Anti-VEGFR2 antibody preparation

Abstract

The invention provides an anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody preparation which comprises an anti-VEGFR2 antibody, a buffer solution, a protein protective agent and a surfactant, and the pH (potential of hydrogen) value of the antibody preparation is 5.0-6.7. The anti-VEGFR2 antibody preparation of the present invention can maintain the stability of the antibody in a liquid form. In addition, the invention also relates to application of the anti-VEGFR2 antibody preparation in preparation of drugs for preventing and / or treating diseases caused by tumor angiogenesis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to an anti-VEGFR2 antibody preparation, and also to use of the antibody preparation in preventing and / or treating diseases. Technical Background

[0002] Studies have shown that the formation of new blood vessels plays an important role in promoting the growth and invasion of local tumors. Specifically, new blood vessels can provide cancer cells with oxygen and nutrients, cytokines and growth factors, as well as escape pathways that tend to metastasize. VEGF and its receptor family play a very important role in this process. Currently known VEGF receptors include VEGFR-1 (Flt-1, fms-like tyrosine kinase), VEGFR-2 (also known as KDR / Flk-1, kinase insert chimeric receptor, fetal liver kinase-1), VEGFR-3 (Flt-4), neuropilin-1, and neuropilin-2. Human VEGF receptor 2 (VEGFR-2), also known as KDR (kinase insert domain-containing receptor, KDR) is the main signal transduction receptor for VEGF, which is involved in the migration, proliferation, and survival of new vascular endothelial cells.

[0003] Recombinant anti-VEGFR2 fully human monoclonal antibody is a humanized VEGFR2 antagonist that specifically binds to VEGFR2, blocks the binding of VEGFR2 to its ligands (VEGF-A, VEGF-C, VEGF-D), thereby inhibiting the activation of VEGFR2 ligands, and ultimately inhibiting the proliferation and migration of ligand-induced human endothelial vascular cells. The primary structure of the recombinant anti-VEGFR2 fully human monoclonal antibody is complex, with poor stability, and it is easy to produce protein aggregates and insoluble particles.

[0004] Therefore, it is necessary to develop a suitable formulation for recombinant anti-VEGFR2 fully human monoclonal antibodies to obtain antibody preparations with good stability. Summary of the invention

[0005] In the present application, the inventors have developed a recombinant anti-VEGFR2 fully human monoclonal antibody liquid preparation with excellent stability, which is beneficial for clinical use and drug storage.

[0006] Liquid preparations

[0007] One aspect of the present invention provides a liquid preparation. In some embodiments, the liquid preparation comprises:

[0008] (1) anti-VEGFR2 antibody at a concentration of 2-20 mg / ml;

[0009] (2) Buffer solution with a concentration of 5-20 mg / ml;

[0010] (3) a protein protectant at a concentration of 50-200 mg / ml;

[0011] (4) a surfactant at a concentration of 0.002% to 0.02%;

[0012] Wherein, the pH value of the liquid preparation is 5.0-6.7.

[0013] In some embodiments, the anti-VEGFR2 antibody is a recombinant anti-VEGFR2 fully human monoclonal antibody produced by a Chinese hamster ovary cell expression system.

[0014] In some embodiments, the heavy chain variable region of the anti-VEGFR2 antibody contains CDR1 as shown in SEQ ID NO:5, CDR2 as shown in SEQ ID NO:6; CDR3 as shown in SEQ ID NO:7; and / or, the light chain variable region of the anti-VEGFR2 antibody contains CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9; CDR3 as shown in SEQ ID NO:10.

[0015] In some embodiments, the anti-VEGFR2 antibody has a heavy chain variable region and a light chain variable region, wherein:

[0016] - the heavy chain variable region has SEQ ID NO: 1 or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% identical thereto; and / or

[0017] - the light chain variable region has SEQ ID NO: 2, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% identical thereto.

[0018] In some embodiments, the anti-VEGFR2 antibody has a heavy chain variable region (VH) as shown in SEQ ID NO:1; and a light chain variable region (VL) as shown in SEQ ID NO:2.

[0019] In some embodiments, the anti-VEGFR2 antibody is an IgG type antibody.

[0020] In some embodiments, the anti-VEGFR2 antibody has a light chain constant region as shown in SEQ ID NO:3, and / or a heavy chain constant region as shown in SEQ ID NO:4.

[0021] In some embodiments, the anti-VEGFR2 antibody has a heavy chain and a light chain, wherein:

[0022] - the heavy chain has SEQ ID NO: 11 or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% identical thereto; and / or

[0023] - The light chain has SEQ ID NO: 12, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% identical thereto.

[0024] In some embodiments, the anti-VEGFR2 antibody has a light chain as shown in SEQ ID NO:11 and a heavy chain as shown in SEQ ID NO:12.

[0025] In some embodiments, the concentration of the anti-VEGFR2 antibody is 1-100 mg / ml, for example, 1-50 mg / ml, 1-20 mg / ml, and preferably 5-15 mg / ml.

[0026] In some embodiments, the anti-VEGFR2 antibody is at a concentration of about 10 mg / ml.

[0027] In some embodiments, the surfactant is selected from polysorbate surfactants, for example, polysorbate-20, polysorbate-80, polysorbate-60, polysorbate-40; or Pluronic.

[0028] In some embodiments, the surfactant is polysorbate 80.

[0029] In some embodiments, the concentration of polysorbate 80 is 0.005%-0.02% (w / w); preferably, the concentration of polysorbate 80 is about 0.005% (w / w), about 0.01%% (w / w) or about 0.02% (w / w); more preferably, the concentration of polysorbate 80 is about 0.005% (w / w).

[0030] In some embodiments, the buffer is selected from the group consisting of histidine buffer, citrate buffer and phosphate buffer; preferably, the histidine buffer is histidine-histidine hydrochloride buffer, the citrate buffer is citric acid / sodium citrate buffer, and the phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer; more preferably, the buffer is histidine-histidine hydrochloride buffer.

[0031] In some embodiments, the concentration of the buffer is 5-25 mM, such as 5-15 mM, preferably, the concentration of the buffer is about 10 mM. Moreover, the buffer is preferably a histidine-histidine hydrochloride buffer of about 10 mM.

[0032] In some embodiments, the protein protectant is selected from glycine, sucrose, sodium chloride and any combination thereof. In some embodiments, the total concentration of the protein protectant is 50-300mM, and preferably the concentration of each protein protectant is 50-150mM. Preferably, the protein protectant is a combination of glycine and sodium chloride. In some embodiments, the concentration of glycine is 100-150mM, such as about 133mM, and the concentration of sodium chloride is 50mM-150mM, such as 75-120mM; preferably, the concentration of sodium chloride is about 75mM.

[0033] In some embodiments, the pH value of the liquid formulation is 5.5-6.5, for example, about 5.5, 5.7, 5.9, 6.1, 6.3, or 6.5; preferably, the pH value of the liquid formulation is about 6.0.

[0034] In some embodiments, preferably, the liquid preparation comprises: about 10 mg / ml anti-VEGFR2 antibody, about 10 mM histidine-histidine hydrochloride buffer, about 133 mM glycine, about 75 mM sodium chloride, about 0.005% (w / w) polysorbate 80, and the pH value of the liquid preparation is about 6.0.

[0035] In some embodiments, the liquid preparation is an injection, more preferably a subcutaneous injection or an intravenous injection. In some embodiments, the liquid preparation is an intravenous infusion.

[0036] On the other hand, the present invention also provides a solid antibody preparation, which is obtained by solidifying the liquid antibody preparation of the present invention, for example, by crystallization, spray drying, freeze drying, from the liquid antibody preparation of the present invention. In some embodiments, the solid antibody preparation is a lyophilized powder injection. The solid antibody preparation can be reconstituted in an appropriate solvent before use to form a reconstituted preparation of the present invention. The reconstituted preparation is also a liquid antibody preparation of the present invention. The solvent that can be used to reconstitute the liquid preparation of the present invention includes, but is not limited to, water for injection.

[0037] On the other hand, the present invention also provides a kind of article, it comprises the container that liquid preparation of the present invention is housed.Suitable container is for example plastic container, metal alloy container or glass container, such as ampoule, vial or syringe.In some preferred embodiments, described article can further include instructions for use.

[0038] The liquid preparation of the present invention is stable. In certain embodiments, the liquid preparation of the present invention is stable for at least 10 days, at least 20 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, at least 36 months, or longer, at about -80°C to about 45°C, such as -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C. In some embodiments, the liquid formulations of the invention are stable for at least 10 days, at least 20 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, or longer when stored at a temperature of about 5°C to 40°C (e.g., at a temperature of about 25°C or a temperature of about 40°C) and shaking.

[0039] In some embodiments, the stability of the preparation after storage can be indicated by detecting one or more indicators selected from the following: the appearance of the preparation, protein concentration, pH value, particle size distribution, insoluble particle count, osmotic pressure, antibody monomer purity, charge variants and biological activity. In some embodiments, the stability of the liquid preparation of the present invention can be detected in a forced experiment of high temperature stress, such as after storage at 40°C ± 2°C for at least 1 week, 2 weeks or 1 month, or in an accelerated experiment, such as after storage at 25°C ± 2°C for at least 1 month or 2 months, or in a long-term experiment, such as after storage at 5°C ± 3°C for at least 2 months or 3 months. In some embodiments, in a storage assay experiment, such as in a forced experiment or an accelerated experiment, the preparation is shaken, such as 100r / min shaken for 3 days, 5 days, 1 week or 2 weeks to detect the stability of the preparation.

[0040] Therapeutic uses of liquid preparations

[0041] The anti-VEGFR2 antibody liquid preparation provided by the present invention can be used for preventing and / or treating tumors and / or diseases caused by tumor angiogenesis by inhibiting tumor angiogenesis.

[0042] Therefore, in another aspect, the present invention also relates to the use of the liquid preparation of the present invention in the preparation of a medicament for preventing and / or treating diseases such as tumors and / or tumor neovascularization. In some embodiments, the tumor includes but is not limited to: gastric cancer, esophageal gastric junction adenocarcinoma, non-small cell lung cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, melanoma, renal cell carcinoma, glioblastoma, ovarian cancer and prostate cancer.

[0043] In some embodiments, the anti-VEGFR2 antibody liquid formulation provided by the present invention can be administered to a subject (e.g., a human) by a suitable administration route, including but not limited to injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), transdermal, mucosal, nasal, respiratory and / or oral administration.

[0044] Definition of terms

[0045] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0046] As used herein, the term "antibody" is used in the broadest sense, including various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity. For example, an antibody can be an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the corresponding antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE. In each case, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region consists of 3 or 4 domains (CH1, CH2 and CH3, and CH4); each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of one domain, CL. The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0047] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0048] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. In addition, the corresponding relationship between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0049] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the IMGT, Kabat, Chothia or AbM numbering systems.

[0050] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homogeneous antibody molecules (i.e., a group of identical antibody molecules except for natural mutations that may occur spontaneously). Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homogeneous antibody population, and is not to be understood as requiring the preparation of the antibody by any particular method.

[0051] As used herein, the term "humanized antibody" refers to an antibody that can be prepared by replacing a portion of a human antibody with a portion of a non-human antibody prepared by immunizing a mammal other than a human. Typically, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). For example, a humanized antibody can be prepared by transplanting a CDR sequence derived from the germline of another mammalian species onto a human framework sequence.

[0052] As used herein, the term "fully human antibody" refers to antibodies that only contain sequences derived from human germline immunoglobulin sequences. It is understood by those skilled in the art that fully human antibodies do not exclude the inclusion of amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced by random or in vitro site-specific mutations or by in vivo somatic mutations. Fully human antibodies can be prepared by using transgenic mice into which human immunoglobulin genes have been transferred (XenoMouse (Chemical Biology (2000), vol.7, issue 8, p.R185-6), HuMAb-Mouse (Infection and Immunity (2002), vol.70, issue 2, p.612-9), TC mouse (Biotechnology and Genetics Enginnering Revew (2002), vol.19, p.73-82), and KM mouse (Cloning Stem Cells (2002), vol.4, issue 1, p.91-102)), and the target antibody can be mass-produced by isolating antibody-producing lymphocytes from mice and forming hybridomas. Fully human antibodies can also be prepared by phage display (FEBS Letter (1998), vol.441, p.20-24). In this method, by using a phage that integrates human antibody genes into a circular single-stranded DNA, a fully human antibody can be expressed on the phage surface in the form of a fusion with the coat protein of the phage. Fully human antibodies can also be prepared by using transgenic mice to which human immunoglobulin variable region genes have been transferred, which can produce chimeric antibodies consisting of fully human variable regions and mouse constant regions, and then fully human variable regions can be connected to human immunoglobulin constant regions using methods known in the art to produce fully human antibodies. For example, the DNA encoding VH can be operably connected to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region.For example, the DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0053] As used herein, the term "IgG-type antibody" refers to an immunoglobulin molecule of the IgG isotype consisting of two heavy chains and two light chains, wherein each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2 and CH3), and each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).

[0054] In this article, "sequence identity" refers to the degree of sequence identity based on nucleotide or amino acid by nucleotide in a comparison window. "Sequence identity percentage ratio" can be calculated in the following manner: the sequences of two optimal comparisons are compared in a comparison window, it is determined that there are identical nucleic acid bases (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in two sequences The number of positions is to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (that is, window size), and the result is multiplied by 100, to produce the sequence identity percentage ratio. In order to determine the optimal comparison carried out for the sequence identity percentage ratio, it can be realized in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for comparing sequences, including any algorithm required for achieving maximum alignment within the full-length sequence being compared or within the target sequence region. In this article, if not otherwise specified, sequence identity is determined over the full length of the sequence to be compared.

[0055] The term "antibody preparation" refers to a preparation that is in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain other components that are unacceptably toxic to the subject to whom the preparation is to be administered. Such antibody preparations are generally sterile. Typically, a pharmaceutically acceptable excipient is included in the antibody preparation. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a subject mammal so that an effective dose of the active ingredient used in the preparation can be delivered to the subject. The concentration of the excipient is compatible with the mode of administration, and may be, for example, acceptable for injection.

[0056] In this article, "stable" antibody formulations refer to antibodies in the formulation that retain an acceptable degree of physical stability and / or chemical stability after being stored under specific conditions. Typically, after a period of storage time, the formulation does not show aggregation, precipitation, turbidity and / or denaturation; or shows very little aggregation, precipitation, turbidity and / or denaturation, then the antibody can be considered to "maintain its physical stability" in the formulation. In some embodiments, the physical stability of the formulation can be measured by measuring the amount of visible aggregates and / or soluble aggregates in the formulation after storage for a specific time under specific temperatures and conditions and / or the purity of the antibody monomer. Typically, after a period of storage time, the antibody in the formulation does not show significant chemical changes, then the antibody can be considered to "maintain its chemical stability" in the formulation. In some embodiments, the stability of the formulation can be indicated by measuring the percentage change value of the charge variant of the antibody in the formulation after storage for a specific time under specific temperatures and conditions.

[0057] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.

[0058] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or is at risk of suffering from a disease.

[0059] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0060] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. In this article, when "about" is used to describe a measurable value (e.g., concentration of a substance, time, temperature, etc.), it is meant to include a range of ±10% of a given value.

[0061] As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0062] Embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become implementable for those skilled in the art.

[0063] Sequence information

[0064] The information of the sequences involved in the present invention is described in the following Table 1:

[0065] Table 1: Sequence information

[0066]

[0067] DETAILED DESCRIPTION

[0068] The present invention is now described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). It is known to those skilled in the art that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention.

[0069] Example

[0070] Material

[0071] The anti-VEGFR2 antibody used in the following examples is an IgG-type fully human anti-VEGFR2 antibody consisting of a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NO:12.

[0072] General method description

[0073] Protein content determination

[0074] Determined by UV spectrophotometry.

[0075] pH determination

[0076] The determination was carried out in accordance with General Chapter 0631 “pH Determination Method” of the 2015 edition of the Chinese Pharmacopoeia.

[0077] Insoluble particle determination

[0078] The light obstruction method was used to determine the content of insoluble particles according to General Chapter 0903 “Insoluble Particles Test Method” of the 2015 edition of the Chinese Pharmacopoeia.

[0079] Osmolality measurement

[0080] The freezing point depression method was used to test the product in accordance with General Chapter 0632 “Osmotic pressure molar concentration determination method” of the 2015 edition of the Chinese Pharmacopoeia.

[0081] Differential Scanning Calorimetry (DSC)

[0082] The Tm values ​​of the samples were analyzed using a microcal PEAQ-DSC.

[0083] Dynamic light scattering (DLS) detection

[0084] Detection was performed using a DLS instrument. Take 50 μl of sample to a clean absorption pool, select "Protein" for the detection mode, select "Water" for the medium, set the temperature to 25°C, preheat for 60s to 180s, select the micro-absorption pool, and use the automatic acquisition mode for 3 acquisitions. Analyze using the DLS instrument analysis software, using the non-negative least squares (NNLS) fitting algorithm, and obtain the Z-average particle size (Z-Average) and distribution coefficient (PDI) of each sample based on the light intensity (Intensity) distribution.

[0085] Size exclusion-high-performance liquid chromatography (SEC-HPLC) was tested according to the general rule 0512 of the 2015 edition of the Chinese Pharmacopoeia. The chromatographic column was filled with a chromatographic gel suitable for separating proteins with a molecular weight of 10 to 500 kD (such as TSKgel G3000SWXL, 300×7.8 mm or other suitable chromatographic columns); the mobile phase was a mixed solution of disodium hydrogen phosphate and sodium chloride (50 mmol / L disodium hydrogen phosphate, 300 mM sodium chloride, pH to 7.0); the flow rate was 1.0 ml per minute; the detection wavelength was 280 nm. Take 10 μl of the test solution and inject it into the liquid chromatograph.

[0086] Cation exchange high performance liquid chromatography (CEX-HPLC) detection

[0087] The determination was carried out according to the general rule 0512 of the 2015 edition of the Chinese Pharmacopoeia. A weak cation exchange column (such as ProPacWCX-10, 4mm×250mm or other suitable columns) was used, with phase A (4mmol / L 2-methylpiperazine, 4mmol / L imidazole, 4mmol / L tris(hydroxymethyl)aminomethane, pH5.0) and phase B (4mmol / L 2-methylpiperazine, 4mmol / L imidazole, 4mmol / L tris(hydroxymethyl)aminomethane, 16mmol / L sodium chloride, pH 10.8) as the mobile phases, the column temperature was set at 30°C, the flow rate was 1.0ml per minute, and the detection wavelength was 280nm. The reference and test samples were diluted with mobile phase A to contain about 1.0mg protein per ml, filtered through a 0.22mm mixed fiber membrane, and used as the test solution and reference solution. Take 100 μl of the test solution and reference solution respectively, inject them into the liquid chromatograph for gradient elution.

[0088] Example 1 Buffer system and pH screening

[0089] This example investigates the effects of different buffer systems on fully human anti-VEGFR2 antibody proteins. According to five preset prescriptions, the anti-VEGF2 antibody was formulated in a histidine / histidine hydrochloride buffer system at pH 5.5, 6.0 or 6.5, or in a phosphate buffer system at pH 6.5 or a citric acid buffer system at pH 6.0, to obtain five samples, in which the anti-VEGFR2 antibody protein (Sichuan Kelun Botai Biotechnology Co., Ltd., batch number 1) had a concentration of 10.0 mg / ml. Take 1.0 ml of the sample, filter and fill it into a 2 ml vial, add a stopper and cap it, and place it at 2-8 ° C and 40 ± 2 ° C for 4 weeks. Analyze the final protein content, pH value, DLS, SEC-HPLC and CEX-HPLC results. The specific formula and test results are shown in Table 2-1, Table 2-2 and Table 2-3.

[0090] The results show that under the set investigation conditions, the protein content and pH of the samples prepared in 5 different formulation buffer systems remain basically unchanged. See Table 2-1.

[0091] DSC was used to characterize the thermal stability of the antibody. The DSC results showed that the TmOnset of the protein in the histidine / histidine hydrochloride buffer system at pH 6.0 and 6.5 was the highest, indicating that the preparation had good thermal stability. See Table 2-3, which shows TmOnset (i.e., the initial thermal transition temperature) and Tm1 (i.e., the median thermal transition temperature).

[0092] The particle size and dispersion coefficient were measured by DLS. The DLS test results showed that the particle size of the sample was the smallest in the histidine / histidine hydrochloride buffer system, and all of them were single peaks. See Table 2-2.

[0093] The results of SEC-HPLC and CEX-HPLC showed that the purity of the samples did not decrease significantly after 4 weeks at 2-8℃. After 4 weeks at 40℃, the purity of the samples showed a significant downward trend, and the change trend was consistent; among them, the main peaks of the samples in the pH 6.0 histidine / histidine hydrochloride buffer system had the smallest decrease in SEC-HPLC and CEX-HPLC, with the main peaks decreasing by about 2.5% and 14.9%, respectively.

[0094] Based on the above screening results, 10 mM histidine / histidine hydrochloride at pH 6.0 was selected as the buffer system for anti-VEGFR2 antibodies.

[0095] Table 2-1 Buffer system / pH screening test results

[0096]

[0097]

[0098] Note: 05 and 40 refer to the test conditions of 2~8℃ and 40±2℃ respectively; T0 is the starting point; W is the time unit week, 4W=4 weeks.

[0099] Table 2-2 Buffer system / pH screening test results

[0100]

[0101] Note: 05 and 40 refer to the test conditions of 2~8℃ and 40±2℃ respectively; T0 is the starting point; W is the time unit week, 4W=4 weeks.

[0102] Table 2-3 Buffer system / pH+ screening DSC test results

[0103]

[0104] Note: T0 is the starting point.

[0105] Example 2 Excipient Screening

[0106] Based on the preparation buffer system / pH screening (10mM histidine / histidine hydrochloride, pH 6.0), 133mM glycine, 133mM sucrose and 150mM sodium chloride were used to conduct preparation stability studies, where, except for the sodium chloride excipient group, 75mM sodium chloride was added to the other two groups of excipients to adjust the osmotic pressure to the isotonic range (the osmotic pressure molar concentration range of normal human blood is 285-310mOsm / kg). See Table 3-1 for specific prescriptions.

[0107] The protein samples were replaced into the three groups of formulations by ultrafiltration and the protein concentration was adjusted to 10 mg / ml. 1 ml of the sample was put into a 2 ml vial, stoppered and capped, and then placed at 25 ± 2 °C for inspection. The inspection plan is shown in Table 3-1.

[0108] Table 3-1 Experimental plan for different excipients

[0109]

[0110] Note: X = appearance, protein concentration, pH, DLS, SEC-HPLC, CEX-HPLC; Y = DSC, osmotic pressure

[0111] DSC was used to analyze the stability of antibodies in the three groups of samples. The results showed that the thermal stability of sample 2-3 (only sodium chloride as excipient) was the lowest, with a TmOnset of 61.8°C; there was no significant difference in thermal stability between sample 2-1 (sodium chloride and glycine as excipients) and sample 2-2 (sodium chloride and sucrose as excipients), with TmOnsets of 63.1°C and 63.5°C, respectively.

[0112] The final appearance, protein content, pH value, DLS, SEC-HPLC and CEX-HPLC results of the three groups of samples were analyzed, as shown in Tables 3-2 and 3-3.

[0113] The results of protein concentration and pH showed that there was no significant difference in protein concentration and pH value among the three groups of samples after being observed at 25±2℃ for 4 weeks.

[0114] Sample 2-1 (sodium chloride and glycine as auxiliary materials) was observed at 25±2°C for 4 weeks, and its appearance was a colorless, slightly opalescent liquid; samples 2-2 (sodium chloride and sucrose as auxiliary materials) and 2-3 (only sodium chloride as auxiliary material) were observed at 25±2°C for 4 weeks, and visible filaments appeared on the surface.

[0115] DLS results showed that after being observed at 25±2℃ for 4 weeks, the protein particle sizes of the three groups of samples were distributed in the range of 11 to 13 nm, showing a single peak.

[0116] The results of SEC-HPLC and CEX-HPLC showed that after being observed at 25±2℃ for 4 weeks, the purity of the main peak of the three groups of samples showed a slight downward trend.

[0117] Based on the above results, 133 mM glycine and 75 mM sodium chloride are preferred as auxiliary materials.

[0118] Table 3-2 Results of screening of different auxiliary materials

[0119]

[0120] Note: 25 refers to the test conditions, which is 25±2℃; T0 is the starting point; W is the time unit, 4W=4 weeks.

[0121] Table 3-3 Results of screening of different auxiliary materials

[0122]

[0123] Note: 25 refers to the test conditions, which is 25±2℃; T0 is the starting point; W is the time unit, 4W=4 weeks.

[0124] Example 3 Surfactant content screening

[0125] Based on the screening of excipients, the content screening of polysorbate 80 was carried out. The concentrations of polysorbate 80 were designed to be 0, 0.005% (w / w), 0.01% (w / w) and 0.02% (w / w), respectively, and the test was conducted at 25±2°C, in the dark, and with shaking at 100 rpm for 4 weeks.

[0126] Adjust the protein concentration to 10mg / ml, take 1.0ml sample and fill it into 2ml vial, stopper and cap it before inspection. The inspection plan is detailed in Table 4-1.

[0127] Table 4-1 Surfactant screening inspection plan

[0128]

[0129] Note: X: appearance, protein concentration, pH value, SEC-HPLC, CEX-HPLC, DLS, insoluble particles, osmotic pressure; Y: biological activity.

[0130] The final appearance, protein content, pH value, DLS, SEC-HPLC and CEX-HPLC results of the three groups of samples were analyzed, as shown in Tables 4-2 and 4-3.

[0131] Samples of four different concentrations of polysorbate 80 were observed at 25±2℃ and 100rpm for 4 weeks. They all appeared as colorless, clear liquids; the protein concentrations were all around 10.0mg / ml; and the pH value was maintained at around 6.0.

[0132] DLS results showed that at 25±2°C and 100 rpm for 4 weeks, the particle sizes of samples with four polysorbate 80 concentrations were all distributed in the range of 11 to 12 nm, with a single peak.

[0133] The results of SEC-HPLC and CEX-HPLC showed that the main peak purity of the samples showed a downward trend when they were observed at 25±2℃ and 100rpm for 4 weeks. The SEC and CEX main peak purity of the samples containing 0.005% polysorbate 80 decreased the least, by 0.8% and 2.7% respectively; the SEC and CEX main peak purity of the samples containing 0.02% polysorbate 80 decreased the most, by 1.4% and 5.3% respectively. According to the results of SEC-HPLC and CEX-HPLC, when the content of polysorbate 80 was as low as 0.005%, the samples of prescription 3-2 still had good antibody physical stability and chemical stability compared with the samples with higher polysorbate 80 content.

[0134] The control of insoluble particles is an important indicator to ensure the safe use of preparations. Therefore, in this study, the increasing trend of insoluble particles in the three groups of samples was also analyzed. The results of the insoluble particle counting showed that: at 25±2℃, 100rpm for 4 weeks, the sample containing 0.005% polysorbate 80 had the least number of insoluble particles, and the sample containing 0.01% polysorbate 80 had a slightly higher number of insoluble particles; while the number of insoluble particles in the samples without polysorbate 80 and containing 0.02% polysorbate 80 was significantly higher. See Table 4-3.

[0135] Table 4-2 Screening results of different surfactant concentrations

[0136]

[0137] Note: The prescription composition is shown in Table 4-1; 25 means the test condition is 25±2℃; A means the test condition is shaking; T0 is the starting point; W is the time unit week, 4W=4 weeks.

[0138] Table 4-3 Screening results of different surfactant concentrations

[0139]

[0140] Note: The prescription composition is shown in Table 4-1; 25 refers to the test condition, which is 25±2℃; A means the test condition is shaking; T0 is the starting point; W is the time unit, 4W=4 weeks.

[0141] Based on the above results, the stability of the formulations containing 0.01% and 0.005% polysorbate 80 was further investigated in a high temperature forced experiment. Specifically, the stability of formulations 3-2 and 3-3 was compared under 2-8°C and 40±2°C, light-proof, and non-shaking conditions, and the results are shown in Tables 4-4 and 4-5.

[0142] After being observed at 2-8°C and 40±2°C for 4 weeks, the samples all appeared as colorless, clear liquids; the protein content was around 10.0 mg / ml; and the pH was maintained at around 6.0.

[0143] The DLS test results showed that the particle size distribution of the samples in the two formulations was 11-13 nm, and the osmotic pressure was maintained in the isotonic range (280-320 mOsm / kg).

[0144] The results of SEC-HPLC and CEX-HPLC showed that: after 4 weeks of observation at 2-8℃, the purity of the samples did not change significantly; after 4 weeks of observation at 40±2℃, the purity of the samples showed a downward trend. Compared with prescription 3-3, the SEC main peak and CEX main peak corresponding to the samples of prescription 3-2 decreased less, the SEC polymer increased less, and the CEX acid peak area increased less. Through comparative tests, it can be seen that prescription 3-2 is more conducive to maintaining the stability of the samples.

[0145] Table 4-4 Stability results at different surfactant concentrations

[0146]

[0147] Note: The prescription composition is shown in Table 4-1. 05 and 40 indicate that the test conditions are 2~8℃ and 40±2℃ respectively; T0 is the starting point; W is the time unit, 2W=2 weeks, 4W=4 weeks

[0148] Table 4-5 Stability results at different surfactant concentrations

[0149]

[0150] Note: The prescription composition is shown in Table 4-1. 05 and 40 are the test conditions of 2~8℃ and 40±2℃ respectively; T0 is the starting point; W is the time unit, 2W=2 weeks, 4W=4 weeks

[0151] From the above screening and comparison experiments, it can be seen that the formulation containing 0.005% polysorbate 80 is more advantageous for the antibody of the present invention. This advantage is obvious, especially considering the possibility of polysorbate 80 producing unstable products during long-term storage of the antibody formulation due to autooxidation or exposure to light.

[0152] Based on the above results, the preferred formulation is 10 mM histidine / histidine hydrochloride buffer system, 133 mM glycine, 75 mM sodium chloride, 0.005% (w / w) polysorbate 80, pH 6.0.

[0153] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A liquid preparation, comprising: (1) anti-VEGFR2 antibody at a concentration of 2-20 mg / ml; (2) Buffer solution with a concentration of 5-20 mg / ml; (3) a protein protectant at a concentration of 50-200 mg / ml; (4) a surfactant at a concentration of 0.002% to 0.02%; in, The pH value of the liquid preparation is 5.0-6.7, Wherein, the heavy chain variable region of the anti-VEGFR2 antibody contains CDR1 as shown in SEQ ID NO:5, CDR2 as shown in SEQ ID NO:6; and CDR3 as shown in SEQ ID NO:7; and the light chain variable region of the anti-VEGFR2 antibody contains CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9; and CDR3 as shown in SEQ ID NO:

10.

2. The liquid preparation according to claim 1, wherein the anti-VEGFR2 antibody is a recombinant anti-VEGFR2 fully human monoclonal antibody produced by a Chinese hamster ovary cell expression system.

3. The liquid preparation according to claim 1 or 2, wherein the anti-VEGFR2 antibody has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO:

2.

4. The liquid preparation according to any one of claims 1 to 3, wherein the anti-VEGFR2 antibody is an IgG antibody, Preferably, the antibody has a light chain constant region as shown in SEQ ID NO: 3, and / or a heavy chain constant region as shown in SEQ ID NO: 4, More preferably, the antibody has a heavy chain as shown in SEQ ID NO:11 and a light chain as shown in SEQ ID NO:

12.

5. The liquid preparation according to any one of claims 1 to 4, wherein the concentration of the anti-VEGFR2 antibody is 5-15 mg / ml; Preferably, the concentration of the anti-VEGFR2 antibody is about 10 mg / ml.

6. The liquid preparation according to any one of claims 1 to 5, wherein the surfactant is polysorbate 80.

7. The liquid preparation according to claim 6, wherein the concentration of polysorbate 80 is 0.005%-0.02% (w / w); Preferably, the concentration of polysorbate 80 is about 0.005% (w / w), about 0.01%% (w / w) or about 0.02% (w / w); More preferably, the concentration of polysorbate 80 is about 0.005% (w / w).

8. The liquid preparation according to any one of claims 1 to 7, wherein the buffer is selected from the group consisting of histidine buffer, citrate buffer and phosphate buffer; Preferably, the histidine buffer is histidine-histidine hydrochloride buffer, the citrate buffer is citric acid / sodium citrate buffer, and the phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer; More preferably, the buffer is histidine-histidine hydrochloride buffer.

9. The liquid formulation according to any one of claims 1 to 8, wherein the concentration of the buffer is 5-15 mM, preferably, the concentration of the buffer is about 10 mM.

10. The liquid preparation according to any one of claims 1 to 9, wherein the protein protective agent is selected from glycine, sucrose, sodium chloride and any combination thereof, and the total concentration of the protein protective agent is 50-300 mM; Preferably, the protein protecting agent is a combination of glycine and sodium chloride.

11. The liquid formulation according to claim 10, wherein the concentration of glycine is about 133 mM, and the concentration of sodium chloride is 75 mM-150 mM; Preferably, the concentration of sodium chloride is about 75 mM.

12. The liquid preparation according to any one of claims 1 to 11, wherein the pH value of the liquid preparation is 5.5-6.5; Preferably, the pH of the liquid formulation is about 6.

0.

13. The liquid preparation according to any one of claims 1-12, wherein the liquid preparation comprises: about 10 mg / ml anti-VEGFR2 antibody, about 10 mM histidine-histidine hydrochloride buffer, about 133 mM glycine, about 75 mM sodium chloride, about 0.005% (w / w) polysorbate 80, and the pH value of the liquid preparation is about 6.

0.

14. An article of manufacture comprising a container containing the liquid formulation according to any one of claims 1 to 13.

15. Use of the liquid preparation according to any one of claims 1 to 13 in the preparation of a medicament for preventing and / or treating: tumors and / or diseases caused by tumor angiogenesis.

16. The use according to claim 15, wherein the tumor is selected from the group consisting of gastric cancer, esophagogastric junction adenocarcinoma, non-small cell lung cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, melanoma, renal cell carcinoma, glioblastoma, ovarian cancer and prostate cancer.

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