Botulinum toxin protein compositions

Abstract

A botulinum toxin protein composition comprising a botulinum toxin protein and a protein stabilizer, the protein stabilizer being selected from either an amino acid or an amino acid-like compound, the amino acid being selected from one or more of L-histidine, L-glycine, L-arginine, L-methionine, and L-lysine, and the amino acid-like compound being levocarnitine, the botulinum toxin protein composition can be applied as a liquid formulation in the medical and cosmetic fields, and the activity of the botulinum toxin protein can be stably maintained in a liquid state.

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical formulations, and in particular to botulinum toxin protein compositions and methods for their preparation. [Background technology]

[0002] Natural botulinum neurotoxins (BoNTs) are toxic proteins produced by the anaerobic Clostridium botulinum bacterium, which cause muscle paralysis in mammals by inhibiting the presynaptic release of the neurotransmitter acetylcholine at the neuromuscular junction. Seven types of BoNTs have been discovered to date, named BoNT / A through BoNT / G. Due to their unique pharmacological properties, including high specificity for the nervous system, limited diffusion upon local injection, and reversibility of action, BoNTs have rapidly developed as a treatment for cholinergic hyperexcitation and are widely applied in the medical and cosmetic fields.

[0003] In general, therapeutic pharmaceutical active proteins are usually prepared in liquid solutions, especially for use as injections. Therapeutic active proteins are usually prepared as compositions and are commercially available in the form of ready-to-use liquids or as lyophilized forms to be reconstituted in liquid solutions. For example, commercially available BoNT / A formulations are all vacuum-dried or lyophilized solid preparations that need to be reconstituted by medical personnel when used, so the volume of reconstitution must be precisely controlled. In addition, due to individual differences in the dosage required for different treatment purposes and for different patients, the dosage required for each treatment varies considerably. The amount of drug contained in commercial products is often reconstituted in a volume specified by the manufacturer, after which clinicians administer a small portion of the bottle's contents to the patient, and the remainder is stored in the refrigerator for later use. Research has shown that reconstituted BoNT / A is estimated to lose at least 44% of its potency when stored in a refrigerator (approximately 4°C) for 12 hours, so in practical operation, it is generally recommended to use it within 4 hours after reconstitution, but this results in drug waste and imposes unnecessary economic burden on patients.

[0004] In addition, because the concentration of botulinum toxin used is extremely low, it is usually formulated with high concentrations of exogenous proteins to maintain the stability of the botulinum toxin protein at extremely low concentrations and to avoid the drug's adsorption to solid surfaces. In botulinum toxin formulations, albumin and gelatin are often used as stabilizers, but they may not only induce immune reactions but also introduce a source of contamination. Summary of the Invention [Problem to be solved by the invention]

[0005] In response to the technical problems described in the "Background Art" section, the present invention provides a botulinum toxin protein composition and a method for producing the same. [Means for solving the problem]

[0006] The technical aspects of the present invention are as follows.

[0007] The present invention provides a botulinum toxin protein composition comprising a botulinum toxin protein and a protein stabilizer.

[0008] The protein stabilizer is selected from either an amino acid or an amino acid-like compound.

[0009] Preferably, the amino acid-like compound is levocarnitine (also known as vitamin Bt, L-levocarnitine).

[0010] More preferably, the molar content of levocarnitine is 5 to 20 mM, and in one specific embodiment, the molar content of levocarnitine is 10 mM.

[0011] Preferably, the molar content of said amino acids is 5-20 mM, and in one specific embodiment, the molar content of said amino acids is 10 mM.

[0012] More preferably, said amino acids are selected from one or more of L-histidine, L-glycine, L-arginine, L-methionine, L-histidine, L-lysine.

[0013] Generally, proteins are easily decomposed and inactivated at low concentrations (<0.1mg / ml). Certain amino acids and amino acid-like compounds of the present invention can increase the solubility and stability of proteins at a specific pH, and amino acids and amino acid structure-like compounds can reduce surface adsorption, protect the conformation of proteins, and prevent protein denaturation and aggregation. Furthermore, when the protein stabilizer of the present invention is levocarnitine, compared with commonly used additives, levocarnitine can impart low viscosity to the formulation, reduce the aggregation and precipitation of botulinum toxin protein, and is advantageous in maintaining the stability of botulinum toxin protein.

[0014] Preferably, the botulinum toxin protein is selected from any one of botulinum toxin type A, botulinum toxin type B, botulinum toxin type C1, botulinum toxin type C2, botulinum toxin type D, botulinum toxin type E, botulinum toxin type F, and botulinum toxin type G, and more preferably, the botulinum toxin protein is botulinum toxin type A.

[0015] More preferably, the botulinum toxin is a recombinant botulinum toxin.

[0016] Even more preferably, the recombinant botulinum toxin protein has at least one dimeric structure in which a single polypeptide chain is cleaved by a protease to form the polypeptide.

[0017] The single chain polypeptide is (I) a first polypeptide fragment, (a) tag protein, (b) a structural region comprising a first protease cleavage site; (c) Linker and a first polypeptide fragment comprising: (II) a second polypeptide fragment, (d) a first functional amino acid structural region comprising a metal ion-dependent protease activity domain; (e) a structural region comprising a second protease cleavage site; (f) a second functional amino acid structural region that includes a receptor binding domain capable of binding to a receptor on the surface of a target cell and / or a translocation domain capable of mediating translocation of the polypeptide across the vesicle membrane. and a second polypeptide fragment comprising Includes.

[0018] The term "tag protein" refers to a type of protein molecule that can bind to a specific ligand.

[0019] Preferably, the tag protein is selected from tag proteins known to those skilled in the art or tag proteins designed by a computer program, said tag protein being capable of specifically binding to a known substrate.

[0020] More preferably, the tag protein is selected from, but is not limited to, proteins such as glutathione S-transferase (GST), C-myc, chitin-binding domain, maltose-binding protein (MBP), SUMO heterophilic moiety, monoclonal antibody or protein A, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, histidine affinity tag (HAT), polyhistidine.

[0021] Preferably, said "tag protein" is capable of increasing the solubility of the toxin polypeptide precursor in the host.

[0022] Preferably, said "tag protein" enables the toxin polypeptide precursor to exist in a soluble state in the host cell.

[0023] In one specific embodiment, the tag protein is located at the N-terminus of the single-chain polypeptide.

[0024] In one specific embodiment, the tag protein is glutathione s-transferase (GST), and more preferably, the glutathione s-transferase has the amino acid sequence shown in SEQ ID NO:2.

[0025] Preferably, neither the first nor the second protease cleavage site can be cleaved by a human protease or a protease itself produced by the host cell expressing said single-chain polypeptide.

[0026] More preferably, said first protease cleavage site and said second protease cleavage site are the same or different.

[0027] Said first and second protease cleavage sites are not recognised and cleaved by endogenous proteases of the host cell during expression or of the organism during use.

[0028] More preferably, the above identified proteases are one or a combination of two selected from, but not limited to, non-human enterokinase, tobacco etch virus protease, Bacillus subtilis derived protease, Bacillus amyloliquefaciens derived protease, rhinovirus derived protease, papain, insect papain homologs, or crustacean papain homologs.

[0029] In one specific embodiment, the proteases that specifically recognize the first protease cleavage site and that specifically recognize the second protease cleavage site are both proteases derived from rhinoviruses.

[0030] Preferably, the second restriction enzyme cleavage site is embedded, partially replaces, or completely replaces the naturally occurring loop region between the first and second functional peptide segments, where "embedded" means inserted between two amino acids in the loop region, "partially replaces" means that the second restriction enzyme cleavage site replaces a part of the amino acid sequence of the loop region, and "completely replaces" means that the naturally occurring amino acid sequence of the loop region is completely replaced by the second restriction enzyme cleavage site.

[0031] More preferably, the structural region comprising a first protease cleavage site and the structural region comprising a second protease cleavage site are selected from, but are not limited to, one or a combination of two restriction enzyme cleavage sites, such as DDDDK, EXXYXQS / G, HY, YH or LEVLFQGP.

[0032] In one specific embodiment, the structural region containing the first protease cleavage site and the structural region containing the second protease cleavage site are both LEVLFQGP, and the cleavage site is between QG.

[0033] Preferably, the linker does not exceed five amino acids.

[0034] Preferably, the linker enables the first protease cleavage site to be readily recognised or bound by the protease.

[0035] More preferably, said linker does not affect the function of the single chain polypeptide.

[0036] More preferably, after said first protease cleavage site is cleaved, the linker retained at the N-terminus of the second polypeptide fragment does not affect the function of the second polypeptide fragment.

[0037] More preferably, the GS portion of the linker does not exceed five amino acid residues.More preferably, the linker is selected from the group consisting of glycine-serine (abbreviated as GS) short peptides, GGS, GGGS, GGGGS, GSGS, GGSGS, GGSGS, GGGSS, and the like linkers.

[0038] In one specific embodiment, the amino acid sequence of the structural region and linker containing the first protease cleavage site is LEVLFQGPLGS.

[0039] Preferably, the metal ion-dependent protease activity domain is Zn 2+ dependent protease activity domain.

[0040] Preferably, the first functional amino acid structural region and / or the second functional amino acid structural region are encoded by a natural sequence and / or a synthetic sequence. More preferably, the first functional amino acid structural region of the single-chain polypeptide is encoded by the ZnS domain of a Clostridial toxin light chain. 2+ Contains the protease activity domain.

[0041] Preferably, the target cell capable of binding to the receptor binding domain of a human cell surface antigen in the second functional amino acid structural region is a target cell in which a SNARE complex is present, such as, for example, a neuronal cell, a pancreatic cell, or other cell in which a SNARE complex is present.

[0042] More preferably, the target cell in the second functional amino acid structural region refers to a human nerve cell or a pancreatic cell, and the receptor-binding domain is a receptor-binding domain capable of specifically binding to a human nerve cell or a pancreatic cell.

[0043] More preferably, the receptor binding domain of the second functional amino acid structural region is a cell surface antigen-binding structural domain of the heavy chain of a clostridial toxin, and the translocation domain of the second functional amino acid structural region that mediates the movement of a polypeptide across a vesicle membrane is a structural domain that mediates the movement of a clostridial toxin across a vesicle membrane.

[0044] More preferably, the Clostridial toxin is a botulinum toxin or a tetanus toxin.

[0045] More preferably, the botulinum toxin is selected from any of the serotypes BoNT / A to BoNT / H and their derivatives known in the prior art.

[0046] More preferably, the Clostridial toxin is selected from a tetanus toxin or any of its derivatives.

[0047] More preferably, the first functional amino acid structural region includes part or all of the light chain of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / H, or tetanus toxin.

[0048] More preferably, the second functional amino acid structural region includes part or all of the heavy chain of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / H, or tetanus toxin.

[0049] In one specific embodiment, the first functional amino acid structural region is the light chain of BoNT- / A, and the second functional amino acid structural region is the heavy chain of BoNT- / A.

[0050] More preferably, the first functional amino acid structural region and the second functional amino acid structural region can be derived from different serotypes. They can be any combination of the serotypes. For example, the first functional amino acid structural region is derived from the light chain of BoNT- / A, and the second functional amino acid structural region is derived from the heavy chain of BoNT- / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G; or the first functional amino acid structural region is derived from the light chain of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G, and the second functional amino acid structural region is derived from the heavy chain of BoNT / A, etc.

[0051] More preferably, the amino acid sequence of said second polypeptide fragment comprises the fragment shown in SEQ ID NO:7.

[0052] In one specific embodiment, the single-chain polypeptide includes, in order from the N-terminus, glutathione S-transferase, LEVLFQGPLGS, the light chain of BoNT / A, LEVLFQGP, and the heavy chain of BoNT / A.

[0053] More preferably, the single chain polypeptide has the amino acid sequence shown in SEQ ID NO:11.

[0054] More preferably, the recombinant botulinum toxin type A protein is produced by cleaving the single-chain polypeptide with a first protease to remove the tag protein, cleaving it with a second protease, and then forming at least one dimer between the first functional amino acid structural region and the second functional amino acid structural region.

[0055] Preferably, the first and second functional amino acid structural regions are linked by a disulfide bond to form a dimeric structure.

[0056] The recombinant botulinum toxin type A protein of the present invention is formed from a specific single-chain polypeptide, which has a specific structure, and the single-chain polypeptide of this structure is characterized by strong specificity and low toxicity. Specifically, from the viewpoint of strong specificity, the tag protein in the single-chain polypeptide can bind to a specific ligand, and by being detached from the ligand, it becomes easy to select a molecule co-expressed with the tag protein from different polypeptides or protein products, and the purity of the product can be significantly improved during the production process, and as a result, a product with higher purity can be obtained in high yield. From the viewpoint of low toxicity, the tag protein in the single-chain polypeptide prevents the protease structural domain of the first functional amino acid structural region of the polypeptide from exerting the function of a protease, and replaces the natural restriction enzyme cleavage site between the first functional amino acid structural region and the second functional amino acid structural region with a site that can only be recognized by a specific enzyme, thereby reducing the molecular activity (neurotoxicity) of the single-chain polypeptide to less than one ten-thousandth of that of the natural toxin, effectively reducing the toxicity, and the toxicity is significantly reduced, improving safety in the production process and making the entire industrialized production easier to carry out. That is, the single-chain polypeptide of the recombinant botulinum toxin protein of the present invention has a higher yield, higher purity, and higher safety than the conventional technology, and is therefore easy to produce and purify industrially.

[0057] Preferably, the composition further comprises a solubilizer and / or a salt compound.

[0058] More preferably, the concentration of the solubilizer is 0.05% by volume to 0.2% by volume.

[0059] More preferably, the concentration of the salt is 0.9% by weight.

[0060] More preferably, the solubilizer is selected from Tweens, Spans, and sodium dodecyl sulfate.

[0061] More preferably, the Tween system is selected from one or more of Tween 20, Tween 40, Tween 60, or Tween 80.

[0062] More preferably, the span system is selected from one or more of Span 20, Span 40, Span 60, Span 80, or Span 85.

[0063] In one specific embodiment, the solubilizing agent is Tween 80.

[0064] Preferably, the salt is selected from either an inorganic salt or an organic salt.

[0065] More preferably, said inorganic salt is selected from one or more of sodium chloride, sodium phosphate, magnesium sulfate, sodium acetate, sodium propionate and potassium phosphate.

[0066] More preferably, the organic salt is selected from one or more of sodium lactate, sodium succinate.

[0067] In one specific embodiment, the inorganic salt is sodium chloride.

[0068] Preferably, the composition comprises a botulinum toxin protein, levocarnitine, Tween 80, and sodium chloride.

[0069] Alternatively, the composition comprises a botulinum toxin protein, an amino acid, Tween 80, and sodium chloride.

[0070] More preferably, the molar content of levocarnitine is 5 to 20 mM, preferably 10 mM.

[0071] Alternatively, the molar content of the amino acid is 5 to 20 mM, and preferably 10 mM.

[0072] More preferably, the concentration of Tween 80 is 0.05% by volume to 0.2% by volume.

[0073] In one specific embodiment, the concentration of Tween 80 is 0.1% by volume.

[0074] More preferably, the concentration of sodium chloride is 0.9% by weight.

[0075] The composition is a liquid formulation.

[0076] The present invention further provides a method for producing a botulinum toxin protein composition, comprising the step of mixing the ingredients.

[0077] Preferably, the method comprises the steps of: (a) producing and purifying a botulinum toxin protein; (b) adding a protein stabilizer solution to the botulinum toxin protein; Equipped with.

[0078] The water is water for injection.

[0079] More preferably, said step (b) further comprises adding a solubilising agent and / or a salt.

[0080] The botulinum toxin protein, protein stabilizer, solubilizer and / or salt are as defined above.

[0081] In one specific embodiment, the method of manufacture comprises: (a) producing and purifying a recombinant botulinum toxin type A protein; (b) adding a protein stabilizer water, Tween 80 and sodium chloride, water to the recombinant botulinum toxin type A protein; Equipped with.

[0082] The water is water for injection.

[0083] The present invention further provides use of the above-mentioned botulinum toxin protein composition in the manufacture of a medicament for the prevention and / or treatment of a disease associated with cholinergic nerves innervating muscles or exocrine glands, or for medical cosmetics.

[0084] Diseases or conditions associated with cholinergic nerves innervating said muscles or exocrine glands include exemplary diseases or conditions of hyperactivity of muscles or exocrine glands associated with cholinergic innervation, such as Frey's syndrome, crocodile tears syndrome, axillary hyperhidrosis, plantar hyperhidrosis, head and neck hyperhidrosis, body hyperhidrosis, rhinorrhea, Parkinson's disease, amyotrophic lateral sclerosis, hypersalivation, drooling, sialorrhea, spasticity, palatal myoclonus, myoclonus, myokymia, rigidity, benign muscle spasms, hereditary jaw tremors, abnormalities of mandibular movement, unilateral masseter muscle spasms, hypertrophic branchial arch muscle disease, masseter hypertrophy, anterior tibial hypertrophy, nystagmus, oscillopsia, nuclear These include: superior gaze palsy, continual partial epilepsy, planning spasmodic torticollis surgery, abductor vocal cord paralysis, refractory variant dysphonia, upper esophageal sphincter dysfunction, vocal cord granuloma, stuttering, Gilles de la Tourette syndrome, middle ear myoclonus, protective laryngeal closure, post-laryngectomy speech disorder, protective ptosis, entropion, sphincter of Oddi dysfunction, pseudoesophageal achalasia, nonachalasia esophageal motility disorder, vaginismus, post-operative bed rest, tremor, bladder dysfunction, hemifacial spasm, reinnervation dyskinesia, stiff-man syndrome, tetanus, benign prostatic hyperplasia, obesity treatment, infantile cerebral palsy, achalasia, anal fissure.

[0085] The medical cosmetic treatments include smoothing and preventing wrinkles, slimming the face, and repairing scars.

[0086] Said wrinkles are regarded as wrinkles, ridges or folds of the skin.Preferably, they are wrinkles that are visible on human face.Examples of wrinkles include tear troughs, frown lines, crow's feet, nasolabial folds, jaw lines, perioral wrinkles, cheek wrinkles, marionette lines, lip wrinkles, forehead wrinkles, vertical frown lines, horizontal nose lines, nasolabial folds, under-eye wrinkles and chin wrinkles. Effect of the Invention

[0087] Compared with the prior art, the present invention has the following beneficial effects:

[0088] (1) The present invention provides a stable botulinum toxin protein composition. This composition uses amino acids or amino acid-like substances instead of human-derived proteins (e.g., human serum albumin) to eliminate the risk of human-derived viruses. In addition, the composition can be stably stored in a liquid state in a standard refrigerator (about 4°C) for more than one year and at room temperature (25°C) for more than six months in liquid state by using Tween 80 in combination to prevent adsorption.

[0089] (2) The present invention provides a recombinant botulinum toxin protein composition containing a highly purified recombinant botulinum toxin type A protein produced using a specific single-chain polypeptide. This composition can increase the purity of the botulinum toxin type A protein in a preparation, and can avoid the immune risks that may occur when the inactivated botulinum protein contained in botulinum toxin type A extracted from Clostridium botulinum is ingested into the human body, thereby providing a new option for expanding the scope of application of the recombinant botulinum toxin type A protein.

[0090] (3) When the botulinum toxin protein composition of the present invention is used as a liquid preparation, the convenience of clinical use is improved and the drawbacks of freeze-dried or vacuum-dried products usually having a short storage period after reconstitution, which can lead to wasteful use, artificial contamination during the reconstitution process, and inaccurate reconstitution, can be improved. [Brief description of the drawings]

[0091] [Figure 1] SDS-PAGE results of initial purification of GST-BoNT / A and further removal of the GST tag. Lane 1 is GST-BoNT / A obtained by initial purification using a GST affinity chromatography column, and lane 2 is BoNT / A after GST removal, in which the GST tag protein of the initial purified protein was removed by enzymatic cleavage with Rinovirus 3C Protease. [Diagram 2]This is an SDS-PAGE result of highly purified BoNT / A protein, which was obtained by further purifying the product after cleaving off the GST tag using an ion exchange column. [Diagram 3] These are the results of verification of double-stranded dissociation of BoNT / A under reducing conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0093] In the present invention, the term "U" refers to the unit of potency, which is a unit of biological potency diversity used pharmacologic-wise. Since the chemical components of these drugs are not constant, or it has never been possible to determine their mass standards by physicochemical methods, their potency is often determined by biological experimental methods and by comparison with standards. Through such bioassays, the smallest potency unit with a certain biological potency is called a "unit" (U), and the standard unit established by international agreement is called an "international unit" (IU).

[0094] The term "BoNT / A" refers to the botulinum toxin type A protein.

[0095] The term "tag protein" refers to a peptide or protein that is expressed as a fusion with a target protein using in vitro recombinant DNA techniques to facilitate expression and detection of the target protein.

[0096] The term "human serum albumin" is abbreviated as "HSA" and refers to a protein in human plasma that transports fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules in body fluids, while also maintaining normal osmotic pressure of the blood. Clinically, HSA can be used to treat shock and burns, to replenish blood lost during surgery, accidents, and hemorrhage, and is also used as a plasma volume expander.

[0097] The term "dimeric structure" generally refers to a polymeric formulation consisting of two non-covalently linked molecules.

[0098] The technical embodiments of the present invention will be described below clearly and completely with reference to the embodiments of the present invention. Of course, the described embodiments are only a part of the embodiments of the present invention, but not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative ingenuity belong to the protection scope of the present invention. EXAMPLES

[0099] Experimental Example 1: Preparation of recombinant botulinum toxin type A protein Specifically, it is divided into the following eight steps:

[0100] 1. Design and construction of nucleic acid molecules encoding single polypeptide chains of modified botulinum toxins The nucleic acid molecule comprises, in order from the 5' end: (a) The nucleotide sequence encoding glutathione S-transferase is shown in SEQ ID NO:1, and the amino acid sequence of the glutathione S-transferase it encodes is shown in SEQ ID NO:2. (b) The nucleotide sequence encoding the first protease cleavage site is shown in SEQ ID NO:3 and the amino acid sequence which it encodes is shown in SEQ ID NO:4. (c) The nucleotide sequence encoding the linker GS is shown in SEQ ID NO:5 and is GGATCC. (d) a nucleotide sequence encoding a second polypeptide fragment comprising a BoNT / A light chain, a second protease cleavage site, and a BoNT / A heavy chain, as set forth in SEQ ID NO:6, which encodes the amino acid sequence set forth in SEQ ID NO:7, wherein the nucleotide sequence encoding the second protease cleavage site is set forth in SEQ ID NO:8, and the amino acid sequence it encodes is set forth in SEQ ID NO:9.

[0101] The natural loop region between the BoNT / A light chain and the BoNT / A heavy chain may be absent, for example by removing the KTKSLDKGYNK linker sequence between the light and heavy chains to reduce non-specific protease cleavage.

[0102] The nucleotide sequence encoding the single chain polypeptide is shown in SEQ ID NO:10, and the amino acid sequence of the single chain polypeptide it encodes is shown in SEQ ID NO:11.

[0103] 2. Construct a plasmid containing the nucleic acid molecule of the sequence of step 1 The synthetic genetically optimized GST-BoNT / A described above was synthetically added with NdeI and NotI restriction enzyme cleavage sites at both ends, digested with NdeI and NotI at 37°C (New England Biolabs), purified using a QIquick gel extraction kit (Qiagen), and inserted into the NdeI and NotI sites of the pET28a (Novagen) plasmid vector using T4 DNA ligase (NEB).

[0104] 3. Transfect the plasmid constructed in step 2 into host cells (a) Production of competent cells One tube of E. coli cells, E. coli BL21 DE3 (New England Biolabs), was inoculated into a test tube containing 3 ml of LB medium and cultured overnight with shaking at 37°C. The next day, 0.5 ml of the bacterial solution was inoculated into a 250 ml flask containing 50 ml of LB medium and cultured with vigorously shaking (250 rpm) at 37°C for about 3 to 5 hours. When the OD at 600 nm of the colony reached 0.3 to 0.4, the flask was removed and left to stand on ice for 10 to 15 minutes. The bacterial solution was poured into a 50 ml centrifuge tube under sterile conditions and centrifuged at 1000 g for 10 minutes at 4°C. The supernatant was discarded, the tube was collected, 10 ml of 0.1 M CaCl2 was added, and the tube was shaken to suspend the cells, and the tube was centrifuged at 1000 g for 10 minutes at 4°C in an ice bath for 30 minutes. The supernatant was discarded, the tube was collected, and 4 ml of 0.1 M CaCl2 pre-cooled on ice was added to resuspend the cells. The tube was dispensed at 0.2 ml per tube, stored at 4°C, and used within 24 hours. The remainder was stored in a low-temperature refrigerator at -70°C.

[0105] (b) Transfection: An appropriate amount of DNA (ng) was mixed with competent E. coli and allowed to stand in an ice bath for 15 minutes, then heat-shocked at 42°C for 30 seconds. After allowing to stand in an ice bath for 5 minutes, the mixture was placed in SOC medium and shaken at 250 rpm for 1 hour. The mixture was then spread onto a plate containing antibiotics and cultured overnight at 37°C.

[0106] 4. Cultivation of host cells and induction of single-chain polypeptide expression Medium ingredients and their ratios: 11.8 g / L tryptone, 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 4 ml / L glycerol.

[0107] Culture conditions: Cells were grown overnight at 37° C. with shaking at 250 rpm.

[0108] Induction expression: When E. coli grows to OD600 = 1, 1 mM IPTG is added and induced expression is performed for 5 hours at 25°C. OD600 is selected from 0.2 to 1.5, and the temperature is selected from 37°C to 10°C. The expression time may be 5 to 16 hours.

[0109] Cell recovery: E. coli was harvested by centrifugation at 3000 rpm for 30 minutes.

[0110] 5. Identification of single polypeptide expression levels A glutathione purification resin chromatography column 1 cm in diameter and 1 cm in height was prepared using glutathione purification resin from GenScript, and 25 ml of the lysate was slowly poured onto the top of the column, being careful not to wash away the resin. The lysate was allowed to pass through the column slowly over the course of 1 hour to adsorb GST-BoNT / A in the lysate. GST-BoNT / A was obtained by elution according to standard methods.

[0111] The usual method is as follows: the chromatography column was washed with 20 column volumes of phosphate buffer, and GST-BoNT / A was eluted with 10 column volumes of freshly prepared 10 mM glutathione elution buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris-HCl (pH 8.0)). The elution of the fusion protein was monitored using absorbance at 280 nm. The GST-tagged protein was excised from the eluted protein using Rinovirus 3C Proteas.

[0112] The products before and after removal of the GST-tagged protein were collected and subjected to a standard SDS-PAGE experiment. As shown in Figure 1, compared to lane 1, in which the tagged protein had not been removed, the GST-tagged protein in lane 1 was excised with Rinovirus 3C Protease, resulting in a BoNT / A molecule without GST.

[0113] Measurement of the proportion of GST-BoNT / A in the total protein: Purified GST-BoNT / A was separated by electrophoresis at 200 volts using 4-12% SDS-PAGE (Biorad), and the major band with a molecular weight of 175 kd was GST-BoNT / A.

[0114] 6. Removal of tagged proteins and purification The product of step 5 was subjected to removal of the GST tagged protein and purification of the toxic peptide.

[0115] When GST-BoNT / A re-adsorbed onto a glutathione purification resin chromatography column was treated with GenScript's 3C enzyme, the 3C enzyme cleaved the first restriction enzyme cleavage site between GST and BoNT / A, releasing GST, and simultaneously cleaved the second restriction enzyme cleavage site between the light chain and heavy chain of BoNT / A.

[0116] When the glutathione purification resin chromatography column was treated with phosphate buffer, the GST-tagged protein remained on the column and was removed, whereas the light and heavy chains of BoNT / A were eluted with the phosphate buffer.

[0117] The GST-removed product was collected and subjected to a conventional SDS-PAGE experiment, and the resulting band no longer contained the GST-tagged protein, indicating that the GST-tagged protein had been completely removed, as shown in Figure 2. Using the scanned image of the SDS-PAGE, the resulting BoNT / A was calculated to be more than 90% pure from the gray scale of the bands.

[0118] Substituting the GS portion of the linker with a GSS, GSGS, or GGSGS polypeptide has the same effect as GS, resulting in better exposure of the tagged protein, which can then be completely excised.

[0119] 7. Dissociation of the dimeric BoNT / A duplex under reducing conditions The product of step 6 was subjected to reduction experiments.

[0120] The samples were reduced with 100 mM dithiothreitol at 100°C for 5 min. The samples were separated by electrophoresis on a 4-12% SDS-PAGE (Biorad) at 200 volts to separate the heavy and light chains. As shown in Figure 3, the product obtained in step 6 was reduced under reducing conditions, and a conventional SDS-PAGE experiment was performed on the resulting product, resulting in two distinct bands with molecular weights of 100 Kda and 50 Kda, respectively, indicating that the product formed in step 6 is a dimeric structure in which two peptide segments are linked by a disulfide bond.

[0121] 8. Toxicity test of GST-BoNT / A LD when the GST-BoNT / A obtained in step 5 was intraperitoneally injected into mice 50 The LD ranged from 45 ng to 450 ng. Considering the purity of the injected botulinum toxin protein, the converted LD 50 The LD was in the range of 22.5 ng to 225 ng, with the median value being 123.75 ng. 50 Considering the purity of the injected botulinum toxin protein, the converted LD 50 ranged from 0.006 ng to 0.015 ng, with a median of 0.0105 ng (see Table 1).

[0122] [Table 1]

[0123] GST-BoNT / A possesses botulinum toxin activity and has a median lethal dose (LD ) of 1.2 mg / kg / day after intraperitoneal injection in mice. 50 ) is the LD of BoNT / A protein 50This indicates that the GST-BoNT / A recombinant proteotoxin polypeptide precursor molecule is approximately 11,786 times less active than the final product BoNT / A. This experiment demonstrates that the GST-BoNT / A recombinant proteotoxin polypeptide precursor molecule has botulinum toxin activity. Because botulinum toxin is extremely toxic, high levels of safety precautions are required during the manufacturing process before the precursor molecule is activated.

[0124] Example 2 Optimization of Recombinant Botulinum Toxin Type A Protein Composition The activity of the botulinum toxin protein (prepared in Example 1) was assessed as the median mouse lethal dose, expressed in mouse units (U), with one unit defined as the amount of recombinant botulinum toxin type A protein injected intraperitoneally required to kill 50% of the total mouse population.

[0125] I. The Animal Testing Process 1. Selection and grouping: Adult mice of the CD-1 strain were selected, divided into groups, weighed, and numbered so that each group contained an equal number of mice of similar weight.

[0126] 2. Drug Administration: The different formulations in Table 2 were administered, and at the time of administration, CD-1 mice were intraperitoneally injected with a single 0.5 mL of the recombinant botulinum toxin type A protein preparation of the different formulations. The amount of recombinant botulinum toxin type A protein administered intraperitoneally to the mice was 2 × LD 50 (2U).

[0127] 3. Observation: The body weight of each animal was measured and recorded once before the start of the study (before the first day of dosing), and once every 24 hours after dosing. Mortality was monitored at 24 (± 1 hour), 48 (± 1 hour), 72 (± 1 hour), and 96 (± 1 hour) hours after dosing.

[0128] II. Experimental Results Each formulation (1-4) containing 2 U of botulinum toxin shown in Table 2 was intraperitoneally injected into mice, and the lethality of mice in each group caused by 2 U of botulinum toxin after each formulation was stored at 4°C for 0-211 days was observed. The experimental process was the same as the animal experiment described above, and the experimental results are shown in Table 2.

[0129] [Table 2]

[0130] As can be seen from the results in Table 2, after the four formulations in the table were stored at 4°C for 0 to 211 days, the lethal effect (drug activity) of the recombinant botulinum toxin type A on mice did not decrease, and the biological activity of the recombinant botulinum toxin type A protein was maintained before storage at 4°C, indicating that the formulations selected in the present invention can maintain the long-term stability of the recombinant botulinum toxin type A.

[0131] Two formulations in Table 2 were selected to measure the median lethal dose (LD) of recombinant botulinum toxin type A in mice after storage at 4°C for 44, 75, and 211 days. 50 The experimental results are shown in Table 3.

[0132] [Table 3]

[0133] As can be seen from Table 3, formulation 1 can maintain the activity of recombinant botulinum toxin type A protein even after being left at 4°C for 211 days, and formulation 2 can maintain the activity of recombinant botulinum toxin type A protein even after being left at 4°C for 70 days. The median mouse lethal dose (LD ) of recombinant botulinum toxin type A calculated from the multiple dose-activity relationship was 1.2 mg / kg / day. 50 ) did not change significantly after 44, 70, and 211 days at 4°C, respectively, and the median lethal dose was essentially unchanged, fully demonstrating that the pharmaceutical formulation of the present invention can effectively maintain the long-term stability of the biological activity of recombinant botulinum toxin type A.

[0134] The present invention uses recombinant botulinum toxin and uses amino acids or amino acid-like compounds as protein stabilizers, and can be stored at low temperatures (e.g., 4° C.) for long periods (e.g., 70 days or more at 4° C.) The pharmaceutical formulation of the present invention can maintain the activity of natural botulinum toxin proteins (type A botulinum toxin protein, type B botulinum toxin protein, type C1 botulinum toxin protein, type C2 botulinum toxin protein, type D botulinum toxin protein, and type E botulinum toxin protein).

[0135] The above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and mechanism of the present invention shall be included in the protection scope of the present invention.

[0136] The embodiments and methods described herein may vary based on the ability, experience, and preferences of those skilled in the art.

[0137] In the present invention, the method steps are described in a certain order, but the order of the method steps is not limited thereto.

Claims

1. comprising a botulinum toxin protein and a protein stabilizer, A botulinum toxin protein composition, wherein the protein stabilizer is selected from any of amino acids and amino acid-like compounds.

2. 2. The composition of claim 1, wherein the amino acid-like compound comprises levocarnitine (L-carnitine).

3. 3. The composition according to claim 2, characterized in that the molar content of levocarnitine is between 5 and 20 mM.

4. 2. The composition according to claim 1, characterized in that the molar content of the amino acid is between 5 and 20 mM.

5. 5. The composition of claim 4, wherein the amino acids are selected from one or more of L-histidine, L-glycine, L-arginine, L-methionine, L-histidine and L-lysine.

6. 2. The composition according to claim 1, wherein the botulinum toxin protein is selected from any one of botulinum toxin type A protein, botulinum toxin type B protein, botulinum toxin type C1 protein, botulinum toxin type C2 protein, botulinum toxin type D protein, botulinum toxin type E protein, botulinum toxin type F protein, botulinum toxin type G protein, and botulinum toxin type H protein, preferably, the botulinum toxin protein is botulinum toxin type A protein, and more preferably, the botulinum toxin is a recombinant botulinum toxin.

7. The composition of claim 1 , further comprising a solubilizer and / or salts.

8. The composition according to claim 7, characterized in that the concentration of the solubilizer is from 0.01% to 0.2% by volume.

9. The composition according to claim 7, characterized in that the concentration of the salts is 0.9% by weight.

10. The solubilizing agent is selected from the group consisting of Tweens, Span, and sodium dodecyl sulfate; Preferably, the tween system is selected from one or more of: tween 20, tween 40, tween 60, or tween 80; Preferably, the span system is selected from one or more of Span 20, Span 40, Span 60, Span 80, or Span 85; More preferably, the composition according to claim 7, characterized in that said solubilizer is Tween 80.

11. the salts are selected from either inorganic salts or organic salts; Preferably, the inorganic salt is selected from one or more of sodium chloride, sodium phosphate, magnesium sulfate, sodium acetate, sodium propionate and potassium phosphate; Preferably, the organic salt is selected from one or more of sodium lactate, sodium succinate; More preferably, the composition according to claim 7, characterized in that said inorganic salt is sodium chloride.

12. The composition comprises a botulinum toxin protein, levocarnitine, Tween 80, and sodium chloride; or 2. The composition of claim 1, wherein the composition comprises a botulinum toxin protein, an amino acid, Tween 80, and sodium chloride.

13. The concentration of Tween 80 is 0.05% by volume to 0.2% by volume; 13. A composition according to claim 12, characterized in that the concentration of Tween 80 is preferably 0.1% by volume.

14. 13. The composition according to claim 12, characterized in that the concentration of sodium chloride is 0.9% by weight.

15. The composition of claim 1 , wherein the composition is a liquid formulation.

16. A method for producing the composition according to claim 1, comprising the step of mixing the components.

17. Use of the composition according to any one of claims 1 to 15 in the manufacture of a medicament for the prevention and / or treatment of a disease associated with cholinergic nerves innervating muscles or exocrine glands. or Use in the manufacture of a medicine for medical cosmetics, comprising: Preferably, the medical cosmetic treatment includes smoothing or preventing wrinkles, slimming the face, body shaping, and scar repair; Preferably, the disease associated with the cholinergic nerve is Frey's syndrome, crocodile tears syndrome, axillary hyperhidrosis, plantar hyperhidrosis, head and neck hyperhidrosis, body hyperhidrosis, rhinorrhea, Parkinson's disease, amyotrophic lateral sclerosis, salivation, drooling, sialorrhea, spasticity, palatal myoclonus, myoclonus, myokymia, rigidity, benign muscle spasm, hereditary jaw tremor, abnormal mandibular movement, unilateral masticatory muscle spasm, hypertrophic branchial arch muscle disease, masseter hypertrophy, tibial anterior hypertrophy, nystagmus, oscillopsis, supranuclear gaze palsy, continuous partial epilepsy, spasmodic torticollis surgery planning, abductor vocal cord paralysis, intractable variant dysphonia, upper esophageal fasciculus sphincter dysfunction, vocal cord granuloma, stuttering, Gilles de la Tourette syndrome, middle ear myoclonus, protective laryngeal closure, post laryngectomy speech disorder, protective ptosis, entropion, sphincter of Oddi dysfunction, pseudoesophageal achalasia, nonachalasia esophageal motility disorder, vaginismus, post-operative bed rest, tremor, bladder dysfunction, hemifacial spasm, reinnervation dyskinesia, stiff man syndrome, tetanus, benign prostatic hyperplasia, obesity treatment, infantile cerebral palsy, achalasia, anal fissure.

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