Formulations of anti-aPC monoclonal antibodies and their uses

Abstract

The present invention provides an anti-aPC monoclonal antibody formulation, which has a buffer system of histidine / histidine hydrochloride or acetic acid / sodium acetate, and which contains at least two of the following protectant components: a. a sugar selected from trehalose and sucrose or a sugar alcohol selected from sorbitol and mannitol, b. an amino acid selected from arginine hydrochloride or aspartic acid, and c. sodium chloride, and which has a pH of 5.0 to 6.3. The anti-aPC monoclonal antibody formulation of the present invention ensures long-term stability of the anti-aPC monoclonal antibody under storage conditions, and a formulation that can maintain this stability can be obtained.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and specifically to the formulation of anti-aPC monoclonal antibodies and their uses. [Background technology]

[0002] The anti-aPC monoclonal antibodies are humanized antibodies obtained by humanizing HAPC 1573 or simultaneously screening for single-site and combined-site mutations. They selectively bind to and inhibit activated protein C, but do not bind to or inhibit unactivated protein C. They selectively bind to human activated protein C, but do not bind to activated protein C or unactivated protein C from other species. The affinities of the antibodies have been significantly improved, with some antibodies having affinities 100-fold higher than those of HAPC 1573. In a Protac-based APTT blood coagulation assay, a very small amount of antibody, e.g., 1 μg / ml, produced a maximal inhibitory effect on aPC anticoagulant activity, potentially enabling the antibodies to block the anticoagulant activity of aPC.

[0003] However, the stability of anti-aPC monoclonal antibodies in practical use is still low, and how to improve the stability of anti-aPC monoclonal antibody formulations in production applications remains a challenge to be resolved in this field. Summary of the Invention

[0004] In view of the above-mentioned drawbacks of the existing technology, the present invention aims to provide an anti-aPC monoclonal antibody formulation to solve the problem of poor stability of the anti-aPC monoclonal antibody system in the existing technology, which has a buffer system of histidine / histidine hydrochloride or acetic acid / sodium acetate, and contains at least the following protectant components: a. a sugar selected from trehalose and sucrose or a sugar alcohol selected from sorbitol and mannitol; b. an amino acid selected from arginine hydrochloride or aspartic acid; c. Sodium chloride and the pH of the formulation is 5.0 to 6.3.

[0005] In one embodiment of the invention, the buffer system is 10 mM histidine / histidine hydrochloride, 20 mM histidine / histidine hydrochloride, or 20 mM acetic acid / sodium acetate. In another embodiment of the invention, the buffer system is 10 mM histidine / histidine hydrochloride, or 20 mM histidine / histidine hydrochloride.

[0006] In one embodiment of the present invention, the buffer system is 10 mM histidine / histidine hydrochloride. In one embodiment of the present invention, the trehalose content is 5 to 15 wt%. In one embodiment of the present invention, the sucrose content is 8 wt%. In one embodiment of the present invention, the sorbitol content is 6 wt%. In one embodiment of the present invention, the mannitol content is 6 wt%. In one embodiment of the present invention, the arginine hydrochloride content is 50 to 150 mmol / L. In one embodiment of the present invention, the aspartic acid content is 10 mmol / L. In one embodiment of the present invention, the sodium chloride content is 50 to 150 mmol / L.

[0007] In one embodiment of the present invention, the protectant component in the formulation is 8% trehalose + 100 mmol / L arginine hydrochloride, or 6% sorbitol + 100 mmol / L arginine hydrochloride, or 6% mannitol + 100 mmol / L arginine hydrochloride, or 10 The mixture is selected from 0 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride, or 100 mmol / L arginine hydrochloride + 10 mmol / L aspartic acid, or 8% trehalose + 100 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 100 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 150 mmol / L sodium chloride.

[0008] Preferably, the protectant component in the formulation is selected from 8% trehalose + 100 mmol / L arginine hydrochloride or 100 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride.

[0009] In one embodiment of the present invention, the formulation is the formulation according to any one of claims 1 to 2, characterized in that the formulation further comprises a surfactant, and the surfactant is selected from polysorbate 80 and / or P188.

[0010] Preferably, the surfactant is 200-500 ppm Polysorbate 80, 200-500 ppm P188, or 200 ppm Polysorbate 80 + 200 ppm P188.

[0011] Preferably, the surfactant is 200 ppm polysorbate 80, 300 ppm polysorbate 80, 500 ppm polysorbate 80, 300 ppm P188, 500 ppm P188, or 200 ppm polysorbate 80 + 200 ppm P188. Preferably, the surfactant is 200 ppm polysorbate 80 + 200 ppm P188.

[0012] In one embodiment of the present invention, the anti-aPC monoclonal antibody has an amino acid sequence of its light chain represented by SEQ ID NO: 1 and an amino acid sequence of its heavy chain represented by SEQ ID NO: 2, or the anti-aPC monoclonal antibody has an amino acid sequence of its light chain represented by SEQ ID NO: 3 and an amino acid sequence of its heavy chain represented by SEQ ID NO: 4.

[0013] Preferably, the anti-aPC monoclonal antibody has a light chain amino acid sequence shown in SEQ ID NO:1 and a heavy chain amino acid sequence shown in SEQ ID NO:2.

[0014] In one embodiment of the present invention, the anti-aPC monoclonal antibody in the formulation is of the IgG2 or IgG4 subtype. Preferably, the anti-aPC monoclonal antibody is of the IgG4 subtype.

[0015] In one embodiment of the present invention, the content of the anti-aPC monoclonal antibody in the formulation is 30 to 70 mg / ml, preferably 27 to 33, 45 to 55, or 63 to 77 mg / ml.

[0016] In one embodiment of the present invention, the formulation comprises 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; alternatively, the formulation comprises 20 mM histidine / histidine hydrochloride, 6% sorbitol, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; alternatively, the formulation comprises 20 mM histidine / histidine hydrochloride, 6% mannitol, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml. The formulation has a content of 30 mg / ml and a pH of 5.3, or the formulation contains 20 mM histidine / histidine hydrochloride, 100 mM arginine hydrochloride, 50 mM sodium chloride, and 200 ppm polysorbate 80, and the content of the anti-aPC monoclonal antibody is 30 mg / ml and the pH is 5.3, or the formulation contains 20 mM histidine / histidine hydrochloride, 100 mM arginine hydrochloride, 10 mM aspartic acid, and 200 ppm polysorbate 80, and the content of the anti-aPC monoclonal antibody is 30 mg / ml. l and a pH of 5.3; alternatively, the formulation comprises 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM sodium chloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and a pH of 5.3; or alternatively, the formulation comprises 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and a pH of 5.6.

[0017] In one embodiment of the present invention, the formulation contains a 10 mM histidine / histidine hydrochloride buffer system, 10% trehalose, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, has a content of the anti-aPC monoclonal antibody of 30 mg / mL, and has a pH of 5.7 to 6.3.

[0018] To achieve the above and other related objects, the present invention provides any one or more of the following uses of the anti-aPC monoclonal antibody formulations:

[0019] i) Use in the manufacture of a drug for the prevention and / or treatment of blood coagulation deficiency or defect diseases; ii) Use in the production of a promoter that enhances the degradation of histones H3 and H4 by aPC; iii) Use in the manufacture of a medicament for inhibiting the anticoagulant activity of activated protein C in an individual; iv) Use in the manufacture of a medicament for treating an individual in need of blood clotting; vi) Use in the manufacture of a medicament for regulating hemostasis in an individual; vii) Use in the manufacture of immunological testing assays or reagents or kits for the detection of aPC for disease or non-disease diagnostic or therapeutic purposes; viii) Use in combination with other agents or drugs.

[0020] In one embodiment of the present invention, the blood coagulation deficiency or defect disease includes a disease in which a blood coagulation disorder occurs due to a deficiency of a blood coagulation factor, causing bleeding, and blood coagulation failure due to an injury or surgery.

[0021] Preferably, the blood clotting deficiency or defect disease comprises hemophilia, sepsis, wound bleeding.

[0022] As described above, the anti-aPC monoclonal antibody formulation of the present invention has the following beneficial effects.

[0023] The anticoagulant activity of aPC can be inhibited with high potency, and the minimum dose of anti-aPC monoclonal antibody that inhibits the anticoagulant activity of aPC in human plasma deficient in several blood coagulation factors was determined using APTT experiments induced by protein C activator (protac), and complete inhibition was achieved at a maximum concentration of 1 μg / ml.

[0024] To summarize the above, a series of screening and optimization was carried out using high-throughput systems for the buffer system, pH, protective agent, and surfactant in the formulation of the drug product. Among the protective agent components, the formulation of 8% trehalose + 100 mM arginine hydrochloride was found to be superior to other formulations. The formulation with 200 ppm polysorbate 80 as a surfactant showed the best stability compared to other formulations. After comprehensive analysis and comparison of physical and chemical properties and appearance, the optimal formulation was found to be a 10 mM histone / histidine hydrochloride buffer system, pH 6.0±0.3, 10% trehalose, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, with a protein content of 30 mg / mL. This not only guaranteed the performance of each physical and chemical property and appearance, but also showed no foreign matter on its appearance under freezing and thawing conditions. [Brief explanation of the drawings]

[0025] [Figure 1A] , [Figure 1B] , [Figure 1C] , [Figure 1D] , [Figure 1E] , [Figure 1F] , [Figure 1G] , [Figure 1H] Figure 1 shows an in vitro pharmacological efficacy study of the mutant antibody, HAPC-14-6, which inhibits the anticoagulant activity of aPC in blood coagulation factor-deficient plasma. Figure 1A shows the effect on blood coagulation factor FVIII-deficient plasma, Figure 1B shows the effect on blood coagulation factor FIX-deficient plasma, Figure 1C shows the effect on blood coagulation factor FXI-deficient plasma, Figure 1D shows the effect on blood coagulation factor FVII-deficient plasma, Figure 1E shows the effect on blood coagulation factor FXII-deficient plasma, Figure 1F shows the effect on blood coagulation factor FV-deficient plasma, Figure 1G shows the effect on blood coagulation factor FX-deficient plasma, and Figure 1H shows the effect on VWF-deficient plasma. HAPC-14-3 in Figures 1A-1H is a monoclonal antibody with a different mutated variable region sequence from HAPC-14-6 and is described in Chinese Patent Application No. 202110791310X. [Figure 2A] , [Figure 2B] , [Figure 2C]Figure 2 shows in vivo drug efficacy experiments, in which Figure 2A shows the effect of injecting FVIII, HAPC1573, and HAPC-14-6 into FVIII-deficient mice on the blood coagulation function of the deficient mice, Figure 2B shows the effect of injecting different doses of HAPC-14-6 into FVIII-deficient mice on the blood coagulation function of the deficient mice, and Figure 2C shows the effect of injecting HAPC1573 and different doses of HAPC-14-6 into FVIII-deficient mice on the blood coagulation function of the deficient mice. [Figure 3] FIG. 3 shows an example of the NR-CE detection results of the IgG2 subtype sample of SR602 in Table 2 of the present invention. [Figure 4] FIG. 4 shows the change trends of the monomers in SEC-HPLC at 40° C. for each formulation in Table 3 of the present invention. [Figure 5] FIG. 5 shows the change tendency of aggregates in SEC-HPLC at 40° C. for each formulation in Table 3 of the present invention. [Figure 6] FIG. 6 shows the change tendency of the acidic peak of iCIEF at 40° C. for each formulation in Table 3 of the present invention. [Figure 7] FIG. 7 shows the change tendency of the main peak of iCIEF at 40° C. for each formulation in Table 3 of the present invention. [Figure 8] FIG. 8 shows the change in the main peak of nrCE-SDS at 40° C. for each formulation in Table 3 of the present invention. [Figure 9] FIG. 9 shows the change tendency of the main peak of rCE-SDS for each formulation in Table 3 of the present invention at 40°C. [Figure 10] FIG. 10 shows the change in the monomer content of each formulation in Table 15 of the present invention at 40° C. as determined by SEC-HPLC. [Figure 11] FIG. 11 shows the change tendency of aggregates in SEC-HPLC at 40° C. for each formulation in Table 15 of the present invention. [Figure 12] FIG. 12 shows the change tendency of the acidic peak of iCIEF at 40° C. for each formulation in Table 15 of the present invention. [Figure 13] FIG. 13 shows the change tendency of the main peak of iCIEF at 40° C. for each formulation in Table 15 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below in conjunction with specific examples. Of course, it should be understood that these examples are merely for the purpose of illustrating the present invention and do not limit the scope of protection of the present invention.

[0027] Although the present invention will be described below with reference to specific examples, those skilled in the art will readily appreciate other advantages and effects of the present invention from the disclosure herein. The present invention may be implemented or applied in other different specific embodiments, and the details herein may be modified or changed in various ways based on different perspectives and applications without violating the spirit of the present invention.

[0028] In the following examples, any process equipment or devices not specifically specified were those commonly used in the art. All pressure values ​​and ranges refer to relative pressures.

[0029] Furthermore, unless otherwise specified, it is understood that one or more method steps referred to in the present invention do not exclude the possibility that other method steps may exist before or after the combined step, or that other method steps may be inserted between these explicitly mentioned steps. Furthermore, it is understood that the combined or connected relationship between one or more equipment / apparatus referred to in the present invention does not exclude the possibility that other equipment / apparatus may exist before or after the combined equipment / apparatus, or that other equipment / apparatus may be inserted between these explicitly mentioned two equipment / apparatus, unless otherwise specified. Furthermore, unless otherwise specified, the numbers of each method step are merely a convenient tool for distinguishing each method step, and do not limit the order of each method step or the feasible scope of the present invention. Any modification or adjustment of their relative relationships is considered within the feasible scope of the present invention if it does not result in a substantial change in the technical content. Example 1 In vitro pharmacodynamic study of anti-aPC monoclonal antibody HAPC-14-6

[0030] 1.1 The anti-aPC monoclonal antibody used in this example is the anti-aPC monoclonal antibody HAPC-14-6, which is described in Chinese Patent Application No. 202110791310X and is the anti-aPC monoclonal antibody HAPC-14-6 in this patent.

[0031] In this example, the anti-aPC monoclonal antibody HAPC-14-6 has a light chain variable region and a heavy chain variable region similar to those of the anti-aPC monoclonal antibodies SR602 and SR604 of the present invention, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 5, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 6.

[0032] HAPC-14-6 was derived from HAPC1573 by humanization followed by further mutation. HAPC-14-6 binds to haPC but not to hPC, and its affinity is nearly 100-fold higher than that of a humanized antibody with unmutated CDR regions. The light chain of HAPC-14-6 is of the kappa subtype.

[0033] Production of anti-aPC monoclonal antibody HAPC-14-6: Seed cells expressing monoclonal antibody HAPC-14-6 were constructed using conventional methods (e.g., by cloning the antibody heavy and light chain coding genes into an expression vector, e.g., pKS001, and then introducing the constructed expression vector into CHO cells to obtain seed cells), and after fermentation and purification, anti-aPC monoclonal antibody HAPC-14-6 was obtained.

[0034] In this example, the effects of different concentrations of HAPC1573 and anti-aPC monoclonal antibody HAPC-14-6 on blood coagulation factor-deficient plasma were measured.

[0035] Using protac-induced APTT experiments, we determined the minimum dose of anti-aPC monoclonal antibody required to inhibit the anticoagulant activity of aPC in human plasma deficient in several blood coagulation factors. A concentration of up to 1 μg / ml was sufficient to inhibit more than 95% of the anticoagulant activity of aPC, whereas HAPC1573 required a concentration of 100 μg / ml or more.

[0036] 1.2 Experimental method and principles Principle of activated partial thromboplastin time (APTT) measurement: Factors XII and XI are activated with kaolin at 37°C, and cephalin (partial thromboplastin) is used instead of platelet factor 3. Ca 2+ The time required for plasma clotting in the presence of thromboplastin was measured and determined as the activated partial thromboplastin time (ACTPT). This is a relatively sensitive and most common screening test for the intrinsic blood coagulation system.

[0037] The newly developed anti-aPC monoclonal antibody, HAPC-14-6, was able to inhibit the anticoagulant activity of aPC at concentrations up to 1 μg / ml in human plasma from individuals deficient in several blood coagulation factors, including those deficient in blood coagulation factors V, VII, VIII, IX, X, XI, XII, and VWF.

[0038] 1.3 Measurement of the effect of antibodies on factor-deficient plasma (1) Experimental results of different antibodies against deficient plasma and different concentrations of blood coagulation factors VIII and VIX Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0039] Blood clotting factor FVIII deficient plasma+ Antibody (final concentration) at each dilution in Table 1 + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0040] Control: 150 μl blood coagulation factor FVIII-deficient plasma + 150 μl OWREN-KOLLER buffer.

[0041] Measurement method: STAGO Compact Max APTT-Program.

[0042] Blood coagulation factor FIX deficient plasma+ Antibody (final concentration) for each dilution in Table 1 + 0.01U / ml Protac (final concentration) + OWREN-KOLLER buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0043] Control: 150 μl of FIX-deficient plasma + 150 μl of Owren-Koller buffer.

[0044] Measurement method: STAGO Compact Max APTT-Program.

[0045] Measurement results: As shown in Table 1, the anti-aPC monoclonal antibody HAPC14-6 was able to highly inhibit the anticoagulant activity of aPC in blood coagulation factor VIII- and VIX-deficient plasma. At similar antibody concentrations, the anti-aPC monoclonal antibody HAPC14-6 was far more effective at inhibiting the anticoagulant activity of aPC than the mouse anti-aPC antibody 1573.

[0046] [Table 1]

[0047] (2) Experimental results of different concentrations of different antibodies against blood coagulation factor FVIII-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0048] Blood clotting factor FVIII deficient plasma+ Antibody (final concentration) at each dilution in Figure 1A + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0049] Control: 150 μl blood coagulation factor FVIII-deficient plasma + 150 μl OWREN-KOLLER buffer.

[0050] Measurement method: STAGO Compact Max APTT-Program.

[0051] Experimental results: As shown in Figure 1A, in FVIII-deficient plasma, anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0052] (3) Experimental results of different concentrations of different antibodies against FIX-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0053] Blood coagulation factor FIX deficient plasma+ Antibody (final concentration) at each dilution in Figure 1B + 0.01 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0054] Control: 150 μl of FIX-deficient plasma + 150 μl of Owren-Koller buffer.

[0055] Measurement method: STAGO Compact Max APTT-Program.

[0056] Measurement results: As shown in Figure 1B, in FIX-deficient plasma, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0057] (4) Experimental results of different concentrations of different antibodies against FXI-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0058] Blood clotting factor FXI deficient plasma+ Antibody (final concentration) at each dilution in Figure 1C + 0.06 U / ml Protac (final concentration) +OWREN-KOLLER buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0059] Control: 150 μl blood clotting factor FXI-deficient plasma + 150 μl OWREN-KOLLER buffer.

[0060] Measurement method: STAGO Compact Max APTT-Program.

[0061] Measurement results: As shown in Figure 1C, in blood coagulation factor FXI-deficient plasma, anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0062] (5) Experimental results of different concentrations of different antibodies against blood coagulation factor FVII-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0063] FVII-deficient plasma + Antibody (final concentration) at each dilution in Figure 1D + 0.1 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0064] Control: 150 μl of FVII-deficient plasma + 150 μl of Owren-Koller buffer.

[0065] Measurement method: STAGO Compact Max APTT-Program.

[0066] Measurement results: As shown in Figure 1D, in blood coagulation factor FVII-deficient plasma, the anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0067] (6) Experimental results of different concentrations of different antibodies against blood coagulation factor FXII-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0068] Blood coagulation factor FXII deficient plasma+ Antibody (final concentration) at each dilution in Figure 1E + 0.025 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0069] Control: 150 μl of blood coagulation factor FXII-deficient plasma + 150 μl of Owren-Koller buffer.

[0070] Measurement method: STAGO Compact Max APTT-Program.

[0071] Measurement results: As shown in Figure 1E, in blood coagulation factor FXII-deficient plasma, the anti-aPC monoclonal antibody The monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0072] (7) Experimental results of different concentrations of different antibodies against FV-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0073] Blood coagulation factor FV deficient plasma (contains 5% standard plasma)+ Antibody (final concentration) at each dilution in Figure 1F + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0074] Control: 150 μl of blood coagulation factor FV-deficient plasma (containing 5% normal plasma) + 150 μl of Owren-Koller buffer.

[0075] Measurement method: STAGO Compact Max APTT-Program.

[0076] Measurement results: As shown in Figure 1F, in blood coagulation factor FV-deficient plasma, anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0077] (8) Experimental results of different concentrations of different antibodies against FX-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0078] Blood coagulation factor FX-deficient plasma (containing 1% standard plasma) Antibody (final concentration) at each dilution in Figure 1G + 0.05 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0079] Control: 150 μl of blood coagulation factor FX-deficient plasma (containing 1% normal plasma) + 150 μl of Owren-Koller buffer.

[0080] Measurement method: STAGO Compact Max APTT-Program.

[0081] Measurement results: As shown in Figure 1G, in blood coagulation factor FX-deficient plasma, anti-aPC monoclonal antibody completely inhibited the anticoagulant activity of aPC.

[0082] (9) Experimental results of different concentrations of different antibodies against VWF-deficient plasma Experimental method: The antibodies were diluted to the appropriate concentrations with Owren-Koller buffer.

[0083] Blood clotting factor VWF deficient plasma+ Antibody (final concentration) at each dilution in Figure 1H + 0.01 U / ml Protac (final concentration) + Owren-Koller buffer The mixture was left at room temperature for 15 minutes. 150μl Total 150μl

[0084] Control: 150 μl of VWF-deficient plasma + 150 μl of Owren-Koller buffer.

[0085] Measurement method: STAGO Compact Max APTT-Program.

[0086] Measurement results: As shown in Figure 1H, in plasma deficient in the blood coagulation factor VWF, the anti-aPC monoclonal antibody HAPC-14-6 sufficiently inhibited the anticoagulant activity of aPC. Example 2: Experiments on the in vivo pharmacodynamics of anti-aPC monoclonal antibodies

[0087] In a humanized protein C hemophilia mouse model (see paragraphs

[0230] to

[0238] of Chinese patent application CN201911027061.6), a certain dose of anti-aPC monoclonal antibody HAPC-14-6 was injected, and the amount of bleeding after the mice's tails were amputated was observed to check the blood coagulation effect of the anti-aPC monoclonal antibody on the inhibition of the anticoagulation function of aPC.

[0088] 2.1 Experimental method The mice were administered intravenously through the corner of the eye. One hour after administration, the mice were anesthetized by intraperitoneal injection of pentobarbital sodium chloride. After anesthesia, the mice's tails were cut 2 mm from the end of the tail, and the wound was placed in 15 ml of 37°C saline to collect blood flow from the mice. After 20 minutes, collection was stopped. The collected blood was centrifuged to lyse red blood cells, and the supernatant was collected. The supernatant was detected by ultraviolet spectrophotometry at 600 nm. The measured OD value represented the amount of bleeding, and the blood coagulation function of the mice was evaluated. WT mice were normal mice, and untreated mice were untreated humanized protein C hemophilia A mice or untreated humanized protein C hemophilia B mice.

[0089] 2.2 Experimental results As shown in Figure 2A, humanized protein C hemophilia A mice were injected with 1 IU / mouse of blood coagulation factor FVIII, 20 μg / mouse of mouse anti-APC monoclonal antibody 1573, and 1 μg / mouse of HAPC-14-6. Mice injected with the new anti-APC monoclonal antibody HAPC-14-6 had their blood coagulation function restored to the level of normal mice, consistent with the effects of mice treated with blood coagulation factor FVIII.

[0090] As shown in Figure 2B, humanized protein C hemophilia A mice were injected with different doses of HAPC-14-6, but up to 1 μg of HAPC-14-6 was able to restore the blood coagulation function of the deficient mice to normal levels.

[0091] As shown in Figure 2C, humanized protein C hemophilia B mice were injected with different doses of HAPC-14-6, but up to 1 μg of HAPC-14-6 was able to restore the blood coagulation function of the deficient mice to normal levels.

[0092] The results show that in this invention, the minimum dose of anti-aPC monoclonal antibody required to inhibit the anticoagulant activity of aPC in human plasma deficient in several blood coagulation factors was determined using an APTT experiment induced by protein C activating enzyme (protac). It was found that complete inhibition was possible at a concentration of up to 1 μg / ml, whereas the mouse antibody HAPC1573 required a concentration of 100 μg / ml or more. Example 3: Monoclonal antibody screening test

[0093] Three minipools of HAPC-14-6 with the same variable region but different IgG subtypes (IgG2 and IgG4 subtypes) were selected. Single clones were plated and subjected to a series of clone screening experiments. Finally, 10 monoclonal cell lines highly expressing full-length monoclonal antibodies were selected. The IgG4 monoclonal antibody had a light chain of the kappa subtype with the sequence shown in SEQ ID NO: 1 and a heavy chain with the amino acid sequence shown in SEQ ID NO: 2, and was designated SR604. The IgG2 monoclonal antibody had a light chain of the kappa subtype with the sequence shown in SEQ ID NO: 3 and a heavy chain with the amino acid sequence shown in SEQ ID NO: 4, and was designated SR602.

[0094] Production of anti-aPC monoclonal antibodies: Monoclonal antibodies were expressed by conventional methods. Seed cells were constructed (for example, genes encoding the full-length heavy and light chains of an antibody were cloned into an expression vector, e.g., pKS001, and the constructed expression vector was then introduced into CHO cells to obtain seed cells), which were then inoculated into a 200 L fermentation tank. After cultivation, the fermented liquid was subjected to affinity chromatography, low-pH virus inactivation and depth filtration of the intermediate product, anion exchange chromatography, cation exchange chromatography, virus removal filtration (NF), and ultrafiltration concentrate replacement (UF / DF), as well as purification processes for producing a raw liquid, and a sample whose quality met the raw liquid standards was obtained.

[0095] The total yield of the production process was 71.5%, and the final purified sample stock solution (SR604) had 0.2% aggregates, 99.6% main peak, and 0.3% debris content by SEC-HPLC. The iCIEF detection results were acid peak 23.9%, main peak 64.7%, base peak 11.3%, HCP residue 14 ppm, HCDNA residue 0 ppb, ProA residue <2 ppm, nrCE-SDS 97.6%, and rCE-SDS(HC+LC) 96.2%.

[0096] The expression level of the IgG2 subtype clone was higher than that of the IgG4 subtype, reaching a maximum of 7.7 g / L. When NR-CE detection was performed on the expressed products, split peaks were observed in all detection results for the IgG2 subtype expressed products. The data for the major peaks for each number are shown in Table 2.

[0097] [Table 2]

[0098] The expression products of 10 monoclonal cell lines selected by IgG4 size exclusion were characterized for their physical and chemical properties. The results were normal. SEC analysis showed no significant antibody aggregates. CIEF analysis showed a PI of 7.2 and no significant abnormalities in the acid and base peaks. LC-MS analysis showed that the molecular weight of the antibody was consistent with the designed results. HPLC quantitative data showed a difference of approximately 10% compared to the Octet Titer analysis data, but this did not affect the screening of the clones. The final IgG4 subtype clone had a maximum expression of over 5 g / L, while the other clones all had expression levels of 3-4 g / L, making them suitable for subsequent production. Figure 3 shows an example of the NR-CE analysis results for the SR602 IgG2 subtype sample listed in Table 2 of the present invention.

[0099] Overall, the anti-aPC monoclonal antibody SR604 (IgG4 subtype) is superior to SR602 (IgG2 subtype). Example 4 Screening experiment for protective agents

[0100] In this example, the anti-aPC monoclonal antibody SR604 tested in Example 3 was selected to screen for protective agents.

[0101] In this example, the protein content of anti-aPC monoclonal antibody was 30 mg / mL, and the basic buffer system was 20 mM histidine / histidine hydrochloride, pH 5.3, and different combinations of protecting agents were screened.

[0102] [Table 3]

[0103] Finally, the stability of each composition sample A to G in Table 3 under each of the influencing factor conditions in Table 4 was examined.

[0104] [Table 4]

[0105] The above combinations A to G were sampled at the time points shown in Table 4, and the pH (only at point 0), viscosity (only at point 0), appearance, protein content, SEC-HPLC, charge distribution (iCIEF), rCE-SDS purity, and nrCE-SDS purity of each sample were examined. The detection results are shown in Tables 5 to 11.

[0106] [Table 5]

[0107] As can be seen from Table 5, the viscosity under all seven formulation conditions was below 3 mPa·s, indicating that the addition of arginine hydrochloride or sodium chloride plays a certain role in reducing the viscosity of the protein.

[0108] [Table 6] TIFF2026501650000008.tif131163TIFF2026501650000009.tif165165TIFF2026501650000010.tif119164TIFF2026501650000011.tif118162

[0109] Note: The number of "+" indicates the number of granules produced.

[0110] As can be seen from Table 6, the proteins in the seven formulations A to G were After being stored at 2°C and 40°C for 6 weeks, foreign matter was observed in all of the formulations, with varying degrees of appearance. At 2°C to 8°C, only formulations C (6% mannitol + 100mM arginine hydrochloride) and D (100mM arginine hydrochloride + 50mM sodium chloride) showed no foreign matter after being stored for 6 weeks, while formulation A (8% trehalose + 100mM arginine hydrochloride) showed a weaker Tyndall effect after being stored at 40°C. All formulations except formulation F (8% trehalose + 100mM sodium chloride) showed foreign matter in varying degrees of appearance after six freeze-thaw cycles. It can be seen that formulations A to G maintain a stable appearance over a certain period of time.

[0111] [Table 7]

[0112] As can be seen from Table 7, the protein content of the samples under the seven formulation conditions A to G did not change significantly compared to the 0 point under each of the conditions examined.

[0113] [Table 8] TIFF2026501650000014.tif231167

[0114] The SEC results of the seven formulation samples (A-G) under conditions of 2-8°C, 25°C, shaking, and freezing / thawing showed no significant changes compared to the zero point, and there were no significant differences between the formulations. In Figures 4 and 5, the SEC of each formulation at 40°C showed a clear increase in aggregates. It was shown that the monomer content clearly tended to decrease.

[0115] [Table 9] TIFF2026501650000016.tif133151TIFF2026501650000017.tif69150

[0116] The iCIEF results for the seven formulations under conditions of 2-8°C, 25°C, shaking, and freezing / thawing showed no significant changes compared to the zero point, and there were no significant differences between the formulations. Figures 6 and 7 show that at 40°C, the iCIEF for each formulation showed a clear increase in the acidic peak and a clear decrease in the main peak, and the range of change between the formulations was similar.

[0117] [Table 10]

[0118] The nrCE-SDS results for the seven formulations under conditions of 2-8°C, 25°C, shaking, and freezing / thawing showed no significant changes compared to the zero point, and there were no significant differences between the formulations. Figure 8 shows that at 40°C, the main peak of the nrCE-SDS for each formulation showed a clear tendency to decrease, with the largest decrease observed for formulations C (6% mannitol + 100 mM arginine hydrochloride), D (100 mM arginine hydrochloride + 50 mM sodium chloride), and E (100 mM arginine hydrochloride + 10 mM aspartic acid).

[0119] [Table 11]

[0120] The results of rCE-SDS for the seven formulation samples under conditions of 2-8°C, 25°C, shaking, and freezing / thawing showed no significant changes compared to the zero point, and there was no significant difference between the formulations. In Figure 9, at 40°C, the main peak of rCE-SDS for each formulation was clearly reduced. There was a tendency for this to occur, but among these, the C (6% mannitol + 100 mM arginine hydrochloride), D (100 mM arginine hydrochloride + 50 mM sodium chloride) and E (100 mM arginine hydrochloride + 10 mM aspartic acid) formulations showed a relatively large decrease.

[0121] A comprehensive comparison of the physical and chemical properties and appearance results showed that adding various combinations of sugars and protective agents such as arginine hydrochloride to the formulations could inhibit protein aggregation and precipitation to some extent and reduce the viscosity of the drug solution to 3 mPa·s or less, within the controllable range for drug production. Among these, formulation A (8% trehalose + 100 mM arginine hydrochloride) had the best stability compared to the other formulations. However, all formulations showed signs of foreign matter after 6 weeks at 25°C and 40°C. Further screening and optimization of the formulations is needed to improve the protein's long-term stability. Example 5 High-throughput screening experiments

[0122] 2.1 Buffer system and pH screening studies In this example, high-throughput rapid screening tools such as UNcle and a microplate reader were used to increase the protein concentration to 50 mg / mL based on the preferred formulation A (8% trehalose + 100 mM arginine hydrochloride). A total of 12 formulations with different buffer types, pH, and types and concentrations of protective agents were selected. Using an isothermal stability method, each formulation was incubated at 55°C for 21 hours, and the denaturation, aggregation, particle size, particle size distribution, and turbidity of the samples were examined to improve the long-term stability of the protein.

[0123] [Table 12]

[0124] The samples in the six formulation conditions in Table 12 were incubated at 55°C for a certain period of time, and the turbidity (OD350) was measured at regular intervals by the turbidity method. The data on protein denaturation, aggregation, and particle size change output from UNcle were then collected. The optimal formulation was determined by screening and sorting each formulation.

[0125] As can be seen from comparing the denaturation, aggregation, particle size, and particle size distribution curves of each formulation in different buffer systems and pH, proteins in the histidine salt system have better thermal stability than those in the acetate salt system, and the higher the pH of the buffer system, the lower the concentration, making the protein less likely to denature and aggregate, and reducing the change in particle size.

[0126] As can be seen by comparing the turbidity changes of each formulation under different buffer systems and pH conditions, proteins in the histidine salt system show smaller changes in turbidity than those in the acetate salt system, and the higher the pH of the buffer system, the lower the concentration and the less likely the protein turbidity is to be denatured.

[0127] The above UNcle and turbidity detection results were analyzed and compared comprehensively, and 10 mM histidine salt, pH 6.0 was selected as the basic buffer system and pH for the formulation of the preparation.

[0128] 2.2 Screening studies for protective agents in pharmaceutical formulations [Table 13]

[0129] Samples under the five formulation conditions in Table 13 were incubated at 55°C for a certain period of time, and the turbidity (OD350) was detected at regular intervals using the turbidity method.The optimal formulation was determined by screening and sorting each formulation in combination with the data on protein denaturation, aggregation, and changes in particle size output from UNcle.

[0130] Comparison of the denaturation, aggregation, particle size, and particle size distribution curves for different concentrations of the protective agent showed that within the 8%-12% trehalose range, the higher the trehalose content, the less likely the protein was to denature and aggregate, and the smaller the change in particle size. Furthermore, when the arginine hydrochloride content in the formulation was 100 mM, the rate of protein denaturation and aggregation was slower than in the 75 mM and 125 mM formulations, indicating better thermal stability.

[0131] Comparing the turbidity changes of different concentrations of the protectant, we found that within the range of 75 mM to 125 mM arginine hydrochloride, the higher the arginine hydrochloride content, the smaller the change in turbidity of the protein. Also, the trehalose content of 10% resulted in a smaller change in turbidity of the protein compared to the trehalose content of 8% and 12%.

[0132] After analyzing and comparing the above UNcle and turbidity detection results and determining the appropriate osmotic pressure level of the formulation, 10% trehalose and 100 mM arginine hydrochloride were selected as the protective agent combination for the formulation of the preparation.

[0133] 2.3 Screening of surfactant concentrations [Table 14]

[0134] Based on the isothermal stability of UNcle, samples of the above three sets of formulation conditions were incubated at 55°C for a certain period of time, and the appropriate concentration of surfactant was screened by comparing the rate of denaturation, aggregation, and particle size increase between formulations.

[0135] As obtained by comparing the denaturation, aggregation, particle size, and particle size distribution curves of surfactants at different concentrations, the rate of protein denaturation and aggregation was slowest when polysorbate 80 was at a concentration of 200 ppm compared with concentrations of 300 ppm and 500 ppm, indicating its better thermal stability. 200 ppm polysorbate 80 was used as the surfactant in the formulation of the drug product.

[0136] Therefore, to summarize the above, a series of screening and optimization was carried out using high-throughput systems for the buffer system, pH, protective agents, and surfactants in the formulation, and the final formulation was determined to be 10 mM histidine / histidine hydrochloride buffer system, pH 6.0, 10% trehalose, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, and with a protein content of 50 g / L. Example 5 Further optimization experiments of the formulation

[0137] In the formulation determination experiment of this example, a lower limit pH of 5.3 and an upper limit pH of 6.3 were simultaneously set based on the pH of 6.0 of the preferred formulation in the high-throughput screening experiment, thereby verifying the stability of the formulation after being left for a long period of time within that pH range and providing a controllable pH range for subsequent formulation production.

[0138] [Table 15]

[0139] The sample from Example 2 was ultrafiltered and replaced with a basic buffer system of 10 mM histidine / histidine hydrochloride, pH 5.3, pH 6.0, or pH 6.3, and concentrated to a final protein content of 50 mg / mL or more. Three different formulations were then prepared by adding different ratios of protective agent, as shown in Table 15. The samples from the three formulations were sterilized and filtered using a 0.22 μm filter, and then divided into vials for stability evaluation.

[0140] [Table 16]

[0141] Sampling was performed for the above three formulations at the times shown in the table, and the sample appearance, SEC-HPLC, charge distribution (iCIEF), rCE-SDS purity, nrCE-SDS purity, etc. were examined. The results are shown in Table 17.

[0142] [Table 17] TIFF2026501650000026.tif164163TIFF2026501650000027.tif99161

[0143] After 6 weeks at 2-8°C, formulations 2-1 and 2-3 showed foreign matter in one duplicate well, while formulation 2-2 showed no foreign matter. After 6 weeks at 25°C, formulations 2-3 showed foreign matter in one duplicate well, while formulations 2-3 showed foreign matter in one duplicate well. After 6 weeks at 40°C, all formulations showed foreign matter, although formulation 2-2 performed slightly better in terms of appearance. After freezing and thawing, all formulations showed foreign matter after three cycles of freezing and thawing, indicating that proteins are more sensitive to freezing and thawing.

[0144] [Table 18]

[0145] Compared to the zero point, the SEC results for the three formulations with different pH levels showed no significant changes after six freeze-thaw cycles or after 6 weeks at 2-8°C and 25°C, and there was no significant difference between the formulations. Figures 10 and 11 show that after 6 weeks at 40°C, the aggregates increased and the main peaks decreased, and the lower the pH, the faster the aggregate growth rate.

[0146] [Table 19]

[0147] Compared to the zero point, the acidic peaks of iCIEF tended to increase and the main peaks tended to decrease for the three formulations with different pH values ​​after six freeze-thaw cycles or after storage for six weeks at 2-8°C and 25°C. Figures 12 and 13 show that after storage for six weeks at 40°C, the acidic peaks of iCIEF tended to increase significantly and the main peaks tended to decrease, but the differences between the formulations were not significant.

[0148] [Table 20]

[0149] Compared to the zero point, the nrCE-SDS results for the three different pH formulations showed no significant changes after six freeze-thaw cycles or after 6 weeks at 2-8°C and 25°C. After 6 weeks at 40°C, the main peak of nrCE-SDS tended to decrease slightly, but the difference between the formulations was not significant.

[0150] [Table 21]

[0151] Compared to the zero point, the rCE-SDS results for the three different pH formulations showed no significant changes after six freeze-thaw cycles or after 6 weeks at 2-8°C and 25°C. After 6 weeks at 40°C, the main peak of rCE-SDS tended to decrease slightly, but the difference between the formulations was not significant.

[0152] Comprehensive analysis and comparison of the physical, chemical, and visual properties revealed that the protein exhibited the best visual performance and stable physical and chemical properties at pH 6.0. Therefore, the final formulation pH range was determined to be 5.7–6.3, with a midpoint of 6.0. Furthermore, because the three different pH formulations still exhibited visible foreign matter after freezing and thawing, the protein concentration was ultimately reduced from 50 mg / mL to 30 mg / mL to improve stability of the protein under freeze-thaw conditions. The final SR604 protein formulation was formulated with a 10 mM histidine / histidine hydrochloride buffer system, pH 6.0 ± 0.3, 10% trehalose, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, resulting in a protein content of 30 mg / mL.

[0153] The above description is merely a preferred embodiment of the present invention and does not impose any formal or substantive limitations on the present invention. Those skilled in the art may make slight improvements and supplements without departing from the method of the present invention, and these improvements and supplements are also considered to be within the scope of protection of the present invention. Those skilled in the art may also make equivalent changes, such as modifications, alterations, and developments, to the technical content disclosed above, as long as they do not deviate from the spirit and scope of the present invention, and these are also embodiments of the present invention with equivalent effects. At the same time, the same changes made to the above embodiments based on the substantial technology of the present invention are also considered to be equivalent to the above embodiments. Any changes, modifications and developments of the above mentioned changes are within the scope of the technical solution of the present invention.

Claims

1. A formulation of an anti-aPC monoclonal antibody, comprising: the buffer system of the formulation is histidine / histidine hydrochloride or acetic acid / sodium acetate; At least the following protectant ingredients: a. a sugar selected from trehalose and sucrose or a sugar alcohol selected from sorbitol and mannitol; b. an amino acid selected from arginine hydrochloride or aspartic acid; c. Sodium chloride and A formulation characterized in that the pH of the formulation is 5.0 to 6.

3.

2. the buffer system is 10 mM histidine / histidine hydrochloride or 20 mM histidine / histidine hydrochloride or 20 mM acetic acid / sodium acetate; Preferably, the buffer system is 10 mM histidine / histidine hydrochloride or 20 mM histidine / histidine hydrochloride; Preferably, the buffer system is 10 mM histidine / histidine hydrochloride; Alternatively, the trehalose content is 5 to 15 wt %; Alternatively, the sucrose content is 8 wt %; Alternatively, the sorbitol content is 6 wt %; Alternatively, the mannitol content is 6 wt %; Alternatively, the content of arginine hydrochloride is 50 to 150 mmol / L, Alternatively, the content of aspartic acid is 10 mmol / L, Alternatively, the formulation according to claim 1, characterized in that the content of sodium chloride is 50 to 150 mmol / L.

3. the protectant component in the formulation is selected from 8% trehalose + 100 mmol / L arginine hydrochloride, or 6% sorbitol + 100 mmol / L arginine hydrochloride, or 6% mannitol + 100 mmol / L arginine hydrochloride, or 100 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride, or 100 mmol / L arginine hydrochloride + 10 mmol / L aspartic acid, or 8% trehalose + 100 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 100 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 100 mmol / L sodium chloride, or 8% trehalose + 100 mmol / L arginine hydrochloride + 150 mmol / L sodium chloride; The formulation according to any one of claims 1 to 2, characterized in that the protectant component in the formulation is preferably selected from 8% trehalose + 100 mmol / L arginine hydrochloride or 100 mmol / L arginine hydrochloride + 50 mmol / L sodium chloride.

4. the formulation further comprises a surfactant, the surfactant being selected from polysorbate 80 and / or P188; Preferably, the surfactant is 200-500 ppm Polysorbate 80 or 200-500 ppm P188 or 200 ppm Polysorbate 80 + 200 ppm P188; Preferably, the surfactant is 200 ppm Polysorbate 80 or 300 ppm Polysorbate 80 or 500 ppm Polysorbate 80 or 300 ppm P188 or 500 ppm P188 or 200 ppm Polysorbate 80 + 200 ppm P188, preferably, the surfactant is 200 ppm Polysorbate 80 + 200 ppm The formulation according to any one of claims 1 to 3, characterized in that it is P188.

5. The anti-aPC monoclonal antibody has an amino acid sequence of its light chain represented by SEQ ID NO: 1 and an amino acid sequence of its heavy chain represented by SEQ ID NO: 2, or the anti-aPC monoclonal antibody has an amino acid sequence of its light chain represented by SEQ ID NO: 3 and an amino acid sequence of its heavy chain represented by SEQ ID NO: 4, Preferably, the anti-aPC monoclonal antibody has an amino acid sequence of its light chain set forth in SEQ ID NO: 1 and an amino acid sequence of its heavy chain set forth in SEQ ID NO:

2.

6. The formulation according to any one of claims 1 to 5, wherein the anti-aPC monoclonal antibody in the formulation is of the IgG2 subtype or the IgG4 subtype, and preferably the anti-aPC monoclonal antibody is of the IgG4 subtype.

7. The formulation according to any one of claims 1 to 6, wherein the content of the anti-aPC monoclonal antibody in the formulation is 30 to 70 mg / ml, preferably 27 to 33, 45 to 55, or 63 to 77 mg / ml.

8. the formulation contains 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or the formulation contains 20 mM histidine / histidine hydrochloride, 6% sorbitol, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or the formulation contains 20 mM histidine / histidine hydrochloride, 6% mannitol, 100 mM arginine hydrochloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or the formulation contains 20 mM histidine / histidine hydrochloride, 100 mM arginine hydrochloride, 50 mM sodium chloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or the formulation contains 20 mM histidine / histidine hydrochloride, 100 mM arginine hydrochloride, 10 mM aspartic acid, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or The formulation contains 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM sodium chloride, 200 ppm polysorbate 80, the content of the anti-aPC monoclonal antibody is 30 mg / ml, and the pH is 5.3; or The formulation according to any one of claims 1 to 7, comprising 20 mM histidine / histidine hydrochloride, 8% trehalose, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, with a content of the anti-aPC monoclonal antibody of 30 mg / ml and a pH of 5.

6.

9. The formulation according to any one of claims 1 to 8, comprising a 10 mM histidine / histidine hydrochloride buffer system, 10% trehalose, 100 mM arginine hydrochloride, and 200 ppm polysorbate 80, with a content of the anti-aPC monoclonal antibody of 30 mg / mL and a pH of 5.7 to 6.

3.

10. Any one or more of the following uses of the formulation of anti-aPC monoclonal antibody according to any one of claims 1 to 9: i) Use in the manufacture of a medicament for the prevention and / or treatment of blood coagulation deficiency or defect diseases; ii) Use in the production of a promoter that enhances the degradation of histones H3 and H4 by aPC; iii) Use in the manufacture of a medicament for inhibiting the anticoagulant activity of activated protein C in an individual; iv) Use in the manufacture of a medicament for treating an individual in need of blood clotting; vi) Use in the manufacture of a medicament for regulating hemostasis in an individual; vii) Use in the manufacture of immunological testing assays or a detection reagent or kit for aPC for disease or non-disease diagnostic or therapeutic purposes; viii) Use in combination with other agents or drugs.

11. The blood coagulation deficiency or defect disease includes diseases in which blood coagulation disorders occur due to a lack of blood coagulation factors, leading to bleeding, and blood coagulation disorders caused by wounds or surgery.

11. The use according to claim 10, wherein the blood coagulation deficiency or defect disease preferably includes hemophilia, sepsis, wound bleeding.

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