Freeze-dried preparations containing botulinum toxin and methods for their preparation.

Abstract

The invention relates to a freeze-dried botulinum toxin formulation capable of long-term preservation, comprising botulinum toxin, protamine sulfate, isotonic and buffering agents, and a freeze-dried botulinum toxin formulation according to the invention that reduces the rate of inactivation of botulinum toxin during freeze-drying, can maintain the stability of botulinum toxin for longer than conventional freeze-dried botulinum toxin formulations, forms a uniform particle size so that the effect at the site of application is the same and can prevent side effects in which the effect of toxin overuse is only manifested at certain sites.

Description

Long-term storage-capable botulinum toxin freeze-dried formulation composition

[0001] The present invention relates to a botulinum toxin lyophilized formulation composition capable of long-term storage, and more particularly, to a botulinum toxin lyophilized formulation composition capable of long-term storage, comprising a complex of botulinum toxin and protamine sulfate, an isotonic agent, and a buffer.

[0002]

[0003] A variety of 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.

[0004] Botulinum toxin, a neurotoxic toxin derived from the above Clostridium strains, is classified into seven types, A to G, based on its serological characteristics. Each toxin has a toxin protein of about 150 kDa, which naturally forms a complex in which it is bound to several non-toxic proteins. The medium complex (300 kDa) is composed of the toxin protein and the non-toxic non-hemagglutinin protein, and the large (450 kDa) complex and the large-large (900 kDa) complex are formed in the form of the medium complex 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 low pH and various proteolytic enzymes in the intestine.

[0005] The above toxin is synthesized as a single polypeptide with a molecular weight of approximately 150 kDa within the cell, and is then cleaved at a position one-third from the N-terminus by the action of intracellular proteases or artificial enzyme treatment such as trypsin, thereby dividing into two units, a light chain (L: light chain) (molecular weight: 50 kDa) and a heavy chain (H: heavy chain) (molecular weight: 100 kDa). The toxicity of the toxin divided in this way is significantly increased compared to when it is a single polypeptide. The two units are linked to each other by a disulfide bond, and each has a different function. The heavy chain binds to the receptor of the target cell and has the function of forming a channel by reacting with the biological membrane at low pH (pH4) (Mantecucco, C et al, TIBS, 18:324, 1993), and the light chain has pharmacological activity and makes the cell permeable using a detergent or inhibits the secretion of neurotransmitters when introduced into the cell through electroporation, etc.

[0006] The toxin inhibits the exocytosis of acetylcholine at cholinergic presynapses at the neuromuscular junction, causing systemic weakness. Given that even trace amounts of the toxin are toxic, it has been suggested that the toxin possesses some enzymatic activity.

[0007] Recent studies have shown that the toxin possesses metallopeptidase activity, and its substrates are synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP25), which are unit proteins that form the exocytosis machinery complex. Each type of toxin uses one of these three proteins as a substrate, with types B, D, F, and G known to cleave synaptobrevin at a specific site, types A and E known to cleave SNAP25, and type C known to cleave syntaxin at a specific site.

[0008] In particular, botulinum toxin type A is known to be soluble in dilute aqueous solutions with a pH of 4.0 to 6.8. At pHs above approximately 7, the stabilized non-toxin protein dissociates from the neurotoxin, resulting in a gradual loss of toxicity, which is known to decrease particularly with increasing pH and temperature.

[0009] The above botulinum toxin is a toxin that can be used as one of the four major biological terrorism weapons, along with Bacillus anthracis, Yersinia pestis, and smallpox virus, because it is lethal to the human body in small amounts and easy to mass-produce. However, it has been revealed that among the above botulinum toxins, type A toxin can paralyze local muscles at the injection site when injected in a dose below that which does not affect the human body systemically. Since this characteristic can be utilized in a wide range of applications such as wrinkle removers, spastic hemiplegia, and cerebral palsy treatments, demand is rapidly increasing, and research on methods for producing botulinum toxin is actively being conducted to meet this demand.

[0010] The currently representative commercialized product is BOTOX from Allergan, USA.® (botulinum toxin type A purified neurotoxin complex), each BOTOX ® A 100-unit vial of botulinum toxin type A complex contains approximately 5 ng of purified botulinum toxin type A complex, 0.5 mg human serum albumin, and 0.9 mg sodium chloride, supplied in a vacuum-dried form and reconstituted with sterile saline without preservatives (0.9% sodium chloride injection). Another commercially available product is Dysport from Ipsen, UK. ® (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 before use) and MyoBloc from Solstice Neurosciences ® (a solution for injection containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride, pH 5.6).

[0011] Botulinum toxin is mainly distributed in lyophilized form, and much research has been conducted to maintain the long-term stability of lyophilized formulations. For example, US 7,744,904 B1 attempted to enhance the stability of botulinum toxin by simply mixing alpha, beta, and gamma cyclodextrins with botulinum toxin in a phosphate buffer to form a cyclodextrin-botulinum toxin complex. However, while alpha, beta, and gamma cyclodextrins are permitted for oral administration, they are not suitable as excipients for botulinum toxin, which is mainly used in injections, due to their limited solubility and renal toxicity. Furthermore, if the particle size of botulinum toxin is not uniform when injected into the body, the effect may not be distributed evenly within the body, failing to achieve the desired therapeutic or cosmetic effect, which may lead to various side effects.

[0012] Accordingly, the present inventors have made efforts to develop a freeze-dried formulation in which botulinum toxin is homogeneously granulated and stability is also improved. As a result, when a freeze-dried formulation of botulinum toxin containing protamine sulfate as an excipient is manufactured, protamine sulfate and botulinum toxin form a complex, thereby improving the efficacy of botulinum toxin to last for more than 6 months compared to the conventional 3 months, and the botulinum toxin particles can be uniformly manufactured with a PDI value of 0.3 or less, thereby completing the present invention.

[0013]

[0014] Prior art literature

[0015] Patent documents

[0016] (Patent Document 1) US 7,744,904 B1

[0017]

[0018] Summary of the invention

[0019] The purpose of the present invention is to provide a novel botulinum toxin freeze-dried formulation composition capable of forming a homogeneous particle size of botulinum toxin and having improved long-term storage stability, and a method for producing the same.

[0020] To achieve the above purpose, the present invention provides a botulinum toxin lyophilized formulation composition comprising a complex of botulinum toxin and protamine sulfate, a tonicity agent, and a buffer.

[0021] The present invention also provides a method for preparing a lyophilized formulation of botulinum toxin, comprising the following steps:

[0022] (a) a step of mixing botulinum toxin and protamine sulfate to form a complex of botulinum toxin and protamine sulfate;

[0023] (b) a step of preparing a botulinum toxin stock solution by adding one or more excipients, isotonic agents, and buffers selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol to the complex of the botulinum toxin and protamine sulfate; and

[0024] (c) A step of freeze-drying the above botulinum toxin stock solution.

[0025]

[0026] Detailed description of the invention and preferred embodiments

[0027] 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 pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0028]

[0029] In the present invention, it was confirmed that when a complex of botulinum toxin and protamine sulfate is prepared and formulated into a lyophilized form, the long-term storage stability of the lyophilized form can be improved while maintaining uniform size.

[0030]

[0031] Accordingly, the present invention relates to a botulinum toxin lyophilized formulation composition comprising a complex of botulinum toxin and protamine sulfate, a tonicity agent and a buffering agent, in one aspect.

[0032] In the present invention, botulinum toxin has a negative charge, and protamine sulfate has a positive charge, and when reacted in a solution, they form an ionic complex, and protamine sulfate binds to a botulinum toxin protein with 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 protamine sulfate is bound has a positive charge and may form an additional complex with the botulinum toxin.

[0033] Therefore, in order to form a complex having improved long-term storage stability of the freeze-dried formulation according to the present invention, the concentration of the protamine sulfate in the present invention may be, 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.

[0034] In addition, in the present invention, the botulinum toxin and protamine sulfate may be characterized by forming a complex at 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.

[0035] In the present invention, the composition may be characterized in that it further comprises one or more excipients selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol, and preferably further comprises hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol, but is not limited thereto.

[0036] In the present invention, the concentration of the hydroxypropyl-betacyclodextrin may be, but is not limited to, 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 %.

[0037] In the present invention, the concentration of the human serum albumin may be, but is 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 %.

[0038] In the present invention, the mannitol functions as a cryopreservative, and sucrose, glycerin, trehalose, or lactose may be used as a substitute for it, but is not limited thereto. The content of the cryopreservative may be 0.1 to 8 w / v % based on the total composition, but is not limited thereto.

[0039] In the present invention, preferably, the cryopreservative may be mannitol, and the concentration of the mannitol may be characterized by including 0.1 to 10 w / v %, preferably 0.1 to 8 w / v %, and more preferably 4 to 8 w / v %, but is not limited thereto.

[0040] In the present invention, the isotonic agent may be sodium chloride, glycerin, mannitol, sucrose, potassium chloride, or dextrose, but is not limited thereto. The content of the isotonic agent may be 0.7 to 0.95 w / v % based on the total composition, but is not limited thereto.

[0041] In the present invention, the isotonic agent may preferably be sodium chloride, and the concentration of the sodium chloride may be, 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%.

[0042] The composition according to the present invention may contain a buffer that exhibits a pH higher than a physiologically suitable isoelectric point, thereby ensuring long-term stability.

[0043] In the present invention, the physiologically suitable pH of the buffer should 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 having a pH of about 6.5 can be used.

[0044] In the present invention, as the physiologically suitable buffer, sodium citrate, succinic acid, phosphoric acid, potassium phosphate monobasic, sodium acetate, or sodium chloride may be used, but is not limited thereto.

[0045] In the present invention, the concentration of the buffer suitable for freeze-drying may be 10 to 35 mM, preferably 10 to 30 mM, and most preferably 15 to 25 mM based on the entire composition.

[0046] Preferably, the pH of the buffer is 6.0 to 7.0, and the concentration is 10 to 35 mM, but is not limited thereto.

[0047] As one embodiment, phosphoric acid having a pH of 6.5 as the physiologically suitable buffer may be contained at a concentration of 20 mM based on the total composition.

[0048] The freeze-dried formulation composition according to the present invention uses protamine sulfate to induce a complex with botulinum toxin, and hydrophobicity is imparted to this by adding hydroxypropyl-betacyclodextrin and human serum albumin. In addition, the solubility of botulinum toxin can be increased using a tonicity agent, preferably sodium chloride.

[0049] In the present invention, the protamine sulfate is a molecule containing a large number of positively charged amino acids, which forms a complex with botulinum toxin and contributes to the stability of the botulinum toxin.

[0050] Meanwhile, in the present invention, the hydroxypropyl-betacyclodextrin (which may be used interchangeably with 'betadex') has a sugar chain in a cyclic structure and has amphiphilicity similar to polysorbate 20 or polysorbate 80, thereby contributing to the stability of botulinum toxin.

[0051] In the present invention, "botulinum toxin" may include neurotoxins (NTXs) produced by Clostridium botulinum strains or variants thereof, as well as modified, recombinant, hybrid, and chimeric botulinum toxins. In the present invention, the recombinant botulinum toxin may have a light chain and / or a heavy chain produced recombinantly by a non-Clostridial species. In the present invention, the botulinum toxin includes both botulinum toxin complexes (i.e., 300, 600, and 900 kDa complexes) as well as pure botulinum toxin (i.e., about 150 kDa neurotoxic molecule).

[0052] In the present invention, the botulinum toxin may be characterized by being selected from the group consisting of serotypes A, B, C, D, E, F, and G, but is not limited thereto.

[0053] 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 may be 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.

[0054] In the present invention, the freeze-dried composition may additionally contain an antioxidant, and the antioxidant may be alpha-tocopherol, but is not limited thereto.

[0055]

[0056] The present invention relates from another aspect to a method for preparing a lyophilized formulation of botulinum toxin, comprising the following steps:

[0057] (a) a step of mixing botulinum toxin and protamine sulfate to form a complex of botulinum toxin and protamine sulfate;

[0058] (b) a step of preparing a botulinum toxin stock solution by adding one or more excipients, isotonic agents, and buffers selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol to the complex of the botulinum toxin and protamine sulfate; and

[0059] (c) A step of freeze-drying the above botulinum toxin stock solution.

[0060]

[0061] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0062]

[0063] Experimental Example 1. Preparation and Size Measurement of Botulinum Toxin / Protamine Sulfate Complex

[0064] To maintain the stability of botulinum toxin, a botulinum toxin / protamine sulfate complex was prepared using protamine sulfate, which has a positive charge. Since botulinum toxin has a relatively negative charge, a buffer solution with a pH of about 6 or higher was used to induce complex formation with protamine sulfate, as PI is about pH 6. The buffer solution used was 20 mM phosphate buffer solution (pH 6.5). Botulinum toxin type A (900 kDa, Daewoong Co., Ltd.) was prepared at 0.1 mg / mL, and protamine sulfate was dissolved in 20 mM phosphate buffer solution (pH 6.5) at concentrations of 0.5, 1.0, and 2.0 mg / mL, respectively, and mixed at a 1:1 (v / v) ratio, and complex formation was induced at room temperature for about 30 minutes.

[0065] To confirm the presence or absence of complex formation, the particle size of botulinum toxin or botulinum toxin / protamine sulfate complex was determined using a nanoparticle size analyzer (Zetasizer ZSP, Malvern). A nanoparticle size analyzer measures the refractive index when light is transmitted through a sample and the light is received again, thereby measuring the particle size. As a result of the test, as shown in Table 1, botulinum toxin type A showed a size of approximately 19 nm, whereas when complexed with protamine sulfate at a ratio of 1:5, it showed a size of approximately 182 nm. In the cases of 1:10 and 1:20, it was confirmed that the sizes were approximately 335 and 481 nm, respectively. Meanwhile, as a result of checking the PDI (polydispersity index), when botulinum toxin type A:protamine sulfate formed a complex at a ratio of 1:5, the PDI value was found to be 0.3 or less, and therefore, the complex at that ratio showed a uniform particle size, indicating that it was the most advantageous composition ratio for each complex to exhibit the same effect when injected into the body.

[0066]

[0067]

[0068]

[0069] Experimental Example 2. Screening of excipients and stress stability evaluation of a freeze-dried formulation containing botulinum toxin as the main ingredient.

[0070] The aim was to screen for excipient compositions that exhibit stability in a lyophilized formulation of botulinum toxin, using a 1:5 complex of botulinum toxin / protamine sulfate as a basic composition. The lyophilized formulation composition uses a 20 mM phosphate buffer solution (pH 6.5) as a basic solvent for forming the botulinum toxin / protamine sulfate complex, and various excipients such as stabilizers, antioxidants, cryopreservatives, and isotonic agents were screened to confirm the stability of botulinum toxin.

[0071] In this case, the botulinum toxin was tested at 40 U / mL, and the final solution was prepared with the excipient composition shown in Table 2 below, dispensed into 50 U / vial, and lyophilized. A stress stability test was conducted to indirectly confirm the long-term stability after lyophilization. The samples were stored in a stress chamber at 40°C for 12 weeks, and the change in potency was measured to confirm the toxin storage stability. Specifically, the stability of the botulinum toxin was evaluated through an animal potency test using 4-week-old ICR mice (Koatech, Korea) at the indicated specific period. The test substance was rehydrated with physiological saline, diluted into 7 concentrations, and the diluted solution was administered at 0.1 mL / mouse. The animals were observed for death for 3 days and the LD50 value was obtained to calculate the potency value. The experiment was conducted with 10 mice for each concentration, and the results are expressed as the average value.

[0072] In this case, the potency of each formulation was set to 100% when preparing the formulation. However, since botulinum toxin is a protein, it is affected by the composition of excipients, external environment, and freeze-drying process parameters. Therefore, the potency on the first day of harshness was converted back to 100%, and the change in potency thereafter was calculated. In this case, the potency was judged to be appropriate within ±20% of the initial potency value of 100%.

[0073]

[0074]

[0075] As a result, in the case where human serum albumin was used as a stabilizer and mannitol was used as a freeze-drying agent (Comparative Example 1) and in the case where Betadex and human serum albumin were used as stabilizers and sodium chloride was used as an isotonic agent (Comparative Example 2), both were determined to be unsuitable in terms of botulinum toxin titer after 12 weeks under harsh conditions. However, in the case where Betadex and human serum albumin were used as stabilizers, sodium chloride was used as an isotonic agent, and mannitol was used as a cryopreservative together (Example 1), it was confirmed that the botulinum toxin titer was the best after 12 weeks under harsh conditions. Meanwhile, in the case where human serum albumin and betadex were added with polysorbate as stabilizers, sodium chloride as a tonicity agent, and mannitol as a freeze-drying agent (Example 2), and in the case where alpha-tocopherol was added and used as an antioxidant (Example 3), it was confirmed that the titer after 12 weeks of harsh conditions was appropriate. However, considering the results of the above experiment and the economic cost of the excipients, Example 1 was selected as the basic excipient composition and further research was conducted.

[0076]

[0077] Experimental Example 3. Optimization of excipient composition and evaluation of harshness stability.

[0078] To confirm the effect in a concentration range containing 0.35-0.5 (w / v)% human serum albumin, 5-20 (w / v)% Betadex, and 4-8 (w / v)% mannitol, a total of three additional compositions were prepared as shown in Table 3, based on a 1:5 complex of botulinum toxin / protamine sulfate as a base composition and fixed in 0.9% sodium chloride and 20 mM phosphate buffer solution (pH 6.5). All compositions were prepared in the same manner except for the concentration of excipients. After preparation, they were dispensed into 10 mL vials, freeze-dried, and the harshness stability of the botulinum toxin was evaluated for 12 weeks.

[0079]

[0080]

[0081] As a result, it was experimentally verified that the stability of botulinum toxin is maintained for more than 8 weeks, specifically for 12 weeks, in the concentration range containing 0.35-0.5% human serum albumin, 5-20% Betadex, and 4-8% mannitol. Based on the results of previous studies, the inventors of the present invention have confirmed that when stability is secured for 2 weeks, stability is confirmed for about 6 months in a refrigerator, and when stability is secured for 8 weeks, stability is confirmed for about 2 years or more in a refrigerator. Therefore, it was found that a freeze-dried formulation of botulinum toxin containing 0.35-0.5% human serum albumin, 10-20% Betadex, and 4-8% mannitol at an optimal concentration that has stability within the titer range of 80-120% for up to 12 weeks can maintain its efficacy for at least 6 months, preferably 12 months, and most preferably 24 months or more during the distribution process.

[0082]

[0083] Experimental Example 4. Evaluation of the Effect of Cyclodextrin-Based Excipients on the Stability of Botulinum Toxin

[0084] Betadex is a derivative of beta-cyclodextrin (β) that has improved hydrophilic properties by introducing hydroxy and propyl functional groups. Betadex is currently used as a pharmaceutical excipient approved by the FDA, EU, etc., and as a stabilizer for the main ingredient of pharmaceuticals. Cyclodextrins are classified into three types according to their structure: alpha (α), beta (β), and gamma (γ), and their internal structure with relatively hydrophobic characteristics stabilizes them through inclusion of the main ingredient. Therefore, we attempted to evaluate the stabilizing effects of botulinum toxin using cyclodextrin excipients similar to Betadex.

[0085] First, α, β, γ-cyclodextrins are evaluated as unsuitable as excipients for pharmaceutical injections by the FDA and KFDA due to their nephrotoxicity, so they were excluded from this experiment. An experiment was conducted to see if methyl-beta-cyclodextrin, beta-cyclodextrin sulfate, beta-cyclodextrin sulfobutyl ether sodium, hydroxyethyl-beta-cyclodextrin, hydroxypropyl-gamma-cyclodextrin, or hydroxypropyl-alpha-cyclodextrin could be used as a substitute for Betadex (hydroxypropyl-beta-cyclodextrin).

[0086]

[0087] To this end, experiments were conducted using a 1:5 botulinum toxin / protamine sulfate complex as the basic composition, fixed at 0.9% sodium chloride, 20 mM phosphate buffer (pH 6.5), 0.5% human serum albumin, and 4% mannitol, and varying only the cyclodextrin-based excipient. In this case, the concentration of the cyclodextrin-based excipient was maintained at the same 5%.

[0088]

[0089]

[0090] As a result, as confirmed in Table 4 above, except for Betadex (hydroxypropyl-beta-cyclodextrin, Example 7) according to the present invention, other cyclodextrin-based excipients were determined to have inadequate storage stability of botulinum toxin in the harsh 4-week stability evaluation, and failed to maintain potency stability for more than 6 weeks. Therefore, it was found that the formulation for cryopreservation of botulinum toxin of the present invention was particularly effective when using Betadex among cyclodextrins.

[0091]

[0092] Experimental Example 5. Test to evaluate the effect of protamine sulfate on botulinum toxin stability.

[0093] Protamine sulfate, a positively charged peptide, can form a complex with the relatively negatively charged botulinum toxin through ionic bonding. This complex formation with botulinum toxin may contribute to the stability of the botulinum toxin protein. Therefore, a comparative study was conducted to determine the effect of the presence or absence of protamine sulfate on the stability of botulinum toxin.

[0094]

[0095] For this purpose, a comparative experiment was conducted using botulinum toxin alone or a 1:5 complex of botulinum toxin / protamine sulfate, fixed with 0.9% sodium chloride, 20 mM phosphate buffer solution (pH 6.5), 0.5% human serum albumin, 4% mannitol, and 10% Betadex.

[0096]

[0097]

[0098] As a result, it was confirmed that the stability of the botulinum toxin lyophilized formulation was maintained only in the formulation containing protamine sulfate, as shown in Table 5.

[0099]

[0100] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0101]

[0102] The botulinum toxin freeze-dried formulation according to the present invention can produce an efficient and stable freeze-dried formulation by lowering the inactivation rate of botulinum toxin generated during the freeze-drying process. In addition, the botulinum toxin freeze-dried formulation according to the present invention can not only maintain the stability of botulinum toxin for a long period of time compared to conventional botulinum toxin freeze-dried formulations, but also form a uniform particle size so that the effect is equally exhibited at the site of administration and the side effect of excessive toxin administration occurring only at some sites can be prevented.

Claims

1. A composition of a botulinum toxin lyophilized formulation comprising botulinum toxin and protamine sulfate complex, a tonicity agent and a buffer.

2. A botulinum toxin freeze-dried formulation composition, characterized in that in claim 1, the concentration of protamine sulfate is 1.72 nM to 0.22 mM.

3. A botulinum toxin freeze-dried formulation composition, characterized in that in claim 1, the botulinum toxin and protamine sulfate form a complex at a mass ratio of 1:5 to 1:

20.

4. A botulinum toxin lyophilized formulation composition according to claim 1, characterized in that the composition further comprises one or more excipients selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol.

5. A botulinum toxin lyophilized formulation composition according to claim 4, characterized in that the composition further comprises hydroxypropyl-beta-cyclodextrin, human serum albumin, and mannitol.

6. A botulinum toxin freeze-dried formulation composition, characterized in that in clause 5, the concentration of the hydroxypropyl-betacyclodextrin is 5 to 20 w / v %.

7. A botulinum toxin freeze-dried formulation composition, characterized in that in clause 5, the concentration of the human serum albumin is 0.35 to 0.5 w / v %.

8. A botulinum toxin freeze-dried formulation composition, characterized in that in clause 5, the concentration of mannitol is 4 to 8 w / v %.

9. A botulinum toxin freeze-dried formulation composition, characterized in that the concentration of the isostatic agent in the first paragraph is 0.7 to 1.0 w / v %.

10. A botulinum toxin freeze-dried formulation composition according to claim 1, characterized in that the pH of the buffer is 6.0 to 7.0 and the concentration is 10 to 35 mM.

11. A botulinum toxin lyophilized formulation 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.

12. A method for manufacturing a lyophilized formulation of botulinum toxin comprising the following steps: (a) a step of mixing botulinum toxin and protamine sulfate to form a complex of botulinum toxin and protamine sulfate; (b) a step of preparing a botulinum toxin stock solution by adding one or more excipients, isotonic agents and buffers selected from the group consisting of polysorbate 20, hydroxypropyl-beta-cyclodextrin, human serum albumin and mannitol to the complex of the botulinum toxin and protamine sulfate; and (c) A step of freeze-drying the above botulinum toxin stock solution.