± 10%. In some embodiments, the viscosity at 25°C is between 1.0 centipoise ± 10% and 20 centipoise ± 10%. In some embodiments, the viscosity of the liquid pharmaceutical formulation is ≤15 centipoise. In some embodiments, the viscosity of the liquid pharmaceutical formulation is ≤20 centipoise. In some embodiments, the viscosity of the liquid pharmaceutical formulation is ≤10 centipoise. In some embodiments, the viscosity at 25°C is 5 centipoise ± 10%, 6.0 centipoise ± 10%, 7.0 centipoise ± 10%, 7.1 centipoise ± 10%, 7.2 centipoise ± 10%, 7.9 centipoise ± 10%, 8.3 centipoise ± 10%, 9.0 centipoise ± 10%, 9.6 centipoise ± 10%, 10.0 centipoise ± 10%, 10.6 centipoise ± 10%, 11.4 centipoise ± 10%, 11.6 centipoise ± 10%, 11.8 centipoise ± 10%, 12.0 centipoise ± 10%, 13.0 centipoise ± 10%, 14.0 centipoise ± 10%, 15.0 centipoise ± 10%, or 16 centipoise ± 10%.

[0024] On one hand, the present invention provides a stable, low-viscosity liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human ANGPTL3 at a concentration of 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml; (ii) 0 mM to 40 ± 8 mM of histidine; (iii) 0% to 0.5% ± 0.25% (w / v) of polysorbate 80; (iv) 50 ± 10 mM to 75 ± 15 mM of arginine-HCl; and (v) 0 to 5% ± 1% of proline, with a pH of about 5.3 to about 6.7; wherein the anti-ANGPTL3 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR / LCVR combination comprising heavy chain and light chain complementarity-determining regions (HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3), which respectively comprise the amino acid sequences SEQ ID NOs:68–70–72 / SEQ ID NOs:76–78–80. In one embodiment, the anti-ANGPTL3 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), which respectively comprise the amino acid sequences of SEQ ID NO: 66 and SEQ ID NO: 74. In some embodiments, the antibody comprises an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 isotypes. In one embodiment, the antibody comprises human IgG4 isotype.

[0025] In some embodiments, the present invention provides a stable, low-viscosity liquid pharmaceutical formulation comprising: (i) 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml of a human antibody specifically binding to human ANGPTL3; (ii) 0 mM to 40 ± 8 mM of histidine; (iii) 0% to 0.5% ± 0.25% (w / v) of polysorbate 80; (iv) 50 ± 10 mM to 75 ± 15 mM of arginine-HCl; and (v) 0 to 5% ± 1% of proline, with a pH of about 5.3 to about 6.7; wherein the anti-ANGPTL3 antibody comprises HCVR and LCVR, wherein HCVR has at least about 90% sequence identity with SEQ ID NO:66 and / or LCVR has at least about 90% sequence identity with SEQ ID NO:74.

[0026] In some embodiments, the present invention provides a stable, low-viscosity liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human ANGPTL3 at a concentration of 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml; (ii) 0 mM to 40 ± 8 mM of histidine; (iii) 0% to 0.5% ± 0.25% (w / v) of polysorbate 80; (iv) 50 ± 10 mM to 75 ± 15 mM of arginine-HCl; and (v) 0 to 5% ± 1% of proline, with a pH of about 5.3 to about 6.7; wherein the anti-ANGPTL3 antibody comprises HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO:66 having no more than five amino acid substitutions, and wherein the LCVR comprises the amino acid sequence of SEQ ID NO:74 having no more than two amino acid substitutions.

[0027] In some embodiments, any of the foregoing formulations has properties selected from the group consisting of: (i) the formulation is stable for 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 is of low viscosity (viscosity less than about 20 centipoise, preferably less than about 15 centipoise); (iii) the formulation is stable even with variations in excipient concentration of up to ±50%, as described herein; (iv) the formulation is isotonic with physiological conditions; (iv) the formulation is stable and compatible with subcutaneous delivery devices and procedures; and (v) the formulation is stable for long-term storage in pre-filled syringes.

[0028] In some embodiments of this invention, a stable liquid formulation is provided comprising: (i) a human antibody specifically binding to human ANGPTL3 at a concentration of 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml; (ii) 5 mM ± 1 mM to 20 ± 4 mM histidine; (iii) 0.05% ± 0.025% to 0.3% ± 0.15% (w / v) polysorbate 80; (iv) 50 ± 5 mM to 75 ± 7.5 mM arginine-HCl; and (v) 1% ± 0.2% to 5% ± 1% proline, at a pH of about 6.0, wherein the antibody comprises HCVR / LCVR, the HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 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.

[0029] In one embodiment of this, the stable liquid formulation comprises (i) 50 ± 7.5 mg / mL of anti-ANGPTL3 antibody; (ii) 10 ± 2 mM of histidine; (iii) 0.1% ± 0.05% (w / v) of polysorbate 80; (iv) 3% ± 0.6% of proline; and (v) 70 ± 5 mM of arginine-HCl, pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, said HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0030] In another embodiment, the stable liquid formulation comprises (i) 100 ± 15 mg / mL of anti-ANGPTL3 antibody; (ii) 10 ± 2 mM of histidine; (iii) 0.1% ± 0.05% (w / v) of polysorbate 80; (iv) 3% ± 0.6% of proline; and (v) 70 ± 5 mM of arginine-HCl at pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, the HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0031] In another embodiment, the stable liquid formulation comprises (i) 150 ± 22.5 mg / mL of anti-ANGPTL3 antibody; (ii) 10 ± 2 mM of histidine; (iii) 0.1% ± 0.05% (w / v) of polysorbate 80; (iv) 70 ± 5 mM of arginine-HCl; and (v) 3% ± 0.6% of proline at pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR containing the amino acid sequence pair of SEQ ID NO: 66 / 74. In one embodiment of the formulation disclosed herein, the viscosity is less than about 20 centipoise, and in another embodiment, it is less than about 15 centipoise.

[0032] In another embodiment of this, the stable liquid formulation comprises (i) 175 ± 26.25 mg / mL of anti-ANGPTL3 antibody; (ii) 10 ± 2 mM of histidine; (iii) 0.1% ± 0.05% (w / v) of polysorbate 80; (iv) 70 ± 5 mM of arginine-HCl; and (v) 3% ± 0.6% of proline at pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, which comprises the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0033] In another embodiment of this aspect, the stable liquid formulation comprises (i) 200 ± 30.00 mg / mL of anti-ANGPTL3 antibody; (ii) 10 ± 2 mM of histidine; (iii) 0.1% ± 0.05% (w / v) of polysorbate 80; (iv) 70 ± 5 mM of arginine-HCl; and (v) 3% ± 0.6% of proline at pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, which comprises the amino acid sequence pair of SEQ ID NO: 66 / 74.

[0034] In one embodiment of the formulation disclosed herein, the formulation further comprises 5% ± 1% (w / v) sucrose.

[0035] In one embodiment, after storage at 45°C for 21 days, ≥95% of the antibodies are natural, and ≥45% of the antibodies are in the major charge form. In one embodiment, after storage at 5°C for 36 months, >98% of the antibodies are natural, and >55% of the antibodies are in the major charge form. In one embodiment, after storage at -20°C for 9 months, >98% of the antibodies are natural, and >61% of the antibodies are in the major charge form. In one embodiment, after storage at -30°C for 36 months, >98% of the antibodies are natural, and >56% of the antibodies are in the major charge form.

[0036] The cation exchange chromatography elution curve of a monoclonal antibody typically includes three peaks: an early elution peak and a late elution peak (i.e., the so-called acidic and basic variants, respectively), and a peak with the highest abundance called the main peak (or the main charge form or variant).

[0037] In one aspect, a liquid pharmaceutical preparation of any of the foregoing 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 microinfuser. In another embodiment, the container is a syringe. In another embodiment, the container is a pre-filled syringe. In one embodiment, the syringe includes a fluorocarbon-coated plunger. In some embodiments, the syringe is a 1 mL or 2.25 mL long glass syringe containing less than about 500 parts per billion of tungsten, equipped with a 27-G needle, a fluorocarbon-coated butyl rubber stopper, and a latex-free, non-cytotoxic rubber needle cap. In a particular embodiment, the syringe is a 1 mL long glass syringe equipped with a 27-G thin-walled needle, a FLUROTEC-coated 4023 / 50 rubber stopper, and an FM 27 rubber needle cap. In another specific embodiment, the syringe is a 1 mL or 3 mL plastic syringe with a 27-G needle. In one embodiment, the plastic syringe is distributed by BECTON DICKINSON. In another embodiment, the container is equipped with... Type 1 clear glass vial with a 4432 / 50 chlorinated butyl stopper.

[0038] On one hand, the present invention provides a medicine box comprising the pharmaceutical composition of any of the foregoing 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 equipped with a 27-G thin-walled needle, a FLUROTEC-coated 4023 / 50 rubber stopper, and an FM 27 rubber needle cap.

[0039] In some embodiments, the present invention provides a pre-filled syringe comprising a stable liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human ANGPTL3 at a concentration of 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml; (ii) histidine at a concentration of 5 mM ± 1 mM to 20 ± 4 mM; (iii) polysorbate 80 at a concentration of 0.05% ± 0.025% to 0.3% ± 0.15% (w / v); (iv) arginine-HCl at a concentration of 50 ± 10 mM to 75 ± 15 mM; and (v) proline at a concentration of 1% ± 0.2% to 5% ± 1%, pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, the HCVR / LCVR comprising SEQ ID NO. The amino acid sequence pair NO:66 / 74; wherein the formulation has the property selected from the group consisting of: (i) after storage at 5°C for 36 months, ≥98% of the antibody is in the natural form; (ii) after storage at 5°C for 36 months, ≥55% of the antibody is the major charge variant; (iii) the formulation is stable to agitation stress, wherein after agitation stress of 120 minutes in a clear glass vial, ≥98% of the antibody is in the natural form.

[0040] In some embodiments, the present invention provides a stable liquid pharmaceutical formulation comprising: (i) a human antibody specifically binding to human ANGPTL3 at a concentration of 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml; (ii) 5 mM ± 1 mM to 20 ± 4 mM histidine; (iii) 0.05% ± 0.025% to 0.3% ± 0.15% (w / v) polysorbate 80; (iv) 50 ± 10 mM to 75 ± 15 mM arginine-HCl; and (v) 1% ± 0.2% to 5% ± 1% proline at pH 6.0 ± 0.3, wherein the antibody comprises HCVR / LCVR, the HCVR / LCVR comprising SEQ ID The amino acid sequence pair NO:66 / 74; wherein the formulation has properties selected from the group consisting of: (i) the formulation is stable and compatible with use in subcutaneous and / or intravenous delivery devices; (ii) the formulation is chemically and physically stable when diluted with standard diluents known in the art (e.g., 0.9% sodium chloride or 5% glucose); (iii) the formulation is stable in the form of pre-filled syringes or auto-injectors; and (iv) the formulation is compatible with standard infusion pumps (e.g., peristaltic pumps, fluid displacement pumps).

[0041] Other implementation schemes will become apparent from the detailed description that follows. Brief description of the attached diagram

[0043] Figure 1 The diagram illustrates the effect of H4H1276S concentration on viscosity.

[0044] Figure 2 The table shown summarizes the effect of pH on the stability of 150 mg / mL H4H1276S incubated at 45°C for 28 days. a. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. b. The report shows the change in purity relative to the starting material. In all five formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0045] Figure 3 The table shown summarizes the effect of polysorbate 80 on the stability of 150 mg / mL H4H1276S after (120 min vortex) agitation. a. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. b. The report represents the change in purity relative to the starting material. In all 12 formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0046] Figure 4 The table shown summarizes the effect of polysorbate 80 concentration on the stability of 150 mg / mL H4H1276S after incubation at 45°C for 28 days. a. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. b. The report shows the change in purity relative to the starting material. In all five formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0047] Figure 5 The table shown summarizes the effect of polysorbate 80 concentration on the formation of subvisible particles of 150 mg / mL H4H1276S after stirring (vortexing for 120 minutes) or incubation at 45°C for 28 days. a. Data were filtered using ECD (μm) ≥ 5.00, aspect ratio < 0.85, and an ignore edge particle filter. 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.

[0048] Figure 6 The table shown summarizes the effects of sucrose and proline on the stability of H4H1276S after incubation at -20°C for 9 months. a. This corresponds to a formulation containing 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, and 70 mM arginine-HCl. b. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. c. The report represents the change in purity relative to the starting material. In all five formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0049] Figure 7 The table shown summarizes the effects of sucrose and proline on the stability of H4H1276S after 8 freeze / thaw cycles. a. This corresponds to a formulation containing 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, and 70 mM arginine-HCl. b. If the sample is translucent to slightly opaque, with virtually no visible particles, and colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. c.The report represents the change in purity relative to the starting material. In all five formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0050] Figure 8 The table shown summarizes the effects of sucrose and proline on the stability of H4H1276S after incubation at 45°C for 21 days. a. This corresponds to a formulation containing 175 mg / mL H4H1276S, 10 mM histidine, pH 6.0, and 70 mM arginine-HCl. b. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. c. The report represents the change in purity relative to the starting material. In all five formulations, the starting material (without incubation) contained ≥98.4% of the SE-UPLC native peak and ≥62.7% of the CEX-UPLC main peak. 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.

[0051] Figure 9 The table shown summarizes the effects of sucrose and proline on the stability of H4H1276S after incubation at -30°C for 36 months. a. If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. b. The report presents the purity variation relative to the starting material. For both formulations, the starting material (without incubation) contained 98.8% of the natural peak as determined by SE-UPLC; and according to CEX-UPLC, 59.2% of the main peak for the sucrose formulation and 60.0% for the proline formulation. 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.

[0052] Figure 10 The table shown summarizes the effects of sucrose and proline on the stability of H4H1276S after incubation at 5°C for 36 months. a.If the sample is translucent to slightly opaque, with virtually no visible particles, and is colorless to pale yellow, then the sample is acceptable in terms of color and visual appearance. b. The report presents the change in purity relative to the starting material. For both formulations, the starting material (without incubation) contained 98.8% of the natural peak by SE-UPLC; and, according to CEX-UPLC determination, 59.2% of the main peak for the sucrose formulation and 60.0% of the main peak for the proline formulation. c. Report the average of three independent samples. 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.

[0053] Figure 11 The diagram illustrates the relationship between the viscosity of the lead formulation and the concentration of H4H1276S.

[0054] Figure 12A and 12B The diagram illustrates the relationship between H4H1276S concentration, temperature, and viscosity. The star and circle dots at 150 mg / mL H4H1276S also correspond to the recommended storage temperature of 5°C (circle) or the recommended application temperature of 25°C (star). The star and circle dots at 165 mg / mL H4H1276S correspond to a formulation of +10% manufacturing specification. Figure 12A The formulation consists of 10 mM histidine, 70 mM arginine-HCl, 5% sucrose and 0.1% polysorbate 80, pH 6. Figure 12B The formulation consists of 10 mM histidine, 70 mM arginine-HCl, 3% proline and 0.1% polysorbate 80, pH 6.

[0055] Figure 13A and 13B The bar chart shows the screening results for viscosity-reducing excipients. Figure 13A In this study, the viscosity of various excipients added to the base formulation was measured, and the pH was adjusted for several options. Figure 13B In this study, the viscosity of various excipients added to different base formulations was measured.

[0056] Figure 14 The bar chart shows the relative increase (quantified as a percentage) of HMW material for various excipients after 21 days of incubation at 45°C.

[0057] Figure 15A and 15B bar chart ( Figure 15A ) and line graph ( Figure 15BThe stability of H4H1276S formulations containing viscosity-reducing excipients was described. Figure 15A In this study, for different concentrations of sucrose and / or L-proline, the relative increase in HMW, acidity, and viscosity was measured in formulations containing 70 mM Mg-HCl vs. 25 mM Mg(OAc)₂. Figure 15B In this study, the stability of frozen storage (-20°C) was measured based on the change in the percentage of HMW substances over time.

[0058] Figure 16A and 16B It is a contour plot showing temperature, H4H1276S concentration, and viscosity, which are interrelated. Figure 16A For formulations containing 10 mM histidine, pH 6, 70 mM Arg-HCl and 3% (w / v) proline; Figure 16B For formulations containing 10 mM histidine, pH 6, 70 mM Arg-HCl and 5% (w / v) sucrose.

[0059] Figure 17 The graph illustrates the relationship between viscosity and H4H1276S concentration at 20°C. Viscosity is plotted as a function of protein concentration. The data is fitted to an exponential curve using GraphPad Prism. This equation can be used to predict viscosity based on known concentrations, which is useful for defining manufacturing specifications. Invention Details

[0061] Before describing the formulations and methods of the present invention, it should be understood that the invention is not limited to the specific formulations, methods, and experimental conditions described, as such formulations, methods, and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; the scope of the invention will be limited only by the appended claims.

[0062] 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 pertains. As used herein, the term "about," when used with a particular numerical value or range of values, indicates that the value may differ from the stated value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.). Preferred methods and materials are described herein, but any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention. All publications mentioned herein are incorporated herein by reference in their entirety.

[0063] definition

[0064] As used herein, the term "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, wherein the inactive ingredient, when combined with the active ingredient or one or more other inactive ingredients, is suitable for therapeutic administration to humans or non-human animals. As used herein, unless otherwise specifically indicated, the term "formulation" means "pharmaceutical formulation." This invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to certain embodiments of the invention, the therapeutic polypeptide is an antibody, or an antigen-binding fragment thereof, that specifically binds to human angiopoietin-like protein 3 (ANGPTL3).

[0065] As used herein, the term "human angiopoietin-like protein 3" or "hANGPTL3" refers to ANGPTL3 having the nucleic acid sequence shown in SEQ ID NO:162 and the amino acid sequence shown in SEQ ID NO:161, or a biologically active fragment thereof.

[0066] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule composed of four polypeptide chains (i.e., two heavy (H) chains and two light (L) chains linked together by disulfide bonds). Each heavy chain consists of a heavy chain variable region (HCVR) and a heavy chain constant region (C). H ; by structural domain C H 1. C H 2 and C H It consists of 3 components. Each light chain consists of a light chain variable region (LCVR) and a light chain constant region (C). L) Composition. HCVR and LCVR can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each HCVR and LCVR consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0067] It is also possible to replace one or more CDR residues or omit one or more CDRs. Scientific literature has described that in antibodies, one or two CDRs may be non-essential for binding. Padlan et al. (1995 FASEBJ.9:133-139), based on published crystal structures, analyzed the contact region between the antibody and its antigen and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found that in many antibodies, one or two CDRs do not have the amino acid that contacts the antigen (see also Vajdos et al., 2002 J Mol Biol 320:415-428).

[0068] CDR residues that do not contact the antigen can be identified from Kabat CDR regions located outside of Chothia CDRs through molecular modeling and / or empirical methods. If a CDR or one or more of its residues can be omitted, it can usually be substituted with an amino acid at the corresponding position in another human antibody sequence or a common sequence of such sequences. The substitution position and the substituted amino acid in the CDRs can also be selected empirically. Empirical substitutions can be conserved or non-conserved.

[0069] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present invention may include 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 mutations in vivo), such as in CDRs, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human FR sequence.

[0070] Compared to corresponding germline sequences, the fully human anti-hANGPTL3 antibody disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The antibodies and their antigen-binding fragments described herein may be derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more frame and / or CDR regions are mutated to corresponding residues of the germline sequence from which the antibody is derived, or mutated to corresponding residues of another human germline sequence, or mutated to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”).

[0071] Those skilled in the art can readily generate various antibody and antigen-binding fragments comprising one or more germline reversion single mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In some embodiments, all frame and / or CDR residues within the VH and / or VL domains are reverted to residues in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are reverted to the original germline sequence, for example, mutated residues are found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues are found only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated).

[0072] Furthermore, the antibodies described herein may contain any combination of two or more germline mutations within the frame and / or CDR region, for example, wherein certain residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once an antibody and antigen-binding fragment containing one or more germline mutations is obtained, one or more desired properties can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibody and antigen-binding fragments obtained in this general manner are included within the scope of this invention.

[0073] The present invention also includes anti-ANGPTL3 antibodies comprising variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, said variants having one or more conserved substitutions. For example, the present invention includes anti-ANGPTL3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or 1 conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein. In one embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 having 10 or fewer conserved amino acid substitutions. In another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 having 8 or fewer conserved amino acid substitutions. In another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 having 6 or fewer conserved amino acid substitutions. In yet another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 having 4 or fewer conserved amino acid substitutions. In yet another embodiment, the HCVR comprises the amino acid sequence of SEQ ID NO: 66 having 2 or 1 conserved amino acid substitutions. In one embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 having 10 or fewer conserved amino acid substitutions. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 having 8 or fewer conserved amino acid substitutions. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 having 6 or fewer conserved amino acid substitutions. In another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 having 4 or fewer conserved amino acid substitutions. In yet another embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 74 having 2 or 1 conserved amino acid substitutions.

[0074] Unless otherwise specified, the term "antibody" as used herein should be understood to encompass an antibody molecule (i.e., a "whole antibody molecule") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as its antigen-binding fragment. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein capable of specifically binding to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from, for example, whole antibody molecules using any suitable standard technique, such as proteolytic digestion, or by recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-displayed antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical or molecular biological techniques, such as arranging one or more variable and / or constant domains into suitable conformations, or introducing codons, generating cysteine ​​residues, modifying, adding or deleting amino acids, etc.

[0075] 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; (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, diabody, triabody, tetrabody, microbody, nanobody (e.g., monovalent nanobody, bivalent nanobody, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also covered in the term "antigen-binding fragment" as used herein.

[0076] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will generally contain at least one core-receptor domain (CDR) adjacent to or in frame with one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0077] In some embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be present in the antigen-binding fragment of the antibody of the present 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 L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or may be connected via full or partial hinge or connector regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise any of the variable and constant domain configurations listed above, non-covalently bound to each other and / or to one or more monomeric VH or VL domains (e.g., via disulfide bonds) as homodimers or heterodimers (or other polymers).

[0078] Similar to whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two distinct variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed herein, can be adapted to the context of the antigen-binding fragment of the antibodies of the present invention using conventional techniques available in the art.

[0079] In some embodiments, the antibodies or antibody fragments described herein may be conjugated to a therapeutic component (“immunoconjugate”), such as cytotoxins, chemotherapeutic agents, immunosuppressants, or radioisotopes.

[0080] The term "specific binding" or similar expressions refer to the formation of a relatively stable complex between an antibody or its antigen-binding fragment and the antigen under physiological conditions. Specific binding can be measured using the equilibrium dissociation constant (K). D Approximately 1x10 -6 M or smaller (i.e., the smaller K) D The tighter the binding (indicating a stronger binding), the more effective the binding. Methods for determining whether two molecules specifically bind are well-known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc. However, isolated antibodies that specifically bind hANGPTL3 may exhibit cross-reactivity with other antigens, such as ANGPTL3 molecules from other species, such as cynomolgus monkey ANGPTL3, mouse ANGPTL3, rat ANGPTL3, and / or hANGPTL4. Furthermore, multispecific antibodies (e.g., bispecific antibodies) that bind hANGPTL3 and one or more other antigens are still considered antibodies that "specifically bind" hANGPTL3, as used herein.

[0081] As used in this article, the term "K" D "It is intended to refer to the equilibrium dissociation constant of a specific antibody-antigen interaction."

[0082] As used herein, “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, isolated antibodies that specifically bind to hANGPTL3 may be cross-reactive with other antigens, such as ANGPTL3 molecules from other species, such as cynomolgus monkeys, mice, rats, and / or other related proteins such as human ANGPTL4.

[0083] As used herein, “neutralizing,” “blocking,” or “eliminating” antibodies (or antibodies that “neutralize,” “block,” or “eliminate” ANGPTL3 activity) are intended to mean antibodies that, when assessed by standard in vitro assays known in the art, result in direct inhibition of at least one biological activity of ANGPTL3 upon binding. The terms “neutralizing,” “inhibiting,” “blocking,” and “eliminating” are used interchangeably herein. A “non-blocking” antibody is an antibody whose binding to ANGPTL3 does not directly block the target activity of ANGPTL3, when assessed by standard in vitro assays, but such antibody may still be an “interfering” antibody, i.e., the binding of said antibody to ANGPTL3 results in indirect inhibition, reduction, attenuation, or other interference with at least one biological activity of ANGPTL3 in vivo, for example, by enhancing the clearance of ANGPTL3 from circulation. The clearance of ANGPTL3 from circulation can be particularly enhanced by a combination of at least two non-blocking antibodies. The neutralization, inhibition, elimination, reduction, attenuation, or interference of the biological activity of ANGPTL3 can be assessed by measuring one or more indicators of ANGPTL3 biological activity using one or more of several standard in vitro or in vivo assays known in the art.

[0084] As used herein, the term "surface plasmon resonance" refers to, for example, the phenomenon that can be described using BIACORE. TM The system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ) allows for the analysis of real-time, biologically specific interactions in optical phenomena by detecting changes in protein concentration within a biosensor matrix.

[0085] The term "epitope" is an antigenic region that binds to an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and contain those residues that directly contribute to the affinity for the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may include determinants of chemically active surface groups constituting the molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge properties.

[0086] The terms “substantially identical” or “substantially the same” when referring to a nucleic acid or a fragment thereof indicate that, when the nucleic acid or a fragment thereof is optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as described below, and can be measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP.

[0087] When applied to peptides, the term "substantially similar" or "substantially similar" means that two peptide sequences, when optimally aligned using, for example, procedures such as GAP or BESTFIT with default vacancy weights, share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99% sequence identity. Preferably, the dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is the substitution of one amino acid residue by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. The means of making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy 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 conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution is any variation that has a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443 45. "Moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0088] Sequence analysis software is commonly used to measure the sequence similarity of peptides. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as GAP and BESTFIT, which can determine sequence homology or sequence identity between closely related peptides using default parameters, such as homologous peptides from different biological species or between wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentages of sequence identity between query and search sequences for the best overlapping regions (Pearson (2000), ibid.). Another preferred algorithm when comparing the sequences of this invention with databases containing large numbers of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, which uses default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol.215:403 410 and (1997) Nucleic Acids Res.25:3389 402.

[0089] The phrase “therapeutic effective amount” refers to the amount that, when applied, produces the desired effect. The exact amount will depend on the therapeutic purpose, the age and size of the patient, the route of administration, etc., and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

[0090] bioequivalent

[0091] The anti-hANGPTL3 antibodies and antibody fragments described herein encompass proteins having an amino acid sequence different from that of the mAb but retaining the ability to bind human ANGPTL3. When compared to the parental sequence, such variant mAbs and antibody fragments contain one or more amino acid additions, deletions, or substitutions, but exhibit biological activity substantially equivalent to that of the mAb. Similarly, the DNA sequences encoding anti-hANGPTL3 antibodies described herein encompass sequences containing one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequences, but encoding anti-hANGPTL3 antibodies or antibody fragments substantially bioequivalent to those described herein. Examples of such variant amino acid and DNA sequences have been discussed above.

[0092] Two antigen-binding proteins or antibodies are considered bioequivalent, for example, if they are pharmaceutical equivalents or substitutes, i.e., their absorption rates and extent of absorption do not show significant differences when administered as a single or multiple dose at the same molar dose under similar experimental conditions. Certain antibodies are considered equivalents or substitutes if they are equivalent in extent of absorption but not in rate of absorption, but can still be considered bioequivalent for reasons where such difference in absorption rate is intentional and reflected in the label, and is not necessary for achieving effective in vivo drug concentrations (e.g., with prolonged use), and is considered medically irrelevant to the specific pharmaceutical product under investigation. In one embodiment, two antigen-binding proteins are bioequivalent if they do not have clinically significant differences in safety, purity, and potency.

[0093] In one implementation, the two antigen-binding proteins are bioequivalent if a patient can switch between the reference product and the biological product once or multiple times without an expected increase in the risk of side effects, including significant clinical changes in immunogenicity, or without a decrease in effectiveness compared to continued treatment without such a switch.

[0094] In one implementation, two antigen-binding proteins are bioequivalent if they act against one or more conditions of use through one or more co-operating mechanisms (to the extent known of these mechanisms).

[0095] Bioequivalence can be demonstrated through in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals, in which the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluids is measured as a function of time; (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals, in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that can establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0096] Bioequivalent variants of the anti-hANGPTL3 antibody of the present invention can be constructed, for example, by various substitutions or deletions of biologically undesirable terminal or internal residues or sequences. 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 bonds during renaturation.

[0097] Antibodies that specifically bind to ANGPTL3

[0098] The pharmaceutical formulations of the present invention may comprise a human antibody or an antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 3 (ANGPTL3). Exemplary anti-human ANGPTL3 antibodies that may be included in the pharmaceutical formulations of the present invention are described in patent applications US 9,018,356B2, WO2008 / 073300 and US 7,935,796, the disclosures of which are incorporated herein by reference in their entirety.

[0099] In some embodiments, the anti-ANGPTL3 antibody comprises an HCVR / LCVR amino acid sequence pair having a sequence number selected from the following SEQ ID Nos: 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”).

[0100] According to certain embodiments of the present invention, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 68, the HCDR 2 of SEQ ID NO: 70, and the HCDR 3 of SEQ ID NO: 72. In some embodiments, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises the HCVR of SEQ ID NO: 66.

[0101] According to certain embodiments of the present invention, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:76, the LCDR2 of SEQ ID NO:78, and the LCDR3 of SEQ ID NO:80.

[0102] In some embodiments, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises the LCVR of SEQ ID NO: 74.

[0103] According to certain embodiments of the present invention, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises HCVR having 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:66.

[0104] According to certain embodiments of the present invention, the anti-human ANGPTL3 antibody or its antigen-binding fragment comprises an LCVR having 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:74.

[0105] According to certain embodiments of the present invention, an anti-human ANGPTL3 antibody or its antigen-binding fragment comprises an HCVR, the HCVR comprising an amino acid sequence of SEQ ID NO:66 having no more than 5 amino acid substitutions.

[0106] According to certain embodiments of the present invention, an anti-ANGPTL3 antibody or its antigen-binding fragment comprises an LCVR, the LCVR comprising an amino acid sequence of SEQ ID NO:74 having no more than two amino acid substitutions.

[0107] Sequence identity can be measured by any method known in the art (e.g., GAP, BESTFIT, and BLAST).

[0108] The present invention also includes formulations comprising anti-ANGPTL3 antibodies, wherein the anti-ANGPTL3 antibodies comprise variants of any one of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, wherein said variants have one or more conserved amino acid substitutions. For example, the present invention includes formulations comprising anti-ANGPTL3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences, wherein said HCVR, LCVR, and / or CDR amino acid sequences have, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or other conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein.

[0109] In some implementations, the anti-ANGPTL3 antibody includes an Fc region selected from a group consisting of human IgG1, IgG2, IgG3 and IgG4 isotypes.

[0110] The non-limiting exemplary antibody used in the embodiments herein is referred to as "H4H1276S" or "mAb1". This antibody is also referred to as H4H1276S in US9,018,356B2. mAb1 (H4H1276S) comprises the HCVR / LCVR amino acid sequence pair having SEQ ID NO:66 / 74 and the HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3 domain represented by SEQ ID NO:68–70–72 / SEQ ID NO:76–78–80.

[0111] The full-length sequence of H4H1276S is as follows:

[0112] Heavy chain sequence (SEQ ID NO:195)

[0113] EVQLVESGGGVIQPGGSLRLSCAASGFTFDDYAMNWVRQGPGKGLEWVSAISGDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAFFYCAKDLRNTIFGVVIPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0114] Light chain sequence (SEQ ID NO:196)

[0115] DIQMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0116] It should be noted that there may be some inaccuracies in the translation due to the complexity of the biological sequence content. It is recommended to double-check with relevant professionals for more accurate understanding.The amount of antibody or its antigen-binding fragment contained in the pharmaceutical formulation of the present invention may vary depending on the specific characteristics required by the formulation and the specific circumstances and purpose of its intended use. In some embodiments, the pharmaceutical formulation is a liquid formulation that may contain 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 of antibody; Antibody concentrations of 100 mg / mL to 220±33 mg / mL; 90±13.5 mg / mL to 210±31.5 mg / mL; 100±15 mg / mL to 200±30 mg / mL; 110±16.5 mg / mL to 190±28.5 mg / mL; 120±18 mg / mL to 180±27 mg / mL; 130±19.5 mg / mL to 170±25.5 mg / mL; 140±21 mg / mL to 160±24 mg / mL; 150±22.5 mg / mL; or 175±26.25 mg / mL.For example, the formulation of the present invention may contain 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 125 mg / mL; about 130 mg / mL; about 135 mg / mL; about 135 mg / mL. Approximately 140 mg / mL; approximately 145 mg / mL; approximately 150 mg / mL; approximately 155 mg / mL; approximately 160 mg / mL; approximately 165 mg / mL; approximately 170 mg / mL; approximately 175 mg / mL; approximately 180 mg / mL; approximately 185 mg / mL; approximately 190 mg / mL; approximately 195 mg / mL; approximately 200 mg / mL; approximately 205 mg / mL; approximately 210 mg / mL; approximately 215 mg / mL; approximately 220 mg / mL; approximately 225 mg / mL; approximately 230 mg / mL; approximately 235 mg / mL; approximately 240 mg / mL; approximately 245 mg / mL; or approximately 250 mg / mL of an antibody or antigen-binding fragment thereof that specifically binds to human ANGPTL3. In one embodiment, the formulation of the present invention comprises approximately 150 mg / mL of an anti-human ANGPTL3 antibody or an antigen-binding fragment thereof.

[0117] Excipients and pH

[0118] The pharmaceutical formulations of the present invention comprise one or more excipients. As used herein, the term "excipient" refers to any non-therapeutic agent added to the formulation to provide a desired consistency, viscosity, or stabilizing effect.

[0119] In some embodiments, the pharmaceutical formulation of the present invention comprises at least one organic cosolvent, the type and amount of which can stabilize the anti-hANGPTL3 antibody under vigorous treatment or agitation (e.g., vortexing). In some embodiments, "stabilization" means preventing the formation of aggregated antibody exceeding 3% (in moles) of the total antibody amount during vigorous treatment. In some embodiments, vigorous treatment is vortexing the solution containing the antibody and the organic cosolvent for about 60 minutes or about 120 minutes.

[0120] In some embodiments, the organic cosolvent is a nonionic surfactant, such as alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbate compounds, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamer compounds, such as poloxamer 181, poloxamer 188, and poloxamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as Tween 20, sorbitol monolaurate, and polyoxyethylene sorbitol monolaurate. Poloxamer 188 is also known as PLURONIC F68. In some embodiments, the organic cosolvent included in the formulations of the present invention is polysorbate 80.

[0121] The amount of nonionic surfactant included in the pharmaceutical formulation of the present invention can vary depending on the specific properties required by the formulation and the specific circumstances and purpose for which the formulation is intended to be applied. In some embodiments, the formulation may contain 0.01% ± 0.005% to 0.5% ± 0.25% surfactant. For example, the formulation of the present invention may 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%; about 0.18%; 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% of polysorbate 20 or polysorbate 80. In some embodiments, the formulation of the present invention comprises about 0.1% (w / v) of polysorbate 80.

[0122] The pharmaceutical formulations of the present invention may also contain one or more stabilizers of a type and amount that can stabilize the anti-hANGPTL3 antibody under heat stress. In some embodiments, "stable" means that a certain significant percentage of the antibody remains in its native conformation when the solution containing the antibody and the heat stabilizer is maintained at about 45°C for up to about 28 days. In some embodiments, "stable" means that a non-significant percentage of the antibody aggregates when the solution containing the antibody and the heat stabilizer is maintained at about 45°C for up to about 28 days. As used herein, "native" refers to the dominant form of the antibody as determined by size exclusion, which is typically the intact monomer of the antibody. The term "native" also refers to the non-aggregated and non-degrading form of the antibody.

[0123] In some embodiments, the heat stabilizer is a sugar, such as sucrose, and the amount contained in the formulation may vary depending on the specific circumstances of its use and intended purpose. In some embodiments, the formulation may 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, the pharmaceutical formulation of the present invention may 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 some embodiments, the formulation of the present invention does not contain sugar.

[0124] The pharmaceutical formulations of this invention may also contain buffers or buffer systems for maintaining a stable pH and aiding in the stabilization of anti-hANGPTL3 antibodies. In some embodiments, "stable" means that the amount of antibody aggregation is minimized when the solution containing the antibody and buffer is maintained at about 45°C for up to about 28 days. In some embodiments, "stable" means that the amount of native conformation antibody is maximized when the solution containing the antibody and buffer is maintained at about 45°C for up to about 28 days, as determined by size exclusion chromatography. "Native" or "native conformation" refers to the antibody fraction that is neither aggregated nor degraded. This is typically determined by an assay measuring the relative size of the antibody entities, such as size exclusion chromatography. Unaggregated and undegraded antibodies elute in a fraction equal to that of the native antibody, which is typically the dominant elution fraction. Aggregated antibodies elute in a fraction showing a size larger than that of the native antibody. Degraded antibodies elute in a fraction showing a size smaller than that of the native antibody.

[0125] In some implementations, "stable" means that, after a solution containing antibody and buffer is kept at about 45°C for up to about 28 days, at least about 46% of the antibody is in its major charge form, as determined by cation exchange chromatography. "Major charge" or "major charge form" refers to the antibody fraction eluted from the ion exchange resin in the main peak, which typically has a more "basic" peak on one side and a more "acidic" peak on the other.

[0126] The pharmaceutical formulations of the present invention can have a pH of about 5.2 to about 6.4. For example, the formulations of the present invention can 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.

[0127] In some embodiments, the buffer or buffering system comprises at least one buffer whose buffering range fully or partially overlaps with the pH range of 5.5–7.4. In some embodiments, the buffer comprises a histidine buffer. In some embodiments, histidine is present at concentrations 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 some embodiments, the buffering system comprises 10 mM ± 2 mM of histidine at a pH of 6.0 ± 0.3.

[0128] The pharmaceutical formulations of the present invention may further comprise one or more excipients for maintaining or reducing the viscosity of the formulation containing a high concentration of anti-ANGPTL3 antibody drug substance (e.g., typically about 150 mg / ml antibody). In some embodiments, the at least one viscosity modifier is selected from: arginine-HCl, sodium chloride, histidine-HCl, sodium acetate (pH 5), calcium chloride, magnesium chloride, calcium acetate, and magnesium acetate. In some embodiments, the formulations of the present invention comprise arginine HCl.

[0129] In some embodiments, the pharmaceutical formulation comprises at least one amino acid. In some embodiments, the amino acid is proline, and the pharmaceutical formulation of the present invention contains proline, preferably 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 comprises an amount of proline sufficient to maintain the viscosity of the liquid formulation at less than 20 ± 3 centipoise, less than 15 ± 2.25 centipoise, or less than 11 ± 1.65 centipoise. In some embodiments, the formulation comprises an amount of proline sufficient to maintain the viscosity at or below 15 ± 2.25 centipoise. In some embodiments, the formulation may contain about 1% to about 5% proline; about 2% to about 4% proline; or about 3% proline. For example, the pharmaceutical formulation of the present 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.

[0130] In antibody purification, it may be necessary or required to exchange one buffer for another to obtain appropriate excipient concentrations, antibody concentrations, pH, etc. Buffer exchange can be accomplished, for example, by ultrafiltration / percolation (UF / DF), using, for example, a semi-percolation tangential flow filtration membrane. However, the use of such techniques may lead to the Gibbs-Donnan effect [Bolton et al., 2011, Biotechnol. Prog. 27(1):140-152]. During protein concentration, the accumulation of positive charge on the product side of the membrane is electrolyzed by the preferential migration of positive ions to the other 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 expected target concentration of these components due to the electrostatic repulsion of the positively charged percolation buffer excipients to the charged antibody proteins in the UF / DF step. Therefore, the present invention includes formulations in which the concentrations of, for example, histidine and / or L-proline differ from the amounts or ranges described herein due to the Gibbs-Donnan effect.

[0131] Volume repulsion describes the behavior of highly concentrated samples, where a significant portion of the total solution volume is occupied by the solute, particularly large molecules such as proteins, thereby excluding the solvent from this space. This, in turn, reduces the total volume of solvent available for dissolving other solutes, potentially leading to unequal distribution across the ultrafiltration membrane. Therefore, the present invention includes formulations in which the concentrations of, for example, histidine and / or L-proline may differ from the amounts or ranges described herein due to volume repulsion effects.

[0132] During the manufacture of the formulations of this invention, the composition of the formulation may change. These changes may involve the concentration of the active ingredient, the concentration of the excipient, and / or the pH of the formulation. Since changes in any of these parameters can affect the stability or potency of the drug, acceptable range (PAR) studies have been conducted to assess whether compositional changes within specified ranges would affect the stability or potency of the antibody. Therefore, this invention includes formulations comprising anti-ANGPTL3 antibodies that are stable and retain potency when the excipient concentration changes by no more than 50%. For example, this document includes anti-ANGPTL3 antibody formulations wherein the stability and potency of the formulation are not affected by changes in the concentrations of the antibody, histidine, arginine-HCl, and / or polysorbate by ±10%, ±20%, ±30%, ±40%, or ±50%.

[0133] Stability and viscosity of pharmaceutical preparations

[0134] The pharmaceutical formulations of this invention generally exhibit a high level of stability. The term "stable," as used herein with respect to pharmaceutical formulations, means that the antibody in the pharmaceutical formulation retains an acceptable degree of chemical structure or biological function after being stored under specified conditions. The formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after a specified period of storage. In some cases, the retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody's structure or function after a specified period of storage may be considered "stable."

[0135] Stability can be measured, for example, by determining the percentage of natural antibodies retained in the formulation after storage at a specified temperature for a specified period of time. The percentage of natural antibodies can be determined, in particular, by size exclusion chromatography (e.g., size exclusion ultra-high performance liquid chromatography [SE-UPLC]), where natural means non-aggregated and non-degraded. "Acceptable level of stability," as used herein, means that at least 90% of the natural form of antibody can be detected in the formulation after storage at a given temperature for a specified period of time. In some embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the natural form of antibody can be detected in the formulation after storage at a specified temperature for a specified period of time. The specified time 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 for storing the drug formulation when assessing stability can be any temperature from about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°-8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, if more than about 95%, 96%, 97%, or 98% of the natural antibodies are detected by SE-UPLC after 6 months of storage at 5°C, the drug formulation can be considered stable. The drug formulation can be considered stable if, after storage at 25°C for 6 months, more than approximately 95%, 96%, 97%, or 98% of natural antibodies are detected by SE-UPLC. Similarly, after storage at 45°C for 28 days, more than approximately 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% of natural antibodies are detected by SE-UPLC. The drug formulation can also be considered stable if, after storage at -20°C for 12 months, more than approximately 96%, 97%, or 98% of natural antibodies are detected by SE-UPLC. Furthermore, after storage at -30°C for 12 months, more than approximately 96%, 97%, or 98% of natural antibodies are detected by SE-UPLC. Finally, after storage at -80°C for 12 months, more than approximately 96%, 97%, or 98% of natural antibodies are detected by SE-UPLC.

[0136] Specifically, stability can be measured by determining the percentage of antibodies forming aggregates in the formulation after storage at a specified temperature for a specified time, where stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated antibodies can be determined, in particular, by size exclusion chromatography (e.g., size exclusion ultra-high performance liquid chromatography [SE-UPLC]). "Acceptable level of stability," as used herein, refers to the detection of up to 5% of antibodies in aggregate form (also referred to as high molecular weight – HMW – form) in the formulation after storage at a specified temperature for a specified period of time. In some embodiments, acceptable level of stability means that up to about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of antibodies can be detected as aggregates in the formulation after storage at a specified temperature for a specified period of time. The specified time 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 drug formulation can be stored to evaluate stability can be any temperature from about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, if less than about 2%, 1%, 0.5%, or 0.1% of antibodies are detected in aggregate form after 12 months of storage at 5°C, the drug formulation can be considered stable. If, after storage at 25°C for 3 months, less than approximately 4%, 3%, 2%, 1%, 0.5%, or 0.1% of antibodies are detected in aggregated form, the drug formulation can also be considered stable. Similarly, if, after storage at 45°C for 28 days, less than approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of antibodies are detected in aggregated form, the drug formulation can also be considered stable. Furthermore, if, after storage at -20°C, -30°C, or -80°C for 3 months, less than approximately 3%, 2%, 1%, 0.5%, or 0.1% of antibodies are detected in aggregated form, the drug formulation can also be considered stable.

[0137] In particular, stability can be measured by determining the percentage of antibody that migrates during ion exchange in a fraction more acidic than the major fraction (“major charged form”) of the antibody (“acidic form”), where stability is inversely proportional to the percentage of the acidic form of the antibody. Not wishing to be bound by theory, deamidation of an antibody can result in the antibody carrying a greater negative charge and thus being more acidic relative to the undeamidated antibody (see, for example, Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). The percentage of “acidified” antibody can be determined, in particular, by ion exchange chromatography (e.g., cation exchange ultra-high performance liquid chromatography [CEX-UPLC]). “Acceptable level of stability,” as used herein, refers to the detection of up to 45% of the antibody in the formulation as the more acidic form after storage at a specified temperature for a specified period of time. In some embodiments, acceptable stability refers to the presence of up to about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in an acidic form in the formulation after storage at a specified temperature for a specified period of time. In one embodiment, acceptable stability refers to the presence of less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in an acidic form in the formulation after storage at a specified temperature for a specified period of time. The specified time for measuring 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 more. The temperature at which a drug formulation can be stored to assess stability can be any temperature from about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C. For example, 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 three months of storage at -80°C, -30°C, or -20°C, the drug formulation can be considered stable.If, after six months of storage at 5°C, less than approximately 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 antibodies are in a more acidic form, the pharmaceutical preparation can be considered stable. If, after six months of storage at 25°C, less than approximately 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 antibodies are in a more acidic form, then the pharmaceutical preparation can also be considered stable. If, after storage at 45°C for 28 days, less than approximately 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 detected in a more acidic form, then the drug formulation can be considered stable.

[0138] Other methods can be used to assess the stability of the formulations of the present invention, such as using differential scanning calorimetry (DSC) to determine thermal stability, using controlled agitation to determine mechanical stability, and measuring solution turbidity by absorbance at about 350 nm or about 405 nm. For example, if the OD of the formulation relative to zero is measured after storage at about 5°C to about 25°C for 6 months or longer. 405 OD of the formulation 405 If the variation is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less), the formulation of the present invention can be considered stable.

[0139] Measuring the biological activity or binding affinity of the antibody to its target can also be used to assess stability. For example, a formulation of the present invention can be considered stable if, after storage at temperatures such as 5°C, 25°C, 45°C, etc., for a specified period of time (e.g., 1 to 12 months), the anti-ANGPTL3 antibody contained in the formulation binds to ANGPTL3 with an affinity of at least 90%, 95%, or more of the binding affinity of the antibody before storage. Binding affinity can be determined by, for example, ELISA or surface plasmon resonance. Biological activity can be determined by an ANGPTL3 activity assay, for example, by contacting cells expressing ANGPTL3 with a formulation containing an anti-ANGPTL3 antibody. The binding of the antibody to such cells can be measured directly, for example, by FACS analysis. Alternatively, the downstream activity of the ANGPTL3 system can be measured in the presence of the antibody and compared with the activity of the ANGPTL3 system in the absence of the antibody. In some embodiments, ANGPTL3 may be endogenous in the cells. In other embodiments, ANGPTL3 may be ectopically expressed in the cells.

[0140] Additional methods for assessing antibody stability in formulations are shown in the examples presented below.

[0141] In some embodiments, the liquid pharmaceutical formulations of the present invention can exhibit low to moderate levels of viscosity. As used herein, “viscosity” can be “kinematic viscosity” or “absolute viscosity.” “Kinematic viscosity” is a measure of the resistance to flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in the same capillary viscometer and allowed to flow under gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. For example, if one fluid takes 200 seconds to complete flow and another takes 400 seconds, the second fluid has twice the kinematic viscosity of the first fluid. “Absolute viscosity,” sometimes called dynamic or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L. 2 / T, where L is length and T is time. Generally, kinematic viscosity is expressed in centistokees (cSt). The SI unit for kinematic viscosity is mm. 2 / s, or 1 cSt, is the unit for absolute viscosity, expressed in centipoise (cP). The SI unit for absolute viscosity is millipascal-second (mPa-s), where 1 cP = 1 mPa-s.

[0142] As used herein, with respect to the fluid formulations of the present invention, a low viscosity level will be characterized by an absolute viscosity of less than about 20 centipoise (cP). For example, the fluid formulations of the present invention will be considered to have “low viscosity” if, when measured using standard viscosity measurement techniques, 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. As used herein, a medium viscosity level, with respect to the fluid formulations of the present invention, will exhibit an absolute viscosity between about 35 cP and about 20 cP. For example, the fluid formulation of the present invention will be considered to have "medium viscosity" if, when measured using standard viscosity measurement techniques, 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 15.1 cP.

[0143] As illustrated in the following examples, the inventors have surprisingly discovered 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 antibody with about 1% to about 5% proline and without the need for stabilizers such as sucrose. Such formulations are stable to stress during processing and storage in a temperature range of 45°C to -80°C (as demonstrated herein) and exhibit a viscosity of less than about 15 cP.

[0144] Exemplary formulation

[0145] According to one aspect of the 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 adequate buffering at about pH 6.0 ± 0.3; (iii) an organic co-solvent that protects the structural integrity of the antibody; and (iv) a viscosity modifier that is an excipient that reduces viscosity. According to another aspect of the 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) specifically binding to human ANGPTL3 at a concentration of about 25 to about 250 mg / ml; (ii) a buffer system that provides adequate buffering at about pH 6.0 ± 0.3; (iii) an organic co-solvent that protects the structural integrity of the antibody; (iv) a viscosity modifier, said viscosity modifier being an excipient that reduces viscosity; and (iv) an amino acid that maintains viscosity for easy injection within a convenient volume for subcutaneous administration.

[0146] According to one embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 25 mg / ml ± 7.5 mg / mL; (ii) 10 mM ± 2 mM of histidine, which acts as a buffer at pH 6.0 ± 0.3; (iii) 0.1% w / v ± 0.05% w / v of polysorbate 80; (iv) 70 ± 5 mM of arginine-HCl; and (v) approximately 3% (w / v) ± 0.6% of L-proline. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO:66 / 74.

[0147] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 50 mg / ml ± 7.5 mg / mL; (ii) 10 mM ± 2 mM histidine; (iii) 0.1% ± 0.05% (w / v) polysorbate 80; (iv) 3% ± 0.6% proline; and (v) 70 ± 5 mM arginine-HCl, pH 6.0 ± 0.3. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pairs of SEQ ID NO:66 / 74.

[0148] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 100 mg / ml ± 15 mg / mL; (ii) 10 mM ± 2 mM histidine; (iii) 0.1% ± 0.05% (w / v) polysorbate 80; (iv) 3% ± 0.6% proline; and (v) 70 ± 5 mM arginine-HCl, pH 6.0 ± 0.3. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO:66 / 74.

[0149] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 150 mg / ml ± 22.5 mg / mL; (ii) 10 mM ± 2 mM histidine; (iii) 0.1% ± 0.05% (w / v) polysorbate 80; (iv) 70 ± 5 mM arginine-HCl; and (v) 3% ± 0.6% proline, at pH 6.0 ± 0.3. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO:66 / 74. In some embodiments of the formulations disclosed herein, the viscosity is less than about 20 centipoise; in still other embodiments, the viscosity of the formulation is less than about 15 centipoise.

[0150] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 175 mg / ml ± 26.25 mg / mL; (ii) 10 mM ± 2 mM histidine; (iii) 0.1% ± 0.05% (w / v) polysorbate 80; (iv) 70 ± 5 mM arginine-HCl; and (v) 3% ± 0.6% proline, at pH 6.0 ± 0.3. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pair of SEQ ID NO:66 / 74.

[0151] According to another embodiment, the stable, low-viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human ANGPTL3, comprising HCDR1 of SEQ ID NO:68, HCDR2 of SEQ ID NO:70, HCDR3 of SEQ ID NO:72, LCDR1 of SEQ ID NO:76, LCDR2 of SEQ ID NO:78, and LCDR3 of SEQ ID NO:80, at a concentration of 200 mg / ml ± 30.00 mg / mL; (ii) 10 mM ± 2 mM histidine; (iii) 0.1% ± 0.05% (w / v) polysorbate 80; (iv) 70 ± 5 mM arginine-HCl; and (v) 3% ± 0.6% proline, at pH 6.0 ± 0.3. In another embodiment, the antibody comprises: HCVR / LCVR comprising the amino acid sequence pairs of SEQ ID NO:66 / 74.

[0152] Other non-limiting examples of pharmaceutical formulations covered by this invention are set forth elsewhere herein, including the examples presented below.

[0153] Containers and application methods

[0154] The pharmaceutical formulations of the present invention can be contained in any container suitable for storing pharmaceuticals and other therapeutic compositions. For example, the pharmaceutical formulations can be contained in sealed and sterilized plastic or glass containers of a prescribed volume, such as vials, ampoules, syringes, cartridges, or bottles. 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 formulations of the present invention.

[0155] The pharmaceutical formulations of this invention can be contained in either a "normal tungsten" syringe or a "low tungsten" syringe. As will be understood by those skilled in the art, the process of manufacturing a glass syringe typically involves using a heated tungsten rod to puncture the glass, creating a hole through which liquid can be drawn from and expelled from the syringe. This process results in the deposition of trace amounts of tungsten on the inner surface of the syringe. Subsequent cleaning and other treatment steps can be used to reduce the tungsten content in the syringe. As used herein, the term "normal tungsten" refers to a syringe containing 500 or more parts per billion (ppb) of tungsten. The term "low tungsten" refers to a syringe containing less than 500 ppb of tungsten. For example, according to the invention, a low tungsten syringe may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 10, 90, 80, 70, 60, 50, 40, 30, 20, 10 ppb or less of tungsten.

[0156] Rubber plungers used in syringes and rubber stoppers used to seal vial openings can be coated to prevent contamination of the pharmaceutical contents of the syringe or vial, or to maintain their stability. Therefore, according to some embodiments, the pharmaceutical formulations of the present invention can be contained in a syringe containing a coated plunger, or in a vial sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for vials and syringes containing the pharmaceutical formulations of the present invention are mentioned, for example, in U.S. Patent 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 entirety. Specific exemplary coated rubber stoppers and plungers that may be used in the context of the present invention may be marketed under trade names. Purchased from West Pharmaceutical Services, Inc. (Lionville, PA). This is an example of a fluorocarbon coating used to minimize or prevent pharmaceutical products from adhering to rubber surfaces.

[0157] According to some embodiments of the present invention, the pharmaceutical preparation may be contained in a low-tungsten syringe comprising a fluorocarbon-coated plunger.

[0158] The pharmaceutical formulation can be administered to the patient via parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneously, via mucous membranes, nose, lungs, or orally. Many reusable pen or auto-injector delivery devices can be used for subcutaneous delivery of the pharmaceutical formulation of this invention. Examples include, but are not limited to, AUTOPEN. TM (Owen Mumford,Inc.,Woodstock,UK),DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen(Eli Lilly and Co.,Indianapolis,IN),NOVOPEN TM I,II and III(Novo Nordisk,Copenhagen,Denmark),NOVOPENJUNIOR TM (Novo Nordisk,Copenhagen,Denmark),BD TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM OPTIPEN PRO TM OPTIPEN STARLET TM , and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen or auto-injector delivery devices that can be used in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR. TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM Pen (Abbott Labs, Abbott Park, IL).

[0159] This document also considers the use of microinfusion devices for delivering the pharmaceutical formulations of the present invention. As used herein, the term “microinfusion device” refers to a subcutaneous delivery device designed to slowly administer a large volume (e.g., up to about 2.5 ml or more) of a therapeutic formulation over a prolonged period of time (e.g., about 10, 15, 20, 25, 30 or more minutes). See, for example, US 6,629,949; US 6,659,982; and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinfusion devices are particularly suitable for delivering large doses of therapeutic proteins contained in high concentrations (e.g., about 100, 125, 150, 175, 200 or more mg / mL) or viscous solutions.

[0160] In some embodiments, the stable liquid pharmaceutical preparation of any of the foregoing aspects is contained in a sterile glass vial and administered as an IV infusion.

[0161] In one embodiment, the container is a 20 mL Type I clear borosilicate glass vial. In some embodiments, the container is equipped with... Type 1 borosilicate glass vials, 2 mL or 3 mL, with coated 4432 / 50 butyl rubber stoppers.

[0162] In one embodiment, the liquid pharmaceutical preparation of the present invention, containing about 25 mg / mL or 50 mg / mL mAb1, is used for intravenous administration, and the pharmaceutical preparation may be contained in a glass vial.

[0163] In some embodiments, the present invention provides an autoinjector that contains any of the liquid formulations described herein.

[0164] In some embodiments, the present 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 mAb1, about 10 mM histidine, pH about 6.0, about 70 mM arginine-HCl, about 3% proline and about 0.1% polysorbate 80.

[0165] In some embodiments, the present invention provides a pre-filled syringe comprising any of the liquid formulations described herein.

[0166] In some embodiments, the present invention provides a pre-filled syringe comprising a stable liquid formulation comprising about 50 mg / mL, about 100 mg / mL, about 150 mg / mL, or about 175 mg / mL mAb1, about 10 mM histidine, pH about 6.0, about 70 mM arginine-HCl, about 3% proline, and about 0.1% polysorbate 80. In some 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 guard.

[0167] In one embodiment, a liquid drug formulation containing approximately 175 mg / mL ± 26.25 mg / mL mAb1 in a pre-filled syringe is administered subcutaneously in a volume not exceeding approximately 2 mL. In some embodiments, the syringe is a 1 mL or 2.25 mL long glass syringe equipped with a 27-gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle guard. In one embodiment, the syringe is equipped with a 27-gauge needle, an FM27 rubber needle guard, and... OMPI 1mL long glass syringe with coated 4023 / 50 rubber plunger.

[0168] In one embodiment, a liquid drug formulation containing about 150 mg / mL ± 22.5 mg / mL mAb1 in a pre-filled syringe in a volume of about 1 to about 2 mL is administered subcutaneously. In one embodiment, the syringe is a 1 mL or 2.25 mL long glass syringe with a 27-gauge thin-walled needle, a fluorocarbon-coated rubber plunger, and a rubber needle guard. In one embodiment, the syringe is equipped with a 27-gauge needle, an FM27 rubber needle guard, and... OMPI 1mL long glass syringe with coated 4023 / 50 rubber plunger.

[0169] Therapeutic applications of pharmaceutical preparations

[0170] The pharmaceutical formulations of the present invention are particularly useful for treating, preventing, or improving any disease or condition associated with ANGPTL3 activity, including ANGPTL3-mediated diseases or conditions. Diseases or conditions that can be treated with the formulations of the present invention are any diseases or conditions (e.g., ANGPTL3-mediated diseases or conditions) that can be improved, alleviated, suppressed, prevented, or have their incidence reduced by removing, inhibiting, reducing, or otherwise interfering with ANGPTL3 activity compared to treatment without anti-ANGPTL3 antibody therapy.

[0171] Examples of diseases or conditions that can be treated with the formulations of the present invention include, but are not limited to, those involving lipid metabolism, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia, including severe hypertriglyceridemia with TG > 1000 mg / dL, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipid malnutrition, lipoatrophy, etc., said diseases or conditions may be caused by, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor (LDLR) activity and / or LDL receptor deficiency (e.g., having LDLR... - / - The invention can prevent or treat diseases or conditions associated with or caused by hyperlipidemia, hyperlipoproteinemia, and / or dyslipidemia, including but not limited to cardiovascular diseases or conditions such as atherosclerosis, aneurysm, hypertension, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, etc.; acute pancreatitis; non-alcoholic steatohepatitis (NASH); glycemic disorders such as diabetes; obesity, etc.

[0172] Other examples of diseases or conditions that can be treated with the formulations of the present invention include cancer / tumor and non-tumor angiogenesis-related diseases or conditions, including ocular angiogenesis diseases or conditions such as age-related macular degeneration, central retinal vein occlusion or branch retinal vein occlusion, diabetic retinopathy, retinopathy of prematurity, etc., and inflammatory diseases or conditions such as arthritis, rheumatoid arthritis (RA), psoriasis, etc. Example

[0173] The following examples are provided to offer a complete disclosure and description to those skilled in the art regarding how to implement and use the methods and compositions of the present invention, and are not intended to limit the scope of the invention. While efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are molar parts, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atm or near atm.

[0174] Example 1: Development of anti-ANGPTL3 antibody formulation

[0175] For later-stage clinical development, higher doses of IV and SC administration are planned. Therefore, formulation development studies were conducted with the aim of developing a 150 mg / mL liquid formulation suitable for IV or SC injection. A high-concentration liquid formulation is necessary to deliver doses up to 450 mg of H4H1276S via one or two SC injections. For IV administration, a high-concentration liquid formulation is also advantageous as it allows for the addition of a smaller volume of DP to the IV infusion bag. This supports dosage administration of up to 15 mg of H4H1276S per kg of patient body weight.

[0176] 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. Based on the knowledge gained from the initial formulation development, formulation development activities for the 150 mg / mL liquid formulation involved evaluating viscosity-reducing excipients, pH, surfactants, and heat stabilizers to identify excipients that can enhance protein stability while maintaining an acceptable solution viscosity at higher protein concentrations of 150-200 mg / mL H4H1276S.

[0177] Throughout the formulation development process, three main protein stress conditions (representing extreme handling conditions beyond what antibody drug products will encounter during processing, manufacturing, transportation, storage, and labeling) were employed to develop and optimize antibody formulations and assess the impact of potential real-world stresses on drug stability. These stress conditions include:

[0178] • Vortex the protein solution at room temperature. The vortex in the glass vial exceeds the stirring that occurs during protein processing and manufacturing.

[0179] • Incubate the protein solution at elevated temperatures (37°C, 40°C, or 45°C) relative to the recommended DP storage conditions (2°C–8°C).

[0180] • Expose the protein to multiple freeze-thaw cycles. Since the protein will undergo at least one freeze-thaw cycle during the manufacturing process of DP, multiple freeze-thaw cycles can simulate and exceed the actual stress that the protein is expected to experience.

[0181] Anti-ANGPTL3 antibody: Anti-ANGPTL3 antibodies are described in US 9,018,356 B2, which are incorporated herein in their entirety. The exemplary antibody used in the following examples is the fully human anti-ANGPTL3 antibody H4H1276S (as disclosed in '356'), which comprises: a heavy chain variable region / light chain variable region HCVR / LCVR amino acid sequence pair containing SEQ ID NO:66 / 74; a heavy chain and light chain complementarity-determining region CDR sequence containing SEQ ID NO:68 / 70 / 72 / 76 / 78 / 80; and is also referred to herein as "mAb1".

[0182] Example 2: Exemplary Formulation

[0183] In some embodiments, mAb1 is formulated as a buffered aqueous solution 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. Exemplary formulations include: 150 mg / mL H4H1276S, 10 mM histidine, pH 6.0, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 3% (w / v) proline.

[0184] Example 3: A method for evaluating formulation stability

[0185] 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) form, low molecular weight (LMW) form, charge variants, and other chemical modifications of the protein. The physical and chemical stability of antibody drug products (e.g., H4H1276S) are assessed using the following assays:

[0186] • Visually inspect the color and appearance (can detect the presence of visible particles in the solution).

[0187] pH

[0188] • Turbidity is measured by the increase in optical density (OD) at 405 nm.

[0189] • Through Micro-Flow Imaging TM (MFI) for subvisible particle analysis

[0190] • Protein concentration was determined by reversed-phase ultra-high performance liquid chromatography (RP-UPLC), and reported as a percentage of protein recovery relative to the starting material.

[0191] Purity is measured using the following methods:

[0192] Size exclusion ultra-high performance liquid chromatography (SE-UPLC)

[0193] -Reduced and Non-Reduced Microchip Capillary Electrophoresis-Sodium Dodecyl Sulfate (MCE-SDS)

[0194] • Charge variant analysis:

[0195] - Cation exchange UPLC (CEX-UPLC)

[0196] -Imaging capillary isoelectric focusing (iCIEF)

[0197] • Efficacy determined by bioassay:

[0198] - The relative power of each sample is determined by bioassay and is defined as: (IC50 reference sample / IC50 sample) * 100%. The measurement power of the storage-stable sample must be within 50% to 150% of the measurement power of the reference standard.

[0199] For the chemical stability of the formulation, the formation of covalently modified forms of the protein (e.g., covalent aggregates, cleavage products, or charge variants) and non-covalently modified forms (e.g., non-covalent aggregates) are assessed. Higher and lower molecular weight degradation products can be separated from the natural antibody using SE-UPLC and MCE-SDS methods.

[0200] Example 4: Selection of viscosity-reducing reagents

[0201] To understand how viscosity is affected by increasing H4H1276S concentration, formulations with different protein concentrations were prepared in 10 mM histidine, pH 6.0, 5% sucrose, and 0.1% polysorbate 80. This formulation is equivalent to the first-in-human (FIH) formulation intended for intravenous administration in initial clinical studies. The viscosity of each sample was measured at 20°C, and the results are as follows: Figure 1 (As shown by the curve above). The viscosity measured at 150 mg / mL is greater than 40 centipoise (cP), which is significantly higher than the target acceptable viscosity of 20 cP. Therefore, the use of viscosity-reducing excipients should be considered to achieve a formulation with a target protein concentration of 150 mg / mL and an acceptable viscosity.

[0202] To identify suitable viscosity-reducing excipients, the effect of selected excipients on the viscosity of the H4H1276S formulation was examined. The excipients included were 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 the viscosity at all tested protein concentrations. Figure 1 (See the curve below). Compared to other excipients, arginine-HCl was found to be the most effective in reducing viscosity. Furthermore, it had minimal impact on stability. Table 1 below summarizes the excipients tested for viscosity reduction. Arginine-HCl was chosen for further development studies because it provided sufficient viscosity reduction. Subsequent formulation development studies included 70-75 mM arginine-HCl to determine the optimal pH, surfactant concentration, and heat stabilizer.

[0203] Table 1: Summary of excipients for viscosity reduction test

[0204] excipient Concentration range Effect on viscosity Impact on stability Arginine-HCl 50-75mM Significantly reduced small Sodium chloride 50-75mM Significantly reduced small Histidine-HCl 40-65mM reduce Untested Sodium acetate, pH 5 40-60mM Significantly reduced Reduced stability Calcium chloride 25mM Significantly reduced small Magnesium chloride 25mM Significantly reduced small Calcium acetate 25mM Significantly reduced small Magnesium acetate 25mM Significantly reduced small

[0205] Example 5: pH Selection

[0206] The effect of pH on the thermal stability of H4H1276S in liquid formulations was examined by incubating 150 mg / mL H4H1276S at 45 °C, at different pH ranges, and in 10 mM histidine with 5% (w / v) sucrose or 2% (w / v) proline for 28 days. Sucrose and proline were considered potential heat stabilizers and were included, thus allowing the effects of buffers and pH to be studied using formulation compositions that are more representative of the final formulation. Results were obtained from SE-UPLC and CEX-UPLC analyses (Table 2, etc.). Figure 2 As shown, higher pH values ​​minimize the formation of HMW substances, while lower pH values ​​minimize the formation of charge variants. Histidine buffer at pH 6.0 was chosen as the formulation buffer because it provides the optimal balance between HMW substance formation and charge variant formation.

[0207] Example 6: Optimization of Surfactant Concentration

[0208] Surfactants are frequently added to antibody formulations to protect proteins from agitation-induced aggregation. During the development of an initial lyophilizable formulation, HMW formation was observed when 50 mg / mL H4H1276S was agitated. Adding 0.1% (w / v) polysorbate 80 protected H4H1276S from agitation-induced instability. However, protein concentration, heat stabilizer content, and the presence of other excipients can affect the protein's sensitivity to agitation stress. Therefore, the minimum amount of polysorbate 80 required to protect 150 mg / mL H4H1276S from agitation stress was evaluated. Concentrations of 0.0%, 0.02%, 0.05%, and 0.1% (w / v) of polysorbate 80 were tested in the presence of 5% sucrose, 2% sucrose, and 1.3% proline or 2% proline (%w / v). Samples were formulated at pH 6 with 10 mM histidine and 70 mM arginine to better represent the final formulation. The results are summarized in Tables 3, 4, and 5 (as shown below). Figure 3 , 4 (as shown in Figure 5).

[0209] Stir for 120 minutes (Table 3, as shown in Table 3). Figure 3 When the surfactant-free formulation was shown, a significant increase in HMW (4.8-6.3%) was observed. Adding 0.02% (w / v) polysorbate 80 was insufficient to protect H4H1276S from agitation-induced instability, while 0.05% (w / v) or higher of polysorbate 80 provided adequate stability regardless of the heat stabilizer contained in the formulation. The data indicate that at least 0.05% (w / v) of polysorbate 80 is required to protect H4H1276S from agitation-induced instability, and that with at least 0.05% (w / v) of polysorbate 80, agitation-induced instability is not affected by the choice of heat stabilizer. At 45°C incubation, the addition of polysorbate 80 did not affect the formation of HMW in H4H1276S, regardless of the heat stabilizer contained in the formulation (Table 4, e.g.). Figure 4 (As shown). For formulations without heat stabilizers, the relative change in HMW from t=0 is comparable to that of formulations containing polysorbate. For formulations containing only sucrose, sucrose and proline, or only proline, the increases in HMW are 3.1-3.2%, 3.5-3.8%, and 3.8-4.1%, respectively.

[0210] When compared between heat stabilizer groups, H4H1276S showed moderately improved stability when formulated with sucrose (compared to proline) and incubated under stress conditions. However, this difference was not considered significant. For all evaluated formulations, the difference in the total relative change in charge variant distribution from t=0 was considered within the variability range of the assay. The effects of agitation and incubation at 45°C on particle formation were assessed using MFI, and the results are shown in Table 5 (e.g.). Figure 5 (As shown). No discernible trend was observed in particle formation, indicating that particle formation is unaffected by the concentration of polysorbate 80 or the heat stabilizer included in the formulation. Although stability was achieved at a polysorbate concentration of 0.05% (w / v), 0.1% (w / v) polysorbate 80 was chosen as the surfactant concentration. This higher concentration of polysorbate ensures the robustness of the formulation, exhibiting stability comparable to the 0.05% (w / v) polysorbate formulation, and provides additional stability when diluted to IV bags.

[0211] Example 7: Effect of heat stabilizers

[0212] Stabilizers can be added to antibody formulations to increase the stability of proteins in liquid formulations and during frozen storage. In previous formulations, sucrose was included as a heat stabilizer. However, sucrose also increases the viscosity of the solution. Therefore, proline was evaluated as a heat stabilizer, with the expectation that it would affect the solubility and colloidal stability of proteins to improve the storage stability of monoclonal antibody product formulations without increasing the viscosity of the final formulation (Table 6 below).

[0213] Table 6: Viscosities of H4H1276S formulations with different concentrations of sucrose and proline at 20°C

[0214]

[0215] To support the selection of components for bulk-formulated drug substance compositions and to assess the need for heat stabilizers in such formulations, the stability of 175 mg / mL H4H1276S in 10 mM histidine at pH 6.0 and 70 mM arginine-HCl (without sucrose or proline) was evaluated by assessing its stability at -20°C for frozen storage and its freeze / thaw stability (frozen at -30°C and thawed at room temperature) (Tables 7 and 8 are shown respectively). Figure 6 and 7 Although -30°C is the expected long-term storage condition for the formulated pharmaceutical substances, the stability of frozen storage at -20°C was evaluated as an accelerated frozen storage condition for formulation development. After 9 months of incubation at -20°C, an increase of 7.2% in HMW substance was observed (Table 7, e.g.). Figure 6 (As shown). Although a small increase in HMW content was observed in this formulation after 8 freeze-thaw cycles (Table 8, as shown). Figure 7 (As shown), however, the stability data for -20°C cryopreservation indicates that heat stabilizers are needed to support long-term cryopreservation of large quantities of pharmaceutical substances.

[0216] To identify and optimize the concentration of one or more heat stabilizers in liquid formulations, 150 mg / mL H4H1276S 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. The formulations were also incubated at -20 °C and subjected to freeze-thaw cycles (-30 °C freezing; room temperature thawing) to compare frozen storage stability (which is required to support the storage of batch-formulated pharmaceutical substances). Polysorbate 80 was included in these formulations to better represent the final product formulation. The viscosity of all samples at 20 °C was measured at t=0 and is summarized in Table 6 above. Table 6 illustrates 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 there is no negative impact on stability.

[0217] Compared to proline, H4H1276S showed moderately improved stability when formulated with sucrose and incubated at 45°C for 21 days (Table 9, e.g.) Figure 8 (As shown). For the 5% sucrose formulation, the total relative change in HMW formation from t=0 was 2.7%, while for the 2% proline formulation, the total relative change in HMW formation from t=0 was 3.3%. This difference was not considered significant. This difference in the total relative change in charge variants from t=0 between the different formulations assessed was considered to be within the range of variability of the assay.

[0218] After incubation at -20°C or 8 freeze-thaw cycles (-30°C freezing; thawing at room temperature), no significant changes in any quality attribute were observed for all formulations containing sucrose and / or proline (Tables 7 and 8, as shown). Figure 6 and 7 (As shown). In summary, when stored under accelerated or stress storage conditions, the proline-containing formulations exhibited lower viscosity and comparable stability compared to the sucrose-containing formulations. Based on these results, two lead formulations were selected: 1) 150 mg / mL H4H1276S, 10 mM histidine pH 6.0, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 5% (w / v) sucrose; 2) 150 mg / mL H4H1276S, 10 mM histidine pH 6.0, 70 mM arginine-HCl, 0.1% (w / v) polysorbate 80, and 3% (w / v) proline. The proline concentration was slightly increased to ensure adequate protein stability under representative long-term storage conditions.

[0219] Example 8: Formulation Selection

[0220] Long-term stability was examined to compare the stability of the two lead formulations (see Example 7 above). Stability data at -30°C were collected to assess the long-term storage of batch-formulated pharmaceutical substances (Table 10, e.g.) Figure 9 (As shown). Stability data at 5°C were collected to assess the long-term storage of the drug product (Table 11, as shown). Figure 10 (As shown). These data indicate that the two lead formulations obtained equivalent stability profiles.

[0221] The relationship between viscosity, protein concentration, and temperature was used to facilitate the selection of formulations that can be delivered at a range of protein concentrations and temperatures. The effect of protein concentration on the final formulation viscosity and how excipients affect this relationship were considered. Similarly, temperature was considered for a) withdrawal and / or administration (approximately room temperature); b) batch manufacturing process steps (typically approximately 15–25 °C); and c) storage.

[0222] To guide the selection of the final formulation and better characterize the effect of the heat stabilizer on viscosity, formulations with different concentrations of H4H1276S were prepared in 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). Viscosities were measured at temperatures ranging from 5 °C to 35 °C. Figure 11 This illustrates the relationship between the viscosity of the two formulations at 20°C and the concentration of H4H1276S. (Compared to sucrose-containing formulations...) Figure 11 Compared to the top curve, proline-containing preparations ( Figure 11 The bottom curve shows that the viscosity remains low at various H4H1276S concentrations.

[0223] This trend is even more pronounced at lower temperatures. Figure 12A and 12B Data shows that formulations containing 3% (w / v) proline... Figure 12B This provides a wider operating range for viscosity values ​​that are considered acceptable. Since the two lead formulations have comparable stability, and the proline-containing formulation has favorable viscosity characteristics, the formulation containing 3% (w / v) proline was chosen for the final drug product formulation.

[0224] The final H4H1276S liquid pharmaceutical product formulation contains 150 mg / mL H4H1276S, 10 mM histidine, 70 mM arginine-HCl, 3% (w / v) proline, and 0.1% (w / v) polysorbate 80, pH 6.0. The primary degradation pathways identified during development were high molecular weight substances and charge variants. The final osmotic pressure of the formulation is approximately 480 mOsm / kg, and the viscosity is approximately 15 centipoise (20°C), suitable for clinical use.

[0225] Table 12 below summarizes the H4H1276S formulations developed for preclinical (GLP toxicology) and clinical applications.

[0226] Table 12: Composition of H4H1276S formulations and H4H1276S clinical DP formulations used in GLP toxicology studies

[0227]

[0228] Example 9: Summary of Stability Studies of H4H1276S Drug Product

[0229] Studies were conducted to evaluate the storage and accelerated stability (data not shown) of the H4H1276S 150 mg / mL pharmaceutical product (DP). Stability was assessed for the following: color and appearance, turbidity (increased OD at 405 nm), pH, particulate matter by MFI, percentage of total protein recovery by RP-UPLC (reversed-phase ultra-high performance liquid chromatography), percentage purity by non-reducing and reducing MCE-SDS (microchip capillary electrophoresis-sodium dodecyl sulfate), percentage purity by SE-UPLC (size exclusion ultra-high performance liquid chromatography), charge variant analysis by CEX-UPLC (cation exchange ultra-high performance liquid chromatography), charge variant analysis by iCIEF (imaging capillary isoelectric focusing), and percentage of relative potency by bioassays.

[0230] The DP used for storage and accelerated stability studies was prepared by incubating 5.0 mL of the prepared drug substance (FDS) in a 20 mL Type 1 clear glass vial. H4H1276S DP was physically and chemically stable after storage at 5°C for at least 12 months. No significant changes in physical or chemical stability were detected in any of the monitored properties.

[0231] Subsequently, incubation was performed at 25°C / 60% RH (accelerated stability) or 45°C (stress stability). These accelerated and stress conditions were chosen to elucidate the degradation pathway of H4H1276S. Regarding the accelerated stability study, significant formation of HMW substances and charge variants was detected after 3 months at 25°C / 60% RH. After 1 month of incubation at 25°C / 60% RH, no significant formation of HMW substances or charge variants was observed, indicating that H4H1276S DP can be exposed to room temperature for short periods. After incubation under accelerated conditions, H4H1276S retained its potency, as determined by bioassay analysis.

[0232] Incubation at 45°C for as short as 7 days resulted in the significant formation of HMW material and charge variants, indicating that the increase of HMW material and the formation of charge variants are the main degradation pathways of H4H1276S DP. H4H1276S DP was found to be physically and chemically stable upon agitation (vortexing at ambient temperature) for 120 minutes or after 8 freeze-thaw cycles (freezing at -30°C and thawing at room temperature). No significant changes in physical or chemical stability were detected in any of the monitored properties.

[0233] Results from accelerated storage and stress stability studies of DP indicate that H4H1276S 150 mg / mL DP is stable during manufacturing and storage. Furthermore, the H4H1276S formulation can withstand short-term exposure to room temperature without compromising its physical or chemical stability. H4H1276S 150 mg / mL DP is preferably stored between 2°C and 8°C, with exposure limited to temperatures above 2°C to 8°C.

[0234] Example 10: Development of a high-concentration liquid drug product based on amino acids, H4H1276S, from excipient screening to rheological characterization.

[0235] H4H1276S was lyophilized and then reconstituted to a higher protein concentration using a solution containing the desired excipient. The pre-lyophilized formulation contained 4 mL of 87.5 mg / mL H4H1276S, 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 solution to produce a final H4H1276S concentration of 160–175 mg / mL (nominal). The reconstitution solution was adjusted so that the final sample contained 20 mM histidine, pH 6, and 5% (w / v) sucrose and the test excipient. To adjust the pH from 6 to 5, acetate buffer was added to the reconstitution solution to achieve a final acetate concentration of 40 mM.

[0236] To investigate the accelerated and frozen storage stability of the H4H1276S formulation containing viscosity-reducing excipients, the viscosity of the test formulation was measured at 20 °C using a Rheosense viscometer. The test formulation was 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 aggregate content of the obtained substances was analyzed by SEC, and the formation of charge variants was analyzed by CEX.

[0237] To understand the relationship between protein concentration, temperature, and viscosity of H4H1276S formulations with reduced viscosity, 200 mg / mL H4H1276S formulations containing the following excipient combinations were prepared. All formulations contained 10 mM histidine pH 6 and 0.1% (w / v) polysorbate 80:

[0238] 5% (w / v) sucrose

[0239] 5% (w / v) sucrose, 70 mM L-Arg-HCl

[0240] • 3% (w / v) sucrose, 1.3% (w / v) proline, 70 mM L-Arg-HCl

[0241] 3% (w / v) proline

[0242] • 3% (w / v) proline, 70 mM L-Arg-HCl.

[0243] The 200 mg / mL formulation was diluted to a protein concentration of 50–200 mg / mL using the appropriate formulation buffer. Viscosities of each formulation were measured on a Rheosense viscometer at temperatures ranging from 5–35 °C. Finally, data analysis was performed using GraphPad Prism and MiniTab.

[0244] The screening results for viscosity-reducing excipients show... Figure 13A and 13B In China. For Figure 13A During reconstruction, 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 solution at pH 5, acetate was added to a final concentration of 40 mM. The equivalent formulation containing 40 mM acetate at pH 6.0 had a viscosity of 64.1 centipoise, indicating that both adding acetate and adjusting the pH to 5.0 reduced the viscosity of the H4H1276S formulation. Figure 13BDuring reconstitution, the base formulation contains 165 mg / mL H4H1276S, 20 mM histidine / 40 mM acetate, pH 5.0, 0.1% (w / v) polysorbate 80 and 5% (w / v) sucrose.

[0245] The addition of monovalent and divalent salts (L-Arg-HCl, NaCl, CaCl2, MgCl2, Ca(OAc)2, Mg(OAc)2) reduced the viscosity of the H4H1276S formulation. Adjusting the pH from 6 to 5 also reduced the viscosity of the H4H1276S formulation. Finally, sucrose increased the viscosity of the H4H1276S formulation, while L-proline did not affect this viscosity.

[0246] The formation of high molecular weight (HMW) substances after incubation at 45°C for 21 days is as follows: Figure 14 As shown. Selected H4H1276S formulations containing various VR (viscosity-reducing) excipients were incubated at 45°C and aggregate content was analyzed by SEC. The relative increase of HMW content from t=0 was plotted as a function of the formulation. Under accelerated conditions (45°C for 21 days), the following was observed:

[0247] L-Arg-HCl has almost no effect on the formation of HMW substances;

[0248] • Lowering the pH of the H4H1276S formulation led to increased formation of HMW substances;

[0249] • The relative increase in HMW substances was similar in formulations containing CaCl2 and MgCl2 compared to formulations containing L-Arg-HCl and NaCl.

[0250] L-proline acts as a heat stabilizer and reduces the formation of high-molecular-weight substances (HMWs); and

[0251] Compared to L-Arg-HCl formulations, formulations containing Ca(OAc)2 and Mg(OAc)2 exhibit reduced HMW formation.

[0252] Therefore, L-Arg-HCl or Mg(OAc)2 (substantial viscosity reduction) in a pH 6 buffer (better accelerated stability) were chosen as lead viscosity reducers for further development.

[0253] The stability of H4H1276S formulations containing viscosity-reducing excipients is summarized in... Figure 15A and 15BIn this study, a 150 mg / mL H4H1276S formulation containing 70 mM L-Arg-HCl or 25 mM Mg(OAc)2 was prepared with various concentrations of sucrose and / or L-proline. The concentration was adjusted to achieve an osmotic pressure of approximately 300 mM to maintain isotonicity. Formulations containing only L-Arg-HCl or Mg(OAc)2 were prepared using 175 mg / mL H4H1276S. Figure 15A (Degradation of H4H1276S after 21 days of incubation at 45°C was depicted). The sample was incubated at 45°C, and aggregate content was analyzed by SEC, while the formation of acidic charge variants was analyzed by CEX. The relative increase in HMW or acidic substance from t=0 was plotted as a function of formulation. For Figure 15B (The cryogenic storage stability of H4H1276S was depicted). Samples were incubated at -20°C for 9 months, and aggregate content was analyzed by SEC. The percentage of HMW material was 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 material was observed at -80°C for any formulation. At 5°C or -30°C, formulations without heat stabilizers showed increased HMW material content.

[0254] The Mg(OAc)2 formulation exhibits slightly lower stability under accelerated storage conditions compared to formulations containing L-Arg-HCl, with increased formation of both high-molecular-weight (HMW) and acidic substances. Replacing sucrose with L-proline reduces viscosity in a concentration-dependent manner. The addition of sucrose, L-proline, or a combination of both excipients adequately protects H4H1276S from HMW formation at -20°C. Therefore, 70 mM L-Arg-HCl was chosen as a lead viscosity reducer for further development. The provided data support the use of Mg(OAc)2 as a backup excipient.

[0255] The relationship between protein concentration, temperature, and viscosity is shown in Figure 16A and 16B These graphs show isopleths of viscosity versus protein concentration and temperature. The isopleths were generated in Minitab. The shapes at the points corresponding to 150 mg / mL and 165 mg / mL (or 150 mg / mL + 10%) H4H1276S represent the recommended storage temperature at 5°C (circles) or the recommended application temperature at 25°C (stars).

[0256] The relationship between viscosity and protein concentration at 20°C is shown in... Figure 17In this study, viscosity was plotted as a function of protein concentration, and the data was fitted to an exponential curve using GraphPad Prism. This formula can be used to predict viscosity based on known concentrations, which is useful for defining the manufacturing specifications of the final product and for informing process development. The formula for a formulation containing 3% L-proline and 70 mM L-Arg-HCl is: Viscosity = 0.444e 0.023[H4H1276S] .

[0257] The contour maps and graphs above support the following observations:

[0258] Compared to sucrose-containing formulations, L-proline-containing formulations consistently exhibited lower viscosity at various H4H1276S concentrations (and temperatures; data not shown) with or without 70 mM L-Arg-HCl.

[0259] The viscosity versus protein concentration curve (at 20°C) of the formulation containing the combination of 5% sucrose and 3% L-proline is very similar to that of the 3% proline formulation. In fact, the difference between the two formulations is more pronounced at lower temperatures and higher protein concentrations. The contour plots of these formulations show subtle but clear differences (data not shown).

[0260] When both temperature and manufacturing specifications are taken into account, L-proline formulations offer a wider operating range for viscosity values ​​considered acceptable for SC administration. Figure 16A ).

[0261] An amino acid-based formulation containing 150 mg / mL H4H1276S, 10 mM histidine, pH 6, 70 mM L-Arg-HCl, and 3% proline was selected for H4H1276S DP development.

[0262] Formulations containing a combination of sucrose and L-proline have been phased out and are no longer being developed because they offer no advantage in terms of stability during frozen storage (see above).

[0263] Therefore, this paper reveals a thorough understanding of the relationship between viscosity, protein concentration, and temperature, which can inform decision-making and facilitate the selection of formulations that are stable during storage and can be delivered in pre-filled syringes or autoinjectors. Considering the manufacturing specifications of the pharmaceutical product (DP) and the process-related manufacturing steps (i.e., batches of drug substances), ideally, viscosity should be considered across a range of protein concentrations, and the target concentration should be selected at the lower end of the viscosity vs. protein concentration curve.

[0264] Example 11: Compatibility with intravenous delivery devices

[0265] For clinical delivery, 150 mg / mL H4H1276S DP can be diluted in intravenous (IV) bags containing normal saline for intravenous (IV) administration at clinical doses of 5 mg / kg and 15 mg / kg. The stability of H4H1276S during use was evaluated to support IV administration at clinical doses. Two mixed concentrations, 0.5 mg / mL H4H1276S and 20 mg / mL H4H1276S, were tested to attempt to cover both low- and high-concentration mixtures that might be administered in a clinical setting.

[0266] To evaluate the delivery of mixtures from IV bags using IV pumps and infusion sets incorporating in-line filters, IV bags containing physiological saline made of polyvinyl chloride (PVC) and di(2-ethylhexyl) phthalate (DEHP) and two commonly used infusion pumps (peristaltic pumps and volumetric pumps) were tested. Several infusion sets incorporating base materials (PVC with DEHP, PVC with TOTM, and polyethylene) and 0.2 μm polyethersulfone in-line filters were also evaluated.

[0267] Measurement test

[0268] The compatibility of the H4H1276S mixture with the materials used in IV delivery devices was evaluated using the following assays:

[0269] • Visually inspect color and appearance

[0270] pH

[0271] • Turbidity is measured by increasing the optical density (OD) at 405 nm.

[0272] • Subvisible particle analysis of the mixture using light-masking (HIAC)

[0273] • Protein concentration determined by reversed-phase high-performance liquid chromatography (RP-UPLC)

[0274] Purity determined by SE-UPLC

[0275] • Power determined by bioassay: The relative power of each sample is determined using a bioassay and defined as: (IC50 reference sample / IC50 sample) × 100%. The measurement power of the storage-stable sample must be within 50-150% of the measurement power of the reference standard.

[0276] Research Plan

[0277] 100 mL saline IV bags containing H4H1276S DP were subjected to various stress conditions to assess the stability of H4H1276S in the mixture and during intravenous delivery. The IV bags containing the mixture were initially held at 5°C for 24 hours; then the bags were incubated at 25°C for at least 8 hours. After these incubations, each infusion set to be evaluated was connected to the IV bag, exposed to the mixture, and held at ambient temperature for 1 hour. Each mixture was then infused through the corresponding infusion set at a rate of 25 mL / hr or 500 mL / hr. Clinically, DP diluted in 100 mL or 250 mL IV bags may be used for administration. For this compatibility study, 100 mL IV bags were used to test each dose.

[0278] Research Results

[0279] The 0.5 mg / mL and 20 mg / mL H4H1276S diluted in saline were physically and chemically stable under all test conditions, including: i) 24 hours in IV bags at 5°C, ii) 8 hours in IV bags at 25°C, and iii) 1 hour in all tested infusion sets at room temperature.

[0280] Furthermore, the H4H1276S mixture was stable when pumped through each evaluated infusion set using various infusion pumps at rates of 25 mL / hr and 500 mL / hr. No precipitates were detected by visual inspection or turbidity measurements. The pH of the solution remained stable, and no significant decrease in protein concentration was observed. No significant changes in the relative percentages of high or low molecular weight substances were observed in this compatibility study, as determined by size exclusion ultra-high performance liquid chromatography (SE-UPLC). Compared to t=0, no significant changes in subvisible particle levels were observed after the sample was pumped through the infusion set at 500 mL / hr, as determined by HIAC analysis. Finally, all tested samples maintained potency as determined by bioassays.

[0281] These data support the following clinical dosage preparation and administration:

[0282] • The saline IV bag, made of PVC with DEHP, is compatible with H4H1276S for IV administration.

[0283] • H4H1276S can be diluted to a concentration as low as 0.5 mg / mL in PVC IV bags containing physiological saline for intravenous administration;

[0284] • H4H1276S can be diluted to concentrations up to 20.0 mg / mL in PVC IV bags containing physiological saline for intravenous administration;

[0285] The H4H1276S mixture in physiological saline is stable in a PVC IV bag at 5°C for up to 24 hours and at 25°C for 8 hours. Diluted H4H1276S mixtures can be administered within four hours of preparation.

[0286] • The H4H1276S mixture in physiological saline can be administered using a standard infusion pump;

[0287] • The H4H1276S mixture can be administered using an infusion set made of DEHP-containing PVC, TOTM-containing PVC, or polyethylene.

[0288] The H4H1276S mixture is compatible with the use of online 0.2μm polyethersulfone filters;

[0289] The H4H1276S mixture can be administered at a flow rate of 25 to 500 mL / hr.

[0290] Example 12: Further stability study of the H4H1276S drug product

[0291] The H4H1276S formulation was stored at -20°C and 5°C for up to 36 months, and its stability was analyzed.

[0292] First, the formulation consisted of 150 mg / mL H4H1276S, 10 mM histidine, pH 6.0, 70 mM arginine-HCl, 3% (w / v) proline, and 0.1% (w / v) polysorbate 80. A 2.0 mL filling volume was achieved in a 5 mL Nalge-Nunc gamma-irradiated polycarbonate vial with a polysiloxane-lined seal.

[0293] Table 13: Stability of the prepared drug substance of H4H1276S at -20℃

[0294]

[0295] Next, the formulation consisted of 150 mg / mL H4H1276S drug product, 10 mM L-histidine, pH 6.0, 70 mM arginine-HCl, 3% (w / v) L-proline, and 0.1% (w / v) polysorbate 80. The volume was filled to 5.0 mL in a 20 mL Type I clear glass vial with a 20 mm... Coated West S2-451 4432 / 50GRY B2-40 bottle stopper.

[0296] Table 14: Stability study of H4H1276S drug product stored at 5°C

[0297]

[0298] The formulated pharmaceutical substances can be stored at -20°C for extended periods (up to 36 months), and the pharmaceutical products can be stored at 5°C for extended periods (up to 24 months). As shown in Tables 13 and 14, the formulated H4H1276S exhibits stability; for example, values ​​in all tests remained within acceptable ranges throughout the entire storage period.

[0299] Example 13: Container

[0300] The primary container for antibody drug products used in clinical development and commercialization is the pre-filled syringe, which can be presented as a standalone syringe for self-injection or integrated into an auto-injector device for self-dosing. Antibody formulations can also be developed in glass vials (for intravenous infusion delivery).

[0301] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications that can be made to this invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. These modifications are intended to fall within the scope of the appended claims.

Claims

1. A liquid pharmaceutical formulation comprising: i) 150 mg / mL ± 22.25 mg / mL anti-ANGPTL3 antibody or an antigen-binding fragment thereof; ii) 5 mM ± 1 mM to 20 mM ± 4 mM histidine; iii) 0.1% w / v ± 0.05% to 0.5% w / v ± 0.25% polysorbate 80; iv) 50 mM to 75 mM arginine-HCl; and v) 1% w / v ± 0.2% to 5% w / v ± 1% w / v proline, wherein the liquid pharmaceutical formulation has a pH of 6.0 ± 0.3, and wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (HCDRs), namely, HCDR1 with an amino acid sequence as shown in SEQ ID NO:68, HCDR2 with an amino acid sequence as shown in SEQ ID NO:70, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

68. HCDR3 as shown in NO:72; and three light chain complementarity-determining regions (LCDRs), namely, LCDR1 with an amino acid sequence as shown in SEQ ID NO:76, LCDR2 with an amino acid sequence as shown in SEQ ID NO:78, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

80.

2. The liquid pharmaceutical formulation of claim 1, 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%.

3. The liquid pharmaceutical preparation of any one of claims 1-2, wherein the viscosity of the liquid pharmaceutical preparation is less than about 20 cP.

4. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 95% of the antibody or its antigen-binding fragment has a natural conformation after 21 days at 45°C.

5. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 45% of the antibody or its antigen-binding fragment is the major charge variant of the antibody or its antigen-binding fragment after 21 days at 45°C.

6. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 98% of the antibody or its antigen-binding fragment has a natural conformation after 36 months at 5°C.

7. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 55% of the antibody or its antigen-binding fragment is the major charge variant of the antibody or its antigen-binding fragment after 36 months at 5°C.

8. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 98% of the antibody or its antigen-binding fragment has a natural conformation after 36 months at -30°C.

9. The liquid pharmaceutical formulation of any one of claims 1-2, wherein at least about 57% of the antibody or its antigen-binding fragment is the major charge variant of the antibody or its antigen-binding fragment after 36 months at -30°C.

10. The liquid pharmaceutical formulation of any one of claims 1-2, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) with an amino acid sequence as shown in SEQ ID NO: 66 and a light chain variable region (LCVR) with an amino acid sequence as shown in SEQ ID NO:

74.

11. The liquid pharmaceutical preparation of any one of claims 1-2, wherein the liquid pharmaceutical preparation is in a pre-filled syringe or an autoinjector.

12. The liquid pharmaceutical preparation of any one of claims 1-2, wherein the liquid pharmaceutical preparation is in a glass vial.

13. A medicine box comprising a liquid pharmaceutical preparation, a container, and instructions for use as claimed in any one of claims 1-12.

14. The medicine box of claim 13, wherein the container is a pre-filled syringe or an auto-injector.

15. Use of the liquid pharmaceutical preparation of any one of claims 1-12 in the preparation of a medicament for treating familial hypercholesterolemia in a subject.

16. The use of claim 15, wherein the liquid pharmaceutical preparation is administered subcutaneously to a subject.

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