Pharmaceutical formulations containing therapeutic antibodies and uses thereof

Abstract

Provided is a stable pharmaceutical formulation comprising a monoclonal antibody that specifically binds to human MASP-2 present in high and low concentrations, as well as a kit, a pharmaceutical composition, a unit dosage form, and a method for preparing the same. The invention also relates to a treatment method for inhibiting MASP-2-dependent complement activation and treating related diseases by using the preparation and the kit, the pharmaceutical composition and the unit dosage form, and application of the preparation and the kit, the pharmaceutical composition and the unit dosage form to prepare corresponding medicaments. Also provided are novel monoclonal antibodies that specifically bind to human MASP-2.

Description

Pharmaceutical preparations containing therapeutic antibodies and uses thereof Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular to the field of therapeutic antibody preparations. More specifically, the present invention relates to pharmaceutical preparations comprising monoclonal antibodies that specifically bind to human MASP-2, and uses thereof. Background Art

[0002] MASP-2 (MBL-associated serine protease 2) participates in the complement system and shows significant homology to MASP-1 and two C1q-related serine proteases, C1r and C1s. Once the lectin recognizes and binds to the pathogen, the zymogen of MASP-2 is cleaved between the CCP2 and SP domains (cleavage between the conserved R444 and I445), becoming an active form consisting of two polypeptide chains (heavy chain / A chain and light chain / B chain) connected by a disulfide bond (C434-C552) (ABW Boldt et al., Human Immunology, 72(9):753-760 (2011)). When MBL binds to pathogens, MASP-2 is activated, cleaving complement components C4 and C2 into C4a, C4b, C2a, and C2b, generating the C3 convertase C4bC2b. C3 is then converted to C3b by C4bC2b, and finally, C5 is converted to C5b by C3b, forming the membrane attack complex (MAC). C3 activation ultimately leads to the formation of the MAC, initiating a cascade of downstream complement activation, stimulating the innate immune response. C4b can actually activate the production of C4d. C4d deposition may be a marker of complement activation. Studies have shown that positive C4d staining is an independent risk factor for ESRD in patients with IgAN (see Clin J Am Soc Nephrol 9:897-904, 2014). In IgAN patients, kidneys with C4d staining have significantly shorter survival times than those without C4d staining. C4d staining has also been detected in the kidneys of patients with lupus nephritis and membranous glomerulonephritis. MASP-2 has been identified as a very promising target for the treatment of autoimmune diseases.

[0003] In addition to its important role in immune defense, the complement system also contributes to tissue damage in many clinical situations. Currently, there remains a need for therapeutically effective complement inhibitors, such as new MASP-2 antibodies, particularly those with advantageously high binding affinity and specificity to prevent adverse reactions.

[0004] Therapeutic antibodies need to be formulated into a preparation suitable for administration to patients, and the preparation can also maintain its stability during storage and subsequent use. For example, if the preparation solution is improperly configured, the therapeutic antibodies therein may be more prone to degradation, aggregation and / or undesirable or even harmful chemical modifications. The stability of the antibody in the liquid preparation depends not only on the type of excipient used in the preparation, but also on the amount and ratio of the excipient relative to each other. In addition, when preparing liquid antibody preparations, in addition to stability, other factors must also be considered, such as the antibody concentration that a given preparation can accommodate and the visual quality or appearance of the preparation, etc. Therefore, when preparing therapeutic antibody preparations, special care must be taken to obtain a preparation that maintains stability, contains sufficient concentrations of antibodies and has other characteristics that allow the preparation to be easily administered to the patient.

[0005] For anti-MASP-2 antibodies, for example, Omeros' monoclonal antibody Narsoplimab (OMS721) has entered Phase III clinical trials. Its Chinese patent application number CN201780051640.7 discloses a MASP-2 antibody formulation containing 185 mg / mL protein, 20 mM L-histidine, 200 mM L-arginine HCl, 0.01% (w / v) polysorbate 80, and a pH of 5.5-6.5. However, the above formulation does not achieve the required stability for the anti-MASP-2 monoclonal antibodies of the present invention. Therefore, for the novel anti-MASP-2 monoclonal antibodies provided by the present invention, there is still a need to develop stable liquid formulations for the anti-MASP-2 antibodies of the present invention.

[0006] Summary of the Invention

[0007] MASP-2 is an important and advantageous therapeutic target. The present invention provides formulations for anti-MASP-2 antibody preparations that maintain good stability in liquid form at both high and low concentrations, meeting the requirements of production processes and clinical administration, and supporting the development of intravenous and subcutaneous injections of the anti-MASP-2 monoclonal antibodies. The present invention also provides antibodies directed against MASP-2.

[0008] 1. Antibody Preparations of the Present Invention

[0009] In one aspect, the present invention provides a stable liquid pharmaceutical formulation, characterized in that the drug is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human MASP-2, and the formulation further comprises a buffer, a surfactant, and an excipient.

[0010] In some embodiments of the present invention, the buffer in the stable liquid pharmaceutical formulation comprises histidine and / or acetate buffer system; the surfactant comprises polysorbate; the excipient comprises sucrose, trehalose and / or proline; and the pH value of the stable liquid pharmaceutical formulation is 4.7-6.1.

[0011] Thus, in some embodiments of the present invention, a stable liquid pharmaceutical formulation is provided, comprising: (a) a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2; (b) a buffer comprising a histidine and / or acetate buffer system; (c) a surfactant comprising a polysorbate; and (d) an excipient comprising sucrose, trehalose, and / or proline; wherein the formulation has a pH of 4.7-6.1.

[0012] In some embodiments, the antibody concentration is 10 mg / ml ± 1 mg / ml to 200 mg / ml ± 20 mg / ml. In some embodiments, the antibody concentration is 20 mg / ml ± 2 mg / ml. In some embodiments, the antibody concentration is 60 mg / ml ± 6 mg / ml. In some embodiments, the antibody concentration is 100 mg / ml ± 10 mg / ml. In some embodiments, the antibody concentration is 150 mg / ml ± 15 mg / ml. In some embodiments, the antibody concentration is about 20 mg / ml to about 100 mg / ml.

[0013] In some embodiments, the buffer comprises histidine at a concentration of 5 mM ± 1 mM to 20 mM ± 4 mM. In some cases, the histidine buffer concentration is 10 mM ± 2 mM. In some embodiments, the histidine buffer comprises L-histidine and L-histidine hydrochloride monohydrate. In some cases, the buffer comprises 0.175 mg / ml of L-histidine and 1.86 mg / ml of L-histidine hydrochloride monohydrate.

[0014] In some embodiments, the buffer comprises acetic acid and its concentration is 5 mM ± 1 mM to 20 mM ± 4 mM. In some cases, the acetate buffer concentration is 10 mM ± 1 mM. In some embodiments, the acetate buffer comprises acetic acid and sodium acetate trihydrate.

[0015] In some embodiments, the polysorbate concentration is 0.025% ± 0.01% to 0.1% ± 0.01% (w / v). In some cases, the polysorbate concentration is 0.05% ± 0.01% (w / v). In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80.

[0016] In some embodiments, the excipient is sucrose. In some embodiments, the sucrose concentration is 5.5% ± 0.5% to 9% ± 0.5% (w / v). In certain embodiments, the sucrose concentration is 5.8% ± 0.5% to 8.6% ± 0.5% (w / v). In some embodiments, the sucrose concentration is 5.8% ± 0.5% (w / v). In some embodiments, the sucrose concentration is 6.0% ± 0.5% (w / v). In some embodiments, the sucrose concentration is 6.5% ± 0.5% (w / v). In some embodiments, the sucrose concentration is 7.0% ± 0.5% (w / v). In some embodiments, the sucrose concentration is 8.6% ± 0.5% (w / v).

[0017] In some embodiments, the excipient is trehalose. In some embodiments, the trehalose concentration is 5.5% ± 0.5% to 9% ± 0.5% (w / v). In certain embodiments, the trehalose concentration is 7.0% ± 0.5% to 9.0% ± 0.5% (w / v). In some embodiments, the trehalose concentration is 7.0% ± 0.5% (w / v). In some embodiments, the trehalose concentration is 8.0% ± 0.5% (w / v). In some embodiments, the trehalose concentration is 9.0% ± 0.5% (w / v).

[0018] In some embodiments, the excipient is proline. In some embodiments, the proline concentration is 200 mM to 300 mM. In certain embodiments, the proline concentration is 200 mM to 250 mM. In some embodiments, the proline concentration is 230 mM.

[0019] In some embodiments, the buffer used in the formulation of the present invention can control the pH of the formulation of the present invention in the range of about 4.7-6.1, preferably about 5.0-6.0, more preferably about 5.0-5.5, and most preferably 5.3±0.1. In some specific embodiments, the antibody formulation of the present invention has a pH value of about 4.7, 5.0, 5.2, 5.3, 5.4, 5.5, 6.0, 6.1, 6.2, or 6.5.

[0020] In a preferred embodiment of the present invention, the surfactant in the liquid preparation of the present invention is a polysorbate, such as polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40; Pluronic, etc. In a preferred embodiment, the liquid preparation of the present invention comprises polysorbate 80 as a surfactant. In another preferred embodiment, the liquid preparation of the present invention comprises polysorbate 20 as a surfactant.

[0021] In one embodiment, the liquid preparation is a parenteral preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid preparation is an intravenous infusion.

[0022] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0023] (a) 20 mg / ml ± 2 mg / ml to 200 mg / ml ± 20 mg / ml antibody,

[0024] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0025] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0026] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, or 200 mM to 300 mM proline,

[0027] pH is 4.7 to 6.0.

[0028] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0029] (a) 20 mg / ml ± 2 mg / ml antibody,

[0030] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0031] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0032] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0033] The pH is 5.3±0.5.

[0034] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0035] (a) 60 mg / ml ± 6 mg / ml antibody,

[0036] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0037] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0038] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0039] The pH is 5.3±0.5.

[0040] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0041] (a) 100 mg / ml ± 10 mg / ml antibody,

[0042] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0043] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0044] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0045] The pH is 5.3±0.5.

[0046] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0047] (a) 150 mg / ml ± 15 mg / ml antibody,

[0048] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0049] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0050] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0051] The pH is 5.3±0.5.

[0052] Liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or longer.In one embodiment, liquid preparation of the present invention can be at about-80 ℃ to about 45 ℃, for example, -80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ conditions, store 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, and be stable.

[0053] In some embodiments, the liquid formulations of the present invention have an average particle size of 2-10 μm, as measured by microfluidics imaging (MFI).

[0054] In some embodiments, the liquid formulations of the present invention have an osmolality between 250 and 350 mOsm / kg H2O.

[0055] In some embodiments, the liquid formulation of the present invention has a viscosity of about 1.0 centipoise to 30 centipoise, for example, a viscosity of about 1.0 centipoise to 10 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0 centipoise to 20 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0 centipoise to 10 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 centipoise. In one embodiment, the viscosity of the liquid formulation at 4-40°C is about 5.0 centipoise to 7.0 centipoise. In one embodiment, the viscosity of the liquid formulation at 4-40°C is about 6.8 centipoise. In one embodiment, the viscosity of the liquid formulation at 25°C is about 6.8 centipoise.

[0056] In one embodiment, the liquid formulation of the present invention can be stably stored for at least 24 months. In another embodiment, the liquid formulation of the present invention is stable at any temperature between -80 ° C and 45 ° C. In another embodiment, the liquid formulation of the present invention remains stable for at least 3 months at about 2 ° C -8 ° C, preferably at least 12 months, more preferably at least 24 months, and most preferably at least 36 months. In one embodiment, the liquid formulation of the present invention remains stable at room temperature or, for example, at about 25 ° C for at least 2 months, preferably at least 3 months, more preferably at least 6 months, and most preferably at least 12 months. In another embodiment, the liquid formulation of the present invention remains stable at about 40 ° C for at least 2 weeks, preferably at least 1 month, more preferably at least 6 weeks, and most preferably at least 8 weeks. The stability is such as shown in that the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%.

[0057] In one embodiment, the stability of the preparation after storage can be indicated by changes in the appearance, visible foreign matter, protein content, monomer purity, and / or charge variants of the preparation. In one embodiment, the stability of the liquid preparation of the present invention can be detected in a forced experiment of high temperature stress, for example, after storage at 40°C ± 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, for example, after storage at 25°C ± 2°C for at least 1 month or 2 months, or in a long-term experiment, for example, after storage at 5°C ± 3°C for at least 6 months or 12 months. The stability is, for example, manifested as an antibody monomer purity of more than 95% in a dissolved state, preferably 98%, and more preferably 99%.

[0058] In one embodiment, after storage, the stability of the liquid formulation of the present invention is visually inspected, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance, is a colorless to light yellow liquid, and is free of foreign matter. In one embodiment, no visible foreign matter is present in the formulation by visual inspection under a clarity detector. In one embodiment, the absorbance of the formulation at about 405 nm or about 350 nm is detected to determine the turbidity of the solution. In one embodiment, the transition temperature T at which the protein undergoes conformational change or aggregation during the temperature increase process, resulting in a change in particle size, is determined. onset , check the stability of the liquid preparation of the present invention, preferably T onset Above 60°C, 63°C, 65°C, 68°C or 70°C. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in protein content, and the rate of change in protein content relative to the initial value on storage day 0 is no more than 10%, preferably no more than 5%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in monomer purity of the antibody in the liquid formulation of the present invention, wherein the change in monomer purity relative to the initial value on storage day 0 by size exclusion high performance liquid chromatography (SEC-HPLC) is no more than 10%, for example, no more than 5%, 4%, 3%, for example, a change of no more than 1-2%, preferably no more than 1%. In another embodiment, after storage, the change in monomer purity by non-reduced and / or reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method does not decrease by more than 10%, for example, no more than 5%, 4%, or 3%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is detected by whole-column imaging capillary isoelectric focusing electrophoresis (icIEF) or cation exchange high performance liquid chromatography (CEX-HPLC), wherein the sum of the change values ​​of the charge variants (main component, acidic component and basic component) of the antibody relative to the initial value on storage day 0 does not exceed 50%, for example, no more than 40%, 30%, 20%, 10%, or 5%.

[0059] In some embodiments, the formulation is stable after storage, for example after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ± 2°C for 1 month, preferably having one or more of the following characteristics:

[0060] (i) the preparation has an antibody monomer purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, or 99%, as measured by SEC-HPLC;

[0061] (ii) the preparation has an antibody monomer purity greater than 90%, preferably greater than 92%, 94%, 96%, or 98%, as measured by reducing or non-reducing CE-SDS (e.g., non-reducing CE-SDS);

[0062] (iii) The sum of the changes in the components of the antibody protein in the formulation (main component, acidic component, and basic component) relative to the initial value on storage day 0, as measured by iCIEF or CEX, does not exceed 50%, for example, no more than 40%, 30%, 20%, 10%, or 5%.

[0063] In some embodiments, the formulation is stable after shaking (e.g., after shaking for 14 days), after stirring (e.g., after stirring for 6 hours), after exposure to light (e.g., after exposure to light for 3 days), and / or after freeze-thaw (e.g., after 5 cycles of freeze-thaw), preferably having one or more of the following characteristics:

[0064] (i) the preparation has an antibody monomer purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, or 99%, as measured by SEC-HPLC;

[0065] (ii) the preparation has an antibody monomer purity greater than 90%, preferably greater than 92%, 94%, 96%, or 98%, as measured by reducing or non-reducing CE-SDS (e.g., non-reducing CE-SDS);

[0066] (iii) The sum of the changes in the components of the antibody protein in the formulation (main component, acidic component, and basic component) relative to the initial value on storage day 0, as measured by iCIEF or CEX, does not exceed 50%, for example, no more than 40%, 30%, 20%, 10%, or 5%.

[0067] In preferred embodiments of the present invention, the formulations have high antibody concentrations, low viscosity suitable for administration, particularly subcutaneous administration, high physical and chemical stability, and are less susceptible to aggregates and particulates during storage and to charge heterogeneity changes. These advantages are highly beneficial for producing complete, effective, and highly consistent clinical drugs.

[0068] In one aspect, the present invention provides a delivery device comprising a liquid antibody formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe comprising a liquid antibody formulation or a solid antibody formulation of the present invention, e.g., for intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.

[0069] In another aspect, the present invention provides a method of administering an antibody to a subject, e.g., a mammal, e.g., a human, comprising administering to the subject a liquid antibody formulation or a solid antibody formulation of the present invention, e.g., by a delivery device using a prefilled syringe.

[0070] In yet another aspect, the present invention provides use of a liquid antibody formulation or solid antibody formulation of the present invention for preparing a delivery device (e.g., a prefilled syringe) or a medicament for treating, preventing, or delaying a condition associated with, for example, ... in a subject.

[0071] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0072] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer comprising 10 mM ± 2 mM histidine, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0073] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0074] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0075] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 10 mM ± 2 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0076] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0077] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0078] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0079] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0080] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0081] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0082] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0083] In any of these embodiments, the polysorbate can be polysorbate 80 or polysorbate 20, with polysorbate 80 being preferred.

[0084] In one aspect, the present invention provides a pharmaceutical composition, wherein the composition comprises a pharmaceutical formulation as discussed above or herein, and the composition is contained in a container. In some embodiments, the container is a vial. In some cases, the vial is a Type 1 clear glass vial of 2 ml, 5 ml, 10 ml, or 20 ml. In some embodiments, the container is a syringe. In some cases, the syringe is low tungsten glass. In some embodiments, the container is a prefilled syringe. In some embodiments, the composition is contained in an autoinjector.

[0085] In one aspect, the present invention provides a kit comprising (i) a container containing a composition comprising a pharmaceutical formulation as discussed above or herein, and instructions for using the composition. In some embodiments, the container is a glass vial. In some embodiments, the container is a prefilled syringe. In some embodiments, the container is an autoinjector.

[0086] In one aspect, the present invention provides a unit dosage form comprising a pharmaceutical formulation as discussed above or herein, wherein the antibody is present in an amount of 0.1 mg to 500 mg. In some cases, the antibody is present in an amount of 200-400 mg. In some embodiments, the unit dosage form is a glass vial, such as a vial. In some embodiments, the unit dosage form is a prefilled syringe. In some embodiments, the unit dosage form is an autoinjector.

[0087] In any of the pharmaceutical compositions, kits, or unit dosage forms discussed above or herein, the container containing the pharmaceutical formulation can comprise a headspace comprising a gas, wherein the gas comprises less than 5% oxygen by volume. In some cases, the gas comprises less than 1% oxygen by volume. In some cases, the gas comprises no more than 0.1% oxygen by volume.

[0088] In one aspect, the present invention provides a container containing a pharmaceutical composition, wherein the composition comprises a pharmaceutical formulation as discussed above or herein. In some cases, the container is a syringe. In some cases, the container is a prefilled syringe. In some cases, the container is an autoinjector. In some cases, the container is a glass vial.

[0089] In various embodiments, any of the features or components of the embodiments discussed above or herein can be combined, and such combinations are encompassed within the scope of the present invention. Any specific value discussed above or herein can be combined with another relevant value discussed above or herein to describe a range, wherein the value represents the upper and lower ends of the range, and such ranges and all values ​​within such ranges are encompassed within the scope of the present invention. Each of the values ​​discussed above or herein can be expressed as having a change of 1%, 5%, 10% or 20%. For example, a 10mM concentration can be expressed as 10mM ± 0.1mM (1% change), 10mM ± 0.5mM (5% change), 10mM ± 1mM (10% change) or 10mM ± 2mM (20% change).

[0090] 2. Anti-MASP-2 Antibodies of the Present Invention

[0091] In one aspect, the invention provides anti-MASP-2 antibodies or antibody fragments (preferably antigen-binding fragments) that bind to MASP-2 or a fragment thereof (preferably a human MASP-2 protein).

[0092] In some embodiments, the present invention provides novel monoclonal anti-MASP-2 antibodies and fragments thereof.

[0093] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to MASP-2, wherein the antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics: a) no cross-reactivity with mice or rats; b) a longer serum half-life in monkeys compared to OMS721; c) no cross-reactivity with C1s, C1r, MASP1, or MASP3; d) the ability to selectively block complement activation via the MBL pathway; e) the ability to specifically bind to human MASP-2 with a K DA value of no more than 27.8 nM (or no more than 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.009 nM, 0.008 nM, 0.007 nM, 0.006 nM, 0.005 nM, 0.004 nM, 0.003 nM, 0.002 nM, or 0.001 nM), as measured by Bio-Layer Interferometry; f) is capable of blocking the activation of complement C3 as determined by ELISA in 1% human serum with an IC 50 No more than 0.08 μg / mL (or no more than 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL or 0.01 μg / mL), or its IC is determined by ELISA in 10% human serum 50 No more than 0.20 μg / mL (or no more than 0.15 μg / mL, 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL); g) capable of blocking complement C3 activation in 50% human serum; h) capable of blocking complement C4 activation, with an IC as determined by ELISA in 2% human serum. 50 No more than 0.11 μg / mL (or no more than 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL), or its IC as determined by ELISA in 10% human serum 50no more than 0.69 μg / mL (or no more than 0.65 μg / mL, 0.6 μg / mL, 0.55 μg / mL, 0.5 μg / mL, 0.45 μg / mL, 0.4 μg / mL, 0.35 μg / mL, 0.3 μg / mL, 0.25 μg / mL, or 0.2 μg / mL, 0.15 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL); i) capable of blocking MAC formation as determined by ELISA in 2% human serum. 50 No more than 0.27 μg / mL (or no more than 0.25 μg / mL, or 0.2 μg / mL, 0.15 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL).

[0094] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof comprising:

[0095] a heavy chain CDR1 comprising the amino acid sequence of DYYIN (SEQ ID NO: 1),

[0096] a heavy chain CDR2 comprising the amino acid sequence of WIFPGSX1SX2YX3X4X5X6FX7X8 (SEQ ID NO: 2), and

[0097] a heavy chain CDR3 comprising the amino acid sequence of GDRSGPFX9Y (SEQ ID NO: 3); and / or

[0098] a light chain CDR1 comprising the amino acid sequence of KSSQSLLYSNGKTYLN (SEQ ID NO: 4),

[0099] a light chain CDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 5), and

[0100] Including VQX 10 The light chain CDR3 has the amino acid sequence of THFPFT (SEQ ID NO: 6).

[0101] Wherein X1 is E, D or G, X2 is A or P, X3 is H or Y, X4 is S or N, X5 is E or Q, X6 is K or N, X7 is K or Q, X8 is A or G, X9 is A or P, and X 10It is V or G.

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0103] A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, and / or

[0104] a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and / or

[0105] a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12; and / or

[0106] A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and / or

[0107] A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and / or

[0108] A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.

[0109] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof comprising:

[0110] a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or

[0111] a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12; or

[0112] a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or

[0113] A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

[0114] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0115] a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or

[0116] A light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:14.

[0117] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0118] A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or.

[0119] a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or

[0120] a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; or

[0121] A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0122] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0123] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 80% identical thereto;

[0124] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 80% identical thereto;

[0125] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 80% identical thereto;

[0126] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 80% identical thereto;

[0127] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 80% identical thereto;

[0128] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 80% identical thereto; or

[0129] A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 80% identical thereto.

[0130] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0131] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or a sequence with at least 80% identity;

[0132] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, or a sequence with at least 80% identity;

[0133] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, or a sequence with at least 80% identity; or

[0134] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, or a sequence having at least 80% identity.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26 and / or a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 30.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0137] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16;

[0138] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16;

[0139] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19;

[0140] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28;

[0141] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30;

[0142] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28;

[0143] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30;

[0144] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28;

[0145] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30;

[0146] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; or

[0147] A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30.

[0148] In certain embodiments, the antibodies or antigen-binding fragments thereof further comprise one or more amino acid residue mutations while retaining specific binding affinity for MASP-2. In certain embodiments, at least one mutation is a conservative substitution, or all mutations are conservative substitutions. In certain embodiments, at least one mutation occurs at one or more positions within a CDR sequence and / or at one or more positions within a non-CDR sequence in a heavy chain variable region or a light chain variable region.

[0149] In certain embodiments, the antibody or its antigen-binding fragment further comprises an immunoglobulin constant region, optionally comprising a heavy chain constant region of IgG, and / or a light chain constant region. In certain embodiments, the constant region comprises a mouse constant region, a rabbit constant region or a human constant region, optionally the constant region comprises a constant region of human IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the heavy chain constant region comprises one or more amino acid substitutions at amino acid residues 252, 254 or 256 relative to a wild-type human IgG constant region, optionally, the amino acid substitution at amino acid residue 252 is substituted with tyrosine, the amino acid substitution at amino acid residue 254 is substituted with threonine, and the amino acid substitution at amino acid residue 256 is substituted with glutamic acid. In certain embodiments, the heavy chain constant region comprises a sequence having at least 80% identity to a wild-type human IgG constant region amino acid sequence, and has amino acid residue 252 substituted with tyrosine, amino acid residue 254 substituted with threonine, and amino acid residue 256 substituted with glutamic acid relative to a wild-type human IgG constant region.

[0150] In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, a dimeric or multimeric antibody, or a modified antibody (e.g., a glycosylated antibody).

[0151] In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelid single domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0152] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to MASP-2 and does not cross-react with C1s, C1r, MASP1, or MASP3.

[0153] In a preferred embodiment of the present invention, the anti-MASP-2 antibody or antigen-binding fragment thereof has the sequence shown in the following table:

[0154] Table 1 CDR sequences of mouse anti-MASP-2 antibody and humanized 129C10 antibody

[0155] Table 2 Antibody sequences of the present invention

[0156] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments have advantages in reducing immunogenicity against humans. Humanized antibodies are chimeric in their variable regions, with non-human CDR sequences grafted onto human FR sequences (or substantially human FR sequences). Humanization of antibodies or antigen-binding fragments can essentially be achieved by replacing corresponding human CDR genes with non-human (e.g., mouse) CDR genes in human immunoglobulin genes (e.g., see Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). In some embodiments, the humanized antibodies or antigen-binding fragments provided herein are substantially entirely composed of human sequences, except that the CDR sequences are non-human. In certain embodiments, the variable region FRs and constant regions (if any) are completely or substantially completely derived from human immunoglobulin sequences. The human FR sequence and the human constant region sequence can be from different human immunoglobulin genes, for example, the FR sequence is from an antibody of one person and the constant region is from an antibody of another person. In some embodiments, the humanized antibody or antigen-binding fragment comprises human heavy / light chain FR1-4.

[0157] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided by the present invention comprise one or more heavy chain FR sequences of the human germline framework sequence VH / 1-2, and / or one or more light chain FR sequences of the human germline framework sequence VK / 2-30, with or without back mutations. Back mutations can be introduced into the human germline framework sequence as needed. In certain embodiments, the humanized antibody 129C10 of the present invention may comprise one or more mutations selected from the group consisting of R71V, A93T, V67A, and M69L (based on Kabat numbering) in the heavy chain framework sequence VH / 1-2, and / or A65G, K64Q, and E61Q (based on Kabat numbering) in the heavy chain CDR2. The humanized antibody 129C10 may comprise one or more back mutations selected from the group consisting of F36L and T69A (based on Kabat numbering) in the light chain framework sequence VK / 2-30.

[0158] In certain embodiments, the humanized heavy and light chains of the antibodies and antigen-binding fragments thereof provided herein are substantially non-immunogenic in humans and retain substantially the same or even higher affinity as the parent antibody to MASP-2.

[0159] In some embodiments, the anti-MASP-2 antibodies described herein are human antibodies. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol 20:450-459 (2008).

[0160] In some embodiments, the anti-MASP-2 antibodies described herein are chimeric antibodies.

[0161] III. Therapeutic Methods and Uses of the Antibodies or Their Preparations of the Present Invention

[0162] The present invention also provides a method of treatment comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment of the invention, or an antibody pharmaceutical preparation of the invention, or a pharmaceutical composition, kit or unit dosage form of the invention to a subject in need thereof, thereby treating or preventing a disease or condition associated with MASP-2-dependent complement activation.

[0163] The present invention provides methods for inhibiting MASP-2-dependent complement activation in a subject in need thereof by inhibiting MASP-2, or methods for treating or preventing a condition or disease associated with MASP-2-dependent complement activation, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment provided herein, or an antibody pharmaceutical formulation of the invention, or a pharmaceutical composition, kit, or unit dosage form of the invention.

[0164] In another aspect, the invention provides a method of treating a condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment provided herein, or an antibody pharmaceutical formulation of the invention, or a pharmaceutical composition, kit, or unit dosage form of the invention.

[0165] In another aspect, the present invention further provides a method for reducing the serum C4 level in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein, or the antibody pharmaceutical preparation of the present invention, or the pharmaceutical composition, kit or unit dosage form of the present invention, thereby reducing the serum C4 level in the subject.

[0166] In another aspect, the present invention further provides a method for treating a condition in a subject, wherein the condition would benefit from a reduction in the subject's serum C4 level, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or an antibody pharmaceutical formulation of the present invention, or a pharmaceutical composition, kit or unit dosage form of the present invention, thereby treating the condition.

[0167] In another aspect, the present invention further provides a method for treating or preventing a condition or disease associated with abnormal (e.g., elevated) serum C4 levels, comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or an antibody pharmaceutical preparation of the present invention, or a pharmaceutical composition, kit or unit dosage form of the present invention, thereby treating the condition or disease.

[0168] In certain embodiments, the method further comprises administering a second therapeutic agent.

[0169] In certain embodiments, the subject is a human. In certain embodiments, administration is by oral, nasogastric, intravenous, subcutaneous, sublingual, or intramuscular injection.

[0170] In one aspect, the present invention further provides the use of the antibody or antigen-binding fragment thereof provided by the present invention, or the antibody pharmaceutical preparation of the present invention, or the pharmaceutical composition, kit or unit dosage form of the present invention in the manufacture of a medicament, wherein the medicament is used for one or more of the following:

[0171] (1) Used to inhibit MASP-2-dependent complement activation;

[0172] (2) for treating or preventing diseases or conditions associated with MASP-2-dependent complement activation;

[0173] (3) for treating a condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation;

[0174] (4) used to reduce the serum C4 level of a subject;

[0175] (5) for treating a condition in a subject that would benefit from a reduction in the subject's serum C4 level;

[0176] (6) For treating or preventing conditions or diseases associated with abnormal (e.g., elevated) serum C4 levels.

[0177] In some embodiments, any of the foregoing diseases or conditions include autoimmune diseases, vascular disorders, ischemia-reperfusion injury, arteriosclerosis, inflammation, pulmonary disorders, extracorporeal reperfusion procedures, musculoskeletal disorders, renal disorders, skin disorders, organ or tissue transplant procedures, nervous system diseases or injuries, blood disorders, genitourinary diseases, non-obese diabetes or complications associated with type 1 or type 2 diabetes, cancer, endocrine diseases, ophthalmic diseases, or COVID-19.

[0178] In some specific embodiments, the autoimmune disease comprises thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy associated with hematopoietic transplantation (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy.

[0179] In some specific embodiments, the vascular disorders include cardiovascular disorders, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renal vascular disorders, mesenteric / intestinal vascular disorders, revascularization of grafts and / or regrafts, vasculitis, Henlein-Schonlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu arteritis, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis following stenting, rotational aneurysm resection, and percutaneous transluminal coronary angioplasty (PTCA).

[0180] In some specific embodiments, the ischemia-reperfusion injury includes ischemia-reperfusion injury associated with aortic aneurysm repair, cardiopulmonary bypass, revascularization associated with organ transplantation and / or limb / digit replantation, stroke, myocardial infarction and hemodynamic resuscitation after shock and / or surgery.

[0181] In some specific embodiments, the inflammation comprises inflammatory gastrointestinal diseases, including pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and diverticulitis.

[0182] In some specific embodiments, the pulmonary disorders include acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease); meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burns, non-cardiogenic pulmonary edema, transfusion-related dyspnea, emphysema, cystic fibrosis, SARS-CoV, MERS-CoV and SARS-CoV-2 (Covid-19) related diseases.

[0183] In some specific embodiments, the extracorporeal circulation reperfusion process includes hemodialysis, plasmapheresis, leukocyte apheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced exfracorporeal membrane oxygenation LDL precipitation (HELP) and cardiopulmonary bypass (CPB).

[0184] In some specific embodiments, the musculoskeletal disorder comprises osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathies, crystalline arthropathy, and systemic lupus erythematosus (SLE).

[0185] In some specific embodiments, the renal disorder comprises mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henlein-Schonlein purpura nephritis, and IgA nephropathy.

[0186] In some specific embodiments, the skin disorder comprises psoriasis, autoimmune bullous skin diseases, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita, herpes gestationis, thermal burns, and chemical burns.

[0187] In some specific embodiments, the organ or tissue transplantation process includes organ allotransplantation, organ xenotransplantation, and organ and tissue transplantation.

[0188] In some specific embodiments, the nervous system disease or injury comprises multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome, reperfusion following stroke, degenerative disc disease, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or hemorrhage, demyelination, and meningitis.

[0189] In some specific embodiments, the blood disease comprises sepsis, severe sepsis, septic shock, acute respiratory distress syndrome caused by sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

[0190] In some specific embodiments, the genitourinary disorders include painful bladder, sensory bladder, chronic nonbacterial cystitis, interstitial cystitis, infertility, placental dysfunction, miscarriage, and pre-eclampsia.

[0191] In some specific embodiments, the endocrine diseases include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the regulated release of prolactin, growth hormone or other insulin-like growth factors, and adrenocorticotropic hormone from the pituitary gland.

[0192] In some specific embodiments, the ophthalmic disease comprises age-related macular degeneration.

[0193] The therapeutic methods of the present invention comprise administering to a subject any formulation, pharmaceutical composition, or unit dosage form comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 as described herein. The subject to whom the pharmaceutical formulation is administered can be, for example, any human or non-human animal in need of such treatment.

[0194] IV. Others

[0195] In one aspect, the present invention provides nucleic acids encoding any of the above anti-MASP-2 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0196] In one embodiment, the present invention provides a method for producing an anti-MASP-2 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing a host cell under conditions suitable for expression of a nucleic acid encoding the antibody or fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-MASP-2 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

[0197] In one aspect, the invention relates to a method for detecting a MASP-2 protein in a sample, the method comprising (a) contacting the sample with any of the anti-MASP-2 antibody preparations described herein or an antibody of the invention; and (b) detecting the formation of a complex between the anti-MASP-2 antibody or fragment thereof and the MASP-2 protein. In one embodiment, the anti-MASP-2 antibody is detectably labeled.

[0198] The present invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, applies to any and all formulations or anti-MASP-2 antibodies or fragments thereof, methods, and uses of the invention described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] FIGURE 1 shows that MASP-2 antibodies block complement C3 activation.

[0200] FIGURE 2 shows that MASP-2 antibodies block complement C4 activation.

[0201] FIGURE 3 shows that MASP-2 antibodies block MAC formation.

[0202] FIGURE 4 shows that chimeric and humanized MASP-2 antibodies block complement C3 activation.

[0203] FIGURE 5 shows that the MASP-2 antibodies 129C10-hu and OMS721-analog block complement C4 activation in 2% human serum.

[0204] FIGURE 6 shows that the MASP-2 antibodies 129C10-hu and OMS721-analog block MAC formation in 2% human serum.

[0205] FIGURE 7 shows that MASP-2 antibodies block C3 activation in 1% human serum.

[0206] FIGURE 8 shows that MASP-2 antibodies block C3 activation in 10% human serum.

[0207] FIGURE 9 shows that MASP-2 antibodies block C3 activation in 50% of human serum.

[0208] FIGURE 10 shows that MASP-2 antibodies block C4 activation in 10% human serum.

[0209] FIGURE 11 shows the affinity kinetics of the MASP-2 antibody 129C10-hu to hMASP-2.

[0210] Figures 12A-12E show the binding of the MASP-2 antibody 129C10-Hu to human C1s (12A), C1r (12B), MASP1 (12C), MASP3 (12D), and MASP2 (12E) as detected by ELISA.

[0211] Figures 13A-13B show the binding of MASP-2 antibodies 129C10-hu (13A) and OMS721-analog (13B) to human, cynomolgus monkey, rat, and mouse MASP2 as detected by ELISA.

[0212] FIGURE 14 shows the cross-reactivity of the MASP-2 antibody 129C10-hu with cynomolgus monkey MASP-2.

[0213] FIGURE 15 shows the selectivity of the MASP-2 antibody 129C10-hu in neutralizing three complement activation pathways.

[0214] Figures 16A-16B show the binding kinetics of the MASP-2 antibodies 129C10-hu-WT (16A) and 129C10-hu-YTE (16B) to human FcRn.

[0215] FIGURE 17 shows the pharmacokinetic (PK) results of the MASP-2 antibodies 129C10-hu-WT and 129C10-hu-YTE in cynomolgus monkey serum.

[0216] FIGURES 18A-18C show the PK and pharmacodynamic (PD) results of the MASP-2 antibodies 129C10-hu-WT and 129C10-hu-YTE in cynomolgus monkey serum.

[0217] FIG19 shows the lowering effect of 129C10-hu on serum C4c in monkeys after the first administration.

[0218] FIG20 shows the lowering effect of 129C10-hu on monkey serum C4c after the fourth dose.

[0219] Figures 21-23 show the results of experiments conducted for screening the optimal pH values ​​of formulations F12-F20 after 14 days at 40°C. Figure 21 shows the results of HMW% analysis by SEC for the nine formulations. Figure 22 shows the results of LMW% analysis by NR CE-SDS for the nine formulations. Figure 23 shows the results of CEX analysis for the percentages of the main peak, acidic peak, and basic peak for the nine formulations.

[0220] Figures 24-29 show the experimental results for formulations F1-F5 used to screen for optimal excipients. Figures 24-26 show the SEC, NR CE-SDS, and CEX results for the five formulations after 14 days at 40°C. Figure 27 shows the CEX results for each formulation after 14 days of shaking. Figures 28-29 show the CEX and NR CE-SDS results for each formulation after 6 hours of stirring.

[0221] Figures 30-32 show the experimental results for formulations F6-F11, used to screen for optimal surfactants. Figure 30 shows the SEC results for the six formulations after 14 days at 40°C. Figures 31-32 show the stability results of each formulation tested by NR CE-SDS and CEX, respectively, after stirring at 100 rpm for 6 hours.

[0222] Figures 33-44 show the experimental results for formulations F31-F37, used to screen for the optimal formulation. Figures 33-35 show the SEC, NR CE-SDS, and CEX results for the seven formulations after 4-6 weeks at 40°C. Figures 36-38 show the SEC, NR CE-SDS, and CEX results for the seven formulations after 4-6 weeks at 25°C. Figures 39-41 show the SEC, NR CE-SDS, and CEX results for the seven formulations after 3 days of illumination. Figures 42-44 show the SEC, NR CE-SDS, and CEX results for the seven formulations after 10 days of shaking at 200 rpm.

[0223] Detailed Description of the Invention

[0224] definition

[0225] Unless otherwise specified, 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. As used herein, when used with respect to a particular numerical value or range of values, the term "about" as used herein will include the numerical value with which it is associated with a fluctuation of ±10%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values ​​therebetween (e.g., 90.5, 95, 101, 105, 109.95, etc.). With respect to ratios, the term "about" is used to qualify each digit of the given ratio. For example, a ratio of about 1:1 means a ratio of 0.9 to 1.1:0.9 to 1.1.

[0226] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0227] The term "patient" as used herein refers to mammals and non-mammalian individuals who can benefit from the poorly soluble anti-tumor active agents of the present invention. Mammals refer to any member of the mammalian family, including but not limited to: humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats and pigs; livestock such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs; and the like. Examples of non-mammals include but are not limited to birds. The term "patient" does not limit the patient to a specific age or gender. In some embodiments, the patient is a human.

[0228] Unless otherwise specified, "administering" and "administering" are used interchangeably herein.

[0229] As used herein, the term "weight percent by volume" or "% w / v" refers to the percentage weight (in grams) of a single component relative to the total volume of the mixture containing that component. For example, 500 mg of a component in a total volume of 8 ml is 6.25% w / v, and 500 mg of a component in a total volume of 5 ml is 10% w / v.

[0230] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A natural intact antibody comprises two heavy chains (H) and two light chains (L). The heavy chains of mammals are divided into α, δ, ε, γ, and μ, each of which consists of a variable region (V H ) and the first, second and third constant regions (C H1 ,CH2 ,C H3 ). The light chains of mammals are divided into λ and κ, and each light chain consists of a variable region (V L stands for lambda light chain, or V K represents kappa light chain) and a constant region (C L Represents lambda light chain or C K The Y represents a light chain). Antibodies are Y-shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains, held together by disulfide bonds. Each arm of the Y consists of the variable region and first constant region of a heavy chain bound to the variable region and constant region of a light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the two chains typically contain three highly variable loops called complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252): 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interspersed between four flanking regions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are divided into different classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into several subclasses, such as 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).

[0231] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment that has two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment that has only a single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are bivalent.

[0232] In this context, a "bispecific" antibody is an artificial antibody that has fragments from two different monoclonal antibodies and is able to bind to two different epitopes. These two epitopes can be present on the same antigen, or they can be present on two different antigens.

[0233] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one, two or three CDRs, or any other antibody fragment capable of binding to an antigen but not comprising the complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds Diabodies). Single-chain antibody molecules (scFv), scFv dimers (divalent diabodies), bispecific antibodies, multispecific antibodies, camelid single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0234] "Fab" with respect to antibodies refers to the portion of an antibody composed of one light chain (including the variable and constant regions) bound to the variable region and first constant region of one heavy chain through disulfide bonds.

[0235] "Fab'" refers to the Fab fragment including part of the hinge region.

[0236] "F(ab')2" refers to a dimer of Fab'. "Fv," for an antibody, refers to the smallest fragment with a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0237] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain and the variable region of a single heavy chain are connected by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: two V H The parts are connected by a peptide linker (e.g., a long flexible linker) and are connected to the two V LIn some embodiments, the dsFv-dsFv' is bispecific, wherein each disulfide-paired heavy and light chain has a different antigenic specificity.

[0238] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of one light chain variable region and one heavy chain variable region linked to each other directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0239] "Fc," with respect to antibodies, refers to the portion of an antibody comprised of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not bind to an antigen.

[0240] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0241] A "camelid single-domain antibody," "heavy-chain antibody," or "HCAb" is a HHeavy chain antibodies are antibodies that contain heavy chain domains but no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Although camelid antibodies lack light chains, they have reliable antigen-binding regions (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain (VHH domain) of a heavy-chain antibody represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0242] "Nanobodies" are antibody fragments consisting of a heavy chain VHH domain and two constant domains (CH2 and CH3).

[0243] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments contain one V H domain and V on the same polypeptide chain L Domain connected (V H -V L or V L -V H(See, for example, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15; 90(14): 6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short, the two domains on the same chain are paired, and the domains are forced to pair with the complementary domains of the other chain, thereby generating two antigen-binding sites. These antigen-binding sites can be directed against the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody directed against two different antigens (or epitopes). In certain embodiments, an "scFv dimer" is a bivalent diabody or a bivalent ScFv (BsFv) comprising V H -V L (connected by a peptide linker) with another V H -V L The molecules dimerize, so that a part of V H With another part of the V L In other embodiments, a "scFv dimer" is a bispecific diabody comprising a V L1 -V H2 (connected by a peptide linker) associated with V H1 -V L2 (connected by a peptide linker), such that V H1 and V L1 Cooperate, V H2 and V L2 Each combination has a different antigen specificity.

[0244] "Domain antibodies" are antibody fragments that contain only the variable region of a heavy chain or a light chain. H The domains are covalently linked with a peptide linker to form a bivalent or multivalent domain antibody. H The domains can be directed against the same or different antigens.

[0245] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chains are derived from one species and the remainder of the heavy and / or light chains are derived from another species. In an illustrative example, a chimeric antibody can comprise a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse or rat. In some embodiments, the non-human animal is a mammal, e.g., a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0246] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs from non-human animals, FR regions from humans, and, when applicable, constant regions from humans.

[0247] As used herein, the term "MASP-2" refers to the mannan-binding lectin-associated serine protease 2, an important member of the MBL pathway of the complement system. The amino acid and nucleic acid sequences of human, mouse, and cynomolgus monkey MASP-2 can be found in public databases such as GenBank, UniProt, and Swiss-Prot. As used herein, the term MASP-2 includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type MASP-2. In certain embodiments, the human MASP-2 protein comprises the amino acid sequence of SEQ ID NO:39. In certain embodiments, the mouse MASP-2 protein comprises the amino acid sequence of SEQ ID NO:40. In certain embodiments, the cynomolgus monkey MASP-2 protein comprises the amino acid sequence of SEQ ID NO:43. In certain embodiments, a chimeric MASP-2 protein is synthesized that includes the mouse MASP-2 CUB1-EGF-CUB2 domain (residues 20-297) and the human MASP-2 CCP1-CCP2-SP domain (residues 298-686) and has the amino acid sequence of SEQ ID NO: 42.

[0248] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human MASP-2 with a binding affinity (K) of D )≤10 -6 M (e.g. ≤5x10 -8 M,≤2x10 -8 M,≤10 -8 M,≤5x10 -9 M,≤2x10 -9 M,≤10 -9 M,≤5x10 -10 M,≤2x10 -10 M,≤10 -10 M,≤5x10 -11 M,≤2x10 -11 M,≤10 -11 M,≤5x10 -12 M,≤4x10 -12 M,≤3x10 -12 M,≤2x10 -12 M,or≤10 -12 M. K used hereD The ratio of the dissociation rate to the association rate (k off / k on ), can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, bio-layer interferometry, and flow cytometry (e.g., FACS). In certain embodiments, K D The values ​​can be suitably determined by using the ForteBio method.

[0249] As used herein, the ability to "block binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding between two molecules (e.g., a MASP-2 protein and an anti-MASP-2 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits binding between the two molecules by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In certain embodiments, such inhibition can be greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0250] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they likely bind to the same or a closely related epitope on the antigen. For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody to an antigen (e.g., human / monkey MASP-2) by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, then the antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as the reference antibody.

[0251] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, whether a human monoclonal antibody binds to the same epitope as an antibody of the present invention by determining whether the test antibody prevents the binding of the test antibody to the MASP-2 antigen polypeptide. If the test antibody competes with the antibody of the present invention, as evidenced by reduced binding of the antibody of the present invention to the MASP-2 antigen polypeptide, then the two antibodies bind to the same or a closely related epitope. Alternatively, if binding of the test antibody to the MASP-2 antigen polypeptide is inhibited by the antibody of the present invention, then the two antibodies bind to the same or a closely related epitope.

[0252] As used herein, the term "OMS721" refers to Omeros' monoclonal antibody, Narsoplimab (OMS721), the complete structure of which is described in U.S. Patent No. 9011860B2. The OMS721 antibody formulation is disclosed in Chinese Patent Application No. CN201780051640.7, which is incorporated herein by reference in its entirety. OMS721 has a human IgG4 constant region. The heavy chain sequence of OMS721 is shown in SEQ ID NO: 32, and the light chain sequence is shown in SEQ ID NO: 33.

[0253] Heavy chain of OMS721 (hIgG4 kappa) (SEQ ID NO: 32):

[0254] Light chain of OMS721 (hIgG4 kappa) (SEQ ID NO: 33):

[0255] "Conservative substitutions" in amino acid sequences refer to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (such as Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (such as Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (such as Asp and Glu), between residues with basic side chains (such as His, Lys, and Arg), or between residues with aromatic side chains (such as Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, thereby preserving the biological activity of the protein.

[0256] As used herein, the terms "homolog" and "homologous" are interchangeable and refer to a nucleic acid sequence (or its complement) or amino acid sequence that is at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to another sequence when optimally aligned.

[0257] "Percent (%) sequence identity" with respect to amino acid sequences (or nucleic acid sequences) refers to the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical with the amino acid (or nucleic acid) residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. To determine the percentage of amino acid (or nucleic acid) sequence identity, alignment can be achieved using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al, J. Mol. Biol., 215:403–410 (1990); Stephen F et al, Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D G et al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool, or customize the parameters according to the comparison situation, for example, by selecting an appropriate algorithm.

[0258] As used herein, "treating" or "treating" a condition includes preventing or ameliorating a condition, slowing the onset or rate of development of a condition, reducing the risk of a disease, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a disease, causing complete or partial regression of a condition, curing a condition, or some combination thereof.

[0259] An "isolated" substance has been altered by the hand of man from its natural state. If an "isolated" component or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it has been sufficiently separated from coexisting materials in its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably to refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or 91% pure. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).

[0260] The term "vector" as used herein refers to a vector into which a polynucleotide encoding a protein can be operably inserted to achieve expression of the protein. A vector can be used to transform, transduce or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids, phages, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda phage or M13 phage, and animal viruses. Animal virus categories used as vectors include retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses and papillomaviruses (such as SV40). The vector can include various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, the vector can also include a replication origin. The vector can also include materials that help it enter the cell, including but not limited to virus particles, liposomes or protein coatings. The vector can be an expression vector or a cloning vector. The vector provided by the present invention (e.g., an expression vector) contains a nucleic acid sequence encoding an antibody or its antigen-binding fragment provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably connected to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40), λ phages, and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p 15TV-L,pPro18,pTD,pRS10,pLexA,pACT2.2,pCMV-SCRIPT.RTM,pCDM8,pCDNA1.1 / amp,pcDNA3.1,pRc / RSV,PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0261] As used herein, a "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0262] The complement system has been implicated in the pathogenesis of numerous acute and chronic diseases and conditions, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, capillary leak following thermal burns, inflammation following cardiopulmonary bypass, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In nearly all of these diseases and conditions, complement is not the cause but rather one of several factors involved in the pathogenesis. Complement activation may be a major pathological mechanism and represents a promising point of clinical control in many of these disease states. MASP-2 participates in the MBL pathway, one of the three major complement activation pathways, and may also contribute to the pathogenesis of many of these diseases and conditions. In some embodiments, the disease or disorder associated with MASP-2-dependent complement activation is an autoimmune disease, a vascular disorder, ischemia-reperfusion injury, arteriosclerosis, inflammation, a pulmonary disorder, extracorporeal reperfusion surgery, a musculoskeletal disorder, a renal disorder, a skin disorder, organ or tissue transplant surgery, a nervous system disease or injury, a blood disorder, a genitourinary disorder, non-obese diabetes or complications associated with type 1 or type 2 diabetes, cancer, an endocrine disorder, or an ophthalmologic disorder.

[0263] As used herein, the term "mannan-binding lectin" ("MBL") is equivalent to mannan-binding protein ("MBP"). The term "membrane attack complex" ("MAC," also known as C5b-9) refers to a complex composed of the five terminal complement components (C5-C9) that inserts into and disrupts cell membranes.

[0264] As used herein, the term "autoimmune disease" refers to a pathophysiological condition in which an immune response is directed against the body's own tissues, causing damage thereto (autoimmunity). Examples of autoimmune diseases include, but are not limited to, thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy associated with hematopoietic transplantation (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy.

[0265] The term "tumor" used herein refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors.

[0266] The term "pharmaceutically acceptable" means that the specified carrier, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient.

[0267] pharmaceutical preparations

[0268] As used herein, the term "pharmaceutical formulation" means a combination of at least one active ingredient (e.g., a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2) and at least one inactive ingredient that, when combined with the active ingredient and / or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. Unless otherwise specifically indicated, the term "formulation" as used herein means "pharmaceutical formulation." The present invention provides pharmaceutical formulations comprising at least one therapeutic antibody. According to certain embodiments of the present invention, the therapeutic antibody is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2. More specifically, the present invention includes pharmaceutical formulations comprising: (i) a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2; (ii) a buffer comprising a histidine and / or acetate buffer system; (iii) a surfactant comprising a polysorbate; and (iv) an excipient comprising sucrose, trehalose, and / or proline. Additional components may be included in the formulations of the present invention if they do not significantly interfere with the stability of the formulation. Specific exemplary components and formulations encompassed by the present invention are described in detail below.

[0269] In certain embodiments, the pharmaceutical formulations of the present invention are liquid formulations. As used herein, the expression "liquid formulation" means a mixture of at least two components that exists primarily in a liquid state at about 2° C. to about 40° C. The liquid formulation may have low viscosity, medium viscosity, or high viscosity depending on its specific ingredients.

[0270] As used herein, the term "buffer" refers to a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid. For example, as used herein, "acetate buffer" refers to a mixture comprising sodium acetate and its conjugate acid, acetic acid. Because a chemical equilibrium is established between the weak acid and its conjugate base, a solution containing a buffer resists sudden changes in pH when a small amount of acid or base is added to the solution.

[0271] As used herein, the term "osmolarity" refers to a measure of the osmotic pressure of dissolved solute particles in an aqueous solution. Solute particles include two ions as well as non-ionized molecules. Osmotic pressure is expressed as the concentration (i.e., osmoles) of osmotically active particles dissolved in 1 kg of solvent (i.e., water). Osmolarity is expressed herein in milliosmoles per kilogram of water (mOsm / kg).

[0272] The term "excipient" herein refers to any non-therapeutic agent added to a formulation to provide desired consistency, viscosity and / or stabilization. Preferred excipients of the present invention include, but are not limited to, trehalose, sucrose, and proline.

[0273] “Particle size (Diameter)” and “mean particle size (Mean Diameter)” refer to the intensity volume diameter corresponding to the microparticle preparation, which can be measured using a dynamic light scattering instrument such as a laser particle size analyzer.

[0274] Antibodies of the present invention

[0275] The amount of antibody or antigen-binding fragment thereof contained in the pharmaceutical formulations of the present invention can vary depending on the specific characteristics desired for the formulation and the specific situation and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation can contain from about 0.1 mg / mL to about 500 mg / mL of antibody; from about 0.5 mg / mL to about 400 mg / mL of antibody; from about 1 mg / mL to about 200 mg / mL of antibody; from about 2 mg / mL to about 100 mg / mL; from about 1 mg / mL to about 5 mg / mL of antibody; from about 10 mg / mL to about 30 mg / mL of antibody; from about 75 mg / mL to about 125 mg / mL; from about 5 mg / mL to about 50 mg / mL; or from about 2 mg / mL to about 150 mg / mL of antibody. For example, the formulation of the present invention may be a liquid formulation comprising about 0.5 mg / mL; about 1 mg / mL; about 2 mg / mL; about 3 mg / mL; about 4 mg / mL; about 5 mg / mL; about 6 mg / mL; about 7 mg / mL; about 8 mg / mL; about 9 mg / mL; about 10 mg / mL; about 11 mg / mL; about 12 mg / mL; about 13 mg / mL; about 14 mg / mL; about 15 mg / mL; about 16 mg / mL; about 17 mg / mL; about 18 mg / mL; about 19 mg / mL; about 20 mg / mL; about 21 mg / mL; about 22 mg / mL; about 23 mg / mL; about 24 mg / mL; about 25 mg / mL; about 26 mg / mL; about 27 mg / mL; about 28 mg / mL; about 29 mg / mL; about 30 mg / mL; about 31 mg / mL; about 32 mg / mL; about 33 mg / mL; about 34 mg / mL; about 36 mg / mL; about 37 mg / mL; about 38 mg / mL; about 39 mg / mL; about 40 mg / mL; about 41 mg / mL; about 42 mg / mL; about 43 mg / mL; about 44 mg / mL; about 45 mg / mL; about 46 mg / mL; about 47 mg / mL; about 48 mg / mL; about 49 mg / mL; about 50 mg / mL; about 51 mg / mL; about 52 mg / mL; about 53 mg / mL; about 54 mg / mL mL; about 19 mg / mL; about 20 mg / mL; about 21 mg / mL; about 22 mg / mL; about 23 mg / mL; about 24 mg / mL; about 25 mg / mL; about 26 mg / mL; about 27 mg / mL; about 28 mg / mL; about 29 mg / mL; about 30 mg / mL; about 35 mg / mL; about 40 mg / mL; about 45 mg / mL; about 50 mg / mL; about 55 mg / mL; about 60 mg / mL; about 65 mg / mL; about 70 mg / mL L; about 75 mg / mL; about 80 mg / mL; about 85 mg / mL; about 90 mg / mL; about 95 mg / mL; about 96 mg / mL; about 97 mg / mL; about 98 mg / mL; about 99 mg / mL; about 100 mg / mL; about 101 mg / mL; about 102 mg / mL; about 103 mg / mL; about 104 mg / mL; about 105 mg / mL; about 110 mg / mL; about 115 mg / mL; about 120 mg / mL; about 125 mg / mL or about 200 mg / mL of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2.In certain embodiments, the pharmaceutical formulation is a liquid formulation that may contain 1±0.1 mg / mL to 200±20 mg / mL of antibody; 2±0.2 mg / mL to 100±10 mg / mL of antibody; 1±0.5 mg / mL to 30±5 mg / mL of antibody; 10±1 mg / mL to 30±3 mg / mL of antibody; 1±0.1 mg / mL to 3±0.3 mg / mL of antibody; 15±1.5 mg / mL to 25±2.5 mg / mL of antibody; 90±5 mg / mL to 110±5 mg / mL of antibody; or 150±7.5 mg / mL to 170±7.5 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 20±2 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 60±6 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 100±10 mg / ml of antibody. In some embodiments, the pharmaceutical formulation contains 150 ± 15 mg / ml of antibody.

[0276] Bioequivalent 11

[0277] The present invention encompasses antibodies or antigen-binding fragments thereof having amino acid sequences that differ from the amino acid sequences of the exemplary molecules disclosed herein, but which retain specific binding ability to human MASP-2. Such variant molecules may comprise one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, yet still exhibit biological activity substantially equivalent to that of the antibodies discussed herein.

[0278] The present invention includes antigen binding molecules that are bioequivalent to any one of the exemplary antibodies described herein. For example, if two antibodies are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in absorption rate and absorption extent when administered under similar experimental conditions with the same dose (single dose or multiple doses), they are considered to be bioequivalent. If some antibodies are equivalent in terms of their absorption extent but not equivalent in terms of their absorption rate, they are considered to be equivalents or pharmaceutical substitutes, and can still be considered to be bioequivalent, because the difference in terms of such absorption rate is established and reflected in the label, it is not necessary for reaching effective in vivo drug concentrations when, for example, long-term use, and is medically insignificant for the specific drug under study.

[0279] In one embodiment, two antibodies are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0280] In one embodiment, two antibodies are bioequivalent if a patient can switch one or more times between the reference product and the biological product without an expected increase in risk of side effects, including clinically significant changes in immunogenicity or diminished effectiveness, compared to continued therapy without such switching.

[0281] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals, in which the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum or other biological fluids; (b) in vitro tests that correlate with and reasonably predict the in vivo bioavailability data in humans; (c) in vivo tests in humans or other mammals, in which the appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) well-controlled clinical trials that establish the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0282] Formulation excipients and pH

[0283] The pharmaceutical formulations of the present invention comprise one or more excipients. As used herein, the term "excipient" means any non-therapeutic agent added to a formulation to provide the desired consistency, viscosity and / or stabilizing effect.

[0284] In certain embodiments, the pharmaceutical preparation of the present invention has a viscosity of less than 30cP, which is conducive to delivering the composition from a pre-filled syringe or an automatic syringe. In some embodiments, the stable liquid formulation of the present invention has a viscosity of about 1.0cP-30cP. In some cases, when measured using a dynamic light scattering instrument (DLS), the viscosity of the pharmaceutical formulation at 25 ° C is less than 28cP, less than 25cP, less than 23cP, less than 20cP, less than 18cP, less than 15cP, less than 13cP, less than 10cP, less than 7cP or less than 5cP, for example, about 1.0cP-10cP. In one embodiment, the viscosity of the liquid formulation at 25 ° C is about 1.0cP-20cP. In some embodiments, when measured using DLS, the viscosity of the pharmaceutical formulation at 25 ° C at an antibody concentration of up to 150mg / ml is less than 15cP. In one embodiment, the viscosity of the liquid formulation at 25 ° C is about 1.0cP-10cP. In one embodiment, the viscosity of the liquid formulation at 25°C is about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 cP. In some embodiments, the viscosity of the pharmaceutical formulation at 25°C at an antibody concentration of up to 150 mg / ml is less than 7 cP when measured using DLS. In one embodiment, the viscosity of the liquid formulation at 4-40°C is about 5.0 cP-7.0 cP. In one embodiment, the viscosity of the liquid formulation at 4-40°C is about 6.8 cP. In one embodiment, the viscosity of the liquid formulation at 25°C is about 6.8 cP.

[0285] The amount of excipients contained in the pharmaceutical formulations of the present invention varies depending on the specific circumstances in which the formulation is used and the intended purpose. In certain embodiments, the formulation may contain from about 0.1% to about 20% excipients; from about 0.5% to about 20% excipients; from about 1% to about 20% excipients; from about 2% to about 15% excipients; from about 5% to about 15% excipients; from about 7.5% to about 12.5% ​​excipients; or from about 9% to about 11% excipients. For example, the pharmaceutical formulations of the present invention may comprise about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%, about 13.5%, about 14.0%, about 14.5%, about 15%, or about 20% excipients (e.g., sucrose). In some embodiments, the formulation contains about 5.8% ± 0.5% excipients (e.g., sucrose). In some embodiments, the formulation contains about 6.0% ± 0.5% excipients (e.g., sucrose). In some embodiments, the formulation contains about 6.5% ± 0.5% excipients (e.g., sucrose). In some embodiments, the formulation contains about 7.0% ± 0.5% excipients (e.g., sucrose). In some embodiments, the formulation contains about 8.6% ± 0.5% excipients (e.g., sucrose). Each of the above percentages corresponds to a weight / volume percentage (w / v). In some cases, the formulation contains 5.5% ± 0.5% to 9% ± 0.5% (w / v) sucrose. In some cases, the formulation contains 5.5% ± 0.5% to 9% ± 0.5% (w / v) trehalose. In some cases, the formulation contains 200 mM to 300 mM proline.

[0286] Surfactants in formulations

[0287] The pharmaceutical formulations of the present invention may also include one or more surfactants of a type and in an amount that stabilizes the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 under conditions of rough handling or agitation, such as orbital shaking. As used herein, the term "surfactant" means a substance that reduces the surface tension of a fluid in which the substance is dissolved and / or reduces the interfacial tension between oil and water. Surfactants can be ionic or nonionic. Exemplary nonionic surfactants that can be included in the formulations of the present invention include, for example, alkyl poly(ethylene oxides), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 188 (also known as Pluronic F68), poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20, preferably polysorbate 80.

[0288] The amount of surfactant contained in the pharmaceutical formulations of the present invention can vary depending on the specific characteristics desired for the formulation and the specific situation and purpose for which the formulation is intended to be used. In certain embodiments, the formulation may contain from about 0.01% to about 1% surfactant; from about 0.01% to about 0.5% surfactant; from about 0.05% to about 0.15%; from about 0.08% to about 0.12%; or from about 0.09% to about 0.11% surfactant. For example, a formulation of the invention may comprise about 0.01%; about 0.02%; about 0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.10%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; or about 0.30% of a surfactant (e.g., polysorbate 80). In some embodiments, the formulation contains about 0.1% surfactant (e.g., polysorbate 80). Each of the above percentages corresponds to a weight / volume percentage (w / v). In some cases, the formulation contains 0.025% ± 0.01% to 0.1% ± 0.01% w / v polysorbate 80. In some cases, the formulation contains 0.05% ± 0.01% w / v polysorbate 80.

[0289] Buffer system and pH of the formulation

[0290] The pharmaceutical formulations of the present invention may also include a buffer or buffer system for maintaining a stable pH and for stabilizing the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2. In some embodiments, the buffer or buffer system comprises at least one buffer whose buffering range fully or partially covers the pH range of 4.7 to 6.1. In certain embodiments, the buffer comprises a histidine buffer and / or an acetate buffer. In certain embodiments, the buffer (e.g., histidine) is present at a concentration of about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM; about 3 mM to about 18 mM, about 5 mM to about 15 mM; or about 8 mM to about 12 mM. In some embodiments, buffer (such as histidine) is with about 1mM;About 2mM;About 3mM;About 4mM;About 5mM;About 6mM;About 7mM;About 8mM;About 9mM;About 10mM;About 11mM;About 12mM;About 13mM;About 14mM;About 15mM;About 16mM;About 17mM;About 18mM;About 19mM;Or the concentration of about 20mM exists.In some cases, preparation contains the histidine buffer that concentration is 5mM ± 1mM to 20mM ± 4mM.In some cases, preparation contains the histidine buffer that concentration is 10mM ± 2mM.In some embodiments, histidine buffer comprises L-histidine and monohydrate L-histidine hydrochloride.In some cases, histidine buffer comprises the L-histidine that concentration is 0.175mg / mL and the monohydrate L-histidine hydrochloride that concentration is 1.86mg / mL.

[0291] In some embodiments, the preparation contains an acetate buffer. In some embodiments, the buffer (e.g., acetic acid) is present in a concentration of about 1mM to about 40mM, about 1mM to about 30mM, about 1mM to about 20mM, about 3mM to about 18mM, about 5mM to about 15mM, or about 8mM to about 12mM. In some embodiments, the buffer (e.g., acetic acid) is present in a concentration of about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, or about 20mM. In some cases, the preparation contains an acetate buffer at a concentration of 5mM ± 1mM to 20mM ± 4mM. In some cases, the formulation contains an acetate buffer at a concentration of 10 mM ± 2 mM. In some embodiments, the acetate buffer comprises acetic acid and sodium acetate trihydrate. In some cases, the histidine buffer comprises a histidine salt at a concentration of 10 mM.

[0292] During the antibody purification process, it may be desirable or necessary to exchange one buffer for another to achieve the appropriate excipient concentration, antibody concentration, pH, etc. Buffer exchange can be accomplished, for example, by ultrafiltration / diafiltration (UF / DF) using, for example, a semipermeable tangential flow filtration membrane. However, the use of such techniques may induce the Gibbs-Donnan effect (Bolton et al., 2011, Biotechnol. Prog. 27(1):140-152). During protein concentration, the accumulation of positive charge on the product side of the membrane is electrically balanced by the preferential migration of positive ions to the opposite side of the membrane. A potential consequence of this phenomenon is that the final concentration of certain components (e.g., histidine) may be lower than the intended target concentration of these components due to electrostatic repulsion of the positively charged diafiltration buffer excipient from the positively charged antibody protein during the UF / DF step. Therefore, the present invention includes formulations in which, due to the Gibbs-Donnan effect, the concentration of, for example, histidine / acetate is varied within the amounts or ranges described herein.

[0293] Size exclusion describes the property of highly concentrated samples in which a significant portion of the total volume of a solution is occupied by solutes, particularly macromolecules such as proteins, forcing solvent out of this space. This, in turn, reduces the total volume of solvent available to dissolve other solutes, which can lead to uneven distribution across ultrafiltration membranes. Thus, the present invention encompasses formulations in which, due to the size exclusion effect, the concentration of, for example, histidine can be varied within the amounts or ranges described herein.

[0294] During manufacture of the formulations of the present invention, the composition of the formulation may vary. These variations may include the concentration of the active ingredient, the concentration of the excipients, and / or the pH of the formulation. The present invention includes formulations comprising monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to human MASP-2 and are stable and retain potency despite variations in excipient concentration of up to at least 10%. For example, formulations of monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to human MASP-2 are encompassed herein, wherein the stability and potency of the formulation are unaffected by variations in the concentration of the antibody, excipients, buffer, and / or surfactant by ±10% or ±20%.

[0295] Stability of pharmaceutical preparations

[0296] The pharmaceutical preparations of the present invention exhibit a high level of stability. As used herein, the term "stable" with respect to pharmaceutical preparations means that the antibody in the pharmaceutical preparation retains an acceptable degree of structure and / or function and / or biological activity after storage for a defined period of time. Even if the antibody contained in the preparation cannot maintain 100% of its structure and / or function and / or biological activity after storage for a defined period of time, the preparation can still be stable. In some cases, the maintenance of the structure and / or function and / or biological activity of about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody after storage for a defined period of time can be considered "stable".

[0297] In some embodiments, the stable liquid formulations of the present invention have an average particle size of 2-10 μm, as measured by microfluidics imaging (MFI).

[0298] In some embodiments, the stable liquid formulations of the present invention have an osmolality between 250 and 350 mOsm / kg H2O.

[0299] In some embodiments, stability can be measured by measuring the protein content (concentration) of the antibody in the preparation after storing a defined time at a given temperature.When " acceptable stability " is used herein, the phrase means that the protein content of the antibody detected in the preparation changes no more than 10% after storing a defined time at a given temperature.In certain embodiments, the protein content of the antibody detected in the preparation changes no more than 10% after storing a defined time at a given temperature, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.The defined time before measuring stability can be at least 3 days, at least 5 days, at least 1 week, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, 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 24 months, at least 36 months or longer. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature between about -80°C and about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if the protein content of the antibody detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 4°C for 3 months. A pharmaceutical formulation can also be considered stable if the protein content of the antibody detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 4°C for 6 months. A pharmaceutical formulation may also be considered stable if the amount of antibody protein detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after 9 months of storage at 4°C. A pharmaceutical formulation may also be considered stable if the amount of antibody protein detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after 12 months of storage at 4°C. A pharmaceutical formulation may also be considered stable if the amount of antibody protein detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after 24 months of storage at 4°C. A pharmaceutical formulation may also be considered stable if the amount of antibody protein detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after 36 months of storage at 4°C. The pharmaceutical formulation may also be considered stable if the protein content of the antibody detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 25°C for 2 weeks.A pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 25°C for 1 month. A pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 25°C for 4 weeks. A pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 25°C for 6 weeks. A pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 40°C for 2 weeks. The pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 40°C for 4 weeks. The pharmaceutical formulation may also be considered stable if the antibody protein content detected in the formulation does not change by more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage at 40°C for 6 weeks.

[0300] In some embodiments, stability can be measured by measuring the monomer purity percentage of the antibody in the formulation after storing a defined time at a given temperature. The monomer purity of the antibody can be measured by size exclusion chromatography (such as size exclusion high performance liquid chromatography (SEC-HPLC)). When "acceptable stability" is used herein, the phrase means that the monomer purity of the antibody can be detected in the formulation at least 90% after storing a defined time at a given temperature. In certain embodiments, the monomer purity of the antibody can be detected in the formulation at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% after storing a defined time at a given temperature. The defined amount of time before stability is measured can be at least 3 days, at least 5 days, at least 1 week, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, 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 24 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation can be stored when stability is assessed can be any temperature from about -80°C to about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if the monomer purity of the antibody is greater than about 90%, 95%, 96%, or 97% as detected by SEC-HPLC after storage at 4°C for 3 months. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 95%, 96%, or 97% after storage at 4° C. for 6 months. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% after storage at 4° C. for 9 months. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% after storage at 4° C. for 12 months. The pharmaceutical formulation may also be considered stable if the monomeric purity of the antibody is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% as detected by SEC-HPLC after storage at 4°C for 24 months.A pharmaceutical formulation may also be considered stable if the monomer purity of the antibody is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% as determined by SEC-HPLC after storage for 36 months at 4° C. A pharmaceutical formulation may also be considered stable if the monomer purity of the antibody is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% as determined by SEC-HPLC after storage for 2 weeks at 25° C. A pharmaceutical formulation may also be considered stable if the monomer purity of the antibody is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% as determined by SEC-HPLC after storage for 1 month at 25° C. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% after storage at 25° C. for 4 weeks. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% after storage at 25° C. for 6 weeks. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody as detected by SEC-HPLC is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% after storage at 40° C. for 2 weeks. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% as determined by SEC-HPLC after storage for 4 weeks at 40° C. The pharmaceutical formulation may also be considered stable if the monomer purity of the antibody is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% as determined by SEC-HPLC after storage for 6 weeks at 40° C.

[0301] In other embodiments, other methods are used to assess the stability of the formulations of the invention, such as absorbance at about 405 nm or about 350 nm to determine the turbidity of the solution, differential scanning calorimetry (DSC) to determine thermal stability, and controlled agitation to determine mechanical stability. For example, a formulation of the invention may be considered stable if the OD405 of the formulation changes by less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less) relative to the OD405 of the formulation at t = 0 after storage at about 4° C. to about 25° C. for 6 weeks or longer.

[0302] In some embodiments, the stability of the formulation after storage can be indicated by inspecting the appearance of the formulation for visible foreign matter. In a specific embodiment, after storage, the stability of the liquid formulation of the present invention is determined by visual inspection, wherein the liquid formulation of the present invention remains clear to slightly opalescent, a colorless to pale yellow liquid, and is free of foreign matter. In one embodiment, no visible foreign matter is present in the formulation as determined by visual inspection using a clarity detector.

[0303] In other embodiments, stability is assessed by measuring the binding affinity of the antibody to its target. For example, a formulation of the invention can be considered stable if, after storage at a temperature such as -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 40°C, or 45°C for a defined amount of time (e.g., 7 days to 12 months, 24 months, or 36 months), the monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 contained within the formulation binds to human MASP-2 with at least 80%, 85%, 90%, 95% or more of the antibody's binding affinity prior to storage. Binding affinity can be determined by any method, such as ELISA or plasmon resonance. Biological activity can be determined by a MASP-2 activity assay, for example, by contacting cells expressing MASP-2 with a formulation comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2. Binding of the antibody to such cells can be measured directly, such as by FACS analysis.

[0304] In some embodiments, stability can be measured by determining the percentage of antibodies that form aggregates (high molecular weight (HMW)) in the formulation after storing a defined amount of time under defined conditions, wherein stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated antibodies can be determined, in particular, by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography (SEC-HPLC)). When "acceptable stability" is used herein, the phrase means that up to 6% of the antibodies are in the aggregated form detected in the formulation after storing a defined amount of time at a given temperature. In certain embodiments, acceptable stability means that up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibodies can be detected in the aggregates in the formulation after storing a defined amount of time at a given temperature. The defined amount of time before measuring stability can be at least 3 days, at least 5 days, at least 1 week, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80°C to about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if, after storage at 40°C for 6 weeks, less than about 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibody is detected in aggregated form. The pharmaceutical formulation can also be considered stable if less than about 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25% or 0.1% of the antibody is detected in aggregated form after storage at 25°C for 6 weeks.

[0305] In some embodiments, stability can be measured, inter alia, by determining the percentage of antibody that migrates in a fraction that is more acidic ("acidic form") than the major elution fraction of the antibody ("major charged form") during ion exchange, wherein stability is inversely proportional to the fraction of antibody that is in the acidic form. The percentage of "acidified" antibody can be determined by ion exchange chromatography, such as cation exchange high performance liquid chromatography (CEX-HPLC) or whole column imaging capillary isoelectric focusing (icIEF). When "acceptable stability" is used herein, the phrase means that no more than 52% of the antibody is in the more acidic form detected in the formulation after storage at a defined temperature for a defined amount of time. In certain embodiments, acceptable stability means that up to about 52%, 50%, 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody can be detected in the acidic form of the formulation after storage for a defined amount of time at a given temperature. The defined amount of time before stability is measured 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, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80°C to about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if less than about 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in the more acidic form after storage at -80°C, -30°C, or -20°C for 36 months. The pharmaceutical formulation may also be considered stable if less than about 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in the more acidic form after storage at 4°C for 12 months, 24 months, or even 36 months.The pharmaceutical formulation may also be considered stable if less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in the more acidic form after storage at 25°C for 4 or 6 weeks. The pharmaceutical formulation may also be considered stable if less than about 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is in the more acidic form after storage at 37°C for 4 or 6 weeks. The pharmaceutical formulation may also be considered stable if less than about 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody is detectable in the more acidic form after storage at 40°C for 4 or 6 weeks.

[0306] In one embodiment, the transition temperature T is determined by measuring the protein undergoing conformational change or aggregation during the temperature increase process, which results in a change in particle size. onset , check the stability of the liquid formulation of the present invention, for example, the liquid formulation of the present invention has a T greater than about 60°C, greater than about 63°C, greater than about 65°C, greater than about 68°C or greater than about 70°C onset .

[0307] Reference to the stability of a pharmaceutical formulation "after a specified period of time" means that the stability parameter measurement is performed at the end of the specified period of time or near the end of the specified period of time, and means that the pharmaceutical formulation does not necessarily maintain the same stability for the parameter measured thereafter. For example, reference to a specific stability after 6 weeks means that the stability measurement is performed at or about 6 weeks after the start of the study. Additional methods for assessing the stability of antibodies in formulations are shown in the Examples presented below.

[0308] In some preferred embodiments of the invention, the formulation is stable after shaking (eg, after 14 days of shaking), as assessed as described above.

[0309] In some preferred embodiments of the invention, the formulation is stable after stirring (eg, after stirring for 6 hours), as assessed above.

[0310] In some preferred embodiments of the present invention, the formulation is stable after exposure to light (eg, after 3 days of exposure to light), as assessed as described above.

[0311] In some preferred embodiments of the present invention, the formulation is stable after freeze-thaw (eg, after 5 cycles of freeze-thaw), as assessed as described above.

[0312] Exemplary Formulations

[0313] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0314] (a) 20 mg / ml ± 2 mg / ml to 200 mg / ml ± 20 mg / ml antibody,

[0315] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0316] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0317] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, or 200 mM to 300 mM proline,

[0318] pH is 4.7 to 6.0.

[0319] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0320] (a) 20 mg / ml ± 2 mg / ml antibody,

[0321] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0322] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0323] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0324] The pH is 5.3±0.5.

[0325] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0326] (a) 60 mg / ml ± 6 mg / ml antibody,

[0327] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0328] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0329] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0330] The pH is 5.3±0.5.

[0331] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0332] (a) 100 mg / ml ± 10 mg / ml antibody,

[0333] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0334] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0335] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0336] The pH is 5.3±0.5.

[0337] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0338] (a) 150 mg / ml ± 15 mg / ml antibody,

[0339] (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate,

[0340] (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and

[0341] (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose,

[0342] The pH is 5.3±0.5.

[0343] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0344] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer comprising 10 mM ± 2 mM histidine, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0345] In some embodiments, the pharmaceutical formulation comprises: (a) 20 mg / ml ± 2 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0346] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0347] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 10 mM ± 2 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0348] In some embodiments, the pharmaceutical formulation comprises: (a) 60 mg / ml ± 6 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0349] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0350] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0351] In some embodiments, the pharmaceutical formulation comprises: (a) 100 mg / ml ± 10 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0352] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetic acid, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, at a pH of 5.3 ± 0.5.

[0353] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0354] In some embodiments, the pharmaceutical formulation comprises: (a) 150 mg / ml ± 15 mg / ml antibody, (b) 0.175 mg / ml L-histidine, (c) 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, at a pH of 5.3 ± 0.1.

[0355] In any of these embodiments, the polysorbate can be polysorbate 80 or polysorbate 20, with polysorbate 80 being preferred.

[0356] In some embodiments, the pharmaceutical formulation does not comprise other buffer systems / buffers.

[0357] In some embodiments, the pharmaceutical formulation does not comprise additional surfactants.

[0358] In some embodiments, the pharmaceutical formulation does not contain other excipients.

[0359] The pharmaceutical formulations comprising the anti-MASP-2 antibodies or antigen-binding fragments thereof of the present invention can maintain good stability of the antibodies in liquid form at relatively high concentrations (e.g., about 100 mg / mL, about 150 mg / mL) and low concentrations (e.g., about 20 mg / mL, about 50 mg / mL), meet the requirements of the production process and the needs of clinical application, and can support the development of the antibodies as intravenous and subcutaneous injections.

[0360] Additional non-limiting examples of pharmaceutical formulations encompassed by the present invention are set forth elsewhere herein, including in the working examples presented below.

[0361] Container and method of application

[0362] The pharmaceutical preparations of the present invention can be contained in any container suitable for storing medicines and other therapeutic compositions. For example, the pharmaceutical preparations can be contained in sealed and sterilized plastic or glass containers with a defined volume, such as vials, phials, ampoules, syringes, cartridges, bottles, or IV bags. Different types of bottles, including, for example, clear and opaque (e.g., amber) glass or plastic vials, can be used to contain the preparations of the present invention. Similarly, any type of syringe can be used to contain and / or administer the pharmaceutical preparations of the present invention. In some embodiments, the pharmaceutical preparations are contained in prefilled syringes. In some embodiments, the pharmaceutical preparations are contained in prefilled staked needle syringes.

[0363] The pharmaceutical preparations of the present invention can be contained in "normal tungsten" syringes or "low tungsten" syringes. As will be understood by those skilled in the art, the method for manufacturing glass syringes generally involves using a hot tungsten rod to pierce the glass, thereby creating a hole from which liquid can be aspirated and discharged from the syringe. This process results in trace amounts of tungsten being deposited on the inner surface of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means a syringe containing greater than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means a syringe containing less than 500 ppb of tungsten. For example, according to the present invention, a low tungsten syringe may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 ppb or less of tungsten.

[0364] The pharmaceutical preparation can be administered to the patient by a parenteral route such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, transmucosal, nasal, pulmonary and / or oral administration. In some embodiments, the pharmaceutical preparation of the present invention is delivered subcutaneously using a reusable pen and / or autoinjector delivery device. In some cases, the pharmaceutical preparation is contained in a syringe that is particularly suitable for use with an autoinjector.

[0365] Also contemplated herein are the use of microinfusion sets to deliver the pharmaceutical formulations of the present invention. As used herein, the term "microinfusion set" refers to a subcutaneous delivery device designed to slowly administer a relatively large volume (e.g., up to about 2.5 mL, about 3.0 mL, or more) of a therapeutic formulation over an extended period of time (e.g., about 10, 15, 20, 25, 30, or more).

[0366] In certain embodiments, the pharmaceutical formulation is administered via IV drip, such that the formulation is diluted in an IV bag containing a physiologically acceptable solution. In one embodiment, the pharmaceutical composition is a compounded sterile formulation in an IV bag, such that a single dose of the drug is diluted to 100 mL, 250 mL (or other similar amount suitable for intravenous drip delivery) of a physiological buffer (e.g., 0.9% saline).

[0367] The pharmaceutical formulations of the present invention may also be contained in unit dosage form. As used herein, the term "unit dosage form" refers to physically discrete units suitable for use as unit dosages for the patient to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier, diluent, or excipient. In various embodiments, the unit dosage form is contained within a container as discussed herein. Actual dosage levels of the active ingredient (e.g., a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2) in the formulations of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without adverse effects on the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or species used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts. The term "diluent" as used herein refers to a solution suitable for altering or achieving an exemplary or appropriate concentration or concentrations as described herein.

[0368] In various embodiments, the unit dosage form contains an amount of the active ingredient (e.g., a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2) intended for a single use. In various embodiments, the amount of the active ingredient in the unit dosage form is from about 0.1 mg to about 5000 mg, from about 100 mg to about 1000 mg, and from about 100 mg to about 500 mg, from about 100 mg to about 400 mg, from about 100 mg to about 200 mg, from about 200 mg to about 400 mg, from about 250 mg to about 350 mg, from about 125 mg to about 175 mg, from about 275 mg to about 325 mg, from about 1 mg to about 250 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 25 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg, or ranges or intervals thereof. Ranges intermediate to the amounts recited above, for example, from about 2 mg to about 100 mg or from 2 mg to 20 mg, are also intended to be part of the present invention. For example, it is intended to include a combination of any one of the values ​​described above (or the value contained in the value described above) as the value range of the upper limit and / or lower limit. In a particular embodiment, the preparation is often supplied in the form of a liquid in a unit dosage form. In some embodiments, the unit dosage form contains 2 to 2.5 mg or 10 to 11 mg, 20 to 25 mg, 80 to 90 mg, 100 to 125 mg, 160 to 180 mg, 200 to 225 or 320 to 360 mg, 200 to 400 mg. In some embodiments, the unit dosage form according to the present invention is suitable for subcutaneous administration to a patient (for example, a unit dosage form containing an antibody with a concentration of about 100 mg / ml or about 150 mg / ml).

[0369] In one embodiment, the invention provides a unit dosage form comprising about 20 mg, about 80 mg, about 160 mg, about 200 mg, about 320 mg, or about 400 mg of an antibody in a stable formulation, wherein the formulation comprises 200 mg or 400 mg of the antibody unit dosage form.

[0370] The present invention also includes methods of preparing unit dosage forms.In exemplary embodiments, a method for preparing a pharmaceutical unit dosage form comprises combining the formulation of any of the foregoing embodiments in a suitable container, such as those discussed herein.

[0371] In various embodiments, the pharmaceutical formulation is contained in a container (e.g., a vial or prefilled syringe) that may contain less than 5% by volume of an oxidizing gas (e.g., oxygen) headspace gas. In various embodiments, the concentration of the oxidizing gas (e.g., oxygen) in the headspace of the container may be less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, or less than 1.5%. In one embodiment, the concentration of the oxidizing gas (e.g., oxygen) in the headspace is less than about 1%. In one embodiment, the concentration of the oxidizing gas (e.g., oxygen) in the headspace does not exceed about 0.5%. In one embodiment, the concentration of the oxidizing gas (e.g., oxygen) in the headspace does not exceed about 0.1%. In various embodiments, the concentration of the oxidizing gas (e.g., oxygen) in the headspace of the pharmaceutical container is less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%. In some cases, the concentration of oxygen in the headspace gas is about 0.01% to about 1.5%. In some cases, the oxygen concentration in the headspace gas is from about 0.75% to about 1.25%. In some cases, the oxygen concentration in the headspace gas is from about 0.05% to about 0.15%. In various embodiments, the oxidizing gas (e.g., oxygen) in the headspace is replaced or substantially replaced by an inert gas such as nitrogen, argon, helium, xenon, neon, krypton, or radon. In one embodiment, the non-oxidizing gas is nitrogen. In one embodiment, the non-oxidizing gas is argon.

[0372] Therapeutic use of pharmaceutical preparations

[0373] The pharmaceutical formulations of the present invention are particularly useful for treating, preventing, and / or ameliorating any disease or condition associated with cells expressing human MASP-2. Exemplary, non-limiting diseases and conditions that can be treated by administering the pharmaceutical formulations of the present invention include diseases or conditions that would benefit from inhibition of MASP-2-dependent complement activation.

[0374] In some embodiments, the disease or condition that would benefit from inhibition of MASP-2-dependent complement activation comprises an autoimmune disease, a vascular disorder, ischemia-reperfusion injury, arteriosclerosis, inflammation, a pulmonary disorder, an extracorporeal reperfusion procedure, a musculoskeletal disorder, a renal disorder, a skin disorder, an organ or tissue transplant procedure, a nervous system disease or injury, a blood disorder, a genitourinary disorder, non-obese diabetes or complications associated with type 1 or type 2 diabetes, cancer, an endocrine disorder, an ophthalmologic disorder, or COVID-19.

[0375] In some specific embodiments, the autoimmune disease comprises thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy associated with hematopoietic transplantation (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy.

[0376] In some specific embodiments, the vascular disorders include cardiovascular disorders, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renal vascular disorders, mesenteric / intestinal vascular disorders, revascularization of grafts and / or regrafts, vasculitis, Henlein-Schonlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu arteritis, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis following stenting, rotational aneurysm resection, and percutaneous transluminal coronary angioplasty (PTCA).

[0377] In some specific embodiments, the ischemia-reperfusion injury includes ischemia-reperfusion injury associated with aortic aneurysm repair, cardiopulmonary bypass, revascularization associated with organ transplantation and / or limb / digit replantation, stroke, myocardial infarction and hemodynamic resuscitation after shock and / or surgery.

[0378] In some specific embodiments, the inflammation comprises inflammatory gastrointestinal diseases, including pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and diverticulitis.

[0379] In some specific embodiments, the pulmonary disorders include acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease); meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burns, non-cardiogenic pulmonary edema, transfusion-related dyspnea, emphysema, cystic fibrosis, SARS-CoV, MERS-CoV and SARS-CoV-2 (Covid-19) related diseases.

[0380] In some specific embodiments, the extracorporeal circulation reperfusion process includes hemodialysis, plasmapheresis, leukocyte apheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced exfracorporeal membrane oxygenation LDL precipitation (HELP) and cardiopulmonary bypass (CPB).

[0381] In some specific embodiments, the musculoskeletal disorder comprises osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathies, crystalline arthropathy, and systemic lupus erythematosus (SLE).

[0382] In some specific embodiments, the renal disorder comprises mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henlein-Schonlein purpura nephritis, and IgA nephropathy.

[0383] In some specific embodiments, the skin disorder comprises psoriasis, autoimmune bullous skin diseases, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita, herpes gestationis, thermal burns, and chemical burns.

[0384] In some specific embodiments, the organ or tissue transplantation process includes organ allotransplantation, organ xenotransplantation, and organ and tissue transplantation.

[0385] In some specific embodiments, the nervous system disease or injury comprises multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome, reperfusion following stroke, degenerative disc disease, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or hemorrhage, demyelination, and meningitis.

[0386] In some specific embodiments, the blood disease comprises sepsis, severe sepsis, septic shock, acute respiratory distress syndrome caused by sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

[0387] In some specific embodiments, the genitourinary disorders include painful bladder, sensory bladder, chronic nonbacterial cystitis, interstitial cystitis, infertility, placental dysfunction, miscarriage, and pre-eclampsia.

[0388] In some specific embodiments, the endocrine diseases include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the regulated release of prolactin, growth hormone or other insulin-like growth factors, and adrenocorticotropic hormone from the pituitary gland.

[0389] In some specific embodiments, the ophthalmic disease comprises age-related macular degeneration.

[0390] In some embodiments of the methods provided herein, a subject is determined to have elevated serum levels of C4, or to have C4d deposits or positive staining for C4d in a sample of interest. In some embodiments, the condition or disease is IgA nephropathy. Positive staining for C4d has been reported to be an independent risk factor for the development of ESRD in IgA nephropathy. C4d can be detected using any suitable method known in the art, for example, by using an ELISA or immunofluorescence microscopy.

[0391] The therapeutically effective amount of the antibodies or antigen-binding fragments provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medications, the subject's health and potential cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression. A person of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage based on these and other circumstances or requirements.

[0392] In certain embodiments, a therapeutically effective dose of an anti-MASP-2 antibody or antigen-binding fragment provided herein is from about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain embodiments, the dosage of the antibody or antigen-binding fragment is about 50 mg / kg or less, in certain embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage can be changed during the course of treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage can be changed during the course of treatment according to the subject's response.

[0393] Dosage regimens may be adjusted to provide the optimum desired response (eg, therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over a period of time.

[0394] The anti-MASP-2 antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0395] In some embodiments, the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with one or more additional therapeutic modalities or agents. For example, an antibody or antigen-binding fragment disclosed herein can be used in combination with another therapeutic agent, such as an anti-autoimmune drug.

[0396] In certain embodiments, an anti-MASP-2 antibody or antigen-binding fragment disclosed herein for administration in combination with one or more additional therapeutic agents can be administered simultaneously with the one or more additional therapeutic agents. In certain embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent can be administered as part of the same pharmaceutical composition. However, for an anti-MASP-2 antibody or antigen-binding fragment to be used "in combination" with another therapeutic agent, it is not necessary that the anti-MASP-2 antibody or antigen-binding fragment be used simultaneously with the therapeutic agent or in the same composition. An anti-MASP-2 antibody or antigen-binding fragment administered before or after another agent is considered to be administered "in combination" with that agent, as the phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with an antibody or antigen-binding fragment disclosed herein is administered according to the schedule listed in the product information sheet for the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or other regimens well known in the art.

[0397] The therapeutic methods of the present invention comprise administering to a subject any formulation comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human MASP-2 as disclosed herein. The subject to whom the pharmaceutical formulation is administered can be, for example, any human or non-human animal in need of such treatment. For example, the subject can be diagnosed with or considered to be at risk for any of the aforementioned diseases or conditions. The present invention further encompasses the use of any of the pharmaceutical formulations disclosed herein in the manufacture of a medicament for treating any disease or condition associated with cells expressing human MASP-2, including any of the exemplary diseases, disorders, and conditions mentioned above.

[0398] In some embodiments, the present invention provides a kit comprising a pharmaceutical formulation (e.g., a container having a formulation or unit dosage form) and packaging or labeling (e.g., a package insert) as discussed herein, and instructions for use of the pharmaceutical formulation to treat a disease or condition as discussed above. In some cases, the instructions provide the use of a unit dosage form as discussed herein for treating a disease or condition. DETAILED DESCRIPTION

[0399] The purpose of the following examples is to facilitate providing those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and is not intended to limit the scope of the invention as claimed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should also be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0400] Note: NT in the tables of the present invention means not detected unless otherwise specified; ND means not detected unless otherwise specified.

[0401] Example 1: Generation of Human, Mouse, and Cynomolgus Monkey MASP-2 Antigens

[0402] 1. Construction of Expression Antigen

[0403] To restrict the antibody's epitope to the complement binding and activation domains of human MASP-2, we designed a chimeric antigen for animal immunization. We synthesized coding sequences expressing the mouse MASP-2 CUB1-EGF-CUB2 domain (residues 20-297 of SEQ ID NO:40) and the human MASP-2 CCP1-CCP2-SP domain (residues 298-686 of SEQ ID NO:39). Furthermore, an IL-2 secretion signal peptide sequence (SEQ ID NO:41) was added to the N-terminus, and a FLAG tag sequence was added to the C-terminus. These elements were combined into an open reading frame (ORF) to create a chimeric antigen expression construct (SEQ ID NO:42). Other constructs expressing full-length human MASP-2 (SEQ ID NO:39), mouse MASP-2 (SEQ ID NO:40), and cynomolgus monkey MASP-2 (SEQ ID NO:43) were synthesized with a C-terminal HIS tag. In addition, another construct expressing full-length human MASP-2 (SEQ ID NO: 39) with a FLAG tag at the C-terminus was generated by PCR. Human MASP-2 protein (SEQ ID NO: 39)

[0404] Mouse MASP-2 protein (SEQ ID NO:40)

[0405] IL-2 secretion signal peptide sequence (SEQ ID NO: 41)

[0406] Chimeric MASP-2 protein (SEQ ID NO:42)

[0407] Cynomolgus monkey MASP-2 protein (SEQ ID NO:43)

[0408] 2. Expression and Purification of MASP-2 Antigen

[0409] The aforementioned MASP-2 expression constructs were transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit. ExpiCHO-S cells were cultured in serum-free ExpiCHO Expression Medium. Fourteen days after transfection, the supernatant was collected. After centrifugation and filtration, the supernatant was loaded onto an anti-FLAG or anti-HIS column and purified using a GE Akta purification system. After washing, the MASP-2 protein was eluted with citric acid (pH 3.5) and used for animal immunization.

[0410] Example 2: Generation of MASP-2 Antibodies

[0411] 1. Immunization and Hybridoma Fusion

[0412] Different strains of mice or rats were immunized with DNA expressing the chimeric MASP-2 antigen described above using the Helios gene gun system (Bio-Rad). Immunized animals were boosted with recombinant MASP-2 protein every two weeks. Four days after the final boost, the animals were sacrificed for hybridoma fusion. Spleen cells were isolated and fused with SP2-0 cells via electrofusion. The resulting hybridoma cells were cultured in DMEM medium supplemented with hypoxanthine, aminopterin, and thymidine.

[0413] 2. Hybridoma Screening

[0414] After 10 days of culture, hybridoma supernatants were collected for antigen binding screening. Full-length human MASP-2 protein was coated onto ELISA plates at a concentration of 0.5 μg / ml, with 100 μl per well. After blocking with PBS containing 1% BSA, 1% normal goat serum, and 0.05% Tween 20, hybridoma supernatants were added to the plates for 1 hour. Specific binding of the hybridoma antibodies to human MASP-2 was detected using horseradish peroxidase (HRP)-linked anti-mouse antibodies. Positive clones that showed ELISA binding were selected for further activity screening.

[0415] 3. Activity Screening

[0416] ELISA plates were coated with 10 μg / ml mannan, with 100 μl per well, overnight at 4°C. After washing three times with PBS + 0.1% Tween 20, the plates were blocked with blocking buffer (10 mM Tris-HCl + 0.1% human serum albumin + 140 mM NaCl) for 1 hour. 50 μl of hybridoma supernatant was mixed with 50 μl of 1% human serum (Quidel, A113) diluted in detection buffer (0.1% human serum albumin + 20 mM Tris-HCl + 2 mM CaCl2 + 140 mM NaCl + 1 mM MgCl2 + 0.05% Tween 20) and incubated on ice for 45 minutes. The blocking buffer was discarded from the mannan-coated plates, and the supernatant-serum mixture was added. The plates were incubated at 37°C for 1.5 hours. After washing three times with wash buffer, C4 activation was monitored by measuring C4b deposition. Deposited C4b is detected using an HRP-linked anti-C4c antibody (Quidel-A211). If MASP-2 activity is inhibited by the antibody, less C4b will be deposited on the bottom of the plate. Using this method, hybridoma antibodies with MASP-2 inhibitory activity are selected.

[0417] Positive antibodies were then subcloned and rescreened by ELISA binding and C4 activation assays.

[0418] Example 3: Characterization of the inhibitory activity of anti-human MASP-2 antibodies.

[0419] 1. Purification of Hybridoma Antibodies

[0420] After subcloning, positive hybridoma clones with neutralizing activity were expanded in 10 cm dishes. After centrifugation and filtration, the antibody-containing supernatant was loaded onto a Protein A column and purified using a GE AKTA purification system. After washing, the antibody was eluted with citric acid (pH 3.5).

[0421] 2. Activity Evaluation of Purified Antibodies

[0422] MASP-2 is a key component of the lectin pathway, cleaving complement factors C4 and C2 to generate the C3 convertase, C4bC2a. Activation of C3 ultimately leads to the formation of the membrane attack complex (MAC). To test whether antibodies that inhibit MASP-2 could reduce lectin pathway activation, activation of C4, C3, and MAC was assessed in the presence of purified antibodies against MASP-2.

[0423] ELISA plates were coated with 10 μg / ml mannan, with 100 μl per well, overnight at 4°C. After washing three times with PBS + 0.1% Tween 20, the plates were blocked with blocking buffer (10 mM Tris-HCl + 0.1% human serum albumin + 140 mM NaCl) for 1 hour. Antibodies were serially diluted in detection buffer (0.1% human serum albumin + 20 mM Tris-HCl + 2 mM CaCl2 + 140 mM NaCl + 1 mM MgCl2 + 0.05% Tween 20) containing 1% human serum (Quidel, A113) and incubated on ice for 45 minutes. The blocking buffer was discarded from the mannan-coated plates, and the antibody-serum mixture was added. The plates were incubated at 37°C for 1.5 hours. Activated complement components should be deposited on the bottom surface of the plate, while inactivated components should remain soluble in the buffer. After washing three times with wash buffer, activation of complement components was monitored using HRP-linked complement antibodies, including anti-C3c antibody (Quidel-A205), anti-C4c antibody (Quidel-A211), and anti-MAC (SC5b-9) antibody (Quidel-A239). The detection antibodies were HRP-linked in-house. MASP-2 antibodies purified from hybridoma cells blocked activation of complement C3 (see Figure 1), complement C4 (see Figure 2), and MAC (see Figure 3) in a dose-dependent manner.

[0424] Example 4: Cloning of V-type genes and generation of chimeric antibodies

[0425] 1. Cloning and sequencing of V-type genes of hybridoma antibodies

[0426] Lead antibodies with ideal characteristics were selected for V-type gene cloning. The sequences of the mouse anti-human MASP-2 light and heavy chain variable regions were obtained by polymerase chain reaction (PCR) amplification. Total RNA from positive hybridoma cells was isolated using the MiniBest Universal RNA Extraction Kit (TaKaRa), and cDNA was synthesized using the 1st Strand cDNA Synthesis Kit (TaKaRa) with oligo(dT) primers. The variable regions of the mouse IgG gene were amplified by PCR using primers of different isotypes for the heavy chain variable region and kappa chain primers for the light chain variable region. The PCR products were subcloned into a TA cloning vector. For each variable gene construct, more than 10 single clones were used for DNA sequencing by Synbio Technologies (Suzhou, China). The amino acid sequences of VH and Vκ were determined from the DNA sequencing results.

[0427] 2. Construction of Chimeric Antibodies

[0428] Three antibodies with different sequences, including 129C10, 160D10, and 125D5, were selected as lead antibodies for chimeric antibody generation; their SEQ IDs are listed in Tables 1 and 2. After sequencing analysis and confirmation, cDNAs for the heavy and light chain variable regions were synthesized and fused to the constant region sequences of human IgG4 and human kappa. To enhance antibody secretion, signal peptide sequences were added to the N-termini of the heavy and light chains, respectively. The resulting chimeric antibody genes were cloned into expression vectors. Large-scale DNA preparation was performed using the Qiagen Plasmid Maxi-prep System.

[0429] 3. Expression and Purification of Chimeric Antibodies

[0430] ExpiFectamine from Invitrogen was used according to the manufacturer's protocol. TM Co-transfection of heavy and light chains was performed using CHO reagent. TM CHO reagent: ExpiCHO-S cells were cultured in ExpiCHO expression medium at a rate of 5-6 x 10 6 The concentration of cells / ml was transfected with equal amounts of heavy chain vector and light chain vector DNA at a final concentration of 0.8μg / ml. TM CHO reagents with cold OptiPRO TM Dilute in culture medium and mix by swirling the tube and / or inverting. TM The CHO / plasmid DNA mixture was incubated at room temperature for 1-5 minutes and then slowly transferred to the shake flask containing cells. The transfected cells were cultured at 37°C in a humidified atmosphere of 5% CO2 on an orbital shaker (shaking speed of 125 rpm). 18 to 22 hours after transfection, the ExpiCHO TM Feed, and harvest the conditioned medium on day 10. The supernatant was centrifuged at 4,000 rpm for 20 minutes and then filtered through a 0.22 μm filter capsule to remove cell debris. The filtered supernatant was loaded into a pre-equilibrated Protein-A affinity column. The Protein-A resin was washed with equilibration buffer (PBS), and the antibody was eluted with 25 mM citrate (pH 3.5). The purified antibody solution was adjusted to pH 6.0-7.0 with 1 M Tris-base (pH 9.0). Endotoxin was controlled below 1 EU / mg. Finally, the purified antibody was identified by SDS-PAGE.

[0431] Example 5: Generation and characterization of humanized antibodies

[0432] 1. Production, Expression, and Purification of Humanized Antibodies

[0433] The sequence of the variable region domains of mouse antibody 129C10 was used to determine the germline sequence with the highest homology to the respective mouse frameworks. Humanized variants with CDR grafting and backmutations were designed using computer modeling.

[0434] 129C10

[0435] The human germline framework sequences VH / 1-2 were used for the heavy chain, and VK / 2-30 for the light chain, respectively, for CDR grafting.

[0436] Heavy chain (HC) variants 1, 2, 3, and 4 were generated by directly grafting three CDRs onto the germline sequence (SEQ ID NO: 37), with the addition of R71V, A93T mutations (SEQ ID NO: 20) for HC variant 1, R71V, A93T, V67A, M69L mutations (SEQ ID NO: 22) for HC variant 2, R71V, A93T, A65G, K64Q, E61Q mutations (SEQ ID NO: 24) for HC variant 3, and R71V, A93T, V67A, M69L, A65G, K64Q, E61Q mutations (SEQ ID NO: 26) for HC variant 4. It should be noted that HC variants 3 and 4 have three mutations (A65G, K64Q, E61Q) introduced into HC CDR2 to further enhance the humanization of the antibody.

[0437] Germline sequence of 129C10HC:

[0438] VH / 1-2 (129C10-HC germline, SEQ ID NO: 37).

[0439] VH / 1-2 variant 1 (Hu129C10_Ha, SEQ ID NO:20).

[0440] VH / 1-2 variant 2 (Hu129C10_Hb, SEQ ID NO: 22).

[0441] VH / 1-2 variant 3 (Hu129C10_Hc, SEQ ID NO: 24).

[0442] VH / 1-2 variant 4 (Hu129C10_Hd, SEQ ID NO: 26).

[0443] Light chain (LC) variants 1 and 2 were generated by direct grafting of three CDRs to the germline sequence (SEQ ID NO: 38), with the addition of an F36L backmutation (SEQ ID NO: 28) for LC variant 1 and an F36L, T69A backmutation (SEQ ID NO: 30) for LC variant 2, respectively.

[0444] Germline sequence of 129C10LC

[0445] VK / 2-30 (129C10-LC-germline, SEQ ID NO: 38)

[0446] VK / 2-30 variant 1 (Hu129C10_La, SEQ ID NO:28).

[0447] VK / 2-30 variant 2 (Hu129C10_Lb, SEQ ID NO:30).

[0448] The cDNAs for the variable regions of the heavy and light chains were synthesized and then fused with sequences for the constant regions of human IgG4 and human kappa. The resulting antibody gene sequences were cloned into an expression vector. Large-scale DNA was prepared using the Qiagen Plasmid Maxiprep system and expressed using Invitrogen's ExpiFectamine according to the manufacturer's protocol. TM Cells were transfected using CHO reagent. The supernatant was harvested when cell viability exceeded 60% and filtered through a 0.22 μm filter capsule to remove cell debris. The filtered supernatant was then loaded onto a pre-equilibrated Protein-A affinity column. The Protein A resin was washed with equilibration buffer (PBS), and the antibody was eluted with 25 mM citrate (pH 3.5). The purified antibody solution was adjusted to pH 6.0-7.0 with 1 M Tris-base (pH 9.0). Endotoxin was controlled below 1 EU / mg. Finally, the purified antibody was identified by SDS-PAGE.

[0449] Benchmark antibodies OMS721-analog and 129C10-hu-YTE (Hu129C10_HaLa-hIgG4 with amino acid substitutions M252Y / S254T / T256E [YTE], see Tables 2-3) were also constructed, and the expression and purification procedures were the same as described above.

[0450] Example 6: Blocking Activity of Chimeric and Humanized MASP-2 Antibodies

[0451] The purified antibody was serially diluted starting at 100 μg / ml to create a concentration gradient. A complement C3 activation assay was used to assess the blocking activity of MASP-2 antibodies, as described in Example 3. 100 μl of 10 μg / ml mannan per well was coated onto an ELISA plate overnight at 4°C. The antibody was incubated with 1% human serum (Quidel, A113) on ice for 45 minutes. After washing and blocking the plate, the antibody-serum mixture was added and incubated at 37°C for 90 minutes. After washing, deposited activated C3 was detected using an HRP-linked anti-C3c antibody (Quidel-A205). Figure 4 shows the blocking activity of chimeric and humanized antibodies against complement C3 activation. Because the humanized 129C10 variant, 129C10HaLa, showed the best affinity and C3 blocking activity, 129C10HaLa was selected as the lead antibody for further in vitro and in vivo studies and was designated 129C10-hu.

[0452] Example 7: Comparison of the blocking activity of complement C4 and MAC between the lead antibody 129C10-hu and the benchmark antibody OMS721-analog

[0453] 1. Comparison of blocking activity of complement C4 and MAC activation

[0454] Using the assay described in Example 3, the blocking activity of the lead MASP-2 antibodies 129C10-hu and OMS721-analog against complement C4 and MAC activation in 2% human serum was compared. ELISA plates were coated with 100 μl of 10 μg / ml mannan per well overnight at 4°C. The antibodies were incubated with 2% human serum (Quidel, A113) on ice for 45 minutes. After washing and blocking the plates, the antibody-serum mixture was added and incubated at 37°C for 90 minutes. After washing, deposited activated C4 was detected using an HRP-linked anti-C4c antibody (Quidel-A211). MAC activation was detected using an HRP-linked anti-MAC (SC5b-9) antibody (Quidel-A239). As shown in Figures 5 and 6, the results showed that 129C10-hu was approximately 10 times more potent than OMS721-analog in blocking the activation of complement C4 (IC50: 0.11 μg / mL vs. 1.70 μg / mL) and MAC (IC50: 0.27 μg / mL vs. 1.97 μg / mL).

[0455] 2. Comparison of activity in serum of different concentrations

[0456] Different concentrations of human serum (1%, 10%, and 50%) were used in the complement C3 activation assay. For 1% and 10% human serum, the assay was performed the same as described in Example 3. For 50% human serum, mannan was coated at a concentration of 1 μg / ml, with 100 μl per well incubated overnight at 4°C. After washing three times with PBS + 0.1% Tween 20, the plates were blocked with blocking buffer (10 mM Tris-HCl + 0.1% human serum albumin + 140 mM NaCl) for 1 hour. Antibodies were serially diluted in assay buffer (0.1% human serum albumin + 20 mM Tris-HCl + 2 mM CaCl2 + 140 mM NaCl + 1 mM MgCl2 + 0.05% Tween 20) containing 50% human serum (Quidel, A113), followed by incubation on ice for 45 minutes. The blocking buffer was removed from the mannan-coated plates, and the antibody-serum mixture was added. The plates were incubated at 37°C for 30 minutes. After washing three times, activated C3 was detected using an HRP-linked anti-C3c antibody (Quidel-A205). As shown in Figure 7, 129C10-hu and OMS721-analog exhibited similar inhibitory efficacy against C3 activation in 1% serum (IC50: 0.08 μg / mL vs. 0.10 μg / mL). Interestingly, 129C10-hu exhibited three-fold greater potency than OMS721-analog at the high concentration of 10% serum (IC50: 0.20 μg / mL vs. 0.69 μg / mL) (see Figure 8). More importantly, at a concentration of 50% serum, 129C10-hu still exhibited blocking activity against C3 activation, with an IC50 of 0.05 μg / mL, while OMS721-analog lost its activity (see Figure 9). 129C10-hu also showed a 2-fold higher potency than OMS721-analog in blocking C4 activation in 10% human serum (IC50: 0.69 μg / mL vs. 1.59 μg / mL) (see FIG10 ).

[0457] Example 8: Detection of the Binding Affinity of MASP-2 Antibodies to Human MASP-2 by Biolayer Interferometry (ForteBio)

[0458] The antibody to be tested was diluted to a concentration of 100 nM in ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween-20). Human MASP-2 protein was diluted in kinetic buffer to create a gradient of 100 nM, 50 nM, and 25 nM. 0 nM was used as a reference control. The antibody was immobilized on a Protein A biosensor. A baseline was measured for 60 seconds, followed by 180 seconds for antibody-MASP-2 binding to obtain a K value. onFactor data. K was obtained by subsequent dissociation in kinetic buffer for 180 seconds. off Factor data were obtained. The biosensor was regenerated in 10 mM glycine, pH 2.0 buffer. All kinetic data were collected at 30°C. Data were acquired using a ForteBio Octet RED96 and analyzed using Octet Data Analysis software. As shown in Table 3, all tested MASP-2 hybridoma antibodies had high binding affinity to human MASP-2, with KD values ​​ranging from 10 -10 M to 10 -8 After humanization, the lead antibody 129C10-hu (129C10-HaLa) maintained the highest binding affinity to hMASP-2, with a KD value of <10 -12 M (reached the detection limit of ForteBio) (Table 4), K on The value is 3.53E+4, K off The value is <1.0E-7 (see Figure 11).

[0459] Table 3 Binding affinity of MASP-2 hybridoma antibodies

[0460] Table 4 Binding affinity of humanized or chimeric antibodies to MASP-2

[0461] Example 9: Detection of binding specificity of 129C10-hu by ELISA

[0462] Human C1s / C1r and MASP-1 / 3 were purchased from R&D, Cusbio, and others. Recombinant human complement components C1s / C1r, MASP-1, or MASP-3 (1 mg / ml) were coated overnight at 4°C, washed three times with wash buffer, added with blocking buffer (200 μL / well) for 1 hour at 4°C, washed three times, added with serially diluted Ab for 1 hour at room temperature, washed three times, added with mouse anti-human IgG4 HRP (1:20,000) for 1 hour at room temperature, washed three times, and detected with tetramethylbenzidine (TMB) at OD 450 nm for 40 minutes. The EC50 values ​​for 129C10-Hu and OMS721-analog binding to C1s, C1r, MASP1, MASP2, or MASP3 are shown in Figures 12A-12E, respectively. The results showed that 129C10-Hu only bound to human MASP2, but not to human C1s, C1r, MASP1 or MASP3.

[0463] Example 10: Cross-reaction of 129C10-hu by ELISA

[0464] Rat / mouse MASP-2 was ordered from Cusbio. Human / cynomolgus monkey MASP-2 was generated in-house. The ELISA assay was performed according to the following protocol: MASP-2 from various species (1 μg / ml) was coated overnight at 4°C; washed three times with wash buffer; blocking buffer (200 μL / well) was added for 2 hours at room temperature; washed three times; serially diluted 129C10-hu or OMS721-analog as a control was added for 1 hour at room temperature; washed three times; mouse anti-human IgG4 Fc HRP (1:20,000) was added for 1 hour at room temperature; washed three times; and detected with TMB at OD 450 nm for 2 minutes. The EC50 values ​​for binding of 129C10-hu and OMS721-analog to MASP-2 from various species are shown in Figures 13A and 13B, respectively.

[0465] Example 11: Cross-reactivity of 129C10-hu with cynomolgus monkey MASP-2, as determined by using C4 activation in cynomolgus monkey serum

[0466] The assay was performed as described in Example 3, except that cynomolgus monkey serum was used. 100 μl of 10 μg / ml mannan was coated on an ELISA plate at 4°C overnight. The antibody was incubated with 1% cynomolgus monkey serum on ice for 45 minutes. After washing and blocking the plate, the antibody-serum mixture was added and then incubated at 37°C for 90 minutes. After washing, the deposited activated C4 was detected with an HRP-linked anti-C4c antibody (Quidel-A211). As shown in Figure 14, 129C10 blocked C4 activation in cynomolgus monkeys with an IC of 1. 50 The concentration of 129C10 was 0.4973 μg / ml, indicating that 129C10 could bind to cynomolgus monkey MASP-2 and block its activity.

[0467] Example 12: Selectivity of 129C10 in Blocking Activation of the MB-Lectin Complement Pathway

[0468] There are three pathways that initiate complement activation: the classical pathway, the MB-Lectin (MBL) pathway, and the alternative pathway. These pathways rely on different molecules for activation, yet they converge to produce a common set of effector molecules, such as the membrane attack complex (MAC). All three pathways are crucial components of innate immunity and play distinct roles in defending against different infections (Noris M, et al. 2013. JM.). Next, we tested the selectivity of 129C10-hu for blocking the MBL pathway.

[0469] The Wieslab Complement System Screening Kit (IBL America, Cat# COMPL 300RUO) was used to determine the selectivity of the lead antibody, 129C10-hu. Plates were pre-coated with mannan as an initiator of the MBL pathway, IgM as an initiator of the classical pathway, and LPS as an initiator of the alternative pathway. 129C10-hu was serially diluted in assay buffer containing human serum (Quidel, A113) and incubated on ice for 45 minutes. The antibody-serum mixture was added to the plate and incubated at 37°C for 60 minutes. After washing, deposited MACs were detected with an AP-linked anti-C5b-9 antibody. EDTA was used as a positive control to block complement activation. As shown in Figures 15a-15C, 129C10-hu only blocked complement activation initiated by the MBL pathway, but not the other two complement pathways, demonstrating that the 129C10-hu antibody selectively blocks complement activation via the MBL pathway.

[0470] Example 13: Prolonging the half-life of 129C10-hu by introducing a YTE mutation at the Fc site.

[0471] Dall'Acqua WF et al. reported that introducing the triple mutation M252Y / S254T / T256E (YTE) into the Fc region of IgG can enhance its binding affinity to FcRn and prolong its half-life in vivo (Dall'Acqua WF, et al. 2006. JBC). Motavizumab-YTE is the first human IgG with a YTE mutation and demonstrated good tolerability and prolonged half-life in a Phase I clinical trial (Robbie, GJ, et al. 2013. AAC).

[0472] We introduced the M252Y / S254T / T256E (YTE) mutation into 129C10-hu, generating 129C10-hu-YTE. Its binding affinity to FcRn was assessed using a biolayer interferometer (ForteBio). 129C10-hu or 129C10-hu-YTE was diluted in ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween-20) to a concentration series of 500 nM, 167 nM, 56 nM, 19 nM, and 0 nM. His-tagged human FcRn (FCGRT & B2M) protein was diluted in kinetic buffer to a concentration gradient of 100 nM. The FcRn protein was immobilized on a Ni-NTA biosensor. Association and dissociation kinetics were recorded and analyzed. As shown in FIG16A , FIG16B and Table 5, the YTE mutation increased the binding affinity of 129C10-hu to human FcRn by 3-fold.

[0473] Table 5 Binding affinity of 129C10-hu and 129C10-hu-YTE to human FcRn

[0474] Example 14: Pharmacokinetic (PK) / Pharmacodynamic (PD) Study of MASP-2 Antibodies 129C10-hu and 129C10-hu-YTE in Cynomolgus Monkeys

[0475] Two cynomolgus monkeys per group received 10 mg / kg of 129C10-hu, 129C10-hu-YTE, or OMS721-analog intravenously. Serum samples were collected at 0, 0.5, 2, 8, 24, 48, 72, 96, 168, 336, 504, 672, and 840 hours after infusion. Serum antibody concentrations and the efficacy of lectin pathway activation were measured.

[0476] Serum concentrations were determined using a developed ELISA method with a detection range of 0.625–40 ng / mL. Microplates were pre-coated with a human IgG-specific anti-IgG antibody [R10z8e6]. After blocking, standards (STD), quality control (QC) samples, matrix blanks, and test samples were added. After washing, biotinylated mouse anti-human IgG4 was added to the microplate wells, followed by HRP-labeled streptavidin. TMB was added to the microplate wells. OD values ​​for the QC and test samples were converted to concentrations by comparison with a simultaneously analyzed standard curve and regressed using a four-parameter logistic model.

[0477] For testing the efficacy of lectin pathway activation, 10 μg / ml mannan was coated onto ELISA plates. Cynomolgus monkey serum samples were diluted to 2% with C4 activation buffer. After washing and blocking the plates, the diluted serum was added and incubated at 37°C for 90 minutes. After washing, deposited activated C4 was detected using an HRP-linked anti-C4c antibody (Quidel-A211).

[0478] As shown in the PK results in Figure 17 and Table 6, the half-life of 129C10-hu in crab-eating monkeys was 164.77 hours, longer than that of OMS721-analog (130.152 hours). The YTE mutation increased the half-life of 129C10-hu to 274.404 hours. As shown in the PD results in Figure 18, the activation efficacy of the lectin pathway was suppressed to the basal level 0.5 hours after administration of the antibody. The inhibitory effect lasted for 2 weeks in the OMS721-analog group, 3 weeks in the 129C10-hu group, and about 4 weeks in the 129C10-hu-YTE group.

[0479] Table 6 Summary of data from cynomolgus monkey PK studies

[0480] In a separate study, male and female cynomolgus monkeys were assigned to five groups of two males and two females each and received either subcutaneous injections of 0 (vehicle control), 15, 20, and 295.8 mg / kg 129C10-hu or intravenous injections of 15 mg / kg 129C10-hu at 3 mL / kg for four weeks (up to five times). The vehicle control was administered in buffered saline.

[0481] Blood samples were collected before dosing and approximately 0.083, 2, 6, 24, 48, 96, and 168 hours after the first (see Figure 19) and fourth (see Figure 20) doses. Serum complement 4c (C4c) was analyzed using an enzyme-linked immunosorbent assay (ELISA). Briefly, mannan (Sigma) was diluted to 10 μg / ml in coating buffer. 100 μl of mannan working solution was added to each well of a 96-well plate. The plate was incubated overnight at 4°C. The plate was washed three times, then 200 μl of blocking buffer was added and incubated at room temperature for 1 hour. Monkey serum was diluted to 2% with C4 activation buffer. 100 μl of the diluted serum was added to the 96-well plate and incubated at 37°C for 70 minutes. The plate was washed three times with wash buffer. 100 μl of HRP-linked anti-C4c antibody (diluted 1:5000 in blocking buffer) was added and incubated at room temperature for 1 hour. The plate was washed three times with wash buffer. Add 100 μl of TMB substrate solution to each well of a 96-well plate and incubate at room temperature for 5-10 minutes. Add 50 μl of stop solution to each well. Read the plate at 450 nm. Optical density (OD450) values ​​at 450 nm are expressed as individual values ​​and averaged for each animal at each time point.

[0482] After the first and fourth doses, serum C4c showed dose-dependent reductions at doses ≥15 mg / kg, beginning 2 hours after dosing in animals administered subcutaneously and 0.083 hours after dosing in animals administered IV. The effects persisted throughout the dosing period at all three dose levels administered SC or IV, with maximal effects observed 24-96 hours after the first dose. Maximum mean reductions from baseline of 88.4% in males and 92.8% in females were noted at a SC dose of 295.8 mg / kg. In conclusion, 129C10-hu significantly reduced serum C4c in monkeys in a dose-dependent manner after five weekly doses of 15, 60, or 295.8 mg / kg administered SC or IV, suggesting that serum C4c may be a potential pharmacodynamic marker.

[0483] Example 15: Materials and methods for antibody formulation studies

[0484] The protein, i.e., antibody molecule, selected for the preparation research of the present invention was examined using 129C10-hu-YTE as an example.

[0485] The instruments and equipment used in the present invention are shown in Table 7 below, the consumables are shown in Table 8 below, and the reagents are shown in Table 9 below.

[0486] Table 7 Instruments and Equipment

[0487] Table 8 Consumables

[0488] Table 9 Reagents

[0489] The method steps involved in the present invention are as follows:

[0490] 1. General Buffer Preparation Method: All buffers are prepared using specific acidic and basic ion pairs. Accurately weigh the excipients for the acidic and basic ion pairs and add approximately 60% of the target buffer volume to Milli-Q water. Mix thoroughly and measure the pH of the solution. If the pH deviates from the target, adjust the pH using the appropriate ion pair. Then, dilute the solution to the target weight or volume with Milli-Q water. Finally, measure the solution's conductivity, osmotic pressure, and pH for verification.

[0491] 2. Sample preparation method: Use dialysis to replace the DS buffer into the target formulation, or directly add high-concentration excipients and surfactants into high-concentration DS, and then dilute DS to the target concentration. The dialysis method can be divided into the following two types according to the consumables used: (1) If the formulation of the target formulation is different from the buffer in the DS, use dialysis to replace the DS buffer with the target formulation (surfactants cannot be added through dialysis). Load the sample into The dialysis bag is sealed and placed in a buffer solution with a volume of approximately 100 to 200 times the sample volume. Dialysis is performed three times, with durations of 4 hours, 4 hours, and overnight, respectively, at a stirring speed of 200 rpm. Then, an appropriate amount of surfactant and other excipient stock solutions are pipetted into the sample solution and the sample solution is diluted to the target concentration. (2) Slide-A-Lyzer dialysis cards can also be used for dialysis. Use a syringe with a needle to load 3-12 ml of sample into the dialysis card, immerse it in 100 to 200 times the target buffer solution, and dialyze the dialysis card three times at room temperature with stirring at 300 rpm for 4 hours, 4 hours, and overnight, respectively.

[0492] 3. Appearance: Check the appearance of the black and white backgrounds using a YB-2 clarity tester. Report clarity, color, and visible particles.

[0493] 4. pH: The pH of the sample is measured by Measure the sample pH using a Seven Compact pH meter with a Micro-Pro-ISM electrode. Calibrate the pH meter before use.

[0494] 5. Protein Concentration: Protein concentration was determined by absorbance at 280 nm on a NanoDrop 2000 spectrophotometer. The extinction coefficient (E1%) used throughout this study was 1.422 L / g / cm. Each sample was measured in duplicate at a loading volume of 2.0 μL, and the average concentration was reported.

[0495] 6. Dynamic Light Scattering (DLS): Protein size distribution was determined by DLS using the following measurement conditions: acquisition time: 5 s, acquisition per measurement: 20 times, temperature: 25°C.

[0496] 6.1. Determination of k using DLS D Value: The samples were diluted with corresponding buffer to concentrations of 1, 2, 4, 8, 12, 16, and 20 mg / mL, and the k D The values ​​were analyzed, with acquisition time of 5 s, 20 acquisitions per measurement, and temperature of 25 °C.

[0497] 6.2. Determination of Viscosity by DLS: The sample to be tested was mixed with standard particles with a diameter of 100 nm. The final concentration of the standard particles was 1%. The apparent particle size of the standard particles was measured by DLS. The acquisition time was 5 s, the acquisition time was 20 times per measurement, and the temperature was 25°C. The acquired data was analyzed using DYNAMICS software to calculate the viscosity of the system.

[0498] 6.3. Determination of T using DLS onset Value: T onset The test measures the transition temperature when the protein undergoes conformational changes or aggregation during the heating process, causing the particle size to change. The sample was diluted to 4 mg / mL with the corresponding buffer, 100 μL was added to a 96-well plate, centrifuged at 3000 rpm for 5 minutes, 10 μL of silicone oil was added to cover the sample, and centrifuged at 3000 rpm for 5 minutes. The instrument linearly heated the sample at 1°C / min within the range of 25-80°C, and a single sample was measured 8 times, with each acquisition lasting 3 seconds. The particle size and temperature data were collected, and the curve was drawn. The linear intersection function provided by the official software of the instrument was used to calculate the T value of the sample. onset .

[0499] 7. Size Exclusion Chromatography (SEC): Protein aggregation was determined by SEC using a Waters H Class ultra-high performance liquid chromatograph and a Waters BEH Protein SEC column (150 × 4.6 mm, 1.7 μm). The mobile phase consisted of 50 mM sodium phosphate buffer, 300 mM NaCl, pH 6.8 ± 0.1. The flow rate was 0.4 mL / min. Samples were diluted to 2 mg / mL, the assay volume was 10 μL, and the detection wavelength was 280 nm.

[0500] 8. Cation Exchange Chromatography (CEX): Protein charge heterogeneity was determined using CEX-HPLC. The liquid chromatography system was an Agilent 1260 Infinity II HPLC system using a Thermo Propac WCX-10 BioLC column (4 × 250 mm, 10 μm). After homogenization, the sample was centrifuged, the supernatant was transferred, and the sample was diluted to 2.0 mg / mL with mobile phase A. Chromatographic conditions are shown in Table 10.

[0501] Table 10 Cation chromatography operating conditions

[0502] 9. Non-reducing capillary electrophoresis (NR-CE-SDS): Protein fragmentation was determined by NR-CE-SDS. The standard or test sample was diluted to 4 mg / mL with phosphate-citrate buffer, and 25 μL was vortexed with 75 μL SDS sample buffer and 5 μL NEM (100 mM N-ethylmaleimide) for denaturation. The denatured sample was centrifuged, incubated at 70 ± 2°C for 10 ± 2 min, cooled at room temperature, and then centrifuged again. Separation was performed on a PA800 plus using an SDS separation gel kit and uncoated fused silica capillaries. High-speed separation mode was used, with an effective capillary length of 10 cm.

[0503] 10. Protein Solution Turbidity Test (OD405): Measure the sample turbidity using an Envision multi-functional microplate reader. Use buffer as a blank control. The difference between the sample and blank control is the sample's OD value at 405 nm.

[0504] 11. Microfluidics Imaging (MFI): The number of subvisible particles in the sample is determined using the Protein Simple MFI 5200. 1 mL of sample is pipetted directly without dilution. The number of particles ≥2 μm, ≥10 μm, and ≥25 μm per mL of sample is reported.

[0505] 12. Whole-column imaging capillary isoelectric focusing (icIEF): The sample is mixed with an appropriate mixture containing a pI marker and ampholytes and then introduced into the FC-coated capillary of the iCE3 system via an autosampler. After a high-voltage separation step, the different charge isomers of the protein migrate to their respective isoelectric points. These separated positions are then captured by a UV detector (UV absorption spectra acquired at 280 nm using a CCD camera with full-column detection). The isoelectric point of the sample peak is calibrated using the iCE system software based on the focus position of the corresponding pI marker. Subsequent data analysis is performed in Empower software. Chromatographic conditions are shown in Table 11 below.

[0506] Table 11 Chromatographic operating conditions

[0507] 13. Binding Activity: This method utilizes an anti-MASP-2 antibody C4 activation inhibition assay. The coating is mannan, the detection antibody is Complement C4c Antibody (HRP), and the substrate for the enzyme reaction is TMB. The mannan coating is adsorbed onto an ELISA solid-phase support adsorption plate. After washing and blocking, a mixture of the test sample and human serum complement is added to bind to the plate. The higher the sample concentration, the greater the inhibition of MASP-2 cleavage of C4 in serum, the lower the C4b content, and the less C4c cleaved. After incubation and washing, the detection antibody is added, followed by further incubation and washing to remove unbound detection antibody. The substrate is added for color development, and finally, the reaction stop solution is added. The absorbance is read on a microplate reader at 450 nm / 650 nm. The logarithm of the anti-MASP-2 antibody protein concentration is plotted on the horizontal axis, with OD values ​​plotted as OD values. 450nm-650nm The IC values ​​of the samples and the standards were obtained by four-parameter fitting. 50 The final result is based on the standard IC 50 Divide the value by the sample IC 50 The relative activity of the values ​​is reported.

[0508] Example 16: Excipient screening experiment

[0509] 1. Experimental Design

[0510] The sample formulation information, stability test storage conditions, and test methods are shown in Table 12. The UFDF sample was prepared by dialysis replacement into the target formulation buffer, followed by dilution to 30 mg / mL with the corresponding excipient stock solution, and finally filtration through a 0.22 μm PVDF membrane.

[0511] Table 12 Excipient screening experimental design

[0512] 2. Experiment

[0513] 2.1. Experimental results at 40°C

[0514] After 14 days at 40°C, there was no significant difference in particle size, concentration, and pH among the five formulations. In terms of purity, the purity of all formulations decreased, with F2 (NaCl) and F3 (Sor) having the most SEC main peaks, and the CEX main peak of F3 (Sor) decreasing the most. The specific degradation trends are shown in Figures 24, 25, and 26.

[0515] 2.2. Shaking test results

[0516] The CEX results of each formulation under shaking conditions in the excipient screening are shown in Figure 27. After shaking for 1 day, 3 days, and 5 days, there was no significant change in the appearance, concentration, OD405, and purity of the five formulations.

[0517] 2.3. Stirring test results

[0518] The appearance of samples from all five formulations changed from opalescent to turbid, but F4 (Suc) remained opalescent after 2 hours of stirring, while the other formulations became turbid after 2 hours of stirring. With the exception of F1 (Arg), the OD405 values ​​of all formulations increased. The concentration and pH of all formulations remained unchanged. After stirring for 2 and 6 hours, the SEC purity of the five formulations remained unchanged. The CEX and NR CE-SDS results of each formulation under stirring conditions during excipient screening are shown in Figures 28 and 29.

[0519] 2.4. Freeze-thaw test results

[0520] After five cycles of freeze-thaw cycles, sample concentration, particle size, OD405, and pH remained unchanged for all five formulations. Regarding insoluble particulate matter, the concentration of F2 (NaCl)-insoluble particles showed a significant increase. The MFI results of each formulation under freeze-thaw conditions during excipient screening are shown in Table 13.

[0521] Table 13 MFI results_freeze-thaw

[0522] Summarize

[0523] For F2 (NaCl) and F3 (Sor), the decrease in the main peaks in SEC and CEX at 40°C was greater than that of the other formulations, significantly inferior to the other formulations. Therefore, these two excipients were excluded. Overall, there were no significant differences between arginine, sucrose, and trehalose, all meeting the experimental requirements. Considering the cost and the wide application of excipients in biologics, sucrose F4 (Suc) was selected for the next step of the experiment.

[0524] Example 17: Surfactant screening experiment

[0525] 1. Experimental Design

[0526] Table 14 lists sample formulation information, stability test conditions, and testing methods. UFDF samples were diluted to 50 mg / mL using the corresponding excipient stock solution and filtered through a 0.22 μm PVDF membrane. 1 mL of sample was transferred to a 2R vial for the 40°C test, 2 mL to a 6R vial for the stirring test, and 2 mL to a 2R vial for the shake test.

[0527] Table 14 Surfactant screening experimental design

[0528] 2. Experiment

[0529] 2.1. Experimental results at 40°C

[0530] (1) Appearance, particle size, Conc., pH and OD405

[0531] As shown in Table 15, the appearance, particle size, Conc., pH and OD405 of the six formulations did not change after treatment at 40°C.

[0532] Table 15 Appearance, particle size, concentration and OD405 of surfactant screening experiment_High temperature test results

[0533] SOC=Slight Opalescence

[0534] (2)SEC

[0535] As shown in Table 16 and Figure 30, the main SEC peaks of all samples decreased slightly after treatment at 40°C, but there was no significant difference among the six formulations.

[0536] Table 16 SEC results of surfactant screening experiment_high temperature experiment

[0537] (3) NR CE-SDS

[0538] As shown in Table 17, the NR CE-SDS MP% of all formulations decreased after treatment at 40°C, but the changes were not large and were all within the acceptable range.

[0539] Table 17 Surfactant screening experiment NR CE-SDS results_high temperature experiment

[0540] (4)CEX

[0541] As shown in Table 18, after treatment at 40°C, the main peak MP% of all formulations decreased moderately, but all were within an acceptable range, and there was no significant difference in the CEX of each formulation.

[0542] Table 18 CEX results_high temperature test

[0543] 2.2. Stirring test results

[0544] The stability results of each formulation in the surfactant screening under 100 rpm stirring conditions are shown in Table 19, Figures 31, and 32. After 6 hours of stirring at 100 rpm, the appearance of F6 (T2), F7 (T5), F9 (W2), and F10 (W5) changed from opalescent to turbid. The OD405 values ​​of all formulations increased, while the concentration and pH remained unchanged. After 2 and 6 hours of stirring, the purity of all six formulations remained unchanged.

[0545] Table 19 Appearance, concentration, OD405, pH, particle size results_Stirring experiment

[0546] SOC = Slightly Opalescent, Turbid = Turbid

[0547] 2.3. Shaking test results

[0548] (1) Appearance, concentration, OD405

[0549] As shown in Table 20, after shaking at 200 rpm for 5 days, there was no significant change in the appearance, concentration and OD405 of each formulation.

[0550] Table 20 Appearance, concentration, OD405 results_shaking experiment

[0551] SOC=Slight Opalescence

[0552] (2) NR CE-SDS

[0553] As shown in Table 21, after shaking treatment, there was no significant difference in the changes of the main peaks of NR CE-SDS for all formulations.

[0554] Table 21 NR CE-SDS results_shaking experiment

[0555] (3)CEX

[0556] As shown in Table 22, after shaking, the main CEX peaks of all formulations did not change significantly.

[0557] Table 22 CEX results_shaking test

[0558] 3. Summary

[0559] At 40°C, there was no significant change in the appearance, OD405, and DLS results of each formulation. The SEC of all formulations decreased slightly, and the main peaks of NR CE-SDS and CEX decreased moderately. The differences between the formulations were small.

[0560] Under stirring conditions, the concentrations of the formulations did not change significantly, but the appearance of F6 (T2), F7 (T5), F8 (W2) and F10 (W5) changed from opalescent to turbid, the OD405 of F7 (T5), F9 (W2) and F10 (W5) increased, the DLS particle size and PDI of all formulations increased, and the purity did not change significantly.

[0561] Under shaking conditions, there was no significant change in the appearance and DLS results of each formulation, no significant change in the purity of all formulations, and no difference between the formulations.

[0562] In the surfactant screening, there was no significant difference between polysorbate 80 and polysorbate 20. All formulations showed some resistance to shaking stress, meeting the experimental requirements. Based on comprehensive cost considerations, polysorbate 80 was selected as the surfactant for the subsequent experiments.

[0563] Example 18: pH screening experiment

[0564] 1. Experimental Design

[0565] The sample formulation information and test method are shown in Table 23. The UFDF sample was prepared by dialysis replacement into the target formulation buffer, then diluted to 30 mg / mL with the corresponding excipient stock solution, and finally filtered through a 0.22 μm PVDF membrane. After the sample preparation, the appearance, concentration, osmotic pressure, viscosity, k D , T onset Perform the measurement.

[0566] Table 23 pH screening experiment design

[0567] 2. Experiment

[0568] Freeze-thaw experiments

[0569] (1) Concentration, pH, particle size and OD405 results

[0570] As shown in Table 24, after five freeze-thaw cycles, the OD405 values ​​of F18 (Cit 5.5), F19 (Cit 6.0), and F20 (Cit 6.5) showed an increasing trend, while the OD405 values ​​of the remaining formulations remained unchanged. The sample concentration and pH values ​​of all nine formulations remained unchanged. The particle size of the samples F18 (Cit 5.5), F19 (Cit 6.0), and F20 (Cit 6.5) was larger, and remained unchanged after freeze-thaw.

[0571] Table 24 Appearance, concentration, pH and OD405 results_freeze-thaw

[0572] (2) MFI results

[0573] As shown in Table 25, at T0, the concentration of insoluble particles ≥25 μm was less than 10 particles / mL for all formulations, and less than 100 particles / mL for insoluble particles ≥10 μm for all formulations. After freeze-thaw, the concentrations of insoluble particles ≥2 μm and ≥10 μm in F19 (Cit6.0) showed a significant increase, while no significant differences were observed among the other formulations.

[0574] Table 25 MFI results_freeze-thaw

[0575] 2.2. Shaking experiment

[0576] (1) Appearance, concentration, pH and OD405 results

[0577] As shown in Table 26, at T0, all samples exhibited opalescence and no visible foreign matter was found. Protein concentration and pH values ​​met the requirements. After shaking at 200 rpm for 5 days, F18 (Cit 5.5) became turbid, and the OD405 values ​​of F18 (Cit 5.5), F19 (Cit 6.0), and F20 (Cit 6.5) increased. The appearance and OD405 values ​​of the remaining formulations remained unchanged. The sample concentrations and pH values ​​of all nine formulations remained unchanged.

[0578] Table 26 Appearance, concentration, pH and OD405 results_Shaking

[0579] SOC=Slight Opalescence

[0580] Turbid

[0581] (2) NR CE-SDS and CEX results

[0582] As shown in Tables 27 and 28, the main peaks of NR CE-SDS and CEX did not change for all formulations.

[0583] Table 27 NR CE-SDS results_shaking

[0584] Table 28 CEX results_shaking

[0585] 2.3. High temperature 40℃ experiment

[0586] (1) Concentration, pH, particle size and OD405 results

[0587] As shown in Table 29, after 14 days at 40°C, there were no significant differences in the concentration, pH, and particle size of the nine formulations. However, the OD405 of F18 (Cit 5.5) increased to a certain extent.

[0588] Table 29 Concentration, pH and OD405 Results_High Temperature

[0589] (2)SEC Results

[0590] As shown in Table 30 and Figure 21, the SEC HMW% content of proteins in the histidine formulation was generally low, while the SEC HMW% content of proteins in the acetic acid formulation and the citric acid formulation increased with increasing pH, with the SEC HMW% of F14 (Ace5.5) increasing to a greater extent.

[0591] Table 30 SEC results_high temperature

[0592] (3) NR CE-SDS results

[0593] As shown in Table 31 and Figure 22, at a high temperature of 40°C, the LMW% of all formulations increased over time, but the results of all formulations were within the acceptable range.

[0594] Table 31 NR CE-SDS results_high temperature

[0595] (4) CEX results

[0596] As shown in Table 32 and Figure 23, the results of all formulations were within an acceptable range, among which the main peak of F17 (His6.0) showed the largest change, and there was no significant difference in the changes of the other formulations.

[0597] Table 32 CEX results_high temperature

[0598] 3. Summary

[0599] In the pH screening experiment at T0, the particle sizes of F18 (Cit5.5), F19 (Cit6.0) and F20 (Cit6.5) were larger, indicating a looser structure. In the shaking experiment, the appearance of F18 (Cit5.5) changed from opalescent to turbid, which was inferior to the other formulations. In the freeze-thaw experiment, the number of insoluble particles in F19 (Cit6.0) and F20 (Cit6.5) changed greatly. At 40°C, the OD405 of F18 (Cit5.5) increased, the SEC high polymer of F14 (Ace5.5) increased significantly, and the CEX main peak of F16 (His5.5) and F17 (His6.0) decreased significantly, and the NR CE-SDS oligomers increased significantly.

[0600] Overall, the stability of the acetic acid and histidine systems is better. In general, pH 4.5 and pH 5.0 in the acetic acid system, and pH 5.0 and pH 5.5 in the histidine system have better stability for proteins. Considering that the acetate buffer system at pH 5.0 and the histidine system at pH 5.5 have better buffering capacity and robustness, there is a better operating space when adjusting the pH in the process, and better risk control for extreme cases that deviate from the target value. In addition, for preparations for subcutaneous injection, the use of histidine is less irritating and has higher patient tolerance. Therefore, F13 (Ace5.0) acetate buffer pH 5.0 and F16 (His5.5) histidine buffer pH 5.5 are preferred buffer systems.

[0601] Example 19: Low-concentration prescription confirmation test

[0602] 1. Experimental Design

[0603] Sample formulation information and testing methods are shown in Table 33. Samples were prepared by dialysis into the target formulation's buffer, then diluted to 20 mg / mL with the corresponding excipient stock solution, and finally filtered through a 0.22 μm PVDF membrane. After sample preparation, their appearance, concentration, osmotic pressure, viscosity, kD, and tonset were measured.

[0604] Table 33 Prescription design scheme

[0605] 2. Experimental Results

[0606] 2.1 Appearance, concentration, pH, particle size and OD405 results

[0607] As shown in Table 34, formulation F21 showed no significant changes in its basic properties (appearance, concentration, pH, and biological activity) after undergoing stirring, freeze-thaw, light exposure, shaking, and incubation at 5°C, 25°C, 40°C, -20°C, and -30°C. Particle size and PD% showed no significant differences under any stability conditions except 40°C; as 40°C is a relatively severe stress condition for protein formulations, significant variations in results are normal and should be evaluated based on the results of SEC, NR-CE, and CEX.

[0608] Table 34 Appearance, concentration, pH and OD405 results

[0609] SOC = Slightly Opalescent, Multimodal = Multimodal, NT = Not Detected

[0610] 2.2 SEC, CEX, and NR CE-SDS results

[0611] As shown in Table 35, the results of SEC, CEX, and NR CE for Formulation F21 remained unchanged after stirring, freeze-thaw, exposure to light, shaking, and incubation at 5°C, 25°C, -20°C, and -30°C. At 40°C, the main peaks for SEC, CEX, and NR CE decreased to some extent, but this did not affect biological activity. There were no significant differences in any of the test items when comparing samples inverted at 40°C for four weeks with those in upright storage, demonstrating good compatibility between the packaging material and the protein formulation.

[0612] Table 15 SEC, CEX, NR CE-SDS results

[0613] ND means not detected

[0614] 3. Summary

[0615] Results from low-concentration formulation confirmation experiments demonstrated that the final formulation, F21, maintained long-term stability at 5°C, 25°C, -20°C, and -30°C, exhibited good tolerance to freeze-thaw and shaking, and remained stable under light and agitation conditions. However, some changes occurred at 40°C, necessitating that this sample be protected from prolonged exposure to high temperatures. The low-concentration formulation development process involved surfactant screening, excipient screening, pH screening, and final formulation confirmation studies. The final formulation was selected as 20 mg / mL protein, 10 mM histidine salts, 8.6% (w / v) sucrose, 0.05% (w / v) polysorbate 80, and a pH of 5.3.

[0616] Example 20: High concentration experiment

[0617] 1. Experimental Design

[0618] The sample prescription information, stability test storage conditions and test methods are shown in Table 36. The UFDF sample was concentrated by ultrafiltration and diluted to 150 mg / mL with the corresponding excipient stock solution, and finally filtered through a 0.22 μm PVDF membrane to prepare the sample. D , T onset and viscosity determination.

[0619] Table 36 High concentration experimental design

[0620] 2. Experimental Results

[0621] Table 37 kD value, Tonset and osmotic pressure results

[0622] (1)k D Value Result

[0623] The kD values ​​for acetate buffers at pH 4.5, 5.0, and 5.5, and histidine buffers at pH 5.0, 5.5, and 6.0, were all positive. In 10 mM acetate buffer at pH 5.0, the kD for a formulation containing 5.8% sucrose and 230 mM proline as excipients was positive, while the kD for a formulation containing 125 mM arginine as excipient was negative. A positive kD indicates strong net repulsive forces between protein molecules in the system, which is beneficial for long-term colloidal stability. A negative kD indicates net attractive forces between protein molecules, which is detrimental to long-term colloidal stability.

[0624] (2)T onset result

[0625] T onset It can predict the thermal stability of proteins. Except for the F29 (ARG) arginine formulation, the T onset The values ​​are all greater than 60°C, indicating that the protein has good thermal stability in these formulations.

[0626] (3) Osmotic pressure

[0627] According to the osmotic pressure results, the concentrations of 125 mM arginine and 5.8% sucrose were lower than the target values ​​and needed to be further adjusted in subsequent experiments.

[0628] 3. Summary

[0629] Through general property studies in different buffers and excipients, the kD is a positive value, T onset The values ​​are all greater than 60℃, which meet the test requirements. D and T onset All are inferior to sucrose and proline. For subcutaneous injections, a histidine buffer system is less irritating to the injection, and the pH should not be too low. Therefore, overall, 10mM Acetate, pH 5.0, 10mM Histidine, pH 5.0, and 10mM Histidine, pH 5.5 are preferred, in combination with sucrose or proline for further research.

[0630] Example 21: 6 Prescription Research and Process Comparison Experiment

[0631] 1. Experimental Design

[0632] Table 38 shows sample formulation information, stability test storage conditions, and testing methods. Fed-batch UFDF samples were first concentrated to an appropriate concentration by ultrafiltration, then diluted to 150 mg / mL with the corresponding excipient stock solution, and finally filtered through a 0.22 μm PVDF membrane. 1 mL of sample was transferred to a 2R vial for 25°C, 40°C, and light testing, 2 mL of sample was transferred to a 6R vial for a stirring test, and 2 mL of sample was transferred to a 2R vial for a shake test.

[0633] Table 38 Best 6-prescription experimental design

[0634] 2. Experiment

[0635] 2.1. High temperature 40℃ experiment

[0636] (1) Appearance, particle size, concentration, OD405 and pH

[0637] As shown in Table 39, the osmolality of all formulations was within the pre-specified target (250-350 mOsm / kg H2O). Formulations containing sucrose as an excipient (F31, F32, F33, and F37) exhibited higher viscosities than those containing proline (F34, F35, and F36), while the histidine formulation exhibited higher viscosity than the acetic acid formulation. The pH values ​​of F34 (Ace50Pro), F35 (His50Pro), and F36 (Fed-Batch) were slightly higher than the target values ​​and require adjustment in subsequent experiments. After treatment at 40°C for 4 weeks, the appearance, particle size, concentration, and pH of all seven formulations remained unchanged. However, a slight increase in OD405 was observed for F31 (Ace50Suc), F32 (His50Suc), F33 (His55Suc), F34 (Ace50Pro), and F36 (His55Pro).

[0638] Table 39 Appearance, particle size, concentration, OD405 and pH results - high temperature 40℃ experiment

[0639] SYC: slightly yellow and clear, no visible foreign matter

[0640] CC: Colorless and clear, no visible foreign matter

[0641] (2)SEC

[0642] As shown in Table 40 and Figure 33, after treatment at 40°C for 4 and 6 weeks, the SEC HMW% increased and the MP% decreased for all formulations. The main peak of F31 (Ace50Suc) and F32 (His50Suc) decreased more significantly, while that of F33 (His55Suc) and F36 (His55Pro) decreased less. Comparing F31 (Ace50Suc) and F37 (Fed-Batch) with the same formulation, the Fed-Batch-derived stock solution showed a smaller decrease in the main SEC peak than the Perfusion solution at 40°C.

[0643] Table 40 SEC results_high temperature experiment

[0644] (3) NR CE-SDS

[0645] As shown in Table 38 and Figure 34, after treatment at 40°C for 4 and 6 weeks, all formulations showed some increase in NR CE-SDS LMW% and a decrease in MP%, with F34 (Ace50Pro) showing the greatest change. Compared to F31 (Ace50Suc) and F37 (Fed-Batch), the Fed-Batch-derived DS showed a smaller decrease in the main NR CE-SDS peak at 40°C than the Perfusion-derived one.

[0646] Table 41 NR CE-SDS results_high temperature experiment

[0647] (4)CEX

[0648] As shown in Table 42 and Figure 35, after 4 weeks of treatment at 40°C, the acidic peaks of all formulations increased and the MP% decreased. The CEX main peak of F31 (Ace50Suc) decreased slightly more than that of F37 (Fed-Batch) of the same formulation.

[0649] Table 42 CEX results_high temperature test

[0650] (5)Activity

[0651] As shown in Table 43, after 4 weeks at 40°C, there was no significant difference in the activity of the formulations.

[0652] Table 43 Activity results_high temperature experiment

[0653] 2.2. 25°C Experiment

[0654] After 4 weeks of treatment at 25°C, all formulations showed no changes in appearance, concentration, pH, or OD405. All samples showed moderate decreases in purity (SEC, NR CE-SDS, and CEX), with F31 (Ace50Suc) showing the greatest changes by SEC and F34 (Ace50Pro) by NR CE-SDS. Overall, the histidine buffer system outperformed the acetate buffer system. Specific degradation trends are shown in Figures 36, 37, and 38.

[0655] 2.3. Lighting

[0656] After three days of light treatment, all formulations showed no changes in appearance, DLS, concentration, pH, or OD405. Purity decreased in all formulations, as shown in Figures 39, 40, and 41. F35 (His50Pro) showed the least increase in SEC HMW% and NR CE-SDS LMW% analysis. F33 (His55Suc) and F36 (His55Pro) showed the least change in CEX analysis.

[0657] 2.4. Shaking

[0658] After 10 days of shaking at 200 rpm, all formulations showed no changes in appearance, DLS, Conc., pH, and OD405. Purity remained unchanged by CEX. However, all formulations showed changes by SEC and NR CE-SDS after 10 days of shaking at 200 rpm. F32 (His50Suc) showed the least increase in LMW%, while the remaining formulations showed no significant differences. Specific trends are shown in Figures 42, 43, and 44. After 10 days of shaking at 200 rpm, the number of insoluble particulates did not differ significantly among the formulations.

[0659] 3. Summary

[0660] Under 25°C conditions for 4 weeks, there was no significant change in the appearance, DLS, OD405 and other results of each formulation, but the SEC of F31 (Ace50Suc) changed more than the other formulations, and the NR CE-SDS of F34 (Ace50Pro) changed more than the other formulations.

[0661] After 4 weeks at 40°C, appearance, concentration, pH, and DLS remained unchanged. However, the OD405 values ​​of F31 (Ace50Suc), F32 (His50Suc), F33 (His55Suc), F34 (Ace50Pro), and F36 (His55Pro) increased slightly. The main peaks of all formulations decreased to some extent by SEC, CEX, and NR CE-SDS. F31 (Ace50Suc) and F32 (His50Suc) showed greater changes in SEC than the other formulations, while F34 (Ace50Pro) showed slightly greater changes in NR CE-SDS than the other formulations. Comparing the perfusion process F31 (Ace50Suc) with the fed-batch process F37 (Fed-Batch) at 40°C, the fed-batch process F37 (Fed-Batch) showed less change in all purity levels than the perfusion process F31 (Ace50Suc).

[0662] After 3 days of illumination, no significant changes were observed in the concentration, appearance, pH, or DLS results for each formulation. The main peaks for SEC, NR CE-SDS, and CEX all decreased, with the decreases for F35 (His50Pro) by SEC and NR CE-SDS being smaller than those for the other formulations. The decreases for F33 (His55Suc) and F36 (His55Pro) by CEX were also smaller than those for the other formulations.

[0663] Under shaking conditions, no significant changes were observed in concentration, appearance, pH, DLS, OD405, or CEX main peak across all formulations. The main peaks for SEC and NR CE-SDS showed a slight decrease, likely due to the shaking experiments being conducted at 25°C. F35 (His50Pro) showed the smallest change in SEC, while F32 (His50Suc) showed the smallest change in NR CE-SDS.

[0664] In this study, acetate buffer at pH 5.0, as well as histidine buffers at pH 5.0 and pH 5.5, were tested with sucrose, proline, and 0.05% polysorbate 80. Light, shaking, and pressure at 25°C and 40°C were examined. Furthermore, the stability of stock solutions produced using perfusion and fed-batch processes was compared. Results showed that the purity of histidine buffer varied less than that of acetate buffer at 40°C, 25°C, and light, and that the fed-batch process was more stable at 40°C than the perfusion process. Given the greater suitability of histidine for subcutaneous administration and the widespread use of sucrose in biologics, 10 mM histidine and sucrose were selected at a pH of 5.3, midway between 5.0 and 5.5, for final formulation confirmation.

[0665] Example 22: Screening experiment of surfactant concentration in high concentration protein

[0666] 1. Experimental Design

[0667] Table 44 lists sample formulation information, stability test storage conditions, and testing methods. UFDF samples were diluted to 100 mg / mL with the corresponding excipient stock solution and filtered through a 0.22 μm PVDF membrane. 2 mL of sample was transferred to a 6R vial for the stirring test, and 2 mL of sample was transferred to a 2R vial for the shaking test.

[0668] Table 44 Second surfactant concentration screening experimental design

[0669] 2. Results

[0670] In this experiment, three concentrations of polysorbate 80, 0.025%, 0.05% and 0.1%, were investigated, and the samples were subjected to stress tests such as shaking and stirring. The results showed that there was no significant difference in the appearance, insoluble particles, particle size, purity (SEC) and other results of the samples at each polysorbate 80 concentration, all of which met the test requirements. The intermediate concentration of 0.05% was selected for the final prescription confirmation test.

[0671] Table 45 Results of the second surfactant concentration screening experiment

[0672] NT = Not Detected

[0673] Example 23: Prescription Confirmation Experiment

[0674] 1. Experimental Design

[0675] Sample prescription information, stability test storage conditions, and testing methods are shown in Table 46. UFDF samples were diluted with the stock solution to the target concentrations of protein, sucrose, and polysorbate 80, then filtered through a 0.22 μm PVDF membrane. 2 mL of sample was transferred to 2R vials for stability tests at -40°C, -20°C, 5°C, 25°C, and 40°C, freeze-thaw tests at -20°C / -40°C, shake tests, and light exposure tests. 2 mL of sample was transferred to 6R vials for a stirring test, and 2 mL of sample was transferred to 5 mL PC vials for a -80°C freeze-thaw test.

[0676] Table 46 Preparation prescription confirmation study plan

[0677] 2. Results

[0678] In the prescription confirmation experiment, the selected prescriptions were subjected to freeze-thaw, stirring, shaking, light, forced degradation (40°C), accelerated (25°C), and long-term (5 / -20 / -40°C) conditions at different temperatures. The results showed that the purity of the samples did not change significantly under freeze-thaw, stirring, shaking, and long-term conditions. The purity decreased under 25°C and light conditions. After 6 weeks at 40°C, the purity of the samples decreased significantly, but it did not affect the binding activity, and the PS80 concentration in the prescription did not decrease.

[0679] The results of the final prescription confirmation experiment are shown in Tables 47, 48, 49 and 50.

[0680] In the long-term stability experiments at 5°C, -20°C and -40°C, there was no significant change in the sample appearance, DLS, SEC, CEX and NR-CE-SDS results.

[0681] At 25°C, no significant changes were observed across all formulations at 6 weeks, with a slight decrease in the main CEX peak after 3 and 6 months. There was no significant decrease in the polysorbate 80 content.

[0682] After being placed at 40℃ for 6 weeks, the main peaks of the samples in SEC, CEX and NR-CE-SDS decreased significantly, but the content of polysorbate 80 did not decrease significantly.

[0683] After 7 days of illumination, there was no significant change in the sample's appearance or DLS results. There was a slight decrease in purity, but this did not affect binding activity.

[0684] The sample's appearance, DLS, SEC, CEX, and NR-CE-SDS results remained unchanged during freeze-thaw cycles. Furthermore, the concentration of insoluble particles did not significantly increase. Freezing at different temperatures had no significant effect on the sample's freeze-thaw stability.

[0685] After stirring at 200 rpm for 2 hours, the appearance of the sample changed from slightly opalescent to turbid, so the insoluble particles were not detected. The other results did not change significantly.

[0686] After shaking at 200 rpm at 25°C for two weeks, there was no significant change in the appearance, DLS, OD405, MFI, SEC, NR-CE-SDS and CEX main peak results of the sample.

[0687] There was no significant difference in any test item between the samples inverted and upright at 40°C for 4 weeks, indicating that the packaging material is compatible with the protein preparation.

[0688] Table 47 Final formula confirmation high temperature 40℃ test results

[0689] Table 48 Accelerated (25°C) stability test results for final formulation confirmation

[0690] Table 49 Final formulation confirmation long-term (5℃ / -20℃ / -40℃) stability test results

[0691] Table 50 Final formulation confirmation stirring, freeze-thaw, light, and shaking test results

[0692] SOC = Slightly Opalescent, Turbid = Turbid, NT = Not Detected

[0693] 3. Summary

[0694] The high-concentration final formulation remains stable for extended periods at -40°C, -20°C, 5°C, and 25°C, with good tolerance to freeze-thaw and shaking. However, changes may occur under light and agitation conditions, with the most significant changes at 40°C. Therefore, this sample should be protected from prolonged light exposure, high temperatures, and vigorous agitation.

[0695] The scope of the present invention is not limited by the specific embodiments described herein. In fact, in addition to those described, various modifications provided herein will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A stable liquid pharmaceutical preparation comprising: (a) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human MASP-2; (b) a buffer comprising histidine and / or acetate buffer system; (c) a surfactant comprising a polysorbate; and (d) excipients comprising sucrose, trehalose and / or proline; The pH of the preparation is 4.7-6.1, preferably 5.0-6.0, more preferably 5.0-5.5, most preferably 5.

3.

2. The pharmaceutical formulation according to claim 1, wherein the formulation has one or more of the following characteristics: (i) average particle size of 2-10 μm; (ii) Osmolality is between 250 and 350 mOsm / kg H2O; (iii) a viscosity of about 1.0 centipoise to 30 centipoise, such as about 1.0 centipoise to 10 centipoise; (iv) after being placed at 40° C. or 25° C. for 6 weeks or shaken for 14 days, the appearance remains or substantially remains slightly opalescent or clear, the average particle size remains unchanged or substantially unchanged, and the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%; (v) after being stored at 4° C. for 1 to 3 years, the appearance remains or substantially remains slightly opalescent or clear, the average particle size remains unchanged or substantially unchanged, and the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%; (vi) after 3 days of illumination, the appearance remains or substantially remains slightly opalescent or clear, the average particle size remains unchanged or substantially unchanged, and the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%; (vii) after stirring for 6 hours, the appearance remains or substantially remains slightly opalescent or clear, the average particle size remains unchanged or substantially unchanged, and the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%; and (viii) after five cycles of freezing and thawing, the appearance remains or substantially remains slightly opalescent or clear, the average particle size remains unchanged or substantially unchanged, and the purity of the antibody monomer in the dissolved state exceeds 95%, preferably 98%, and more preferably 99%.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the antibody concentration is 10 mg / ml±1 mg / ml to 200 mg / ml±20 mg / mL. The pharmaceutical preparation according to claim 3 , wherein the antibody concentration is 20 mg / ml±2 mg / mL. The pharmaceutical preparation according to claim 3 , wherein the antibody concentration is 60 mg / ml±6 mg / ml. The pharmaceutical preparation according to claim 3 , wherein the antibody concentration is 100 mg / ml±10 mg / ml.

7. The pharmaceutical preparation according to claim 3, wherein the antibody concentration is 150 mg / ml±15 mg / ml.

8. The pharmaceutical formulation of claim 3, wherein the antibody concentration is about 20 mg / ml to about 100 mg / ml.

9. The pharmaceutical formulation according to any one of claims 1 to 8, wherein the buffer comprises histidine, and the histidine concentration is 5 mM ± 1 mM to 20 mM ± 4 mM.

10. The pharmaceutical preparation according to claim 9, wherein the buffer concentration is 10 mM ± 2 mM. The pharmaceutical preparation according to claim 10 , wherein the buffer comprises L-histidine and L-histidine hydrochloride monohydrate.

12. The pharmaceutical formulation of claim 11, wherein the buffer comprises about 0.175 mg / ml of L-histidine and about 1.86 mg / ml of L-histidine hydrochloride monohydrate.

13. The pharmaceutical formulation according to any one of claims 1 to 12, wherein the polysorbate concentration is 0.025% ± 0.01% to 0.1% ± 0.01% (w / v).

14. The pharmaceutical formulation according to claim 13, wherein the polysorbate concentration is 0.05%±0.01% (w / v).

15. The pharmaceutical formulation according to any one of claims 1 to 14, wherein the polysorbate is polysorbate 80 or polysorbate 20, preferably polysorbate 80.

16. The pharmaceutical formulation according to any one of claims 1 to 15, wherein the excipient comprises sucrose.

17. The pharmaceutical formulation according to claim 16, wherein the sucrose concentration is 5.5%±0.5% to 9%±0.5% (w / v).

18. The pharmaceutical formulation according to claim 17, wherein the sucrose concentration is 5.8%±0.5% to 8.6%±0.5% (w / v).

19. The pharmaceutical formulation according to claim 18, wherein the sucrose concentration is 5.8%±0.5% (w / v).

20. The pharmaceutical formulation according to claim 18, wherein the sucrose concentration is 6.0% ± 0.5% (w / v).

21. The pharmaceutical formulation according to claim 18, wherein the sucrose concentration is 6.5%±0.5% (w / v).

22. The pharmaceutical formulation according to claim 18, wherein the sucrose concentration is 7.0% ± 0.5% (w / v).

23. The pharmaceutical formulation according to claim 18, wherein the sucrose concentration is 8.6% ± 0.5% (w / v).

24. The pharmaceutical formulation according to claim 1, comprising: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate 80, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, The pH is 5.3±0.

5.

25. The pharmaceutical preparation according to claim 24, comprising: (a) 20 mg / ml ± 2 mg / ml antibody, (b) a buffer containing 10 mM ± 2 mM histidine, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

26. The pharmaceutical formulation according to claim 25, comprising: (a) 20 mg / ml ± 2 mg / ml antibody, (b) about 0.175 mg / ml L-histidine, (c) about 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

27. The pharmaceutical formulation according to claim 1, comprising: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, The pH is 5.3±0.

5.

28. The pharmaceutical formulation according to claim 27, comprising: (a) 60 mg / ml ± 6 mg / ml antibody, (b) a buffer comprising 10 mM ± 2 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 8.6% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

29. The pharmaceutical preparation according to claim 28, comprising: (a) 60 mg / ml ± 6 mg / ml antibody, (b) about 0.175 mg / ml L-histidine, (c) about 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 8.6% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

30. The pharmaceutical formulation according to claim 1, comprising: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, The pH is 5.3±0.

5.

31. The pharmaceutical preparation according to claim 30, comprising: (a) 100 mg / ml ± 10 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

32. The pharmaceutical formulation according to claim 31, comprising: (a) 100 mg / ml ± 10 mg / ml antibody, (b) about 0.175 mg / ml L-histidine, (c) about 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

33. The pharmaceutical formulation according to claim 1, comprising: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 5 mM ± 1 mM to 20 mM ± 4 mM histidine or acetate, (c) 0.025% ± 0.01% to 0.1% ± 0.01% (w / v) polysorbate, and (d) 6.5% ± 0.5% to 8.6% ± 0.5% (w / v) sucrose or trehalose, The pH is 5.3±0.

5.

34. The pharmaceutical formulation according to claim 33, comprising: (a) 150 mg / ml ± 15 mg / ml antibody, (b) a buffer comprising 10 mM ± 1 mM histidine buffer system, (c) 0.05% ± 0.01% (w / v) polysorbate, and (d) 7.0% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

35. The pharmaceutical formulation according to claim 34, comprising: (a) 150 mg / ml ± 15 mg / ml antibody, (b) about 0.175 mg / ml L-histidine, (c) about 1.86 mg / ml L-histidine monohydrochloride monohydrate, (d) 0.05% ± 0.01% (w / v) polysorbate, and (e) 7.0% ± 0.5% (w / v) sucrose, The pH is 5.3±0.

1.

36. The pharmaceutical formulation according to any one of claims 21 to 35, wherein the polysorbate is polysorbate 20 or polysorbate 80, preferably polysorbate 80.

37. The pharmaceutical formulation of any one of claims 1 to 36, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein: The amino acid sequence of HCDR1 is shown as DYYIN (SEQ ID NO: 1), the amino acid sequence of HCDR2 is shown as WIFPGSX1SX2YX3X4X5X6FX7X8 (SEQ ID NO: 2), and the amino acid sequence of HCDR3 is shown as GDRSGPFX9Y (SEQ ID NO: 3); and / or The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: the amino acid sequence of LCDR1 is shown as KSSQSLLYSNGKTYLN (SEQ ID NO: 4), the amino acid sequence of LCDR2 is shown as LVSKLDS (SEQ ID NO: 5), and the amino acid sequence of LCDR3 is shown as VQX 10 THFPFT (SEQ ID NO: 6), wherein X1 is E, D or G, X2 is A or P, X3 is H or Y, X4 is S or N, X5 is E or Q, X6 is K or N, X7 is K or Q, X8 is A or G, X9 is A or P, and X 10 It is V or G.

38. The pharmaceutical formulation of claim 37, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11 or SEQ ID NO: 12; and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 13 or SEQ ID NO:

14.

39. The pharmaceutical formulation of any one of claims 1 to 36, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12; or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:8, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

11.

40. The pharmaceutical formulation of claim 39, wherein the antibody or antigen-binding fragment thereof further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: The amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13; or The amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

14.

41. The pharmaceutical preparation according to any one of claims 37 to 40, wherein: the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:7, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:12, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13; or the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:14; or The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:8, the amino acid sequence of HCDR3 is shown in SEQ ID NO:11, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

13.

42. The pharmaceutical formulation of any one of claims 37-41, wherein the heavy chain variable region comprises a sequence selected from any one of SEQ ID NOs: 15, 17, 18, 20, 22, 24 and 26, or a sequence having at least 80% sequence identity thereto.

43. The pharmaceutical preparation of claim 42, wherein the light chain variable region comprises a sequence selected from any one of SEQ ID NOs: 16, 19, 28 and 30, or a sequence having at least 80% sequence identity thereto.

44. The pharmaceutical formulation of any one of claims 37-41, wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17, and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16 a light chain variable region having an amino acid sequence of A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:22, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:30; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 30; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:26, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

30.

45. The pharmaceutical formulation according to any one of claims 37-44, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region, optionally a heavy chain constant region of IgG, and / or a light chain constant region.

46. ​​The pharmaceutical formulation of claim 45, wherein the constant region comprises a mouse constant region, a rabbit constant region or a human constant region, optionally, the constant region comprises a constant region of human IgG1, IgG2, IgG3 or IgG4.

47. A pharmaceutical formulation according to claim 45, wherein the heavy chain constant region comprises one or more amino acid substitutions at amino acid residues 252, 254 or 256 relative to a wild-type human IgG constant region, optionally, the amino acid substitution at amino acid residue 252 is substituted with tyrosine, the amino acid substitution at amino acid residue 254 is substituted with threonine, and the amino acid substitution at amino acid residue 256 is substituted with glutamic acid.

48. The pharmaceutical preparation of claim 47, wherein the heavy chain constant region comprises a sequence that is at least 80% identical to a wild-type human IgG constant region amino acid sequence, and has amino acid residue 252 substituted by tyrosine, amino acid residue 254 substituted by threonine, and amino acid residue 256 substituted by glutamic acid relative to the wild-type human IgG constant region.

49. A pharmaceutical composition, wherein the composition comprises the pharmaceutical formulation according to any one of claims 1 to 48, and the composition is contained in a container.

50. The pharmaceutical composition according to claim 49, wherein the container is a vial, such as a vial.

51. The pharmaceutical composition of claim 49, wherein the container is a syringe.

52. The pharmaceutical composition of claim 49, wherein the container is a prefilled syringe.

53. The pharmaceutical composition of claim 49 contained in an automatic injector.

54. A pharmaceutical composition according to any one of claims 49 to 53, wherein the container comprises a headspace comprising a gas, wherein the gas comprises less than 5% by volume oxygen, preferably comprises less than 1% by volume oxygen, more preferably comprises no more than 0.1% by volume oxygen.

55. A kit comprising: (i) a container containing a composition comprising a pharmaceutical formulation according to any one of claims 1 to 48, and instructions for use of the composition; or (ii) comprising a container comprising a pharmaceutical composition according to any one of claims 49 to 54, and instructions for use of said composition.

56. A unit dosage form comprising a pharmaceutical formulation according to any one of claims 1 to 48, wherein the antibody is present in an amount of 0.1 mg to 500 mg.

57. The unit dosage form of claim 56, wherein said antibody is present in an amount of 200 mg or said antibody is present in an amount of 400 mg.

58. The unit dosage form according to claim 56 or 57, which is a glass vial, such as a vial.

59. The unit dosage form according to claim 56 or 57, which is a pre-filled syringe.

60. The unit dosage form according to claim 56 or 57, which is an automatic injector.

61. The unit dosage form according to claim 56 or 57, wherein the glass vial comprises a headspace comprising a gas, wherein the gas comprises less than 5% oxygen by volume, preferably comprises less than 1% oxygen by volume, and more preferably comprises no more than 0.1% oxygen by volume.

62. A method of inhibiting MASP-2-dependent complement activation, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical formulation of any one of claims 1 to 48, or the pharmaceutical composition of any one of claims 59-54, or the kit of claim 55, or the unit dosage form of claims 56-61.

63. A method for reducing serum C4 levels in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical formulation of any one of claims 1 to 48, or the pharmaceutical composition of any one of claims 59-54, or the kit of claim 55, or the unit dosage form of claims 56-61.

64. A method of treating a disease or condition in a subject that (1) would benefit from a reduction in serum C4 levels in the subject; (2) is associated with abnormal (e.g., elevated) serum C4 levels; (3) would benefit from inhibition of MASP-2-dependent complement activation; and / or (4) is associated with MASP-2-dependent complement activation, The method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical formulation of any one of claims 1 to 48, or the pharmaceutical composition of any one of claims 59-54, or the kit of claim 55, or the unit dosage form of claims 56-61.

65. Use of the pharmaceutical formulation of any one of claims 1 to 48, or the pharmaceutical composition of any one of claims 59 to 54, or the kit of claim 55, or the unit dosage form of claims 56 to 61 in the preparation of a medicament for use in one or more of: (1) Used to inhibit MASP-2-dependent complement activation; (2) for treating or preventing diseases or conditions associated with MASP-2-dependent complement activation; (3) for treating a condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation; (4) Used to reduce the serum C4 level of subjects; (5) for treating a condition in a subject that would benefit from a reduction in the subject's serum C4 level; (6) For treating or preventing conditions or diseases associated with abnormal (e.g. elevated) serum C4 levels.

66. The use of claim 65, wherein the disease or condition is an autoimmune disease, a vascular disorder, ischemia-reperfusion injury, arteriosclerosis, inflammation, a pulmonary disorder, an extracorporeal reperfusion process, a musculoskeletal disorder, a kidney disorder, a skin disorder, an organ or tissue transplant process, a nervous system disease or injury, a blood disease, a genitourinary disease, non-obese diabetes or complications associated with type 1 or type 2 diabetes, cancer, an endocrine disease, an ophthalmic disease, or COVID-19.

67. The use of claim 66, wherein: The autoimmune diseases include thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), hematopoietic transplantation-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy; The vascular disorders include cardiovascular disorders, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renal vascular disorders, mesenteric / intestinal vascular disorders, revascularization of grafts and / or regrafts, vasculitis, Henlein-Schonlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu arteritis, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis following stenting, rotational aneurysm resection, and percutaneous transluminal coronary angioplasty (PTCA), The ischemia-reperfusion injury includes ischemia-reperfusion injury associated with aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis associated with organ transplantation and / or limb / finger replantation, stroke, myocardial infarction, and hemodynamic resuscitation after shock and / or surgery; The inflammation includes inflammatory gastrointestinal diseases, which include pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and diverticulitis; The pulmonary diseases include acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, Chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease); meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burns, non-cardiogenic pulmonary edema, transfusion-related dyspnea, emphysema, cystic fibrosis, SARS-CoV, MERS-CoV, and SARS-CoV-2 (Covid-19)-related diseases; The extracorporeal circulation reperfusion process includes hemodialysis, plasmapheresis, leukocyte removal, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation LDL precipitation (HELP) and cardiopulmonary bypass (CPB); The musculoskeletal disorders include osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathies, crystalline arthropathy, and systemic lupus erythematosus (SLE); The renal disorders include mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranous proliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinaemic glomerulonephritis, lupus nephritis, Henlein-Schonlein purpura nephritis, and IgA nephropathy; Such skin conditions include psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita, herpes gestationis, thermal burns, and chemical burns; The organ or tissue transplantation process includes organ allotransplantation, organ xenotransplantation, organ and tissue transplantation; The nervous system diseases or injuries include multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome, reperfusion following stroke, degenerative disc, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or hemorrhage, demyelination and meningitis; The blood diseases include sepsis, severe sepsis, septic shock, acute respiratory distress syndrome caused by sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura and hemolytic uremic syndrome; The genitourinary disorders include painful bladder disease, sensory bladder disease, chronic nonbacterial cystitis, interstitial cystitis, infertility, placental dysfunction and miscarriage and pre-eclampsia; Such endocrine disorders include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the regulated release of prolactin, growth hormone or other insulin-like growth factors, and adrenocorticotropic hormone from the pituitary gland; The ophthalmic disease includes age-related macular degeneration.

68. The use of claim 65, wherein the medicament is for subcutaneous administration, parenteral administration and / or intravenous administration.