Separated anti-MASP2 antibody preparation and application

Abstract

The anti-MASP2 antibody preparation provided by the invention is high in long-term storage stability under the conditions of freezing and thawing, high temperature, acceleration and refrigeration, good stability of the preparation in the processes of preparation, transportation and storage can be ensured, and clinical medication safety and quality controllability are ensured.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an isolated anti-MASP2 antibody preparation and application. Background Art

[0002] The complement system is a major branch of the immune response, interacting with multiple immune mechanisms in both innate and adaptive immunity. It primarily activates through three distinct cascades: the classical pathway (CP), the lectin pathway (LP), or the alternative pathway (AP). Although each pathway has distinct initiating factors and unique, specific factors, they all involve the activation of C3 and C5 and lead to a common pathway, the formation of the membrane attack complex (MAC, C5b-9), which binds to the target cell membrane and causes cell lysis. Unique factors associated with LP activation include collectins (mannose-binding lectin, collectin-10, collectin-11), ficolins (ficolin 1, ficolin 2, ficolin 3), and proteins of the mannose-binding lectin-associated serine protease (MASP) family (MASP-1, MASP-2, MASP-3, MAp19, MAp44). Mannose-binding lectins, collectins, and ficolins are pattern recognition molecules (PRMs) that react with pathogen-associated pattern molecules (PAMPs) or damage-associated pattern molecules (DAMPs). DAMPs include endogenous ligands, such as abnormally glycosylated host cell surface structures. Although complement activation relies on direct lysis of pathogens / abnormal self-cells, these factors can act as opsonins, thereby facilitating phagocytosis (Cedzyński, Maciej, and Anna S Cancers vol.12,7 1792.4Jul.2020).

[0003] Dimers, trimers and / or higher oligomers of these PRMs trimeric structural subunits form complexes with homodimers of MASPs, wherein the homodimers of MASPs initially exist in the form of enzyme precursors (zymogens) (Kjaer, TroelsR et al. Molecular immunology vol.56, 4 (2013): 413-22). When PRMs bind to targets, MASPs are cleaved and activated (Dahl, MR et al. Immunity vol.15, 1 (2001): 127-35.; Degn, E etal. Journal of immunology (Baltimore, Md.: 1950) vol. 183, 11 (2009): 7371-8.; Héja, Dávid et al. Proceedings of the National Academy of Sciences of the United States of America vol. 109, 26 (2012): 10498-503.). Although MASP-2 can self-activate and has long been considered the central activator of the lectin pathway (Ambrus, Géza et al. Journal of immunology (Baltimore, Md.: 1950) vol. 170, 3 (2003): 1374-82.; Gál, Péter et al. The Journal of biological chemistry vol. 280, 39 (2005): 33435-44.; Matsushita, M et al. Journal of immunology (Baltimore, Md.: 1950) vol. 165, 5 (2000): 2637-42), recent evidence from different authors suggests that MASP-1 can also self-activate and plays an important role in activating MASP-2 (Degn, E etal. Journal of immunology (Baltimore, Md.: 1950) vol. 189, 8 (2012): 3957-69.; Héja, Dávid et al. Proceedings of the National Academy of Sciences of the United States of America vol. 109, 26 (2012): 10498-503.; Kjaer, Troels R et al. al. Molecularimmunology vol.56,4(2013):413-22.).

[0004] Activated MASP-2 cleaves complement components C4 and C2, generating the C3 convertase C4bC2a. MASP-1 can also cleave C2. The C3 convertase cleaves C3 into C3b, amplifying the cascade and mediating phagocytosis and adaptive immune responses. Additional C3b molecules are added to the C3 convertase to form the C5 convertase (C4bC2aC3b). C3b serves as a binding site for C5, initiating the assembly of the membrane attack complex (MAC) by cleaving C5 into C5a and C5b. C5a is a potent anaphylatoxin, while C5b forms a complex with C6 and C7 and inserts into the cell membrane. Subsequently, C8 and 10-18 C9 molecules bind to this complex to form a fully functional MAC (C5b-9), ultimately leading to cell lysis and death (Beltrame, Marcia H et al. Molecular Immunology vol. 67, 1 (2015): 85-100). In addition, it can activate prothrombin, thereby participating in the activation of the coagulation system (Garred, Peter et al. Immunological reviews vol. 274, 1 (2016): 74-97.)

[0005] Several groundbreaking studies have shown that MASP2 is important not only in the activation of the lectin pathway but also in the formation of the fibrin clot in the coagulation cascade. MASP-2 levels are also associated with many diseases, including schizophrenia (Mayilyan, Karine R et al. Neuroscience letters vol. 404, 3 (2006): 336-41), septic shock (Charchaflieh, Jean et al. Clinical & developmental immunology vol. 2012 (2012): 407324.), acute lymphoblastic leukemia, non-Hodgkin's lymphoma, central nervous system tumors (Fisch, UrsP et al. Swiss medical weekly vol. 141w13191. 29Apr. 2011.), colorectal cancer (Ytting, Henriette et al. Clinical cancer research: an official journal of the American Association for Cancer Research vol. 11, 4 (2005): 1441-6.; Ytting, Henriette et al. Human immunology vol.69,7(2008):414-20.), thrombotic microangiopathy (Elhadad,S et al.Clinical and experimental immunology vol.203,1(2021):96-104), systemic lupus erythematosus (Xu,Wang-Dong et al.Journal of cellular and molecular medicine vol.24,18(2020):10432-10443), IgA nephropathy (Lafayette,Richard A et al.Kidney internationalreports vol.5,11 2032-2041.13Aug.2020), 2019-nCoV (Rambaldi,Alessandro et al.Immunobiology vol.225,6(2020):152001), etc. Therefore, the development of inhibitors against MASP2 is of great importance.

[0006] Currently, there are reports of antibodies and antibody preparations targeting MASP2. For example, Chinese Patent CN103687620B discloses the anti-MASP2 antibody OMS721 and its applications, and Chinese Patent CN109890367A discloses a MASP2 inhibitory antibody preparation. In the current therapeutic field, there is still a need for anti-MASP2 antibody preparations with excellent stability to ensure stability during preparation, transportation, and storage, thereby ensuring clinical safety and quality control.

[0007] The present invention provides an isolated anti-MASP2 antibody preparation for use in treating diseases and conditions associated with MASP2-dependent complement activation. Summary of the Invention

[0008] In one aspect, the present invention provides an anti-MASP2 antibody preparation comprising an isolated anti-MASP2 antibody, a stabilizer, a surfactant, and a buffer, wherein the buffer is acetate buffer and the stabilizer is sucrose.

[0009] In some embodiments, the antibody concentration is 80 mg / ml-160 mg / ml, preferably 100 mg / ml-140 mg / ml. In some specific embodiments, the antibody concentration is 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml or 160 mg / ml.

[0010] In some embodiments, the concentration of the stabilizer is 50 mg / ml-110 mg / ml, preferably 60 mg / ml-90 mg / ml. In some specific embodiments, the concentration of the stabilizer is 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 105 mg / ml or 110 mg / ml.

[0011] In some embodiments, the concentration of the buffer is 10 mM-30 mM; preferably, the concentration of the buffer is 15 mM-25 mM. In some specific embodiments, the concentration of the buffer is 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM.

[0012] In some embodiments, the pH of the formulation is 4.5-5.4, preferably 4.6-5.2, more preferably 4.7-5.0. In some specific embodiments, the pH of the formulation is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4.

[0013] In some embodiments, the surfactant is polysorbate; preferably, the polysorbate is Tween-80.

[0014] In some embodiments, the concentration of the surfactant is 0.05 mg / ml-1.0 mg / ml; preferably, the concentration of the surfactant is 0.2 mg / ml-0.5 mg / ml. In some specific embodiments, the concentration of the surfactant is 0.20 mg / ml, 0.25 mg / ml, 0.30 mg / ml, 0.35 mg / ml, 0.40 mg / ml, 0.45 mg / ml or 0.50 mg / ml.

[0015] In some embodiments, the antibody formulation is any one of the following formulations:

[0016] (1) The antibody concentration is 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml or 160 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80;

[0017] (2) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 100 mg / ml or 110 mg / ml of sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml of Tween-80;

[0018] (3) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80;

[0019] (4) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80;

[0020] (5) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.25 mg / ml, 0.30 mg / ml, 0.35 mg / ml, 0.40 mg / ml, 0.45 mg / ml, 0.50 mg / ml, 0.60 mg / ml, 0.70 mg / ml, 0.80 mg / ml, 0.90 mg / ml or 1.0 mg / ml of Tween-80.

[0021] In some embodiments, the antibody formulation is any one of the following formulations:

[0022] (1) The antibody concentration is 100 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 15 mM acetate buffer; the pH value of the preparation is 4.6; and the surfactant is 0.20 mg / ml Tween-80;

[0023] (2) The antibody concentration is 120 mg / ml; the stabilizer is 80 mg / ml sucrose; the buffer is 20 mM acetate buffer; the pH value of the preparation is 4.8; and the surfactant is 0.30 mg / ml Tween-80;

[0024] (3) The antibody concentration is 140 mg / ml; the stabilizer is 90 mg / ml sucrose; the buffer is 25 mM acetate buffer; the pH value of the preparation is 5.2; and the surfactant is 0.50 mg / ml Tween-80.

[0025] In some embodiments, the formulation may further comprise a preservative and / or an antioxidant. The preservative or antioxidant is one commonly used in antibody formulations. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof; and the antioxidant is methionine.

[0026] In some embodiments, the concentration of the preservative is 0-0.2 mg / ml, preferably 0.02-0.1 mg / ml. In some specific embodiments, the concentration of the preservative is 0 mg / ml, 0.02 mg / ml, 0.04 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.10 mg / ml, 0.12 mg / ml, 0.14 mg / ml, 0.16 mg / ml, 0.18 mg / ml or 0.20 mg / ml.

[0027] In some embodiments, the concentration of the antioxidant is 0-20 mM, the concentration of the antioxidant is 5 mM-15 mM. In some embodiments, the concentration of the antioxidant is 0, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM or 20 mM.

[0028] In some embodiments, the antibody comprises a VH comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0029] In some embodiments, the antibody comprises a V H , which comprises the amino acid sequence SEQ ID NO: 7, and V L , which comprises the amino acid sequence SEQ ID NO:8.

[0030] In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:9 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:10.

[0031] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0032] In some embodiments, the antibody preparation described above is a liquid preparation or a powder for injection.

[0033] In another aspect, the present invention provides use of any of the antibodies described above, or any of the antibody preparations described above, in the preparation of a medicament for treating or preventing a disease or condition;

[0034] Preferably, the disease or condition is an autoimmune disease, inflammatory disease, blood disease, coagulopathy, angiogenesis-dependent disease and / or viral infectious disease associated with MASP2-dependent complement activation; more preferably, the disease or condition is selected from ischemia-reperfusion injury, atherosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, IgA nephropathy, severe sepsis, septic shock, acute respiratory distress syndrome or systemic inflammatory response syndrome caused by sepsis, hemorrhagic shock, hemolytic anemia, autoimmune thrombosis Transplant thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), TMA secondary to transplantation (TA-TMA), Upshaw-Schulman syndrome, catastrophic antiphospholipid syndrome (CAPS), Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancers, age-related macular degeneration, proliferative diabetic retinopathy, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection;

[0035] More preferably, the coronavirus is SARS-CoV, MERS-CoV and / or SARS-CoV-2.

[0036] In another aspect, the present invention provides a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of any of the anti-MASP2 antibodies or antibody formulations described above. Administration includes intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalational, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, mucosal, or transdermal administration. In some embodiments, the formulation is administered intravenously. In some embodiments, the formulation is administered subcutaneously. In some embodiments, the formulation is administered intramuscularly.

[0037] In some embodiments, any of the isolated anti-MASP2 antibodies described above comprises an Fc fragment. In some embodiments, the isolated anti-MASP2 antibody is a full-length IgG antibody. In some embodiments, the isolated anti-MASP2 antibody is a full-length IgG1 or IgG4 antibody. In some embodiments, the isolated anti-MASP2 antibody is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the isolated anti-MASP2 antibody is a chimeric, murine, fully human, or humanized antibody. In some embodiments, the isolated anti-MASP2 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain Fv (scFv), Fv fragment, dAb, Fd, nanobody, diabody, and linear antibody.

[0038] The "antibodies" described herein include full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains typically include three hypervariable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified using the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of the full-length antibodies described in the present invention are IgA, IgD, IgE, IgG and IgM, which are characterized by having α, δ, ε, γ and μ heavy chains, respectively. Furthermore, the several major antibody classes described in the present invention can be further divided into subclasses, including: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0039] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized two-chain antibodies (dsdiabodies), single-chain antibodies (scFv), scFv dimers (divalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure.

[0040] The present invention uses MASP2 as a buffer, acetate as a buffer, and sucrose as a stabilizer, so that the anti-MASP2 antibody preparation has excellent stability under high temperature, light, oscillation, freeze-thaw, accelerated and long-term conditions, which can ensure the stability of the preparation during preparation, transportation and storage, and ensure clinical drug safety and quality controllability. DETAILED DESCRIPTION

[0041] In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention clearer, the present invention will be described in detail below with reference to specific embodiments. The following embodiments are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0042] Unless otherwise specified, the reagents used in the following examples were prepared by conventional methods or obtained from commercial channels. The experimental methods used were conventional methods unless otherwise specified. The materials and instruments used were obtained from commercial channels unless otherwise specified.

[0043] In the present examples, a mutant of the anti-MASP2 antibody humM2mc67-9 is used as an example. The amino acid sequence of the humM2mc67-9 antibody is disclosed in International Patent Application WO2023103789A1, the disclosure of which is incorporated herein by reference in its entirety. This mutant humM2mc67-9 differs from humM2mc67-9 only in having an S228P mutation (EU numbering) in the heavy chain constant region, and is hereinafter referred to as humM2mc67-9(S228P).

[0044] The amino acid sequence of the anti-MASP2 antibody humM2mc67-9 (S228P) used in the examples of the present invention is shown in Tables 1a and 1b:

[0045] Table 1a Amino acid sequences of the CDRs of the anti-MASP2 antibody humM2mc67-9(S228P)

[0046]

[0047] Table 1b. Amino acid sequence of anti-MASP2 antibody humM2mc67-9(S228P)

[0048]

[0049]

[0050] Example 1. Preparation of anti-MASP2 antibody preparation

[0051] The anti-MASP2 antibody formulation is formulated as follows:

[0052] Table 2: Prescription of anti-MASP2 antibody preparations

[0053]

[0054]

[0055] Preparations of the anti-MASP2 antibody humM2mc67-9(S228P) were prepared referring to the formulations in Table 2 for subsequent stability testing.

[0056] Example 2: Stability Test of Anti-MASP2 Antibody Formulation

[0057] The stability of the various formulations listed in Table 2 was investigated under freeze-thaw, high-temperature, accelerated, and long-term refrigeration conditions. Testing included appearance, visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, thermal stability, insoluble particulate matter, aggregation, and charge heterogeneity. Aggregate characteristics were assessed using SEC, and charge heterogeneity was assessed using iCIEF.

[0058] 1. Viscosity and osmotic pressure testing

[0059] After the anti-MASP2 antibody preparation was completed, the viscosity and osmotic pressure of the antibody preparation were tested. The results are shown in Table 3:

[0060] Table 3: Viscosity and osmotic pressure test results of anti-MASP2 antibody formulations

[0061] prescription 1 2 3 4 5 6 7 Viscosity (cP) 6.65 8.14 10.6 20.05 15.73 8.61 5.68 Osmolality (mOsm / kg) 284 347 368 342 400 230 198

[0062] As shown in the results in Table 3, the antibodies in Prescription Groups 1-3 still have low viscosity at concentrations of 100 mg / m-140 mg / ml, and osmotic pressures are between 284-368 mOsm / kg, which are more advantageous when injected subcutaneously than other groups.

[0063] The preparations of prescriptions 1-7 were placed at 4°C for 14 days. No phase change occurred in prescription groups 1-3 and 6-7; a phase change occurred in prescription group 4 on the 3rd day and recovered within 2 hours at room temperature; a small phase change occurred in prescription group 5 on the 14th day and recovered within 30 minutes at room temperature.

[0064] 2. Stability test under freeze-thaw conditions

[0065] The stability of each antibody preparation was tested under freeze-thaw conditions (freeze: -75°C ± 10°C overnight; thaw at room temperature for 2 hours, freeze-thaw 3 times).

[0066] (1) The test results of each formulation of the humM2mc67-9 (S228P) antibody are shown in Table 4. Overall, after three freeze-thaw cycles, the visible foreign matter, antibody concentration, pH value, osmotic pressure, and viscosity of each antibody formulation remained essentially unchanged compared to before freeze-thaw (freeze-thaw cycle 0). The aggregates detected by SEC showed minimal changes, all within 0.30%.

[0067] The number of insoluble particles varied significantly across formulations. Table 4 shows that before freeze-thaw cycles (0 freeze-thaw cycles), the number of insoluble particles larger than 10 μm in formulations 1-3 was approximately 30-100 particles / ml, the number of insoluble particles larger than 25 μm was approximately 5-32 particles / ml, and the total number of insoluble particles was approximately 500-1125 particles / ml. After three freeze-thaw cycles, the number of insoluble particles of various types increased: the number of insoluble particles larger than 10 μm increased to 64-164 particles / ml, the number of insoluble particles larger than 25 μm increased to 8-51 particles / ml, and the total number of insoluble particles increased to approximately 1000-1986 particles / ml. However, in contrast, the number of insoluble particles of various types in formulations 4-7 before and after three freeze-thaw cycles, as well as the magnitude of the increase after three freeze-thaw cycles relative to the pre-freeze-thaw level, were significantly higher than in formulations 1-3.

[0068] The results of each test item in the above freeze-thaw test showed that the humM2mc67-9(S228P) antibody preparations in prescription groups 1-3 had excellent stability under freeze-thaw conditions and were significantly better than those in other prescription groups.

[0069] Table 4: Freeze-thaw test results of humM2mc67-9(S228P) antibody formulations

[0070]

[0071] 3. Stability test under high temperature conditions

[0072] The stability of each antibody formulation was tested under high temperature conditions (40°C ± 2°C) after 0 days, 7 days, 14 days, and 1 month.

[0073] (1) The test results for each formulation of the humM2mc67-9(S228P) antibody are shown in Tables 5a and 5b. The results show that after one month of storage, the visible foreign matter, concentration, pH, osmotic pressure, and viscosity of each antibody formulation remained essentially unchanged compared to day 0. However, the main peak detected by SEC in formulations 1-3 decreased slightly, with a change of 3.4%-4.1%. The main peak detected by SEC in formulations 4-7 also decreased, but the change was as high as 6.2%-8.9%. The iCIEF detected acidic and alkaline peaks in all prescription groups increased, while the main peak decreased. The acidic peaks in prescription groups 1-3 changed by 1.9%-3.0%, the main peak by only 8.8%-9.7%, and the alkaline peak by only 6.6%-7.0%. However, in comparison, the peaks in prescription groups 4-7 changed significantly more, with acidic peaks changing by 3.2%-4.3%, main peaks by 10.5%-12.8%, and alkaline peaks by 6.6%-9.2%.

[0074] The above results indicate that the humM2mc67-9(S228P) antibody preparations in prescription groups 1-3 can maintain basic stability under high temperature conditions, and their stability is significantly better than that in prescription groups 4-7.

[0075] Table 5a: humM2mc67-9(S228P) Antibody High Temperature Test Results (SEC)

[0076]

[0077] Table 5b: hM2mc67-9(S228P) antibody high temperature test results (iCIEF)

[0078]

[0079]

[0080] 4. Stability test under accelerated conditions

[0081] The stability of each antibody formulation was tested after 0 days, 14 days, and 1 month under accelerated conditions (25°C ± 2°C).

[0082] (1) The test results for each formulation of the humM2mc67-9 (S228P) antibody are shown in Table 6. The results show that after one month of storage, the visible foreign matter, concentration, pH value, osmotic pressure, and viscosity of each antibody formulation remained essentially unchanged compared to day 0. However, the main peak detected by SEC in each formulation group decreased slightly, with the main peak change in formulations 1-3 ranging from 1.0-1.1% and in formulations 4-7 ranging from 1.2-1.8%. These results indicate that each formulation of the humM2mc67-9 antibody can maintain basic stability under high temperature conditions, with formulations 1-3 showing particularly excellent stability.

[0083] Table 6: Accelerated test results of humM2mc67-9 antibody formulations

[0084]

[0085] 5. Stability test under long-term refrigeration conditions

[0086] The stability of each antibody formulation was tested after long-term refrigeration (5°C ± 3°C) for 0 days, 14 days, 1 month, 3 months, and 6 months.

[0087] (1) The test results of humM2mc67-9 (S228P) formulations 1-3 are shown in Table 7. The results show that after 6 months of storage, the visible foreign matter, concentration, pH value, osmotic pressure, and viscosity of each antibody formulation remained essentially unchanged compared to day 0. SEC detection showed a slight decrease in the main peak, with a change range of 0.1-0.2%. iCIEF detection showed a slight increase in the acidic peak and alkaline peak, and a slight decrease in the main peak, with a change range of 0.9-1.7% for the acidic peak, 2.1-2.6% for the main peak, and 0.6-1.2% for the alkaline peak. These results demonstrate the excellent stability of each formulation of the humM2mc67-9 (S228P) antibody under long-term refrigerated conditions.

[0088] Table 7: Stability results of humM2mc67-9(S228P) antibody formulations under long-term refrigeration conditions

[0089]

[0090] In summary, the anti-MASP2 antibody preparation of the present invention has strong stability under freeze-thaw, high temperature, accelerated and long-term refrigeration conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, ensuring clinical drug safety and quality controllability.

Claims

1. An anti-MASP2 antibody preparation, characterized in that The preparation comprises an isolated anti-MASP2 antibody, a stabilizer, a surfactant, and a buffer; the buffer is acetate buffer, and the stabilizer is sucrose.

2. The preparation according to claim 1, characterized in that The antibody concentration is 80 mg / ml-160 mg / ml, preferably 100 mg / ml-140 mg / ml.

3. The preparation according to claim 1 or 2, characterized in that The concentration of the stabilizer is 50 mg / ml-110 mg / ml; preferably 60 mg / ml-90 mg / ml.

4. The preparation according to any one of claims 1 to 3, characterized in that The concentration of the buffer solution is 10 mM-30 mM; preferably, the concentration of the buffer solution is 15 mM-25 mM.

5. The preparation according to any one of claims 1 to 4, characterized in that The pH value of the preparation is 4.5-5.4; preferably 4.6-5.

2.

6. The preparation according to any one of claims 1 to 5, characterized in that The surfactant is polysorbate; preferably, the polysorbate is Tween-80.

7. The preparation according to any one of claims 1 to 6, characterized in that The concentration of the surfactant is 0.05 mg / ml-1.0 mg / ml; preferably, the concentration of the surfactant is 0.2 mg / ml-0.5 mg / ml.

8. The preparation according to claim 1, characterized in that The antibody preparation is any one of the following preparations: (1) The antibody concentration is 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml or 160 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80; (2) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 100 mg / ml or 110 mg / ml of sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml of Tween-80; (3) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 10 mM, 15 mM, 20 mM, 25 mM or 30 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80; (4) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3 or 5.4; the surfactant is 0.2 mg / ml-0.5 mg / ml Tween-80; (5) The antibody concentration is 100 mg / ml-140 mg / ml; the stabilizer is 60 mg / ml-90 mg / ml sucrose; the buffer is 15 mM-25 mM acetate buffer; the pH value of the preparation is 4.6-5.2; the surfactant is 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.25 mg / ml, 0.30 mg / ml, 0.35 mg / ml, 0.40 mg / ml, 0.45 mg / ml, 0.50 mg / ml, 0.60 mg / ml, 0.70 mg / ml, 0.80 mg / ml, 0.90 mg / ml or 1.0 mg / ml of Tween-80.

9. The preparation according to claim 1, characterized in that The antibody preparation is any one of the following preparations: (1) The antibody concentration is 100 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 15 mM acetate buffer; the pH value of the preparation is 4.6; and the surfactant is 0.20 mg / ml Tween-80; (2) The antibody concentration is 120 mg / ml; the stabilizer is 80 mg / ml sucrose; the buffer is 20 mM acetate buffer; the pH value of the preparation is 4.8; and the surfactant is 0.30 mg / ml Tween-80; (3) The antibody concentration is 140 mg / ml; the stabilizer is 90 mg / ml sucrose; the buffer is 25 mM acetate buffer; the pH value of the preparation is 5.2; and the surfactant is 0.50 mg / ml Tween-80.

10. The preparation according to any one of claims 1 to 9, characterized in that The formulation further comprises a preservative and / or an antioxidant.

11. The preparation according to claim 10, characterized in that The preservative is ethylenediaminetetraacetic acid and / or diethylenetriaminepentaacetic acid; and the antioxidant is methionine.

12. The preparation according to claim 10 or 11, characterized in that The concentration of the preservative is 0-0.2 mg / ml, preferably 0.02-0.1 mg / ml.

13. The preparation according to any one of claims 10 to 12, characterized in that The concentration of the antioxidant is 0-20 mM, preferably 5 mM-15 mM.

14. The preparation according to any one of claims 1 to 13, characterized in that The antibody comprises a VH comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

15. The preparation according to any one of claims 1 to 14, characterized in that The antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and a VL comprising the amino acid sequence of SEQ ID NO:

8.

16. The preparation according to any one of claims 1 to 15, characterized in that The antibody further comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:

10.

17. The preparation according to any one of claims 1 to 16, characterized in that The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:

12.

18. The preparation according to any one of claims 1 to 17, characterized in that The preparation is a liquid preparation or a powder for injection.

19. Use of the preparation according to any one of claims 1 to 18 in the preparation of a medicament for treating or preventing a disease or condition; Preferably, the disease or condition is an autoimmune disease, inflammatory disease, blood disease, coagulopathy, angiogenesis-dependent disease and / or viral infectious disease associated with MASP2-dependent complement activation; more preferably, the disease or condition is selected from ischemia-reperfusion injury, atherosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, IgA nephropathy, severe sepsis, septic shock, acute respiratory distress syndrome or systemic inflammatory response syndrome caused by sepsis, hemorrhagic shock, hemolytic anemia, autoimmune thrombosis Transplant thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), TMA secondary to transplantation (TA-TMA), Upshaw-Schulman syndrome, catastrophic antiphospholipid syndrome (CAPS), Degos disease, disseminated intravascular coagulation (DIC), angiogenesis-dependent cancers, age-related macular degeneration, proliferative diabetic retinopathy, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neovascular glaucoma, corneal neovascularization, retinopathy of prematurity, and respiratory distress syndrome or pneumonia caused by coronavirus infection; More preferably, the coronavirus is SARS-CoV, MERS-CoV and / or SARS-CoV-2.

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