Low-viscosity, high-concentration evolocumab formulations and methods of making the same

Abstract

Formulations of PCSK9-binding polypeptides containing N-acetyl arginine and having reduced viscosity compared to formulations lacking N-acetyl arginine, e.g., formulations containing evolocumab, are provided herein. Additionally, a method for formulating such compositions is provided herein, which is advantageous in that it saves on certain components. Such formulations containing PCSK9-binding polypeptides may be administered to patients to treat and / or prevent PCSK9-related diseases, conditions, and disorders.

Description

Technology Field

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 462,266 filed on February 22, 2017, the entirety of which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application is filed with a sequence list in electronic format. The sequence list is provided with the filename "A-2112-WO-PCT_sequence_listing_ST25.txt", was created on January 31, 2018, and has a size of 21 KB. The information in the electronic format of the sequence list is incorporated herein by reference in its entirety.

[0005] Technology field

[0006] The subject matter presented relates to pharmaceutical compositions of evolocumab and other PCSK9-binding polypeptides and methods for reducing the viscosity of such compositions. Specifically, the subject matter presented relates to pharmaceutical compositions of evolocumab and other PCSK9-binding polypeptides comprising N-acetyl arginine, and the use of N-acetyl arginine for reducing the viscosity of high-concentration evolocumab and other PCSK9-binding polypeptide formulations. Additionally, the disclosed subject matter presents methods for preparing such pharmaceutical compositions. Background Technology

[0007] Therapeutic antibodies are formulated as solutions for administration, such as parenteral injections. For products administered subcutaneously by self-administration, formulations requiring a delivery dose of more than 1 to 2 milliliters are poorly tolerable. To address this problem, antibodies can be formulated at high concentrations (e.g., 70 mg / mL to 210 mg / mL or more), thereby reducing the dosage amount.

[0008] However, some highly concentrated antibody formulations can be difficult to manufacture and administer. For example, evolocumab (REPATHA), a monoclonal antibody that binds to PCSK9. ® In the formulations, evolocumab concentrations of approximately 70 mg / mL or higher have increased viscosity. However, the effective dose of evolocumab is 210 mg Q2W or 420 mg Q4W. High-viscosity formulations are not only difficult to handle during manufacturing, including the bulk and filling stages, but also difficult to draw out and inject with a syringe, making administration to patients difficult and unpleasant.

[0009] To reduce the viscosity of the antibody formulation, unmodified arginine, glycine, serine, or proline amino acids were added to the antibody composition. For example, an antibody formulation containing 80 mg / mL of antibody and 75 mg / mL to about 125 mg / mL of arginine can be lyophilized and reconstituted to 120 to 200 mg / mL; the final arginine concentration can be 431 mM to 718 mM (Morichika & Kameoka, 2007). Arginine reduced the viscosity of the formulation compared to the control (Morichika & Kameoka, 2007). Additionally, the effect of arginine was insufficient to reduce the viscosity of evolocumab to the desired level.

[0010] There is a need in the industry to reduce the viscosity of evolocumab- and other PCSK9-binding polypeptide-containing formulations with compounds more efficient than arginine. The problem to be solved

[0011] In a first embodiment, a pharmaceutical composition comprising the following is provided herein:

[0012] a. PCSK9-binding polypeptides selected from the group consisting of the following:

[0013] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0014] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0016] iii. Monoclonal antibodies comprising the following:

[0017] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0018] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0019] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0020] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0021] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0022] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0023] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0024] and

[0025] b. N-acetyl arginine,

[0026] Herein, the pharmaceutical composition has a viscosity of at least less than about 80 cP. In this first embodiment, the PCSK9-binding polypeptide may be a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0027] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0028] b. Light chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. Additionally, in this first embodiment, the pharmaceutical composition may have a viscosity of at least less than about 50 cP. The pharmaceutical composition may have an osmolality of about 250 to about 400 mOsm / kg, e.g. about 300 mOsm / kg, or is isotonic with human blood cells. The concentration of PCSK9-binding polypeptide may be about 140 mg / mL to about 260 mg / mL, e.g. 210 mg / mL. N-acetylarginine may be present at a concentration of about 25 mM to about 230 mM, e.g. 140 mM to about 170 mM, or 140 mM. The pharmaceutical composition of this embodiment may further comprise a buffer selected from the group consisting of, for example, acetate, glutamate, histidine, and phosphate buffers, or a combination thereof. The buffer may be present at a concentration of about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of about 10 mM. The pH of this pharmaceutical composition may be about 4.8 to about 6.9, for example, about 5.4. The pharmaceutical composition of this embodiment may comprise a surfactant, for example, polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), a polyoxyethylene-polyoxypropylene block copolymer (poloxamer, for example, Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®The composition may further comprise a surfactant selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of about 0.0001% (w / v) to about 1% (w / v). In some pharmaceutical compositions of this embodiment, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v). Additionally, the pharmaceutical composition of this embodiment may further comprise proline, which may be present at a concentration of about 50 mM to about 150 mM, e.g., 90 to 120 mM, or about 120 mM. In some cases, the pharmaceutical composition of this first embodiment may further comprise an arginine salt, which may be present at a concentration of about 25 mM to about 150 mM, for example, about 50 mM to about 100 mM. The arginine salt may be, for example, arginine-HCl, arginine acetate, or arginine glutamate. In some cases, the arginine salt is arginine HCl and is present at a concentration of about 50 mM. The PCSK9-binding polypeptide in the pharmaceutical composition of this first embodiment may be stable for at least about 2 years or even more than 5 years when stored at about -30°C or lower. At 5°C, the PCSK9-binding polypeptide may be stable in this pharmaceutical composition for at least about 6 months to about 24 months. At 25°C, the PCSK9-binding polypeptide may be stable for at least about 1 month, more than 3 months, or even more than 6 months. At 40℃, PCSK9-binding polypeptides can be stable for at least one month.The pharmaceutical composition of this first embodiment may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides in an amount of less than about 3%, for example, 2.5% or less of the total PCSK9-binding polypeptide concentration.

[0029] In a second aspect, a pharmaceutical composition comprising the following is disclosed herein:

[0030] a. PCSK9-binding polypeptides selected from the group consisting of the following:

[0031] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0032] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0033] iii. Monoclonal antibodies comprising the following:

[0034] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0035] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0036] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0037] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0038] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0039] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0040] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0041] and

[0042] b. N-acetyl arginine;

[0043] c. Arginine salt;

[0044] d. buffer; and

[0045] e. Surfactants,

[0046] Here, the pharmaceutical composition has a viscosity of at least about 80 cP or less.

[0047] In this second embodiment, the PCSK9-binding polypeptide is a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0048] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0049] b. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0050] The pharmaceutical composition of this second embodiment may have a viscosity of at least less than about 50 cP. The pharmaceutical composition may have an osmolality of about 250 to about 400 mOsm / kg, for example about 300 mOsm / kg, or is isotonic with human blood cells. The concentration of PCSK9-binding polypeptide may be about 140 mg / mL to about 260 mg / mL, for example 210 mg / mL. N-acetylarginine may be present at a concentration of about 25 mM to about 230 mM, for example 140 mM to about 170 mM, or 140 mM. The pharmaceutical composition of this embodiment may further comprise a buffer selected from the group consisting of, for example, acetate, glutamate, histidine, and phosphate buffers, or a combination thereof. The buffer may be present at a concentration of about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of about 10 mM. The pH of such pharmaceutical compositions may be about 4.8 to about 6.9, for example, about 5.4. Pharmaceutical compositions of this embodiment may contain a surfactant, for example, polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), a polyoxyethylene-polyoxypropylene block copolymer (poloxamer, for example, Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®The composition may further comprise a surfactant selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of about 0.0001% (w / v) to about 1% (w / v). In some pharmaceutical compositions of this embodiment, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v). Additionally, the pharmaceutical composition of this embodiment may further comprise proline; the proline may be present at a concentration of about 50 mM to about 150 mM, e.g., 90 to 120 mM, or about 120 mM. In some cases, the pharmaceutical composition of this second embodiment may further comprise an arginine salt, which may be present at a concentration of about 25 mM to about 150 mM, for example, about 50 mM to about 100 mM. The arginine salt may be, for example, arginine-HCl, arginine acetate, or arginine glutamate. In some cases, the arginine salt is arginine HCl and is present at a concentration of about 50 mM. The PCSK9-binding polypeptide in the pharmaceutical composition of this second embodiment may be stable for at least about 2 years or even more than 5 years when stored at about -30°C or lower. At 5°C, the PCSK9-binding polypeptide may be stable in this pharmaceutical composition for at least about 6 months to about 24 months. At 25°C, the PCSK9-binding polypeptide may be stable for at least about 1 month, more than 3 months, or even more than 6 months. At 40℃, PCSK9-binding polypeptides can be stable for at least one month.The pharmaceutical composition of this second embodiment may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides in an amount of less than about 3%, for example, 2.5% or less of the total PCSK9-binding polypeptide concentration.

[0051] In a third embodiment, a pharmaceutical composition comprising the following is disclosed herein:

[0052] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0053] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0054] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0055] iii. Monoclonal antibodies comprising the following:

[0056] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0057] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0058] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0059] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0060] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0061] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0062] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0063] b. N-acetyl arginine present at a concentration of approximately 140 mM;

[0064] c. Arginine HCl present at a concentration of approximately 50 mM;

[0065] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0066] e. Sodium acetate at a concentration of approximately 10 mM.

[0067] In this third embodiment, the pharmaceutical composition may have a pH of about 5.1 to about 5.7, for example, about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 50 cP.

[0068] In a fourth aspect, a pharmaceutical composition comprising the following is disclosed herein:

[0069] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0070] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0071] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0072] iii. Monoclonal antibodies comprising the following:

[0073] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0074] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0075] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0076] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0077] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0078] 2. Sequence No. 2, the variable region of the light chain of the amino acid sequence,

[0079] and

[0080] b. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0081] c. N-acetyl arginine present at a concentration of approximately 140 mM;

[0082] d. Arginine HCl present at a concentration of approximately 63 mM;

[0083] e. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0084] f. Sodium acetate at a concentration of approximately 10 mM.

[0085] In this fourth embodiment, the pharmaceutical composition may have a pH of about 5.1 to about 5.7, for example, about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 80 cP.

[0086] In a fifth aspect, a pharmaceutical composition comprising the following is disclosed herein:

[0087] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0088] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0089] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0090] iii. Monoclonal antibodies comprising the following:

[0091] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0092] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0093] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0094] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0095] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0096] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0097] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0098] b. N-acetyl arginine present at a concentration of approximately 155 mM;

[0099] c. Arginine HCl present at a concentration of approximately 70 mM;

[0100] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0101] e. Sodium acetate at a concentration of approximately 10 mM.

[0102] In this fifth embodiment, the pharmaceutical composition may have a pH of about 5.1 to about 5.7, for example, about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 45 cP.

[0103] In a sixth aspect, a pharmaceutical composition comprising the following is disclosed herein:

[0104] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0105] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0106] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0107] iii. Monoclonal antibodies comprising the following:

[0108] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0109] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0111] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0112] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0113] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0114] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0115] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0116] b. N-acetyl arginine present at a concentration of approximately 170 mM;

[0117] c. Arginine HCl present at a concentration of approximately 63 mM;

[0118] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0119] e. Sodium acetate at a concentration of approximately 10 mM.

[0120] In this sixth embodiment, the pharmaceutical composition may have a pH of about 5.1 to about 5.7, for example, about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 60 cP.

[0121] In a seventh aspect, a pharmaceutical composition comprising the following is disclosed herein:

[0122] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0123] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0124] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0125] iii. Monoclonal antibodies comprising the following:

[0126] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0127] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0129] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0130] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0131] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0132] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0133] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0134] b. N-acetyl arginine present at a concentration of approximately 155 mM;

[0135] c. Proline present at a concentration of approximately 120 mM;

[0136] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0137] e. Sodium acetate at a concentration of approximately 10 mM.

[0138] In this seventh embodiment, the pharmaceutical composition may have a pH of about 5.1 to about 5.7, for example, about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 60 cP.

[0139] In these third to seventh embodiments, the PCSK9-binding polypeptide may be stable in the pharmaceutical composition of these embodiments for at least about 2 years or even 5 years when stored at about -30°C or lower. At 5°C, the PCSK9-binding polypeptide may be stable in these pharmaceutical compositions for at least about 6 months to about 24 months. At 25°C, the PCSK9-binding polypeptide may be stable for at least about 1 month, 3 months, or even 6 months. At 40°C, the PCSK9-binding polypeptide may be stable for at least 1 month. The pharmaceutical composition of these embodiments may contain high molecular weight aggregates or oligomers of the PCSK9-binding polypeptide in an amount of less than about 3%, for example 2.5% or less of the total PCSK9-binding polypeptide concentration.

[0140] In any of the aforementioned embodiments, the pharmaceutical composition may be a liquid.

[0141] In the eighth embodiment, a method for treating a subject in need thereof is disclosed herein, comprising the step of administering any of the seven embodiments described above.

[0142] In a ninth embodiment, a kit comprising any of the pharmaceutical compositions of the first to seventh embodiments and a delivery device is disclosed herein. The delivery device may be selected from the group consisting of a syringe, an injection pen, a body syringe, and a self-injector. The kit may further include instructions for administering the pharmaceutical composition using the delivery device.

[0143] In a 10th embodiment, a method for preparing a pharmaceutical composition of a PCSK9-binding polypeptide comprising at least 140 mg / mL of PCSK9-binding polypeptide is disclosed herein, comprising the step of adding an effective amount of N-acetyl arginine to a pharmaceutical composition comprising a PCSK9-binding polypeptide such that the viscosity of the pharmaceutical composition is reduced compared to a pharmaceutical composition lacking N-acetyl arginine, wherein the PCSK9-binding polypeptide is selected from the group consisting of the following:

[0144] a. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0145] b. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0146] c. Monoclonal antibodies containing the following:

[0147] i. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0148] ii. Light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0149] d. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238; and

[0150] e. A monoclonal antibody that binds to PCSK9 at an epitope in PCSK9 that overlaps with the epitope bound by the antibody comprising the following:

[0151] i. The variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0152] ii. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0153] iii. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR.

[0154] In this tenth embodiment, the viscosity of the pharmaceutical composition is less than about 80 cP, or less than about 50 cP. The pharmaceutical composition of the method of this embodiment may have an osmolality of about 250 to about 400 mOsm / kg, for example about 300 mOsm / kg, or is isotonic with human blood cells. The concentration of PCSK9-binding polypeptide may be about 140 mg / mL to about 260 mg / mL, for example 210 mg / mL. N-acetyl arginine may be present at a concentration of about 25 mM to about 230 mM, for example 140 mM to about 170 mM, or 140 mM. The pharmaceutical composition of this embodiment may further comprise a buffer selected from the group consisting of, for example, acetate, glutamate, histidine, and phosphate buffers, or a combination thereof. The buffer may be present at a concentration of about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of about 10 mM. The pH of this pharmaceutical composition may be about 4.8 to about 6.9, for example, about 5.4. The pharmaceutical composition of this embodiment may contain a surfactant, for example, polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), a polyoxyethylene-polyoxypropylene block copolymer (poloxamer, for example, Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®The composition may further comprise a surfactant selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of about 0.0001% (w / v) to about 1% (w / v). In some pharmaceutical compositions of this embodiment, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v). Additionally, the pharmaceutical composition of this embodiment may further comprise proline; the proline may be present at a concentration of about 50 mM to about 150 mM, e.g., 90 to 120 mM, or about 120 mM. In some cases, the pharmaceutical composition of this 10th embodiment may further comprise an arginine salt, which may be present at a concentration of about 25 mM to about 150 mM, for example, about 50 mM to about 100 mM. The arginine salt may be, for example, arginine-HCl, arginine acetate, or arginine glutamate. In some cases, the arginine salt is arginine HCl and is present at a concentration of about 50 mM. The PCSK9-binding polypeptide in the pharmaceutical composition of this 10th embodiment may be stable for at least about 2 years or even more than 5 years when stored at about -30°C or lower. At 5°C, the PCSK9-binding polypeptide may be stable in this pharmaceutical composition for at least about 6 months to about 24 months. At 25°C, the PCSK9-binding polypeptide may be stable for at least about 1 month, more than 3 months, or even more than 6 months. At 40℃, PCSK9-binding polypeptides can be stable for at least one month.The pharmaceutical composition of this 10th embodiment may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides in an amount of less than about 3%, for example, 2.5% or less of the total PCSK9-binding polypeptide concentration.

[0155] In the eleventh aspect, a method for formulating a therapeutic polypeptide is disclosed herein, comprising:

[0156] a. A first concentration step in which polypeptides are concentrated in a first solution;

[0157] b. A first solution exchange step in which a polypeptide concentrated in a first solution is exchanged with a second solution containing N-acetyl arginine using dialysis filtration;

[0158] c. A second concentration step in which polypeptides are concentrated in the second solution;

[0159] d. A second solution exchange step in which the polypeptide in the concentrated second solution is exchanged with a third solution containing N-acetyl arginine using dialysis filtration; and

[0160] e. A third concentration step in which polypeptides are concentrated in the third solution;

[0161] Herein, the therapeutic polypeptide blocks the binding of PCSK9 to LDLR and comprises a PCSK9-binding polypeptide selected from the group consisting of the following:

[0162] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0163] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0164] iii. Monoclonal antibodies comprising the following:

[0165] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0166] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0167] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0168] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0169] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0170] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0171] 3. Here, the epitope of the monoclonal antibody additionally overlaps with the site in the EGFa domain of LDLR.

[0172] In the method of this eleventh embodiment, the PCSK9-binding polypeptide that blocks the binding of PCSK9 to LDLR may be a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0173] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0174] b. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0175] Additionally, in this eleventh embodiment, prior to the third concentration step, the temperature of the solution containing the polypeptide may be increased from about 25°C to about 37°C. Also, the first solution exchange step may be achieved using at least 3 diavolumes of the second solution. In some sub-embodies of this eleventh embodiment, the second solution exchange step is achieved using at least 4 diavolumes of the third solution. In other sub-embodies, the initial concentration of the therapeutic protein is about 11 mg / mL or less. Additionally, the therapeutic polypeptide concentration may be increased by about 3 to about 7 times, for example, when the increased concentration of the polypeptide is about 35 mg / mL to about 70 mg / mL. In some sub-embodies, in the second concentration step, the therapeutic polypeptide concentration is increased by about 2 to 4 times from the first concentration step, for example, to about 140 mg / mL. In the third concentration step, the therapeutic polypeptide concentration may be increased by about 1.5 to about 2 times from the second concentration step, such as about 260 mg / mL. Thus, the therapeutic polypeptide may have a final concentration that is at least about 19 to 20 times more concentrated than the initial therapeutic polypeptide concentration, such as about 210 mg / mL. The concentration step may include fed-batch ultrafiltration; additionally, the second solution and the third solution may be identical. For example, the second or third solution containing N-acetyl arginine may contain an arginine salt and a buffer, wherein, for example, N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM; the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and is present at a concentration of about 25 mM to about 150 mM; The buffer is sodium acetate buffer at a concentration of about 5 mM to about 30 mM.In another sub-modality, N-acetyl arginine is present at a concentration of about 140 to about 170 mM; Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 to about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In yet another sub-modality, N-acetyl arginine is present at a concentration of about 140 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In a further sub-modality, N-acetyl arginine is present at a concentration of about 155 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In another sub-module, N-acetyl arginine is present at a concentration of about 170 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM. Additionally, the composition may further comprise proline, wherein proline is present at a concentration of about 50 mM to about 150 mM. The second or third solution may have a pH of about 4.8 to about 6.9, e.g., 5.4. In the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following may be used:

[0176] a. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0177] b. An opening area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

[0178] c. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0179] d. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0180] e. Standard weight exceeding approximately 160 g / ㎡ but 180 g / ㎡ or less;

[0181] f. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0182] g. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0183] h. Maximum supply pressure of approximately 60 psi.

[0184] Additionally, surfactants, e.g., polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ® A surfactant selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS) may be added to the third solution after the third solution has been concentrated. The surfactant may be present at a concentration of about 0.0001% (w / v) to about 1% (w / v). In some pharmaceutical compositions of this embodiment, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v).

[0185] In the 12th aspect, a method for formulating a therapeutic polypeptide is disclosed herein, comprising the following:

[0186] a. A first concentration step in which polypeptides in a first solution are concentrated using fed-batch ultrafiltration;

[0187] b. A first solution exchange step in which the polypeptide concentrated in the first solution is exchanged with a second solution containing N-acetyl arginine, an arginine salt, and a buffer using dialysis filtration and three dialysis volumes of the second solution;

[0188] c. A second concentration step in which polypeptides in the second solution are concentrated using fed-batch ultrafiltration;

[0189] d. A second solution exchange step in which the polypeptide in the concentrated second solution is exchanged with a third solution containing N-acetyl arginine, an arginine salt, and a buffer using dialysis filtration and 4 dialysis volumes of the third solution;

[0190] e. A step in which the temperature of the solution containing the polypeptide increases from about 25°C to about 37°C after the second solution exchange step; and

[0191] f. A third concentration step in which the polypeptide is further concentrated using fed-batch ultrafiltration concentration;

[0192] Here, in the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following is used:

[0193] a. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0194] b. An opening area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

[0195] c. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0196] d. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0197] e. Standard weight exceeding approximately 160 g / ㎡ but 180 g / ㎡ or less;

[0198] f. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0199] g. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0200] h. Maximum supply pressure of approximately 60 psi;

[0201] Herein, the therapeutic polypeptide blocks the binding of PCSK9 to LDLR and comprises a PCSK9-binding polypeptide selected from the group consisting of the following:

[0202] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0203] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0204] iii. Monoclonal antibodies comprising the following:

[0205] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0206] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0207] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0208] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0209] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0210] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0211] 3. Here, the epitope of the monoclonal antibody additionally overlaps with the site in the EGFa domain of LDLR.

[0212] Additionally, in this 12th embodiment, the first solution exchange step may be achieved using at least 3 dialysis volumes of the second solution. In some sub-embodies of this 12th embodiment, the second solution exchange step is achieved using at least 4 dialysis volumes of the third solution. In other sub-embodies, the initial concentration of the therapeutic protein is about 11 mg / mL or less. Additionally, the therapeutic polypeptide concentration may be increased by about 3 to about 7 times, for example, when the increased concentration of the polypeptide is about 35 mg / mL to about 70 mg / mL. In some sub-embodies, in the second concentration step, the therapeutic polypeptide concentration is increased by about 2 to 4 times from the first concentration step, for example, to about 140 mg / mL. In the third concentration step, the therapeutic polypeptide concentration may be increased by about 1.5 to about 2 times from the second concentration step, for example, to about 260 mg / mL. Accordingly, the therapeutic polypeptide may have a final concentration that is at least about 19 to 20 times more concentrated than the initial therapeutic polypeptide concentration, such as about 210 mg / mL. The concentration step may include fed-batch ultrafiltration; additionally, the second solution and the third solution may be identical. N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM; the arginine salt may be Arg HCl, Arg acetate, or Arg glutamate, wherein Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 25 mM to about 150 mM; and the buffer is sodium acetate buffer at a concentration of about 5 mM to about 30 mM. In another sub-modality, N-acetyl arginine is present at a concentration of about 140 to about 170 mM; Arg HCl, Arg acetate, or Arg glutamate are present at a concentration of about 63 to about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM.In another sub-modality, N-acetyl arginine is present at a concentration of about 140 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In a further sub-modality, N-acetyl arginine is present at a concentration of about 155 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In yet another sub-modality, N-acetyl arginine is present at a concentration of about 170 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM. Additionally, the composition may further comprise proline, wherein proline is present at a concentration of about 50 mM to about 150 mM. The second or third solution may have a pH of about 4.8 to about 6.9, for example, 5.4. Additionally, a surfactant, for example, polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), a polyoxyethylene-polyoxypropylene block copolymer (poloxamer, for example, Pluronic). ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®A surfactant selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS) may be added to the third solution after the third solution has been concentrated. The surfactant may be present at a concentration of about 0.0001% (w / v) to about 1% (w / v). In some pharmaceutical compositions of this embodiment, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v).

[0213] In the 13th aspect, a method for formulating a therapeutic polypeptide is disclosed herein, comprising:

[0214] a. A first concentration step in which polypeptides in a first solution are concentrated using fed-batch ultrafiltration;

[0215] b. A first solution exchange step in which the polypeptide concentrated in the first solution is exchanged to the second solution using dialysis filtration and three dialysis volumes of the second solution;

[0216] c. A second concentration step in which polypeptides in the second solution are concentrated using fed-batch ultrafiltration;

[0217] d. A second solution exchange step in which polypeptides in the concentrated second solution are exchanged with the third solution using dialysis filtration and 4 dialysis volumes of the third solution;

[0218] e. A step in which the temperature of the solution containing the polypeptide increases from about 25°C to about 37°C after the second solution exchange step; and

[0219] f. A third concentration step in which the polypeptide is further concentrated using fed-batch ultrafiltration concentration;

[0220] g. Alternatively, a step of adding polyoxyethylene sorbitan monooleate (polysorbate 80) to the product solution of the third concentration step at a concentration of about 0.01% (w / v).

[0221] Herein, the second and third solutions comprise a solution selected from the group consisting of: a solution containing about 140 mM N-acetylarginine, about 50 mM Arg HCl, and about 10 mM sodium acetate and having a pH of about 5.2; a solution containing about 155 mM N-acetylarginine, about 70 mM Arg HCl, and about 10 mM sodium acetate and having a pH of about 5.4; and a solution containing about 170 mM N-acetylarginine and about 10 mM sodium acetate and having a pH of about 5.6;

[0222] Here, in the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following is used:

[0223] a. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0224] b. An opening area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

[0225] c. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0226] d. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0227] e. Standard weight exceeding approximately 160 g / ㎡ but 180 g / ㎡ or less;

[0228] f. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0229] g. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0230] h. Maximum supply pressure of approximately 60 psi;

[0231] Herein, the therapeutic polypeptide blocks the binding of PCSK9 to LDLR and comprises a PCSK9-binding polypeptide selected from the group consisting of the following:

[0232] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0233] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0234] iii. Monoclonal antibodies comprising the following:

[0235] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0236] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0237] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0238] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0239] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0240] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0241] 3. Here, the epitope of the monoclonal antibody additionally overlaps with the site in the EGFa domain of LDLR. means of solving the problem

[0242] The inventors have surprisingly discovered that N-acetyl arginine (NAR), a derivative of arginine, reduces the viscosity of pharmaceutical compositions containing evolocumab at high concentrations (greater than 100 mg / mL, e.g., 140 mg / mL or more) more effectively than non-acetylated arginine. While pharmaceutical compositions with a viscosity of 50 cP or less are easy to manufacture and administer to patients without significant problems, formulations with higher viscosity are difficult to handle (e.g., when filling syringes) and difficult to administer. While unmodified arginine is known to reduce the viscosity of high-concentration protein formulations, arginine glutamate reduced the viscosity of a similar proline-containing evolocumab (210 mg / mL) formulation by only 50 cP (from 159 cP to 109 cP), which is significantly higher than the target of 50 cP or less. Additionally, the addition of arginine monohydrochloride (Arg HCl) alone did not achieve this goal, reducing the viscosity of the proline-containing formulation from 159 cP to 70 cP. However, NAR achieved a remarkable effect in further reducing viscosity to 58 cP (a reduction of over 101 cP compared to the proline formulation), and when combined with Arg HCl (to increase NAR solubility and achieve an isotonic formulation), a viscosity below 50 cP was achieved; in fact, exceeding the target by 7 cP, the formulation had a viscosity of 43 cP. See Figure 2. Since NAR solubility is limited to less than 230 mM, other excipients are required to achieve an isotonic formulation for subcutaneous administration. The unexpected finding that arginine chloride salts are more effective at reducing the viscosity of evolocumab than other arginine salts such as glutamate was crucial for minimizing formulation viscosity.

[0243] Another unexpected finding was the pH-dependent stability and viscosity effects of evolocumab in the presence of NAR and arginine HCl. A significant increase in evolocumab aggregation rate was observed at elevated temperatures below pH 5.0. Additionally, a pH-dependent decrease in viscosity was observed when the pH increased from 5.1 to 6.9.

[0244] The inventors have further discovered that a two-step ultrafiltration / dialysis filtration (UF / DF) process can produce pharmaceutical formulations of such NAR-containing, high evolocumab concentrations with significantly reduced NAR material compared to a conventional one-step process. Since NAR is about 10 times more expensive than Arg HCl, significant cost savings can be achieved by this method.

[0245] definition

[0246] The preceding general description and the following detailed description are illustrative and for illustrative purposes only and are not restrictive. The use of the singular form includes the plural form unless specifically stated otherwise. The use of "or" means "and / or" unless otherwise stated. The use of the term "containing," as well as other forms such as "include" and "included," is not restrictive. Terms such as "element" or "component" encompass both elements and components containing one unit and elements and components containing more than one subunit, unless specifically stated otherwise. The use of the term "part" may include a part of a moiety or the entire moiety. When a numerical range, e.g., 1 through 5, is referred to, it explicitly includes all values ​​in between, such as 1, 2, 3, 4, and 5, as well as decimals thereof, such as 1.5, 2.2, 3.4, and 4.1.

[0247] When “about” or “~” is used to modify a quantity (e.g., “about” 3 mM), it implies that there may be deviations around the modified quantity. These deviations may be caused by various factors such as typical measurement and processing procedures, unintended errors, and ingredient purity.

[0248] "N-acetyl arginine (NAR) refers to the molecule of chemical formula 1.

[0249] [Chemical Formula 1]

[0250]

[0251] In the context of a pharmaceutical composition, an "additive" is not a naturally occurring part of a substance (e.g., a drug substance) but rather for a specific purpose (e.g. , It refers to a substance intentionally added to satisfy preservation, viscosity reduction, and stabilization.

[0252] "An analogue" refers to an amino acid sequence that has insertions, deletions, or substitutions compared to a parent sequence but substantially retains the biological activity of the parent sequence as determined using biological analysis. Analogues include polypeptides having modified glycosylation and polypeptides without glycosylation. Formulations may also include analogue polypeptides chemically modified to attach, for example, water-soluble polymers (e.g., PEGylated), radionuclides, or other diagnostic, targeted, or therapeutic moiety, or derivatives of naturally occurring polypeptides.

[0253] "Antibody" refers to any isoform intact immunoglobulin, including, for example, chimeric, humanized, human, and bispecific antibodies. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains. The antibody sequence may be derived from only a single species or may be "chimeric," that is, different parts of the antibody may be derived from two different species. "Antibody" also comprises an antibody comprising two substantial full-length heavy chains and two substantial full-length light chains, provided that this antibody possesses the same or similar binding and / or function as an antibody consisting of two full-length light chains and heavy chains. For example, antibodies having 1, 2, 3, 4, or 5 amino acid residue substitutions, insertions, or deletions at the N-terminus and / or C-terminus of a heavy chain and / or light chain are included in the definition, provided that these antibodies possess the same or similar binding and / or function as antibodies comprising two full-length heavy chains and two full-length light chains. Antibodies include, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies.

[0254] A typical antibody structural unit comprises a tetramer. Each of these tetramers typically consists of two identical pairs of polypeptide chains, each pair having one full-length light chain (about 25 kDa) and one full-length heavy chain (about 50 to 70 kDa). The amino-terminal end of each chain typically contains a variable region of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxy-terminal end of each chain typically defines a constant region capable of performing effector functions. Light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and the isoforms of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG antibodies have several subclasses, including IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including IgM1 and IgM2. IgA is similarly subdivided into subclasses including IgA1 and IgA2. Within the full-length light and heavy chains, the variable and constant regions are typically connected by a “J” region of about 12 or more amino acids, and the heavy chain also includes a “D” region of about 10 or more amino acids. The variable region of each light / heavy chain pair typically forms an antigen-binding site.

[0255] The variable region typically exhibits the same general structure as a relatively conserved framework region (FR) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework region, which can enable binding with specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain typically follow Kabat (Kabat, Wu, Perry, Gottesman, & Foeller, 1991; Kabat, Wu, Reid-Miller, Perry, & Gottesman, 1987) or Chothia (Chothia & Lesk, 1987; Chothia et al., 1989).

[0256] Instead of full-length antibodies, "fragments" or "antigen-binding fragments" of antibodies may be used. "Antibody fragments" refer to Fab, Fab', F(ab')2, and Fv fragments containing at least one CDR of immunoglobulin sufficient to confer specific antigen binding to a target protein such as PCSK9.

[0257] The antibody heavy chain can bind to an antigen in the absence of the antibody light chain. The antibody light chain can bind to an antigen in the absence of the antibody heavy chain. The antibody binding domain can bind to an antigen in the absence of the antibody light chain. The antibody binding domain can bind to an antigen in the absence of the antibody heavy chain. Individual variable domains can specifically bind to an antigen in the absence of other variable domains.

[0258] In the heavy chain, the CDR regions are typically designated as H1, H2, and H3 and are numbered sequentially from the amino terminus to the carboxyl terminus. In the light chain, the CDR regions are designated as L1, L2, and L3 and are numbered sequentially from the amino terminus to the carboxyl terminus.

[0259] The term "light chain" includes a full-length light chain and a fragment thereof having a variable region sequence sufficient to confer binding specificity. The full-length light chain includes a variable region domain VL and an invariant region domain CL. The variable region domain of the light chain is located at the amino-terminus of the polypeptide. The light chain includes a kappa chain and a lambda chain.

[0260] The term “heavy chain” includes the full-length heavy chain and fragments thereof having variable region sequences sufficient to confer binding specificity. The full-length heavy chain includes a variable region domain VH and three constant region domains CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, the CH domain is at the carboxyl-terminus, and CH3 is closest to the carboxyl-terminus of the polypeptide. The heavy chain may be any isoform including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0261] Each individual immunoglobulin chain typically consists of several "immunoglobulin domains," each composed of approximately 90 to 110 amino acids and possessing a characteristic folding pattern. These domains are the basic units of antibody polypeptides. In humans, IgA and IgD isoforms contain four heavy chains and four light chains; IgG and IgE isoforms contain two heavy chains and two light chains; and IgM isoforms contain five heavy chains and five light chains. The heavy chain C region typically contains one or more domains capable of performing effector functions. The number of heavy chain constant region domains depends on the isoform. For example, the IgG heavy chain contains three C region domains known as CH1, CH2, and CH3. In certain cases, the anti-PCSK9 antibody is an IgG1, IgG2, or IgG4 subtype.

[0262] The term “variable region” or “variable domain” refers to a portion of the light chain and / or heavy chain of an antibody, typically comprising approximately 120 to 130 amino-terminal amino acids in the heavy chain and about 100 to 110 amino-terminal amino acids in the light chain. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

[0263] "Antigen" means a molecule or part of a molecule that can be bound by a selective binding agent (e.g., including an antibody or a binding fragment thereof), such as a PCSK9-binding polypeptide. In some cases, an antigen may be used in animals to produce an antibody that can bind to that antigen. An antigen may possess one or more epitopes that can interact with different PCSK9-binding polypeptides.

[0264] "Arginine salts" refers to salts of arginine. Examples include arginine monohydrochloride (Arg HCl), arginine acetate (Arg acetate), and arginine glutamate (Arg glutamate).

[0265] "Buffer" means any pharmaceutically acceptable buffer, including acetate, glutamate, histidine, and phosphate buffers, and salts thereof.

[0266] When used in the context of antibodies competing for the same epitope, "compete" refers to competition between antibodies in which the antibody under test prevents or inhibits (e.g., reduces) the specific binding of a reference antibody (e.g., ligand, or reference antibody) to a common antigen (e.g., PCSK9 or a fragment thereof), as determined by the analysis. To determine whether one antibody competes with another, many types of competitive binding analyses may be used, e.g., solid-phase direct or indirect immunoassays using various industry-accepted reagents and labels. Competitive inhibition is measured by determining the amount of label that binds to a solid surface or cell in the presence of the test antibody. Usually, the test antibody is present in excess. Antibodies identified by the competitive analysis include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody to cause steric hindrance. Usually, when a competitive antibody is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antibody to the common antigen by at least 40 to 45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, or 75% or more. In some cases, binding is inhibited by at least 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, or 97% or more.

[0267] "Diafiltration," "DF," and similar terms refer to the use of an ultrafiltration membrane (i.e., a semipermeable membrane capable of distinguishing molecules of different shapes and sizes) to remove, replace, or reduce the concentration of salts or solvents from a solution or mixture containing, for example, polypeptides or other biomolecules.

[0268] "Diavolume (DV)" refers to the volume of dialysis buffer introduced in a unit operation compared to the volume of the dialysis fluid in the context of filtration.

[0269] An "epitope" comprises any determinant that can be bound by a PCSK9-binding polypeptide, such as an antibody. The epitope is a region of the antigen bound by a PCSK9-binding polypeptide targeting that antigen, and when the antigen is a protein, it comprises a specific amino acid that comes into direct contact with the PCSK9-binding polypeptide. The epitope determinant may comprise chemically active surface groups of the molecule, such as amino acids, sugar side chains, phosphoryl groups, and sulfonyl groups, and may possess specific three-dimensional structural features and specific charge features. Generally, antibodies specific to a specific target antigen preferentially recognize epitopes on the target antigen in a mixture of complexes of proteins or other macromolecules.

[0270] "Excipient" refers to a somewhat inert substance added to a prescription as a diluent or vehicle, or to provide form or viscosity when a therapeutic drug is administered in tablet form; for example, it means single-ingredient syrups, vegetable gums, aromatic powders, honey, and various elixirs.

[0271] "Supply cross-flow" refers to the supply flow rate (L / hour) divided by the membrane area (m²).

[0272] "Flux (LMH)" in the context of filtration refers to the volume of liters per hour per square meter of membrane area (L / h / ㎡).

[0273] "High molecular weight species" or "HMW species" refers to a therapeutic protein larger than the original therapeutic polypeptide, as determined by field-acceptance analysis, in the context of pharmaceutical formulations containing a therapeutic polypeptide. HMW species include oligomers of the therapeutic polypeptide and aggregates of the therapeutic polypeptide.

[0274] "Holdup volume (HUV)" refers to the line capacity of a TFF system, including cartridges, in the context of filtration.

[0275] “Identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by the alignment and comparison of sequences. “Identity percentage” refers to the percentage of identical residues between amino acids or nucleotides in the molecules being compared and is calculated based on the minimum size of the molecules being compared. In these calculations, gaps in alignment (if any) are preferably addressed by a specific mathematical model or algorithm. These techniques are well known in the art.

[0276] In calculating the percentage of identity, the sequences being compared are typically aligned to maximize the largest match between the sequences.

[0277] A specific alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and even if there is no significant relationship between the two full-length sequences, this small aligned region may have very high sequence identity. Therefore, the selected alignment method can be adjusted to result in alignment covering a desired number of adjacent amino acids (e.g., 50 amino acids) of the target polypeptide if desired.

[0278] Twenty common amino acids, non-natural amino acids, e.g., α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and stereoisomers of other non-conventional amino acids (e.g., D-amino acids) may be suitable components of the PCSK9-linked polypeptide. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).

[0279] Conserved amino acid substitutions can encompass non-naturally existing amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimics and other inverted or reversed forms of amino acid moiety.

[0280] In making modifications to antigen-binding proteins or PCSK9 proteins, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobic and charge characteristics. The importance of amino acid hydropathic indices in conferring interacting biological functions within proteins is understood in the art. A specific amino acid can be substituted with another amino acid having a similar hydropathic index or score and can still retain similar biological activity.

[0281] Substitution of similar amino acids can be effectively achieved based on hydrophilicity. Additionally, epitopes can be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0282] "Low molecular weight species" or "LMW species" refers to a polypeptide smaller than the original therapeutic polypeptide, as determined by field-acceptance analysis, in the context of pharmaceutical formulations containing a therapeutic polypeptide. LMW species include fragments of the therapeutic polypeptide.

[0283] "Neutralizing antibody" or "antibody that neutralizes a target," as used in "anti-PCSK9 neutralizing antibody," refers to an antibody that binds to a target and prevents or reduces the biological activity of that target. This can be accomplished, for example, by directly blocking a binding site on the target, or by binding to the target and altering its binding ability through indirect means, for example, structural or energetic changes in the target. In evaluating the binding and / or specificity of an antibody or its immunological functional fragment, when an excess of antibody reduces the amount of binding partners binding to the ligand (as measured by an in vitro competitive binding assay) by at least about 1 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, 97 to 98%, 98 to 99% or more, the antibody or fragment may substantially inhibit the target from binding to its binding partners. In the case of a PCSK9 antibody, such neutralizing molecules may attenuate the ability of PCSK9 to bind to LDLR. In some cases, neutralizing ability is characterized or described through a competitive assay. In some cases, neutralizing ability is described in terms of IC50 or EC50 values. In some cases, the antibody neutralizes PCSK9 by binding to it and preventing PCSK9 from binding to LDLR, or by reducing PCSK9's ability to bind to LDLR. In some cases, the antibody neutralizes PCSK9 by binding to it, thereby preventing or reducing PCSK9-mediated degradation of LDLR while allowing PCSK9 to still bind to LDLR. Thus, in some cases, the neutralizing antibody may still allow PCSK9 / LDLR binding, but prevents or reduces subsequent PCSK9-involved degradation of LDLR. Neutralization results in a reduction of LDL-C (and / or other lipids, e.g., ApoB, Lp(a), etc.).PCSK9-binding polypeptides beyond antibodies, including variants of these PSK9-binding polypeptides, can have the same activity.

[0284] "PCSK9-binding polypeptide" refers to a polypeptide that binds to the proprotein convertase subtilisin / kexin type 9 (PCSK9) protein. In some cases, PCSK9-binding polypeptides block the binding of PCSK9 to the low-density lipid receptor (LDLR). The PCSK9-binding polypeptides that block in this way may be monoclonal antibodies (mAbs) and may be one of the following:

[0285] a. An mAb (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0286] b. An mAb competing with evolocumab for binding to PCSK9;

[0287] c. mAb containing the following:

[0288] i. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0289] ii. Light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0290] d. mAb binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ No. 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0291] e. mAb binding to PCSK9 at an epitope in PCSK9 that overlaps with the epitope bound by the antibody including the following:

[0293] i. The variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0294] ii. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0295] iii. The epitope of the mAb further overlaps with a site in the epidermal growth factor-like repeat A (EGF-A) domain of LDLR; or

[0296] f. mAb comprising a heavy chain polypeptide containing the following complementarity determining region (CDR):

[0297] i. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0298] ii. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0299] The indicated amino acid sequences are presented in Table 1, which also presents the heavy chain variable region and light chain variable region of evolocumab. The full-length nucleotide sequences of the evolocumab heavy and light chains are provided in Table 2, along with the nucleotide sequences for evolocumab HCVR and LCVR.

[0300]

[0301]

[0302]

[0304]

[0305] Evolocumab is CAS registration number 1256937-27-5.

[0306] Variants of evolocumab that do not affect PCSK9-binding and inhibitory properties are shown in Table 3.

[0307]

[0308] "Pharmaceutical composition" or "pharmaceutical formulation," etc., means a generally sterile composition of a pharmaceutically active drug, such as a biologically active protein (e.g., PCSK9-binding polypeptide), suitable for administration to a subject requiring it, e.g., parenteral administration (including intravenous, intramuscular, subcutaneous, nebulized, pulmonary, nasal, or spinal), and comprises only pharmaceutically acceptable excipients, diluents, and other additives considered safe by the Federal Drug Administration or other foreign national authorities. A pharmaceutical formulation is a liquid that can be administered directly, e.g. It includes an aqueous solution and a freeze-dried powder that can be reconstituted into a solution by adding a diluent before administration.

[0309] “Polypeptide” or “protein” means a macromolecule having the amino acid sequence of the original protein, i.e., a protein produced by naturally occurring cells and non-recombinant cells; or a protein produced by genetically engineered cells or recombinant cells, and includes molecules having the amino acid sequence of the original protein, or molecules having deletions, additions, and / or substitutions thereof from one or more amino acids of the original sequence. The term also encompasses amino acid polymers, which are chemical analogs of naturally occurring amino acids and polymers in which one or more amino acids correspond. “Polypeptide fragment” refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to the original full-length protein. Such fragments may also contain amino acids modified compared to the original protein. Fragments may be about 5 to 500 amino acid lengths. For example, the fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. A useful polypeptide fragment comprises an immunologically functional fragment of an antibody that includes a binding domain. In the case of a PCSK9-binding antibody, a useful fragment comprises, but is not limited to, a CDR region, a variable domain of the heavy chain and / or light chain, a part of the antibody chain, or a variable region thereof that includes two CDRs.

[0310] "Preventing" (such as the onset of symptoms, disease, or disorder) does not require the elimination of the possibility of an event. Rather, it indicates that the possibility of the event occurring has been reduced in the presence of the compound or method. In a therapeutic sense, prevention includes preventive measures and applications that reduce the risk of a disorder or other risk factors developing in the subject.

[0311] "Stable pharmaceutical formulation," "stable formulation," or "pharmaceutical formulation is stable" refers to a pharmaceutical formulation of PCSK9-binding polypeptide that exhibits a loss of reduced biological activity of 5% or less and / or limited increase in aggregation compared to a control formulation sample when stored at about -30°C (or below) to about 5°C to about 40°C for at least 1 month, or 2 months, or 3 months, or 6 months, or 1 year, or 2 years, or 5 years or more. Formulation stability may be determined by a person skilled in the art using any number of standard analyses, including size-exclusion HPLC (SEC-HPLC), cation-exchange HPLC (CEX-HPLC), detection of particles invisible to the naked eye under light shielding (“HIAC”), and / or visual inspection. Typically, the shelf life of the formulation decreases as the storage temperature increases.

[0312] Techniques for evaluating degradation vary depending on the identity of the protein in the pharmaceutical formulation. Exemplary techniques include, for example, size-exclusion chromatography (SEC)-HPLC for detecting aggregation; reverse phase (RP)-HPLC for detecting protein fragmentation; ion exchange-HPLC for detecting changes in the charge of the protein; mass spectrometry; fluorescence spectroscopy; circular dichroism (CD) spectroscopy; Fourier transform infrared spectroscopy (FT-IR); and Raman spectroscopy for detecting changes in protein conformation. All of these techniques may be used alone or in combination to evaluate the degradation of the protein in the pharmaceutical formulation and to determine the shelf life of the formulation. The pharmaceutical formulations disclosed herein typically exhibit an increase in degradation (e.g., fragmentation, aggregation, or unraveling) of about 2% to about 3% or less over two years when stored at 2 to 8°C.

[0313] "Subject" or "patient" is used interchangeably and includes human and non-human animal subjects, as well as those with officially diagnosed disabilities, those without officially recognized disabilities, those under medical attention, and those at risk of developing disabilities.

[0314] "Surfactant" means a surface-active agent and includes substances commonly referred to as wetting agents, interfacial tension inhibitors, detergents, dispersants, emulsifiers, and quaternary ammonium disinfectants. Surfactants are discussed further below.

[0315] "Tangential flow filtration," or "TFF," refers to a process in which a solution passes tangentially across an ultrafiltration membrane (i.e., a semipermeable membrane capable of distinguishing molecules of different sizes and shapes) through which low molecular weight salts and / or solutes pass under pressure.

[0316] "Therapeutic effective dose" refers to the amount of PCSK9 antigen-binding polypeptide determined to produce a therapeutic response in a subject. Such therapeutic effective dose is readily identified by those skilled in the art.

[0317] "Trans Membrane Pressure (TMP)" refers to the average pressure difference from the feed side to the filtrate side of the membrane in the context of filtration and can be expressed by Equation 1:

[0318] [Equation 1]

[0319]

[0320] "To treat" and "to provide treatment" include providing therapeutic interventions. Treatment encompasses cases where symptoms or inherent risk factors are reduced without requiring a complete cure of the disorder.

[0321] "Ultrafiltration," "ultrafiltration," "UF," and similar terms refer to the use of a semipermeable membrane that distinguishes molecules of different shapes and sizes to separate molecules from other molecules or to concentrate similar or substantially identical molecules.

[0322] A "variant" of PCSK9-binding refers to an amino acid sequence in which one or more amino acid residues are inserted, deleted, or / or substituted compared to other polypeptide sequences. Variants include fusion proteins.

[0323] "Viscosity" refers to the resistance to fluid flow, measured in centipoise (cP) or millipascal-seconds (mPa). · It can be measured in units of s), where 1 cP = 1 mPa·s at a given shear rate. Viscosity can be measured using a viscometer, e.g., a Brookfield Engineering (Middleboro, MA) dial reading viscometer, or a flowmeter, e.g. m -VROC TM Viscosity may be measured using a flow meter or a TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter. Viscosity may be measured using any other method known in the art and in any other unit (e.g., absolute, kinematic, or dynamic viscosity). Regardless of the method used to determine viscosity, the percentage reduction in viscosity between the excipient formulation and the control formulation will remain largely the same at a given shear rate.

[0324] Components of the composition and method

[0325] PCSK9-binding polypeptide

[0326] PCSK9-binding polypeptides including evolocumab are well described (Chan et al., 2012).

[0327] The pre-protein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of low-density lipoprotein receptor (LDLR) protein (Horton, Cohen, & Hobbs, 2007; Seidah & Prat, 2007). PCSK9 is a prohormone-pre-protein convertase of the subtilisin (S8) family of serine proteases (Seidah et al., 2003). An exemplary human PCSK9 amino acid sequence is shown as SEQ No. 3 in Table 1, which is the (raw) pre-protein form of PCSK9. PCSK9 proteins may also include fragments of the full-length PCSK9 protein. The structure of the PCSK9 protein is known (Cunningham et al., 2007; Piper et al., 2007). PCSK9 includes a signal sequence, an N-terminal prodomain, a subtilisin-like catalytic domain, and a C-terminal domain.

[0328] PCSK9-binding polypeptides are polypeptides containing one or more complementarity determining regions (CDRs). In some PCSK9-binding polypeptides, the CDR is embedded into a framework region, which orients the CDR to achieve appropriate PCSK9 binding characteristics. PCSK9-binding polypeptides can interfere with, block, reduce, or modulate the interaction between PCSK9 and LDLR. Such PCSK9-binding polypeptides are referred to as "neutralizing" polypeptides. Even if a PCSK9-binding polypeptide neutralizes PCSK9 and binds to it, binding between PCSK9 and LDLR can still occur. For example, PCSK9-binding polypeptides prevent or reduce the negative effects of PCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Therefore, PCSK9-binding polypeptides can modulate or alter the ability of PCSK9 to degrade LDLR without preventing binding between PCSK9 and LDLR. These PCSK9-binding polypeptides can be described as "non-competitive neutralizing." Neutralizing PCSK9-binding polypeptides can bind to PCSK9 at a site or in a manner that prevents PCSK9 from binding to LDLR. These PCSK9-binding polypeptides can be described as "competitive neutralizing." PCSK9 neutralizers can cause a greater amount of free LDLR to be present in the subject, resulting in more LDLR binding to LDL, thereby reducing the amount of LDL in the subject. Ultimately, this leads to a decrease in the amount of serum cholesterol present in the subject.

[0329] Some PCSK9-binding polypeptides can inhibit PCSK9-mediated activity (including binding). PCSK9-binding polypeptides can also inhibit interactions between PCSK9 and LDLR and other physiological effects mediated by PCSK9. PCSK9-binding polypeptides can be human, for example, fully human antibodies against PCSK9.

[0330] Some PCSK9-binding polypeptides can bind to the catalytic domain of PCSK9. PCSK9-binding polypeptides can also bind to the mature form of PCSK9. In other cases, PCSK9-binding polypeptides can bind to the prodomain of PCSK9. In some cases, PCSK9-binding polypeptides can selectively bind to the mature form of PCSK9. In some cases, PCSK9-binding proteins bind to the catalytic domain, either effectively binding PCSK9 to LDLR or preventing it from binding. Some PCSK9-binding polypeptides do not bind to the C-terminus of the catalytic domain. In other cases, PCSK9-binding polypeptides do not bind to the N-terminus of the catalytic domain. In other cases, PCSK9-binding polypeptides do not bind to the N- or C-terminus of the PCSK9 protein. In some cases, PCSK9-binding polypeptides bind to any one of the epitopes bound by the anti-PCSK9 antibody. In some cases, this can be determined by competitive analysis between candidate antibodies and reference antibodies such as evolocumab. In some cases, PCSK9-binding polypeptides bind to specific conformational states of PCSK9 to prevent PCSK9 from interacting with LDLR. In some cases, PCSK9-binding polypeptides bind to the V domain of PCSK9. In some cases, PCSK9-binding polypeptides bind to the V domain of PCSK9 and prevent (or reduce) PCSK9 from binding to LDLR. In some cases, PCSK9-binding polypeptides bind to the V domain of PCSK9 and do not prevent (or reduce) PCSK9 binding to LDLR, while PCSK9-binding polypeptides prevent or reduce negative activity mediated by PCSK9 against LDLR.

[0331] In some cases, the PCSK9-binding polypeptide comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some cases, the PCSK9-binding polypeptide comprises (a) a polypeptide structure and (b) one or more CDRs inserted into and / or linked to this polypeptide structure. The polypeptide structure may take various different forms. For example, it may be a framework of a naturally occurring antibody, or a fragment or variant thereof, or may include such a framework, or may be synthetic.

[0332] The polypeptide structure of the PCSK9-binding polypeptide may be an antibody or derived from an antibody, which includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (antibody mimics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and parts or fragments thereof. In some cases, the PCSK9-binding polypeptide is a fragment of an antibody (e.g., Fab, Fab', F(ab')2, or scFv).

[0333] Certain PCSK9-binding polypeptides bind specifically or selectively to human PCSK9. In some cases, PCSK9-binding polypeptides bind specifically or selectively to human PCSK9 proteins having or consisting of residues 153 to 692 of SEQ ID NO: 3. In some cases, PCSK9-binding polypeptides bind specifically to at least a fragment of a PCSK9 protein with or without a signal sequence and / or a full-length PCSK9 protein.

[0334] In some cases, the antibody comprises at least one variable heavy chain and one variable light chain. In other cases, the antibody comprises two identical light chains and two identical heavy chains. For example, the antibody or PCSK9-binding polypeptide may comprise a heavy chain and a light chain, two heavy chains, or two light chains. In some cases, the PCSK9-binding polypeptide comprises (or consists of) 1, 2, and / or 3 heavy chain and / or light chain CDRs from at least one sequence listed in Table 1 (SEQ NOs: 4 to 9). In some cases, all 6 CDRs (CDRs 1 to 3 from the light chain (CDRL1, CDRL2, CDRL3) and CDRs 1 to 3 from the heavy chain (CDRH1, CDRH2, and CDRH3)) are part of the PCSK9-binding polypeptide. In some cases, 1, 2, 3, 4, or 5 or more CDRs are included in the PCSK9-binding polypeptide. In some cases, one heavy chain and one light chain CDR from the CDRs in the sequences of Table 1 are included in the PCSK9-binding polypeptide. In some cases, additional sections are included in the PCSK9-binding polypeptide.

[0335] PCSK9-binding polypeptides can be encoded by the nucleic acid sequences shown in Table 2 for evolocumab.

[0336] In some cases, the PCSK9-binding polypeptide binds to variants of PCSK9 that are at least 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 95, or 95 to 99 percent identical to or have a greater percentage of identity with respect to the PCSK9 of SEQ NO: 3 (but does not block). In some cases, the PCSK9-binding polypeptide binds to these variants (but does not block). In some cases, the PCSK9-binding polypeptide binds to these variants of PCSK9 and prevents them from interacting with LDLR. In some cases, the PCSK9-binding polypeptide binds to variants of PCSK9 and prevents them from interacting with LDLR. In some cases, the variants of PCSK9 are human variants, e.g., variants at position 474, E620G, and / or E670G. In some cases, the amino acid at position 474 is valine.

[0337] Humanized PCSK9-binding polypeptide (e.g., antibody)

[0338] PCSK9-binding polypeptides may include humanized antibodies and / or parts thereof.

[0339] Humanized antibodies are substantially non-immunogenic in humans and have substantially the same target affinity as antibodies from other species from which humanized antibodies are derived.

[0340] Antibody modifications can be designed to achieve increased binding affinity to a target and / or to reduce the immunogenicity of the antibody in the recipient. In certain cases, humanized antibodies are modified to remove glycosylation sites to increase the antibody's affinity for its cognate antigens. Techniques such as "reshaping," "hyperchimerization," or "veneering / resurfacing" may be used to produce humanized antibodies. These techniques typically reduce antibody immunogenicity by reducing the number of foreign residues, but they do not prevent anti-genotype and anti-allotype reactions following repeated administration of the antibody. Other methods for reducing immunogenicity are known in the art.

[0341] The CDRs of the light and heavy chain variable regions of antibodies against PCSK9 can be transplanted into framework regions (FRs) from the same or different species. The CDRs of the light and heavy chain variable regions can be transplanted into common human FRs. In some cases, the FR of the antibody against the PCSK9 heavy or light chain can be replaced with an FR from a different heavy or light chain. The variable region transplanted from the antibody can be used in conjunction with a constant region different from the constant region of the antibody against PCSK9. The transplanted variable region may be part of a single-chain Fv antibody.

[0342] PCSK9-binding polypeptide variant

[0343] Other useful antibodies are variants of the PCSK9-binding polypeptide listed above formed by combinations or sub-parts of variable heavy chains and variable light chains shown in Table 1, each comprising a variable light chain and / or variable heavy chain having at least 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, 97 to 99%, or more than 99% identity with the amino acid sequence of the sequence in Table 1 (the whole sequence or a sub-part of the sequence, e.g., one or more CDRs). In some cases, these antibodies comprise at least one heavy chain and one light chain, while in other cases, variant forms comprise two identical light chains and two identical heavy chains (or sub-parts thereof).

[0344] In certain cases, the PCSK9-binding polypeptide comprises a heavy chain comprising a variable region having an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0345] In some cases, the PCSK9-binding polypeptide comprises a sequence that is at least 90%, 90% to 95%, and / or 95% to 99% identical to one or more CDRs from at least one of the sequences of SEQ NOs: 4 to 9. In some cases, 1, 2, 3, 4, 5, or 6 CDRs (each identical to the above sequence by at least 90%, 90% to 95%, and / or 95% to 99%) are present.

[0346] In certain cases, the PCSK9-binding polypeptide comprises a light chain comprising a variable region having an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11 or 15.

[0347] In other cases, the PCSK9-binding polypeptide comprises a heavy chain comprising a variable region having an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10 or 14.

[0348] Persons skilled in the art can determine suitable variants of PCSK9-binding polypeptides using well-known techniques. In certain cases, persons skilled in the art can identify suitable regions of the molecule that can be altered without impairing activity by targeting regions not considered important for activity. In certain cases, conserved portions and residues of the molecule among similar polypeptides can be identified. In certain cases, even regions that may be important for biological activity or structure can undergo conserved amino acid substitutions without impairing biological activity or negatively affecting the polypeptide structure.

[0349] Additionally, those skilled in the art may review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. By considering these comparisons, the importance of amino acid residues in a protein corresponding to amino acid residues important for activity or structure in a similar protein can be predicted. Those skilled in the art may select chemically similar amino acid substitutions for these predicted important amino acid residues.

[0350] Persons skilled in the art may also analyze the three-dimensional structure and amino acid sequence of similar PCSK9-binding polypeptides. Taking this information into account, persons skilled in the art may predict the alignment of amino acid residues of the antibody with respect to its three-dimensional structure. In certain cases, persons skilled in the art may choose not to create radical changes in amino acid residues predicted to be on the surface of the protein, as these residues may be implicated in important interactions with other molecules. Furthermore, persons skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. Next, variants may be screened using activity assays known in the art. These variants may be used to gather information regarding suitable variants. For example, if it is observed that a change at a specific amino acid residue leads to disruption, undesirable reduction, or unsuitable activity, variants having such changes may be avoided. That is, based on the information gathered from these routine experiments, persons skilled in the art may easily determine the amino acids for which additional substitutions should be avoided, either alone or in combination with other mutations.

[0351] In certain cases, PCSK9-binding polypeptide variants include glycosylated variants, wherein the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain cases, protein variants include more or fewer N-linked glycosylation sites than the original protein. Additional preferred antibody variants include cysteine ​​variants, wherein one or more cysteine ​​residues are deleted or substituted with other amino acids (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody needs to be refolded into a biologically active conformation, such as after the separation of an insoluble inclusion body. Cysteine ​​variants generally have fewer cysteine ​​residues than the original protein and typically have an even number to minimize interactions caused by unpaired cysteine.

[0352] Competing PCSK9-binding polypeptides

[0353] For specific binding to evolocumab, or to PCSK9, a PCSK9-binding polypeptide that competes with a functional fragment binding to an epitope bound by evolocumab may be used. Such PCSK9-binding polypeptides may also bind to the same epitope PCSK9-binding polypeptide or an overlapping epitope. PCSK9-binding polypeptides and fragments that bind to or compete with the same epitope as evolocumab exhibit similar functional characteristics. Thus, as specific examples, the provided PCSK9-binding polypeptides include those that compete with each of the six CDRs of evolocumab (SEQ NOs: 4 to 9) or with antibodies having two light chains and two heavy chains of SEQ NOs: 2 and 1, or with PCSK9-binding polypeptides.

[0354] Exemplary epitope

[0355] An epitope of sequence number 3 (human PCSK9 polypeptide) to which an anti-PCSK9 antibody binds is provided. In the case of evolocumab (and evolocumab variants having HCVR of SEQ ID NO: 14 and LCVR of SEQ ID NO: 15), these are S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and It is D238.

[0356] Preparation of PCSK9-binding polypeptides (e.g., antibodies)

[0357] Specific strategies may be used to manipulate intrinsic properties of antibodies, such as their affinity for a target. These strategies involve the use of site-specific or random mutagenesis of the polynucleotide molecule encoding the antibody to generate antibody variants. In certain cases, this generation is followed by the selection of antibody variants exhibiting desired changes, such as increased or decreased affinity.

[0358] In mutagenic strategies, the amino acid residues targeted may be amino acid residues in CDR or FR.

[0359] In certain cases, a library of smaller and more effectively screened antibody variants is produced by restricting random or site-directed mutagenesis to hypermutation sites in the CDR, which correspond to regions prone to mutation during the somatic affinity maturation process.

[0360] Antibodies may be expressed in cell lines. Sequences encoding specific antibodies (e.g., polynucleotides encoding the polypeptides of SEQ NOs. 1 and 2, e.g., polynucleotides of SEQ NOs. 12 and 13) may be used to transform suitable mammalian host cells. Transformation may be carried out by any known method for introducing polynucleotides into host cells. The transformation procedure used depends on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, plasmofusion, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus.

[0361] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and many other cell lines.

[0362] In certain cases, antibodies are produced by the 21B12 hybridoma cell line (Jackson et al., 2009). In certain cases, PCSK9-binding polypeptide has a dissociation constant (K) of approximately less than 1 nM, e.g., 1000 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, and / or 1 pM to 0.1 pM or less. D Combine with PCSK9 using ).

[0363] The PCSK9-binding polypeptide may comprise at least one immunoglobulin molecule selected from IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isoforms. In certain cases, the PCSK9-binding polypeptide comprises a human kappa light chain and / or a human heavy chain. In certain cases, the heavy chain is of IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isoform. In certain cases, the PCSK9-binding polypeptide is cloned for expression in mammalian cells. In certain cases, the PCSK9-binding polypeptide comprises an invariant region other than any invariant region of IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isoforms.

[0364] In certain cases, the PCSK9-binding polypeptide comprises a human lambda light chain and a human IgG2 heavy chain. In certain cases, the PCSK9-binding polypeptide comprises a human lambda light chain and a human IgG4 heavy chain. In certain cases, the PCSK9-binding polypeptide comprises a human lambda light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In other embodiments, the PCSK9-binding polypeptide comprises a human kappa light chain and a human IgG2 heavy chain. In certain cases, the PCSK9-binding polypeptide comprises a human kappa light chain and a human IgG4 heavy chain. In certain cases, the PCSK9-binding polypeptide comprises a human kappa light chain and a human IgG1, IgG3, IgE, IgA, IgD, or IgM heavy chain. In certain cases, the PCSK9-binding polypeptide comprises a variable region of an antibody linked to a constant region that is neither the constant region in the IgG2 isoform nor the constant region in the IgG4 isoform. In certain cases, PCSK9-binding polypeptides are cloned for expression in mammalian cells.

[0365] In the case of evolocumab, the antibody is an IgG2-lambda human monoclonal antibody; a disulfide with gamma 2 heavy chain-lambda light chain tetrakisdisulfide. Evolocumab is glycosylated at Asn-291 and Asn-291" and has disulfide crosslinks between residues 22'-90', 22"-90", 22"-96', 22"-96", 129"-214', 129"-214", 137'-196', 137"-196", 142"-198, 142"-198", 217-217', 218-218', 221-221", 224-224", 255-315, 255"-315", 361-419, and 361"-419".

[0366] In certain cases, conservative modifications to the heavy and light chains of evolocumab, or to the heavy and light chains of antibodies with HCVR and LCVR of SEQ ID NOs: 14 and 15, can produce antibodies against PCSK9 having functional and chemical characteristics similar to antibodies from hybridoma family 21B12. In contrast, substantial modifications to the functional or chemical characteristics of antibodies against PCSK9 can be achieved by selecting substitutions in the amino acid sequences of the heavy and light chains that differ significantly in their effect on maintaining the size of the side chains, such as (a) the structure of the molecular backbone in the region of substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect of these on maintaining the size of the side chains.

[0367] For example, conservative amino acid substitution may include replacing the original amino acid residue with a non-original residue at that position such that there is no or minimal effect on the polarity or charge of the amino acid residue.

[0368] PCSK9-binding polypeptides often contain one or more polypeptides. Any variety of expression vectors / host systems may be used to express polynucleotide molecules encoding polypeptides containing one or more PCSK9-binding polypeptide components or the PCSK9-binding polypeptide itself. Such systems include microorganisms, e.g., bacteria transformed with recombinant bacteriophages, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., CaMV, the cauliflower mosaic virus; TMV, the tobacco mosaic virus) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0369] A polypeptide comprising one or more PCSK9-binding polypeptide components or the PCSK9-binding polypeptide itself can be purified from various expression systems; such techniques are well known to those skilled in the art.

[0370] Pharmaceutical formulation components

[0371] In some embodiments, a pharmaceutical formulation comprising a PCSK9-binding polypeptide comprises one or more different PCSK9-binding polypeptides. In certain embodiments, the pharmaceutical formulation comprises one or more PCSK9-binding polypeptides, wherein the antigen-binding protein for PCSK9 binds to one or more epitopes. In some embodiments, the various antigen-binding proteins will not compete with each other for binding to PCSK9. In some embodiments, the pharmaceutical formulation comprises evolocumab.

[0372] PCSK9-binding polypeptides can be linked to half-life extending vehicles. Such vehicles include polyethylene glycol (PEG), glycogen (e.g., glycosylation of PCSK9-binding polypeptides), and dextran.

[0373] Acceptable formulation components are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical formulation may include, for example, agents to modify, maintain, or preserve the pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissociation or release rate, adsorption, or penetration of the composition.

[0374] For example, suitable formulation materials include amino acids (e.g., proline, arginine, lysine, methionine, taurine, glycine, glutamine, or asparagine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, sodium phosphate, sodium acetate ("NaOAC"), Tris-HCl, Tris buffer, citrate, phosphate buffer, phosphate-buffered saline (i.e., PBS buffer) or other organic acids); extenders (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, sucrose, fructose, lactose, mannose, trehalose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); coloring agents, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; It comprises surfactants or wetting agents (e.g., Pluronic, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tiloxapal); stability enhancers (e.g., sucrose or sorbitol); enteric enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0375] In one embodiment, the pharmaceutical formulation comprises a high concentration of PCSK9-binding polypeptide. In a specific embodiment, the PCSK9-binding polypeptide concentration is in the range of about 70 mg / mL to about 260 mg / mL, e.g., about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, or about 260 mg / mL. In some embodiments, the concentration of evolocumab is in the range of about 140 mg / mL to about 210 mg / mL, e.g., about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, about 250 mg / mL, or about 260 mg / mL.

[0376] In another embodiment, the pharmaceutical formulation comprises at least one buffer, such as sodium acetate, phosphate, phosphate-buffered saline ("PBS"), histidine, and / or Tris buffer, having a pH of about 7.0 to 8.5. The buffer acts to maintain a physiologically appropriate pH. Additionally, the buffer can improve the isotonicity and chemical stability of the pharmaceutical formulation. In certain embodiments, the buffer is in the range of about 5 mM to about 100 mM. ,For example, it is a buffer of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 nM. In a specific embodiment, the buffer is NaOAC. In a specific embodiment, the buffer is NaOAC and is present at a concentration of about 10 mM. In another embodiment, the buffer is sodium glutamate. In a specific embodiment, the buffer is sodium glutamate and is present at a concentration of about 10 mM. In yet another embodiment, the buffer is a phosphate buffer. In a specific embodiment, the phosphate buffer is present at a concentration of about 10 mM. Also, in additional embodiments, the buffer is histidine. In some of these embodiments, the histidine buffer is present at a concentration of about 10 mM. Useful pH values ​​for pharmaceutical formulations include about 4 to about 7, or about 4.8 to about 6.9, or about 5.0 to about 5.5, or about 5, or about 5.4.

[0377] In a specific embodiment, the pharmaceutical formulation is isotonic with an osmotic concentration in the range of about 250 to about 400 mOsm / kg, e.g., about 250 mOsm / kg, about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, about 290 mOsm / kg, about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, about 350 mOsm / kg, about 360 mOsm / kg, about 370 mOsm / kg, about 380 mOsm / kg, about 390 mOsm / kg, or about 400 mOsm / kg. Osmolality is a measure of the ratio of solute to the volume of fluid. That is, it is the number of molecules and ions (or molecules) per kilogram of solution. In a specific embodiment, the osmolality is 300 mOsm / kg. Osmolality can be measured by an osmometer, for example, the Advanced Instruments 2020 Multi-Sample Osmometer (Norwood, MA). The Advanced Instruments 2020 Multi-Sample Osmometer measures osmolality by using the freezing point depression method. The higher the osmolality in a solution, the lower the freezing point. Osmolality can also be measured in any other unit using any other method known in the art, such as linear extrapolation. In another embodiment, the pharmaceutical formulation is isotonic with human blood cells, for example, red blood cells.

[0378] In another aspect, the pharmaceutical formulation comprises at least one surfactant polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), a polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic). ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®)) Polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). In certain embodiments, the pharmaceutical formulation contains a surfactant in a volume weight (w / v) range of about 0.0001% to about 10% of the formulation, for example, about 0.0001%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, or about 10% of the surfactant (w / v) of the formulation. In a specific embodiment, the pharmaceutical formulation contains polysorbate 80 at a concentration ranging from about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation contains polysorbate 80 at a concentration of about 0.01% w / v of the formulation. In another embodiment, the formulation contains Pluronic ® ) F-68 is contained in a concentration ranging from about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation is Pluronic ® ) F-68 is included at a concentration of about 0.01% w / v of the formulation. In another embodiment, the formulation includes vitamin E TPGS at a concentration ranging from about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation includes vitamin E TPGS at a concentration of about 0.01% w / v of the formulation.

[0379] A pharmaceutical formulation may comprise at least one stabilizer, for example, a polyhydroxy hydrocarbon (including sorbitol, mannitol, glycerol, and dulcitol) and / or a disaccharide (including sucrose, lactose, maltose, and trehalose) and / or an amino acid (beyond these formulations including arginine salts, for example, arginine monohydrochloride, and acetyl derivatives of arginine, for example, N-acetyl arginine, may include, for example, proline, lysine, methionine, and taurine) and / or benzyl alcohol; the total of the polyhydroxy hydrocarbon and / or disaccharide and / or amino acid and / or benzyl alcohol is about 0.5% to about 10% w / v of the formulation. The pharmaceutical formulation may contain proline in an amount of, for example, about 10 mM to about 200 mM, for example, about 50 mM to about 150 mM, for example, about 90 mM to about 120 mM, for example, about 120 mM.

[0380] In one embodiment, the pharmaceutical formulation has a viscosity level of less than about 80 centipoise (cP) when measured at room temperature (i.e., 25°C). In a specific embodiment, the pharmaceutical formulation has a viscosity level of about 70 cP, about 60 cP, about 50 cP, about 40 cP, about 30 cP, about 25 cP, about 20 cP, about 18 cP, about 15 cP, about 12 cP, about 10 cP; about 8 cP, about 6 cP, about 4 cP; about 2 cP; or less than about 1 cP.

[0381] In one embodiment, the pharmaceutical formulation is stable when measured by at least one stability analysis, e.g., an analysis examining the biophysical or biochemical properties of PCSK9-binding polypeptides over time. The stability of the pharmaceutical formulation can be measured using SEC-HPLC. SEC-HPLC separates proteins based on differences in their hydrodynamic volumes. Molecules with larger hydrodynamic protein volumes elute faster than molecules with smaller volumes. In the case of SEC-HPLC, a stable pharmaceutical formulation exhibits an increase of about 5% or less in HMW species compared to a control sample, e.g., an increase of about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less in HMW species compared to a control sample.

[0382] Alternatively, or additionally, stability can be measured using cation-exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based on differences in their surface charges. At a set pH, charged isoforms of anti-PCSK9 ABP are separated on a cation-exchange column and eluted using a salt gradient. The eluent is monitored by ultraviolet (UV) absorbance. The distribution of charged isoforms is evaluated by determining the peak area of ​​each isoform as a percentage of the total peak area. For CEX-HPLC, a stable pharmaceutical formulation exhibits a degradation of about 5% or less in the major isoform peak compared to a control sample, for example, a degradation of about 3% or less to about 5% in the major isoform peak compared to a control sample; a degradation of about 4% or less, about 3% or less, about 2% or less, about 1% or less, or about 0.5% or less in the major isoform peak compared to a control sample.

[0383] Alternatively, or additionally, formulation stability can be measured using light-shielded particle detection (HIAC). An electronic liquid-transfer particle counting system (HIAC / Royco 9703 (Hach Company; Loveland, CO) or equivalent) comprising a light-shielded sensor with a liquid sampler (HIAC / Royco HRLD-150 or equivalent) quantifies the number of particles and their size range in a given test sample. As particles in the liquid pass between the light source and the detector, they attenuate or "shield" the beam of light falling on the detector. When the concentration of particles is within the normal range of the sensor, these particles are detected one by one. The path of each particle passing through the detection zone reduces the incident light on the light-detector, and the voltage output of the light-detector is instantaneously reduced. The change in voltage is recorded as an electrical pulse, which is converted by the instrument into the number of particles present. This method is non-specific and measures particles regardless of their origin. The particle sizes monitored are typically 10 μm and 25 μm. For HIAC, a stable pharmaceutical formulation indicates 6,000 or fewer 10 μm particles per container (or unit) compared to a control sample, e.g., 5,000 or fewer, 4,000 or fewer, 3,000 or fewer, 2,000 or fewer, and 1,000 or fewer 10 μm particles per container (or unit) compared to a control sample. In other cases, a stable pharmaceutical formulation indicates 600 or fewer 25 μm particles per container (or unit) compared to a control sample, e.g., 500 or fewer, 400 or fewer, 300 or fewer, 200 or fewer, 100 or fewer, and 50 or fewer 25 μm particles per container (or unit) compared to a control sample.

[0384] Pharmaceutical formulation stability can also be evaluated using visual evaluation. Visual evaluation is a qualitative method used to describe the visible physical characteristics of a sample. Samples are viewed against a black and / or white background of an inspection booth according to the characteristics being evaluated (e.g., color, transparency, presence of particles or foreign matter). Samples are also viewed against an opaque reference standard and a color reference standard. In the case of visual evaluation, a stable pharmaceutical formulation does not show significant changes in color, transparency, or the presence of particles or foreign matter compared to a control sample.

[0385] Exemplary pharmaceutical formulation

[0386] Exemplary pharmaceutical formulations of PCSK9-binding polypeptides are shown in Table 4. Some formulations are given as ranges, and sub-examples (e.g., 1.1) are given as specific examples.

[0387]

[0389]

[0390] Therapeutic application

[0391] PCSK9-binding polypeptides, such as evolocumab, can be used for various therapeutic applications. For example, PCSK9-binding polypeptides are useful for treating PCSK9-associated conditions, e.g., cholesterol-related disorders (serum cholesterol-related disorders), e.g., hypercholesterolemia. PCSK9-binding polypeptides may be useful for treating outcomes, symptoms, and / or pathologies associated with PCSK9 activity.

[0392] Disorders associated with, including, or affected by elevated levels of a molecule or group of molecules, including cholesterol (including serum cholesterol), LDL, LDLR, PCSK9, VLDL-C, apoprotein B (“ApoB”), lipoprotein A (“Lp(a”)), triglycerides, HDL-C, non-HDL-C, and total cholesterol levels, may be addressed by a method of using the evolocumab pharmaceutical composition disclosed herein to treat, / or prevent, or / or reduce the risk of such disorders in subjects. The disclosed evolocumab composition may be used to regulate the levels of these molecules or groups of molecules, such as by reducing the amount of these molecules or groups of molecules. For example, the disclosed evolocumab composition may be used in a method of lowering the amount of these molecules or groups of molecules from abnormally high levels, or even from normal levels. That is, the levels of cholesterol (including serum cholesterol), LDL, LDLR, PCSK9, VLDL-C, ApoB, Lp(a), triglycerides, HDL-C, non-HDL-C, and total cholesterol may be reduced.

[0393] "Cholesterol-related disorders" (including "serum cholesterol-related disorders") include any one or more of the following: familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes mellitus, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and generally, dyslipidemia which may be manifested by, for example, elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and / or low HDL. Some non-limiting examples of primary and secondary dyslipidemia include metabolic syndrome, diabetes mellitus vera, familial mixed hyperlipidemia, familial hypertriglyceridemia, heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial apolipoprotein B-100 deficiency; familial hypercholesterolemia including polygenic hypercholesterolemia; remnant removal disease, hepatic lipase deficiency; Secondary dyslipidemia caused by any of the following: dietary indiscretion, hypothyroidism, estrogen and progestin therapy, beta-blockers, and drugs including thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage disease, liver cancer, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia. The disclosed evolocumab composition may also be used to treat and / or prevent and / or reduce the risk thereof of atherosclerotic diseases, e.g., cardiovascular death, non-cardiovascular or all-cause death, coronary heart disease, coronary artery disease, peripheral artery disease, stroke (ischemic and hemorrhagic), angina pectoris, or cerebrovascular disease and acute coronary syndrome, myocardial infarction, and unstable angina pectoris.The disclosed evolocumab composition may also be useful for reducing the risk of chest pain and / or chest pain with evidence of blocked arteries and / or graft-related vascular disease in patients with cardiovascular events due to established heart disease, such as fatal and non-fatal heart attack, fatal and non-fatal stroke, certain types of heart surgery, hospitalization due to heart failure, heart disease, and / or previous heart attack or previous heart surgery. In some cases, the disclosed evolocumab composition may be used in a method to prevent or reduce cardiovascular risk due to elevated CRP or hsCRP. In some embodiments, the ABP and the method may be used to reduce the risk of recurrent cardiovascular events.

[0394] Diseases or disorders that can generally be managed (treated or prevented) through the use of statins may also benefit from the application of the disclosed evolocumab composition. Additionally, disorders or diseases that may benefit from the prevention of cholesterol synthesis or increased LDLR expression may also be treated using the disclosed evolocumab composition. Furthermore, the use of the disclosed evolocumab composition may be particularly useful for the treatment of diabetes. Diabetes is not only a risk factor for coronary heart disease, but insulin also increases the expression of PCSK9. That is, individuals with diabetes have elevated plasma lipid levels (which may be associated with high PCSK9 levels) and may benefit from lowering these levels.

[0395] When PCSK9-binding polypeptides are used for therapeutic applications, PCSK9-binding polypeptides may inhibit, interfere with, or modulate one or more biological activities of PCSK9. For example, PCSK9-binding polypeptides may specifically bind to human PCSK9 and / or may substantially inhibit the binding of human PCSK9 to LDLR by at least about 20% to 40%, 40% to 60%, 60% to 80%, 80% to 85%, or more (e.g., by measuring binding in an in vitro competitive binding assay). In some cases, PCSK9-binding polypeptides 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 K less than M (more strongly bound) d In some cases, the PCSK9-binding polypeptide has an IC50 of less than 1 μM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, and 10 pM to 1 pM for blocking the binding of LDLR to PCSK9.

[0396] Pharmaceutical formulations may be administered as combination therapy, that is, in combination with other agents. Combination therapy may include a PCSK9-binding polypeptide in combination with at least one anti-cholesterol agonist. The agonist comprises chemical formulations, antibodies, antigen-binding domains, and combinations and conjugates thereof prepared by in vitro synthesis. In certain embodiments, the agonist may act as an agonist, antagonist, allosteric modifier, or toxin. In certain embodiments, the agonist may act to inhibit or stimulate its target (e.g., a receptor for enzyme activation or inhibition), thereby promoting increased expression of LDLR or lowering serum cholesterol levels.

[0397] PCSK9-binding polypeptides may be administered prior to, concurrently with, and after treatment with cholesterol-lowering (serum and / or total cholesterol) agents. For example, PCSK9-binding polypeptides may be administered prophylactically to prevent or alleviate the onset of hypercholesterolemia, heart disease, diabetes, and / or any cholesterol-related disorder. Additionally, PCSK9-binding polypeptides may be administered for the treatment of pre-existing hypercholesterolemic conditions. In some cases, administration of PCSK9-binding polypeptides may delay the onset of the disorder and / or symptoms associated with the disorder. In some cases, PCSK9-binding polypeptides are provided to subjects lacking any symptoms of a cholesterol-related disorder or any subset thereof.

[0398] PCSK9-binding polypeptides can be used in combination with specific therapeutic agents to treat various cholesterol-related disorders, such as hypercholesterolemia. Depending on the pathological condition and the level of treatment required, two or more agents may be administered. These agents may be provided together in the same formulation. Alternatively, these agents may be formulated separately and provided together in a treatment kit if desired. In other examples, these agents may be provided separately.

[0399] Dosage and Medication Therapy

[0400] The amount of PCSK9-binding polypeptide, e.g., mAb, e.volocumab, administered to the patient is a therapeutically effective amount. Typical doses of PCSK9-binding protein may range from about 0.1 µg / kg to about 100 mg / kg or more. In certain cases, the dose may be PCSK9-binding polypeptide in the range of 0.1 µg / kg to about 100 mg / kg; or 1 µg / kg to about 100 mg / kg; or 5 µg / kg to about 100 mg / kg; or 1 mg / kg to about 50 mg / kg; or 2 mg / kg to about 20 mg / kg; or 2 mg / kg to about 10 mg / kg.

[0401] The amount (or dose) of PCSK9-binding polypeptide is at least about 10 mg to about 1400 mg; or about 14 mg to about 1200 mg; or about 14 mg to about 1000 mg; or about 14 mg to about 800 mg; or about 14 mg to about 700 mg; or about 14 mg to about 480 mg; or about 20 mg to about 480 mg; or about 70 mg to about 480 mg; or about 80 mg to about 480 mg; or about 90 mg to about 480 mg; or about 100 mg to about 480 mg, or about 105 mg to about 480 mg; or about 110 mg to about 480 mg; or about 115 mg to about 480 mg; or about 120 mg to about 480 mg; or about 125 mg to about 480 mg; or about 130 mg to about 480 mg; or about 135 mg to about 480 mg; or about 140 mg to about 480 mg; or about 145 mg to about 480 mg; or about 150 mg to about 480 mg; or about 160 mg to about 480 mg; or about 170 mg to about 480 mg; or about 180 mg to about 480 mg or about 190 mg to about 480 mg or about 200 mg to about 480 mg; or about 210 mg to about 480 mg; or about 220 mg to about 480 mg; or about 230 mg to about 480 mg; or about 240 mg to about 480 mg; or about 250 mg to about 480 mg; or about 260 mg to about 480 mg; or about 270 mg to about 480 mg; or about 280 mg to about 480 mg; or about 290 mg to about 480 mg; or about 300 mg to about 480 mg; or about 310 mg to about 480 mg; or about 320 mg to about 480 mg; or about 330 mg to about 480 mg;or about 340 mg to about 480 mg; or about 350 mg to about 480 mg; or about 360 mg to about 480 mg; or about 370 mg to about 480 mg; or about 380 mg to about 480 mg; or about 390 mg to about 480 mg; or about 400 mg to about 480 mg; or about 410 mg to about 480 mg; or about 420 mg to about 480 mg; or about 430 mg to about 480 mg; or about 440 mg to about 480 mg; or about 450 mg to about 480 mg; or about 460 mg to about 480 mg; or about 470 mg to about 480 mg may be a PCSK9-binding polypeptide in the range of about 470 mg to about 480 mg.;

[0402] The frequency of administration will consider the pharmacokinetic parameters of the PCSK9-binding polypeptide and / or any additional therapeutic agent in the formulation. Clinicians may administer the formulation until a dose is reached that achieves the desired effect. The formulation may be administered as a single dose, or over time as two, three, or four or more doses (which may or may not contain the same amount of PCSK9-binding polypeptide), or as a continuous infusion via an implantable device or catheter. The formulation may also be delivered subcutaneously or intravenously using a needle and syringe. Regarding subcutaneous delivery, pen delivery devices, as well as body syringes and self-injector delivery devices, may deliver pharmaceutical formulations containing PCSK9-binding polypeptide.

[0403] In certain cases, PCSK9-binding polypeptides in doses of at least about 10 mg; or up to about 14 mg; or up to about 20 mg; or up to about 35 mg; or up to about 40 mg; or up to about 45 mg; or up to about 50 mg; or up to about 70 mg are administered once a week (QW) to patients who require it.

[0404] In other cases, PCSK9-binding polypeptide at doses of at least about 70 mg, or up to about 100 mg; or up to about 105 mg, or up to about 110 mg; or up to about 115 mg, or up to about 120 mg; or up to about 140 mg; or up to about 160 mg; or up to about 200 mg; or up to about 250 mg; or up to 280 mg; or up to 300 mg; or up to 350 mg; or up to 400 mg; or up to 420 mg is administered to patients who require it once every two weeks (or once every two weeks; "Q2W:").

[0405] In other specific cases, PCSK9-binding polypeptides at doses of at least about 250 mg; or up to about 280 mg; or up to about 300 mg; or up to about 350 mg; or up to about 400 mg; or up to about 420 mg; or up to about 450 mg; or up to 480 mg are administered to patients who require it once every 4 weeks ("Q4W") (or once a month).

[0406] For example, evolocumab can be administered Q2W at a dose of 210 mg. Alternatively, evolocumab can be administered Q4W at a dose of 420 mg. Depending on the condition being treated, both of these drug doses may be administered weekly.

[0407] In some cases, serum LDL cholesterol levels are reduced by at least about 15% compared to serum LDL cholesterol levels before administration. In some embodiments, serum LDL cholesterol levels are reduced by at least about 20%, or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even more.

[0408] Storage and Kit

[0409] A formulation comprising a PCSK9-binding polypeptide, with or without at least one additional therapeutic agent, can be prepared for storage by mixing a selected formulation having a desired degree of purity with a selective formulation agent in the form of a lyophilized cake or an aqueous solution. Additionally, a formulation comprising a PCSK9-binding polypeptide, with or without at least one additional therapeutic agent, can be formulated as a lyophilized product using a suitable excipient.

[0410] Once a pharmaceutical formulation is formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. These formulations may be stored in a ready-to-use form or in a form that is reconstituted before administration (e.g., in a lyophilized form). In some cases, PCSK9-binding polypeptide formulations may be stored in containers, e.g., suitable storage bags (e.g., those manufactured by Sartorius (Gottingen, DE)) or polycarbonate carboys. Once a pharmaceutical formulation is formulated, it may also be stored in glass vials (e.g., 5 cc glass vials) as well as in pre-filled syringes (PFS; e.g., 2.25 mL PFS) in a ready-to-use form as a solution or suspension.

[0411] In a specific embodiment, a kit is provided for producing a single-dose dosage unit. In a specific embodiment, the kit may include both a first container having a dry protein and a second container having an aqueous formulation. In a specific embodiment, a kit is provided that includes single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0412] Ultrafiltration / dialysis of PCSK9-binding polypeptide formulations containing N-acetyl arginine

[0413] The PCSK9-binding polypeptide can be formulated at about 210 g / L in the disclosed UF / DF process. For example, in the disclosed method, a first dialysis filtration (DF1) concentration of 70 g / L (or 35 g / L) is dialysis filtration three times and concentrated to 140 g / L. Next, a second dialysis filtration (DF2) concentration of 140 g / L is dialysis filtration four times and concentrated to a concentration of 260 g / L. Next, the concentrated pool is recovered from the system as a formulation flush at a final concentration of 210 g / L.

[0414] In the disclosed method, the examples show that first ultrafiltration (UF1) concentrations of about 70 mg / mL and about 35 mg / mL do not have a significant effect on the HWM (%) in the final drug substance (DS). Additionally, in the disclosed method, only 7 dialysis volumes of DF are required in the DF step to ensure that dialysis filtration is completed.

[0415] The disclosed UF / DF processes are summarized in Tables 5 and 6.

[0416]

[0417]

[0418]

[0419]

[0420] Accordingly, a method for formulating a PCSK9-binding polypeptide, such as a PCSK9-binding polypeptide that blocks the binding of PCSK9 to LDLR, is disclosed herein:

[0421] a. A first concentration step in which polypeptides are concentrated in a first solution;

[0422] b. A first solution exchange step in which a polypeptide concentrated in a first solution is exchanged with a second solution containing N-acetyl arginine using dialysis filtration;

[0423] c. A second concentration step in which polypeptides are concentrated in the second solution;

[0424] d. A second solution exchange step in which the polypeptide in the concentrated second solution is exchanged with a third solution containing N-acetyl arginine using dialysis filtration; and

[0425] e. A third concentration step in which polypeptides are concentrated in the third solution.

[0426] Before the third concentration step, the temperature of the solution containing the polypeptide may be increased from about 25°C to about 37°C, for example, about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and about 37°C. Additionally, the first solution exchange step may be achieved using at least 3 dialysate volumes of the second solution; in some cases, additional dialysate volumes, for example, 4, 5, or 6 dialysate volumes, may be used. The second solution exchange step may be achieved using at least 4 dialysate volumes of the third solution; additional dialysate volumes may be used, including 5, 6, or 7 dialysate volumes. The initial concentration of the PCSK9-binding polypeptide may be about 11 mg / mL or less, for example, less than 1 mg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or about 11 mg / mL. Additionally, the PCSK9-binding polypeptide concentration may be increased by about 3 to about 7 times, for example, about 3, 4, 5, 6, or about 7 times. For example, the increased concentration of polypeptide may be about 35 mg / mL to about 70 mg / mL or more, for example about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mg / mL or more.In the second concentration step, the PCSK9-binding polypeptide concentration increases by about 2 to 4 times (e.g., about 2, 3, or about 4 times) from the first concentration step, e.g., about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, It increases to 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 250, or about 300 mg / mL. In the third concentration step, the concentration of the PCSK9-binding polypeptide may be increased by about 1.5 to about 2 times from the second concentration step, for example, to about 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 250, or about 300 mg / mL, for example, to about 260 mg / mL. Thus, the PCSK9-binding polypeptide may have a final concentration, for example, about 210 mg / mL, which is at least about 19 to 20 times more concentrated than the initial concentration of the therapeutic polypeptide. The concentration step may include fed-batch ultrafiltration; additionally, the second solution and the third solution may be identical.

[0427] A second or third solution comprising N-acetyl arginine (e.g., "NAR solution") may comprise an arginine salt and a buffer, wherein, for example, N-acetyl arginine is about 25 mM to about 230 mM, e.g., about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, Present at a concentration of 215, 220, 225, or about 230 mM; the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and is present at a concentration of about 25 mM to about 150 mM, e.g., about 25, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or about 150 mM; The buffer is sodium acetate buffer at a concentration of about 5 mM to about 30 mM, e.g., about 5, 10, 15, 20, 25, or about 30 mM. In another sub-modification, N-acetyl arginine is present at a concentration of about 140 to about 170 mM; Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 to about 70 mM (e.g., about 63, 64, 65, 66, 67, 68, 69, or about 70 mM) and sodium acetate buffer is present at a concentration of about 10 mM. For example, N-acetyl arginine may be present at a concentration of about 140 mM, Arg HCl, Arg acetate, or Arg glutamate may be present at a concentration of about 63 mM, and sodium acetate buffer may be present at a concentration of about 10 mM.In a further sub-modality, N-acetyl arginine is present at a concentration of about 155 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM. In another example, N-acetyl arginine is present at a concentration of about 170 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0428] Additionally, the composition (including the NAR solution) may further include proline, wherein the proline is present at a concentration of about 50 mM to about 150 mM, for example, about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or about 150 mM. The second or third solution may have a pH of about 4.8 to about 6.9, for example, about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or about 6.9, for example, a pH of 5.3, 5.4, or 5.5. In the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following may be used:

[0429] a. Mesh opening greater than about 350 μm but less than or equal to about 500 μm, e.g., about 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 μm;

[0430] b. An opening area that is greater than about 32% of the membrane area but less than or equal to about 36% (e.g., about 32, 33, 34, 35, or about 36%);

[0431] c. A number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm, e.g., approximately 16.2, 16, 15.8, 15.6, 15.4, 15.2, 15, 14.8, 14.6, 14.4, 14.2, 14, 13.8, 13.6, 13.4, 13.2, 13, 12.8, 12.6, 12.4, or approximately 12.2 n / cm;

[0432] d. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm, e.g., approximately 270, 280, 290, 300, 310, 320, 330, or 340 μm;

[0433] e. Exceeding approximately 160 g / m² but not exceeding 180 g / m², e.g., approximately 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, or 180 g / m² 2 Person standard weight;

[0434] f. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0435] g. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0436] h. Maximum supply pressure of approximately 60 psi.

[0437] Additionally, polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®Surfactants such as polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS) may be added to the third solution after the third solution has been concentrated. In some cases, the surfactant is a polyoxyethylene-polyoxypropylene block copolymer (Pluronic ® ) F-68) or D-α-tocopherol polyethylene glycol succinate (vitamin E TPGS). The surfactant may be in a concentration range of about 0.0001% to about 10% by volume ("w / v") of the formulation, for example, about 0.0001%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, or about 10% surfactant (w / v) of the formulation. In a specific embodiment, the pharmaceutical formulation contains polysorbate 80 in a concentration range of about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation contains polysorbate 80 at a concentration of about 0.01% w / v of the formulation. In another embodiment, the formulation contains Pluronic ® ) F-68 is contained in a concentration ranging from about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation is Pluronic ® ) F-68 is included at a concentration of about 0.01% w / v of the formulation. In another embodiment, the formulation includes vitamin E TPGS at a concentration ranging from about 0.0001% to about 1% w / v of the formulation. In a specific embodiment, the pharmaceutical formulation includes vitamin E TPGS at a concentration of about 0.01% w / v of the formulation. Effects of the invention

[0438] The present invention provides a formulation of a PCSK9-binding polypeptide comprising N-acetyl arginine and having reduced viscosity compared to a formulation lacking N-acetyl arginine, e.g., a formulation comprising evolocumab. Furthermore, the present invention provides a method for formulating such a composition which is advantageous in that it saves on certain components. The formulation of the present invention comprising a PCSK9-binding polypeptide may be administered to a patient to treat and / or prevent PCSK9-related diseases, conditions, and disorders. Brief explanation of the drawing

[0439] Fig. 1 This shows a graph of data from size-exclusion chromatography-high-pressure liquid chromatography (SE-HPLC) of high-concentration evolocumab samples in various formulations containing N-acetyl arginine after 1 month of incubation at 40°C. x - Description of the formulation shown on the axis: [Acetate (mM)] / [N-Acetyl Arginine (mM)] / [Proline (mM)]. Y- The axis represents the percentage of samples that are high molecular weight (HMW) species (aggregates and oligomers of evolocumab). Fig. 2 Evolocumab 210 mg / mL at 25℃ - 1000 sec -1 It shows a graph comparing formulation viscosity at shear rate. Fig. 3a This shows a graph generated by the JMP Prediction Profiler software of SE-HPLC data for an evolocumab formulation maintained at 40°C for one month. Fig. 3b It shows similar data except that the sample was maintained at 25℃ for 3 months. Fig. 4 This shows a bar graph of the percentage of HMW species by SE-HPLC for a 210 mg / mL evolocumab formulation maintained at 40°C for 1 month. The formulations differ in the concentration of N-acetyl arginine and arginine HCl at various pH values. FIGS. 5a to 5cFigure 5 shows SE-HPLC chromatographs of selected pH 4.8 and pH 5.4 evolocumab samples maintained at the indicated temperatures and times (5°C for 6 months (Fig. 5a); 25°C for 3 months (Fig. 5b); and 40°C for 1 month (Fig. 5c)) compared to an evolocumab proline formulation control. Description of formulation: [Acetate (mM)] / [N-acetyl arginine (mM)] / [Arginine HCl (mM)]. Figures 6a and 6b This shows a graph of the percentage of HMW species occurring over time in the indicated evolocumab formulations maintained at the indicated temperatures (5°C (Fig. 6a); 25°C (Fig. 6b)). The tested 210 mg / mL evolocumab N-acetyl arginine formulation was compared to a 140 mg / mL evolocumab proline formulation control. Description of formulation: [Acetate (mM)] / [N-acetyl arginine (mM)] / [Arginine HCl (mM)]. Fig. 7a This shows a graph generated by the JMP Prediction Profiler software of cation exchange chromatography-high pressure liquid chromatography (CEX-HPLC) data for an evolocumab formulation maintained at 40°C for one month. Fig. 7b It shows similar data except that the sample was maintained at 25°C for 3 months. For more specific details, refer to the examples. Fig. 8 This shows a graph of the percentage of acid peaks by CEX-HPLC of a 210 mg / mL evolocumab formulation maintained at 40℃ for 1 month. Figures 9a and 9b It shows a graph of invisible particles per milliliter (greater than 10 μM, Fig. 9a; or greater than 25 μM, Fig. 9b) determined by the light-shielding liquid delivery particle count of various evolocumab formulations maintained at 5°C, 25°C, and 40°C for 3 months. Fig. 9cIt shows a graph of particles invisible to the naked eye (10 μM or 25 μM or more) determined by the light-shielding liquid delivery particle count of various evolocumab formulations maintained at 5°C or 25°C for 6 months. Fig. 10 It shows a graph of invisible particles of various evolocumab formulations determined by Micro Flow Imaging (MFI) analysis, filtered by an aspect ratio (AR) of less than 0.70. Fig. 11 It shows a summary graph of the viscosity of various evolocumab formulations maintained at the indicated temperature and for the specified time, where T0 represents the initial viscosity value. Fig. 12 This shows a table and graph representing pH values ​​over time up to 3 months for evolocumab formulations with different pH and buffers. Samples were maintained at 40°C. FIGS. 13a to 13c This shows graphs of SE-HPLC data illustrating the effect of pH on the percentage of HMW species of 210 mg / mL evolocumab formulations with different pH and buffers up to 3 months at 4°C (Fig. 13a), 25°C (Fig. 13b), and 40°C (Fig. 13c). In addition to 10 mM of the listed buffers, each sample contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 14 It shows a graph of oligomer levels of the evolocumab formulation at various pH values ​​immediately after formulation. Fig. 15 ) shows a chromatogram displaying protein molecules of various sizes of evolocumab formulations with different pH and buffer levels after being maintained at 40°C for 3 months. (LMW = low molecular weight species). FIGS. 16a to 16cFigure 16 shows graphs of data from CEX-HPLC analysis (% major peaks) over time for 210 mg / mL evolocumab formulations with different pH and buffers. Figure 16a shows the percentage of major peaks over time for samples maintained at 5°C, while Figure 16b shows the same type of data for samples maintained at 25°C and Figure 16c shows the same type of data for samples maintained at 40°C. In addition to the 10 mM buffers listed for each, all samples contain 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 17 This shows a graph of data from a CEX-HPLC analysis measuring the percentage of acidic species in 210 mg / mL evolocumab formulations over time, with different pH and buffers maintained at 40°C. In addition to 10 mM of each listed buffer, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 18 This shows a chromatogram of CEX-HPLC data showing the acidity and basicity, and the analysis of major peak species of 210 mg / mL evolocumab formulations with different pH and buffers, maintained at 25°C for 3 months. In addition to 10 mM of each listed buffer, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 19 is a chromatogram of peptide mapping data for various evolocumab formulations with different pH and buffers, and the samples were maintained at 40°C for 1 month. In addition to 10 mM of each listed buffer, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 20The graph shows the results of several experiments analyzing evolocumab in formulations with different pH and buffers after 1 month at 40°C. All samples contained 10 mM buffer, 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. For more specific details, refer to the drawings and examples. Figures 21a and 21b It shows a graph of invisible particles per milliliter (greater than 10 μM, Fig. 21a; or greater than 25 μM, Fig. 21b) determined by the light-shielding liquid delivery particle count of various evolocumab formulations with different pH and buffer maintained at 5°C, 25°C, and 40°C for 2 months. FIGS. 21c and 21d This shows graphs of invisible particles (greater than 10 μM, Fig. 21c; greater than 25 μM, Fig. 21d) of evolocumab formulations with different pH and buffers maintained at 5°C or 25°C for 6 months. In addition to the 10 mM buffer listed for each sample, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 22 This shows a graph of invisible particles identified by Micro Flow Imaging (MFI) of evolocumab formulations with different pH and buffers. In addition to 10 mM of each listed buffer, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 23This shows a graph of the results of reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) assays on 210 mg / mL evolocumab formulations with different pH and buffers maintained at 25°C for 6 months. In addition to 10 mM of each listed buffer, all samples contained 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Fig. 24 This shows a graph of the relationship between viscosity and pH of evolocumab formulations with different pH and buffers. All samples contain 10 mM buffer, 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80. FIGS. 25a to 25c This shows a graph of the percentage of HMW species measured by SE-HPLC for different evolocumab formulations maintained for the indicated periods at 5°C (Fig. 25a), 25°C (Fig. 25b), and 40°C (Fig. 25c). Description of formulation: [Acetate (mM)] / [N-acetyl arginine (mM)] / [Arginine HCl (mM)]. FIGS. 26a to 26c Figure 26 shows a graph of the percentage of acid peaks measured by CEX-HPLC for different evolocumab formulations maintained for the indicated periods at 5°C (Fig. 26a), 25°C (Fig. 26b), and 40°C (Fig. 26c). Description of formulation: [Acetate (mM)] / [N-acetyl arginine (mM)] / [Arginine HCl (mM)]. Fig. 27 Figure 1 shows a graph of the percentage of Pre -LC + LC + HC (LC = light chain, HC = heavy chain) by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) for different evolocumab formulations maintained at 5°C, 25°C, and 40°C for 3 months compared to the initial level (T0). Description of formulation: [Acetate (mM)] / [N-acetyl arginine (mM)] / [Arginine HCl (mM)]. Fig. 28It shows a graph of the viscosity of different evolocumab formulations of evolocumab at three different concentrations; viscosity data at 90,000 sec at the indicated temperature -1 It was determined using a flow meter at the shear rate up to. Description of formulation: [Acetate (mM)] / [N-Acetyl Arginine (mM)] / [Arginine HCl (mM)]. Fig. 29 This shows a graph of evolocumab UF / DF flux data in three types of NAR formulation buffers. Fig. 30 Figure 1 shows a graph of the percentage of HMW species formation in the evolocumab UF / DF process (UF / DF-70 and UF / DF-35) with evolocumab at 35 mg / mL and 70 mg / mL in UF1 / DF1. Figure 2 also shows the concentration of evolocumab in mg / mL at each stage of the process for the two initial concentrations of evolocumab. FIGS. 31a to 31f Figures 31 and 31 show the SE-HPLC (Figures 31a, 31c, and 31e) and CEX-HPLC (Figures 31b, 31d, and 31f) graphs of evolocumab in NAR-containing formulations at pH 5.2 (Figs. 31a and 31b), pH 5.4 (Figs. 31c and 31d), and pH 5.6 (Figs. 31e and 31f). Figures also show the percentages of HMW species, LMW species, and evolocumab at various stages during the UF / DF process. Fig. 32 It shows the percentage of HMW species formation of evolocumab NAR DS samples from a full-hold study at 2 to 8°C and room temperature. Fig. 33 It shows a graph of the percentage of HMW species formation in evolocumab NAR OC samples from a full-hold study at elevated temperatures. Fig. 34 It shows a graph of viscosity measurements of the evolocumab NAR formulation at different temperatures. FIGS. 35a to 35c It shows graphs of SE-HPLC data for all study formulations used in Example 9 after incubation for up to 6 months at 4°C (Fig. 35a), 25°C (Fig. 35b), and 40°C (Fig. 35c). Fig. 35dIt displays 40℃ SE-HPLC data (shown as a line graph in Fig. 35c) as a bar chart, making it more identifiable to compare the aggregation levels between formulations. FIGS. 36a to 36f This shows a graph of CEX-HPLC data for the study formulation used in Example 9, showing the change in the % acid peak and % basic peak over time after incubation up to 3 months at 4°C (Fig. 36a (% acidic), Fig. 36b (% basic)), 25°C (Fig. 36c (% acidic), Fig. 36d (% basic)), and 40°C (Fig. 36e (% acidic), Fig. 36f (% basic)). FIGS. 37a to 37d It shows graphs of rCE-SDS data for the major peak % and LMW species % for the study formulation used in Example 9 over time after incubation up to 3 months at 30°C (Fig. 37a (% major peak), Fig. 37b (% LMW species)) and 40°C (Fig. 37c (% major peak), Fig. 37d (% LMW species)). FIGS. 38a to 38d Figure 38 shows graphs of data on visually invisible particles by light-shielding particle counting using HIAC for the study formulation used in Example 9 after incubation for up to 3 months at 4°C and 40°C. Fig. 38a: HIAC - ≥10 μm - 4°C; Fig. 38b: HIAC - ≥25 μm - 4°C; Fig. 38c: HIAC - ≥10 μm - 40°C; and HIAC - ≥25 μm - 40°C. Specific details for implementing the invention

[0440] The following exemplary sections are provided merely as examples and are not presented to limit the scope of the disclosure or claims in any way.

[0441] Examples

[0442] Viscosity measurements were performed using TA Instruments (New Castle, DE) AESR-G2 cone and plate flowmeters unless otherwise noted.

[0443] Example 1 - Design of Experiment (DOE) Study; Optimization of Evolocumab Formulations Containing N-Acetyl Arginine

[0444] A Design of Experiment (DOE) study was initiated to optimize evolocumab formulations containing N-acetyl arginine. Viscosity, pre-filled syringe ejection force, stability, pH, and osmolality were tested for 11 formulations. Data for the initial formulation parameters are shown in Table 7.

[0445]

[0447] Acetate concentration was minimized to reduce aggregation rate based on the 1-month 40°C data shown in Figure 1. Proline concentration was increased to 120 mM to make the formulation isotonic. N-acetyl arginine (155 mM) was selected to balance viscosity reduction and the crystallization of 170 mM N-acetyl arginine at 4°C. In the data, polysorbate-80 (TWEEN ® ) 80; Polyoxyethylene sorbitan monooleate), Pluronic ® It was shown that using different surfactants between F-68 (polyoxyethylene-polyoxypropylene block copolymer), vitamin E TPGS (D-α-tocopherol polyethylene glycol succinate) did not lead to a significant difference in viscosity or stability.

[0448] Based on these data, a formulation of 10 mM acetate, 155 mM N-acetyl arginine, 120 mM proline, 0.01% polysorbate-80, pH 5.4 was found to be suitable for a reduced viscosity formulation of high concentrations (e.g., 190 to 210 mg / mL) of evolocumab. The data presented in Table 8 indicate that this formulation is isotonic and has low viscosity.

[0449]

[0450] Example 2 - DOE study to evaluate the effects of excipient concentration, pH, and buffer

[0451] Examples 2 to 4 relate to formulations optimized to reduce viscosity as much as possible while minimizing any effect on aggregation, deamidation, and other stability indicator factors.

[0452] N-acetyl arginine (NAR) was selected based on its superior viscosity-reducing effect in excipient selection studies (e.g., see Example 1). The concentration of NAR was limited by its solubility at 2 to 8°C. A NAR concentration of 155 mM in the dialysis filtration (DF) buffer was selected based on stability studies showing that NAR did not exhibit crystallization at concentrations as high as 175 mM. Arginine HCl (70 mM) was added to the DF buffer to achieve an isotonic drug product formulation with the lowest viscosity and to improve the solubility of NAR. A formulation pH of 5.4 was selected to minimize deamidation and aggregation of evolocumab. The buffer and surfactant (acetate / polysorbate-80) showed minimal effect on the viscosity and stability of the evolocumab formulation.

[0453] Three major studies were conducted to investigate the safety of evolocumab in NAR / arginine HCl formulations. These are as follows:

[0454] 1. DOE study to evaluate the effects of excipient concentration, pH, and buffer (acetate versus glutamate) (Example 2).

[0455] 2. A wide-range pH study designed to evaluate the effect of pH (5.1 to 6.9) on viscosity / stability (Example 3).

[0456] 3. A scale-down study to evaluate the stability of a best formulation candidate, including the effects of drug product manufacturing unit manipulation (Example 4).

[0457] result

[0458] A viscosity target of 50 cP or less was established as a goal. The formulation viscosity screening indicated that NAR was the most effective excipient for reducing viscosity. However, due to the limited solubility of NAR, a combination of NAR and arginine HCl was used to achieve an isotonic formulation and meet the objective of a 210 mg / mL evolocumab formulation with a viscosity of 50 cP or less. A summary of the selected formulations evaluated during screening is shown in Figure 2.

[0459] A DOE study was designed to evaluate the effects of NAR / arginine HCl concentration, pH, and buffer species on the stability and viscosity of evolocumab at 210 mg / mL. Formulation variables for each sample are shown in Table 9. The study design included NAR concentrations of 155 to 175 mM, arginine HCl concentrations of 50 to 100 mM, and pH 4.8 to 5.4. The study also included a comparison between acetate and glutamate buffers at 10 mM. The study included a 140 mg / mL proline formulation control prepared from starting materials of the same lot as the NAR / Arg HCl samples and filled into a PFS. Samples were sterile filtered using a 0.2 μm PVDF filter and manually filled into a glass PFS with a filling capacity of approximately 2.0 mL.

[0460]

[0461] The data are tabled in Table 10 for pH, concentration, osmolality, and viscosity. pH values ​​were observed to be close to the target for all samples. In the study, osmolality ranged from 256 to 392 mOsm / kg. Viscosity was found to exhibit an inconsistent trend toward lower viscosity at higher pH.

[0462]

[0463] Analysis of size-exclusion high-pressure liquid chromatography (SE-HPLC) data using JMP statistical analysis software (SAS; Cary, NC; Figs. 3a and 3b) revealed the significant effects of pH and arginine HCl concentration on the loss of the major peak percentage over one month at 40°C (Fig. 3a). Lower pH and higher arginine HCl concentrations caused a greater loss of the major peak, attributed primarily to an increase in the aggregate peak and, to a lesser extent, the oligomer peak. The two peaks are combined and classified as high molecular weight (HMW) species. Fig. 4 shows the effects of pH and arginine HCl on the percentage of HMW species at the one-month, 40°C time point. Significantly higher levels of HMW species percentage were observed in the pH 4.8 sample compared to the pH 5.4 sample. Furthermore, the effect of higher arginine HCl concentrations leading to higher HMW species percentages was significantly more pronounced at pH 4.8 compared to pH 5.4.

[0464] Figures 5a to 5c show the SE-HPLC chromatograms of selected pH 4.8 and pH 5.4 samples compared to a 140 mg / mL proline control for 6 months at 5°C, 3 months at 25°C, and 1 month at 40°C, respectively. The chromatograms show a tendency for the level of aggregates to increase at pH 4.8 compared to pH 5.4. The chromatograms also indicate that, despite the faster aggregation rate observed in the 210 mg / mL NAR / Arg HCl sample, the degradation profile in the 210 mg / mL NAR / Arg HCl sample was similar to that of the 140 mg / mL proline control.

[0465] Despite the differences in pH and Arg HCl concentration dependence shown at 40°C, all formulations studied in this experiment showed similar percentages of HMW species at 5°C for 6 months and at 25°C for 1 month (Figs. 6a, 6b).

[0466] Trypsin-degraded peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis was performed on selected samples. The HPLC-UV chromatograms of the peptide maps were visually compared with the proline evolocumab formulation control and the evolocumab reference standard. No new peaks were observed in the NAR / Arg HCl samples compared to the control (Fig. 8). Furthermore, analysis of chemical modifications detected by mass spectrometry showed no significant changes between the NAR / Arg HCl samples and the control. Comparative quantification was performed, and the percentages of N55 and N33 deamidation, as well as M246 and M422 oxidation, are shown in Table 11. Slightly higher levels of N55 deamidation were observed in the pH 5.4 NAR / Arg HCl samples compared to the pH 5.1 NAR / Arg HCl and pH 5.0 proline samples, consistent with an increase in the acid peak observed by CEX. N33 (potential deamidation site in the complementarity determining region (CDR)) did not appear to show a significant increase in deamidation in the evaluated pH range and conditions. The oxidation rate in both M246 and M422 was higher in the 140 mg / mL proline control sample compared to the 210 mg / mL NAR / Arg HCl sample after incubation in ASC.

[0467]

[0468] The number of particles invisible to the naked eye, as determined by light-shielded liquid-transferred particle counts and micro-flow imaging (MFI), did not show a significant trend associated with the formulation variables studied (Figs. 9a to 9c and Fig. 10). The number of invisible particles was similar between the 210 mg / mL NAR / Arg HCl sample and the 140 mg / mL proline control.

[0469] Figure 11 compiles viscosity data from this study at zero, 3 months, and 6 months. The data indicate that viscosity remained stable for all formulations at 5°C and 25°C up to 6 months. The 40°C samples showed a pH-dependent increase in viscosity, which is associated with an increase in the percentage of HMW species observed by SE-HPLC at 40°C (Figure 4).

[0470] observation

[0471] Approximately isotonic formulation (about 300 mOsm / kg) was achieved in the study midpoint formulation (10 mM buffer, 165 mM NAR, 75 mM arginine HCl).

[0472] The increase in pH led to a decrease in the percentage of aggregates after accelerated storage conditions, as observed using size exclusion chromatography (SEC), as well as a higher percentage of acidic species and an increase in deamidation, as detected by cation exchange.

[0473] Arginine HCl concentration had a greater effect on stability at pH 4.8 (leading to increased aggregation at 25°C and 40°C), and this effect was minimized at pH 5.4.

[0474] Acetate and glutamate had similar effects on stability and viscosity.

[0475] A new peak in the NAR / Arg HCl versus proline control was not observed by peptide mapping of the selected samples during time points of 3 months at 5°C and 25°C and 1 month at 40°C.

[0476] The viscosity remains stable for up to 6 months at 5°C and 25°C.

[0477] NAR is maintained as soluble in formulations having DF buffer concentrations of up to 175 mM and Arg HCl concentrations of 50 to 100 mM at 5°C.

[0478] Example 3 - pH Study

[0479] The pH study was designed to further investigate the effects of pH over a wider range. The study included formulations with a target pH range of 5.1 to 6.9. The pH and buffer used for each sample are listed in Table 12. All samples were formulated at 210 mg / mL with 10 mM buffer, 165 mM NAR, 75 mM arginine HCl, and 0.01% polysorbate-80. Samples were sterile filtered using a 0.2 μm PVDF filter and manually filled into glass PFS with a filling capacity of 2.0 mL.

[0480]

[0481] result

[0482] The pH values ​​for each sample over a period of 3 months at zero time and 40°C are shown in Fig. 12.

[0483] Figures 13a to 13c show the effect of pH on HMW species percentage (oligomer percentage + aggregate percentage) by SE-HPLC at 4°C, 25°C, and 40°C for up to 3 months, respectively. The 5°C and 25°C data showed minimal distinction between formulations at different pH values. The most significant difference associated with pH was the higher levels of HMW species percentage from zero time in the pH 6.6 and pH 6.9 formulations. Figure 14 shows a plot of oligomer levels versus pH at zero time. A slight upward trend in oligomers was observed with increasing pH, and a sharper increase was observed above pH 6.3. Oligomer levels were minimized at lower pH values.

[0484] As previously shown in the DOE study (Example 2), the percentage of HMW species increased over time at each pH studied at 40°C, and lower pH was associated with a higher initial rate of increase.

[0485] From a comparison of SE-HPLC chromatograms at 40°C and 3 months, increased levels of aggregate peaks occurred along with a decrease in pH (Fig. 15).

[0486] Figures 16a to 16c show the effect of pH on the percentage of major peaks by CEX-HPLC at 5°C (Fig. 16a), 25°C (Fig. 16b), and 40°C (Fig. 16c) up to 6 months. At 5°C and 6 months, there was no significant change in the percentage of major peaks for samples with a pH of 6.0 or lower, while a decrease was observed in the percentage of major peaks for samples with a pH of 6.3 or higher (Fig. 16a). In Figs. 16b and 16c, CEX-HPLC data from ASC indicated that the percentage of major peaks decreased with increasing pH, and the rate of decrease was significantly accelerated at pH 6.0 or higher.

[0487] Figure 17 shows that the decrease in the pH dependence of the percentage of the major CEX peaks at 40°C is attributed to the increase in the percentage of acidic species.

[0488] A comparison of CEX-HPLC chromatograms at 25°C and 3 months is shown in Fig. 18. The percentage of the acid peak increased with increasing pH, and there was a significant change in the chromatographic profile of the sample with a pH greater than 6.3.

[0489] Trypsin-degraded peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis was performed on samples after storage at 40°C for one month. The peptide map HPLC-UV chromatograms (Fig. 19) were visually compared with reference standards across the pH range. Furthermore, analysis of chemical modifications by mass spectrometry showed no significant changes associated with pH, ​​except for N55 and N33 deamidation. Comparative quantitative levels of N55 and N33 deamidation, as well as M246 and M422 oxidation, are shown in Table 13. Increases in the levels of N55 deamidation percentage and N33 deamidation percentage were associated with an increase in the level of the acid peak percentage observed in the CEX-HPLC data along with increasing pH. For the oxidation levels in M246 or M422, pH did not appear to have a significant effect.

[0490]

[0491] The results of the biological analysis for the 1-month 40℃ sample are shown in Fig. 20 along with the initial major peak percentage (CEX-HPLC) and the N55 percentage and N33 percentage (not deamidated).

[0492] Particles invisible to the naked eye, as determined by the light-shielding liquid transfer particle count (Figs. 21a to 21c) and MFI (Fig. 22), did not show a significant trend associated with pH.

[0493] The data in Fig. 23 show that pH has minimal effect on fragmentation or other degradation as measured by reduction capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) analysis up to 6 months at 25°C. In Fig. 23, the percentage of precursor light chain plus light chain + heavy chain (pre LC + LC + HC) shows a slight decrease at the edges of the pH range. Lower pH samples contained a slightly higher percentage of medium molecular weight (MMW) species, while higher pH samples contained a slightly higher percentage of high molecular weight (HMW) species. The percentage of low molecular weight (LMW) species is similar across the pH range.

[0494] Finally, as shown in Fig. 24, there is a good linear relationship between viscosity and pH with a decrease of 6.6 cP per pH unit.

[0495] observation

[0496] Increased pH led to (1) a decrease in aggregation rate at 40°C, (2) higher initial levels of oligomers, (3) increased deamidation rates at 25°C and 40°C, and (4) lower viscosity. pH did not appear to have a significant effect on visible or invisible particles, and pH did not appear to have a significant effect on fragmentation or other degradation as measured by rCE-SDS.

[0497] Example 4 - Scale-down Study

[0498] Three formulations underwent commercial manufacturability and stability evaluation. The formulation candidates underwent various unit operations and simulated commercial manufacturing prior to submitting them for stability evaluation.

[0499] result

[0500] Physical properties were determined for each formulation of the drug substance.

[0501] The pH was determined to be about 0.17 higher than the pH of the DF buffer in each formulation. The DF buffer for formulation 3 was 0.14th from the target, which resulted in a DS pH of 0.31st from the target.

[0502] SE-HPLC (HMW species percentage) results for drug product stability at 5°C, 25°C, and 40°C are shown in Figures 25a to 25c. While the rate of increase in HMW species percentage was similar for all three samples at 5°C and 25°C, the aggregation rate at 40°C was slightly higher in Sample 1 (which had a lower pH).

[0503] CEX-HPLC data are shown in Figures 26a to 26c and show the expected increase in pH and temperature dependence of the percentage of acidic peaks observed in previous studies (see previous examples).

[0504] The rCE-SDS data shown in Figure 27 indicates similar levels of PreLC + HC + LC percentages for each scale-reduction formulation at all tested temperatures and time points.

[0505] The turbidity level did not change significantly for up to 4 months at 5℃ and 25℃, but increased after 4 months at 40℃.

[0506] The sample concentrations for the reduced scale were adjusted to 200, 210, and 220 mg / mL for each formulation. The samples m -VROC TM 90,000 sec by flow meter (RheoSense; San Ramon, CA) -1 Tests were conducted at shear rates up to and temperatures from 18°C ​​to 28°C. The data in Figure 28 covered a viscosity range of 22 to 52 cP and showed the effects of protein concentration, temperature, and formulation changes on viscosity.

[0507] observation

[0508] The levels of coagulation rate and HMW species percentage by SE-HPLC were consistent with those observed in previous studies (e.g., see previous examples).

[0509] The levels and rates of change of the major, acidic, and basic peak percentages by CEX-HPLC were consistent with those observed in previous studies.

[0510] rCE-SDS data did not show significant fragmentation or other degradation in RSC and ASC up to 3 months.

[0511] Example 5 - Ultrafiltration / dialysis filtration of evolocumab in a formulation containing N-acetyl arginine

[0512] Materials and Methods

[0513] Small-scale ultrafiltration / dialysis filtration (UF / DF) development experiments were conducted using a Pellicon equipped with an Ultracel PLCTK membrane and a 30 kD molecular weight cut-off (D screen, 0.11 m²; EMD Millipore; Billerica, MA). ® ) 3 cassettes were used. The experiment was conducted in a tangential flow filtration (TFF) process system (PendoTECH; Princeton, NJ). The experiment was performed at room temperature (22.2±2℃).

[0514] UF / DF development experiments were conducted on a small scale to evaluate three NAR formulation buffers with different pH values, and permeation flux data for their concentrations were compared. Two DF stages were used to reduce costs by decreasing NAR consumption. Additional experiments were performed in the UF1 / DF1 stage to evaluate two target concentrations (35 mg / mL and 70 mg / mL) in high molecular weight formulations during the UF / DF process. Other UF / DF operating parameters were not evaluated. The general procedure for the UF / DF experiments is described in Table 14.

[0515]

[0516] A280 Measurements were taken using a variable path length spectrophotometer (SoloVPE system; SoloVPE; Bridgewater, NJ) with an absorption coefficient of 1.5 (cm) -1 (g / L) -1 It was performed as follows.

[0517] The analytical methods used to evaluate the quality of the product pool included SE-HPLC, rCE-SDS, and CEX-HPLC.

[0518] NAR: Ac-Arg-OH, Biochem Catalog No.- E-1025

[0519] NAR formulation buffer research results

[0520] This experiment evaluated three NAR formulation buffers in the UF / DF process. The UF / DF process followed the following general guidelines: (1) Evolocumab was concentrated to 70 mg / mL via fed-batch concentration (UF1) and dialyzipped with NAR formulation buffer using 3 dialyzed volumes (DF1); (2) Protein was further concentrated to 140 mg / mL (UF2) and dialyzipped with NAR formulation buffer using 4 dialyzed volumes (DF2); (3) Protein was superconcentrated to the target of approximately 260 mg / mL and recovered from the system at 37°C. The protein loading / member area was 1468 g / m² at NAR pH 5.4. 2 It was 800 g / m² at NAR pH 5.2 and 5.6. 2 was.

[0521] Flux data were plotted against concentration and compared across all NAR formulation buffers. Additionally, samples were taken from each dialysis filtration step to analyze excipient levels, thereby studying dialysis filtration performance and determining the minimum dialysis volume.

[0522] Results and Observations

[0523] Flux data from the UF / DF study were used to generate the filtrate flux versus concentration plot shown in Figure 29. The flux profiles were similar across the three NAR formulation buffers. Flux increased as the protein buffered into the NAR formulation buffer (DF1 and DF2), but decreased significantly with increasing protein concentration (UF1, UF2, and OC). The total process time for UF / DF was approximately 20 hours at NAR pH 5.4 and 10 hours at NAR pH 5.2 and 5.6 due to different membrane areas. The similar flux profile results demonstrated that the NAR formulation buffer had no significant effect on process flux. A summary of the small-scale UF / DF study of evolocumab drug substance (DS) is shown in Table 15.

[0524] To test dialysis filtration performance and determine the minimum dialysis volume, samples were taken at each dialysis volume (1 to 7 DV) during the dialysis filtration stages (DF1 and DF2). Representative sample analyses in Table 16 are from the evolocumab NAR pH 5.4 UF / DF study. The results showed that after 5 dialysis volumes, dialysis filtration to NAR pH 5.4 was essentially completed.

[0525]

[0526]

[0527] Example 6 - Monitoring of DS concentration and HMW formation during UF / DF of evolocumab formulations

[0528] The purpose of the experiment was to evaluate target concentrations in fed-batch concentration / dialysis filtration (UF1 / DF1) and to determine target concentrations that minimize HMW formation during UF / DF operation. The UF / DF process followed the general guidelines listed in Table 17. Evolocumab (11-mg / mL) was concentrated to 35 mg / mL or 70 mg / mL via fed-batch concentration (UF1). The protein loading / member area was 800 g / m². 2The NAR formulation buffer contained 10 mM acetate, 155 mM N-acetyl arginine, 70 mM arginine HCl, and pH 5.3.

[0529] In addition, to evaluate UF / DF operation, protein samples were collected from each UF / DF stage for product pool quality analysis. Using product pool quality data, the effect of different UF1 / DF1 product concentrations on HMW formation was studied.

[0530] Materials and Methods

[0531] See Example 5.

[0532] Results and Observations

[0533] Figure 30 shows a plot comparing HWM (%) formation in the evolocumab UF / DF process (UF / DF-70 and UF / DF-35) at 35 mg / mL and 70 mg / mL in UF1 / DF1. Initially, the HWM species (%) in UF / DF-70-UF1 was 0.2% higher than in UF / DF-35-UF1, but after UF1, the HMW (%) was similar and identical at the final DS. The results indicated that HWM species are reversible and that the target concentration at UF1 had no significant effect on HWM species formation at the final DS. It is recommended to set the target concentration to 70 mg / mL to reduce the pool dose.

[0534] Example 7 - Full hold study of 3 NAR DS from small UF / DF

[0535] In this example, the stability of three batches of evolocumab formulated with three NAR formulation buffers was monitored. Each 10 mL DS sample was maintained at 2 to 8°C. At each time point shown in Figures 31a to 31f, approximately 1 mL of sample was taken for analysis and sent to 2 to 8°C.

[0536] Results and Observations

[0537] SE-HPLC and CEX results for evolocumab NAR at 2 to 8°C are shown in Figures 36 and 37. The CEX and rCE-SDS results yielded similar results. The DS batches produced from NAR pH 5.2, 5.4, and pH 5.6 contained 1.4%, 1.5%, and 1.7% HMW species, respectively, during UF / DF operation compared to 1.1% in the feed material. The percentage of HMW species is shown in Figure 32 compared to the evolocumab feed material and NAR DS over 11 weeks. All three formulation batches showed an increase of 0.4% to 0.5% in HMW (%) species. These DS batches were stable for at least 7 days at 2 to 8°C.

[0538] Example 8 - In-process full hold study for evolocumab HMP CPD1 NAR overconcentration (260 g / L) at 37°C, 39°C, 42°C, and 45°C

[0539] The stability of evolocumab superconcentrate (260 g / L) during processing was evaluated at 37°C, 39°C, 42°C, and 45°C. 30 mL of OC samples were incubated in a water bath in a room set to a controlled temperature. At each time point shown in Figures 33 and 34, approximately 1 mL of sample was taken, frozen, and placed on dry ice for analysis. Viscosity values ​​were measured for the OC samples at higher temperatures and compared with those maintained at 23°C and 25°C.

[0540] Results and Observations

[0541] SE-HPLC results showed that, as shown in Figure 33, evolocumab NAR OC was stable for 3 hours at 37°C, 39°C, and 42°C, but was not stable at 45°C. CEX and rCE-SDS results were similar. Figure 34 shows that the viscosity of OC decreased with increasing temperature, and that the viscosity dropped by 19% (14.7 cP) when the temperature increased from 37°C to 42°C.

[0542] Section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described. All literature, or parts of literature, cited in this application, including patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0543] Example 9 - Formulation Robustness DOE Study

[0544] The Design of Formulation Robustness (DOE) study was designed to investigate the effects of formulation variables within a specific design space for evolocumab stability. The study variables included evolocumab concentration, NAR concentration, Arg HCl concentration, and pH. The 18 formulations listed in Table 17 were sterile filtered using a 0.2 μm PVDF filter and filled into 2.0 mL glass pre-filled syringes. Samples were incubated at various temperatures and then tested by an array of stability indication assay methods. All formulations contained 10 mM sodium acetate and 0.01% (w / v) polysorbate-80 in addition to the components listed in Table 17.

[0545]

[0546] Table 18 shows the evolocumab concentration, pH value, and excipient concentration measured for each study formulation. All are close to the target levels listed in Table 17.

[0547]

[0548] Results and Observations

[0549] To evaluate aggregation, SE-HPLC analysis was performed on the formulations shown in Table 18 after 0 to 6 months of incubation at 4°C (Fig. 35a), 25°C (Fig. 35b), and 40°C (Figs. 35c and 35d). The data indicated that while the growth rate in HMW species % was similar for all formulations under the 4°C and 25°C conditions, a pH-dependent effect on aggregation was observed under the accelerated 40°C stress condition. It was observed that the lower pH sample at pH 5.1 had the fastest aggregation rate at 40°C, while the sample with pH 5.7 had the slowest aggregation rate (Figs. 35c and 35d). The pH 5.4 sample was observed to have an intermediate aggregation rate between pH 5.1 and 5.7 at 40°C, but these rates were more similar to the lower aggregation rates observed in the pH 5.7 sample (Figs. 35c and 35d).

[0550] The effect of pH on the increase in the % acidic peak and % basic peak over time (up to 3 months) was examined using CEX-HPLC, and the data are presented in Figs. 36a (acidic peak) and 36b (basic peak) for 4°C; Figs. 36c (acidic peak) and 36d (basic peak) for 25°C; and Figs. 36e (acidic peak) and 36f (basic peak) for 40°C. As shown in the data for the 25°C and 40°C samples (Figs. 36c to 36f), it was observed that pH affects the % acidic and % basic peaks in the formulation. Data for the pH 5.7 sample were closely grouped with a higher rate of increase in the % acidic peak, while data for the pH 5.1 sample showed the lowest rate of increase. Two samples at pH 5.4 were observed to have a decomposition rate just between the observed rates of these samples at pH 5.1 and pH 5.7. Trends in the % change of the basic peak appeared less distinct, but a trend toward higher % levels of the basic peak at lower pH was observed from the samples incubated at 40°C.

[0551] Fragmentation or other degradation of evolocumab in the formulation at 30°C or 40°C for up to 3 months was analyzed using rCE-SDS and SE-HPLC analysis; Figures 37a to 37b present data for samples incubated at 30°C, and Figures 37c to 37d show data for samples incubated at 40°C. Within the design space of this study, no significant trends related to the formulation composition were observed in the rCE-SDS data.

[0552] The presence and amount of particles invisible to the naked eye were determined in the formulations over time up to 3 months, as determined by light-shielding particle counts using HIAC after incubation at 4°C and 40°C; these data are shown in Figures 38a to 38d. (Figures 38a (particles larger than 10 μm) and 38b (particles larger than 25 μm) show results for samples maintained at 4°C; Figures 38c (particles larger than 10 μm) and 38d (particles larger than 25 μm) show results for samples maintained at 40°C). From these data, no significant trend in the number of particles invisible to the naked eye was observed in relation to the formulation variables studied.

[0553] From this example, the data demonstrated that no significant effect was observed on stability during storage at 4°C or 25°C within the narrow formulation design space studied. Data related to pH at 40°C suggested that pH affects the aggregation rate detected by SE-HPLC, as well as the percentage of acidic species detected by CEX-HPLC and, to a lesser extent, the percentage of basic species.

[0554] Exemplary Implementation Example

[0555] Exemplary embodiments of pharmaceutical compositions comprising evolocumab are disclosed herein, wherein the composition comprises N-acetyl arginine and the pharmaceutical composition has a viscosity of at least about 80 cP (measured by a flow meter, e.g., a TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter). Additionally, a method for formulating a therapeutic polypeptide such as evolocumab is disclosed herein, wherein the composition comprises NAR.

[0556] Example 1 of Implementation : In a pharmaceutical composition comprising the following:

[0557] a. PCSK9-binding polypeptides selected from the group consisting of the following:

[0558] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0559] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0560] iii. Monoclonal antibodies comprising the following:

[0561] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0562] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0563] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0564] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0565] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0566] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0567] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0568] and

[0569] b. N-acetyl arginine,

[0570] A pharmaceutical composition having a viscosity of at least about 80 cP or less.

[0571] Implementation Example 2 : In Embodiment 1, the pharmaceutical composition wherein the PCSK9-binding polypeptide is a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0572] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0573] b. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0574] Implementation Example 3 : In Embodiment 2 or 3, the pharmaceutical composition is a pharmaceutical composition having a viscosity of at least less than about 50 cP.

[0575] Implementation Example 4 : In Embodiment 2 or 3, the pharmaceutical composition has an osmotic concentration of about 250 to about 400 mOsm / kg.

[0576] Implementation Example 5: In Embodiment 4, the pharmaceutical composition is a pharmaceutical composition having an osmotic concentration of about 300 mOsm / kg.

[0577] Implementation Example 6 : In Embodiment 5, the pharmaceutical composition is an isotonic pharmaceutical composition with human blood cells.

[0578] Implementation Example 7 : A pharmaceutical composition in Embodiment 1 in which the PCSK9-binding polypeptide is present at a concentration of about 140 mg / mL to about 260 mg / mL.

[0579] Example 8 : A pharmaceutical composition in which the PCSK9-binding polypeptide concentration in Embodiment 2 is about 210 mg / mL.

[0580] Implementation Example 9 : A pharmaceutical composition in any one of embodiments 1 to 8, wherein N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM.

[0581] Example 10 : A pharmaceutical composition in Embodiment 9 in which N-acetyl arginine is present at a concentration of about 140 mM to about 170 mM.

[0582] Example 11 of implementation : A pharmaceutical composition in Embodiment 10 in which N-acetyl arginine is present at a concentration of about 140 mM.

[0583] Example 12 : A pharmaceutical composition comprising a buffer additionally in any one of embodiments 1 to 11.

[0584] Example 13 : In Embodiment 13, the buffer is a pharmaceutical composition selected from the group consisting of acetate, glutamate, histidine, and phosphate buffers, or a combination thereof.

[0585] Example 14 : A pharmaceutical composition in Embodiment 13, wherein the buffer is present at a concentration of about 5 mM to about 30 mM.

[0586] Example 15 of implementation : In Embodiment 14, the buffer is sodium acetate and is a pharmaceutical composition present at a concentration of about 10 mM.

[0587] Example 16 : A pharmaceutical composition in which, in any one of Embodiments 1 to 15, the pH is about 4.8 to about 6.9.

[0588] Example 17 of implementation : A pharmaceutical composition in which the pH of Embodiment 11 is approximately 5.4.

[0589] Example 18 : A pharmaceutical composition comprising a surfactant additionally in any one of Embodiments 1 to 17.

[0590] Example 19 : In Embodiment 18, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ® A pharmaceutical composition selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

[0591] Example 20 : A pharmaceutical composition in which the surfactant is present at a concentration of about 0.0001% (w / v) to about 1% (w / v) in Embodiment 19.

[0592] Example 21 of implementation : A pharmaceutical composition in Embodiment 20, wherein the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of about 0.01% (w / v).

[0593] Example 22 : A pharmaceutical composition comprising additionally proline in any one of Embodiments 1 to 21.

[0594] Implementation Example 23 : A pharmaceutical composition in Embodiment 22 in which proline is present at a concentration of about 50 mM to about 150 mM.

[0595] Example 24 : A pharmaceutical composition in Embodiment 23 in which proline is present at a concentration of about 90 mM to about 120 mM.

[0596] Example 25 : A pharmaceutical composition in Embodiment 22 or 23 in which proline is present at a concentration of about 120 mM.

[0597] Example 26 : A pharmaceutical composition comprising an arginine salt further comprising, in any one of Embodiments 1 to 25.

[0598] Example 27 : A pharmaceutical composition in Embodiment 26, wherein the arginine salt is present at a concentration of about 25 mM to about 150 mM.

[0599] Example 28 : A pharmaceutical composition in Embodiment 27, wherein the arginine salt is present at a concentration of about 50 mM to about 100 mM.

[0600] Example 29 : A pharmaceutical composition in Embodiment 26, wherein the arginine salt is arginine-HCl, arginine acetate, or arginine glutamate.

[0601] Example 30 : A pharmaceutical composition in Embodiment 29 in which arginine HCl is present at a concentration of about 50 mM.

[0602] Implementation Example 31 : A pharmaceutical composition in any one of Embodiments 1 to 30 in which the PCSK9-binding polypeptide is stable for at least about 2 years when stored at about -30℃ or lower.

[0603] Implementation Example 32 : In Embodiment 31, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 5 years.

[0604] Implementation Example 33 : A pharmaceutical composition in any one of Embodiments 1 to 30 in which the PCSK9-binding polypeptide is stable for at least about 6 months when stored at about 5°C.

[0605] Example 34 : In Embodiment 33, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 24 months.

[0606] Example 35 of implementation : A pharmaceutical composition in any one of Embodiments 1 to 30 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 25°C.

[0607] Example 36 : In Embodiment 35, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 3 months.

[0608] Example 37 : In Embodiment 35, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 6 months.

[0609] Example 38 : A pharmaceutical composition in any one of Embodiments 1 to 30 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 40°C.

[0610] Example 39 : A pharmaceutical composition in any one of embodiments 1 to 38, wherein the composition comprises a high molecular weight aggregate or oligomer of PCSK9-linked polypeptide in an amount of less than about 3% of the PCSK9-linked polypeptide concentration.

[0611] Example 40 : A pharmaceutical composition in which, in Embodiment 39, the high molecular weight aggregate or oligomer of PCSK9-linked polypeptide is present at a concentration of less than about 2.5% of PCSK9-linked polypeptide.

[0612] Example 41 of implementation : In a pharmaceutical composition comprising the following:

[0613] a. PCSK9-binding polypeptides selected from the group consisting of the following:

[0614] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0615] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0616] iii. Monoclonal antibodies comprising the following:

[0617] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0618] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0619] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0620] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0621] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0622] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0623] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0624] and

[0625] b. N-acetyl arginine;

[0626] c. Arginine salt;

[0627] d. buffer; and

[0628] e. Surfactants,

[0629] A pharmaceutical composition having a viscosity of at least about 80 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0630] Implementation Example 42: In Embodiment 41, the pharmaceutical composition wherein the PCSK9-binding polypeptide is a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0631] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0632] b. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0633] Implementation Example 43 : In Embodiment 41 or 42, the pharmaceutical composition is a pharmaceutical composition having a viscosity of at least less than about 50 cP.

[0634] Implementation Example 44 : In Embodiment 41 or 42, the pharmaceutical composition has an osmotic concentration of about 250 to about 400 mOsm / kg.

[0635] Example 45 : In Embodiment 44, the pharmaceutical composition is a pharmaceutical composition having an osmotic concentration of about 300 mOsm / kg.

[0636] Implementation Example 46 : In Embodiment 45, the pharmaceutical composition is an isotonic pharmaceutical composition with human blood cells.

[0637] Example 47 : A pharmaceutical composition according to Embodiment 41 or 42, wherein the PCSK9-binding polypeptide is present at a concentration of about 140 mg / mL to about 260 mg / mL.

[0638] Example 48 : A pharmaceutical composition in which the PCSK9-binding polypeptide concentration in Embodiment 47 is about 210 mg / mL.

[0639] Example 49 : A pharmaceutical composition in any one of embodiments 41 to 48, wherein N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM.

[0640] Example 50 : A pharmaceutical composition in Embodiment 49 in which N-acetyl arginine is present at a concentration of about 140 mM to about 170 mM.

[0641] Example 51 of implementation : A pharmaceutical composition in Embodiment 50 in which N-acetyl arginine is present at a concentration of about 140 mM.

[0642] Example 52 : In Embodiment 41 or 42, the buffer is a pharmaceutical composition selected from the group consisting of acetate, glutamate, histidine, and phosphate buffers, or a combination thereof.

[0643] Example 53 : A pharmaceutical composition in Embodiment 52, wherein the buffer is present at a concentration of about 5 mM to about 30 mM.

[0644] Example 54 : A pharmaceutical composition in Embodiment 53, wherein the buffer is sodium acetate and is present at a concentration of about 10 mM.

[0645] Example 55 : A pharmaceutical composition according to Embodiment 41 or 42, wherein the pH is about 4.8 to about 6.9.

[0646] Example 56 : A pharmaceutical composition in which the pH of Embodiment 55 is approximately 5.4.

[0647] Example 57 : In Embodiment 41 or 42, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ®A pharmaceutical composition selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

[0648] Example 58 : A pharmaceutical composition in which the surfactant is present at a concentration of about 0.0001% (w / v) to about 1% (w / v) in Embodiment 57.

[0649] Example 59 : A pharmaceutical composition in Embodiment 57, wherein the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of about 0.01% (w / v).

[0650] Example 60 : A pharmaceutical composition comprising additionally proline in Embodiment 41 or 42.

[0651] Example 61 of implementation : A pharmaceutical composition in Embodiment 60, wherein proline is present at a concentration of about 50 mM to about 150 mM.

[0652] Example 62 : A pharmaceutical composition in Embodiment 61 in which proline is present at a concentration of about 90 to about 120 mM.

[0653] Example 63 : A pharmaceutical composition in which proline is present at a concentration of about 120 mM in Embodiment 62.

[0654] Example 64 : A pharmaceutical composition according to Embodiment 41 or 42, wherein the arginine salt is present at a concentration of about 25 mM to about 150 mM.

[0655] Example 65 : A pharmaceutical composition in Embodiment 64, wherein the arginine salt is present at a concentration of about 50 mM to about 100 mM.

[0656] Example 66: A pharmaceutical composition in Embodiment 64, wherein the arginine salt is arginine-HCl, arginine acetate, or arginine glutamate.

[0657] Example 67 : A pharmaceutical composition in Embodiment 66 in which arginine HCl is present at a concentration of about 50 mM.

[0658] Example 68 : A pharmaceutical composition in any one of embodiments 41 to 67 in which the PCSK9-binding polypeptide is stable for at least about 2 years when stored at about -30°C or lower.

[0659] Example 69 : In Embodiment 68, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 5 years.

[0660] Example 70 : A pharmaceutical composition in any one of embodiments 41 to 67 in which the PCSK9-binding polypeptide is stable for at least about 6 months when stored at about 5°C.

[0661] Example 71 of implementation : In Embodiment 70, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 24 months.

[0662] Example 72 : A pharmaceutical composition in any one of embodiments 41 to 67 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 25°C.

[0663] Example 73 : In Embodiment 72, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 3 months.

[0664] Example 74 : In Embodiment 73, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 6 months.

[0665] Example 75: A pharmaceutical composition in any one of embodiments 41 to 67 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 40°C.

[0666] Example 76 : A pharmaceutical composition in any one of embodiments 41 to 75, wherein the composition comprises a high molecular weight aggregate or oligomer of PCSK9-linked polypeptide at a concentration of less than about 3% of PCSK9-linked polypeptide.

[0667] Example 77 : A pharmaceutical composition in which, in Embodiment 76, the high molecular weight aggregate or oligomer of PCSK9-linked polypeptide is present at a concentration of less than about 2.5% of PCSK9-linked polypeptide.

[0668] Example 78 : Pharmaceutical composition comprising the following:

[0669] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0670] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0671] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0672] iii. Monoclonal antibodies comprising the following:

[0673] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0674] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0675] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0676] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0677] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0678] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0679] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0680] b. N-acetyl arginine present at a concentration of approximately 140 mM;

[0681] c. Arginine HCl present at a concentration of approximately 50 mM;

[0682] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0683] e. Sodium acetate at a concentration of approximately 10 mM.

[0684] Example 79 : In Embodiment 78, the pharmaceutical composition is a pharmaceutical composition having a pH of about 5.1 to about 5.7.

[0685] Example 80 : In Embodiment 78 or 79, the pharmaceutical composition has a viscosity of at least about 50 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0686] Example 81 : Pharmaceutical composition comprising the following:

[0687] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0688] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0689] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0690] iii. Monoclonal antibodies comprising the following:

[0691] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0692] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0694] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0695] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0696] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0697] 2. Sequence No. 2, the variable region of the light chain of the amino acid sequence,

[0698] and

[0699] b. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0700] c. N-acetyl arginine present at a concentration of approximately 140 mM;

[0701] d. Arginine HCl present at a concentration of approximately 63 mM;

[0702] e. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0703] f. Sodium acetate at a concentration of approximately 10 mM.

[0704] Example 82 : In Embodiment 81, the pharmaceutical composition is a pharmaceutical composition having a pH of about 5.1 to about 5.7.

[0705] Example 83 : In Embodiment 81 or 82, the pharmaceutical composition has a viscosity of at least about 80 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0706] Example 84 : Pharmaceutical composition comprising the following:

[0707] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0708] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0709] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0710] iii. Monoclonal antibodies comprising the following:

[0711] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0712] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0714] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0715] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0716] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0717] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0718] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0719] b. N-acetyl arginine present at a concentration of approximately 155 mM;

[0720] c. Arginine HCl present at a concentration of approximately 70 mM;

[0721] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0722] e. Sodium acetate at a concentration of approximately 10 mM.

[0723] Example 85 : In Embodiment 84, the pharmaceutical composition is a pharmaceutical composition having a pH of about 5.1 to about 5.7.

[0724] Example 86: In Embodiment 84 or 85, the pharmaceutical composition has a viscosity of at least about 45 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0725] Example 87 : Pharmaceutical composition comprising the following:

[0726] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0727] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0728] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0729] iii. Monoclonal antibodies comprising the following:

[0730] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0731] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0733] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0734] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0735] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0736] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0737] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0738] b. N-acetyl arginine present at a concentration of approximately 170 mM;

[0739] c. Arginine HCl present at a concentration of approximately 63 mM;

[0740] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0741] e. Sodium acetate at a concentration of approximately 10 mM.

[0742] Example 88 : In Embodiment 87, the pharmaceutical composition is a pharmaceutical composition having a pH of about 5.1 to about 5.7.

[0743] Example 89: In Embodiment 87 or 88, the pharmaceutical composition has a viscosity of at least about 60 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0744] Example 90 : Pharmaceutical composition comprising the following:

[0745] a. PCSK9-binding polypeptide at a concentration of about 195 to 225 mg / mL selected from the group consisting of the following:

[0746] i. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0747] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0748] iii. Monoclonal antibodies comprising the following:

[0749] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0750] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0752] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

[0753] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0754] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0755] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0756] 3. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR;

[0757] b. N-acetyl arginine present at a concentration of approximately 155 mM;

[0758] c. Proline present at a concentration of approximately 120 mM;

[0759] d. Polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of about 0.005% (w / v) to about 0.015% (w / v); and

[0760] e. Sodium acetate at a concentration of approximately 10 mM.

[0761] Example 91 of implementation : In Embodiment 90, the pharmaceutical composition is a pharmaceutical composition having a pH of about 5.1 to about 5.7.

[0762] Example 92: In Embodiment 90 or 91, the pharmaceutical composition has a viscosity of at least about 60 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0763] Example 93 : A pharmaceutical composition in any one of embodiments 78 to 92 in which the PCSK9-binding polypeptide is stable for at least about 2 years when stored at about -30°C or lower.

[0764] Example 94 : In Embodiment 93, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 5 years.

[0765] Example 95 of implementation : A pharmaceutical composition in any one of embodiments 78 to 92 in which the PCSK9-binding polypeptide is stable for at least about 6 months when stored at about 5°C.

[0766] Example 96 : In Embodiment 95, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 24 months.

[0767] Example 97 : A pharmaceutical composition in any one of embodiments 78 to 92 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 25°C.

[0768] Example 98 : In Embodiment 97, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 3 months.

[0769] Example 99 : In Embodiment 98, the PCSK9-binding polypeptide is a pharmaceutical composition that is stable for at least about 6 months.

[0770] Example 100: A pharmaceutical composition in any one of embodiments 78 to 92 in which the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 40°C.

[0771] Example 101 : A pharmaceutical composition in any one of embodiments 78 to 92, wherein the composition comprises a high molecular weight aggregate or oligomer of PCSK9-linked polypeptide at a concentration of less than about 3% of the PCSK9-linked polypeptide.

[0772] Example 102 : A pharmaceutical composition in which, in Embodiment 101, the high molecular weight aggregate or oligomer of PCSK9-linked polypeptide is present at a concentration of less than about 2.5% of PCSK9-linked polypeptide.

[0773] Example 103 : A pharmaceutical composition in any one of Embodiments 1 to 102, wherein the pharmaceutical composition is a liquid.

[0774] Example 104 : A method for treating a target requiring the same, comprising the step of administering a pharmaceutical composition of any one of Embodiments 1 to 103.

[0775] Example 105 A kit comprising a pharmaceutical composition of any one of embodiments 1 to 103, and a delivery mechanism selected from the group consisting of a syringe, an injection pen, a body syringe, and a self-injector.

[0776] Example 106 : A kit according to Embodiment 105, further comprising instructions for administering a pharmaceutical composition using a delivery mechanism.

[0777] Example 107A method for preparing a PCSK9-binding polypeptide in a pharmaceutical composition comprising at least 140 mg / mL of PCSK9-binding polypeptide, comprising the step of adding an effective amount of N-acetyl arginine to a pharmaceutical composition comprising a PCSK9-binding polypeptide such that the viscosity of the pharmaceutical composition is reduced compared to a pharmaceutical composition lacking N-acetyl arginine, wherein the PCSK9-binding polypeptide is selected from the group consisting of the following:

[0778] a. A monoclonal antibody (evolocumab) comprising a heavy chain polypeptide having the amino acid sequence of SEQ ID NO: 1 and a light chain polypeptide having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0779] b. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0780] c. Monoclonal antibodies containing the following:

[0781] i. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0782] ii. Light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0783] d. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238; and

[0784] e. A monoclonal antibody that binds to PCSK9 at an epitope in PCSK9 that overlaps with the epitope bound by the antibody comprising the following:

[0785] iii. The variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0786] iv. The light chain variable region of the amino acid sequence in SEQ ID NO: 2, and

[0787] v. Here, the epitope of the monoclonal antibody further overlaps with the site in the EGF-A domain of LDLR.

[0788] Example 108 : In Embodiment 107, the viscosity of the pharmaceutical composition is less than 80 cP (measured by a flow meter, e.g., TA Instruments (New Castle, DE) AESR-G2 cone and plate flow meter).

[0789] Example 109 : A method in Embodiment 108 in which the viscosity of the pharmaceutical composition is less than 50 cP.

[0790] Example 110 : A method in any one of embodiments 107 to 109, wherein the pharmaceutical composition has an osmotic concentration of about 250 to about 400 mOsm / kg.

[0791] Example 111: In Embodiment 110, the pharmaceutical composition has an osmotic concentration of about 300 mOsm / kg.

[0792] Example 112 : In Embodiment 111, the pharmaceutical composition is isotonic with human blood cells.

[0793] Example 113 : A method in which, in Embodiment 107, the PCSK9-binding polypeptide is present at a concentration of about 180 mg / mL to about 260 mg / mL.

[0794] Example 114 : A method in which, in Embodiment 113, the PCSK9-binding polypeptide concentration is about 210 mg / mL.

[0795] Example 115 : A method in any one of embodiments 107 to 114, wherein N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM.

[0796] Example 116 : A method in which, in Embodiment 115, N-acetyl arginine is present at a concentration of about 140 mM to about 170 mM.

[0797] Example 117 : In Embodiment 115, a method in which N-acetyl arginine is present at a concentration of about 140 mM.

[0798] Example 118 : A method comprising an additional buffer in any one of embodiments 107 to 117.

[0799] Example 119 : In Embodiment 118, the buffer is selected from the group consisting of acetate, glutamate, histidine, and phosphate buffers, or a combination thereof.

[0800] Example 120 : In Embodiment 119, the buffer is present at a concentration of about 5 mM to about 30 mM.

[0801] Example 121: In Embodiment 120, the buffer is sodium acetate and is present at a concentration of about 10 mM.

[0802] Example 122 : A method in any one of embodiments 107 to 121, wherein the pH is about 4.8 to about 6.9.

[0803] Implementation Example 123 : In Embodiment 122, the method in which the pH is approximately 5.4.

[0804] Example 124 : A method comprising a surfactant additionally in any one of embodiments 107 to 123.

[0805] Example 125 : In Embodiment 124, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ® A method selected from the group consisting of polyethylene glycol octylphenyl ether (Triton X-100), polyethylene glycol alkyl ether (Brij), polypropylene glycol alkyl ether, glucoside alkyl ether, and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

[0806] Example 126 : In Embodiment 125, the surfactant is present at a concentration of about 0.0001% (w / v) to about 1.0% (w / v).

[0807] Example 127 : In Embodiment 126, the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of about 0.01% (w / v).

[0808] Example 128: A method comprising additionally proline in any one of embodiments 107 to 127.

[0809] Example 129 : In Embodiment 128, the method in which proline is present at a concentration of about 50 mM to about 150 mM.

[0810] Example 130 : In Embodiment 129, the method in which proline is present at a concentration of about 90 mM to about 120 mM.

[0811] Example 131 : In Embodiment 130, the method in which proline is present at a concentration of about 120 mM.

[0812] Example 132 : A method comprising additionally an arginine salt in any one of embodiments 107 to 131.

[0813] Example 133 : In Embodiment 132, the method in which the arginine salt is present at a concentration of about 25 mM to about 150 mM.

[0814] Example 134 : In Embodiment 133, the method in which the arginine salt is present at a concentration of about 50 mM to about 100 mM.

[0815] Example 135 : In Embodiment 133, the arginine salt is arginine-HCl, arginine acetate, or arginine glutamate.

[0816] Example 136 : In Embodiment 135, a method in which arginine HCl is present at a concentration of about 50 mM.

[0817] Example 137 : A method in which, in any one of embodiments 107 to 136, the PCSK9-binding polypeptide is stable for at least about 2 years when stored at about -30°C or lower.

[0818] Example 138 : In Embodiment 137, the PCSK9-binding polypeptide is stable for at least about 5 years.

[0819] Example 139 : A method in which, in any one of embodiments 107 to 136, the PCSK9-binding polypeptide is stable for at least about 6 months when stored at about 5°C.

[0820] Example 140 : In Embodiment 139, the PCSK9-binding polypeptide is stable for at least about 24 months.

[0821] Example 141 : A method in which, in any one of embodiments 107 to 136, the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 25°C.

[0822] Example 142 : In Embodiment 141, the PCSK9-binding polypeptide is stable for at least about 3 months.

[0823] Example 143 : In Embodiment 142, the PCSK9-binding polypeptide is stable for at least about 6 months.

[0824] Example 144 : A method in which, in any one of embodiments 107 to 136, the PCSK9-binding polypeptide is stable for at least about 1 month when stored at about 40°C.

[0825] Example 145 : A method in any one of embodiments 107 to 136, wherein the composition comprises a high molecular weight aggregate or oligomer of PCSK9-linked polypeptide in an amount of less than about 3% of the PCSK9-linked polypeptide concentration.

[0826] Example 146 : In Embodiment 145, the high molecular weight aggregate or oligomer of PCSK9-linked polypeptide is present in an amount of less than about 2.5% of the PCSK9-linked polypeptide concentration.

[0827] Example 147 : A method for formulating a therapeutic polypeptide comprising the following:

[0828] a. A first concentration step in which polypeptides are concentrated in a first solution;

[0829] b. A first solution exchange step in which a polypeptide concentrated in a first solution is exchanged with a second solution containing N-acetyl arginine using dialysis filtration;

[0830] c. A second concentration step in which polypeptides are concentrated in the second solution;

[0831] d. A second solution exchange step in which the polypeptide in the concentrated second solution is exchanged with a third solution containing N-acetyl arginine using dialysis filtration; and

[0832] e. A third concentration step in which polypeptides are concentrated in the third solution;

[0833] A method comprising a therapeutic polypeptide that blocks the binding of PCSK9 to LDLR and a PCSK9-binding polypeptide selected from the group consisting of the following:

[0834] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0835] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0836] iii. Monoclonal antibodies comprising the following:

[0837] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0838] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0839] i. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0840] ii. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0841] 3. The variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0842] 4. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0843] 5. Here, the epitope of the monoclonal antibody additionally overlaps with the site in the EGFa domain of LDLR.

[0844] Example 148 : In Embodiment 147, the PCSK9-binding polypeptide that blocks the binding of PCSK9 to LDLR is a monoclonal antibody comprising a heavy chain polypeptide having the following complementarity determining region (CDR):

[0845] a. Heavy chains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and

[0846] b. Light chains CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0847] Example 149: A method in which, prior to the third concentration step, the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C.

[0848] Example 150 : In Embodiment 147, the first solution exchange step is achieved using at least 3 diavolumes of the second solution.

[0849] Example 151 : In Embodiment 147, the second solution exchange step is achieved by using at least 4 diavolumes of the third solution.

[0850] Example 152 : A method in which, in Embodiment 147, the initial concentration of the therapeutic protein is about 11 mg / mL or less.

[0851] Example 153 : A method in which, in the first concentration step of Embodiment 147, the therapeutic polypeptide concentration is increased by about 3 to about 7 times.

[0852] Example 154 : A method in Embodiment 153 in which the increased concentration of polypeptide is about 35 mg / mL to about 70 mg / mL.

[0853] Example 155 : A method in which, in the second concentration step, the therapeutic polypeptide concentration is increased by about 2 to 4 times from the first concentration step in the second concentration step in the embodiment 147.

[0854] Example 156 : A method in which, in Embodiment 155, the increased polypeptide concentration is about 140 mg / mL.

[0855] Example 157 : A method in which, in Embodiment 147, the therapeutic polypeptide concentration in the third concentration step is increased by about 1.5 to about 2 times from the second concentration step.

[0856] Example 158 : A method in which, in Embodiment 157, the increased polypeptide concentration is about 260 mg / mL.

[0857] Example 159 : In Embodiment 147, a method wherein the therapeutic polypeptide has a final concentration that is at least about 19 to 20 times more concentrated than the initial concentration of the therapeutic polypeptide.

[0858] Example 160 : A method in which, in Embodiment 159, the final concentration of the therapeutic polypeptide is about 210 mg / mL.

[0859] Example 161 : In Embodiment 147, the concentration step comprises a fed-batch ultrafiltration method.

[0860] Example 162 : In Embodiment 147, the second solution and the third solution are the same method.

[0861] Example 163 : In Embodiment 147, the second or third solution containing N-acetyl arginine comprises an arginine salt and a buffer.

[0862] Example 164 : In Embodiment 163, N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM; the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and is present at a concentration of about 25 mM to about 150 mM; and the buffer is sodium acetate buffer at a concentration of about 5 mM to about 30 mM.

[0863] Example 165 : A method according to Embodiment 164, wherein N-acetyl arginine is present at a concentration of about 140 to about 170 mM; Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 to about 70 mM; and sodium acetate buffer is present at a concentration of about 10 mM.

[0864] Example 166: A method in Embodiment 164 in which N-acetyl arginine is present at a concentration of about 140 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0865] Example 167 : A method in which, in Embodiment 164, N-acetyl arginine is present at a concentration of about 155 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0866] Example 168 : A method in which, in Embodiment 164, N-acetyl arginine is present at a concentration of about 170 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0867] Example 169 : A method according to Embodiment 165 or 166, further comprising proline, wherein the proline is present at a concentration of about 50 mM to about 150 mM.

[0868] Example 170 : A method in any one of embodiments 163 to 169, wherein the second or third solution has a pH of about 4.8 to about 6.9.

[0869] Example 171 : In Embodiment 170, the method in which the pH is approximately 5.4.

[0870] Example 172 : In Embodiment 147, a method in which a dialysis filtration membrane having at least one feature selected from the group consisting of the following is used in the first and second solution exchange steps:

[0871] a. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0872] b. An opening area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

[0873] c. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0874] d. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0875] e. Standard weight exceeding approximately 160 g / ㎡ but 180 g / ㎡ or less;

[0876] f. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0877] g. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0878] h. Maximum supply pressure of approximately 60 psi.

[0879] Example 173 : A method in which a surfactant is added to a third solution after the third solution is concentrated, in any one of embodiments 147 to 172.

[0880] Example 174 : In Embodiment 173, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ® A method comprising a polyethylene glycol octylphenyl ether (Triton X-100), a polyethylene glycol alkyl ether (Brij), a polypropylene glycol alkyl ether, a glucoside alkyl ether, and a D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS), present at a concentration of about 0.0001% to about 1%.

[0881] Example 175: In Embodiment 175, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v).

[0882] Example 176 : A method for formulating a therapeutic polypeptide comprising the following:

[0883] a. A first concentration step in which polypeptides in a first solution are concentrated using fed-batch ultrafiltration;

[0884] b. A first solution exchange step in which the polypeptide concentrated in the first solution is exchanged with a second solution containing N-acetyl arginine, an arginine salt, and a buffer using dialysis filtration and three dialysis volumes of the second solution;

[0885] c. A second concentration step in which polypeptides in the second solution are concentrated using fed-batch ultrafiltration;

[0886] d. A second solution exchange step in which the polypeptide in the concentrated second solution is exchanged with a third solution containing N-acetyl arginine, an arginine salt, and a buffer using dialysis filtration and 4 dialysis volumes of the third solution;

[0887] e. A step in which the temperature of the solution containing the polypeptide increases from about 25°C to about 37°C after the second solution exchange step; and

[0888] f. A third concentration step in which the polypeptide is further concentrated using fed-batch ultrafiltration concentration;

[0889] In the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following is used:

[0890] g. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0891] h. An opening area that is greater than approximately 32% of the membrane area but less than or equal to approximately 36%;

[0892] i. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0893] j. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0894] k. Standard weight exceeding approximately 160 g / ㎡ but not exceeding 180 g / ㎡;

[0895] l. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0896] m. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0897] n. Maximum supply pressure of approximately 60 psi;

[0898] A method comprising a therapeutic polypeptide that blocks the binding of PCSK9 to LDLR and a PCSK9-binding polypeptide selected from the group consisting of the following:

[0899] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0900] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0901] iii. Monoclonal antibodies comprising the following:

[0902] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0903] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0904] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0905] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0906] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0907] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0908] 3. Here, the epitope of the monoclonal antibody additionally overlaps with the site in the EGFa domain of LDLR.

[0909] Example 177 : A method in which, prior to the third concentration step, the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C.

[0910] Example 178 : A method in which, in Embodiment 176, the initial concentration of the therapeutic protein is 11 mg / mL or less.

[0911] Example 179 : A method in Embodiment 176 in which the therapeutic polypeptide concentration is increased by about 3 to about 7 times in the first concentration step.

[0912] Example 180: A method in Embodiment 179 in which the increased concentration of polypeptide is about 35 mg / mL to about 70 mg / mL.

[0913] Example 181 : A method in Embodiment 176 in which the therapeutic polypeptide concentration in the second concentration step is increased by about 2 to 4 times from the first concentration step.

[0914] Example 182 : A method in which, in Embodiment 181, the increased polypeptide concentration is about 140 mg / mL.

[0915] Example 183 : A method in which, in the third concentration step, the therapeutic polypeptide concentration is increased by about 1.5 to about 2 times from the second concentration step.

[0916] Example 184 : A method in which, in Embodiment 183, the increased polypeptide concentration is about 260 mg / mL.

[0917] Example 185 : In Embodiment 176, a method wherein the therapeutic polypeptide has a final concentration that is at least about 19 to 20 times more concentrated than the initial concentration of the therapeutic polypeptide.

[0918] Example 186 : A method in which, in Embodiment 185, the final concentration of the therapeutic polypeptide is about 210 mg / mL.

[0919] Example 187 : In Embodiment 176, N-acetyl arginine is present at a concentration of about 25 mM to about 230 mM; the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and is present at a concentration of about 25 mM to about 150 mM; and the buffer is sodium acetate buffer at a concentration of about 5 mM to about 30 mM.

[0920] Example 188: A method according to Embodiment 188, wherein N-acetyl arginine is present at a concentration of about 140 to about 170 mM; Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 to about 70 mM; and sodium acetate buffer is present at a concentration of about 10 mM.

[0921] Example 189 : In Embodiment 188, a method in which N-acetyl arginine is present at a concentration of about 140 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0922] Example 190 : In Embodiment 188, a method in which N-acetyl arginine is present at a concentration of about 155 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 70 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0923] Example 191 : In Embodiment 188, a method in which N-acetyl arginine is present at a concentration of about 170 mM, Arg HCl, Arg acetate, or Arg glutamate is present at a concentration of about 63 mM, and sodium acetate buffer is present at a concentration of about 10 mM.

[0924] Example 192 : A method according to Embodiment 176, further comprising proline, wherein the proline is present at a concentration of about 50 mM to about 150 mM.

[0925] Example 193 : A method in any one of embodiments 176 to 192, wherein the second or third solution has a pH of about 4.8 to about 6.9.

[0926] Example 194 : In Embodiment 193, the method in which the pH is approximately 5.4.

[0927] Example 195: A method in which a surfactant is added to a third solution after the third solution is concentrated, in any one of embodiments 176 to 194.

[0928] Example 196 : In Embodiment 195, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamer, e.g., Pluronic ® ) F-68 and other Pluronic ® )), sorbitan alkyl ester (Span(Span ® A method comprising a polyethylene glycol octylphenyl ether (Triton X-100), a polyethylene glycol alkyl ether (Brij), a polypropylene glycol alkyl ether, a glucoside alkyl ether, and a D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS), present at a concentration of about 0.0001% to about 1%.

[0929] Example 197 : In Embodiment 196, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of about 0.01% (w / v).

[0930] Example 198 : A method for formulating a therapeutic polypeptide comprising the following:

[0931] a. A first concentration step in which polypeptides in a first solution are concentrated using fed-batch ultrafiltration;

[0932] b. A first solution exchange step in which the polypeptide concentrated in the first solution is exchanged to the second solution using dialysis filtration and three dialysis volumes of the second solution;

[0933] c. A second concentration step in which polypeptides in the second solution are concentrated using fed-batch ultrafiltration;

[0934] d. A second solution exchange step in which polypeptides in the concentrated second solution are exchanged with the third solution using dialysis filtration and 4 dialysis volumes of the third solution;

[0935] e. A step in which the temperature of the solution containing the polypeptide increases from about 25°C to about 37°C after the second solution exchange step; and

[0936] f. A third concentration step in which the polypeptide is further concentrated using fed-batch ultrafiltration concentration;

[0937] g. Alternatively, a step of adding polyoxyethylene sorbitan monooleate to the product solution of the third concentration step at a concentration of about 0.01% (w / v),

[0938] The second and third solutions comprise a solution selected from the group consisting of: a solution containing about 140 mM N-acetylarginine, about 50 mM Arg HCl, and about 10 mM sodium acetate and having a pH of about 5.2; a solution containing about 155 mM N-acetylarginine, about 70 mM Arg HCl, and about 10 mM sodium acetate and having a pH of about 5.4; and a solution containing about 170 mM N-acetylarginine and about 10 mM sodium acetate and having a pH of about 5.6;

[0939] In the first and second solution exchange steps, a dialysis filtration membrane having at least one feature selected from the group consisting of the following is used:

[0940] h. Mesh opening greater than approximately 350 μm but less than or equal to approximately 500 μm;

[0941] i. An opening area that is greater than approximately 32% of the membrane area but less than or equal to approximately 36%;

[0942] j. Number of meshes less than approximately 16.2 n / cm but greater than or equal to approximately 12.2 n / cm;

[0943] k. Wire diameter greater than approximately 270 μm but less than or equal to approximately 340 μm;

[0944] l. Standard weight exceeding approximately 160 g / ㎡ but not exceeding 180 g / ㎡;

[0945] m. Thickness greater than approximately 515 μm but less than or equal to approximately 610 μm;

[0946] n. Membrane load greater than approximately 1138.1 g / m² but less than or equal to approximately 1919.3 g / m²; and

[0947] o. Maximum supply pressure of approximately 60 psi;

[0948] A method comprising a therapeutic polypeptide that blocks the binding of PCSK9 to LDLR and a PCSK9-binding polypeptide selected from the group consisting of the following:

[0949] i. A monoclonal antibody (evolocumab) comprising a heavy chain having the amino acid sequence of SEQ ID NO: 1 and a light chain having the amino acid sequence of SEQ ID NO: 2, or an antigen-binding fragment thereof;

[0950] ii. A monoclonal antibody competing with evolocumab for binding to PCSK9;

[0951] iii. Monoclonal antibodies comprising the following:

[0952] 1. A heavy chain polypeptide comprising the following complementarity determining region (CDR): heavy chain CDR1, which is CDR1 at SEQ ID NO: 14 or 16; heavy chain CDR2, which is CDR2 at SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 at SEQ ID NO: 14 or 16, and

[0953] 2. A light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 at SEQ ID NO: 15 or 17; light chain CDR2, which is CDR2 at SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 at SEQ ID NO: 15 or 17;

[0954] iv. Monoclonal antibody binding to at least one of the following residues of PCSK9 comprising the amino acid sequence of SEQ ID NO: 3: S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;

[0955] v. Monoclonal antibodies that bind to PCSK9 at epitopes in PCSK9 that overlap with epitopes bound by antibodies including the following:

[0956] 1. A variable region of the heavy chain of the amino acid sequence in SEQ ID NO: 1; and

[0957] 2. The variable region of the light chain of the amino acid sequence in SEQ ID NO: 2, and

[0958] Here, the epitope of the monoclonal antibody further overlaps with the site in the EGFa domain of LDLR.

[0959] Example 199 : In Embodiment 104, the subject has a disease or disability selected from the group consisting of the following:

[0960] Familial hypercholesterolemia (including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial apolipoprotein B-100 deficiency; polygenic hypercholesterolemia), non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes mellitus, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and dyslipidemia (primary and secondary dyslipidemia, e.g., metabolic syndrome, diabetes mellitus vera, familial mixed hyperlipidemia, familial hypertriglyceridemia; remnant removal disease, hepatic lipase deficiency; dietary indiscretion, hypothyroidism, secondary dyslipidemia caused by drugs including estrogen and progestin therapy, beta-blockers, and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage Cholesterol-related diseases or disorders selected from the group consisting of disease, liver cancer, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, or alcohol-induced hypertriglyceridemia.

[0961] Atherosclerotic disease selected from the group consisting of cardiovascular death, non-cardiovascular or all-cause death, coronary heart disease, coronary artery disease, peripheral artery disease, stroke (ischemic and hemorrhagic), angina pectoris, cerebrovascular disease and acute coronary syndrome, myocardial infarction and unstable angina pectoris; and

[0962] Diseases or disorders that can be treated with statins.

[0963] Example 200: In Embodiment 104, the method of treating the subject comprises reducing the risk of a condition selected from the group consisting of a cardiovascular condition due to elevated CRP or hsCRP, a cardiovascular condition due to heart disease, a cardiovascular event due to heart disease, and / or a cardiovascular event due to heart disease in a patient with heart disease.

[0964] Example 201 of implementation : A pharmaceutical composition of any one of embodiments 1 to 103 for use as a medicine.

[0965] Example 202 : In Embodiment 201, the pharmaceutical composition is intended for use in the treatment of a disease or disorder selected from the group consisting of the following:

[0966] Familial hypercholesterolemia (including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial apolipoprotein B-100 deficiency; polygenic hypercholesterolemia), non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes mellitus, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and dyslipidemia (primary and secondary dyslipidemia, e.g., metabolic syndrome, diabetes mellitus vera, familial mixed hyperlipidemia, familial hypertriglyceridemia; remnant removal disease, hepatic lipase deficiency; dietary indiscretion, hypothyroidism, secondary dyslipidemia caused by drugs including estrogen and progestin therapy, beta-blockers, and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage Cholesterol-related diseases or disorders selected from the group consisting of disease, liver cancer, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, or alcohol-induced hypertriglyceridemia.

[0967] Atherosclerotic disease selected from the group consisting of cardiovascular death, non-cardiovascular or all-cause death, coronary heart disease, coronary artery disease, peripheral artery disease, stroke (ischemic and hemorrhagic), angina pectoris, cerebrovascular disease and acute coronary syndrome, myocardial infarction and unstable angina pectoris; and

[0968] Diseases or disorders that can be treated with statins.

[0969] Example 203 : In Embodiment 202, the agent is a method for reducing the risk of a condition selected from the group consisting of a patient with a cardiovascular event due to heart disease, a fatal and non-fatal heart attack, a fatal and non-fatal stroke, heart surgery, hospitalization due to heart failure, heart disease, and / or a pre-existing heart disease, a cardiovascular condition due to elevated CRP or hsCRP, and a recurrent cardiovascular event.

[0970] abbreviation

[0971]

[0972]

[0973] References

[0974] Chan, J., Gibbs, J., Dias, C., Wasserman, S., Scott, R., Clogston, C., … Stein, E. (2012). WO Patent No. WO2012154999.

[0975] Chothia, C., & Lesk, A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol, 196 (4), 901-917.

[0976] Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill, S. J., Air, G., … et al. (1989). Conformations of immunoglobulin hypervariable regions. Nature, 342 (6252), 877-883. doi: 10.1038 / 342877a0

[0977] Cunningham, D., Danley, D. E., Geoghegan, K. F., Griffor, M. C., Hawkins, J. L., Subashi, T. A.,… Qiu, X. (2007). Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol, 14 (5), 413-419. doi: 10.1038 / nsmb1235

[0978] Horton, J. D., Cohen, J. C., & Hobbs, H. H. (2007). Molecular biology of PCSK9: its role in LDL metabolism. Trends Biochem Sci, 32 (2), 71-77. doi: 10.1016 / j.tibs.2006.12.008

[0979] Jackson, S. M., Walker, N. P., Piper, D. E., Shan, B., Shen, W., Chan, J.,… Carabeo, T. (2009). WO Patent No. WO 2009 / 026558.

[0980] Kabat, E., Wu, T., Perry, H., Gottesman, K., & Foeller, C. (1991). Sequences of proteins of immunological interest(Vol. Publication No. 91-3242). Bethesda, MD: National Institutes of Health.

[0981] Kabat, E., Wu, T., Reid-Miller, M., Perry, H., & Gottesman, K. (1987). Sequences of proteins of immunological interest (4th ed. Vol. No. 165-492). Bethesda, MD: US Government Printing Office.

[0982] Morichika, T., & Kameoka, D. (2007). WO Patent No. WO2007074880.

[0983] Piper, D. E., Jackson, S., Liu, Q., Romanow, W. G., Shetterly, S., Thibault, S. T.,… Walker, N. P. (2007). The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol. Structure, 15 (5), 545-552. doi: 10.1016 / j.str.2007.04.004

[0984] Seidah, N. G., Benjannet, S., Wickham, L., Marcinkiewicz, J., Jasmin, S. B., Stifani, S.,… Chretien, M. (2003). The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci USA, 100 (3), 928-933. doi: 10.1073 / pnas.0335507100

[0985] Seidah, N. G., & Prat, A. (2007). The proprotein convertases are potential targets in the treatment of dyslipidemia. J Mol Med (Berl), 85 (7), 685-696. doi: 10.1007 / s00109-007-0172-7

Claims

Claim 1 A pharmaceutical composition comprising a. evolocumab at a concentration of 210 mg / mL; b. 155 mM to 175 mM N-acetyl arginine; and c. 50 mM to 100 mM arginine hydrochloride, wherein the pharmaceutical composition has a viscosity of less than 50 cp, an osmolality of 256 to 392 mOsm / kg, and a pH of 4.8 to 5.

33. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete

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