RU2782792C2 - Pharmaceutical composition containing antibodies to pcsk-9, and its use - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to the field of composition of pharmaceutical preparations. A pharmaceutical composition is proposed, containing an antibody to PCSK-9 or its antigen-binding fragment at a concentration from 1 mg/ml to 150 mg/ml, a histidine buffer at a concentration from 5 mM to 30 mM, trehalose or sucrose at a concentration from 10 mg/ml to 75 mg/ml, polysorbate 80 or polysorbate 20 at a concentration from 0.05 mg/ml to 0.6 mg/ml, pH is from 5.5 to 6.5. The antibody to PCSK-9 or its antigen-binding fragment contains HCDR1, HCDR2, and HCDR3 having SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and LCDR1, LCDR2, and LCDR3 having SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. A lyophilized composition and a reduced composition based on the specified composition are also proposed.

EFFECT: inventions provide for the production of stable compositions, which are used in the treatment of PCSK-9-mediated disease or disorder.

28 cl, 2 dwg, 14 tbl, 14 ex

Description

This application claims priority from Chinese Patent Application No. CN201710519829.6 filed on June 30, 2017. The entire content of the above application is incorporated herein by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the field of pharmaceutical formulation, in particular to a pharmaceutical composition containing an anti-PCSK-9 antibody and an antigen-binding fragment thereof and their use as a drug.

BACKGROUND OF THE INVENTION

Hypercholesterolemia is an abnormal lipid metabolism disorder characterized by elevated serum cholesterol levels. Its main manifestation is an increased level of cholesterol in the blood serum, which causes the aggregation of cholesterol in the blood vessels and, consequently, leads to the occurrence of atherosclerosis. Numerous results of clinical and experimental studies have proven that abnormal lipid metabolism is closely associated with the onset and development of coronary heart disease. Consequently, lowering the concentration of cholesterol in the blood has become the main means of treating and preventing atherosclerosis.

With the rapid improvement in living standards, dyslipidemia has also become a major threat to urban and rural residents in China. According to 2012 statistics, approximately 40% of deaths per year in China were related to cardiovascular diseases. Currently, the mortality rate from dyslipidemia among adults in China is 18.6%, with an estimated 160 million people suffering from dyslipidemia. The incidence rates for different types of dyslipidemia are as follows: 2.9% for hypercholesterolemia, 11.9% for hypertriglyceridemia, 7.4% for HDL-deficiency lipoproteinemia, and 3.9% for slightly elevated blood cholesterol. In the "Chinese Expert Consensus on Prevention and Control of Chronic Diseases" reached by the Industry Committee for the Prevention and Control of Chronic Diseases, Committee of Experts on Disease Prevention and Control, Ministry of Health, 2012, it was stated that in China, 33 million people suffer from hypercholesterolemia, however, in terms of local areas, the incidence of dyslipidemia is much more serious than the above data.

Currently, drugs used clinically to control lipid levels are mainly directed towards statins. Lipitor®, the most widely used and best-selling cholesterol-lowering drug, reduces cholesterol production by blocking the effect of an enzyme that produces cholesterol in the liver and increases the absorption of cholesterol from the blood by the liver, thereby lowering blood cholesterol levels. However, Lipitor® has its drawbacks. First, it will be clear from the data that Lipitor® can reduce LDL levels by 30-40%, but many patients still fail to achieve effective blood lipid lowering (LDL < 50 mg/dL). ). Secondly, there is a difference in the percentage of positive clinical responses to Lipitor® among patients of different races. For these reasons, patients still need a more effective medication to lower blood lipids.

As an autosomal dominant monogenic hereditary disease, hereditary hypercholesterolemia (FM) is clinically characterized by a significant increase in the level of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) in the blood, the presence of xanthelasma, corneal arch and the development of premature cardiovascular disease. Mutations in the low-density lipoprotein receptor (LDL receptor, LDLR) gene cause LDLR deficiency or absence. Consequently, LDL-c is not transported to the liver for degradation, resulting in an increase in LDL-c levels in the blood. Three genes have now been determined to correlate with the onset of FM: the LDLR gene, the apolipoprotein B100 gene, and the subtilisin-kexin-type proprotein convertase 9 (PCSK9) gene.

As a proprotein convertase, proprotein convertase subtilisin-kexin type 9 (PCSK9), belongs to the protease K subfamily in the secretory family of Bacillus subtilis. The encoded protein is synthesized as a soluble proenzyme and undergoes intramolecular processing in the endoplasmic reticulum by autocatalysis. According to experimental results, PCSK9 can promote degradation of the LDL receptor and thus increase plasma LDL cholesterol levels. The LDL receptor mediates the process of LDL endocytosis in the liver, which is the main route for removing LDL from the circulatory system. It was found that mutations in the PCSK9 gene were identified in 12.5% of patients with hypercholesterolemia (ADH). There are different types of PCSK9 mutations. According to the different effects of mutations on the level of LDL-c regulated by PCSK9, mutations can be divided into two types: a loss-of-function type and a gain-of-function type. Among them, loss-of-function mutations are associated with low blood cholesterol levels and prevent the onset of atherosclerotic heart disease. The incidence of PCSK9 mutations associated with low cholesterol levels is higher in African populations than in other races. PCSK9 mutations, which are gain-of-function mutations, increase plasma cholesterol levels by increasing PCSK9 function and decreasing LDLR expression, which can lead to severe hypercholesterolemia and premature ischemic atherosclerotic heart disease. Gain-of-function mutations in PCSK9 have now been found to include D374Y, S127R, F216L, N157K, R306S, and so on. Compared to wild-type PCSK9, in D374Y mutants, the content of LDLR on the cell surface decreased by 36%, and in S127R mutants it decreased by 10%.

However, antibodies become unstable due to their large molecular weights, complex structures, and susceptibility to degradation, polymerization, or unwanted chemical modification. In order to create antibodies that are suitable for administration, retain their stability during storage and subsequent use, and have better effects, it is especially important to study stable formulations based on antibodies.

Currently, several companies are developing anti-PCSK-9 antibodies and pharmaceutical formulations such as formulations based on CN 103717237 A, CN 104364266 A, etc. However, for novel anti-PCSK-9 antibodies, it still remains necessary to develop a pharmaceutical composition containing an anti-PCSK-9 antibody that is more suitable for administration.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising an anti-PCSK9 antibody or antigen binding fragment thereof and a buffer, wherein the buffer is preferably a histidine buffer or a succinate buffer or a phosphate buffer or a citrate buffer, more preferably a histidine buffer, most preferably a histidine hydrochloride buffer.

In an alternative embodiment, wherein the concentration of the PCSK-9 antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 1 mg/mL to 150 mg/mL, preferably from 30 mg/mL to 100 mg/mL, more preferably from 50 mg/mL. ml up to 60 mg/ml, most preferably 50 mg/ml. Non-limiting examples of such concentrations of the PCSK-9 antibody or antigen-binding fragment thereof include 45 mg/mL, 46 mg/mL, 47 mg/mL, 48 mg/mL, 49 mg/mL, 50 mg/mL, 51 mg/mL, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml or 60 mg/ml.

In an alternative embodiment, the buffer concentration is from about 5 mM to 50 mM, preferably from 5 mM to 30 mM, non-limiting examples of buffer concentrations include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM and 30 mM, more preferably 10 mM to 15 mM, most preferably 10 mM.

In an alternative embodiment, the pH of the buffer in the pharmaceutical composition is from about 5.5 to 6.5, preferably from about 6.0 to 6.5, non-limiting examples of the pH of the buffer include about 6.0, about 6.1, about 6 .2, about 6.3, about 6.4, or about 6.5.

Moreover, in an alternative embodiment, the pharmaceutical composition further comprises a saccharide. "Saccharide" as used herein includes the conventional compound (CH ₂ O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, melesitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc. The preferred saccharide herein is a non-reducing disaccharide, more preferably trehalose or sucrose.

In an alternative embodiment, where the concentration of saccharide in the pharmaceutical preparation is from about 10 mg/ml to 75 mg/ml, preferably from 20 mg/ml to 60 mg/ml, more preferably from about 20 mg/ml to 40 mg/ml, most preferably 25 mg/ml. Non-limiting examples of saccharide concentration include 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml or 40 mg/ml.

In an alternative embodiment, the pharmaceutical composition further comprises a surfactant. As used herein, "surfactant" may be selected from the group consisting of polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glycoside, lauryl sulfo betaine, myristyl sulfo betaine, linoleyl sulfo betaine, stearyl sulfo betaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, стеарилсаркозина, линолеилбетаина, миристилбетаина, цетилбетаина, лаурамидопропилбетаина, кокамидопропилбетаина, линолеамидопропилбетаина, миристамидопропилбетаина, пальмидопропилбетаина, изостеарамидопропилбетаина, миристамидопропилдиметиламина, пальмидопропилдиметиламина, изостеарамидопропилдиметиламина, метил-кокоил таурата натрия, метил-олеилтаурата натрия, полиэтиленгликоля, полипропиленгликоля и сополимеров этилена и пропиленгликоля и т.д. The preferred surfactant herein is Polysorbate 20 or Polysorbate 80, more preferably Polysorbate 80.

In an alternative embodiment, the concentration of surfactant in the pharmaceutical composition is from about 0.05 mg/mL to 1.0 mg/mL, preferably from 0.1 mg/mL to 0.4 mg/mL, non-limiting examples of surfactant concentration active substances include 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.25 mg/ml, 0.3 mg/ml, 0.35 mg/ml, 0.4 mg/ml ml, even more preferably 0.1 mg/ml to 0.3 mg/ml, more preferably 0.1 mg/ml to 0.2 mg/ml, most preferably 0.2 mg/ml.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 1-150 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-30 mM histidine buffer, pH about 5.5-6.5, more preferably about 6.0-6.5;

(c) 10-75 mg/ml sucrose; and

(d) 0.05-0.6 mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-20 mM histidine buffer;

(c) 25 mg/ml sucrose; and

(d) 0.1-0.3 mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 1-150 mg/ml of an anti-PCSK-9 antibody or antigen-binding fragment thereof, and

(b) 5-30 mM histidine buffer; and the pH of the pharmaceutical composition is about 6.0-6.5.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 1-150 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-30 mM histidine buffer;

(c) 10-75 mg/ml sucrose; and

(d) 0.05-0.6 mg/ml polysorbate 80; and the pH of the pharmaceutical composition is about 6.0-6.5, and the histidine buffer is preferably a histidine hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof,

(b) 5-20 mM histidine buffer,

(c) 25 mg/ml sucrose and

(d) 0.1-0.3 mg/ml polysorbate 80; and the pH of the pharmaceutical composition is about 6.0-6.5, and the histidine buffer is preferably a histidine hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 10 mM histidine buffer;

(c) 25 mg/ml sucrose; and

(d) 0.2 mg/ml polysorbate 80 and the pH of the pharmaceutical composition is 6.3 ± 0.1 and the histidine buffer is preferably a histidine hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 150 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 30 mM histidine hydrochloride buffer;

(c) 75 mg/ml sucrose; and

(d) 0.6 mg/ml polysorbate 80; the final pH value of the pharmaceutical composition is 6.3.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 10 mM histidine hydrochloride buffer, pH 6.0;

(c) 25 mg/ml sucrose; and

(d) 0.2 mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 150 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 20 mM histidine hydrochloride buffer, pH 6.5; and

(c) 70 mg/ml sucrose.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 150 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 20 mM histidine hydrochloride buffer, pH 6.5; and

(c) 70 mg/ml α,α-trehalose dihydrate.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 20 mM histidine hydrochloride buffer, pH 6.0;

(c) 25 mg/ml sucrose; and

(d) 0.2 mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition contains:

(a) 50 mg/ml of anti-PCSK-9 antibody or antigen-binding fragment thereof;

(b) 20 mM histidine hydrochloride buffer, pH 6.5;

(c) 25 mg/ml sucrose; and

(d) 0.2 mg/ml polysorbate 80.

In an alternative embodiment, the PCSK-9 antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises HCDR1, HCDR2, and HCDR3 having the sequences under SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and

LCDR1, LCDR2 and LCDR3 having the sequences under SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

In an alternative embodiment, the PCSK-9 antibody or antigen-binding fragment thereof in the pharmaceutical composition is selected from the group consisting of a mouse antibody, a chimeric antibody, and a humanized antibody, a humanized antibody being preferred.

In an alternative embodiment, the light chain of the PCSK-9 antibody in the pharmaceutical composition is characterized by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the light chain of the h001-4-YTE antibody, while the heavy chain of the anti-PCSK-9 antibody in the pharmaceutical composition is characterized by at least 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the antibody heavy chain h001-4-YTE. The light chain of the h001-4-YTE antibody has the amino acid sequence of SEQ ID NO: 30 and the heavy chain of the h001-4-YTE antibody has the amino acid sequence of SEQ ID NO: 32.

The present invention further provides a method for preparing the pharmaceutical composition described above, comprising the step of replacing the stock solution for an anti-PCSK-9 antibody or antigen-binding fragment thereof with a buffer. Preferably the buffer is a histidine buffer, more preferably a histidine hydrochloride buffer. The buffer concentration is preferably from about 5 mM to 30 mM, and non-limiting examples of the buffer concentration include 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM , 22 mm, 24 mm, 26 mm, 28 mm and 30 mm, more preferably from 10 mm to 15 mm; while the pH of the buffer is from about 6.0 to 6.5, non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5.

In addition, the method for preparing the pharmaceutical composition described above further comprises the step of adding sucrose and polysorbate 80 to the solution obtained in the replacement step, and then adjusting to a final volume with a buffer, where the buffer concentration is preferably from 10 mM to 20 mM, non-limiting examples of concentration buffers include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM and 20 mM; wherein the pH of the buffer is approximately 6.0-6.5, non-limiting examples of the pH of the buffer include 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5.

The present invention further provides a method for preparing a lyophilized formulation comprising an anti-PCSK-9 antibody, which includes the step of lyophilizing the pharmaceutical composition described above.

In an alternative embodiment, the process for preparing a lyophilized formulation comprising an anti-PCSK-9 antibody comprises the sequential steps of pre-freeze, primary dry, and post-dry, wherein the pre-freeze is freezing from 5°C to -40°C to -50°C, most preferably -45°C, with no requirement for maintaining a vacuum. The primary drying temperature is -14°C to 0°C, most preferably -5°C; the vacuum pressure is from 0.1 mbar to 0.5 mbar, most preferably 0.3 mbar. The secondary drying temperature is from 20°C to 30°C, preferably 25°C, the vacuum pressure is from 0.1 mbar to 0.5 mbar, most preferably 0.3 mbar, and the vacuum pressure is reduced to a value from 0.005 mbar to 0. 02 mbar, most preferably 0.01 mbar.

The present invention further provides a freeze-dried formulation containing an anti-PCSK-9 antibody obtained by the method for preparing a freeze-dried formulation containing an anti-PCSK9 antibody described above.

In some embodiments, the lyophilized formulation is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the freeze-dried formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

The present invention further provides a method for preparing a solution reconstituted from a lyophilized formulation containing an anti-PCSK-9 antibody, which includes the step of reconstituting the lyophilized composition described above, wherein the reconstituting solvent is selected, without limitation, from water for injection, saline, or glucose solution.

The present invention further provides a solution reconstituted from a freeze-dried formulation containing an anti-PCSK-9 antibody obtained by the method for preparing a solution reconstituted from a freeze-dried formulation containing an anti-PCSK-9 antibody described above.

In an alternative embodiment, the reconstituted solution containing the PCSK-9 antibody of the present invention contains this component at a concentration of 2-5 times the concentration of this component in the pharmaceutical composition before lyophilization, preferably 3 times.

In an alternative embodiment, the reconstituted solution contains an anti-PCSK-9 antibody of the present invention, wherein the concentration of the anti-PCSK9 antibody or antigen-binding fragment is from about 120 mg/mL to 200 mg/mL, most preferably 150 mg/mL.

In an alternative embodiment, in the case of a reconstituted solution containing an anti-PCSK-9 antibody of the present invention, the pH of the pharmaceutical composition is about 6.0-6.5, preferably 6.4. The pH value of the reconstituted solution depends on the pH value of the buffer used in formulating the medicinal product. If the pH of the drug is 6.0, the final pH of the reconstituted solution is 6.3±1.

In an alternative embodiment, in the case of a reconstituted solution containing an anti-PCSK-9 antibody of the present invention, the buffer concentration is from about 15 mM to 45 mM, preferably 30 mM.

In an alternative embodiment, the reconstituted solution containing the PCSK-9 antibody of the present invention further comprises a disaccharide, wherein the disaccharide is preferably selected from the group consisting of trehalose and sucrose, most preferably sucrose.

In an alternative embodiment, in the case of a reconstituted solution containing an anti-PCSK-9 antibody of the present invention, the disaccharide concentration is from about 55 mg/mL to 95 mg/mL, preferably about 75 mg/mL.

In an alternative embodiment, the reconstituted solution containing the anti-PCSK-9 antibody of the present invention further comprises a surfactant, wherein the surfactant is preferably polysorbate, more preferably polysorbate 80.

In an alternative embodiment, in the case of a reconstituted solution containing an anti-PCSK-9 antibody of the present invention, the concentration of surfactant is from about 0.4 mg/mL to 0.8 mg/mL, preferably 0.6 mg/mL.

The present invention further provides a product or kit containing a container containing any of the stable pharmaceutical compositions described herein. In some embodiments, this is a glass vial.

The present invention further provides for the use of a pharmaceutical composition, or a lyophilized formulation, or a solution reconstituted from a lyophilized formulation, as described above, in formulating a medicament for the treatment of a PCSK9-mediated disease or disorder, wherein the disease or disorder is preferably a cholesterol-related disease; more preferably selected from the group consisting of hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, apoplexy, cardiovascular disease, Alzheimer's disease and general dyslipidemia; most preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention further provides a method for treating and preventing a PCSK-9 related disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition, or a lyophilized formulation, or a solution reconstituted from a lyophilized formulation, as described above, wherein the disease or disorder preferably is a disease associated with cholesterol; more preferably selected from the group consisting of hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, apoplexy, cardiovascular disease, Alzheimer's disease and general dyslipidemia; most preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention further provides a product containing a container containing a pharmaceutical composition, or a lyophilized formulation, or a solution reconstituted from a lyophilized formulation, as described above.

It should be understood that one, more or all features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to a person skilled in the art. These and other embodiments of the present invention are further described in the detailed description below.

DETAILED DESCRIPTION OF THE INVENTION

I. Terminology

To facilitate understanding of the present invention, some technical and scientific terms are specifically defined below. Except for terms specifically defined elsewhere in this document, all other technical and scientific terms used in this document should be considered to have the same meaning as is generally known to a person skilled in the art to which the present invention pertains.

Used in this document, the term "buffer" refers to a buffer solution that resists changes in pH due to the action of its components, which are acids and their conjugate bases. Examples of buffers that control pH within a suitable range include acetate buffer, succinate buffer, gluconate buffer, histidine buffer, oxalate buffer, lactate buffer, phosphate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, and other organic acid buffers. .

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include a histidine hydrochloride buffer, a histidine acetate buffer, a histidine phosphate buffer and a histidine sulfate buffer, etc., preferably a histidine hydrochloride buffer. The histidine hydrochloride buffer is prepared using histidine and hydrochloric acid or histidine and histidine hydrochloride.

"Citrate buffer" is a buffer that contains citrate ions. Examples of the citrate buffer include citric acid-sodium citrate buffer, citric acid-potassium citrate buffer, citric acid-calcium citrate buffer, and citric acid-magnesium citrate buffer, etc. The preferred citrate buffer is a buffer based on citric acid and sodium citrate.

"Succinate buffer" is a buffer that contains succinate ions. Examples of the succinate buffer include succinic acid-sodium succinate buffer, succinic acid-potassium succinate buffer, succinic acid-calcium succinate buffer, and so on. A preferred succinate buffer is a succinic acid-sodium succinate buffer.

"Phosphate buffer" is a buffer that contains phosphate ions. Examples of the phosphate buffer solution include a buffer based on disodium hydrogen phosphate and sodium dihydrogen phosphate and a buffer based on disodium hydrogen phosphate and potassium dihydrogen phosphate, etc. The preferred phosphate buffer is a buffer based on disodium hydrogen phosphate and sodium dihydrogen phosphate.

"Acetate buffer" is a buffer that contains acetate ions. Examples of the acetate buffer include acetic acid-sodium acetate buffer, acetic acid-histidine buffer, acetic acid-potassium acetate buffer, acetic acid-calcium acetate buffer, and acetic acid-magnesium acetate buffer, etc. d. The preferred acetate buffer is an acetic acid-sodium acetate buffer.

"Pharmaceutical composition" refers to a pharmaceutical composition containing a mixture of one or more compounds according to the present invention, or a physiologically/pharmaceutically acceptable salt or product thereof, with other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to maintain the stability of the active ingredient, which is an antibody, and allow the introduction into the body by promoting the absorption of the active ingredient and thereby the implementation of the biological effect. Used in this document, the terms "pharmaceutical composition" and "composition" are not mutually exclusive.

"Lyophilized formulation" means a formulation or pharmaceutical composition obtained by a vacuum freeze-drying step from a pharmaceutical composition in the form of a liquid or solution, or a formulation in the form of a liquid or solution.

Lyophilization of the present invention includes pre-freezing, primary drying and secondary drying. The purpose of pre-freezing is to freeze the product to obtain a crystalline solid. Pre-freezing temperature and pre-freezing speed are two important process parameters. In the present invention, the pre-freeze temperature is set to -45°C and the pre-freeze speed is set to 1°C/min. Primary drying is also known as freeze drying, which is the main lyophilization step. Its purpose is to maintain the shape of the product while removing ice from the product, and to minimize damage to the product. If the temperature and degree of vacuum of the first drying are not suitable, the product will be destroyed. Higher temperatures and higher vacuum levels increase the efficiency of lyophilization, but at the same time increase the risk of product degradation. The primary drying temperature of the present invention may be a standard temperature in the art, eg -27°C to 0°C, preferably -14°C to -5°C. Secondary drying is also known as desorption, which is the main step for removing bound water from the product by creating a deep vacuum (0.01 mbar) and increasing the temperature (20-40°C). Since most biological products are temperature sensitive, the secondary drying temperature is set at the lower end of the temperature range, i.e. 25°C. The lyophilization time depends on the freezer, the dosage of the lyophilized composition and the container of the lyophilized composition. Such adjustment of the lyophilization time is well known to those skilled in the art.

The pharmaceutical composition of the present invention is able to provide a stable effect: the antibody in the composition of the pharmaceutical composition essentially retains its physical stability and/or chemical stability and/or biological activity during storage, preferably, the pharmaceutical composition essentially retains its physical stability, chemical stability and biological activity during storage. The storage time is in most cases chosen based on the predetermined shelf life of the pharmaceutical composition. Currently, there are a number of analytical methods for measuring the stability of proteins, which measure the stability after storage for a selected period of time at a selected temperature.

A "stable" antibody pharmaceutical formulation is an antibody pharmaceutical formulation that does not show any significant change when stored at refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, and more preferably 1 year, and even more preferably up to 2 years. Additionally, a "stable" liquid formulation is one that exhibits the desired properties after storage for periods including 1 month, 3 months, or 6 months at temperatures including 25°C and 40°C. Typical acceptance criteria for determining stability are as follows: antibody monomer degradation, as measured by SEC-HPLC, is typically no more than about 10%, preferably about 5%. The antibody-based pharmaceutical formulation is colorless or clear to slightly opalescent when viewed visually. The concentration, pH and osmolality of the composition are characterized by an error of no more than +/-10%. Typically, a cut-off of no more than about 10%, preferably about 5%, is observed. Typically, no more than about 10%, preferably about 5%, is aggregated.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation and/or denaturation when assessed visually for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.

An antibody "retains chemical stability" in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by identifying and quantifying chemically altered forms of a protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or cutoff (assessed by methods such as size exclusion chromatography and SDS-PAGE, etc.), oxidation (assessed by methods such as peptide mapping combined with mass spectroscopy or MALDI /TOF/MS, etc.), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, measurement of isoaspartic acid, etc.) and isomerization (assessed by measurement of isoaspartic acid, peptide mapping, etc.).

An antibody "retains its biological activity" in a pharmaceutical composition if the value of the biological activity of the antibody at the considered point in time is in a predetermined range of biological activity values demonstrated at the time of preparation of the pharmaceutical composition. The biological activity of an antibody can be determined, for example, using an antigen binding assay.

As used herein, the one-letter code and the three-letter code for amino acids are as described in J. Biol. Chem, 243, (1968) p. 3558.

As used herein, the term "antibody" refers to an immunoglobulin, a four peptide chain structure in which two identical heavy chains are disulfide-linked to two identical light chains.

In the present invention, the antibody light chain referred to herein also contains a light chain constant region that provides for a human or mouse κ, λ chain, or a variant thereof.

In the present invention, the heavy chain of the antibody referred to herein also contains a heavy chain constant region that provides human or mouse IgG1, 2, 3, 4, or a variant thereof.

The variable region (Fv region) contains approximately 110 amino acids near the N-terminus of the heavy and light chain of an antibody. The constant region (C-region) containing the remaining amino acids near the C-terminus of the antibody is relatively stable. The variable region contains three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine the specificity of an antibody are also referred to as complementarity determining regions (CDRs). Each of the light chain variable regions (LCVR) and heavy chain variable regions (HCVR) consists of three CDR regions and four FR regions arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDRs are referred to as LCDR1, LCDR2 and LCDR3; the three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3. The number and location of CDR amino acid residues in the LCVR and HCVR regions of an antibody or antigen-binding fragment herein follow known Kabat numbering criteria (LCDR1-3, HCDE2-3) or follow Kabat and Chothia numbering criteria (HCDR1).

An antibody of the present invention includes a mouse antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody.

The term "mouse antibody" in the present invention refers to an anti-human PCSK9 monoclonal antibody produced in accordance with knowledge and skill in the art. Upon receipt, the subject to be tested is injected with the PCSK9 antigen, and then a hybridoma is isolated expressing an antibody that has the desired sequences or functional characteristics.

The term "chimeric antibody" means an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can attenuate the immune response elicited by mouse antibodies. To construct a chimeric antibody, a hybridoma is first created that secretes specific mouse monoclonal antibodies, then the variable region gene is cloned from mouse hybridoma cells. Then, if necessary, the human antibody constant region gene is cloned. The mouse variable region gene is ligated to a human constant region gene to form a chimeric gene, which is then inserted into a vector for the expression of human sequences, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. In a preferred embodiment of the present invention, the light chain of the chimeric anti-PCSK9 antibody further comprises light chain constant regions from human κ, λ, or a variant thereof. The heavy chain of the chimeric anti-PCSK9 antibody further comprises heavy chain constant regions from human IgG1, IgG2, IgG3 or IgG4 or variants thereof. The human antibody constant region can be selected from a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably a heavy chain constant region of human IgG2 or IgG4 or human IgG4 devoid of ADCC toxicity (antibody dependent cellular cytotoxicity) as a result of amino acid mutation.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody obtained by grafting mouse CDR sequences into a human antibody variable region framework, namely an antibody derived from different types of human antibody framework sequences. The humanized antibody overcomes the disadvantage of a strong antibody-mediated response induced by a chimeric antibody that carries a large amount of mouse protein components. Such framework sequences can be obtained from a public DNA database covering germline antibody gene sequences or published references. For example, the DNA sequences of the human germline heavy and light chain genes can be found in the VBase human germline gene sequence database (available at www.mrccpe.com.ac.uk/vbase) and also in Kabat, EA, et al, 1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid a decrease in activity with a decrease in immunogenicity, framework sequences in the variable region of a human antibody are subjected to minimal backmutations to maintain activity. The humanized antibody of the present invention also includes a humanized antibody for which CDR affinity maturation was achieved using phage display.

"Anti-PCSK-9 antibody" is an antibody that specifically binds to PCSK-9, including, without limitation, the h001 series of humanized anti-PCSK-9 antibodies from PCT/CN2016/111053. Meanwhile, in the case of the h001 series of humanized anti-PCSK-9 antibodies, mice immunized with human PCSK-9 were screened and converted by humanization.

"Antigen-binding fragment" in the present invention refers to a Fab fragment, Fab' fragment or F(ab')2 fragment having antigen-binding activity, as well as a scFv fragment that binds to human PCSK9, and other fragments capable of binding to PCSK9, which are formed by the VH and VL regions of antibodies that bind PCSK9; it contains one or more CDR regions selected from the group consisting of antibodies under SEQ ID NO: 12 - SEQ ID NO: 17 described in the present invention. Lacking a constant region, the Fv fragment contains a heavy chain variable region and a light chain variable region, which is the minimum antibody fragment that has all of the antigen-binding sites. Typically, the Fv of an antibody further comprises a polypeptide linker between the VH and VL domains and is capable of forming the structure necessary for antigen binding. Also, different linkers can be used to join the variable regions of two antibodies to form a polypeptide chain called a single chain antibody or single chain Fv (scFv). The term "binding to PCSK-9" in this invention means the ability to interact with human PCSK-9. The term "antigen-binding sites" in the present invention refers to continuous or discontinuous, three-dimensional sites on an antigen recognized by an antibody or antigen-binding fragment of the present invention.

Methods for preparing and performing purification of antibodies and antigen binding fragments are well known in the art and can be found, for example, in the Antibody Experimental Technology Guide of Cold Spring Harbor, sections 5-8 and 15. The antibody or antigen binding fragments of the present invention are subjected to genetic engineering. with the introduction of one or more human framework regions (FRs) into a CDR region derived from a non-human organism. Human germline FR sequences can be obtained from the ImMunoGeneTics (IMGT) human germline variable region gene database using IMGT's MOE software on their website or from The Immunoglobulin FactsBook, 2001ISBN012441351.

The engineered antibody or antigen-binding fragments of the present invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy chain and light chain can be cloned into a GS expression vector and recombined. The recombinant immunoglobulin expression vector can be stably transfected into the CHO cell line. As a more preferred method, well known in the art, mammalian cell-based expression systems provide antibody glycosylation, typically at a highly conserved N-terminus in the Fc region. Clones can be obtained that stably express antibodies that specifically bind to PCSK-9. Positive clones can be expanded in serum-free culture medium to produce antibodies in bioreactors. The culture medium into which the antibody has been secreted can be purified using conventional techniques. For example, purification can be performed using an FF column packed with Protein A or G Sepharose that has been equilibrated with an appropriate buffer. The column is washed to remove components with non-specific binding. Bound antibody is eluted with a pH gradient and then pooled and antibody fragments are determined by SDS-PAGE. The antibody can be filtered and concentrated using conventional techniques. Soluble aggregate and multimers can be effectively removed using conventional techniques, including size exclusion or ion exchange chromatography. The resulting product may be subjected to immediate freezing, for example at -70°C, or may be lyophilized.

"Conservative modifications" or "conservative substitution or substitution" refers to the substitution of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, conformation and backbone rigidity, etc.), whereby changes can often be made without changing the biological activity of the protein. It is known to those skilled in the art that, in general, a single amino acid substitution in non-essential regions of a polypeptide does not substantially alter the biological activity of the polypeptide (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub Co., ^p.224 (4th Ed.)). In addition, substitutions for structurally or functionally similar amino acids are less likely to impair biological activity.

"Identity" of amino acids refers to the sequence similarity between two protein sequences or between two polypeptide sequences. If a position in both of the two compared sequences is occupied by the same amino acid residue, then the molecules are the same at that position. Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al, (1977) Nucleic Acids Res. 25:3389-3402, respectively. BLAST analysis software is available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

"Administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to bringing an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition into contact with an animal, human, subject, cell, tissue, organ, or biological fluid. "Introduction" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Processing a cell encompasses bringing the reagent into contact with the cell, as well as bringing the reagent into contact with a liquid, where the liquid is in contact with the cells. "Introduction" and "treatment" also means in vitro and ex vivo treatment of, for example, a cell with a reagent, diagnostic agent, binding compound, or other cell. "Treatment" when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treat" means to administer a therapeutic agent, such as a composition containing any of the binding compounds of the present invention, internally or externally to a patient who has one or more symptoms of a disease for which the agent has known therapeutic activity. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated, so as to cause regression or suppress the progression of such symptom(s) to any clinically measurable degree. The amount of a therapeutic agent that is effective in reducing any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors such as the stage of the disease, the age, weight of the patient, and the ability of the drug to elicit the desired response in the patient. . Whether a symptom of a disease has improved can be assessed by any clinical measurement commonly used by physicians or other qualified healthcare professionals to assess the severity or progression status of that symptom. Although embodiments of the present invention (for example, a method of treatment or industrial products) may not be effective in reducing the symptom(s) of interest of the disease in each patient, it should reduce the target(s) of interest (- symptom(s) of the disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis test (H -test), the Jonkheer-Terpstra test and the Wilcoxon test.

An "effective amount" encompasses an amount sufficient to provide improvement in relation to a symptom or sign of a disease or to prevent them. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage protocol to avoid significant side effects or toxic effects.

"Tm value" refers to the thermal denaturation temperature of the protein, namely the temperature at which half of the protein unfolds and the spatial structure of the protein is destroyed at this time, therefore, the higher the Tm value, the higher the thermal stability of the protein.

"Substitution" refers to the replacement of a solvent system that dissolves antibody proteins. For example, a stable composition buffer system is used to replace a high salt solvent system or a hypertonic solvent system containing an antibody protein in physical modes of operation. While physical modes of operation include, without limitation, ultrafiltration, dialysis or recovery after centrifugation.

EXAMPLES AND TESTS

Hereinafter, the present invention is further described in detail with reference to the following examples, however, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

In examples of the present invention, where no specific conditions are described, the experiments are usually carried out under standard conditions, or under conditions suggested by the manufacturers of materials or products. Unless the source of the reagents is specifically indicated, the reagents are commercially available standard reagents.

EXAMPLES

Obtaining PCSK-9 antigen and anti-PCSK-9 antibody

Example 1 Preparation of PCSK9 antigen and test protein

Protein design and expression

UniProt subtilisin/kexin-type proprotein convertase 9 (human PCSK9, Uniprot number: Q8MBP7) was used as a template in designing the amino acid sequences of the PCSK9 antigen and test protein of the present invention. PCSK9 proteins were fused to different tags such as a His tag or an immunostimulatory peptide such as the PADRE peptide, then cloned into pTT5 vectors (Biovector, Cat #: 102762) or pTargeT vectors (Promega, A1410), respectively. PCSK9 proteins were then transiently expressed in 293 cells or stably expressed in CHO-S cells and purified. Finally, the antigen and test protein of the present invention were obtained.

His-tagged PCSK9, PCSK9-His6, used as an immunogen to immunize mice or a reagent to detect:

MGTVSSRRSWWPLPLLLLLLLLGPAGARA

SEQ ID NO: 1

Note: The underlined sequence is the signal peptide and the italicized part is the His tag (His6 tag) sequence.

PCSK9 with PADRE peptide and His tag, PCSK9-PADRE-His6, which is used as an immunogen containing PADRE peptide that can induce immunization:

MGTVSSRRSWWPLLLLLLLLGPAGARA GSGAKFVAAWTLKAAA HHHHHH

SEQ ID NO: 2

Note: The underlined sequence is the signal peptide, the double underlined sequence is the linker, the dotted line sequence is the PADRE peptide, and the italicized portion is the His6 tag.

A fusion protein based on His-tag and PCSK9 with a TEV cleavage site, PCSK9-TEV-His6, N-PCSK9 (PCSK9 N-terminal domain), can be obtained by TEV cleavage and can be used as an immunogen:

MGTVSSRRSWWPLPLLLLLLLLLGPAGARA QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTH ENLYFQ GAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQHHHHHH

SEQ ID NO: 3

Note: The underlined sequence is the signal peptide, the double underlined sequence is the TEV cleavage site, and the italicized portion is the His6 tag.

His-tagged mutant PCSK9-D374Y protein, PCSK9-D374Y-His6, which is used as test reagent:

MGTVSSRRSWWPLPLLLLLLLLGPAGARA

SEQ ID NO: 4

Note: The underlined sequence is the signal peptide and the italicized part is the His6 tag.

PCSK9 protein fused to biotin acceptor BP15 peptide and His tag, PCSK9-BP15-His6, as test reagent; the BP15 peptide can be biotinylated during expression, avoiding the need for in vitro biotin labeling and possible conformational changes.

MGTVSSRRSWWPLLLLLLLLGPAGARA GSTSGSGLNDIFEAQKIEWHE HHHHHH

SEQ ID NO: 5

Note: The underlined sequence is the signal peptide, the double underlined sequence is the biotin acceptor peptide, and the italicized part is the His6 tag.

PCSK9-Y is the abbreviation for the D374Y mutant PCSK9 protein fused to the BP15 biotin acceptor peptide and His tag, PCSK9-D374Y-BP15-His6, which is used as the test protein:

MGTVSSRRSWWPLLLLLLLLGPAGARA GSTSGSGLNDIFEAQKIEWHE HHHHHH

SEQ ID NO: 6

Note: The underlined sequence is the signal peptide, the double underlined sequence is the biotin acceptor peptide, and the italicized portion is the His6 tag.

The extracellular domain of the LDLR protein, which is a PCSK9 receptor, with a Flag tag and a His tag, LDLR-ECD-Flag-His6, used as a test reagent:

MGPWGWKLRWTVALLLAAAGTAVG DYKDDDDK HHHHHH

SEQ ID NO: 7

Note: The underlined sequence is the signal peptide, the double underlined sequence is the Flag tag, and the italicized portion is the His6 tag.

LCDR-Fc, LDLR truncated extracellular domain fusion protein with hIgG1 Fc (with PCSK9 binding activity), LDLR-sECD-Fc (hIgG1), used as test reagent:

MEFGLSWLFLVAILKGVQCGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACK EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 8

Note: The underlined sequence is the signal peptide, the double underlined sequence is the truncated LDLR extracellular domain with PCSK9 binding activity (LDLR-sECD), and the italicized part is hIgG1-Fc.

Highly truncated LDLR extracellular domain fusion protein with hIgG1 Fc fragment (with PCSK9 binding activity), LDLR-ssECD-Fc (hIgG1), used as test reagent:

MEFGLSWLFLVAILKGVQCGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDID EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 9

Note: The underlined sequence is the signal peptide, the double underlined sequence is the largely truncated LDLR extracellular domain with PCSK9 binding activity (LDLR-ssECD), and the italicized portion is hIgG1-Fc.

Example 2 Purification of Recombinant PCSK9 Proteins and Recombinant LDLR Proteins and Purification of Hybridoma Recombinant Antibodies

1. Purification steps for His-tagged recombinant proteins:

The supernatant samples from expressing cells were centrifuged at a high centrifugation speed and impurities were removed. The buffer was changed to PBS and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with 2-5 column volumes of a PBS solution containing 5 mM imidazole. After buffer exchange, the supernatant sample was loaded onto an immobilized metal ion affinity chromatography (IMAC) column. The column was washed with PBS containing 5 mM imidazole until the reading at A ₂₈₀ decreased to baseline. The chromatographic column was then washed with PBS+ 10 mM imidazole to remove non-specific binding proteins and the eluate was collected. The target protein was eluted with a PBS solution containing 300 mM imidazole and the eluted fractions corresponding to its peak were collected. The collected eluate was concentrated and further purified by Superdex 200 (GE) size exclusion chromatography using PBS as mobile phase. Fractions corresponding to the multimer peak were discarded and eluted fractions corresponding to the target protein peak were collected. The resulting proteins were identified by electrophoresis, peptide mapping and LC-MS. Got PCSK9-His6 (SEQ ID NO:1), PCSK9-PADRE-His6 (SEQ ID NO: 2), PCSK9-TEV-His6 (SEQ ID NO: 3), PCSK9-D374Y-His6 (SEQ ID NO: 4) , PCSK9-BP15-His6 (SEQ ID NO: 5) and PCSK9-D374Y-BP15-His6 (SEQ ID NO: 6) and used as immunogens or test protein of the present invention. PCSK9-TEV-His6 was purified and digested with the TEV enzyme. The TEV enzyme incompletely cleaved with PCSK9-TEV-His6 or the His-tagged C-terminal domain fragment was removed from the resulting product on an IMAC column. The IMAC eluate was concentrated to yield a PCSK9 segment with only the N-terminal domain (abbreviated as N-PCSK9) and used as an immunogen to immunize mice.

2. Purification steps of recombinant LDLR-ECD-Flag-His6 protein (SEQ ID NO: 7) with His tag and Flag tag

Samples were centrifuged at high speed to remove impurities and then concentrated to the required volume. The anti-Flag affinity chromatography column was equilibrated with 2-5 column volumes of 0.5×PBS buffer. After removal of impurities, the supernatant samples from the expressing cells were loaded onto the column. The column was washed with 0.5×PBS until the index at A ₂₈₀ decreased to baseline. The column was washed with PBS containing 0.3 M NaCl and contaminant proteins were eluted and collected. Target proteins were eluted with 0.1 M acetic acid (pH 3.5-4.0) and collected, followed by pH neutralization. The collected eluate was concentrated and further purified by Superdex 200 (GE) size exclusion chromatography using PBS as mobile phase. The fraction corresponding to the peak of the multimer was removed and the eluted fractions corresponding to the peak of the target protein were collected. The resulting proteins were identified by electrophoresis, peptide mapping and LC-MS and aliquoted for later use. LDLR-ECD-Flag-His6 (SEQ ID NO: 7) with FLAG/His6 tags was generated and used to test the performance of the antibody of the present invention.

3. Purification steps for Fc and LDLR based fusion protein

Supernatant samples from expressing cells were centrifuged at high speed to remove impurities, and then the samples were concentrated to volume and loaded onto a protein A _column . . Target proteins were eluted with 100 mM sodium acetate, pH 3.0 and then neutralized with 1 M Tris-HCl. The eluted samples were suitably concentrated and further purified by Superdex 200 (GE) size exclusion chromatography pre-equilibrated with PBS. Collected fractions corresponding to peaks without multimers. This method was used to purify LDLR-sECD-Fc (hIgG1) (SEQ ID NO: 8) and LDLR-ssECD-Fc (hIgG1) (SEQ ID NO: 9), both of which can be used to test the performance of anti-PCSK9 antibodies.

Example 3 Preparation of hybridoma monoclonal antibodies to human PCSK9

1. Immunization

Human monoclonal antibodies to PCSK9 were obtained by immunization of mice. Female SJL white mice, 6 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., livestock license number: SCXK (Beijing) 2012-0001) were used in this experiment and reared in the SPF laboratory. After purchase, the mice were kept in the laboratory for 1 week using a 12/12 hour light/dark cycle at 20-25°C and 40-60% humidity. Mice that have adapted to the environment, were immunized in accordance with the following two schemes, 6-10 individuals per group. The immunogens were His-tagged PCSK9-His6 (SEQ ID NO: 1), human PCSK9-PADRE-His6 (SEQ ID NO: 2) and N-PCSK9 (SEQ ID NO: 3).

Scheme A: emulsification with Freund's adjuvant (Sigma lot number: F5881/F5506). Complete Freund's adjuvant (CFA) was used for primary immunization and incomplete Freund's adjuvant (IFA) was used for booster immunization. The ratio of antigen to adjuvant was 1:1, 100 μg/mouse for primary immunization, and 50 μg/mouse for booster immunization. On day 0, each mouse was injected intraperitoneally (IP) with 100 μg of emulsified antigens, after primary immunization once every two weeks, for a total of 6-8 weeks.

Scheme B Mice were cross-immunized with Titermax (Sigma lot number: T2684) and alum (Thremo lot number: 77161). The ratio of antigen to adjuvant (Titermax) was 1:1 and the ratio of antigen to adjuvant (alum) was 3:1, 10-20 μg/mouse in the primary immunization, and 5 μg/mouse in the booster immunization. On day 0, each mouse was injected intraperitoneally (IP) with 20/10 μg of emulsified antigens, following weekly primary immunization, with Titermax and alum alternated for a total of 6-11 weeks. Four weeks after immunization, the antigen was injected dorsally or intraperitoneally, according to the state of swelling of the dorsal and abdominal region.

2. Cell fusion

Mice were selected for splenocyte fusion with high serum antibody titers (see trials 1 and 2, PCSK9 binding ELISA method) and with antibody titers trending towards a stable level. 72 hours before the fusion, selected mice were immunized by intraperitoneal injection of PCSK9-His6 at 10 μg/mouse. To obtain hybridoma cells, spleen lymphocytes and Sp2/0 myeloma cells (ATCC® CRL-8287™) were fused using an optimized PEG-mediated fusion procedure. Merged hybridoma cells were resuspended in complete HAT medium (RPMI-1640 medium containing 20% FBS, 1×HAT and 1×OPI) and then aliquoted into 96-well cell culture plates (1×10 ⁵ /150 μl/well) followed by incubation at 37°C and 5% CO ₂ . On day 5 post-fusion, complete HAT medium was added at 50 μl/well and incubated at 37° C. and 5% CO ₂ . From day 7 to day 8 post-fusion, depending on cell growth density, all medium was changed to complete HT medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI), 200 μl/well , and incubated at 37 C and 5% CO ₂ .

3. Screening for hybridoma cells

Between day 10 and day 11 post-fusion, depending on cell growth density, ELISA assays were performed to detect binding to PCSK9 or PCSK9-Y (see trials 1 and 2). Positive cells in the ELISA test were identified as those that blocked the binding of PCSK9 or PCSK9-Y to LDLR in the ELISA (see tests 3 and 4). The medium in the positive wells was changed and the cells were distributed into 24-well plates according to cell growth density. After retesting, the cell strains transferred to the 24-well plate were saved and their first subcloning was performed. Positive cells identified by screening after the first subcloning (see trials 1 and 2) were retained and subjected to their second subcloning. Positive cells identified by screening after the second subcloning (see trials 1 and 2) were maintained and allowed to express the protein. After several fusions, hybridoma cells capable of blocking PCSK9 or PCSK9-Y binding to LDLR were obtained.

Hybridoma clone mAb-001 was obtained by screening based on a blocking assay and a binding assay. Antibodies were further obtained by a serum-free cell culture method, and the antibody was purified according to the purification example for use in the test example.

The sequence of the variable region of the mouse antibody from hybridoma clone mAb-001 was as follows:

> mAb-001 VH

QVHLQQSGAELAKPGASVKLSCKASGYTFN DYWMH WVKERPGQGLEWIG YINPSSGFTKYHQNFKD KATLTADKSSSTAYMQLSSLTYDDSAVYYCAR QYDYDEDWYFDV WGTGTTVTVSS

SEQ ID NO: 10

> mAb-001VL

DIVMSQSPSSLAVSAGEKVTMSC KSSQSLLNSRTRKNFLA WYQQKPGQSPKLLIY WASTRES GVPDRFTGRGSGTDFTLTISSVQAEDLAVYYC KQSFNLFT FGSGTKLEIK

SEQ ID NO: 11

Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the italicized portion is the FR sequence and the underlined portion is the CDR sequence.

Table 1. Sequences of the CDR region of the heavy chain and light chain of the anti-PCSK-9 antibody mAb-001

Example 4 Humanization of an Anti-Human PCSK9 Hybridoma Monoclonal Antibody

1. Selection of the scaffold to be humanized for hybridoma clone mAb-001

By database alignment of the IMGT human germline heavy and light chain variable region genes using IMGT's MOE software, heavy and light chain variable region genes with high homology to mAb-001, respectively, were selected as templates. The CDRs of these two mouse antibodies were respectively grafted into the respective human antibody templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were numbered and annotated according to the Kabat numbering system.

The mAb-001 humanized light chain templates are IGKV1-39*01 and hjk2.1, and the humanized heavy chain templates are IGHV1-2*02 and hjh2. The sequence of the variable region of the humanized h001-1 humanized antibody is as follows:

> h001-1 VH

QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYWMH WVRQAPGQGLEWMG YINPSSGFTKYHQNFKD RVMTRDTSISTAYMELSRLRSDDTAVYYCAR QYDYDEDWYFDV WGQGTTVTVSS

SEQ ID NO: 18

> h001-1 VL

DIQMTQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNFLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC KQSFNLFT FGQGTKLEIK

SEQ ID NO: 24

Note: The order in the variable region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the italicized portion is the FR sequence and the underlined portion is the CDR sequence.

2. Template selection and backmutation design for hybridoma clone mAb-001 are shown in Table 2. Combinations of humanized sequences from hybridoma clones after backmutation are shown in Table 4.

Table 2. Template selection and backmutation design

Note: For example, according to the Kabat numbering system, S66D means that the S at position 66 was replaced by a D as a result of a backmutation.

"Grafted" means that the CDRs from the mouse antibody were grafted into the sequence of the human germline FR region, and the specific sequences of the mutated variable regions are shown in Table 3.

Table 3. Variable region sequences of each mutant

Note: The underlined portions of the sequences are the CDR regions.

Table 4 Combinations of humanized mAb-001 mouse antibody sequences

Note: The above table shows combinations of humanized antibody variable regions obtained by combining different sequences and mutated sequences based on them. For example, h001-1 indicates that the variable region of the humanized h001-1 antibody consists of the h001_VL1 light chain and the h001_VH.1A heavy chain, and so on.

3. The above humanized sequences were combined to form an antibody wherein the heavy chain constant region is derived from human IgG1 and the light chain constant region is derived from the human kappa chain. An appropriate humanized antibody was prepared, and the resulting anti-PCSK9 antibodies of the present invention had a relatively high binding activity for PCSK9 and PCSK9-, and could also effectively block the binding of PCSK9/PCSK9-Y to LDLR.

Example 5 Construction and expression of humanized anti-human PCSK9 antibodies in IgG1 and IgG1-YTE formats

A method for constructing and expressing humanized anti-human PCSK9 antibodies includes the following.

1. Development of primers. Several primers were designed using the online DNAWorks software (v3.2.2, http://helixweb.nih.gov/dnaworks/) to synthesize VH/VK-containing gene fragments required for recombination: a 5'-signal peptide of length - 30 b.p. + VH/VK +CH1/CL 30 bp long - 3'. The principle of primer design is as follows: if target gene 2 differs from target gene 1 by 2 amino acids, design another primer specific to the mutation site as shown in FIG. one.

2. Fragment splicing: According to TaKaRa's PrimeSTAR GXL DNA polymerase manual, two-step PCR amplification was carried out with several primers designed above, and gene fragments containing VH/VK required for recombination were obtained.

3. Construction of pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)) and enzymatic digestion with restriction enzymes.

The pHr expression vector (with signal peptide and constant region gene fragment (CH1-FC/CL)) was designed and constructed using the properties of certain site-specific restriction enzymes, such as BsmBI, whose recognition sequence differs from the cleavage site, as shown in FIG. . 2. The vector was digested with BsmBI and the sticky ends were reconstituted for later use.

4. Construction of the recombinant expression vector VH-CH1-FC-pHr/VK-CL-pHr.

VH/VK containing the gene fragments required for recombination and the restored expression vector pHr (with the signal peptide and the constant region gene fragment (CH1-FC/CL)) were digested with the BsmBI enzyme and added to competent DH5 alpha cells in a ratio of 3:1 , incubated in an ice bath at 0°C for 30 min., and subjected to heat shock for 90 seconds at 42°C. Then a 5-fold volume of LB medium was added, incubated at 37°C for 45 minutes, added to an LB-Amp plate, and cultured at 37°C overnight. Individual clones were selected and sent for sequencing to obtain target clones.

The antibody of this invention can be designed and constructed in accordance with the above method, but is not limited to them. Taking h001-4 as an example, the antibody and variants thereof have been designed to produce: 1) h001-4-WT: h001-4 in IgG format, ie. a combination of a humanized h001-4 sequence combining a heavy chain constant region derived from human IgG1 with a light chain constant region derived from a human kappa chain; 2) h001-4-YTE: h001-4 in IgG1-YTE format, i.e. a combination of a humanized h001-4 sequence combining a mutant human IgG1 heavy chain constant region (YTE mutation) with a light chain constant region derived from the human kappa chain. Mutant human IgG1 may also include forms with other mutations.

The sequences of engineered and expressed humanized anti-human PCSK9 antibodies (in their IgG1 and IgG1-YTE formats) are as follows.

H001-4 in IgG1 format, its heavy chain constant region is derived from human IgG1, and its light chain constant region is derived from human kappa light chain.

The amino acid sequence of the heavy chain (human IgG1) is as follows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGYINPSSGFTKYHQNFKDRVTMTRDTSISTAYMELSRLRSDDTAVYYCARQYDYDEDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 28

The heavy chain DNA sequence is as follows:

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCCAGTGC

SEQ ID NO: 29

h001-4-kappa

The amino acid sequence of the light chain is as follows:

DIVMSQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNFLAWYQQKPGKSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCKQSFNLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKTHNKSSVY

SEQ ID NO: 30

The light chain DNA sequence is as follows:

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATTGC

SEQ ID NO: 31

h001-4-IgG1-YTE light chain is h001-4-kappa: SEQ ID NO: 30)

The amino acid sequence of the IgG1-YTE heavy chain is as follows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGYINPSSGFTKYHQNFKDRVTMTRDTSISTAYMELSRLRSDDTAVYYCARQYDYDEDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 32

The heavy chain DNA sequence is as follows:

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCCAGTGC

SEQ ID NO: 33

Note: The underlined sequence is the signal peptide DNA sequence.

A typical process for obtaining a pharmaceutical composition (composition) based on an antibody

Step 1: A sample of purified anti-PCSK-9 antibody (such as h001-4-YTE) is taken and the solvent is replaced (preferably by ultrafiltration) with 6 times the volume of buffer containing no antibody (such as 10 mM histidine hydrochloride buffer). , pH 6.0) with an ultrafiltration membrane until the protein was concentrated to 60 mg/mL (±2 mg/mL). A volume of sucrose stock solution was added and mixed until a final sucrose concentration of 25 mg/mL was reached. A volume of Tween-80 stock solution was added and mixed until a final Tween-80 concentration of 0.2 mg/mL was reached. Added 10 mm histidine buffer (pH 6.0) to bring the protein to a concentration of 50 mg/ml (±5 mg/ml). The pH value of the final product was approximately 6.3 ± 0.1 (other formulations to be tested or stable formulations should be prepared in the same way).

After the product had been filtered, in-process control samples were taken to determine its sterility. The drug was passed through a 0.22 µm PVDF filter element and the filtrate was collected.

Stage 2. The loading volume was adjusted to 3.6 ml, the filtrate was introduced into 6 ml vials, covered with a stopper, the loading difference was determined by taking samples for control during production at the beginning, middle and end of the filling stage, respectively.

Step 3: After filling and capping, the solution was placed in a lyophilization chamber and lyophilized. The lyophilization process included pre-freezing, primary drying and secondary drying. After the lyophilization process was completed, the lyophilized powder was sealed under vacuum.

The lyophilization time can be adjusted depending on the specific situation. The type of lyophilizer, the loading volume of the lyophilized drug, and the container that contains the lyophilized drug will affect the lyophilization time. Such timing is widely known to those skilled in the art.

Step 4: The capping machine was turned on, aluminum caps were added, and thus capping was performed.

Stage 5. Visual inspection was used to confirm that the product did not have such defects as destruction and inaccurate loading volume. Printed and pasted the label on the bottle; they printed a label on the box, the box was folded, packed and a label was stuck on the box.

Example 6 Buffer System Screening

Received samples of the composition based on antibodies to PCSK9 with a protein concentration of 1 mg/ml with the following buffer solutions:

1) 20 mM citric acid-sodium citrate, pH 4.0;

2) 20 mM citric acid-sodium citrate, pH 4.5;

3) 20 mM citric acid-sodium citrate, pH 5.0;

4) 20 mM citric acid-sodium citrate, pH 5.5;

5) 20 mM citric acid-sodium citrate, pH 6.0;

6) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.0;

7) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.5;

8) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.0;

9) 20 mM sodium dihydrogen phosphate disodium hydrogen phosphate, pH 7.5;

10) 20 mM Tris-HCl, pH 7.5;

11) 20 mM Tris-HCl, pH 8.0;

12) 20 mM Tris-HCl, pH 8.5.

The thermal stability of the anti-PCSK9 antibody in each formulation sample was determined by differential scanning calorimetry (DSC). Analysis of the mean thermal denaturation temperature (Tm) of the anti-PCSK-9 antibody showed that it exhibited greater thermal stability at pH ≥ 6.0. The results are shown in Table 5. Therefore, buffers with pH values of 6.0-6.5, such as histidine hydrochloride buffer, sodium dihydrogen phosphate and disodium hydrogen phosphate buffer, succinic acid and sodium succinate buffer, and buffer based on citric acid and sodium citrate.

Table 5. Screening results using DSC antibody H001-4-YTE in different buffer systems and at different pH values

Note: n.p. means that the ingredient was not added.

Received samples of the composition based on antibodies to PCSK9 with a protein concentration of 1 mg/ml with the following buffer solutions:

1) 20 mM histidine hydrochloride, pH 6.0;

2) 20 mM histidine hydrochloride, pH 6.5;

3) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.0;

4) 20 mM sodium dihydrogen phosphate disodium hydrogen phosphate, pH 6.5;

5) 20 mM succinic acid-sodium succinate, pH 6.0;

6) 20 mM citric acid-sodium citrate, pH 6.0;

7) 20 mM citric acid-sodium citrate, pH 6.5.

The thermal stability of the anti-PCSK9 antibody in each formulation sample was determined by differential scanning calorimetry (DSC). Analysis of the mean thermal denaturation temperature (Tm) of the anti-PCSK-9 antibody showed (see Table 6) that the anti-PCSK-9 antibody was slightly more stable in the histidine, phosphate and succinate buffer systems than in the citrate buffer system.

Table 6. Screening results using DSC antibody H001-4-YTE in different buffer systems and at different pH values

Note: n.p. means that the ingredient was not added.

Example 7 Screening for Sugars in a Formula

Received samples of the composition based on antibodies to PCSK9 with a protein concentration of 150 mg/ml with the following buffer solutions:

1) 20 mM histidine hydrochloride, 70 mg/ml sucrose, pH 6.5;

2) 20 mM histidine hydrochloride, 70 mg/ml α,α-trehalose dihydrate, pH 6.5.

Each formulation was filtered and filled into 2 ml vials, 0.5 ml per vial, and the vial was stoppered. Samples were alternately exposed to high temperature 40°C, low temperature, light and freeze-thaw cycles (samples were placed at -20°C for 24 hours, and then removed and placed at room temperature until completely thawed and stirred sample, which was one cycle). The results showed that sucrose and trehalose had a similar effect on the stability of the PCSK-9 antibody. Sucrose was chosen as a stabilizer for the anti-PCSK-9 antibody, the results are shown in Table 7.

Table 7. Results of accelerated experiment for H001-4-YTE with different sugars

Received samples of the composition based on antibodies to PCSK9 with a protein concentration of 150 mg/ml with the following buffer solutions:

1) 20 mM histidine hydrochloride, 0 mg/ml sucrose, pH 6.5;

2) 20 mM histidine hydrochloride, 10 mg/ml sucrose, pH 6.5;

3) 20 mM histidine hydrochloride, 40 mg/ml sucrose, pH 6.5;

4) 20 mM histidine hydrochloride, 70 mg/ml sucrose, pH 6.5.

The osmotic pressure value indicates that the osmotic pressure meets the minimum requirement for subcutaneous injection if the sucrose concentration is ≥70 mg/mL. The anti-PCSK-9 antibody formulation of the present invention was prepared by a method involving lyophilization at low concentration and reconstitution at high concentration (3 times the volume of the stock solution was lyophilized, one volume of water for injection was used for reconstitution, and unless otherwise indicated, in the present invention the above ratio is taken to perform lyophilization). If the sucrose concentration in the final formulation (reconstituted formulation) is 75 mg/mL, this not only meets the injection osmotic pressure requirement, but also makes it easier to obtain that sucrose concentration from the stock solution concentration of 25 mg/mL, and still sucrose concentration of 25 mg/ml is easily lyophilized.

Table 8. The results of determining the ratio of saccharide concentration and osmotic pressure for the composition based on H001-4-YTE

Note: n.p. means that the ingredient was not added.

Example 8 Screening for Surfactants in Formulations

Received samples of the composition based on antibodies to PCSK9 with a protein concentration of 150 mg/ml with the following buffer solutions:

1) 20 mM histidine hydrochloride, pH 6.5;

2) 20 mM histidine hydrochloride, 0.2 mg/ml polysorbate 20, pH 6.5;

3) 20 mM histidine hydrochloride, 0.4 mg/ml polysorbate 80, pH 6.5.

A sample of each formulation was filtered and filled into 2 ml vials at 0.5 ml per vial, and the vial was stoppered. The samples were placed in a shaker maintained at a constant temperature of 25°C and shaken at 300 rpm. The results of the appearance analysis showed that the surfactants were effective in preventing the aggregation of anti-PCSK-9 antibodies. For H001-4-YTE, no significant differences were observed between Polysorbate 20 and Polysorbate 80. Polysorbate 80 was chosen as the formulation stabilizer based on the anti-PCSK-9 antibody.

Table 9. Effect of surfactant on aggregation of H001-4-YTE with shaking at 300 rpm, 25°C

Example 9 Screening and confirmation of the buffer system in the composition

Formulation samples containing 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 50 mg/ml antibody were prepared using a buffer solution containing 20 mM histidine hydrochloride or 20 mM succinic acid-sodium succinate at pH 6.0 or 6.5. Each formulation was filtered and filled into 6 ml neutral borosilicate glass vials, 3.6 ml per lyophilization vial, and the vial was stoppered with the halogenated butyl rubber stopper used for the lyophilized sterile powder. The lyophilized product was stored at 2-8°C, 25°C and 40°C and then reconstituted with water for injection for stability analysis. The results showed that the PCSK9 antibody was very stable at 2-8°C and 25°C. However, the appearance of the reconstituted composition on day 0 showed that the succinic acid buffer at pH 6.0 had a significant level of opalescence compared to the His-based system (not shown in the table), so the histidine system was chosen as the buffer system for the antibody to PCSK-9.

Table 10. Stability of H001-4-YTE Lyophilized Powder at Different Temperatures

Example 10 Comprehensive Screening of Composition Components

Anti-PCSK-9 formulation samples containing 50 mg/mL of anti-PCSK-9 antibody and 25 mg/mL of sucrose were prepared with the following buffers with different pH values, different ionic strengths and different concentrations of surfactants:

1) 10 mM histidine hydrochloride, 0.1 mg/ml polysorbate 80, pH 6.5;

2) 10 mM histidine hydrochloride, 0.3 mg/ml polysorbate 80, pH 6.0;

3) 10 mM histidine hydrochloride, 0.2 mg/ml polysorbate 80, pH 5.5;

4) 15 mM histidine hydrochloride, 0.3 mg/ml polysorbate 80, pH 5.5;

5) 10 mM histidine hydrochloride, 0.2 mg/ml polysorbate 80, pH 6.0;

6) 15 mM histidine hydrochloride, 0.3 mg/ml polysorbate 80, pH 6.5;

7) 15 mM histidine hydrochloride, 0.2 mg/ml polysorbate 80, pH 6.0;

8) 5 mM histidine hydrochloride, 0.3 mg/ml polysorbate 80, pH 5.5;

9) 15 mM histidine hydrochloride, 0.1 mg/ml polysorbate 80, pH 5.5;

10) 5 mM histidine hydrochloride, 0.1 mg/ml polysorbate 80, pH 6.0;

11) 5 mM histidine hydrochloride, 0.2 mg/ml polysorbate 80, pH 6.5.

A sample of each formulation was filtered and filled into 6 ml neutral borosilicate glass vials of 3.6 ml per lyophilization vial, and the vial was stoppered with the halogenated butyl rubber stopper used for the lyophilized sterile powder. The lyophilized product was stored at 40° C. for stability analysis. Analysis data based on SEC monomer composition differences showed that PCSK-9 antibody formulated with 10 mM histidine hydrochloride buffer pH 6.0, 0.2 mg/mL polysorbate 80 was more stable at a protein concentration of 50 mg/ml and a sucrose concentration of 25 mg/ml. After lyophilization and reconstitution, the concentration of each component of the final formulation (150 mg/ml protein, 75 mg/ml sucrose, 30 mM histidine hydrochloride buffer and 0.6 mg/ml polysorbate 80) was determined as three times the drug concentration before lyophilization, pH 6.3 ± 0.1.

Table 11. Results of the experiment with exposing H001-4-YTE to high temperature

Example 11 Optimizing the Primary Drying Temperature

Samples of an anti-PCSK9 antibody formulation with 50 mg/ml anti-PCSK9 antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80 formulation were prepared using 10 mM histidine hydrochloride buffer at pH 6.0. The antibody was added to a 6 ml vial at 3.6 ml per vial and lyophilized at a primary drying temperature of −14° C. and −5° C., respectively, and the vial was stoppered for lyophilization. The reconstituted samples were compared before and after lyophilization. The results showed that -5°C is the preferred temperature for primary drying in lyophilization.

Table12. Comparison of H001-4-YTE samples obtained using different drying methods before and after lyophilization.

Example 12 Determining Compatibility Between Formulation and Containers of Different Materials

H001-4-YTE was obtained at a concentration of 50 mg/ml in a solution containing 10 mM histidine hydrochloride, pH 6.0, with 25 mg/ml sucrose and 0.2 mg/ml polysorbate 80. Samples of the composition were poured into glass flasks, bags for storing liquids and stainless steel containers with a capacity of 316 liters and left at 2-8°C for 24 hours. Analysis of the content and purity of the protein showed that H001-4-YTE is stable for 24 hours. The formulation was compatible with a 316 L stainless steel container, glass bulb and liquid storage bag.

Table 13. Stability of H001-4-YTE in contact with various materials

Example 13 - Determination of the compatibility of the composition with various filters

Received H001-4-YTE at a concentration of 50 mg/ml in a solution containing 10 mM histidine hydrochloride buffer, pH 6.0, with 25 mg/ml sucrose and 0.2 mg/ml polysorbate 80. A sample of the composition was passed through PES -filters and PVDF filters with a pore size of 0.22 μm, and samples were collected after 30 min. and after 1 hour for testing. Analysis of protein content, appearance and purity showed that H001-4-YTE was stable for 1 hour from contact with the filter. The formulation is compatible with both PES and PVDF filters.

Table 14. Stability of H001-4-YTE with Different Filter Membrane Materials

Example 14 Other Compounds Alternative to This Compound

The pharmaceutical composition of the present invention can be used as a stock solution for preparing a pharmaceutical formulation or directly as an injection solution. When the pharmaceutical composition of the present invention is used as a stock solution for preparing a pharmaceutical preparation, a lyophilized formulation is obtained by a lyophilization method and reconstituted in an injection solution for clinical use. The lyophilized formulation of the present invention is prepared according to a method of low concentration lyophilization and high concentration reconstitution, i.e., a low concentration pharmaceutical formulation solution is lyophilized to obtain a lyophilized formulation, and the lyophilized formulation is reconstituted to obtain a pharmaceutical composition with a higher concentration for clinical use. applications. The lyophilized formulation has greater storage stability than the liquid formulation. The higher the concentration of each component of the initial solution for obtaining a pharmaceutical composition used to obtain a lyophilized preparation, the longer the lyophilization time. When considering the performance of the lyophilization process as a whole, the concentration of each component of the stock solution for obtaining a pharmaceutical composition after reconstitution is usually 2-5 times the concentration of the component in the injection solution before reconstitution, and in the preferred embodiment of the present invention, the excess is 3-fold.

The stable pharmaceutical formulation of the present invention comprises an anti-PCSK-9 antibody protein (non-limiting embodiment such as h001-4-YTE) in combination with any of the following stable buffers:

(1) 150 mg/ml h001-4-YTE antibody, 75 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 30 mM histidine hydrochloride buffer, pH 6.4;

(2) 150 mg/ml h001-4-YTE antibody, 75 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 30 mM histidine hydrochloride buffer, pH 6.2;

(3) 150 mg/ml h001-4-YTE antibody, 75 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 30 mM histidine hydrochloride buffer, pH 6.3;

(4) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.4;

(5) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.3;

(6) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.2;

(7) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, final pH 6.1;

(8) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.0;

(9) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.5;

(10) 50 mg/ml h001-4 antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.3;

(11) 50 mg/ml h001-4 antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.2;

(12) 50 mg/ml h001-4 antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.4;

(13) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.1 mg/ml polysorbate 80, and 20 mM histidine hydrochloride buffer, pH 6.3;

(14) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 15 mM histidine hydrochloride buffer, pH 6.2;

(15) 50 mg/ml h001-4-YTE antibody, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 20 mM histidine hydrochloride buffer, pH 6.4.

(16) 30 mg/ml h001-4-YTE antibody, 10 mg/ml sucrose, 0.05 mg/ml polysorbate 80, and 5 mM histidine hydrochloride buffer, pH 5.5;

(17) 70 mg/ml h001-4-YTE antibody, 75 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 30 mM histidine hydrochloride buffer, pH 6.5;

(18) 45 mg/ml h001-4-YTE antibody, 20 mg/ml sucrose, 0.1 mg/ml polysorbate 80, and 8 mM histidine hydrochloride buffer, pH 6.0;

(19) 55 mg/ml h001-4-YTE antibody, 40 mg/ml sucrose, 0.3 mg/ml polysorbate 80, and 15 mM histidine hydrochloride buffer, pH 6.2.

The pharmaceutical composition of the present invention can be obtained in the form of an appropriate lyophilized formulation using a lyophilization process, and the lyophilized formulation can be reconstituted with water for injection to obtain the following pharmaceutical composition for clinical use:

(1) 150 mg/ml anti-PCSK-9 protein, 75 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 20 mM histidine hydrochloride buffer, pH 6.3 ± 0.1;

(2) 120 mg/ml anti-PCSK-9 protein, 55 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 10 mM histidine hydrochloride buffer, pH 6.0;

(3) 200 mg/ml anti-PCSK-9 protein, 95 mg/ml sucrose, 0.8 mg/ml polysorbate 80, and 30 mM histidine hydrochloride buffer, pH 6.5.

While specific embodiments of the present invention have been described above, those skilled in the art will appreciate that they are illustrative only. Many changes or modifications can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, the scope of the present invention is defined by the appended claims.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO.,LTD; SHANGHAI HENGRUI

PHARMACEUTICAL CO.,LTD

<120> Pharmaceutical composition containing antibodies to PCSK-9, and its

application

<130> P19413698EN

<150> CN201710519829.6

<151> 2017-06-30

<160> 33

<170> PatentIn version 3.5

<210> 1

<211> 698

<212> PROTEIN

<213> Artificial sequence

<220>

<223> His-tagged PCSK9, PCSK9-His6, used as an immunogen for

immunization of mice or used as a test reagent

<400> 1

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln His His His His His His His

690 695

<210> 2

<211> 714

<212> PROTEIN

<213> Artificial sequence

<220>

<223> PCSK9 with PADRE peptide and His tag, PCSK9-PADRE-His6, applied

as an immunogen, where the PADRE peptide it contains can

induce immunization

<400> 2

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Gly Ala Lys Phe Val Ala Ala Trp Thr Leu

690 695 700

Lys Ala Ala Ala His His His His His His His

705 710

<210> 3

<211> 704

<212> PROTEIN

<213> Artificial sequence

<220>

<223> PCSK9 fusion protein with TEV cleavage site and

His-tag, PCSK9-TEV-His6, N-PCSK9 (N-terminal domain of PCSK9),

used as an immunogen, which can be obtained by

splitting by TEV

<400> 3

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Glu Asn Leu Tyr Phe Gln Gly Ala Gly Trp Gln Leu Phe Cys Arg

450 455 460

Thr Val Trp Ser Ala His Ser Gly Pro Thr Arg Met Ala Thr Ala Val

465 470 475 480

Ala Arg Cys Ala Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser

485 490 495

Arg Ser Gly Lys Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys

500 505 510

Leu Val Cys Arg Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala

515 520 525

Ile Ala Arg Cys Cys Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr

530 535 540

Ala Pro Pro Ala Glu Ala Ser Met Gly Thr Arg Val His Cys His Gln

545 550 555 560

Gln Gly His Val Leu Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp

565 570 575

Leu Gly Thr His Lys Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn

580 585 590

Gln Cys Val Gly His Arg Glu Ala Ser Ile His Ala Ser Cys Cys His

595 600 605

Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro

610 615 620

Gln Glu Gln Val Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly

625 630 635 640

Cys Ser Ala Leu Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val

645 650 655

Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val Ser Thr Thr Gly Ser

660 665 670

Thr Ser Glu Gly Ala Val Thr Ala Val Ala Ile Cys Cys Arg Ser Arg

675 680 685

His Leu Ala Gln Ala Ser Gln Glu Leu Gln His His His His His His

690 695 700

<210> 4

<211> 698

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutant protein PCSK9-D374Y with His tag, PCSK9-D374Y-His6,

used as test reagent

<400> 4

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Tyr Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln His His His His His His His

690 695

<210> 5

<211> 719

<212> PROTEIN

<213> Artificial sequence

<220>

<223> PCSK9 protein fused to BP15 biotin acceptor peptide,

and His-tag

<400> 5

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Thr Ser Gly Ser Gly Leu Asn Asp Ile Phe

690 695 700

Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His His

705 710 715

<210> 6

<211> 719

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutant PCSK9 D374Y protein fused to BP15 peptide,

being a biotin acceptor, and His-tag, PCSK9-D374Y-BP15-His6,

as test protein

<400> 6

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Tyr Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Thr Ser Gly Ser Gly Leu Asn Asp Ile Phe

690 695 700

Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His His

705 710 715

<210> 7

<211> 802

<212> PROTEIN

<213> Artificial sequence

<220>

<223> The extracellular domain of the LDLR protein, which is the PCSK9 receptor,

with Flag-tag and His-tag, LDLR-ECD-Flag-His6, as

test reagent

<400> 7

Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu

1 5 10 15

Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe

20 25 30

Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly

35 40 45

Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu

50 55 60

Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn

65 70 75 80

Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp

85 90 95

Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp

100 105 110

Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys

115 120 125

Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro

130 135 140

Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys

145 150 155 160

Ile Pro Gln Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly

165 170 175

Ser Asp Glu Trp Pro Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly

180 185 190

Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu

195 200 205

Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp

210 215 220

Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu

225 230 235 240

Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp

245 250 255

Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn

260 265 270

Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu

275 280 285

Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp

290 295 300

Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp

305 310 315 320

Asn Asn Gly Gly Cys Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr

325 330 335

Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys

340 345 350

Glu Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys

355 360 365

Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln

370 375 380

Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr

385 390 395 400

Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met Thr Leu Asp Arg

405 410 415

Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu

420 425 430

Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln

435 440 445

Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser

450 455 460

Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala

465 470 475 480

Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly

485 490 495

Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe

500 505 510

Arg Glu Asn Gly Ser Lys Pro Arg Ala Ile Val Val Asp Pro Val His

515 520 525

Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys

530 535 540

Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile

545 550 555 560

Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr

565 570 575

Trp Val Asp Ser Lys Leu His Ser Ile Ser Ser Ile Asp Val Asn Gly

580 585 590

Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro

595 600 605

Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp Thr Asp Ile Ile

610 615 620

Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn

625 630 635 640

Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met Val Leu Phe His

645 650 655

Asn Leu Thr Gln Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu

660 665 670

Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn

675 680 685

Pro His Ser Pro Lys Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu

690 695 700

Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala

705 710 715 720

Thr Gln Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val

725 730 735

Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu

740 745 750

Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser

755 760 765

His Gln Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro

770 775 780

Ser Ser Val Arg Asp Tyr Lys Asp Asp Asp Asp Lys His His His His

785 790 795 800

His His

<210> 8

<211> 331

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR-Fc, LDLR truncated extracellular domain fusion protein

with Fc hIgG1 (with PCSK9 binding activity), LDLR -sECD CFc

(hIgG1), as test reagent

<400> 8

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser

20 25 30

His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp

35 40 45

Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu Cys

50 55 60

Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly Gly

65 70 75 80

Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys

85 90 95

Ala Cys Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

100 105 110

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

115 120 125

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

130 135 140

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

145 150 155 160

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

165 170 175

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

180 185 190

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

195 200 205

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

210 215 220

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

225 230 235 240

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

245 250 255

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

260 265 270

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

275 280 285

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

290 295 300

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

305 310 315 320

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 9

<211> 294

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fusion protein based on even more truncated LDLR extracellular domain

with Fc hIgG1 (with PCSK9 binding activity), LDLR-ssECD CFc

(hIgG1), as test reagent

<400> 9

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser

20 25 30

His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp

35 40 45

Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu Pro

50 55 60

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

65 70 75 80

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

85 90 95

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

100 105 110

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

115 120 125

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

130 135 140

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

145 150 155 160

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

165 170 175

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

180 185 190

Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr Lys Asn

195 200 205

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

210 215 220

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

225 230 235 240

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

245 250 255

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

260 265 270

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

275 280 285

Ser Leu Ser Pro Gly Lys

290

<210> 10

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 10

Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Lys Glu Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Tyr Asp Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Thr Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 11

<211> 112

<212> PROTEIN

<213> Mus musculus

<400> 11

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 12

<211> 5

<212> PROTEIN

<213> Mus musculus

<400> 12

Asp Tyr Trp Met His

fifteen

<210> 13

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 13

Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe Lys

1 5 10 15

asp

<210> 14

<211> 12

<212> PROTEIN

<213> Mus musculus

<400> 14

Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val

1 5 10

<210> 15

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 15

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Phe Leu

1 5 10 15

Ala

<210> 16

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 16

Trp Ala Ser Thr Arg Glu Ser

fifteen

<210> 17

<211> 8

<212> PROTEIN

<213> Mus musculus

<400> 17

Lys Gln Ser Phe Asn Leu Phe Thr

fifteen

<210> 18

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ch-001

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 19

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VH.1A

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 20

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VH.1B

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 21

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VH.1C

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 22

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VH.1D

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 23

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VH.1E

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ch-001 hVL.1 (with grafted CDRs)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VL.1A

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 26

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VL.1B

<400> 26

Asp Ile Gln Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-VL.1C

<400> 27

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 28

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-4-IgG1

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 29

<211> 1413

<212> DNA

<213> Artificial sequence

<220>

<223> h001-4-IgG1

<400> 29

atggagtttg ggctgagctg gctttttctt gtcgcgattc ttaagggtgt ccagtgccag 60

gtgcagctgg tgcagagcgg cgctgaggtg aagaagcccg gagcgagcgt aaaggtgagc 120

tgcaaggcca gcggatacac cttcaccgac tactggatgc actgggtgag gcaggcccca 180

ggacagggcc tggagtggat gggctacatc aaccccagca gcggctttac caagtatcac 240

cagaacttca aagacagggt gaccatgacc agggacacca gcatcagcac cgcctacatg 300

gagctgagca ggctgaggag cgacgacacc gccgtgtact actgcgccag gcaatacgac 360

tacgacgagg actggtactt cgacgtgtgg ggccaaggaa ccaccgtgac tgtgagcagc 420

gcttcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcggggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tcggggtaaa tga 1413

<210> 30

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> h001-4-kappa

<400> 30

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> h001-4-kappa

<400> 31

atggacatgc gcgtgcccgc ccagctgctg ggcctgctgc tgctgtggtt ccccggctcg 60

cgatgcgaca tcgtgatgtc tcagagccca tctagcctga gcgccagcgt gggcgacagg 120

gtaaccatca cctgcaagag cagccaaagc ctgctgaaca gcaggacccg caagaacttc 180

ctggcttggt atcagcagaa gcccggcaag tctcccaagt tgctgatcta ctgggccagc 240

accagggaga gcggcgtgcc cgacaggttc agcggctccg gcagcggcac cgacttcacc 300

ctgaccatct ctagtctgca gcccgaggac ttcgccacct actactgcaa gcagagcttc 360

aatctgttca ccttcggcca gggcaccaag ctggagatca agcgtacggt ggctgcacca 420

tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 480

tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 540

ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 600

agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 660

tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag 720

tgttga 726

<210> 32

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain h001-4-IgG1-YTE

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr

245 250 255

Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 33

<211> 1413

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain h001-4-IgG1-YTE

<400> 33

atggagtttg ggctgagctg gctttttctt gtcgcgattc ttaagggtgt ccagtgccag 60

gtgcagctgg tgcagagcgg cgctgaggtg aagaagcccg gagcgagcgt aaaggtgagc 120

tgcaaggcca gcggatacac cttcaccgac tactggatgc actgggtgag gcaggcccca 180

ggacagggcc tggagtggat gggctacatc aaccccagca gcggctttac caagtatcac 240

cagaacttca aagacagggt gaccatgacc agggacacca gcatcagcac cgcctacatg 300

gagctgagca ggctgaggag cgacgacacc gccgtgtact actgcgccag gcaatacgac 360

tacgacgagg actggtactt cgacgtgtgg ggccaaggaa ccaccgtgac tgtgagcagc 420

gcttcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tctacatcac ccgggagcct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcggggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tcggggtaaa tga 1413

<---

Claims (60)

1. Pharmaceutical composition for the treatment of PCSK-9-mediated disease or disorder, containing: an antibody to PCSK-9 or its antigen-binding fragment at a concentration of 1 mg/ml to 150 mg/ml; buffer at a concentration of from 5 mm to 30 mm, where the buffer is a histidine buffer; a disaccharide at a concentration of 10 mg/ml to 75 mg/ml, wherein the disaccharide is trehalose or sucrose; and a surfactant at a concentration of 0.05 mg/ml to 0.6 mg/ml, where the surfactant is Polysorbate 80 or Polysorbate 20; wherein the pH value of the pharmaceutical composition is from 5.5 to 6.5; and the anti-PCSK-9 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively. 2. The pharmaceutical composition of claim 1, wherein the buffer is a histidine hydrochloride buffer. 3. Pharmaceutical composition according to claim 1 or 2, wherein the concentration of the anti-PCSK-9 antibody or antigen-binding fragment thereof is between 30 mg/ml and 100 mg/ml. 4. The pharmaceutical composition according to claim 3, wherein the concentration of the anti-PCSK-9 antibody or antigen-binding fragment thereof is between 45 mg/mL and 55 mg/mL. 5. Pharmaceutical composition according to claim 4, wherein the concentration of the anti-PCSK-9 antibody or antigen-binding fragment thereof is 50 mg/ml. 6. A pharmaceutical composition according to any one of the preceding claims, wherein the pH value of the pharmaceutical composition is between 6.0 and 6.5. 7. Pharmaceutical composition according to claim 6, wherein the pH value of the pharmaceutical composition is 6.0. 8. A pharmaceutical composition according to any one of the preceding claims, wherein the concentration of the histidine buffer is between 5 mM and 20 mM. 9. The pharmaceutical composition according to claim 8, wherein the concentration of the histidine buffer is between 5 mM and 15 mM. 10. Pharmaceutical composition according to claim 9, wherein the concentration of histidine buffer is 10 mM. 11. A pharmaceutical composition according to any one of the preceding claims, wherein the disaccharide concentration is between 20 mg/ml and 40 mg/ml. 12. Pharmaceutical composition according to claim 11, wherein the disaccharide concentration is 25 mg/ml. 13. A pharmaceutical composition according to any of the preceding claims, wherein the disaccharide is sucrose. 14. A pharmaceutical composition according to any one of the preceding claims, wherein the concentration of the surfactant is between 0.1 mg/ml and 0.4 mg/ml. 15. Pharmaceutical composition according to claim 14, wherein the concentration of the surfactant is from 0.1 mg/ml to 0.3 mg/ml. 16. Pharmaceutical composition according to claim 15, wherein the concentration of the surfactant is 0.2 mg/ml. 17. A pharmaceutical composition according to any one of the preceding claims, wherein the surfactant is polysorbate 80. 18. A pharmaceutical composition according to any one of the preceding claims, wherein the PCSK-9 antibody, or antigen-binding fragment thereof, comprises the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 27. 19. A pharmaceutical composition according to any one of the preceding claims, wherein the anti-PCSK-9 antibody comprises a light chain of SEQ ID NO: 30 and a heavy chain of SEQ ID NO: 28 or 32. 20. Pharmaceutical composition according to any one of the preceding paragraphs, containing: an antibody to PCSK-9, where the antibody to PCSK-9 contains a heavy chain under SEQ ID NO: 32 and a light chain under SEQ ID NO: 30; while the concentration of antibodies to PCSK-9 is from 45 mg/ml to 55 mg/ml; buffer based on histidine hydrochloride, while the concentration of the buffer based on histidine hydrochloride is from 5 mm to 15 mm; a disaccharide, where the disaccharide is sucrose, wherein the concentration of sucrose is from 20 mg/ml to 40 mg/ml; a surfactant, where the surfactant is polysorbate 80 and the concentration of polysorbate 80 is from 0.1 mg/ml to 0.4 mg/ml; and the pH value of the pharmaceutical composition is from 6.0 to 6.5. 21. Pharmaceutical composition according to any one of the preceding paragraphs, containing: an antibody to PCSK-9, where the antibody to PCSK-9 contains a heavy chain under SEQ ID NO: 32 and a light chain under SEQ ID NO: 30; while the concentration of antibodies to PCSK-9 is 50 mg/ml; buffer based on histidine hydrochloride, while the concentration of buffer based on histidine hydrochloride is 10 mm; a disaccharide, where the disaccharide is sucrose, wherein the concentration of sucrose is 25 mg/ml; a surfactant, where the surfactant is polysorbate 80 and the concentration of polysorbate 80 is 0.2 mg/ml; and the pH value of the pharmaceutical composition is 6.0. 22. A lyophilized formulation for the treatment of a PCSK-9-mediated disease or disorder, comprising an anti-PCSK-9 antibody obtained by a method comprising a freeze-drying step of a pharmaceutical composition according to any one of claims 1 to 21, wherein the freeze-drying includes successive pre-freezing steps , primary drying and secondary drying. 23. A reconstituted solution for treating a PCSK-9 mediated disease or disorder, comprising an anti-PCSK-9 antibody obtained by a method comprising the step of reconstituting a lyophilized formulation according to claim 22. 24. A reconstituted solution containing an antibody to PCSK-9 according to claim 23, containing: an antibody to PCSK-9, where the concentration of the antibody to PCSK-9 is from 120 mg/ml to 200 mg/ml; buffer, where the buffer is a histidine buffer, while the concentration of histidine buffer is from 15 mm to 40 mm; a disaccharide, where the disaccharide is trehalose or sucrose, wherein the concentration of the disaccharide is from 55 mg/ml to 95 mg/ml; a surfactant, where the surfactant is polysorbate 20 or polysorbate 80, the concentration of the surfactant is from 0.4 mg/ml to 0.8 mg/ml; and the pH value of the reconstituted solution is between 6.0 and 6.5. 25. A reconstituted solution containing an antibody to PCSK-9 according to claim 23, containing: an antibody to PCSK-9, wherein the concentration of the antibody to PCSK-9 is 150 mg/ml; buffer, where the buffer is a histidine buffer, while the concentration of histidine buffer is 30 mm; a disaccharide, where the disaccharide is sucrose, the concentration of sucrose being 75 mg/ml; a surfactant, where the surfactant is polysorbate 80, wherein the concentration of polysorbate 80 is 0.6 mg/ml; and the pH value of the reconstituted solution is 6.3. 26. A reconstituted solution containing an antibody to PCSK-9 according to claim 23, containing: an antibody to PCSK-9, where the antibody to PCSK-9 contains a heavy chain under SEQ ID NO: 32 and a light chain under SEQ ID NO: 30; while the concentration of antibodies to PCSK-9 is from 120 mg/ml to 200 mg/ml; buffer based on histidine hydrochloride, while the concentration of the buffer based on histidine hydrochloride is from 15 mm to 40 mm; a disaccharide, where the disaccharide is sucrose, wherein the concentration of sucrose is from 55 mg/ml to 95 mg/ml; a surfactant, where the surfactant is a polysorbate 80, while the concentration of polysorbate 80 is from 0.4 mg/ml to 0.8 mg/ml; and the pH value of the reconstituted solution is between 6.0 and 6.5. 27. The use of a pharmaceutical composition according to any one of claims 1-21, or a lyophilized formulation according to claim 22, or a solution reconstituted from a lyophilized formulation according to any one of claims 23-26 in the treatment of a PCSK-9-mediated disease or disorder, wherein the disease or disorder is a disease associated with cholesterol. 28. Use according to claim 27, wherein the disease or disorder is selected from the group consisting of hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, apoplexy, cardiovascular disease (CVD), Alzheimer's disease, and general dyslipidemia.

Images