阅读说明：本技术 抗pcsk9抗体的药物递送装置 (Drug delivery device for anti-PCSK 9 antibody ) 是由 陈海涛 舒怡 谢皖 冯辉 于 2021-04-23 设计创作，主要内容包括：本发明提供一种药物递送装置,其包含自动注射器和抗PCSK9(人类前蛋白转化酶枯草溶菌素9)抗体的药物制剂。本发明提供的药物制剂在储存数月之后,抗体仍具有高稳定性。(The present invention provides a drug delivery device comprising an autoinjector and a pharmaceutical formulation of an anti-PCSK 9 (human proprotein convertase subtilisin/kexin 9) antibody. The antibody of the pharmaceutical preparation provided by the invention still has high stability after being stored for months.)

1. A drug delivery device, comprising:

(a) an auto-injector, and

(b) a pharmaceutical formulation of an anti-PCSK 9 antibody;

wherein the pharmaceutical formulation of the anti-PCSK 9 antibody comprises: an anti-PCSK 9 antibody or antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2.

2. The drug delivery device of claim 1, wherein the auto-injector isAn auto-injector.

3. The drug delivery device of claim 1, wherein the auto-injector is equipped with a pre-filled syringe, the pharmaceutical formulation of the anti-PCSK 9 antibody being pre-filled within the pre-filled syringe.

4. The drug delivery device of claim 1, wherein the anti-PCSK 9 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID No. 3 and a light chain having the amino acid sequence set forth in SEQ ID No. 4.

5. The drug delivery device of any one of claims 1 to 4, wherein the buffer is a histidine buffer, the buffer concentration is 15-25mM, and the pH of the buffer is 5.5-6.5.

6. The drug delivery device of claim 5, wherein the buffer has a concentration of about 20mM and the pH of the buffer is 5.5 to 6.5.

7. The drug delivery device of any one of claims 1-4, wherein the stabilizer is arginine hydrochloride at a concentration of 50mM to 200mM in the pharmaceutical formulation.

8. The drug delivery device of claim 7, wherein the stabilizer is arginine hydrochloride at a concentration of 130-200mM in the drug formulation.

9. The drug delivery device of any one of claims 1 to 4, wherein the anti-PCSK 9 antibody or antigen-binding fragment thereof is at a concentration of about 100mg/mL to about 200 mg/mL.

10. The drug delivery device of any one of claims 1 to 4, wherein the surfactant is polysorbate 20 at a concentration of about 0.01% (w/v) to about 0.05% (w/v).

11. The drug delivery device of claim 10, wherein the surfactant is polysorbate 20 at a concentration of about 0.02% (w/v).

12. The drug delivery device of any one of claims 1 to 4, wherein the pharmaceutical formulation of the anti-PCSK 9 antibody comprises:

(1) about 100mg/mL to about 200mg/mL of an antibody or antigen-binding fragment thereof to PCSK 9;

(2) about 10-50mM histidine buffer, at a pH of about 5.5-6.5;

(3) about 50mM to about 200mM arginine salt; and

(4) from about 0% to about 0.1% (w/v) of a nonionic surfactant.

13. The drug delivery device of claim 12, wherein the pharmaceutical formulation of the anti-PCSK 9 antibody comprises:

(A) (a) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof against PCSK 9; (b) about 20mM histidine buffer, at a pH of about 5.5-6.5; (c) arginine salt at about 60 ± 5mM, 130 ± 5mM, or 160 ± 5 mM; (d) from about 0% to about 0.1% (w/v) of a nonionic surfactant; or

(B) (a) about 20mM histidine buffer at a pH of about 5.5-6.5; (b) about 160mM arginine hydrochloride; (c) about 0.02% (w/v) polysorbate 20; and (d) about 150mg/mL of an antibody or antigen-binding fragment thereof against PCSK 9; or

(C) (a) about 20mM histidine buffer at a pH of about 5.5-6.5; (b) about 130mM arginine hydrochloride; (c) about 0.02% (w/v) polysorbate 20; and (d) about 150mg/mL of an antibody or antigen-binding fragment thereof directed to PCSK 9.

14. The drug delivery device of any one of claims 1-4, wherein a single administered dose of the pharmaceutical formulation is a fixed dose of 150mg, 300mg, or 450mg of the antibody or antigen-binding fragment thereof.

15. The drug delivery device of any of claims 1-4, wherein the drug formulation is administered weekly, biweekly, triweekly, every four weeks, or every five weeks.

16. The drug delivery device of claim 14, wherein the drug formulation is administered by subcutaneous injection in a single administration dose of 150mg fixed dose administered once every two weeks or in a single administration dose of 450mg fixed dose administered once every four weeks.

17. The drug delivery device of claim 16, wherein the administration period of the pharmaceutical formulation is two weeks, one month, two months, three months, four months, five months, half a year or more.

18. Use of a drug delivery device of any one of claims 1-17 in the manufacture of a medicament or kit for treating, preventing, or ameliorating a disease associated with PCSK9 activity.

19. The use of claim 18, wherein the disease associated with PCSK9 activity is selected from familial hypercholesterolemia, non-familial hypercholesterolemia, elevated lipoproteins, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, alzheimer's disease, peripheral arterial disease, hyperlipidemia, and dyslipidemia.

20. A kit comprising one or more prefilled syringes prefilled with 1mL of a pharmaceutical formulation, wherein the pharmaceutical formulation is according to any one of claims 1 and 4-13.

Technical Field

The present invention relates to the field of therapeutic antibody formulations. In particular, the present invention relates to a drug delivery device comprising an autoinjector and a pharmaceutical formulation of an anti-PCSK 9 (human proprotein convertase subtilisin/kexin 9) antibody.

Background

Proprotein convertase subtilisin/kexin type 9(PCSK9) is a proprotein convertase with the effect of promoting the degradation of low-density lipoprotein (LDL) receptors on the surface of liver cells to increase the LDL cholesterol level in plasma, and its expression increase is closely related to human dyslipidemia and cardiovascular related diseases. The patent with publication number WO2017088782 discloses that various anti-PCSK 9 antibodies can significantly reduce LDL concentration in blood by antagonizing PCSK9 bioactivity, and have important prospects in the field of treatment of diseases related to hypercholesterolemia and the like. Like any protein therapeutic, therapeutic anti-PCSK 9 antibodies are subject to physical and chemical instabilities, such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shearing during manufacture or storage (Wang et al, j.pharm.sci.96:1-26, 2007(Wang et al, journal of pharmaceutical science, 96, pages 1-26, 2007)). Therefore, it is very challenging to develop an antibody formulation that can maintain the stability of the physicochemical properties of the antibody.

Hypercholesterolemia and other related diseases are generally chronic diseases and it is important to provide the convenience of the patient to take medication out of hospital or by self-administration. Protein therapeutics can generally only be administered by parenteral routes, where Subcutaneous (SC) or Intramuscular (IM) routes of administration can reduce treatment costs and improve convenience for patients and healthcare providers during administration. The small volume required for SC or IM injections (typically 0.5 to 2mL) presents an additional formulation challenge, as administration requires high concentration antibody formulations, typically between 100mg to 1g protein per dose, to achieve therapeutic levels. Highly concentrated protein formulations often increase protein aggregation, precipitation and viscosity, which can have negative consequences during processing, manufacture and storage, where increased viscosity can also have negative effects on formulation administration, such as sensory pain and burning symptoms and limitations on drug delivery device options (Shire et al, j.pharm.sci.93:1390-1402, 2004(Shire et al, journal of pharmaceutical sciences 93, 1390-.

Thus, there is a need in the art for a high concentration protein formulation that provides, inter alia, low viscosity and reduces pain in patients.

Drugs may be administered by using drug delivery devices, such as auto-injectors and in vivo injectors, that provide several benefits in delivering drugs and/or therapeutic agents. One of the benefits may include ease of use as compared to conventional delivery methods using, for example, conventional syringes.

However, even with autoinjectors, patients may experience challenges during use of the drug delivery device. For example, a user may be uncertain whether the medication within the drug delivery device is the correct medication prescribed for them. The user may also be uncertain whether the actions and their sequence are operating the drug delivery device correctly. Even if the actions are performed in the proper sequence, the user may not be certain whether the medication has been completely delivered and the injection process is complete. Furthermore, users often wish to perform as few steps as possible during drug administration. When injecting drugs subcutaneously, patients may require a quick but smooth and stable injection.

In addition to these mechanical considerations, the design of autoinjectors may require user-friendly considerations. Furthermore, it may be desirable that operation of the injector be limited to only those times when the injector is properly positioned for injection and/or when the user performs an appropriate sequence of actions.

Disclosure of Invention

The present invention provides a drug delivery device comprising an autoinjector and a pharmaceutical formulation of an anti-PCSK 9 (human proprotein convertase subtilisin/kexin 9) antibody. The drug delivery device provided by the invention is characterized in that the drug preparation containing the anti-PCSK 9 antibody has high stability and low viscosity; meanwhile, the automatic syringe is used, and the operation is simple and convenient.

In one aspect, the invention provides a drug delivery device comprising (1) an autoinjector, (2) a pharmaceutical formulation of an anti-PCSK 9 antibody, wherein the pharmaceutical formulation of an anti-PCSK 9 antibody comprises: an anti-PCSK 9 antibody or antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2.

In one embodiment, the automatic injector isAn auto-injector.

In one embodiment, the auto-injector is equipped with a pre-filled syringe into which the pharmaceutical formulation of the anti-PCSK 9 antibody is pre-filled.

In one embodiment, wherein the buffer is a histidine buffer. In one embodiment, the histidine buffer is made from L-histidine and L-histidine monohydrochloride. In one embodiment, the concentration of the buffer is 15 to 25 mM. In one embodiment, the histidine buffer is at a concentration of about 20 mM. In one embodiment, the pH of the buffer is 5.5 to 6.5.

In one embodiment, the stabilizer comprises one or more selected from arginine or a salt thereof, sorbitol, mannitol, or sucrose. In one embodiment, the above stabilizer is an arginine salt. In one embodiment, the concentration of arginine, or a salt thereof, is about 50mM to about 200 mM. In some specific embodiments, the concentration of arginine, or a salt thereof, is about 60mM, 130mM, 160mM, or 165 mM.

In one embodiment, the anti-PCSK 9 antibody comprises a Heavy Chain (HC) and a Light Chain (LC), wherein the amino acid sequence of HC is SEQ ID No. 3 and the amino acid sequence of LC is SEQ ID No. 4.

In one embodiment, the anti-PCSK 9 antibody or antigen-binding fragment thereof is present at a concentration of about 100mg/mL to about 200 mg/mL. In a specific embodiment, the concentration of the anti-PCSK 9 antibody or antigen-binding fragment thereof is about 150 mg/mL.

In one embodiment, the above surfactant comprises polysorbate 20 or polysorbate 80 at a concentration of about 0.01% to about 0.05% (w/v). In a specific embodiment, the surfactant comprises polysorbate 20 at a concentration of about 0.02% (w/v).

In one embodiment, the above pharmaceutical formulation of an anti-PCSK 9 antibody comprises: (1) about 20mM histidine buffer, pH 5.5-6.5; (2) about 50mM to about 200mM arginine or a salt thereof; (3) about 100mg/mL to about 200mg/mL of an anti-PCSK 9 antibody or antigen-binding fragment thereof; and (4) about 0.02% (w/v) polysorbate 20; the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

In one embodiment, the above pharmaceutical formulation of an anti-PCSK 9 antibody comprises: (1) about 20mM histidine buffer, pH 5.5-6.5; (2) about 160mM arginine hydrochloride; (3) about 150mg/mL of an anti-PCSK 9 antibody or antigen-binding fragment thereof; and (4) about 0.02% (w/v) polysorbate 20; the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

In one embodiment, the single administered dose of the above pharmaceutical formulation is a fixed dose of 150mg, 300mg, or 450mg of the anti-PCSK 9 antibody or antigen-binding fragment thereof.

In one embodiment, the above drug delivery device contains a single administered dose of a drug formulation; preferably, the drug delivery device contains one, two or three injections, wherein the injection size is 150mg (1mL) per injection.

In one embodiment, the pharmaceutical formulation is administered weekly, biweekly, every three weeks, every four weeks, or every five weeks.

In one embodiment, the above pharmaceutical formulation is administered in a single administration dose of 150mg fixed dose administered once every two weeks, or in a single administration dose of 450mg fixed dose administered once every four weeks administered by subcutaneous injection.

In one embodiment, the pharmaceutical formulation is administered for a period of one week, two weeks, three weeks, one month, two months, three months, four months, five months, half a year or more.

In one embodiment, the invention provides that the anti-PCSK 9 antibody has at least 94% of the antibody in native conformation after 28 days of storage at 40 ℃.

In one embodiment, the invention provides a pharmaceutical formulation having at least 45% of the antibodies with the predominantly charged variant after 28 days of storage at 40 ℃.

In one embodiment, the pharmaceutical formulation provided herein has at least 98% of the antibody in its native conformation after 12 months of storage at 2-8 ℃.

In one embodiment, the pharmaceutical formulation provided by the present invention has at least 87% of the antibodies with the predominantly charged variant after 12 months of storage at 2-8 ℃.

In another aspect, the invention provides the use of a drug delivery device for the treatment, prevention or amelioration of a disease associated with PCSK9 activity, or for the manufacture of a medicament for the treatment, prevention or amelioration of a disease associated with PCSK9 activity.

In one embodiment, the above-mentioned diseases associated with PCSK9 activity are selected from familial hypercholesterolemia (including heterozygote familial hypercholesterolemia and homozygote familial hypercholesterolemia), non-familial hypercholesterolemia, lipoprotein elevation, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, alzheimer's disease, peripheral artery disease, hyperlipidemia, and dyslipidemia.

In a third aspect, the present invention also provides a kit comprising one or more prefilled syringes pre-filled with 1mL of the pharmaceutical formulation described in any of the above embodiments.

In one embodiment, the kit further comprises an automatic injector for assembling the prefilled syringe and performing the administration function.

Drawings

FIG. 1: the JS 002-containing formulation inhibited LDL binding and uptake by cells.

FIG. 2: JS002 was administered several times by subcutaneous injection to observe the effect on hyperlipidemic rhesus monkey LDL-C for 78 days.

FIG. 3: assembly process diagram of single-use recombinant humanized anti-PCSK 9 monoclonal antibody prefilled automatic injector.

Detailed Description

It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for performing the disclosed methods.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those disclosed herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

By "therapeutically active antibody" or "therapeutic antibody" is meant an antibody that can be used for therapeutic purposes, i.e., for treating a disorder in a subject. It should be noted that although therapeutic proteins may be used for therapeutic purposes, the invention is not limited to such use as the proteins may also be used for in vitro studies.

The term "pharmaceutical formulation" or "formulation" is an article of manufacture in a form such that the biological activity of the active ingredient is effective and free of other ingredients having unacceptable toxicity to the subject to which the formulation is administered. The formulation is sterile.

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In one embodiment, the aqueous liquid formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that can be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

Herein, the term "buffer providing a pH of about 5.5 to about 6.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.5 to about 6.0. In one embodiment, the pH is about 6.0.

Examples of "buffers" that control pH within this range herein include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine and/or salts thereof, methionine, citrates, phosphates, citrate/phosphates, imidazole, combinations thereof, and other organic acid buffers. In one embodiment, the buffer is not a protein. In one embodiment, the buffer is histidine and/or a salt thereof, preferably L-histidine and/or a salt thereof. Typically, the concentration of the buffer in the formulation may be in the range of 5-100mM, or about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM, or any two of these as endpoints, such as 10-30mM or 15-25 mM. In one embodiment, the buffer concentration is about 20 mM.

A "histidine buffer" is a buffer comprising histidine and/or a salt thereof. Salts of histidine include one or more of histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate. In one embodiment of the invention, the histidine buffer is: histidine buffer made from 1-10mM L-histidine and 10-20mM L-histidine monohydrochloride. In one embodiment, the histidine preparation is: histidine buffer pH 6.0 made from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In one embodiment, the histidine preparation is: histidine buffer pH 5.5 was prepared from 9.5mM L-histidine and 10.5mM L-histidine monohydrochloride. In some embodiments, the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4.

Herein, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" both refer to mass-to-volume concentrations (w/v), such as "0.02%" in "about 0.02% polysorbate 20" which means "0.02 g of solute in 100mL of liquid".

As used herein, the term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), Sodium Dodecyl Sulfate (SDS). In one embodiment, the nonionic surfactant is polysorbate 20. In one embodiment, the concentration of polysorbate 20 is about 0 to 0.1% (w/v). In one embodiment, the concentration of polysorbate 20 is about 0.01% to 0.05% (w/v). In one embodiment, the concentration of polysorbate 20 is about 0.02% (w/v). In one embodiment, the nonionic surfactant is polysorbate 80. In one embodiment, the concentration of polysorbate 80 is about 0 to 0.1% (w/v). In one embodiment, the concentration of polysorbate 80 is about 0.01% to 0.05% (w/v). In one embodiment, the concentration of polysorbate 80 is about 0.02% (w/v).

As used herein, the term "stabilizer" can reduce aggregation of antibodies and other proteins. Exemplary stabilizers include, but are not limited to: human Serum Albumin (HSA), Bovine Serum Albumin (BSA), alpha-casein, globulin, alpha-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Stabilizers also include amino acids and their metabolites and their salts such as hydrochlorides, for example: arginine, glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opioids) (alanine, octopine, glycinol (strombine)) and N-oxide of Trimethylamine (TMAO). Stabilizers also include sugars, polyols, their metabolites, and the like, such as NaCl, KCl, MgCl₂、CaCl₂Sucrose, mannitol, sorbitol, and the like. In one embodimentThe stabilizer is mannitol. In one embodiment, the stabilizing agent is sucrose. In one embodiment, the stabilizer is sorbitol. In one embodiment, the stabilizer is an amino acid or salt thereof. In one embodiment, the amino acid is arginine or arginine hydrochloride. In one embodiment, the concentration of arginine or arginine hydrochloride is about 20 to 200 mM. In one embodiment, the concentration of arginine or arginine hydrochloride is about 50 to 200 mM. In one embodiment, the concentration of arginine or arginine hydrochloride is about 60mM, 130mM, 160mM, or 165 mM.

The term "viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity". "kinematic viscosity" is a measure of the resistance of a fluid to flow under the influence of gravity. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity ═ kinematic viscosity X density). Kinematic viscosity is measured as L2/T, where L is the length and T is the time. Typically, kinematic viscosities are expressed in centistokes (cSt). The international system of kinematic viscosity is in mm2/s, lcSt. Absolute viscosity is expressed in units of centipoise (cP). The international unit system of absolute viscosity is millipascal-seconds (mPa-s), where 1cP is lmPa-s.

For liquid-type formulations of the present invention, the term "low level viscosity" as used herein will mean an absolute viscosity of less than about 15 centipoise (cP). For example, if the formulation exhibits an absolute viscosity of about 15cP, about 14cP, about 13cP, about 12cP, about 11cP, about 10cP, about 9cP, about 8cP, or less, when measured using standard viscosity measurement techniques, the liquid-type formulation of the present invention will be considered to have a "low viscosity". For the liquid-type formulations of the present invention, the term "intermediate level viscosity" as used herein will mean an absolute viscosity of between about 35cP and about 15 cP. For example, if the formulation exhibits an absolute viscosity of about 34cP, about 33cP, about 32cP, about 31cP, about 30cP, about 29cP, about 28cP, about 27cP, about 26cP, about 25cP, about 24cP, about 23cP, about 22cP, about 21cP, about 20cP, about 19cP, 18cP, about 17cP, about 16cP, or about 15.lcP, the inventive liquid-type formulation will be considered to have a "medium viscosity". The liquid pharmaceutical formulations of the present invention may exhibit low to moderate levels of viscosity in certain embodiments. In one embodiment, the present invention has made the surprising discovery that a liquid-type formulation with low viscosity can be obtained by formulating an antibody at a concentration of about 100 and 200mM with 60-165mM arginine or a salt thereof. In one embodiment, it has further been found that arginine or a salt thereof significantly reduces the viscosity of formulations containing other tonicity agents such as sucrose, sorbitol, mannitol and the like.

By "isotonic" is meant that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or subfreezing type.

A "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may also be considered "stable". Various analytical techniques for measuring Protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody may be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SE-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In one embodiment, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

Stability can be measured by determining (among other methods) the percentage of antibody ("acidic form") that migrates during ion exchange in a fraction that is acidic relative to the main fraction of antibody ("predominantly charged form"), where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In one embodiment, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in acidic form is detectable in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical formulation if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements correlate with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).

The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), Strasbourg, France). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

An antibody "retains its chemical stability" in a pharmaceutical formulation if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical formulation "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation for a period of time (e.g., 1 to 12 months) at a temperature, e.g., 5 ℃, 25 ℃, 45 ℃, etc., the formulation comprises an anti-PCSK 9 antibody that binds PCSK9 with at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

anti-PCSK 9 antibodies

The formulations of the invention comprise an antibody or antigen-binding fragment thereof that specifically binds to human PCSK 9. The term "PCSK 9" as used herein is a human proprotein convertase belonging to the proteinase K subfamily of the secreted subtilase family. The literature has demonstrated that PCSK9 can increase plasma LDL levels by binding to and promoting degradation of low density lipoprotein particle receptors.

The term "antibody" as used herein is to be understood as including whole antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to human PCSK9 or an epitope thereof.

The term "full length antibody", as used herein, refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in total length) and two light (L) chains (about 25kDa in total length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently for different systems. The system described by Kabat (supra) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also provides an accurate residue boundary defining 3 CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al found that some of the subsections within the Kabat CDRs adopt an almost identical peptide backbone configuration despite large diversity at the amino acid sequence level (Chothia et al, (1987) mol.biol.196: 901-917; Chothia et al, (1989) Nature342: 877-883). Other boundaries defining CDRs which overlap with the Kabat CDRs have been described by Padlan (1995) FASEB J.9: 133-. Still other CDR boundary definitions may not strictly follow one of the systems described herein, but still overlap with the Kabat CDRs, although they may be shortened or lengthened, since specific residues or groups of residues, or even entire CDRs, were not found to significantly affect antigen binding according to prediction or experimentation. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

An "antigen-binding fragment" as used herein includes a fragment or derivative of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.

The term "single" or "one-time" administration of a pharmaceutical formulation of the present invention means one round of administration, including, but not limited to, one, two, three, four or more pre-filled syringes per round, so long as the total drug dosage for that round of administration reaches the prescribed dosage.

The anti-PCSK 9 antibody or antigen-binding fragment thereof according to the present invention includes any one of the anti-PCSK 9 antibodies described in international publication No. WO 2017088782. In some embodiments, the 6 CDRs of the antibody used in the methods, uses, pharmaceutical formulations and delivery devices of the invention are derived from the 6 CDRs of the antibody JS002 disclosed in WO 2017088782.

In one embodiment, the anti-PCSK 9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence of SEQ ID No. 2.

The non-limiting, exemplary antibody used in the examples herein is referred to as "JS 002", which is a humanized complete antibody that specifically binds to human PCSK9, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID No. 3 and the amino acid sequence of the light chain is SEQ ID No. 4.

Pharmaceutical preparation

The preparation of the invention is a liquid preparation which contains high-concentration active antibody and has high stability and low viscosity. In particular, the present inventors have found that formulations containing arginine salts have a significantly lower viscosity than formulations containing tonicity agents. In addition, the arginine salt contained in the formulation significantly reduces the formulation containing tonicity agent.

The formulations of the invention comprise an antibody or antigen-binding fragment thereof that specifically binds human PCSK9, a buffer, and a stabilizer. Preferably, the pH of the formulation of the invention is in the range of 5.5 to 6.5.

In the formulations of the invention, the anti-PCSK 9 antibody or antigen-binding fragment thereof typically ranges from 100mg/mL to about 200 mg/mL. In some embodiments, the anti-PCSK 9 antibody or antigen-binding fragment thereof is contained at 150 ± 10mg/mL in a formulation of the invention. In a preferred embodiment, the anti-PCSK 9 antibody or antigen-binding fragment thereof contained in the formulations of the invention comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2. More preferably, the antibody or antigen-binding fragment comprises a Heavy Chain (HC) and a Light Chain (LC), wherein HC has the amino acid sequence set forth in SEQ ID NO. 3 and LC has the amino acid sequence set forth in SEQ ID NO. 4.

In the formulation of the present invention, the buffer may be an acetate buffer or a histidine buffer, preferably a histidine buffer. Preferably, the pH of the buffer is in the range of 5.5-6.0. Preferably, the histidine buffer of the present invention comprises histidine and a salt of histidine as described herein. Typically, the histidine buffer used in the present invention contains 1-10mM histidine and 10-20mM histidine salt (e.g., monohydrochloride). Preferably, the molar ratio of histidine to salt of histidine in the histidine buffer is in the range of 1:1 to 1: 4. In the formulations of the invention, the buffer may be present at a concentration of 10-50mM, preferably 15-25mM, such as 20 mM.

In the formulation of the present invention, the stabilizer may be selected from one or more of arginine or a salt thereof, sorbitol, mannitol and sucrose, preferably arginine salt. Exemplary arginine salts include arginine hydrochloride. Other arginine salts useful as stabilizers in pharmacy may also be useful in the present invention. In general, the concentration of the stabilizer in the formulations of the invention may be in the range of 50mM to 300mM, such as 50-200mM, 130-200mM or 160-250 mM. When arginine salt is used alone, the concentration thereof in the preparation may be in the range of 50-200mM, such as 130-200 mM. In some embodiments, the stabilizer is a mixture of an arginine salt and mannitol or sorbitol. When arginine salts are used in combination with mannitol or sorbitol, the concentration of arginine salts in the formulation may be in the range of 50-150mM, mannitol or sorbitol in the range of 50-200mM, and the total concentration of the two stabilizers may be in the range of 50mM to 300mM, such as 50mM to 250 mM.

In some embodiments, the formulations of the present invention comprise: (1) an antibody or antigen-binding fragment thereof against PCSK 9; (2) histidine buffer at a pH of about 5.5 to 6.5; and (3) arginine salts.

In some embodiments of the invention, the formulations of the invention further comprise a nonionic surfactant as described herein. Typically, when included, the concentration of nonionic surfactant in the formulation does not exceed 0.1% (w/v), such as 0.01-0.05% (w/v). Preferred nonionic surfactants include polysorbate 20 and/or polysorbate 80, with a particularly preferred concentration of about 0.02% (w/v).

In one embodiment, the formulation of the present invention comprises: (1) about 100mg/mL to about 200mg/mL of an antibody or antigen-binding fragment thereof to PCSK 9; (2) about 10-50mM histidine buffer, pH about 5.5-6.5; (3) about 50mM to about 200mM arginine salt; and (4) from about 0% to about 0.1% (w/v) of a nonionic surfactant.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof to PCSK 9; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) arginine salt at about 60 ± 5mM, 130 ± 5mM, or 160 ± 5 mM; and (4) from about 0% to about 0.1% (w/v) of a nonionic surfactant.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof to PCSK 9; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 130 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof against PCSK9, wherein the antibody comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 130 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention has a pH of 5.5 to 6.5 and comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof against PCSK9, wherein the antibody comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 130 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof to PCSK9, wherein the antibody is a full-length antibody, wherein the amino acid sequence of the heavy chain is SEQ ID No. 3 and the amino acid sequence of the light chain is SEQ ID No. 4; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 130 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof to PCSK 9; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 160 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof against PCSK9, wherein the antibody comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 160 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention has a pH of 5.5 to 6.5 and comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof against PCSK9, wherein the antibody comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 160 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

In one embodiment, the formulation of the present invention comprises: (1) about 150 ± 10mg/mL of an antibody or antigen-binding fragment thereof to PCSK9, wherein the antibody is a full-length antibody, wherein the amino acid sequence of the heavy chain is SEQ ID No. 3 and the amino acid sequence of the light chain is SEQ ID No. 4; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 160 ± 5mM arginine salt; and (4) about 0.02% (w/v) polysorbate 20.

Drug delivery device and method of administration

Examples of disposable pen and/or autoinjector delivery devices useful for subcutaneous delivery of the drug formulations of the present invention include, but are not limited to(Ypsomed)、S0L0STAR^TMPen (sanofi-aventis) and FLEXPEN^TM(Novo Nordisk) and KWIKPEN^TM(Eli Lilly)、SURECLICK^TMAutomatic injector (Amgen, T)housand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA^TMPens (Abbott Labs, Abbott Park, IL).

Selected by the invention(Ypsmmed AG) autoinjector has a number of significant advantages, greatly meeting the needs of patients. The method mainly comprises the following steps:

2, the operation is simple in step;

the needle head protective cap is easy to remove;

displaying a large window;

a skin pressing operation trigger mechanism;

a "click" cue for start and end of injection;

the locked needle shield provides complete protection of the needle from needle stick injuries.

In some embodiments, the present invention provides a drug delivery device comprising (1) an autoinjector, (2) a pharmaceutical formulation of an anti-PCSK 9 antibody, wherein the pharmaceutical formulation of an anti-PCSK 9 antibody is a pharmaceutical formulation according to any of the embodiments of the present invention.

In one embodiment, the automatic injector isAn auto-injector.

In one embodiment, the automatic injector is equipped with a prefilled syringe into which the pharmaceutical formulation of the anti-PCSK 9 antibody is prefilled.

In some embodiments, the present invention provides a kit comprising one or more prefilled syringes pre-filled with 1mL of a pharmaceutical formulation according to any one of the embodiments of the present invention.

Preferably, the pharmaceutical formulation for use in the drug delivery device or kit comprises: an anti-PCSK 9 antibody or antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 1 and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2; more preferably, in the pharmaceutical preparation, the anti-PCSK 9 antibody comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is shown as SEQ ID No. 3 and the light chain amino acid sequence is shown as SEQ ID No. 4. Preferably, in the pharmaceutical preparation, the buffer is histidine buffer, the concentration of the buffer is 15-25mM, and the pH of the buffer is 5.5-6.5; preferably, the stabilizer is arginine hydrochloride at a concentration of 50mM to 200mM in the formulation; preferably, the surfactant is polysorbate 20 at a concentration of about 0.01% to about 0.05% (w/v). More preferably, the pharmaceutical formulation of an anti-PCSK 9 antibody comprises: (a) about 20mM histidine buffer; (b) about 160mM arginine hydrochloride; (c) about 0.02% (w/v) polysorbate 20; and (d) about 150mg/mL of the anti-PCSK 9 antibody or antigen-binding fragment thereof.

Preferably, the prefilled syringe is a 1mL pre-filled glass or polymer syringe.

Preferably, the kit contains two, four, six, eight, ten or twelve of said prefilled syringes for administration by subcutaneous injection at a frequency of once every two weeks at a dose of 150mg of said anti-PCSK 9 antibody or antigen-binding fragment thereof administered at a time over a dosing period of 1 month, 2 months, 3 months, 4 months, 5 months or 6 months.

Preferably, the kit contains three, six, nine, twelve, fifteen or eighteen of said prefilled syringes for administration by subcutaneous injection at a dosing frequency of once every four weeks, at a dose of three (i.e., 450mg of said antibody or antigen-binding fragment thereof against PCSK9) doses per administration over a dosing period of 1 month, 2 months, 3 months, 4 months, 5 months or 6 months.

Preferably, the kit contains the number of said prefilled syringes that the patient can administer once every two weeks, once a single dose and/or three times every four weeks, three doses consecutively for 1-6 months.

Preferably, the prefilled syringe comprises a needle cannula (containing a needle), a needle shield and a piston, wherein the pharmaceutical formulation is pre-loaded within the needle cannula. Preferably, the prefilled syringe is a disposable prefilled syringe.

Preferably, the cartridge further contains an auto-injector for assembling the prefilled syringe and performing the administration function. Preferably, the automatic injector consists of an upper part and a lower part, wherein the upper half part comprises a spring pushing device, and the lower half part comprises a prefilled injector storage bin, a drug delivery trigger button and a protective cap.

Use and dosage of drug delivery devices

Adult primary hypercholesterolemia (including heterozygote-type familial hypercholesterolemia) and mixed dyslipidemia:

can be used as dietary adjuvant therapy for treating adult primary hypercholesterolemia (familial and non-familial heterozygote) or mixed dyslipidemia patients to reduce low density lipoprotein cholesterol (LDL-C) level: in patients who have failed to achieve LDL-C targets on statin therapy at the maximum tolerated dose, either in combination with statins, or with statins and other lipid lowering therapies, or in patients who are not statin-tolerant or contraindicated for use, either alone or in combination with other lipid lowering therapies.

Reducing the risk of cardiovascular events:

reducing the risk of myocardial infarction, stroke and coronary revascularization in adult patients with atherosclerotic cardiovascular disease (ASCVD) by combination with maximum tolerated doses of statins, with or without other lipid lowering therapies, or in patients with statin intolerance or contraindication, alone or in combination with other lipid lowering therapies.

Homozygous type familial hypercholesterolemia (HoFH):

is used for treating homozygous familial hypercholesterolemia of adults or adolescents of 12 years old or older. Can be used in combination with diet therapy and other treatments for reducing Low Density Lipoprotein (LDL), such as statins, for patients with homozygous familial hypercholesterolemia (HoFH) and a need for further reduction of low density lipoprotein cholesterol (LDL-C).

For primary hypercholesterolemia (including heterozygous familial hypercholesterolemia), mixed dyslipidemia, and adult patients already suffering from ASCVD, a subcutaneous dose of 150mg once every 2 weeks or 450mg once every 4 weeks can be administered. Based on patient preference in terms of dosing frequency and dose per injection. When the dosage regimen is changed, the first administration of the new regimen is performed on the planned date of the next original dosing regimen.

For HoFH patients, a dose of 450mg can be administered subcutaneously 1 time every 4 weeks. Since the response to treatment depends on the level of LDL-receptor function, LDL-C levels should be measured in HoFH patients 4-8 weeks after administration.

The pharmaceutical formulations of the present invention are useful, inter alia, for treating, preventing or ameliorating any disease or disorder associated with PCSK9 activity, including PCSK 9-mediated diseases or disorders. Exemplary, non-limiting diseases and disorders that may be treated or prevented by the pharmaceutical formulations of the present invention include various dyslipidemia disorders, such as hypercholesterolemia, familial hypercholesterolemia, 13/4 dyslipidemia, familial 13/4 dyslipidemia, dys3 lipoproteinemia, familial dys3 lipoproteinemia, 13 x 4 triglyceridemia and familial hypertriglyceridemia.

The invention also includes a method of treating any of the above disorders comprising administering to a subject in need thereof a pharmaceutical formulation according to any of the embodiments of the invention via a drug delivery device according to any of the embodiments of the invention. Most preferably, the pharmaceutical formulation administered comprises: (a) about 20mM histidine buffer; (b) about 160mM arginine hydrochloride; (c) about 0.02% (w/v) polysorbate 20; and (d) about 150mg/mL of the anti-PCSK 9 antibody or antigen-binding fragment thereof; administering to a subject in need thereof a dosing frequency of once every two weeks at a fixed dose of 150mg of said antibody or antigen-binding fragment thereof per time over a dosing period of one week, two weeks, three weeks, one month, two months, three months, four months, five months, one half year or more, or administering to a subject in need thereof a dosing frequency of once every four weeks at a fixed dose of 450mg of said antibody or antigen-binding fragment thereof per time over a dosing period of one month, two months, three months, four months, five months, one half year or more. Administration by subcutaneous injection or intravenous injection is preferred.

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Example 1: buffer system and pH screening assay

In the liquid type antibody preparation, the buffer system and pH closely affect the stability of the antibody, and each antibody having unique physicochemical properties has the optimum kind and pH of the buffer. An optimal buffer system and pH are screened to ensure that the anti-PCSK 9 antibody disclosed by the invention has optimal stability so as to be suitable for clinical application.

The study was carried out with JS002 concentration of about 150mg/mL, with the adjuvant 130mM arginine hydrochloride. And (4) carrying out dialysis liquid change by using a dialysis bag so that the JS002 protein is in the corresponding prescription, and placing the sample in a sealed centrifuge tube for buffer liquid screening. We screened both sodium acetate buffer and histidine buffer (histidine to histidine hydrochloride molar ratio 1: 1) types, with pH levels from 5.0 to 6.0 (as shown in table 1). The samples were placed at 40 ℃/ambient RH and removed for analytical testing at weeks 2 and 4, respectively. The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The percentage of the natural form (protein monomer) to the aggregated form JS002 was determined by size exclusion chromatography (SEC-HPLC) and the percentage of the acidic and basic forms mAb by cation exchange chromatography (CEX-HPLC). The influence of different buffer systems and pH on the stability of JS002 antibody is examined by fitting a straight line and calculating the descending slope (%/week) with the SEC-HPLC monomer content and the CEX-HPLC main peak content at the start of the test (0W), at two weeks (2W) and at four weeks (4W), and the results are summarized in Table 2.

Table 1: buffer system and prescription information in pH screening experiments

Prescription number	pH	Buffer system	Auxiliary materials
				1	5.0	20mM sodium acetate buffer	130mM arginine hydrochloride
2	5.5	20mM sodium acetate buffer	130mM arginine hydrochloride
				3	6.0	20mM histidine buffer	130mM arginine hydrochloride

Table 2: summary of stability results in buffer systems and pH screening experiments

As shown in Table 2, in SEC-HPLC experimental detection, the antibody can be kept relatively stable within the pH range of 5.5-6.0, and after the antibody is placed at the high temperature of 40 ℃ for 4 weeks, the monomer content of a sample is more than 94%, and the monomer purity reduction rate is less than 1.5%/week; the main charge of the sample accounts for more than 45%, and the rate of decrease of the main charge is less than 10% per week. When the buffer system was histidine buffer and the pH was 6.0 (recipe No. 3), the monomer purity of the sample was the highest (about 97%) after 4 weeks at high temperature 40 ℃ and the rate of decrease in monomer purity was only 0.57%/week. Based on these results, 20mM histidine buffer with pH 5.5-6.0 was selected for the development of liquid JS002 formulation.

Example 2: stabilizer screening experiments

To further explore the effect of different excipients on antibody stability and viscosity, we selected formulations of one or a combination of sucrose, arginine hydrochloride, sorbitol or mannitol for comparative testing. Namely, the above different excipients or their combination were added to 20mM histidine buffer (pH 5.5 or 6.0) containing JS002 at about 150mg/mL, respectively, and the specific prescription information is shown in Table 3. The preparations of all the prescriptions are subpackaged and placed at 40 ℃, and are taken out at 2 weeks and 4 weeks respectively for analysis and detection of the stability of the antibody and the viscosity of the preparation solution. Detecting the change of JS002 monomer content by molecule exclusion high performance liquid chromatography (SEC-HPLC), detecting the main peak of JS002 charge content by weak cation high performance liquid chromatography (CEX-HPLC), and detecting the viscosity of the preparation by standard method at the fourth week. As shown in Table 4, when the formulation contains one or a combination of sucrose, arginine hydrochloride, sorbitol or mannitol, the antibody has strong thermal stability, namely, after the preparation is placed at high temperature of 40 ℃ for 4 weeks, the content of the monomer in the sample is more than 98%, and the main charge is more than 51%.

In particular, the stability of the antibody is the strongest and the viscosity is the lowest when the formulation contains only arginine salt, as a result of a combination of data analysis. Specifically, after standing at high temperature of 40 ℃ for 4 weeks, the formulation group containing only arginine salt: (1) for the stability of the antibody structure, the reduction rate of the monomer purity of the antibody is obviously lowest, as low as 0.1%/week, about 25-30% of the highest sucrose group, the monomer purity of the antibody is as high as 98.8%, and the influence of sucrose, sorbitol or mannitol on the stability of the antibody can be improved to a certain extent; (2) for stability of e.g. antibody charge, the rate of decrease of the antibody's main charge is lowest, as low as 6.25%/week, with main charge as high as 61.2%; (3) with respect to the viscosity of the formulations, the arginine-only formulation viscosity was below 5cP (Table 5), significantly below the other respective group prescriptions, and in particular was only about 20-25% of the sucrose-containing formulation group. In addition, arginine salts significantly improved the viscosity of sucrose, sorbitol or mannitol containing formulations by about 50%.

High concentration antibody solutions are generally more likely to cause antibody aggregation, precipitation, etc., resulting in decreased antibody stability, and increased solution viscosity leading to difficulty in administration by injection (particularly subcutaneous or intramuscular injection). Through the above experiments, we found that arginine salt not only can ensure the stability of the antibody JS002, but also can significantly reduce the liquid viscosity in the preparation liquid preparation. In particular, antibody stability and solution viscosity effects are best when the buffer is a histidine buffer (pH 5.5 or 6.0), containing only one stabilizer of arginine salt.

Table 3: prescription information in stabilizer screening experiments

Table 4: summary of stabilizer screening test results

Table 5: viscosity of each formulation

Prescription number	4	5	6	7	8	9	10	11	12	13	14	15	16	17
															Viscosity (cP)	25.3	22.5	4.8	4.3	16.0	18.4	14.1	17.9	12.2	11.9	7.8	8.1	8.0	8.0

Example 3: surfactant screening experiments

The addition of surfactants to liquid formulations is commonly used as an agent to protect proteins such as antibodies from air/solution interface-induced stress, solution/surface-induced stress during storage to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation, which facilitates the stabilization of the physicochemical properties of the antibodies. Polysorbate 20 or polysorbate 80 was added to each of the formulations containing 20mM histidine buffer (pH 6.0), 130mM arginine hydrochloride and 150mg/ml JS002 at different concentrations (0-0.5%), and the mixture was allowed to stand at 40 ℃ for 2 weeks and then analyzed. As shown in table 6, the SEC-HPLC monomer content and CEX-HPLC main peak content measurements show no significant difference in thermal stability of the antibody JS002 in formulations containing different concentrations (0-0.5%) of polysorbate 20 or polysorbate 80, but all maintain high stability.

Table 6: surfactant screening

Example 4: long term stability study of formulations

Liquid pharmaceutical preparations containing therapeutic antibodies are typically stored at 2-8 ℃, so it is important that the formulations maintain high stability over long term storage. Based on the screening results above, we designed 4 formulations for long term stability studies of the formulations.

4 formulations of the formulations shown in Table 7 were stored in clear vials, and after standing at 2-8 ℃ for several months, each sample was analyzed. Stability was assessed by the following parameters: (a) visual appearance; (b) detection of insoluble particles by light blocking (OD405 nm); (c) the pH value; (d) detecting the molecular weight of the antibody by a CE-SDS method; (d) SEC-HPLC measures the content of antibody monomer (mass standard:noless than 97.0%), polymer (mass standard:nomore than 3.0%) or fragment (mass standard:nomore than 1.0%); (e) measuring the main charge (mass standard: more than or equal to 70.0 percent), the acidic charge (mass standard: less than or equal to 30.0 percent) or the alkaline charge content (mass standard: less than or equal to 15.0 percent) of the antibody by CEX-HPLC; (f) detecting the antibody binding activity by an ELISA method (mass standard: 70-130% of a reference substance); and (g) the biological activity of the antibody (HepG2 cell uptake LDL assay, quality standard: 70% -130% of reference). As shown in Table 8, the 4 formulations showed very good stability during storage at 2-8 deg.C for 1-12 months.

Table 7: preparation formula for long-term stability research of preparation

Table 8: long term stability data for formulation formulations

Example 5: room temperature storage stability of high concentration anti-PCSK 9 antibodies

Liquid pharmaceutical preparations containing therapeutic antibodies typically need to be stored at 2-8 ℃ until the end of the storage period. Thus, refrigeration by the patient is required between the time of purchase and use of the drug. Depending on the proposed dosing regimen, this may lead to storage times up to several weeks being taken up by the patient in the case of self-administered drugs. Thus, drugs that do not need to be stored under refrigerated conditions show a significant increase in patient convenience for home care products and a reduction in the impact of drug quality in case of incorrect storage, thereby reducing the monitoring of complaints rates and temperature excursions.

As shown in Table 9, the formulations disclosed herein (recipe number: 20) have a higher stability against protein degradation, with the resulting degradation kinetic parameters measured at 25 ℃ meeting the requirements of environmental storage for up to 6 months.

Table 9: summary of formulation accelerated stability data (25. + -. 2 ℃ C.)

Example 6: ForteBio affinity assay (biological light interferometry)

ForteBio affinity assay was performed according to the current method. Briefly, 4mg of JS002 (recipe No. 29) and Evolcumab (140 mg/mL/count, available from AMGEN, lot No. 1063135) were exchanged by 100-fold with a phosphate buffer solution in a 10KD ultrafiltration tube, and the protein content was measured at an absorbance of 280nm after exchange, and the concentration was adjusted to 2 mg/mL. The Biotin was taken out, equilibrated to room temperature, and 2mg of Sulfo-NHS-Biotin was weighed and added to 300. mu.L of ultrapure water, to obtain a 10mM Biotin mother liquor. 2mg/mL JS002 and Evolcumab 1mL each were placed in a new EP tube, following the protein: biotin 1:6, add 8 μ L of biotin stock solution. Mixing, and reacting at room temperature for 1-2 h. After the biotin reaction is finished, performing 100-fold liquid change in a 10KD ultrafiltration tube by using a phosphate buffer solution, measuring the protein content by using an absorption value of 280nm after the liquid change, and adjusting the concentration to be within the range of 1-2 mg/mL. The biotinylated protein is packaged into 0.1mL per tube, stored at-80 ℃ and frozen and thawed no more than 1 time. Respectively coupling biotinylated JS002 and Evolocumab antibodies (5 mu g/mL) to Streptavidin (SA) bioprobes, balancing experimental buffer solutions (0.1% BSA, 0.02% Tween-20 and 1xPBS) for 300s, then sequentially injecting diluted PCSK9 with different concentrations into the JS002 plate and the Evolocumab plate respectively, combining for 300s, and dissociating for 1800 s. The affinity constant was calculated according to the formula KD ═ koff/kon.

The test results are shown in table 10. Fortebio results show that the JS002 is obviously superior to Evolcumab which is a medicine with the same target point in the binding affinity with PCSK 9.

Table 10: fortebio comparison of JS002 and Repatha affinity for human PCSK9

	KD(M)	kon(1/Ms)	kdis(1/s)
				JS002	2.15-11	6.02E+04	1.30E-06
Evolocumab	6.51-10	4.92E+04	3.21E-05

Example 7: JS002 cell biological activity study (HepG2 cell LDL uptake method)

The true bookThe assay assesses the uptake of LDL from HepG2 cells exposed to human PCSK9-D347Y by JS002 (prescription No.: 29) at the cellular level and compared with the marketed co-targeted drug Evolcumab (140 mg/ml/count, from AMGEN, batch No.: 1063135). Briefly, human hepatoma cell line (HepG2) cells (ATCC, batch No.: 62591368) were plated at 2.0 × 10⁴Density of cells/well plating (80. mu.L per well), 37 ℃, 7% CO₂The culture was carried out overnight. Respectively carrying out concentration gradient dilution on JS002 antibody and Evolcumab (initial concentration is 20 mu g/mL, 2 times concentration gradient dilution), adding 10 mu L of antibody diluent into HepG2 cells, incubating for half an hour, simultaneously diluting the antigen to 1 mu g/mL, adding 10 mu L of antigen diluent into HepG2 cells, and incubating the antigen antibody and the cells for 4-6 h. Fluorescently labeled LDL (3. mu.g/mL) was added, incubated with the cells for 16-18h, and the amount of intracellular uptake of fluorescence was detected by a microplate reader.

The experimental result is shown in figure 1, and the result shows that JS002 can be combined with cell surface human PCSK9-D347Y, so that the combination of the JS002 and LDLR is inhibited, and the combination and the uptake of LDLR to LDL are increased. Moreover, JS002(EC50 ═ 92.68ng/mL) has a significantly better effect in promoting LDL endocytosis than Evolcumab (EC50 ═ 151.1 ng/mL).

Example 8: study on hyperlipidemia rhesus monkey JS002 lipid-lowering effect

19 hyperlipidemic rhesus monkeys (LDL is more than or equal to l.3mmol/L) were selected for the experiment and divided into 4 groups: atorvastatin calcium groups (1-4 weeks with l.2mg/kg, 5-8 weeks with 2.4mg/kg, 4), JS002 high dose groups (12mg/kg, 5), JS002 low dose groups (4mg/kg, 5) and placebo groups (5), atorvastatin calcium groups were orally administered for 56 days continuously, and the elution period was 21 days, JS002 high dose groups and JS002 low dose groups were administered once every 4 weeks for 2 times, and 78 days were continuously observed, and main efficacy analysis indexes (LOL-C, TC, HDL-C, ApoAL, B and TG), secondary efficacy indexes (Apobody weight), and changes in safety indexes (blood, blood routine indexes and clinical observation) were periodically observed during the administration period and the elution period, and JS002 (prescription No. 20) was analyzed and evaluated for the blood lipid-lowering effect on Hosta monkeys.

The results of the experiment are shown in FIG. 2. In the experiment, the blood lipid level of the placebo group is stable, the drug effect result of the positive control atorvastatin calcium group is similar to that reported in clinical literatures, and the experiment system is stable and reliable.

Under the experimental system, the JS002 high-dose test sample of 12mg/kg (equivalent to 420mg/70kg of human body with clinical planned dose, one injection every 4 weeks and subcutaneous injection for 2 times) has extremely obvious effect of reducing the LOL-C of the hyperlipidemic rhesus monkey, and the reduction level of D2-D71 after the administration of the test sample compared with the baseline LDL-C can be maintained at 40% -70%; the JS002 low dose group of 4mg/kg (equivalent to the clinically planned dose of 140mg/70kg for a human, one injection every 4 weeks, subcutaneous injection for 2 times), compared with the baseline, the D2-D57 reduced level can be maintained at 20% -70%, and the D57-D78 drug effect is obviously weakened and rebounded. The intensity and duration of action of the JS002 high dose group were superior to those of the JS002 low dose group (fig. 2). No significant changes were seen in TG, FPG, ApoAl and HDL-C throughout the dosing period; the safety indexes are not changed related to the administration.

Example 9: cynomolgus pharmacokinetic study

The pharmacokinetic properties of healthy cynomolgus monkeys after a single or multiple administration of different doses of JS002 (prescription No. 20) were observed in the experiment. The cynomolgus monkey experimental groups and dosing are shown in table 11. Wherein A, B, C, D groups were administered in single doses of 2mg/kg, 10mg/kg, 50mg/kg and 10mg/kg, respectively, wherein A, B and C groups were administered subcutaneously and group D was administered intravenously. Group E was given as a 10mg/kg continuous dose once a week four times a week.

Table 11: animal grouping and dosing

Note:

sc subcutaneous injection.

Iv intravenous injection.

QW once weekly.

Serum samples were prepared after sampling and quantitative detection was performed by a methodologically validated enzyme-linked immunosorbent assay, with the results shown in table 12.

Table 12: cynomolgus pharmacokinetic parameters (mean. + -. standard deviation)

Note: NA indicates no detection.

The results show that after a single subcutaneous injection of JS002, a single dose of the test drug (2, 10, 50mg/kg), the serum drug exposure level increased with increasing dose, showing a non-linear pharmacokinetic profile. JS002 eliminating phase half-life period t_1/256-74h, and the effective half-life period within 336h is 50-139 h.

The bioavailability of the tested drug JS002 in 10mg/kg after single subcutaneous injection of the cynomolgus monkey is 89.15%.

After the cynomolgus monkey is continuously injected with JS002 of 10mg/kg (1 time per week and 4 times per week) for multiple times, the exposure amount of the medicine in the body is obviously higher than that of a single administration group with the same dose, and the medicine is obviously accumulated in the body.

Example 10: cynomolgus monkey immunotoxicity and immunogenicity studies

This test was conducted by repeating the toxicity test of JS002 (prescription No. 20) administered to a test drug by subcutaneous injection to cynomolgus monkeys for four weeks. According to the GLP specification requirement of CFDA, the possible immune toxicity and immunogenicity of the recombinant humanized anti-PCSK 9 monoclonal antibody injection are evaluated. Four dose groups, namely an excipient control group (0mg/kg), a low dose group (30mg/kg), a medium dose group (100mg/kg) and a high dose group (000 mg/kg), are set in the experiment, and 10 animals and male and female are respectively arranged in each group. The administration was l times per week for 4 weeks with a recovery period of 4 weeks. Within the study dose range, after the cynomolgus monkey is injected with excipient and the test drug JS002 at different doses (30, 100, 300mg/kg) for multiple times of subcutaneous injection, no anti-drug antibody can be detected in blood samples of all individuals at different time points, and the sample positive rate and the individual positive rate of each group are 0.0%. The results suggest that the test drug JS002 was less immunogenic in cynomolgus monkeys after multiple subcutaneous injections at different doses (30, 100, 300 mg/kg). The kidney tissues of all groups of animals are subjected to immune complex detection, and no immune complex deposition phenomenon is found. Other immune related indexes show that the absolute count and the classified count of the white blood cells, the immunoglobulin level and the AIG ratio of each group of animals, the gross anatomical observation of lymph organs/tissues, the weight/coefficient of thymus and spleen organs and the like have no abnormal change of the mutual honoring of the medicines, and the distribution of lymphocyte subgroups has no change of the regularity related to the medicines. At the same time, histopathological examination showed normality.

Example 11: recombinant humanized anti-PCSK 9 monoclonal antibody injection (prefilling automatic injector)

[ PRODUCT NAME ]

Recombinant humanized anti-PCSK 9 monoclonal antibody injection (prefilling automatic injector)

[ COMPOSITION ] A

Recombinant humanized anti-PCSK 9 monoclonal antibody injection (pre-filled auto-injector): shanghai Junshi bioengineering, Inc., the components are detailed as follows:

1. recombinant humanized anti-PCSK 9 monoclonal antibody injection (pre-filled syringe):

1.1. recombinant humanized anti-PCSK 9 monoclonal antibody injection: shanghai Junshi Biotechnology Co., Ltd

1.2. Prefilled syringe barrel (containing needle and cap): becton, Dickinson and Company

1.3. Pre-filled syringe piston: west Pharmaceutical Services, Inc.

2. An automatic injector: YpsoMate (Ypsomerd AG).

Wherein, the product is sterile water injection with the specification of 150mg (1mL) per injection for subcutaneous injection. Each 1mL of the disposable prefilled automatic syringe comprises: the recombinant humanized anti-PCSK 9 monoclonal antibody (JS002, prescription No. 20) or the recombinant humanized anti-PCSK 9 monoclonal antibody (JS002, prescription No. 29).

The prefilled syringe is an inner packing material of medicine, and is directly contacted with liquid medicine, and its components mainly include needle tube (including needle head), needle head protective cap and piston.

The automatic injector consists of an upper part and a lower part, wherein the upper half part comprises a spring pushing device, and the lower half part comprises a prefilled injector storage bin, a drug delivery trigger button and a protective cap. The fitting is not in direct contact with the medical fluid.

[ Combined means ]

The drug manufacturers fill the recombinant humanized anti-PCSK 9 monoclonal antibody injection liquid in the prefilled syringe (figures 3, a and b), then assemble the prefilled syringe in the automatic syringe (figure 3, c), assemble the recombinant humanized anti-PCSK 9 monoclonal antibody (prefilled automatic syringe) (figure 3, d) for one-time use. The product can be directly used by patients without any combination.

[ METHOD OF USE ]

The administration is subcutaneous.

In application, the abdomen (except 5 cm area around umbilicus), thigh or upper arm (only when other people assist injection) can be selected, and the injection site is cleaned with alcohol cotton ball, and the skin is kept dry. In preparation for injection, the cap of the auto-injector is removed immediately. The injection site is then stretched or pinched to form a tight surface. The auto-injector is then placed on the skin at a 90 degree angle and pressed down hard until an audible "click" is heard, indicating the start of the injection. The automatic injector continues to be pressed against the skin until a second "click" is heard, while a change in the colour of the viewing window is visible, which signifies the end of the injection. After injection is finished, the automatic injector is pulled out of the skin, and the needle head protection device automatically returns to the original position to shield the needle head. The injection time is generally no more than 15 seconds. The prefilled automatic injector is disposable.

Sequence listing

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SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

SHANGHAI JUNSHI BIOENGINEERING Co.,Ltd.

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