Formulation for fusion protein comprising extracellular domain of ige fc receptor subunit alpha

Abstract

This invention relates to a formulation optimized for a fusion protein dimer comprising the extracellular region of the α-subunit of an IgE Fc receptor. Specifically, this invention relates to an aqueous pharmaceutical formulation comprising the extracellular region of the α-subunit of an IgE Fc receptor, histidine, proline, methionine, and poloxamer 188, wherein the formulation has a pH of 6.0 to 7.0. Compared to conventional therapeutic agents containing anti-IgE antibodies, the fusion protein contained in the formulation according to this invention is included in a high concentration, exhibiting improved stability, convenient administration via subcutaneous injection, and excellent IgE binding capacity. Therefore, it can be effectively used as an injectable treatment for IgE-mediated allergic diseases.

Description

Technical Field

[0001] Invention Field This invention relates to formulations optimized for fusion protein dimers comprising the extracellular region of the α subunit of an IgE Fc receptor. Prior Technology

[0002] Background of the Invention In modern industrialized and Westernized society, allergic diseases, such as allergic rhinitis, atopic dermatitis, and food allergies, including asthma, are rapidly increasing, as are systemic anaphylactic reactions and severe allergic diseases. These chronic immune diseases severely impair an individual's quality of life, and the socioeconomic costs are correspondingly soaring. Therefore, there is an urgent need to take measures to overcome these diseases.

[0003] Most allergic diseases are caused by an excessive immune response to immunoglobulin E (IgE). IgE is an antibody that exists in serum at very low concentrations under normal conditions. IgE is also usually produced by harmless antigens. An increase in the number of IgE cells without any specific stimulus can lead to allergic diseases. The abnormally increased number of IgE cells can bind to high-affinity IgE Fc receptors (FcεRIs) on the surface of mast cells, basophils, and similar cells. This binding causes mast cells or basophils to release chemical mediators, such as histamine, leukotrienes, prostaglandins, bradykinin, and platelet-activating factor. The release of these chemical mediators leads to allergic symptoms. Specifically, allergic diseases can present with worsening symptoms due to the binding between IgE and FcεRIs.

[0004] Currently, various methods have been proposed to treat allergic diseases, such as avoiding allergens, administering anti-allergy medications, regulating IgE synthesis in the body, and developing anti-IgE antibodies. However, the treatments known to date have many drawbacks, such as failing to address the root cause of allergies, insufficient drug efficacy, and serious side effects.

[0005] In addition, immunoglobulin components (KR 10-1783272 B1) that can bind to IgE and FcγRIIb with high affinity and inhibit IgE-expressing cells are under investigation. Such components have been reported for the treatment of IgE-mediated diseases, including allergies and asthma.

[0006] Meanwhile, omalizumab (trade name: Xolair), which targets the Fc portion of IgE antibodies, has been developed and used as a treatment for refractory severe asthma and refractory urticaria. However, administering high doses of omalizumab to maintain therapeutic efficacy leads to high costs and side effects such as angioedema and systemic allergic reactions. Furthermore, post-marketing results have reported allergic granulomatous vasculitis and idiopathic severe thrombocytopenic purpura. Therefore, there is an increasing need to develop therapeutic agents that can effectively treat allergic diseases without side effects.

[0007] To develop therapeutic agents that can effectively treat allergic diseases without side effects, a fusion protein containing the extracellular region of the α subunit of the IgE Fc receptor was developed, and the therapeutic effect of this fusion protein on allergic diseases was demonstrated (KR 10-2038675 B1). To effectively apply this protein to the treatment of allergic diseases, it is necessary to develop a stable, high-concentration protein formulation that provides dosage and delivery advantage. Summary of the Invention

[0008] Invention Summary [Technical Issues] One objective of this invention is to provide an optimized formulation for the commercial use of a fusion protein dimer for the treatment of IgE-mediated allergic diseases. Specifically, the inventors have developed a high-concentration aqueous pharmaceutical formulation optimized for subcutaneous injection to commercialize the fusion protein dimer, thereby completing this invention. [Solution to the Problem]

[0009] To address the aforementioned problems, the present invention provides an aqueous pharmaceutical formulation comprising a fusion protein dimer, the fusion protein dimer comprising the extracellular region of the α subunit of the IgE Fc receptor (FcεRIα ECD) and having a pH of 6.0 to 7.0. [Invention Effects]

[0010] The aqueous pharmaceutical formulation according to the present invention exhibits excellent stability. This formulation comprises a fusion protein dimer containing the extracellular region (FcεRIα ECD) of the α-subunit of the IgE Fc receptor and has a pH of 6.0 to 7.0. Furthermore, the formulation containing a high concentration of the fusion protein dimer according to the present invention can be administered rapidly and conveniently via subcutaneous injection. Additionally, the formulation exhibits excellent stability, and its shelf life can be increased by reducing aggregate formation. Therefore, the formulation containing the fusion protein dimer comprising the extracellular region (FcεRIα ECD) of the α-subunit of the IgE Fc receptor can be effectively used as an injectable treatment for allergic diseases. Simple Explanation of the Diagram

[0011] Figure 1 shows photographs of samples (#1 to #4) subjected to light stress for 7.5 hours at 750 W / m2. Figure 2 shows photographs of samples (#1 to #6) subjected to stress for 5 days at 35°C and 200 rpm. Figure 3 shows photographs of samples (#1 to #6) subjected to stress for 14 days at 35°C and 200 rpm. Figure 4 shows the recovery rate (%) of samples (#1 to #6) subjected to stress for 14 days at 35°C and 200 rpm. Figure 5 shows the peak area of ​​the samples under various stress conditions. Figure 6 is a diagram showing the aggregate area of ​​the samples under various stress conditions. Figure 7 is a diagram showing the segment area of ​​the sample under various stress conditions. Figure 8 shows a syringe filled with the initial sample (#1 or #2) and a photograph of the syringe after 14 days of stress exposure at 35°C and 200 rpm. Figure 9 shows the force / length diagram. In this case, it is launched at a speed of 190 mm / min. Figure 10 is a graph showing the main peak area of ​​samples (#1 and #2) under various stress conditions. Implementation

[0012] Detailed Description of Preferred Embodiments [Formulation optimized for fusion protein dimers] In one embodiment of the present invention, an aqueous pharmaceutical formulation is provided comprising a fusion protein dimer containing the extracellular region (FcεRIα ECD) of the α subunit of an IgE Fc receptor. Here, the pH of the aqueous pharmaceutical formulation may be in the range of 6.0 to 7.0, 6.1 to 7.0, 6.2 to 6.9, 6.3 to 6.8, 6.4 to 6.8, or 6.4 to 6.6.

[0013] As used herein, the term "medicinal compound" refers to a formulation which is present in a form that allows the biological activity of the active ingredient to be clearly effective and does not include any ingredient that would cause side effects in the individual to whom the compound is administered.

[0014] The term "individual" can refer to mammals such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, and rats, but is more preferably humans, dogs, or cats.

[0015] As used herein, the term "aqueous pharmaceutical formulation" refers to a pharmaceutical formulation that uses a suitable aqueous solvent, such as water or an aqueous / oil mixture (e.g., a mixture of water and alcohol). The formulation may maintain stability, such as chemical or physical stability, or biological activity.

[0016] The term "stability" refers to a property of maintaining a constant state and is generally related to minimizing the degradation, denaturation, aggregation, or unfolding of bioactive substances such as proteins, peptides, or bioactive macromolecules.

[0017] Meanwhile, the formulation can be a liquid formulation. The liquid formulation is an aqueous solution or suspension that can remain stable at room temperature and can be refrigerated (e.g., 2°C to 8°C) or frozen (e.g., -20°C or -70°C) during storage.

[0018] The aqueous pharmaceutical formulation of this invention can be administered non-enterally. In this case, non-enteral administration can be carried out by methods such as subcutaneous administration, intravenous administration, mucosal administration, and intramuscular administration. In one embodiment of this invention, the formulation can preferably be administered by subcutaneous injection.

[0019] In one embodiment of the present invention, the aqueous pharmaceutical formulation may contain high concentrations, specifically 50 mg / mL or higher, 60 mg / mL or higher, or 70 mg / mL or higher, of fusion protein dimers. In one embodiment of the present invention, the concentration of the fusion protein dimer may be 50 mg / mL to 150 mg / mL, 50 mg / mL to 130 mg / mL, 50 mg / mL to 120 mg / mL, 50 mg / mL to 110 mg / mL, 50 mg / mL to 100 mg / mL, 50 mg / mL to 90 mg / mL, 50 mg / mL to 80 mg / mL, 60 mg / mL to 150 mg / mL, 60 mg / mL to 130 mg / mL, 60 mg / mL to 120 mg / mL, 60 mg / mL to 110 mg / mL, 60 mg / mL to 100 mg / mL, 60 mg / mL to 90 mg / mL, 60 mg / mL to 80 mg / mL, 70 mg / mL to 150 mg / mL, 70 mg / mL to 130 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 1 ...20 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 120 mg / mL, The concentrations are from 100 mg / mL to 70 mg / mL to 90 mg / mL or from 70 mg / mL to 80 mg / mL, with a preference for 75 mg / mL, but not limited thereto.

[0020] In addition, aqueous pharmaceutical formulations may further contain buffers and stabilizers. Here, the buffer may be histidine. Histidine may be present in the formulation at a concentration of 10 mM to 100 mM. In one embodiment of the invention, histidine, as a buffer, may be present in the formulation at the following concentrations: 10 mM to 90 mM, 10 mM to 80 mM, 10 mM to 70 mM, 10 mM to 60 mM, 20 mM to 90 mM, 20 mM to 80 mM, 20 mM to 70 mM, 20 mM to 60 mM, 30 mM to 90 mM, 30 mM to 80 mM, 30 mM to 70 mM, 30 mM to 60 mM, 40 mM to 90 mM, 40 mM to 80 mM, 40 mM to 70 mM, 40 mM to 60 mM, 50 mM to 90 mM, 50 mM to 80 mM, 50 mM to 70 mM, or 50 mM to 60 mM. Preferably, it may be present at a concentration of 55 mM.

[0021] Histidine buffers may comprise solutions of histidine chloride, histidine acetate, histidine phosphate, or histidine sulfate, but are not limited thereto. The pH of the histidine buffer or histidine-HCl buffer may be in the range of 5.5 to 7.5, 6.0 to 7.0, 6.1 to 7.0, 6.2 to 6.9, 6.3 to 6.8, 6.4 to 6.8, or 6.4 to 6.6, and is preferably pH 6.5.

[0022] The stabilizer may be proline. Proline may be present in the formulation at a concentration of 200 mM to 300 mM. In one embodiment of the present invention, proline may be present in the formulation at the following concentrations: 200 mM to 290 mM, 200 mM to 280 mM, 200 mM to 270 mM, 200 mM to 260 mM, 210 mM to 290 mM, 210 mM to 280 mM, 210 mM to 270 mM, 210 mM to 260 mM, 220 mM to 290 mM, 220 mM to 280 mM, 220 mM to 270 mM, 220 mM to 260 mM, 230 mM to 290 mM, 230 mM to 280 mM, 230 mM to 270 mM, 230 mM to 260 mM, 240 mM to 290 mM, 240 mM to 280 mM, 240 mM to 270 mM. mM or 240 mM to 260 mM. Preferably, it can be present at a concentration of 250 mM.

[0023] The aqueous pharmaceutical formulation of the present invention may further contain an antioxidant. Here, the antioxidant may be methionine. Methionine may be present in the formulation at a concentration of 10 mg / mL to 30 mg / mL. In one embodiment of the present invention, methionine may be present in the formulation at the following concentrations: 10 mg / mL to 28 mg / mL, 10 mg / mL to 26 mg / mL, 10 mg / mL to 24 mg / mL, 10 mg / mL to 22 mg / mL, 12 mg / mL to 28 mg / mL, 12 mg / mL to 26 mg / mL, 12 mg / mL to 24 mg / mL, 12 mg / mL to 22 mg / mL, 14 mg / mL to 28 mg / mL, 14 mg / mL to 26 mg / mL, 14 mg / mL to 24 mg / mL, 14 mg / mL to 22 mg / mL, 16 mg / mL to 28 mg / mL, 16 mg / mL to 26 mg / mL, 16 mg / mL to 24 mg / mL, 16 mg / mL to 22 mg / mL, 18 mg / mL to 28 mg / mL, 18 mg / mL to 26 mg / mL, 18 mg / mL to 24 mg / mL. mg / mL or 18 mg / mL to 22 mg / mL. Preferably, it may be present at a concentration of 20 mg / mL.

[0024] Antioxidants are agents that inhibit the oxidation of other molecules and can reduce the formation of aggregates in formulations by removing oxygen.

[0025] Furthermore, aqueous pharmaceutical formulations may further include surfactants. Surfactants are agents that reduce the surface tension of liquids and control the formation of soluble and insoluble aggregates. Surfactants may be nonionic surfactants and may be polysorbates, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 181, poloxamer 188, and poloxamer 407; or polyethylene glycol (PEG), but are not limited thereto. Preferably, the surfactant may be poloxamer.

[0026] Specifically, poloxamer can be poloxamer 188. Poloxamer 188 can be present in formulations at concentrations from 0.01 w / v% to 1 w / v%. In one embodiment of the invention, poloxamer 188 may be present in the formulation at the following concentrations: 0.02 w / v% to 0.8 w / v%, 0.02 w / v% to 0.6 w / v%, 0.02 w / v% to 0.4 w / v%, 0.02 w / v% to 0.2 w / v%, 0.02 w / v% to 0.15 w / v%, 0.04 w / v% to 0.8 w / v%, 0.04 w / v% to 0.6 w / v%, 0.04 w / v% to 0.4 w / v%, 0.04 w / v% to 0.15 w / v%, 0.06 w / v% to 0.8 w / v%, 0.06 w / v% to 0.6 w / v%, 0.06 w / v% to 0.4 w / v%, 0.06 w / v% to 0.2 w / v%. w / v%, 0.06 w / v% to 0.15 w / v%, 0.08 w / v% to 0.8 w / v%, 0.08 w / v% to 0.6 w / v%, 0.08 w / v% to 0.4 w / v%, 0.08 w / v% to 0.2 w / v%, 0.08 w / v% to 0.15 w / v%, 0.09 w / v% to 0.8 w / v%, 0.09 w / v% to 0.6 w / v%, 0.09 w / v% to 0.4 w / v%, 0.09 w / v% to 0.2 w / v%, or 0.09 w / v% to 0.15 w / v%. Preferably, it can be present at a concentration of 0.1 w / v%.

[0027] In another embodiment of the invention, an aqueous pharmaceutical formulation is provided, comprising i) a fusion protein dimer comprising FcεRIα ECD at a concentration of 50 mg / mL to 150 mg / mL; ii) histidine at a concentration of 10 mM to 100 mM; iii) proline at a concentration of 200 mM to 300 mM; iv) methionine at a concentration of 10 mg / mL to 30 mg / mL; and v) poloxamer 188 at a concentration of 0.01 w / v% to 1 w / v%. Here, the pH of the aqueous pharmaceutical formulation may be in the range of 6.0 to 7.0, 6.1 to 7.0, 6.2 to 6.9, 6.3 to 6.8, 6.4 to 6.8, or 6.4 to 6.6. Preferably, it may be pH 6.5.

[0028] In one embodiment of the present invention, the aqueous pharmaceutical formulation may comprise i) a fusion protein dimer comprising FcεRIα ECD at a concentration of 75 mg / mL; ii) histidine at a concentration of 55 mM; iii) proline at a concentration of 250 mM; iv) methionine at a concentration of 20 mg / mL; and v) poloxamer 188 at a concentration of 0.1 w / v%.

[0029] Aqueous pharmaceutical preparations may be stored in containers selected from the following groups: vials, cartridges, syringes, and auto-injectors.

[0030] In addition, containers for storing the preparations may be stored at room temperature, between 2°C and 8°C, or between 25°C and 40°C until administered to the individual in need of treatment.

[0031] The preparation can be administered via non-enteral methods, such as subcutaneous, intravenous, mucosal, intramuscular, or intraperitoneal administration, but not limited to these, using an 18G to 32G needle in a volume of 5 mL or less, 3 mL or less, or 2 mL or less. Preferably, it can be administered subcutaneously using a 27G needle in a volume of 1 mL or less. [Include] [IgE Fc] [Receptor] [α] [Fusion protein of the extracellular region of the subunit]

[0032] The fusion protein dimer described above is as follows. Specifically, the dimer comprises two monomers, each of which contains the extracellular region of the α subunit of the IgE Fc receptor (FcεRIα ECD).

[0033] The monomer may contain a modified Fc region, and the modified Fc region and FcεRIα ECD may be linked via the hinge of the IgD antibody.

[0034] As used herein, the term "IgE" refers to the antibody protein known as immunoglobulin E. IgE has an affinity for mast cells, basophils in the blood, or their analogues. Furthermore, the reaction between IgE antibodies and their corresponding antigens (allergens) causes an inflammatory response. Additionally, IgE is known to be an antibody that causes a systemic allergic reaction, which occurs due to the rapid secretion by mast cells or basophils.

[0035] As used herein, the term "IgE Fc receptor" is also called the Fcε receptor, and it binds to the Fc portion of IgE. There are two types of receptors. Receptors with high affinity for IgE Fc are called Fcε receptor I (FcεRI). Receptors with low affinity for IgE Fc are called Fcε receptor II (FcεRII). FcεRI are found in mast cells and basophils. When IgE antibodies bound to FcεRI are cross-linked with multivalent antigens, degranulation occurs in mast cells or basophils, releasing various neurotransmitters, including histamine. This release leads to an immediate allergic reaction.

[0036] FcεRI is a membrane protein composed of one α chain, one β chain, and two γ chains linked by disulfide bonds. The portion of these chains that binds to IgE is the α chain (FcεRIα), and FcεRIα is approximately 60 kDa in size, consisting of a hydrophobic region within the cell membrane and a hydrophilic region outside the cell membrane. Specifically, IgE binds to the extracellular region of the α chain.

[0037] Specifically, the α-subunit of the IgE Fc receptor may have the amino acid sequence described in NP_001992.1. Additionally, the extracellular region (FcεRIα ECD) of the α-subunit of the IgE Fc receptor may have the amino acid sequence of SEQ ID NO: 1. In this specification, the extracellular region of the α-subunit of the IgE Fc receptor may be a fragment or variant of the extracellular region of the α-subunit of the IgE Fc receptor, as long as the fragment or variant can bind to IgE.

[0038] Variants can be prepared by substituting, deleting, or adding one or more proteins to the wild-type FcεRIα ECD (extracellular region), as long as the method does not alter the function of the α chain of FcεRI. These various proteins or peptides may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the amino acid sequence of SEQ ID NO: 1. Additionally, the FcεRIα ECD of SEQ ID NO: 1 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 5.

[0039] Additionally, as used herein, the term "modified Fc region" refers to a region in which a portion of the Fc portion of an antibody has been modified. Here, the term "Fc region" refers to a protein containing the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin but not the heavy chain variable region, light chain variable region, light chain constant region, or heavy chain constant region 1 (CH1) of an immunoglobulin. Specifically, a modified Fc region means a region obtained by substituting some amino acids in an Fc region or by combining different types of Fc regions. Specifically, a modified Fc region may have the amino acid sequence of SEQ ID NO: 2. Furthermore, the modified Fc region of SEQ ID NO: 2 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 6.

[0040] Furthermore, the "modified Fc region" of this invention may be in the form of a glycan chain in its natural form, a glycan chain with an increase in number relative to the natural form, or a glycan chain with a reduction in number relative to the natural form, or a form in which the glycan chain has been removed. Immunoglobulin Fc glycan chains may be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0041] Here, the "modified Fc region" of the present invention can be a region lacking antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions due to the absence of FcγR or C1q binding sites. Furthermore, the modified Fc region and FcγRα ECD can be linked via the hinge of an IgD antibody. The hinge of the IgD antibody consists of 64 amino acids and may selectively include 20 to 60 consecutive amino acids, or 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one embodiment, the hinge of the IgD antibody may consist of 30 or 49 amino acids, as described below. Additionally, the hinge of the IgD antibody can be a hinge variant obtained by modifying the hinge region, wherein the hinge may include at least one cysteine. Here, the hinge variant can be obtained by modifying some of the hinge sequence of the IgD antibody to minimize the generation of truncated forms during protein production. In one embodiment, the hinge may include the following sequence:

[0042] Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17), wherein Xaa1 can be Lys or Gly, and Xaa2 can be Glu, Gly, or Ser. Specifically, the hinge may have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19, thereby minimizing the generation of truncated forms during protein production. In another embodiment, the hinge may include the following sequence:

[0043] Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 18), wherein Xaa3 can be Lys or Gly, and Xaa4 can be Glu, Gly, or Ser. Specifically, the hinge may have the amino acid sequence of SEQ ID NO: 4, thereby minimizing the generation of truncated forms during protein production.

[0044] Specifically, at least one Thr in the hinge having the sequence of SEQ ID NO: 4 may be glycosylated. Specifically, the 13th, 14th, 18th, and 19th Thr of the amino acid in SEQ ID NO: 18 may be glycosylated. Preferably, all four amino acids may be glycosylated. Here, glycosylation may be O-glycosylation.

[0045] Additionally, as described above, the fusion protein dimer provided by the present invention can be in the form of two monomers bound together, each monomer being obtained by binding between the extracellular region of the α subunit of the IgE Fc receptor and the modified Fc region. The fusion protein dimer can also be in the form of two identical monomers bound together by cysteine ​​located at the hinge site. Furthermore, the fusion protein dimer can also be in the form of two different monomers bound together. For example, when the two monomers are different from each other, the polypeptide dimer can be in the form of one monomer comprising the extracellular region of the α subunit of the IgE Fc receptor, and the other monomer comprising a fragment of the extracellular region of the α subunit of the IgE Fc receptor. Here, the monomer examples may have the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0046] In addition, the fusion protein dimer provided by the present invention exhibits a binding affinity to IgE that is 10 to 100 times, 20 to 90 times, 20 to 70 times, 30 to 70 times, or 40 to 70 times higher than that of omalizumab (an anti-IgE antibody), and can better exhibit a binding affinity to IgE that is 70 times higher than that of omalizumab. [Therapeutic applications of formulations optimized for fusion protein dimers]

[0047] In another embodiment of the invention, an aqueous pharmaceutical formulation comprising a fusion protein dimer is provided for use in the prevention or treatment of allergic diseases.

[0048] In this specification, the term "allergic disease" refers to pathological symptoms caused by mast cell activation, such as mast cell degranulation-mediated allergic reactions. Such allergic diseases include food allergies, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, systemic anaphylactic reactions, urticaria, pruritus, insect allergies, chronic idiopathic urticaria, chronic spontaneous urticaria, drug allergies, and similar conditions. Specifically, allergic diseases can be IgE-mediated.

[0049] The aqueous pharmaceutical formulation of this invention can be administered to individuals according to dosing regimens and dosages effective for treating allergic diseases. The formulation of this invention may contain any amount (effective amount) of fusion protein dimer, as long as the active ingredient exhibits anti-allergic activity. Based on the total weight of the composition, the typical effective amount of the active ingredient will be determined to be in the range of 0.001% by weight to 20.0% by weight. Here, "effective amount" refers to the amount capable of inducing an anti-allergic effect. Such effective amounts can be determined experimentally using the normal skills of a person skilled in this art.

[0050] Depending on the patient's condition, weight, sex, age, disease severity, or route of administration, the preferred daily dose range of the formulation is 0.01 μg / kg to 10 g / kg, and more preferably 0.01 mg / kg to 1 g / kg. Administration may be performed once or several times a day. Such dosages should in no way be construed as limiting the scope of the invention.

[0051] On the other hand, the present invention provides a method for treating or preventing allergic diseases, comprising administering to an individual an aqueous pharmaceutical formulation containing a fusion protein dimer.

[0052] The individual can be a mammal, preferably a human. In this case, administration can be via non-enteral routes. In this case, non-enteral administration can be performed by subcutaneous administration, intravenous administration, mucosal administration, intramuscular administration, or similar methods. [Methods of Implementing the Invention]

[0053] The invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative and the scope of the invention is not limited thereto. [Preparation Example] [1.] [Preparation includes] [FcεRIα ECD] [And after modification] [Fc] [District] [Fusion protein dimer] [:] [GI-301]

[0054] The C-terminus modified polypeptide of the extracellular region (FcεRIα-ECD) of the α subunit of the IgE Fc receptor was prepared according to the method disclosed in U.S. Patent No. 7,867,491.

[0055] First, to express proteins (FcεRIαECD-Fc1), (FcεRIαECD-Fc2), and (FcεR1αECD-Fc3), the extracellular region of the α chain of FcεRI containing the amino acid sequence of SEQ ID NO: 1 and the modified immunoglobulin Fc of SEQ ID NO: 2 were connected via hinges of SEQ ID NO: 19, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The cartridges obtained by connecting the genes encoding each protein were then colonized into vectors to construct the FcεRIα ECD-Fc protein expression vector. Next, each of the expression vectors was transfected into CHO cells.

[0056] Here, during transduction into cell lines, expression vectors obtained by selecting the α-2,6-sialyltransferase gene into a vector were simultaneously transfected to prepare cell lines capable of expressing the sialylated FcεRIα ECD-Fc2ST and FcεRIα ECD-Fc3ST proteins, respectively. The fusion protein dimer produced by the cell lines was named "GI-301". [Example] [1.] [Production of highly concentrated products] [GI-301] [Of] [Buffer solution and] [pH] [conditional] [optimization]

[0057] Testing was conducted to develop a liquid formulation for subcutaneous injection containing approximately 100 mg / mL of GI-301. The formulation should be stable for storage at 2°C to 8°C and have a viscosity low enough for injection. First, the buffer solution and pH conditions were optimized.

[0058] To determine the appropriate buffer formulation, a total of 25 different buffers with different pH, buffer components, ionic strength and stabilizers were selected by design of experiment (DOE) and pre-screening (steps 1 and 2) and final screening (step 3). i) Pre-screening (Steps 1 and 2):

[0059] - Dynamic laser light scattering was measured in a DLS disk reader to confirm the increase in the colloidal and thermodynamic stability of the sample.

[0060] - For each condition, dialyze the sample against the formulation buffer.

[0061] Colloidal stability was determined by measuring the hydrodynamic radius of samples at various concentrations in each formulation buffer to confirm protein-protein interactions. These results were used to predict formulation candidates with a low risk of protein aggregation during handling and storage.

[0062] Denaturation temperature (thermodynamic stability) is determined by measuring the hydrodynamic radius of samples at various temperatures in each formulation buffer. These results are used to predict formulation candidates with a low risk of protein denaturation during processing and storage. The hydrodynamic radius of a protein increases significantly as denaturation begins. The unfolding initiation temperature can be considered an indicator of protein secondary structure stability. ii) Final screening (step 3):

[0063] - Colloidal stability (intermolecular interactions, A2) is measured using CG-MALS (composition gradient multi-angle light scattering).

[0064] - Thermodynamic stability was measured using Nano DSC (Nano Differential Scanning Calorimetry).

[0065] - The aggregation state of proteins was confirmed by SE-HPLC.

[0066] - Relative performance is measured by ELISA.

[0067] Based on the above results, select the four most suitable ingredients for blending.

[0068] Specifically, the general physical properties of GI-301 regarding pH, buffer composition, and ionic strength were evaluated in the first 14 samples listed in Table 1. Samples were prepared by dialyzing GI-301 against each formulation buffer.

[0069] The pH was adjusted from 6.0 to 7.4, which is within the acceptable pH range for subcutaneous injection. For each pH value, appropriate buffer components were selected to achieve sufficient buffering capacity. The buffer components were selected from pharmaceutically acceptable excipients. Sodium chloride was added to adjust the ionic strength of the formulation (maximum ionic strength was set to isotonic). The results of the first pre-screening are summarized in Table 1 below. [Table 1] [#] [pH] [Buffer Components] [Salt] [ / ] [Stabilizer] [T, onset ] [[]℃ []] [kd[mL / mg×10, -2 , ], 1) ] [actual] [pH] 1 7.4 10 mM sodium phosphate 150 NaCl 65 -0.1 7.36 2 7.4 10 mM sodium phosphate none 68 3.1 7.36 3 7.4 10 mM Tris / HCl 150 NaCl 65 -0.1 7.40 [4] [7.4] [10 mM Tris / HCl] [none]

[64] [5.7] [7.38] 5 6.7 10 mM sodium phosphate 150 NaCl 66 -1.2 6.72 6 6.7 10 mM sodium phosphate none 60 3.3 6.68 7 6.7 10 mM His / HCl 150 NaCl 63 -1.1 6.71 [8] [6.7] [10 mM His / HCl] [none]

[69] [14.2] [6.53] 9 6.0 10 mM His / HCl 150 NaCl 54 -1.4 6.03

[10] [6.0] [10 mM His / HCl] [none]

[59] [5.8] [6.01] 11 6.7 10 mM sodium citrate 150 NaCl 65 -1.2 6.62 12 6.7 10 mM sodium citrate none 66 0.6 6.66 13 6.0 10 mM sodium citrate 150 NaCl 64 -1.4 6.00 14 6.0 10 mM sodium citrate none 63 0.1 6.00 1) Example of sample #1: kd = -0.1 mL / mg × 10-2 = -0.001 mL / mg

[0070] Generally, the colloidal stability of GI-301 is low at high ionic strengths and is independent of the buffer system used and the pH of the formulation. At low ionic strengths, the buffer components show the greatest influence on the colloidal stability of GI-301. The effect of pH is not significant. The best colloidal stability was observed in the His / HCl buffer system, followed by Tris / HCl, sodium phosphate, and sodium citrate. Generally, the higher the acidity of the formulation, the lower the colloidal stability in a buffer group. Excellent colloidal stability was observed in formulation sample #8 (10 mM His / HCl, pH 6.7), sample #10 (10 mM His / HCl, pH 6.0), and sample #4 (10 mM Tris / HCl, pH 7.4).

[0071] The thermodynamic stability of GI-301 was similar in all formulation samples except for samples #9 and #10. GI-301 exhibited high thermodynamic stability at normal drug storage temperatures.

[0072] The actual pH values ​​of the dialysis samples met the required pH values, except for formulation sample #8, in which a slight Donnan effect was observed.

[0073] Considering these results, His / HCl at pH 6.7 and Tris / HCl at pH 7.4 were selected as the most suitable buffer solutions for GI-301. Stabilizers and nonionic isotropic agents suitable for liquid and / or lyophilization were tested in samples #15 to #20. The excipients used in the tests were trehalose (a cryoprotectant used in lyophilization), mannitol (a build-up agent and nonionic isotropic agent used in lyophilization), and proline (an amino acid stabilizer used in liquid formulations, nonionic). The results of the two-step screening of excipients are summarized in Table 2 below. [Table 2] [#] [pH] [Buffer Components] [Salt] [ / ] [Stabilizer] [T, onset ] [[]℃ []] [kd[mL / mg×10, -2 , ]] [actual] [pH] 15 6.7 10 mM His / HCl 250 mM Trehalose 67 6.3 6.44

[16] [6.7] [10 mM His / HCl] [250 mM] Mannitol

[66] [13.5] [6.52]

[17] [6.7] [10 mM His / HCl] [250 mM] [Proline]

[64] [20.4] [6.55] 18 7.4 10 mM Tris / HCl 250 mM Trehalose 65 5.2 7.34

[19] [7.4] [10 mM Tris / HCl] [250 mM] Mannitol

[68] [6.1] [7.40]

[20] [7.4] [10 mM Tris / HCl] [250 mM] [Proline]

[68] [7.3] [7.39]

[0074] Increased colloidal stability was observed with the addition of mannitol and proline to both His / HCl and Tris / HCl buffer systems. A slight decrease in colloidal stability was observed with the addition of trehalose. Excipients did not show a significant effect on the thermodynamic stability of GI-301 or on the actual pH. In the final screening experiments, CG-MALS (A2), Nano DSC (Tonset), SE-HPLC (relative monomer content), and ELISA (relative efficacy) were used to study the most promising formulation samples in both liquid and lyophilized forms. The results of the final screening are summarized in Table 3 below. [Table 3] [#] [pH] [Buffer Components] [Stabilizer] [T, onset ] [Nano DSC] [[]℃ []] [A, 2] [[10, -4 , mol*ml*g, -2 , ]] [according to] [SE-HPLC] [Relative monomer content] [[%]] [ELISA] [efficacy] [[%]] twenty one 6.7 10 mM His / HCl 250 mM Trehalose 51 4.8 96.2 103 twenty two 6.7 10 mM His / HCl 250 mM mannitol 52 7.8 96.1 98 twenty three 6.7 10 mM His / HCl 250 mM proline 52 8.7 96.1 101 twenty four 7.4 10 mM Tris / HCl 250 mM mannitol 51 4.4 96.1 92 25 7.4 10 mM Tris / HCl 250 mM proline 49 4.8 96.1 106

[0075] The relative ranking of colloidal stability and tonset for the five samples was similar to the results previously obtained using DLS. CG-MALS results showed strong repulsive protein-protein interactions in all formulation samples. Compared to DLS measurements, nanoDSC measurements showed lower tonset values ​​for thermal defolding. DLS measurements showed an increase in the hydrodynamic radius of proteins, a result of aggregation due to thermal defolding. In contrast, nanoDSC measured the thermodynamic transition from folded to unfolded proteins and therefore showed lower tonset values. The relative efficacy measured by ELISA was almost 100% in all formulation samples.

[0076] Based on the above results, and taking into account the items described in Table 4 below, a "concentration challenge test" was conducted. [Table 4] [#] [pH] [Buffer Components] [Stabilizer] [describe] twenty one 6.7 10 mM His / HCl 250 mM Trehalose The actual pH of the liquid formulation is 6.5; a backup is used for freeze-drying. twenty two 6.7 10 mM His / HCl 250 mM mannitol Liquid formulations with a pH of 6.5; commonly used excipients twenty three 6.7 10 mM His / HCl 250 mM proline Liquid formulations with a pH of 6.5 may exhibit a low tendency to aggregate. 25 7.4 10 mM Tris / HCl 250 mM proline A liquid formulation with a pH of 7.4; perfectly isotonic, but may have lower chemical stability. [Example] [2.] [Regarding high concentrations] [GI-301] [A test of probability]

[0077] Of the 25 samples tested in Example 1, four of the most suitable samples were selected for the concentration challenge test. The four candidate formulation samples selected based on the results of Example 1 are summarized in Table 5 below. [Table 5] [#] [pH] [Buffer Components] [Stabilizer] 1 6.7 10 mM His / HCl 250 mM Trehalose 2 6.7 10 mM His / HCl 250 mM mannitol 3 6.7 10 mM His / HCl 250 mM proline 4 7.4 10 mM Tris / HCl 250 mM proline The following tests were conducted on the four formulation candidates:

[0078] - Confirm that the target concentration of approximately 100 mg / mL has been achieved in each formulation sample (using ultrafiltration and process-related time intervals).

[0079] - Measure the pH of concentrated and mixed samples to monitor pH shifts caused by the Donnan effect.

[0080] - Filter the sample using a 0.22 μm membrane filter and aseptically fill it into a 2R vial using a multichannel pipette with a sterile plunger tip (target filling volume of 1.2 mL for 100 mg / mL).

[0081] - Challenge tests were conducted on all highly concentrated samples by storing them at 4°C and 35°C for 72 hours.

[0082] - After 72 hours, the turbidity and sediment of the sample were analyzed by visual inspection.

[0083] - Viscosity and Injectability: Injectability of each formulation sample was assessed by measuring the ejection force. The contents of the syringe were dispensed via a connected needle at a specified lateral velocity using a force / displacement measuring device. The following parameters of the sample were analyzed: visual appearance, concentration according to UV 280, pH, viscosity using a capillary viscometer, turbidity measured by turbidimetry, osmotic concentration by weight moles, relative efficacy according to ELISA, and aggregation state according to SE-HPLC.

[0084] As a result, the four formulations in Table 5 above could be successfully concentrated to a concentration of 100 mg / mL. After storage at 35°C for 72 hours, the highly viscous (100 mg / mL) solution in the vial did not show precipitation or significant turbidity.

[0085] Injectability testing confirmed that using a 30G cannula resulted in an ejection force exceeding the 20 N limit for all 100 mg / mL samples. The ejection force was acceptable when using a 27G cannula. The 30G cannula was acceptable when diluted to a concentration of 50 mg / mL.

[0086] As a result of the concentration, the pH decreased by approximately 0.4 to 0.8 (Donnan effect). To compensate for the Donnan effect, the pH was adjusted back to its original concentration using highly concentrated L-histamine and TRIS base.

[0087] The final protein concentration was approximately 65 mg / mL. A 1 mL syringe containing 1 mL of sample was passed through the tensile testing machine, and the shear stress material (the contents ejected from the syringe) was analyzed.

[0088] All results are shown in Table 6 below. [Table 6] [sample] [GI-301] [Concentration] [Primary Packaging Materials] [method] [#1] [#2] [#3] [#4] 100 mg / mL 2R small bottle Visual appearance / precipitation After 72 hours at 5°C √ √ √ √ 100 mg / mL 2R small bottle Visual appearance / precipitation After 72 hours at 35°C √ √ √ √ 100 mg / mL 1 mL syringe with 30G cannula Thrust X X X X Visual appearance / precipitation √ √ √ √ 100 mg / mL 1 mL syringe 27G cannula Thrust √ √ √ √ Visual appearance / precipitation √ √ √ √ 50 mg / mL 1 mL syringe with 30G cannula Thrust √ √ √ √ Visual appearance / precipitation √ √ √ √ pH - Effects of the release √ √ √ √ 65 mg / mL (Donnan effect; compensated samples #2-#4) 1 mL syringe 27G cannula Thrust √ √ √ Visual appearance / precipitation √ √ √ pH - Effects of the release √ √ √ SEC √ √ √ ELISA √ √ √ viscosity √ √ √ Turbidity √ √ √ Weight mole osmotic concentration √ √ √ Acceptable result: √ Unacceptable result: X

[0089] When using protein at concentrations of 65 mg / mL or lower, the injectability of all four formulation samples was confirmed, even with the use of 30G tubing.

[0090] In all four formulation samples, the GI-301 protein was extremely stable during short-term storage at concentrations of 100 mg / mL or lower.

[0091] Since long-term stability cannot be predicted based on short-term data, accelerated stability testing was conducted separately.

[0092] The four formulation candidates showed similar results, as shown in Table 6 above. [Example] [3.] [Accelerated Stability Test] [(] [Forced Degradation Test] [I)]

[0093] In Example 2, the protein was concentrated at high concentrations in four formulation buffers, and injectability was also verified. Analysis of the four samples confirmed no significant differences among them. Therefore, forced degradation tests were performed on the four samples from Example 2 (see Table 5) using 2R vials.

[0094] Four candidate formulations with a target protein concentration of 50 mg / mL were prepared, and the following steps were performed: - Ten 2R vials are aseptically filled for each sample. - Forced thermal stress and stirring: 200 rpm at 35°C for 2 weeks or 5 days. - Light exposure: 750 W / m² for 7.5 hours. - Analyze the following items of the sample: visual appearance, concentration according to UV 280, pH, turbidity measured by turbidimetric method, osmotic concentration by weight moles, relative efficacy according to ELISA and aggregation state according to SE-HPLC.

[0095] Four formulation samples were successfully concentrated to approximately 50 mg / mL with appropriate pH offset compensation. Subsequently, accelerated stress stability tests were performed using 2R vials.

[0096] The best (√) and worst (X) results are shown in Table 7 below. [Table 7] [Experimental Methods] [sample] [#1] [#2] [#3] [#4] Visual inspection 5 days 35℃, 200 rpm 14 days, 35℃, 200 rpm Light 5 days 35℃, 200 rpm 14 days 35℃, 200 rpm Light 5 days 35℃ 200 rpm 14 days 35℃ 200 rpm Light 5 days 35℃, 200 rpm 14 days, 35℃, 200 rpm Light X X √ √ X √ √ X √ √ X √ SE-HPLC 14 days 35℃ 200 rpm Light 14 days 35℃ 200 rpm Light 14 days 35℃ 200 rpm Light 14 days 35℃ 200 rpm Light SE- HPLC Main Peak (General) √ √ X X Aggregates √ √ X X Excerpt √ X X √

[0097] Based on the results, sample #3 (proline, histidine / HCl, pH 6.7) was the most suitable buffer for the next development step. Additionally, this sample was used for a 150 mg / mL concentration test via cross-flow filtration. Meanwhile, as a result of SE-HPLC analysis, all formulation samples could be frozen / thawed three times without significant negative impact. [Example] [4.] [Feasibility Analysis of High Concentration Filtration via Cross-Flow Filtration]

[0098] Based on the results of Example 3, a formulation containing 250 mM proline, 10 mM histidine / HCl, and pH 6.7 was selected as the formulation for a feasibility analysis of high concentration (150 mg / mL) via cross-flow filtration. The goal was to obtain a concentrated protein solution (150 mg / mL). [Example] [4.1.] [Cross-flow filtration and] [pH] [adjust]

[0099] In the first step, 1,400 g of the 7.5 mg / mL substance was concentrated to approximately 200 mL. Concentration was carried out at a transmembrane pressure (TMP) of 1.0 bar.

[0100] Following this step, buffer exchange (each perfiltration) begins in an isochoric manner.

[0101] The target buffer solution was used to replace the permeate discharge, and this buffer was fed into the stirred vessel of the cross-flow apparatus. The total feed volume of the target buffer solution was 950 g. The concentration of the original DS buffer solution was less than 1% at the end of the dialysis step.

[0102] Next, a second concentration step was performed until a volume of approximately 110 g was achieved. The pH was adjusted to a target of 6.70 using a 150 mM histidine / 250 mM proline buffer. Afterward, the concentration step was continued.

[0103] It was confirmed that the maximum concentration was reached when the permeate volume decreased and the cross-flow filtration device issued a peak pressure alarm. The pH was finely adjusted, and the concentrate was collected. The resulting solution was pre-filtered with a 1 μm filter, then filtered aseptically with a 0.2 μm filter, and stored at 2°C to 8°C until further processing. [Example] [4.2.] [Visual Inspection]

[0104] After filtration and concentration, the solution is slightly yellow and exhibits turbidity.

[0105] The solution obtained after filtration through 1.0 μm and 0.2 μm filters was transparent and slightly yellow. [Example] [4.3. UV 280]

[0106] After filtration, the concentration of the resulting solution was 106.3 mg / mL ± 2.6 mg / mL (n=3). [Example] [4.4.] [Injectability]

[0107] Table 8 shows the maximum ejection force during the measurement. The 27G cannula is suitable for GI-301 at a concentration of approximately 100 mg / mL. The 30G cannula exhibits excessively high ejection force at a concentration of approximately 100 mg / mL. Each of the above experiments was repeated twice (n=2). [Table 8] casing Fmax [N] 27G (0.4 mm OD) 12 mm 10.6±0.5 30G (0.3 mm OD) 12 mm 23.5±1.1

[0108] Cross-flow concentration of GI-301 was confirmed to be feasible. The target concentration (150 mg / mL) could not be achieved due to the high viscosity of the solution at high concentrations. The concentration after dialysis was approximately 106 mg / mL. Injectability was demonstrated with a moderate push force of approximately 11 N using a 27G cannula. [Example] [5.] [Accelerated Stability Test] [(] [Forced Degradation Test] [II)]

[0109] In Example 4, the buffer composition (250 mM proline, histidine / HCl, pH 6.7) was concentrated to a maximum concentration of approximately 106 mg / mL via cross-flow filtration. Therefore, a forced degradation test II was performed at a target concentration of 100 mg / mL GI-301. The surfactant and / or antioxidant methionine were added to sample #3 obtained in Example 2 to confirm the benefits of these additives (Table 9). 2R vials were filled with 1.5 mL of the corresponding solution, and the samples were analyzed after exposure to thermal / mechanical stress and light stress. [Table 9] Basic components 250 mM proline, histidine / HCl, pH 6.7 [additive] [#1] (No additives) [#2] [#3] [#4] [#5]1) [#6]1) Methionine 20 mg / mL 20 mg / mL 20 mg / mL 20 mg / mL Tween 80 0.1 w / v% 0.1 w / v% Polosham 0.1 w / v% Tween 20 0.1 w / v% 1) Prepare samples after obtaining results #1 to #4.

[0110] The following are samples of formulations subjected to forced degradation tests using surfactants and methionine: #1: Additive-free formulation (no PS80, no methionine) #2: Formula containing methionine (20 mg / mL) #3: Formula containing Tween 80 (0.1 w / v%) #4: A formulation containing Tween 80 (0.1 w / v%) and methionine (20 mg / mL). #5: A formulation containing poloxamer 188 (0.1 w / v%) and methionine (20 mg / mL). #6: A formulation containing Tween 20 (0.1 w / v%) and methionine (20 mg / mL). [Example] [5.1.] [Visual Inspection]

[0111] As a result of visual inspection, the solution became more yellow due to light stress. After storage at 35°C and 200 rpm for 14 days, the turbidity of samples #1, #3, and #4 increased, sample #6 showed slight turbidity, and samples #2 and #5 showed a clear solution state (Figures 1 to 3). Table 10 shows the results of visual inspection of each sample. [Table 10] sample #1 #2 #3 #4 #5 #6 Optical stress 7.5 hours, 750 W / m2 Clear solution, pale yellow Clear solution, pale yellow Clear solution, pale yellow Clear solution, pale yellow not applicable not applicable 35℃ 200 rpm, 5 days Visible turbidity, pale yellow Clear solution, pale yellow Visible turbidity, pale yellow Visible turbidity, pale yellow Clear solution, pale yellow Clear solution, pale yellow 35℃ 200 rpm 14 days High turbidity, pale yellow Clear solution, pale yellow High turbidity, pale yellow High turbidity, pale yellow Clear solution, pale yellow Slightly turbid, pale yellow [Example] [5.2. UV 280]

[0112] Table 11 shows the concentrations of the samples after the forced degradation test. There were no significant differences based on stress exposure. [Table 11] Samples (n=2) #1 #2 #3 #4 #5 #6 c [mg / mL] c [mg / mL] c [mg / mL] c [mg / mL] c [mg / mL] c [mg / mL] 35℃ 200 rpm, 5 days 96.8±0.7 98.9±2.1 96.2±0.3 95.5±0.3 93.9±1.5 95.3±2.8 35℃ 200 rpm, 14 days 102.1±1.6 94.3±1.7 96.5±2.5 94.5±1.5 95.6±1.0 96.0±2.5 Optical stress 7.5 hours 750 W / m2 97.6±1.6 96.1±1.1 95.2±0.8 95.8±2.4 - - [Example] [5.3. pH] [Measurement]

[0113] Table 12 shows the pH of the samples after the forced degradation test. There was no significant change in pH. [Table 12] Samples (n=2) #1 #2 #3 #4 #5 #6 pH pH pH pH pH pH 35℃ 200 rpm, 5 days 6.82±0.01 6.74±0.00 6.74±0.00 6.72±0.00 not applicable not applicable 35℃ 200 rpm, 14 days 6.76±0.00 6.71±0.00 6.73±0.01 6.70±0.00 not applicable not applicable Optical stress 7.5 hours 750 W / m2 6.72±0.01 6.72±0.00 6.71±0.01 6.70±0.01 not applicable not applicable [Example] [5.4.] Size exclusion chromatography [(] [SE-HPLC] [)]

[0114] SE-HPLC is a standard method for detecting aggregated and fragmented proteins. SEC measurement results are shown in Table 13 and Figures 5 to 7. [Table 13] Samples (n=2) Stress type #1 #2 #3 #4 #5 #6 Main peak area / percentage (%) 35℃ 200 rpm 5 days 53.15±1.14 90.07±0.29 89.84±0.00 90.27±0.02 90.15±0.17 89.20±0.08 35℃ 200 rpm 14 days 36.83±0.08 88.07±0.05 87.52±0.04 87.89±0.05 87.78±0.03 86.19±0.01 Optical stress 92.97±0.07 94.08±0.45 93.12±0.43 93.74±0.52 not applicable not applicable Aggregate area / percentage (%) 35℃ 200 rpm 5 days 5.78±0.23 8.20±0.08 8.72±0.04 8.24±0.06 8.26±0.11 9.23±0.05 35℃ 200 rpm 14 days 4.90±0.07 9.38±0.06 9.85±0.05 9.42±0.01 9.46±0.05 10.97±0.01 Optical stress 6.06±0.03 5.28±0.00 6.26±0.02 5.63±0.06 not applicable not applicable Fragment area / percentage (%) 35℃ 200 rpm 5 days 41.07±1.37 1.73±0.21 1.44±0.03 1.50±0.04 1.59±0.05 1.58±0.13 35℃ 200 rpm 14 days 58.27±0.01 2.55±0.01 2.63±0.01 2.69±0.05 2.77±0.02 2.84±0.01 Optical stress 0.98±0.04 0.64±0.45 0.62±0.45 0.63±0.46 not applicable not applicable Recovery rate / percentage (%) 35℃ 200 rpm 14 days 97.17±0.26 100.25±0.07 98.61±0.14 98.56±0.04 100.17±0.14 100.43±0.12

[0115] As shown in Table 13, the recovery rates under stress conditions of 35°C and 200 rpm for 14 days were comparable to those observed visually: samples #2, #5 and #6 showed high recovery rates (Figure 4). Furthermore, as shown in Figure 5, samples #2, #4, and #5, under applied thermal / mechanical stress, yielded better results in measuring the main peak, while samples #1 and #6 showed poorer results. Meanwhile, as shown in Figure 6, protein aggregation was observed in sample #6. Under photostress conditions, sample #2 showed the minimum amount of protein aggregation. As shown in Figure 7, sample #2 demonstrated the best results regarding protein fragments. [Example] [5.5. GI-301] [Based on] [ELISA] [Effectiveness Analysis]

[0116] The relative potency of GI-301 was determined based on the results of an ELISA-based potency analysis. The principle of this analysis is to quantify the GI-301 molecules that can bind to human IgE.

[0117] The relative power of the ELISA analysis is shown in Table 14. All samples maintained high binding affinity. [Table 14] Samples (n=2) #1 #2 #3 #4 35℃, 200 rpm, 5 days 79.7%±3.8% 112.7%±1.6% 92.1% ± 5.2% 94.3% ± 30.4 35℃, 200 rpm, 14 days 104.9%±0.0% 74.2%±6.5% 80.4%±7.5% 110.9%±12.6% Optical stress 110.2%±6.6% 112.2%±5.1% 110.1%±6.8% 111.2%±3.3% [Example] [5.6.] [Turbidity measured using turbidimetry]

[0118] Table 15 presents the results of turbidity measurements. There was no significant correlation between visual appearance and turbidity measurements (compare samples #3 and #4 after 5 days of storage at 35°C). One potential reason for this difference could be the increased milky white light in concentrated protein solutions, which increases the baseline turbidity. Consequently, the visible turbidity may be higher than the baseline turbidity due to protein aggregation. Therefore, the samples were stored at 35°C for 14 days and then analyzed by DLS. [Table 15] Samples (n=2) #1 #2 #3 #4 NTU NTU NTU NTU 35℃, 200 rpm, 5 days 35.05±0.07 11.52±3.80 11.50±0.14 10.70±0.14 Optical stress 11.65±1.48 9.38±1.59 7.37±0.20 6.84±0.06 [Example] [5.7.] [Osmotic Concentration by Weight in Moles]

[0119] The results for weight mole osmotic concentration are shown in Table 16. As expected, methionine increased the weight mole osmotic concentration. [Table 16] Samples (n=2) #1 #2 #3 #4 mOSMOL / kg mOSMOL / kg mOSMOL / kg mOSMOL / kg 35℃, 200 rpm, 5 days 351±1 500±2 338±0 498±1 35℃, 200 rpm, 14 days 363±1 488±3 333±1 492±0 Optical stress 338±2 496±1 338±1 495±0 [Example] [5.8.] [Analysis of Measurement Results]

[0120] The results are summarized in Table 17 below. [Table 17] sample #1 #2 #3 #4 #5 #6 Visual appearance after 14 days at 35℃ / 200 rpm △ ○ × × ○ △ SEC recovery rate after 14 days at 35°C / 200 rpm × ○ △ △ ○ ○ SEC main peak after 14 days at 35℃ / 200 rpm × ○ △ ○ ○ × DLS after 14 days at 35℃ / 200 rpm △ △ × × △ △ ×: Unfavorable result, △: Moderate result, ○: Good result

[0121] Based on these results, the most suitable formulation sample is as follows: Sample #2: His / proline pH 6.7; 20 mg / mL methionine Sample #5: His / proline pH 6.7; 20 mg / mL methionine; 0.1 w / v% poloxamer 188

[0122] As a result of the test, samples #2 and #5 are suitable among the formulation samples in Table 17 above. [Example] [6.] [Forced Degradation Test Using a Syringe]

[0123] Following Example 5, two formulations were selected using 2R vials: 250 mM proline and histidine / HCl, pH 6.7, 0.1 w / v% poloxamer 188, with / without 20 mg / mL methionine (antioxidant). Based on these results, the antioxidant and surfactant were added to the sample (Table 18). The target concentration of the sample was 100 mg / mL. A 2.25 mL syringe was filled with 1.75 mL of the corresponding solution, and the sample was analyzed after 14 days of thermal / mechanical stress exposure.

[0124] The samples used for the forced degradation study are as follows: #1: Formula containing methionine (20 mg / mL) #2: A formulation containing poloxamer 188 (0.1 w / v%) and methionine (20 mg / mL). [Table 18] Basic ingredients 250 mM proline, histidine / HCl, pH 6.7 additive #1 #2 Methionine 20 mg / mL 20 mg / mL Polosham 188 - 0.1 w / v% [Example] [6.1.] [Visual Inspection]

[0125] The results of the visual inspection are shown in Table 19. As shown in Figure 8, there was no significant difference after storage at 35°C for 14 days. [Table 19] sample #1 #2 initial state Clear solution, pale yellow Clear solution, pale yellow 35℃, 200 rpm, 14 days Clear solution, pale yellow Clear solution, pale yellow [Example] [6.2.] [Measurement of Push Force] []

[0126] Table 20 shows the results of the stretching test. When using a 27G cannula, an extrusion force exceeding 20 N was observed in both samples (Figure 9). Therefore, syringes with larger inner diameters or larger needle sizes (e.g., 25G) should be selected to withstand protein concentrations of 100 mg / mL. [Table 20] 27G (0.4 mm OD) 18 mm sample 27G (0.4 mm OD) 18 mm #1 #2 27G (0.4 mm OD) 18 mm Fmax[N] Fmax[N] n1 43.7 43.3 n2 42.6 41.0 [Example] [6.3. UV 280]

[0127] Table 21 shows the concentrations of the samples after the forced degradation test. No significant differences were observed after stress exposure. [Table 21] Samples (n=2) initial state After 14 days at 35℃ and 200 rpm #1 #2 #1 Syringe 1 #1 Syringe 2 #2 Syringe 1 #2 Syringe 2 c[mg / mL] c[mg / mL] c[mg / mL] c[mg / mL] c[mg / mL] c[mg / mL] 98.9±2.9 99.6±1.6 100.2±1.2 101.3±0.8 98.2±1.0 98.9±0.7 [Example] [6.4. pH] [Measurement]

[0128] Table 22 shows the pH of the samples after the forced degradation test. There was no significant change in pH. [Table 22] Samples (n=2) initial state After 14 days at 35℃ and 200 rpm #1 #2 #1 Syringe 1 #1 Syringe 2 #2 Syringe 1 #2 Syringe 2 pH pH pH pH pH pH 6.74±0.00 6.77±0.01 6.75±0.01 6.73±0.03 6.75±0.00 6.75±0.00 [Example] [6.5.] Size exclusion chromatography [(SE-HPLC)]

[0129] The SEC measurement results are shown in Table 23 and Figure 10. [Table 23] Samples (n=2) Initial state (t0) After 14 days at 35℃ and 200 rpm #1 #2 #1 Syringe 1 #1 Syringe 2 #2 Syringe 1 #2 Syringe 2 Main peak area / percentage (%) 93.87±0.14 93.73±0.03 83.83±0.04 83.74±0.16 84.44±0.41 84.10±0.11 Aggregate area / percentage (%) 4.36±0.04 4.49±0.01 9.60±0.04 9.61±0.08 9.40±0.16 9.48±0.05 Fragment area / percentage (%) 1.76±0.18 1.79±0.02 6.57±0.00 6.66±0.08 6.16±0.26 6.41±0.06 Recovery rate / percentage (%) 98.19±0.02 98.79±0.03 101.56±0.28 102.99±0.34 101.44±0.72 102.72±0.14

[0130] After storage at 35°C and 200 rpm for 14 days, there was no significant difference between the two samples. However, the main peak area in sample #2 (containing poloxamer) was better than that in sample #1 (without poloxamer). The fragment area increased by approximately 4% compared to the results from the vials. This is consistent with the main peak results (Figure 10). [Example] [6.6.] [Osmotic Concentration by Weight in Moles]

[0131] The results for weight molar osmotic concentration are shown in Table 24. Even after stress exposure, the weight molar osmotic concentration remained at the same level. [Table 24] Samples (n=2) initial state After 14 days at 35℃ and 200 rpm #1 #2 #1 Syringe 1 #1 Syringe 2 #2 Syringe 1 #2 Syringe 2 mOSMOL / kg mOSMOL / kg mOSMOL / kg mOSMOL / kg mOSMOL / kg mOSMOL / kg 504±1 507±2 515±8 514±6 514±2 516±1 [Example] [6.7.] [Subvisible particles]

[0132] The amount of subvisible particles was assessed using fluid imaging microscopy. The pressure injector filled with water exhibited a low particle load. This indicates that the effect of the silicate injector on particle load is limited.

[0133] Samples #1 and #2 showed comparable particle loading. Most of the observed particles were polysiloxane particles, and no particles indicated protein aggregation / precipitation. [Example] [6.8.] [Analysis of Measurement Results]

[0134] The results are summarized in Table 25. [Table 25] sample #1 #2 Visual appearance after 14 days at 35℃ / 200 rpm ○ ○ SEC recovery rate after 14 days at 35°C / 200 rpm ○ ○ SEC main peak after 14 days at 35℃ / 200 rpm ○ ○ DLS after 14 days at 35℃ / 200 rpm △ △ Flow imaging microscopy (qualitative) ○ ○ ×: Unfavorable result, △: Moderate result, ○: Good result

[0135] The two samples showed almost identical results. The only difference was that sample #2 showed slightly better results in the SEC analysis (relative peak area).

[0136] Based on the above results, the optimal formulation for GI 301 is a buffer solution containing 100 mg / mL GI-301, 20 mg / mL methionine as an antioxidant, and 0.1 w / v% poloxamer 188 as a surfactant, consisting of 250 mM proline, 10 mM histidine / HCl, and pH 6.7.

[0137] However, under the above conditions, due to the Donnan effect occurring during ultrafiltration, the pH drops below the target pH of 6.7 to achieve a final concentration of 100 mg / mL. To compensate for the pH decrease, a process of adjusting the pH by diluting with a high concentration of histamine after ultrafiltration was added. Finally, the preparation conditions for GI-301 were established as follows: 75 mg / mL GI-301, 20 mg / mL methionine, 250 mM proline, 55 mM histamine / HCl, 0.1 w / v% poloxamer 188, and pH 6.5.

[0138] (none)

[0139] <![CDATA[ <110> GI INNOVATION, INC., a South Korean company <![CDATA[ <120> A formulation containing the extracellular region of the IgE Fc receptor subunit α. <![CDATA[ <140> TW 111108408]]> <![CDATA[ <141> 2022-03-08 <![CDATA[ <150> KR 10-2021-0030501]]> <![CDATA[ <151> 2021-03-09 <![CDATA[ <160> 22]]> <![CDATA[ <170> KopatentIn 2.0]]> <![CDATA[<210> 1]]> <![CDATA[<211> 180]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> FceRI1 ECD]]> <![CDATA[<400> 1]]> Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln 180 <![CDATA[ <210> 2]]> <![CDATA[ <211> 215]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Modified Fc]]> <![CDATA[ <400> 2 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <![CDATA[<210> 3]]> <![CDATA[ <211> 30]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> IgD hinge variant]]> <![CDATA[ <400> 3]]> Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <![CDATA[ <210> 4]]> <![CDATA[ <211> 49]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> IgD hinge variant]]> <![CDATA[ <400> 4]]> Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro 1 5 10 15 Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser 20 25 30 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45 Pro <![CDATA[ <210> 5]]> <![CDATA[ <211> 540]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[<223> Nucleotide sequence of FceRI1 ECD]]> <![CDATA[<400> 5]]> gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt caagggcgag 60 aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg tgagcagcac caagtggttc 120 cacaatggca gcctgagcga ggagaccaac agctccctga acatcgtgaa cgccaagttc 180 gaggacagcg gcgagtacaa gtgccagcac cagcaggtga acgagagcga gcccgtgtac 240 ctggaggtgt tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc 300 cagcccctgt tcctgagatg ccacggctgg agaaactggg acgtgtacaa ggtgatctac 360 [[ID=​​​​ gccaccgtgg aggacagcgg cacctactac tgcacaggca aggtgtggca gctggactac 480 gagagcgagc ccctgaacat caccgtgatc aaggctccca gagagaagta ctggctgcag 540 540 <![CDATA[ <210> 6]]> <![CDATA[ <211> 561]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Modified Fc nucleotide sequence <![CDATA[ <400> 6]]> tgcgtggtcg tggatgtgag ccaggaagat cccgaagtgc agttcaactg gtacgtggat 60 ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac 120 agagtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 180 tgcaaggtgt ccaacaaagg cctgcccagc tccatcgaga agaccatcag caaagccaaa 240 ggccagccca gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag 300 aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgtggag 360 tgggaaagca acggccagcc cgagaacaat tacaagacaa cccctcccgt gctggatagc 420 gatggcagct tctttctgta cagcagactg accgtggaca agagcagatg gcaggaaggc 480 aacgtgttca gctgcagcgt gatgcacgaa gccctgcaca accactacac ccagaagagc 540 ctgtccctga gcctgggcaa g 561 <![CDATA[ <210> 7]]> <![CDATA[ <211> 174]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Nucleotide sequence of IgD hinge variant]]> <![CDATA[ <400> 7]]> aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag 60 gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg 120 ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc 174 <![CDATA[ <210> 8]]> <![CDATA[ <211> 231]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> IgD hinge variant nucleoside acid sequence <![CDATA[ <400> 8]]> gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc caccaccagg 60 aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg agaaggagga gcaggaggaa 120 agagaaacca agacccccga gtgccccagc cacacccagc ccctgggcgt gttcctgttc 180 ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac c 231 <![CDATA[ <210> 9]]> <![CDATA[ <211> 25]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Signal peptide <![CDATA[ <400> 9]]> Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala 20 25 <![CDATA[ <210> 10]]> <![CDATA[ <211> 75]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> [nucleotide sequence of signal peptide] <![CDATA[<400> 10]]> atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60 tcccctagcc acgcc 75 <![CDATA[<210> 11]]> <![CDATA[<211> 450]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> [[ID=--]] <![CDATA[<220>]]> [[ID=3--]] <![CDATA[<223> FceRIa ECD-Hinge-Fc2]]> <![CDATA[<400> 11]]> Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val 20 25 30 It should be noted that in the translation, for the tags like and which seem to be incomplete in the original numbering, they are kept as they are according to the requirement. And the Chinese "鉸鏈" is translated as "Hinge" in the text "FceRIa ECD-Hinge-Fc2". If there are more specific requirements or corrections needed for these parts, please adjust accordingly. Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr 35 40 45 Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp 50 55 60 Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile 65 70 75 80 Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln 85 90 95 Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp 100 105 110 Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu 115 120 125 Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile 130 135 140 Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn 145 150 155 160 Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys 165 170 175 Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile 180 185 190 Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Arg Asn Thr 195 200 205 Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln 210 215 220 Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro 225 230 235 240 Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <![CDATA[ <210> 12]]> <![CDATA[ <211> 1350]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Nucleotide sequence of FceRIa ECD-Hydroxylene-Fc2]]> <![CDATA[ <400> 12]]> atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60 tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120 atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180 agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240 gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag 300 agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360 gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420 tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac 480 atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg 540 tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaggc tcccagagag 600 aagtactggc tgcagagaa caccggcaga ggaggcgag aaagaaagg aagcaggag 660 aaggaggagc aggaggaag aggaaccaag accccgagt gccccagcca cacccagcccc 720 ctgggcgtgt tcctgttccc cccaagccc aaggacaccc tgatgatcag cagaaccccc 780 gaggtgacct gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg 840 tacgtggatg gcgtggaagt gcacaacgcc agaccaagc ccagagaga gcagttcaac 900 tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960 gagtacaagt gcaaggtgtc cacaaggc ctgcccagct ccatcgagaa gaccacagc 1020 aaagccaaag gccagcccag agaaccccag gtgtacaccc tgcctcccag ccaggaagag 1080 atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc 1140 gccgtggagt gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg 1200 ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg 1260 caggaaggca acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc 1320 [[ID=2..23]] cagaagagcc tgtccctgag cctgggcaag 1350 <![CDATA[<210> 13]]> <![CDATA[<211> 469]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> FceRIa ECD-Hinge-Fc3]]> <![CDATA[<400> 13]]> Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val 20 25 30 Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr 35 40 45 Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp 50 55 60 Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile 65 70 75 80 Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln 85 90 95 Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp 100 105 110 Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu 115 120 125 Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile 130 135 140 Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn 145 150 155 160 Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys 165 170 175 Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile 180 185 190 Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Ala Gln Pro 195 200 205 Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr 210 215 220 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 225 230 235 240 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 245 250 255 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 340 345 350 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Leu Gly Lys 465 <![CDATA[ <210> 14]]> <![CDATA[ <211> 1407]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> FceRIa ECD-hinge-Fc3 nucleotide sequence]]> <![CDATA[ <400> 14]]> atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60 tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120 atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180 agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240 300 agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360 gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420 480 atctccatca ccaacgccac cgtgaggac agcggcacct actactgcac aggcaaggtg 540 tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag 600 aagtactggc tgcaggccca gcccaggcc gagggcagcc tggctaaggc caccacagct 660 cccgccacca ccaggaacac cggcagagga ggcgagaaa agaaaggaag caaggaag 720 gaggagcagg aggaaagaga aaccaagacc cccgagtgcc ccagccacac ccagcccctg 780 ggcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840 gtgacctgcg tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac 900 gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca gttcaactcc 960 acctacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020 tacaagtgca aggtgtccaa caaaggcctg cccagctcca tcgagaagac catcagcaaa 1080 gccaaaggcc agcccagaga accccaggtg tacaccctgc ctcccagcca ggagagatg 1140 accaagaacc aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc 1200 gtggagtggg aaagcaacgg ccagcccgag aacaattaca agacaacccc tcccgtgctg 1260 gatagcgatg gcagcttctt tctgtacagc agactgaccg tggacaagag cagatggcag 1320 gaaggcaacg tgttcagctg cagcgtgatg cacgaagccc tgcacaacca ctacacccag 1380 aagagcctgt ccctgagcct gggcaag 1407 <![CDATA[ <210> 15]]> <![CDATA[ <211> 406]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human α-2,6-sialic acid transferase <![CDATA[ <400> 15]]> Met Ile His Thr Asn Leu Lys Lys Lys Phe Ser Cys Cys Val Leu Val 1 5 10 15 Phe Leu Leu Phe Ala Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly 20 25 30 Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val Leu 35 40 45 Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser Gln Ser Val Ser 50 55 60 Ser Ser Ser Thr Gln Asp Pro His Arg Gly Arg Gln Thr Leu Gly Ser 65 70 75 80 Leu Arg Gly Leu Ala Lys Ala Lys Pro Glu Ala Ser Phe Gln Val Trp 85 90 95 Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile 100 105 110 Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly 115 120 125 Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu Arg Cys His Leu 130 135 140 Arg Asp His Val Asn Val Ser Met Val Glu Val Thr Asp Phe Pro Phe 145 150 155 160 Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr 165 170 175 Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala Gly Ser 180 185 190 Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp Asp His Asp Ala Val 195 200 205 Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln Gln Asp Val Gly 210 215 220 Thr Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu 225 230 235 240 Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile Val 245 250 255 Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln Asn 260 265 270 Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu His 275 280 285 Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu 290 295 300 Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn Pro 305 310 315 320 Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met Thr Leu Cys Asp 325 330 335 Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys Thr Asp Val 340 345 350 Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly Ala 355 360 365 Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln 370 375 380 Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro Gly 385 390 395 400 Phe Arg Thr Ile His Cys 405 <![CDATA[ <210> 16]]> <![CDATA[ <211> 1218]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Nucleotide sequence of human α-2,6-sialyltransferase <![CDATA[ <400> 16]]> atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt cctgctgttc 60 gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct actacgacag cttcaagctg 120 cagaccaagg agttccaggt gctgaagagc ctgggcaagc tggccatggg cagcgacagc 180 cagagcgtgt ccagctcctc cacccaggat ccccacagag gcacagac cctgggcagc 240 ctgagaggcc tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc 300 agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct gagcatgaac 360 aagtacaagg tgagctacaa aggacccgga cccggcatca agttcagcgc cgaggccctg 420 aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc 480 aacaccagcg agtgggaagg ctacctgccc aaggagca tcaggaccaa ggctggcccc 540 tggggcagat gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga 600 gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc caacttccag 660 caggacgtgg gcacaagac caaatccgg ctgatgaaca gccagctggt gcaaccgag 720 aagcggttcc tgaaggacag cctgtacaac gagggcatcc tgatcgtgtg ggatcccagc 780 gtgtaccaca gcgacatccc caagtggtac cagaatcccg actacaactt cttcaacaac 840 tacaagacct atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg 900 ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca gcccaaccct 960 ccctccagcg gcatgctggg cattatcatc atgatgaccc tgtgcgacca ggtggacatc 1020 tacgagttcc tgcccagcaa gagaaagacc gacgtgtgct actactatca gaagttcttc 1080 gacagcgcct gcaccatggg cgcctaccac cccctgctgt acgagaagaa cctggtgaag 1140 cacctgaacc agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc 1200 ttcagaacca tccactgc 1218 <![CDATA[ <210> 17]]> <![CDATA[ <211> 30]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> IgD hinge chain variant]]> <![CDATA[ <220> ]]> <![CDATA[ <221> VARIANT <![CDATA[ <222> (12)]]> <![CDATA[ <223> X]]>aa is a group composed of Lys and Gly. <![CDATA[ <220> ]]> <![CDATA[ <221> VARIANT <![CDATA[ <222> (13)]]> <![CDATA[ <223> Xaa is a group composed of Glu, Gly, and Ser. <![CDATA[ <400> 17]]> Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <![CDATA[ <210> 18]]> <![CDATA[ <211> 49]]> <![CDATA[ <212> PR]]>T <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> IgD hinge variant]]> <![CDATA[ <220> ]]> <![CDATA[ <221> VARIANT <![CDATA[ <222> (31)]]> <![CDATA[ <223> Xaa selected the group composed of Lys and Gly. <![CDATA[ <220> ]]> <![CDATA[ <221> VARIANT <![CDATA[ <222> (32)]]> <![CDATA[ <223> Xaa can be selected from groups composed of Glu, Gly, or Ser. <![CDATA[ <400> 18]]> Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro 1 5 10 15 Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa 20 25 30 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys 35 40 45 Pro <![CDATA[<210> 19]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> IgD Hinge]]> <![CDATA[<400> 19]]> Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 ​​​​​​​​ <![CDATA[<210> 20]]> <![CDATA[<211> 425]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> FceRIa ECD-Hinge-Fc1]]> <![CDATA[<400> 20]]> Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile > 1 5 10 15 > Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe >20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu >35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 180 185 190 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Lys 420 425 <![CDATA[<210> 21]]> <![CDATA[<211> 425]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> FceRIa ECD-Hinge-Fc2]]> <![CDATA[<400> 21]]> Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 [[ID=·31]]Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly 180 185 190 Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Lys 420 425 <![CDATA[ <210> 22]]> <![CDATA[ <211> 444]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> FceRIa ECD-Hex Chain-Fc3]]> <![CDATA[<400> 22]]> Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile 1 5 10 15 Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30 Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45 Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55 60 Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr 65 70 75 80 Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val 85 90 95 Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn 100 105 110 Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys 115 120 125 Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu 130 135 140 Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr 145 150 155 160 Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175 Tyr Trp Leu Gln Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala 180 185 190 Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu 195 200 205 Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys 210 215 220 Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440

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Claims

1. An aqueous pharmaceutical formulation comprising: a fusion protein dimer comprising an extracellular region (FcεRIα ECD) of an α subunit of an IgE Fc receptor, histidine, and proline, wherein the formulation has a pH of 6.0 to 7.0, wherein the fusion protein dimer comprises two monomers, each comprising the extracellular region of the α subunit of the IgE Fc receptor consisting of the amino acid sequence of SEQ ID NO: 1, wherein each monomer comprises a modified Fc region consisting of SEQ ID NO: 2, and the modified Fc region and the extracellular region of the α subunit of the IgE Fc receptor are connected by a hinge, wherein the hinge comprises the following sequence: Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17), wherein Xaa1 is Lys or Gly and Xaa2 is Glu, Gly or Ser, or Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 18), wherein Xaa3 is Lys or Gly and Xaa4 is Glu, Gly or Ser.

2. The aqueous pharmaceutical preparation of claim 1, wherein the preparation is intended for subcutaneous injection.

3. The aqueous pharmaceutical formulation of claim 1, wherein one of the fusion protein dimers has a concentration of 50 mg / mL to 150 mg / mL.

4. The aqueous pharmaceutical preparation of claim 1, wherein one of the histidines is present at a concentration of 10 mM to 100 mM.

5. The aqueous pharmaceutical formulation of claim 1, wherein the concentration of one of the proline acids is 200 mM to 300 mM.

6. The aqueous pharmaceutical formulation of claim 1, wherein the formulation further comprises an antioxidant.

7. The aqueous pharmaceutical formulation of claim 6, wherein the antioxidant is methionine.

8. The aqueous pharmaceutical preparation of claim 7, wherein the concentration of one of the methionines is from 10 mg / mL to 30 mg / mL.

9. The aqueous pharmaceutical formulation of claim 1, wherein the formulation further comprises a surfactant.

10. The aqueous pharmaceutical formulation of claim 9, wherein the surfactant is poloxamer 188.

11. The aqueous pharmaceutical formulation of claim 10, wherein the concentration of poloxamer 188 is from 0.01 w / v% to 1 w / v.

12. An aqueous pharmaceutical formulation comprising: i) a fusion protein dimer at a concentration of 50 mg / mL to 150 mg / mL, comprising an extracellular region of an α subunit of an IgE Fc receptor; ii) a histidine at a concentration of 10 mM to 100 mM; iii) a proline at a concentration of 200 mM to 300 mM; iv) methionine at a concentration of 10 mg / mL to 30 mg / mL; and v) poloxamer 188 at a concentration of 0.01 w / v% to 1 w / v%, wherein the formulation has a pH of 6.0 to 7.0, wherein the fusion protein dimer comprises two monomers, each comprising the extracellular region of the α subunit of the IgE Fc receptor consisting of the amino acid sequence of SEQ ID NO: 1, wherein the monomer comprises the amino acid sequence of SEQ ID NO:

1. A modified Fc region consisting of 2, wherein the modified Fc region and the extracellular region of the α subunit of the IgE Fc receptor are connected by a hinge, wherein the hinge comprises the following sequence: Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17), wherein Xaa1 is Lys or Gly and Xaa2 is Glu, Gly or Ser, or Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17) 18), where Xaa3 is Lys or Gly and Xaa4 is Glu, Gly or Ser.

13. The aqueous pharmaceutical preparation of claim 12, wherein the preparation is contained in a container selected from the group consisting of: a vial, a cartridge, a syringe, and an auto-injector.

14. The aqueous pharmaceutical preparation of claim 12, wherein the preparation is intended for subcutaneous administration.

15. The aqueous pharmaceutical preparation of claim 12, wherein the preparation is used for the prevention or treatment of an allergic disease.

16. The aqueous pharmaceutical preparation of claim 15, wherein the allergic disease is a disease selected from the group consisting of: food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria, chronic spontaneous urticaria and allergic contact dermatitis.

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