Peptide for inhibiting protein aggregation and protecting protein activity

Abstract

In the present invention, cytosolic abundant heat soluble protein (CAHS), which is over-expressed by water bears (scientific name: tardigrades), living organisms that survive even in extreme environments (high temperature: 151 °C, low temperature: -273 °C, vacuum, radioactive environment), is used to increase the stability of a protein. That is, the present invention specifies a key sequence in the CAHS protein, and thus has improved physical properties while continuing to comprise an effect of stabilizing other proteins, and, thus, the present invention can be used under various conditions.

Description

Peptides that inhibit protein aggregation and protect activity

[0001] The present invention relates to a peptide that inhibits protein aggregation and protects its activity.

[0002]

[0003] Biologics, such as antibodies, and industrial enzymes are often unstable. This reduces product stability and increases the cost of storage at extremely low temperatures (-80°C). While excipients and formulation modifications can stabilize the product, they still require storage at low temperatures. Trehalose, an excipient found in organisms tolerant of dry environments, can increase the temperature at which proteins denature, but it can also have the side effect of reducing drug efficacy or inducing autophagy. Attempts have been made to protect protein-based drugs and enzymes using recombinant human serum albumin, but even with this approach, storage at extremely low temperatures (-80°C) is still necessary. The problems with prior art can be summarized as follows: First, the cost of storing protein-based drugs and enzymes at extremely low temperatures increases the price of the products. Second, trehalose, a conventional excipient, can impair the efficacy of the drug. Third, even if recombinant human serum albumin, a conventional excipient, is utilized, it still requires ultra-low temperature storage, which does not reduce the storage costs of pharmaceuticals.

[0004]

[0005] One object of the present invention is to provide a composition for stabilizing a biological material comprising CAHS (Cytosolic Abundant Heat Soluble protein).

[0006] Another object of the present invention is to provide a method for stabilizing a biological material, comprising the step of adding a composition for stabilizing a biological material comprising a CAHS (Cytosolic Abundant Heat Soluble protein) protein to the biological material.

[0007] Another object of the present invention is to provide a composition for stabilizing the biological material; a pharmaceutical formulation comprising the target biological material.

[0008] More specifically, the purpose of the present invention is to provide a composition for stabilizing biological substances that not only protects the activity of a target protein to a higher level than existing excipients even when a smaller amount is added by specifying a certain region of a CAHS (Cytosolic Abundant Heat Soluble protein), but also has improved physical properties.

[0009]

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011]

[0012] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, specific embodiments may be practiced with one or more of these specific details, or with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of “one embodiment” or “an embodiment” in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0014]

[0015] 1. Composition for stabilizing biological substances containing CAHS (Cytosolic Abundant Heat Soluble protein) protein

[0016] In one specific embodiment of the present invention, a composition for stabilizing a biological material comprising a CAHS (Cytosolic Abundant Heat Soluble protein) protein is provided.

[0017] In the present invention, the "CAHS (Cytosolic Abundant Heat-Soluble Protein)" or "CAHS protein" is a type of protein specific to tardigrades (scientific name: Tardigrade) and is a specific type of intrinsically disordered protein found in tardigrades. They are most commonly used to help them survive desiccation conditions, which confers extreme tolerance. Due to their flexibility, CAHS proteins are highly sensitive to their environment and undergo significant changes in extreme abiotic environments. Furthermore, the protective mechanism in water bears was originally thought to be due to their high levels of trehalose. Trehalose has been shown to work with heat shock proteins and help other organisms, such as yeast, survive desiccation by helping them maintain desiccation-sensitive proteins in solution. However, this theory has been disputed in water bears, as low or no trehalose levels have been found in these animals. Trehalose appears to accumulate in water bears at low or undetectable levels, suggesting it is insufficient to provide protection. Water bears require water bear-specific proteins, including CAHS proteins, for survival. These water bear-specific proteins are a type of intrinsically disordered protein. This means they lack a specific shape, unlike conventional proteins, which rely on folding to perform specific tasks. These proteins utilize a variety of conformations, called ensembles, to utilize a variety of structures. This makes these intrinsically disordered proteins (IDPs) highly responsive to their environment. Furthermore, there are three families of water bear-specific proteins, each named after its location within the cell.These three families include the Cytoplasmic-, Secreted-, and Mitochondrial-Abundant Heat Soluble (CAHS, SAHS, and MAHS) proteins, collectively referred to as Tardigrade Disordered Proteins (TDPs) or Tardigrade-specific proteins. At the level of sequence conservation, TDPs are unique to tardigrades, and beyond the high degree of disorder, the CAHS, SAHS, and MAHS families are dissimilar. All three families are constitutively highly expressed or significantly enriched in response to desiccation. In vivo, ex vivo, and in vitro experiments indicate a functional role for each TDP family member in mitigating cellular perturbations induced by various abiotic stresses. This protein, with the exception of its specificity for tardigrades, resembles abundant proteins from late embryonic development. The three families are dissimilar and have been found to be expressed or enriched during desiccation. Unlike traditional proteins, these intrinsically disordered proteins do not precipitate or denature from solution at high temperatures. Water bears rely on these proteins to survive in extreme environments, allowing them to survive many abiotic stresses such as freezing and heat. While dehydration poses a challenge to cells, which typically rely on a hydrated environment for their proteins to perform many functions, water bear-specific proteins help prevent aggregation of their contents upon initial dehydration and maintain membrane integrity upon rehydration.

[0018] According to recent research results, the sequences of eight CAHS (Cytosolic Abundant Heat Soluble protein) proteins (CAHS1, CAHS2, CAHS3 from R. varieornatus and five proteins from H. dujardini and M. tardigradum) were compared, and four conserved regions were found: HTD / E, ASAARIS, CAHS-c1, and CAHS-c2. Among them, two 19mers, CAHS-motif 1 and CAHS-motif 2, were reported to be conserved in the CAHS-c2 region. However, it is unknown which of these various conserved regions affects the core function of CAHS proteins.

[0019] Water is often considered essential for life, as it is necessary for all metabolic processes. However, many organisms across all biological kingdoms challenge this claim by somehow surviving the loss of all or nearly all of the water in their bodies and cells. To achieve this, specialized organisms enter a state of suspended animation known as anhydrobiosis. In this anhydrobiosis, desiccated organisms can persist for weeks, years, or even centuries in extreme cases, and can reactivate their metabolism and resume life processes upon rehydration. One such anhydrobiotic organism is the tardigrade, colloquially known as a water bear. Water bears (Tardigrades) are microscopic eight-legged animals phylogenetically related to arthropods and nematodes. Currently, ~1,400 species of tardigrades have been described, many of which can tolerate numerous abiotic stresses, including desiccation. How tardigrades survive desiccation remains one of the enduring mysteries of organismal physiology. Biochemical and molecular studies have begun to elucidate how tardigrades survive desiccation, implicated in antioxidant enzymes and reactive oxygen species scavengers, conservation and tardigrade-specific disordered proteins, and DNA repair mechanisms that mediate desiccation tolerance. These pioneering studies have been bolstered by the adoption of various -omics approaches, primarily derived from next-generation sequencing of the genomes and transcriptomes of various tardigrade species. While the genetic and transcriptomic complement of tardigrades under desiccation conditions has been assessed to some extent, other -omics-level analyses, such as metabolomics, are lacking for tardigrades.

[0020] In the present invention, the “biological material” may be selected from the group including DNA, RNA, peptides, proteins, polysaccharides, and polypeptides, or mixtures thereof. More specifically, it may be a protein or a nucleic acid. Preferred peptides and proteins include enzymes, antibodies, blood clotting factors, insulin, interferons, hormones, cytokines, transcription factors, and other regulatory proteins or fragments thereof, or mixtures thereof. The nucleic acids (DNA and RNA) may be single-stranded or double-stranded, high-molecular-weight or low-molecular-weight (short molecules) (e.g., miRNA, siRNA, or highly degraded nucleic acids), and are specifically represented by viral RNA, bacterial RNA, fungal RNA, or cellular RNA, PCR products, genomic DNA or cDNA, viral DNA or cDNA, bacterial DNA or cDNA, fungal DNA or cDNA, and plasmid DNA or cDNA. Novel biological materials include supramolecular structures (e.g., viral particles (e.g., adenovirus, adeno-associated virus, retrovirus, lentis virus, varicella-zoster virus, HIV, or perpesvirus), prokaryotic cells, or eukaryotic cells). The aforementioned biological materials may be collected from natural sources or biotechnically engineered sources, or may be synthesized by recombinant techniques and chemical synthesis, etc.

[0021]

[0022] In another specific embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 1.

[0023] In another specific embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 2.

[0024] In the present invention, the CAHS protein represented by sequence number 2 is a protein corresponding to the 124th to 142nd sequence of the entire CAHS protein sequence, and is a CAHS motif 1. 124-142 Also shown in the specification. In addition, the CAHS protein plays various roles in enabling the water bear to survive in dry conditions, high temperatures, low temperatures, vacuum, and radioactive environments, and in particular, the CAHS protein represented by the above sequence number 2 is a protein for sequences 124 to 142 of the entire CAHS protein sequence, and the corresponding portion may be an essential part that enables the water bear to survive in the above extreme environments (dry conditions, high temperatures, low temperatures, vacuum, and radioactive environments).

[0025] In another specific embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the biological material is any one selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

[0026] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides, and enzymes.

[0027] In another specific embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the stabilizing the biological material prevents denaturation or aggregation of the biological material.

[0028] In the present invention, the above “modification” or “denaturation” or “denaturation” means that a biological material including a protein or nucleic acid loses its secondary structure, tertiary structure, or quaternary structure existing in its original state by applying some external stimulus or compound such as a strong acid or base, heat, concentrated inorganic salt, organic solvent (alcohol or chloroform), or radiation. When a protein in a living cell is denatured, it causes destruction of cell activity and cell suicide. Protein denaturation is a result of cell suicide. Denatured proteins can exhibit a wide range of characteristics, from adaptive changes and loss of solubility to aggregation due to exposure of hydrophobic groups. Denatured proteins cannot function because they lose their three-dimensional structure.

[0029] In the present invention, the term "aggregation" refers to the phenomenon in which biological substances, such as proteins that are inherently disordered or misfolded, aggregate (i.e., accumulate and clump together) either intracellularly or extracellularly. Protein aggregates have been implicated in various diseases known as amyloidosis, including ALS, Alzheimer's disease, Parkinson's disease, and prion diseases. Furthermore, after synthesis, proteins typically fold into a specific thermodynamically most favorable three-dimensional structure. This protein structure is stabilized by non-covalent interactions and disulfide bonds between two cysteine ​​residues. Non-covalent interactions include ionic interactions and weak van der Waals interactions. Ionic interactions form between anions and cations, forming salt bridges that help stabilize the protein. Van der Waals interactions include non-polar interactions (i.e., London dispersion forces) and polar interactions (i.e., hydrogen bonds, dipole-dipole bonds). These play an important role in the secondary and tertiary structures of proteins, such as the formation of alpha helices or beta sheets. Interactions between amino acid residues in a specific protein are crucial to its final structure. When noncovalent interactions are altered, as can occur with changes in amino acid sequence, the protein is prone to misfolding or unfolding. In such cases, if cells do not facilitate protein refolding or degrade unfolded proteins, unfolded / misfolded proteins can aggregate, where exposed hydrophobic regions of the protein can interact with exposed hydrophobic patches of other proteins.

[0030] In another specific embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the deformation or agglomeration of the biological material occurs during a dehydration-rehydration process or a freeze-thaw process.

[0031]

[0032] 2. Method for stabilizing a biological substance containing CAHS (Cytosolic Abundant Heat Soluble protein) protein.

[0033] In one embodiment of the present invention, a method for stabilizing a biological material is provided, comprising the step of adding a composition for stabilizing a biological material comprising a CAHS (Cytosolic Abundant Heat Soluble protein) protein to the biological material.

[0034] Here, the description of CAHS protein and biological material is omitted to avoid duplication as it is described previously in 1. Composition for stabilizing biological material containing CAHS (Cytosolic Abundant Heat Soluble protein).

[0035] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 1. In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 2.

[0036] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the mass ratio of the biological material to the CAHS protein is 1:2 or greater. In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the mass ratio of the biological material to the CAHS protein is 1:2 to 1:100.

[0037] The present invention can protect the activity of a target biological substance at a high level in a dehydration-rehydration process when the mass ratio of the target biological substance and the CAHS protein is 1:2 or more, and can protect the activity of a target biological substance at a high level in a freeze-thaw process when the mass ratio of the biological substance and the CAHS protein is 1:4 or more. In this way, the mass ratio of the target biological substance and the CAHS protein may be 1:2 or more, preferably 1:2 to 1:100, 1:2 to 1:80, or 1:70, 1:2.5 or more, 1:2.5 to 1:100, or 1:2.5 to 1:50, but is not limited thereto.

[0038]

[0039] 3. A pharmaceutical formulation comprising the composition for stabilizing the biological substance and the target biological substance.

[0040] In one embodiment of the present invention, a composition for stabilizing a biological material as described above in 1. 2. is provided; a pharmaceutical formulation comprising a target biological material is provided.

[0041] Here, the description of CAHS protein and biological material is omitted to avoid duplication as it is described previously in 1. Composition for stabilizing biological material containing CAHS (Cytosolic Abundant Heat Soluble protein).

[0042] In one embodiment of the present invention, a pharmaceutical formulation is provided, wherein the pharmaceutical formulation further comprises a stabilizing buffer.

[0043] In one specific embodiment of the present invention, “stabilizer” means any additive added to increase the stability of an active ingredient and prevent the active ingredient from being arbitrarily changed, oxidized, crystallized, or degraded into a compliant substance, and is not particularly limited as long as it is pharmaceutically acceptable. The evaluation of the stabilizing effect of a stabilizer can be performed without limitation on the temperature, but it is generally understood that the stabilizing effect is maintained for a long time at low temperatures rather than at high temperatures. Therefore, the evaluation of long-term stabilizing effect at low temperatures can be replaced with an “accelerated experiment” performed for a short time at high temperatures. For example, the result of evaluating the stabilizing effect of a specific stabilizer at 37°C for 9 days can be understood to be the same as the result of evaluating at 4°C for 3 months, and the result of evaluating at 37°C for 74 days can be understood to be the same as the result of evaluating at 4°C for 24 months (http: / iso-inc.com / medical-package-testing / accelerated-aging.html).

[0044] In one specific embodiment of the present invention, the “stabilizing buffer” or “stabilizing buffering agent” refers to a substance that simultaneously has the effect of a stabilizing agent and the effect of a buffer, and can be prepared by adding a substance having a stabilizing effect to a general buffer, and a synergistic stabilizing effect can be expected by including an additional stabilizing agent in the stabilizing buffer. In the present invention, the stabilizing buffer is preferably a gluconolactone buffer and / or a tartaric acid buffer, but is not limited thereto.

[0045]

[0046] The above “prevention” of the present invention may include, without limitation, any act that can block, suppress or delay symptoms caused by a disease by using the composition of the present invention.

[0047] The above “treatment” and “improvement” of the present invention may include, without limitation, any act that can improve or benefit symptoms caused by a disease by using the composition of the present invention.

[0048] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being for human use.

[0049] The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0050] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0051] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0052] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0053] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0054] A food composition comprising the composition of the present invention as an active ingredient can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts with almost no toxicity or side effects, it can be used with confidence even when taken for long periods of time for preventive purposes.

[0055] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0056] Here, when the food composition is manufactured in the form of a beverage, there are no special restrictions other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like in a typical beverage. That is, as a natural carbohydrate, it may include a monosaccharide such as glucose, a disaccharide such as fructose, a polysaccharide such as sucrose, a dextrin, a cyclodextrin, a typical sugar such as dextrin, a cyclodextrin, a sugar alcohol such as xylitol, sorbitol, and erythritol. As the flavoring agent, there may be mentioned a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

[0057] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0058] These components can be used independently or in combination. The proportions of these additives are not particularly critical, but are typically selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0059] The appropriate total daily dosage may be determined by the treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purpose of the present invention, it is preferable to apply a specific therapeutically effective amount for a specific patient differently depending on various factors such as the type and degree of the response to be achieved, whether other preparations are used in some cases, the composition containing the specific effective ingredient, the patient's age, weight, general health condition, sex and diet, the time of administration, the route of administration and the secretion rate of the composition containing the effective ingredient, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0060] In the present invention, the term "combination" should be understood to refer to simultaneous, separate, or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost.

[0061] In the present invention, the dosage of the binding molecule or antibody-drug conjugate may be about 0.0001 μg to 500 mg per 1 kg of patient body weight, but is not limited thereto.

[0062]

[0063] In one embodiment of the present invention, a composition for stabilizing a biological material comprising a CAHS (Cytosolic Abundant Heat Soluble protein) protein is provided.

[0064] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 1.

[0065] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 2.

[0066] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is a composition in which one sequence of CAHS motif 1 represented by SEQ ID NO: 2 is mutated to alanine.

[0067] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the CAHS protein is any one selected from the group consisting of SEQ ID NOs: 3 to 8.

[0068]

[0069] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the biological material is any one selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

[0070] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides, and enzymes.

[0071] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the stabilizing of the biological material prevents denaturation or aggregation of the biological material.

[0072] In another embodiment of the present invention, a composition for stabilizing a biological material is provided, wherein the deformation or agglomeration of the biological material occurs during a dehydration-rehydration process or a freeze-thaw process.

[0073] In another embodiment of the present invention, a method of stabilizing a biological material is provided, comprising the step of adding a composition for stabilizing a biological material comprising a CAHS (Cytosolic Abundant Heat Soluble protein) protein to the biological material.

[0074] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 1.

[0075] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is represented by SEQ ID NO: 2.

[0076] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is a CAHS motif 1 sequence represented by SEQ ID NO: 2, in which one sequence is mutated to alanine.

[0077] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the CAHS protein is any one selected from the group consisting of SEQ ID NOs: 3 to 8.

[0078] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the mass ratio of the biological material and the CAHS protein is 1:2 or more.

[0079] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the mass ratio of the biological material to the CAHS protein is from 1:2 to 1:100.

[0080] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the biological material is any one selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

[0081] In another embodiment of the present invention, a method of stabilizing a biological material is provided, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides, and enzymes.

[0082] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the stabilizing the biological material prevents denaturation or aggregation of the biological material.

[0083] In another embodiment of the present invention, a method for stabilizing a biological material is provided, wherein the deformation or agglomeration of the biological material occurs during a dehydration-rehydration process or a freeze-thaw process.

[0084] In another embodiment of the present invention, a pharmaceutical formulation is provided, comprising the composition for stabilizing a biological material; and a target biological material.

[0085] In another embodiment of the present invention, a pharmaceutical formulation is provided in which the mass ratio of the target biological material and the CAHS protein is 1:2 to 1:100.

[0086] In another embodiment of the present invention, a pharmaceutical formulation is provided in which the target biological material is any one selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

[0087] In another embodiment of the present invention, a pharmaceutical formulation is provided, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides, and enzymes.

[0088] In another embodiment of the present invention, a pharmaceutical formulation is provided, wherein the pharmaceutical formulation further comprises a stabilizing buffer.

[0089] In another embodiment of the present invention, a pharmaceutical formulation is provided, wherein the pharmaceutical formulation is in a liquid form.

[0090]

[0091] Hereinafter, the present invention will be described in detail based on examples.

[0092]

[0093] The present invention improves the physical properties, which were a shortcoming of existing inventions, while increasing the stability of other proteins when selecting a specific motif among CAHS (Cytosolic Abundant Heat Soluble protein) proteins, thereby achieving the remarkable effect of enabling the storage of other proteins even at temperatures other than extremely low (-80°C). In other words, while existing CAHS proteins have been confirmed to increase the stability of other proteins, their poor physical properties have limited their use.

[0094] The present invention is an invention that not only protects the activity of a target protein to a higher level than existing excipients even when a smaller amount is added by specifying a core sequence among the CAHS proteins, but also improves the physical properties so that it can be used under various conditions.

[0095]

[0096] Figure 1 shows the entire sequence of CAHS (Cytosolic Abundant Heat Soluble protein) protein and the protein sequence of motif 1 among CAHS protein.

[0097] Figure 2 compares the entire sequence of the CAHS protein and the solubility of motif 1 among the CAHS proteins.

[0098] Figures 3(a) to 3(c) show motif 1 of the CAHS protein and the entire CAHS protein. 1-229 The activity protection rate of LDH was compared after dehydration-rehydration of BSA, another excipient.

[0099] Figures 3(d) to 3(f) show motif 1 of the CAHS protein and the entire CAHS protein. 1-229 The activity protection rate of LDH after freeze-thaw with BSA, another excipient, was compared.

[0100] Figure 4 shows motif 1 of CAHS protein and the entire CAHS protein. 1-229 This is the result of measuring the extent to which BSA, another excipient, inhibits the aggregation of LDH during (A) dehydration-rehydration and (B) freeze-thaw processes.

[0101] Figure 5(a) is a graph showing the results of an LDH assay, showing that when CAHS motif 1 according to the present invention is included at -20°C, the activity of LDH, which is a target protein, is well preserved for 1 to 4 weeks.

[0102] Figure 5(b) is a graph showing the results of an LDH assay, showing that when CAHS motif 1 according to the present invention is included at 20°C, the activity of LDH, which is a target protein, is well preserved for 1 to 4 weeks.

[0103] Fig. 5(c) is a graph showing the results of an LDH assay, showing that when CAHS motif 1 according to the present invention is included at 37°C, the activity of LDH, which is a target protein, is well preserved for 1 to 4 weeks. Therefore, Figs. 5(a) and 5(b) are graphs showing that CAHS motif 1 according to the present invention not only well preserves the activity of the target protein at all temperatures, but also well preserves the activity of the target protein over time.

[0104] Figures 6(a) and 6(b) are LDH assay results, which confirm whether a protein in which a part of the sequence of CAHS motif 1 provided by the present invention is converted to a different amino acid preserves LDH activity during the dehydration-rehydration process (dehydration-rehydration) and (B) the freeze-thaw process (freeze-thaw). Specifically, the amino acids Tyr, Gln, Glu, Val, Arg, and Leu of CAHS motif 1 were each substituted with Ala, and it was confirmed that all substitutions exhibited a protective effect on LDH activity.

[0105]

[0106] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0107]

[0108] Example

[0109]

[0110] 1. Selection of specific motifs among CAHS (Cytosolic Abundant Heat Soluble protein) proteins

[0111] The present invention utilizes CAHS (Cytosolic Abundant Heat Soluble Protein), which is overexpressed by the water bear (scientific name: Tardigrade), a living organism that can survive in extreme environments (high temperature: 151℃, low temperature: -273℃, vacuum, and radiation environment), to increase protein stability. To confirm whether CAHS increases protein stability in environments where proteins become unstable, experiments were conducted using LDH (Lactate dehydrogenase) as a model.

[0112] CAHS full sequence including full amino acid sequence1-229 In the case of protein (Fig. 1), although it has been confirmed in previous studies that it protects various types of proteins, its use is limited due to the disadvantage of poor solubility of the CAHS protein itself. Therefore, CAHS protein has various functions, and among them, CAHS motif 1, a sequence predicted to be a core and conserved region where CAHS functions and an essential part for survival, is 124-142 was specified.

[0113]

[0114] 2. Comparison of physical properties (solubility)

[0115] First, CAHS motif 1 124-142 was obtained through peptide synthesis and CAHS motif 1 124-142 and CAHS as a whole 1-229 The proteins were dialyzed at a concentration of 100 uM each in 20 mM potassium phosphate, 150 mM NaCl, pH 7.0 buffer at 4°C for 18 hours and their solubility was compared and observed.

[0116] CAHS Motif 1 124-142 Silver CAHS whole protein 1-229 Compared to the previous one, the protein solubility was improved and it was found that it could be used under various conditions (buffer pH, protein concentration).

[0117] That is, as can be seen in Figure 2, the entire CAHS protein 1-229 While gelation occurs and shows low properties, CAHS motif 1 according to the present invention 124-142 CAHS total protein 1-229 It was found that it dissolves well in water and exhibits excellent physical properties.

[0118]

[0119] 3. Comparison of protein activity protection rates

[0120] CAHS motif 1 of the present invention in the model protein LDH (Lactate dehydrogenase) 124-142 After adding BSA (Bovine Serum Albumin), a well-known excipient, and performing dehydration-rehydration and freeze-thaw, the experiment was conducted to determine how much LDH activity was protected. As a result, CAHS motif 1 was found to be more effective than the condition without other excipients. 124-142 It was confirmed that this LDH activity was protected at a high level. In addition, compared to BSA, a well-known excipient, CAHS motif 1 124-142 This LDH activity was protected at a higher level. Furthermore, the entire CAHS protein 1-229 Compared to CAHS motif 1 124-142 It was confirmed that this LDH activity was protected to a higher level. In particular, CAHS whole protein 1-229 Since it was not as effective as the existing well-known excipient BSA, CAHS motif 1 124-142 We were able to confirm once again that this protein's activity was best protected (Fig. 3(a) to Fig. 3(f)).

[0121]

[0122] In particular, as can be seen in Figure 3, the model protein and CAHS motif 1 according to the present invention 124-142 For dehydration-rehydration, a mass ratio of 1:2.5 was effective, and for freeze-thaw, a mass ratio of 1:5 was effective.

[0123] This is up to 1 / 10 times less than the existing well-known excipient BSA, which must be added at a mass ratio of 1:25 for dehydration-rehydration and 1:25 for freeze-thaw to protect activity, CAHS motif 1. 124-142 Even when added, it showed a protection rate of 70-100%, and compared to the existing well-known excipient, BSA, which showed a protection rate of approximately 50-60%, it was confirmed that it showed a higher activity protection rate even with a smaller amount.

[0124]

[0125] 4. Comparison of inhibition of protein aggregation

[0126] CAHS motif 1 of the present invention in the model protein LDH (Lactate dehydrogenase) 124-142 After adding BSA (Bovine Serum Albumin), a well-known excipient, dehydration-rehydration and freeze-thaw were performed, and the degree of protein aggregation was confirmed through the degree of absorption at 340 nm through protein aggregation based on the light scattering effect using UV. In other words, protein aggregation is the main cause of loss of protein activity. Protein aggregation is induced in the process of freezing and thawing the protein (freeze and thaw) or in an environment without water (dehydration), which causes loss of protein activity. Through the present invention, CAHS motif 1 according to the present invention 124-142 It was confirmed that this effectively inhibits the aggregation of LDH during the freezing and thawing process and the dehydration and rehydration process.

[0127]

[0128] In particular, as shown in Fig. 4, BSA hardly prevented aggregation during the dehydration and rehydration process, whereas the present invention confirmed that aggregation was reduced by more than half. Furthermore, the CAHS whole protein 1-229 Rather, it was found through Figures 4(a) and 4(b) that the protein aggregation phenomenon became more severe in stress situations (dehydration-rehydration and freeze-thaw).

[0129]

[0130] 5. Comparison of protein activity protection rates according to temperature and time

[0131] CAHS motif 1, a stabilizer provided by the present invention, is added to LDH (Lactate dehydrogenase), a target candidate protein for active protection experiments. 124-142 After mixing in a mass ratio of 1:50, the activity of LDH protein was examined under different temperature and time conditions to determine the protein activity protection rate according to temperature and time.

[0132] Specifically, the temperature conditions were -20℃, 20℃, and 37℃, and the time conditions were 0, 1, 2, and 4 weeks, and the activity of LDH protein was confirmed, as shown in Figures 5(a) to 5(c).

[0133]

[0134] As a result, the CAHS motif 1 provided by the present invention is effective under all temperature conditions and all time conditions. 124-142 It was confirmed that the protein activity was well preserved.

[0135]

[0136] 6. CAHS Motif 1 124-142 Comparison of protein activity protection rates of sequence variants

[0137] As shown in Table 1 below, CAHS motif 1, a stabilizer provided by the present invention 124-142After mutating some of the sequences to alanine (Ala), dehydration-rehydration and freeze-thaw were performed to compare the protein activity preservation efficiency.

[0138] Specifically, as shown in Figure 6(c), CAHS motif 1 124-142 Among the sequences, considering the charge, hydrophobicity, and hydrophilicity of each amino acid, among the amino acid sequences of CAHS motif 1, represented by sequence number 2, Y (Tyr), Q (Gln), E (Glu), V (Val), R (Arg), and L (Leu) were each mutated to alanine at one site.

[0139] Sequence information Sequence number CAHS motif 1 YRKQQEVEADKIRKELEKQ SEQ ID NO. 2 Candidate 1 (Y124A) ARKQQEVEADKIRKELEKQ SEQ ID NO. 3 Candidate 2 (Q128A) YRKQAEVEADKIRKELEKQ SEQ ID NO. 4 Candidate 3 (E129A) YRKQQAVEADKIRKELEKQ SEQ ID NO. 5 Candidate 4 (V130A) YRKQQEAEADKIRKELEKQ SEQ ID NO. 6 Candidate 5 (R136A) YRKQQEVEADKIAKELEKQ SEQ ID NO. 7 Candidate 6 (L139A) YRKQQEVEADKIRKEAEKQ SEQ ID NO. 8

[0140]

[0141] As a result, as shown in FIG. 6(a) and FIG. 6(b), all candidates 1 to 6 are CAHS motif 1 according to the present invention. 124-142 It was confirmed that they showed similar levels of protective efficacy. Specifically, although the difference was not large, candidates 1, 3, and 4 showed higher protective efficacy in dehydration-rehydration, and candidates 1, 2, 3, and 4 showed higher protective efficacy in freeze-thaw. 124-142 It was confirmed that it showed a similar protective effect.

[0142]

[0143] conclusion

[0144] 1. CAHS motif 1 according to the present invention compared to BSA, a previously known excipient 124-142 It was confirmed that the activity of the model protein used in the experiment was better protected during dehydration-rehydration and freeze-thaw processes.

[0145] 2. CAHS motif 1 according to the present invention 124-142 It was confirmed that the aggregation of LDH, a model protein, was effectively inhibited during the dehydration-rehydration and freeze-thaw processes.

[0146] 3. CAHS total protein 1-229 CAHS motif 1 according to the present invention, which has a key function in protecting protein-based materials 124-142 The function and solubility of CAHS protein were improved by specifying .

[0147] 4. In addition, CAHS motif 1 provided by the present invention 124-142 It was confirmed that the protein activity was well protected under all time and temperature conditions.

[0148] 5. Also, CAHS motif 1 is provided even when one sequence is mutated to alanine. 124-142 Similarly, it was confirmed that protein activity was well protected.

Claims

1. A composition for stabilizing a biological material containing CAHS (Cytosolic Abundant Heat Soluble protein).

2. In paragraph 1, A composition for stabilizing a biological material, wherein the CAHS protein is represented by sequence number 1.

3. In paragraph 2, A composition for stabilizing a biological material, wherein the CAHS protein is represented by sequence number 2.

4. In paragraph 3, A composition for stabilizing a biological material, wherein the CAHS protein is a composition in which one sequence of CAHS motif 1 represented by sequence number 2 is mutated to alanine.

5. In paragraph 4, A composition for stabilizing a biological material, wherein the CAHS protein is any one selected from the group consisting of SEQ ID NOs: 3 to 8.

6. In paragraph 3, The above biological material is a composition for stabilizing a biological material selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

7. In paragraph 6, A composition for stabilizing a biological material, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides and enzymes.

8. In paragraph 7, A composition for stabilizing a biological material, wherein the stabilizing biological material prevents denaturation or aggregation of the biological material.

9. In paragraph 8, A composition for stabilizing a biological material, wherein the deformation or agglomeration of the biological material occurs during a dehydration-rehydration process or a freeze-thaw process.

10. A method for stabilizing a biological material, comprising the step of adding a composition for stabilizing a biological material containing a CAHS (Cytosolic Abundant Heat Soluble protein) protein to the biological material.

11. In paragraph 10, A method for stabilizing a biological material, wherein the CAHS protein is represented by sequence number 1.

12. In paragraph 11, A method for stabilizing a biological material, wherein the CAHS protein is represented by sequence number 2.

13. In paragraph 12, A method for stabilizing a biological material, wherein the mass ratio of the biological material and the CAHS protein is 1:2 or more.

14. In paragraph 13, A method for stabilizing a biological material, wherein the mass ratio of the biological material and the CAHS protein is 1:2 to 1:

100.

15. In paragraph 14, A method for stabilizing a biological material, wherein the biological material is any one selected from the group consisting of DNA, RNA, peptides, proteins, polysaccharides, and polypeptides.

16. In paragraph 15, A method for stabilizing a biological material, wherein the protein comprises at least one selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides and enzymes.

17. In paragraph 16, The above biological material stabilization is a method for stabilizing a biological material, which prevents denaturation or aggregation of the biological material.

18. In paragraph 17, A method for stabilizing a biological material, wherein the deformation or agglomeration of the biological material occurs during a dehydration-rehydration process or a freeze-thaw process.

19. A composition for stabilizing a biological substance according to any one of claims 1 to 9; a pharmaceutical formulation comprising a target biological substance.

20. In paragraph 19, A pharmaceutical formulation wherein the mass ratio of the biological substance to be targeted and the CAHS protein is 1:2 to 1:

100.

21. In paragraph 20, The biological material to be targeted above is a pharmaceutical formulation selected from the group consisting of DNA, RNA, peptide, protein, polysaccharide, and polypeptide.

22. In paragraph 21, A pharmaceutical formulation comprising at least one protein selected from the group consisting of growth factors, hormones, interferons, vaccines, recombinant proteins, recombinant peptides and enzymes.

23. In paragraph 22, The pharmaceutical formulation above further comprises a stabilizing buffer.

24. In paragraph 22, The above pharmaceutical formulation is a liquid pharmaceutical formulation.

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