Vacuum drying method of botulinum toxin

Abstract

The present invention relates to a vacuum drying method of botulinum toxin, in detail, to a vacuum drying method of botulinum toxin, which comprises: step a) drying a solution containing botulinum toxin for the first time under the conditions of a temperature of 5 to 30°C and a pressure of 0.01 to 2 mmHg; step b) drying the first dried product obtained through step a) for the second time under the conditions of a temperature of 5 to 20°C and a pressure of 0.01 to 1 mmHg; and step c) final drying of the second dried product obtained through step b) under the conditions of a temperature of 5 to 30°C and a pressure of 0.01 to 0.5 mmHg. The present invention has the advantages of improving yield, stability and preparation efficiency compared to the existing method of vacuum drying botulinum toxin by freeze drying.

Description

Technical Field

[0001] This invention relates to the drying process in the preparation of botulinum toxin preparations, specifically to a vacuum drying method that dries botulinum toxin using vacuum drying instead of freeze drying. Therefore, compared to existing methods, this method can improve yield, stability, and preparation efficiency. Background Technology

[0002] Botulinum toxin is a neurotoxic protein produced by Clostridium botulinum and related species. It is known to block the release of acetylcholine, a neurotransmitter secreted from the axonal terminals at the neuromuscular junction. Although there are eight serologically associated natural botulinum toxins, types A to H, their effects on humans vary depending on the type, but all are known to be fatal. Type H botulinum toxin can cause death with an injection of only one two-billionth of a gram, while types A and B botulinum toxin can induce disease. In the case of type A botulinum toxin, since 1978, with its safety and efficacy confirmed, it has been used for strabismus correction. In 1987, after injection of this toxin to treat patients with blepharospasm, wrinkles between the eyebrows disappeared, thus raising interest in its potential for cosmetic purposes. Typically, botulinum toxin type A and botulinum toxin type B are drugged and injected into muscles to treat muscle spasms and diseases caused by excessive muscle activity. However, considering the impact of injection dosage on the human body, guidelines for the use of botulinum toxin and guidelines for the approval of botulinum toxin have been developed.

[0003] Currently, in the drug formulation process of botulinum toxin, due to problems such as protein instability and loss of active ingredients, additional components such as stabilizers are required. Relatedly, Korean Patent No. 10-1087017 discloses a method for preparing a liquid composition using polysorbate 20, methionine, and / or isoleucine.

[0004] To prevent the decomposition of botulinum toxin preparations, further simplify the handling of botulinum toxin preparations, and reduce transportation costs, commercially available botulinum toxin preparations are distributed in powder form after freeze-drying or vacuum drying. Before use, they can be reconstituted using liquid carriers such as water or saline solution.

[0005] However, freeze-drying, a commonly used drying technique in the prior art, suffers from drawbacks such as loss of botulinum toxin due to its instability and the long drying time required. Therefore, the inventors focused on improving the drying method for botulinum toxin, resulting in the discovery of a vacuum drying method, which, by applying the vacuum drying process of this invention consisting of specific steps, not only reduces the loss of active substances but also significantly shortens the processing time while producing products with excellent properties. Summary of the Invention

[0006] Technical issues

[0007] The purpose of this invention is to provide a vacuum drying method for botulinum toxin that significantly improves yield, stability, preparation efficiency, and product performance through vacuum drying, thereby solving problems such as long drying time and low yield of botulinum toxin when preparing botulinum toxin by existing methods of drying botulinum toxin by freeze drying.

[0008] Problem-solving methods

[0009] To achieve the above objectives, the present invention provides a vacuum drying method for botulinum toxin, comprising: step a), performing a first drying of a solution containing botulinum toxin at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 2 mmHg; step b), performing a second drying of the first dried product obtained in step a), at a temperature of 5°C to 20°C and a pressure of 0.01 mmHg to 1 mmHg; and step c), performing a third drying of the second dried product obtained in step b), at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 0.5 mmHg.

[0010] In one embodiment of the present invention, in step a) above, the first drying can be carried out at an initial temperature of 5°C or above, while the temperature is increased at a rate of 0.1°C / min to 2°C / min.

[0011] In one embodiment of the present invention, the upper limit of pressure in step a) can be 1.7 mmHg. Furthermore, in a specific embodiment of the present invention, the upper limit of pressure in step b) can be 0.8 mmHg. Furthermore, in a specific embodiment of the present invention, the upper limit of pressure in step c) can be 0.4 mmHg. In a more specific embodiment of the present invention, the upper limit of pressure in step c) can be 0.2 mmHg.

[0012] In one embodiment of the present invention, the drying time in steps a), b), and c) can be 30 minutes to 120 minutes, respectively.

[0013] In one specific embodiment of the present invention, the total drying time of the above-mentioned vacuum drying method for botulinum toxin can be less than 6 hours, more specifically, less than 5 hours, or less than 4 hours.

[0014] In one embodiment of the present invention, the titer recovery rate of the above-mentioned vacuum drying method for botulinum toxin can be 80% or more, and more specifically, the titer recovery rate can be 85% or more or 90% or more.

[0015] In one embodiment of the present invention, the botulinum toxin in step a) is selected from one or more of the group consisting 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, botulinum toxin type G and serotype.

[0016] In one embodiment of the present invention, the solution containing the botulinum toxin of step a) above may contain one or more of the following: i) the group consisting of excipients, stabilizers, preservatives, buffers, thickeners, suspending agents, emulsifiers, flavoring agents, coloring agents and cosolvents; ii) the group consisting of sodium chloride, calcium chloride, sodium phosphate, calcium phosphate, sodium acetate, ethanol, propylene glycol, polyethylene glycol and aqueous solutions thereof; and iii) the group consisting of albumin, gelatin, sugar and sugar alcohols.

[0017] The effects of the invention

[0018] This invention relates to a vacuum drying method for botulinum toxin. Because it is prepared by vacuum drying, there is no need to adjust the temperature below zero degrees Celsius. Compared with existing technologies, this method not only allows for drying in a shorter time but also improves the overall yield due to the high titer recovery rate. Furthermore, the botulinum toxin formulation prepared by the drying method of this invention is thin and hard, thus exhibiting advantages such as strong impact resistance and excellent product properties. Attached Figure Description

[0019] Figure 1 The graph shows the results of the titer recovery rate of the drying method for determining botulinum toxin.

[0020] Figure 2 A diagram illustrating the finished dosage form prepared by vacuum drying of botulinum toxin.

[0021] Figure 3 A diagram illustrating the finished dosage form prepared by freeze-drying botulinum toxin. Detailed Implementation

[0022] The best embodiment of the present invention

[0023] This invention relates to a vacuum drying method for botulinum toxin, comprising: step a), performing a first drying of a solution containing botulinum toxin at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 2 mmHg; step b), performing a second drying of the first dried product obtained in step a), at a temperature of 5°C to 20°C and a pressure of 0.01 mmHg to 1 mmHg; and step c), performing a final drying of the second dried product obtained in step b), at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 0.5 mmHg.

[0024] The present invention will now be described in detail.

[0025] This invention relates to a vacuum drying method for botulinum toxin, comprising: step a), performing a first drying of a solution containing botulinum toxin at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 2 mmHg; step b), performing a second drying of the first dried product obtained in step a), at a temperature of 5°C to 20°C and a pressure of 0.01 mmHg to 1 mmHg; and step c), performing a final drying of the second dried product obtained in step b), at a temperature of 5°C to 30°C and a pressure of 0.01 mmHg to 0.5 mmHg.

[0026] Throughout this specification, when a part is said to "contain" a structural element, it means, unless specifically stated otherwise, that other structural elements are also contained, and not excluded.

[0027] Throughout this manual, terms such as "upper limit," "lower limit," and "X to Y" indicate that the corresponding value is included, not excluded.

[0028] In this invention, botulinum toxin is a neurotoxic polypeptide produced by Clostridium botulinum and related species. It includes not only substances inherently generated by Clostridium but also all substances generated through modification, recombination, etc. Furthermore, in this invention, unless otherwise defined, botulinum toxin includes all substances recognizable by a person skilled in the art, including but not limited to type A botulinum toxin, type B botulinum toxin, type C1 botulinum toxin, type C2 botulinum toxin, type D botulinum toxin, and type E botulinum toxin. Botulinum toxin can be obtained by any known method or commercial channel, and there are no particular restrictions on the method of acquisition.

[0029] In one embodiment of the present invention, the botulinum toxin in step a) is selected from one or more of the group consisting 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, botulinum toxin type G and serotype.

[0030] In one embodiment of the present invention, the solution containing the botulinum toxin of step a) above may contain one or more of the following: i) the group consisting of excipients, stabilizers, preservatives, buffers, thickeners, suspending agents, emulsifiers, flavoring agents, coloring agents and cosolvents; ii) the group consisting of sodium chloride, calcium chloride, sodium phosphate, calcium phosphate, sodium acetate, ethanol, propylene glycol, polyethylene glycol and aqueous solutions thereof; and iii) the group consisting of albumin, gelatin, sugar and sugar alcohols.

[0031] In this invention, additives may include excipients, stabilizers, preservatives, buffers, thickeners, suspending agents, emulsifiers, flavoring agents, colorants, solubilizers, solvents, dispersants, disintegrants, plasticizers, lubricants, analgesics, diluents, masking agents, etc. Additives are substances other than the pharmacologically active ingredients contained in the formulation, used for additional purposes beyond pharmacological activity. Examples include additives for improving the stability and bioavailability of drug compositions, additives for maintaining the quality of the formulation, such as for the preservation or use of the drug composition, and additives for adjusting the physical properties of the drug composition. Additives can generally be used within a range that does not directly affect the pharmacological effect.

[0032] For example, excipients may be included to increase stability. Excipients may include sodium bicarbonate, sodium chloride, calcium phosphate, dextrin, etc.

[0033] Furthermore, a co-solvent may be included to increase the solubility of the liquid substance. Co-solvents may include sodium salicylate, urea, monoethylacetamide, propylene glycol, dimethylacetamide, hexamine, ethyl carbamate, sodium acetate, etc.

[0034] Furthermore, a stabilizer may be included to prevent decomposition by acids, moisture, etc. Stabilizers may include nitrogen, carbon dioxide, sugars, sugar alcohols, human serum albumin, weak acids and their salts, weak bases and their salts, proteins, etc. However, this is exemplary, and the present invention is not limited thereto.

[0035] In this invention, when the temperature of the step is below the upper limit, a stable formulation can be formed without destroying the active ingredients of botulinum toxin.

[0036] In one specific embodiment of the present invention, in step a) above, the first drying can be performed simultaneously with heating at an initial temperature of 5°C or above at a rate of 0.1°C / min to 2°C / min. The initial temperature may be within the range described above.

[0037] Specifically, step a) above can be divided into up to 14 steps by changing the pressure conditions as the temperature increases. In each step, the first drying can be carried out by changing the temperature, pressure, and holding time.

[0038] In one specific embodiment of the present invention, step a) above may be a first drying step performed as follows: divided into up to 14 steps by changing the temperature, pressure, and time, with the conditions of each step selected from the following ranges: temperature from 5°C to 30°C, heating rate from 0.1°C / min to 2°C / min, pressure from 0.01 mmHg to 2 mmHg, and time from 0.1 minutes to 40 minutes. More specifically, step a) above may be divided into 6 to 14 steps, 7 to 14 steps, 8 to 14 steps, 9 to 14 steps, or 10 to 14 steps by changing the temperature, pressure, and time. In each step, the set temperature may increase sequentially. However, the pressure and time do not increase or decrease sequentially.

[0039] In this invention, the first drying process is divided into a maximum of 14 steps. In each step, the active ingredients of botulinum toxin can be prevented from being destroyed to the greatest extent by adjusting the temperature, pressure, and time. If the pressure and temperature are kept constant below 4 steps during the first drying process, the active ingredients of botulinum toxin will be relatively reduced because the final botulinum toxin stock solution will form different internal and external states.

[0040] As a specific example, when step a) above is divided into 10 steps, steps 1 to 10 are performed sequentially. Step 1 can be performed at an initial set temperature of 5°C, a heating rate of 0.1°C / min to 1°C / min, a pressure of 0.01 mmHg to 2 mmHg, and for 5 minutes. Step 2 can be performed at a set temperature of 10°C, a heating rate of 0.5°C / min, a pressure of 0.01 mmHg to 1.5 mmHg, and for 5 minutes. Step 3 can be performed at a set temperature of 5°C, a heating rate of 0.5°C / min, a pressure of 0.01 mmHg to 1.0 mmHg, and for 10 minutes. Step 4 can be performed at a set temperature of 5°C, a heating rate of 0.75°C / min, a pressure of 0.01 mmHg to 1.0 mmHg, and for 20 minutes. Furthermore, step 5 can be performed at a set temperature of 15°C, a heating rate of 0.75°C / min, a pressure of 0.01 mmHg to 1.0 mmHg, and for 10 minutes. Steps 6, 7, 8, 9, and 10 can be performed under the same conditions (temperature, heating rate, and pressure) as step 5 or lower. However, this is exemplary, and the present invention is not limited thereto.

[0041] In one specific embodiment of the present invention, step a) above may be a first drying step performed in the following manner: divided into 10 to 14 steps by changing the temperature, pressure and time, wherein the conditions of each step are selected from the following range: temperature from 5°C to 30°C, heating rate from 0.1°C / min to 1°C / min, pressure from 0.01 mmHg to 2 mmHg and time from 0.1 minutes to 30 minutes.

[0042] Furthermore, in one embodiment of the present invention, step a) above may be a first drying step performed in the following manner: divided into 10 to 14 steps by changing the temperature, pressure and time, wherein the conditions of each step are selected from the following range: temperature from 5°C to 30°C, heating rate from 0.1°C / min to 0.8°C / min, pressure from 0.01 mmHg to 2 mmHg and time from 0.1 minutes to 30 minutes.

[0043] More specifically, step a) above can be a first drying step performed in the following manner: divided into 10 to 14 steps, with the conditions for steps 1 to 4 selected from the following range: temperature of 5°C to 10°C, heating rate of 0.1°C / min to 1°C / min, pressure of 0.01 mmHg to 2 mmHg, and time of 5 to 20 minutes, and the conditions for the remaining steps selected from the following range: temperature of 5°C to 30°C, heating rate of 0.1°C / min to 1°C / min, pressure of 0.01 mmHg to 1.7 mmHg, and time of 5 to 20 minutes.

[0044] More specifically, step a) above can be a first drying step performed in the following manner: divided into 10 to 14 steps, with the conditions for steps 1 to 3 selected from the following range: temperature of 5°C to 20°C, heating rate of 0.1°C / min to 0.5°C / min, pressure of 0.01 mmHg to 2 mmHg, and time of 5 to 20 minutes; the conditions for steps 4 to 7 selected from the following range: temperature of 5°C to 20°C, heating rate of 0.1°C / min to 0.5°C / min, pressure of 0.01 mmHg to 1 mmHg, and time of 5 to 20 minutes; and the conditions for steps 8 to 14 selected from the following range: temperature of 5°C to 20°C, heating rate of 0.1°C / min to 0.8°C / min, pressure of 0.01 mmHg to 0.5 mmHg, and time of 5 to 20 minutes.

[0045] In one embodiment of the present invention, step b) above may be a second drying step performed in the following manner: divided into two steps by changing the temperature, pressure and time, with the conditions of each step selected from the following range: temperature from 5°C to 30°C, pressure from 0.01 mmHg to 1 mmHg and time from 0.1 minutes to 80 minutes.

[0046] In one specific embodiment of the present invention, step b) above may be a second drying step performed in the following manner: divided into two steps by changing the temperature, pressure and time, with the conditions of each step selected from the following range: temperature of 20°C to 30°C, pressure of 0.05 mmHg to 1 mmHg and time of 20 minutes to 50 minutes.

[0047] In this invention, the second drying is carried out in two steps, which can produce the same effect as the first drying.

[0048] In one embodiment of the present invention, in steps a), b), and c), the lower pressure limit for each step can be from 0.01 mmHg to 0.5 mmHg. Specifically, in one embodiment of the present invention, the lower pressure limit for each step can be 0.01 mmHg, 0.05 mmHg, 0.09 mmHg, 0.13 mmHg, 0.17 mmHg, 0.20 mmHg, 0.25 mmHg, or 0.30 mmHg.

[0049] In one specific embodiment of the present invention, the upper limit of pressure in step a) above can be 1.7 mmHg. In a specific example of the present invention, the upper limit of pressure in step a) above can be 1.6 mmHg, 1.5 mmHg, 1.4 mmHg, 1.3 mmHg, 1.2 mmHg, 1.1 mmHg, 1.05 mmHg, or 1.0 mmHg.

[0050] Furthermore, in one specific embodiment of the present invention, in step b) above, the upper limit of pressure can be from 0.6 mmHg to 2.0 mmHg. In one specific embodiment of the present invention, the upper limit of pressure in step b) above can be 2.0 mmHg, 1.5 mmHg, 1.0 mmHg, 0.8 mmHg, 0.75 mmHg, 0.7 mmHg, or 0.6 mmHg.

[0051] Furthermore, in one specific embodiment of the present invention, the upper limit of pressure in step c) above can be 0.4 mmHg. In a specific example of the present invention, the upper limit of pressure in step c) above can be 0.48 mmHg, 0.45 mmHg, 0.42 mmHg, 0.40 mmHg, 0.38 mmHg, 0.35 mmHg, 0.32 mmHg, 0.30 mmHg, 0.28 mmHg, 0.25 mmHg, 0.22 mmHg, 0.20 mmHg, or 0.10 mmHg.

[0052] In this invention, when vacuum drying is performed within the aforementioned pressure range in each step, the purity of the botulinum toxin is not reduced, thus providing botulinum toxin with higher purity. When botulinum toxin is administered to the human body through methods such as surgery, it typically lasts for about 3 months, thus requiring repeated administration. This can potentially lead to resistance issues caused by antibody formation in the body and stability issues due to high-dose administration. However, the vacuum drying method of this invention achieves high purity by performing vacuum drying within the aforementioned pressure range, thereby enabling effective results at low doses.

[0053] Furthermore, in this invention, if vacuum drying is performed within the aforementioned pressure range, the botulinum toxin preparation exhibits thin and hard characteristics, thus demonstrating impact resistance.

[0054] In one embodiment of the present invention, the drying time in steps a), b), and c) can be 30 minutes to 120 minutes, respectively.

[0055] In a more specific embodiment of the present invention, the drying time of step b) above can be 50 to 80 minutes.

[0056] In a more specific embodiment of the present invention, the drying time in step c) above can be 40 minutes to 100 minutes.

[0057] In one specific embodiment of the present invention, the total drying time of the vacuum drying method for botulinum toxin described above can be 6 hours or less. In a specific example of the present invention, the total drying time of the vacuum drying method for botulinum toxin described above can be 5 hours, 4.8 hours, 4.5 hours, 4.2 hours, or 4 hours or less.

[0058] In this invention, the specific drying time can be adjusted within the aforementioned temperature and pressure range to minimize botulinum toxin loss, stabilize formulation formation, and improve preparation efficiency. Furthermore, compared to the at least 18 hours of drying time required by existing freeze-drying methods for botulinum toxin, this invention significantly reduces the drying time, thereby demonstrating excellent preparation efficiency.

[0059] In the vacuum drying method for botulinum toxin of the present invention, the total drying time refers to the total drying time including the first drying time in step a), the second drying time in step b), and the final drying time in step c), and not the total time required to prepare the botulinum toxin preparation. In the present invention, by utilizing vacuum drying, the total drying time is shortened, thereby inducing an improvement in the preparation efficiency of the botulinum toxin preparation.

[0060] In one embodiment of the present invention, the titer recovery rate of the above-described botulinum toxin drying method can be 80% or higher. Specifically, the titer recovery rate can be 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, or 90% or higher. In the present invention, the titer recovery rate is expressed as follows: the titers of the final stock solution, the vacuum-dried product, and the freeze-dried product are calculated, and the titers of the vacuum-dried product and the freeze-dried product are compared with those of the final stock solution, with the titer of the final stock solution being taken as 100%, to express the recovery rate.

[0061] The recovery rate of the titer of the freeze-dried product (%) = (average titer of the freeze-dried product / average titer of the final original solution) × 100

[0062] Vacuum-dried product titer recovery rate (%) = (Average titer of vacuum-dried product / Average titer of final original solution) × 100

[0063] Because of the high titer recovery rate of the present invention, the loss of botulinum toxin protein is minimal when preparing botulinum toxin formulations, and the therapeutic or cosmetic effects of the active botulinum toxin ingredient can be effectively demonstrated even at low doses.

[0064] The present invention will now be described in detail through examples and experimental cases.

[0065] However, the following embodiments and experimental examples are merely examples of the present invention, and the content of the present invention is not limited to the following embodiments and experimental examples.

[0066] Example 1. Botulinum toxin final stock solution

[0067] The final botulinum toxin stock solution was prepared using Clostridium botulinum toxin type A, sodium chloride as an excipient, and human serum albumin as a stabilizer. Detailed composition is shown in Table 1. A 50 ml final stock solution was prepared, with a target final stock solution volume of 100 units / 0.1 mL to 120 units / 0.1 mL, taking into account potential losses.

[0068] Table 1

[0069]

[0070] Example 2. Vacuum drying method for botulinum toxin

[0071] The final botulinum toxin stock solution from Example 1 was vacuum dried to produce the finished product. The final botulinum toxin stock solution from Example 1 was filled into vials, and half of the rubber stopper was inserted. The vials containing the final botulinum toxin stock solution were then placed in a freeze dryer.

[0072] Subsequently, drying preparation steps, a first drying, a second drying, and a third drying are performed, each step being carried out by varying the temperature and pressure. The temperature and pressure ranges for each step are as follows.

[0073] In the drying preparation step, the shelf temperature of the freeze dryer is maintained within the range of 5℃ to 30℃ for 0 to 10 minutes or more. Subsequently, drying begins within the range of shelf temperature of the freeze dryer (5℃ to 30℃) and minimum chamber pressure (0.01 mmHg to 2 mmHg).

[0074] The first drying process is divided into a maximum of 14 steps, with each step involving drying for 0 to 40 minutes within a chamber pressure of 0.01 mmHg to 2 mmHg and a shelf temperature of 5°C to 30°C (heating rate: minimum 0.1°C / min to 0.5°C / min).

[0075] The second drying process is divided into at least two steps, with each step involving drying for 0 to 80 minutes within a chamber pressure of 0.01 mmHg to 1 mmHg and a shelf temperature of 5°C to 20°C.

[0076] The final third drying was carried out in one step, and the final drying was completed by drying for at least 30 minutes at a chamber pressure of 0.01 mmHg to 0.5 mmHg and a shelf temperature range of 5°C to 30°C.

[0077] After drying, the solution is maintained at -0.3 bar to -0.7 bar using nitrogen (N2), and a rubber stopper is inserted. The vacuum is then released, the dried vials are removed from the freeze dryer shelf, sealed with aluminum caps, and stored under refrigeration. The finished product prepared according to Example 2 is as follows: Figure 2 As shown.

[0078] Example 2-1. Vacuum drying of botulinum toxin (1)

[0079] The final botulinum toxin stock solution from Example 1 was vacuum-dried to produce the finished product. The final botulinum toxin stock solution from Example 1 was filled into vials, with half of the rubber stopper inserted. The vials containing the final botulinum toxin stock solution were placed in a freeze dryer, and the shelf temperature was set to 5°C–10°C and maintained for 10 minutes for drying. Subsequently, the shelf temperature was maintained at 5°C–10°C, and the chamber pressure was set to 0.1 mmHg to begin drying.

[0080] The first drying process is carried out in 10 steps by changing the temperature and pressure, and is conducted according to the conditions shown in Table 2 below.

[0081] Table 2

[0082] step temperature heating rate pressure time 1 5℃ 0.5℃ / minute 0.1 mmHg 10 minutes 2 5℃ 0.3℃ / minute 0.5 mmHg 15 minutes 3 5℃ 0.3℃ / minute 0.7 mmHg 15 minutes 4 5℃ 0.4℃ / minute 0.7 mmHg 10 minutes 5 15℃ 0.4℃ / minute 0.1 mmHg 10 minutes 6 15℃ 0.4℃ / minute 0.2 mmHg 5 minutes 7 15℃ 0.8℃ / minute 0.2 mmHg 5 minutes 8 15℃ 0.8℃ / minute 0.3 mmHg 5 minutes 9 15℃ 0.8℃ / minute 0.3 mmHg 10 minutes 10 20℃ 0.3℃ / minute 0.4 mmHg 10 minutes

[0083] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.3 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes for the second drying process.

[0084] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 30°C, and drying for more than 40 minutes.

[0085] After drying, maintain the pressure at -0.3 bar to -0.7 bar using nitrogen (N2), insert rubber stoppers, release the vacuum, remove the dried vials from the freeze dryer shelf, seal them with aluminum caps, and store them under refrigeration.

[0086] Example 2-2. Vacuum drying of botulinum toxin (2)

[0087] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the first, second, and third drying processes were changed.

[0088] The first drying process is carried out in 13 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 3 below.

[0089] Table 3

[0090] step temperature heating rate pressure time 1 5℃ 0.5℃ / minute 0.1 mmHg 5 minutes 2 5℃ 0.3℃ / minute 1.5mmHg 15 minutes 3 5℃ 0.3℃ / minute 1.8 mmHg 10 minutes 4 5℃ 0.5℃ / minute 1mmHg 5 minutes 5 15℃ 0.5℃ / minute 0.8 mmHg 5 minutes 6 15℃ 0.5℃ / minute 0.7 mmHg 10 minutes 7 15℃ 0.5℃ / minute 0.3 mmHg 10 minutes 8 15℃ 0.5℃ / minute 0.4 mmHg 10 minutes 9 15℃ 0.5℃ / minute 0.5 mmHg 15 minutes 10 20℃ 0.3℃ / minute 0.5 mmHg 5 minutes 11 20℃ 0.3℃ / minute 0.4 mmHg 5 minutes 12 20℃ 0.3℃ / minute 0.3 mmHg 10 minutes 13 20℃ 0.3℃ / minute 0.3 mmHg 10 minutes

[0091] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 10°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.35 mmHg and the shelf temperature is set to 10°C and maintained for 25 minutes for the second drying process.

[0092] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 25°C, and drying for more than 40 minutes.

[0093] Examples 2-3. Vacuum drying of botulinum toxin (3)

[0094] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the first, second, and third drying processes were changed.

[0095] The first drying process is carried out in 14 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 4 below.

[0096] Table 4

[0097] step temperature heating rate pressure time 1 5℃ 0.5℃ / minute 0.1 mmHg 5 minutes 2 5℃ 0.4℃ / minute 0.5 mmHg 10 minutes 3 5℃ 0.4℃ / minute 1mmHg 10 minutes 4 5℃ 0.5℃ / minute 0.5 mmHg 10 minutes 5 15℃ 0.5℃ / minute 0.8 mmHg 5 minutes 6 15℃ 0.5℃ / minute 0.9 mmHg 5 minutes 7 15℃ 0.5℃ / minute 1mmHg 5 minutes 8 15℃ 0.5℃ / minute 0.3 mmHg 10 minutes 9 15℃ 0.5℃ / minute 0.4 mmHg 10 minutes 10 25℃ 0.3℃ / minute 0.5 mmHg 5 minutes 11 25℃ 0.3℃ / minute 0.2 mmHg 5 minutes 12 25℃ 0.3℃ / minute 0.3 mmHg 5 minutes 13 25℃ 0.3℃ / minute 0.4 mmHg 10 minutes 14 25℃ 0.3℃ / minute 0.5 mmHg 10 minutes

[0098] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 15°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.35 mmHg and the shelf temperature is set to 15°C and maintained for 30 minutes for the second drying process.

[0099] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 30°C, and drying for more than 40 minutes.

[0100] Examples 2-4. Vacuum drying of botulinum toxin (4)

[0101] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the first, second, and third drying processes were changed.

[0102] The first drying process is carried out in 10 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 5 below.

[0103] Table 5

[0104]

[0105]

[0106] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 15°C and maintained for 30 minutes; ii) the chamber pressure is set to 0.4 mmHg and the shelf temperature is set to 15°C and maintained for 30 minutes for the second drying process.

[0107] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 30°C, and drying for more than 40 minutes.

[0108] Examples 2-5. Vacuum drying of botulinum toxin (5)

[0109] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the drying preparation, the first drying, the second drying, and the third drying were changed.

[0110] Set the shelf temperature of the freeze dryer to 0℃~5℃ and maintain it for 10 minutes. Then, maintain the shelf temperature of the freeze dryer at 0℃~5℃, set the chamber pressure to 1.6mmHg, and begin drying.

[0111] The first drying process is carried out in 11 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 6 below.

[0112] Table 6

[0113] step temperature heating rate pressure time 1 5℃ 0.3℃ / minute 1.6 mmHg 5 minutes 2 5℃ 0.3℃ / minute 1.6 mmHg 5 minutes 3 5℃ 0.3℃ / minute 1.4 mmHg 10 minutes 4 5℃ 0.4℃ / minute 1.2 mmHg 10 minutes 5 15℃ 0.4℃ / minute 1.2 mmHg 5 minutes 6 15℃ 0.4℃ / minute 1.0 mmHg 5 minutes 7 15℃ 0.4℃ / minute 1.0 mmHg 10 minutes 8 15℃ 0.8℃ / minute 0.5 mmHg 15 minutes 9 15℃ 0.8℃ / minute 0.4 mmHg 5 minutes 10 20℃ 0.3℃ / minute 0.3 mmHg 15 minutes 11 20℃ 0.3℃ / minute 0.1 mmHg 20 minutes

[0114] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.3 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.4 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes for the second drying process.

[0115] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 30°C, and drying for more than 40 minutes.

[0116] Examples 2-6. Vacuum drying of botulinum toxin (6)

[0117] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the drying preparation, the first drying, the second drying, and the third drying were changed.

[0118] Set the shelf temperature of the freeze dryer to 0℃~5℃ and maintain it for 10 minutes. Then, maintain the shelf temperature of the freeze dryer at 0℃~5℃, set the chamber pressure to 1.6mmHg, and begin drying.

[0119] The first drying process is carried out in 14 steps by varying the temperature and pressure, and is conducted under the conditions shown in Table 7 below.

[0120] Table 7

[0121] step temperature heating rate pressure time 1 5℃ 0.4℃ / minute 1.6 mmHg 5 minutes 2 5℃ 0.4℃ / minute 1.0 mmHg 5 minutes 3 5℃ 0.5℃ / minute 0.8 mmHg 10 minutes 4 5℃ 0.5℃ / minute 0.5 mmHg 20 minutes 5 10℃ 0.4℃ / minute 1mmHg 5 minutes 6 10℃ 0.4℃ / minute 0.9 mmHg 5 minutes 7 10℃ 0.5℃ / minute 0.8 mmHg 5 minutes 8 10℃ 0.5℃ / minute 0.5 mmHg 10 minutes 9 15℃ 0.3℃ / minute 0.5 mmHg 5 minutes 10 15℃ 0.3℃ / minute 0.4 mmHg 5 minutes 11 15℃ 0.3℃ / minute 0.3 mmHg 10 minutes 12 15℃ 0.3℃ / minute 0.2 mmHg 10 minutes 13 20℃ 0.3℃ / minute 0.2 mmHg 5 minutes 14 20℃ 0.3℃ / minute 0.1 mmHg 10 minutes

[0122] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.35 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 20°C and maintained for 25 minutes for the second drying process.

[0123] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 30°C, and drying for more than 40 minutes.

[0124] Examples 2-7. Vacuum drying of botulinum toxin (7)

[0125] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the drying preparation, the first drying, the second drying, and the third drying were changed.

[0126] Set the shelf temperature of the freeze dryer to 0℃~5℃ and maintain it for 10 minutes. Then, maintain the shelf temperature of the freeze dryer at 0℃~5℃, set the chamber pressure to 1.6mmHg, and begin drying.

[0127] The first drying process is carried out in 10 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 8 below.

[0128] Table 8

[0129] step temperature heating rate pressure time 1 5℃ 0.5℃ / minute 1.6 mmHg 5 minutes 2 5℃ 0.3℃ / minute 1.5mmHg 5 minutes 3 5℃ 0.3℃ / minute 1mmHg 10 minutes 4 5℃ 0.3℃ / minute 0.9 mmHg 15 minutes 5 10℃ 0.5℃ / minute 0.8 mmHg 5 minutes 6 10℃ 0.5℃ / minute 0.7 mmHg 10 minutes 7 10℃ 0.5℃ / minute 0.6 mmHg 10 minutes 8 10℃ 0.5℃ / minute 0.5 mmHg 10 minutes 9 15℃ 0.5℃ / minute 0.4 mmHg 15 minutes 10 15℃ 0.5℃ / minute 0.1 mmHg 20 minutes

[0130] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.4 mmHg and the shelf temperature is set to 15°C and maintained for 25 minutes; ii) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 15°C and maintained for 25 minutes for the second drying process.

[0131] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 25°C, and drying for more than 40 minutes.

[0132] Examples 2-8. Vacuum drying of botulinum toxin (8)

[0133] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the drying preparation, the first drying, the second drying, and the third drying were changed.

[0134] Set the shelf temperature of the freeze dryer to 0℃~5℃ and maintain it for 10 minutes. Then, maintain the shelf temperature of the freeze dryer at 0℃~5℃, set the chamber pressure to 1.6mmHg, and begin drying.

[0135] The first drying process is carried out in 12 steps by changing the temperature and pressure, and is conducted under the conditions shown in Table 9 below.

[0136] Table 9

[0137] step temperature heating rate pressure time 1 5℃ 0.3℃ / minute 1.6 mmHg 5 minutes 2 5℃ 0.3℃ / minute 1.5mmHg 5 minutes 3 5℃ 0.3℃ / minute 1.0 mmHg 10 minutes 4 5℃ 0.3℃ / minute 1.0 mmHg 5 minutes 5 10℃ 0.3℃ / minute 0.9 mmHg 5 minutes 6 10℃ 0.5℃ / minute 0.8 mmHg 10 minutes 7 10℃ 0.5℃ / minute 0.5 mmHg 10 minutes 8 15℃ 0.8℃ / minute 0.5 mmHg 5 minutes 9 15℃ 0.8℃ / minute 0.4 mmHg 5 minutes 10 20℃ 0.3℃ / minute 0.3 mmHg 10 minutes 11 20℃ 0.3℃ / minute 0.2 mmHg 10 minutes 12 20℃ 0.3℃ / minute 0.1 mmHg 15 minutes

[0138] The second drying process is carried out in two steps: i) the chamber pressure is set to 0.4 mmHg and the shelf temperature is set to 15°C and maintained for 30 minutes; ii) the chamber pressure is set to 0.5 mmHg and the shelf temperature is set to 15°C and maintained for 30 minutes for the second drying process.

[0139] The third drying process is completed by setting the chamber pressure to 0.01 mmHg, the shelf temperature to 25°C, and drying for more than 40 minutes.

[0140] Examples 2-9. Vacuum drying of botulinum toxin (9)

[0141] The process was carried out in the same manner as in Example 2-1, but with the difference that the temperature and pressure of the drying preparation, the first drying, the second drying, and the third drying were changed.

[0142] Set the shelf temperature of the freeze dryer to 0℃~5℃ and maintain it for 10 minutes. Then, maintain the shelf temperature of the freeze dryer at 0℃~5℃, set the chamber pressure to 1.6mmHg, and begin drying.

[0143] The first drying process is carried out in 14 steps by varying the temperature and pressure, and is conducted under the conditions shown in Table 10 below.

[0144] Table 10

[0145] step temperature heating rate pressure time 1 10℃ 0.1℃ / minute 1.0 5 minutes 2 20℃ 0.2℃ / minute 0.9 mmHg 5 minutes 3 20℃ 0.2℃ / minute 0.8 mmHg 10 minutes 4 20℃ 0.2℃ / minute 0.1 mmHg 5 minutes 5 20℃ 0.2℃ / minute 0.2 mmHg 5 minutes 6 20℃ 0.2℃ / minute 0.3 mmHg 10 minutes 7 20℃ 0.2℃ / minute 0.4 mmHg 10 minutes 8 20℃ 0.5℃ / minute 0.1 mmHg 5 minutes 9 20℃ 0.5℃ / minute 0.15mmHg 5 minutes 10 20℃ 0.5℃ / minute 0.2 mmHg 10 minutes 11 25℃ 0.5℃ / minute 0.1 mmHg 10 minutes 12 25℃ 0.6℃ / minute 0.2 mmHg 10 minutes 13 25℃ 0.7℃ / minute 0.2 mmHg 10 minutes 14 25℃ 0.8℃ / minute 0.2 mmHg 10 minutes

[0146] The second drying process is carried out in two steps: i) setting the chamber pressure to 0.2 mmHg and the shelf temperature to 25°C and maintaining this temperature for 30 minutes; ii) setting the chamber pressure to 0.2 mmHg and the shelf temperature to 25°C and maintaining this temperature for 30 minutes to perform the second drying process.

[0147] The third drying process is completed by setting the chamber pressure to 0.05 mmHg, the shelf temperature to 25°C, and drying for more than 40 minutes.

[0148] Comparative Example 1. Freeze-drying method for botulinum toxin

[0149] The final botulinum toxin stock solution from Example 1 was freeze-dried to produce the finished product. The final botulinum toxin stock solution from Example 1 was filled into vials, and half of the rubber stopper was inserted. The shelf temperature of the freeze dryer was set to 0°C–5°C. After placing the vials containing the final botulinum toxin stock solution into the freeze dryer, the shelf temperature was set to approximately -45°C and maintained for approximately 30 minutes. Subsequently, the shelf temperature of the freeze dryer was maintained at approximately -45°C, and the chamber pressure was set to approximately 0.1 mmHg to begin drying.

[0150] The first drying process involves setting the chamber pressure to approximately 10 mmHg and the shelf temperature to approximately -45°C (heating rate: 1°C / 4 minutes) and maintaining this temperature for approximately 60 minutes. The second drying process involves setting the chamber pressure to approximately 10 mmHg and the shelf temperature to approximately -10°C (heating rate: 1°C / 4 minutes) and maintaining this temperature for approximately 30 minutes. Finally, the drying process is completed by setting the chamber pressure to approximately 10 mmHg and the shelf temperature to approximately 30°C (heating rate: minimum 1°C / 4 minutes) and performing the drying for at least 10 hours.

[0151] After drying, rubber stoppers are inserted, the vacuum is released, the dried vials are removed from the freeze dryer shelf, sealed with aluminum caps, and stored under refrigeration. The prepared product appears as follows. Figure 3 As shown.

[0152] Experimental Example 1. Animal Titer Test

[0153] 1-1. Determination Method

[0154] The titer recovery rate determination method is as follows: Calculate the titers of the final stock solution, the vacuum-dried product, and the freeze-dried product. Taking the titer of the final stock solution as 100%, compare the titers of the vacuum-dried and freeze-dried products with the final stock solution to express the recovery rate as a percentage (%). In the case of the freeze-dried product titer, it was confirmed that the recovery rate of the freeze-dried product titer (average) relative to the final stock solution titer (average) is approximately 72%, and the recovery rate of the vacuum-dried product titer (average) relative to the final stock solution titer (average) is approximately 93%.

[0155] The recovery rate of the titer of the freeze-dried product (%) = (average titer of the freeze-dried product / average titer of the final original solution) × 100

[0156] Vacuum-dried product titer recovery rate (%) = (Average titer of vacuum-dried product / Average titer of final original solution) × 100

[0157] 1-2. Experimental Results

[0158] Three animal titer tests were conducted on the final stock solution of Example 1, the vacuum-dried product of Example 2, and the freeze-dried product of Comparative Example 1. The results are shown in Table 11.

[0159] Table 11

[0160]

[0161]

[0162] As shown in Table 11, the final animal titer results of the stock solution showed a minimum of 113 units / 0.1 ml, a maximum of 132 units / 0.1 ml, and an average of 122 units / 0.1 ml. Considering the preparation deviation and the experimental deviation, the preparation target was set at 100 units / 0.1 mL ± 10% (100 units / 0.1 mL ~ 120 units / 0.1 mL), and it was confirmed that there were no abnormalities in the preparation process.

[0163] Animal titer tests on the freeze-dried product showed a minimum of 83 units / vial, a maximum of 94 units / vial, and an average of 86 units / vial, with a recovery rate of only 72% relative to the final stock solution. Animal titer tests on the vacuum-dried product showed a minimum of 108 units / vial, a maximum of 123 units / vial, and an average of 114 units / vial, confirming a recovery rate of 93% relative to the final stock solution.

[0164] Therefore, it was confirmed that the titer recovery rate of the product prepared by vacuum drying was approximately 21% higher than that prepared by freeze drying, thus confirming less loss of botulinum toxin protein. Furthermore, as... Figure 2 As shown, the finished product is thin and hard, highly impact-resistant, and highly stable. Furthermore, compared to freeze drying, vacuum drying takes significantly less time, thus offering an advantage in efficiency.

[0165] Industrial availability

[0166] This invention relates to the preparation of botulinum toxin, which can be effectively applied in the fields of medicine and cosmetics.

Claims

1. A vacuum drying method for botulinum toxin, characterized in that, include: Step a) The solution containing botulinum toxin is dried for the first time under the conditions of temperature of 5℃~30℃ and pressure of 0.01mmHg~2mmHg; Step b), the first dried product obtained in step a) is subjected to a second drying process at a temperature of 5℃ to 20℃ and a pressure of 0.01 mmHg to 1 mmHg; and Step c), the second dried product obtained in step b) is subjected to a third drying process at a temperature of 5℃ to 30℃ and a pressure of 0.01 mmHg to 0.5 mmHg. In step a) above, the first drying is carried out while the temperature is increased at an initial temperature above 5°C at a rate of 0.1°C / min to 2°C / min. The method does not include the step of freeze-drying botulinum toxin.

2. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, In step a) above, the upper limit of pressure is 1.7 mmHg.

3. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, In step b) above, the upper limit of pressure is 0.8 mmHg.

4. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, In step c) above, the upper limit of pressure is 0.4 mmHg.

5. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, In step c) above, the upper limit of pressure is 0.2 mmHg.

6. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, In steps a), b), and c) above, the drying time is 30 minutes to 120 minutes, respectively.

7. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The total drying time for the vacuum drying method of the above-mentioned botulinum toxin is less than 6 hours.

8. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The total drying time for the above-mentioned vacuum drying method for botulinum toxin is less than 5 hours.

9. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The total drying time for the above-mentioned vacuum drying method for botulinum toxin is less than 4 hours.

10. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The titer recovery rate of the above-mentioned vacuum drying method for botulinum toxin is over 80%.

11. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The titer recovery rate of the above-mentioned vacuum drying method for botulinum toxin is over 85%.

12. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The titer recovery rate of the above-mentioned vacuum drying method for botulinum toxin is over 90%.

13. The vacuum drying method for botulinum toxin according to claim 1, characterized in that, The botulinum toxin in step a) above is selected from one or more of the group consisting 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, botulinum toxin type G, and serotype botulinum toxin.

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