Novel liquid dosage form for plasma proteins

Abstract

The present invention relates to a pharmaceutical dosage form composition for a plasma protein, specifically an ADAMTS-13 protein, and a composition for preventing or treating a thrombotic disease containing the same. The present invention significantly improves the stability of plasma proteins, which are highly at risk of denaturation and contamination from the moment of separation and purification from blood and whose pharmacological activity and quality deteriorate during long-term storage. In particular, not only is the colloidal stability, refrigeration stability, purity, and aggregation inhibition rate maintained at a high level, but also the cake properties after lyophilization are maintained well for a long time, so that the ADAMTS-13 protein, which is an important therapeutic resource used for various thrombotic diseases, can be usefully used to maintain and store it for a long time without loss of a therapeutically effective amount.

Description

Technical Field

[0001] The present invention relates to a liquid dosage form composition of a plasma protein, specifically, ADAMTS-13 protein.

Background Art

[0002] In the development of protein pharmaceuticals, in order to ensure sufficient storage stability of proteins that are vulnerable to stability, the development of an efficient lyophilized dosage form is an important issue. Before the lyophilization process starts, it is necessary to store in a liquid state for a certain period of time. In order to prevent quality degradation during this storage period, a certain degree of stability must be ensured even in the liquid dosage form.

[0003] Additives (Excipients) used in lyophilized dosage forms are classified into two types: crystalline and amorphous according to the properties of the substances. Crystalline additives play the role of a bulking agent in the lyophilized dosage form, providing mechanical strength to the lyophilized cake and making the cake stronger. Representative crystalline additives include mannitol, glycine, etc. On the other hand, amorphous additives play the role of a protein stabilizer in the lyophilized dosage form, and by being present adjacent to the protein, they play a role in stabilizing the protein from the stress generated during the lyophilization process.

[0004] On the other hand, in the case of sodium chloride mainly used in protein pharmaceutical dosage forms, it can have both amorphous and crystalline forms depending on the added concentration. Generally, when it is 150 mM or less of NaCl, it mainly exhibits amorphous properties, and when it is 200 mM or more, it is reported that most of it crystallizes.

[0005] Stabilizers in the form of lyophilized desiccants protect proteins from stress generated during the lyophilization process, stabilize them, and at the same time contribute to the stabilization of proteins during storage after drying and reconstitution. Therefore, in particular, in protein formulations intended for long-term storage by lyophilization, searching for optimal stabilizer components and their optimal content ratios is an important issue that is no less important than discovering new pharmacological components in terms of the value and utility of proteins as therapeutic resources.

[0006] Numerous papers and patent documents are referred to throughout this specification, and their incorporation is indicated. The disclosures of the incorporated papers and patent documents are hereby incorporated by reference in their entirety into this specification to more clearly explain the level in the technical field to which the present invention pertains and the content of the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The inventors have made intensive research efforts to develop an excellent liquid dosage form composition that can improve the stability of plasma proteins, which are at high risk of contamination and denaturation from the moment of separation and purification from blood, and in particular, can maintain their physical properties, biological activities, and pharmacological effects for a long time even during lyophilization. As a result, by containing 0.2 mg / ml to 1.2 mg / ml of plasma proteins, which are pharmacological components, in the dosage form, when 40 mM to 200 mM of an amino acid stabilizer, specifically arginine, is contained, it has been found that it exhibits significantly excellent stability in terms of various indicators such as colloidal stability, refrigerated stability, suppression of unfolding, blocking of aggregation, and cake properties after lyophilization, thus completing the present invention.

[0008] Therefore, an object of the present invention is to provide a pharmaceutical dosage form composition for plasma proteins.

[0009] Another object of the present invention is to provide a composition for preventing or treating thrombotic diseases.

[0010] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

Means for Solving the Problems

[0011] According to one aspect of the present invention, the present invention provides a pharmaceutical dosage form composition containing 0.2 mg / ml to 1.2 mg / ml of plasma protein and 40 mM to 200 mM of an amino acid stabilizer.

[0012] The inventors have improved the stability of plasma proteins, which are at high risk of contamination and denaturation from the moment of separation and purification from blood, and in particular, have developed an excellent liquid dosage form composition that can maintain its physical properties, biological activity, and pharmacological effects for a long period even during lyophilization. As a result, when the dosage form contains 0.2 mg / ml to 1.2 mg / ml of the plasma protein as a pharmacological component and 40 mM to 200 mM of the amino acid stabilizer, specifically arginine, it has been found to exhibit significantly excellent stability in terms of various indicators such as colloidal stability, refrigerated stability, suppression of unfolding, prevention of aggregation, and cake properties after lyophilization.

[0013] As used herein, the term "Plasma protein" generically refers to water-soluble proteins present in human or animal plasma, encompassing all protein components in blood other than those contained in white blood cells and red blood cells among the proteins contained in blood. Plasma proteins account for approximately 8% of whole plasma and are responsible for hemostatic functions (prothrombin, fibrinogen), hormone transport (such as serum albumin and lipid proteins), and immune functions (immunoglobulins and complement proteins). Plasma proteins can be obtained by various fractionation and purification methods known in the art, but the problems of chemical and physical instability due to long-term storage and environmental changes must be overcome. Physical instability induces deformations that do not cause covalent bond changes in proteins, namely adsorption, aggregation, and precipitation formation, and chemical instability involves deformations such as deamidation, racemization, hydrolysis, oxidation, beta-elimination, and disulfide exchange. Such instabilities lead to distortion of the inherent biological activity and reduction of the pharmacological effect.

[0014] As used herein, the term "stabilizer" means any additive added to a dosage form to increase the stability of an active ingredient and prevent the active ingredient from being arbitrarily denatured, oxidized, aggregated, or crystallized, or denatured into a flexible substance, ultimately resulting in loss or reduction of pharmacological activity, and is not overly limited as long as it is pharmaceutically acceptable. The term "amino acid stabilizer" means a stabilizer that induces the above-described stabilizing effect by adding an amino acid as a main component or an auxiliary component to a dosage form.

[0015] According to a specific embodiment of the present invention, the amino acid is one or more selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.

[0016] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases can include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium, among others.

[0017] According to specific embodiments of the present invention, the composition of the present invention contains 0.25 mg / ml to 1.0 mg / ml of plasma protein, more specifically 0.3 mg / ml to 0.7 mg / ml of plasma protein, even more specifically 0.3 mg / ml to 0.4 mg / ml of plasma protein, and most specifically approximately 0.36 mg / ml of plasma protein.

[0018] According to specific embodiments of the present invention, the composition of the present invention contains 60 mM to 180 mM of an amino acid stabilizer, more specifically 60 mM to 160 mM of an amino acid stabilizer, even more specifically 60 mM to 140 mM of an amino acid stabilizer, even more specifically 80 mM to 135 mM of an amino acid stabilizer, even more specifically 100 mM to 130 mM of an amino acid stabilizer, and most specifically approximately 120 mM of an amino acid stabilizer.

[0019] According to specific embodiments of the present invention, the composition additionally contains 0 - 1.5 w / v% of a sugar stabilizer based on the total composition.

[0020] More specifically, it additionally contains 0.2 - 1.5 w / v%, even more specifically 0.3 - 1.5 w / v%, even more specifically 0.5 - 1.5 w / v%, even more specifically 0.7 - 1.3 w / v%, even more specifically 0.9 - 1.1 w / v%, and most specifically approximately 1 w / v% of a sugar stabilizer.

[0021] According to a specific embodiment of the present invention, the sugar is one or more selected from the group consisting of sucrose, trehalose, and pharmaceutically acceptable salts thereof.

[0022] According to a specific embodiment of the present invention, the composition additionally contains 100 mM to 400 mM of inorganic salts.

[0023] As used herein, the term "inorganic salt" means a salt in which cations and anions are combined with each other by ionic bonds in an aqueous solution, and is a salt derived from an inorganic substance that does not contain a C-H bond. The inorganic salts that can be used in the present invention include, for example, NaCl, CaCl 2 , KCl, MgCl 2 , and combinations thereof, but are not limited thereto. Specifically, the inorganic salt of the present invention is a mixture of NaCl and CaCl 2 .

[0024] According to a specific embodiment of the present invention, the NaCl in the inorganic salt may be contained in an amount of 140 mM to 370 mM, more specifically 180 to 340 mM, even more specifically 220 to 310 mM, more specifically 260 to 300 mM, and most specifically about 280 mM.

[0025] According to a specific embodiment of the present invention, the CaCl 2 in the inorganic salt may be contained in an amount of 2 mM to 6 mM, more specifically 3 to 5 mM, and most specifically about 4 mM.

[0026] According to a specific embodiment of the present invention, the composition of the present invention contains 10 mM to 30 mM of histidine, more specifically 15 mM to 25 mM of histidine, and most specifically about 20 mM of histidine.

[0027] According to a specific embodiment of the present invention, the composition additionally contains a nonionic surfactant in an amount of 0.01 to 0.1 v / v% based on the total composition.

[0028] As used herein, the term "surfactant" means a soluble compound used to increase the water solubility of hydrophobic substances or to increase the miscibility of a plurality of substances having different hydrophobicities. The term "nonionic surfactant" means a surfactant that does not contain an ionizable functional group or atomic group in the whole molecule and remains undissociated and dissolved even in an aqueous solution state.

[0029] According to a specific embodiment of the present invention, the nonionic surfactant that can be used in the present invention is one or more selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40, and more specifically, it is polysorbate 80.

[0030] According to a specific embodiment of the present invention, the composition of the present invention contains a nonionic surfactant in an amount of 0.03 to 0.08 v / v%, and most specifically, about 0.05 v / v% of the nonionic surfactant.

[0031] According to a specific embodiment of the present invention, the plasma protein to which the dosage form composition of the present invention is applied is ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protein, a variant thereof, or a functional partial fragment thereof.

[0032] As used herein, the term "functional part" means a fragment in which some amino acid residues are deleted from the full-length protein, and is an analog of the full-length protein that maintains its original biological activity and function.

[0033] According to the present invention, the first sequence of the sequence list is the amino acid sequence of the ADAMTS13 protein consisting of 1427 amino acids. Therefore, the ADAMTS13 mutant protein of the present invention or a functional partial fragment thereof may be a mutant of the full-length (1427 a.a.) ADAMTS13 protein or a partial fragment thereof containing the region from positions 75 to 685 in which an amino acid mutation is introduced. The partial fragment containing the region from positions 75 to 685 may be, for example, 1-685 (685 a.a.) or 75-685 (611 a.a.).

[0034] The first sequence of the sequence list, which is an amino acid sequence essentially containing the ADAMTS13 protein of the present invention and a functional partial fragment thereof, also includes an amino acid sequence showing substantial identity with the above sequence. Substantial identity means that when the amino acid sequence and any other sequence are aligned to maximize correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art, it means an amino acid sequence showing a homology of at least 70%, specifically at least 80%, more specifically at least 90%, and most specifically at least 95%.

[0035] According to a specific embodiment of the present invention, the mutant of the ADAMTS13 protein includes substitution of one or more amino acid residues selected from the group consisting of the 85th, 93rd, 126th, 135th, 278th, 282nd, 308th, 314th, 317th, 334th, 364th, 376th, 413th, 427th, 452nd, 465th, 567th, 578th, 585th, 589th, 607th, 608th, 609th, 612th, 618th, 624th, 630th, 635th, 643rd, 650th, 651st, 654th, 655th, 656th, 658th, 664th and 672nd residues of the first sequence of the sequence list.

[0036] The inventors of the present invention have identified the core regions recognized by autoantibodies against ADAMTS13, and when substitutions are made to some of the amino acids within these regions, the binding to autoantibodies is blocked and the vWF degrading activity and thrombus inhibitory activity are maintained, thereby finding that it can be used as an effective therapeutic composition for various diseases caused by excessive thrombus formation, including thrombotic thrombocytopenic purpura (TTP).

[0037] As used herein, the term "autoantibody" refers to one of the immunoglobulin proteins that contains one or more variable domains that bind to the epitope of an antigen and specifically recognize the antigen, and is produced by an individual's own immune system to recognize and target the individual's own proteins. The presence of autoantibodies causes a decrease or loss of the inherent function or biological activity of the protein specifically recognized by the autoantibodies, thus causing various diseases.

[0038] According to a specific embodiment of the present invention, the mutant of the ADAMTS13 protein is selected from the group consisting of each mutant protein containing a substitution of an amino acid residue at the following positions: - residues 85 and 317; residue 612; two or more of residues 282, 465 and 672; residue 635; residues 452 and 612; two or more of residues 278, 334 and 427; residue 618; residue 135; two or more of residues 126, 567 and 651; residue 413; residue 334; residue 314; two or more of residues 93, 364 and 376; residue 308; residue 656; residue 607; residues 612 and 624; residue 589; residues 650 and 656; residue 643; residues 585 and 658; two or more of residues 630, 654 and 664; four or more of residues 589, 608, 609, 624 and 655; residue 578; residue 585; residues 314 and 635; and residues 314 and 612.

[0039] More specifically, the substitution of the amino acid residue is substitution of the 85th residue with Phe, substitution of the 93rd residue with Val, substitution of the 126th residue with Met, substitution of the 135th residue with Ile, substitution of the 278th residue with Ile, substitution of the 282nd residue with Ala, substitution of the 308th residue with Lys, substitution of the 314th residue with Thr, substitution of the 317th residue with His, substitution of the 334th residue with Thr or Val, substitution of the 364th residue with Arg, substitution of the 376th residue with Asp, substitution of the 413th residue with Asp, substitution of the 427th residue with Asn, substitution of the 452nd residue with Ile, substitution of the 465th residue with Asp, substitution of the 567th residue with Ser, substitution of the 578th residue with Leu, substitution of the 585th residue with Asn or Met, substitution of the 589th residue with Gln, substitution of the 607th residue with Arg, substitution of the 608th residue with Met, substitution of the 609th residue with Leu, substitution of the 612th residue with Phe or Tyr, substitution of the 618th residue with Ser, substitution of the 624th residue with Asp or Cys, substitution of the 630th residue with Leu, substitution of the 635th residue with Val, substitution of the 643rd residue with Phe, substitution of the 650th residue with His, substitution of the 651st residue with Asp, substitution of the 654th residue with Gly, substitution of the 655th residue with Val, substitution of the 656th residue with Arg or His, substitution of the 658th residue with His, substitution of the 664th residue with Asn, and substitution of the 672nd residue with Val, and is one or more selected from the group consisting of these.

[0040] According to a specific embodiment of the present invention, the plasma protein of the present invention described above has the Fc region of IgG4 immunoglobulin conjugated thereto.

[0041] When the present inventors conjugate the Fc region derived from IgG4 immunoglobulin to the ADAMTS13 mutant protein discovered in the present invention, while maintaining the inherent vWF cleavage activity and neutralizing antibody avoidance activity as they are, the in vivo stability is significantly increased. In particular, it has been found that the structural instability appearing in the open form fragment in which a part of the C-terminus of ADAMTS13 is removed is remarkably improved.

[0042] According to a more specific embodiment of the present invention, the Fc region includes substitution of one or more amino acid residues selected from the group consisting of the 22nd, 24th, and 26th residues of the second sequence in the sequence listing. More specifically, the substitution of the amino acid residues is one or more selected from the group consisting of substitution of the 22nd residue with Tyr, substitution of the 24th residue with Thr, and substitution of the 26th residue with Glu.

[0043] According to the present invention, the second sequence in the sequence listing is the Fc region (217 a.a.) derived from IgG4 immunoglobulin. The present inventors have found that when the Fc region [IgG4(YTE)] derived from IgG4 immunoglobulin in which the 22nd, 24th, and 26th residues are substituted with Tyr, Thr, and Glu, respectively, is fused to the aforementioned ADAMTS13 mutant protein or a functional partial fragment thereof, the blood half-life is maximized and the physiological activity after administration can be maintained for a long period.

[0044] According to a specific embodiment of the present invention, the hinge region of IgG1 immunoglobulin is additionally included between the plasma protein and the Fc region of IgG4 immunoglobulin.

[0045] According to the present invention, the hinge region derived from IgG1 immunoglobulin may be represented by the third sequence (15 a.a.) in the sequence listing.

[0046] According to still another aspect of the present invention, the present invention provides a composition for preventing or treating thrombotic diseases, which contains, as an active ingredient, the pharmaceutical dosage form composition of the present invention for an ADAMTS13 protein, a variant thereof, or a functional partial fragment thereof as a plasma protein.

[0047] As used herein, the term "thrombotic disease" means a systemic disease in which blood flow is reduced or blocked by a thrombus formed by platelet aggregation in the microcirculation system of blood vessels, thereby inducing ischemic injury in various organs such as the kidney, heart, and brain.

[0048] When the ADAMTS13 enzyme is inhibited in activity by a neutralizing antibody and cannot properly degrade von Willebrand factor (vWF), excessive platelet aggregation and excessive thrombus formation occur. Therefore, the ADAMTS13 mutant protein of the present invention, which maintains or improves vWF-degrading activity while efficiently avoiding neutralizing antibodies, can be used as an efficient preventive or therapeutic composition for various thrombotic diseases.

[0049] As used herein, the term "prevention" means suppressing the occurrence of a disease or disorder in a subject who has not been diagnosed as having the disease or disorder but is at risk of developing such a disease or disorder.

[0050] As used herein, the term "treatment" means (a) suppressing the development of a disease, disorder or symptom; (b) alleviating a disease, disorder or symptom; or (c) eliminating a disease, disorder or symptom. When the composition of the present invention is administered to a subject, it specifically recognizes and degrades vWF regardless of the presence or absence of neutralizing antibodies, thereby blocking the formation of excessive thrombi, and plays a role in suppressing the progression of thrombotic diseases, or removing or alleviating them. Therefore, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological components and applied as a therapeutic adjuvant for treating the diseases. For this reason, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant agent".

[0051] As used herein, the term "administer" or "administering" means directly administering a therapeutically effective amount of the composition of the present invention to a subject such that the same amount is formed in the subject's body.

[0052] In the present invention, the term "therapeutically effective amount" means the content of a composition in which the pharmacological component in the composition is contained to such an extent that it provides a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and thus includes the meaning of "prophylactically effective amount".

[0053] As used herein, the term "subject" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or macaques. Specifically, the subject of the present invention is a human.

[0054] According to a specific embodiment of the present invention, the thrombotic disease is thrombotic microangiopathy (TMA). More 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, and even more specifically, it is thrombocytopenic purpura or sickle cell disease, and most specifically, it is thrombocytopenic purpura.

[0055] According to still another aspect of the present invention, the present invention provides a method for preventing or treating a thrombotic disease, which includes the step of administering to a subject the pharmaceutical dosage form composition of the present invention as described above against ADAMTS13 protein, its variant or a functional partial fragment thereof as a plasma protein.

[0056] Since the plasma proteins used in the present invention, their dosage form components, and the thrombotic diseases that can be prevented or treated thereby have already been described above, the description thereof is omitted to avoid excessive duplication.

Advantages of the Invention

[0057] Summarizing the features and advantages of the present invention, they are as follows: (a) The present invention provides a pharmaceutical dosage form composition for a plasma protein, specifically, an ADAMTS-13 protein, and a composition for preventing or treating a thrombotic disease containing the same. (b) The present invention significantly improves the stability of plasma proteins, which are highly at risk of denaturation and contamination from the moment of separation and purification from blood and whose pharmacological activity and quality deteriorate during long-term storage. In particular, it not only maintains high levels of colloidal stability, refrigerated stability, purity, and aggregation inhibition rate, but also the cake properties after lyophilization remain good for a long time, making it useful for maintaining and storing the ADAMTS-13 protein, an important therapeutic resource used for various thrombotic diseases, without loss of therapeutically effective amounts for a long time.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0059] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for explaining the present invention more specifically, and it will be obvious to those with ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0060] Example

Example

[0061] Example 1: Selection of Formulation Components of ADAMTS13 Protein The components and contents of the liquid formulation composition for ADAMTS13 protein described later in Example 3 below were determined by the following process.

[0062] Freeze-drying process Each sample solution after the completion of formulation was dispensed into glass vials (3 ml) at 1.0 ml each. After half-capping with a rubber stopper, it was loaded onto the shelf of a freeze dryer (Freeze dryer, Lyostar3, SP scientific). Thereafter, freeze-drying was carried out under the conditions shown in Table 1 below. The manufactured freeze-dried preparation was capped with an aluminum cap after the completion of freeze-drying.

[0063]

Table 1

[0064] The stability of the manufactured freeze-dried preparation was analyzed after reconstitution with 1.0 ml of distilled water.

[0065] Size-exclusion liquid chromatography (SE-HPLC) To perform size exclusion chromatography, the sample was diluted to 1.0 mg / ml with the mobile phase (1xPBS, Lonza) (if it was below 1.0 mg / ml, it proceeded without dilution), sterilization filtered, and after injecting 200 μL of the filtered sample into a vial insert, it was inserted into a screw top vial for preparation. Subsequently, after connecting the mobile phase to the pump, the mobile phase was flowed at a flow rate of 0.5 mL / min through Waters e2695 and Waters 2489 instruments (Waters Japan), and an analytical column (TSKgel G3000SWXL, Tosoh) was installed. The mobile phase was flowed at a speed of 0.5 mL / min for more than 30 minutes until the detector signal stabilized to reach equilibrium. When the temperature of the autosampler dropped to 4°C, the sample was inserted into the sampler. After injecting 30 μL of the sample, it was flowed with the mobile phase for 35 minutes, and the detection peak was confirmed at 280 nm. Subsequently, analysis was performed using the Empower Pro software on the PC.

[0066] Colloidal stability (B 22 ) Since the degree of protein-protein interaction affects aggregation and solubility, etc., colloidal stability is an important item that must be considered in the development of protein dosage forms. Using B 22 (second virial coefficient) as a representative colloidal stability index, generally when the B 22 value shows a large positive value, the repulsive force between proteins is strong and the probability of aggregation occurring decreases.

[0067] To evaluate the colloidal stability against five stabilizers, as a common composition, a composition with pH 7.4 containing 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl 2 , 120 mM of NaCl, and 1.0% sucrose was included with 100 mM each of glycine, lysine, proline, alanine, or arginine as amino acids.

[0068] Liquid samples of each composition with completed dosage form were analyzed for colloidal stability (B 22 ) using the UNCLE device.

[0069] B 22 The larger the positive value of the B value, the better the protein is dispersed without aggregating with each other. As shown in Figure 1, it was confirmed that the colloidal stability is in the order of arginine > glycine > proline > alanine > lysine > control group.

[0070] Turbidity analysis Turbidity was analyzed using Lunatic (Unchained Labs). 2.0 μL of the sample was injected into a Lunatic plate (Unchained Labs), the turbidity at 350 nm was measured, and the turbidity of the placebo buffer was subtracted from the turbidity value of the sample to calculate the final value.

[0071] Thermal unfolding and thermal aggregation analysis When the protein is unfolded due to an increase in temperature (unfolding), the degree of protein unfolding can be measured by detecting the emission wavelength of tryptophan exposed on the surface. For differential scanning fluorimetry analysis based on such intrinsic fluorescence intensity, the UNCLE (Unchained Labs) device was used. To analyze the thermal stability of the protein using the said device, Tm (Thermal unfolding) and Tagg (Thermal aggregation) were measured as follows:

[0072] After injecting 8.8 μL of the sample into the Uni Sample Loader (Unchained Labs) twice repeatedly, the temperature was increased from 25 °C to 95 °C at a rate of 1 °C / min. While increasing the temperature, the intensity of the wavelength (250 - 720 nm) emitted by the excitation wavelength of 266 nm was measured. Fluorescence data analysis was performed using UNCLE Analysis software (Unchained Labs).

[0073] By the above analysis, the temperature at the time of the maximum value of the fluorescence emission peak was defined as Tm, and the static light scattering of the protein at 266 nm (SLS266) was measured to define the temperature at the time when protein aggregation starts (protein aggregation onset temperature) as Tagg.

[0074] SE-HPLC evaluation for 7 stabilizers As a common composition, 0.9 mg / ml of ADAMTS protein, 20 mM histidine, 2.0 mM of CaCl 2 and 120 mM of NaCl were included in a composition of pH 7.4, and 100 mM each of arginine, serine, valine, threonine, proline or glycine was included as an amino acid stabilizer, or 1.0 w / v% of sucrose was included as a sugar stabilizer.

[0075] After storing the liquid samples of each composition after dosage form formation at room temperature for 7 hours, SE-HPLC analysis was performed. As a result, as shown in Figure 2, the liquid stability by the 7 stabilizers for which the comparative experiment was carried out was also the best for arginine, and the stability and purity were in the order of arginine > sucrose > proline > glycine > valine, threonine > control group > serine.

[0076] Liquid stability evaluation by addition of arginine To evaluate the liquid stability of a composition at pH 7.4 containing 0.049 mg / mL of ADAMTS protein, 15 mM sodium phosphate, and 50 mM NaCl with or without the addition of 20 mM arginine, after storing the formulated liquid samples at room temperature for 3 hours and 19 hours, SE-HPLC analysis was performed at each analysis time point. As a result, as shown in Figure 3, it was confirmed that when 20 mM arginine was added, the refrigerated stability at 5°C was significantly improved compared to the control group.

[0077] Evaluation of Liquid Stability According to the Concentration of the Target Protein and the Concentration of Arginine SE-HPLC analysis As a common composition, 20 mM histidine, 4.0 mM CaCl 2 and 160 mM NaCl were included in a composition at pH 7.4, and liquid samples containing 0.2, 0.6, and 1.0 mg / ml of ADAMTS protein and 20, 80, and 140 mM of arginine were evaluated for stability.

[0078] After storing the formulated samples at room temperature for 18 hours and performing SE-HPLC analysis, as shown in Figure 4A, it was confirmed that the lower the concentration of the target protein and the higher the concentration of arginine, the more the purity was improved.

[0079] DOE design On the other hand, in order to more precisely explore the optimal concentrations of arginine and the target protein, DOE (Design Of Experiments) was performed. After setting two operating parameters, the concentration of arginine and the concentration of ADAMTS protein, as factors (X values), and purity (SE-HPLC) as the response (Y value), a DOE design was implemented using the JMP (registered trademark) 10.0 statistical program. Specifically, using RSM (Response Surface Model), the axial points were set in the CCD (Central Composite Design) type. Finally, a total of 9 Run conditions including "2-level full factorial design 4 Runs + center point 1 Run + axial points for 2 factors 4 Runs" were designed.

[0080] The stock sample solution (1.1 mg / mL) was added with arginine according to the DOE conditions, diluted, left standing at room temperature (about 15 - 25 °C) for 18 hours, and then subjected to SE-HPLC analysis.

[0081] The DOE statistical analysis was performed by multiple regression analysis using stepwise regression, and the model establishment followed the response surface modeling method. The analysis used the Fit model method in JMP, and the effects used in the model included two main effects, second-order interactions, and the squared terms of each main effect. The stopping rule for removing non-significant factors and selecting significant terms used the P-value threshold (P-value ≤ 0.25, direction: Mixed).

[0082] As a result of the analysis, the R square of the model equation was 0.96, and the adjusted R square was 0.94. The P-value of the ANOVA (analysis of variance) of the model equation was 0.0006, which was smaller than the significance level of 0.05, and the significance of this model could be confirmed.

[0083] The factors determined to be significant with P-values lower than the significance level α = 0.05 were the concentration of the protein, which is a main effect, and the concentration of arginine, which is a main effect. The P-value for the curvature of arginine * arginine was 0.0765, showing a value close to the significance level α = 0.05 (Figure 4B).

[0084] As a result of the prediction profiler analysis, it was found that the monomer % value was the highest around 120 mM of arginine, and 100 - 140 mM was the optimal concentration of arginine (Figure 4C).

[0085] Quality evaluation of the lyophilized composition according to the concentration of NaCl As a common composition, 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl2 NaCl was added to the composition at pH 7.4 containing 0.05% PS80 at concentrations of 50, 100, 120, 150, 200 and 300 mM. The samples with completed formulation were lyophilized to observe the cake properties and subjected to SE-HPLC analysis. As a result, as the concentration of NaCl increased, stronger and better cake properties were shown (Figure 5A), while no remarkable purity difference due to the concentration of NaCl was observed in the SE-HPLC results (Figure 5B).

[0086] Evaluation of liquid stability according to the concentration of NaCl NaCl was added to the composition at pH 7.4 containing 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 and 20 mM arginine at concentrations of 0, 40, 80, 120 and 160 mM. The liquid samples with completed formulation were stored at room temperature, and SE-HPLC analysis was performed at 4, 8 and 12 hours. As a result, as shown in Figure 6, it was confirmed that the higher the concentration of NaCl, the better the liquid stability.

[0087] Quality evaluation of lyophilized composition according to the concentration of sugar (NaCl 120 mM) Sucrose or trehalose at concentrations of 0.0, 0.5, 1.0 and 2.0% (w / v) or trehalose at a concentration of 1.0% (w / v) was added to the composition at pH 7.4 containing 1.2 mg / ml of ADAMTS protein, 120 mM of NaCl, 20 mM histidine, 4.0 mM CaCl 2 and 0.05% PS80. The samples with completed formulation were lyophilized to observe the cake properties and subjected to SE-HPLC analysis. As a result, at NaCl 120 mM, no collapse occurred when 1.0% sucrose was added, but collapse occurred when 2.0% sucrose was added, and the concentration of sucrose at which collapse started was predicted to be between 1.0 - 2.0% (Figure 7A). The formulation with 1.0% trehalose additive showed properties similar to those of the formulation with 1.0% sucrose (Figure 7A). On the other hand, as a result of SE-HPLC, no significant purity difference due to the concentration of the added sugar was observed, and it was found that the purity was maintained even when collapse occurred.

[0088] Quality evaluation of freeze-dried compositions based on the content ratio of sucrose and a bulking agent (NaCl 120 mM) As a common composition, 1.2 mg / ml of ADAMTS protein, 120 mM of NaCl , 20 mM histidine, 4.0 mM of CaCl 2 To a composition at pH 7.4 containing 0.05% PS80, sucrose and a bulking agent (mannitol, glycine) were added at 1.0 / 3.0 (w / v) (content ratio 1:3) and 2.0 / 2.0% (w / v) (content ratio 1:1), respectively. The samples after formulation were freeze-dried to observe the cake properties, and as a result of SE-HPLC analysis, when NaCl was fixed at 120 mM and the content ratio of the bulking agent to sucrose was added at 1:3 and 2:2, collapse occurred (Figure 8A), and as a result of SE-HPLC, no significant difference was observed among the samples (Figure 8B).

[0089] Quality evaluation of freeze-dried products based on the concentration of glycine (NaCl 120 mM) As a common composition, 1.2 mg / ml of ADAMTS protein, 120 mM of NaCl , 1.0% sucrose, 20 mM histidine, 4.0 mM of CaCl 2 To a composition at pH 7.4 containing 0.05% PS80, glycine was added at concentrations of 0, 20, 40, 60, 80, and 100 mM. The samples after formulation were freeze-dried to observe the cake properties, and as a result of SE-HPLC analysis, when NaCl 120 mM and sucrose 1.0% were fixedly added, increasing the concentration of glycine had a negative impact on the cake properties, and it was found that complete collapse occurred when glycine was added at 60 mM or more (Figure 9).

[0090] Quality evaluation of freeze-dried products based on the concentration of arginine (NaCl 120 mM) As a common composition, 1.2 mg / ml of ADAMTS protein, 120 mM of NaCl , 1.0% sucrose, 20 mM histidine, 4.0 mM of CaCl 2Arginine was added to a composition at pH 7.4 containing 0.05% PS80 at concentrations of 0, 20, 60, and 100 mM. The samples that had been formulated were lyophilized to observe the cake properties, and as a result of performing SE-HPLC analysis, 120 mM NaCl and 1.0% sucrose were fixedly added, and increasing the concentration of arginine had a negative effect on the cake properties, and it completely collapsed when 60 mM or more of arginine was added (Figure 10).

[0091] Quality evaluation of lyophilized products based on the content ratio of NaCl and sucrose (without arginine) As a common composition, 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 NaCl was added to a composition at pH 7.4 containing 0.05% PS80 at concentrations of 120, 160, and 200 mM, and sucrose was added at concentrations of 1.0, 1.5, and 2.0% (w / v). The samples that had been formulated were lyophilized to observe the cake properties, and as a result of performing SE-HPLC analysis, the higher the concentration of NaCl and the lower the concentration of sucrose, the better the cake properties were shown. Specifically, i) when 120 mM NaCl was added, the collapse start point was within the range of 1.0 - 1.5% sucrose, ii) when 160 mM NaCl was added, in the section of 1.5 - 2.0% sucrose, and iii) when 200 mM NaCl was added, it was confirmed that the collapse started in the section exceeding 2.0% sucrose (Figure 11A).

[0092] On the other hand, as a result of SE-HPLC, no significant change was observed depending on the content ratio of NaCl and sucrose when arginine was not added (Figure 11B).

[0093] Quality evaluation of lyophilized products based on the content ratio of NaCl and sucrose (120 mM arginine) As a common composition, 0.5 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 0.05% PS80 and 120 mM arginineTo a composition with a pH of 7.4 containing [substance], NaCl was added at concentrations of 160, 200, 240, and 280 mM, and sucrose was added at concentrations of 0.0, 0.5, and 1.0% (w / v). The samples that had completed formulation were lyophilized to observe the cake properties, and as a result of SE-HPLC analysis, when arginine was added at a fixed concentration of 120 mM, the stronger the cake properties appeared as the concentration of NaCl increased and the concentration of sucrose decreased. Specifically, i) when adding 160 mM NaCl, in the addition range of 0.0 - 0.5% or more of sucrose; ii) when adding 200 mM NaCl, in the addition range exceeding 1.0% of sucrose; iii) when adding 240 mM NaCl, in the addition range exceeding 1.0% of sucrose; iv) when adding 280 mM NaCl, it was found that there was a disintegration start point in the addition range exceeding 1.0% of sucrose respectively (Figure 12A).

[0094] On the other hand, as a result of SE-HPLC, when 120 mM of arginine was included, no notable purity change due to the concentrations of NaCl and sucrose was confirmed. However, in the formulation with 160 mM NaCl and no added sucrose, the purity was 97.4%, showing relatively lower results compared to other samples (Figure 12B).

[0095] Evaluation of lyophilization stability according to the mixing ratio of sucrose to NaCl (arginine 120 mM) As a common composition, 0.5 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl 2 , 0.05% PS80 and 120 mM arginine To a composition with a pH of 7.4 containing [substance], NaCl was added at concentrations of 200, 240, and 280 mM, and sucrose was added at concentrations of 0.0, 0.5, and 1.0% (w / v). The samples that had completed formulation were lyophilized to evaluate the accelerated stability (40 °C). As a result, in the presence of 120 mM arginine, the purity at the first month at 40 °C did not show a significant change compared to the initial value depending on the mixing ratio of sucrose to NaCl (Figure 13).

[0096] Prevention of agitation-induced aggregation by the concentration of polysorbate 80 As a common composition, 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl 2 , 120 mM of NaCl and 1.0% sucrose in a composition of pH 7.4 were each added with polysorbate 80 at concentrations of 0.0, 0.001, 0.005, 0.01, 0.05 and 0.1% (v / v). After the liquid samples with completed formulation were vortexed to generate artificial shear stress, analysis (properties, turbidity, SE-HPLC) was performed, and sampling and analysis were carried out at 30 seconds and 2 minutes after the start of stirring. As a result of the property analysis after 2 minutes of stirring, it was confirmed that the sample became clear and transparent when 0.005% or more of polysorbate 80 was added (Figure 14A).

[0097] On the other hand, when 0.005% or more of polysorbate 80 was added, it was found that no agitation-induced aggregation occurred by confirming that there was no change in turbidity compared to the initial stage (Figure 14B). At the same time, as a result of SE-HPLC, it was confirmed that when 0.05% or more of polysorbate 80 was added, a purity decrease of around 5% occurred compared to the initial stage (Figure 14C).

[0098] Evaluation of liquid stability according to the concentration of polysorbate 80 As a common composition, 0.5 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl 2 , 120 mM of NaCl and 1.0% sucrose in a composition of pH 7.4 were added with polysorbate 80 at concentrations of 0.0, 0.005, 0.01, 0.05 and 0.09% (v / v). After the liquid samples with completed formulation were stored at room temperature for 6 hours and then subjected to SE-HPLC analysis, it was confirmed that as the concentration of polysorbate 80 increased, the liquid stability (purity) decreased slightly (Figure 15).

[0099] Evaluation of lyophilized product quality according to the concentration of polysorbate 80 As a common composition, 0.5 mg / ml of ADAMTS protein, 20 mM histidine, 4.0 mM of CaCl 2, Polysorbate 80 was added to a composition with a pH of 7.4 containing 120 mM NaCl and 1.0% sucrose at concentrations of 0.0, 0.005, 0.01, 0.05, and 0.09% (v / v). After freeze-drying the liquid samples after the formulation was completed and performing SE-HPLC analysis, as the concentration of Polysorbate 80 increased, the effect of preventing purity loss during the freeze-drying process became greater, and a recovery of 94% or more of the monomer was confirmed when added at 0.01% or more (Figure 16A). At the same time, as the concentration of Polysorbate 80 increased, the formation of higher-order aggregates was efficiently blocked (Figure 16B).

[0100] Thermal unfolding and heat-aggregation evaluation by pH As a common composition, 20 mM histidine, 4 mM CaCl 2 and 0.05% PS80 were included. After preparing different diafiltration buffers in advance at pH 6.0, 6.5, 7.0, 7.2, and 7.4 only, samples were prepared through a buffer exchange process with a solution containing the ADAMTS protein. Using Amicon® Ultra-15 Centrifugal Filter Units 30K, centrifugation was performed at 3000 rpm under refrigerated conditions.

[0101] The common composition of the samples after the buffer exchange was 10 mg / ml ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 and 0.05% PS80, and the pH was 6.0, 6.5, 7.0, 7.2, and 7.4 respectively.

[0102] The liquid samples after the formulation was completed were used with the UNCLE device to measure T m and T aggAs a result of the analysis, it was confirmed that the Tm (thermal unfolding) value, which is proportional to the structural thermal stability of the protein, increases as the pH increases, and shows a lower Tm value at pH 6.0 compared to other experimental groups (Figure 17A). In the case of thermal aggregation (T agg ), which reflects the degree of unstable aggregation occurrence, it was confirmed that the T agg value increases as the pH increases, and shows a lower T agg value at pH 6.0 and pH 6.5 compared to other experimental groups (Figure 17B).

[0103] Quality evaluation of freeze-dried products according to the concentration of CaCl 2 1.2 mg / ml of ADAMTS protein, 20 mM histidine, 120 mM NaCl, 1.0% sucrose and 0.05% PS80 were included as common components in a composition with pH 7.4, and CaCl at concentrations of 2.0, 4.0 and 8.0 mM were added. The samples after formulation were freeze-dried and subjected to SE-HPLC analysis. As a result, they showed a partially disintegrated property regardless of the concentration of CaCl 2 (Figure 18A). As a result of SE-HPLC analysis, it was confirmed that the purity was improved as the concentration of CaCl 2 increased (Figure 18B). 2

Example

Example

[0104] Example 2: Further optimization of the formulation components of ADAMTS13 protein Quality evaluation of freeze-dried products according to the mixing ratio of sucrose to NaCl 0.36 mg / ml of ADAMTS protein, 20 mM histidine, CaCl 2To a composition at pH 7.4 containing 4 mM, 120 mM L-Arg and 0.05% PS80, NaCl was added at concentrations of 100, 150, 200, 250, 300, 350 and 400 mM, and sucrose was added at concentrations of 0, 0.5, 1 and 1.5% (w / v). The samples with completed formulation were lyophilized to observe the cake properties, and the purity before and after the process was analyzed by SE-HPLC. As a result, as shown in Table 2 below, no significant purity change was observed due to the content ratio of NaCl and sucrose, and it was found that excellent purity was maintained in all the ranges tested.

[0105]

Table 2

[0106] However, as shown in FIGS. 19A and 19B, it was confirmed that as the concentration of NaCl increased and the concentration of sucrose decreased, better cake properties were shown.

[0107] Quality evaluation of lyophilized products by high-concentration treatment of sucrose As a common composition, 0.36 mg / ml of ADAMTS protein, 20 mM histidine, CaCl 2 To a composition at pH 7.4 containing 4 mM, 120 mM L-Arg and 0.05% PS80, NaCl was added at concentrations of 200, 250 and 300 mM, and sucrose was added at concentrations of 1.5 and 2.5%. The samples with completed formulation were lyophilized to observe the cake properties. As a result, as shown in FIG. 20, it was confirmed that as the concentration of NaCl increased and the concentration of sucrose decreased, better cake properties were shown.

[0108] Evaluation of colloidal stability according to the concentration of arginine (B 22 , kD) As a common composition, 0.36 mg / ml of ADAMTS protein, 20 mM histidine, CaCl 2Arginine was added to a pH 7.4 composition containing 4 mM, 280 mM NaCl, 1% sucrose and 0.05% PS80 at concentrations of 40, 80, 120, 160 and 200 mM. The formulated liquid samples were analyzed for colloidal stability (B 22 , kD) using the UNCLE instrument. B 22 The larger the positive value of B

[0109] and kD, the better the dispersion without aggregation between proteins. However, as shown in Figure 21, it was confirmed that the closer to 120 mM arginine, the better the colloidal stability was exhibited. Quality evaluation of lyophilized products by arginine concentration 2 Arginine was added to a pH 7.4 composition containing 0.36 mg / ml ADAMTS protein, 20 mM histidine, CaCl

[0110] Storage time and temperature stability of the final liquid formulation of GC1126A 0.36 mg / ml ADAMTS protein, 20 mM histidine, CaCl 2 4 mM, 120 mM L-Arg, 280 mM NaCl, 1% Sucrose and 0.05% PS80. After the formulated liquid samples were stored at room temperature (25 °C) and refrigerated (4 °C) respectively, changes over time were confirmed by analyzing purity (SE-HPLC), titer (activity of ADAMTS13), protein concentration (UV) and turbidity. As a result, in the case of purity, a purity decrease of about 4% was shown compared to the initial stage from the third day of storage at room temperature, and a purity decrease of about 15% was shown compared to the initial stage on the seventh day of storage, and no change over time was observed until the seventh day of refrigerated storage (Figure 23A).

[0111] As a result of observing the titer change, on the 7th day of storage at room temperature, a titer decrease of about 50% was shown compared to the initial stage. When considering the titer analysis deviation, no significant change over time was observed until the 7th day of storage in the refrigerator (Figure 23B).

[0112] In the case of the protein concentration, from the 3rd day of storage at room temperature, the protein concentration increased compared to the initial stage. This is judged to be the result of an increase in UV absorbance due to an increase in the opacity of the sample caused by particle aggregation. No change over time was observed until the 7th day of storage in the refrigerator (Figure 23C).

[0113] On the other hand, the turbidity of the final liquid dosage form showed an increasing trend from the 3rd day of storage at room temperature, but no change over time was observed until the 7th day of storage in the refrigerator (Figure 23D).

[0114] Optimization of the concentrations of NaCl and sucrose As a common composition, 0.5 mg / ml of ADAMTS protein, 20 mM of histidine, CaCl 2 4 mM, 120 mM of L-Arg and 0.05% of PS80 were added to the composition at pH 7.4 with NaCl at concentrations of 200, 240 and 280 mM and sucrose at concentrations of 0, 0.5 and 1%.

[0115] In order to explore the optimal concentrations of NaCl and sucrose, DOE (Design Of Experiments) was conducted. After setting two operating parameters, the concentrations of NaCl and sucrose, as factors (X values), and purity (SE-HPLC) and titer (activity of ADAMTS13) as responses (Y values), the DOE design was implemented using the JMP (registered trademark) 10.0 statistical program. For the experimental design, the full factorial design method was used, including two center points. Finally, a total of six conditions were designed, including "2-level full factorial design 4 Runs + center point 2 Runs".

[0116]

Table 3

[0117] The dosage form buffer solution was prepared according to pre-designed conditions and mixed with the stock solution to prepare the final stock solution. The prepared dosage form was dispensed into 3-ml vials at 1 ml each and lyophilized. The lyophilized specimens were stored refrigerated (5 °C), and quality analysis was performed after 6 months.

[0118] The DOE statistical analysis was carried out by performing multiple regression analysis using stepwise regression. The analysis was performed using the "Fit model" method of JMP, and the effects used in the model included two main effects, second-order interactions, and the square terms of each main effect. A stopping rule for removing non-significant factors and selecting significant terms was used with a P-value threshold (P-value ≤ 0.25, direction: Mixed).

[0119] As a result of the analysis, good purity was shown for 6 months under all experimental conditions, and no purity difference due to the concentrations of NaCl and sucrose was confirmed (Figure 24A). Along with this, the titer after 6 months compared to the initial titer of each experimental group was within the analysis deviation and showed no significant difference (Figure 24B).

[0120] Example 3: Preparation of ADAMTS13 protein variants The inventors attempted to prepare a human ADAMTS13 variant that can efficiently avoid the ADAMTS13 neutralizing antibody possessed by aTTP patients using the random mutagenesis method. To confirm whether variants having mutations in the MDTCS portion or the S domain can avoid the ADAMTS13 neutralizing antibody, Fabs were prepared using the HuCAL system of Bio-Rad for antibodies that recognize different epitopes for human ADAMTS13, and 16 types with excellent binding ability to human ADAMTS13 were selected. As shown in Figure 25C, the binding regions of each antibody are such that Ab4-16 specifically binds to the S domain, Ab67 to the MDTCS portion, and Ab66 to the C-terminal portion (TSP-2 to TSP-8 domains).

[0121] The evaluation of mutants was performed by analyzing the relative results against non-mutated or silent mutants (hereinafter, wild-type clones) having the same amino acid sequence as wild-type ADAMTS13 among the mutants created by generating wild-type ADAMTS13 constructs and random mutations. The activity and the binding ability to Ab4-16 and Ab67 were analyzed for 59 wild-type clones in total, and the analysis results of each wild-type clone were relativized based on the result value of the wild-type ADAMTS13 construct and shown in FIG. 26. As shown in FIG. 26, the avoidance rates and relative activities against Ab4-16 and Ab67 antibodies for wild-type ADAMTS13 (59 types) containing conservative amino acid substitutions, mutated ADAMTS13 (304 types), and selected ADAMTS13 (26 types) were shown. Although the wild-type clones had no mutations, they showed a difference in relative results from the result value of the wild-type ADAMTS13 construct. Among the 304 mutated ADAMTS13, 26 mutated ADAMTS13 having values above a specific numerical avoidance rate or relative activity were selected as shown in Table 4 below.

[0122] Specifically, the avoidance rate of Ab4-16 for mutated ADAMTS13 showed a range of -29.5% to 33.4%, the avoidance rate of Ab67 showed a range of -24.4% to 30.5%, and the relative activity showed a range of 62.7% to 128.9% (Table 4). As a result of applying the normal distribution using the three-sigma rule, it was predicted that almost all the results of the WT clones were within this distribution range in the case of the avoidance rate of Ab4-16 in the range of -34.1% to 38.5%, the avoidance rate of Ab67 in the range of -38.5% to 42.3%, and the relative activity in the range of 47.0% to 145.2%. Therefore, in the selection of mutants, the avoidance rate of Ab4-16 exceeding 38.5%, the avoidance rate of Ab67 exceeding 42.3%, or the relative activity of 47.0% or more was applied as the selection criteria based on the maximum value of three-sigma.

[0123]

Table 4

[0124] Selection of mutants that avoid ADAMTS13-neutralizing antibodies and have activity above the level of wild-type ADAMTS13 After transfecting cells with WT clones and mutants having amino acid mutations, the amount of protein present in the culture medium was measured, and neutralizing antibody binding and activity assays were performed on 304 mutants with an expression concentration of 50 ng / mL or higher. It was found that mutants with amino acid mutations had diverse distributions in terms of neutralizing antibody binding or relative activity compared to the WT clones. Among these, 26 mutants that satisfied the selection criteria were finally selected. It was confirmed that 18 of the 26 mutants had mutations in the S domain. In particular, 13 of the mutants with mutations in the S domain (1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02, 8C04, 8D01, 8F01) had a high avoidance rate for Ab4-16, and 5 (7E01, 7G08, 8C02, 8D01, 8D05) showed the characteristic of having high relative activity (Table 5). Six mutants had mutations in the D domain, and among these, 5 (46, 3A06, 4C07, 4E11, 4H07) showed the characteristic of having a high avoidance rate for Ab67. In addition, 6 mutants with mutations in the M domain, 2 in the C domain, and 2 in the T domain were confirmed. The repeatability of the results was confirmed by performing three repeated experiments on the 26 selected mutants, and 12 mutants that continuously maintained superiority in repeated tests compared to the WT clone were selected for in vitro efficacy testing. Among the 12 mutants, 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02, and 8D01 were selected because of their excellent avoidance rate for the Ab4-16 antibody, and 4C07, 4E11, and 4H07 were selected because of their excellent avoidance rate for Ab67. Finally, 8D05 was selected based on its excellent relative activity.

[0125] In order to confirm the ability to evade additional neutralizing antibodies in the selected 12 mutants, in addition to the Ab4-16 and Ab67 antibodies used in the screening, we also tried to confirm whether it was possible to evade the Ab4-20, Ab60, Ab61, Ab64, and Ab65 neutralizing antibodies. Among the 12 mutants, in the case of 8 mutants 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02, 8D01 excluding 8D05 out of the 9 mutants with amino acid mutations in the S domain, they showed excellent evasion rates against Ab4-16 and Ab4-20 prepared based on the ADAMTS13 autoantibody sequence of aTTP patients, indicating that they were also excellent in the ability to evade Ab60 and Ab61. In contrast, in the case of mutants 4C07, 4E11, 4H07 with amino acid mutations in the D domain, they had an excellent evasion rate against Ab67 (Figure 27, Table 6).

[0126]

Table 5-1

[0127]

Table 5-2

[0128]

Table 6

[0129] On the other hand, in structural-functional studies, it has been reported that the MDTCS domain, in which the CUB2 domain was removed from the C-terminal TSP-2 among a total of 14 domains of ADAMTS13, has a function of a metalloprotease similar to ADAMTS13 and can cleave vWF (Shelat et al., 2005). This suggests that even the MDTCS fragment, which is not full-length ADAMTS13, may function as a therapeutic agent for TTP disease, meaning that it is in a truncated form and can evade neutralizing antibodies present in the plasma of patients that bind to the C-terminal portion.

[0130] Therefore, the selected 12 mutants were sliced into MDTCS, and DM1 and DM2 mutants having two amino acid mutations were additionally prepared by combining the selected mutant amino acid residues among them. Using the culture solution and purified solution expressed in cells, the avoidance rate and relative activity of single or mixed neutralizing antibodies were measured by the method mentioned above to try to select the final candidate substances.

[0131] As a result of measuring the binding avoidance rate and relative activity of 8 single neutralizing antibodies using the culture solution, in the cases of 2B01, 3B05, 4H07, 5G08, 8D05 and DM1, all neutralizing antibodies were avoided at a similar level (Figure 28, Table 7). In the cases of 1C03, 2B02, 5C09, 7A02, 8D01 and DM2, excellent binding avoidance rates against 3-01 and Ab60 were shown. All 6 candidates have amino acid mutations in the S domain, and except for 8D01, they commonly have mutations at the 612th amino acid. In the case of 4E11 and DM2, they have excellent binding avoidance rates against Ab67, and all have mutations at the 314th amino acid which is in the D domain. In terms of relative activity, DM1 was the lowest at 57.9%, and 1C03 was the highest at 133.1%. All candidates except the above 2 showed relative activities of 78.2 - 125.1% and showed activities similar to the MDTCS-Fc control group (Table 7). Under the condition of mixing 9 neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67) at the same ratio, in order to confirm the avoidance ability of 12 candidate mutants except 4C07 and 5C09 compared to MDTCS-Fc as the control group, 7.5 nM of the mixed neutralizing antibody and 4 nM of the expression culture solution of each mutant were mixed, and then reacted at room temperature for 1 hour to measure the residual activity. It was shown that MDTCS-Fc maintained a residual activity of 3.5%, and it was confirmed that 3B05, 4E11, 4H07, 5G08 and 8D05 had residual activities of 1.24 - 2.42%, indicating that the residual activity was lower than that of MDTCS-Fc (Figure 29). In contrast, in the cases of 1C03, 2B01, 2B02, 7A02, 8D01, DM1 and DM2, the residual activity was maintained at 7.69 - 18.81%, and it was confirmed that they were superior in the avoidance ability by the mixed neutralizing antibody compared to MDTCS-Fc (Figure 29).

[0132] To confirm the neutralizing antibody evasion ability of candidate substance variants using the purified solution, protein purification was carried out in each culture solution using the Phytip system (protein A resin), and the evasion rate of a single neutralizing antibody was confirmed using the obtained purified solution. As a result, it was confirmed that most of the variants were similar in tendency to the results evaluated in the culture solution state (Figure 30).

[0133] It was confirmed that 2B01, 5G08, 8D05, and the DM1 variant evaded more than 24.6% of all eight neutralizing antibodies. In the cases of 1C03, 2B02, 7A02, 8D01, and DM2, similar to the results of the culture solution, they showed excellent binding evasion rates against 3-01 and Ab60. In the cases of 4E11 and DM2, similar to the results of the culture solution, it was confirmed that they were excellent in the binding evasion rate against Ab67. The relative activities of each variant are shown in Figure 30 and Table 8. It was confirmed that the residual activity of MDTCS-Fc was maintained at 3.76% under the mixed neutralizing antibody condition, and the 3B05, 4E11, 4H07, and 8D05 variants were at 2.05 - 3.50%, indicating that their activities were more suppressed compared to MDTCS-Fc. In contrast, in the cases of 1C03, 2B01, 2B02, 5G08, 7A02, 8D01, DM1, and DM2, it was confirmed that 6.34 - 12.8% of the residual activity was maintained, indicating that they were excellent in the evasion ability of the mixed neutralizing antibody compared to MDTCS-Fc (Figure 31).

[0134] Based on the above results, comprehensively considering the relative activity or the evasion rate of the mixed neutralizing antibody, 1C03, 2B02, 7A02, DM2, and 5C09 which has the 612th amino acid mutation and is judged to be excellent in the evasion ability of the mixed neutralizing antibody were selected as five candidates for further research.

[0135]

Table 7

[0136]

Table 8

[0137] DNA was prepared by conjugating IgG1-YTE to the five finally selected MDTCS variants slices, and the binding avoidance ability and relative activity of eight single neutralizing antibodies were measured in the culture solution expressed in cells. As a result, 1C03, 2B02, 5C09 and 7A02 showed excellent binding avoidance rates against Ab3-01 and Ab60, and in the case of DM2, it was confirmed that they were excellent in the binding avoidance rates against Ab3-01, Ab60 and Ab67 (Figure 33). In the case of relative activity, it showed an activity similar to MDTCS-IgG1-YTE at 98.6 - 127.7% (Figure 33). Under the condition that nine neutralizing antibodies were mixed at the same ratio, in order to confirm the avoidance ability with five variant substances compared to the control MDTCS-IgG1-YTE, after mixing the mixed neutralizing antibodies (nine kinds) with the expression culture solution of each variant, they were reacted at room temperature for 1 hour and the residual activity was measured. In the case of MDTCS-IgG1-YTE, it was shown that 5.5% of the residual activity was maintained, and in the case of the five substances, the residual activity was maintained at 8.18 - 13.42%, and it was confirmed that they were excellent in the avoidance ability by neutralizing antibodies compared to the control group (Figure 34).

[0138] In the expression culture solution obtained by conjugating IgG1-YTE to the above five MDTCS variant slices, proteins were purified using the Phytip system (protein A resin), and the residual activity and specific titer of the mixed neutralizing antibody in the purified solution were measured. The concentration of the purified solution was confirmed by Fc ELISA, and it was confirmed by silver staining that the target protein was eluted. As a result of measuring the residual activity under the condition of the mixed neutralizing antibody, it was confirmed that MDTCS-IgG1-YTE was maintained at 0.4%, and it was revealed that 1C03, 2B02, 5C09, 7A02, and DM2 maintained residual activities of 5.7%, 1.7%, 8.8%, 2.6%, and 7.9%, respectively, and it was confirmed that the mixed antibody had superior avoidance ability compared to MDTCS-IgG1-YTE. As a result of measuring the specific titer, it was confirmed that the four variants except 7A02 (10,288 IU / mg) showed specific titers of 19,091 - 22,379 IU / mg similar to those of the control group (18,030 IU / mg) (Figure 35). As a result, it was confirmed that the IgG1-YTE conjugate substances of the five variant slices were superior in avoidance ability with the mixed neutralizing antibody compared to the control group, and that the four variants except 7A02 had specific titers at levels similar to those of the control group.

[0139] Evaluation of the presence or absence of an increase in the half-life of MDTCS slice variants due to Fc attachment To confirm whether the Fc(IgG1-YTE) attached to MDTCS actually brings about an increase in the half-life, pharmacokinetic analysis was performed in mice. For this purpose, MDTCS or MDTCS attached with IgG1-YTE, and four final candidate (1C03, 5C09, 7A02, DM2) mutant section proteins were administered to mice via the tail vein, and plasma was secured at each time point. Administration was carried out so that the specific activity of each substance would be 160 IU / kg based on the reference, and the activity of the substance remaining in the plasma secured at each time point was measured by an activity assay. The secured results were summarized by the naive pooled method, and pharmacokinetic analysis was performed by noncompartmental analysis using this. The half-life of MDTCS was measured to be 2.898 hours, MDTCS-IgG1-YTE was 11.51 hours, and in the case of each mutant it was 5.184 - 9.902 hours. Compared with MDTCS as the control group, the half-life was extended 1.79 - 3.97 times by the conjugation of IgG1-YTE. Not only that, the MRT (Mean Residence Time) value indicating the mean residence time of the substance in the body was also 7.189 - 11.67 hours by the conjugation of IgG1-YTE, showing an increased residence time compared to 3.743 hours (Table 9, Figure 32). As a result, it was confirmed that the IgG1-YTE fusion is effective in maintaining the activity by the blood half-life and the mean duration in the body.

[0140]

Table 9

[0141] As described above, the inventors have discovered 26 novel mutants that avoid ADAMTS13-neutralizing antibodies that bind to the MDTCS portion or the S domain, or exhibit more activity than wild-type ADAMTS13. Among these, 12 mutants with the best avoidance ability or significantly superior comparative activity against 9 neutralizing antibodies were selected, and by preparing an MDTCS fragment essential for vWF cleavage while efficiently avoiding neutralizing antibodies that bind to the C-terminus, mutants with a greatly improved avoidance rate of autoantibodies that bind to the D, C, or S domain of aTTP patients were identified. The mutants of the present invention can be usefully utilized as effective pharmacological components with improved stability and persistence of physiological activity by increasing the blood half-life through IgG1-YTE conjugation.

[0142] Confirmation results of mutant PoC in an aTTP mimic disease mouse model For the final candidate selection in the established aTTP-mimic mouse model, after administration of the control substance (MDTCS-IgG1-YTE) or the selected five mutant candidate substances (1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, 7A02-IgG1-YTE, DM2-IgG1-YTE), the neutralizing antibody avoidance rate was evaluated (Figure 36A). Among the five mutants, DM2-IgG1-YTE showed the highest residual activity of human ADAMTS13 at all administered doses of 5,000 or 7,000 IU / kg (Figure 36B). The DM2-IgG1-YTE substance, which showed the best neutralizing antibody avoidance rate, was finally selected and administered at different concentrations to confirm the ability to maintain the residual activity of human ADAMTS13 and the degree of alleviation of clinical symptoms. As a result (Figure 37A), it was confirmed that as the dose of DM2 increased, the degree of alleviation of clinical symptoms increased. In the 7000 IU / kg administration group, it was observed that the improvement in the values of platelets and LDH was relatively high with the administration of DM2-IgG1-YTE compared to MDTCS-IgG1-YTE (Figure 37B). Consistent with the improvement in the values of platelets and LDH, as a result of the human ADAMTS13 activity test, an increase in residual activity proportional to the administered dose of the control substance or candidate substance was observed (Figure 37B). From the observation results of clinical symptoms, it was confirmed that the deaths and hematuria that appeared in the aTTP-mimic mouse model tended to be alleviated by the administration of the control substance or candidate substance. In particular, in the case of the DM2-IgG1-YTE treatment group, there were no dead individuals at all administered doses, and no individuals showing hematuria symptoms were observed when administered at 7000 IU / kg or more (Figure 37C). When administered at 7000 IU / kg, the average residual activity was 0.32 IU / mL for DM2-IgG1-YTE, which was more than 9.6 times higher than 0.03 IU / mL for MDTCS-IgG1-YTE. The difference in the observation results of the above clinical symptoms was judged to be due to such a difference in residual activity. From the observation results of general blood tests, clinical chemistry tests and clinical symptoms, the administration of MDTCS-IgG1-YTE and DM2-IgG1-YTE tended to improve the clinical symptoms of aTTP that appeared in the aTTP-mimic mouse model, thereby confirming the in vivo PoC.In particular, when administered at 7000 IU / kg, it was confirmed that DM2-IgG1-YTE was superior in its improvement effect compared to MDTCS-IgG1-YTE.

[0143] Confirmation results of the mutant PoC in the cTTP disease mouse model Using DM2-IgG1-YTE, which showed the most excellent potency among the five mutant candidates in the aTTP mimic mouse model, the presence or absence of improvement in the hematological and clinical symptoms seen in TTP disease and the degree of recovery of human ADAMTS13 activity were confirmed in the cTTP mouse model (Figure 38A). It was confirmed that the improvement of platelet and LDH values occurred in a concentration-dependent manner with an increase in the administration dose of DM2-IgG1-YTE. A significant increase in platelets was observed in the DM2-IgG1-YTE 180 and 360 IU / kg administration groups compared to the control group. In particular, in the case of the 360 IU / kg administration group, it was found that it only recovered to the normal level (Figure 38B). In the case of the LDH value, it was confirmed that the LDH recovered to a level similar to that of the control group in the 180 IU / kg administration group (Figure 38B). In the case of the activity of ADAMTS13, the activity of 0.1 IU / mL or more was shown on average in the DM2-IgG1-YTE 60 IU / kg administration group, and the activity of 1.08 IU / mL was measured at the administration dose of 360 IU / kg (Figure 38B). Therefore, it was found that the DM2-IgG1-YTE mutant effectively improved the clinical symptoms and recovered the activity of ADAMTS13 in cTTP disease mice. For this reason, the present inventors selected and optimized the ADAMTS13 protein agent components of Examples 1 and 2 described above using DM2-IgG1-YTE.

[0144] As described above, specific parts of the present invention have been described in detail. However, for those with ordinary knowledge in the art, such specific descriptions are merely preferred embodiments, and thus it is obvious that the scope of the present invention is not limited. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical dosage form composition comprising plasma protein at 0.2 mg / ml to 1.2 mg / ml and an amino acid stabilizer at 40 mM to 200 mM.

2. The composition according to claim 1, wherein the amino acid is one or more selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.

3. The dosage form composition according to claim 1, wherein the composition additionally comprises a sugar stabilizer at 0 - 1.5 w / v% based on the total composition.

4. The composition according to claim 3, wherein the sugar is one or more selected from the group consisting of sucrose, trehalose, and pharmaceutically acceptable salts thereof.

5. The dosage form composition according to claim 1, wherein the composition additionally comprises inorganic salts at 100 mM to 400 mM.

6. The inorganic salt is one or more selected from the group consisting of NaCl, CaCl ２ , KCl and MgCl ２ The composition according to claim 5, characterized in that it is one or more selected from the group consisting of

7. The inorganic salt is a mixture of NaCl and CaCl ２ The composition according to claim 6, characterized in that it is a mixture of

8. The dosage form composition according to claim 1, wherein the composition additionally comprises a nonionic surfactant at 0.01 - 0.1 v / v% based on the total composition.

9. The composition according to claim 8, wherein the nonionic surfactant is one or more selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40.

10. The composition according to claim 1, wherein the plasma protein is an ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protein, a variant thereof, or a functional partial fragment thereof.

11. The composition according to claim 10, wherein the variant of the ADAMTS13 protein comprises substitution of one or more amino acid residues selected from the group consisting of the 85th, 93rd, 126th, 135th, 278th, 282nd, 308th, 314th, 317th, 334th, 364th, 376th, 413th, 427th, 452nd, 465th, 567th, 578th, 585th, 589th, 607th, 608th, 609th, 612th, 618th, 624th, 630th, 635th, 643rd, 650th, 651st, 654th, 655th, 656th, 658th, 664th, and 672nd residues of the first sequence in the sequence listing.

12. The variant of the ADAMTS13 protein is characterized by being selected from the group consisting of each mutant protein containing substitution of an amino acid residue at the following positions: - residues at positions -85 and 317; residue at position 612; two or more of residues at positions 282, 465 and 672; residue at position 635; residues at positions 452 and 612; two or more of residues at positions 278, 334 and 427; residue at position 618; residue at position 135; two or more of residues at positions 126, 567 and 651; residue at position 413; residue at position 334; residue at position 314; two or more of residues at positions 93, 364 and 376; residue at position 308; residue at position 656; residue at position 607; residues at positions 612 and 624; residue at position 589; residues at positions 650 and 656; residue at position 643; residues at positions 585 and 658; two or more of residues at positions 630, 654 and 664; four or more of residues at positions 589, 608, 609, 624 and 655; residue at position 578; residue at position 585; residues at positions 314 and 635; and residues at positions 314 and 612. **Claim 13** The substitution of the amino acid residue is one or more selected from the group consisting of substitution of the 85th residue with Phe, substitution of the 93rd residue with Val, substitution of the 126th residue with Met, substitution of the 135th residue with Ile, substitution of the 278th residue with Ile, substitution of the 282nd residue with Ala, substitution of the 308th residue with Lys, substitution of the 314th residue with Thr, substitution of the 317th residue with His, substitution of the 334th residue with Thr or Val, substitution of the 364th residue with Arg, substitution of the 376th residue with Asp, substitution of the 413th residue with Asp, substitution of the 427th residue with Asn, substitution of the 452nd residue with Ile, substitution of the 465th residue with Asp, substitution of the 567th residue with Ser, substitution of the 578th residue with Leu, substitution of the 585th residue with Asn or Met, substitution of the 589th residue with Gln, substitution of the 607th residue with Arg, substitution of the 608th residue with Met, substitution of the 609th residue with Leu, substitution of the 612th residue with Phe or Tyr, substitution of the 618th residue with Ser, substitution of the 624th residue with Asp or Cys, substitution of the 630th residue with Leu, substitution of the 635th residue with Val, substitution of the 643th residue with Phe, substitution of the 650th residue with His, substitution of the 651th residue with Asp, substitution of the 654th residue with Gly, substitution of the 655th residue with Val, substitution of the 656th residue with Arg or His, substitution of the 658th residue with His, substitution of the 664th residue with Asn, and substitution of the 672nd residue with Val, the composition according to claim 11.

14. The composition according to claim 10, wherein the plasma protein is characterized in that the Fc region of IgG4 immunoglobulin is conjugated.

15. The composition according to claim 14, wherein the Fc region comprises substitution of one or more amino acid residues selected from the group consisting of the 22nd, 24th, and 26th residues of the second sequence in the sequence listing.

16. The composition according to claim 15, wherein the substitution of the amino acid residue is one or more selected from the group consisting of substitution of the 22nd residue with Tyr, substitution of the 24th residue with Thr, and substitution of the 26th residue with Glu.

17. The composition according to claim 14, wherein a hinge region of IgG1 immunoglobulin is additionally included between the plasma protein and the Fc region of the IgG4 immunoglobulin.

18. A composition for preventing or treating a thrombotic disease, comprising, as an active ingredient, the composition according to any one of claims 10 to 17.

19. The composition according to claim 18, wherein the thrombotic disease is thrombotic microangiopathy (TMA).

20. The composition according to claim 19, wherein the thrombotic microangiopathy is selected from the group consisting of thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count), preeclampsia, and sickle cell disease.

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