EA045816B1 - LOW-VISCOSITY, HIGHLY CONCENTRATED COMPOSITIONS BASED ON EVOLOCUMAB AND METHODS FOR THEIR PRODUCTION - Google Patents

Description

Related applications

The present invention claims benefit from U.S. Provisional Patent Application No. 62/462,266, filed February 22, 2017, which is incorporated herein by reference in its entirety.

List of sequences

This application is submitted with a sequence listing in electronic form. The sequence listing is provided in the form of a file called A-2112-WO-PCT sequence listing ST25.txt, created on January 31, 2018 and having a size of 21 kilobytes. The electronic sequence listing information is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to the field of pharmaceutical compositions based on evolocumab and other PCSK9-binding polypeptides and methods for reducing the viscosity of such compositions. In particular, the present invention relates to pharmaceutical compositions based on evolocumab and other PCSK9-binding polypeptides containing N-acetylarginine, and to the use of N-acetylarginine to reduce the viscosity of highly concentrated compositions based on evolocumab and other PCSK9-binding polypeptides. Moreover, the disclosed subject matter of the invention provides methods associated with the preparation of such pharmaceutical compositions.

State of the art

The therapeutic antibodies are formulated in the form of a solution for administration, such as parenteral injection. For products that are administered subcutaneously via self-administration, formulations that require administration volumes greater than 1 to 2 milliliters are poorly tolerated. To overcome this problem, antibodies can be formulated at high concentrations (eg, such as 70 mg/ml to 210 mg/ml or more), thereby reducing the dose size.

However, some highly concentrated antibody formulations can be difficult to manufacture and administer. For example, in a formulation based on evolocumab (REPATHA®), a monoclonal antibody that binds PCSK9, evolocumab concentrations above approximately 70 mg/mL exhibit increased viscosity. However, effective doses of evolocumab are 210 mg Q2W or 420 mg Q4W. Highly viscous formulations are not only difficult to handle during manufacturing, including the bulk and filling stages, but they are also difficult to syringe and inject, making administration to the patient cumbersome and unpleasant.

To reduce the viscosity of the antibody formulations, the unmodified amino acids arginine, glycine, serine or proline were added to the antibody formulations. For example, antibody formulations containing 80 mg/ml antibody and 75 mg/ml to approximately 125 mg/ml arginine can be lyophilized and reconstituted to 120-200 mg/ml; however, final concentrations of arginine can range from 431 to 718 mM (Morichika & Kameoka, 2007). Although arginine reduced the viscosity of the formulations compared to controls (Morichika & Kameoka, 2007). Moreover, the effect of arginine was insufficient to reduce the viscosity of evolocumab to the required levels.

There is a need in the art to reduce the viscosity of formulations containing evolocumab and other PCSK9-binding polypeptides with compounds that are more effective than arginine.

Short description

In a first aspect, provided herein is a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at a location on the PCSK9 epi

- 1 045816 top that overlaps with an epitope that is bound by an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds; And

b) N-acetylarginine, wherein the pharmaceutical composition has a viscosity of at least less than about 80 cP. In such a first aspect, the PCSK9-binding polypeptide may be a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively. Moreover, in this first aspect, the pharmaceutical composition may have a viscosity of at least less than about 50 cP. The pharmaceutical composition may have an osmolality of from about 250 to about 400 mOsm/kg, such as about 300 mOsm/kg, or is isotonic to a human blood cell. The concentration of the PCSK9 binding polypeptide can be from about 140 mg/ml to about 260 mg/ml, such as 210 mg/ml. N-acetylarginine may be present at a concentration of from about 25 mM to about 230 mM, such as from 140 mM to about 170 mM, or at 140 mM. The pharmaceutical composition of this aspect may further comprise a buffer, such as a buffer selected from the group consisting of acetate, glutamate, histidine and phosphate buffers or combinations thereof. The buffer may be present at a concentration ranging from about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of approximately 10 mM. The pH value of such pharmaceutical compositions can be from about 4.8 to about 6.9, for example, the pH value is about 5.4. The pharmaceutical compositions of this aspect may further comprise a surfactant, such as a surfactant selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types of Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of from about 0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions of this aspect, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v). Moreover, the pharmaceutical compositions of this aspect may further contain proline, which may be present at a concentration of from about 50 mM to about 150 mM, for example, from 90 to 120 mM, or at about 120 mM. In some cases, the pharmaceutical composition of this first aspect may further comprise an arginine salt, which may be present at a concentration of from about 25 mM to about 150 mM, such as from 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 approximately 50 mM. The PCSK9 binding polypeptide may be stable for at least about two years, or even five years or more, if stored at about -30° C. or lower in the pharmaceutical compositions of this first aspect. At 5°C, the PCSK9 binding polypeptide may be stable for from at least about six months to about 24 months or more in such pharmaceutical compositions. At 25°C, the PCSK9 binding polypeptide may be stable for at least about one month or longer, three months or longer, or even six months or longer. At 40°C, the PCSK9-binding polypeptide may be stable for at least one month or longer. Pharmaceutical compositions of this first aspect may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides at a level of less than about 3%, such as 2.5% or less, of the total concentration of PCSK9-binding polypeptide.

In a second aspect, this document discloses a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is selected from the group consisting of:

i) a monoclonal antibody 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

- 2 045816 activity under SEQ ID NO: 2 (evolocumab), or an antigen-binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds; And

b) N-acetylarginine;

c) arginine salt;

d) buffer and

e) a surfactant, wherein the pharmaceutical composition has a viscosity of at least less than about 80 cP.

In this second aspect, the PCSK9-binding polypeptide is a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

Pharmaceutical compositions of this second aspect may have a viscosity of at least less than about 50 cP. The pharmaceutical composition may have an osmolality of from about 250 to about 400 mOsm/kg, such as about 300 mOsm/kg, or is isotonic to a human blood cell. The concentration of the PCSK9 binding polypeptide can be from about 140 mg/ml to about 260 mg/ml, such as 210 mg/ml. N-acetylarginine may be present at a concentration of from about 25 mM to about 230 mM, such as from 140 mM to about 170 mM, or at 140 mM. The pharmaceutical composition of this aspect may further comprise a buffer, such as a buffer selected from the group consisting of acetate, glutamate, histidine and phosphate buffers or combinations thereof. The buffer may be present at a concentration ranging from about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of approximately 10 mM. The pH value of such pharmaceutical compositions can be from about 4.8 to about 6.9, for example, the pH value is about 5.4. The pharmaceutical compositions of this aspect may further comprise a surfactant, such as a surfactant selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F- 68 and other types of Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of from about 0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions of this aspect, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v). Moreover, the pharmaceutical compositions of this aspect may further comprise proline; wherein proline may be present at a concentration of from about 50 mM to about 150 mM,

- 3 045816 such as from 90 to 120 mM, or at approximately 120 mM. In some cases, the pharmaceutical composition of this second aspect may further comprise an arginine salt, which may be present at a concentration of from about 25 mM to about 150 mM, such as from 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 approximately 50 mM. The PCSK9 binding polypeptide may be stable for at least about two years, or even five years or more, if stored at about -30° C. or below in the pharmaceutical compositions of this second aspect. At 5°C, the PCSK9 binding polypeptide may be stable for from at least about six months to about 24 months or more in such pharmaceutical compositions. At 25°C, the PCSK9 binding polypeptide may be stable for at least about one month or longer, three months or longer, or even six months or longer. At 40°C, the PCSK9-binding polypeptide may be stable for at least one month or longer. The pharmaceutical compositions of this second aspect may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides at a level of less than about 3%, such as 2.5% or less, of the total concentration of PCSK9-binding polypeptide.

In a third aspect, this document discloses a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, 1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I 369 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 140 mM;

c) arginine-HCl present at a concentration of approximately 50 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

In this third aspect, the pharmaceutical composition may have a pH value of from about 5.1 to about 5.7, such as a pH value of about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 50 cP.

In a fourth aspect, this document discloses a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; CDR2 severe

- 4 045816 chain, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

b) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

c) N-acetylarginine, present at a concentration of approximately 140 mM;

d) arginine-HCl present at a concentration of approximately 63 mM;

e) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015%; And

f) sodium acetate at a concentration of approximately 10 mM.

In this fourth aspect, the pharmaceutical composition may have a pH value of from about 5.1 to about 5.7, such as a pH value of about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 80 cP.

In a fifth aspect, this document discloses a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 155 mM;

c) arginine-HCl present at a concentration of approximately 70 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

In this fifth aspect, the pharmaceutical composition may have a pH value of from about 5.1 to about 5.7, such as a pH value of about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 45 cP.

In a sixth aspect, this document discloses a pharmaceutical composition comprising:

a) PCSK9-binding polypeptide, which is present at a concentration of

- 5 045816 approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 170 mM;

c) arginine-HCl present at a concentration of approximately 63 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015%; And

e) sodium acetate at a concentration of approximately 10 mM.

In this sixth aspect, the pharmaceutical composition may have a pH value of from about 5.1 to about 5.7, such as a pH value of about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 60 cP.

In a seventh aspect, this document discloses a pharmaceutical composition comprising:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I1 96 , M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 155 mM;

c) proline, present at a concentration of approximately 120 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration ranging from

- 6 045816 approximately 0.005% (w/v) to approximately 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

In this seventh aspect, the pharmaceutical composition may have a pH value of from about 5.1 to about 5.7, such as a pH value of about 5.4. The pharmaceutical composition may have a viscosity of at least less than about 60 cP.

In these first to seventh aspects, the PCSK9 binding polypeptide may be stable for at least about two years, or even five years or more, if stored at about -30° C. or lower in the pharmaceutical compositions of these aspects. At 5°C, the PCSK9 binding polypeptide may be stable for from at least about six months to about 24 months or more in such pharmaceutical compositions. At 25°C, the PCSK9 binding polypeptide may be stable for at least about one month or longer, three months or longer, or even six months or longer. At 40°C, the PCSK9-binding polypeptide may be stable for at least one month or longer. Pharmaceutical compositions in these aspects may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides at a level of less than about 3%, such as 2.5% or less, of the total concentration of PCSK9-binding polypeptide.

In any of the previous aspects, the pharmaceutical composition may be a liquid.

In an eighth aspect, this document discloses a method of treating a subject in need thereof, comprising administering a pharmaceutical composition according to any of the previous seven aspects.

In a ninth aspect, this document discloses a kit comprising a pharmaceutical composition according to any one of the first to seventh aspects and a delivery device. The delivery device may be selected from the group consisting of a syringe, a pen, a body injector, and an auto-injector. The kit may further contain instructions for administering the pharmaceutical composition using a delivery device.

In a tenth aspect, this document discloses a method for preparing a pharmaceutical composition based on a PCSK9-binding polypeptide containing at least 140 mg/ml of a PCSK9-binding polypeptide, comprising adding to the pharmaceutical composition containing the PCSK9-binding polypeptide an effective amount of N-acetylarginine, thereby causing the viscosity pharmaceutical composition is reduced compared to a pharmaceutical composition not containing N-acetylarginine, and wherein the PCSK9-binding polypeptide is selected from the group consisting of:

a) a monoclonal antibody containing 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 (evolocumab), or an antigen binding fragment thereof;

b) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

c) a monoclonal antibody containing:

i) a heavy chain polypeptide comprising the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and ii) a light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

d) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238; And

e) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

i) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1; and ii) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and iii) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds.

In this tenth aspect, 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 aspect may have an osmolality of from about 250 to about 400 mOsm/kg, such as about 300 mOsm/kg, or is isotonic to a human blood cell. The concentration of the PCSK9 binding polypeptide can be from about 140 mg/ml to about 260 mg/ml, such as 210 mg/ml. N-acetylarginine may be present at a concentration of from about 25 mM to about 230 mM, such as from 140 mM

- 7 045816 to approximately 170 mM, or at 140 mM. The pharmaceutical composition of this aspect may further comprise a buffer, such as a buffer selected from the group consisting of acetate, glutamate, histidine and phosphate buffers or combinations thereof. The buffer may be present at a concentration ranging from about 5 mM to about 30 mM. In some cases, the buffer is sodium acetate and is present at a concentration of approximately 10 mM. The pH value of such pharmaceutical compositions can be from about 4.8 to about 6.9, for example, the pH value is about 5.4. The pharmaceutical compositions of this aspect may further comprise a surfactant, such as a surfactant selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F- 68 and other types of Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of from about 0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions of this aspect, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v). Moreover, the pharmaceutical compositions of this aspect may further comprise proline; wherein the proline may be present at a concentration of from about 50 mM to about 150 mM, such as from 90 to 120 mM, or at about 120 mM. In some cases, the pharmaceutical composition of this tenth aspect may further comprise an arginine salt, which may be present at a concentration of from about 25 mM to about 150 mM, such as from 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 approximately 50 mM. The PCSK9 binding polypeptide may be stable for at least about two years, or even five years or more, if stored at about -30° C. or lower in the pharmaceutical compositions of this tenth aspect. At 5°C, the PCSK9 binding polypeptide may be stable for from at least about six months to about 24 months or more in such pharmaceutical compositions. At 25°C, the PCSK9 binding polypeptide may be stable for at least about one month or longer, three months or longer, or even six months or longer. At 40°C, the PCSK9-binding polypeptide may be stable for at least one month or longer. The pharmaceutical compositions of this tenth aspect may contain high molecular weight aggregates or oligomers of PCSK9-binding polypeptides at a level of less than about 3%, such as 2.5% or less, of the total concentration of PCSK9-binding polypeptide.

In an eleventh aspect, this document discloses a method for formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated;

b) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine using diafiltration;

c) a second concentration step, in which the polypeptide in the second solution is concentrated;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution containing N-acetylarginine using diafiltration; And

e) a third concentration step, in which the polypeptide in the third solution is concentrated;

wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID

- 8 045816

NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) where the epitope for the monoclonal antibody additionally overlaps with the region to which the EGFa domain of the LDLR binds.

In the methods of this eleventh aspect, the PCSK9-binding polypeptide that blocks PCSK9 binding to the LDLR may be a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

Moreover, in this eleventh aspect, before the third concentration step, the temperature of the solution containing the polypeptide can be increased from about 25°C to about 37°C. Also, the first stage of replacing the solution can be carried out using at least three diavolumes of the second solution. In some sub-aspects of this eleventh aspect, the second solution replacement step is carried out using at least four dia-volumes of the third solution. In other sub-aspects, the initial concentration of the therapeutic protein is approximately 11 mg/ml or less. In addition, the concentration of the therapeutic polypeptide can be increased by about 3 to about 7 times, such as, for example, when the increased concentration of the polypeptide is from about 35 mg/ml to about 70 mg/ml. In some sub-aspects, the second concentration step increases the concentration of the therapeutic polypeptide by about 2 to 4 times the first concentration step, to, for example, about 140 mg/ml. In the third concentration step, the concentration of the therapeutic polypeptide may be increased by about 1.5 to about 2 times the second concentration step, to about 260 mg/ml. Accordingly, the therapeutic polypeptide may have a final concentration that is at least about 19 to 20 times the initial concentration of the therapeutic polypeptide, such as about 210 mg/ml. Concentration steps may include batch ultrafiltration; furthermore, the second solution and the third solution may be identical. For example, the second or third solution containing N-acetylarginine may contain an arginine salt and a buffer, where, for example, N-acetylarginine is present at a concentration of from 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 from about 25 mM to about 150 mM; and the buffer is a sodium acetate buffer at a concentration of from about 5 mM to about 30 mM. In other sub-aspects, N-acetylarginine is present at a concentration of from 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 still other sub-aspects, N-acetylarginine is present at a concentration of approximately 140 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, and sodium acetate buffer is present at a concentration of approximately 10 mM. In additional sub-aspects, N-acetylarginine is present at a concentration of approximately 155 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 70 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. In still other sub-aspects, N-acetylarginine is present at a concentration of approximately 170 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. Moreover, the compositions may further contain proline, where proline is present at a concentration of from about 50 mM to about 150 mM. The second or third solution may have a pH value of from about 4.8 to about 6.9, such as 5.4. In the first and second stages of solution replacement, a diafiltration membrane having at least one characteristic selected from the group consisting of:

a) the size of the mesh openings, which is greater than approximately 350 µm but less than or equal to

- 9 045816 approximately 500 microns;

b) a clear cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

c) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

d) a filament diameter that is greater than approximately 270 µm but less than or equal to approximately 340 µm;

e) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

f) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

g) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

h) maximum supply pressure of approximately 60 psi.

Moreover, a surfactant may be added to the third solution after concentration, such as a surfactant selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and Dα-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The surfactant may be present at a concentration of from about 0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions of this aspect, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v).

In a twelfth aspect, this document discloses a method for formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated using batch ultrafiltration;

b) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine, an arginine salt and a buffer using diafiltration and three dia volumes of the second solution;

c) a second concentration step in which the polypeptide in the second solution is concentrated using batch ultrafiltration;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution containing N-acetylarginine, an arginine salt and a buffer using diafiltration and four dia volumes of the third solution;

e) the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C after the second solution replacement step; And

f) a third concentration step in which the polypeptide is further concentrated using batch ultrafiltration concentration;

wherein the first and second stages of solution replacement may employ a diafiltration membrane having at least one characteristic selected from the group consisting of:

a) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm;

b) a clear cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

c) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

d) a filament diameter that is greater than approximately 270 µm but less than or equal to approximately 340 µm;

e) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

f) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

g) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

h) maximum supply pressure of approximately 60 psi; and wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof;

- 10 045816 ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

ii i) monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) where the epitope for the monoclonal antibody additionally overlaps with the region to which the EGFa domain of the LDLR binds.

Moreover, in this twelfth aspect, the first solution replacement step can be carried out using at least three diavolumes of the second solution. In some sub-aspects of this twelfth aspect, the second solution replacement step is carried out using at least four dia-volumes of the third solution. In other sub-aspects, the initial concentration of the therapeutic protein is approximately 11 mg/ml or less. In addition, the concentration of the therapeutic polypeptide can be increased by about 3 to about 7 times, such as, for example, when the increased concentration of the polypeptide is from about 35 mg/ml to about 70 mg/ml. In some sub-aspects, the second concentration step increases the concentration of the therapeutic polypeptide by about 2 to 4 times the first concentration step, to, for example, about 140 mg/ml. In the third concentration step, the concentration of the therapeutic polypeptide may be increased by about 1.5 to about 2 times the second concentration step, to about 260 mg/ml. Accordingly, the therapeutic polypeptide may have a final concentration that is at least about 19 to 20 times the initial concentration of the therapeutic polypeptide, such as about 210 mg/ml. Concentration steps may include batch ultrafiltration; furthermore, the second solution and the third solution may be identical. N-acetylarginine is present at a concentration ranging from 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 from about 25 mM to about 150 mM; and the buffer is a sodium acetate buffer at a concentration of from about 5 mM to about 30 mM. In other sub-aspects, N-acetylarginine is present at a concentration of from 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 still other sub-aspects, N-acetylarginine is present at a concentration of approximately 140 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. In additional sub-aspects, N-acetylarginine is present at a concentration of approximately 155 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 70 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. In still other sub-aspects, N-acetylarginine is present at a concentration of approximately 170 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. Moreover, the compositions may further contain proline, where proline is present at a concentration of from about 50 mM to about 150 mM. The second or third solution may have a pH value of from about 4.8 to about 6.9, such as 5.4. Moreover, a surfactant, such as a surfactant selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F- 68 and other types of Pluronic®), complex

- 11 045816 sorbitan alkyl ethers (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers and D-a-tocopherol polyethylene glycol succinate (vitamin E TPGS). The surfactant may be present at a concentration of from about 0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions of this aspect, the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v).

In a thirteenth aspect, this document discloses a method for formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated using batch ultrafiltration;

b) a first solution replacement step, in which the concentrated polypeptide in the first solution is replaced by a second solution using diafiltration and three diavolumes of the second solution;

c) a second concentration step in which the polypeptide in the second solution is concentrated using batch ultrafiltration;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution using diafiltration and four diavolumes of the third solution;

e) the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C after the second solution replacement step; And

f) a third concentration step in which the polypeptide is further concentrated using batch ultrafiltration concentration;

g) alternatively, the step of adding polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of approximately 0.01% (w/v) to the solution obtained in the third concentration step, wherein the second and third solutions comprise a solution selected from the group consisting of a solution , containing approximately 140 mm N-acetylarginine, approximately 50 mm Arg-HCl and approximately 10 mm sodium acetate, the solution having a pH value of approximately 5.2; a solution containing approximately 155 mM N-acetylarginine, approximately 70 mM Arg-HCl and approximately 10 mM sodium acetate, the solution having a pH value of approximately 5.4; and a solution containing approximately 170 mM N-acetylarginine, approximately 10 mM sodium acetate, the solution having a pH value of approximately 5.6;

wherein the first and second stages of solution replacement may employ a diafiltration membrane having at least one characteristic selected from the group consisting of:

a) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm;

b) a clear cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

c) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

d) a filament diameter that is greater than approximately 270 µm but less than or equal to approximately 340 µm;

e) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

f) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

g) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

h) maximum supply pressure of approximately 60 psi; and wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

ii i) monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

- 12 045816

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) where the epitope for the monoclonal antibody additionally overlaps with the region to which the EGFa domain of the LDLR binds.

Brief description of graphic materials

In fig. Figure 1 shows a graph of size exclusion chromatography-high pressure liquid chromatography (SE-HPLC) data for evolocumab samples at high concentrations in various N-acetylarginine-containing formulations after 1 month of incubation at 40°C. Symbols for compounds shown on the x-axis: [acetate (mM)]/N-acetylarginine (mM)] / [proline (mM)]. The Y-axis indicates the percentage of the sample that is high molecular weight (HMW) species (aggregates and oligomers of evolocumab).

In fig. Figure 2 shows a diagram comparing the viscosity of formulations based on evolocumab with a concentration of 210 mg/ml at a shear rate of 1000 s ^-1 at 25°C.

In fig. 3A shows graphs generated by JMP Prediction Profiler software based on SE-HPLC data for an evolocumab formulation held at 40°C for one month. In fig. Figure 3B shows similar data, except that the samples were kept at 25°C for three months.

In fig. Figure 4 shows a bar graph of the percentage of HMW species by SE-HPLC for 210 mg/mL evolocumab formulations held at 40°C for one month. The formulations varied N-acetylarginine concentrations and arginine-HCl concentrations at different pH values.

In fig. 5A-5C show SE-HPLC chromatograms for selected evolocumab samples at pH 4.8 and pH 5.4 maintained at the indicated temperatures and for the indicated periods of time (5°C for six months (Fig. 5A); 25° C for three months (Fig. 5B) and 40°C for one month (Fig. 5C)), and compared with a proline-containing evolocumab control formulation. Composition symbols: [acetate (mM)]/[N-acetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 6A and 6B are graphs of the percentage of HMW species that emerged over time in the indicated evolocumab formulations maintained at the indicated temperatures (5° C. (Fig. 6A); 25° C. (Fig. 6B)). Test formulations of 210 mg/mL evolocumab containing N-acetylarginine are compared to a control formulation of 140 mg/mL evolocumab containing proline. Designations for compositions: [acetate (mM)]/N-acetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 7A shows cation exchange chromatography-high pressure liquid chromatography (CEX-HPLC) graphs generated by JMP Prediction Profiler software for evolocumab formulations held at 40° C. for one month. In fig. 7B shows similar data, except that the samples were kept at 25°C for three months. See the Examples section for more details.

In fig. Figure 8 shows a graph of the percent acid peak magnitude as measured by CEX-HPLC for evolocumab formulations at a concentration of 210 mg/mL maintained at 40°C for one month.

In fig. 9A-9B show graphs of the number of particles invisible to the naked eye, as determined by counting suspension particles using light blocking (greater than or equal to 10 μm, FIG. 9A; or greater than or equal to 25 μm, FIG. 9B), per milliliter of various formulations based on evolocumab, maintained at 5, 25 and 40°C for three months. In fig. 9C shows a graph of the number of particles not visible to the naked eye, as determined by light blocking suspension particle counting (greater than or equal to 10 μm or 25 μm), in various evolocumab formulations maintained at 5°C or 25°C for six months. .

In fig. 10 is a plot of the number of particles not visible to the naked eye in various evolocumab formulations, as determined by microflow imaging (MFI) analysis, filtered by an aspect ratio (AR) of less than 0.70.

In fig. Figure 11 shows the resulting viscosity chart of various evolocumab formulations held for specified periods of time and at specified temperatures, where TO denotes

- 13 045816 indicates the initial viscosity values.

In fig. 12 is a chart and table showing the pH values of evolocumab formulations differing in pH and buffer over time up to three months. The samples were kept at 40°C.

In fig. 13A-13C are SE-HPLC graphs demonstrating the effect of pH on the percentage of HMW species in 210 mg/mL evolocumab formulations that differ in pH and buffer for up to three months at 4 °C (Fig. 13A), 25°C (Fig. 13B) and 40°C (Fig. 13C). In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 14 shows a graph of oligomer levels in evolocumab formulations at varying pH immediately after formulation.

In fig. 15 shows chromatograms showing the different protein molecular sizes in evolocumab formulations that differ in pH and buffer after incubation at 40°C for three months.

(LMW=low molecular weight species).

In fig. 16A-16C show graphs of data from CEX-HPLC analyzes (% major peak) of 210 mg/mL evolocumab formulations that differ in pH and buffer over time. In fig. 16A shows the percentage of the main peak for samples held at 5°C over time, while FIG. 16B shows the same type of data for samples held at 25°C, and FIG. 16C shows the same type of data for samples held at 40°C. In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 17 shows a graph of data from CEX-HPLC analyzes of 210 mg/mL evolocumab formulations that differ in pH and buffer maintained at 40° C. over time, measuring the percentage of acidic species in the formulations . In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 18 shows CEX-HPLC chromatograms for 210 mg/mL evolocumab formulations that differ in pH and buffer maintained at 25°C for three months, with analysis showing acidic, basic, and major peaks for varieties. In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. Figure 19 shows peptide mapping chromatograms for various evolocumab formulations differing in pH and buffer, with samples maintained at 40°C for one month. In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 20 shows a graph summarizing the results of several experiments in which evolocumab was analyzed in formulations that differed in pH and buffer after 1 month at 40°C. All samples contained 10 mM buffer, 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80. For additional details, see the figure and the Examples section.

In fig. 21A-21B are graphs of the number of particles invisible to the naked eye, as determined by counting suspension particles using light blocking (greater than or equal to 10 μm, FIG. 21A; or greater than or equal to 25 μm, FIG. 21B), per milliliter of various formulations based on evolocumab, differing in pH value and buffer, maintained at 5°C, 25°C and 40°C for two months. In fig. 21C-21D show graphs of the number of particles not visible to the naked eye (greater than or equal to 10 μm, FIG. 21C; greater than or equal to 25 μm, FIG. 21D) in evolocumab formulations differing in pH and buffer maintained at 5°C. or 25°C for six months. In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 22 shows a graph of the number of particles not visible to the naked eye in evolocumab-based formulations that differ in pH and buffer, as determined by microflow imaging (MFI). In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 23 shows a graph of reducing sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) assays performed on 210 mg/mL evolocumab formulations of varying pH and buffer maintained at 25° C. for 6 months. In addition to 10 mM of each buffer listed, all samples contained 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

In fig. 24 shows a graph of the relationship between viscosity and pH of evolocumab formulations that differ in pH and buffer. All samples contained 10 mM buffer, 165 mM N-acetylarginine, 75 mM arginine-HCl, and 0.01% polysorbate-80.

- 14 045816

In fig. 25A-25C show graphs of the percentage of HMW species as measured by SE-HPLC for different evolocumab formulations held for the indicated periods at 5°C (FIG. 25A), 25°C (FIG. 25B), and 40°C (Fig. 25C). Composition symbols: [acetate (mM)]/[Nacetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 26A-26C show graphs of the percent acid peak magnitude as measured by CEX-HPLC for different evolocumab formulations held for the indicated periods at 5°C (FIG. 26A), 25°C (FIG. 26B), and 40°C (Fig. 26C). Composition symbols: [acetate (mM)]/[I-acetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 27 shows a graph of the percentage of LC+LC+HC precursors (LC=light chain, HC=heavy chain) measured by reducing sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) for different evolocumab formulations incubated for three months at 5°C, 25°C and 40°C, compared with initial levels (T0). Composition symbols: [acetate (mM)]/[N-acetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 28 shows a chart of the viscosity of different evolocumab formulations with three different concentrations of evolocumab, with viscosity data determined using a rheometer at shear rates up to 90,000 s ^-1 at the indicated temperatures. Composition symbols: [acetate (mM)]/[I-acetylarginine (mM)]/[arginine-HCl (mM)].

In fig. 29 shows a graph of UF/DF flow data for evolocumab in three NAR formulation buffers.

In fig. 30 shows a graph of the percentage of HMW species formation during the UF/DF process of evolocumab at 35 mg/ml and 70 mg/ml evolocumab at the UF1/DF1 stage (UF/DF-70 and UF/DF-35). The figure also shows the concentration of evolocumab in mg/ml at each stage of the process for two initial concentrations of evolocumab.

In fig. 31A-31F show SE-HPLC (FIGS. 31A, 31C and 31E) and CEX-HPLC (FIGS. 31B, 31D and 31F) data plots for evolocumab in NAR-containing formulations at pH 5.2 (FIG. 31A and 31B), a pH value of 5.4 (FIGS. 31C and 31D) and a pH value of 5.6 (FIGS. 31E and 31F). The figures also show the percentage of HMW species, the percentage of LMW species and the percentage of evolocumab at various stages during the UF/DF process.

In fig. 32 shows the percentage of HMW species formation in a evolocumab DS sample with NAR from pool studies at 2-8° C. and room temperature.

In fig. 33 shows a graph of the percentage formation of HMW species in OS samples of evolocumab with NAR from elevated temperature pool studies.

In fig. 34 shows a chart of viscosity measurements of evolocumab formulations with NAR at different temperatures.

In fig. 35A-35C show SE-HPLC data plots for all test formulations used in Example 9 after incubation at 4°C (Fig. 35A), 25°C (Fig. 35B) and 40°C (Fig. 35C) up to up to 6 months. In fig. 35D displays the 40° C. SE-HPLC data (shown as a line graph in FIG. 35C) as a histogram, making comparisons of aggregation levels between formulations more clear.

In fig. 36A-36F are CEX-HPLC plots for the test formulations used in Example 9, showing changes in % acidic and % basic peaks over time after incubation at 4°C (Figure 36A (% acidic), Figure 36B ( % basic)), 25°C (Figure 36C (% acidic), Fig. 36D (% basic)) and 40°C (Figure 36E (% acidic), Fig. 36F (% basic)) for up to three months.

In fig. 37A-37D show rCE-SDS plots of % main peak and % LMW species for the test formulations used in Example 9 over time after incubation at 30°C (Fig. 37A (% main peak), Fig. 37B (% LMW varieties)) and 40°C (Fig. 37C (% main peak), Fig. 37D (% LMW varieties)) for up to three months.

In fig. 38A-38D show graphs of NEA particle count data obtained by light-blocking particle counts using HIAC for the test formulations used in Example 9 after incubation at 4°C and 40°C for up to three months. Fig. 38A: HIAC - > 10 µm - 4°C; fig. 38V: HIAC - > 25 µm - 4°C; fig. 38C: HIAC - > 10 µm 40°C; and HIAC - > 25 µm - 40°C.

- 15 045816

Detailed Description of the Invention

The inventors of the present invention have surprisingly discovered that the arginine derivative, Nacetylarginine (NAR), effectively reduces the viscosity of pharmaceutical compositions containing high concentrations (more than 100 mg/ml, such as 140 mg/ml and more) of evolocumab, to a greater extent than non-acetylated arginine. Pharmaceutical compositions having a viscosity of 50 cP or less are easy to manufacture and administer to a patient without significant difficulty, whereas formulations with a higher viscosity are difficult to handle (eg, when filling syringes) and to administer. Although unmodified arginine is known to reduce the viscosity of high protein formulations, only arginine glutamate reduced the viscosity of a comparable evolocumab formulation (210 mg/mL) containing proline by 50 cP (from 159 cP to 109 cP), well above target. value of 50 cP or less. Moreover, 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. At the same time, NAR had the unexpected effect of further reducing viscosity to 58 cP - a reduction of over 101 cP relative to the proline containing formulation, and when combined with Arg-HCl (to increase the solubility of NAR and obtain an isotonic formulation) resulted in a viscosity below 50 cP, and the target was actually exceeded by 7 cP because the formulation had a viscosity of 43 cP. See fig. 2. Since the solubility of NAR is limited to less than 230 mM, another excipient is required to obtain an isotonic formulation for subcutaneous administration. An unexpected finding was that arginine chloride salt was more effective in reducing the viscosity of evolocumab than other arginine salts such as glutamate, which was critical to minimizing the viscosity of the formulation.

Another unexpected finding was the pH-dependent effects on evolocumab stability and viscosity observed in the presence of NAR and arginine-HCl. A significant increase in the rate of aggregation of evolocumab was observed at pH values less than 5.0 at elevated temperatures. Also, a pH-dependent decrease in viscosity was observed as the pH value increased from pH 5.1 to 6.9.

The present inventors have further discovered that by using a two-step ultrafiltration/diafiltration (UF/DF) process, such NAR-containing highly concentrated evolocumab pharmaceutical formulations can be prepared with significant savings in NAR material compared to traditional one-step processes. Such methods provide significant cost savings since NAR is approximately ten times more expensive than Arg-HCl.

Definitions.

Both the above general description and the following detailed description are provided by way of example and explanation only and are not limiting. The use of singular forms includes plural forms unless specifically stated otherwise. The use of or means and/or, unless otherwise stated. The use of the term including, as well as other forms such as includes and included, is not limiting. Terms such as element or component cover both elements and components containing a single unit and elements and components that contain more than one subunit, unless specifically stated otherwise. The use of the term part may include part of a fragment or the entire fragment. When referring to a numeric range such as 1-5, all intermediate values such as 1, 2, 3, 4 and 5, as well as their fractional values such as 1, 5, 2.2, 3.4 and 4.1.

Approximately or ~ means, in the case of modification of the amount (eg, approximately 3 mM), that fluctuations around the modified amount may occur. These fluctuations can be due to a variety of reasons, such as typical measuring and processing procedures, inevitable errors, purity of ingredients, etc.

N-acetylarginine (NAR) refers to the molecule of formula 1.

In the context of a pharmaceutical composition, an additive means a substance that is not naturally part of the material (eg, drug substance) but is added intentionally to perform some specific purpose (eg, preservation, viscosity reduction, stabilization).

Analog refers to an amino acid sequence that contains insertions, deletions, or

- 16 045816 substitution relative to the original sequence, while it substantially retains the biological activity of the original sequence, as determined using biological assays. Analogues include polypeptides with modified glycosylation, polypeptides without glycosylation. The compositions may also contain derivatives of naturally occurring polypeptides or analogs thereof that have been chemically modified, for example, to attach water-soluble polymers (eg, PEGylated), radionuclides, or other diagnostic or targeting or therapeutic moieties.

Antibody refers to an intact immunoglobulin of any isotype and includes, for example, chimeric, humanized, human and bispecific antibodies. An intact antibody will typically contain at least two full length heavy chains and two full length light chains.

The antibody sequences may be derived exclusively from one species or may be chimeric, that is, different parts of the antibody may be derived from two different species. An antibody also includes antibodies containing two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibodies retain the same or similar binding and/or function as an antibody consisting of two full-length light and heavy chains. For example, antibodies having substitutions, insertions or deletions of 1, 2, 3, 4 or 5 amino acid residues at the N-terminus and/or C-terminus of the heavy and/or light chains are included in the definition provided that the antibodies retain the same or similar binding and/or function as antibodies containing 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.

Typical antibody structural units include a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, with each pair having one full-length light chain (approximately 25 kDa) and one full-length heavy chain (approximately 50-70 kDa). The amino terminal portion 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 portion of each chain typically defines a constant region that may be responsible for effector function. Light chains are usually classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and they respectively define the antibody isotype as IgM, IgD, IgG, IgA, and IgE. IgG antibodies have several subclasses, including IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including IgM1 and IgM2. IgA is similarly divided into subclasses including IgA1 and IgA2. Within full-length light and heavy chains, typically the variable and constant regions are connected by a J region of about 12 or more amino acids, with the heavy chain also containing a D region of about ten or more amino acids. The variable regions of each light/heavy chain pair typically form an antigen-binding region.

Variable regions are typically characterized by the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two strands of each pair are typically aligned to framework regions that can mediate binding to a specific epitope. From N-terminus to C-terminus, the variable regions of both the light and heavy chains typically contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The assignment of amino acids to each domain is usually carried out according to the definitions of Rabat (Rabat, Wu, Perry, Gottesman, & Foeller, 1991; Rabat, Wu, Reid-Miller, Perry, & Gottesman, 1987) or Chothia (Chothia & Lesk, 1987 ; Chothia et al., 1989).

Instead of a full-length antibody, a fragment or antigen-binding fragment of an antibody can be used. An antibody fragment refers to Fab, Fab', F(ab')2 and Fv fragments that contain at least one immunoglobulin CDR sufficient to provide specific antigenic binding to a target protein, such as PCSR9.

The heavy chain of an antibody can bind to an antigen in the absence of the light chain of the antibody. The light chain of an antibody can bind to an antigen in the absence of the heavy chain of the antibody. The binding region of an antibody can bind to an antigen in the absence of the antibody light chain. The binding region of an antibody can bind to an antigen in the absence of the antibody heavy chain. A single variable region can specifically bind to an antigen in the absence of other variable regions.

The CDR regions in the heavy chain are usually designated H1, H2 and H3 and are numbered sequentially from the amino terminus to the carboxy terminus. The CDR regions in the light chain are designated L1, L2, and L3 and are numbered sequentially from the amino terminus to the carboxy terminus.

The term light chain includes the full-length light chain and fragments thereof having a variable region sequence sufficient to provide binding specificity.

The full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The light chain variable region domain is located at the amino terminus of the polypeptide. Lungs

- 17 045816 pi includes kappa chains and lambda chains.

The term heavy chain includes the full-length heavy chain and fragments thereof having a variable region sequence sufficient to provide binding specificity.

The full-length heavy chain contains a variable region domain, VH, and three constant region domains, CH1, CH2 and CH3. The VH domain is located at the amino terminus of the polypeptide, and the CH domains are located at the carboxyl terminus, with CH3 closest to the carboxyl terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

Each individual immunoglobulin chain usually consists of several immunoglobulin domains, each of which consists of approximately 90-110 amino acids and has a characteristic folding pattern. These domains are the basic units of antibody polypeptides. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that may be responsible for effector function. The number of heavy chain constant region domains depends on the isotype. For example, IgG heavy chains contain three C-region domains known as CH1, CH2 and CH3. In certain cases, the anti-PCSK9 antibody is of the IgG1, or IgG2, or IgG4 subtype.

The term variable region or variable domain refers to a portion of the light and/or heavy chain of an antibody, typically comprising about 120-130 amino-terminal amino acids in the heavy chain and about 100-110 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.

Antigen refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as a PCSK9-binding polypeptide (including, for example, an antibody or binding fragment thereof). In some cases, an antigen can be used in an animal to produce antibodies that can bind to that antigen. An antigen may contain one or more epitopes that can interact with different PCSK9-binding polypeptides.

Arginine salt refers to the salt of arginine. Examples include arginine monohydrochloride (Arg-HCl), arginine acetate (Arg acetate), and arginine glutamate (Arg glutamate).

Buffer means any pharmaceutically acceptable buffer, including acetate, glutamate, histidine and phosphate buffers and their salts.

Compete, when used in the context of antibodies that compete for the same epitope, means competition between antibodies, as determined by an assay in which the test antibodies prevent or inhibit (e.g., reduce) the specific binding of a reference antibody (e.g., ligand or reference antibodies) with a common antigen (for example, PCSK9 or a fragment thereof). To determine whether one antibody competes with another, many types of competitive binding assays can be used, for example: solid-phase direct or indirect immunoassays using a variety of reagents and labels accepted in the art. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of a test antibody. Typically the antibody being tested is present in excess. Antibodies identified by a competition assay include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope close enough to the epitope that the reference antibody binds to cause steric hindrance. Most often, if a competing 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 40-45%, 45-50%, 50-55%, 55-60%, 60 -65%, 65-70%, 70-75% or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 9597%, or 97% or more.

Diafiltration, DF and similar terms mean the use of an ultrafiltration membrane (i.e. a semi-permeable membrane that can distinguish between molecules of different shapes and sizes) to remove, replace or reduce the concentration of salts or solvents in solutions or mixtures containing, for example, polypeptides or other biomolecules.

In the context of filtration, diavolume (DV) refers to the volume of diafiltration buffer introduced into a typical process relative to the volume of concentrate.

An epitope includes any determinant that a PCSK9-binding polypeptide, such as an antibody, can bind. An epitope is a region of an antigen that is bound by a PCSK9-binding polypeptide that targets such antigen and, when the antigen is a protein, contains specific amino acids that directly contact the PCSK9-binding polypeptide. Determinant epitopes may contain reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups and sulfonyl groups, and may have specific three-dimensional structural characteristics and specific charge characteristics. In general, antibodies specific for a particular target antigen preferentially recognize an epitope on an anti

- 18 045816 target gene in a complex mixture of proteins or other macromolecules.

Excipient means a more or less inert substance added to a formulation as a diluent or carrier medium or to obtain appearance or consistency if the drug is administered in pill form; for example, sugar syrup, vegetable gums, aromatic powders, honey and various tinctures.

Cross feed flow denotes the feed flow rate (l/h) divided by the membrane area (m ² ).

In the context of filtration, flow (LMH) refers to liters per hour per square meter of membrane area (l/h/ ^m2 ).

In the context of a pharmaceutical composition containing a therapeutic polypeptide, high molecular weight species or HMW species refer to therapeutic proteins that are larger than the original therapeutic polypeptide, as determined by assays conventional in the art. HMW species include therapeutic polypeptide oligomers and therapeutic polypeptide aggregates.

In the context of filtration, bypass volume (HUV) refers to the volume of product in the TFF system piping, including the volume contained in the cartridge.

Identity refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence alignment and comparison. Percent identity refers to the percentage of identical amino acid or nucleotide residues in the molecules being compared and is calculated based on the size of the smallest molecule being compared. In these calculations, gaps in alignments (if any) are preferably taken into account using a specific mathematical model or algorithm. Such techniques are well known in the art.

When calculating percent identity, the sequences to be compared are usually aligned to maximize the highest match between sequences.

The use of certain alignment schemes to align two amino acid sequences may result in a short region match in the two sequences, and the small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method can be adjusted if desired to result in an alignment that spans the desired number of contiguous amino acids (eg, 50 amino acids) of the target polypeptide.

Stereoisomers (eg, D-amino acids) of twenty traditional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-traditional amino acids may also be suitable components for PCSK9-binding polypeptides. Examples of non-traditional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline).

Conservative amino acid substitutions may involve unnatural amino acid residues that are typically introduced through chemical peptide synthesis rather than through synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid fragments.

When making changes to the antigen binding protein or PCSK9 protein, the amino acid hydrophobicity index can be taken into account. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. The importance of an amino acid's hydrophobicity index in conferring a protein's biological interaction function is obvious in the art. Certain amino acids can be replaced by other amino acids characterized by a similar index or hydrophobicity index, and the proteins retain similar biological activity.

Substitution of such amino acids can be carried out effectively on the basis of hydrophilicity. Epitopes in primary amino acid sequences can also be identified based on hydrophilicity. Such regions are also called epitope core regions.

In the context of a pharmaceutical composition containing a therapeutic polypeptide, small molecule species or LMW species refer to polypeptides that are smaller than the original therapeutic polypeptide, as determined by assays conventional in the art. LMW varieties include therapeutic polypeptide fragments.

A neutralizing antibody or an antibody that neutralizes a target, as used in the expression PCSK9 neutralizing antibody, refers to an antibody that binds to a target and prevents or reduces the biological activity of such target. This can be done, for example, by directly blocking the binding site on the target or by binding to the target

- 19 045816 and changes in the ability of the target to bind through indirect mechanisms, such as structural or energetic changes in the target. When assessing the binding and/or specificity of an antibody or immunologically functional fragment thereof, the antibody or fragment may substantially inhibit binding of the target to its binding partner when excess antibody reduces the amount of binding partner bound to the ligand by at least about 120%, 20 -30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97 -98%, 9899% or greater (as measured in an in vitro competitive binding assay). In the case of anti-PCSK9 antibodies, such a neutralizing molecule may reduce the ability of PCSK9 to bind LDLR. In some cases, neutralizing capacity is characterized or described using competitive analysis. In some cases, neutralization capacity is reported as an IC50 or EC50 value. In some cases, antibodies provide neutralization by binding to PCSK9 and preventing PCSK9 from binding to the LDLR or by reducing the ability of PCSK9 to bind to the LDLR. In some cases, antibodies provide neutralization by binding to PCSK9, while still allowing PCSK9 to bind to the LDLR, preventing or reducing PCSK9-mediated destruction of the LDLR. Thus, in some cases, in the presence of a neutralizing antibody, PCSK9/LDLR binding is still possible, but subsequent PCSK9-mediated destruction of LDLR is prevented or reduced. Neutralization results in a decrease in the level of LDL-C (and/or other lipids such as ApoB, Lp(a), etc.). Non-antibody PCSK9-binding polypeptides, including variants of such PCSK-binding polypeptides, may have the same activities.

PCSK9-binding polypeptide refers to a polypeptide that binds the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein. In some cases, PCSK9-binding polypeptide blocks PCSK9 binding to low-density lipoprotein receptors (LDLRs). Such blocking PCSK9-binding polypeptides may be monoclonal antibodies (mAbs) and may be one of the following:

a) mAb containing 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 (evolocumab), or an antigen binding fragment thereof;

b) a mAb that competes with evolocumab for binding to PCSK9;

c) mAb containing:

i) a heavy chain polypeptide comprising the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and ii) a light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

d) an mAb that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, A239, G244, M247, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, IA S372, C375, C378, R237, and D238;

e) an mAb that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

i) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1; and ii) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and iii) wherein the mAb epitope further overlaps with a region to which the epidermal growth factor-like repeat A (EGF-A) domain of LDLR binds; or

f) an mAb that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

i) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; and ii) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

The indicated amino acid sequences are presented in table. 1, which also includes the heavy chain variable region and the light chain variable region of evolocumab. The full-length nucleotide sequences of the heavy chain and light chain of evolocumab are shown in Table. 2, as well as the nucleotide sequences of HCVR and LCVR of evolocumab.

- 20 045816

Table 1

Sequences of PCSK9 and PCSK9-binding polypeptide

Evolocumab NS sequence (USAN ; SEQ ID NO:1) EVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60 AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSSASTKG 120 PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 180 SSWTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA GPSVFLFPPK 240 PKDTLMISRT PEVTCVWDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF NSTFRWSVL 300 TWHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT 360 CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTDKSR WQQGNVFSCS 420 VMHEALHNHY TQKSLSLSPG K 441 LC sequence of evolocumab (USAN ; SEQ ID NO:2) ESALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV 60 SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVLG QPKAAPSVTL 120 FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTTPSKQ SNNKYAASSY 180 LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTECS 215 Pre-Protein PCSK9 (human; SEQ ID NO: 3) MGTVSSRRSW WPLPLLLLLL LLLGPAGAR QEDEDGDYEE LVLALRSEED

- 21 045816

GLAEAPEHGT 60

TATFHRCAKD PWRLPGTYW VLKEETHLSQ SERTARRLQA QAARRGYLTK ILHVFHGLLP 120 GFLVKMSGDL LELALKLPHV DYIEEDSSVF AQSIPWNLER ITPPRYRADE YQPPDGGSLV 180 EVYLLDTSIQ SDHREIEGRV MVTDFENVPE EDGTRRFHRQA SKCDSHGTHL AGWSGRDAG 240 VAKGASMRSL RVLNCQGKGT VSGTLIGLEF IRKSQLVQPV GPLWLLPLA GGYSRVLNAA 300 CQRLARAGW LVTAAGNFRD DACLYSPASA PEVITVGATN AQDQPVTLGT LGTNFGRCVD 360 LFAPGEDIG ASDCSTCFV SQSGTSQAAA HVAGIAAMML SAEPELTLAE LRQRLIHFSA 420 KDVINEAWFP EDQRVLTPNL VAALPPSTHG AGWQLFCRTV WSAHSGPTRM ATAIARCAPD 480 EELLSCSSFS RSGKRRGERM EAQGGKLVCR AHNAFGGEGV YAIARCCLLP QANCSVHTAP 540 PAEASMGTRV HCHQQGHVLT GCSSHWEVED LGTHKPPVLR PRGQPNQCVG HREASIHASC 600 CHAPGLECKV KEHGIPAPQG QVTVACEEGW TLTGCSALPG TSHVLGAYAV DNTCWRSRD 660 VSTTGSTSEE AVTAVAICCR SRHLAQASQE LQ 692 HCVR sequence evolocumab (SEQ ID NO:14) QVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60

AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSS 115

LCVR sequence of evolocumab (SEQ ID NO:15)

QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI

YEVSNRPSGV 60

SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVL 109

CDR1 LC (SEQ ID NO:4)

TGTSSDVGGY NSVS 14

CDR2 LC (SEQ ID NO:5)

EVSNRPS 7

CDR3 LC (SEQ ID NO:6)

NSYTSTSMV 9

CDR1 HC (SEQ ID NO:7)

GYTLTSYGIS 10

CDR2HC (SEQ ID NO:8)

WVSFYNGNTN YAQKLQ 16

CDR3HC (SEQ ID NO:9)

GYGMDV 6

- 22 045816

table 2

Polynucleotide sequences of evolocumab

Evolocumab NS sequence (SEQ ID NO:12) (please note attention, peptide) what are nucleotides 1-57 code native signal atggactgga ccactccgag cctggaggat 60 ccttttcttg gtggcagcag ccacaggtgt gttcagctgg gaaggtctcc tgcagtctgg 120 agctgaggtg aagaagcctg gggcctcagt tgcaaggctt acaggcccct ctggttacac 180 cttaaccagc tatggtatca gctgggtgcg ggacaagggc aaactatgca ttgagtggat 240 gggatgggtc agtttttata atggtaacac cagaagctcc agcctacatg agggcagagg 300 caccatgacc acagacccat ccacgagcac gagctgagga aggctacggt gcctgagatc 360 tgacgacacg gccgtgtatt actgtgcgag atggacgtct caagggccca ggggccaagg 420 gaccacggtc accgtctcct ctgcctccac tcggtcttcc ggccctggggc ccctggcgcc 480 ctgctccagg agcacctccg agagcacagc tgcctggtca aggcgctctg aggactactt 540 ccccgaaccg gtgacggtgt cgtggaactc accagcggcg ctccctcagc tgcacacctt 600 cccagctgtc ctacagtcct caggactcta agcgtggtga ccgtgccctc cagcaacttc ggcaccaga cctacacctg

caacgtagat 660

- 23 045816

cacaagccca gcaacaccaa ggtggacag acagttgagc gcaaatgttg tgtcgagtgc 720 ccaccgtgcc cccaaaaccc cagcaccacc 780 tgtggcagga ccgtcagtct tcctcttccc aaggacacc ggacgtgagc tcatgatctc 840 ccggacccct gaggtcacgt gcgtggtggt cacgaagacc gcataatgcc ccgaggtcca 900 gttcaactgg tacgtggacg gcgtggaggt aagacaaagc cgtcctcacc caggggaga 960 gcagttcaac agcacgttcc gtgtggtcag gttgtgcacc caacaaaggc aggactggct 1020 gaacggcaag gagtacaagt gcaaggtctc ctcccagccc agaaccacag ccatcgagaa 1080 aaccatctcc aaaaccaaag ggcagccccg gtgtacacc cctgacctgc tgcccccatc 1140 ccggggagg atgaccaaga accaggtcag ctggtcaaag tggggcagccg gcttctaccc 1200 cagcgacatc gccgtggagt ggggagagcaa gagaacaact cttcctctac acaagaccac 1260 acctcccatg ctggactccg acggctcctt agcaagctca atgctccgtg ccgtggacaa 1320 gagcaggtgg cagcagggga acgtcttctc atgcatgagg tccgggtaaa ctctgcacaa 1380 ccactacacg cagaagagcc tctccctgtc Evolocumab LC sequence (SEQ ID NO:13) atggacatga gagaggtgcc gggtgcccgc 60 tcagctcctg gggctcctgc tgctgtggct agatgtgagt acagtcgatc ctgccctgac 120 tcagcctgcc tccgtgtctg ggtctcctgg accatctcct ctcctggtac gcactggaac 180 cagcagtgac gttggtggtt ataactctgt caacagcacc tcggccctca caggcaaagc 240 ccccaaactc atgatttatg aggtcagtaa ggggtttcta gaccatctct atcgcttctc 300 tggctccaag tctggcaaca cggcctccct

- 24 045816

gggctccagg ctgaggacga ggctgattat tactgcaatt catatacaag caccagcatg 360 gtattcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 420 actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 480 ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 540 aaggcggggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 600 agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag

ctgccaggtc 660 acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 711

Evolocumab HCVR sequence (SEQ ID NO:10)

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta caccttaacc agctatggta tcagctggggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg gtcagttttt ataatggtaa cacaaactat 180 gcacagaagc tccaggggcag aggcaccatg accacagacc catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtgt attackgtgc

gagaggctac 300 ggtatggacg tctggggcca agggaccacg gtcaccgtct cctct 345

LCVR sequence of evolocumab (SEQ ID NO:11)

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctggggctc 240 caggctgagg acgaggctga ttattactgc aattcatata caagcaccag

catggtattc 300 ggcggaggga ccaagctgac cgtccta 327

Evolocumab has CAS registration number 1256937-27-5. A variant of evolocumab that does not alter its PCSK9-binding and inhibitory properties is shown in Table 1. 3.

Table 3

HCVR and LCVR sequences of the evolocumab variant HCVR sequence of the evolocumab variant (SEQ ID NO:16) QVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60 AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSS 115 LC variant e sequence Volocumab (SEQ ID NO:17) QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV 60 SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVL 109

Pharmaceutical composition or pharmaceutical composition, etc. means a composition, usually sterile, of a pharmaceutically active drug, such as a biologically active

- 25 045816 tive protein (for example, PCSK9-binding polypeptide), which is suitable for administration, such as parenteral administration (including intravenous, intramuscular, subcutaneous, aerosol, intrapulmonary, intranasal or intrathecal), to a subject in need thereof, and it contains only pharmaceutically acceptable excipients, diluents and other additives recognized as safe by the Federal Drug Administration or other foreign national regulatory authorities.

Pharmaceutical formulations include liquids, such as aqueous solutions, which can be administered directly, and lyophilized powders, which can be reconstituted into solutions by adding a diluent prior to administration.

Polypeptide or protein means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally occurring and non-recombinant cell; or produced by a genetically engineered or recombinant cell, and includes molecules having the amino acid sequence of a native protein, or molecules having deletions, additions and/or substitutions of one or more amino acids in the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogues of the corresponding naturally occurring amino acid and polymers. A polypeptide fragment refers to a polypeptide that has a deletion at the amino terminus, a deletion at the carboxyl terminus, and/or an internal deletion from the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. The length of the fragments can range from approximately five to 500 amino acids. For example, the length of the fragments may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids. Useful polypeptide fragments include immunologically functional antibody fragments, including binding domains. In the case of a PCSK9 binding antibody, useful fragments include, but are not limited to, a CDR region, a heavy and/or light chain variable domain, a portion of an antibody chain, or just a variable region thereof containing two CDRs.

Prevention (eg, the occurrence of a symptom, disease or disorder) does not require eliminating the possibility of the occurrence of the phenomenon. It rather means that the likelihood of the phenomenon occurring was reduced in the presence of the compound or method. Prevention in the therapeutic sense refers to types of prophylactic treatment and routes of administration that reduce the risk that a subject will develop a disorder or other risk factor.

Stable pharmaceutical formulation, stable formulation, or pharmaceutical formulation is stable refers to a pharmaceutical formulation based on PCSK9-binding polypeptides that exhibits a limited increase in aggregation and/or reduced loss of biological activity of no more than 5% when stored at a temperature of about -30°C From (or below) to about 5°C to about 40°C for at least 1 month, or 2 months, or three months, or 6 months, or 1 year, or 2 years, or 5 years, or longer according to compared with the control sample. Formulation stability can be determined by one of ordinary skill in the art using any number of standard assays, including size exclusion HPLC (SEC-HPLC), cation exchange HPLC (CEX-HPLC), light-blocking detection of particles invisible to the naked eye (HIAC), and/or visual inspection. Typically, the higher the storage temperature, the shorter the shelf life of the composition.

Methodologies for assessing degradation vary depending on the nature of the protein in the pharmaceutical formulation. Exemplary techniques include size exclusion chromatography (SEC)-HPLC to detect, for example, aggregation, reverse phase (RP)-HPLC to detect, for example, protein fragmentation, ion exchange HPLC to detect, for example, protein charge changes, mass spectrometry, fluorescence spectroscopy, spectroscopy circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used individually or in combination to evaluate protein degradation in a pharmaceutical formulation and determine the shelf life of such formulation. The pharmaceutical compositions disclosed herein generally exhibit an increase in degradation (eg, fragmentation, aggregation, or unfolding) of no more than about 2% to about 3% over two years when stored at 2-8°C.

Subject or patient is used interchangeably and includes human subjects and non-human animal subjects, as well as subjects with recognized disorders, subjects without officially recognized disorders, subjects receiving medical care, and subjects at risk of developing disorders.

Surfactant means a surfactant, including substances commonly called wetting agents, surface tension reducers, detergents, dispersants, emulsifiers and quaternary ammonium antiseptics. Surfactants are further discussed below.

Tangential flow filtration or TFF refers to a process in which a solution passes

- 26 045816 dit tangentially along the ultrafiltration membrane (ie, a semi-permeable membrane that can distinguish between molecules of different shapes and sizes), with salts and/or solutes of lower molecular weight passing through it under pressure.

A therapeutically effective amount refers to that amount of PCSK9 antigen binding polypeptide that is determined to result in a therapeutic response in a subject. Such therapeutically effective amounts can be readily determined by one of ordinary skill in the art.

In the context of filtration, transmembrane pressure (TMP) denotes the average pressure difference between the feed side and the filtrate side of the membrane and can be expressed using equation (1):

I feed pressure + concentrate pressure

TMP = -------------2-------------- filtrate pressure (Equation (1))

Treat and provide treatment include providing types of therapeutic treatment. Treatment does not require complete cure of the disorder and covers cases in which symptoms or underlying risk factors are reduced.

Ultrafiltration, ultrafiltration implementation, UF and similar terms refer to the use of a semi-permeable membrane that distinguishes between molecules of different shapes and sizes, separating molecules from other molecules or concentrating similar or substantially identical molecules.

A PCSK9 binding polypeptide variant refers to an amino acid sequence where one or more amino acid residues in the amino acid sequence are inserted, deleted and/or substituted from another polypeptide sequence. Variants include fusion proteins.

Viscosity refers to a fluid's resistance to movement and can be measured in units of centipoise (cP) or millipascal seconds (mPa-s), where 1 cP = 1 mPa-s at a given shear rate. Viscosity can be measured using a viscometer, such as the analog viscometer from Brookfield Engineering (Middleboro, MA), or a rheometer, such as the m-VROC™ rheometer, or the AESR-G2 cone-plane rheometer from TA Instruments (New Castle, DE ). Viscosity can be measured using any other methods and in any other units known in the art (eg, absolute, kinematic or dynamic viscosity). Regardless of the method used to determine viscosity, the percentage reduction in viscosity in excipient formulations compared to control formulations will remain approximately the same for a given shear rate.

Components of the compositions and methods of PCSK9-binding polypeptides PCSK9-binding polypeptides, including evolocumab, have been well described (Chan et al., 2012).

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease involved in the regulation of low-density lipoprotein receptor (LDLR) protein levels (Horton, Cohen, & Hobbs, 2007; Seidah & Prat, 2007). PCSK9 is a prohormone proprotein convertase in the subtilisin (S8) family of serine proteases ( Seidah et al., 2003 ). An exemplary amino acid sequence of human PCSK9 is shown under SEQ ID NO: 3 in table. 1, which is the (full-length) precursor protein form of PCSK9. PCSK9 proteins may also include fragments of the full-length PCSK9 protein. The structure of the PCSK9 protein has been revealed (Cunningham et al., 2007; Piper et al., 2007). PCSK9 contains a signal sequence, an N-terminal prodomain, a subtilisin-like catalytic domain, and a C-terminal domain.

PCSK9-binding polypeptides are polypeptides that contain one or more complementarity determining regions (CDRs). In some PCSK9-binding polypeptides, the CDRs are embedded in a framework region that orients the CDRs such that the CDRs have the proper PCSK9 binding properties. PCSK9-binding polypeptides can interfere with, block, reduce or modulate the interaction between PCSK9 and LDLR. Such PCSK9-binding polypeptides are designated as neutralizing polypeptides. However, binding between PCSK9 and LDLR can occur even though the PCSK9-binding polypeptide is neutralizing and bound to PCSK9. For example, a PCSK9-binding polypeptide prevents or reduces the deleterious effects of PCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Thus, PCSK9-binding polypeptide may modulate or alter the ability of PCSK9 to degrade LDLR without preventing binding between PCSK9 and LDLR. Such PCSK9-binding polypeptides can be described as neutralizing in a non-competitive manner.

A neutralizing PCSK9-binding polypeptide can bind to PCSK9 at a location or in a manner that prevents PCSK9 from binding to the LDLR. Such PCSK9-binding polypeptides can be described as neutralizing in a competitive manner. PCSK9 neutralizing agents may cause more free LDLR to be present in the subject, resulting in greater binding of LDLR to LDL and thereby reducing the amount of LDL in the subject.

- 27 045816 ect. In turn, this results in a decrease in the amount of cholesterol present in the subject's blood serum.

Some PCSK9-binding polypeptides can inhibit PCSK9-mediated activity (including binding). PCSK9-binding polypeptides can also suppress interactions between PCSK9 and LDLR and other physiological effects mediated by PCSK9. The PCSK9-binding polypeptides may be human, such as fully human, anti-PCSK9 antibodies.

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 the PCSK9 prodomain. 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 in such a way that PCSK9 cannot bind or bind as efficiently to the LDLR. Some PCSK9-binding polypeptides do not bind to the C-terminus of the catalytic domain. In other cases, the PCSK9-binding polypeptide does not bind to the N-terminus of the catalytic domain. In other cases, the PCSK9-binding polypeptide does not bind to the N- or C-terminus of the PCSK9 protein. In some cases, the PCSK9-binding polypeptide binds to any of the epitopes that anti-PCSK9 antibodies bind. In some cases, this can be determined using competitive binding assays performed with a candidate antibody and a reference antibody, such as evolocumab. In some cases, PCSK9-binding polypeptides bind to PCSK9 with a specific conformational structure in such a way as to prevent PCSK9 from interacting with the LDLR. In some cases, the PCSK9-binding polypeptide binds to the PCSK9 V domain. In some cases, PCSK9 binding polypeptide binds to the PCSK9 V domain and prevents (or reduces) PCSK9 binding to the LDLR. In some cases, the PCSK9 binding polypeptide binds to the PCSK9 V domain, and although it does not prevent (or reduce) the binding of PCSK9 to the LDLR, the PCSK9 binding polypeptide prevents or reduces the deleterious activities mediated by the effects of PCSK9 on the LDLR.

In some cases, PCSK9-binding polypeptides contain one or more CDRs (eg, 1, 2, 3, 4, 5, or 6 CDRs). In some cases, a PCSK9-binding polypeptide comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or linked to the polypeptide structure. The polypeptide structure can take a number of different forms. For example, it may be or contain the framework region of a naturally occurring antibody or fragment or variant thereof, or may be synthetic.

The polypeptide structure of PCSK9-binding polypeptides can be an antibody or derived from an antibody, including monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies (mimetic antibodies), chimeric antibodies, humanized antibodies, fusion antibodies (sometimes called antibody conjugates ) and parts or fragments of each of them, respectively. In some cases, the PCSK9-binding polypeptide is an antibody fragment (eg, Fab, Fab', F(ab') ₂ or scFv).

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

In some cases, the antibodies contain at least one heavy chain variable region and one light chain variable region. In other cases, antibodies contain two identical light chains and two identical heavy chains. By way of example, an 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 contains (or consists of) 1, 2 and/or 3 CDR heavy and/or light chains from at least one of the sequences (SEQ ID NO: 4-9) listed in table. 1. In some cases, all six CDRs (light chain CDR1-3 (CDRL1, CDRL2, CDRL3) and heavy chain CDR1-3 (CDRH1, CDRH2 and CDRH3)) are part of the PCSK9-binding polypeptide. In some cases, 1, 2, 3, 4, 5 or more CDRs are contained in the PCSK9 binding polypeptide. In some cases, one heavy chain CDR and one light chain CDR from the CDRs in the sequences in Table 1 are contained in a PCSK9-binding polypeptide. In some cases, additional regions are contained in the PCSK9-binding polypeptide.

The PCSK9-binding polypeptide can be encoded by the nucleic acid sequence shown in table. 2 for evolocumab.

In some cases, a PCSK9-binding polypeptide binds to (but does not block) PCSK9 variants that are at least 50, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99 percent identical, or are characterized by a high percentage of identity with PCSK9 under SEQ ID NO: 3. In some cases

- 28 045816 x PCSK9-binding polypeptide binds to (but does not block) such variants. In some cases, the PCSK9-binding polypeptide binds to such PCSK9 variants and prevents their interaction with the LDLR. In some cases, PCSK9-binding polypeptide binds to PCSK9 variants and prevents them from interacting with the LDLR. In some cases, the PCSK9 variant is a human protein variant, such as the variants at position 474, E620G and/or E670G. In some cases, the amino acid at position 474 is valine.

Humanized PCSK9-binding polypeptides (eg antibodies).

The PCSK9 binding polypeptide may comprise a humanized antibody and/or a portion thereof.

A humanized antibody is substantially non-immunogenic in humans and has substantially the same affinity for its target as an antibody from another species from which the humanized antibody is derived.

The antibody can be modified to provide increased binding affinity to the target and/or to reduce the immunogenicity of the antibody in the recipient. In certain cases, humanized antibodies are modified to eliminate sites of glycosylation to increase the affinity of the antibody for the antigen it recognizes. Techniques such as reconstitution, hyperchimerization, or veining/surface modification can be used to produce humanized antibodies. Such techniques generally reduce the immunogenicity of the antibody by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-alotypic reactions after repeated administration of the antibody. Other methods for reducing immunogenicity are known in the art.

The CDRs of the anti-PCSK9 antibody light and heavy chain variable regions can be grafted onto framework regions (FRs) from an antibody of the same or a different species. The light and heavy chain variable region CDRs can be grafted onto consensus human FRs. In some cases, FRs from the heavy chain or light chain of an anti-PCSK9 antibody may be replaced by FRs from another heavy chain or light chain. The grafted variable regions from the antibody can be used with a constant region that is different from the constant region of the anti-PCSK9 antibody. The grafted variable regions may be part of a single chain Fv antibody.

PCSK9-binding polypeptide variants.

Other antibodies that are useful are variants of the above PCSK9-binding polypeptides formed by the combination of heavy chain variable regions and light chain variable regions shown in Table. 1, or components thereof, and containing a light chain variable region and/or a heavy chain variable region, each of which is characterized by at least 50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 9597%, 97-99%, or more than 99% identity with the amino acid sequences from the sequences in the table. 1 (either as a whole sequence or as a component of a sequence, such as one or more CDRs). In some cases, such antibodies contain at least one heavy chain and one light chain, while in other cases, variant forms contain two identical light chains and two identical heavy chains (or components thereof).

In certain cases, the PCSK9-binding polypeptide contains a heavy chain containing a variable region containing 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.

In some cases, the PCSK9 binding polypeptide contains a sequence that is at least 90%, 90-95% and/or 95-99% identical to one or more CDRs of a CDR in at least one of the sequences of SEQ ID NO: 4- 9. In some cases, 1, 2, 3, 4, 5 or 6 CDRs are present (each being at least 90%, 90-95% and/or 95-99% identical to the above sequences).

In certain cases, the PCSK9-binding polypeptide contains a light chain containing a variable region containing 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.

In other cases, the PCSK9-binding polypeptide comprises a heavy chain containing a variable region containing 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.

One skilled in the art can determine suitable variants of PCSK9-binding polypeptides using well known techniques. In certain cases, one skilled in the art may be able to identify suitable regions of the molecule that can be modified without interfering with its activity by targeting regions that are not considered important for activity. In certain cases, it is possible to identify residues and portions of molecules that are conserved among similar polypeptides. In certain cases, even areas that may be important for

- 29 045816 biological activity or for structure, can be subjected to conservative amino acid substitutions without impairing the biological activity or adversely affecting the structure of the polypeptide.

In addition, one of ordinary skill in the art can review structure-function studies identifying residues in such polypeptides that are important for activity or structure. Given this comparison, it is possible to predict the importance of amino acid residues in a protein that correspond to amino acid residues that are important for activity or structure in similar proteins.

One skilled in the art may select chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in connection with such structure of similar PCSK9-binding polypeptides. Given such information, one skilled in the art can predict the alignment of amino acid residues of the antibody, taking into account its three-dimensional structure. In certain cases, one skilled in the art may decide not to make radical changes to amino acid residues that are believed to be on the surface of a protein because such residues may be involved in important interactions with other molecules. In addition, one skilled in the art can create test variants containing one amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known in the art. Such options can be used to collect information about suitable options. For example, if it is observed that a change to a particular amino acid residue has resulted in disrupted, undesirably reduced or inappropriate activity, variants with such a change may be avoided. In other words, based on the information collected in such standard experiments, one skilled in the art can easily identify amino acids in which additional substitutions should be avoided, either alone or in combination with other mutations.

In certain cases, variants of a PCSK9-binding polypeptide include glycosylation variants in which the number and/or type of glycosylation sites have been changed compared to the amino acid sequences of the parent polypeptide. In certain cases, protein variants contain more or fewer N-linked glycosylation sites than the native protein. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced with a different amino acid (eg, serine) from the original amino acid sequence. Cysteine variants may be useful when antibodies must be refolded into a biologically active conformation, for example, after isolation of insoluble inclusion bodies. Cysteine variants typically contain fewer cysteine residues than the native protein and usually contain an even number to minimize interactions due to unpaired cysteine residues.

Competing PCSK9-binding polypeptides.

PCSK9-binding polypeptides that compete with evolocumab or functional moieties that bind to an epitope that binds evolocumab for specific binding to PCSK9 can be used. Such PCSK9-binding polypeptides may also bind to the same epitope as the PCSK9-binding polypeptides, or to an overlapping epitope. PCSK9-binding polypeptides and fragments that compete with evolocumab or bind to the same epitope exhibit similar functional properties. Thus, by specific example, contemplated PCSK9-binding polypeptides include those that compete with an antibody or PCSK9-binding polypeptide having all six CDRs of evolocumab (SEQ ID NOs: 4-9) or two light chains and two heavy chains of SEQ ID NO: 2 and 1 respectively.

Illustrative epitopes.

Epitopes are provided from SEQ ID NO: 3 (human PCSK9 polypeptide) to which anti-PCSK9 antibodies bind. In the case of evolocumab (and the evolocumab variant 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, A239, G244, M247, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, I369, IA S372, C375, C378, R237 and D238.

Production of PCSK9-binding polypeptides (eg antibodies).

Certain strategies can be used to control the inherent properties of an antibody, such as the affinity of the antibody for its target. Such strategies include the use of site-specific or random mutagenesis of a polynucleotide molecule encoding an antibody to create a variant antibody. In certain cases, such creation is followed by screening for antibody variants that exhibit the desired change, for example, increased or decreased affinity.

The amino acid residues targeted by mutagenesis strategies may be residues in the CDR or FR.

- 30 045816

In certain cases, smaller and more efficiently screened libraries of antibody variants are generated by limiting random or site-directed mutagenesis to hypermutated regions in the CDRs, which are regions that correspond to regions prone to mutation during the somatic affinity maturation process.

Antibodies can be expressed in cell lines. Sequences (such as polynucleotides encoding the polypeptides of SEQ ID NOs: 1 and 2, such as the polynucleotides of SEQ ID NOs: 12 and 13) encoding specific antibodies can be used to transform a suitable host cell, which is a mammalian cell. Transformation can be carried out using any known method of introducing polynucleotides into a host cell. The transformation procedure used depends on the host to be 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, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei.

Mammalian cell lines suitable for expression as hosts 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 cells ( BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), human kidney epithelial cells 293 and a number of other cell lines.

In certain cases, antibodies are produced by the 21B12 hybridoma cell line (Jackson et al., 2009). In certain cases, PCSK9-binding polypeptides bind to PCSK9 with a dissociation constant (KD) of less than about 1 nM, such as 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.

PCSK9-binding polypeptides may comprise an immunoglobulin molecule of at least one of the isotypes IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD and IgM. In certain cases, PCSK9-binding polypeptides contain a human kappa light chain and/or a human heavy chain. In certain cases, the heavy chain is of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD or IgM isotype. In certain cases, PCSK9-binding polypeptides have been cloned for expression in mammalian cells. In certain cases, PCSK9-binding polypeptides contain a constant region different from any of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD and IgM isotype constant regions.

In certain cases, PCSK9-binding polypeptides contain human lambda light chain and human IgG2 heavy chain. In certain cases, PCSK9-binding polypeptides contain human lambda light chain and human IgG4 heavy chain. In certain cases, PCSK9 binding polypeptides contain a human lambda light chain and a human IgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In other embodiments, the PCSK9-binding polypeptides comprise a human kappa light chain and a human IgG2 heavy chain. In certain cases, PCSK9 binding polypeptides contain human kappa light chain and human IgG4 heavy chain. In certain cases, PCSK9-binding polypeptides contain human kappa light chain and human IgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In certain cases, PCSK9-binding polypeptides contain antibody variable regions ligated to a constant region that is neither an IgG2 isotype constant region nor an IgG4 isotype constant region. In certain cases, PCSK9-binding polypeptides have been cloned for expression in mammalian cells.

In the case of evolocumab, the antibody is a human monoclonal antibody with an IgG2 lambda chain; gamma 2 heavy chain disulfide with lambda light chain tetrakis disulfide. Evolocumab is glycosylated at Asn-291 and Asn-291 and has disulfide bridges 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.

In certain cases, conservative modifications in the heavy and light chains of evolocumab, or in such antibody chains having HCVR and LCVR of SEQ ID NOs: 14 and 15, can result in anti-PCSK9 antibodies having functional and chemical characteristics similar to those of the antibodies produced by line hybrid 21B12. Conversely, significant modifications in the functional or chemical characteristics of anti-PCSK9 antibodies can be achieved by selecting heavy and light chain substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the molecular backbone structure at the substitution site, e.g. or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain.

For example, a conservative amino acid substitution may involve replacing a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.

PCSK9-binding polypeptides often comprise one or more polypeptides. Liu

- 31 045816 A variety of expression vector/host systems can be used to express polynucleotide molecules encoding polypeptides comprising one or more components of a PCSK9 binding polypeptide or a PCSK9 binding polypeptide itself. Such systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; systems based on insect cells infected with viral expression vectors (for example, baculovirus); systems based on plant cells transfected with viral expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg, Ti plasmid or pBR322); or animal cell based systems.

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

Components of pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprising a PCSK9 binding polypeptide contains more than one different PCSK9 binding polypeptide. In certain embodiments, the pharmaceutical compositions contain more than one PCSK9 binding polypeptide, wherein the PCSK9 antigen binding proteins bind more than one epitope. In some embodiments, different antigen binding proteins will not compete with each other for binding to PCSK9. In some embodiments, the pharmaceutical composition contains evolocumab.

The PCSK9-binding polypeptide may be associated with a carrier medium, prolonging the half-life. Such carrier media include polyethylene glycol (PEG), glycogen (eg, glycosylation of PCSK9-binding polypeptide), and dextran.

Acceptable components of the composition are preferably non-toxic to recipients at the dosages and concentrations used. Pharmaceutical formulations may contain means to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition.

For example, suitable formulation materials include amino acids (such as proline, arginine, lysine, methionine, taurine, glycine, glutamine or asparagine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, sodium phosphate, sodium acetate (NaOAC), Tris-HCl, Tris buffer, citrate, phosphate buffer, phosphate-buffered saline (i.e. PBS buffer) or other organic acid buffers ); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, sucrose, fructose, lactose, mannose, trehalose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); dyes, flavors and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); agents that normalize tonicity (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery media; thinners; excipients and/or pharmaceutical adjuvants.

In one aspect, the pharmaceutical composition contains high concentrations of a PCSK9-binding polypeptide. In certain embodiments, the concentration of PCSK9 binding polypeptide ranges from 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 ml, approximately 110 mg/ml, approximately 120 mg/ml, approximately 130 mg/ml, approximately 140 mg/ml, approximately 150 mg/ml, approximately 160 mg/ml, approximately 170 mg/ml, approximately 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 ranges from about 140 mg/mL to about 210 mg/mL, such as about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about

- 32 045816 specifically 170 mg/ml, approximately 180 mg/ml, approximately 190 mg/ml, approximately 200 mg/ml, approximately 210 mg/ml, approximately 220 mg/ml, approximately 230 mg/ml, approximately 240 mg/ml , approximately 250 mg/ml or approximately 260 mg/ml.

In another aspect, the pharmaceutical composition contains at least one buffer agent, such as, for example, sodium acetate, phosphate, phosphate saline (PBS), histidine buffer and/or Tris buffer with a pH value of approximately 7.0-8, 5. The buffer serves to maintain a physiologically appropriate pH value. In addition, the buffer can improve the isotonicity and chemical stability of the pharmaceutical formulation. In certain embodiments, the buffering agent content ranges from about 5 mM to about 100 mM, for example, 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 buffer agent. In certain embodiments, the buffering agent is NaOAC. In certain embodiments, the buffering agent is NaOAC and is present at a concentration of approximately 10 mM. In other embodiments, the buffer is monosodium glutamate. In certain embodiments, the buffering agent is monosodium glutamate and is present at a concentration of approximately 10 mM. In yet other embodiments, the buffering agent is a phosphate buffer. In certain embodiments, the phosphate buffer is present at a concentration of approximately 10 mM. In still further embodiments, the buffering agent is histidine. In some of these embodiments, the histidine buffer is present at a concentration of approximately 10 mM. Suitable pH values for the pharmaceutical composition include from about 4 to about 7, or from about 4.8 to about 6.9, or from about 5.0 to about 5.5, or about 5, or about 5.4.

In certain embodiments, the pharmaceutical composition is isotonic, with an osmolality ranging from about 250 to about 400 mOsm/kg, for example, about 250 mOsm/kg, about 260 mOsm/kg, about 270 mOsm/kg, about 280 mOsm /kg, approximately 290 mOsm/kg, approximately 300 mOsm/kg, approximately 310 mOsm/kg, approximately 320 mOsm/kg, approximately 330 mOsm/kg, approximately 340 mOsm/kg, approximately 350 mOsm/kg, approximately 360 mOsm/kg , approximately 370 mOsm/kg, approximately 380 mOsm/kg, approximately 390 mOsm/kg, or approximately 400 mOsm/kg. Osmolality is a measure of the ratio of solutes to fluid volume. In other words, it represents the number of molecules and ions (or molecules) per kilogram of solution. In certain embodiments, the osmolality is 300 mOsm/kg.

Osmolality can be measured using an osmometer, such as the Model 2020 Multi-Sample Osmometer from Advanced Instruments, Norwood, MA. The Advanced Instruments Model 2020 Multi-Sample Osmometer measures osmolality using the freezing point depressing method. The higher the osmolyte content in the solution, the more its freezing point decreases. Osmolality can also be measured using any other methods and apparatus known in the art, such as linear extrapolation. In other embodiments, the pharmaceutical composition is isotonic to a human blood cell, such as a red blood cell.

In yet another aspect, the pharmaceutical composition contains at least one surfactant such as polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types of Pluronic®), sorbitan alkyl complex esters (Span®) polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers and D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS). In certain embodiments, the pharmaceutical composition contains a surfactant at a concentration that ranges from about 0.0001% to about 10 wt. % by volume (w/v) of the formulation, for example, surfactant is 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% (w/v) of the formulation. In certain embodiments, the pharmaceutical formulation contains polysorbate 80 at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical composition contains polysorbate 80 at a concentration of approximately 0.01% w/v of the composition. In other embodiments, the formulation contains Pluronic® F-68 at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical composition contains Pluronic® F-68 at a concentration of approximately

- 33 045816 significant 0.01% w/v composition. In yet other embodiments, the formulation contains Vitamin E TPGS at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical formulation contains Vitamin E TPGS at a concentration of approximately 0.01% w/v of the formulation.

The pharmaceutical composition may contain at least one stabilizing agent, such as a polyhydroxyhydrocarbon (including sorbitol, mannitol, glycerol and dulcitol) and/or a disaccharide (including sucrose, lactose, maltose and trehalose), and/or an amino acid (in addition to contain an arginine salt, such as arginine monohydrochloride, and an acetyl derivative of arginine, such as N-acetylarginine, and they may contain, for example, proline, lysine, methionine and taurine), and/or benzyl alcohol; wherein the total content of said polyhydroxyhydrocarbon and/or disaccharide and/or amino acid and/or benzyl alcohol is from about 0.5% to about 10% w/v of the composition. The pharmaceutical composition may contain proline, for example, at a level of from about 10 mM to about 200 mM, such as from about 50 mM to about 150 mM, such as from about 90 mM to about 120 mM, such as about 120 mM.

In one aspect, the pharmaceutical composition has a viscosity level of less than about 80 centipoise (cP) as measured at room temperature (ie, 25°C). In certain embodiments, the pharmaceutical composition has a viscosity level of less than 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, approximately 10 cP; approximately 8 cP, approximately 6 cP, approximately 4 cP; approximately 2 cP or approximately 1 cP.

In one aspect, the pharmaceutical composition is stable, as measured by at least one stability assay, such as an assay that examines the biophysical or biochemical characteristics of the PCSK9-binding polypeptide over time. The stability of a pharmaceutical formulation can be measured using SEC-HPLC. SEC-HPLC separates proteins based on differences in their hydrodynamic volumes. Molecules with large hydrodynamic protein volumes elute earlier than molecules with smaller volumes. In the case of SEC-HPLC, the stable pharmaceutical formulation exhibits no more than about 5% increase in HMW species relative to the control sample, such as, for example, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than approximately 0.5% increase in the content of HMW species compared to the control sample.

Alternatively or in addition, stability can be measured using cation exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based on differences in their surface charge. At a given pH, charged anti-PCSK9 ABP isoforms are separated on a cation exchange column and eluted using a salt concentration gradient. The eluent is monitored by ultraviolet light (UV) absorption. The distribution of charged isoforms is assessed by determining the peak area of each isoform as a percentage of the total peak area. In the case of CEX-HPLC, the stable pharmaceutical formulation exhibits no more than about 5% reduction in the major isoform peak compared to a control sample, such as, for example, no more than about 3% to about 5% reduction in the major isoform peak compared to the control sample; no more than about 4% reduction, no more than about 3% reduction, no more than about 2% reduction, no more than about 1% reduction, no more than about 0.5% reduction in the major isoform peak compared to the control sample.

Also, as an alternative or addition, formulation stability can be measured using light blocking (HIAC) detection of particles invisible to the naked eye. An electronic suspension particle counting system (HIAC/Royco 9703 (Hach Company; Loveland, CO) or equivalent) containing a light blocking sensor (HIAC/Royco HRLD-150 or equivalent) with a liquid sampler quantifies the number of particles and their size range in a given test sample. sample. When particles in a liquid pass between the light source and the detector, they weaken or block the beam of light that hits the detector. When the particle concentration is within the sensor's normal detection range, the particles are detected individually. The passage of each particle through the detection zone reduces the intensity of light incident on the photodetector, and the photodetector output voltage decreases momentarily. Changes in voltage are recorded as electrical pulses, which are converted by the device into the number of particles present. The method is non-specific and measures particles regardless of their origin. The monitored particle sizes are typically 10 µm and 25 µm. In the case of HIAC, the stable pharmaceutical formulation exhibits no more than 6000 10 µm particles per container (or unit) compared to the control sample, such as no more than 5000, no more than 4000, no more than 3000, no more than 2000, no more than 1000 10 µm particles per container (or unit) compared to the control sample. In other cases, a stable pharmaceutical composition has no increase in

- 34 045816 more than 600 particles of 25 microns per container (or unit) compared to a control sample, such as not more than 500, not more than 400, not more than 300, not more than 200, not more than 100, not more than 50 particles of size 25 µm per container (or unit) compared to control sample.

The stability of a pharmaceutical formulation can also be assessed using visual assessment. Visual evaluation is a qualitative technique used to describe the visible physical characteristics of a sample. The sample is viewed against a black and/or white background in a viewing booth, depending on the characteristic being assessed (e.g., color, clarity, presence of particles or foreign matter). Samples are also reviewed against a turbidity reference standard and color reference standards. When visually assessed, the stable pharmaceutical formulation shows no significant change in color, clarity, presence of particles or foreign matter compared to the control sample.

Illustrative Pharmaceutical Formulations.

In table 4 shows exemplary pharmaceutical formulations based on PCSK9 binding polypeptides. For some compositions, ranges are given, and for compositions from the subparagraphs of the examples (for example, 1.1), the specific composition from the example is given.

Table 4

Exemplary Pharmaceutical Formulations Based on PCSK9-Binding Polypeptides

Example PCSK9- Final binding polypeptide Buffer Auxiliary substances Surface-active substance meaning pH Viscosity 1000/s at (sp) Osmolality 1 195-227 mg/ml 10 mM NaOAc 140 mM NAR 63 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.2 -80 -27 0 mOsm/kg eleven 227 mg/ml 10 mM NaOAc 140 mM NAR 63 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.2 77.4 269 2 195-227 mg/ml 10 mM NaOAc 155 mM NAR 70 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.4 -50 -300 2.1 218 mg/ml 10 mM NaOAc 170 mM NAR 63 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.6 49.6 cP 302 3 195-227 mg/ml 10 mM NaOAc 170 mM NAR 63 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.6 -50 cP -300 mOsm/kg 3, 1 222 mg/ml 10 mM NaOAc 170 mM NAR 63 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.6 52.3 cP 296 mOsm/kg 4 195-227 mg/ml 10 mM NaOAc 140 mM NAR 50 mM Arg-HCl 0.005% - 0.015% polysorbate 80 5.1-5.7 4.1 210 mg/ml 10 mM NaOAc 140 mM NAR 50 mM Arg-HCl 0.01% (w/v) polysorbate 80 5.4 -40 cP 5 188-190 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 290 -23 5.1 190 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 290 22.8 6 200-201 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 295 -35 6, 1 200 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 295 34.5 7 210-214 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 298 -50 7.1 210 mg/ml 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 298 51, 4

* Expected variability in concentration measurements, formulation process, and viscosity measurements was observed.

Routes of therapeutic use.

PCSK9-binding polypeptides, such as evolocumab, can be used in a variety of therapeutic applications. For example, PCSK9-binding polypeptides are useful for treating conditions associated with PCSK9, such as high cholesterol disorders (serum cholesterol disorders), such as hypercholesterolemia. PCSK9-binding polypeptides can be used in the treatment of effects, symptoms and/or pathology associated with PCSK9 activity.

With disorders that are associated with elevated levels, which are based on, or may be influenced by elevated levels of molecules or groups of molecules, including cholesterol levels (including serum cholesterol), LDL, LDLR, PCSK9, VLDL-C , apolipoprotein B (ApoB), lipoprotein A (Lp(a)), triglycerides, HDLC other than HDL-C and total cholesterol can be controlled using methods that use the evolocumab-based pharmaceutical compositions disclosed herein to treating, and/or preventing, and/or reducing the risk of such disorders occurring in the subject. Ras

- 35 045816 Covered compositions based on evolocumab can be used to modulate the levels of such molecules or groups of molecules, such as reducing the amount of such molecules or groups of molecules. For example, the disclosed evolocumab compositions can be used in methods for reducing the amount of such molecules or groups of such molecules from an abnormally high level or even from a normal level, that is, the amount of cholesterol (including serum cholesterol), LDL, LDLR, PCSK9, can be reduced. VLDL-C, ApoB, Lp(a), triglycerides, HDL-C other than HDL-C and total cholesterol levels.

A high cholesterol disorder (which includes disorders associated with high serum cholesterol) includes any one or more of the following: familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary artery disease heart disease, stroke, cardiovascular disease, Alzheimer's disease and dyslipidemias in a broad sense, which may manifest, for example, as increased serum total cholesterol, increased LDL, increased triglycerides, increased VLDL and/or low HDL content. Some non-limiting examples of primary and secondary dyslipidemias include metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, types of familial hypercholesterolemia including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial hypercholesterolemia with defective apolipoprotein B-100; polygenic hypercholesterolemia; familial dysbetalipoproteinemia, hepatic lipase deficiency; dyslipidemia secondary to any of the following: indiscriminate eating, hypothyroidism, medications, including estrogen and progestin therapy, beta blockers and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency and alcoholic hypertriglyceridemia. The disclosed evolocumab compositions may also be used to treat and/or prevent and/or reduce the risk of atherosclerotic diseases, such as cardiovascular death, non-cardiovascular death, or all-cause ischemic death. heart disease, coronary insufficiency, peripheral artery disease, stroke (ischemic and hemorrhagic), angina or cerebrovascular accident, as well as acute coronary syndrome, myocardial infarction and unstable angina. The disclosed evolocumab compositions may also be useful in reducing the risk of fatal and non-fatal heart attacks, fatal and non-fatal strokes, certain types of cardiac surgery, hospitalization for heart failure, chest pain in patients with heart and/or cardiovascular disease complications due to established heart disease, such as a previous heart attack, previous heart surgery, and/or chest pain with signs of blocked arteries, and/or transplant-related vascular disease. In some cases, the disclosed evolocumab compositions may be used in methods of preventing or reducing the risk of cardiovascular disease caused by elevated levels of CRP or hsCRP. In some embodiments, the AVR and methods can be used to reduce the risk of recurrent cardiovascular events.

Diseases or disorders that typically can be controlled (either treated or prevented) through the use of statins may also benefit from the use of the disclosed evolocumab-based compositions. Moreover, disorders or diseases that may benefit from prevention of cholesterol synthesis or increased expression of LDLR can also be treated using the disclosed evolocumab-based compositions. In addition, the use of the disclosed evolocumab-based compositions may be particularly useful in the treatment of diabetes. Not only is diabetes a risk factor for coronary heart disease, but insulin also increases PCSK9 expression. That is, people with diabetes have elevated plasma lipid levels (which may be associated with high PCSK9 levels) and may benefit from lowering these levels.

When a PCSK9-binding polypeptide is used for therapeutic applications, the PCSK9-binding polypeptide can inhibit, interfere with, or modulate one or more biological activities of PCSK9. For example, a PCSK9-binding polypeptide may specifically bind to human PCSK9 and/or significantly inhibit the binding of human PCSK9 to the LDLR by at least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (eg, as measured by binding in an in vitro competitive binding assay). In some cases, the PCSK9-binding polypeptide has a Kd of less (stronger binding) 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , ^10-11 , 10 ^-12 , 10 ^-13 M. In some cases, PCSK9 -binding polypeptide has an IC50 when blocking LDLR binding to PCSK9 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

- 36 045816

200 pM, less than 200 pM, from 200 pM to 150 pM, from 200 pM to 100 pM, from 100 pM to 10 pM, from 10 pM to 1 pM.

Pharmaceutical compositions can be administered as combination therapy, i.e. in combination with other means. The combination therapy may include a PCSK9-binding polypeptide in combination with at least one anti-cholesterol agent. The agents include in vitro synthetically produced chemical compounds, antibodies, antigen-binding regions, and combinations and conjugates thereof. In certain embodiments, the agent may act as an agonist, antagonist, allosteric modulator, or toxin. In certain embodiments, the agent may act to inhibit or stimulate its target (eg, by activating or inhibiting a receptor or enzyme) and thereby stimulate an increase in LDLR expression or a decrease in serum cholesterol levels.

The PCSK9-binding polypeptide can be administered before, concomitantly, or after treatment with a cholesterol-lowering agent (serum and/or total cholesterol). For example, the PCSK9 binding polypeptide can be administered prophylactically to prevent or control the occurrence of hypercholesterolemia, heart disease, diabetes and/or any disorder associated with elevated cholesterol levels. Moreover, the PCSK9-binding polypeptide can be administered to treat an existing condition of hypercholesterolemia. In some cases, administration of a PCSK9-binding polypeptide may delay the onset of the disorder and/or symptoms associated with the disorder. In some cases, the PCSK9-binding polypeptide is administered to a subject who does not have any symptoms of any of the disorders associated with high cholesterol, or a subclass thereof.

The PCSK9-binding polypeptide can be used with specific therapeutic agents to treat various disorders associated with elevated cholesterol levels, such as hypercholesterolemia. Depending on the condition and level of treatment required, two, three or more agents may be administered. Such agents can be supplied together by being included in one formulation. Alternatively, such agents may be formulated separately and, if desired, supplied together by inclusion in a treatment kit. In another example, such agents may be supplied separately.

Dosage and dosage regimens.

The amount of PCSK9-binding polypeptide, such as mAb, such as evolocumab, administered to a patient is a therapeutically effective amount. A typical dosage of PCSK9 binding protein may range from about 0.1 μg/kg up to about 100 mg/kg or more. In certain cases, the dosage may vary from 0.1 mcg/kg up to approximately 100 mg/kg; or from 1 μg/kg up to about 100 mg/kg; or from 5 μg/kg up to about 100 mg/kg; or from 1 mg/kg to about 50 mg/kg; or from 2 mg/kg to about 20 mg/kg; or from 2 mg/kg to about 10 mg/kg PCSK9-binding polypeptide.

The amount (or dose) of PCSK9-binding polypeptide can range from at least about 10 mg to about 1400 mg; or from about 14 mg to about 1200 mg; or from about 14 mg to about 1000 mg; or from about 14 mg to about 800 mg; or from about 14 mg to about 700 mg; or from about 14 mg to about 480 mg; or from about 20 mg up to about 480 mg; or from about 70 mg up to about 480 mg; or from about 80 mg to about 480 mg; or from about 90 mg to about 480 mg; or from about 100 mg to about 480 mg, or from about 105 mg to about 480 mg; or from about 110 mg to about 480 mg; or from about 115 mg to about 480 mg; or from about 120 mg to about 480 mg; or from about 125 mg to about 480 mg; or from about 130 mg to about 480 mg; or from about 135 mg to about 480 mg; or from about 140 mg to about 480 mg; or from about 145 mg to about 480 mg; or from about 150 mg to about 480 mg; or from about 160 mg to about 480 mg; or from about 170 mg to about 480 mg; or from about 180 mg to about 480 mg; or from about 190 mg to about 480 mg; or from about 200 mg to about 480 mg; or from about 210 mg to about 480 mg; or from about 220 mg to about 480 mg; or from about 230 mg to about 480 mg; or from about 240 mg to about 480 mg; or from about 250 mg to about 480 mg; or from about 260 mg to about 480 mg; or from about 270 mg to about 480 mg; or from about 280 mg to about 480 mg; or from about 290 mg to about 480 mg; or from about 300 mg to about 480 mg; or from about 310 mg to about 480 mg; or from about 320 mg to about 480 mg; or from about 330 mg to about 480 mg; or from about 340 mg to about 480 mg; or from about 350 mg to about 480 mg; or from about 360 mg to about 480 mg; or from about 370 mg to about 480 mg; or from about 380 mg to about 480 mg; or from about 390 mg to about 480 mg; or from about 400 mg to about 480 mg;

- 37 045816 or from about 410 mg to about 480 mg; or from about 420 mg to about 480 mg; or from about 430 mg to about 480 mg; or from about 440 mg to about 480 mg; or from about 450 mg to about 480 mg; or from about 460 mg to about 480 mg; or from about 470 mg to about 480 mg of PCSK9-binding polypeptide.

When determining dosing frequency, the pharmacokinetic parameters of the PCSK9-binding polypeptide and/or any additional therapeutic agents in the formulation will be considered. The clinician may administer the formulation until a dosage is achieved that provides the desired effect. The composition can be administered as a single dose or as two, three, four or more doses (which may or may not contain the same amount of PCSK9-binding polypeptide) over time, or as a continuous infusion through an implantable device or catheter. The formulation can also be delivered subcutaneously or intravenously using a needle and syringe. With respect to subcutaneous delivery, pen delivery devices, as well as body injector and auto-injector delivery devices can be used to deliver pharmaceutical formulations containing PCSK9-binding polypeptides.

In certain cases, a dose 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 of PCSK9-binding polypeptide is administered once per week (QW) to a patient in need thereof.

In other cases, a dose 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 of PCSK9-binding polypeptide is administered once every two weeks (or every two weeks; Q2W:) to a patient in need thereof.

In other specified cases, a dose 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 of PCSK9-binding polypeptide is administered once every four weeks (Q4W) (or once per calendar month) to a patient in need thereof.

For example, evolocumab can be administered Q2W in 210 mg doses. Alternatively, evolocumab can be administered as Q4W in 420 mg doses. Depending on the characteristics of the condition being treated, both of these doses of the drug may be administered once a week.

In some cases, the serum LDL cholesterol level is reduced by at least about 15% compared to the predose serum LDL cholesterol level. In some embodiments, the serum LDL cholesterol level is reduced by at least about 20%, or by at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90% or even more.

Storage and kits.

Formulations containing a PCSK9-binding polypeptide, with or without at least one additional therapeutic agent, can be prepared for storage by mixing the selected composition, characterized by the desired degree of purity, with optional agents to obtain the composition in the form of a lyophilized mass or an aqueous solution. In addition, a composition containing a PCSK9-binding polypeptide, with or without at least one additional therapeutic agent, can be formulated as a lyophilisate using appropriate excipients.

Once the pharmaceutical formulation has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (eg, lyophilized) that is reconstituted before administration. In some cases, PCSK9 binding polypeptide formulations can be stored in containers such as suitable storage bags (eg, those manufactured by Sartorius (Gottingen, Germany)) or in polycarbonate bottles. Once the pharmaceutical formulation has been formulated, it can also be stored in pre-filled syringes (PFS; such as 2.25 ml PFS) as a solution or suspension in ready-to-use form, as well as in glass vials (such as glass vials volume 5 cm ³ ).

In certain embodiments, kits are provided to provide a unit of administration as a single dose. In certain embodiments, the kit may contain both a first container containing a dried protein and a second container containing an aqueous formulation. In certain embodiments, kits are provided containing single and multi-chamber

- 38 045816 pre-filled syringes (e.g. liquid syringes and lyophilized syringes).

Ultrafiltration/diafiltration of PCSK9-binding polypeptide-based formulations containing N-acetylarginine.

Using the disclosed UF/DF methods, formulations containing approximately 210 g/L of PCSK9-binding polypeptides can be prepared. For example, in the disclosed methods, the first diafiltration solution (DF1) at a concentration of 70 g/L (or 35 g/L) is diafiltered three times and concentrated to 140 g/L. The second diafiltration solution (DF2) at a concentration of 140 g/L is then diafiltered four times and concentrated to a concentration of 260 g/L. The concentrated pool is then removed from the system using a bleed solution to give a final concentration of 210 g/L.

In the disclosed methods, the first ultrafiltration (UF1) concentration of approximately 70 mg/ml and approximately 35 mg/ml, as shown in the examples, does not have a significant effect on the HWM content (%) of the final drug substance (DS). Moreover, in the disclosed methods, only seven diavolumes of DF are needed in the DF step to ensure completion of diafiltration.

The disclosed UF/DF method is summarized in Table. 5 and 6.

Table 5

General UF/DF procedure and operating parameters

Process description Conditions

General information Membrane/Temperature/Dimensional characteristics of the membrane

Equalization (EQ) buffer/buffer for DF Concentration 1 (UF1) Diafiltration 1 (DF1) Concentration 2 (UF2) Diafiltration 2 (DF2) Buffers for NAR formulations Concentrate to target concentration for DF Transmembrane pressure (TMP) is 18 psi Cross feed flow rate is 300 LMH (liters/m ² /h) 3 dia volume TMP is 18 psi Cross feed flow rate is 300 LMH Concentrate to target concentration for DF TMP is 18 psi Cross flow rate is 300 LMH 4 dia.

- 39 045816

TMP is 18 psi

Cross feed flow rate is 300 LMH

Concentration 3 Concentrate to target concentration (overconcentration TMP is initially 18 psi; e; OS) control valve is fully open The cross feed flow rate is 60 LMH Operating temperature 37 °C Recycling 10 minute recirculation The cross feed flow rate is 60 LMH The path for filtrate is closed (no TMP) Extraction Extract the protein solution through the low point or concentrate channel Run with buffer through concentrate channel Cleaning >20 l/ ^m2 single pass 30-minute recirculation, 20 l/ ^m2 Storage >20 l/m ² one run

Table 6

Stage

Leveling up

Operating parameters of the UF/DF method

Parameter

Unit

Target value

Working range

Bleeding solution PW/water for injection (WFI) or DIW n. d Feed Flow LMH 300 n. D Feed volume l/m ² 20 n. D Filtrate volume Worker control strategy l/m ² >10 Single pass filtrate valve open n. n. d d

- 40 045816

Solution for PW/WFI or n. d. integrity testing DIWW Integrity testing Diffusion flow ml/min 0.11 m ² =14 0.57 m ² =60 1.14 m ² =117 n. d. Pressure at PYa ί sg thirty n. P testing m Testing time MIN. 10 n. d. Solution for PW/WFI or n. d. NWP D.I.W. NWP LMH/psig NWP 8-14, >70% new membrane for n. . d. NWP Balancing Feed Flow LMH 300 n. . d. membranes Solution for EQ NAR solution n. . d. Feed volume l/m ² 20 n. . d. Filtrate volume l/m ² >10 n. . d. TMR psig 20 n. . d. Valve open n. . d. Strategy filtrate worker o stumps GP control O ,2-1, p. o ± o run Target g/l 70 n. . d. meaning Concentration 1 concentrations Feed Flow LMH <300 n. . d. TMR psig 18 n. . d. Working temperature °C 20 + 50 Buffer for diafiltration NAR solution n. . D. And Number IT/TG'' ΠΓΊ h π Diafiltration 1 diavolumes Feed Flow LMH <300 n. D · . d. TMR psig 18 + 50 Working temperature °C 20 + 50 Concentration 2 Target concentration value g/l 140 n. . D. Feed Flow LMH <300 n. . d. TMR psig 18 + 50

- 41 045816

Working °C 20 ± 50 temperature Buffer for Diafiltration Number Diafiltration 2 dia volumes Feed flow number LMH NAR solution 4 <300 n. n. n. D. D. D. TMR psig 18 n. D. Working °C 20 n. D. temperature Concentration Target g/l 260 n. D. 3 value concentration Feed flow LMH <60 n. . D. TMR psig Initially it is 18. Once the TMP valve is fully open, TMP control is no longer possible. n. . D.

Operating temperature ±2.0

Recirculation at General valve closed n. . d. Flow mode description low recirculating pressure its filtrate LMH speed 60 n. . D. flow Time 10 n. d. (Tj minutes Yong recycling Target g/l 240 n. . D. C O meaning WITH concentrations CD Buffer for NAR solution n. . D. S extraction Ή CD Volume Sliding 1 n. . D. CD leveling up AND through the bypass ω S concentrate noah volume Temperature 20 reservoir for _OS ± 2.0 Concentrate extracted not after extraction product

- 42 045816

I'm the starter Stream mode Filtrate valve open n. d. pumping one run Solution for 0.5 M NaOH n. d. cleaning Feed volume l/m ² 20 n. d. AND Volume l/m ² >10 n. D. f filtrate Qi Yu Feed Flow LMH 300 n. D. S f TMR psig 20 n. D. Recycling Stream mode Valve open n. D. she Y ation general Ei o recirculating its filtrate O Solution for 0.5 M NaOH n. D. cleaning Time minutes thirty n. D - Recirculation Volume 20 n. D. recycling Feed Flow LMH 300 n. D. TMR psig 20 n. D.

Storage Valve open filtrate Flow mode of one pass Solution for 0.1 M NaOH storage Supply volume l/m ^{2 20} Volume , ₉ >10 l/m ² filtrate Feed flow LMH ²⁰⁰ TMP psig ²⁰

Thus, disclosed herein is a method for constructing a PCSK9-binding polypeptide, such as a PCSK9-binding polypeptide that blocks PCSK9 binding to an LDLR, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated;

b) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine using diafiltration;

c) a second concentration step, in which the polypeptide in the second solution is concentrated;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution containing N-acetylarginine using diafiltration; And

f) a third concentration step, in which the polypeptide in the third solution is concentrated;

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, to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , and approximately 37°C. Also, the first stage of solution replacement can be carried out using at least three diavolumes of the second solution; in some cases additional diavolumes can be used, for example four, five or six diavolumes. The second stage of solution replacement can be carried out using at least four diavolumes of the third solution; however, additional diameters may be used, including five, six, or seven diameters. The initial concentration of the PCSK9 binding polypeptide may be about 11 mg/ml or less, such as less than 1 mg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or about 11 mg/ml. In addition, the concentration of the PCSK9 binding polypeptide can be increased by about 3 to about 7 times, such as about 3, 4, 5, 6, or about 7 times. For example, the increased concentration of the polypeptide is from about 35 mg/ml to about 70 mg/ml or greater, 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 approximately 70 mg/ml or more. In the second concentration step, the concentration of the PCSK9 binding polypeptide is increased by about 2 to 4 times the first concentration step (e.g., about 2, 3, or about 4 times), for example, to 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, 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 approximately 300 mg/ml. In the third concentration step, the concentration of PCSK9 binding polypeptide can be increased by about 1.5 to about 2

- 43 045 816 times relative to the second concentration step, for example, to approximately 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 250 or approximately 300 mg/ml, for example, to approximately 260 mg/ml. Therefore, the PCSK9-binding polypeptide may have a final concentration that is at least about 19-20 times the initial concentration of the therapeutic polypeptide, for example, about 210 mg/ml. Concentration steps may include batch ultrafiltration; furthermore, the second solution and the third solution may be identical.

The second or third solution containing N-acetylarginine (for example, a NAR solution) may contain an arginine salt and a buffer, where, for example, N-acetylarginine is present at a concentration of from about 25 mM to about 230 mM, such as 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, 215, 220, 225, or approximately 230 mM; the arginine salt is Arg-HCl, Arg acetate or Arg glutamate and is present at a concentration of from about 25 mM to about 150 mM, for example, 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 approximately 150 mM; and the buffer is a sodium acetate buffer at a concentration of from about 5 mM to about 30 mM, such as about 5, 10, 15, 20, 25 or about 30 mM. In other sub-aspects, N-acetylarginine is present at a concentration of from 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 approximately 10 mM. For example, N-acetylarginine may be present at a concentration of approximately 140 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. In additional sub-aspects, N-acetylarginine is present at a concentration of approximately 155 mM, ArgHCl, Arg acetate or Arg glutamate is present at a concentration of approximately 70 mM, sodium acetate buffer is present at a concentration of approximately 10 mM. In yet another example, N-acetylarginine is present at a concentration of approximately 170 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Moreover, the compositions (including NAR solutions) may further contain proline, wherein proline is present at a concentration of from 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 approximately 150 mM. The second or third solution may have a pH value of from about 4.8 to about 6.9, such as 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 approximately 6.9, such as 5.3, 5.4 or 5.5. In the first and second stages of solution replacement, a diafiltration membrane having at least one characteristic selected from the group consisting of:

a) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm, for example approximately 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 , 490, or approximately 500 µm;

b) a free cross-sectional area that is greater than about 32% but less than or equal to about 36% (eg, about 32, 33, 34, 35, or about 36%) of the membrane area;

c) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm, for example about 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 threads/cm;

d) a filament diameter that is greater than about 270 microns but less than or equal to about 340 microns, such as approximately 270, 280, 290, 300, 310, 320, 330 or 340 microns;

e) a basis weight that is greater than about 160 g/ ^m2 but less than or equal to 180 g/ ^m2 , for example about 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, or 180 g/ ^m2 ;

f) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

g) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

h) maximum supply pressure of approximately 60 psi.

Moreover, a surfactant such as polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other vi

- 44 045816 dy Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and D-a-tocopherol polyethylene glycol succinate (vitamin E TPGS ). In some cases, the surfactant is a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® F-68) or D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS). The concentration of surfactant can vary in the range from about 0.0001% to about 10 wt. % by volume (w/v) of the formulation, for example, surfactant is 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% (w/v) of the formulation. In certain embodiments, the pharmaceutical formulation contains polysorbate 80 at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical composition contains polysorbate 80 at a concentration of approximately 0.01% w/v of the composition. In other embodiments, the formulation contains Pluronic® F-68 at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical formulation contains Pluronic® F-68 at a concentration of approximately 0.01% w/v of the formulation. In yet other embodiments, the formulation contains Vitamin E TPGS at a concentration that ranges from about 0.0001% to about 1% w/v of the formulation. In certain embodiments, the pharmaceutical formulation contains Vitamin E TPGS at a concentration of approximately 0.01% w/v of the formulation.

The following Examples section is provided by way of example only and is not intended to limit the present invention or claims in any way.

Examples

Viscosity measurements were performed using an AESR-G2 cone-plane rheometer from TA Instruments (New Castle, DE) unless otherwise noted.

Example 1: Experimental Design (DOE); optimization of evolocumab-based formulations containing N-acetylarginine.

A design of experimental design (DOE) is proposed to optimize evolocumab formulations containing N-acetylarginine. Eleven formulations were tested for viscosity, prefilled syringe pressure, stability, pH and osmolality. Data on the initial parameters of the compositions are shown in table. 7.

Table 7

Experimental design for evolocumab formulations with NAR, data for time zero

Force

No. Target pH value Target Value [evoloku mab] (mg/ml) Target Value [NAR] Surface-active substance Target Value [acetate] pH value Osmolality [evoloku mab] (mg/ml) Viscosity at 1000/s (sp) Pressing force at pressing time 10 s (N) pressing at pressing time 12 s (H) Pressing force at pressing time 20 s (N) 1 5 190 140 0.05% F68 thirty 4.94 257 192 29.5 34 28 17 2 5 210 140 0.05% thirty 4.94 261 204 42.7 47 38 23 TPGS 3 5.4 190 140 0.01% 10 5.35 243 192 26, 8 29 24 15 PS80 4 5.4 210 140 0.05% F68 10 5.36 254 207 49.0 51 41 25 5 5.2 200 155 0.01% 20 5.11 265 198 33.5 37 thirty 19 PS80 6 5.2 200 155 0.05% F68 20 5.10 261 197 34.6 38 31 19 7 5.2 200 155 0.05% 20 5.10 266 201 32.3 38 31 19 TPGS 8 5 210 170 0.01% thirty 4.99 299 213 45.2 47 38 23 PS80 9 5 210 170 0.05% F68 thirty 5.01 290 213 46.7 48 39 24 10 5 210 170 0.05% thirty 4.98 292 214 49.4 49 40 25 TPGS eleven 5.4 190 170 0.05% 10 5.34 286 189 24.3 29 24 15 TPGS

The acetate concentration was minimized to reduce the rate of aggregation based on the data obtained after one month incubation at 40° C. shown in FIG. 1. The proline concentration was increased to 120 mM to make the composition isotonic. N-acetylarginine (155 mM) was chosen to maintain a balance between viscosity reduction and N-acetylarginine crystallization,

- 45 045816 observed at a concentration of 170 mM at 4°C. The data also indicated that varying the surfactant between Polysorbate 80 (TWEEN® 80; polyoxyethylene sorbitan monooleate), Pluronic® F-68 (polyoxyethylene-polyoxypropylene block copolymer), and Vitamin E TPGS (D-αtocopherol polyethylene glycol succinate) did not result in a significant difference in viscosity or stability.

Based on these data, it was found that a formulation containing 10 mM acetate, 155 mM N-acetylarginine, 120 mM proline, 0.01% polysorbate-80, with a pH value of 5.4, is suitable for reducing the viscosity of the evolocumab-based formulation at high concentrations (eg 190-210 mg/ml). The data presented in Table 8 indicate that this composition is isotonic and has low viscosity.

Table 8

Time zero data for 190-210 mg/mL evolocumab formulations containing 10 mM acetate, 155 mM N-acetylarginine, 120 mM proline, 0.01% polysorbate-80, pH 5.4

Compound [evolocumab] pH value Osmolality Viscosity 10/155/120-0.01% 188 5.38 290 22. PS-80-190 mg/ml 10/155/120-0.01% 201 5.40 295 34.5 PS-80-200 mg/ml 10/155/120-0.01% 214 5.43 298 51, 4 PS-80-210 mg/ml

Example 2: DOE Study to Evaluate the Effect of Excipient Concentration, pH, and Buffer.

Examples 2-4 are directed toward formulations that have been optimized to maximize viscosity reduction while minimizing any impact on aggregation, deamidation, and other factors that indicate stability.

N-acetylarginine (NAR) was selected based on its superior effect on reducing viscosity in excipient screening studies (see, for example, Example 1). The concentration of NAR was limited by its solubility at 2-8°C. The NAR concentration of 155 mM in diafiltration (DF) buffer was chosen based on stability studies showing no crystallization of NAR at concentrations up to 175 mM. Arginine-HCl (70 mM) was added to the DF buffer to ensure an isotonic formulation of the drug with the lowest viscosity and to improve the solubility of NAR. The pH 5.4 formulation was selected to minimize aggregation and deamidation of evolocumab. The buffer and surfactant (acetate/polysorbate 80) had minimal effect on the viscosity and stability of the evolocumab formulation.

Three main studies were conducted to investigate the stability of evolocumab in NAR/arginine-HCl-containing formulations. They were next.

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

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

3. Downscaled study to evaluate the stability of lead candidate formulations, including the effects of typical drug product manufacturing processes (Example 4).

Results.

The target viscosity value was set to <50 cP. Viscosity screening of the formulations showed that NAR was the most effective excipient in reducing viscosity. However, due to the limited solubility of NAR, a combination of NAR and arginine-HCl was used to provide an isotonic formulation and achieve the goal of obtaining a 210 mg/mL evolocumab formulation with a viscosity less than or equal to 50 cP. A summary of the selected formulations that were evaluated during the screening is shown in FIG. 2.

The 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. For each sample, the formulation parameters varied are shown in Table 9. The study design included NAR concentrations ranging from 155-175 mM, arginine-HCl concentrations ranging from 50-100 mM, and pH values ranging from 4.8 to 5.4. The study also included a comparison of acetate and glutamate buffers at 10 mM. The study involved a 140 mg/mL proline control formulation prepared from the same batch of starting material as the NAR/Arg-HCl containing samples and loaded into PFS. Samples were subjected to sterilization filtration using PVDF filters with a pore size of 0.2 μm and manually filled into glass PFS at

- 46 045816 filling volume of ~2.0 ml.

Table 9

POE study plan

pH value NAR (mm) Arg-HCl (mm) Buffer PS-80 Evolocumab (mg/ml) Sample code 4.8 155 50 10 mM acetate 0.01% 210 DOE 1-10/155/50, pH value 4.8 5, 4 155 50 10 mM glutamate 0.01% 210 DOE 2-10/155/50, pH value 5.4 4.8 175 50 10 mM glutamate 0.01% 210 DOE 3-10/175/50, pH value 4.8 5, 4 175 50 10 mM acetate 0.01% 210 DOE 4-10/175/50, pH value 5.4 5, 1 165 75 10 mM acetate 0.01% 210 DOE 5-10/165/75, pH value 5.1 5, 1 165 75 10 mM glutamate 0.01% 210 DOE 6-10/165/75, pH value 5.1 4.8 155 100 10 mM glutamate 0.01% 210 DOE 7-10/155/100 pH value 4.8 5, 4 155 100 10 mM acetate 0.01% 210 DOE 8-10/155/100 pH value 5, 4 4.8 175 100 10 mM acetate 0.01% 210 DOE 9-10/175/100 pH value 4.8 5, 4 175 100 10 mM glutamate 0.01% 210 DOE 10-10/175/100 pH value 5, 4 50 0 0 20 mM acetate 0.01% 140 Control with a concentration of 140 mg/ml containing proline

Data on pH values, concentration, osmolality and viscosity are presented in tabular form in table. 10. The observed pH values were close to the target values for all samples. Osmolality in the study covered the range of 256-392 mOsm/kg. The viscosity was found to show an inconsistent trend towards decreasing viscosity with increasing pH value.

Table 10

Data on pH, concentration, osmolality and viscosity values based on DOE

Sample Meaning rn Concentration mOsm/kg Viscosity (cP 1000 s ^-1.25 ' at ^E S) 1-10/155/50, pH value 4.8 4.83 209.0 256 45, 3 2-10/155/50, pH value 5.4 5.29 209.0 271 43, 8 3-10/175/50, pH value 4.8 4, 81 210, 6 301 46, 6 4-10/175/50, pH value 5.4 5, 31 212.9 297 41, 0 5-10/165/75, pH value 5.1 5, 07 211.9 326 48.2 6-10/165/75, pH value 5.1 5, 03 209, 8 340 47.5 7-10/155/100, pH value 4.8 4.80 208.5 369 44.9 8-10/155/100, pH value 5.4 5.29 210, 3 366 38.8 9-10/175/100, pH value 4.8 4.84 208.7 376 43, 9 10-10/175/100, pH value 5.4 5, 33 209, 2 392 36, 6

Analysis of size exclusion liquid chromatography (SE-HPLC) data using JMP Statistical Analysis Software (SAS; Cary, NC; Figures 3A and 3B) revealed significant effects of pH and arginine-HCl concentration on lead loss.

- 47 045816 ranks of the main peak as a percentage at 40°C for 1 month (Fig. 3A). Lower pH and higher arginine-HC1 concentration caused greater loss of the main peak, primarily due to an increase in the aggregate peak and, to a lesser extent, the oligomer peak. Both peaks together were categorized as high molecular weight (HMW) species. In fig. Figure 4 shows the effects of pH and arginine-HCl on the percentage of HMW species for a time point of 1 month at 40°C. Significantly higher percentage levels of HMW species were observed in samples with a pH value of 4.8 compared to samples with a pH value of 5.4. In addition, the effect of higher arginine-HCl concentration resulting in a higher percentage of HMW species was significantly more pronounced at pH 4.8 compared to pH 5.4.

In fig. 5A-5C show SE-HPLC chromatograms for selected samples with a pH value of 4.8 and a pH value of 5.4 compared to a control with a concentration of 140 mg/ml containing proline, maintained at 5°C for 6 months, 25°C c for three months and 40°C for 1 month, respectively. The chromatograms show a trend toward higher levels of aggregates at pH 4.8 relative to pH 5.4. The chromatograms also showed that the degradation profile was comparable for the 210 mg/mL samples containing NAR/Arg-HCl compared to the 140 mg/mL control containing proline, despite the higher rate of aggregation observed for the samples with a concentration of 210 mg/ml containing NAR/Arg-HCl.

Despite the differences associated with pH and Arg-HCl concentration observed at 40°C, all formulations tested in this experiment showed comparable percentages of HMW species when held for up to 6 months at 5°C and up to up to 1 month at 25°C (Fig. 6A, 6B).

Tryptic peptide mapping using liquid chromatography-mass spectrometry (LC-MS) analysis was performed on selected samples. Peptide map chromatograms obtained using an HPLC ultraviolet (UV) detector were visually compared to control evolocumab formulations containing proline and an evolocumab reference standard. No new peaks were detected in samples containing NAR/Arg-HCl compared to the control (Fig. 8). In addition, analysis of chemical modifications by mass spectrometry showed no significant changes between NAR/Arg-HCl-containing samples and controls. Relative quantitation was carried out, and in table. Figure 11 shows the percentages of deamidation at N55 and N33, as well as oxidation at M246 and M422. Slightly higher levels of deamidation at N55 were observed in samples containing NAR/Arg-HCl, pH 5.4, compared to samples containing NAR/Arg-HCl, pH 5.1, and samples containing proline, with a pH value of 5.0, which is consistent with the increase in the acidic peak observed by CEX. It turned out that N33 (a potential deamidation site in the complementarity determining region (CDR)) did not show a significant increase in deamidation over the range of pH values and conditions assessed. Oxidation rates for both M246 and M422 were higher for the 140 mg/mL proline control samples compared to the 210 mg/mL NAR/Arg-HCl samples after incubation with ASC.

Table 11

Deamidation and oxidation of evolocumab (selected samples)

Sample % deamidation % deamidation % oxidation % oxidation M24 6 on M422 by N55 by N33 By 10/165/75, acetate, pH value 5.1, 11, 6 0.8 5, 4 3.7 5°C, 3 months 10/165/75, acetate, pH value 5.1, 11, 8 0.7 6, 0 3, 8 25°C, 3 months 10/165/75, acetate, pH value 5.1, 13, 6 0.9 8.4 5, 1 40°C, 1 month 10/155/100, acetate, pH value 5.4, 12.3 0.9 5, 4 3.7 5°C, 3 months 10/155/100, acetate, pH value 5.4, 12.1 0.6 6, 1 3, 9 25°C, 3 months 10/155/100, acetate, pH value 5.4, 40°C, 1 month 15, 2 0.8 8.4 5, 2 Control with a concentration of 140 mg/ml containing proline, pH value 5.0, 10, 6 0.6 5, 6 3.7 5°C, 3 months Control with a concentration of 140 mg/ml containing proline, pH value 5.0, 10, 8 0.7 6, 7 3, 8 25°C, 3 months Control with a concentration of 140 mg/ml containing proline, pH value 5.0, 11.8 0.7 11.0 6, 5 40°C, 1 month

- 48 045816

The number of particles not visible to the naked eye, which was determined by counting particles of the suspension using light blocking and microflow imaging (MFI), did not show significant trends that correlated with the studied variable parameters of the compositions (Fig. 9A-9C and Fig. 10). The amounts of particles invisible to the naked eye were comparable between the 210 mg/mL samples containing NAR/Arg-HCl and the 140 mg/mL control containing proline.

In fig. 11 collects viscosity data from this study at time zero, time points three months and six months. Data indicate that viscosity remained constant for up to six months at 5°C and 25°C for all formulations. At 40°C, the samples showed a pH-dependent increase in viscosity, which correlated with the increase in the percentage of HMW species observed by SE-HPLC at 40°C (Fig. 4).

Observations.

An almost isotonic composition (~300 mOsm/kg) was achieved with the medium formulation in this study (10 mM buffer, 165 mM NAR, 75 mM arginine-HCl).

Increasing the pH results in a decrease in the percentage of aggregates after accelerated aging as observed by size exclusion chromatography (SEC) and also an increase in deamidation with a higher percentage for acidic species as observed by cation exchange chromatography.

Arginine-HCl concentration had a greater effect on stability (leading to increased aggregation at 25°C and 40°C) at pH 4.8 and its effect was minimized at pH 5.4.

Acetate and glutamate are comparable in their effects on stability and viscosity.

No new peaks were observed in samples containing NAR/Arg-HCl compared to controls containing proline when peptide mapping selected samples for time points up to three months at 5°C and 25°C and one month at 40°C .

Viscosity remains stable for up to six months at 5°C and 25°C.

NAR remains soluble in formulations at 5°C at concentrations in DF buffer up to 175 mM with Arg-HCl concentrations in the range of 50-100 mM.

Example 3 Study on pH values.

The pH study was designed to further examine the influence of pH values over a wider range. The study included formulations with a target pH range of 5.1-6.9. The pH value and buffer used for each sample are given in Table. 12. All samples were formulated at a concentration of 210 mg/ml with 10 mM buffer, 165 mM NAR, 75 mM arginine-HCl, 0.01% polysorbate-80. Samples were subjected to sterilization filtration using 0.2 μm PVDF filters and manually filled into glass PFS at a fill volume of 2.0 mL.

Table 12

Variable pH and buffer parameters for evolocumab formulations (210 mg/mL) containing 165 mM NAR, 75 mM Arg-HCl, and 0.01% (w/v) polysorbate-80

Sample pH value Buffer

1 5, 1 Acetate 2 5, 4 Acetate 3 5, 7 Acetate 4 6, 0 Histidine 5 6, 3 Histidine 6 6, 6 Phosphate 7 6, 9 Phosphate

Results.

The pH values for each sample at time zero and after three months at 40°C are shown in FIG. 12.

In fig. 13A-13C show the effect of pH on the percentage of HMW species (percentage oligomers+percentage aggregates) as measured by SE-HPLC for up to three months at 4°C, 25°C and 40°C, respectively. Data obtained at 5°C and 25°C showed minimal differences between formulations with different pH values. The most significant difference correlated with pH was the higher percentage levels of HMW species at time zero for the pH 6.6 and pH 6.9 formulations. In fig. 14 shows a graph of oligomer levels at time zero as a function of pH. A slight upward trend in oligomer levels was observed with increasing pH values, with a sharp increase observed for pH values greater than 6.3. Oligomer levels dropped to a minimum at lower pH.

As previously observed in the DOE study (Example 2), the percentage of HMW

- 49 045816 species increased over time at 40°C for each pH value tested, with lower pH values correlating with higher initial rates of increase.

Comparing SE-HPLC chromatograms for a three month time point at 40°C, increased peak aggregate levels increased with decreasing pH (Figure 15).

In fig. 16A-16C show the effect of pH on the percentage of major peak magnitude from CEX-HPLC results for up to six months at 5°C (Fig. 16A), 25°C (Fig. 16B) and 40°C (Fig. 16C). ). At the time point of six months at 5°C, there was no significant change in the magnitude of the main peak as a percentage for samples with a pH value <6.0, while at the same time a decrease in the magnitude of the main peak as a percentage was observed for samples with a pH value >6.3 (Fig. 16A). CEX-HPLC data at ASC in Fig. 16B and FIG. 16C indicated that the percentage magnitude of the main peak decreased with increasing pH, and that the rate of decrease increased significantly at pH >6.0.

In fig. 17 shows that the pH-dependent decrease in the magnitude of the main peak in percent CEX at 40°C was due to an increase in the percentage value for the species in acidic conditions.

A comparison of CEX-HPLC chromatograms for a time point of three months at 25°C can be seen in FIG. 18. The percent acid peak magnitude increased with increasing pH value, and a significant change in the chromatographic profile was observed for samples with a pH value greater than 6.3.

Tryptic peptide mapping using liquid chromatography-mass spectrometry (LC-MS) analysis was performed on samples after storage for 1 month at 40°C. Peptide map chromatograms obtained using the HPLC-UV detector (Figure 19) were visually compared to a reference standard and over a range of pH values. In addition, analysis of chemical modifications using mass spectrometry demonstrated the absence of significant changes correlating with pH value, with the exception of deamidation at N55 and N33. The relative quantitative levels of deamidation at N55 and N33, as well as oxidation at M246 and M422 are shown in Table. 13. Increasing levels of percent deamidation at N55 and percent deamidation at N33 correlated with the increased levels of percent acid peak magnitude observed in the CEX-HPLC data with increasing pH. It turned out that there is no significant effect of pH value on the oxidation levels of M246 or M422.

Table 13

Deamidation and oxidation of evolocumab

Sample % desamidirov N55 guide % desamidirov N33 code % oxidation according to M246 % oxidation according to M422 Meaning pH 5.1-5°С, 4 10.7 0.7 5, 8 1.5 months Meaning pH 5.1-40°С, 1 13.7 eleven 7.1 1.9 month Meaning pH 5.4-40°C, 1 15.5 0.9 6, 5 1.5 month Meaning pH 5.7-40°C, 1 19, 6 1.3 6, 9 16 month Meaning pH 6.0-40°С, 1 24.8 2.0 6, 3 1.5 month Meaning pH 6.3-40°C, 1 31.9 2.4 5, 8 1.1 month Meaning pH 53.3 4.9 6, 7 1.3 6.6-40°C, 1 month Meaning pH 6.9-40°C, 1 70.5 100 6, 9 1.4 month

The results of biological analyzes of samples incubated for one month at 40°C were plotted in FIG. 20 along with the initial percentage of the main peak (CEX-HPLC) and the percentage of N55 and the percentage of N33 (without deamidation).

- 50 045816

The number of particles invisible to the naked eye, which was determined by counting particles of the suspension using light blocking (Fig. 21A-21C) and MFI (Fig. 22), showed no significant trends that correlated with the pH value.

Data in Fig. 23 show that pH had minimal effect on fragmentation or other degradation as measured by reducing sodium dodecyl sulfate capillary electrophoresis (rCE-SDS) analysis for up to six months at 25°C. Percentage of Light Chain Precursors Plus Light Chains+Heavy Chains (LC+LC+HC Precursors) In FIG. 23 shows a slight decrease at the edges of the pH range. Samples with lower pH contained a slightly higher percentage of medium molecular weight (MMW) species, while samples with higher pH contained a slightly higher percentage of HMW species. The percentage of low molecular weight (LMW) species is comparable across the pH range.

Finally, as shown in FIG. 24, there is a well-defined linear relationship between viscosity and pH value, with a decrease of 6.6 cP per pH unit observed.

Observations.

Increasing the pH resulted in (1) decreased aggregation rates at 40°C, (2) higher initial oligomer content levels, (3) increased rates of deamidation at 25°C and 40°C, and (4) lower viscosity. The pH value did not appear to have a significant effect on particles visible or invisible to the naked eye, nor did the pH value appear to have a significant effect on fragmentation or other degradation as measured by rCE-SDS.

Example 4: Study on a reduced scale.

The three formulations were evaluated for commercialization and stability. The candidate formulations were subjected to various typical processes simulating commercial production before being subjected to stability testing.

Results.

Physical properties were determined for each formulated drug substance.

Each formulation was determined to have a pH value ~0.17 higher than the pH value of the DF buffer. In the case of Formulation 3, the DF buffer had a pH value that was 0.14 above target, resulting in a DS pH value that was 0.31 above target.

SE-HPLC results for drug stability (percentage of HMW species) at 5°C, 25°C and 40°C are shown in FIG. 25A-25C. The rates of increase in the percentage of HMW species were comparable for all three samples at 5°C and 25°C, with the rate of aggregation being slightly higher for sample 1 (lower pH) at 40°C.

The CEX-HPLC data are shown in Figures 26A-26C and show the expected pH- and temperature-dependent percentage increases in acid peak magnitude observed in previous studies (see previous examples).

The rCE-SDS data shown in FIG. 27 demonstrate comparable percentage levels of LC+HC+LC precursors for each downscaled formulation at all temperatures and time points tested.

Turbidity levels varied little for up to four months at 5°C and 25°C, but increased after 4 months at 40°C.

Downscaled sample concentrations were adjusted to 200, 210, and 220 mg/mL for each formulation. Samples were tested using an m-VROC™ rheometer (RheoSense; San Ramon, CA) at shear rates up to 90,000 s ^-1 and at temperatures from 18°C to 28°C. Data in Fig. 28 covered the viscosity range of 22–52 cP and illustrated the effects of protein concentration, temperature, and composition changes on viscosity.

Observations.

Levels of percentage HMW species and aggregation rates from SEHPLC results were consistent with those observed in previous studies (e.g., see previous examples).

The levels and rates of change in percentage values of the main, acidic, and basic peaks determined by CEX-HPLC were consistent with those observed in previous studies.

rCE-SDS data demonstrated no significant fragmentation or other degradation up to three months in RSC and ASC.

Example 5: Ultrafiltration/diafiltration of evolocumab in formulations containing N-acetylarginine.

Materials and methods.

Small scale ultrafiltration/diafiltration (UF/DF) development experiments used a Pellicon® 3 cassette with a 30 kDa molecular weight cutoff Ultracel PLCTK membrane (D Screen, 0.11 m ² ; EMD Millipore; Billerica, MA). The experiments were carried out on a technological

- 51 045816 tangential flow filtration (TFF) system (PendoTECH; Princeton, NJ). Experiments were carried out at room temperature (22.2±2°C).

UF/DF development experiments were performed on a small scale to evaluate three NAR formulation buffers at different pH values and compared their filtrate flow data as a function of concentration. Two stages of DF were used to save on NAR consumption. Additional experiments were performed for the UF1/DF1 stage to evaluate the effect of two target concentrations (35 mg/ml and 70 mg/ml) on the formation of high molecular weight species during the UF/DF process. Other UF/DF performance parameters were not evaluated. The general procedure for UF/DF experiments is described in Table. 14.

Table 14

General UF/DF procedure and operating parameters

Process description Conditions Have you assessed general information Membrane/Temperature/Membrane Dimensions No Equalization (EQ) buffer/buffer for DF Buffers for formulations with NAR Yes Concentrate to target concentrations for DF Yes Concentration 1 Transmembrane pressure (TMP) No (UF1) is 18 psi Cross flow rate is 300 LMH (liters/ ^m2 /h) 3 diameters Yes Diafiltration 1 (DF1) TMP is 18 psi Cross flow speed feed is 300 LMH No Concentration 2 Concentrate to target Yes (UF2) concentrations for DF TMP is 18 psi Cross flow speed feed is 300 LMH No Diafiltration 2 (DF2) 4 diameters Yes TMP is 18 psi Cross flow speed feed is 300 LMH No Concentration 3 Concentrate to target Yes (overconcentration concentrations e; OS) TMP is initially 18 psi; control valve fully open Cross flow speed flow rate is 60 LMH Operating temperature 37 °C No Recycling 10 minute recirculation Cross flow speed feed rate is 60 LMH The path for filtrate is closed (no TMP) No Extraction Extract the protein solution through No

- 52 045816 lower point or channel for concentrate

Run with a buffer through the concentrate channel >2 0 l/m ² one pass No

Cleaning 30-minute recirculation, 20 l/ ^m2

Storage Y2 0 l/m ² one run No

A ₂₈₀ measurements were performed using a variable path length spectrophotometer (SoloVPE system; SoloVPE; Bridgewater, NJ) with an extinction coefficient of 1.5 (cm) ^-1 (g/L) ^-1 .

Analytical methods used to assess the quality of the product pool included SE-HPLC, rCE-SDS, and CEX-HPLC.

NAR: Ac-Arg-OH, Biochem catalog number E-1025.

Results of a buffer study for formulations with NAR.

In this experiment, three buffers for NAR formulations were evaluated in the UF/DF method. The UF/DF method followed general guidelines (1) evolocumab was concentrated to 70 mg/mL using batch concentration (UF1) and diafiltration was performed with NAR formulation buffer using 3 diavolumes (DF1); (2) the protein was further concentrated to 140 mg/ml (UF2) and diafiltration was performed with NAR formulation buffer using 4 diavolumes (DF2); (3) the protein was overconcentrated to a target value of ∼260 mg/mL and recovered from the system at 37°C. Protein loading/membrane area was 1468 g/m ² for NAR at pH 5.4 and 800 g/m ² for NAR at pH 5.2 and 5.6.

Flux data was plotted against concentration and comparisons were made across all buffers for NAR formulations. In addition, excipient levels were analyzed by sampling at each diafiltration stage to examine diafiltration efficiency and determine minimum diafiltration volumes.

Results and observations.

The flux data from the UF/DF study was used to create the filtrate flux versus concentration plot shown in FIG. 29. Flux profiles were comparable among the three NAR formulation buffers. The flux increased when the protein buffer was replaced with 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 for NAR pH 5.4 and 10 hours for NAR pH 5.2 and 5.6 due to different membrane areas. Results with a similar flow profile demonstrated that the NAR formulation buffer did not have a significant effect on process flow. A summary of small-scale UF/DF drug substance (DS) studies of evolocumab is shown in Table. 15.

Samples were collected at diafiltration stages (DF1 and DF2) at each diavolume (1 to 7 DV) to test diafiltration efficiency and determine the minimum number of diavolumes. The results of analyzes of representative samples are in table. 16 are from a UF/DF study of evolocumab with NAR at pH 5.4. The results showed that after five diavolumes, diafiltration with replacement by NAR with a pH value of 5.4 was almost complete.

Table 15

Summary of Small Scale UF/DF DS Studies on Evolocumab Variable Parameter pH Value pH Value pH Value

5.2 5.4 5.6 Original materials evolocumab NMP VFP, 11 mg/ml Buffer for composition 10 mM 10 mM 10 mM acetate, acetate, 155 acetate, 170 14 0 mM NAR, mM NAR, 7 0 mM NAR, 63 63 mM Agd- mM Agd-NS1 mM Agd-NS1 NS1 Loading membranes 800 1467.5 800 (g/ ^m2 ) OS (g/l) 262 270 268 DS concentration (g/l) 226 217 222 Viscosity DS in cP at 77.4 49, 6 52.3 1000 s-1

- 53 045816

Table 16 Excipient levels at each DF stage for UF/DF evolocumab with NAR at pH 5.4 Sample Acetate Arginine NAR Na C1 Tris (mm) (mm) (mm) (mm) (mm) (mm) DF0 91.243 N. d. N. d. 155.97 99, 9 1.29 DF1-1DV 38,271 34,193 115,805 25, 94 69, 8 0.21 DF1-2DV 20,435 52,299 153.178 26.78 60.4 0.22 DF1-3DV 13,903 44,951 163,453 9, 88 56, 8 0.08 DF2-4DV 11,354 46.28 167,072 6, 08 56, 4 0.05 DF2-5DV 10,287 45,819 157.11 4, 69 55.5 0.04 DF2-6DV 10,354 46, 283 166,749 3, 80 61.7 0.03 DF2-7DV 10,134 46, 488 162,536 4.86 58.1 0.04

Example 6 Monitoring DS concentration and HMW species formation during UF/DF formulations based on evolocumab.

The purpose of this experiment was to evaluate the target concentration for batch concentration/diafiltration (UF1/DF1) and to determine the target concentration to minimize the formation of HMW species during the UF/DF process. When performing the UF/DF process, the general recommendations listed in Table 1 were followed. 17. Evolocumab (11 mg/ml) was concentrated to 35 mg/ml or 70 mg/ml using batch concentration (UF1). The protein loading/membrane area was 800 g/m ² and the NAR formulation buffer contained 10 mM acetate, 155 mM N-acetylarginine, 70 mM arginine-HCl, pH 5.3.

In addition, protein samples were collected at each UF/DF stage to determine the quality of the product pool to evaluate the UF/DF process. Product pool quality data were used to investigate the effect of different concentrations of UF1/DF1 product on the formation of HMW species.

Materials and methods.

Refer to example 5.

Results and observations.

In fig. 30 shows a graph comparing the formation of HMW species (%) during the UF/DF process of evolocumab at a concentration of 35 mg/ml and 70 mg/ml in the UF1/DF1 stage (UF/DF-70 and UF/DF-35). Initially, the HMW species content (%) in UF/DF-70-UF1 was 0.2% higher than in UF/DF35-UF1, but the HMW species content (%) was comparable after UF1 and the same in the final DS . The result indicated that the HWM species were reversible and the target concentration at UF1 stage did not have a significant effect on the formation of HWM species in the final DS. It is recommended that the target concentration be 70 mg/mL to reduce pool volume.

Example 7: Pooling studies for three DS with NAR obtained by UF/DF at small scale.

In this example, the stability of three batches of evolocumab formulated with three NAR formulation buffers was monitored. Ten ml of each DS sample was kept at 2-8°C. At each time point shown in Figures 31-A-31F, a sample of approximately 1 ml was taken and held at 2-8° C. for analytical testing.

Results and observations.

The SE-HPLC and CEX results for evolocumab in evolocumab with NAR formulations held at 2-8°C are shown in Figures 36 and 37. The SE-HPLC and rCE-SDS results gave comparable results. DS batches produced by NAR at pH 5.2, 5.4 and pH 5.6 contained 1.4%, 1.5% and 1.7% HMW species, respectively, during the UF/DF process compared to 1 .1% in the original material. The % HMW species was plotted to compare evolocumab and DS parent material with NAR over 11 weeks, which is depicted in FIG. 32. All three batches of the composition were characterized by an increase in the content (%) of HMW varieties by 0.4% - 0.5%. These DS batches were stable for at least 7 days at 2-8°C.

Example 8. Pool holding studies during the production of superconcentrated (260 g/L) evolocumab HMP CPD1 with NAR at 37°C, 39°C, 42°C and 45°C.

The stability of superconcentrated (260 g/L) evolocumab during production was assessed at 37°C, 39°C, 42°C and 45°C. Thirty ml of OS samples were incubated in a water bath in a room with controlled temperatures. At each point in time shown in FIG. 33 and 34, a sample of approximately 1 mL was collected, frozen, and transferred to dry ice for analysis. Viscosity values were measured in OC samples at higher temperatures and compared with values during aging at 23°C and 25°C.

Results and observations.

SE-HPLC results showed that the OS of evolocumab with NAR was stable over three hours at 37°C, 39°C, and 42°C, but not at 45°C, as shown in Fig. 33. The results of CEX and rCE-SDS were comparable. In fig. 34 shows that as the temperature increased, the viscosity of the OS decreased, and with an increase in temperature from 37°C to 42°C, the viscosity dropped by 19% (by 14.7 cP).

The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter of the invention described. All documents or portions of documents cited in this application, including patents, patent applications, articles, books and treatises, are hereby expressly incorporated herein by reference in their entirety for all purposes.

Example 9. Study of stability of DOE composition.

A formulation stability experimental design (DOE) was developed to examine the effect of variable formulation parameters within a defined design envelope on the stability of evolocumab. Variable parameters studied included evolocumab concentration, NAR concentration, Arg-HCl concentration, and pH value. Eighteen compounds listed in table. 17 were subjected to sterilization filtration using PVDF filters with a pore size of 0.2 μm and manually filled into glass pre-fill syringes at a fill volume of 2.0 ml. Samples were tested using a battery of analytical methods that measure stability after incubation at various temperatures. All formulations contained 10 mM sodium acetate and 0.01% (w/v) polysorbate-80 in addition to the components listed in Table. 17.

Table 17

Study plan

image C Sample code [ evolocumab ] (mg/ml) Auxiliary substances* pH value 1 210A54NARRT80 210 14 0 mM NAR, mM Arg-HCl 50 5, 4 2 210A54NARRT80 210 14 0 mM NAR, mM Arg-HCl 50 5, 4 3 197A51NAR ₁₅₄ R ₅₅ T8 0 197 154 mM NAR, mM Arg-HCl 55 5, 1 4 1 97A51NARI ₂₆ R45T8 0 197 12 6 mM NAR, mM Arg-HCl 45 5, 1 5 22 3A51NARI ₅₄ R ₄₅ T8 0 223 154 mM NAR, mM Arg-HCl 45 5, 1 6 22 3A51NARi ₂₆ R45T8 0 223 12 6 mM NAR, mM Arg-HCl 45 5, 1 7 1 97A57NARi ₂₆ R55T8 0 197 12 6 mM NAR, mM Arg-HCl 55 5, 7 8 1 97A51NARI ₂₆ R55T8 0 197 12 6 mM NAR, mM Arg-HCl 55 5, 1 9 1 97A51NARI ₅₄ R ₄₅ T8 0 197 154 mM NAR, mM Arg-HCl 45 5, 1 10 22 3A51NARi ₂₆ R55T8 0 223 12 6 mM NAR, mM Arg-HCl 55 5, 1 eleven 22 3A57NAR ₁₂₆ R ₄ 5T8 0 223 12 6 mM NAR, mM Arg-HCl 45 5, 7 12 223A57NAR ₁₅₄ R ₅₅ T8 0 223 154 mM NAR, mM Arg-HCl 55 5, 7 13 223A57NAR ₁₅₄ R ₄₅ T8 0 223 154 mM NAR, mM Arg-HCl 45 5, 7 14 197A57NAR ₁₂₆ R4 ₅ T8 0 197 12 6 mM NAR, mM Arg-HCl 45 5, 7 15 197A57NAR ₁₅₄ R ₅₅ T8 0 197 154 mM NAR, mM Arg-HCl 55 5, 7 16 223A57NAR ₁₂₆ R55T8 0 223 12 6 mM NAR, mM Arg-HCl 55 5, 7 17 1 97A57NARi ₅₄ R ₄₅ T8 0 197 154 mM NAR, mM Arg-HCl 45 5, 7 18 22 3A51NARI ₅₄ R ₅₅ T8 0 223 154 mM NAR, mM Arg-HCl 55 5, 1

*formulations contained 10 mM sodium acetate and 0.01% (w/v) polysorbate-80 as noted in the text.

- 55 045816

In table Figure 18 shows the measured evolocumab concentrations, pH values, and excipient concentrations for each of the formulations studied. All are close to the target levels listed in the table. 17.

Table 18

Initial data for the composition of the medicinal product

Sample No. [evolocumab] (mg/ml) pH value [NAR] (mm) [Arginine] (mm) 1 209 5, 51 145, 0 56, 7 2 211 5.46 143.5 56, 2 3 191 5, 14 156, 7 60, 9 4 201 5, 17 126, 3 46, 9 5 226 5, 13 160.2 48.2 6 225 5, 13 127.8 47.9 7 198 5, 67 135, 8 63, 1 8 199 5, 14 125, 1 59, 1 9 202 5, 13 161.8 49, 9 10 224 5, 12 136, 1 64.5 eleven 220 5.71 132.1 51.5 12 223 5, 73 161.7 63.3 13 218 5, 75 159, 7 50.7 14 197 5.76 128.5 50.5 15 194 5.79 157, 6 61.7 16 227 5, 72 141.0 67.3 17 196 5, 72 154.2 58.1 18 226 5, 17 153, 6 57.8

Results and observations.

To assess aggregation, SE-HPLC analysis was performed on the formulations shown in Table 1. 18, after 0-6 months of incubation at 4°C (Fig. 35A), 25°C (Fig. 35B) and 40°C (Figs. 35C and 35D). The data indicated that the rates of increase in % HMW species were similar for all formulations at 4°C and 25°C, but a pH-dependent effect on aggregation was observed under accelerated aging conditions at 40°C. It was observed that samples with a lower pH value, which were characterized by a pH value of 5.1, exhibited the fastest aggregation rate at 40°C, while it was observed that samples with a pH value of 5.7 exhibited the slowest aggregation rate (Fig. 35C and 35D). Samples with a pH value of 5.4 at 40°C were observed to exhibit aggregation rates intermediate between the samples with pH values of 5.1 and 5.7, but these rates more closely resembled the lower aggregation rates observed for samples with a pH value of 5. 7 (FIGS. 35C and 35D).

The effect of pH on the increase in % acidic peak and % basic peak over time (up to three months) was tested using CEX-HPLC analysis, the data of which is shown in FIG. 36A (acidic peak) and 36B (basic peak) for 4°C; in fig. 36C (acidic peak) and 36D (basic peak) for 25°C; and in fig. 36E (acidic peak) and 36F (basic peak) for 40°C. As shown by the data for samples at 25°C and 40°C (Figs. 36C-36F), pH was observed to influence the % acidic and basic peaks in these formulations. The data for the pH 5.7 samples were clearly associated with higher rates of increase in acid peak %, while the data for the pH 5.1 samples showed the slowest rate of increase. The two pH 5.4 samples were observed to have degradation rates that were directly between the observed degradation rates of the pH 5.1 and pH 5.7 samples. The main peak % trends were found to be less pronounced, but a trend towards higher main peak % levels was observed at lower pH values for samples incubated at 40°C.

Fragmentation or other degradation of evolocumab in formulations for up to three months at both 30°C and 40°C was analyzed using rCE-SDS and SE-HPLC assays; Figures 37A-37B show data for samples incubated at 30°C, and Figures 37C-37D show data for samples incubated at 40°C. No significant trend was observed for rCE-SDS data related to formulation composition within the design envelope of this study.

For the formulations, the presence and amounts of particles not visible to the naked eye were determined over time up to three months, as determined by light blocking particle counts using HIAC after incubation at 4°C and 40°C; these data are shown in Fig. 38A-38D. (Figures 38A (particles greater than or equal to 10 µm) and 38B (particles greater than or equal to 25 µm) show results for samples held at 4°C; Figures 38C (particles greater than or equal to 10 µm)

- 56 045816 and 38D (particles greater than or equal to 25 µm) results are shown for samples kept at 40°C). Based on these data, no significant trend was observed in the amounts of particles not visible to the naked eye associated with the formulation variables studied.

The data obtained in this example shows that within the narrow design window studied, no significant impact on stability was observed during storage at 4°C or 25°C. Data associated with pH at 40°C suggested that pH affected the rate of aggregation as detected by SE-HPLC, as well as the percentage value for species in acidic conditions and, to a lesser extent, the percentage value for species in alkaline conditions as detected by CEX-HPLC.

Exemplary Embodiments

Disclosed herein are exemplary embodiments of pharmaceutical compositions containing evolocumab, wherein such compositions contain N-acetylarginine and the pharmaceutical composition has a viscosity of at least less than about 80 cP (measured, for example, using a rheometer such as an AESR-rheometer). G2 with cone plane system from TA Instruments (New Castle, DE)). Moreover, methods for formulating therapeutic polypeptides, such as evolocumab, where such compositions contain NAR are disclosed herein.

Embodiment 1. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds; And

b) N-acetylarginine, wherein the pharmaceutical composition has a viscosity of at least less than about 80 cP.

Embodiment 2. The pharmaceutical composition of Embodiment 1, wherein the PCSK9-binding polypeptide is a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

Embodiment 3. The pharmaceutical composition of embodiment 2 or 3, wherein the pharmaceutical composition has a viscosity of at least less than about 50 cP.

Embodiment 4. The pharmaceutical composition of embodiment 2 or 3, wherein the pharmaceutical composition has an osmolality of from about 250 to about 400 mOsm/kg.

Embodiment 5: The pharmaceutical composition of Embodiment 4, wherein the pharmaceutical composition has an osmolality of approximately 300 mOsm/kg.

Embodiment 6. The pharmaceutical composition according to Embodiment 5, wherein

- 57 045816 maceutical composition is isotonic for human blood cells.

Embodiment 7. The pharmaceutical composition of Embodiment 1, wherein the PCSK9-binding polypeptide is present at a concentration of from about 140 mg/ml to about 260 mg/ml.

Embodiment 8. The pharmaceutical composition of Embodiment 2, wherein the concentration of the PCSK9-binding polypeptide is approximately 210 mg/ml.

Embodiment 9. The pharmaceutical composition according to any one of embodiments 1-8, wherein N-acetylarginine is present at a concentration of from about 25 mM to about 230 mM.

Embodiment 10. The pharmaceutical composition of Embodiment 9, wherein N-acetylarginine is present at a concentration of from about 140 mM to about 170 mM.

Embodiment 11. The pharmaceutical composition of Embodiment 10, wherein N-acetylarginine is present at a concentration of approximately 140 mM.

Embodiment 12: The pharmaceutical composition according to any one of embodiments 1 to 11, further comprising a buffer.

Embodiment 13. The pharmaceutical composition of Embodiment 13, wherein the buffer is selected from the group consisting of acetate, glutamate, histidine and phosphate buffers or a combination thereof.

Embodiment 14. The pharmaceutical composition of Embodiment 13, wherein the buffer is present at a concentration of from about 5 mM to about 30 mM.

Embodiment 15. The pharmaceutical composition of Embodiment 14, wherein the buffer is sodium acetate and is present at a concentration of approximately 10 mM.

Embodiment 16. The pharmaceutical composition according to any one of embodiments 1-15, wherein the pH value is from about 4.8 to about 6.9.

Embodiment 17: The pharmaceutical composition of Embodiment 11, wherein the pH value is approximately 5.4.

Embodiment 18. The pharmaceutical composition according to any one of embodiments 1 to 17, further comprising a surfactant.

Embodiment 19. The pharmaceutical composition of Embodiment 18, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types Pluronic®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

Embodiment 20. The pharmaceutical composition of Embodiment 19, wherein the surfactant is present at a concentration of from about 0.0001% (w/v) to about 1% (w/v).

Embodiment 21. The pharmaceutical composition of Embodiment 20, wherein the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of approximately 0.01% (w/v).

Embodiment 22. The pharmaceutical composition according to any one of embodiments 1-21, further containing proline.

Embodiment 23. The pharmaceutical composition of Embodiment 22, wherein proline is present at a concentration of from about 50 mM to about 150 mM.

Embodiment 24. The pharmaceutical composition of Embodiment 23, wherein proline is present at a concentration of from about 90 mM to about 120 mM.

Embodiment 25. The pharmaceutical composition of Embodiment 22 or 23, wherein proline is present at a concentration of approximately 120 mM.

Embodiment 26. The pharmaceutical composition according to any one of embodiments 1-25, further comprising an arginine salt.

Embodiment 27. The pharmaceutical composition of Embodiment 26, wherein the arginine salt is present at a concentration of from about 25 mM to about 150 mM.

Embodiment 28. The pharmaceutical composition of Embodiment 27, wherein the arginine salt is present at a concentration of from about 50 mM to about 100 mM.

Embodiment 29. The pharmaceutical composition of Embodiment 26, wherein the arginine salt is arginine HCl, arginine acetate or arginine glutamate.

- 58 045816

Embodiment 30. The pharmaceutical composition of Embodiment 29, wherein arginine-HCl is present at a concentration of approximately 50 mM.

Embodiment 31. The pharmaceutical composition of any one of embodiments 1-30, wherein the PCSK9-binding polypeptide is stable for at least about 2 years if stored at about -30°C or lower.

Embodiment 32. The pharmaceutical composition of Embodiment 31, wherein the PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 33. The pharmaceutical composition of any one of embodiments 1-30, wherein the PCSK9-binding polypeptide is stable for at least about 6 months if stored at about 5°C.

Embodiment 34. The pharmaceutical composition of Embodiment 33, wherein the PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 35. The pharmaceutical composition of any one of embodiments 1-30, wherein the PCSK9-binding polypeptide is stable for at least about 1 month if stored at about 25°C.

Embodiment 36. The pharmaceutical composition of Embodiment 35, wherein the PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 37. The pharmaceutical composition of Embodiment 35, wherein the PCSK9-binding polypeptide is stable for at least about 6 months.

Embodiment 38. The pharmaceutical composition of any one of embodiments 1-30, wherein the PCSK9-binding polypeptide is stable for at least about 1 month if stored at about 40°C.

Embodiment 39. The pharmaceutical composition of any one of embodiments 1-38, wherein the composition contains high molecular weight aggregates or oligomers of PCSK9-binding polypeptide at a level of less than about 3% of the concentration of PCSK9-binding polypeptide.

Embodiment 40. The pharmaceutical composition of Embodiment 39, wherein the high molecular weight aggregates or oligomers of the PCSK9 binding polypeptide are present at a level of less than about 2.5% of the concentration of the PCSK9 binding polypeptide.

Embodiment 41. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237, and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds; And

b) N-acetylarginine;

c) arginine salt;

d) buffer and

e) surfactant,

- 59 045816 wherein the pharmaceutical composition has a viscosity of at least less than about 80 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware)).

Embodiment 42. The pharmaceutical composition of Embodiment 41, wherein the PCSK9-binding polypeptide is a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

Embodiment 43. The pharmaceutical composition of embodiment 41 or 42, wherein the pharmaceutical composition has a viscosity of at least less than about 50 cP.

Embodiment 44. The pharmaceutical composition of embodiment 41 or 42, wherein the pharmaceutical composition has an osmolality of from about 250 to about 400 mOsm/kg.

Embodiment 45. The pharmaceutical composition of Embodiment 44, wherein the pharmaceutical composition has an osmolality of approximately 300 mOsm/kg.

Embodiment 46. The pharmaceutical composition of Embodiment 45, wherein the pharmaceutical composition is isotonic to a human blood cell.

Embodiment 47. The pharmaceutical composition of Embodiment 41 or 42, wherein the PCSK9 binding polypeptide is present at a concentration of from about 140 mg/ml to about 260 mg/ml.

Embodiment 48. The pharmaceutical composition of Embodiment 47, wherein the concentration of the PCSK9-binding polypeptide is approximately 210 mg/ml.

Embodiment 49. The pharmaceutical composition of any one of embodiments 41-48, wherein N-acetylarginine is present at a concentration of from about 25 mM to about 230 mM.

Embodiment 50. The pharmaceutical composition of Embodiment 49, wherein N-acetylarginine is present at a concentration of from about 140 mM to about 170 mM.

Embodiment 51. The pharmaceutical composition of Embodiment 50, wherein N-acetylarginine is present at a concentration of approximately 140 mM.

Embodiment 52. The pharmaceutical composition of Embodiment 41 or 42, wherein the buffer is selected from the group consisting of acetate, glutamate, histidine and phosphate buffers or a combination thereof.

Embodiment 53. The pharmaceutical composition of Embodiment 52, wherein the buffer is present at a concentration of from about 5 mM to about 30 mM.

Embodiment 54. The pharmaceutical composition of Embodiment 53, wherein the buffer is sodium acetate and is present at a concentration of approximately 10 mM.

Embodiment 55: The pharmaceutical composition of any one of Embodiments 41 or 42, wherein the pH is from about 4.8 to about 6.9.

Embodiment 56: The pharmaceutical composition of Embodiment 55, wherein the pH value is approximately 5.4.

Embodiment 57. The pharmaceutical composition of Embodiment 41 or 42, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types of Pluronics®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl esters and D-α-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

Embodiment 58. The pharmaceutical composition of Embodiment 57, wherein the surfactant is present at a concentration of from about 0.0001% (w/v) to about 1% (w/v).

Embodiment 59. The pharmaceutical composition of Embodiment 57, wherein the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of approximately 0.01% (w/v).

Embodiment 60. The pharmaceutical composition of Embodiment 41 or 42 further comprising proline.

- 60 045816

Embodiment 61. The pharmaceutical composition of Embodiment 60, wherein proline is present at a concentration of from about 50 mM to about 150 mM.

Embodiment 62. The pharmaceutical composition of Embodiment 61, wherein proline is present at a concentration of from about 90 mM to about 120 mM.

Embodiment 63. The pharmaceutical composition of Embodiment 62, wherein proline is present at a concentration of approximately 120 mM.

Embodiment 64. The pharmaceutical composition of Embodiment 41 or 42, wherein the arginine salt is present at a concentration of from about 25 mM to about 150 mM.

Embodiment 65. The pharmaceutical composition of Embodiment 64, wherein the arginine salt is present at a concentration of from about 50 mM to about 100 mM.

Embodiment 66. The pharmaceutical composition of Embodiment 64, wherein the arginine salt is arginine HCl, arginine acetate or arginine glutamate.

Embodiment 67. The pharmaceutical composition of Embodiment 66, wherein arginine-HCl is present at a concentration of approximately 50 mM.

Embodiment 68. The pharmaceutical composition of any one of embodiments 41-67, wherein the PCSK9 binding polypeptide is stable for at least about 2 years if stored at about -30° C. or lower.

Embodiment 69. The pharmaceutical composition of Embodiment 68, wherein the PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 70. The pharmaceutical composition of any one of embodiments 41-67, wherein the PCSK9 binding polypeptide is stable for at least about 6 months when stored at about 5°C.

Embodiment 71. The pharmaceutical composition of Embodiment 70, wherein the PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 72. The pharmaceutical composition of any one of embodiments 41-67, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 25°C.

Embodiment 73. The pharmaceutical composition of Embodiment 72, wherein the PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 74. The pharmaceutical composition of Embodiment 73, wherein the PCSK9-binding polypeptide is stable for at least about 6 months.

Embodiment 75. The pharmaceutical composition of any one of embodiments 41-67, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 40°C.

Embodiment 76. The pharmaceutical composition of any one of embodiments 41-75, wherein the composition contains high molecular weight aggregates or oligomers of PCSK9-binding polypeptide at a level of less than about 3% of the concentration of PCSK9-binding polypeptide.

Embodiment 77. The pharmaceutical composition of Embodiment 76, wherein the high molecular weight aggregates or oligomers of the PCSK9 binding polypeptide are present at a level of less than about 2.5% of the concentration of the PCSK9 binding polypeptide.

Embodiment 78. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues

- 61 045816 kov PCSK9, while PCSK9 contains the amino acid sequence under 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 D23 8;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 140 mM;

c) arginine-HCl present at a concentration of approximately 50 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

Embodiment 79. The pharmaceutical composition of Embodiment 78, wherein the pharmaceutical composition has a pH value of from about 5.1 to about 5.7.

Embodiment 80. The pharmaceutical composition of Embodiment 78 or 79, wherein the pharmaceutical composition has a viscosity of at least less than about 50 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware) )).

Embodiment 81. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237 and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

b) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

c) N-acetylarginine, present at a concentration of approximately 140 mM;

d) arginine-HCl present at a concentration of approximately 63 mM;

e) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015%; And

f) sodium acetate at a concentration of approximately 10 mM.

Embodiment 82. The pharmaceutical composition of Embodiment 81, wherein the pharmaceutical composition has a pH value of from about 5.1 to about 5.7.

Embodiment 83. The pharmaceutical composition of Embodiment 81 or 82, wherein the pharmaceutical composition has a viscosity of at least less than about 80 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware) )).

Embodiment 84. Pharmaceutical composition containing:

a) PCSK9-binding polypeptide that is present at a concentration of

- 62 045816 approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237 and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 155 mM;

c) arginine-HCl present at a concentration of approximately 70 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

Embodiment 85. The pharmaceutical composition of Embodiment 84, wherein the pharmaceutical composition has a pH value of from about 5.1 to about 5.7.

Embodiment 86. The pharmaceutical composition of Embodiment 84 or 85, wherein the pharmaceutical composition has a viscosity of at least less than about 45 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware) )).

Embodiment 87. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237 and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

- 63 045816

b) N-acetylarginine, present at a concentration of approximately 170 mM;

c) arginine-HCl present at a concentration of approximately 63 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015%; And

e) sodium acetate at a concentration of approximately 10 mM.

Embodiment 88. The pharmaceutical composition of Embodiment 87, wherein the pharmaceutical composition has a pH value of from about 5.1 to about 5.7.

Embodiment 89. The pharmaceutical composition of Embodiment 87 or 88, wherein the pharmaceutical composition has a viscosity of at least less than about 60 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware) )).

Embodiment 90. Pharmaceutical composition containing:

a) a PCSK9-binding polypeptide that is present at a concentration of approximately 195-225 mg/ml, selected from the group consisting of:

i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237 and D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGF-A domain of the LDLR binds;

b) N-acetylarginine, present at a concentration of approximately 155 mM;

c) proline, present at a concentration of approximately 120 mM;

d) polyoxyethylene sorbitan monooleate (polysorbate 80) at a concentration of from about 0.005% (w/v) to about 0.015% (w/v); And

e) sodium acetate at a concentration of approximately 10 mM.

Embodiment 91. The pharmaceutical composition of Embodiment 90, wherein the pharmaceutical composition has a pH value of from about 5.1 to about 5.7.

Embodiment 92. The pharmaceutical composition of Embodiment 90 or 91, wherein the pharmaceutical composition has a viscosity of at least less than about 60 cP (measured, for example, using an AESR-G2 cone plane rheometer from TA Instruments (New Castle, Delaware) )).

Embodiment 93. The pharmaceutical composition of any one of embodiments 78-92, wherein the PCSK9 binding polypeptide is stable for at least about 2 years if stored at about -30° C. or lower.

Embodiment 94. The pharmaceutical composition of Embodiment 93, wherein the PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 95. The pharmaceutical composition of any one of embodiments 78-92, wherein the PCSK9 binding polypeptide is stable for at least about 6 months if stored at about 5°C.

Embodiment 96. The pharmaceutical composition of Embodiment 95, wherein the PCSK9-binding polypeptide is stable for at least about 24 months.

- 64 045816

Embodiment 97. The pharmaceutical composition of any one of embodiments 78-92, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 25°C.

Embodiment 98. The pharmaceutical composition of Embodiment 97, wherein the PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 99. The pharmaceutical composition of Embodiment 98, wherein the PCSK9-binding polypeptide is stable for at least about six months.

Embodiment 100. The pharmaceutical composition of any one of embodiments 78-92, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 40°C.

Embodiment 101. The pharmaceutical composition of any one of embodiments 78-92, wherein the composition contains high molecular weight aggregates or oligomers of PCSK9 binding polypeptide at a level of less than about 3% of the concentration of PCSK9 binding polypeptide.

Embodiment 102. The pharmaceutical composition of Embodiment 101, wherein the high molecular weight aggregates or oligomers of the PCSK9 binding polypeptide are present at a level of less than about 2.5% of the concentration of the PCSK9 binding polypeptide.

Embodiment 103: The pharmaceutical composition according to any one of embodiments 1-102, wherein the pharmaceutical composition is a liquid.

Embodiment 104. A method of treating a subject in need thereof, comprising administering a pharmaceutical composition according to any one of embodiments 1-103.

Embodiment 105. A kit comprising the pharmaceutical composition of any one of embodiments 1-103 and a delivery device selected from the group consisting of a syringe, a pen, a body injector, and an auto-injector.

Embodiment 106. The kit of Embodiment 105 further comprising instructions for administering a pharmaceutical composition using a delivery device.

Embodiment 107. A method of producing a PCSK9-binding polypeptide in a pharmaceutical composition containing at least 140 mg/ml of a PCSK9-binding polypeptide, comprising adding to the pharmaceutical composition containing the PCSK9-binding polypeptide an effective amount of N-acetylarginine, thereby causing the viscosity of the pharmaceutical composition is reduced compared to a pharmaceutical composition not containing N-acetylarginine, and wherein the PCSK9-binding polypeptide is selected from the group consisting of:

a) a monoclonal antibody containing 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 (evolocumab), or an antigen binding fragment thereof;

b) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

c) a monoclonal antibody containing:

i) a heavy chain polypeptide comprising the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and ii) a light chain polypeptide comprising the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

d) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I3 69 , S372, C375, C378, R237 and D238; And

e) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

iii) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1; and iv) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

v) wherein the epitope for the monoclonal antibody further overlaps the region to which the EGF-A domain of the LDLR binds.

Embodiment 108. The method of Embodiment 107, wherein the viscosity of the pharmaceutical composition is less than 80 cP (measured, for example, using a rheometer such as the AESR-G2 cone-plane rheometer from TA Instruments (New Castle, DE)).

Embodiment 109. The method of Embodiment 108, wherein the viscosity is pharmaceutical grade.

- 65 045816 composition is less than 50 cP.

Embodiment 110. The method of any one of embodiments 107-109, wherein the pharmaceutical composition has an osmolality of from about 250 to about 400 mOsm/kg.

Embodiment 111. The method of Embodiment 110, wherein the pharmaceutical composition has an osmolality of approximately 300 mOsm/kg.

Embodiment 112. The method of Embodiment 111, wherein the pharmaceutical composition is isotonic to a human blood cell.

Embodiment 113. The method of Embodiment 107, wherein the PCSK9 binding polypeptide is present at a concentration of from about 180 mg/ml to about 260 mg/ml.

Embodiment 114. The method of Embodiment 113, wherein the concentration of the PCSK9 binding polypeptide is approximately 210 mg/ml.

Embodiment 115. The method of any one of embodiments 107-114, wherein N-acetylarginine is present at a concentration of from about 25 mM to about 230 mM.

Embodiment 116. The method of Embodiment 115, wherein N-acetylarginine is present at a concentration of from about 140 mM to about 170 mM.

Embodiment 117. The method of Embodiment 115, wherein N-acetylarginine is present at a concentration of approximately 140 mM.

Embodiment 118. The method of any one of embodiments 107-117, further including a buffer.

Embodiment 119. The method of Embodiment 118, wherein the buffer is selected from the group consisting of acetate, glutamate, histidine, and phosphate buffers, or a combination thereof.

Embodiment 120. The method of Embodiment 119, wherein the buffer is present at a concentration of from about 5 mM to about 30 mM.

Embodiment 121 The method of Embodiment 120, wherein the buffer is sodium acetate and is present at a concentration of approximately 10 mM.

Embodiment 122. The method of any one of embodiments 107-121, wherein the pH is from about 4.8 to about 6.9.

Embodiment 123 The method of Embodiment 122, wherein the pH value is approximately 5.4.

Embodiment 124. The method of any one of embodiments 107-123, further comprising a surfactant.

Embodiment 125. The method of Embodiment 124, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers, such as Pluronic® F-68 and other types of Pluronic ®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers and D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS).

Embodiment 126. The method of Embodiment 125, wherein the surfactant is present at a concentration of from about 0.0001% (w/v) to about 1.0% (w/v).

Embodiment 127. The method of Embodiment 126, wherein the surfactant is polyoxyethylene sorbitan monooleate and is present at a concentration of approximately 0.01% (w/v).

Embodiment 128. The method of any one of embodiments 107-127, further comprising proline.

Embodiment 129. The method of Embodiment 128, wherein the proline is present at a concentration of from about 50 mM to about 150 mM.

Embodiment 130. The method of Embodiment 129, wherein the proline is present at a concentration of from about 90 mM to about 120 mM.

Embodiment 131 The method of Embodiment 130, wherein proline is present at a concentration of approximately 120 mM.

Embodiment 132. The method of any one of embodiments 107-131, further comprising an arginine salt.

Embodiment 133. The method of Embodiment 132, wherein the arginine salt is present at a concentration of from about 25 mM to about 150 mM.

Embodiment 134. The method of Embodiment 133, wherein the arginine salt is present at a concentration of from about 50 mM to about 100 mM.

- 66 045816

Embodiment 135. The method of Embodiment 133, wherein the arginine salt is arginine HCl, arginine acetate or arginine glutamate.

Embodiment 136. The method of Embodiment 135, wherein the arginine-HCl is present at a concentration of approximately 50 mM.

Embodiment 137. The method of any one of embodiments 107-136, wherein the PCSK9 binding polypeptide is stable for at least about 2 years if stored at about -30°C or lower.

Embodiment 138. The method of Embodiment 137, wherein the PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 139. The method of any one of embodiments 107-136, wherein the PCSK9 binding polypeptide is stable for at least about 6 months if stored at about 5°C.

Embodiment 140. The method of Embodiment 139, wherein the PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 141. The method of any one of embodiments 107-136, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 25°C.

Embodiment 142. The method of Embodiment 141, wherein the PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 143. The method of Embodiment 142, wherein the PCSK9 binding polypeptide is stable for at least about 6 months.

Embodiment 144. The method of any one of embodiments 107-136, wherein the PCSK9 binding polypeptide is stable for at least about 1 month if stored at about 40°C.

Embodiment 145. The method of any one of embodiments 107-136, wherein the composition contains high molecular weight aggregates or oligomers of PCSK9-binding polypeptide at a level of less than about 3% of the concentration of PCSK9-binding polypeptide.

Embodiment 146. The method of Embodiment 145, wherein the high molecular weight aggregates or oligomers of the PCSK9 binding polypeptide are present at a level of less than about 2.5% of the concentration of the PCSK9 binding polypeptide.

Embodiment 147: A method for formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated;

b) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine using diafiltration;

c) a second concentration step, in which the polypeptide in the second solution is concentrated;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution containing N-acetylarginine using diafiltration; And

e) a third concentration step, in which the polypeptide in the third solution is concentrated;

wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

i) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

ii) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

- 67 045816

3) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1; And

4) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

5) where the epitope for the monoclonal antibody further overlaps with the region to which the EGFa domain of the LDLR binds.

Embodiment 148. The method of Embodiment 147, wherein the PCSK9-binding polypeptide that blocks PCSK9 binding to the LDLR is a monoclonal antibody that contains a heavy chain polypeptide containing the following complementarity determining regions (CDRs):

a) heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 7, 8 and 9, respectively; And

b) light chain CDR1, CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively.

Embodiment 149. The method of Embodiment 147, wherein prior to the third concentration step, the temperature of the solution containing the polypeptide is raised from about 25°C to about 37°C.

Embodiment 150. The method of Embodiment 147, wherein the first solution replacement step is carried out using at least three volumes of the second solution.

Embodiment 151. The method of Embodiment 147, wherein the second solution replacement step is carried out using at least four diavolumes of the third solution.

Embodiment 152. The method of Embodiment 147, wherein the initial concentration of the therapeutic protein is approximately 11 mg/ml or less.

Embodiment 153. The method of Embodiment 147, wherein the first concentration step increases the concentration of the therapeutic polypeptide by about 3-fold to about 7-fold.

Embodiment 154. The method of Embodiment 153, wherein the increased concentration of the polypeptide is from about 35 mg/ml to about 70 mg/ml.

Embodiment 155. The method of Embodiment 147, wherein the second concentration step increases the concentration of the therapeutic polypeptide by about 2 to 4 times the first concentration step.

Embodiment 156. The method of Embodiment 155, wherein the increased concentration of polypeptide is approximately 140 mg/ml.

Embodiment 157 The method of Embodiment 147, wherein the third concentration step increases the concentration of the therapeutic polypeptide by a factor of about 1.5 to about 2 times the second concentration step.

Embodiment 158. The method of Embodiment 157, wherein the increased polypeptide concentration is approximately 260 mg/ml.

Embodiment 159. The method of Embodiment 147, wherein the therapeutic polypeptide has a final concentration that is at least about 19 to 20 times the initial concentration of the therapeutic polypeptide.

Embodiment 160. The method of Embodiment 159, wherein the final concentration of the therapeutic polypeptide is approximately 210 mg/ml.

Embodiment 161. The method of Embodiment 147, wherein the concentration steps include batch ultrafiltration.

Embodiment 162. The method of Embodiment 147, wherein the second solution and the third solution are identical.

Embodiment 163. The method of Embodiment 147, wherein the second or third solution containing N-acetylarginine contains an arginine salt and a buffer.

Embodiment 164. The method of Embodiment 163, wherein N-acetylarginine is present at a concentration of from 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 from about 25 mM to about 150 mM; and the buffer is a sodium acetate buffer at a concentration of from about 5 mM to about 30 mM.

Embodiment 165. The method of Embodiment 164, wherein N-acetylarginine is present at a concentration of from 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.

Embodiment 166. The method of Embodiment 164, wherein N-acetylarginine is present at a concentration of approximately 140 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 167. The method of Embodiment 164, wherein N-acetylarginine is present

- 68 045816 is present at a concentration of approximately 155 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 70 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 168. The method of Embodiment 164, wherein N-acetylarginine is present at a concentration of approximately 170 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 169. The method of Embodiment 165 or 166, further comprising proline, wherein the proline is present at a concentration of from about 50 mM to about 150 mM.

Embodiment 170. The method of any one of embodiments 163-169, wherein the second or third solution has a pH value of from about 4.8 to about 6.9.

Embodiment 171 The method of embodiment 170, wherein the pH value is approximately 5.4.

Embodiment 172. The method of Embodiment 147, wherein the first and second solution replacement steps employ a diafiltration membrane having at least one characteristic selected from the group consisting of:

a) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm;

b) a clear cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

c) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

d) a filament diameter that is greater than approximately 270 µm but less than or equal to approximately 340 µm;

e) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

f) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

g) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

h) maximum supply pressure of approximately 60 psi.

Embodiment 173. The method as in any one of embodiments 147-172, wherein the surfactant is added to the third solution after concentration is performed.

Embodiment 174. The method of Embodiment 173, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types of Pluronic ®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers, and D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS), and is present at concentrations ranging from about 0 .0001% to approximately 1%.

Embodiment 175 The method of Embodiment 175, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v).

Embodiment 176. A method of formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated using batch ultrafiltration;

b) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine, an arginine salt and a buffer using diafiltration and three dia volumes of the second solution;

c) a second concentration step in which the polypeptide in the second solution is concentrated using batch ultrafiltration;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution containing N-acetylarginine, an arginine salt and a buffer using diafiltration and four dia volumes of the third solution;

e) the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C after the second solution replacement step; And

f) a third concentration step in which the polypeptide is further concentrated using batch ultrafiltration concentration;

- 69 045816 wherein in the first and second stages of solution replacement a diafiltration membrane having at least one characteristic selected from the group consisting of:

g) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm;

h) a clear cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

i) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

j) a filament diameter that is greater than about 270 microns but less than or equal to approximately 340 microns;

k) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

l) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

m) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

n) maximum supply pressure of approximately 60 psi; and wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and

3) where the epitope for the monoclonal antibody additionally overlaps with the region to which the EGFa domain of the LDLR binds.

Embodiment 177 The method of Embodiment 176, wherein prior to the third concentration step, the temperature of the solution containing the polypeptide is raised from about 25°C to about 37°C.

Embodiment 178. The method of Embodiment 176, wherein the initial concentration of the therapeutic protein is 11 mg/ml or less.

Embodiment 179. The method of Embodiment 176, wherein the first concentration step increases the concentration of the therapeutic polypeptide by about 3-fold to about 7-fold.

Embodiment 180. The method of Embodiment 179, wherein the increased concentration of the polypeptide is from about 35 mg/ml to about 70 mg/ml.

Embodiment 181. The method of Embodiment 176, wherein the second concentration step increases the concentration of the therapeutic polypeptide by about 2 to 4 times the first concentration step.

Embodiment 182. The method of Embodiment 181, wherein the increased polypeptide concentration is approximately 140 mg/ml.

Embodiment 183. The method of Embodiment 176, wherein the third concentration step increases the concentration of the therapeutic polypeptide by a factor of about 1.5 to about 2 times the second concentration step.

Embodiment 184. The method of Embodiment 183, wherein the increased polypeptide concentration is approximately 260 mg/ml.

Embodiment 185. The method of Embodiment 176, wherein the therapeutic poly

- 70 045816 the peptide has a final concentration that is at least about 19-20 times the initial concentration of the therapeutic polypeptide.

Embodiment 186. The method of Embodiment 185, wherein the final concentration of the therapeutic polypeptide is approximately 210 mg/ml.

Embodiment 187. The method of Embodiment 176, wherein N-acetylarginine is present at a concentration of from 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 from about 25 mM to about 150 mM, and the buffer is a sodium acetate buffer at a concentration of from about 5 mM to about 30 mM.

Embodiment 188 The method of Embodiment 188, wherein N-acetylarginine 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.

Embodiment 189. The method of Embodiment 188, wherein N-acetylarginine is present at a concentration of approximately 140 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 190. The method of Embodiment 188, wherein N-acetylarginine is present at a concentration of approximately 155 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 70 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 191. The method of Embodiment 188, wherein N-acetylarginine is present at a concentration of approximately 170 mM, Arg-HCl, Arg acetate or Arg glutamate is present at a concentration of approximately 63 mM, sodium acetate buffer is present at a concentration of approximately 10 mM.

Embodiment 192. The method of Embodiment 176 further comprising proline, wherein the proline is present at a concentration of from about 50 mM to about 150 mM.

Embodiment 193. The method of any one of embodiments 176-192, wherein the second or third solution has a pH value of from about 4.8 to about 6.9.

Embodiment 194 The method of Embodiment 193, wherein the pH value is approximately 5.4.

Embodiment 195. The method as in any one of embodiments 176-194, wherein the surfactant is added to the third solution after concentration is performed.

Embodiment 196. The method of Embodiment 195, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan monooleate (polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer (poloxamers such as Pluronic® F-68 and other types of Pluronic ®), sorbitan alkyl esters (Span®), polyethylene glycol octyl phenyl ethers (Triton X-100), polyethylene glycol alkyl ethers (Brij), polypropylene glycol alkyl ethers, glucosidalkyl ethers, and D-a-tocopherol polyethylene glycol succinate (Vitamin E TPGS), and is present at a concentration of about 0 .0001% to approximately 1%.

Embodiment 197. The method of Embodiment 196, wherein the surfactant is polyoxyethylene sorbitan monooleate (polysorbate 80) and is present at a concentration of approximately 0.01% (w/v).

Embodiment 198: A method of formulating a therapeutic polypeptide, comprising:

a) a first concentration step, in which the polypeptide in the first solution is concentrated using batch ultrafiltration;

b) a first solution replacement step, in which the concentrated polypeptide in the first solution is replaced by a second solution using diafiltration and three diavolumes of the second solution;

c) a second concentration step in which the polypeptide in the second solution is concentrated using batch ultrafiltration;

d) a second solution replacement step in which the polypeptide in the concentrated second solution is replaced by a third solution using diafiltration and four diavolumes of the third solution;

e) the temperature of the solution containing the polypeptide is increased from about 25°C to about 37°C after the second solution replacement step; And

f) a third concentration step, in which the polypeptide is further concentrated at

- 71 045816 by changing concentration using ultrafiltration with batch loading;

g) alternatively, the step of adding polyoxyethylene sorbitan monooleate at a concentration of approximately 0.01% (w/v) to the solution obtained in the third concentration step, wherein the second and third solutions comprise a solution selected from the group consisting of a solution containing approximately 140 mM N-acetylarginine, about 50 mM Arg-HCl and about 10 mM sodium acetate, the solution having a pH of about 5.2; a solution containing approximately 155 mM N-acetylarginine, approximately 70 mM Arg-HCl and approximately 10 mM sodium acetate, the solution having a pH value of approximately 5.4; and a solution containing approximately 170 mM N-acetylarginine, approximately 10 mM sodium acetate, the solution having a pH value of approximately 5.6;

wherein the first and second stages of solution replacement may employ a diafiltration membrane having at least one characteristic selected from the group consisting of:

h) a mesh opening size that is greater than approximately 350 µm but less than or equal to approximately 500 µm;

i) a free cross-sectional area that is greater than approximately 32% but less than or equal to approximately 36% of the membrane area;

j) a mesh density less than about 16.2 threads/cm but greater than or equal to about 12.2 threads/cm;

k) a filament diameter that is greater than approximately 270 µm but less than or equal to approximately 340 µm;

l) a basis weight that is greater than approximately 160 g/ ^m2 but less than or equal to 180 g/ ^m2 ;

m) a thickness that is greater than approximately 515 µm but less than or equal to approximately 610 µm;

n) membrane loading greater than about 1138.1 g/m ² but less than or equal to about 1919.3 g/m ² ; And

o) maximum supply pressure of approximately 60 psi; and wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of:

i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof;

ii) a monoclonal antibody that competes with evolocumab for binding to PCSK9;

iii) a monoclonal antibody containing:

1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, which is CDR1 in SEQ ID NO: 14 or 16; Heavy chain CDR2, which is CDR2 in SEQ ID NO: 14 or 16; Heavy chain CDR3, which is CDR3 in SEQ ID NO: 14 or 16, and

2) a light chain polypeptide containing the following CDRs: light chain CDR1, which is CDR1 in SEQ ID NO: 15 or 17; Light chain CDR2, which is CDR2 in SEQ ID NO: 15 or 17; and light chain CDR3, which is CDR3 in SEQ ID NO: 15 or 17;

iv) a monoclonal antibody that binds to at least one of the following residues of PCSK9, wherein PCSK9 contains 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, I36 9, S372, C375, C378, R237, D238;

v) a monoclonal antibody that binds to PCSK9 at the location of an epitope on PCSK9 that overlaps with an epitope that binds to an antibody containing:

1) a heavy chain variable region with the amino acid sequence from SEQ ID NO: 1; And

2) a light chain variable region having the amino acid sequence of SEQ ID NO: 2, and wherein the epitope for the monoclonal antibody further overlaps with the region to which the EGFa domain of the LDLR binds.

Embodiment 199. The method of Embodiment 104, wherein the subject has a disease or disorder selected from the group consisting of a disease or disorder associated with elevated cholesterol selected from the group consisting of familial hypercholesterolemia (including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial hypercholesterolemia with defective apolipoprotein B-100; polygenic hypercholesterolemia), non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease and types of dyslipidemia (including primary and secondary dyslipidemia, such such as metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertrigism

- 72 045816 hyporidemia; familial dysbetalipoproteinemia, hepatic lipase deficiency; dyslipidemia secondary to indiscriminate eating, hypothyroidism, medications, including estrogen and progestin therapy, beta-blockers and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage disease, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency or alcoholic hypertriglyceridemia; atherosclerotic disease selected from the group consisting of death due to cardiovascular disease, death due to non-cardiovascular disease or death from all causes, coronary heart disease, coronary insufficiency, peripheral artery disease, stroke (ischemic and hemorrhagic), angina pectoris , cerebrovascular accidents and acute coronary syndrome, myocardial infarction and unstable angina; and diseases or disorders that can be treated with statins.

Embodiment 200. The method of embodiment 104, wherein treating the subject involves reducing the risk of developing a condition selected from the group consisting of fatal and non-fatal heart attack, fatal and non-fatal stroke, heart surgery, hospitalization for heart failure, chest pain in a subject having heart disease and/or cardiovascular complications due to established heart disease, a cardiovascular condition due to elevated levels of CRP or hsCRP, and a recurrent cardiovascular complication.

Embodiment 201. The pharmaceutical composition according to any one of embodiments 1-103 for use as a medicinal product.

Embodiment 202. The pharmaceutical composition of Embodiment 201, wherein the medicinal product is for use in the treatment of a disease or disorder selected from the group consisting of a disease or disorder associated with elevated cholesterol selected from the group consisting of familial hypercholesterolemia (including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial hypercholesterolemia with defective apolipoprotein B-100; polygenic hypercholesterolemia), non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease and types of dyslipidemia (including primary and secondary dyslipidemias, such as metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia; familial dysbetalipoproteinemia, hepatic lipase deficiency; dyslipidemia secondary to dietary indiscriminateness, hypothyroidism, medications, including estrogen and progestin therapy, beta blockers and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage disease, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency or alcoholic hypertriglyceridemia; atherosclerotic disease selected from the group consisting of death due to cardiovascular disease, death due to non-cardiovascular disease or death from all causes, coronary heart disease, coronary insufficiency, peripheral artery disease, stroke (ischemic and hemorrhagic), angina pectoris , cerebrovascular accidents and acute coronary syndrome, myocardial infarction and unstable angina; and diseases or disorders that can be treated with statins.

Embodiment 203. The method of embodiment 202, wherein the medicinal product is intended to reduce the risk of developing a condition selected from the group consisting of fatal and non-fatal heart attack, fatal and non-fatal stroke, heart surgery, hospitalization for heart failure, chest pain in a subject having heart disease and/or cardiovascular complications due to established heart disease, a cardiovascular condition due to elevated levels of CRP or hsCRP, and a recurrent cardiovascular complication.

- 73 045816

Abbreviations Reduction Definition Arg-HCl Arginine-NS1 AS C Storage conditions at ambient temperature

air CDR Complementarity determining region SEX Cation exchange DF Diafil tradition D.I.W. Deionized water DOE Experimental plan D.S. Medicinal substance D.V. Diavolume EGF-A Repeat A epidermal growth factor-like EQ Equilibrium FR Wireframe area NS Heavy chain (antibodies) HCVR Heavy chain variable region (antibodies) HDLC High-density lipoprotein cholesterol ΗI AC Detecting particles invisible to the naked eye using light blocking H.M.W. High molecular weight HPLC High Pressure Liquid Chromatography HPLC-UV High pressure liquid chromatography with ultraviolet detector L.C. Light chain (antibodies) LCVR Light chain variable region (antibodies) LDL Low density lipoprotein LDLR Low-density lipoprotein receptors LMH liters/ ^m2 /h. H.M. Low molecular weight mAb Monoclonal antibody MFI Microflow visualization NaOAC Sodium acetate NAR N-acetylarginine NTU Nephelometric unit of turbidity

- 74 045816

OS Super concentrated, super concentrated

PEG Polyethylene glycol

RES Pre-filled syringe

PVDF Polyvinylidene fluoride

PW Purified water rCE-SDS Capillary electrophoresis with sodium dodecyl sulfate under reducing conditions

room temperature

SEC Size Exclusion Chromatography

SE-HPLC Size exclusion liquid chromatography high pressure

TEE Filtration in tangential flow

TMP Transmembrane pressure

UF Ultrafiltration

UFDF Ultrafiltration/Diafiltration

UV Ultraviolet

VF Virus filtering

VLDL-C Very Low Density Lipoprotein Cholesterol

WFI Water for injection

Bibliography

Chan, J., Gibbs, J., Dias, C., Wasserman, S., Scott, R., Clogston, C., . . . Stein, E. (2012). Patent document

World Intellectual Property Organization No. WO2012154999.

Chothia, S., & Lesk, A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol, 196(4), 901-917.

Chothia, C., Lesk, A. M., Tramontane, A., Levitt, M., Smith-Gill, S. J., Air, G., . . . et al. (1989) Conformations of immunoglobulin hypervariable regions. Nature, 342(6252), 877883. doi: 10.1038/342877a0

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/nsmbl235

Horton, J. D., Cohen, J. C., & Hobbs, H. H. (2007).

-

Claims (16)

Molecular biology of PCSR9: its role in LDL metabolism. Trends Biochem Sci, 32(2), 11-11. doi: 10.1016/j.tibs.2006.12.008 Jackson, S. M., Walker, N. P., Piper, D. E., Shan, B., Shen, W., Chan, J., . . . Carabeo, T. (2009). World Intellectual Property Organization Patent Document No. WO 2009/026558. Rabat, E., Wu, T., Perry, N., Gottesman, K., & Foeller, S. (1991). Sequences of proteins of immunological interest (Vol. Publication No. 91-3242). Bethesda, MD: National Institutes of Health. Rabat, E., Wu, T., Reid-Miller, M., Perry, H., & Gottesman, R. (1987). Sequences of proteins of immunological interest (4th ed. Vol. No. 165-492). Bethesda, MD: US Government Printing Office. Morichika, T., & Rameoka, D. (2007). World Intellectual Property Organization Patent Document No. WO2007074880. Piper, D. E., Jackson, S., Liu, Q., Romanow, W. G., Shetterly, S., Thibault, S. T., . . . Walker, N. P. (2007). The crystal structure of PCSR9: a regulator of plasma LDLcholesterol. Structure, 15(5), 545-552. doi: 10.1016/j.str.2007.04.004 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 Seidah, N. G., & Prat, A. (2007) . The proprotein convertases are potential targets in the treatment of dyslipidemia. J Mol Med (Berl), 85(1), 685-696. doi: 10.1007/s00109-007-0172-7 FORMULA OF THE INVENTION 1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 7, 8 and 9, respectively, and 1) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 7, 8 and 9, respectively, and 1. Pharmaceutical composition containing: a) at least 140 mg/ml PCSK9-binding polypeptide, which is selected from the group consisting of: i) a monoclonal antibody 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 (evolocumab), or an antigen binding fragment thereof; and ii) a monoclonal antibody containing: 2) a light chain polypeptide containing the following CDRs: light chain CDR1, CDR2 and CDR3, having the amino acid sequence SEQ ID NO: 4, 5 and 6, respectively. 2. The pharmaceutical composition according to claim 1, wherein the PCSK9-binding polypeptide is present at a concentration ranging from 140 to 260 mg/ml. 2) a light chain polypeptide containing the following CDRs: light chain CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 4, 5 and 6, respectively; And b) from about 25 mM to about 230 mM N-acetylarginine, and c) at least one component of an arginine or proline salt, where the pharmaceutical composition has a viscosity of less than 80 cP. 3. Pharmaceutical composition according to claim 1 or 2, where the concentration of PCSK9-binding polypeptide is 210 mg/ml. 4. Pharmaceutical composition according to any one of claims 1-3, additionally containing a buffer. 5. Pharmaceutical composition according to any one of claims 1-4, additionally containing a surfactant. 6. Pharmaceutical composition according to any one of claims 1-5, additionally containing proline. 7. Pharmaceutical composition according to any one of claims 1-6, additionally containing an arginine salt. 8. Pharmaceutical composition according to any one of claims 1 to 7, where the composition contains high molecular weight aggregates or oligomers of PCSK9-binding polypeptide at a level of less than 3% of the concentration - 76 045816 tion of PCSK9-binding polypeptide. 9. Pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition is liquid. 10. Use of a pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for lowering serum cholesterol in a human subject. 11. Use of a pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for the treatment of a cholesterol-related disorder in a human subject, wherein the cholesterol-related disorder includes one or more of the following: familial hypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, dyslipidemia, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemia, heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial apolipoprotein defect B-100, polygenic hypercholesterolemia syndrome, nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, alcoholic hypertriglyceridemia or dyslipidemia secondary to any of the following factors: poor diet, hypothyroidism, medications, beta blockers and thiazide diuretics. 12. A kit for the treatment of a cholesterol-related disorder in a human subject, comprising a pharmaceutical composition according to any one of claims 1 to 9 and a delivery device selected from the group consisting of a syringe, a pen, a body injector and an auto-injector . 13. A method of producing a PCSK9-binding polypeptide in a pharmaceutical composition containing at least 140 mg/ml PCSK9-binding polypeptide, comprising adding to the pharmaceutical composition containing the PCSK9-binding polypeptide from about 25 mm to about 230 mm N-acetylarginine and at least one component of an arginine or proline salt, whereby the viscosity of the pharmaceutical composition is reduced compared to a pharmaceutical composition not containing N-acetylarginine, and wherein the PCSK9-binding polypeptide is selected from the group consisting of: a) a monoclonal antibody containing 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 (evolocumab), or an antigen binding fragment thereof; And b) a monoclonal antibody containing: i) a heavy chain polypeptide containing the following complementarity determining regions (CDRs): heavy chain CDR1, CDR2 and CDR3 having the amino acid sequence SEQ ID NO: 7, 8 and 9, respectively, and ii) a light chain polypeptide containing the following CDRs: CDR1, Light chain CDR2 and CDR3 having the amino acid sequence of SEQ ID NO: 4, 5 and 6, respectively; wherein the pharmaceutical composition has a viscosity of less than 80 cP. 14. A method for producing a therapeutic polypeptide, including: a) a first solution replacement step in which the concentrated polypeptide in the first solution is replaced by a second solution containing N-acetylarginine using diafiltration; b) a second concentration step, in which the polypeptide in the second solution is concentrated; c) a second solution exchange step in which the polypeptide in the concentrated second solution is exchanged for a third solution containing from about 25 mM to about 230 mM N-acetylarginine using diafiltration; And d) a third concentration step, in which the polypeptide in the third solution is concentrated; wherein the therapeutic polypeptide comprises a PCSK9-binding polypeptide that blocks binding of PCSK9 to the LDLR and is selected from the group consisting of: i) a monoclonal antibody containing 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 (evolocumab), or an antigen-binding fragment thereof; and ii) a monoclonal antibody containing: 15. The method according to claim 14, where before the third stage of concentration, the temperature of the solution containing the polypeptide is increased from 25°C to 37°C. 16. The method according to claim 14, where in the first stage of concentration the concentration of the therapeutic polypeptide increases from 3 to 7 times. -