Novel liquid formulations of plasma proteins

Abstract

The invention relates to a pharmaceutical preparation of plasma protein, especially ADAMTS-13 protein, and a composition composed of the pharmaceutical preparation and used for preventing or treating thrombotic diseases. According to the pharmaceutical preparation, the stability of the plasma protein can be remarkably improved, and the risk that the plasma protein is immediately polluted and denatured after being separated and purified from blood is very high, so that the pharmacological activity and quality of the plasma protein can be reduced in the long-term storage process. Particularly, the pharmaceutical preparation disclosed by the invention can keep the colloidal stability, the refrigeration stability, the purity and the aggregation inhibition property of the plasma protein at a relatively high level, and the appearance of the pressed powder obtained after freeze drying can be kept good for a long time. Therefore, the pharmaceutical preparation disclosed by the invention can be well used for long-term preservation and storage of the ADAMTS-13 protein without losing the effective treatment dose of the protein, and the ADAMTS-13 protein is an important treatment resource for various thrombotic diseases.

Description

Technical Field

[0001] The present invention relates to a plasma protein, in particular to a liquid preparation of ADAMTS-13 protein. Background Art

[0002] In the development of protein drugs, how to develop an efficient lyophilized formulation to ensure sufficient storage stability for low-stability proteins is an important issue. Before the freeze-drying process begins, it is necessary to store the formulation in a liquid state for a period of time. In order to prevent quality degradation during this period, stability must be ensured to a certain extent even in liquid formulations.

[0003] Excipients used in freeze-dried preparations are divided into two categories: crystalline and amorphous, depending on the characteristics of the material. Crystalline excipients act as bulking agents in freeze-dried preparations, providing mechanical strength to the freeze-dried powder cake and making it more solid. Typical crystalline excipients include mannitol and glycine. On the other hand, amorphous excipients can be used as protein stabilizers in freeze-dried preparations, existing adjacent to the protein to stabilize the protein and protect it from the pressure generated during the freeze-drying process.

[0004] Meanwhile, sodium chloride is mainly used in protein drug preparations, and it may exist in two forms, amorphous and crystalline, depending on the added concentration. Generally speaking, it is reported that when sodium chloride is added at a concentration of 150mM or less, it is mainly in an amorphous state, while when it is added at a concentration of 200mM or more, it is mainly in a crystalline state.

[0005] Stabilizers in lyophilized formulations protect and stabilize proteins from stresses generated during the freeze-drying process and also help stabilize proteins during storage and reconstitution after drying is complete. Therefore, particularly in protein formulations that are intended to be stored for long periods of time via freeze-drying, finding the optimal stabilizer components and the optimal ratio between them is as important as discovering new pharmacological components from the perspective of the value and utility of proteins as therapeutic resources.

[0006] Some publications and patent documents are mentioned and cited throughout this specification. In order to more clearly describe the current status of the relevant technology and the content of the present invention, the disclosures of the cited publications and patent documents are fully incorporated herein by reference. Summary of the invention

[0007] Technical issues

[0008] The inventors have conducted in-depth research and developed a liquid preparation with improved plasma protein stability, wherein the plasma protein is separated from the blood and purified immediately after being contaminated and denatured with a high risk, and the pharmaceutical preparation can maintain the physical properties, biological activity and pharmacological effects of the plasma protein for a long time, especially during the freeze-drying process. Therefore, the inventors found that when the preparation contains 0.2 mg / ml to 1.2 mg / ml of plasma protein as a therapeutic active ingredient, and 40 mM to 200 mM of an amino acid stabilizer, particularly arginine, the preparation shows very outstanding stability in various indicators such as colloidal stability, cold storage stability, inhibition of protein depolymerization, prevention of protein aggregation, and appearance of the powder after freeze-drying. On this basis, the present invention has been completed.

[0009] Therefore, the object of the present invention is to provide a pharmaceutical preparation of plasma protein.

[0010] Another object of the present invention is to provide a composition for preventing or treating thrombotic diseases.

[0011] Other objects and advantages of the present invention will become apparent from the following detailed description of the invention, the appended claims and the accompanying drawings.

[0012] Technical Solution

[0013] In a first aspect of the present invention, a pharmaceutical preparation is provided, comprising 0.2 mg / ml to 1.2 mg / ml of plasma protein and 40 mM to 200 mM of an amino acid stabilizer.

[0014] The inventors have conducted in-depth research and developed a liquid pharmaceutical preparation with improved plasma protein stability, wherein the plasma protein has a high risk of being contaminated and denatured immediately after separation and purification from blood, and the pharmaceutical preparation can maintain the physical properties, biological activity and pharmacological effects of the plasma protein for a long time, especially during the freeze-drying process. Therefore, the inventors found that when the preparation contains 0.2 mg / ml to 1.2 mg / ml of plasma protein as a therapeutic active ingredient and 40 mM to 20 mM of an amino acid stabilizer, especially arginine, the preparation exhibits very outstanding stability in various indicators such as colloidal stability, cold storage stability, inhibition of protein depolymerization, prevention of protein aggregation, and appearance of the powder after freeze-drying.

[0015] As used herein, the term "plasma protein" refers to water-soluble proteins present in human or animal plasma, including all protein components in the blood except proteins contained in white blood cells and red blood cells. Plasma proteins account for about 8% of the total plasma and are responsible for hemostasis (prothrombin and fibrinogen), hormone transport (serum albumin, lipoproteins, etc.) and immune action (immunoglobulins and complement proteins). Plasma proteins can be obtained by various fractionation and purification methods known in the art, but the chemical and physical instability problems caused by long-term storage and environmental changes must be overcome. Physical instability involves modifications that do not cause covalent changes in proteins, namely adsorption, aggregation and precipitation; chemical instability involves modifications such as deamination, racemization, hydrolysis, oxidation, β-elimination and disulfide exchange. These instabilities can lead to changes in intrinsic biological activity and reduced pharmacological effects.

[0016] As used herein, the term "stabilizer" refers to any additive added to a pharmaceutical preparation, which functions to increase the stability of the active ingredient, prevent the active ingredient from arbitrarily denaturing, oxidizing, aggregating, crystallizing or denaturing into related substances, thereby preventing the pharmacological activity of the active ingredient from being lost or reduced. The stabilizer is not subject to any particular limitation, as long as it is a pharmaceutically acceptable stabilizer. The term "amino acid stabilizer" refers to a stabilizer that produces the above-mentioned stabilizing effect by adding amino acids as a main ingredient or auxiliary ingredient to a pharmaceutical preparation.

[0017] According to a specific embodiment of the present invention, the amino acid is selected from at least one of the following groups: arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.

[0018] As used herein, the term "pharmaceutically acceptable salt" includes salts extracted from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluenesulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Examples of salts derived from suitable bases include salts of alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium, etc.

[0019] According to a specific embodiment of the present invention, the preparation of the present invention contains 0.25 mg / ml to 1.0 mg / ml plasma protein, preferably 0.3 mg / ml to 0.7 mg / ml plasma protein, more preferably 0.3 mg / ml to 0.4 mg / ml plasma protein, and optimally about 0.36 mg / ml plasma protein.

[0020] According to a specific embodiment of the present invention, the preparation of the present invention contains 60mM to 180mM amino acid stabilizer, preferably 60mM to 160mM amino acid stabilizer, more preferably 60mM to 140mM amino acid stabilizer, more preferably 80mM to 135mM amino acid stabilizer, more preferably 100mM to 130mM amino acid stabilizer, and optimally about 120mM amino acid stabilizer.

[0021] According to a specific embodiment of the present invention, the preparation further comprises a sugar stabilizer in an amount of 0-1.5 w / v % of the total volume of the preparation.

[0022] Preferably, the preparation further comprises a sugar stabilizer in an amount of 0.2-1.5 w / v%, more preferably 0.3-1.5 w / v%, more preferably 0.5-1.5 w / v%, more preferably 0.7-1.3 w / v%, more preferably 0.9-1.1 w / v%, and most preferably about 1 w / v%.

[0023] According to a specific embodiment of the present invention, the sugar is selected from the following group: sucrose, trehalose, or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0024] According to a specific embodiment of the present invention, the preparation further comprises 100 mM to 400 mM of an inorganic salt.

[0025] As used herein, the term "inorganic salt" refers to a salt extracted from an inorganic substance that does not contain a CH bond, which is a salt composed of an ionic combination of cations and anions in an aqueous solution. Examples of inorganic salts that can be used in the present invention include, but are not limited to, NaCl, CaCl2, KCl, MgCl2, and combinations thereof. Preferably, the inorganic salt of the present invention is a mixture of NaCl and CaCl2.

[0026] According to a specific embodiment of the present invention, the concentration of NaCl in the inorganic salt is 140mM to 370mM, preferably 180mM to 340mM, more preferably 220mM to 310mM, more preferably 260mM to 300mM, and most preferably about 280mM.

[0027] According to a specific embodiment of the present invention, the concentration of CaCl2 in the inorganic salt is 2mM to 6mM, preferably 3mM to 5mM, and most preferably about 4mM.

[0028] According to a specific embodiment of the present invention, the preparation of the present invention contains 10 mM to 30 mM histidine, preferably 15 mM to 25 mM histidine, and most preferably about 20 mM histidine.

[0029] According to a specific embodiment of the present invention, the preparation further comprises a non-ionic surfactant in an amount of 0.01-0.1 v / v% of the total volume of the preparation.

[0030] As used herein, the term "surfactant" refers to a soluble compound used to increase the water solubility of a hydrophobic material or to increase the compatibility of various materials with different hydrophobicities. The term "nonionic surfactant" refers to a surfactant that does not dissociate even in an aqueous solution because its entire molecular structure does not contain an ionizable functional group or atomic group.

[0031] According to a specific embodiment of the present invention, the nonionic surfactant of the present invention is selected from the following group: polysorbate 80, polysorbate 60, polysorbate 40, or a combination thereof; preferably polysorbate 80.

[0032] According to a specific embodiment of the present invention, the preparation of the present invention comprises 0.03-0.08 v / v% of a non-ionic surfactant, preferably about 0.05 v / v% of a non-ionic surfactant.

[0033] According to a specific embodiment of the present invention, the plasma protein applicable to the preparation of the present invention is selected from the following group: ADAMTS13 (disintegrant and metalloproteinase with thrombin type 1 motif, member 13) protein, its variant, or its functional fragment.

[0034] As used herein, the term "functional fragment" refers to a fragment produced by deleting one or more amino acid residues from a full-length protein, which is a full-length protein analog that retains the biological activity and function of the full-length protein.

[0035] In the present invention, SEQ ID NO: 1 is an amino acid sequence of an ADAMTS13 protein consisting of 1,427 amino acids. Therefore, the ADAMTS13 variant protein or its functional fragment described in the present invention is an ADAMTS13 variant protein or its fragment including the 75-685 amino acid region in which amino acid mutations are introduced into the full-length (1,427 amino acids) ADAMTS13 protein. For example, the fragment containing the 75-685a.a. region can be the 1-685a.a. (685a.a) or 75-685a.a. (611a.a) region.

[0036] SEQ ID NO: 1 is the amino acid sequence of ADAMTS13 protein, which basically includes the functional fragment of ADAMTS13 protein, and it also includes the amino acid sequence showing substantial identity with the above sequence. As used herein, the term "substantially identical" means that when the amino acid sequence is aligned with any other sequence so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art, it shows at least 70% homology, preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

[0037] According to a specific embodiment of the present invention, the variant of ADAMTS13 protein includes a substitution of at least one amino acid residue, and the amino acid residue is selected from the residues at the following positions: 85, 93, 126, 135, 278, 282, 308, 314, 317, 334, 364, 376, 413, 427, 452, 465, 567, 578, 585, 589, 607, 608, 609, 612, 618, 624, 630, 635, 643, 650, 651, 654, 655, 656, 658, 664, 672 of SEQ ID NO:1.

[0038] The inventors have identified the core regions recognized by anti-ADAMTS13 autoantibodies and found that when certain amino acids in these regions are substituted, the binding of these regions to autoantibodies can be blocked while maintaining their vWF degradation activity and thrombosis inhibition activity. Therefore, ADAMTS13 proteins mutated at the above positions can be used as an effective therapeutic ingredient for treating various diseases caused by excessive thrombosis, including thrombotic thrombocytopenic purpura (TTP).

[0039] As used herein, the term "autoantibody" refers to an antibody produced by an individual's own immune system that recognizes and targets an individual's own proteins. An autoantibody is an immunoglobulin that contains at least one variable domain that binds to an antibody epitope and specifically recognizes an antigen. The presence of autoantibodies can lead to degradation or loss of the intrinsic function or biological activity of the protein specifically recognized by the autoantibody, thereby causing various diseases.

[0040] According to a specific embodiment of the present invention, the variant of the ADAMTS13 protein is selected from the following group of protein variants, and the variant protein comprises a substitution of an amino acid residue at the following position:

[0041] Position 85 and 317, position 612, two or more of positions 282, 465 and 672, position 635, position 452 and 612, two or more of positions 278, 334 and 427, position 618, position 135, position 126, two or more of positions 567 and 651, position 413, position 334, position 314, two or more of positions 93, 364 and 376, position 308, position 656, position 607, positions 612 and 624, position 589, position 650 and 656, position 643, positions 585 and 658, two or more of positions 630, 654 and 664, four or more of positions 589, 608, 609, 624 and 655, position 578, position 585, positions 314 and 635, positions 314 and 612.

[0042] Specifically, the amino acid residue is substituted by at least one selected from the group consisting of: position 85 is substituted by Phe, position 93 is substituted by Val, position 126 is substituted by Met, position 135 is substituted by Ile, position 278 is substituted by Ile, position 282 is substituted by Ala, position 308 is substituted by Lys, position 314 is substituted by Thr, position 317 is substituted by His, position 334 is substituted by Thr or Val, position 364 is substituted by Arg, position 376 is substituted by Asp, position 413 is substituted by Asp, position 427 is substituted by Asn, position 452 is substituted by Ile, position 465 is substituted by Asp, position 567 is substituted by Ser, position 578 is substituted by Leu, Position 585 is substituted with Asn or Met, position 589 is substituted with Gln, position 607 is substituted with Arg, position 608 is substituted with Met, position 609 is substituted with Leu, position 612 is substituted with Phe or Tyr, position 618 is substituted with Ser, position 624 is substituted with Asp or Cys, position 630 is substituted with Leu, position 635 is substituted with Val, position 643 is substituted with Phe, position 650 is substituted with His, position 651 is substituted with Asp, position 654 is substituted with Gly, position 655 is substituted with Val, position 656 is substituted with Arg or His, position 658 is substituted with His, position 664 is substituted with Asn, and position 672 is substituted with Val.

[0043] According to a specific embodiment of the present invention, the plasma protein of the present invention binds to the Fc region derived from IgG4 immunoglobulin.

[0044] The inventors found that when the Fc region derived from IgG4 immunoglobulin is combined with the ADAMTS13 variant protein of the present invention, while maintaining its inherent vWF cleavage activity and neutralizing antibody escape activity, the in vivo stability of the variant protein is greatly improved, especially the structural instability presented in the open fragment with the C-terminus of the ADAMTS13 portion removed is significantly reduced.

[0045] According to a specific embodiment of the present invention, the Fc region includes at least one amino acid residue substitution, and the amino acid residue is selected from the residues at the following positions: residues 22, 24, and 26 of SEQ ID NO: 2. Specifically, the amino acid residue substitution is at least one selected from the following group: position 22 is substituted by Tyr, position 24 is substituted by Thr, and position 26 is substituted by Glu.

[0046] According to the present invention, SEQ ID NO: 2 is an amino acid sequence of the Fc region (217a.a.) derived from IgG4 immunoglobulin. The inventors have found that when the Fc region [IgG4(YTE)] derived from IgG4 immunoglobulin in which residues 22, 24 and 26 are substituted by Tyr, Thr and Glu, respectively, is fused with the above-mentioned ADAMTS13 variant protein, or a functional fragment thereof, the blood half-life of the protein can be extended to the maximum extent, and its physiological activity can be maintained for a long time after administration.

[0047] According to a specific embodiment of the present invention, the plasma protein further comprises a hinge region derived from IgG1 immunoglobulin between the Fc region derived from IgG4 immunoglobulin.

[0048] According to the present invention, the hinge region derived from an IgG1 immunoglobulin can be represented by SEQ ID NO: 3 (15 a.a.).

[0049] In another aspect of the present invention, a composition for preventing or treating thrombotic diseases is provided, wherein the composition comprises the above-mentioned pharmaceutical preparation of the present invention as an active ingredient, wherein the pharmaceutical preparation contains ADAMTS13 protein, its variant or its functional fragment as a plasma protein.

[0050] As used herein, the term "thrombotic disease" refers to a systemic disease in which platelets aggregate in the vascular microcirculatory system to form thrombi, resulting in reduced or blocked blood flow, thereby causing ischemic damage to organs such as the kidneys, heart, and brain.

[0051] If the activity of the ADAMTS13 enzyme is inhibited by neutralizing antibodies and cannot degrade Willebrand factor (vWF) normally, excessive platelet aggregation and excessive thrombosis will occur. Therefore, the ADAMTS13 variant protein of the present invention can efficiently evade neutralizing antibodies while maintaining or improving its activity of degrading vWF, and can be used as an effective preventive or therapeutic component for various thrombotic diseases.

[0052] As used herein, the term "preventing" or "preventing" refers to inhibiting the occurrence of a disorder or disease in a subject who has not yet been diagnosed with the disorder or disease but is at risk of developing the disorder or disease.

[0053] As used herein, the term "treatment" means: (a) inhibiting the development of a disorder, disease or condition; (b) alleviating a disorder, disease or condition; or (c) eliminating a disorder, disease or condition. When the composition of the present invention is administered to a subject, whether or not neutralizing antibodies are present, it can specifically recognize and degrade vWF, thereby blocking the formation of blood clots, thereby inhibiting the development of thrombotic diseases, or eliminating or alleviating thrombotic diseases. Therefore, the composition of the present invention itself can be a composition for treating these diseases, or it can be used as a therapeutic adjuvant in combination with other pharmacological ingredients to treat the above-mentioned diseases. Therefore, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0054] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of the composition of the present invention directly to a subject, thereby forming the same amount of the composition of the present invention in the body of the subject.

[0055] As used herein, the term "therapeutically effective amount" refers to the content of a pharmaceutical composition sufficient to allow the pharmacological ingredients contained in the composition to produce a therapeutic or preventive effect on a subject to which the composition is administered, and thus also includes a "prophylactically effective amount".

[0056] As used herein, the term "subject" includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Preferably, the subject of the present invention is a human.

[0057] According to a specific embodiment of the present invention, the thrombotic disease is thrombotic microangiopathy (TMA). Specifically, the thrombotic microangiopathy is selected from the group consisting of thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (hemolysis, elevated liver enzymes, low platelet count), preeclampsia and sickle cell disease. Preferably, the thrombotic microangiopathy is thrombocytopenic purpura or sickle cell disease. Most preferably, the thrombotic microangiopathy is thrombocytopenic purpura.

[0058] In another aspect of the present invention, a method for preventing or treating thrombotic diseases is provided, the method comprising administering the above-mentioned pharmaceutical preparation of the present invention to a subject, the pharmaceutical preparation comprising ADAMTS13 protein, its variant or its functional fragment as a plasma protein.

[0059] Since the plasma proteins used in the present invention, the components for preparing the plasma proteins, and the thrombotic diseases that can be prevented or treated are as described above, they are omitted here to avoid excessive overlap.

[0060] Advantages

[0061] The features and advantages of the present invention are summarized as follows:

[0062] (a) The present invention provides a pharmaceutical preparation of plasma protein, in particular ADAMTS-13 protein, and a composition composed thereof for preventing or treating thrombotic diseases.

[0063] (b) The pharmaceutical preparation of the present invention significantly improves the stability of plasma proteins. Plasma proteins are at high risk of being contaminated and denatured immediately after separation and purification from blood, so their pharmacological activity and quality will decrease during long-term storage. In particular, the colloidal stability, cold storage stability, purity and aggregation inhibition of plasma proteins can be maintained at a high level, and the appearance of the freeze-dried powder can be maintained well for a long time. Therefore, the pharmaceutical preparation of the present invention can be well used for long-term preservation and storage of ADAMTS-13 protein without losing the therapeutically effective amount of the protein. ADAMTS-13 protein is an important therapeutic resource for various thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The measurements of B 22 The values ​​were used to evaluate the colloidal stability of formulations containing five amino acid stabilizers.

[0065] Figure 2 Shown are the results of evaluating the monomer recovery at room temperature after adding seven stabilizers (six amino acids and sucrose) by size exclusion liquid chromatography (SE-HPLC).

[0066] Figure 3 Shown are the results of the evaluation of refrigerated stability by SE-HPLC, compared with the control group, depending on the amount of 20 mM arginine added.

[0067] FIG4 shows the results of SE-HPLC evaluation of purity and stability according to the concentration of the medicinal ingredient protein (ADAMTS-13) and the concentration of arginine ( Figures 4a to 4c ).

[0068] Figure 5 shows the powder appearance at different NaCl concentrations ( Figure 5a ) and the results of SE-HPLC evaluation of purity at different NaCl concentrations ( Figure 5b ).

[0069] Figure 6 The graph shows the results of SE-HPLC in liquid preparations containing 20 mM arginine, depending on the NaCl concentration.

[0070] FIG. 7 shows the appearance of the powder cake produced in the presence of 120 mM NaCl according to the sucrose concentration and with or without the addition of trehalose ( Figure 7a ) and SE-HPLC results ( Figure 7b ).

[0071] FIG8 shows the appearance of powder cakes according to the mixing ratio of sucrose to mannitol or glycine (a leavening agent). Figure 8a ) and SE-HPLC results ( Figure 8b ).

[0072] Fig. 9 The appearance of the powder cake was compared depending on the concentration of glycine in the presence of 120 mM NaCl.

[0073] Fig.10 The appearance of the powder cake was compared according to the concentration of arginine in the presence of 120 mM NaCl.

[0074] Figure 11 shows the appearance of the powder cake in the absence of arginine ( Fig.11a ) and SE-HPLC results ( Fig.11b ) depends on the mixing ratio of NaCl and sucrose.

[0075] FIG. 12 shows the appearance of the powder cake in the absence of 120 mM arginine ( Fig.12a ) and SE-HPLC results ( Figure 12b ) depends on the mixing ratio of NaCl and sucrose.

[0076] Fig.13 The results of measuring the change in purity after standing at 40°C for 1 month depending on the mixing ratio of NaCl to sucrose are shown.

[0077] FIG. 14 shows the concentration of polysorbate 80, as measured by observing the appearance ( Fig.14a ), measuring turbidity ( Fig.14b ) and SE-HPLC( Fig.14c ) to evaluate the effect of preventing aggregation caused by stirring.

[0078] Fig.15 The results of the evaluation of the liquid phase stability by SE-HPLC are shown to be dependent on the polysorbate 80 concentration.

[0079] FIG. 16 shows the results of evaluating the quality of the freeze-dried formulation by SE-HPLC ( Fig.16a ), and the results of determining the generation of higher-order aggregates ( Fig.16b ) depends on the concentration of polysorbate 80.

[0080] Figure 17 shows the measurement of thermal folding (T m )( Fig.17a ) and evaluate heat accumulation (T agg )( Fig.17b ) depends on the pH value.

[0081] Figure 18 shows the appearance of the freeze-dried powder cake ( Fig.18a ) and SE-HPLC analysis results ( Fig.18b ) depends on the concentration of CaCl2.

[0082] FIG. 19 shows the results of the appearance evaluation according to the NaCl and sucrose concentrations ( Fig.19a ), and shows the appearance of the powder at each concentration ( Fig.19b ).

[0083] Fig. 20 The dependence of the powder cake appearance on the NaCl and sucrose concentrations is shown.

[0084] Fig.21 Results of evaluating colloidal stability according to arginine concentration are shown.

[0085] Figure 22 shows the appearance of the pressed powder ( Fig.22a ) and purity ( Figure 22b ) depends on the arginine concentration.

[0086] FIG. 23 shows the physical properties of the final selected liquid formulation of the present invention, specifically showing the change in purity over time ( Fig.23a ), changes in potency over time ( Figure 23b ), protein concentration changes over time ( Fig.23c ) and the change of turbidity over time ( Fig.23d ).

[0087] FIG24 shows the effect of NaCl and sucrose concentration on long-term storage stability, specifically showing the purity ( Fig.24a ) and potency ( Figure 24b ) changes.

[0088] Figure 25 shows the results of determining the binding sites of each domain of ADAMTS13 that bind to the neutralizing antibody. Specifically, Fig.25aSchematic diagram showing six types of ADAMTS13 fragments (or wild-type full-length ADAMTS13) with various domain combinations, which were constructed to confirm the expression rate or neutralizing antibody binding site of ADAMTS13. Fig.25b Results of Western blot analysis using anti-V5 antibody after immunoprecipitation with a mixture of protein A-Sepharose and each neutralizing antibody are shown. The input shows the protein expression level of each domain. Fig.25c The schematic diagram shows the neutralizing antibody binding sites of ADAMTS13 and shows that Ab 4-16 antibodies specifically bind to domain S, Ab 67 antibodies bind to domain D, and Ab66 antibodies bind to domains T2 to T8.

[0089] Figure 26 shows the results of screening for variants that can escape anti-ADAMTS13 antibodies or have higher activity than wild-type ADAMTS13. Fig.26a ) and Ab 67 antibody ( Figure 26b ) escape rate, and the ADAMTS13 activity of the wild type and ADAMTS13 variants. The relative activity was calculated by measuring the specific activity of the wild type and the variant, and substituting the measured value into the following formula: relative activity (%) = specific activity of the variant / specific activity of the wild type ADAMTS13 × 100.

[0090] Fig.27a and 27b The results of measuring the escape rates of individual neutralizing antibodies of 12 full-length ADAMTS13 variants are shown. The antibody escape rates of 12 full-length ADAMTS13 variants against 7 neutralizing antibodies (Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured. The neutralizing antibody escape rate was calculated by measuring the relative binding level with WT ADAMTS13 to determine the relative escape rate, and then substituting the relative escape rate into the following formula: Escape rate (%) = (1-binding affinity of variant / binding affinity of WT ADAMTS13) × 100

[0091] Fig.28a and 28bThe results of measuring the escape rate of a single neutralizing antibody under the conditions of expressing each Fc-binding MDTCS fragment variant are shown. One MDTCS fragment is obtained from each of the 12 selected variants, and Fc is combined with it to obtain 12 MDTCS fragment variants. In addition, two variants (DM1 and DM2) with two amino acid mutations are obtained by combining the amino acid residues of the selected variants. Using the culture medium expressing 14 variants (including 12 MDTCS fragment variants and two variants), the neutralizing antibody escape rate of the variant to 8 single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65 and Ab67) and the relative ADAMTS13 activity of the variant are measured. Escape rate (%) = (1-binding affinity / binding affinity with variant) × 100; relative activity (%) = specific activity of variant / specific activity of WT ADAMTS13 × 100.

[0092] Fig.29 The results of evaluating the ability of each Fc-bound MDTCS fragment variant to escape mixed neutralizing antibodies under medium conditions in which each Fc-bound MDTCS fragment variant has been expressed are shown. Nine neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67) mixed in equal proportions were cultured with 4 nM medium expressing MDTCS-Fc and 12 candidate variants, and the residual activity of each candidate variant was calculated using the following formula: Residual activity (%) = A ÷ B x 100 (A: activity under mixed neutralizing antibody treatment conditions; B: activity under no neutralizing antibody treatment conditions).

[0093] Fig.30a and 30b The results of evaluating the escape rate of a single neutralizing antibody under the conditions of culture medium and purified solution in which each Fc-bound MDTCS fragment variant has been expressed are shown. Using culture medium expressing 12 variants (1C03, 2B01, 2B02, 3B05, 4E11, 4H07, 5G08, 7A02, 8D01, 8D05, DM1 and DM2) or purified solution purified using the Phytip system, the neutralizing antibody escape rate of each variant against 8 single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65 and Ab67) and the relative activity of each variant ( Fig.30a and 30b). The concentration in the corresponding purified solution was determined by Fc ELISA. Escape rate (%) = (1-binding affinity to variant / binding affinity to MDTCS-Fc) x 100; Relative activity (%) = specific activity of variant / specific activity of MDTCS-Fc x 100. The blue bars represent the median of the purified variant proteins, and the gray bars represent the median of the expressed variant proteins.

[0094] Fig.31 The results of measuring the escape rate of mixed neutralizing antibodies under the conditions of culture medium and purified solution in which each Fc-bound MDTCS fragment variant has been expressed are shown. 8 kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65 and Ab67) mixed in equal proportions were cultivated together with 4nM culture medium or purified solution in which control MDTCS-Fc and 12 kinds of candidate variants have been expressed, and then the residual activity of each variant was measured. The residual activity was calculated using the following formula. Residual activity (%) = A ÷ B x 100 (A: activity under mixed neutralizing antibody treatment conditions, B: activity under unused neutralizing antibody treatment conditions).

[0095] Fig.32 The pharmacokinetic results of the Fc-bound MDTCS fragment variants are shown. Each of the four final candidate variants (1C03, 5C09, 7A02, and DM2) fragment proteins bound to IgG1-YTE, MDTCS, Fc (IgG1-YTE), and IgG1-YTE was injected into mice via the tail vein, and then plasma was collected over time. Based on the specific activity, the dosage of each substance reached 160 IU / kg, and then the activity of the residual substance in the plasma at each time point was measured by an activity assay.

[0096] Figure 33 shows the results of evaluating the escape rate of a single neutralizing antibody under the conditions of culture medium in which each IgG1-YTE-bound MDTCS fragment variant has been expressed. The MDTCS fragment was obtained from five selected variants and IgG1-YTE was bound to it. Using the culture medium in which each variant bound to IgG1-YTE has been expressed, the neutralizing antibody escape rate of each variant bound to IgG1-YTE against 8 single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65 and Ab67) and the relative activity of each variant bound to IgG1-YTE were measured ( Fig.33a and 33b ). Escape rate (%) = (1-binding affinity to variant / binding affinity to MDTCS-IgG1-YTE) × 100; Relative activity (%) = specific activity of variant / specific activity of MDTCS-IgG1-YTE × 100.

[0097] Fig.34 The results of evaluating the ability to escape mixed neutralizing antibodies under medium conditions in which each MDTCS fragment variant bound to IgG1-YTE has been expressed are shown. Nine neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66 and Ab67) mixed in equal proportions were cultured with 4nM medium in which the control MDTCS-IgG1-YTE and five candidate variants were expressed, and the residual activity of each candidate variant was calculated using the following formula: Residual activity (%) = A ÷ B x 100 (A: activity under mixed neutralizing antibody treatment conditions; B: activity under no neutralizing antibody treatment conditions).

[0098] Fig.35 The results of evaluating the ability of each MDTCS fragment variant bound to IgG1-YTE to escape mixed neutralizing antibodies under purified solution conditions are shown. Nine neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67) mixed in equal proportions were incubated with a 4 nM purified solution of the control MDTCS-IgG1-YTE and five candidate variants, and the residual activity of each candidate variant was calculated using the following formula: Residual activity (%) = A ÷ B x 100 (A: activity under mixed neutralizing antibody treatment conditions; B: activity under conditions without neutralizing antibody treatment).

[0099] FIG36 schematically shows the process of using aTTP to simulate the mouse model to test the neutralizing antibody escape rate ( Fig.36a ), and the dose-dependent residual ADAMTS13 activity of each candidate variant was shown ( Fig.36b ).

[0100] FIG37 shows a schematic diagram of the process of using the TTP-simulated mouse model to test the dose-dependent residual ADAMTS13 activity of DM2-IgG1-YTE that exhibits the highest neutralizing antibody escape rate and clinical symptoms ( Fig.37a ), observed the results of platelet improvement, LDH level and ADAMTS13 activity ( Figure 37b ) and observed clinical symptoms ( Fig.37c ).

[0101] FIG38 shows a schematic diagram of the process of testing whether the administration of DM2-IgG1-YTE to the cTTP mouse model alleviates hematological and clinical symptoms, and testing the degree of restoration of human ADAMTS13 activity ( Fig.38a ), and observed the results of platelet improvement and LDH level and the results of ADAMTS13 activity recovery ( Figure 38b ). DETAILED DESCRIPTION

[0102] The present invention will be described in more detail below with reference to examples. These examples are only used to explain the present invention in more detail, and it is obvious to those skilled in the art that according to the subject matter of the present invention, the scope of the present invention is not limited by these examples.

[0103] Example

[0104] Example 1: Selection of ADAMTS13 protein preparation ingredients

[0105] The components and contents of the ADAMTS13 protein liquid preparation described in Example 3 below were determined by the following method.

[0106] Freeze drying process

[0107] After preparation, 1.0 ml of each sample solution was dispensed into each glass bottle (3 ml), which was then partially sealed with a rubber stopper and loaded into the rack of a freeze dryer (Lyostar 3, SP Scientific). Subsequently, freeze drying was performed according to the conditions shown in Table 1 below, and the prepared freeze-dried preparations were covered with aluminum caps after freeze drying.

[0108] [Table 1]

[0109]

[0110]

[0111] The stability of the prepared lyophilized formulations was analyzed after reconstitution with 1.0 ml of distilled water.

[0112] Size Exclusion Liquid Chromatography (SE-HPLC)

[0113] For size exclusion liquid chromatography, the sample was diluted to 1.0 mg / ml with mobile phase (1x PBS, Lonza) (samples of 1.0 mg / mml or less were not diluted) and then sterile filtered. 200 μL of the filtered sample was placed in the vial cannula, which was then inserted into the screw cap vial. Subsequently, the mobile phase was connected to the pump, and the analytical column (TSKgelG3000SWXL, Tosoh) was installed in the Waters e2695 & Waters 2489 system (Waters, Japan) while the mobile phase was allowed to flow at a flow rate of 0.5 mL / min. The mobile phase was allowed to flow at a flow rate of 0.5 mL / min for more than 30 minutes to reach equilibrium until the detector signal was stable. When the temperature of the autosampler dropped to 4 °C, the sample was placed in the injector. 30 μL of sample was injected, and then the mobile phase flowed for 35 minutes, and the detection peak at 280 nm was measured. Then the analysis was performed using Empower Pro software on the computer.

[0114] Colloidal stability (B 22 )

[0115] Since the extent of protein-protein interactions can affect aggregation and solubility, colloidal stability is an important factor that must be considered during protein formulation development. 22 The value (second virial coefficient) is a representative indicator of colloid stability. 22 When the value is a larger positive value, the repulsion between proteins is stronger and the probability of aggregation is reduced.

[0116] To evaluate the colloidal stability of the five stabilizers, 100 mM glycine, lysine, proline, alanine or arginine was added as amino acids to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2, 120 mM NaCl and 1.0% sucrose.

[0117] Liquid samples of each formulated composition were analyzed using the UNCLE instrument (B 22 )'s colloidal stability.

[0118] B 22 The higher the positive value of , the better the protein dispersion and the less aggregation will occur. Figure 1 As shown, it can be confirmed that the order of colloidal stability from high to low is arginine>glycine>proline>alanine>lysine>control group.

[0119] Turbidity analysis

[0120] Turbidity was analyzed using Lunatic (Unchained Labs). 2.0 μL of sample was injected into a Lunatic disk (Unchained Labs) and the turbidity was measured at 350 nm. The final value was calculated by subtracting the turbidity value of the placebo buffer from the turbidity value of the sample.

[0121] Thermal unfolding and thermal aggregation analysis

[0122] The degree of protein unfolding can be measured by measuring the emission wavelength of tryptophan exposed on the surface as the protein folds as the temperature increases. The UNCLE (Unchained Labs) system is used for differential scanning fluorimetry analysis, which is based on this intrinsic fluorescence intensity. When using the above system to analyze the thermal stability of proteins, T m (thermal decomposition) and T agg The measurement method of (heat accumulation) is as follows:

[0123] 8.8 μL of sample was injected twice into the Uni sample loader (Unchained Labs), and then the temperature was raised from 25°C to 95°C at a rate of 1°C / min. While the temperature was raised, the intensity of the emission wavelength (250-720 nm) with an excitation wavelength of 266 nm was measured. Fluorescence data analysis was performed using UNCLE analysis software (Unchained Labs).

[0124] Through the above analysis, the temperature when the fluorescence emission peak is at its maximum is defined as T m The static light scattering of proteins at 266 nm (SLS266) was measured, and the temperature at which the proteins began to aggregate (protein aggregation onset temperature) was defined as T agg .

[0125] SE-HPLC evaluation of seven stabilizers

[0126] 100 mM arginine, serine, valine, threonine, proline or glycine was added as an amino acid stabilizer to an alkaline composition (pH 7.4) containing 0.9 mg / ml ADAMTS protein, 20 mM histidine, 2.0 mM CaCl2 and 120 mM NaCl, or 1.0 w / v% sucrose was added to the alkaline composition as a sugar stabilizer.

[0127] After preparation, the liquid samples of each composition were stored at room temperature for 7 hours and then analyzed by SE-HPLC. Figure 2 As shown, it has been confirmed that among the seven stabilizers tested for comparison, arginine exhibits the best liquid phase stability, and the stability and purity are arginine>sucrose>proline>glycine>valine, threonine>control group>serine.

[0128] Evaluation of liquid phase stability according to arginine addition

[0129] To evaluate whether liquid phase stability depends on the addition of 20 mM arginine to a composition containing 0.049 mg / mL ADAMTS protein, 15 mM sodium phosphate, and 50 mM NaCl (pH 7.4), the formulated liquid samples were stored at room temperature for 3 hours and 19 hours and then analyzed by SE-HPLC at each analytical time point. Figure 3 As shown, it was confirmed that when 20 mM arginine was added, the refrigerated stability at 5°C was significantly improved compared with the control group.

[0130] Evaluation of liquid phase stability according to target protein concentration and arginine concentration

[0131] SE-HPLC analysis

[0132] Liquid samples were prepared by adding 0.2, 0.6 and 1.0 mg / ml of ADAMTS protein and 20, 80 and 140 mM of arginine to an alkaline composition (pH 7.4) containing 20 mM histidine, 4.0 mM CaCl2 and 160 mM NaCl, respectively. The stability of each liquid sample was evaluated.

[0133] Each prepared sample was stored at room temperature for 18 hours and then subjected to SE-HPLC analysis. Figure 4a As shown, it was confirmed that the purity increased with decreasing target protein concentration and increasing arginine concentration.

[0134] DOE Design

[0135] At the same time, in order to more closely explore the optimal concentrations of arginine and target protein, DOE (experimental design) was performed. The two operating parameters, arginine concentration and ADAMTS protein concentration, were set as factors (X values), and purity (SE-HPLC) was set as the response (Y value), and then 10.0 statistical program was used for DOE design. Specifically, the response surface model (RSM) was used, and the axial point was set to the central composite design (CCD) type. Finally, a total of 9 running conditions were designed, including "full factorial design 2 levels 4 runs + center point 1 run + axial point 2 factors 4 runs".

[0136] According to the DOE conditions, arginine was added to the sample stock solution (1.1 mg / mL), the sample solution was diluted, placed at room temperature (about 15-25° C.) for 18 hours, and then subjected to SE-HPLC analysis.

[0137] DOE statistical analysis was performed by multiple regression analysis using stepwise regression, and the model was established following the response surface model method. The Fit model method of JMP was used for analysis, and the effects used in the model included two main effects: two-factor interactions, and square terms for each main effect. As a termination rule for removing insignificant factors and selecting valid terms, a P value critical value (P value ≤ 0.25, direction: mixed) was used.

[0138] The analysis results show that the R square of the model is 0.96, and the R square corrected value is 0.94. The ANOVA (analysis of variance) P value of the model is 0.0006, which is less than the effective level of 0.05, indicating that the model is effective.

[0139] The factors that were considered significant based on P values ​​below the significant level α = 0.05 were the main effect protein concentration and the main effect arginine concentration. The P value for arginine*arginine curvature was 0.0765, which is close to the significant level α = 0.05 ( Figure 4b ).

[0140] As a result of the prediction analyzer analysis, the monomer % value was the highest when the arginine concentration was about 120 mM, indicating that 100 to 140 mM was the optimal concentration of arginine ( Figure 4c ).

[0141] Evaluation of the quality of freeze-dried compositions according to NaCl concentration

[0142] NaCl at concentrations of 50, 100, 120, 150, 200 and 300 mM was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2 and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder was observed, and SE-HPLC analysis was performed. The results confirmed that with the increase in NaCl concentration, the appearance of the powder became harder and better looking ( Figure 5a ). However, the results of SEP-HPLC showed that the purity did not vary significantly with the NaCl concentration ( Figure 5b ).

[0143] Evaluation of liquid phase stability according to NaCl concentration

[0144] NaCl at a concentration of 0, 40, 80, 120 and 160 mM was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2 and 20 mM arginine. Each prepared liquid sample was stored at room temperature for 4, 8 and 12 hours before SE-HPLC analysis. The results are shown in FIG. Figure 6 As shown, it is confirmed that the higher the NaCl concentration, the better the liquid phase stability.

[0145] Evaluation of the quality of the lyophilized composition according to the sugar concentration (120 mM NaCl)

[0146] Sucrose at a concentration of 0.0, 0.5, 1.0 or 2.0% (w / v) or 1.0% (w / v) trehalose was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 120 mM NaCl, 20 mM histidine, 4.0 mM CaCl2 and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder cake was observed, and SE-HPLC analysis was performed. The results confirmed that under the condition of 120 mM NaCl, collapse did not occur when 1.0% sucrose was added, but collapse occurred when 2.0% sucrose was added, indicating that the sucrose concentration at which collapse began was between 1.0% and 2.0% ( Figure 7a The formulation containing 1.0% trehalose showed a similar appearance to the formulation containing 1.0% sucrose ( Figure 7a ). At the same time, the results of SE-HPLC confirmed that there was no significant difference in purity with different concentrations of added sugar, indicating that purity can be maintained even if collapse occurs.

[0147] Evaluation of the quality of the freeze-dried composition based on the content ratio of sucrose and bulking agent (120 mM NaCl)

[0148] Sucrose and a leavening agent (mannitol or glycine) at a ratio of 1.0 / 3.0% (w / v) (a content ratio of 1:3) or 2.0 / 2.0% (w / v) (a content ratio of 1:1) were added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 120 mM NaCl, 20 mM histidine, 4.0 mM CaCl2, and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder cake was observed, and SE-HPLC analysis was performed. The results confirmed that when NaCl was fixed at 120 mM and sucrose and a leavening agent were added at a content ratio of 1:3 or 2:2, collapse occurred in all cases ( Figure 8a ), SE-HPLC results showed no significant differences between different samples ( Figure 8b ).

[0149] Evaluation of lyophilization quality based on glycine concentration (120 mM NaCl)

[0150] Glycine at concentrations of 0, 20, 40, 60, 80 and 100 mM was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 120 mM NaCl, 1.0% sucrose, 20 mM histidine, 4.0 mM CaCl2 and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder was observed, and SE-HPLC analysis was performed. The results showed that when 120 mM NaCl and 1.0% sucrose were fixed and the concentration of glycine was increased, the appearance of the powder was adversely affected, and when the added concentration of glycine was 60 mM or higher, the powder collapsed completely ( Fig. 9 ).

[0151] Evaluation of lyophilization quality based on arginine concentration (120 mM NaCl)

[0152] Arginine at concentrations of 0, 20, 60 and 100 mM was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 120 mM NaCl, 1.0% sucrose, 20 mM histidine, 4.0 mM CaCl2 and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder cake was observed, and SE-HPLC analysis was performed. The results confirmed that when 120 mM NaCl and 1.0% sucrose were fixed and the arginine concentration was increased, the appearance of the powder cake was adversely affected, and when the arginine concentration was 60 mM or higher, the powder cake collapsed completely ( Fig.10 ).

[0153] Evaluation of freeze-dried product quality based on the ratio of NaCl to sucrose (without arginine)

[0154] In an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2 and 0.05% PS80, sodium chloride at concentrations of 120, 160 and 200 mM, and sucrose at concentrations of 1.0, 1.5 and 2.0% (w / v) were added. Each formulated sample was freeze-dried, the appearance of the powder was observed, and SE-HPLC analysis was performed. The results showed that the appearance of the powder was better with increasing sodium chloride concentration and decreasing sucrose concentration. Specifically, it was confirmed that: i) when 120 mM NaCl was added, it began to collapse in the range of sucrose concentration of 1.0% to 1.5%; ii) when 160 mM NaCl was added, it began to collapse in the range of sucrose concentration of 1.5% to 2.0%; iii) when 200 mM NaCl was added, it began to collapse in the range of sucrose concentration above 2.0% ( Fig.11a ).

[0155] Meanwhile, it was confirmed that when arginine was not added, the SE-HPLC results depending on the content ratio of NaCl and sucrose did not change significantly ( Fig.11b ).

[0156] Evaluation of freeze-dried product quality based on the ratio of NaCl to sucrose (120 mM arginine)

[0157] In an alkaline composition (pH 7.4) containing 0.5 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2, 0.05% PS80 and 120 mM arginine, 160, 200, 240 and 280 mM NaCl and 0.0, 0.5 and 1.0% (w / v) sucrose were added. Each prepared sample was freeze-dried, the appearance of the powder was observed, and SE-HPLC analysis was performed. The results confirmed that when the fixed addition amount of arginine was 120 mM, the appearance of the powder became firm as the NaCl concentration increased and the sucrose concentration decreased. Specifically, it can be seen that when i) 160 mM NaCl is added, collapse begins at sucrose concentrations ranging from 0.0 to 0.5%; ii) when 200 mM NaCl is added, collapse begins at sucrose concentrations above 1.0%; iii) when 240 mM NaCl is added, collapse begins at sucrose concentrations above 1.0%; and iv) when 280 mM NaCl is added, collapse begins at sucrose concentrations above 1.0% ( Fig.12a ).

[0158] Meanwhile, the results of SE-HPLC confirmed that when 120 mM arginine was added, the purity did not change significantly due to the different concentrations of NaCl and sucrose, but the purity of the preparation containing 160 mM NaCl and no sucrose was 97.4%, which was relatively low compared with the other samples ( Figure 12b ).

[0159] Evaluation of freeze-drying stability according to the mixing ratio of NaCl to sucrose (120 mM arginine)

[0160] In an alkaline composition (pH 7.4) containing 0.5 mg / ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl2, 0.05% PS80 and 120 mM arginine, 200, 240 and 280 mM NaCl and 0.0, 0.5 and 1.0% (w / v) sucrose were added. Each prepared sample was freeze-dried and its accelerated stability (40°C) was evaluated. The results confirmed that in the presence of 120 mM arginine, after being placed at 40°C for 1 month, the purity depending on the mixing ratio of sodium chloride and sucrose did not change significantly compared with the initial stage ( Fig.13 ).

[0161] Prevention of agitation-induced aggregation depending on the concentration of polysorbate 80

[0162] Polysorbate 80 with a concentration of 0.0, 0.001, 0.005, 0.01, 0.05 and 0.1% (v / v) was added to an alkaline composition (pH 7.4) containing 1.2mg / ml ADAMTS protein, 20mM histidine, 4.0mM CaCl2, 120mM NaCl and 1.0% sucrose. Each prepared liquid sample was vortexed to generate artificial shear stress and then analyzed (appearance, turbidity and SE-HPLC). Samples were taken for analysis after 30 seconds and 2 minutes of stirring. The result of analyzing the appearance after stirring for 2 minutes showed that when the added concentration of polysorbate 80 was 0.005% or higher, the sample became clear and transparent ( Fig.14a ).

[0163] It can also be seen that when the addition concentration of polysorbate 80 is 0.005% or higher, the turbidity does not change compared with the initial stage, indicating that no aggregation caused by stirring occurs ( Fig.14b ). In addition, the results of SE-HPLC confirmed that when the added concentration of polysorbate 80 was 0.05% or more, the purity decreased by 5% or less than that of the initial stage ( Fig.14c ).

[0164] Evaluation of liquid phase stability depending on polysorbate 80 concentration

[0165] The polysorbate 80 that concentration is 0.0, 0.005, 0.01, 0.05 and 0.09% (v / v) is added in the alkaline composition (pH 7.4) containing 0.5mg / ml ADAMTS protein, 20mM histidine, 4.0mM CaCl2, 120mM NaCl and 1.0% sucrose. Each prepared liquid sample is stored at room temperature for 6 hours, and is then analyzed with SE-HPLC. The result confirms that along with the increase of polysorbate 80 concentration, liquid phase stability (purity) decreases slightly ( Fig.15 ).

[0166] Assessment of lyophilization quality based on polysorbate 80 concentration

[0167] Polysorbate 80 was added to an alkaline composition (pH 7.4) containing 0.5mg / ml ADAMTS protein, 20mM histidine, 4.0mM CaCl2, 120mM NaCl and 1.0% sucrose at a concentration of 0.0, 0.005, 0.01, 0.05 and 0.09% (v / v). Each prepared liquid sample was processed by freeze drying and then analyzed by SE-HPLC. The results confirmed that, as the concentration of polysorbate 80 increased, the effect of preventing purity loss during freeze drying also increased, and when the concentration of adding polysorbate 80 was 0.01% or higher, it was found that the monomer recovery rate reached 94% or higher ( Fig.16a In addition, the formation of higher-order aggregates was also effectively prevented with the increase of polysorbate 80 concentration ( Fig.16b ).

[0168] Evaluation of pH-dependent thermal unfolding and thermal aggregation

[0169] Diafiltration buffers containing an alkaline composition (containing 20 mM histidine, 4 mM CaCl2, and 0.05% PS80) and having pH values ​​of 6.0, 6.5, 7.0, 7.2, and 7.4 were prepared, and then buffer exchange was performed with a solution containing an ADAMTS protein to prepare the sample. Ultra-15 Centrifugal Filter Unit 30K, centrifuged at 3,000 rpm under refrigerated conditions.

[0170] The buffer exchange samples had a common composition containing 10 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2 and 0.05% PS80, and the pH values ​​were 6.0, 6.5, 7.0, 7.2 and 7.4, respectively.

[0171] The prepared liquid samples were subjected to T m and T agg The results of the analysis showed that thermal unfolding (T m ) value increases with the increase of pH value. The T m The value is lower than that of other samples ( Fig.17a It has been confirmed that thermal aggregation (T agg ) value increases with the increase of pH value. The T agg The value is lower than that of other samples ( Fig.17b ).

[0172] Evaluation of freeze-drying quality based on CaCl2 concentration

[0173] CaCl2 at concentrations of 2.0, 4.0 and 8.0 mM was added to an alkaline composition (pH 7.4) containing 1.2 mg / ml ADAMTS protein, 20 mM histidine, 120 mM NaCl, 1.0% sucrose and 0.05% PS80. Each prepared sample was freeze-dried and analyzed by SE-HPLC. The results confirmed that regardless of the concentration of CaCl2, the samples all showed a partially collapsed appearance ( Fig.18a ). SE-HPLC analysis results confirmed that the purity increased with the increase of CaCl2 concentration ( Fig.18b ).

[0174] Example 2: Further optimization of the composition of ADAMTS13 protein preparation

[0175] Evaluation of freeze-drying quality based on the mixing ratio of NaCl to sucrose

[0176] In an alkaline composition (pH 7.4) containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 120 mM L-Arg and 0.05% PS80, 100, 150, 200, 250, 300, 350 and 400 mM NaCl and 0, 0.5, 1 and 1.5% (w / v) sucrose were added. Each prepared sample was freeze dried, the powder appearance was observed, and the purity before and after freeze drying was analyzed by SE-HPLC. As shown in Table 2 below, the results confirmed that the purity did not change significantly due to the content ratio of sodium chloride and sucrose, indicating that high purity was maintained within all test ranges.

[0177] [Table 2] Purity recovery rate after freeze drying (%)

[0178]

[0179] However, if Fig.19a and 19b As shown, it has been confirmed that with the increase of NaCl concentration and the decrease of sucrose concentration, the appearance of the powder cake will be better.

[0180] Evaluation of freeze-drying quality based on high sucrose treatment

[0181] In an alkaline composition (pH 7.4) containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 120 mM L-Arg and 0.05% PS80, 200, 250 and 300 mM NaCl and 1.5 and 2.5% sucrose were added. Each prepared sample was freeze-dried and its powder appearance was observed. The results are shown in FIG. Fig. 20As shown, it is confirmed that with the increase of sodium chloride concentration and the decrease of sucrose concentration, the appearance of the powder cake is better.

[0182] According to the arginine concentration (B 22 , kD) to evaluate colloidal stability

[0183] Arginine at concentrations of 40, 80, 120, 160 and 200 mM was added to an alkaline composition (pH 7.4) containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 280 mM NaCl, 1% sucrose and 0.05% PS80. The colloidal stability of each formulated liquid sample was analyzed using the UNCLE system (B 22 , kD) B 22 The higher the positive value and kD value, the better the protein dispersion and the less aggregation. Fig.21 As shown, it has been confirmed that the closer the arginine concentration is to 120 mM, the better the colloidal stability.

[0184] Assessment of lyophilization quality based on arginine concentration

[0185] Arginine at concentrations of 40, 80, 120, 160 and 200 mM was added to an alkaline composition (pH 7.4) containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 280 mM NaCl, 1% sucrose and 0.05% PS80. Each prepared sample was freeze-dried, the appearance of the powder was observed, and its purity was analyzed by SE-HPLC. The results confirmed that when the arginine concentration was 80 mM and 120 mM, the powder had a good appearance ( Fig.22a ), but there was no significant difference in purity according to the arginine concentration ( Figure 22b ).

[0186] Storage time and temperature stability of the final liquid formulation of GC1126A

[0187] A prepared liquid sample (pH 7.4) containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 120 mM L-Arg, 280 mM NaCl, 1% sucrose and 0.05% PS80 was stored at room temperature (25°C) or refrigerated (4°C), and then the changes in the samples over time were detected by analyzing the purity (SE-HPLC), potency (ADAMTS13 activity), protein concentration (ultraviolet light) and turbidity. The results of the purity analysis showed that after 3 days and 3 days of storage at room temperature, the purity decreased by about 4% compared with the initial stage. After 7 days of storage, the purity decreased by about 15% compared with the initial stage. Until the 7th day of refrigerated storage, no changes over time were observed ( Fig.23a ).

[0188] By observing the change in potency, it was confirmed that when stored at room temperature for 7 days, the potency decreased by about 50% compared with the initial stage. Considering the deviation of potency analysis, no significant change over time was observed until 7 days of refrigerated storage ( Figure 23b ).

[0189] At day 3 of storage at room temperature, the protein concentration increased compared to the initial stage, which is considered to be the result of increased UV absorbance and increased sample opacity due to particle aggregation. No changes over time were observed after refrigerated storage for up to 7 days ( Fig.23c ).

[0190] Meanwhile, the turbidity of the liquid preparation tended to increase when finally stored at room temperature for 3 days, but no change over time was observed until refrigerated storage for 7 days ( Fig.23d ).

[0191] Optimization of NaCl and sucrose concentrations

[0192] To an alkaline composition (pH 7.4) containing 0.5 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl2, 120 mM L-Arg and 0.05% PS80, 200, 240 and 280 mM NaCl and 0, 0.5 and 1% sucrose were added.

[0193] Experimental design (DOE) was performed to explore the optimal concentrations of NaCl and sucrose. After setting the two operating parameters of NaCl and sucrose concentration as factors (X values), and setting purity (SE-HPLC) and potency (ADAMTS13 activity) as responses (Y values), we used 10.0 statistical program for DOE design. When conducting DOE design, a full factorial design was used with two center points. Finally, a total of 6 conditions were designed, including "full factorial design 2 levels 4 runs + center point 2 runs".

[0194] [Table 3]

[0195]

[0196] The formulation buffer solution was prepared according to the pre-designed conditions and mixed with the stock solution to prepare the final stock solution. 1 ml of the prepared formulation was dispensed into each 3 ml vial and freeze-dried. The freeze-dried samples were refrigerated (5°C) and quality analyzed after 6 months.

[0197] DOE statistical analysis was performed by multiple regression analysis using stepwise regression. The "Fit Model" method of JMP was used for analysis, and the effects used in the model included two main effects: a two-factor interaction and a square term for each main effect. As a stopping rule for removing insignificant factors and selecting valid terms, a P value critical value (P value ≤ 0.25, direction: mixed) was used.

[0198] The results of the analysis showed that the purity was good at 6 months under all experimental conditions, and there was no difference in purity due to the concentration of NaCl and sucrose ( Fig.24a In addition, compared with the initial titer of each sample, the titer after 6 months was within the analytical deviation range and there was no significant difference ( Figure 24b ).

[0199] Example 3: Construction of ADAMTS13 protein variants

[0200] The present inventors used a random mutagenesis method to construct human ADAMTS13 variants that can effectively escape ADAMTS13 neutralizing antibodies in aTTP patients. In order to examine whether variants with mutations in the MDTCS portion or domain S can escape ADAMTS13 neutralizing antibodies, antibody Fabs that recognize different antigenic determinants of human ADAMTS13 were constructed using the HuCAL system (Bio-Rad), and 16 Fabs with good binding affinity to human ADAMTS13 were selected. Regarding the binding region of each antibody, as shown in FIG. Fig.25c As shown, Ab 4-16 specifically binds to domain S, Ab 67 specifically binds to the MDTCS portion, and Ab 66 specifically binds to the C-terminal portion (domain TSP-2 to domain TSP-8).

[0201] The evaluation of the variants was performed by analyzing the relative results of the wild-type ADAMTS13 construct and non-mutagenized or silent mutagenized variants (hereinafter referred to as wild-type clones). Among the variants produced by random mutagenesis, the non-mutagenized or silent mutagenized variants had the same amino acid sequence as the wild-type ADAMTS13. The activity and binding affinity of Ab 4-16 and Ab 67 were analyzed for a total of 59 wild-type clones. The analysis results of each wild-type clone were relative to the result values ​​of the wild-type ADAMTS13 construct, and the results are shown in Figure 26. Figure 26 shows the escape rates and relative activities of wild-type ADAMTS13 (n=59), mutant ADAMTS13 (n=304) and selected ADAMTS13 (n=26) containing conservative amino acid substitutions to antibodies Ab 4-16 and Ab 67. Although the wild-type clones have no mutations, they differ from the result values ​​of the wild-type ADAMTS13 constructs. Among the mutant ADAMTS13 (n=304), 26 mutant ADAMTS13 having values ​​equal to or higher than the specific escape rates or relative activities shown in Table 4 below were selected.

[0202] Specifically, the escape rate of mutant ADAMTS13 to Ab 4-16 ranged from -29.5% to 33.4%, its escape rate to Ab 67 ranged from -24.4% to 30.5%, and its relative activity ranged from 62.7% to 128.9% (Table 4). According to the results of the application of the normal distribution according to the 3σ principle, the escape rate of Ab 4-16 was between -34.1% and 38.5%, the escape rate of Ab 67 was -38.5% to 42.3%, the relative activity was between 47.0% and 145.2%, and almost all the results of WT clones were expected to be within this distribution range. Therefore, when selecting variants according to the maximum value of 3σ, the escape rate of Ab 4-16 exceeded 38.5%, the escape rate of Ab 67 exceeded 42.3%, or the relative activity reached or exceeded 47.0% as the selection criteria.

[0203] [Table 4] Escape rate and relative activity of neutralizing antibodies Ab4-16 and Ab-67

[0204] category Ab 4-16 escape rate Ab 67 escape rate Relative activity Minimum -29.5％ -24.4％ 62.7％ Maximum 33.4％ 30.5％ 128.9％ average value 2.2％ 1.9％ 96.1％ Standard Deviation 12.1％ 13.5％ 16.4％ Mean - 3 standard deviations -34.1％ -38.5％ 47.0％ Mean + 3 standard deviations 38.5％ 42.3％ 145.2％

[0205] Screening for variants that can escape neutralizing antibodies against ADAMTS13 and have higher activity than wild-type ADAMTS13

[0206] After the WT clone and the variants with amino acid mutations were transfected into the cells, the protein content present in each culture medium was measured, and the neutralizing antibody binding affinity and activity were determined for 304 variants with protein expression concentrations of 50 ng / ml or more. The results confirmed that the variants with amino acid mutations had different distributions in terms of neutralizing antibody binding affinity or relative activity compared to the WT clone. Among these variants, 26 variants that met the selection criteria were finally selected. It has been confirmed that 18 of the 26 variants have mutations in domain S, especially 13 variants (1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02, 8C04, 8D01 and 8F01) that have mutations in domain S have higher Ab4-16 escape rates, and 5 variants (7E01, 7G08, 8C02, 8D01 and 8D05) show higher relative activity (Table 5). There are 6 variants with mutations in domain D, of which 5 variants (46, 3A06, 4C07, 4E11 and 4H07) show higher Ab 67 escape rates. In addition, 6 variants with mutations in domain M, 2 variants with mutations in domain C and 2 variants with mutations in domain T were found. After three repeated experiments, the reproducibility of the results was tested for 26 selected variants, and 12 variants that continued to be superior to WT clones in repeated experiments were selected as the objects of in vitro efficacy testing. Among these 12 variants, 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02 and 8D01 were selected for their excellent Ab4-16 antibody escape rates, and 4C07, 4E11 and 4H07 were selected for their excellent Ab67 escape rates. Finally, 8D05 was selected based on excellent relative activity.

[0207] In order to test the ability of the selected 12 variants to escape other neutralizing antibodies, in addition to testing the Ab4-16 and Ab67 antibodies used for screening, the inventors also tested whether these variants can escape neutralizing antibodies Ab4-20, Ab60, Ab61, Ab64 and Ab65. Among these 12 variants, 8 variants except 8D05 in 9 variants have amino acid mutations in domain S (1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02 and 8D01), which show a higher escape rate in Ab4-16 and Ab4-20 constructed based on the ADAMTS13 autoantibody sequence possessed by aTTP patients, and also show excellent ability to escape Ab60 and Ab61. On the other hand, variants 4C07, 4E11 and 4H07 with amino acid mutations in domain D have a high escape rate to Ab67 (Figure 27 and Table 6).

[0208] [Table 5]

[0209] Selection of 26 variants with high escape rates or excellent relative activity against Ab4-16 and Ab67 neutralizing antibodies

[0210]

[0211]

[0212]

[0213] [Table 6]

[0214] Evaluation of single neutralizing antibody escape rates and relative activities of the 12 selected variants

[0215]

[0216] At the same time, structure-function studies reported that among the total 14 domains of ADAMTS13, the MDTCS domain remaining after the CUB2 domain of TSP-2 was removed from the C-terminus had a metalloprotease function similar to ADAMTS13 and was able to cleave VWF (Shelat et al., 2005). This suggests that only the MDTCS fragment, rather than the full-length ADAMTS13, can be used as a drug for the treatment of TTP disease, and that this truncated form can escape the neutralizing antibodies that bind to the C-terminus in the patient's plasma.

[0217] Therefore, the MDTCS fragment was obtained from the selected 12 variants, and DM1 and DM2 variants with two amino acid mutations were additionally constructed by combining the selected mutant amino acid residues. According to the above method, the escape rate and relative activity of single or mixed neutralizing antibodies were determined using the cell expression culture medium and purified solution to screen out the final candidates.

[0218] The binding escape rates and relative activities of 8 single neutralizing antibodies were measured using culture medium, and the results confirmed that 2B01, 3B05, 4H07, 5G08, 8D05 and DM1 escaped all neutralizing antibodies at similar levels (Figure 28 and Table 7 below). 1C03, 2B02, 5C09, 7A02, 8D01 and DM2 had higher escape rates for the binding of 3-01 and Ab60. The domain S of these six candidates all had amino acid mutations, and except for 8D01, the 612th amino acid residue of the other five candidates had mutations. DM1 had the lowest relative activity (57.9%), while 1C03 had the highest relative activity (133.1%). All candidates except the above two candidates showed relative activities of 78.2 to 125.1%, which was similar to the relative activity of the MDTCS-Fc control group (Table 7). In order to detect the antibody escape ability of 12 candidate variants other than 4C07 and 5C09 compared to the control MDTCS-Fc under the treatment conditions of 9 neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66 and Ab67) mixed together in equal proportions, 7.5nM of the neutralizing antibody mixture was mixed with 4nM culture medium in which each variant was expressed. Then, the resulting mixture was incubated at room temperature for 1 hour, and the residual activity of each variant was measured. The results have confirmed that MDTCS-Fc has a residual activity of 3.5%, and 3B05, 4E11, 4H07, 5G08 and 8D05 have a residual activity of 1.24 to 2.42%, which is lower than the residual activity of MDTCS-Fc ( Fig.29 On the other hand, 1C03, 2B01, 2B02, 7A02, 8D01, DM1 and DM2 were confirmed to have residual activity ranging from 7.69 to 18.81%, indicating that these candidate variants were better than MDTCS-Fc in escaping mixed neutralizing antibodies ( Fig.29 ).

[0219] In order to detect the neutralizing antibody escape ability of candidate variants using purified solution, the protein was purified from each culture medium using the Phytip system (protein A resin), and the single neutralizing antibody escape rate of each variant was measured using the purified solution obtained. The results showed that the trend of most variants was similar to the evaluation results in the culture medium state (Figure 30).

[0220] It was confirmed that 2B01, 5G08, 8D05 and DM1 variants showed 24.6% or higher neutralizing antibody escape rates for all 8 neutralizing antibodies, and 1C03, 2B02, 7A02, 8D01 and DM2 showed higher escape rates for the binding of 3-01 and Ab60, similar to the results obtained using the culture medium. It was confirmed that 4E11 and DM2 showed higher escape rates for the binding of Ab67, similar to the results obtained using the culture medium. The relative activity of each variant is shown in Figure 30 and Table 8 below. The results of measuring the residual activity under the conditions of treatment with mixed neutralizing antibodies confirmed that MDTCS-Fc had 3.76% residual activity, and 3B05, 4E11, 4H07 and 8D05 variants had 2.05 to 3.50% residual activity, which was lower than the residual activity of MDTCS-Fc. On the other hand, the results showed that 1C03, 2B01, 2B02, 5G08, 7A02, 8D01, DM1 and DM2 had 6.34 to 12.8% residual activity, indicating that these variants have superior ability to escape mixed neutralizing antibodies compared to MDTCS-Fc ( Fig.31 ).

[0221] Based on the above results, five candidates were selected for further study, including 1C03, 2B02, 7A02 and DM2, which were selected considering the relative activity or escape rate of mixed neutralizing antibodies, and 5C09, which was considered to have excellent ability to escape mixed neutralizing antibodies due to the amino acid mutation at position 612.

[0222] [Table 7]

[0223] Escape rate and relative activity of single neutralizing antibodies against MDTCS fragments including 14 variants (culture medium conditions)

[0224]

[0225]

[0226] [Table 8]

[0227] Measurement of single neutralizing antibody escape rates and relative activity of MDTCS fragments including 14 variants (purified solution conditions)

[0228]

[0229] DNA was formed by combining IgG1-YTE with each of the five finally selected MDTCS variant fragments, and the escape rate of each DNA combined with eight single neutralizing antibodies and the relative activity of each DNA were determined in the culture medium expressed by the cells. The results confirmed that 1C03, 2B02, 5C09 and 7A02 had a higher escape rate when combined with Ab3-01 and Ab60, and DM2 had a higher escape rate when combined with Ab3-01, Ab60 and Ab67 (Figure 33). The relative activity was 98.6% to 127.7%, similar to MDTCS-IgG1-YTE (Figure 33). In order to detect the escape ability of the five variants compared with the control MDTCS-IgG1-YTE under the conditions of equal proportions of the treatment of nine neutralizing antibodies, the mixed neutralizing antibodies (n=9) were mixed with the culture medium expressing each variant. Then, the resulting mixture was cultured at room temperature for 1 hour, and the residual activity of each variant was measured. The results showed that the residual activity of MDTCS-IgG1-YTE was 5.5%, and the residual activities of the five variants ranged from 8.18% to 13.42%, indicating that the variants had a very strong ability to escape neutralizing antibodies compared with the control group ( Fig.34 ).

[0230] The Phytip system (Protein A resin) was used to purify proteins from the culture medium of the expressed IgG1-YTE bound to each of the five MDTCS variant fragments. The residual activity and specific activity of the neutralizing antibodies in the purified solution were determined. The concentration in the purified solution was determined by Fc enzyme-linked immunosorbent assay, and the elution of the target protein was confirmed by silver staining. By measuring the residual activity under mixed neutralizing antibody treatment conditions, it was confirmed that the residual activity of MDTCS-IgG1-YTE was 0.4%, while the residual activities of 1C03, 2B02, 5C09, 7A02 and DM2 were 5.7%, 1.7%, 8.8%, 2.6% and 7.9%, respectively, indicating that they have excellent ability to escape mixed neutralizing antibodies compared with MDTCS-IgG1-YTE. By measuring the specific activity, it was confirmed that except for 7A02 (10,288 IU / mg), the specific activities of the other four variants ranged from 19,091 to 22,379 IU / mg, which were similar to the control (18,030 IU / mg) ( Fig.35 ). Therefore, compared with the control group, the binding of IgG1-YTE to each of the five variant fragments has an excellent ability to escape mixed neutralizing antibodies, and the four variants except 7A02 have similar specific activities to the control group.

[0231] Evaluation of whether Fc binding prolongs the half-life of MDTCS fragment variants

[0232] To confirm whether Fc (IgG1-YTE) bound to MDTCS actually leads to an increase in half-life, pharmacokinetic analysis was performed in mice. To this end, mice were injected with MDTCS, MDTCS bound to IgG1-YTE, and each of the four final candidates (1C03, 5C09, 7A02, and DM2) variant fragment proteins via the tail vein, and plasma was collected over a period of time. Based on the specific activity of each substance, the dosage of each substance was brought to 160 IU / kg, and the activity of the remaining substance in the plasma collected at each time point was then measured by an activity assay. The results obtained were summarized using the original pooling method, and these results were used for pharmacokinetic analysis using a non-compartmental analysis method. It was determined that the half-life of MDTCS was 2.898 hours, the half-life of MDTCS-IgG1-YTE was 11.51 hours, and the half-life of each variant ranged from 5.184 to 9.902 hours. Compared with the control group MDTCS, the half-life of the IgG1-YTE conjugate was extended by 1.79 to 3.97 times. In addition, the IgG1-YTE conjugate resulted in a mean retention time (MRT) value representing the mean retention time of the candidate in vivo of 7.189 to 11.67 hours, which was increased compared with the control group (3.743 hours) (Table 9 and Fig.32 ). The results confirmed that the IgG1-YTE conjugate was able to effectively maintain activity based on blood half-life and mean in vivo residence time.

[0233] [Table 9]

[0234] Pharmacokinetic results

[0235]

[0236] As described above, the inventors have found 26 novel variants that escape ADAMTS13 neutralizing antibodies that bind to the MDTCS portion or domain S, or exhibit the same or higher activity as wild-type ADAMTS13. Among these variants, 12 variants were selected to have the best ability to escape 9 neutralizing antibodies or have significantly superior relative activity, and an MDTCS fragment was constructed, which is essential for vWF cleavage and effectively escapes neutralizing antibody binding to the C-terminus, thereby identifying variants with significantly enhanced ability to escape autoantibodies that bind to the D, C or S domains of ADAMTS13 in aTTP patients. When the variants of the present invention increase their blood half-life through IgG1-YTE conjugates, they can be advantageously used as an effective pharmacological component with better stability and lasting physiological activity.

[0237] Evaluation of the Poc of variants in aTTP mimetic disease mouse model

[0238] To select the final candidates using the established aTTP mimetic mouse model, a control (MDTCS-IgG1-YTE) or each of the selected five variant candidates (1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, 7A02-IgG1-YTE, and DM2-IgG1-YTE) was administered, and then the escape rate of the neutralizing antibody was evaluated ( Fig.36a Among the five variants, DM2-IgG1-YTE exhibited the highest human ADAMTS13 activity at doses of 5,000 or 7,000 IU / kg ( Fig.36b Finally, DM2-IgG1-YTE with the highest neutralizing antibody escape rate was selected and administered at different doses. Its activity against human ADAMTS13 and the degree of clinical symptom relief were tested ( Fig.37a The results confirmed that the degree of clinical symptom relief increased with the increase of DM2 dose, and the platelet and LDH levels in the DM2-IgG1-YTE group at a dose of 7,000 IU / kg were higher than those in the MDTCS-IgG1-YTE group at a dose of 7,000 IU / kg ( Figure 37b ). By detecting the results of human ADAMTS13 activity, it can be observed that ADAMTS13 activity is proportional to the dose of the control substance or candidate substance, which is consistent with the improvement of platelet and LDH content ( Figure 37b ). By observing clinical symptoms, it was confirmed that the death or hematuria that occurred in the aTTP mimic mouse model was alleviated after the administration of the control substance or the candidate substance. In particular, in the DM2-IgG1-YTE treatment group, no death occurred at all doses, and no hematuria symptoms were observed in mice when 7,000 IU / kg or higher doses were administered ( Fig.37c ). When administered at 7,000 IU / kg, the average residual activity of DM2-IgG1-YTE was 0.32 IU / mL, which is 9.6 times that of MDTCS-IgG1-YTE 0.03 IU / mL. The difference in the above-mentioned clinical symptom observation results is believed to be caused by this difference in residual activity. According to the results of general blood tests, clinical chemistry tests and clinical symptom observations, the administration of MDTCS-IgG1-YTE and DM2-IgG1-YTE tended to alleviate the clinical symptoms of aTTP in the aTTP mimic mouse model, indicating that it has in vivo efficacy. In particular, when administered at 7,000 IU / kg, DM2-IgG1-YTE had a better effect on alleviating the clinical symptoms of aTTP than MDTCS-IgG1-YTE.

[0239] Evaluation of the in vivo efficacy of variants in a mouse model of cTTP disease

[0240] The efficacy of DM2-IgG1-YTE in the aTTP mimetic mouse model was the highest among the five candidate variants. The inventors evaluated whether DM2-IgG1-YTE could alleviate the hematological and clinical symptoms of TTP disease in the cTTP mouse model and evaluated its restoration of human ADAMTS13 activity ( Fig.38a The results confirmed that the increase in platelet and LDH levels was concentration-dependent with the increase in DM2-IgG1-YTE dose. In the DM2-IgG1-YTE 180 group at a dose of 360 IU / kg, platelet levels increased significantly compared with the control group, especially in the group at a dose of 360 IU / kg, where platelet levels returned to normal levels ( Figure 38b ). It was confirmed that the LDH level of the group given 180 IU / kg had returned to a level similar to that of the control group ( Figure 38b ). In terms of ADAMTS13 activity, the average ADAMTS13 activity of the DM2-IgG1-YTE group with a dose of 60 IU / kg or more was 0.1 IU / mL or more, and the average ADAMTS13 activity measured at a dose of 360 IU / kg was 1.08 IU / mL ( Figure 38b ). Thus, the DM2-IgG1-YTE variant can effectively alleviate the clinical symptoms of mice with cTTP disease and restore ADAMTS13 activity. Therefore, the present inventors screened and optimized the ADAMTS13 protein preparation components using DM2-IgG1-YTE as described in Examples 1 and 2 above.

[0241] After describing a specific embodiment of the present invention, it will be apparent to those skilled in the art that this description is only a preferred embodiment thereof and does not limit the scope of the present invention. Therefore, the true scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical preparation comprising 0.2 mg / ml to 1.2 mg / ml plasma protein and 40 mM to 200 mM amino acid stabilizer.

2. The pharmaceutical preparation according to claim 1, characterized in that The amino acid is at least one selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.

3. The pharmaceutical preparation according to claim 1, characterized in that It further comprises a sugar stabilizer, wherein the amount of the sugar stabilizer is 0-1.5 w / v% of the total volume of the pharmaceutical preparation.

4. The pharmaceutical preparation according to claim 3, characterized in that The sugar is at least one selected from the group consisting of sucrose, trehalose, and pharmaceutically acceptable salts thereof.

5. The pharmaceutical preparation according to claim 1, characterized in that It further comprises 100 mM to 400 mM of an inorganic salt.

6. The pharmaceutical preparation according to claim 5, characterized in that The inorganic salt is at least one selected from the group consisting of NaCl, CaCl2, KCl and MgCl2.

7. The pharmaceutical preparation according to claim 6, characterized in that The inorganic salt is a mixture of NaCl and CaCl2.

8. The pharmaceutical preparation according to claim 1, characterized in that It further comprises a nonionic surfactant, wherein the amount of the nonionic surfactant is 0.01-0.1 v / v% of the total volume of the preparation.

9. The pharmaceutical preparation according to claim 8, characterized in that The nonionic surfactant is at least one selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40.

10. The pharmaceutical preparation according to claim 1, characterized in that The plasma protein is ADAMTS13 (disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) protein, a variant thereof, or a functional fragment thereof.

11. The pharmaceutical preparation according to claim 10, characterized in that The variant of the ADAMTS13 protein comprises a substitution of at least one amino acid residue selected from the residues at the following positions: 85, 93, 126, 135, 278, 282, 308, 314, 317, 334, 364, 376, 413, 427, 452, 465, 567, 578, 585, 589, 607, 608, 609, 612, 618, 624, 630, 635, 643, 650, 651, 654, 655, 656, 658, 664, 672 of SEQ ID NO:

1.

12. The pharmaceutical preparation according to claim 11, characterized in that The variant of the ADAMTS13 protein is selected from the following group of variant proteins, which contain substitutions of amino acid residues at the following positions: position 85 and 317, position 612, two or more of position 282, 465 and 672, position 635, position 452 and 612, two or more of position 278, 334 and 427, position 618, position 135, two or more of position 126, 567 and 651, position 413, position 334, position 314, two or more of positions 93, 364 and 376, position 308, position 656, position 607, positions 612 and 624, position 589, positions 650 and 656, position 643, positions 585 and 658, two or more of positions 630, 654 and 664, four or more of positions 589, 608, 609, 624 and 655, position 578, position 585, positions 314 and 635, and positions 314 and 612.

13. The pharmaceutical preparation according to claim 11, characterized in that The amino acid residues are substituted with at least one selected from the group consisting of: position 85 is substituted with Phe, position 93 is substituted with Val, position 126 is substituted with Met, position 135 is substituted with Ile, position 278 is substituted with Ile, position 282 is substituted with Ala, position 308 is substituted with Lys, position 314 is substituted with Thr, position 317 is substituted with His, position 334 is substituted with Thr or Val, position 364 is substituted with Arg, position 376 is substituted with Asp, position 413 is substituted with Asp, position 427 is substituted with Asn, position 452 is substituted with Ile, position 465 is substituted with Asp, position 567 is substituted with Ser, position 578 is substituted with Leu, position position 650 is substituted by His, position 651 is substituted by Asp, position 654 is substituted by Gly, position 655 is substituted by Val, position 656 is substituted by Arg or His, position 658 is substituted by His, position 664 is substituted by Asn, and position 672 is substituted by Val.

14. The pharmaceutical preparation according to claim 10, characterized in that The plasma protein binds to the Fc region derived from the IgG4 immunoglobulin.

15. The pharmaceutical preparation according to claim 14, characterized in that The Fc region comprises a substitution of at least one amino acid residue selected from the group consisting of residues at positions 22, 24, and 26 of SEQ ID NO:

2.

16. The pharmaceutical preparation according to claim 15, characterized in that The substitution of the amino acid residue is at least one selected from the following group: position 22 is substituted by Tyr, position 24 is substituted by Thr, and position 26 is substituted by Glu.

17. The pharmaceutical preparation according to claim 14, characterized in that The plasma protein further comprises a hinge region derived from IgG1 immunoglobulin between the Fc region derived from IgG4 immunoglobulin.

18. A composition for preventing or treating thrombotic diseases, characterized in that: The composition comprises the pharmaceutical preparation according to any one of claims 10 to 17 as an active ingredient.

19. The composition according to claim 18, characterized in that The thrombotic disease is thrombotic microangiopathy (TMA).

20. The composition according to claim 19, characterized in that The thrombotic microangiopathy is selected from the group consisting of thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (hemolysis, elevated liver enzymes, low platelet count), preeclampsia, and sickle cell disease.

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