Botulinum toxin freeze-dried preparation composition capable of long-term storage

Abstract

The present invention relates to a botulinum toxin freeze-dried preparation composition capable of long-term storage, which contains a botulinum toxin, protamine sulfate, an isotonicity agent, and a buffering agent. The botulinum toxin freeze-dried preparation according to the present invention not only reduces the inactivation rate of the botulinum toxin that occurs during the freeze-drying process, and can maintain the stability of the botulinum toxin for a longer period of time than conventional freeze-dried botulinum toxin preparations, but also forms a uniform particle size, thereby providing the same effect at all administration sites, and preventing side effects such as excessive toxin administration effects in only some areas.

Description

[Technical field]

[0001] The present invention relates to a freeze-dried preparation composition of a botulinum toxin capable of being stored for a long period of time, and more particularly to a freeze-dried preparation composition of a botulinum toxin capable of being stored for a long period of time, which contains a complex of a botulinum toxin and protamine sulfate, an isotonicity agent, and a buffering agent. [Background technology]

[0002] Various Clostridium strains secreting neurotoxic toxins have been discovered since the 1890s, and the toxins secreted by these strains have been characterized over the past 70 years.

[0003] The neurotoxic toxins derived from the Clostridium strains, i.e., botulinum toxins, are classified into seven types, A to G, based on their serological characteristics. Each toxin has a toxin protein of about 150 kDa, but naturally consists of a complex bound to multiple non-toxic proteins. The medium complex (300 kDa) is composed of a toxin protein and a non-toxic non-hemagglutinin protein, while the large (450 kDa) complex and the large-large (900 kDa) complex are in a form in which the intermediate complex is bound to hemagglutinin (Sugiyama, H, Microbiol Rev, 44:419, 1980). These non-toxic non-hemagglutinin proteins are known to function to protect the toxin from the low pH in the intestine and various proteolytic enzymes.

[0004] The toxin is synthesized in cells as a single polypeptide with a molecular weight of about 150 kDa, and then is cleaved at a position one-third from the N-terminus by the action of intracellular proteases or artificial enzyme treatments such as trypsin, and separated into two units, a light chain (L) (molecular weight: 50 kDa) and a heavy chain (H) (molecular weight: 100 kDa). The toxicity of the toxin thus separated is significantly increased compared to when it is a single polypeptide. The two units are connected to each other by disulfide bonds, and each has a different function. The medium chain binds to a receptor on a target cell and reacts with a biological membrane at low pH (pH 4) to form a channel (Mantecucco, C et al, TIBS, 18:324, 1993), while the light chain has pharmacological activity, making cells permeable using detergents or interfering with the secretion of neurotransmitters when introduced into cells by electroporation or other methods.

[0005] The toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of the neuromuscular junction, causing generalized asthenia. Since toxicity appears even when treated with extremely small amounts of the toxin, it has been thought that the toxin has some enzymatic activity.

[0006] It has been recently revealed that the toxins have metallopeptidase activity, and their substrates are the unit proteins of the exocytosis machinary complex, such as synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP25). Each type of toxin has one of these three proteins as its substrate, and types B, D, F, and G are known to cleave synaptobrevin, types A and E to cleave SNAP25, and type C to cleave syntaxin at specific sites.

[0007] In particular, botulinum toxin type A is known to be soluble in dilute aqueous solutions with a pH between 4.0 and 6.8. It is known that at a pH of about 7 or higher, stabilizing non-toxin proteins are separated from the neurotoxin, resulting in a gradual loss of toxicity, especially as the pH and temperature increase.

[0008] Botulinum toxin is lethal to the human body in small doses and easy to mass-produce, so it is one of the four major biological terrorism weapons along with Bacillus anthracis, Yersinia pestis, and smallpox virus. However, it has been revealed that type A of botulinum toxin can paralyze local muscles at the injection site when injected at a dose that does not affect the human body systemically. Taking advantage of this characteristic, it can be widely used as a wrinkle remover and a treatment for hemiplegia rigidus and cerebral palsy, so demand is rapidly increasing and research on methods of producing botulinum toxin to meet demand is being actively conducted.

[0009] The most representative product currently on the market is BOTOX, manufactured by Allergan, Inc. (R) (botulinum toxin type A purified neurotoxin complex), each BOTOX (R)Each 100-unit vial of botulinum toxin type A complex contains approximately 5 ng of purified botulinum toxin type A complex, 0.5 mg of human serum albumin, and 0.9 mg of sodium chloride, and is supplied in vacuum-dried form without preservatives (0.9% sodium chloride injection) and reconstituted with sterile saline. Another commercially available product is Dysport, manufactured by Ipsen, UK. (R) (Clostridium botulinum type A toxin hemagglutinin complex with lactose and human serum albumin in a botulinum toxin pharmaceutical composition, reconstituted with 0.9% sodium chloride prior to use) and MyoBloc from Solstice Neurosciences (R) (An injectable solution containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride at approximately pH 5.6).

[0010] Botulinum toxin is mainly distributed as a freeze-dried preparation, and many studies have been conducted to keep freeze-dried preparations stable for a long period of time. For example, in US 7,744,904 B1, alpha, beta, and gamma cyclodextrins were simply mixed with botulinum toxin in a phosphate buffer solution to form a cyclodextrin-botulinum toxin complex form, which was used to promote the stability of botulinum toxin. Although oral administration of alpha, beta, and gamma cyclodextrins is permitted, they have problems in that they are not suitable as excipients for botulinum toxin, which is mainly used as an injection, due to their limited solubility and nephrotoxicity. In addition, if the particle size of botulinum toxin is not uniform when it is injected into the body, the effect will not work evenly in the body, and the desired therapeutic or cosmetic effect will not be achieved, which may cause various side effects.

[0011] Therefore, the present inventors endeavored to develop a freeze-dried preparation in which botulinum toxin is homogeneously granulated and also has improved stability. As a result, they confirmed that when a freeze-dried botulinum toxin preparation containing protamine sulfate as an excipient is produced, protamine sulfate and botulinum toxin form a complex, which improves the duration of the efficacy of botulinum toxin from the conventional approximately three months to more than six months, and that the botulinum toxin particles can be produced uniformly with a PDI value of 0.3 or less, thereby completing the present invention. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] US 7,744,904 B1 Summary of the Invention

[0013] An object of the present invention is to provide a novel freeze-dried botulinum toxin preparation composition which allows the production of a uniform particle size of botulinum toxin and has improved long-term storage stability, and a method for producing the same.

[0014] In order to achieve the above object, the present invention provides a botulinum toxin freeze-dried preparation composition comprising a complex of a botulinum toxin and protamine sulfate, an isotonicity agent, and a buffering agent.

[0015] The present invention also provides a method for producing a lyophilized formulation of botulinum toxin, comprising the steps of: (a) mixing a botulinum toxin with protamine sulfate to form a botulinum toxin-protamine sulfate complex; (b) adding one or more excipients selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin and mannitol, an isotonicity agent and a buffer to the complex of botulinum toxin and protamine sulfate to prepare a botulinum toxin stock solution; and (c) freeze-drying the botulinum toxin stock solution. BEST MODE FOR CARRYING OUT THEINVENTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0017] In the present invention, it has been confirmed that when a complex of botulinum toxin and protamine sulfate is produced and then freeze-dried into a preparation, the size is maintained uniform and the long-term storage stability of the freeze-dried preparation can be improved.

[0018] Therefore, in one aspect, the present invention relates to a botulinum toxin lyophilized preparation composition comprising a complex of a botulinum toxin and protamine sulfate, an isotonicity agent, and a buffering agent.

[0019] In the present invention, the botulinum toxin has a negative charge, and the protamine sulfate has a positive charge, and when reacted in a solution, an ionic complex is formed, and the protamine sulfate is bound to the botulinum toxin protein having a large molecular weight in the form of an ionic bond. In this case, the amount of protamine sulfate bound to the botulinum toxin varies depending on the concentration of the added protamine sulfate, and the surface of the botulinum toxin to which the protamine sulfate is bound becomes positively charged, and can form an additional complex with the botulinum toxin.

[0020] Therefore, in order to form a complex with improved long-term storage stability of the lyophilized preparation according to the present invention, the concentration of the protamine sulfate in the present invention is, but is not limited to, 1.72 nM to 0.22 mM, preferably 1.72 nM to 17.2 nM, more preferably 1.72 nM to 6.98 nM, and even more preferably 1.72 nM to 3.44 nM.

[0021] In the present invention, the botulinum toxin and protamine sulfate form a complex in a mass ratio of 1:3 to 1:20, preferably 1:5 to 1:20, more preferably 1:5 to 1:10, and most preferably 1:5, but is not limited thereto.

[0022] In the present invention, the composition further comprises one or more excipients selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin and mannitol, preferably, but not limited to, hydroxypropyl-beta-cyclodextrin, human serum albumin and mannitol.

[0023] In the present invention, the concentration of the hydroxypropyl-beta-cyclodecane is characterized by being 1 to 30 w / v%, preferably 2 to 25 w / v%, more preferably 5 to 20 w / v%, for example, 5 to 15 w / v%, 5 to 10 w / v%, 8 to 15 w / v%, 8 to 10 w / v%, 10 to 20 w / v% or 15 to 20 w / v%, but is not limited thereto.

[0024] In the present invention, the concentration of the human serum albumin is characterized by being, but not limited to, 0.1 to 1.0 w / v%, preferably 0.2 to 0.7 w / v%, more preferably 0.2 to 0.5 w / v%, and most preferably 0.35 to 0.5 w / v%.

[0025] In the present invention, the mannitol functions as a cryopreservation agent, and can be replaced by, but is not limited to, sucrose, glycerin, trehalose, lactose, etc. The content of the cryopreservation agent is, but is not limited to, 0.1 to 8 w / v% based on the total composition.

[0026] In the present invention, the cryopreservation agent is preferably mannitol, and the concentration of the mannitol is, but is not limited to, 0.1 to 10 w / v%, preferably 0.1 to 8 w / v%, and more preferably 4 to 8 w / v%.

[0027] In the present invention, the tonicity agent may be, but is not limited to, sodium chloride, glycerin, mannitol, sucrose, potassium chloride, dextrose, etc. The content of the tonicity agent may be, but is not limited to, 0.7 to 0.95 w / v% based on the total composition.

[0028] In the present invention, the isotonicity agent is preferably sodium chloride, and the concentration of the sodium chloride is, but is not limited to, 0.7-1.0 w / v%, preferably 0.7-0.95 w / v%, more preferably 0.8-0.95 w / v%, and most preferably 0.9 w / v%.

[0029] The composition according to the present invention contains a buffer that exhibits a pH equal to or higher than the physiologically relevant isoelectric point, thereby ensuring long-term stability.

[0030] In the present invention, the physiologically suitable pH of the buffer must be able to be maintained in the range of 6.0 to 7.0, preferably in the range of pH 6.4 to 6.6, and most preferably, a buffer that maintains a pH of about 6.5 can be used.

[0031] In the present invention, the physiologically suitable buffer may be, but is not limited to, sodium citrate, succinic acid, phosphoric acid, potassium phosphate monobasic, sodium acetate, or sodium chloride.

[0032] In the present invention, the concentration of the buffer suitable for lyophilization can be 10 to 35 mM, preferably 10 to 30 mM, and most preferably 15 to 25 mM, based on the total composition.

[0033] Preferably, the buffer has a pH of 6.0-7.0 and a concentration of 10-35 mM, but is not limited thereto.

[0034] In one embodiment, the physiologically suitable buffer comprises phosphate at pH 6.5 at a concentration of 20 mM based on the total composition.

[0035] The freeze-dried formulation composition of the present invention uses protamine sulfate to induce a complex with botulinum toxin, and adds hydroxypropyl-beta cyclodextrin and human serum albumin to impart hydrophobicity to it. Also, an isotonicity agent, preferably sodium chloride, can be used to increase the solubility of botulinum toxin.

[0036] In the present invention, the protamine sulfate is a molecule containing many positively charged amino acids, and forms a complex with botulinum toxin, thereby contributing to the stability of the botulinum toxin.

[0037] On the other hand, in the present invention, the hydroxypropyl-beta cyclodextrin (which can be used in combination with "betadex") has a cyclic sugar chain structure and has amphiphilicity similar to polysorbate 20 and polysorbate 80, and contributes to the stability of botulinum toxin.

[0038] "Botulinum toxin" in the present invention can include both mutant, recombinant, hybrid and chimeric botulinum toxins as well as neurotoxins (NTXs) produced by Clostridium botulinum strains or mutants thereof. In the present invention, recombinant botulinum toxins can have light and / or medium chains recombinantly produced by non-Clostridium species. In the present invention, said botulinum toxins include both pure botulinum toxins (i.e., the approximately 150 kDa neurotoxic molecule) as well as botulinum toxin complexes (i.e., the 300, 600 and 900 kDa complexes).

[0039] In the present invention, the botulinum toxin is characterized in that it is selected from the group consisting of serotypes A, B, C, D, E, F and G, but is not limited thereto.

[0040] Preferably, the botulinum toxin contained in the freeze-dried composition of the present invention is botulinum toxin type A. The composition according to the present invention is characterized in that it contains about 10 to 200 units / ml of botulinum toxin, preferably about 20 to 150 units / ml, and more preferably about 40 to 100 units / ml, but is not limited thereto.

[0041] In the present invention, the freeze-dried composition further contains an antioxidant, which is, but is not limited to, α-tocopherol.

[0042] In another aspect, the present invention relates to a method for producing a lyophilized preparation of botulinum toxin, comprising the steps of: (a) mixing a botulinum toxin with protamine sulfate to form a botulinum toxin-protamine sulfate complex; (b) adding one or more excipients selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin and mannitol, an isotonicity agent and a buffer to the complex of botulinum toxin and protamine sulfate to produce a botulinum toxin stock solution; and (c) freeze-drying the botulinum toxin stock solution.

[0043] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are provided solely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0044] Experimental Example 1 Preparation and size measurement of botulinum toxin / protamine sulfate complex To maintain the stability of botulinum toxin, protamine sulfate, which has a positive charge, was used to prepare a botulinum toxin / protamine sulfate complex. Botulinum toxin, which has a relatively negative charge, has a PI of about pH 6, and a buffer with a pH of 6 or higher was used to induce complex formation with protamine sulfate. The buffer used was 20 mM phosphate buffer (pH 6.5). Botulinum toxin type A (900 kDa, Daio Co., Ltd.) was prepared at 0.1 mg / mL, and protamine sulfate was dissolved at 0.5, 1.0, and 2.0 mg / mL in 20 mM phosphate buffer (pH 6.5), respectively, and then mixed 1:1 (v / v) and complex formation was induced at room temperature for about 30 minutes.

[0045] To confirm whether a complex was formed, a nanoparticle size analyzer (Zetasizer ZSP, Malvern) was used to confirm the particle size of the botulinum toxin or the botulinum toxin / protamine sulfate complex. The nanoparticle size analyzer is a device that measures the size of particles by measuring the refractive index when light is transmitted through a sample and then received again. As a result of the test, as shown in Table 1, it was confirmed that botulinum toxin type A showed a size of about 19 nm, while when it was complexed with protamine sulfate at a ratio of 1:5, it showed a size of about 182 nm. It was confirmed that when it was 1:10 and 1:20, it showed sizes of about 335 and 481 nm, respectively. Meanwhile, when the PDI (polydispersity index) was confirmed, when botulinum toxin type A:protamine sulfate was complexed at a ratio of 1:5, the PDI value was 0.3 or less, and therefore, it was found that the complex at that ratio showed a uniform particle size, which was the most favorable composition ratio for each complex to exert the same effect when injected into the body.

[0046] [Table 1]

[0047] Experimental Example 2 Excipient screening and severe stability evaluation of freeze-dried formulation composition containing botulinum toxin as the main component We tried to screen for an excipient composition that would be stable as a botulinum toxin freeze-dried formulation using a botulinum toxin / protamine sulfate 1:5 complex as the basic composition. The freeze-dried formulation composition used 20 mM phosphate buffer (pH 6.5) as the basic solvent for the formation of the botulinum toxin / protamine sulfate complex, and screened various excipients such as stabilizers, antioxidants, cryopreservatives, and isotonicity agents to confirm the stability of the botulinum toxin.

[0048] In this case, the botulinum toxin was used at 40 U / mL for the experiment, and the final stock solution was prepared with the excipient composition shown in Table 2 below, and then dispensed at 50 U / vial and freeze-dried. After freeze-drying, a harsh stability test was performed to indirectly confirm the long-term stability. Samples were stored in a harsh chamber at 40°C for 12 weeks, and the storage stability of the toxin was confirmed by measuring the change in potency. Specifically, the stability of the botulinum toxin was evaluated through an animal potency test using 4-week-old ICR mice (Koatech, Korea) for the specified period shown. The test substance was rehydrated with saline and then diluted to seven different concentrations, and the diluted solution was administered at 0.1 mL / mouse to observe whether the animals died for three days, and the LD50 value was obtained to calculate the potency. Ten mice were used for each concentration, and the results were shown as the average value.

[0049] In this case, the potency of each formulation was set to 100% when preparing the formulations, but since botulinum toxin is a protein and is affected by the composition of excipients, the external environment, parameters of the freeze-drying process, etc., the potency on day 1 was converted back to 100% and the subsequent potency change was calculated. In this case, it was determined that the potency could be varied up to ±20% based on the initial potency value of 100%.

[0050] [Table 2]

[0051] As a result, first, when human serum albumin was used as a stabilizer and mannitol as a freeze-drying agent (Comparative Example 1), and when betadex, human serum albumin, and sodium chloride as an isotonicity agent were used (Comparative Example 2), the botulinum toxin potency was determined to be incompatible after 12 weeks under harsh conditions. However, when betadex and human serum albumin were used as stabilizers, sodium chloride as an isotonicity agent, and mannitol as a cryopreservation agent were used together (Example 1), the botulinum toxin potency after 12 weeks under harsh conditions was confirmed to be the best. On the other hand, it was confirmed that the potency was appropriate after 12 weeks under harsh conditions when polysorbate was added to human serum albumin and betadex as stabilizers, sodium chloride was used as an isotonicity agent, and mannitol was used as a lyophilization agent (Example 2), and when alpha-tocopherol was added as an antioxidant (Example 3). However, taking into consideration the results of the above experiments and the economic costs of the excipients, Example 1 was selected as the basic excipient composition and future research was carried out.

[0052] Experimental Example 3 Optimization of excipient composition and evaluation of severe stability A total of three additional compositions were prepared as shown in Table 3 to confirm the effect of a range of concentrations of botulinum toxin / protamine sulfate complex, fixed in 0.9% sodium chloride and 20 mM phosphate buffer (pH 6.5) containing human serum albumin 0.35-0.5 (w / v), betadex 5-20 (w / v), and mannitol 4-8 (w / v). All compositions were prepared in the same manner except for the concentration of excipients. After preparation, the compositions were dispensed into 10 mL vials and lyophilized to evaluate the harsh stability of botulinum toxin for 12 weeks.

[0053] [Table 3]

[0054] As a result, it was experimentally verified that the stability of botulinum toxin is maintained for 8 weeks or more, specifically 12 weeks, in a concentration range containing 0.35-0.5% human serum albumin, 5-20% betadex, and 4-8% mannitol. Based on the results of previous research, the present inventors confirmed that when the stability is ensured under 2 weeks of harsh conditions, the stability is confirmed for up to about 6 months under refrigeration, and when the stability is ensured under 8 weeks of harsh conditions, the stability is confirmed for more than about 2 years under refrigeration. Therefore, it was found that the freeze-dried preparation of botulinum toxin containing 0.35-0.5% human serum albumin, 10-20% betadex, and 4-8% mannitol at an optimal concentration having stability within a potency range of 80-120% up to 12 weeks of harsh conditions can maintain its efficacy for at least 6 months or more, preferably 12 months or more, and most preferably 24 months or more during distribution.

[0055] Experimental Example 4: Evaluation test of the effect of cyclodextrin-based excipients on the stability of botulinum toxin Betadex is a derivative of β-cyclodextrin (β) that has been improved with hydrophilic properties by introducing hydroxyl and propyl functional groups. Betadex is currently approved as a pharmaceutical excipient by the FDA and EU, and is used as a stabilizer for the main ingredient of pharmaceuticals. Cyclodextrins are classified into three types according to structure: alpha (α), beta (β), and gamma (γ), and their relatively hydrophobic internal structure stabilizes the main ingredient through inclusion. Therefore, we attempted to evaluate the stabilizing effect of botulinum toxin using a cyclodextrin-based excipient similar to betadex.

[0056] First, α-, β-, and γ-cyclodextrins are nephrotoxic and have been evaluated by the FDA and KFDA as unsuitable as pharmaceutical injection excipients. Therefore, they were excluded from this experiment, and an experiment was conducted to see whether methyl-β-cyclodextrin, beta-cyclodextrin sulfate, beta-cyclodextrin sulfobutylisosorbium, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, or hydroxypropyl-alpha-cyclodextrin could be used instead of Betadex (hydroxypropyl-β-cyclodextrin).

[0057] For this purpose, the botulinum toxin / protamine sulfate 1:5 complex was fixed as the basic composition with 0.9% sodium chloride, 20 mM phosphate buffer (pH 6.5), 0.5% human serum albumin, and 4% mannitol, and experiments were performed by changing only the cyclodextrin excipient, in which the concentration of the cyclodextrin excipient was kept the same at 5%.

[0058] [Table 4]

[0059] As a result, as seen in Table 4, except for betadex (hydroxypropyl-β-cyclodextrin, Example 7) according to the present invention, the other cyclodextrin excipients were judged to be inappropriate for the storage stability of botulinum toxin in the harsh stability evaluation over a period of 4 weeks, and could not maintain potency stability over a harsh period of 6 weeks or more. Therefore, it was found that the use of betadex, among cyclodextrins, as a preparation for frozen storage of botulinum toxin according to the present invention is particularly effective.

[0060] Experimental Example 5: Evaluation test of the effect of protamine sulfate on the stability of botulinum toxin Protamine sulfate is a positively charged peptide, and can form a complex with the relatively negatively charged botulinum toxin through ionic bonds. The formation of a complex with the botulinum toxin can act as a factor in maintaining the stability of the botulinum toxin protein. Therefore, a comparative study was conducted to confirm the effect of the presence or absence of protamine sulfate on the stability of the botulinum toxin.

[0061] For this purpose, the tissue was fixed in 0.9% sodium chloride, 20 mM phosphate buffer (pH 6.5), 0.5% human serum albumin, 4% mannitol, and 10% betadex, and comparative experiments were performed using botulinum toxin alone or a 1:5 botulinum toxin / protamine sulfate complex.

[0062] [Table 5]

[0063] As a result, as shown in Table 5, it was confirmed that only the formulation containing protamine sulfate maintained the stability of the freeze-dried botulinum toxin formulation.

[0064] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0065] The freeze-dried botulinum toxin preparation according to the present invention can reduce the inactivation rate of the botulinum toxin that occurs during the freeze-drying process, and can produce an efficient and stable freeze-dried preparation. In addition, the freeze-dried botulinum toxin preparation according to the present invention can maintain the stability of the botulinum toxin for a longer period of time than conventional freeze-dried botulinum toxin preparations, and can form a uniform particle size, so that the effect is uniform at the administration site, and can prevent side effects caused by excessive toxin administration in only some areas.

Claims

1. Botulinum toxin and protamine sulfate complex, 5-20 w / v% hydroxypropyl-beta-cyclodextrin, 0.35-0.5 w / v% human serum albumin, 0.1-8 w / v% mannitol, 0.7-0.95 w / v% isotonicity agent, and Buffer A botulinum toxin lyophilized formulation composition comprising: The botulinum toxin and protamine sulfate are complexed in a mass ratio of 1:5 to 1:20, and The pH of the buffer is 6.0-7.0 and the concentration is 10-30 mM. Botulinum toxin lyophilized formulation composition.

2. The botulinum toxin freeze-dried preparation composition according to claim 1, wherein the concentration of the protamine sulfate is 1.72 nM to 0.22 mM.

3. The botulinum toxin freeze-dried formulation composition described in claim 1, wherein the buffer is a phosphate buffer or a citrate buffer.

4. The botulinum toxin freeze-dried preparation composition according to claim 1, characterized in that the botulinum toxin is selected from the group consisting of serotypes A, B, C, D, E, F and G.

5. A method for producing a lyophilized formulation of botulinum toxin comprising the steps of: (a) combining in a solution a botulinum toxin and protamine sulfate to form a botulinum toxin-protamine sulfate complex, wherein the botulinum toxin and protamine sulfate form a complex in a mass ratio of 1:5 to 1:20; (b) adding to said botulinum toxin and protamine sulfate complex, 5-20 w / v% hydroxypropyl-beta-cyclodextrin, 0.35-0.5 w / v% human serum albumin, 0.1-8 w / v% mannitol 0.7-0.95 w / v% isotonicity agent, and adding a buffer to prepare a botulinum toxin stock solution, wherein the buffer has a pH of 6.0-7.0 and a concentration of 10-30 mM; (c) freeze-drying the botulinum toxin stock solution.

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