WO2023219379A1 - Novel liquid formulation for lyophilization of plasma protein - Google Patents
Novel liquid formulation for lyophilization of plasma protein Download PDFInfo
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- WO2023219379A1 WO2023219379A1 PCT/KR2023/006256 KR2023006256W WO2023219379A1 WO 2023219379 A1 WO2023219379 A1 WO 2023219379A1 KR 2023006256 W KR2023006256 W KR 2023006256W WO 2023219379 A1 WO2023219379 A1 WO 2023219379A1
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- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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Definitions
- the present invention relates to a liquid formulation composition of plasma proteins, specifically ADAMTS-13 protein.
- Excipients used in freeze-dried formulations are divided into crystalline and amorphous types depending on the characteristics of the material.
- Crystalline additives act as bulking agents in freeze-dried formulations and provide mechanical strength to freeze-dried cakes, making them more robust.
- Representative crystalline additives include mannitol and glycine.
- amorphous additives serve as protein stabilizers in freeze-drying formulations, and by existing adjacent to proteins, they play a role in stabilizing proteins from stress generated during the freeze-drying process.
- sodium chloride which is mainly used in protein pharmaceutical formulations, can have both amorphous and crystalline forms depending on the added concentration. In general, it is reported that when NaCl is less than 150mM, it mainly exhibits amorphous properties, and when it is more than 200mM, it is mostly crystallized.
- Stabilizers in freeze-drying formulations not only protect and stabilize proteins from stress occurring during the freeze-drying process, but also contribute to protein stabilization during storage and reconstitution after drying, especially for long-term storage through freeze-drying.
- the search for optimal stabilizer components and their optimal content ratio is as important as the discovery of new pharmacological ingredients in terms of the value and utility of proteins as therapeutic resources.
- the present inventors have developed an excellent liquid formulation composition that improves the stability of plasma proteins, which have a high risk of contamination and denaturation from the moment they are separated and purified from blood, and can maintain their physical properties, biological activity, and pharmacological effects for a long period of time, especially when freeze-dried.
- We made diligent research efforts to do so As a result, when the sugar stabilizer, specifically sucrose, is included in the formulation at 0 to 1.5 w/v% and the inorganic salt, specifically NaCl, is included at 100 to 400 mM, colloidal stability, refrigeration stability, and unfolding are improved.
- the present invention was completed by discovering that it exhibits significantly excellent stability in various indicators, including inhibition of aggregation, blocking of aggregation, and cake properties after freeze-drying.
- the object of the present invention is to provide a pharmaceutical formulation composition for plasma proteins.
- Another object of the present invention is to provide a composition for preventing or treating thrombotic diseases.
- the present invention provides a pharmaceutical formulation composition for plasma protein comprising 0 - 1.5 w/v% of a sugar stabilizer and 100mM to 400mM of an inorganic salt with respect to the entire composition. do.
- the present inventors have developed an excellent liquid formulation composition that improves the stability of plasma proteins, which have a high risk of contamination and denaturation from the moment they are separated and purified from blood, and can maintain their physical properties, biological activity, and pharmacological effects for a long period of time, especially when freeze-dried.
- We made diligent research efforts to do so As a result, when the sugar stabilizer, specifically sucrose, is included in the formulation at 0 to 1.5 w/v% and the inorganic salt, specifically NaCl, is included at 100 to 400 mM, colloidal stability, refrigeration stability, and unfolding are improved. ) It was found that it showed remarkably excellent stability in various indicators, including inhibition of aggregation, blocking of aggregation, and cake properties after freeze-drying.
- plasma protein refers to the general term for water-soluble proteins present in the plasma of humans or animals, and includes all protein components other than those contained in white blood cells and red blood cells among the proteins contained in the blood. . Plasma proteins account for approximately 8% of total plasma and are responsible for hemostatic function (prothrombin, fibrinogen), transport of hormones (serum albumin and lipoprotein, etc.), and immune function (immunoglobulin and complement protein). Plasma proteins can be obtained from blood by various fractionation and purification methods known in the art, but problems of chemical instability and physical instability due to long-term storage and environmental changes must be overcome.
- Physical instability involves modifications that do not induce covalent changes in the protein, such as adsorption, aggregation, and precipitation, while chemical instability involves modifications such as deamidation, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. do. This instability leads to distortion of the intrinsic biological activity and reduction of the pharmacological effect.
- stabilizer is used to increase the stability of the active ingredient and to prevent the active ingredient from being arbitrarily denatured, oxidized, aggregated, crystallized, or transformed into related substances, ultimately resulting in loss or deterioration of pharmacological activity. It refers to any additive added to the formulation, and is not particularly limited as long as it is pharmaceutically acceptable.
- sugar stabilizer refers to a stabilizer that induces the above-mentioned stabilizing effect by adding sugar as a main or auxiliary ingredient to the formulation.
- the sugar is one or more selected from the group consisting of sucrose, trehalose, and pharmaceutically acceptable salts thereof.
- the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
- 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, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methane.
- 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, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methane.
- sulfonic acid formic acid, benzoic acid
- inorganic salt refers to a salt in which cations and anions are combined through ionic bonds in an aqueous solution and is derived from an inorganic substance and does not contain a CH bond.
- inorganic salts that can be used in the present invention include, but are not limited to, for example NaCl, CaCl 2 , KCl, MgCl 2 and combinations thereof.
- the inorganic salt of the present invention is a mixture of NaCl and CaCl 2 .
- the sugar stabilizer is contained at 0.5 - 1.3 w/v%, more specifically at 0.7 - 1.3 w/v%, and even more specifically at 0.9 w/v%, based on the total composition. - Included at 1.1 w/v%, most specifically at about 1 w/v%.
- the inorganic salt is included in 150-350mM.
- the inorganic salt is included at 120 mM to 200 mM.
- the inorganic salt is included in an amount of 160mM to 200mM.
- the inorganic salt is included at about 200mM.
- CaCl 2 may be included in an amount of 2mM to 6mM, more specifically, 3 to 5mM, and most specifically, about 4mM. .
- the composition of the present invention contains 10mM to 30mM histidine, more specifically 15mM to 25mM histidine, and most specifically about 20mM histidine.
- the composition of the present invention additionally includes 40mM to 200mM of an amino acid stabilizer.
- amino acid stabilizer refers to a stabilizer that induces the above-described stabilizing effect by adding amino acids to the formulation as a main or auxiliary ingredient.
- the composition of the present invention contains 60mM to 180mM of the amino acid stabilizer, more specifically 60mM to 160mM of the amino acid stabilizer, and even more specifically 60mM to 180mM of the amino acid stabilizer. It contains an amino acid stabilizer of 100 mM to 140 mM, more specifically, it contains an amino acid stabilizer of 80 mM to 135 mM, and even more specifically it contains an amino acid stabilizer of 100 mM to 130 mM, and most specifically, it contains an amino acid stabilizer of 100 mM to 130 mM. It includes about 120mM of amino acid stabilizer.
- the amino acid is at least one selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.
- the composition of the present invention additionally contains 40mM to 200mM of an amino acid stabilizer
- the above-described sugar stabilizer is included in 0-1.5 w/v% of the total composition and the inorganic salt is included at 200 - 300mM.
- the sugar stabilizer is contained at 0.5 - 1.3 w/v%, more specifically at 0.7 - 1.3 w/v%, and even more specifically at 0.9 - 1.1 w/v%. , most specifically at about 1 w/v%.
- the inorganic salt is included in an amount of 200 mM to 280 mM.
- the inorganic salt is included in an amount of 240mM to 280mM.
- the present inventors found that the amino acid stabilizer, sugar stabilizer, and inorganic salt each have a mutual influence on each other's optimal content, and in particular, the condition for maintaining good cake properties after freeze-drying the liquid formulation depends on the addition of the amino acid stabilizer. It was established that they were different. Therefore, if the formulation ingredients essentially contain amino acids, specifically 100 to 140mM of arginine, up to 280mM of inorganic salts can be added even when 1.0% of the sugar stabilizer is added, so for the stability of the product, sucrose or arginine must be added. If the content of the amorphous stabilizer needs to be increased, the cake properties can be maintained by increasing the content of the inorganic salt.
- the composition additionally includes 0.01 to 0.1 v/v % of a nonionic surfactant based on the total composition.
- surfactant used in the specification of the present invention refers to a soluble compound used to increase the water solubility of a hydrophobic substance or to increase the miscibility of a plurality of substances having different hydrophobicities.
- nonionic surfactant refers to a surfactant that does not contain ionized functional groups or atomic groups in the entire molecule and thus dissolves without dissociation even in aqueous solution.
- the nonionic surfactant that can be used in the present invention is at least one selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40, and more specifically, polysorbate It's 80.
- compositions of the invention comprise 0.03 to 0.08 v/v % of a nonionic surfactant, most specifically about 0.05 v/v % of a nonionic surfactant.
- the plasma protein to which the formulation composition of the present invention is applied is the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protein, a variant thereof, or a functional fragment thereof.
- ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13
- the term “functional portion” refers to a fragment in which some amino acid residues have been deleted from a full-length protein and an analogue of the full-length protein that maintains its original biological activity and function.
- the first sequence in the sequence list is the amino acid sequence of the ADAMTS13 protein consisting of 1427 amino acids.
- the ADAMTS13 mutant protein of the present invention or a functional partial fragment thereof may be a full-length (1427a.a) ADAMTS13 protein or an ADAMTS13 variant in which an amino acid mutation is introduced into a partial fragment thereof including the region 75-685.
- Some fragments containing region 75-685 may be, for example, 1-685 (685 a.a) or 75-685 (611 a.a).
- the first sequence of the sequence listing which is the amino acid sequence essentially included in the ADAMTS13 protein and some functional fragments thereof of the present invention, also includes an amino acid sequence showing substantial identity with the above sequence.
- Substantial identity means that the amino acid sequence and any other sequence are aligned to correspond as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, homology is at least 70%, specifically, It refers to an amino acid sequence that exhibits at least 80% homology, more specifically at least 90% homology, and most specifically at least 95% homology.
- the variant of the ADAMTS13 protein is 85, 93, 126, 135, 278, 282, 308, 314, 317, 334, 364, 376, 413, 427, 452 of the first sequence of the sequence listing. , 465, 567, 578, 585, 589, 607, 608, 609, 612, 618, 624, 630, 635, 643, 650, 651, 654, 655, 656, 658, 664 and 672 from the group consisting of residues and substitution of one or more amino acid residues of choice.
- the present inventors identified the core regions recognized by autoantibodies of ADAMTS13, and when substitutions were made to some amino acids in these regions, binding to autoantibodies was blocked and vWF decomposition activity and thrombosis inhibitory activities were maintained, thereby preventing thrombotic thrombocytopenia. It was discovered that it can be used as an effective treatment composition for various diseases caused by excessive blood clot formation, including purpura (TTP).
- TTP purpura
- autoantibody refers to an immunoglobulin protein that includes one or more variable domains that bind to an epitope of an antigen and specifically recognizes the corresponding antigen. It is produced by the individual's own immune system and is transmitted to the individual. It refers to an antibody that recognizes and targets its own protein. The presence of autoantibodies causes a decrease or loss of the intrinsic function or biological activity of the protein specifically recognized by the autoantibody, thereby causing various diseases.
- the variant of the ADAMTS13 protein is selected from the group consisting of mutant proteins each containing a substitution of an amino acid residue at the following position:
- substitution of the amino acid residues includes 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, and substitution of the 278th residue with Substitution with Ile, substitution of residue 282 with Ala, substitution of residue 308 with Lys, substitution of residue 314 with Thr, substitution of residue 317 with His, substitution of residue 334 with Thr or Val.
- substitution of residue 364 with Arg substitution of residue 376 with Asp, substitution of residue 413 with Asp, substitution of residue 427 with Asn, substitution of residue 452 with Ile, substitution of residue 465 substitution of Asp, substitution of residue 567 with Ser, substitution of residue 578 with Leu, substitution of residue 585 with Asn or Met, substitution of residue 589 with Gln, substitution of residue 607 with Arg.
- substitution of residue 608 with Met substitution of residue 609 with Leu, substitution of residue 612 with Phe or Tyr, substitution of residue 618 with Ser, substitution of residue 624 with Asp or Cys.
- substitutions substitution of residue 630 with Leu, substitution of residue 635 with Val, substitution of residue 643 with Phe, substitution of residue 650 with His, substitution of residue 651 with Asp, substitution of residue 654 substitution of Gly, substitution of residue 655 with Val, substitution of residue 656 with Arg or His, substitution of residue 658 with His, substitution of residue 664 with Asn, substitution of residue 672 with Val It is one or more selected from the group consisting of the substitution of.
- the above-described plasma protein of the present invention is conjugated to the Fc region of IgG4 immunoglobulin.
- the present inventors found that when an Fc region derived from IgG4 immunoglobulin is conjugated to the ADAMTS13 mutant protein discovered in the present invention, in vivo stability is greatly increased while maintaining the inherent vWF cleavage activity and neutralizing antibody evasion activity, and in particular, the C of ADAMTS13 -It was found that structural instability in open form fragments with some of the ends removed was significantly improved.
- the Fc region includes the substitution of one or more amino acid residues selected from the group consisting of residues 22, 24, and 26 of the second sequence of the sequence listing. More specifically, 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.
- the second sequence in the sequence list is the Fc region (217a.a) derived from IgG4 immunoglobulin.
- the present inventors found that when fusing the above-described ADAMTS13 mutant protein or a functional fragment thereof with an Fc region derived from an IgG4 immunoglobulin [IgG4 (YTE)] in which residues 22, 24, and 26 are substituted with Tyr, Thr, and Glu, respectively, the blood half-life It was found that the physiological activity could be maintained for a long period of time after administration.
- a hinge region of IgG1 immunoglobulin is additionally included between the plasma protein and the Fc region of the IgG4 immunoglobulin.
- the hinge region derived from IgG1 immunoglobulin may be represented by sequence number 3 (15a.a) in the sequence listing.
- the present invention is a plasma protein for the prevention or treatment of thrombotic diseases comprising the above-described pharmaceutical formulation composition of the present invention for ADAMTS13 protein, a variant thereof, or a functional fragment thereof as an active ingredient.
- a composition is provided.
- thrombotic disease refers to a systemic disease in which blood flow is reduced or blocked due to blood clots formed when platelets agglomerate in the microcirculatory system of blood vessels, resulting in ischemic damage to each organ such as the kidneys, heart, and brain. means.
- the ADAMTS13 mutant protein of the present invention which evades neutralizing antibodies with high efficiency and maintains or improves vWF decomposition activity, can be used as an efficient prevention or treatment composition for various thrombotic diseases.
- prevention refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.
- the term “treatment” refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom.
- the composition of the present invention When the composition of the present invention is administered to a subject, it specifically recognizes and decomposes VWF, regardless of the presence of neutralizing antibodies, and blocks the formation of excessive blood clots, thereby suppressing the progression of thrombotic disease, eliminating or alleviating it. do.
- the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for these diseases.
- the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”
- administer refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.
- the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological ingredients in the composition are contained in a sufficient amount to provide a therapeutic or preventive effect to the individual to whom the pharmaceutical composition of the present invention is to be administered. It is meant to include a “prophylactic effective amount.”
- the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is a human.
- the thrombotic disease is thrombotic microangiopathy (TMA). More specifically, the thrombotic microangiopathy includes thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count), It is selected from the group consisting of preeclampsia and sickle cell disease, more specifically thrombotic thrombocytopenic purpura or sickle cell disease, and most specifically thrombotic thrombocytopenic purpura.
- TTP thrombocytopenic purpura
- HUS hemolytic uremic syndrome
- HELLP Hemolysis, Elevated Liver enzymes, Low Platelet count
- the present invention provides prevention of thrombotic disease comprising administering to a subject the above-described pharmaceutical formulation composition for ADAMTS13 protein, a variant thereof, or a functional partial fragment thereof as a plasma protein. Or provide a treatment method.
- the plasma protein used in the present invention Since the plasma protein used in the present invention, its formulation components, and the thrombotic diseases that can be prevented or treated through it have already been described in detail, their description is omitted to avoid excessive duplication.
- the present invention provides a pharmaceutical formulation composition for plasma proteins, specifically ADAMTS-13 protein, and a composition for preventing or treating thrombotic diseases containing the same.
- the present invention significantly improves the stability of plasma proteins, which have a high risk of denaturation and contamination from the moment they are separated and purified from blood, and whose pharmacological activity and quality deteriorate during long-term storage, especially colloidal stability, refrigeration stability, In addition to maintaining high purity and aggregation blocking rate, the cake properties after freeze-drying remain good for a long period of time, making it possible to maintain and store ADAMTS-13 protein, an important therapeutic resource used in various thrombotic diseases, for a long period of time without losing the therapeutically effective amount. It can be usefully used.
- Figure 1 is a diagram showing the cake properties according to changes in NaCl concentration (Figure 1a) and the difference in purity evaluated through SE-HPLC ( Figure 1b).
- Figure 2 is a diagram showing SE-HPLC results according to NaCl concentration in a liquid formulation in the presence of 20 mM arginine.
- Figure 3 is a diagram showing the cake properties (Figure 3a) and SE-HPLC results (Figure 3b) according to the concentration of sucrose and the addition of trehalose in the presence of 120 mM NaCl.
- Figure 4 is a diagram showing the cake properties (Figure 4a) and SE-HPLC results (Figure 4b) according to the mixing ratio of sucrose and mannitol or glycine, which are bulking agents.
- Figure 5 is a diagram comparing cake properties according to glycine concentration in the presence of 120 mM NaCl.
- Figure 6 is a diagram comparing cake properties according to arginine concentration in the presence of 120 mM NaCl.
- Figure 7 is a diagram showing the cake properties (Figure 7a) and SE-HPLC results (Figure 7b) according to the NaCl to sucrose mixing ratio when arginine was not added.
- Figure 8 is a picture showing the cake properties (Figure 8a) and SE-HPLC results (Figure 8b) according to the NaCl to sucrose mixing ratio in the presence of 120mM arginine.
- Figure 9 shows the results of measuring the change in purity at 1 month at 40°C according to the NaCl to sucrose mixing ratio.
- Figure 10 is a diagram showing the results of measuring B 22 values to evaluate the colloidal stability of the formulation according to the addition of five amino acid stabilizers.
- Figure 11 is a picture showing the results of size exclusion liquid chromatography (SE-HPLC) to evaluate the degree of monomer recovery at room temperature after adding 7 types of stabilizers (6 types of amino acids and sucrose).
- SE-HPLC size exclusion liquid chromatography
- Figure 12 is a diagram showing the results of evaluating refrigeration stability by SE-HPLC compared to the control group according to the addition of 20 mM arginine.
- Figure 13 is a diagram showing the results of evaluating purity and stability according to the concentration of the pharmacological ingredient protein (ADAMTS-13) and arginine concentration by SE-HPLC.
- Figure 14 is a diagram showing the effect of preventing stirring-induced aggregation depending on the concentration of polysorbate 80 through property observation ( Figure 14a), turbidity measurement ( Figure 14b), and SE-HPLC ( Figure 14c).
- Figure 15 shows the results of evaluating liquid stability according to polysorbate 80 concentration by SE-HPLC.
- Figure 16 shows the results of evaluating the quality of the formulation after freeze-drying according to polysorbate 80 concentration through SE-HPLC ( Figure 16a) and whether higher-order aggregates were formed ( Figure 16b).
- Figure 17 is a diagram showing the thermal unfolding (T m ) measurement results ( Figure 17a) and thermal aggregation (T agg ) evaluation results ( Figure 17b) according to pH, respectively.
- Figure 18 is a diagram showing the freeze-dried cake properties (Figure 18a) and SE-HPLC analysis results (Figure 18b) according to CaCl 2 concentration.
- Figure 19 is a diagram schematically illustrating the property evaluation results according to NaCl and sucrose concentrations (Figure 19a) and a figure showing the cake properties at each concentration ( Figure 19b).
- Figure 20 is a picture showing cake properties according to NaCl and sucrose concentrations.
- Figure 21 is a graph showing the results of colloidal stability evaluation according to arginine concentration.
- Figure 22 is a diagram showing cake properties (Figure 22a) and purity ( Figure 22b) according to arginine concentration.
- Figure 23 is a picture showing the physical properties of the liquid formulation composition finally selected in the present invention, showing the change in purity over time (Figure 23a), change in titer (Figure 23b), change in protein concentration (Figure 23c), and change in turbidity (Figure 23b). Figure 23d) is shown, respectively.
- Figure 24 is a diagram showing long-term storage stability according to NaCl and sucrose concentrations, showing changes in purity ( Figure 24a) and titer ( Figure 24b) at the start of storage and after 6 months, respectively.
- Figure 25 is a picture showing the results of confirming the binding site of each domain of ADAMTS13 that binds to a neutralizing antibody.
- Figure 25a shows a schematic diagram of six types of ADAMTS13 fragments (or wild-type full-length ADAMTS13) combining various domains, prepared to confirm the expression rate of ADAMTS13 or the binding site of a neutralizing antibody.
- Figure 25b shows the results of immunoprecipitation using proteins expressed in each ADAMTS13 domain by mixing protein A-Sepharose and each neutralizing antibody, followed by Western blotting with anti-V5 antibody. Input shows the expression level of protein for each domain.
- Figure 25c is a schematic diagram of the ADAMTS13 neutralizing antibody binding site, showing that the Ab 4-16 antibody specifically binds to the S domain, the Ab 67 antibody to the D domain, and the Ab 66 antibody to the T2-T8 domain.
- Figure 26 is a diagram showing the results of selection of variants that avoid anti-ADAMTS13 antibodies or have better activity than wild-type ADAMTS13.
- the evasion rate and ADAMTS13 activity against Ab 4-16 antibody ( Figure 26a) and Ab 67 antibody ( Figure 26b) were measured in wild-type clones and ADAMTS13 mutants.
- Figures 27a-27b show the results of confirming the single neutralizing antibody evasion rate of 12 full-length ADAMTS13 variants.
- the antibody evasion rate against 7 types of neutralizing antibodies (Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67) was measured in 12 full-length ADAMTS13 variants.
- Figures 28a-28b show the results of confirming the evasion ability of a single neutralizing antibody under culture conditions expressing Fc-attached MDTCS fragment variants.
- Fc-attached variants and selected variant amino acid residues were combined to produce a total of 14 variants, including 2 DM1 and DM2 variants with 2 amino acid mutations, using culture media expressing them.
- the neutralizing antibody evasion rate and ADAMTS13 relative activity of eight single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured.
- Figure 29 shows the results of confirming the evasion ability of mixed neutralizing antibodies under culture conditions expressing Fc-attached MDTCS fragment variants.
- Figures 30a-30b show the results of confirming the evasion ability of a single neutralizing antibody in the culture and purification conditions of the Fc-attached MDTCS fragment variant.
- 8 types of single neutralizing antibodies Neutralizing antibody evasion rate and relative activity for (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67) were measured ( Figures 30a and 30b). The concentration of the purified solution was confirmed through Fc ELISA.
- Evasion rate (%) (1- variant binding capacity/MDTCS-Fc binding capacity) x 100
- relative activity (%) variant specific titer/MDTCS-Fc specific titer x 100. Blue bars represent purified mutant proteins, gray bars The median value of expressed mutant proteins is shown, respectively.
- Figure 31 shows the results of confirming the ability of the Fc-attached MDTCS fragment variant to evade mixed neutralizing antibodies in culture and purification conditions.
- Figure 32 is a graph showing the pharmacokinetic results of Fc-attached MDTCS fragment variants.
- MDTCS or Fc (IgG1-YTE)-attached MDTCS and four final candidate variants (1C03, 5C09, 7A02, DM2) fragment proteins were administered to mice through the tail vein, and plasma was obtained over time. Based on the specific titer of each substance, it was administered to reach 160 IU/kg, and the activity of substances remaining in the plasma obtained at each time was measured through an activity assay.
- Figure 33 shows the results of confirming the evasion ability against a single neutralizing antibody under culture conditions expressing MDTCS fragment variants attached to IgG1-YTE.
- the five selected variants were subjected to MDTCS fragmentation, and 8 types of single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, The neutralizing antibody evasion rate and relative activity of Ab67) were measured ( Figures 33a and 33b).
- Evasion rate (%) (1-variant binding capacity/MDTCS-IgG1-YTE binding capacity) x 100
- relative activity (%) variant specific titer/MDTCS-IgG1-YTE specific titer x 100.
- Figure 34 shows the results of confirming the evasion ability against mixed neutralizing antibodies under culture conditions expressing MDTCS fragment variants attached to IgG1-YTE.
- Figure 35 shows the results of confirming the evasion ability of the MDTCS fragment variant attached to IgG1-YTE to mixed neutralizing antibodies under purified solution conditions.
- Figure 36 is a schematic diagram of the neutralizing antibody evasion rate test procedure using the aTTP-mimic mouse model ( Figure 36a) and a diagram showing the ADAMTS13 residual activity by dose of variant candidate substances ( Figure 36b).
- Figure 37 is a schematic diagram of the test procedure for ADAMTS13 residual activity maintenance and clinical symptoms by concentration of DM2-IgG1-YTE, which showed the best neutralizing antibody evasion rate using the TTP-mimic mouse model (Figure 37a), platelet and LDH levels This figure shows the improvement and ADAMTS13 activity ( Figure 37b) and clinical symptom observation results (Figure 37c), respectively.
- Figure 38 is a schematic diagram of the test procedure for improvement of hematological and clinical symptoms and degree of recovery of human ADAMTS13 activity by administering DM2-IgG1-YTE to a cTTP mouse model (Figure 38a), and improvement of platelet and LDH levels and ADAMTS13 activity. This figure shows the results of observing recovery ( Figure 38b).
- Example 1 Selection of formulation components of ADAMTS13 protein
- step division Rate/hold shelf temperature Time (min) Vacuum degree (mTorr) One loading Hold 5°C 20 N/A 2 freezing Rate -55°C 200 N/A 3 freezing Hold -55°C 300 N/A 4 1st warming Rate -25°C 100 60 5 1st drying Hold -25°C 2000 60 7 2nd heating Rate 25°C 167 50 8 Secondary drying Hold 25°C 600 50
- the stability of the prepared freeze-dried preparation was analyzed after reconstitution with 1.0 ml distilled water.
- the sample was diluted to 1.0 mg/ml with a mobile phase (1 It was prepared by inserting it into a screw top vial. After connecting the mobile phase to the pump, the mobile phase was flowed through Waters e2695 and Waters 2489 instruments (Waters, Japan) at a flow rate of 0.5 mL/min, and an analytical column (TSKgel G3000SWXL, Tosoh) was installed. The mobile phase was flowed at a rate of 0.5 mL/min for more than 30 minutes to reach equilibrium until the detector signal was stabilized, and when the temperature of the autosampler fell to 4°C, the sample was inserted into the sampler. After injecting 30 ⁇ L of the sample, the mobile phase was run for 35 minutes to confirm the detection peak at 280 nm. Afterwards, analysis was performed using Empower Pro software on a PC.
- B 22 second virial coefficient
- a representative colloidal stability index was used. In general, when the B 22 value is large and positive, the repulsion force between proteins is strong, reducing the probability of aggregation occurring.
- the sample to be analyzed was prepared by diluting it to 5 concentrations (1.2, 0.96, 0.72, 0.48, and 0.24 mg/ml), and then 8.8 ⁇ L of the diluted sample was repeatedly injected into the Uni sample loader (Unchained Labs) three times. The scattered light intensities at each corresponding concentration were measured, the scattered light intensity of the placebo buffer was subtracted, and the final B 22 value was calculated using the Zimm formula (4). Fluorescence data analysis was performed using UNcle Analysis software (Unchained Labs).
- Turbidity was analyzed with Lunatic (Unchained Labs). 2.0 ⁇ L of the sample was injected into a Lunatic plate (Unchained Labs) and the turbidity was measured at 350 nm. The turbidity of the placebo buffer was subtracted from the turbidity value of the sample to calculate the final value.
- Tm thermal unfolding
- Tagg thermal aggregation
- Tm the temperature at the maximum of the fluorescence emission peak
- SLS266 static light scattering at 266 nm
- NaCl was added to a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 and 0.05% PS80 at concentrations of 50, 100, 120, 150, 200 and 300 mM.
- the formulation-completed sample was freeze-dried to observe the cake properties and SE-HPLC analysis was performed.
- Figure 1a As the NaCl concentration increased, the cake properties were more solid and excellent (Figure 1a), while the SE-HPLC results showed that the cake properties increased depending on the NaCl concentration. No significant purity differences were observed (Figure 1b).
- NaCl was added to a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20mM histidine, 4.0mM CaCl 2 and 20mM arginine at concentrations of 0, 40, 80, 120 and 160mM.
- the formulated liquid sample was stored at room temperature, and SE-HPLC analysis was performed at 4, 8, and 12 hours. As shown in Figure 2, it was confirmed that the higher the concentration of NaCl, the better the liquid stability.
- trehalose was added at a concentration of 1.0% (w/v).
- Sucrose and extenders ( 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) were added.
- the formulated sample was lyophilized to observe the cake properties and SE-HPLC analysis was performed. As a result, all samples collapsed when NaCl was fixed at 120mM and the sucrose to extender content ratio was added at 1:3 and 2:2. occurred ( Figure 4a), and no significant difference was observed between samples as a result of SE-HPLC ( Figure 4b).
- Arginine was added at 0, 20, 60 and 100 mM in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 120mM NaCl , 1.0% sucrose , 20mM histidine, 4.0mM CaCl 2 and 0.05% PS80 in common composition. It was added in concentration. As a result of freeze-drying the formulated sample to observe the cake properties and performing SE-HPLC analysis, it was found that when NaCl 120mM and sucrose 1.0% were added fixedly and the arginine concentration was increased, it had a negative effect on the cake properties, and arginine When more than 60mM was added, it completely collapsed (Figure 6).
- NaCl was added at concentrations of 160, 200, 240 and 280 mM and sucrose in a composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 0.05% PS80 and 120 mM arginine as a common composition. It was added at concentrations of 0.0, 0.5, and 1.0 % (w/v).
- concentrations of 0.0, 0.5, and 1.0 % (w/v) As a result of freeze-drying the formulated sample to observe the cake properties and performing SE-HPLC analysis, when arginine was fixedly added at 120mM, solid cake properties appeared as the NaCl concentration increased and the sucrose concentration decreased.
- sucrose in the section where more than 0.0-0.5% of sucrose was added when 160mM of NaCl was added, ii) in the section where more than 1.0% of sucrose was added when 200mM of NaCl was added, iii) in the section where more than 1.0% of sucrose was added when 240mM of NaCl was added. In, iv) when 280mM of NaCl was added, it was found that there was a collapse initiation point in each section where more than 1.0% sucrose was added (FIG. 8a).
- a composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 0.05% PS80, and 120 mM arginine as a common composition was added with NaCl at concentrations of 200, 240, and 280 mM, sucrose at 0.0, and It was added at concentrations of 0.5 and 1.0% (w/v).
- accelerated stability 40°C
- glycine and lysine were used as amino acids in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 120mM NaCl, and 1.0% sucrose as a common composition.
- proline, alanine, or arginine were included at 100mM each.
- Liquid samples of each composition after completion of formulation were analyzed for colloidal stability (B 22 ) using the UNCLE instrument.
- a composition at pH 7.4 containing 0.9 mg/ml ADAMTS protein, 20 mM histidine, 2.0 mM CaCl 2 and 120 mM NaCl as a common composition includes 100 mM each of arginine, serine, valine, threonine, proline or glycine as amino acid stabilizers. Alternatively, 1.0 w/v% of sucrose was included as a sugar stabilizer.
- ADAMTS protein was included at 0.2, 0.6 and 1.0 mg/ml, respectively, and arginine at concentrations of 20, 80 and 140 mM in a composition at pH 7.4 containing 20 mM histidine, 4.0 mM CaCl 2 and 160 mM NaCl as a common composition. The stability of the prepared liquid samples was evaluated.
- DOE Design Of Experiments
- arginine concentration and ADAMTS protein concentration were set as factors (X values) and purity (SE-HPLC) was set as response (Y value), and then a DOE design was performed using the JMP® 10.0 statistical program.
- RSM Response Surface Model
- CCD Central Composite Design
- sample stock solution (1.1 mg/mL) was diluted with arginine according to DOE conditions and left at room temperature (approximately 15-25°C) for 18 hours before SE-HPLC analysis.
- DOE statistical analysis was performed through multiple regression analysis using stepwise regression, and the establishment of the model followed response surface modeling.
- the analysis used JMP's Fit model method, and the effects used in the model included two main effects, a two-way interaction, and the square term of each main effect.
- the P-value threshold was used as a stopping rule to remove non-significant factors and select significant terms (P value ⁇ 0.25, direction: Mixed).
- the R square of the model equation is 0.96 and the R square correction is 0.94.
- the P-value of the model's ANOVA analysis of variance
- the monomer % value was highest around 120 mM arginine, indicating that 100 to 140 mM was the optimal concentration of arginine (FIG. 13c).
- Polysorbate 80 was added at 0.0, 0.001, 0.005, 0.01, 0.05 and 0.1% in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20mM histidine, 4.0mM CaCl 2 , 120mM NaCl and 1.0% sucrose in common composition. Each was added at a concentration of (v/v). Analysis (appearance, turbidity, SE-HPLC) was performed after vortexing the formulated liquid sample to generate artificial shear stress, and sampling was performed at 30 seconds and 2 minutes after the start of stirring. As a result of property analysis after 2 minutes of stirring, it was confirmed that the sample became clear and transparent when more than 0.005% of polysorbate 80 was added (FIG. 14a).
- Polysorbate 80 was added at 0.0, 0.005, 0.01, 0.05 and 0.09% ( It was added at a concentration of v/v). As a result of performing SE-HPLC analysis on the formulated liquid sample after storing it at room temperature for 6 hours, it was confirmed that as the concentration of polysorbate 80 increased, the liquid stability (purity) decreased slightly (FIG. 15).
- Polysorbate 80 was added at 0.0, 0.005, 0.01, 0.05 and 0.09% (v. It was added at a concentration of /v).
- concentration of polysorbate 80 increases, the effect of preventing loss of purity due to the freeze-drying process increases, and when more than 0.01% is added, more than 94% of monomer recovery was confirmed.
- Figure 16a In addition, as the polysorbate 80 concentration increased, the formation of higher-order aggregates was efficiently blocked (FIG. 16b).
- Diafiltration buffers with common compositions of 20mM histidine, 4mM CaCl2, and 0.05% PS80 and different pH values of 6.0, 6.5, 7.0, 7.2, and 7.4 were prepared in advance, and the samples were then subjected to a buffer exchange process with a solution containing ADAMTS protein.
- Manufactured. (Amicon® Ultra-15 Centrifugal Filter Units 30K used, centrifugation performed at 3000 rpm under refrigerated conditions)
- the common composition of the sample after buffer exchange was 10mg/ml ADAMTS protein, 20mM histidine, 4mM CaCl2, and 0.05% PS80, and the pH was 6.0, 6.5, 7.0, 7.2, and 7.4, respectively.
- the Tm (thermal unfolding) value which is proportional to the structural thermal stability of the protein, increases as the pH increases, and at pH 6.0, it is lower than other experimental groups. It was confirmed that the Tm value was observed (FIG. 17a). In the case of thermal aggregation (T agg ), which reflects the degree of unstable aggregation, it was confirmed that the higher the pH, the higher the T agg value, and at pH 6.0 and pH 6.5, the T agg value was lower than that of other experimental groups ( Figure 17b) ).
- NaCl was added at 100, 150 , 200, 250, 300, 350, and To a concentration of 400mM, sucrose was added at concentrations of 0, 0.5, 1 and 1.5% (w/v). The formulation-completed sample was freeze-dried 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 change in purity was observed depending on the NaCl and sucrose content ratio, indicating that excellent purity was maintained in all tested ranges.
- Arginine was added at 40, 80, 120, 160 and 200 in a composition at pH 7.4 containing 0.36 mg/ml ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 280 mM NaCl, 1% sucrose and 0.05% PS80. It was added at a concentration of mM.
- the colloidal stability (B 22 , kD) of the formulated liquid sample was analyzed using the UNCLE instrument. As the B 22 and kD values become more positive, it means that the proteins are well dispersed without aggregation. As shown in Figure 21, it was confirmed that the closer the arginine content is to 120 mM, the better the colloidal stability.
- Arginine was added at 40, 80, 120, 160 and 200 in a composition at pH 7.4 containing 0.36 mg/ml ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 280 mM NaCl, 1% sucrose and 0.05% PS80. It was added at a concentration of mM.
- the formulated sample was freeze-dried, the cake properties were observed, and the purity was analyzed by SE-HPLC. As a result, it was confirmed that the cake properties were excellent when the arginine concentration was 80mM and 120mM (FIG. 22a), but no significant difference in purity was observed depending on the arginine concentration (FIG. 22b).
- a fully formulated liquid sample at pH 7.4 containing 0.36 mg/ml of ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 120 mM L-Arg, 280 mM NaCl, 1% Sucrose, and 0.05% PS80 was stored at room temperature (25°C). Changes over time were confirmed by analyzing purity (SE-HPLC), titer (ADAMTS13 activity), protein concentration (UV), and turbidity after storage under ) and refrigerated (4°C) conditions, respectively.
- purity showed a decrease of about 4% compared to the initial level from the 3rd day of storage at room temperature, a decrease of about 15% compared to the initial level on the 7th day of storage, and no change over time was observed until the 7th day of refrigerated storage (FIG. 23a) .
- the titer decreased by about 50% compared to the initial level after 7 days of storage at room temperature, and considering the deviation of the titer analysis, no clear change over time was observed until the 7th day of refrigerated storage (FIG. 23b).
- a composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4mM CaCl 2 , 120mM L-Arg, and 0.05% PS80 was mixed with NaCl at concentrations of 200, 240, and 280 mM, and sucrose. was added at concentrations of 0, 0.5 and 1%.
- DOE Design Of Experiments
- a formulation buffer was prepared according to pre-designed conditions and mixed with the stock solution to prepare the final stock solution.
- the prepared formulation was freeze-dried by dispensing 1 ml into 3 ml vials, and the freeze-dried sample was stored in the refrigerator (5°C) and quality analysis was performed 6 months later.
- DOE statistical analysis was conducted through multiple regression analysis using stepwise regression. Analyze was done using JMP's 'Fit model' method, and the effects used in the model included two main effects, a two-way interaction, and the square term of each main effect.
- the p-value threshold was used as a stopping rule to remove non-significant factors and select significant terms (P value ⁇ 0.25, direction: Mixed).
- the present inventors attempted to create a human ADAMTS13 variant that can efficiently evade ADAMTS13 neutralizing antibodies possessed by aTTP patients using a random mutation method.
- Fabs were produced using Bio-Rad's HuCAL system for antibodies that recognize different epitopes for human ADAMTS13. 16 species with excellent binding ability to human ADAMTS13 were selected. The binding region of each antibody is shown in the figure. As shown in Figure 25c, Ab 4-16 specifically binds to the S domain, Ab 67 to the MDTCS portion, and Ab 66 to the C-terminal portion (TSP-2 to TSP-8 domains).
- variants were conducted through comparative results analysis of the wild-type ADAMTS13 construct and non-mutant or silent mutant variants (hereinafter referred to as wild-type clones) having the same amino acid sequence as wild-type ADAMTS13 among variants produced through random mutagenesis.
- a total of 59 wild-type clones were analyzed for the activity and binding capacity of Ab 4-16 and Ab 67, and the analysis results of each wild-type clone were relativized based on the results of the wild-type ADAMTS13 construct and are shown in Figure 26.
- the Ab 4-16 evasion rate of mutated ADAMTS13 ranged from -29.5% to 33.4%
- the Ab 67 evasion rate ranged from -24.4% to 30.5%
- the relative activity ranged from 62.7% to 128.9%.
- the range is shown (Table 4).
- the Ab 4-16 evasion rate ranged from -34.1% to 38.5%
- the Ab 67 evasion rate ranged from -38.5% to 42.3%
- the relative activity ranged from 47.0% to 145.2%.
- variants with amino acid mutations had a diverse distribution in neutralizing antibody binding ability or relative activity compared to the WT clone, and among these, 26 variants that satisfied the selection criteria were finally selected. It was confirmed that 18 of the 26 variants had mutations in the S domain, and in particular, 13 of the variants had mutations in the S domain (1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02).
- the MDTCS domain with the C-terminal TSP-2 to CUB2 domains removed has a metalloprotease function similar to ADAMTS13 and is capable of cleaving VWF (Shelat et al. ., 2005).
- the MDTCS fragment, rather than the full-length ADAMTS13 may function as a treatment for TTP disease, and that it can avoid neutralizing antibodies present in patient plasma that bind to the C-terminal portion in a truncated form. .
- the 12 selected variants were fragmented with MDTCS, and DM1 and DM2 variants with two amino acid mutations were additionally created by combining the selected mutant amino acid residues.
- DM1 had the lowest at 57.9% and 1C03 had the highest at 133.1%. All candidates except the above two showed relative activities of 78.2 - 125.1%, similar to the MDTCS-Fc control group (Table 7). Under conditions where 9 types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67) were mixed in equal proportions, 12 types excluding 4C07 and 5C09 compared to MDTCS-Fc, the control group. To confirm the evasion ability of the candidate variants, 7.5 nM of mixed neutralizing antibodies and 4 nM of each variant expression culture medium were mixed and reacted at room temperature for 1 hour to measure the residual activity.
- DNA was prepared by conjugating IgG1-YTE to the five finally selected MDTCS variant fragments, and the binding evasion and relative activity of eight types of single neutralizing antibodies were measured in culture medium expressed in cells.
- 1C03, 2B02, 5C09 and 7A02 showed excellent binding evasion rates for Ab3-01 and Ab60, and in the case of DM2, it was confirmed that the binding evasion rates for Ab3-01, Ab60 and Ab67 were excellent (FIG. 33).
- relative activity it showed similar activity to MDTCS-IgG1-YTE at 98.6-127.7% (Figure 33).
- Proteins were purified from the expression culture medium in which IgG1-YTE was conjugated to the above five MDTCS variant fragments using the Phytip system (protein A resin), and the residual activity and specific titer of mixed neutralizing antibodies were measured in the purification liquid.
- the concentration of the purified solution was confirmed through Fc ELISA, and it was confirmed that the target protein was eluted through silver staining.
- MDTCS-IgG1-YTE was maintained at 0.4%, and 1C03, 2B02, 5C09, 7A02, and DM2 were 5.7%, 1.7%, 8.8%, 2.6%, and 7.9%, respectively.
- the half-life of MDTCS was measured at 2.898 hours, MDTCS-IgG1-YTE at 11.51 hours, and for each variant between 5.184 and 9.902 hours. The half-life was extended by 1.79 to 3.97 times by IgG1-YTE conjugation compared to the control MDTCS.
- the MRT (Mean Residence Time) value which represents the average residence time of the material in the body, also showed an increased residence time of 7.189 - 11.67 hours compared to 3.743 hours due to IgG1-YTE conjugation (Table 9, Figure 32). As a result, it was confirmed that the IgG1-YTE fusion was effective in maintaining activity through blood half-life and average duration in vivo.
- the present inventors discovered 26 new variants that evade ADAMTS13 neutralizing antibodies that bind to the MDTCS portion or S domain or exhibit activity higher than that of wild-type ADAMTS13.
- 12 types with the best evasion or significantly superior comparative activity against 9 types of neutralizing antibodies were selected to produce MDTCS fragments essential for vWF cleavage while efficiently evading neutralizing antibodies that bind to the C-terminus, thereby producing D
- a variant with a significantly improved evasion rate of autoantibodies binding to the C or S domain was identified.
- the variant of the present invention can be usefully used as an effective pharmacological ingredient with improved stability and persistence of physiological activity by increasing the half-life in the blood through IgG1-YTE conjugation.
- a control substance (MDTCS-IgG1-YTE) or five selected variant candidates (1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, After administration (7A02-IgG1-YTE, DM2-IgG1-YTE), the neutralizing antibody evasion rate was evaluated (Figure 36a).
- DM2-IgG1-YTE showed the highest human ADAMTS13 residual activity at all doses of 5,000 or 7,000 IU/kg ( Figure 36b).
- the DM2-IgG1-YTE material that showed the best neutralizing antibody evasion rate was finally selected and administered at different concentrations to confirm the ability to maintain human ADAMTS13 residual activity and the degree of relief of clinical symptoms (Figure 37a). As the DM2 dose increased, clinical symptoms decreased. It was confirmed that the degree of relief increased, and in the 7000 IU/kg administration group, a relatively high improvement in platelet and LDH levels was observed by administration of DM2-IgG1-YTE compared to MDTCS-IgG1-YTE ( Figure 37b) ).
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Abstract
The present invention relates to a pharmaceutical formulation composition for plasma proteins, specifically ADAMTS-13 protein, and a composition for preventing or treating thrombotic diseases comprising same. The present invention significantly improves the stability of plasma proteins, which, from the moment of being isolated and purified from blood, have a high risk of denaturation and contamination, thereby resulting in a decrease in pharmacological activity and quality during long-term storage, and, in particular, maintains colloidal stability, refrigeration stability, purity, and aggregation inhabitation at a high level, as well as maintaining good cake properties after lyophilization for a long period of time, such that the present invention can be effectively used for maintaining and storing ADAMTS-13 protein, which is an important therapeutic resource used for various thrombotic diseases, for a long period of time without loss of a therapeutically effective amount.
Description
본 발명은 혈장 단백질, 구체적으로는 ADAMTS-13 단백질의 액상 제형 조성물에 관한 것이다.The present invention relates to a liquid formulation composition of plasma proteins, specifically ADAMTS-13 protein.
단백질 의약품 개발에 있어 안정성이 취약한 단백질의 충분한 보관 안정성 확보를 위해 효율적인 동결건조 제형의 개발은 중요한 문제이다. 동결건조 공정이 시작되기 이전 액상인 상태로 일정 시간 보관이 필요하며, 이 보관 기간 동안의 품질 저하를 방지하기 위해 액상 제형에서도 어느 정도 안정성을 확보하여야 한다. In the development of protein medicines, the development of efficient freeze-dried formulations is an important issue to ensure sufficient storage stability of proteins with weak stability. Before the freeze-drying process begins, it must be stored in a liquid state for a certain period of time, and a certain degree of stability must be secured in the liquid formulation to prevent quality deterioration during this storage period.
동결건조 제형에 사용되는 첨가제(Excipient)는 물질의 특성에 따라 결정형(Crystalline) 및 무결정형(Amorphous) 두 가지로 나뉜다. 결정형 첨가제는 동결건조 제형에서 증량제(bulking agent)의 역할을 하며 동결건조 케익에 기계적 강도 를 제공하여 케익을 보다 견고하게 만든다. 대표적인 결정형 첨가제는 만니톨, 글라이신 등이 있다. 반면 무결정형 첨가제는 동결건조 제형에 있어 단백질 안정화제 역할을 하며, 단백질과 인접하여 존재함으로써 동결건조 공정 중 발생되는 응력(stress)으로부터 단백질을 안정화시키는 역할을 한다. Excipients used in freeze-dried formulations are divided into crystalline and amorphous types depending on the characteristics of the material. Crystalline additives act as bulking agents in freeze-dried formulations and provide mechanical strength to freeze-dried cakes, making them more robust. Representative crystalline additives include mannitol and glycine. On the other hand, amorphous additives serve as protein stabilizers in freeze-drying formulations, and by existing adjacent to proteins, they play a role in stabilizing proteins from stress generated during the freeze-drying process.
한편 단백질 의약품 제형에 주로 사용되는 염화나트륨의 경우 첨가된 농도에 따라 무결정형 또는 결정형 두 가지 형태를 모두 가질 수 있다. 일반적으로 NaCl 150mM 이하의 경우 무결정형 성질을 주요하게 띄며, 200mM 이상의 경우 대부분 결정화된다고 보고되고 있다. Meanwhile, sodium chloride, which is mainly used in protein pharmaceutical formulations, can have both amorphous and crystalline forms depending on the added concentration. In general, it is reported that when NaCl is less than 150mM, it mainly exhibits amorphous properties, and when it is more than 200mM, it is mostly crystallized.
동결건조 제형에 안정화제는 동결건조 공정 중 발생되는 스트레스로부터 단백질을 보호하고 안정화시킴과 동시에 건조 완료 후 보관 기간 및 재구축(reconstitution) 시에도 단백질 안정화에 기여하므로, 특히 동결건조를 통한 장기 보관이 예정된 단백질 제제에 있어 최적의 안정화제 성분 및 이들의 최적의 함량비탐색은 단백질의 치료자원으로서의 가치와 효용성 차원에서 신규 약리성분의 발굴 못지않게 중요한 문제가 된다. Stabilizers in freeze-drying formulations not only protect and stabilize proteins from stress occurring during the freeze-drying process, but also contribute to protein stabilization during storage and reconstitution after drying, especially for long-term storage through freeze-drying. In a planned protein preparation, the search for optimal stabilizer components and their optimal content ratio is as important as the discovery of new pharmacological ingredients in terms of the value and utility of proteins as therapeutic resources.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.
본 발명자들은 혈액으로부터 분리 정제되는 순간부터 오염 및 변성의 위험이 높은 혈장 단백질의 안정성을 개선하고, 특히 동결 건조시에도 그 물성, 생물학적 활성 및 약리 효과를 장기간 유지시킬 수 있는 우수한 액상 제형 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 제형 내에 당 안정화제, 구체적으로는 수크로스가 0 내지 1.5 w/v%로 포함되면서 무기염, 구체적으로는 NaCl이 100 mM 내지 400 mM로 포함될 경우 콜로이드 안정성, 냉장 안정성, 풀림(unfolding) 억제, 응집 차단 및 동결 건조 후의 케익 성상을 비롯한 다각적인 지표에서 현저히 우수한 안정성을 나타냄을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have developed an excellent liquid formulation composition that improves the stability of plasma proteins, which have a high risk of contamination and denaturation from the moment they are separated and purified from blood, and can maintain their physical properties, biological activity, and pharmacological effects for a long period of time, especially when freeze-dried. We made diligent research efforts to do so. As a result, when the sugar stabilizer, specifically sucrose, is included in the formulation at 0 to 1.5 w/v% and the inorganic salt, specifically NaCl, is included at 100 to 400 mM, colloidal stability, refrigeration stability, and unfolding are improved. ) The present invention was completed by discovering that it exhibits significantly excellent stability in various indicators, including inhibition of aggregation, blocking of aggregation, and cake properties after freeze-drying.
따라서 본 발명의 목적은 혈장 단백질용 약제학적 제형 조성물을 제공하는 데 있다.Therefore, the object of the present invention is to provide a pharmaceutical formulation composition for plasma proteins.
본 발명의 다른 목적은 혈전성 질환의 예방 또는 치료용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for preventing or treating thrombotic diseases.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.
본 발명의 일 양태에 따르면, 본 발명은 전체 조성물에 대해 0 - 1.5 w/v%의 당(sugar) 안정화제 및 100 mM 내지 400 mM의 무기염을 포함하는 혈장 단백질용 약제학적 제형 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical formulation composition for plasma protein comprising 0 - 1.5 w/v% of a sugar stabilizer and 100mM to 400mM of an inorganic salt with respect to the entire composition. do.
본 발명자들은 혈액으로부터 분리 정제되는 순간부터 오염 및 변성의 위험이 높은 혈장 단백질의 안정성을 개선하고, 특히 동결 건조시에도 그 물성, 생물학적 활성 및 약리 효과를 장기간 유지시킬 수 있는 우수한 액상 제형 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 제형 내에 당 안정화제, 구체적으로는 수크로스가 0 내지 1.5 w/v%로 포함되면서 무기염, 구체적으로는 NaCl이 100 mM 내지 400 mM로 포함될 경우 콜로이드 안정성, 냉장 안정성, 풀림(unfolding) 억제, 응집 차단 및 동결 건조 후의 케익 성상을 비롯한 다각적인 지표에서 현저히 우수한 안정성을 나타냄을 발견하였다. The present inventors have developed an excellent liquid formulation composition that improves the stability of plasma proteins, which have a high risk of contamination and denaturation from the moment they are separated and purified from blood, and can maintain their physical properties, biological activity, and pharmacological effects for a long period of time, especially when freeze-dried. We made diligent research efforts to do so. As a result, when the sugar stabilizer, specifically sucrose, is included in the formulation at 0 to 1.5 w/v% and the inorganic salt, specifically NaCl, is included at 100 to 400 mM, colloidal stability, refrigeration stability, and unfolding are improved. ) It was found that it showed remarkably excellent stability in various indicators, including inhibition of aggregation, blocking of aggregation, and cake properties after freeze-drying.
본 명세서에서 용어“혈장 단백질(Plasma protein)”은 인간 또는 동물의 혈장에 존재하는 수용성 단백질을 총칭하는 의미로서, 혈액 속에 포함된 단백질 중 백혈구와 적혈구에 포함된 것 이외의 모든 단백질 성분을 포괄한다. 혈장단백질은 전체 혈장의 약 8% 정도를 차지하며, 지혈작용(프로트롬빈, 피브리노겐), 호르몬의 수송(혈청알부민과 지질단백질 등), 면역작용(면역글로불린과 보체 단백질)을 담당한다. 혈장 단백질은 당업계에 알려진 다양한 분획 및 정제 방법에 의해 혈액으로부터 수득될 수 있으나, 장기간 보관 및 환경 변화에 따른 화학적 불안정성 및 물리적 불안정성 문제가 극복되어야 한다. 물리적 불안정성은 단백질에서 공유결합 변화를 유도하지 않는 변형, 즉 흡착, 응집 및 침전을 형성하며, 화학적 불안정성은 탈아미드화, 라세미체화, 가수분해, 산화, 베타 제거 및 디설파이드 교환 등의 변형을 수반한다. 이러한 불안정성은 고유의 생물학적 활성의 왜곡 및 약리 효과의 감소로 이어진다.As used herein, the term “plasma protein” refers to the general term for water-soluble proteins present in the plasma of humans or animals, and includes all protein components other than those contained in white blood cells and red blood cells among the proteins contained in the blood. . Plasma proteins account for approximately 8% of total plasma and are responsible for hemostatic function (prothrombin, fibrinogen), transport of hormones (serum albumin and lipoprotein, etc.), and immune function (immunoglobulin and complement protein). Plasma proteins can be obtained from blood by various fractionation and purification methods known in the art, but problems of chemical instability and physical instability due to long-term storage and environmental changes must be overcome. Physical instability involves modifications that do not induce covalent changes in the protein, such as adsorption, aggregation, and precipitation, while chemical instability involves modifications such as deamidation, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. do. This instability leads to distortion of the intrinsic biological activity and reduction of the pharmacological effect.
본 명세서에서 용어“안정화제(stabilizer)”는 활성성분의 안정성을 증가시키고, 활성성분이 임의로 변성, 산화, 응집 또는 결정화되거나 유연물질로 변성되어 종국적으로 약리 활성이 상실되거나 저하되는 것을 방지하기 위하여 제형 내에 첨가되는 임의의 첨가제를 의미하며, 약학적으로 허용 가능한 것이라면 크게 제한되지 않는다. In this specification, the term “stabilizer” is used to increase the stability of the active ingredient and to prevent the active ingredient from being arbitrarily denatured, oxidized, aggregated, crystallized, or transformed into related substances, ultimately resulting in loss or deterioration of pharmacological activity. It refers to any additive added to the formulation, and is not particularly limited as long as it is pharmaceutically acceptable.
용어“당 안정화제(sugar stabilizer)”란 당을 주성분 또는 보조적 성분으로 제형에 첨가하여 상술한 안정화 효과를 유도하는 안정화제를 의미한다. The term “sugar stabilizer” refers to a stabilizer that induces the above-mentioned stabilizing effect by adding sugar as a main or auxiliary ingredient to the formulation.
본 발명의 구체적인 구현예에 따르면, 상기 당은 수크로스, 트레할로스 및 이들의 약제학적으로 허용되는 염으로 구성된 군으로부터 선택되는 하나 이상이다.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.
본 명세서에서 용어“약제학적으로 허용되는 염”은 약학적으로 허용되는 무기산, 유기산, 또는 염기로부터 유도된 염을 포함한다. 적합한 산의 예로는 염산, 브롬산, 황산, 질산, 과염소산, 푸마르산, 말레산, 인산, 글리콜산, 락트산, 살리실산, 숙신산, 톨루엔-p-설폰산, 타르타르산, 아세트산, 트리플루로초산, 시트르산, 메탄설폰산, 포름산, 벤조산, 말론산, 나프탈렌-2-설폰산, 벤젠설폰산 등을 들 수 있다. 적합한 염기로부터 유도된 염은 나트륨 등의 알칼리 금속, 마그네슘 등의 알칼리 토금속, 및 암모늄 등을 포함할 수 있다.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, hydrobromic 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, methane. Examples include sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium, and the like.
본 명세서에서 용어“무기염”은 수용액 상에서 양이온 및 음이온이 서로 이온 결합을 통해 조합된 염으로서 C-H 결합을 포함하지 않는 무기물 유래의 염을 의미한다. 본 발명에서 사용될 수 있는 무기염은 예를 들어 NaCl, CaCl2, KCl, MgCl2 및 이들의 조합을 포함하나, 이에 제한되는 것은 아니다. 구체적으로는, 본 발명의 무기염은 NaCl 및 CaCl2의 혼합물이다.As used herein, the term “inorganic salt” refers to a salt in which cations and anions are combined through ionic bonds in an aqueous solution and is derived from an inorganic substance and does not contain a CH bond. Inorganic salts that can be used in the present invention include, but are not limited to, for example NaCl, CaCl 2 , KCl, MgCl 2 and combinations thereof. Specifically, the inorganic salt of the present invention is a mixture of NaCl and CaCl 2 .
본 발명의 구체적인 구현예에 따르면, 상기 당 안정화제는 전체 조성물에 대해 0.5 - 1.3 w/v%로 포함되고, 보다 구체적으로는 0.7 - 1.3 w/v%로 포함되며, 보다 더 구체적으로는 0.9 - 1.1 w/v%로 포함되며, 가장 구체적으로는 약 1 w/v%로 포함된다.According to a specific embodiment of the present invention, the sugar stabilizer is contained at 0.5 - 1.3 w/v%, more specifically at 0.7 - 1.3 w/v%, and even more specifically at 0.9 w/v%, based on the total composition. - Included at 1.1 w/v%, most specifically at about 1 w/v%.
본 발명의 구체적인 구현예에 따르면, 상기 무기염은 150 - 350 mM로 포함된다.According to a specific embodiment of the present invention, the inorganic salt is included in 150-350mM.
보다 구체적으로는, 상기 당 안정화제가 전체 조성물에 대해 0 w/v% 내지 1 w/v%로 포함된 경우, 상기 무기염은 120 mM 내지 200 mM으로 포함된다.More specifically, when the sugar stabilizer is included at 0 w/v% to 1 w/v% based on the entire composition, the inorganic salt is included at 120 mM to 200 mM.
보다 구체적으로는, 상기 당 안정화제가 전체 조성물에 대해 1.4 w/v% 내지 1.6 w/v%로 포함된 경우, 상기 무기염은 160 mM 내지 200 mM으로 포함된다.More specifically, when the sugar stabilizer is included in an amount of 1.4 w/v% to 1.6 w/v% based on the total composition, the inorganic salt is included in an amount of 160mM to 200mM.
보다 구체적으로는, 상기 당 안정화제는 전체 조성물에 대해 약 1.9 w/v% 내지 2.1 w/v%로 포함된 경우, 상기 무기염은 약 200 mM로 포함된다.More specifically, when the sugar stabilizer is included at about 1.9 w/v% to 2.1 w/v% based on the total composition, the inorganic salt is included at about 200mM.
본 발명의 구체적인 구현예에 따르면, 상기 무기염 중 CaCl2은 2 mM 내지 6 mM이 포함될 수 있으며, 보다 구체적으로는 3 내지 5 mM이 포함될 수 있고, 가장 구체적으로는 약 4 mM이 포함될 수 있다. According to a specific embodiment of the present invention, among the inorganic salts, CaCl 2 may be included in an amount of 2mM to 6mM, more specifically, 3 to 5mM, and most specifically, about 4mM. .
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 10mM 내지 30mM의 히스티딘을 포함하며, 보다 구체적으로는 15mM 내지 25mM의 히스티딘을 포함하며, 가장 구체적으로는 약 20mM의 히스티딘을 포함한다. According to a specific embodiment of the present invention, the composition of the present invention contains 10mM to 30mM histidine, more specifically 15mM to 25mM histidine, and most specifically about 20mM histidine.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 40 mM 내지 200 mM의 아미노산 안정화제(stabilizer)를 추가적으로 포함한다.According to a specific embodiment of the present invention, the composition of the present invention additionally includes 40mM to 200mM of an amino acid stabilizer.
본 명세서에서 용어 “아미노산 안정화제”란 아미노산을 주성분 또는 보조적 성분으로 제형에 첨가하여 상술한 안정화 효과를 유도하는 안정화제를 의미한다. As used herein, the term “amino acid stabilizer” refers to a stabilizer that induces the above-described stabilizing effect by adding amino acids to the formulation as a main or auxiliary ingredient.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 60 mM 내지 180 mM의 아미노산 안정화제를 포함하며, 보다 구체적으로는 60 mM 내지 160 mM의 아미노산 안정화제를 포함하고, 보다 더 구체적으로는 60 mM 내지 140 mM의 아미노산 안정화제를 포함하며, 보다 더 구체적으로는 80 mM 내지 135 mM의 아미노산 안정화제를 포함하고, 보다 더 구체적으로는 100 mM 내지 130 mM의 아미노산 안정화제를 포함하며, 가장 구체적으로는 약 120 mM의 아미노산 안정화제를 포함한다.According to a specific embodiment of the present invention, the composition of the present invention contains 60mM to 180mM of the amino acid stabilizer, more specifically 60mM to 160mM of the amino acid stabilizer, and even more specifically 60mM to 180mM of the amino acid stabilizer. It contains an amino acid stabilizer of 100 mM to 140 mM, more specifically, it contains an amino acid stabilizer of 80 mM to 135 mM, and even more specifically it contains an amino acid stabilizer of 100 mM to 130 mM, and most specifically, it contains an amino acid stabilizer of 100 mM to 130 mM. It includes about 120mM of amino acid stabilizer.
본 발명의 구체적인 구현예에 따르면, 상기 아미노산은 아르기닌(Arg), 프롤린(Pro) 및 이들의 약제학적으로 허용되는 염으로 구성된 군으로부터 선택되는 하나 이상이다.According to a specific embodiment of the present invention, the amino acid is at least one selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물에 40 mM 내지 200 mM의 아미노산 안정화제가 추가적으로 포함된 경우, 전술한 당 안정화제는 전체 조성물에 대해 0 - 1.5 w/v%로 포함되고 무기염은 200 - 300 mM로 포함된다.According to a specific embodiment of the present invention, when the composition of the present invention additionally contains 40mM to 200mM of an amino acid stabilizer, the above-described sugar stabilizer is included in 0-1.5 w/v% of the total composition and the inorganic salt is included at 200 - 300mM.
보다 구체적으로는 상기 당 안정화제는 0.5 - 1.3 w/v%로 포함되고, 보다 구체적으로는 0.7 - 1.3 w/v%로 포함되며, 보다 더 구체적으로는 0.9 - 1.1 w/v%로 포함되며, 가장 구체적으로는 약 1 w/v%로 포함된다.More specifically, the sugar stabilizer is contained at 0.5 - 1.3 w/v%, more specifically at 0.7 - 1.3 w/v%, and even more specifically at 0.9 - 1.1 w/v%. , most specifically at about 1 w/v%.
보다 구체적으로는, 상기 당 안정화제가 전체 조성물에 대해 약 0 w/v 내지 0.6%로 포함된 경우, 상기 무기염은 200 mM 내지 280 mM으로 포함된다.More specifically, when the sugar stabilizer is included in an amount of about 0 w/v to 0.6% based on the total composition, the inorganic salt is included in an amount of 200 mM to 280 mM.
보다 구체적으로는, 상기 당 안정화제가 전체 조성물에 대해 0.9 w/v% 내지 1.1 w/v%로 포함된 경우, 상기 무기염은 240 mM 내지 280 mM으로 포함된다.More specifically, when the sugar stabilizer is included in an amount of 0.9 w/v% to 1.1 w/v% based on the total composition, the inorganic salt is included in an amount of 240mM to 280mM.
본 발명자들은 아미노산 안정화제, 당 안정화제 및 무기염이 각각 서로의 최적 함량에 상호 영향을 미치며, 특히 액상 제형의 동결 건조 후 케익 성상이 양호하게 지속되는 조건이 아미노산 안정화제의 첨가 여부에 따라 각각 상이하다는 사실을 규명하였다. 이에, 제형 성분에 아미노산, 구체적으로는 아르기닌 100mM 내지 140mM가 필수적으로 포함될 경우 당 안정화제가 1.0% 첨가된 상황에서도 무기염은 최대 280mM까지 첨가될 수 있어, 제품의 안정성을 위해 수크로스나 아르기닌과 같은 무결정형 안정제의 함량을 증가시켜야 할 경우, 무기염의 함량을 늘림으로써 케익 성상을 유지할 수 있다. The present inventors found that the amino acid stabilizer, sugar stabilizer, and inorganic salt each have a mutual influence on each other's optimal content, and in particular, the condition for maintaining good cake properties after freeze-drying the liquid formulation depends on the addition of the amino acid stabilizer. It was established that they were different. Therefore, if the formulation ingredients essentially contain amino acids, specifically 100 to 140mM of arginine, up to 280mM of inorganic salts can be added even when 1.0% of the sugar stabilizer is added, so for the stability of the product, sucrose or arginine must be added. If the content of the amorphous stabilizer needs to be increased, the cake properties can be maintained by increasing the content of the inorganic salt.
본 발명의 구체적인 구현예에 따르면, 상기 조성물은 전체 조성물에 대해 0.01 내지 0.1 v/v %의 비이온성 계면활성제를 추가적으로 포함한다.According to a specific embodiment of the present invention, the composition additionally includes 0.01 to 0.1 v/v % of a nonionic surfactant based on the total composition.
본 발명의 명세서에서 사용되는 용어 “계면활성제”는 소수성 물질의 수용해도를 증가시키거나 상이한 소수성을 갖는 복수의 물질의 혼화성을 증가시키기 위해 사용되는 가용성 화합물을 의미한다. 용어“비이온성 계면활성제”는 전체 분자 내에 이온화되는 작용기 또는 원자단이 포함되지 않아 수용액 상태에서도 해리되지 않은 채 용해되는 계면활성제를 의미한다.The term “surfactant” used in the specification of the present invention refers to a soluble compound used to increase the water solubility of a hydrophobic substance or to increase the miscibility of a plurality of substances having different hydrophobicities. The term “nonionic surfactant” refers to a surfactant that does not contain ionized functional groups or atomic groups in the entire molecule and thus dissolves without dissociation even in aqueous solution.
본 발명의 구체적인 구현예에 따르면 본 발명에서 사용될 수 있는 비이온성 계면활성제는 폴리소르베이트 80, 폴리소르베이트 60 및 폴리소르베이트 40으로 구성된 군으로부터 선택되는 하나 이상이며, 보다 구체적으로는 폴리소르베이트 80이다.According to a specific embodiment of the present invention, the nonionic surfactant that can be used in the present invention is at least one selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40, and more specifically, polysorbate It's 80.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물은 0.03 내지 0.08 v/v %의 비이온성 계면활성제를 가장 구체적으로는 약 0.05 v/v %의 비이온성 계면활성제를 포함한다.According to specific embodiments of the invention, the compositions of the invention comprise 0.03 to 0.08 v/v % of a nonionic surfactant, most specifically about 0.05 v/v % of a nonionic surfactant.
본 발명의 구체적인 구현예에 따르면, 본 발명의 제형 조성물이 적용되는 혈장 단백질은 ADAMTS13(a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) 단백질, 이의 변이체 또는 이의 기능적 일부 절편이다.According to a specific embodiment of the present invention, the plasma protein to which the formulation composition of the present invention is applied is the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protein, a variant thereof, or a functional fragment thereof.
본 명세서에서 용어“기능적 일부”는 전장 단백질에서 일부 아미노산 잔기가 삭제된 절편으로서 그 본연의 생물학적 활성 및 기능을 유지하는 전장 단백질의 유사체를 의미한다. As used herein, the term “functional portion” refers to a fragment in which some amino acid residues have been deleted from a full-length protein and an analogue of the full-length protein that maintains its original biological activity and function.
본 발명에 따르면, 서열목록 제1서열은 1427개 아미노산으로 구성된 ADAMTS13 단백질의 아미노산 서열이다. 이에, 본 발명의 ADAMTS13 변이 단백질 또는 이의 기능적 일부 절편은 전장(1427a.a) ADAMTS13 단백질 또는 75-685번 영역을 포함하는 이의 일부 절편 내에 아미노산 변이가 도입된 ADAMTS13 변이체일 수 있다. 75-685번 영역을 포함하는 일부 절편은 예를 들어 1-685 (685 a.a) 또는 75-685 (611 a.a)일 수 있다. According to the present invention, the first sequence in the sequence list is the amino acid sequence of the ADAMTS13 protein consisting of 1427 amino acids. Accordingly, the ADAMTS13 mutant protein of the present invention or a functional partial fragment thereof may be a full-length (1427a.a) ADAMTS13 protein or an ADAMTS13 variant in which an amino acid mutation is introduced into a partial fragment thereof including the region 75-685. Some fragments containing region 75-685 may be, for example, 1-685 (685 a.a) or 75-685 (611 a.a).
본 발명의 ADAMTS13 단백질 및 이의 기능적 일부 절편이 필수적으로 포함하는 아미노산 서열인 서열목록 제1서열은 상기 서열과 실질적인 동일성(substantial identity)을 나타내는 아미노산 서열도 포함한다. 실질적인 동일성이란, 상기 아미노산 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석할 시, 최소 70%의 상동성, 구체적으로는 최소 80%의 상동성, 보다 구체적으로는 최소 90%의 상동성, 가장 구체적으로는 최소 95%의 상동성을 나타내는 아미노산 서열을 의미한다.The first sequence of the sequence listing, which is the amino acid sequence essentially included in the ADAMTS13 protein and some functional fragments thereof of the present invention, also includes an amino acid sequence showing substantial identity with the above sequence. Substantial identity means that the amino acid sequence and any other sequence are aligned to correspond as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, homology is at least 70%, specifically, It refers to an amino acid sequence that exhibits at least 80% homology, more specifically at least 90% homology, and most specifically at least 95% homology.
본 발명의 구체적인 구현예에 따르면, 상기 ADAMTS13 단백질의 변이체는 서열목록 제1서열의 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 및 672번째 잔기로 구성된 군으로부터 선택되는 하나 이상의 아미노산 잔기의 치환을 포함한다.According to a specific embodiment of the present invention, the variant of the ADAMTS13 protein is 85, 93, 126, 135, 278, 282, 308, 314, 317, 334, 364, 376, 413, 427, 452 of the first sequence of the sequence listing. , 465, 567, 578, 585, 589, 607, 608, 609, 612, 618, 624, 630, 635, 643, 650, 651, 654, 655, 656, 658, 664 and 672 from the group consisting of residues and substitution of one or more amino acid residues of choice.
본 발명자들은 ADAMTS13의 자가항체가 인식하는 핵심 영역을 동정하고, 이들 영역 내 일부 아미노산에 치환을 가할 경우 자가항체와의 결합이 차단되고 vWF 분해 활성 및 혈전 억제 활성이 유지됨으로써, 혈전성 혈소판 감소성 자반증(TTP)을 비롯하여 과도한 혈전 형성을 원인으로 하는 다양한 질환에 대해 효율적인 치료 조성물로 사용될 수 있음을 발견하였다. The present inventors identified the core regions recognized by autoantibodies of ADAMTS13, and when substitutions were made to some amino acids in these regions, binding to autoantibodies was blocked and vWF decomposition activity and thrombosis inhibitory activities were maintained, thereby preventing thrombotic thrombocytopenia. It was discovered that it can be used as an effective treatment composition for various diseases caused by excessive blood clot formation, including purpura (TTP).
본 명세서에서 용어“자가 항체(autoantibody)”는 항원의 에피토프에 결합하는 하나 이상의 가변 도메인을 포함하여 해당 항원을 특이적으로 인식하는 면역글로불린 단백질의 하나로써, 개체 자신의 면역 시스템에 의해 생성되어 개체 자신의 단백질을 인식하고 표적화하는 항체를 의미한다. 자가항체의 존재는 해당 자가항체에 의해 특이적으로 인식되는 단백질의 고유 기능 또는 생물학적 활성의 저하 또는 상실을 야기하므로, 다양한 질환의 원인이 된다.As used herein, the term “autoantibody” refers to an immunoglobulin protein that includes one or more variable domains that bind to an epitope of an antigen and specifically recognizes the corresponding antigen. It is produced by the individual's own immune system and is transmitted to the individual. It refers to an antibody that recognizes and targets its own protein. The presence of autoantibodies causes a decrease or loss of the intrinsic function or biological activity of the protein specifically recognized by the autoantibody, thereby causing various diseases.
본 발명의 구체적인 구현예에 따르면, 상기 ADAMTS13 단백질의 변이체는 다음 위치에서의 아미노산 잔기의 치환을 포함하는 각 변이 단백질들로 구성된 군으로부터 선택된다: According to a specific embodiment of the present invention, the variant of the ADAMTS13 protein is selected from the group consisting of mutant proteins each containing a substitution of an amino acid residue at the following position:
- 85 및 317번째 잔기; 612번째 잔기; 282, 465 및 672번째 잔기 중 둘 이상; 635번째 잔기; 452 및 612번째 잔기; 278, 334 및 427번째 잔기 중 둘 이상; 618번째 잔기; 135번째 잔기; 126, 567 및 651번째 잔기 중 둘 이상; 413번째 잔기; 334번째 잔기; 314번째 잔기; 93, 364 및 376번째 잔기 중 둘 이상; 308번째 잔기; 656번째 잔기; 607번째 잔기; 612 및 624번째 잔기; 589번째 잔기; 650 및 656번째 잔기; 643번째 잔기; 585 및 658번째 잔기; 630, 654 및 664번째 잔기 중 둘 이상; 589, 608, 609, 624 및 655번째 잔기 중 넷 이상; 578번째 잔기; 585번째 잔기; 314 및 635번째 잔기; 및 314 및 612번째 잔기.- 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.
보다 구체적으로는, 상기 아미노산 잔기의 치환은 85번째 잔기의 Phe로의 치환, 93번째 잔기의 Val으로의 치환, 126번째 잔기의 Met으로의 치환, 135번째 잔기의 Ile으로의 치환, 278번째 잔기의 Ile으로의 치환, 282번째 잔기의 Ala으로의 치환, 308번째 잔기의 Lys으로의 치환, 314번째 잔기의 Thr으로의 치환, 317번째 잔기의 His으로의 치환, 334번째 잔기의 Thr 또는 Val으로의 치환, 364번째 잔기의 Arg으로의 치환, 376번째 잔기의 Asp으로의 치환, 413번째 잔기의 Asp으로의 치환, 427번째 잔기의 Asn으로의 치환, 452번째 잔기의 Ile으로의 치환, 465번째 잔기의 Asp으로의 치환, 567번째 잔기의 Ser으로의 치환, 578번째 잔기의 Leu으로의 치환, 585번째 잔기의 Asn 또는 Met으로의 치환, 589번째 잔기의 Gln으로의 치환, 607번째 잔기의 Arg으로의 치환, 608번째 잔기의 Met으로의 치환, 609번째 잔기의 Leu으로의 치환, 612번째 잔기의 Phe 또는 Tyr으로의 치환, 618번째 잔기의 Ser으로의 치환, 624번째 잔기의 Asp 또는 Cys으로의 치환, 630번째 잔기의 Leu으로의 치환, 635번째 잔기의 Val으로의 치환, 643번째 잔기의 Phe으로의 치환, 650번째 잔기의 His으로의 치환, 651번째 잔기의 Asp으로의 치환, 654번째 잔기의 Gly으로의 치환, 655번째 잔기의 Val으로의 치환, 656번째 잔기의 Arg 또는 His으로의 치환, 658번째 잔기의 His으로의 치환, 664번째 잔기의 Asn으로의 치환, 672번째 잔기의 Val으로의 치환으로 구성된 군으로부터 선택되는 하나 이상이다.More specifically, the substitution of the amino acid residues includes 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, and substitution of the 278th residue with Substitution with Ile, substitution of residue 282 with Ala, substitution of residue 308 with Lys, substitution of residue 314 with Thr, substitution of residue 317 with His, substitution of residue 334 with Thr or Val. Substitution, substitution of residue 364 with Arg, substitution of residue 376 with Asp, substitution of residue 413 with Asp, substitution of residue 427 with Asn, substitution of residue 452 with Ile, substitution of residue 465 substitution of Asp, substitution of residue 567 with Ser, substitution of residue 578 with Leu, substitution of residue 585 with Asn or Met, substitution of residue 589 with Gln, substitution of residue 607 with Arg. Substitution of, substitution of residue 608 with Met, substitution of residue 609 with Leu, substitution of residue 612 with Phe or Tyr, substitution of residue 618 with Ser, substitution of residue 624 with Asp or Cys. Substitutions, substitution of residue 630 with Leu, substitution of residue 635 with Val, substitution of residue 643 with Phe, substitution of residue 650 with His, substitution of residue 651 with Asp, substitution of residue 654 substitution of Gly, substitution of residue 655 with Val, substitution of residue 656 with Arg or His, substitution of residue 658 with His, substitution of residue 664 with Asn, substitution of residue 672 with Val It is one or more selected from the group consisting of the substitution of.
본 발명의 구체적인 구현예에 따르면, 전술한 본 발명의 혈장 단백질은 IgG4 면역글로불린의 Fc 영역이 접합되어 있다. According to a specific embodiment of the present invention, the above-described plasma protein of the present invention is conjugated to the Fc region of IgG4 immunoglobulin.
본 발명자들은 본 발명에서 발굴된 ADAMTS13 변이 단백질에 IgG4 면역글로불린 유래의 Fc 영역을 접합시킬 경우, 고유의 vWF 절단 활성 및 중화항체 회피 활성을 그대로 유지하면서도 생체 내 안정성이 크게 증가하며, 특히 ADAMTS13의 C-말단 일부가 제거된 개방형(open form) 절편들에서 나타나는 구조적 불안정성이 현저히 개선됨을 발견하였다. The present inventors found that when an Fc region derived from IgG4 immunoglobulin is conjugated to the ADAMTS13 mutant protein discovered in the present invention, in vivo stability is greatly increased while maintaining the inherent vWF cleavage activity and neutralizing antibody evasion activity, and in particular, the C of ADAMTS13 -It was found that structural instability in open form fragments with some of the ends removed was significantly improved.
본 발명의 보다 구체적인 구현예에 따르면, 상기 Fc 영역은 서열목록 제2서열의 22, 24 및 26번째 잔기로 구성된 군으로부터 선택되는 하나 이상의 아미노산 잔기의 치환을 포함한다. 보다 구체적으로는, 상기 아미노산 잔기의 치환은 22번째 잔기의 Tyr으로의 치환, 24번째 잔기의 Thr으로의 치환 및 26번째 잔기의 Glu으로의 치환으로 구성된 군으로부터 선택되는 하나 이상이다.According to a more specific embodiment of the present invention, the Fc region includes the substitution of one or more amino acid residues selected from the group consisting of residues 22, 24, and 26 of the second sequence of the sequence listing. More specifically, 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.
본 발명에 따르면, 서열목록 제2서열은 IgG4 면역글로불린 유래의 Fc 영역(217a.a)이다. 본 발명자들은 전술한 ADAMTS13 변이 단백질 또는 이의 기능적 일부 절편에 22, 24 및 26번째 잔기가 각각 Tyr, Thr 및 Glu으로 치환된 IgG4 면역글로불린 유래의 Fc 영역[IgG4 (YTE)]을 융합시킬 경우 혈중 반감기가 극대화되어 투여 후 생리 활성이 장기간 지속될 수 있음을 발견하였다.According to the present invention, the second sequence in the sequence list is the Fc region (217a.a) derived from IgG4 immunoglobulin. The present inventors found that when fusing the above-described ADAMTS13 mutant protein or a functional fragment thereof with an Fc region derived from an IgG4 immunoglobulin [IgG4 (YTE)] in which residues 22, 24, and 26 are substituted with Tyr, Thr, and Glu, respectively, the blood half-life It was found that the physiological activity could be maintained for a long period of time after administration.
본 발명의 구체적인 구현예에 따르면, 상기 혈장 단백질과 상기 IgG4 면역글로불린의 Fc 영역 사이에 IgG1 면역글로불린의 힌지(hinge) 영역이 추가적으로 포함된다.According to a specific embodiment of the present invention, a hinge region of IgG1 immunoglobulin is additionally included between the plasma protein and the Fc region of the IgG4 immunoglobulin.
본 발명에 따르면, IgG1 면역글로불린 유래의 힌지(hinge) 영역은 서열목록 제3서열(15a.a)로 표시될 수 있다. According to the present invention, the hinge region derived from IgG1 immunoglobulin may be represented by sequence number 3 (15a.a) in the sequence listing.
본 발명의 또 다른 양태에 따르면, 본 발명은 혈장 단백질로서 ADAMTS13 단백질, 이의 변이체 또는 이의 기능적 일부 절편에 대한 전술한 본 발명의 약제학적 제형 조성물을 유효성분으로 포함하는 혈전성 질환의 예방 또는 치료용 조성물을 제공한다. According to another aspect of the present invention, the present invention is a plasma protein for the prevention or treatment of thrombotic diseases comprising the above-described pharmaceutical formulation composition of the present invention for ADAMTS13 protein, a variant thereof, or a functional fragment thereof as an active ingredient. A composition is provided.
본 명세서에서 용어“혈전성 질환”은 혈관의 미세순환계에 혈소판이 응집되면서 생성된 혈전으로 인해 혈류가 감소 또는 차단되고, 이로 인해 신장, 심장, 뇌 등의 각 기관에 허혈성 손상이 유발되는 전신 질환을 의미한다.As used herein, the term “thrombotic disease” refers to a systemic disease in which blood flow is reduced or blocked due to blood clots formed when platelets agglomerate in the microcirculatory system of blood vessels, resulting in ischemic damage to each organ such as the kidneys, heart, and brain. means.
ADAMTS13 효소가 중화항체에 의해 활성이 억제되어 폰빌레란트인자(vWF)를 제대로 분해하지 못할 경우, 과도한 혈소판 응집 및 혈전 과생성이 발생한다. 따라서, 중화항체를 높은 효율로 회피하면서도 vWF 분해 활성이 유지 또는 개선된 본 발명의 ADAMTS13 변이 단백질은 다양한 혈전성 질환에 대한 효율적인 예방 또는 치료 조성물로 이용될 수 있다.When the ADAMTS13 enzyme is inhibited by neutralizing antibodies and cannot properly decompose von Willerant factor (vWF), excessive platelet aggregation and thrombus formation occur. Therefore, the ADAMTS13 mutant protein of the present invention, which evades neutralizing antibodies with high efficiency and maintains or improves vWF decomposition activity, can be used as an efficient prevention or treatment composition for various thrombotic diseases.
본 명세서에서 용어“예방”은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸릴 가능성이 있는 대상체에서 질환 또는 질병의 발생을 억제하는 것을 의미한다. As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.
본 명세서에서 용어“치료”는 (a) 질환, 질병 또는 증상의 발전의 억제; (b) 질환, 질병 또는 증상의 경감; 또는 (c) 질환, 질병 또는 증상을 제거하는 것을 의미한다. 본 발명의 조성물을 대상체에 투여하면 중화항체의 존재 여부와 무관하게 vWF를 특이적으로 인식 및 분해하여 과도한 혈전의 생성을 차단함으로써 혈전성 질환의 진행을 억제하거나, 이를 제거하거나 또는 경감시키는 역할을 한다. 따라서, 본 발명의 조성물은 그 자체로 이들 질환 치료의 조성물이 될 수도 있고, 혹은 다른 약리성분과 함께 투여되어 상기 질환에 대한 치료 보조제로 적용될 수도 있다. 이에, 본 명세서에서 용어“치료”또는“치료제”는“치료 보조”또는“치료 보조제”의 의미를 포함한다. As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom. When the composition of the present invention is administered to a subject, it specifically recognizes and decomposes VWF, regardless of the presence of neutralizing antibodies, and blocks the formation of excessive blood clots, thereby suppressing the progression of thrombotic disease, eliminating or alleviating it. do. Accordingly, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for these diseases. Accordingly, in this specification, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”
본 명세서에서 용어“투여”또는“투여하다”는 본 발명의 조성물의 치료적 유효량을 대상체에 직접적으로 투여함으로써 대상체의 체내에서 동일한 양이 형성되도록 하는 것을 말한다.As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.
본 발명에서 용어“치료적 유효량”은 본 발명의 약제학적 조성물을 투여하고자 하는 개체에게 조성물 내의 약리성분이 치료적 또는 예방적 효과를 제공하기에 충분한 정도로 함유된 조성물의 함량을 의미하며, 이에“예방적 유효량”을 포함하는 의미이다. In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological ingredients in the composition are contained in a sufficient amount to provide a therapeutic or preventive effect to the individual to whom the pharmaceutical composition of the present invention is to be administered. It is meant to include a “prophylactic effective amount.”
본 명세서에서 용어 “대상체”는 제한없이 인간, 마우스, 래트, 기니아 피그, 개, 고양이, 말, 소, 돼지, 원숭이, 침팬지, 비비 또는 붉은털 원숭이를 포함한다. 구체적으로는, 본 발명의 대상체는 인간이다. As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is a human.
본 발명의 구체적인 구현예에 따르면, 상기 혈전성 질환은 혈전성 미세 혈관병증(thrombotic microangiopathy, TMA)이다. 보다 구체적으로는, 상기 혈전성 미세 혈관병증은 혈전성 혈소판 감소성 자반증(thrombocytopenic purpura, TTP), 용혈성 요독성 증후군(Hemolytic uremic syndrome, HUS), HELLP(Hemolysis, Elevated Liver enzymes, Low Platelet count), 자간전증(Preeclampsia) 및 겸상적혈구질환(sickle cell disease)으로 구성된 군으로부터 선택되며, 보다 더 구체적으로는 혈전성 혈소판 감소성 자반증 또는 겸상적혈구질환이며, 가장 구체적으로는 혈전성 혈소판 감소성 자반증이다.According to a specific embodiment of the present invention, the thrombotic disease is thrombotic microangiopathy (TMA). More specifically, the thrombotic microangiopathy includes thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count), It is selected from the group consisting of preeclampsia and sickle cell disease, more specifically thrombotic thrombocytopenic purpura or sickle cell disease, and most specifically thrombotic thrombocytopenic purpura.
본 발명의 또 다른 양태에 따르면, 본 발명은 혈장 단백질로서 ADAMTS13 단백질, 이의 변이체 또는 이의 기능적 일부 절편에 대한 전술한 본 발명의 약제학적 제형 조성물을 대상체에 투여하는 단계를 포함하는 혈전성 질환의 예방 또는 치료방법을 제공한다.According to another aspect of the present invention, the present invention provides prevention of thrombotic disease comprising administering to a subject the above-described pharmaceutical formulation composition for ADAMTS13 protein, a variant thereof, or a functional partial fragment thereof as a plasma protein. Or provide a treatment method.
본 발명에서 이용되는 혈장 단백질, 이의 제형 성분 및 이를 통해 예방 또는 치료될 수 있는 혈전성 질환에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위해 그 기재를 생략한다.Since the plasma protein used in the present invention, its formulation components, and the thrombotic diseases that can be prevented or treated through it have already been described in detail, their description is omitted to avoid excessive duplication.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 혈장 단백질, 구체적으로는 ADAMTS-13 단백질에 대한 약제학적 제형 조성물 및 이를 포함하는 혈전성 질환의 예방 또는 치료용 조성물을 제공한다.(a) The present invention provides a pharmaceutical formulation composition for plasma proteins, specifically ADAMTS-13 protein, and a composition for preventing or treating thrombotic diseases containing the same.
(b) 본 발명은 혈액으로부터 분리 정제되는 순간부터 변성 및 오염의 위험성이 높아 장기간 저장 시 약리활성 및 품질의 저하가 발생하는 혈장 단백질에 있어 그 안정성을 현저히 개선하고, 특히 콜로이드 안정성, 냉장 안정성, 순도 및 응집 차단율을 높은 수준으로 유지할 뿐 아니라 동결 건조 후의 케익 성상이 장기간 양호하게 지속됨으로써 다양한 혈전성 질환에 사용되는 중요한 치료 자원인 ADAMTS-13 단백질을 치료학적 유효량의 손실 없이 장기간 유지, 보관하는 데에 유용하게 사용될 수 있다.(b) The present invention significantly improves the stability of plasma proteins, which have a high risk of denaturation and contamination from the moment they are separated and purified from blood, and whose pharmacological activity and quality deteriorate during long-term storage, especially colloidal stability, refrigeration stability, In addition to maintaining high purity and aggregation blocking rate, the cake properties after freeze-drying remain good for a long period of time, making it possible to maintain and store ADAMTS-13 protein, an important therapeutic resource used in various thrombotic diseases, for a long period of time without losing the therapeutically effective amount. It can be usefully used.
도 1은 NaCl 농도 변화에 따른 케익 성상(도 1a) 및 SE-HPLC를 통해 평가한 순도 차이(도 1b)를 각각 보여주는 그림이다. Figure 1 is a diagram showing the cake properties according to changes in NaCl concentration (Figure 1a) and the difference in purity evaluated through SE-HPLC (Figure 1b).
도 2는 20 mM의 아르기닌이 존재하는 경우 액상 제형에서 NaCl 농도에 따른 SE-HPLC 결과를 나타내는 그림이다.Figure 2 is a diagram showing SE-HPLC results according to NaCl concentration in a liquid formulation in the presence of 20 mM arginine.
도 3은 120 mM의 NaCl 존재 하에서 수크로스의 농도 및 트레할로스의 첨가 여부에 따른 케익 성상(도 3a)과 SE-HPLC 결과(도 3b)를 각각 보여주는 그림이다. Figure 3 is a diagram showing the cake properties (Figure 3a) and SE-HPLC results (Figure 3b) according to the concentration of sucrose and the addition of trehalose in the presence of 120 mM NaCl.
도 4는 수크로스와 증량제(bulking agents)인 만니톨 또는 글리신의 배합비에 따른 케익 성상(도 4a) 및 SE-HPLC 결과(도 4b)를 각각 보여주는 그림이다. Figure 4 is a diagram showing the cake properties (Figure 4a) and SE-HPLC results (Figure 4b) according to the mixing ratio of sucrose and mannitol or glycine, which are bulking agents.
도 5는 120 mM의 NaCl 존재 하에 글리신 농도에 따른 케익 성상을 비교한 그림이다. Figure 5 is a diagram comparing cake properties according to glycine concentration in the presence of 120 mM NaCl.
도 6은 120 mM의 NaCl 존재 하에 아르기닌 농도에 따른 케익 성상을 비교한 그림이다. Figure 6 is a diagram comparing cake properties according to arginine concentration in the presence of 120 mM NaCl.
도 7은 아르기닌이 첨가되지 않은 경우 NaCl 대 수크로스 배합비에 따른 케익 성상(도 7a) 및 SE-HPLC 결과(도 7b)를 각각 보여주는 그림이다.Figure 7 is a diagram showing the cake properties (Figure 7a) and SE-HPLC results (Figure 7b) according to the NaCl to sucrose mixing ratio when arginine was not added.
도 8은 120mM의 아르기닌 존재 하에 NaCl 대 수크로스 배합비에 따른 케익 성상(도 8a) 및 SE-HPLC 결과(도 8b)를 각각 보여주는 그림이다.Figure 8 is a picture showing the cake properties (Figure 8a) and SE-HPLC results (Figure 8b) according to the NaCl to sucrose mixing ratio in the presence of 120mM arginine.
도 9는 NaCl 대 수크로스 배합비에 따른 40℃에서 1개월차의 순도 변화를 측정한 결과를 나타낸다.Figure 9 shows the results of measuring the change in purity at 1 month at 40°C according to the NaCl to sucrose mixing ratio.
도 10은 5가지 아미노산 안정화제의 첨가에 따른 제형의 콜로이드 안정성을 평가하기 위해 B22 값을 측정한 결과를 나타내는 그림이다.Figure 10 is a diagram showing the results of measuring B 22 values to evaluate the colloidal stability of the formulation according to the addition of five amino acid stabilizers.
도 11은 7가지 안정화제(아미노산 6종 및 수크로스)를 첨가 후 상온에서의 단량체 회복 정도를 평가하기 위해 크기배제 액체 크로마토그래피(SE-HPLC)를 수행한 결과를 보여주는 그림이다.Figure 11 is a picture showing the results of size exclusion liquid chromatography (SE-HPLC) to evaluate the degree of monomer recovery at room temperature after adding 7 types of stabilizers (6 types of amino acids and sucrose).
도 12는 20 mM의 아르기닌 첨가에 따른 대조군 대비 냉장 안정성을 SE-HPLC로 평가한 결과를 나타내는 그림이다.Figure 12 is a diagram showing the results of evaluating refrigeration stability by SE-HPLC compared to the control group according to the addition of 20 mM arginine.
도 13은 약리 성분인 단백질(ADAMTS-13)의 농도 및 아르기닌 농도에 따른 순도 및 안정성을 SE-HPLC로 평가한 결과를 나타낸 그림이다.Figure 13 is a diagram showing the results of evaluating purity and stability according to the concentration of the pharmacological ingredient protein (ADAMTS-13) and arginine concentration by SE-HPLC.
도 14는 폴리소르베이트 80 농도에 따른 교반-유도 응집의 방지효과를 성상 관찰(도 14a), 탁도(Turbidity) 측정(도 14b) 및 SE-HPLC(도 14c)를 통해 각각 보여주는 그림이다.Figure 14 is a diagram showing the effect of preventing stirring-induced aggregation depending on the concentration of polysorbate 80 through property observation (Figure 14a), turbidity measurement (Figure 14b), and SE-HPLC (Figure 14c).
도 15는 폴리소르베이트 80 농도에 따른 액상 안정성을 SE-HPLC로 평가한 결과이다.Figure 15 shows the results of evaluating liquid stability according to polysorbate 80 concentration by SE-HPLC.
도 16은 폴리소르베이트 80 농도에 따른 동결건조 후의 제형 품질을 SE-HPLC(도 16a) 및 고차 응집체(Higher-order aggregates) 생성 여부(도 16b)를 통해 평가한 결과이다.Figure 16 shows the results of evaluating the quality of the formulation after freeze-drying according to polysorbate 80 concentration through SE-HPLC (Figure 16a) and whether higher-order aggregates were formed (Figure 16b).
도 17은 pH에 따른 열 풀림(thermal unfolding, Tm) 측정 결과(도 17a) 및 열 응집(thermal aggregation, Tagg) 평가 결과(도 17b)를 각각 보여주는 그림이다. Figure 17 is a diagram showing the thermal unfolding (T m ) measurement results (Figure 17a) and thermal aggregation (T agg ) evaluation results (Figure 17b) according to pH, respectively.
도 18은 CaCl2 농도에 따른 동결 건조 케익 성상(도 18a) 및 SE-HPLC 분석 결과(도 18b)를 각각 보여주는 그림이다.Figure 18 is a diagram showing the freeze-dried cake properties (Figure 18a) and SE-HPLC analysis results (Figure 18b) according to CaCl 2 concentration.
도 19는 NaCl 및 수크로스 농도에 따른 성상 평가 결과를 도식화한 그림(도 19a) 및 각 농도별 케익 성상(도 19b)를 각각 보여주는 그림이다.Figure 19 is a diagram schematically illustrating the property evaluation results according to NaCl and sucrose concentrations (Figure 19a) and a figure showing the cake properties at each concentration (Figure 19b).
도 20은 NaCl 및 수크로스 농도에 따른 케익 성상을 보여주는 그림이다.Figure 20 is a picture showing cake properties according to NaCl and sucrose concentrations.
도 21은 아르기닌 농도에 따른 콜로이드 안정성 평가 결과를 보여주는 그래프이다.Figure 21 is a graph showing the results of colloidal stability evaluation according to arginine concentration.
도 22는 아르기닌 농도에 따른 케익 성상(도 22a) 및 순도(도 22b)를 각각 보여주는 그림이다. Figure 22 is a diagram showing cake properties (Figure 22a) and purity (Figure 22b) according to arginine concentration.
도 23은 본 발명에서 최종적으로 선정된 액상 제형 조성물의 물성을 보여주는 그림으로, 시간의 경과에 따른 순도 변화(도 23a), 역가 변화(도 23b), 단백질 농도 변화(도 23c) 및 탁도 변화(도 23d)를 각각 나타낸다.Figure 23 is a picture showing the physical properties of the liquid formulation composition finally selected in the present invention, showing the change in purity over time (Figure 23a), change in titer (Figure 23b), change in protein concentration (Figure 23c), and change in turbidity (Figure 23b). Figure 23d) is shown, respectively.
도 24는 NaCl 및 수크로스 농도에 따른 장기 보관 안정성을 보여주는 그림으로, 보관 개시 시점과 6개월 경과 후의 순도(도 24a) 및 역가(도 24b) 변화를 각각 보여준다. Figure 24 is a diagram showing long-term storage stability according to NaCl and sucrose concentrations, showing changes in purity (Figure 24a) and titer (Figure 24b) at the start of storage and after 6 months, respectively.
도 25는 중화항체와 결합하는 ADAMTS13의 각 도메인의 결합부위를 확인한 결과를 보여주는 그림이다. 도 25a는 ADAMTS13의 발현율 혹은 중화 항체의 결합 부위 확인을 위해 제작한, 다양한 도메인이 조합된 6종의 ADAMTS13 절편(혹은 야생형 전장 ADAMTS13)의 모식도를 나타낸다. 도 25b는 각 ADAMTS13 도메인에서 발현하는 단백질을 이용하여 단백질 A-세파로스와 각 중화항체를 섞어 면역침강한 뒤 항-V5 항체로 웨스턴 블롯팅을 수행한 결과를 보여준다. Input은 도메인별 단백질의 발현 정도를 보여준다. 도 25c는 ADAMTS13 중화항체 결합 부위의 모식도로 Ab 4-16 항체는 S 도메인, Ab 67 항체는 D 도메인, Ab 66 항체는 T2-T8 도메인에 각각 특이적으로 결합함을 도식화한 그림이다.Figure 25 is a picture showing the results of confirming the binding site of each domain of ADAMTS13 that binds to a neutralizing antibody. Figure 25a shows a schematic diagram of six types of ADAMTS13 fragments (or wild-type full-length ADAMTS13) combining various domains, prepared to confirm the expression rate of ADAMTS13 or the binding site of a neutralizing antibody. Figure 25b shows the results of immunoprecipitation using proteins expressed in each ADAMTS13 domain by mixing protein A-Sepharose and each neutralizing antibody, followed by Western blotting with anti-V5 antibody. Input shows the expression level of protein for each domain. Figure 25c is a schematic diagram of the ADAMTS13 neutralizing antibody binding site, showing that the Ab 4-16 antibody specifically binds to the S domain, the Ab 67 antibody to the D domain, and the Ab 66 antibody to the T2-T8 domain.
도 26은 항-ADAMTS13 항체를 회피하거나 야생형 ADAMTS13보다 우수한 활성을 가지는 변이체의 선별 결과를 보여주는 그림이다. 야생형 클론과 ADAMTS13 변이체에서 Ab 4-16 항체(도 26a) 및 Ab 67 항체(도 26b)에 대한 회피율과 ADAMTS13 활성을 측정하였다. 상대활성은 야생형 클론 및 변이체의 비역가를 확인하여 다음수식에 대입함으로써 산출하였다: 상대활성 (%) = 변이체 비역가 / 야생형 ADAMTS13 비역가 X 100 Figure 26 is a diagram showing the results of selection of variants that avoid anti-ADAMTS13 antibodies or have better activity than wild-type ADAMTS13. The evasion rate and ADAMTS13 activity against Ab 4-16 antibody (Figure 26a) and Ab 67 antibody (Figure 26b) were measured in wild-type clones and ADAMTS13 mutants. Relative activity was calculated by checking the specific titers of wild-type clones and variants and substituting them into the following formula: Relative activity (%) = specific titer of variant / specific titer of wild-type ADAMTS13
도 27a-27b는 12종의 전장 ADAMTS13 변이체의 단일 중화항체 회피율을 확인한 결과이다. 12종의 전장 ADAMTS13 변이체에서 중화항체 7종(Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67)에 대한 항체 회피율을 측정하였다. 중화항체 회피율은 WT ADAMTS13 수치와 비교하여 상대적 결합 수준을 확인한 후, 이를 이용하여 상대 회피율을 다음 수식에 대입하여 산출하였다: 회피율(%) = (1- 변이체 결합능/WT ADAMTS13 결합능) x 100 Figures 27a-27b show the results of confirming the single neutralizing antibody evasion rate of 12 full-length ADAMTS13 variants. The antibody evasion rate against 7 types of neutralizing antibodies (Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67) was measured in 12 full-length ADAMTS13 variants. The neutralizing antibody evasion rate was calculated by checking the relative binding level by comparing it with the WT ADAMTS13 value and then using this to substitute the relative evasion rate into the following formula: evasion rate (%) = (1- variant binding capacity/WT ADAMTS13 binding capacity) x 100
도 28a-28b는 Fc 부착 MDTCS 절편 변이체를 발현하는 배양액 조건에서의 단일 중화항체 회피력을 확인한 결과이다. 선정된 12종 변이체를 MDTCS 절편화하여 Fc를 부착한 변이체와 선별된 변이 아미노산 잔기를 조합하여 2개의 아미노산 변이를 갖는 DM1, DM2 변이 2종을 포함한 총 14종의 변이체가 발현된 배양액을 이용하여 단일 중화항체 8종(Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67)의 중화항체 회피율과 ADAMTS13 상대 활성을 측정하였다. 회피율(%)=(1- 변이체 결합능/결합능) x 100; 상대활성(%)=변이체 비역가/WT ADAMTS13 비역가 x 100Figures 28a-28b show the results of confirming the evasion ability of a single neutralizing antibody under culture conditions expressing Fc-attached MDTCS fragment variants. By MDTCS fragmentation of the 12 selected variants, Fc-attached variants and selected variant amino acid residues were combined to produce a total of 14 variants, including 2 DM1 and DM2 variants with 2 amino acid mutations, using culture media expressing them. The neutralizing antibody evasion rate and ADAMTS13 relative activity of eight single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured. Evasion rate (%) = (1- variant binding capacity/binding capacity) x 100; Relative activity (%)=variant specific titer/WT ADAMTS13 specific titer x 100
도 29는 Fc 부착 MDTCS 절편 변이체를 발현하는 배양액 조건에서의 혼합 중화항체 회피력을 확인한 결과이다. 대조군인 MDTCS-Fc와 12종의 후보 변이체를 발현하는 배양액 4nM에 동일 비율로 혼합된 9종의 중화항체(Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67)를 혼합하여 반응시킨 후, 각 후보 변이체의 잔존 활성을 다음 수식에 대입하여 산출하였다: 잔존활성(%) = A ÷ B x 100 (A: 혼합 중화항체 조건에서의 활성, B: 중화항체 처리하지 않은 조건에서의 활성)Figure 29 shows the results of confirming the evasion ability of mixed neutralizing antibodies under culture conditions expressing Fc-attached MDTCS fragment variants. 9 types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, After reacting by mixing Ab67), the residual activity of each candidate variant was calculated by substituting the following formula: Residual activity (%) = A ÷ B x 100 (A: Activity under mixed neutralizing antibody conditions, B: Neutralizing antibody activity under untreated conditions)
도 30a-30b는 Fc 부착 MDTCS 절편 변이체 배양액과 정제액 조건에서의 단일 중화항체 회피력을 확인한 결과이다. 12종의 변이체(1C03, 2B01, 2B02, 3B05, 4E11, 4H07, 5G08, 7A02, 8D01, 8D05, DM1, DM2)가 발현된 배양액 혹은 Phytip system으로 정제한 정제액을 이용하여 단일 중화항체 8종(Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67)에 대한 중화항체 회피율과 상대 활성을 측정하였다(도 30a 및 30b). 해당 정제액의 농도는 Fc ELISA를 통해 확인하였다. 회피율(%)=(1- 변이체 결합능/MDTCS-Fc 결합능) x 100, 상대활성 (%) = 변이체 비역가/ MDTCS-Fc 비역가 x 100. 파란색 바(bar)는 정제된 변이 단백질을, 회색 바는 발현된 변이 단백질의 중앙값을 각각 나타낸다. Figures 30a-30b show the results of confirming the evasion ability of a single neutralizing antibody in the culture and purification conditions of the Fc-attached MDTCS fragment variant. 8 types of single neutralizing antibodies ( Neutralizing antibody evasion rate and relative activity for (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67) were measured (Figures 30a and 30b). The concentration of the purified solution was confirmed through Fc ELISA. Evasion rate (%) = (1- variant binding capacity/MDTCS-Fc binding capacity) x 100, relative activity (%) = variant specific titer/MDTCS-Fc specific titer x 100. Blue bars represent purified mutant proteins, gray bars The median value of expressed mutant proteins is shown, respectively.
도 31은 Fc 부착 MDTCS 절편 변이체의 배양액과 정제액 조건에서의 혼합 중화항체 회피력을 확인한 결과이다. 대조군인 MDTCS-Fc와 12종의 후보 변이체를 발현하는 배양액 혹은 정제액 4 nM에 동일 비율로 혼합된 8종의 중화항체(Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67)를 혼합하여 반응 시킨 후, 각 후보 변이체의 잔존활성을 측정하였다. 잔존 활성은 아래 수식에 대입하여 산출하였다. 잔존활성 (%) = A ÷ B x 100 (A: 혼합 중화항체 조건에서의 활성, B: 중화항체 처리하지 않은 조건에서의 활성)Figure 31 shows the results of confirming the ability of the Fc-attached MDTCS fragment variant to evade mixed neutralizing antibodies in culture and purification conditions. Eight types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, After reacting by mixing Ab65, Ab67), the residual activity of each candidate variant was measured. Residual activity was calculated by substituting the formula below. Residual activity (%) = A ÷ B x 100 (A: Activity under mixed neutralizing antibody conditions, B: Activity under conditions without neutralizing antibody treatment)
도 32는 Fc 부착 MDTCS 절편 변이체의 약동학 결과를 보여주는 그래프이다. MDTCS 혹은 Fc(IgG1-YTE)가 부착 되어있는 MDTCS 및 4개 최종 후보 변이체(1C03, 5C09, 7A02, DM2) 절편 단백질을 마우스에 꼬리 정맥에 투여한 후 시간 별로 혈장을 확보하였다. 각 물질의 비역가를 기준으로 160 IU/kg가 되도록 투여하였고 활성 어세이를 통해 각 시간별로 확보한 혈장 내 잔존하는 물질의 활성을 측정하였다.Figure 32 is a graph showing the pharmacokinetic results of Fc-attached MDTCS fragment variants. MDTCS or Fc (IgG1-YTE)-attached MDTCS and four final candidate variants (1C03, 5C09, 7A02, DM2) fragment proteins were administered to mice through the tail vein, and plasma was obtained over time. Based on the specific titer of each substance, it was administered to reach 160 IU/kg, and the activity of substances remaining in the plasma obtained at each time was measured through an activity assay.
도 33은 IgG1-YTE 부착 MDTCS 절편 변이체를 발현하는 배양액 조건에서의 단일 중화항체에 대한 회피력을 확인한 결과이다. 선정된 5종 변이체를 MDTCS 절편화하여 IgG1-YTE를 부착한 변이체가 발현된 배양액을 이용하여 단일 중화항체 8종 (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab67)의 중화항체 회피율과 상대 활성을 측정하였다(도 33a 및 33b). 회피율(%)=(1-변이체 결합능/ MDTCS-IgG1-YTE 결합능) x 100, 상대활성(%) = 변이체 비역가/MDTCS-IgG1-YTE 비역가 x 100.Figure 33 shows the results of confirming the evasion ability against a single neutralizing antibody under culture conditions expressing MDTCS fragment variants attached to IgG1-YTE. The five selected variants were subjected to MDTCS fragmentation, and 8 types of single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, The neutralizing antibody evasion rate and relative activity of Ab67) were measured (Figures 33a and 33b). Evasion rate (%) = (1-variant binding capacity/MDTCS-IgG1-YTE binding capacity) x 100, relative activity (%) = variant specific titer/MDTCS-IgG1-YTE specific titer x 100.
도 34는 IgG1-YTE 부착 MDTCS 절편 변이체를 발현하는 배양액 조건에서의 혼합 중화항체에 대한 회피력을 확인한 결과이다. 대조군인 MDTCS-IgG1-YTE와 5종의 후보 변이체를 발현하는 배양액 4nM에 동일 비율로 혼합된 9종의 중화항체 (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67)를 혼합하여 반응 시킨 후, 각 후보 변이체의 잔존활성을 다음 수식으로 산출하였다: 잔존활성(%)= A ÷ B x 100 (A: 혼합 중화항체 조건에서의 활성, B: 중화항체 처리하지 않은 조건에서의 활성).Figure 34 shows the results of confirming the evasion ability against mixed neutralizing antibodies under culture conditions expressing MDTCS fragment variants attached to IgG1-YTE. 9 types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, After reacting by mixing Ab66, Ab67), 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 conditions, B: Neutralizing antibody activity under untreated conditions).
도 35는 IgG1-YTE 부착 MDTCS 절편 변이체의 정제액 조건에서의 혼합 중화항체에 대한 회피력을 확인한 결과를 나타낸다. 대조군인 MDTCS-IgG1-YTE와 5종의 후보 변이체를 정제액 4nM에 동일 비율로 혼합된 9종의 중화항체 (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67)를 혼합하여 반응 시킨 후, 각 후보 변이체의 잔존활성을 다음 수식으로 산출하였다: 잔존활성(%) = A ÷ B x 100 (A: 혼합 중화항체 조건에서의 활성, B: 중화항체 처리하지 않은 조건에서의 활성).Figure 35 shows the results of confirming the evasion ability of the MDTCS fragment variant attached to IgG1-YTE to mixed neutralizing antibodies under purified solution conditions. 9 types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66) mixed in equal proportions of control MDTCS-IgG1-YTE and 5 candidate variants in 4nM purified solution , Ab67), 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 conditions, B: Neutralizing antibody treatment activity under no conditions).
도 36은 aTTP-mimic 마우스 모델을 이용한 중화항체 회피율 시험절차에 대한 모식도(도 36a) 및 변이체 후보물질들의 용량 별 ADAMTS13 잔존 활성(도 36b)을 각각 보여주는 그림이다.Figure 36 is a schematic diagram of the neutralizing antibody evasion rate test procedure using the aTTP-mimic mouse model (Figure 36a) and a diagram showing the ADAMTS13 residual activity by dose of variant candidate substances (Figure 36b).
도 37은 TTP-mimic 마우스 모델을 이용하여 가장 우수한 중화항체 회피율을 보인 DM2-IgG1-YTE의 농도별 ADAMTS13 잔존활성 유지력과 임상학적 증상의 시험절차에 대한 모식도(도 37a), 혈소판과 LDH 수치의 개선 및 ADAMTS13 활성(도 37b) 및 임상 증상 관찰 결과(도 37c)를 각각 보여주는 그림이다.Figure 37 is a schematic diagram of the test procedure for ADAMTS13 residual activity maintenance and clinical symptoms by concentration of DM2-IgG1-YTE, which showed the best neutralizing antibody evasion rate using the TTP-mimic mouse model (Figure 37a), platelet and LDH levels This figure shows the improvement and ADAMTS13 activity (Figure 37b) and clinical symptom observation results (Figure 37c), respectively.
도 38은 cTTP 마우스 모델에 DM2-IgG1-YTE를 투여하여 혈액학적 그리고 임상학적 증상의 개선 여부와 인간 ADAMTS13 활성 회복 정도의 시험절차에 대한 모식도(도 38a) 및 혈소판과 LDH 수치의 개선 및 ADAMTS13 활성 회복을 관찰한 결과(도 38b)를 각각 보여주는 그림이다. Figure 38 is a schematic diagram of the test procedure for improvement of hematological and clinical symptoms and degree of recovery of human ADAMTS13 activity by administering DM2-IgG1-YTE to a cTTP mouse model (Figure 38a), and improvement of platelet and LDH levels and ADAMTS13 activity. This figure shows the results of observing recovery (Figure 38b).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
실시예 1: ADAMTS13 단백질의 제형 성분의 선별 Example 1: Selection of formulation components of ADAMTS13 protein
하기 실시예 3에서 후술하는 ADAMTS13 단백질에 대한 액상 제형 조성물의 각 성분과 함량을 아래와 같은 과정을 통해 결정하였다.Each component and content of the liquid formulation composition for ADAMTS13 protein, described later in Example 3, were determined through the following process.
동결건조 공정 Freeze drying process
제형화가 완료된 각 샘플 용액을 글래스 바이알(3 ml)에 1.0ml씩 분주하고, 고무마개로 반타전 후 동결 건조기(Freeze dryer, Lyostar 3, SP scientific) 선반 위에 로딩하였다. 이후, 하기 표 1의 조건으로 동결 건조를 진행하고, 제조된 동결 건조 제제는 동결 건조 완료 후 알루미늄 캡(Aluminum cap)으로 캡핑하였다.1.0 ml of each sample solution whose formulation was completed was dispensed into a glass vial (3 ml), half-closed with a rubber stopper, and then loaded onto the shelf of a freeze dryer (Lyostar 3, SP scientific). Thereafter, freeze-drying was performed under the conditions shown in Table 1 below, and the prepared freeze-dried preparation was capped with an aluminum cap after freeze-drying was completed.
단계step | 구분division | Rate/holdRate/hold | 선반온도shelf temperature | 시간(min)Time (min) | 진공도(mTorr)Vacuum degree (mTorr) | |
1One |
로딩 | HoldHold | 5℃5℃ | 2020 |
N/AN/ |
|
22 | 동결freezing | RateRate | -55℃-55℃ | 200200 |
N/AN/ |
|
33 | 동결freezing | HoldHold | -55℃-55℃ | 300300 |
N/AN/ |
|
44 | 1차 가온1st warming | RateRate |
-25℃-25 |
100100 | 6060 | |
55 | 1차 건조1st drying | HoldHold |
-25℃-25 |
20002000 | 6060 | |
77 |
2차 가온 | RateRate | 25℃25℃ | 167167 | 5050 | |
88 |
2차 건조 | HoldHold | 25℃25℃ | 600600 | 5050 |
제조된 동결 건조 제제의 안정성은 1.0 ml 증류수로 재구축(reconstitution) 한 후 분석을 수행하였다.The stability of the prepared freeze-dried preparation was analyzed after reconstitution with 1.0 ml distilled water.
크기 배제 액체 크로마토그래피(SE-HPLC)Size exclusion liquid chromatography (SE-HPLC)
크기 배제 크로마토그래피 수행을 위해 검체를 이동상(1 x PBS, Lonza)으로 1.0 mg/ml가 되도록 희석한 후(1.0 mg/mml 이하 시 희석없이 진행) 멸균 여과하고, 여과된 검체 200μL를 바이알 인서트(vial insert)에 주입 후 스크류탑 바이알(screw top vial)에 끼워서 준비하였다. 이후 이동상을 펌프에 연결한 뒤, Waters e2695와 Waters 2489 기기(일본 Waters사)에 0.5 mL/min의 유속으로 이동상을 흘려주며 분석 컬럼(TSKgel G3000SWXL, Tosoh)을 장착하였다. 0.5mL/min 속도로 이동상을 30분 이상 흘려주어 검출 신호(detector signal)가 안정화 될 때까지 평형에 도달시키고, 오토샘플러의 온도가 4℃로 떨어지면 검체를 샘플러에 꽂았다. 검체 30μL를 주입한 뒤 이동상으로 35분 간 흘려주어 280 nm에서 검출 피크(detection peak)를 확인하였다. 이후 PC의 Empower Pro software로 분석을 수행하였다.To perform size exclusion chromatography, the sample was diluted to 1.0 mg/ml with a mobile phase (1 It was prepared by inserting it into a screw top vial. After connecting the mobile phase to the pump, the mobile phase was flowed through Waters e2695 and Waters 2489 instruments (Waters, Japan) at a flow rate of 0.5 mL/min, and an analytical column (TSKgel G3000SWXL, Tosoh) was installed. The mobile phase was flowed at a rate of 0.5 mL/min for more than 30 minutes to reach equilibrium until the detector signal was stabilized, and when the temperature of the autosampler fell to 4°C, the sample was inserted into the sampler. After injecting 30 μL of the sample, the mobile phase was run for 35 minutes to confirm the detection peak at 280 nm. Afterwards, analysis was performed using Empower Pro software on a PC.
콜로이드 안정성 (B22 ) Colloidal stability ( B 22 )
단백질-단백질 상호작용 정도가 응집과 용해도 등에 영향을 미치기 때문에 콜로이드 안정성은 단백질 제형의 개발에 있어 반드시 고려되어야 하는 중요한 항목이다. 대표적인 콜로이드 안정성 지표인 B22(second virial coefficient)를 활용하였으며, 일반적으로 B22값이 큰 양의 값을 띌 경우, 단백질 간 반발력(repulsion force)이 강하여 응집이 발생한 확률이 줄어들게 된다. Because the degree of protein-protein interaction affects aggregation and solubility, colloidal stability is an important item that must be considered in the development of protein formulations. B 22 (second virial coefficient), a representative colloidal stability index, was used. In general, when the B 22 value is large and positive, the repulsion force between proteins is strong, reducing the probability of aggregation occurring.
분석하고자 하는 검체를 5가지 농도(1.2, 0.96, 0.72, 0.48 및 0.24 mg/ml)로 희석하여 준비한 뒤, 희석된 검체 8.8 μL를 Uni sample loader(Unchained Labs)에 3회 반복하여 주입하였다. 각 해당 농도에서의 산란광 세기(scattered light intensities)를 측정하고 플라시보 완충액의 산란광 세기를 빼준 뒤 Zimm 공식을 통해 최종적인 B22값을 계산하였다(4). 형광 데이터 분석은 UNcle Analysis 소프트웨어(Unchained Labs)를 사용하였다.The sample to be analyzed was prepared by diluting it to 5 concentrations (1.2, 0.96, 0.72, 0.48, and 0.24 mg/ml), and then 8.8 μL of the diluted sample was repeatedly injected into the Uni sample loader (Unchained Labs) three times. The scattered light intensities at each corresponding concentration were measured, the scattered light intensity of the placebo buffer was subtracted, and the final B 22 value was calculated using the Zimm formula (4). Fluorescence data analysis was performed using UNcle Analysis software (Unchained Labs).
탁도(Turbidity) 분석Turbidity analysis
탁도는 Lunatic(Unchained Labs)으로 분석하였으며, 샘플 2.0 μL를 Lunatic 플레이트(Unchained Labs)에 주입하고 350nm에서의 탁도를 측정하였으며, 플라시보 완충액의 탁도는 샘플의 탁도 값에서 빼서 최종값을 계산하였다.Turbidity was analyzed with Lunatic (Unchained Labs). 2.0 μL of the sample was injected into a Lunatic plate (Unchained Labs) and the turbidity was measured at 350 nm. The turbidity of the placebo buffer was subtracted from the turbidity value of the sample to calculate the final value.
열 풀림 및 열 응집체 형성 (Thermal unfolding and thermal aggregation) 분석Thermal unfolding and thermal aggregation analysis
온도가 높아짐에 따라 단백질이 풀릴 때(unfolding) 표면에 노출된 트립토판에 의한 방출(emission) 파장을 검출함으로써 단백질이 풀린 정도를 측정할 수 있다. 이와 같은 고유 형광 강도에 기반한 시차 주사 형광측정법(differential scanning fluorimetry) 분석을 위해 UNCLE(Unchained Labs) 장비를 사용하였다. 상기 장비를 이용하여 단백질의 열 안정성을 분석하기 위해 Tm(Thermal unfolding) 및 Tagg(Thermal aggregation)를 다음과 같이 측정하였다: As the temperature increases, the degree to which a protein is unfolded can be measured by detecting the emission wavelength caused by tryptophan exposed on the surface when the protein unfolds. UNCLE (Unchained Labs) equipment was used to analyze differential scanning fluorimetry based on this intrinsic fluorescence intensity. To analyze the thermal stability of proteins using the above equipment, Tm (thermal unfolding) and Tagg (thermal aggregation) were measured as follows:
검체 8.8 μL를 Uni 샘플 로더(Unchained Labs)에 2회 반복 주입 후 온도를 25℃부터 1℃/min 속도로 95℃까지 증가시켰다. 온도를 증가시키면서 266nm의 여기 파장(Excitation wavelength)에 의해 방출되는 파장(250-720nm)의 강도를 측정하였다. 형광 데이터 분석(Fluorescence data analysis)은 UNCLE Analysis 소프트웨어(Unchained Labs)를 사용하였다. After repeatedly injecting 8.8 μL of the sample into the Uni sample loader (Unchained Labs) twice, the temperature was increased from 25°C to 95°C at a rate of 1°C/min. As the temperature was increased, the intensity of the wavelength (250-720nm) emitted by the excitation wavelength of 266nm was measured. Fluorescence data analysis used UNCLE Analysis software (Unchained Labs).
상기 분석을 통해 형광 방출 피크의 최대값 시점의 온도를 Tm으로 정의하고, 단백질의 266 nm (SLS266)에서의 정적 광산란(static light scattering)을 측정하여 단백질 응집이 시작되는 시점의 온도(protein aggregation onset temperature)를 Tagg으로 정의하였다.Through the above analysis, the temperature at the maximum of the fluorescence emission peak was defined as Tm, and the temperature at the start of protein aggregation was determined by measuring static light scattering at 266 nm (SLS266) of the protein. temperature) was defined as Tagg.
NaCl 농도에 따른 동결건조 조성물의 품질 평가Quality evaluation of freeze-dried composition according to NaCl concentration
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80를 포함하는 pH 7.4의 조성물에 NaCl을 50, 100, 120, 150, 200 및 300 mM 농도로 첨가하였다. 제형화가 완료된 샘플을 동결 건조하여 케익 성상을 관찰하고 SE-HPLC 분석을 수행g나 결과, NaCl 농도가 증가될수록 보다 견고하고 우수한 케익 성상을 보인 반면(도 1a), SE-HPLC 결과 NaCl 농도에 따른 주요할 만한 순도 차이는 관찰되지 않았다(도 1b).NaCl was added to a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 and 0.05% PS80 at concentrations of 50, 100, 120, 150, 200 and 300 mM. The formulation-completed sample was freeze-dried to observe the cake properties and SE-HPLC analysis was performed. As a result, as the NaCl concentration increased, the cake properties were more solid and excellent (Figure 1a), while the SE-HPLC results showed that the cake properties increased depending on the NaCl concentration. No significant purity differences were observed (Figure 1b).
NaCl 농도에 따른 액상 안정성 평가Liquid stability evaluation according to NaCl concentration
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2 및 20mM 아르기닌을 포함하는 pH 7.4의 조성물에 NaCl을 0, 40, 80, 120 및 160 mM 농도로 첨가하였다. 제형화가 완료된 액상 샘플을 상온에서 보관하며, 4, 8, 12시간이 되는 시점마다 SE-HPLC 분석을 수행한 결과, 도 2에서 보는 바와 같이 NaCl의 농도가 높을수록 액상 안정성이 개선됨을 확인하였다.NaCl was added to a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20mM histidine, 4.0mM CaCl 2 and 20mM arginine at concentrations of 0, 40, 80, 120 and 160mM. The formulated liquid sample was stored at room temperature, and SE-HPLC analysis was performed at 4, 8, and 12 hours. As shown in Figure 2, it was confirmed that the higher the concentration of NaCl, the better the liquid stability.
당 농도에 따른 동결건조 조성물의 품질 평가 (NaCl 120mM)Quality evaluation of freeze-dried composition according to sugar concentration (NaCl 120mM)
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 120 mM NaCl, 20mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80을 포함하는 pH 7.4의 조성물에 0.0, 0.5, 1.0 및 2.0 %(w/v) 농도의 수크로스 또는 1.0 %(w/v) 농도의 트레할로스를 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 SE-HPLC 분석을 수행한 결과, NaCl 120mM에서는 수크로스 1.0% 첨가 시엔 붕괴(collapse)가 발생하지 않았지만 2.0% 첨가 시 붕괴가 발생하여 붕괴가 개시되는 수크로스 농도는 1.0 - 2.0% 사이로 예측되었다(도 3a). 트레할로스 1.0% 첨가 제형은 수크로스 1.0% 첨가 제형과 유사한 성상을 보였다(도 3a). 한편, SE-HPLC 결과 첨가되는 당 농도에 따른 유의한 순도 차이는 관찰되지 않아 붕괴가 발생하여도 순도는 유지됨을 알 수 있었다(도 3b).Sucrose at concentrations of 0.0, 0.5, 1.0 and 2.0% (w/v) in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 120 mM NaCl, 20mM histidine, 4.0 mM CaCl 2 and 0.05% PS80 in common composition. Alternatively, trehalose was added at a concentration of 1.0% (w/v). As a result of freeze-drying the formulated sample, observing the cake properties, and performing SE-HPLC analysis, it was found that in NaCl 120mM, no collapse occurred when 1.0% sucrose was added, but collapse occurred when 2.0% was added, and collapse began. The resulting sucrose concentration was predicted to be between 1.0 and 2.0% (Figure 3a). The formulation with 1.0% trehalose showed similar properties to the formulation with 1.0% sucrose (Figure 3a). Meanwhile, SE-HPLC results No significant difference in purity was observed depending on the added sugar concentration, showing that purity was maintained even when collapse occurred (Figure 3b).
수크로스와 증량제(bulking agent)의 함량비에 따른 동결건조 조성물의 품질 평가(NaCl 120mM)Quality evaluation of freeze-dried composition according to the content ratio of sucrose and bulking agent (NaCl 120mM)
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 120 mM NaCl, 20mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80을 포함하는 pH 7.4의 조성물에 수크로스와 증량제(만니톨, 글라이신)를 각각 1.0 / 3.0(w/v) (함량비 1:3)과 2.0 / 2.0 %(w/v)(함량비 1:1)로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 및 SE-HPLC 분석을 수행한 결과, NaCl을 120mM로 고정하고 수크로스 대 증량제의 함량비를 1:3 및 2:2로 첨가한 경우 모두 붕괴가 발생하였으며(도 4a), SE-HPLC 결과 시료 간 유의한 차이가 관찰되지 않았다(도 4b).Sucrose and extenders ( 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) were added. The formulated sample was lyophilized to observe the cake properties and SE-HPLC analysis was performed. As a result, all samples collapsed when NaCl was fixed at 120mM and the sucrose to extender content ratio was added at 1:3 and 2:2. occurred (Figure 4a), and no significant difference was observed between samples as a result of SE-HPLC (Figure 4b).
글라이신 농도에 따른 동결건조 품질 평가 (NaCl 120mM)Freeze-drying quality evaluation according to glycine concentration (NaCl 120mM)
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 120mM NaCl, 수크로스 1.0%, 20 mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80을 포함하는 pH 7.4의 조성물에 글라이신을 0, 20, 40, 60, 80 및 100 mM 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 및 SE-HPLC 분석을 수행한 결과, NaCl 120mM과 수크로스 1.0%가 고정적으로 첨가되고 글라이신의 농도를 증가시킬 경우 케익 성상에 부정적인 영향을 미치며, 글라이신을 60mM 이상 첨가 시 완전히 붕괴됨을 알 수 있었다(도 5).Glycine was added at 0, 20 , 40, 60, 80 and It was added at a concentration of 100 mM. As a result of observing the cake properties by freeze-drying the formulated sample and performing SE-HPLC analysis, it was found that when NaCl 120mM and sucrose 1.0% were added fixedly and the concentration of glycine was increased, it had a negative effect on the cake properties; It was found that when 60mM or more of glycine was added, it completely collapsed (Figure 5).
아르기닌 농도에 따른 동결건조 품질 평가 (NaCl 120mM)Freeze-dried quality evaluation according to arginine concentration (NaCl 120mM)
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 120mM NaCl, 수크로스 1.0%, 20 mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80을 포함하는 pH7.4의 조성물에 아르기닌을 0, 20, 60 및 100 mM의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 SE-HPLC 분석을 수행한 결과, NaCl 120mM 및 수크로스 1.0%가 고정적으로 첨가되고 아르기닌 농도를 증가시킬 경우 케익 성상에 부정적인 영향을 미치며, 아르기닌을 60mM 이상 첨가 시 완전히 붕괴되었다(도 6).Arginine was added at 0, 20, 60 and 100 mM in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 120mM NaCl , 1.0% sucrose , 20mM histidine, 4.0mM CaCl 2 and 0.05% PS80 in common composition. It was added in concentration. As a result of freeze-drying the formulated sample to observe the cake properties and performing SE-HPLC analysis, it was found that when NaCl 120mM and sucrose 1.0% were added fixedly and the arginine concentration was increased, it had a negative effect on the cake properties, and arginine When more than 60mM was added, it completely collapsed (Figure 6).
NaCl과 수크로스의 함량비에 따른 동결건조 품질 평가 (w/o 아르기닌)Freeze-dried quality evaluation according to the content ratio of NaCl and sucrose (w/o arginine)
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2 및 0.05% PS80을 포함하는 pH 7.4의 조성물에 NaCl을 120, 160 및 200 mM의 농도로, 수크로스를 1.0, 1.5 및 2.0 %(w/v)의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 SE-HPLC 분석을 수행한 결과, NaCl 농도가 증가될수록, 수크로스 농도가 감소될수록 양호한 케익 성상을 보였는데, 구체적으로 i) NaCl 120mM 첨가한 경우 수크로스 1.0-1.5% 범위 내에 붕괴 개시 시점이 존재하며, ⅱ) NaCl 160mM 첨가한 경우 수크로스 1.5-2.0% 구간에서, ⅲ) NaCl 200mM 첨가한 경우 수크로스 2.0% 초과 구간에서 각각 붕괴가 개시되는 것으로 확인되었다(도 7a).NaCl at concentrations of 120, 160 and 200 mM and sucrose at 1.0, 1.5 and 2.0 in a composition at pH 7.4 containing in common composition 1.2 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 and 0.05% PS80. It was added at a concentration of % (w/v). As a result of freeze-drying the formulated sample to observe the cake properties and performing SE-HPLC analysis, the cake properties were shown to be better as the NaCl concentration increased and the sucrose concentration decreased. Specifically, i) when 120mM of NaCl was added. There is a start point of collapse within the range of 1.0-1.5% of sucrose, ii) when 160mM of NaCl is added, in the range of 1.5-2.0% of sucrose, and iii) when 200mM of NaCl is added, collapse begins in the range of more than 2.0% of sucrose. It was confirmed that (Figure 7a).
한편 SE-HPLC 결과 아르기닌이 첨가되지 않은 경우에는 NaCl과 수크로스 함량비에 따른 유의한 변화는 관찰되지 않았다(도 7b).Meanwhile, as a result of SE-HPLC, when arginine was not added, no significant change was observed depending on the NaCl and sucrose content ratio (Figure 7b).
NaCl과 수크로스의 함량비에 따른 동결건조 품질 평가 (아르기닌 120mM)Freeze-dried quality evaluation according to the content ratio of NaCl and sucrose (arginine 120mM)
공통 조성으로 0.5 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2, 0.05% PS80 및 120mM 아르기닌을 포함하는 pH 7.4의 조성물에 NaCl을 160, 200, 240 및 280 mM의 농도로, 수크로스를 0.0, 0.5 및 1.0 %(w/v)의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결 건조하여 케익 성상을 관찰하고 SE-HPLC 분석을 수행한 결과, 아르기닌 120mM로 고정 첨가한 경우, NaCl 농도는 증가될수록, 수크로스 농도는 감소될수록 견고한 케익 성상이 나타났으며, 구체적으로 i) NaCl 160mM 첨가한 경우 수크로스 0.0-0.5% 이상 첨가 구간에서, ⅱ) NaCl 200mM 첨가한 경우 수크로스 1.0% 초과 첨가 구간에서, ⅲ) NaCl 240mM 첨가한 경우 수크로스 1.0% 초과 첨가 구간에서, ⅳ) NaCl 280mM 첨가한 경우 수크로스 1.0% 초과 첨가 구간에서 각각 붕괴 개시 시점이 존재함을 알 수 있었다(도 8a).NaCl was added at concentrations of 160, 200, 240 and 280 mM and sucrose in a composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 0.05% PS80 and 120 mM arginine as a common composition. It was added at concentrations of 0.0, 0.5, and 1.0 % (w/v). As a result of freeze-drying the formulated sample to observe the cake properties and performing SE-HPLC analysis, when arginine was fixedly added at 120mM, solid cake properties appeared as the NaCl concentration increased and the sucrose concentration decreased. Specifically, i) in the section where more than 0.0-0.5% of sucrose was added when 160mM of NaCl was added, ii) in the section where more than 1.0% of sucrose was added when 200mM of NaCl was added, iii) in the section where more than 1.0% of sucrose was added when 240mM of NaCl was added. In, iv) when 280mM of NaCl was added, it was found that there was a collapse initiation point in each section where more than 1.0% sucrose was added (FIG. 8a).
한편, SE-HPLC 결과 아르기닌 120mM가 포함된 경우 NaCl 및 수크로스 농도에 따른 주목할 만한 순도 변화는 확인되지 않았으나, NaCl 160mM 및 수크로스 미첨가 제형에서 순도 97.4%로 다른 시료 대비 상대적으로 낮은 결과를 보였다(도 8b).Meanwhile, SE-HPLC results showed no notable change in purity depending on NaCl and sucrose concentration when arginine 120mM was included, but the purity was 97.4% in the formulation without NaCl 160mM and sucrose, which was relatively low compared to other samples. (Figure 8b).
NaCl 대 수크로스 배합비에 따른 동결건조 안정성 평가 (아르기닌 120mM)Evaluation of freeze-drying stability according to NaCl to sucrose mixing ratio (arginine 120mM)
공통 조성으로 0.5 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2, 0.05% PS80 및 120mM 아르기닌을 포함하는 pH 7.4의 조성물에 NaCl을 200, 240 및 280 mM의 농도로, 수크로스를 0.0, 0.5 및 1.0 %(w/v)의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결 건조하여 가속 안정성(40℃)를 평가한 결과, 120mM 아르기닌 존재 하에서 NaCl 대 수크로스 배합비에 따라 40℃ 1개월차 순도는 초기 대비 유의한 변화가 나타나지 않았다(도 9).A composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 0.05% PS80, and 120 mM arginine as a common composition was added with NaCl at concentrations of 200, 240, and 280 mM, sucrose at 0.0, and It was added at concentrations of 0.5 and 1.0% (w/v). As a result of evaluating the accelerated stability (40°C) by freeze-drying the formulated sample, there was no significant change in purity at 40°C for 1 month compared to the initial period depending on the NaCl to sucrose mixing ratio in the presence of 120mM arginine (FIG. 9).
안정제 5종에 대한 콜로이드 안정성 평가(UNCLE, B22 ) Colloidal stability evaluation of five types of stabilizers ( UNCLE, B 22 )
안정제 5종에 대한 콜로이드 안정성을 평가하기 위해 공통조성으로서 1.2 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 4.0 mM CaCl2, 120mM NaCl, 1.0% 수크로스를 포함하는 pH 7.4의 조성물에 아미노산으로서 글라이신, 라이신, 프롤린, 알라닌 또는 아르기닌을 각 100mM 포함시켰다.To evaluate the colloidal stability of five types of stabilizers, glycine and lysine were used as amino acids in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20 mM histidine, 4.0 mM CaCl 2 , 120mM NaCl, and 1.0% sucrose as a common composition. , proline, alanine, or arginine were included at 100mM each.
제형화가 완료된 각 조성물의 액상 샘플을 UNCLE 기기를 활용하여 콜로이드 안정성(B22)을 분석하였다. Liquid samples of each composition after completion of formulation were analyzed for colloidal stability (B 22 ) using the UNCLE instrument.
B22 값이 양의 값으로 커질수록 단백질끼리 서로 뭉치지 않고 잘 분산되어 있음을 의미하므로, 도 10에서 보는 바와 같이 콜로이드 안정성은 아르기닌 > 글라이신 > 프롤린 > 알라닌 > 라이신 > 대조군 순으로 확인되었다. As the B 22 value increases to a positive value, it means that the proteins do not clump together and are well dispersed. As shown in Figure 10, colloidal stability was confirmed in the order of arginine > glycine > proline > alanine > lysine > control.
안정제 7종에 대한 SE-HPLC평가 SE-HPLC evaluation of 7 types of stabilizers
공통 조성으로서 0.9 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 2.0 mM CaCl2 및 120 mM NaCl를 포함하는 pH 7.4의 조성물에 아미노산 안정화제로서 아르기닌, 세린, 발린, 트레오닌, 프롤린 또는 글라이신을 각 100mM 포함시키거나 또는 당 안정화제로서 수크로스를 1.0 w/v%를 포함시켰다.A composition at pH 7.4 containing 0.9 mg/ml ADAMTS protein, 20 mM histidine, 2.0 mM CaCl 2 and 120 mM NaCl as a common composition includes 100 mM each of arginine, serine, valine, threonine, proline or glycine as amino acid stabilizers. Alternatively, 1.0 w/v% of sucrose was included as a sugar stabilizer.
제형화가 완료된 각 조성물의 액상 샘플을 상온에서 7시간 보관 후 SE-HPLC 분석을 수행하였다. 그 결과, 도 11에서 보는 바와 같이 비교실험을 진행한 7종의 안정화제에 의한 액상 안정성은 역시 아르기닌이 가장 우수하였으며, 안정성 및 순도는 아르기닌 > 수크로스 > 프롤린 > 글라이신 > 발린, 트레오닌 > 대조군 > 세린의 순서로 높았다.Liquid samples of each composition whose formulation was completed were stored at room temperature for 7 hours and then subjected to SE-HPLC analysis. As a result, as shown in Figure 11, arginine had the best liquid stability among the seven stabilizers in the comparative experiment, and the stability and purity were arginine > sucrose > proline > glycine > valine, threonine > control > It was ranked highest in the order of Serine.
아르기닌 첨가에 따른 액상 안정성 평가Liquid stability evaluation according to arginine addition
0.049 mg/mL ADAMTS 단백질, 15 mM 인산나트륨, 50mM NaCl을 포함하는 pH 7.4의 조성물에 20mM 아르기닌의 첨가 여부에 따른 액상 안정성을 평가하기 위해 제형화가 완료된 액상 샘플을 상온에서 3시간 및 19시간 보관 후 각 분석 시점마다 SE-HPLC 분석을 수행하였다. 그 결과, 도 12에서 보는 바와 같이 아르기닌 20mM 첨가 시 대조군 대비 5℃에서의 냉장 안정성이 현저히 개선됨을 확인하였다.To evaluate the liquid stability according to the addition of 20mM arginine to a pH 7.4 composition containing 0.049 mg/mL ADAMTS protein, 15mM sodium phosphate, and 50mM NaCl, the formulated liquid sample was stored 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 12, it was confirmed that when 20mM of arginine was added, the refrigeration stability at 5°C was significantly improved compared to the control group.
목적 단백질 농도 및 아르기닌 농도에 따른 액상 안정성 평가Liquid stability evaluation according to objective protein concentration and arginine concentration
SE-HPLC 분석SE-HPLC analysis
공통 조성으로서 20 mM 히스티딘, 4.0 mM CaCl2 및 160 mM NaCl를 포함하는 pH 7.4의 조성물에 ADAMTS 단백질을 각각 0.2, 0.6 및 1.0 mg/ml로 포함시키고, 아르기닌을 20, 80 및 140 mM 농도로 포함시킨 액상 샘플의 안정성을 평가하였다.ADAMTS protein was included at 0.2, 0.6 and 1.0 mg/ml, respectively, and arginine at concentrations of 20, 80 and 140 mM in a composition at pH 7.4 containing 20 mM histidine, 4.0 mM CaCl 2 and 160 mM NaCl as a common composition. The stability of the prepared liquid samples was evaluated.
제형화가 완료된 시료를 상온에서 18시간 보관 후 SE-HPLC 분석을 수행한 결과, 도 13a에서 보는 바와 같이 목적 단백질의 농도가 감소될수록, 아르기닌 농도가 증가될수록 순도가 개선됨을 확인하였다.As a result of performing SE-HPLC analysis on the formulated sample after storing it at room temperature for 18 hours, it was confirmed that the purity improved as the concentration of the target protein decreased and the arginine concentration increased, as shown in Figure 13a.
DOE 설계DOE design
한편, 아르기닌과 목적 단백질의 최적 농도를 보다 면밀하게 탐색하기 위해 DOE (Design Of Experiments)를 수행하였다. 아르기닌 농도 및 ADAMTS 단백질 농도라는 2개의 작동 파라미터를 요인(X 값)으로 설정하고 순도(SE-HPLC)를 반응(Y 값)으로 설정한 후 JMP® 10.0 통계 프로그램을 사용하여 DOE 설계를 실시하였다. 구체적으로, RSM(Response Surface Model)을 이용하였으며, CCD(Central Composite Design) 타입으로 축점을 설정하였다. 최종적으로 “2수준의 full factorial design 4 Runs + 중심점 1 Run + 2개의 인자에 대한 축점 4 Runs”을 포함한 총 9 Run의 조건을 설계하였다. Meanwhile, DOE (Design Of Experiments) was performed to more closely explore the optimal concentration of arginine and the target protein. The two operating parameters, arginine concentration and ADAMTS protein concentration, were set as factors (X values) and purity (SE-HPLC) was set as response (Y value), and then a DOE design was performed using the JMP® 10.0 statistical program. Specifically, RSM (Response Surface Model) was used, and the axis point was set to CCD (Central Composite Design) type. Finally, a total of 9 Run conditions were designed, including “2-level full factorial design 4 Runs + central point 1 Run + axis point 4 Runs for 2 factors”.
시료 원액(1.1 mg/mL)을 DOE 조건에 맞게 아르기닌을 첨가하고 희석한 뒤 상온(약 15~25℃)에서 18시간 방치한 후, SE-HPLC 분석을 수행하였다. The sample stock solution (1.1 mg/mL) was diluted with arginine according to DOE conditions and left at room temperature (approximately 15-25°C) for 18 hours before SE-HPLC analysis.
DOE 통계분석은 단계적 회귀분석(Stepwise regression)을 활용한 다중회귀분석을 통해 수행하였으며, 모델의 확립은 반응표면 모델링(Response surface modeling) 방식을 따랐다. 분석은 JMP의 Fit model 방법을 이용하였으며, 모델에 사용되는 효과(effect)로는 2개의 주효과, 2차 교호작용, 각 주효과의 제곱항을 포함시켰다. 유의하지 않은 요인을 제거하고 유의한 항을 선정하기 위한 정지 규칙(Stopping Rule)은 P-값 역치를 사용하였다(P 값≤0.25, direction: Mixed). DOE statistical analysis was performed through multiple regression analysis using stepwise regression, and the establishment of the model followed response surface modeling. The analysis used JMP's Fit model method, and the effects used in the model included two main effects, a two-way interaction, and the square term of each main effect. The P-value threshold was used as a stopping rule to remove non-significant factors and select significant terms (P value ≤ 0.25, direction: Mixed).
분석 결과, 모델식의 R square는 0.96이며 R square 수정은 0.94이다. 모델식의 ANOVA(analysis of variance)의 P-값은 유의수준 0.05보다 작은 0.0006 로 본 모델의 유의성을 확인할 수 있었다.As a result of the analysis, the R square of the model equation is 0.96 and the R square correction is 0.94. The P-value of the model's ANOVA (analysis of variance) was 0.0006, which is less than the significance level of 0.05, confirming the significance of this model.
유의 수준 α=0.05 보다 낮은 P-값을 가져서 유의하다고 판단되는 요인은 주효과인 단백질 농도와 주효과인 아르기닌 농도이다. 아르기닌*아르기닌 곡률에 대한 P-값은 0.0765로써 유의 수준 α=0.05에 근접한 수치를 보였다(도 13b). The factors that are considered significant because they have a P-value lower than the significance level α=0.05 are the main effect, protein concentration, and the main effect, arginine concentration. The P-value for arginine*arginine curvature was 0.0765, which was close to the significance level of α=0.05 (FIG. 13b).
예측 프로파일러(Prediction profiler) 분석 결과 아르기닌 120 mM 부근에서 단량체 % 값이 가장 높아 100 내지 140 mM가 아르기닌의 최적 농도임을 알 수 있었다(도 13c).As a result of prediction profiler analysis, the monomer % value was highest around 120 mM arginine, indicating that 100 to 140 mM was the optimal concentration of arginine (FIG. 13c).
폴리소르베이트 80 농도에 따른 교반(agitation)-유도 응집 방지 Prevention of agitation-induced aggregation depending on polysorbate 80 concentration
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 20mM 히스티딘, 4.0mM CaCl2, 120mM NaCl 및 1.0% 수크로스를 포함하는 pH 7.4의 조성물에 폴리소르베이트 80을 0.0, 0.001, 0.005, 0.01, 0.05 및 0.1 %(v/v)의 농도로 각각 첨가하였다. 제형화가 완료된 액상 샘플을 볼택싱하여 인위적인 전단 응력를 발생시킨 후 분석(성상, 탁도, SE-HPLC)을 수행하였으며, 교반 개시 후 30초 및 2분에 샘플링하여 분석하였다. 교반 2분 경과 후 성상 분석 결과 폴리소르베이트 80을 0.005% 이상 첨가 시 시료가 맑고 투명해짐을 확인하였다(도 14a). Polysorbate 80 was added at 0.0, 0.001, 0.005, 0.01, 0.05 and 0.1% in a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20mM histidine, 4.0mM CaCl 2 , 120mM NaCl and 1.0% sucrose in common composition. Each was added at a concentration of (v/v). Analysis (appearance, turbidity, SE-HPLC) was performed after vortexing the formulated liquid sample to generate artificial shear stress, and sampling was performed at 30 seconds and 2 minutes after the start of stirring. As a result of property analysis after 2 minutes of stirring, it was confirmed that the sample became clear and transparent when more than 0.005% of polysorbate 80 was added (FIG. 14a).
한편, 폴리소르베이트 80을 0.005% 이상 첨가 시 초기 대비 탁도 변화가 없음을 확인함으로써 교반-유도 응집이 발생하지 않았음을 알 수 있었다(도 14b). 아울러 SE-HPLC 결과 폴리소르베이트 80을 0.05% 이상 첨가 시 초기 대비 5% 이내 수준의 순도 감소가 발생함을 확인하였다(도 14c).Meanwhile, when 0.005% or more of polysorbate 80 was added, it was confirmed that there was no change in turbidity compared to the initial stage, indicating that stirring-induced aggregation did not occur (FIG. 14b). In addition, SE-HPLC results It was confirmed that when more than 0.05% of polysorbate 80 was added, the purity decreased by less than 5% compared to the initial level (FIG. 14c).
폴리소르베이트 80 농도에 따른 액상 안정성 평가 Liquid stability evaluation according to polysorbate 80 concentration
공통 조성으로 0.5mg/ml ADAMTS 단백질, 20mM 히스티딘, 4.0mM CaCl2, 120 mM NaCl 및 1.0% 수크로스를 포함하는 pH 7.4의 조성물에 폴리소르베이트 80을 0.0, 0.005, 0.01, 0.05 및 0.09 %(v/v) 농도로 첨가하였다. 제형화가 완료된 액상 샘플을 상온에서 6시간 보관 후 SE-HPLC 분석을 수행한 결과, 폴리소르베이트 80 농도가 증가될수록 액상 안정성(순도)은 소폭 감소됨을 확인하였다(도 15). Polysorbate 80 was added at 0.0, 0.005, 0.01, 0.05 and 0.09% ( It was added at a concentration of v/v). As a result of performing SE-HPLC analysis on the formulated liquid sample after storing it at room temperature for 6 hours, it was confirmed that as the concentration of polysorbate 80 increased, the liquid stability (purity) decreased slightly (FIG. 15).
폴리소르베이트 80 농도에 따른 동결건조 품질 평가 Freeze-drying quality evaluation according to polysorbate 80 concentration
공통 조성으로 0.5mg/ml ADAMTS 단백질, 20mM 히스티딘, 4.0mM CaCl2, 120mM NaCl 및 1.0% 수크로스를 포함하는 pH 7.4의 조성물에 폴리소르베이트 80을 0.0, 0.005, 0.01, 0.05 및 0.09 %(v/v) 농도로 첨가하였다. 제형화가 완료된 액상 샘플을 동결건조 후 SE-HPLC 분석을 수행한 결과, 폴리소르베이트 80 농도가 증가 될수록 동결건조 공정에 의한 순도 손실 방지 효과가 커지고, 0.01% 이상 첨가 시 94% 이상의 단량체 회복을 확인하였다(도 16a). 아울러, 폴리소르베이트 80 농도가 증가할수록 고차 응집체(Higher-order aggregates) 생성이 효율적으로 차단되었다(도 16b). Polysorbate 80 was added at 0.0, 0.005, 0.01, 0.05 and 0.09% (v. It was added at a concentration of /v). As a result of performing SE-HPLC analysis after freeze-drying the formulated liquid sample, as the concentration of polysorbate 80 increases, the effect of preventing loss of purity due to the freeze-drying process increases, and when more than 0.01% is added, more than 94% of monomer recovery was confirmed. (Figure 16a). In addition, as the polysorbate 80 concentration increased, the formation of higher-order aggregates was efficiently blocked (FIG. 16b).
pH에 따른 열 풀림(Thermal unfolding) 및 열-응집 평가 Thermal unfolding and thermo-coagulation evaluation according to pH
공통 조성으로 20mM 히스티딘, 4mM CaCl2 및 0.05% PS80을 포함하며 pH만 6.0, 6.5, 7.0, 7.2 및 7.4으로 상이한 Diafiltration 버퍼를 미리 제조한 후, ADAMTS 단백질이 포함된 용액과 버퍼교환 과정을 거쳐 샘플을 제조하였다. (Amicon® Ultra-15 Centrifugal Filter Units 30K 사용, 원심분리는 냉장조건에서 3000rpm으로 수행)Diafiltration buffers with common compositions of 20mM histidine, 4mM CaCl2, and 0.05% PS80 and different pH values of 6.0, 6.5, 7.0, 7.2, and 7.4 were prepared in advance, and the samples were then subjected to a buffer exchange process with a solution containing ADAMTS protein. Manufactured. (Amicon® Ultra-15 Centrifugal Filter Units 30K used, centrifugation performed at 3000 rpm under refrigerated conditions)
버퍼교환이 완료된 샘플의 공통조성은 10mg/ml ADAMTS 단백질, 20mM 히스티딘, 4mM CaCl2 및 0.05% PS80이며 pH는 각각 6.0, 6.5, 7.0, 7.2 및 7.4이다.The common composition of the sample after buffer exchange was 10mg/ml ADAMTS protein, 20mM histidine, 4mM CaCl2, and 0.05% PS80, and the pH was 6.0, 6.5, 7.0, 7.2, and 7.4, respectively.
제형화가 완료된 액상 샘플을 UNCLE 기기를 활용하여 Tm및 Tagg분석 수행한 결과, 단백질의 구조적 열안정성과 비례하는 Tm(thermal unfolding) 값은 pH가 높을수록 증가되며, pH 6.0에서는 타 실험군 대비 낮은 Tm값을 보임을 확인하였다(도 17a). 불안정한 응집 발생 정도를 반영하는 열 응집(thermal aggregation, Tagg)의 경우, pH가 높을수록 Tagg 값이 증가됨을 확인하였으며, pH 6.0 및 pH 6.5에서는 타 실험군 대비 낮은 Tagg 값을 보였다(도 17b).As a result of performing T m and T agg analysis on the formulated liquid sample using UNCLE equipment, the Tm (thermal unfolding) value, which is proportional to the structural thermal stability of the protein, increases as the pH increases, and at pH 6.0, it is lower than other experimental groups. It was confirmed that the Tm value was observed (FIG. 17a). In the case of thermal aggregation (T agg ), which reflects the degree of unstable aggregation, it was confirmed that the higher the pH, the higher the T agg value, and at pH 6.0 and pH 6.5, the T agg value was lower than that of other experimental groups (Figure 17b) ).
CaCl2
농도에 따른 동결건조 품질 평가
Freeze-drying quality evaluation according to CaCl 2 concentration
공통 조성으로 1.2 mg/ml ADAMTS 단백질, 20 mM 히스티딘, 120mM NaCl, 1.0% 수크로스 및 0.05% PS80을 포함하는 pH 7.4의 조성물에 2.0, 4.0 및 8.0 mM 농도의 CaCl2를 첨가하였다. 제형화가 완료된 샘플을 동결건조 하여 SE-HPLC 분석 수행한 결과, CaCl2 농도에 관계없이 부분적으로 붕괴된 성상을 보였으며(도 18a), SE-HPLC 분석 결과 CaCl2 농도가 증가될수록 순도가 개선됨을 확인하였다(도 18b). CaCl 2 at concentrations of 2.0, 4.0 and 8.0 mM was added to a composition at pH 7.4 containing 1.2 mg/ml ADAMTS protein, 20 mM histidine, 120mM NaCl, 1.0% sucrose and 0.05% PS80 as a common composition. As a result of freeze-drying the formulated sample and performing SE-HPLC analysis, it showed partially collapsed properties regardless of CaCl 2 concentration (FIG. 18a), and SE-HPLC analysis showed that purity improved as CaCl 2 concentration increased. Confirmed (FIG. 18b).
실시예 2: ADAMTS13 단백질의 제형 성분의 추가 최적화 Example 2: Further optimization of formulation components of ADAMTS13 protein
NaCl 대 수크로스 배합비에 따른 동결건조 품질 평가Evaluation of freeze-dried quality according to NaCl to sucrose mixing ratio
공통조성으로 0.36 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, L-Arg 120mM 및 0.05%의 PS80을 포함하는 pH 7.4의 조성물에 NaCl을 100, 150, 200, 250, 300, 350 및 400 mM의 농도로, 수크로스를 0, 0.5, 1 및 1.5 %(w/v)의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 공정 전, 후의 순도를 SE-HPLC로 분석하였다. 그 결과, 하기 표 2에서 보는 바와 같이 NaCl과 수크로스 함량비에 따른 유의한 순도 변화는 관찰되지 않아 실험한 모든 범위에서 우수한 순도를 유지함을 알 수 있었다.NaCl was added at 100, 150 , 200, 250, 300, 350, and To a concentration of 400mM, sucrose was added at concentrations of 0, 0.5, 1 and 1.5% (w/v). The formulation-completed sample was freeze-dried 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 change in purity was observed depending on the NaCl and sucrose content ratio, indicating that excellent purity was maintained in all tested ranges.
구분division | NaCl (mM)NaCl (mM) | |||||||
100100 | 150150 | 200200 | 250250 | 300300 | 350350 | 400400 | ||
수크로스sucrose
(%)(%) |
00 | 100.6100.6 | 100.8100.8 | 100.3100.3 | 100.1100.1 | 99.499.4 | 99.899.8 | 100.0100.0 |
0.50.5 | 100.7100.7 | 100.9100.9 | 100.3100.3 | 100.2100.2 | 99.999.9 | 99.999.9 | 99.999.9 | |
1One | 100.7100.7 | 100.9100.9 | 100.4100.4 | 100.2100.2 | 100.2100.2 | 99.999.9 | 99.999.9 | |
1.51.5 | 100.1100.1 | 101.0101.0 | 100.1100.1 | 100.2100.2 | 100.2100.2 | 100.0100.0 | 100.0100.0 |
단, 도 19a 및 도 19b에서 보는 바와 같이, NaCl 농도가 증가할수록, 수크로스가 농도가 감소할수록 양호한 케이크 성상을 보임을 확인할 수 있었다. However, as shown in Figures 19a and 19b, it was confirmed that as the NaCl concentration increased and the sucrose concentration decreased, good cake properties were observed.
수크로스 고농도 처리에 따른 동결건조 품질 평가 Freeze-dried quality evaluation according to high-concentration sucrose treatment
공통조성으로 0.36 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, L-Arg 120mM 및 0.05%의 PS80을 포함하는 pH 7.4의 조성물에 NaCl을 200, 250 및 300mM 농도로, 수크로스를 1.5 및 2.5 %의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰한 결과, 도 20에서 보는 바와 같이 NaCl 농도가 증가할수록, 수크로스가 농도가 감소할수록 양호한 케이크 성상을 나타냄을 확인할 수 있었다. In a composition at pH 7.4 containing 0.36 mg/ml ADAMTS protein, 20 mM histidine, 4mM CaCl 2 , 120mM L-Arg, and 0.05% PS80, NaCl was added at concentrations of 200, 250, and 300mM, and sucrose at 1.5%. and was added at a concentration of 2.5%. As a result of observing the cake properties by freeze-drying the formulated sample, it was confirmed that as the NaCl concentration increased and the sucrose concentration decreased, good cake properties were observed, as shown in FIG. 20.
아르기닌 농도에 따른 콜로이드 안정성 평가 (B22,kD) Colloidal stability evaluation according to arginine concentration ( B 22 ,kD )
공통조성으로 0.36 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, NaCl 280mM, 수크로스 1% 및 0.05%의 PS80을 포함하는 pH 7.4의 조성물에 아르기닌을 40, 80, 120, 160 및 200 mM의 농도로 첨가하였다. 제형화가 완료된 액상 샘플을 UNCLE 기기를 활용하여 콜로이드 안정성(B22, kD)을 분석하였다. B22와 kD 값이 양의 값으로 커질수록 단백질 간 응집 없이 양호하게 분산되어 있음을 의미하는데, 도 21에서 보는 바와 같이 아르기닌 120 mM에 가까울수록 우수한 콜로이드 안정성을 나타냄을 확인하였다. Arginine was added at 40, 80, 120, 160 and 200 in a composition at pH 7.4 containing 0.36 mg/ml ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 280 mM NaCl, 1% sucrose and 0.05% PS80. It was added at a concentration of mM. The colloidal stability (B 22 , kD) of the formulated liquid sample was analyzed using the UNCLE instrument. As the B 22 and kD values become more positive, it means that the proteins are well dispersed without aggregation. As shown in Figure 21, it was confirmed that the closer the arginine content is to 120 mM, the better the colloidal stability.
아르기닌 농도에 따른 동결건조 품질 평가 Freeze-dried quality evaluation according to arginine concentration
공통조성으로 0.36 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, NaCl 280mM, 수크로스 1% 및 0.05%의 PS80을 포함하는 pH 7.4의 조성물에 아르기닌을 40, 80, 120, 160 및 200 mM의 농도로 첨가하였다. 제형화가 완료된 샘플을 동결건조하여 케익 성상을 관찰하고 SE-HPLC로 순도를 분석하였다. 그 결과, 아르기닌 농도가 80 mM 및 120 mM의 경우 케잌 성상이 우수함을 확인할 수 있었으나(도 22a), 아르기닌 농도에 따른 주요할 만한 순도 차이는 관찰되지 않았다(도 22b). Arginine was added at 40, 80, 120, 160 and 200 in a composition at pH 7.4 containing 0.36 mg/ml ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 280 mM NaCl, 1% sucrose and 0.05% PS80. It was added at a concentration of mM. The formulated sample was freeze-dried, the cake properties were observed, and the purity was analyzed by SE-HPLC. As a result, it was confirmed that the cake properties were excellent when the arginine concentration was 80mM and 120mM (FIG. 22a), but no significant difference in purity was observed depending on the arginine concentration (FIG. 22b).
GC1126A 최종 액상 제형에 대한 보관 시간 및 온도 안정성Storage time and temperature stability for GC1126A final liquid formulation
0.36 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, L-Arg 120 mM, NaCl 280 mM, Sucrose 1 % 및 0.05%의 PS80를 포함하는 pH 7.4의 제형화 완료된 액상 샘플을 상온(25℃)과 냉장(4℃) 조건에서 각각 보관 후 순도(SE-HPLC), 역가(ADAMTS13 활성), 단백질 농도(UV) 및 탁도 분석을 수행함으로써 경시 변화를 확인하였다. 그 결과, 순도의 경우 상온 보관 3일부터 초기 대비 약 4% 순도 감소를 보였으며, 보관 7일에는 초기 대비 약 15% 순도 감소를 보였고, 냉장 보관 7일까지 경시 변화 관찰되지 않았다(도 23a). A fully formulated liquid sample at pH 7.4 containing 0.36 mg/ml of ADAMTS protein, 20 mM histidine, 4 mM CaCl 2 , 120 mM L-Arg, 280 mM NaCl, 1% Sucrose, and 0.05% PS80 was stored at room temperature (25°C). Changes over time were confirmed by analyzing purity (SE-HPLC), titer (ADAMTS13 activity), protein concentration (UV), and turbidity after storage under ) and refrigerated (4°C) conditions, respectively. As a result, purity showed a decrease of about 4% compared to the initial level from the 3rd day of storage at room temperature, a decrease of about 15% compared to the initial level on the 7th day of storage, and no change over time was observed until the 7th day of refrigerated storage (FIG. 23a) .
역가 변화를 관찰한 결과 상온 보관 7일에서 초기 대비 약 50%의 역가 감소를 보였으며, 역가 분석 편차를 고려했을 때, 냉장 보관 7일까지 뚜렷한 경시 변화가 관찰되지 않았다(도 23b).As a result of observing the change in titer, the titer decreased by about 50% compared to the initial level after 7 days of storage at room temperature, and considering the deviation of the titer analysis, no clear change over time was observed until the 7th day of refrigerated storage (FIG. 23b).
단백질 농도의 경우, 상온 보관 3일부터 초기 대비 단백질 농도가 증가하였는데, 이는 입자의 응집으로 인해 시료의 불투명도가 증가함으로서 UV 흡광도가 증가한 결과로 판단된다. 냉장 보관 7일까지 경시 변화가 관찰되지 않았다(도 23c).In the case of protein concentration, the protein concentration increased from the initial 3 days of storage at room temperature, which is believed to be the result of increased UV absorbance as the opacity of the sample increased due to particle aggregation. No changes were observed over time until 7 days of refrigerated storage (Figure 23c).
한편, 최종 액상 제형의 탁도는 상온 보관 3일부터 증가하는 양상을 보였으나 냉장 보관 7일까지 경시 변화가 관찰되지 않았다(도 23d). Meanwhile, the turbidity of the final liquid formulation showed an increase from 3 days of room temperature storage, but no change over time was observed until 7 days of refrigerated storage (Figure 23d).
NaCl 및 수크로스 농도의 최적화 Optimization of NaCl and sucrose concentrations
공통조성으로 0.5 mg/ml의 ADAMTS 단백질, 히스티딘 20 mM, CaCl2 4mM, L-Arg 120mM 및 0.05%의 PS80을 포함하는 pH 7.4의 조성물에 NaCl을 200, 240 및 280 mM의 농도로, 수크로스를 0, 0.5 및 1 %의 농도로 첨가하였다.A composition at pH 7.4 containing 0.5 mg/ml ADAMTS protein, 20 mM histidine, 4mM CaCl 2 , 120mM L-Arg, and 0.05% PS80 was mixed with NaCl at concentrations of 200, 240, and 280 mM, and sucrose. was added at concentrations of 0, 0.5 and 1%.
NaCl 및 수크로스의 최적 농도 탐색을 위해 DOE (Design Of Experiments)를 수행하였다. NaCl 및 수크로스 농도라는 2개의 작동 파라미터를 요인(X 값)으로 설정하고 순도(SE-HPLC)와 역가(ADAMTS13 활성)를 반응(Y 값)으로 설정한 후 JMP® 10.0 통계 프로그램을 사용하여 DOE 설계를 실시하였다. 실험설계를 위해 완전요인배치법(full factorial design)을 이용하였으며, 중심점 2개를 포함하였다. 최종적으로“2 수준의 full factorial design 4 Runs + 중심점 2 Runs”를 포함하여 총 6개의 조건을 설계하였다. Design Of Experiments (DOE) was performed to explore the optimal concentrations of NaCl and sucrose. After setting the two operating parameters NaCl and sucrose concentration as factors (X values) and purity (SE-HPLC) and titer (ADAMTS13 activity) as responses (Y values), the DOE was calculated using the JMP ® 10.0 statistical program. The design was carried out. For the experimental design, a full factorial design was used and two central points were included. Ultimately, a total of 6 conditions were designed, including “2-level full factorial design 4 Runs + center point 2 Runs”.
Run Run | XX | |
NaCl (mM)NaCl (mM) | 수크로스sucrose | |
1One | 280280 | 00 |
2 (중심점)2 (center point) | 240240 | 0.50.5 |
33 | 280280 | 1One |
44 | 200200 | 00 |
55 | 200200 | 1One |
6 (중심점)6 (center point) | 240240 | 0.50.5 |
사전에 설계된 조건에 맞게 제형 완충액을 제조하고 원액과 혼합하여 최종원액을 제조하였다. 제조된 제형은 3 ml 바이알에 1 ml씩 분주하여 동결건조하였으며, 동결건조 완료된 검체는 냉장보관(5℃)하고 6개월 뒤 품질 분석을 실시하였다. A formulation buffer was prepared according to pre-designed conditions and mixed with the stock solution to prepare the final stock solution. The prepared formulation was freeze-dried by dispensing 1 ml into 3 ml vials, and the freeze-dried sample was stored in the refrigerator (5°C) and quality analysis was performed 6 months later.
DOE 통계 분석은 단계적 회귀분석(Stepwise regression)을 활용한 다중회귀분석을 진행하였다. 분석(Analyze)은 JMP의 'Fit model' 방법을 이용하였으며, 모델에 사용되는 효과(effect)로는 2개의 주효과, 2차 교호작용, 각 주효과의 제곱항을 포함시켰다. 유의하지 않은 요인을 제거하고 유의한 항을 선정하기 위한 정지규칙(stopping rule)은 p-값 역치를 사용하였다(P 값≤0.25, direction: Mixed).DOE statistical analysis was conducted through multiple regression analysis using stepwise regression. Analyze was done using JMP's 'Fit model' method, and the effects used in the model included two main effects, a two-way interaction, and the square term of each main effect. The p-value threshold was used as a stopping rule to remove non-significant factors and select significant terms (P value ≤ 0.25, direction: Mixed).
분석 결과, 모든 실험 조건에서 6개월 간 양호한 순도를 보여 NaCl, sucrose 농도에 따른 순도 차이는 확인되지 않았다(도 24a). 아울러, 각 실험군의 초기 역가 대비 6개월 경과 뒤의 역가는 분석 편차 이내로서 유의한 차이를 보이지 않았다(도 24b).As a result of the analysis, good purity was observed for 6 months under all experimental conditions, and no difference in purity depending on NaCl and sucrose concentrations was confirmed (FIG. 24a). In addition, compared to the initial titer of each experimental group, the titer after 6 months was within the analysis deviation and did not show a significant difference (FIG. 24b).
실시예 3: ADAMTS13 단백질 변이체의 제작 Example 3: Construction of ADAMTS13 protein variants
본 발명자들은 무작위 돌연변이법을 이용하여 aTTP 환자가 가지고 있는 ADAMTS13 중화항체를 효율적으로 회피할 수 있는 인간 ADAMTS13 변이체 제작하고자 하였다. MDTCS 부분 혹은 S 도메인에 돌연변이를 갖는 변이체가 ADAMTS13의 중화항체를 회피할 수 있는지 여부를 확인하기 위해서 인간 ADAMTS13에 대한 서로 다른 에피토프를 인식하는 항체에 대해 Bio-Rad사 HuCAL 시스템을 이용하여 Fab을 제작하였으며, 인간 ADAMTS13에 대한 결합력이 우수한 16종을 선별하였다. 각 항체의 결합 영역은 도 25c에 나타낸 바와 같이 Ab 4-16은 S 도메인, Ab 67은 MDTCS 부분, Ab 66은 C-말단부(TSP-2 부터 TSP-8 도메인)에 특이적으로 결합한다. The present inventors attempted to create a human ADAMTS13 variant that can efficiently evade ADAMTS13 neutralizing antibodies possessed by aTTP patients using a random mutation method. To determine whether variants with mutations in the MDTCS region or S domain can evade neutralizing antibodies of ADAMTS13, Fabs were produced using Bio-Rad's HuCAL system for antibodies that recognize different epitopes for human ADAMTS13. 16 species with excellent binding ability to human ADAMTS13 were selected. The binding region of each antibody is shown in the figure. As shown in Figure 25c, Ab 4-16 specifically binds to the S domain, Ab 67 to the MDTCS portion, and Ab 66 to the C-terminal portion (TSP-2 to TSP-8 domains).
변이체의 평가는 야생형 ADAMTS13 컨스트럭트와 무작위 돌연변이 생성을 통해 제작된 변이체 중 야생형 ADAMTS13과 동일한 아미노산 서열을 갖는 비-돌연변이 혹은 침묵 돌연변이 변이체(이하 야생형 클론)에 대한 상대적 결과 분석을 통해 진행되었다. 총 59개의 야생형 클론에 대해 활성 및 Ab 4-16, Ab 67의 결합력을 분석하였고, 각 야생형 클론의 분석 결과를 야생형 ADAMTS13 컨스트럭트의 결과값을 기준으로 상대화하여 도 26에 나타내었다. 도 26에서 보는 바와 같이, 보전적 아미노산 치환을 포함하는 야생형 ADAMTS13(59 종), 돌연변이된 ADAMTS13(304 종) 및 선정된 ADAMTS13(26 종)에 대한 Ab 4-16 및 Ab 67 항체에 대한 회피율 및 상대활성을 나타내었다. 야생형 클론은 변이가 없음에도 불구하고 야생형 ADAMTS13 컨스트럭트 결과 값과 상대적인 결과 차이를 보였고, 돌연변이된 ADAMTS13(304 종) 중에서 하기의 표 4에 나타낸 바와 같이 특정 수치의 회피율 또는 상대활성 이상의 값을 갖는 26 종의 돌연변이 ADAMTS13이 선별되었다. The evaluation of variants was conducted through comparative results analysis of the wild-type ADAMTS13 construct and non-mutant or silent mutant variants (hereinafter referred to as wild-type clones) having the same amino acid sequence as wild-type ADAMTS13 among variants produced through random mutagenesis. A total of 59 wild-type clones were analyzed for the activity and binding capacity of Ab 4-16 and Ab 67, and the analysis results of each wild-type clone were relativized based on the results of the wild-type ADAMTS13 construct and are shown in Figure 26. As shown in Figure 26, the escape rates for Ab 4-16 and Ab 67 antibodies against wild-type ADAMTS13 (59 species), mutated ADAMTS13 (304 species) and selected ADAMTS13 (26 species) containing conservative amino acid substitutions, and Relative activity was shown. Although the wild-type clone had no mutation, it showed a difference in results relative to the wild-type ADAMTS13 construct result, and among the mutated ADAMTS13 (304 species), as shown in Table 4 below, it had a value above a certain level of evasion rate or relative activity. Twenty-six mutant ADAMTS13 strains were selected.
구체적으로, 돌연변이된 ADAMTS13의 Ab 4-16 회피율은 -29.5% 내지 33.4%의 범위를 나타내었고, Ab 67 회피율은 -24.4% 내지 30.5%의 범위를 나타내었으며, 상대활성의 경우 62.7% 내지 128.9%의 범위를 나타내었다(표 4). Three-sigma rule을 이용한 정규분포를 적용한 결과, Ab 4-16 회피율의 경우 -34.1% 내지 38.5%, Ab 67 회피율의 경우-38.5% 내지 42.3% 그리고 상대 활성의 경우 47.0% 내지 145.2%의 범위로 WT 클론은 거의 모든 결과가 이 분포도 내에 존재할 것이라 예상하였다. 따라서 변이체 선별에 있어 three-sigma의 최대값을 기준으로 Ab 4-16 회피율이 38.5% 초과, Ab 67 회피율이 42.3% 초과 또는 상대활성이 47.0% 이상을 선정 기준으로 적용하였다. Specifically, the Ab 4-16 evasion rate of mutated ADAMTS13 ranged from -29.5% to 33.4%, the Ab 67 evasion rate ranged from -24.4% to 30.5%, and the relative activity ranged from 62.7% to 128.9%. The range is shown (Table 4). As a result of applying the normal distribution using the three-sigma rule, the Ab 4-16 evasion rate ranged from -34.1% to 38.5%, the Ab 67 evasion rate ranged from -38.5% to 42.3%, and the relative activity ranged from 47.0% to 145.2%. For WT clones, it was expected that almost all results would fall within this distribution. Therefore, in the selection of variants, based on the maximum value of three-sigma, Ab 4-16 evasion rate of more than 38.5%, Ab 67 evasion rate of more than 42.3%, or relative activity of more than 47.0% were applied as selection criteria.
카테고리category |
Ab 4-16 회피율Ab 4-16 | Ab 67 회피율Ab 67 Evasion Rate | 상대적 활성relative activity | |
최소값 minimum value | -29.5%-29.5% | -24.4%-24.4% | 62.7%62.7% | |
최대값maximum value | 33.4%33.4% | 30.5%30.5% | 128.9%128.9% | |
평균average | 2.2%2.2% | 1.9%1.9% | 96.1%96.1% | |
표준편차Standard Deviation | 12.1%12.1% | 13.5%13.5% | 16.4%16.4% | |
평균 - 3표준편차Mean - 3 standard deviations | -34.1%-34.1% | -38.5%-38.5% | 47.0%47.0% | |
평균 + 3표준편차Mean + 3 standard deviations | 38.5%38.5% | 42.3%42.3% | 145.2%145.2% |
ADAMTS13 중화항체를 회피 및 야생형 ADAMTS13 수준 이상의 활성을 갖는 변이체의 선별 Selection of variants that avoid ADAMTS13 neutralizing antibodies and have activity higher than that of wild-type ADAMTS13
WT 클론과 아미노산 변이를 갖는 변이체를 세포에 형질주입한 후, 배양액에 존재하는 단백질의 양을 측정하여 발현 농도가 50 ng/mL 이상인 304개의 변이체에 대해 중화항체 결합력과 활성 어세이를 수행하였다. 아미노산 변이를 갖는 변이체는 WT 클론 대비 중화항체 결합력 혹은 상대활성에 있어 다양한 분포를 갖고 있음을 알 수 있었으며, 이 중 선정 기준을 만족하는 26종의 변이체를 최종 선별하였다. 26종의 변이체 중 18종이 S 도메인에 대한 변이를 지닌 것을 확인하였으며, 특히 S 도메인에 대한 변이를 지닌 변이체 중 13종(1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02, 8C04, 8D01, 8F01)은 Ab 4-16에 대한 회피율이 높았으며 5종(7E01, 7G08, 8C02, 8D01, 8D05)은 높은 상대 활성을 지니는 특성을 보였다(표 5). 6종의 변이체는 D 도메인에 대한 변이를 지니며 이 중 5종(46, 3A06, 4C07, 4E11, 4H07)은 Ab 67에 대한 회피율이 높은 특성을 보였다. 이 밖에 M 도메인에 대한 변이를 지닌 변이체는 6종, C 도메인은 2종, T 도메인은 2종이 확인되었다. 선별한 26종의 변이체에 대하여 3회 반복 실험을 통해 결과의 반복 재현성을 확인하였으며 WT 클론 대비 우수성을 반복 시험에서도 연속적으로 유지하는 변이체 12종을 인 비트로 효력 시험의 대상으로 선정하였다. 12종 변이체 중 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02 및 8D01은 Ab4-16 항체 회피율이 우수하여 선정하였으며, 4C07, 4E11 그리고 4H07은 Ab67 회피율이 우수하여 선정하였다. 마지막으로 8D05는 우수한 상대 활성에 기반하여 선정되었다. After the WT clone and variants with amino acid mutations were transfected into cells, the amount of protein present in the culture medium was measured, and neutralizing antibody binding and activity assays were performed on 304 variants with an expression concentration of 50 ng/mL or more. It was found that variants with amino acid mutations had a diverse distribution in neutralizing antibody binding ability or relative activity compared to the WT clone, and among these, 26 variants that satisfied the selection criteria were finally selected. It was confirmed that 18 of the 26 variants had mutations in the S domain, and in particular, 13 of the variants had mutations in the S domain (1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02). , 8C04, 8D01, 8F01) had a high evasion rate against Ab 4-16, and five species (7E01, 7G08, 8C02, 8D01, 8D05) showed characteristics of high relative activity (Table 5). Six variants have mutations in the D domain, and five of them (46, 3A06, 4C07, 4E11, 4H07) have a high evasion rate against Ab 67. In addition, six types of variants with mutations in the M domain, two types in the C domain, and two types in the T domain were identified. The reproducibility of the results was confirmed through three repeated experiments for the 26 selected variants, and 12 variants that continuously maintained superiority over the WT clone even in repeated tests were selected as the subject of in vitro efficacy testing. Among the 12 variants, 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02, and 8D01 were selected because of their excellent Ab4-16 antibody evasion rate, and 4C07, 4E11, and 4H07 were selected because of their excellent Ab67 evasion rate. Finally, 8D05 was selected based on its excellent relative activity.
선정한 12종 변이체에서 추가 중화항체에 대한 회피력을 확인하기 위해서 스크리닝에 이용한 Ab4-16과 Ab67 항체 이외에 Ab4-20, Ab60, Ab61, Ab64 그리고 Ab65 중화항체에 대해서도 회피가 가능한지를 확인하고자 하였다. 12종의 변이체 중에 S 도메인에 아미노산 변이가 존재하는 9종 중 8D05를 제외한 8종의 변이체 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02, 8D01의 경우 aTTP 환자가 지닌 ADAMTS13 자가항체 서열을 바탕으로 제작된 Ab4-16, Ab4-20에 대한 우수한 회피율을 보였으며, Ab60과 Ab61에 대한 회피력도 우수함을 보여주었다. 반면에 D 도메인에 아미노산 변이가 있는 변이체 4C07, 4E11, 4H07의 경우 Ab67에 대한 회피율이 우수하였다(도 27, 표 6). In order to confirm the ability of the selected 12 variants to evade additional neutralizing antibodies, we wanted to check whether evasion was possible against Ab4-20, Ab60, Ab61, Ab64, and Ab65 neutralizing antibodies in addition to the Ab4-16 and Ab67 antibodies used in screening. Among the 12 variants, 8 variants 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7A02, and 8D01, excluding 8D05, out of 9 with amino acid mutations in the S domain, were based on the ADAMTS13 autoantibody sequence from aTTP patient. It showed excellent evasion rate against Ab4-16 and Ab4-20 produced by , and also showed excellent evasion ability against Ab60 and Ab61. On the other hand, variants 4C07, 4E11, and 4H07 with amino acid mutations in the D domain had excellent evasion rates against Ab67 (Figure 27, Table 6).
변이체 IDVariant ID |
돌연변이 적용 도메인 mutation Applicable domain |
변이 아미노산 잔기variant amino acid residues | Ab4-16 항체의 회피율 Evasion rate of Ab4-16 antibody | Ab67 항체의 회피율 Evasion rate of Ab67 antibody | 상대적 활성relative activity |
4646 | M,DM,D |
L85F P317HL85F P317H |
46.30%46.30% | 53.90%53.90% | 66.80%66.80% |
1C031C03 | SS | S612YS612Y | 48.00%48.00% | 10.40%10.40% | 104.60%104.60% |
1G071G07 | M,C,SM,C,S |
V282A A465D D672VV282A A465D D672V |
41.40%41.40% | 57.90%57.90% | 63.80%63.80% |
2B012B01 | SS | D635VD635V | 87.40%87.40% | 5.40%5.40% | 62.20%62.20% |
2B022B02 | C,SC, S |
R452I S612YR452I S612Y |
69.80%69.80% | 15.30%15.30% | 88.80%88.80% |
3A063A06 | M,D,TM,D,T |
R278I A334T D427NR278I A334T D427N |
42.60%42.60% | 58.50%58.50% | 62.90%62.90% |
3B053B05 | SS | P618SP618S | 66.00%66.00% | 48.40%48.40% | 71.20%71.20% |
3E013E01 | MM | T135IT135I | 52.30%52.30% | 36.00%36.00% | 60.00%60.00% |
3G043G04 | M,SM, S |
V126M A567S E651DV126M A567S E651D |
42.00%42.00% | 29.40%29.40% | 103.50%103.50% |
3H123H12 | TT | N413DN413D | -69.00%-69.00% | -104.60%-104.60% | 162.40%162.40% |
4C074C07 | DD | A334VA334V | -6.60%-6.60% | 72.30%72.30% | 153.10%153.10% |
4E114E11 | DD | A314TA314T | 10.30%10.30% | 96.40%96.40% | 71.90%71.90% |
4H034H03 | M, DM,D |
F93V K364R E376DF93V K364R E376D |
40.30%40.30% | 37.60%37.60% | 65.70%65.70% |
4H074H07 | DD | N308KN308K | 30.20%30.20% | 64.60%64.60% | 106.40%106.40% |
5C095C09 | SS | S612FS612F | 60.90%60.90% | -10.50%-10.50% | 88.50%88.50% |
5G085G08 | SS | Q656HQ656H | 41.40%41.40% | 26.00%26.00% | 91.90%91.90% |
6B126B12 | SS | G607RG607R | 43.90%43.90% | -101.60%-101.60% | 93.80%93.80% |
7A027A02 | SS |
S612F G624DS612F G624D |
57.00%57.00% | -80.50%-80.50% | 128.50%128.50% |
7E017E01 | SS | R589QR589Q | -36.60%-36.60% | -89.70%-89.70% | 149.90%149.90% |
7G087G08 | SS |
Q650H Q656RQ650H Q656R |
-25.20%-25.20% | -47.60%-47.60% | 160.70%160.70% |
8C028C02 | SS | I643FI643F | -19.20%-19.20% | -50.90%-50.90% | 168.10%168.10% |
8C048C04 | SS |
I585N Y658HI585N Y658H |
51.30%51.30% | 34.80%34.80% | 69.00%69.00% |
8C128C12 | SS |
V630L D654G E664NV630L D654G E664N |
-12.00%-12.00% | 73.20%73.20% | 48.10%48.10% |
8D018D01 | SS |
R589Q K608M M609L G624C I655VR589Q K608M M609L G624C I655V |
85.10%85.10% | -78.30%-78.30% | 193.60%193.60% |
8D058D05 | SS | P578LP578L | -1.20%-1.20% | -5.70%-5.70% | 172.30%172.30% |
8F018F01 | SS | I585MI585M | 45.20%45.20% | 34.40%34.40% | 99.40%99.40% |
변이체 IDVariant ID | 변이 위치mutation location | 중화항체 회피율 (%)Neutralizing antibody evasion rate (%) | 상대적활성relative activity | ||||||
Ab4-16Ab4-16 | Ab4-20Ab4-20 | Ab60Ab60 | Ab61Ab61 | Ab64Ab64 | Ab65Ab65 | Ab67Ab67 | |||
1C031C03 | S612YS612Y | 57.5%57.5% | 59.4%59.4% | 97.2%97.2% | 94.7%94.7% | 5.2%5.2% | -11.0%-11.0% | 15.1%15.1% | 103.7%103.7% |
2B012B01 | D635VD635V | 82.9%82.9% | 88.6%88.6% | 92.4%92.4% | 97.4%97.4% | 96.3%96.3% | 8.1%8.1% | 7.9%7.9% | 66.8%66.8% |
2B022B02 |
R452I S612YR452I S612Y |
65.4%65.4% | 61.1%61.1% | 97.3%97.3% | 95.5%95.5% | 0.9%0.9% | -15.0%-15.0% | 15.1%15.1% | 112.6%112.6% |
3B053B05 | P618SP618S | 51.3%51.3% | -6.5%-6.5% | 24.9%24.9% | 39.4%39.4% | 19.4%19.4% | -63.8%-63.8% | 37.4%37.4% | 77.3%77.3% |
4C074C07 | A334VA334V | 4.2%4.2% | -5.0%-5.0% | -3.3%-3.3% | -6.9%-6.9% | -2.2%-2.2% | -4.7%-4.7% | 78.5%78.5% | 152.2%152.2% |
4E114E11 | A314TA314T | 16.6%16.6% | -1.3%-1.3% | 2.7%2.7% | 3.2%3.2% | 5.4%5.4% | -0.8%-0.8% | 98.9%98.9% | 78.3%78.3% |
4H074H07 | N308KN308K | 27.9%27.9% | 1.5%1.5% | 11.0%11.0% | 16.4%16.4% | 17.0%17.0% | 12.8%12.8% | 61.3%61.3% | 94.7%94.7% |
5C095C09 | S612FS612F | 64.6%64.6% | 54.7%54.7% | 97.3%97.3% | 95.2%95.2% | 7.8%7.8% | -1.6%-1.6% | 11.8%11.8% | 104.8%104.8% |
5G085G08 | Q656HQ656H | 47.0%47.0% | 7.2%7.2% | 32.1%32.1% | 46.2%46.2% | 27.4%27.4% | -51.4%-51.4% | 30.3%30.3% | 95.9%95.9% |
7A027A02 |
S612F G624DS612F G624D |
57.1%57.1% | 55.4%55.4% | 97.6%97.6% | 95.5%95.5% | 1.9%1.9% | -26.2%-26.2% | -12.9%-12.9% | 125.3%125.3% |
8D018D01 |
R589Q K608M M609L G624C I655VR589Q K608M M609L G624C I655V |
90.1%90.1% | 98.5%98.5% | 95.2%95.2% | 94.1%94.1% | 20.0%20.0% | -95.9%-95.9% | 7.3%7.3% | 127.0%127.0% |
8D058D05 | P578LP578L | -9.3%-9.3% | -43.1%-43.1% | 3.2%3.2% | 15.9%15.9% | -0.9%-0.9% | -116.4%-116.4% | 11.5%11.5% | 154.7%154.7% |
한편, 구조-기능적 연구에서 ADAMTS13의 총 14개의 도메인 중에 C-말단의 TSP-2부터 CUB2 도메인이 제거된 MDTCS 도메인이 ADAMTS13와 유사한 메탈로프로테아제 기능을 가지고 있어 VWF 절단 가능함이 보고 되었다(Shelat et al., 2005). 이는 전장 ADAMTS13이 아닌 MDTCS 절편만으로도 TTP 질환 치료제로써 기능을 할 가능성을 시사하며 말단이 제거(truncated)된 형태로서 C-말단 부분에 결합하는 환자 혈장에 존재하는 중화항체를 회피할 수 있다는 것을 의미한다. Meanwhile, in a structural-functional study, it was reported that among the 14 domains of ADAMTS13, the MDTCS domain with the C-terminal TSP-2 to CUB2 domains removed has a metalloprotease function similar to ADAMTS13 and is capable of cleaving VWF (Shelat et al. ., 2005). This suggests the possibility that the MDTCS fragment, rather than the full-length ADAMTS13, may function as a treatment for TTP disease, and that it can avoid neutralizing antibodies present in patient plasma that bind to the C-terminal portion in a truncated form. .
이에, 선정된 12종의 변이체를 MDTCS로 절편화하고, 이중 선별된 변이 아미노산 잔기를 조합하여 2개의 아미노산 변이를 가지는 DM1, DM2 변이체를 추가로 제작하였다. 앞서 언급한 방법으로 세포에서 발현한 배양액과 정제액을 이용하여 단일 혹은 혼합 중화항체 회피율과 상대 활성을 측정하여 최종 후보물질을 선별하고자 하였다. Accordingly, the 12 selected variants were fragmented with MDTCS, and DM1 and DM2 variants with two amino acid mutations were additionally created by combining the selected mutant amino acid residues. We attempted to select final candidates by measuring the evasion rate and relative activity of single or mixed neutralizing antibodies using culture and purification solutions expressed in cells using the previously mentioned method.
배양액을 이용한 단일 중화항체 8종의 결합 회피율과 상대 활성을 측정한 결과, 2B01, 3B05, 4H07, 5G08, 8D05 및 DM1의 경우 모든 중화항체를 유사한 수준으로 회피하였다(도 28, 표 7). 1C03, 2B02, 5C09, 7A02, 8D01 및 DM2의 경우 3-01과 Ab60에 대한 우수한 결합 회피율을 보였다. 6종의 후보는 모두 S 도메인에 아미노산 변이를 가지고 있으며, 8D01를 제외하고는 공통적으로 612번째 아미노산에 변이를 가지고 있다. 4E11과 DM2의 경우 Ab67에 대한 결합 회피율이 우수하였으며, 모두 D 도메인인 314번째 아미노산에 변이를 가지고 있다. 상대 활성의 경우 DM1이 57.9%로 가장 낮았으며 1C03이 133.1%로 가장 높았다. 위의 2종을 제외한 모든 후보는 78.2 - 125.1%의 상대활성을 보여 MDTCS-Fc 대조군과 유사한 활성을 보였다(표 7). 9종 중화항체(Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67)를 동일 비율로 혼합한 조건에서 대조군인 MDTCS-Fc 대비 4C07과 5C09를 제외한 12종의 후보 변이체의 회피력을 확인하기 위해 혼합 중화항체 7.5 nM과 각 변이체 발현 배양액 4nM를 혼합한 후, 상온에서 1시간 동안 반응시켜 잔존활성을 측정하였다. MDTCS-Fc의 경우 3.5%의 잔존활성을 유지함을 보였고, 3B05, 4E11, 4H07, 5G08 그리고 8D05는 1.24 - 2.42%의 잔존활성을 가지는 것으로 확인되어 MDTCS-Fc 대비 잔존활성이 낮음을 보여주었다(도 29). 반면에 1C03, 2B01, 2B02, 7A02, 8D01, DM1 및 DM2의 경우 7.69-18.81%로 잔존활성이 유지되어 MDTCS-Fc 대비 혼합 중화항체에 의한 회피력이 우수함을 확인하였다(도 29).As a result of measuring the binding evasion rate and relative activity of eight types of single neutralizing antibodies using culture medium, 2B01, 3B05, 4H07, 5G08, 8D05, and DM1 evaded all neutralizing antibodies at a similar level (Figure 28, Table 7). 1C03, 2B02, 5C09, 7A02, 8D01, and DM2 showed excellent binding avoidance rates for 3-01 and Ab60. All six candidates have amino acid mutations in the S domain, and except for 8D01, they have a mutation at the 612th amino acid in common. In the case of 4E11 and DM2, the binding evasion rate against Ab67 was excellent, and both had mutations at amino acid 314 of the D domain. In terms of relative activity, DM1 had the lowest at 57.9% and 1C03 had the highest at 133.1%. All candidates except the above two showed relative activities of 78.2 - 125.1%, similar to the MDTCS-Fc control group (Table 7). Under conditions where 9 types of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, Ab67) were mixed in equal proportions, 12 types excluding 4C07 and 5C09 compared to MDTCS-Fc, the control group. To confirm the evasion ability of the candidate variants, 7.5 nM of mixed neutralizing antibodies and 4 nM of each variant expression culture medium were mixed and reacted at room temperature for 1 hour to measure the residual activity. In the case of MDTCS-Fc, it was shown to maintain a residual activity of 3.5%, and 3B05, 4E11, 4H07, 5G08 and 8D05 were confirmed to have a residual activity of 1.24 - 2.42%, showing lower residual activity compared to MDTCS-Fc (Figure 29). On the other hand, in the case of 1C03, 2B01, 2B02, 7A02, 8D01, DM1 and DM2, the residual activity was maintained at 7.69-18.81%, confirming that the evasion by mixed neutralizing antibodies was superior to MDTCS-Fc (Figure 29).
정제액을 이용한 후보물질 변이체의 중화항체 회피력을 확인하기 위하여 Phytip system(protein A resin)을 이용하여 각 배양액에서 단백질 정제를 진행하고, 수득된 정제액을 이용하여 단일 중화항체의 회피율을 확인한 결과, 대부분의 변이체가 배양액 상태에서 평가한 결과와 경향성이 유사한 것을 확인하였다(도 30). In order to confirm the neutralizing antibody evasion ability of the candidate variant using purification solution, protein purification was performed from each culture medium using the Phytip system (protein A resin), and the evasion rate of a single neutralizing antibody was confirmed using the obtained purification solution. It was confirmed that most variants had similar trends to the results evaluated in culture (FIG. 30).
2B01 5G08, 8D05 그리고 DM1 변이체는 8종 모든 중화항체에 대해 24.6% 이상 중화항체를 회피함을 확인하였고, 1C03, 2B02, 7A02, 8D01 그리고 DM2의 경우는 배양액 결과와 유사하게 3-01과 Ab60에 대한 우수한 결합 회피율을 보였다. 4E11과 DM2의 경우는 배양액 결과와 동일하게 Ab67에 대한 결합 회피율이 우수함을 확인하였다. 각 변이체의 상대 활성은 도 30 및 표 8에 나타내었다. 혼합 중화항체 조건에서의 잔존활성 측정 결과, MDTCS-Fc는 3.76% 유지되는 것을 확인하였으며, 3B05, 4E11, 4H07 그리고 8D05 변이체는 2.05 - 3.50%으로 MDTCS-Fc 대비 활성이 더 많이 억제된 것을 확인하였다. 반면에 1C03, 2B01, 2B02, 5G08, 7A02, 8D01, DM1 그리고 DM2의 경우 6.34-12.8%의 잔존활성이 유지되는 것을 확인하였으며, 이는 MDTCS-Fc 대비 혼합 중화항체 회피력이 우수함을 보였다 (도 31).It was confirmed that 2B01 5G08, 8D05 and DM1 variants evaded neutralizing antibodies by more than 24.6% for all eight types of neutralizing antibodies, and in the case of 1C03, 2B02, 7A02, 8D01 and DM2, similar to the culture results, 3-01 and Ab60 showed excellent binding avoidance rate. In the case of 4E11 and DM2, it was confirmed that the binding evasion rate for Ab67 was excellent, consistent with the results of the culture medium. The relative activity of each variant is shown in Figure 30 and Table 8. As a result of measuring the residual activity under mixed neutralizing antibody conditions, it was confirmed that 3.76% of MDTCS-Fc was maintained, and the activity of 3B05, 4E11, 4H07 and 8D05 variants was 2.05 - 3.50%, which was confirmed to be more inhibited compared to MDTCS-Fc. . On the other hand, it was confirmed that 6.34-12.8% of residual activity was maintained for 1C03, 2B01, 2B02, 5G08, 7A02, 8D01, DM1 and DM2, which showed superior mixed neutralizing antibody evasion compared to MDTCS-Fc (Figure 31) .
이상의 결과를 바탕으로 상대활성 또는 혼합 중화항체 회피율을 종합적으로 고려하여 1C03, 2B02, 7A02, DM2와 612번 아미노산 변이를 지니고 있어 혼합 중화항체 회피력이 우수할 것으로 판단되는 5C09를 추가 연구를 위한 5종의 후보로 선정하였다. Based on the above results, considering the relative activity or mixed neutralizing antibody evasion rate comprehensively, 1C03, 2B02, 7A02, DM2, and 5C09, which is believed to have excellent ability to evade mixed neutralizing antibodies due to its amino acid mutation at 612, were selected as five types for further research. was selected as a candidate.
변이체IDMutant ID |
변이transition
위치location |
중화항체 회피율 (%)Neutralizing antibody evasion rate (%) |
상대적relative
활성activation |
|||||||
3-013-01 | 4-164-16 | 4-204-20 | Ab60Ab60 | Ab61Ab61 | Ab64Ab64 | Ab65Ab65 | Ab67Ab67 | |||
1C031C03 | S612YS612Y | 100.0%100.0% | 2.0%2.0% | -11.0%-11.0% | 91.3%91.3% | 3.9%3.9% | -5.9%-5.9% | -16.9%-16.9% | 1.1%1.1% | 133.1%133.1% |
2B012B01 | D635VD635V | 25.1%25.1% | 28.2%28.2% | 38.1%38.1% | 38.4%38.4% | 36.2%36.2% | 26.8%26.8% | 11.0%11.0% | 24.9%24.9% | 78.2%78.2% |
2B022B02 |
R452I S612YR452I S612Y |
99.0%99.0% | 1.2%1.2% | 4.2%4.2% | 93.5%93.5% | 2.9%2.9% | -2.1%-2.1% | -12.9%-12.9% | 3.1%3.1% | 116.5%116.5% |
3B053B05 | P618SP618S | 35.3%35.3% | 18.9%18.9% | 25.5%25.5% | 23.6%23.6% | 19.1%19.1% | 21.3%21.3% | 17.9%17.9% | 17.8%17.8% | 92.7%92.7% |
4C074C07 | A334VA334V | -5.9%-5.9% | -11.2%-11.2% | -9.6%-9.6% | -11.3%-11.3% | -3.3%-3.3% | -12.4%-12.4% | -14.6%-14.6% | 14.5%14.5% | 118.2%118.2% |
4E114E11 | A314TA314T | 15.3%15.3% | 7.3%7.3% | 11.9%11.9% | 8.1%8.1% | 9.0%9.0% | 9.7%9.7% | 8.8%8.8% | 83.5%83.5% | 89.7%89.7% |
4H074H07 | N308KN308K | 36.1%36.1% | 20.4%20.4% | 25.8%25.8% | 19.5%19.5% | 22.5%22.5% | 25.1%25.1% | 21.0%21.0% | 53.8%53.8% | 93.6%93.6% |
5C095C09 | S612FS612F | 100.0%100.0% | 15.7%15.7% | 18.7%18.7% | 100.0%100.0% | 12.4%12.4% | 13.0%13.0% | 0.5%0.5% | 8.5%8.5% | 108.2%108.2% |
5G085G08 | Q656HQ656H | 48.7%48.7% | 38.3%38.3% | 41.2%41.2% | 44.2%44.2% | 41.9%41.9% | 37.2%37.2% | 27.8%27.8% | 47.6%47.6% | 85.4%85.4% |
7A027A02 |
S612F G624DS612F G624D |
100.0%100.0% | 7.5%7.5% | 17.4%17.4% | 97.5%97.5% | 11.2%11.2% | 7.2%7.2% | -7.2%-7.2% | 12.3%12.3% | 125.1%125.1% |
8D018D01 |
R589Q K608M M609L G624C I655VR589Q K608M M609L G624C I655V |
100.0%100.0% | 38.9%38.9% | 49.1%49.1% | 71.2%71.2% | 31.6%31.6% | 36.3%36.3% | 18.1%18.1% | 41.8%41.8% | 78.4%78.4% |
8D058D05 | P578LP578L | 39.6%39.6% | 29.9%29.9% | 38.3%38.3% | 32.6%32.6% | 32.4%32.4% | 32.5%32.5% | 21.5%21.5% | 43.7%43.7% | 82.1%82.1% |
DM1DM1 |
A314T D635VA314T D635V |
52.3%52.3% | 48.2%48.2% | 56.9%56.9% | 60.6%60.6% | 54.7%54.7% | 51.6%51.6% | 38.1%38.1% | 86.8%86.8% | 57.9%57.9% |
DM2DM2 | A314TS612FA314TS612F | 100.0%100.0% | 12.0%12.0% | 22.0%22.0% | 95.1%95.1% | 21.6%21.6% | 13.0%13.0% | 7.3%7.3% | 84.8%84.8% | 97.7%97.7% |
변이체 IDVariant ID |
변이transition
위치location |
중화항체 회피율 (%)Neutralizing antibody evasion rate (%) |
상대적 relative
활성activation |
|||||||
3-013-01 | 4-164-16 | 4-204-20 | Ab60Ab60 | Ab61Ab61 | Ab64Ab64 | Ab65Ab65 | Ab67Ab67 | |||
1C031C03 | S612YS612Y | 100.0%100.0% | 15.6%15.6% | 21.1%21.1% | 100.0%100.0% | 16.3%16.3% | 7.3%7.3% | -7.1%-7.1% | 10.9%10.9% | 81.4%81.4% |
2B012B01 | D635VD635V | 46.0%46.0% | 37.8%37.8% | 49.6%49.6% | 65.5%65.5% | 43.7%43.7% | 49.7%49.7% | 28.5%28.5% | 28.2%28.2% | 56.3%56.3% |
2B022B02 | R452IR452I | 100.0%100.0% | 8.4%8.4% | 15.0%15.0% | 100.0%100.0% | 10.3%10.3% | 4.4%4.4% | -9.0%-9.0% | 0.2%0.2% | 91.1%91.1% |
S612YS612Y | ||||||||||
3B053B05 | P618SP618S | 25.5%25.5% | 11.9%11.9% | 13.3%13.3% | 14.9%14.9% | 10.5%10.5% | 13.8%13.8% | 7.9%7.9% | 10.2%10.2% | 83.7%83.7% |
4E114E11 | A314TA314T | 19.6%19.6% | 6.2%6.2% | 11.5%11.5% | 15.7%15.7% | 1.8%1.8% | 8.8%8.8% | 12.6%12.6% | 84.4%84.4% | 77.5%77.5% |
4H074H07 | N308KN308K | 32.6%32.6% | 12.80%12.80% | 16.8%16.8% | 29.0%29.0% | 9.4%9.4% | 16.9%16.9% | 17.0%17.0% | 47.1%47.1% | 77.5%77.5% |
5G085G08 | Q656HQ656H | 89.6%89.6% | 64.5%64.5% | 70.2%70.2% | 81.9%81.9% | 57.90%57.90% | 68.9%68.9% | 59.0%59.0% | 37.7%37.7% | 18.3%18.3% |
7A027A02 | S612FG624DS612FG624D | 100.0%100.0% | 10.4%10.4% | 12.8%12.8% | 100.0%100.0% | 8.5%8.5% | 3.4%3.4% | -7.3%-7.3% | 14.4%14.4% | 97.8%97.8% |
8D018D01 |
R589QK608M M609L G624C I655VR589QK608M M609L G624C I655V |
100.0%100.0% | 29.5%29.5% | 48.2%48.2% | 91.7%91.7% | 20.5%20.5% | 17.1%17.1% | 14.2%14.2% | 31.5%31.5% | 65.1%65.1% |
8D058D05 | P578LP578L | 51.0%51.0% | 27.4%27.4% | 35.1%35.1% | 49.5%49.5% | 27.2%27.2% | 33.8%33.8% | 29.9%29.9% | 46.0%46.0% | 56.3%56.3% |
DM1DM1 | A314TD635VA314TD635V | 50.2%50.2% | 34.0%34.0% | 54.0%54.0% | 66.50%66.50% | 45.3%45.3% | 52.9%52.9% | 24.6%24.6% | 85.1%85.1% | 50.3%50.3% |
DM2DM2 | A314TS612FA314TS612F | 100.0%100.0% | 16.2%16.2% | 24.2%24.2% | 100.0%100.0% | 24.0%24.0% | 11.5%11.5% | 11.0%11.0% | 86.5%86.5% | 73.1%73.1% |
최종 선정된 5종 MDTCS 변이체 절편에 IgG1-YTE를 접합한 DNA를 제작하여 세포에서 발현한 배양액에서 단일 중화항체 8종의 결합 회피력과 상대활성을 측정하였다. 그 결과, 1C03, 2B02, 5C09 및 7A02는 Ab3-01, Ab60에 대한 우수한 결합 회피율을 보였으며, DM2의 경우, Ab3-01, Ab60 및 Ab67에 대한 결합 회피율이 우수함을 확인하였다(도 33). 상대활성의 경우, 98.6-127.7%로 MDTCS-IgG1-YTE와 유사한 활성을 보였다(도 33). 9종 중화항체를 동일 비율로 혼합한 조건에서 대조군인 MDTCS-IgG1-YTE 대비 5종 변이체 물질에서 회피력을 확인하기 위해 혼합 중화항체 (9종)와 각 변이체 발현 배양액을 혼합한 후, 상온에서 1 시간 동안 반응하여 잔존활성을 측정하였다. MDTCS-IgG1-YTE의 경우 5.5%의 잔존활성을 유지함을 보였고, 5종 물질의 경우는 8.18 - 13.42%로 잔존활성이 유지되어 대조군 대비 중화항체에 의한 회피력이 우수함을 확인하였다(도 34). DNA was prepared by conjugating IgG1-YTE to the five finally selected MDTCS variant fragments, and the binding evasion and relative activity of eight types of single neutralizing antibodies were measured in culture medium expressed in cells. As a result, 1C03, 2B02, 5C09 and 7A02 showed excellent binding evasion rates for Ab3-01 and Ab60, and in the case of DM2, it was confirmed that the binding evasion rates for Ab3-01, Ab60 and Ab67 were excellent (FIG. 33). In the case of relative activity, it showed similar activity to MDTCS-IgG1-YTE at 98.6-127.7% (Figure 33). In order to check the evasion ability of 5 types of mutant substances compared to the control group MDTCS-IgG1-YTE under the condition of mixing 9 types of neutralizing antibodies in equal proportions, mixed neutralizing antibodies (9 types) and each variant expression culture were mixed, and then 1 ml at room temperature. Residual activity was measured by reacting over time. In the case of MDTCS-IgG1-YTE, the residual activity was maintained at 5.5%, and in the case of the five substances, the residual activity was maintained at 8.18 - 13.42%, confirming that the evasion by neutralizing antibodies was superior compared to the control group (Figure 34).
위의 5종 MDTCS 변이체 절편에 IgG1-YTE를 접합한 발현 배양액에서 Phytip system(protein A resin)을 이용하여 단백질을 정제하여 정제액에서 혼합 중화항체의 잔존활성과 비역가를 측정하였다. 해당 정제액의 농도는 Fc ELISA를 통해 확인하였고, 은 염색을 통해 목적 단백질이 용출된 것을 확인하였다. 혼합 중화항체 조건에서의 잔존활성 측정 결과, MDTCS-IgG1-YTE는 0.4% 유지되는 것을 확인하였으며, 1C03, 2B02, 5C09, 7A02 그리고 DM2는 각각 5.7%, 1.7%, 8.8%, 2.6%, 7.9%의 잔존활성이 유지되는 것으로 나타나 MDTCS-IgG1-YTE 대비 혼합항체 회피력이 우수함을 확인하였다. 비역가 측정결과, 7A02(10,288 IU/mg)를 제외한 4종 변이체는 대조군(18,030 IU/mg)과 유사한 19,091-22,379 IU/mg의 비역가를 보임을 확인하였다(도 35). 결과적으로 5종 변이체 절편의 IgG1-YTE 접합 물질은 대조군 대비 혼합 중화항체에서의 회피력이 우수하며, 7A02를 제외한 4종 변이체에서는 대조군과 유사한 수준의 비역가를 가지고 있음을 확인하였다. Proteins were purified from the expression culture medium in which IgG1-YTE was conjugated to the above five MDTCS variant fragments using the Phytip system (protein A resin), and the residual activity and specific titer of mixed neutralizing antibodies were measured in the purification liquid. The concentration of the purified solution was confirmed through Fc ELISA, and it was confirmed that the target protein was eluted through silver staining. As a result of measuring the residual activity under mixed neutralizing antibody conditions, it was confirmed that MDTCS-IgG1-YTE was maintained at 0.4%, and 1C03, 2B02, 5C09, 7A02, and DM2 were 5.7%, 1.7%, 8.8%, 2.6%, and 7.9%, respectively. It was confirmed that the residual activity was maintained and that the mixed antibody evasion ability was superior to MDTCS-IgG1-YTE. As a result of specific titer measurement, 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 the control group (18,030 IU/mg) (Figure 35). As a result, it was confirmed that the IgG1-YTE conjugate material of the 5 variant fragments had excellent evasion ability against mixed neutralizing antibodies compared to the control group, and that the 4 variants excluding 7A02 had a specific titer similar to that of the control group.
Fc 부착에 의한 MDTCS 절편 변이체의 반감기 증가 여부 평가Evaluating whether the half-life of MDTCS fragment variants is increased by Fc attachment
MDTCS에 부착된 Fc(IgG1-YTE)가 실제로 반감기 증가를 가져올지를 확인하고자 마우스에서 약동학 분석을 수행하였다. 이를 위해 MDTCS 혹은 IgG1-YTE가 부착 되어있는 MDTCS, 및 4종 최종 후보(1C03, 5C09, 7A02, DM2) 변이체 절편 단백질을 마우스에 꼬리 정맥 투여한 후 시간 별로 혈장을 확보하였다. 각 물질의 비역가를 기준으로 160 IU/kg가 되도록 투여하였고 활성 어세이를 통하여 각 시간별로 확보한 혈장 내에 잔존하는 물질의 활성을 측정하였다. 확보한 결과는 단순병합법 (naive pooled method)으로 정리하였고, 이를 이용하여 비구획 분석법 (noncompartmental analysis)으로 약동학 분석을 수행하였다. MDTCS의 반감기는 2.898시간, MDTCS-IgG1-YTE는 11.51 시간, 그리고 각 변이체의 경우 5.184 - 9.902 시간으로 측정되었다. 대조군인 MDTCS 대비 IgG1-YTE 접합에 의해 1.79 - 3.97배로 반감기가 연장되었다. 뿐만 아니라, 물질의 생체 평균 체류 시간을 나타내는 MRT(Mean Residence Time) 수치도 IgG1-YTE 접합에 의해 7.189 - 11.67시간으로 3.743시간 대비 증가된 체류 시간을 나타내었다(표 9, 도 32). 결과적으로 IgG1-YTE 융합체가 혈중 반감기 및 생체 내 평균 지속시간을 통한 활성을 유지하는데 효과적임을 확인하였다. Pharmacokinetic analysis was performed in mice to confirm whether Fc (IgG1-YTE) attached to MDTCS would actually increase the half-life. For this purpose, MDTCS or MDTCS with IgG1-YTE attached, and the four final candidate (1C03, 5C09, 7A02, DM2) mutant fragment proteins were administered to mice through the tail vein, and plasma was obtained over time. Based on the specific titer of each substance, it was administered to reach 160 IU/kg, and the activity of substances remaining in the plasma obtained at each time was measured through an activity assay. The obtained results were summarized using the naive pooled method, and pharmacokinetic analysis was performed using noncompartmental analysis. The half-life of MDTCS was measured at 2.898 hours, MDTCS-IgG1-YTE at 11.51 hours, and for each variant between 5.184 and 9.902 hours. The half-life was extended by 1.79 to 3.97 times by IgG1-YTE conjugation compared to the control MDTCS. In addition, the MRT (Mean Residence Time) value, which represents the average residence time of the material in the body, also showed an increased residence time of 7.189 - 11.67 hours compared to 3.743 hours due to IgG1-YTE conjugation (Table 9, Figure 32). As a result, it was confirmed that the IgG1-YTE fusion was effective in maintaining activity through blood half-life and average duration in vivo.
후보물질candidate substance | Parameter of PK 분석 파라미터Parameter of PK analysis parameters | ||
t1/2,terminal (hr)t 1/2 ,terminal (hr) | AUCInf (IU*hr/mL)AUC Inf (IU*hr/mL) | MRT (hr)MRT (hrs) | |
MDTCSMDTCS | 2.8982.898 | 5.4675.467 | 3.7433.743 |
MDTCS-IgG1-YTEMDTCS-IgG1-YTE | 11.5111.51 | 16.8916.89 | 11.6711.67 |
1C03-IgG1-YTE1C03-IgG1-YTE | 6.0876.087 | 12.3312.33 | 8.6958.695 |
5C09-IgG1-YTE5C09-IgG1-YTE | 5.1845.184 | 12.0612.06 | 7.9827.982 |
7A02-IgG1-YTE7A02-IgG1-YTE | 9.9029.902 | 14.4914.49 | 10.3510.35 |
DM2-IgG1-YTEDM2-IgG1-YTE | 5.9865.986 | 11.3911.39 | 7.1897.189 |
이상 살펴본 바와 같이, 본 발명자들은 MDTCS 부분 혹은 S 도메인과 결합하는 ADAMTS13 중화항체를 회피하거나 혹은 야생형 ADAMTS13 이상의 활성을 보이는 26종의 신규 변이체를 발굴하였다. 이 중 중화항체 9종에 대하여 가장 회피력이 우수하거나 비교 활성이 현저히 우수한 12종을 선정하여 C-말단과 결합하는 중화항체를 효율적으로 회피하면서 vWF 절단에 필수적인 MDTCS 절편을 제작함으로써 aTTP 환자의 D, C 혹은 S 도메인과 결합하는 자가항체 회피율이 크게 향상된 변이체를 동정하였다. 본 발명의 변이체는 IgG1-YTE 접합을 통하여 혈중 반감기를 증가시킴으로써 안정성 및 생리 활성의 지속성이 개선된, 효과적인 약리성분으로 유용하게 이용될 수 있다. As discussed above, the present inventors discovered 26 new variants that evade ADAMTS13 neutralizing antibodies that bind to the MDTCS portion or S domain or exhibit activity higher than that of wild-type ADAMTS13. Among these, 12 types with the best evasion or significantly superior comparative activity against 9 types of neutralizing antibodies were selected to produce MDTCS fragments essential for vWF cleavage while efficiently evading neutralizing antibodies that bind to the C-terminus, thereby producing D, A variant with a significantly improved evasion rate of autoantibodies binding to the C or S domain was identified. The variant of the present invention can be usefully used as an effective pharmacological ingredient with improved stability and persistence of physiological activity by increasing the half-life in the blood through IgG1-YTE conjugation.
aTTP mimic 질환 마우스 모델에서 변이체 PoC 확인 결과Results of variant PoC confirmation in aTTP mimic disease mouse model
확립된 aTTP-mimic 마우스 모델에서 최종 후보물질 선별을 위해 대조물질 (MDTCS-IgG1-YTE) 혹은 선정된 5종 변이체 후보물질(1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, 7A02-IgG1-YTE, DM2-IgG1-YTE) 투여 후, 중화항체 회피율을 평가하였다(도 36a). 5종 변이체 중 DM2-IgG1-YTE가 5,000 혹은 7,000 IU/kg의 모든 투여 용량에서 가장 높은 인간 ADAMTS13 잔존 활성을 보였다(도 36b). 가장 우수한 중화항체 회피율을 보인 DM2-IgG1-YTE 물질을 최종 선정하여 농도별로 투여하여 인간 ADAMTS13 잔존활성 유지력과 임상학적 증상의 완화 정도를 확인한 결과(도 37a), DM2 용량이 증가할 수록 임상 증상의 완화 정도가 증가하는 것을 확인할 수 있었으며, 7000 IU/kg 투여 그룹에서 MDTCS-IgG1-YTE 대비 DM2-IgG1-YTE 투여에 의해 혈소판과 LDH의 수치의 개선이 상대적으로 높음을 관찰할 수 있었다(도 37b). 혈소판 및 LDH 수치의 개선과 일치하게 인간 ADAMTS13 활성 검사 결과, 대조물질 또는 후보물질 투여 용량과 비례하는 잔존 활성 증가를 관찰할 수 있었다(도 37b). 임상 증상 관찰 결과, aTTP-mimic 마우스 모델에서 나타나는 폐사나 혈뇨가 대조물질 또는 후보물질의 투여에 의해 완화되는 경향을 확인할 수 있었다. 특히 DM2-IgG1-YTE 처리군의 경우 모든 투여 용량에서 사망 개체 없었으며, 7000 IU/kg 이상 투여 시 혈뇨 증상을 보인 개체를 관찰할 수 없었다 (도 37c). 7000 IU/kg 투여 시 평균 잔존 활성은 DM2-IgG1-YTE 0.32 IU/mL로 MDTCS-IgG1-YTE 0.03 IU/mL 대비 9.6 배 더 높았으며, 앞선 임상 증상 관찰 결과의 차이는 이러한 잔존 활성 차이에서 기인한 것으로 판단되었다. 일반 혈액검사, 임상화학검사 및 임상증상의 관찰 결과, MDTCS-IgG1-YTE와 DM2-IgG1-YTE의 투여는 aTTP-mimic 마우스 모델에서 나타나는 aTTP 임상 증상을 개선시키는 경향을 보였고, 이를 통해 인 비보 PoC를 확인할 수 있었다. 특히 7000 IU/kg 투여 시, DM2-IgG1-YTE가 MDTCS-IgG1-YTE 대비 그 개선 효과가 우수함을 확인하였다. To select the final candidate in the established aTTP-mimic mouse model, a control substance (MDTCS-IgG1-YTE) or five selected variant candidates (1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, After administration (7A02-IgG1-YTE, DM2-IgG1-YTE), the neutralizing antibody evasion rate was evaluated (Figure 36a). Among the five variants, DM2-IgG1-YTE showed the highest human ADAMTS13 residual activity at all doses of 5,000 or 7,000 IU/kg (Figure 36b). The DM2-IgG1-YTE material that showed the best neutralizing antibody evasion rate was finally selected and administered at different concentrations to confirm the ability to maintain human ADAMTS13 residual activity and the degree of relief of clinical symptoms (Figure 37a). As the DM2 dose increased, clinical symptoms decreased. It was confirmed that the degree of relief increased, and in the 7000 IU/kg administration group, a relatively high improvement in platelet and LDH levels was observed by administration of DM2-IgG1-YTE compared to MDTCS-IgG1-YTE (Figure 37b) ). Consistent with the improvement in platelet and LDH levels, 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 (FIG. 37b). As a result of observing clinical symptoms, it was confirmed that death and hematuria in the aTTP-mimic mouse model tended to be alleviated by administration of the control or candidate substances. In particular, in the DM2-IgG1-YTE treatment group, there were no deaths at all doses, and no subjects showing hematuria were observed when administered at more than 7000 IU/kg (FIG. 37c). When 7000 IU/kg was administered, the average residual activity was 0.32 IU/mL for DM2-IgG1-YTE, which was 9.6 times higher than 0.03 IU/mL for MDTCS-IgG1-YTE, and the difference in the results observed in the preceding clinical symptoms was due to this difference in residual activity. It was judged that it was done. As a result of general blood tests, clinical chemistry tests, and observation of clinical symptoms, administration of MDTCS-IgG1-YTE and DM2-IgG1-YTE showed a tendency to improve aTTP clinical symptoms in the aTTP-mimic mouse model, and through this, in vivo PoC was able to confirm. In particular, when administered at 7000 IU/kg, it was confirmed that DM2-IgG1-YTE had a superior improvement effect compared to MDTCS-IgG1-YTE.
cTTP 질환 마우스 모델에서 변이체 PoC 확인 결과Results of variant PoC confirmation in cTTP disease mouse model
aTTP mimic 마우스 모델에서 5종 변이체 후보 중 가장 우수한 효력을 보였던 DM2-IgG1-YTE를 이용하여 cTTP 마우스 모델에서 TTP 질환에서 보이는 혈액학적 그리고 임상학적 증상의 개선 여부와 인간 ADAMTS13 활성 회복 정도를 확인하였다 (도 38a). DM2-IgG1-YTE의 투여 용량 증가에 따라 혈소판과 LDH 수치의 개선이 농도 의존적으로 일어남을 확인할 수 있었으며, DM2-IgG1-YTE 180, 360 IU/kg 투여군에서 대조군 대비 유의한 혈소판 증가가 관찰되었고, 특히 360 IU/kg 투여군의 경우 정상 수준만큼으로 회복됨을 볼 수 있었다(도 38b). LDH 수치의 경우, 180 IU/kg 투여군에서 대조군과 유사한 수준으로 LDH가 회복됨을 확인하였다(도 38b). ADAMTS13 활성의 경우, DM2-IgG1-YTE 60 IU/kg 투여군부터 평균 0.1 IU/mL이상의 활성을 보였으며 360 IU/kg 투여 용량에서는 평균 1.08 IU/mL의 활성이 측정되었다 (도 38b). 따라서 DM2-IgG1-YTE 변이체가 cTTP 질환 마우스에서 효과적인 임상증상의 개선과 ADAMTS13의 활성을 회복시킴을 알 수 있었다. 이에, 본 발명자들은 DM2-IgG1-YTE를 이용하여 전술한 실시예 1 및 2의 ADAMTS13 단백질 제형 성분의 선별 및 최적화를 수행하였다Using DM2-IgG1-YTE, which showed the best efficacy among the five variant candidates in the aTTP mimic mouse model, the improvement of 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 improvement in platelet and LDH levels occurred in a concentration-dependent manner as the administered dose of DM2-IgG1-YTE increased, and 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 360 IU/kg administered group, recovery to normal levels was seen (Figure 38b). In the case of LDH levels, it was confirmed that LDH was recovered to a level similar to that in the control group in the 180 IU/kg administered group (FIG. 38b). In the case of ADAMTS13 activity, an average activity of more than 0.1 IU/mL was observed in the DM2-IgG1-YTE 60 IU/kg administered group, and an average activity of 1.08 IU/mL was measured at the 360 IU/kg administered dose (FIG. 38b). Therefore, it was found that the DM2-IgG1-YTE variant effectively improved clinical symptoms and restored the activity of ADAMTS13 in mice with cTTP disease. Accordingly, the present inventors performed selection and optimization of the ADAMTS13 protein formulation components of Examples 1 and 2 described above using DM2-IgG1-YTE.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (21)
- 전체 조성물에 대해 0 - 1.5 w/v%의 당(sugar) 안정화제 및 100 mM 내지 400 mM의 무기염을 포함하는 혈장 단백질용 약제학적 제형 조성물.A pharmaceutical formulation composition for plasma proteins comprising 0 - 1.5 w/v% of a sugar stabilizer and 100mM to 400mM of an inorganic salt relative to the total composition.
- 제 1 항에 있어서, 상기 당은 수크로스, 트레할로스 및 이들의 약제학적으로 허용되는 염으로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The composition according to claim 1, wherein the sugar is at least one selected from the group consisting of sucrose, trehalose, and pharmaceutically acceptable salts thereof.
- 제 1 항에 있어서, 상기 무기염은 NaCl, CaCl2, KCl 및 MgCl2로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The composition according to claim 1, wherein the inorganic salt is at least one selected from the group consisting of NaCl, CaCl 2 , KCl and MgCl 2 .
- 제 3 항에 있어서, 상기 무기염은 NaCl 및 CaCl2의 혼합물인 것을 특징으로 하는 조성물.The composition according to claim 3, wherein the inorganic salt is a mixture of NaCl and CaCl 2 .
- 제 1 항에 있어서, 상기 당 안정화제는 전체 조성물에 대해 0.7 - 1.3 w/v%로 포함되고 상기 무기염은 150 - 350 mM로 포함되는 것을 특징으로 하는 조성물.The composition according to claim 1, wherein the sugar stabilizer is contained in an amount of 0.7 - 1.3 w/v% and the inorganic salt is contained in an amount of 150 - 350mM based on the total composition.
- 제 1 항에 있어서, 상기 조성물은 40 mM 내지 200 mM의 아미노산 안정화제(stabilizer)를 추가적으로 포함하는 것을 특징으로 하는 조성물.The composition of claim 1, wherein the composition additionally contains 40 to 200 mM of an amino acid stabilizer.
- 제 6 항에 있어서, 상기 아미노산은 아르기닌(Arg), 프롤린(Pro) 및 이들의 약제학적으로 허용되는 염으로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The composition according to claim 6, wherein the amino acid is at least one selected from the group consisting of arginine (Arg), proline (Pro), and pharmaceutically acceptable salts thereof.
- 제 6 항에 있어서, 상기 무기염은 200 - 300 mM로 포함되는 것을 특징으로 하는 조성물.The composition according to claim 6, wherein the inorganic salt is contained in an amount of 200-300mM.
- 제 1 항에 있어서, 상기 조성물은 전체 조성물에 대해 0.01 내지 0.1 v/v %의 비이온성 계면활성제를 추가적으로 포함하는 것을 특징으로 하는 조성물.The composition of claim 1, wherein the composition additionally comprises 0.01 to 0.1 v/v % of a nonionic surfactant based on the total composition.
- 제 9 항에 있어서, 상기 비이온성 계면활성제는 폴리소르베이트 80, 폴리소르베이트 60 및 폴리소르베이트 40으로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The composition of claim 9, wherein the nonionic surfactant is at least one selected from the group consisting of polysorbate 80, polysorbate 60, and polysorbate 40.
- 제 1 항에 있어서, 상기 혈장 단백질은 ADAMTS13(a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) 단백질, 이의 변이체 또는 이의 기능적 일부 절편인 것을 특징으로 하는 조성물.The composition of 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 fragment thereof.
- 제 11 항에 있어서, 상기 ADAMTS13 단백질의 변이체는 서열목록 제1서열의 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 및 672번째 잔기로 구성된 군으로부터 선택되는 하나 이상의 아미노산 잔기의 치환을 포함하는 것을 특징으로 하는 조성물.The method of claim 11, wherein the variant of the ADAMTS13 protein is 85, 93, 126, 135, 278, 282, 308, 314, 317, 334, 364, 376, 413, 427, 452, 465 of the first sequence of the sequence listing. one selected from the group consisting of residues 567, 578, 585, 589, 607, 608, 609, 612, 618, 624, 630, 635, 643, 650, 651, 654, 655, 656, 658, 664 and 672 A composition comprising substitution of one or more amino acid residues.
- 제 12 항에 있어서, 상기 ADAMTS13 단백질의 변이체는 다음 위치에서의 아미노산 잔기의 치환을 포함하는 각 변이 단백질들로 구성된 군으로부터 선택되는 것을 특징으로 하는 조성물: The composition of claim 12, wherein the variant of the ADAMTS13 protein is selected from the group consisting of mutant proteins each containing a substitution of an amino acid residue at the following position:- 85 및 317번째 잔기; 612번째 잔기; 282, 465 및 672번째 잔기 중 둘 이상; 635번째 잔기; 452 및 612번째 잔기; 278, 334 및 427번째 잔기 중 둘 이상; 618번째 잔기; 135번째 잔기; 126, 567 및 651번째 잔기 중 둘 이상; 413번째 잔기; 334번째 잔기; 314번째 잔기; 93, 364 및 376번째 잔기 중 둘 이상; 308번째 잔기; 656번째 잔기; 607번째 잔기; 612 및 624번째 잔기; 589번째 잔기; 650 및 656번째 잔기; 643번째 잔기; 585 및 658번째 잔기; 630, 654 및 664번째 잔기 중 둘 이상; 589, 608, 609, 624 및 655번째 잔기 중 넷 이상; 578번째 잔기; 585번째 잔기; 314 및 635번째 잔기; 및 314 및 612번째 잔기.- 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.
- 제 12 항에 있어서, 상기 아미노산 잔기의 치환은 85번째 잔기의 Phe로의 치환, 93번째 잔기의 Val으로의 치환, 126번째 잔기의 Met으로의 치환, 135번째 잔기의 Ile으로의 치환, 278번째 잔기의 Ile으로의 치환, 282번째 잔기의 Ala으로의 치환, 308번째 잔기의 Lys으로의 치환, 314번째 잔기의 Thr으로의 치환, 317번째 잔기의 His으로의 치환, 334번째 잔기의 Thr 또는 Val으로의 치환, 364번째 잔기의 Arg으로의 치환, 376번째 잔기의 Asp으로의 치환, 413번째 잔기의 Asp으로의 치환, 427번째 잔기의 Asn으로의 치환, 452번째 잔기의 Ile으로의 치환, 465번째 잔기의 Asp으로의 치환, 567번째 잔기의 Ser으로의 치환, 578번째 잔기의 Leu으로의 치환, 585번째 잔기의 Asn 또는 Met으로의 치환, 589번째 잔기의 Gln으로의 치환, 607번째 잔기의 Arg으로의 치환, 608번째 잔기의 Met으로의 치환, 609번째 잔기의 Leu으로의 치환, 612번째 잔기의 Phe 또는 Tyr으로의 치환, 618번째 잔기의 Ser으로의 치환, 624번째 잔기의 Asp 또는 Cys으로의 치환, 630번째 잔기의 Leu으로의 치환, 635번째 잔기의 Val으로의 치환, 643번째 잔기의 Phe으로의 치환, 650번째 잔기의 His으로의 치환, 651번째 잔기의 Asp으로의 치환, 654번째 잔기의 Gly으로의 치환, 655번째 잔기의 Val으로의 치환, 656번째 잔기의 Arg 또는 His으로의 치환, 658번째 잔기의 His으로의 치환, 664번째 잔기의 Asn으로의 치환 및 672번째 잔기의 Val으로의 치환으로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The method of claim 12, wherein 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, and substitution of the 278th residue. substitution of Ile, substitution of residue 282 with Ala, substitution of residue 308 with Lys, substitution of residue 314 with Thr, substitution of residue 317 with His, substitution of residue 334 with Thr or Val. Substitution of, 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, 465th Substitution of residues with Asp, substitution of residue 567 with Ser, substitution of residue 578 with Leu, substitution of residue 585 with Asn or Met, substitution of residue 589 with Gln, Arg at residue 607. Substitution, substitution of residue 608 with Met, substitution of residue 609 with Leu, substitution of residue 612 with Phe or Tyr, substitution of residue 618 with Ser, substitution of residue 624 with Asp or Cys. Substitution of, 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, 654th Substitution of residues with Gly, substitution of residue 655 with Val, substitution of residue 656 with Arg or His, substitution of residue 658 with His, substitution of residue 664 with Asn and substitution of residue 672 with Val. A composition characterized in that it is one or more selected from the group consisting of substitution with .
- 제 11 항에 있어서, 상기 혈장 단백질은 IgG4 면역글로불린의 Fc 영역이 접합된 것을 특징으로 하는 조성물. The composition according to claim 11, wherein the plasma protein is conjugated with the Fc region of an IgG4 immunoglobulin.
- 제 15 항에 있어서, 상기 Fc 영역은 서열목록 제2서열의 22, 24 및 26번째 잔기로 구성된 군으로부터 선택되는 하나 이상의 아미노산 잔기의 치환을 포함하는 것을 특징으로 하는 조성물. The composition of claim 15, wherein the Fc region comprises a substitution of one or more amino acid residues selected from the group consisting of residues 22, 24, and 26 of SEQ ID NO:2.
- 제 16 항에 있어서, 상기 아미노산 잔기의 치환은 22번째 잔기의 Tyr으로의 치환, 24번째 잔기의 Thr으로의 치환 및 26번째 잔기의 Glu으로의 치환으로 구성된 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 조성물.The method of claim 16, wherein the substitution of the amino acid residue is at least one 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. Composition.
- 제 15 항에 있어서, 상기 혈장 단백질과 상기 IgG4 면역글로불린의 Fc 영역 사이에 IgG1 면역글로불린의 힌지(hinge) 영역이 추가적으로 포함되는 것을 특징으로 하는 조성물.The composition of claim 15, wherein a hinge region of an IgG1 immunoglobulin is additionally included between the plasma protein and the Fc region of the IgG4 immunoglobulin.
- 제 11 항 내지 제 18 항 중 어느 한 항의 조성물을 유효성분으로 포함하는 혈전성 질환의 예방 또는 치료용 조성물. A composition for preventing or treating thrombotic diseases comprising the composition of any one of claims 11 to 18 as an active ingredient.
- 제 19 항에 있어서, 상기 혈전성 질환은 혈전성 미세 혈관병증(thrombotic microangiopathy, TMA)인 것을 특징으로 하는 조성물. The composition according to claim 19, wherein the thrombotic disease is thrombotic microangiopathy (TMA).
- 제 20 항에 있어서, 상기 혈전성 미세 혈관병증은 혈전성 혈소판 감소성 자반증(thrombocytopenic purpura, TTP), 용혈성 요독성 증후군(Hemolytic uremic syndrome, HUS), HELLP(Hemolysis, Elevated Liver enzymes, Low Platelet count), 자간전증(Preeclampsia) 및 겸상적혈구질환(sickle cell disease)으로 구성된 군으로부터 선택되는 것을 특징으로 하는 조성물.The method of claim 20, wherein the thrombotic microangiopathy is thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count) , a composition selected from the group consisting of preeclampsia and sickle cell disease.
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US20150044171A1 (en) * | 2011-12-21 | 2015-02-12 | The Children's Hospital Of Philadelphia | Gain-of-Function ADAMTS13 Variants Resistant to Auto Antibody Inhibition and Methods of Use Thereof |
US9109217B2 (en) * | 2007-06-22 | 2015-08-18 | The Chemo-Sero-Therapeutic Research Institute | ADAMTS-13 mutant |
JP2016516722A (en) * | 2013-03-15 | 2016-06-09 | バイオジェン・エムエイ・インコーポレイテッドBiog | Factor VIII polypeptide preparation |
JP2018203781A (en) * | 2009-07-31 | 2018-12-27 | バクスアルタ ゲーエムベーハー | Methods of purifying recombinant adamts13 and other proteins, and compositions thereof |
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US9109217B2 (en) * | 2007-06-22 | 2015-08-18 | The Chemo-Sero-Therapeutic Research Institute | ADAMTS-13 mutant |
JP2018203781A (en) * | 2009-07-31 | 2018-12-27 | バクスアルタ ゲーエムベーハー | Methods of purifying recombinant adamts13 and other proteins, and compositions thereof |
US20150044171A1 (en) * | 2011-12-21 | 2015-02-12 | The Children's Hospital Of Philadelphia | Gain-of-Function ADAMTS13 Variants Resistant to Auto Antibody Inhibition and Methods of Use Thereof |
JP2016516722A (en) * | 2013-03-15 | 2016-06-09 | バイオジェン・エムエイ・インコーポレイテッドBiog | Factor VIII polypeptide preparation |
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