Methods of preparing toxins

Abstract

The present invention relates to a culture medium composition for culturing Clostridium botulinum and a method for preparing botulinum toxin using the culture medium composition, wherein the culture medium composition comprises: potato peptone, yeast extract and glucose. According to the present invention, the culture medium is a culture medium excluding animal-derived products and major allergens, and induces a strain producing botulinum toxin to reach a maximum growth level in a short time, thereby having the effect of shortening the production time of the botulinum toxin production process and reducing the unit production cost.

Description

Background of the Invention

[0001] Field of the Invention

[0002] The present invention relates to a culture medium composition for culturing Clostridium botulinum and a method for preparing botulinum toxin using the culture medium composition, and more specifically, to a culture medium composition for culturing Clostridium botulinum, the culture medium composition comprising potato peptone, yeast extract and glucose, and the culture medium composition can effectively shorten the culture time of Clostridium botulinum while excluding animal-derived products and major allergens; and a method for preparing botulinum toxin using the culture medium composition.

[0003] Related technologies

[0004] Botulinum toxin is a neurotoxin produced by bacteria such as Clostridium butyricum, Clostridium baraffi and Clostridium botulinum. Botulinum toxin blocks neuromuscular conduction and causes neuroparalysis in humans and animals. In particular, type A botulinum toxin is known to be highly lethal to humans. In addition to type A botulinum toxin, six other botulinum toxins, type B, type C1, type D, type E, type F, type G and type H, have been identified. Each type of botulinum toxin can be distinguished by a corresponding type-specific antibody, and the severity of the paralysis caused by different types of botulinum toxins and the types of animals affected by them are different.

[0005] The molecular weight of the protein molecule of botulinum toxin is about 150 kD, including a light chain of about 50 kD and a heavy chain of about 100 kD conjugated to the light chain. However, the botulinum toxin released from Clostridium botulinum is released in the form of a complex of 150 kD toxin protein and at least one non-toxin protein. For example, botulinum toxin is released in the form of a complex of 900 kD, 500 kD and 300 kD.

[0006] Botulinum toxins can be very lethal to humans, but have recently been developed to treat a variety of conditions, including neuromuscular disorders characterized by skeletal muscle hyperactivity. is the trademark for botulinum toxin A commercially developed by Allergan for the relief or treatment of blepharospasm, strabismus, cervical dystonia and glabellar (facial) wrinkles, and is currently being investigated for the development of applications for other serotypes and their clinical utilization.

[0007] Botulinum toxin for clinical use is generally isolated from cell culture. Traditionally, botulinum toxin is mainly isolated from animal-derived products through culture, fermentation and purification processes. However, when animal-derived products are used to produce botulinum toxin, there is a concern that when botulinum toxin is administered to a patient, a variety of pathogens or infectious substances from animals may also be administered to the patient. For example, a produced botulinum toxin composition may contain prions. Prions are disease infectious agents that are completely different from bacteria, viruses, fungi and parasites. Animals (including humans) infected with prions experience perforation of the brain like a sponge and death of nerve cells, resulting in corresponding loss of brain function. Prions can generate abnormal conformational isoforms from the same nucleic acid sequence as that producing normal proteins, and infectivity exists during the "recruitment reaction", and normal isomers become prion protein isoforms in the post-translational stage. Normal intrinsic cell proteins induce misfolding and pathogenic prion virus structures. Creutzfeldt-Jakob disease is a rare neurodegenerative disease of human transmissible spongiform encephalopathy, and the infectious agent is an abnormal isoform of prion protein. Subjects with Creutzfeldt-Jakob disease can deteriorate from health status to akinetic mutism within 6 months. Therefore, the administration of pharmaceutical compositions containing biological agents obtained using animal-derived products (such as botulinum toxin) has the risk of causing prion-mediated diseases (such as Creutzfeldt-Jakob disease).

[0008] In order to remove this risk, for example, attempts have been made to exclude animal-derived components from the culture medium during the production of botulinum toxin. As an illustrative example, Allergan has devised a method of fermenting in a culture medium containing soybeans as a plant-derived component, in which no animal-derived components are used (Korean Patent Publication No. 10-2006-0102330), but this method has the following problem, namely, it requires a long time to culture the strain to produce a sufficient amount of botulinum toxin.

[0009] Food allergies are abnormal responses to digested food caused by the immune system. Food allergies often involve symptoms that cause severe discomfort in daily life, such as hives, angioedema, atopic dermatitis, and can lead to anaphylaxis, a severe and dangerous allergic reaction that can be life-threatening. The eight major causes of food allergies announced by the FDA are milk, eggs, fish, crustaceans, tree nuts, peanuts, wheat, and soy, which account for 85% to 90% of the total causes of food allergies.

[0010] Therefore, through extensive efforts to develop a component capable of effectively culturing a strain producing botulinum toxin while replacing a conventional culture medium containing animal-derived components and excluding possible allergens, the present inventors have found that when Clostridium butyricum is cultured in a culture medium composition containing potato peptone, yeast extract and glucose, the culture speed can be significantly improved, thereby allowing Clostridium butyricum to reach a maximum growth level in a very short time. Based on this finding, the present invention has been completed. Summary of the invention

[0011] Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for preparing botulinum toxin, which method prepares botulinum toxin using a culture medium composition that can improve the growth rate of a strain producing botulinum toxin compared to a conventional culture medium composition, as a composition excluding animal-derived products and major allergens.

[0012] According to one aspect of the present invention, the above and other objects can be achieved by providing a method for preparing botulinum toxin, the method comprising: (a) producing botulinum toxin by culturing Clostridium botulinum in a culture medium composition for culturing Clostridium botulinum, the culture medium composition comprising potato peptone, yeast extract and glucose; and (b) recovering the botulinum toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0015] Figure 1 The normal distribution analysis results of the factor experiments based on 8 concentration ranges of various ingredients according to the present invention are shown;

[0016] Figure 2 The results of the Pareto chart analysis of the factor experiment results based on 8 concentration ranges of various ingredients according to the present invention are shown;

[0017] Figure 3 The analysis results of the model suitability of various components according to the present invention are shown based on the results of factor experiments, central point experiments and axial point experiments;

[0018] Figure 4 shows surface analysis results based on the results of factor experiments, center point experiments, and axial point experiments conducted on various components according to the present invention;

[0019] Figure 5 The results of the optimal ratio determined based on the surface analysis results of various components according to the present invention are shown;

[0020] Figure 6 showing the results of determination of absorbance according to the toxin level and strain growth with variable glucose concentration in the optimal ratio according to the present invention; and

[0021] Figure 7 Shown are the results of absorbance assays in terms of toxin levels and strain growth over time when Clostridium botulinum was cultured in the optimized medium composition of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the invention relates. Generally speaking, the nomenclature used herein is well known and commonly used in the art.

[0024] In the present invention, it was determined that when the Clostridium botulinum strain was cultured in a medium containing potato peptone, yeast extract and glucose (the medium does not contain animal-derived components and allergens), the strain reached the highest growth level in a very short time and the culture speed was significantly improved.

[0025] Therefore, in one aspect, the present invention relates to a culture medium composition for culturing Clostridium botulinum, the culture medium composition comprising potato peptone, yeast extract and glucose.

[0026] The botulinum toxin producing strain used in the present invention can be botulinum toxin or its variant, and the most preferred is type A botulinum toxin, NCTC13319, but is not limited to the above strains. For those skilled in the art, it is obvious that any strain capable of producing botulinum toxin can be used.

[0027] The present invention is characterized in that the culture medium composition comprises 2 to 5% (w / v) potato peptone, 0.5 to 2% (w / v) yeast extract and 0.75 to 1.5% (w / v) glucose. Most preferably, the culture medium composition comprises 3% (w / v) potato peptone, 1% (w / v) yeast extract and 1% (w / v) glucose, but is not limited thereto.

[0028] At the same time, in the present invention, it was found that when the culture medium composition is used to culture the botulinum strain, the culture speed is increased during the production of botulinum toxin, and the recovery time can therefore be significantly shortened. That is, in the culture of botulinum using soy peptone, botulinum shows the maximum growth level after 24 hours. On the other hand, the botulinum according to the present invention shows the maximum growth level in about 14 to 18 hours. Regarding the recovery time of the toxin, in the case of conventional botulinum culture, the maximum toxin yield is obtained after at least 48 hours of culture, while in the present invention, the maximum toxin yield is obtained within 48 hours of culture, that is, at about 37 hours of culture (see Figure 7 ). The above-mentioned effects are considered to be obtained when potato peptone is used as peptone.

[0029] Therefore, according to another aspect of the present invention, the present invention relates to a method for preparing a botulinum toxin, the method comprising: (a) producing a botulinum toxin by culturing Clostridium botulinum in a culture medium composition for culturing Clostridium botulinum, the culture medium composition comprising potato peptone, a yeast extract and glucose; and (b) recovering the botulinum toxin.

[0030] In the present invention, the botulinum toxin may be type A botulinum toxin, preferably type A botulinum toxin, NCTC13319, but is not limited thereto.

[0031] In the present invention, the medium composition used for culturing does not include animal-derived products and allergens.

[0032] In the present invention, the medium composition includes 2 to 5% (w / v) potato peptone, 0.5 to 2% (w / v) yeast extract and 0.75 to 1.5% (w / v) glucose.

[0033] When the toxin production in a medium composition containing 3% (w / v) potato peptone, 1% (w / v) yeast extract and 1% (w / v) glucose (which is the optimal mode of the composition ratio of the components of the present invention) is assumed to be 100%, the combination ratio of the plurality of components as described above is the combination ratio of the components for obtaining a 90% toxin production level, which is obtained by a surface analysis method based on the optimization concentration setting program of the Minitab program. That is, when the medium composition contains 2 to 5% (w / v) potato peptone, 0.5 to 2% (w / v) yeast extract and 0.75 to 1.5% (w / v) glucose, a toxin production level corresponding to 90% of the toxin production level in the case of a medium composition containing 3% (w / v) potato peptone, 1% (w / v) yeast extract and 1% (w / v) glucose can be obtained.

[0034] In the present invention, the potato peptone may be present in an amount of 3% (w / v), the yeast extract may be present in an amount of 1% (w / v), and the glucose may be present in an amount of 1% (w / v), but are not limited thereto.

[0035] In the present invention, the botulinum toxin is recovered within 48 hours after the start of the culturing in step (b). Preferably, the botulinum toxin is recovered within 40 hours after the start of the culturing in step (b).

[0036] In the present invention, the term "excluding animal-derived ingredients" means substantially excluding animal-derived ingredients or substantially excluding animal proteins, and in particular refers to excluding or substantially excluding blood-derived, blood-pooled and other animal-derived products or compounds. The term "animal" refers to mammals such as humans), birds, reptiles, fish, insects, spiders or other types of animals. "Animal" does not include microorganisms, such as bacteria. Therefore, a culture medium or method that does not include animal products or a culture medium or method that does not substantially include animal products within the scope of the present invention may include botulinum toxin or botulinum toxin. For example, a method that does not include animal products or a method that does not substantially include animal products refers to a method that does not substantially, substantially or completely include animal-derived proteins, such as immunoglobulins, meat digestive juices, meat by-products and milk or dairy products or decomposition products. Therefore, multiple examples of methods that do not include animal products include methods that do not include meat and dairy products or meat and dairy by-products (such as bacterial culture methods or bacterial fermentation methods).

[0037] As used herein, the term "botulinum toxin" refers not only to neurotoxins produced by Clostridium botulinum, but also to botulinum toxin (or light chain or heavy chain) produced by recombinant non-botulinum strains. As used herein, the term "botulinum toxin" refers to botulinum toxin subtypes A, B, C, D, E, F, G and H (Weller C (October 15, 2013), "New Botulinum Toxin Deemed Deadliest Substance Ever: Sniffing 13-Billionths of a Gram Can Kill", Medical Daily) and G. As used herein, botulinum toxin also includes: botulinum toxin complexes (i.e., 300, 600 and 900 kDa complexes) and pure botulinum toxin (i.e., about 150 kDa). The term "pure botulinum toxin" is defined as a botulinum toxin separated or substantially separated from other proteins, including proteins that form the botulinum toxin complex. Pure botulinum toxin may have a purity of 95% or greater, preferably 99% or greater.

[0038] The present invention provides a culture medium comprising at least reduced levels of animal or dairy by-products, being substantially free of animal or dairy by-products. The term "animal or dairy by-products" refers to a compound or composition of compounds produced in vivo or in vitro in animal (excluding bacterial) cells or by animal (excluding bacterial) cells. Preferred non-animal derived culture medium components such as proteins, amino acids and nitrogen include plants, microorganisms (such as yeast) and synthetic compounds.

[0039] Culture media according to the present invention include, but are not limited to, a culture medium for fermenting small or large quantities of Clostridium botulinum, a culture medium for growing and culturing Clostridium botulinum for inoculation into seed (primary) culture media and fermentation (secondary) culture media, and a culture medium for long-term storage of Clostridium botulinum cultures (e.g., stock cultures).

[0040] As a particularly preferred embodiment of the present invention, the culture medium for growing Clostridium botulinum and producing botulinum toxin may include a potato-derived component, preferably potato peptone, thereby replacing the animal-derived component.

[0041] The present invention provides a method for growing Clostridium botulinum, which can maximize the production of botulinum toxin in the shortest time using a culture medium that is substantially free of animal-derived components. Clostridium botulinum can be grown using a culture medium composition containing potato peptone instead of using animal-derived components.

[0042] In a preferred embodiment of the present invention, the growth of Clostridium botulinum is carried out in two steps (i.e., seed growth and fermentation). Preferably, these two steps are carried out in an anaerobic environment. Seed growth is generally used to "expand" the microbial content in the stored culture. The purpose of seed growth is to increase the microbial content that can be used for fermentation. In addition, the growth of seeds rejuvenates relatively dormant microorganisms in the stored culture and grows into an actively growing culture. In addition, compared to the stored culture, the volume and number of surviving microorganisms used to inoculate the fermentation medium can be more accurately controlled in the more actively growing culture. Therefore, the growth of the seed culture inoculated into the fermentation medium is preferred. In addition, multiple consecutive steps including growth in the seed culture medium can be used to expand the amount of Clostridium botulinum inoculated into the fermentation medium. The growth of Clostridium botulinum in the fermentation step can also be carried out by direct inoculation from the stored culture medium.

[0043] In the fermentation step, part or all of the seed culture medium containing Clostridium botulinum from the seed-grown product can be used to inoculate the fermentation medium. Preferably, about 1 to 10% of the seed culture medium containing Clostridium botulinum from the seed-grown product is used to inoculate the fermentation medium. Fermentation is used to produce the maximum amount of microorganisms in a large-scale anaerobic environment.

[0044] The botulinum toxin contained in the culture of the strain cultured by the method of the present invention can be separated and purified using a protein purification method known to those skilled in the art of protein purification.

[0045] The growth of Clostridium botulinum can be carried out in one or more steps. Preferably, the growth is carried out in two steps. In the first step, seed growth, Clostridium botulinum is suspended in a culture medium composition according to the present invention and cultured at 34±1°C under anaerobic conditions for 10 to 24 hours. Preferably, the seeds are grown for about 14 hours. Preferably, the growth in the seed culture medium at any step does not result in cell autolysis before the final growth product in the seed culture medium is inoculated into the fermentation medium.

[0046] Subsequently, in order to further grow Clostridium botulinum and recover the botulinum toxin, the second step, fermentation, is carried out by inoculating the medium composition of the present invention with part or all of the medium grown from the seeds. After inoculation, Clostridium botulinum is cultured at 34±1°C under anaerobic conditions for about 4 days, and the growth of Clostridium botulinum is monitored by measuring the optical density (OD) of the medium. Preferably, in the fermentation step using the medium composition according to the present invention, the maximum growth level is obtained after about 14 hours and 18 hours, the cells are lysed, and the OD value decreases, thereby recovering the botulinum toxin from Clostridium botulinum within 48 hours, more preferably within 40 hours after incubation in the fermentation step.

[0047] In a preferred embodiment of the present invention, when the culture of Clostridium botulinum is stored at 4°C for long-term storage of Clostridium botulinum and subsequently inoculated with the seed medium, preferably, Clostridium botulinum is stored in a storage medium (3% potato peptone, 1% yeast extract, 1% glucose and 25% glycerol) so that the culture medium is substantially free of animal by-products throughout the production process of botulinum toxin. Example

[0048] Hereinafter, the present invention will be described in more detail with reference to a plurality of embodiments. However, it is obvious to those skilled in the art that these embodiments are provided only to illustrate the present invention and should not be interpreted as limiting the scope of the present invention.

[0049] Example 1: Sample preparation and bacterial culture

[0050] 1-1. Sample preparation

[0051] The botulinum strain used in the present invention is Clostridium botulinum type A, NCTC13319, and the samples used to prepare the culture medium of the experimental group and the control group are as follows: plant peptone (BD, 211906), tryptophan (BD, 211705), yeast extract (BD, 212750), potato peptone E210 (Organotechnie, 19425), pea peptone A482 (Organotechnie, AI275), plant peptone E1 (Organotechnie, 19025), selected plant ketone UF (BD, 210931), peptone from soybean (Sigma, 70 178), Hy-Soy (Kerry, 5X59022), Hy-peptone (Kerry, 5X01111), Soy peptone A2SC (Organotechnie, 19649), Soy peptone A3SC (Organotechnie, 19685), Soy peptone E110 (Organotechnie, 19885), Soy peptone K200 (Organotechnie, 19749), Glucose (Sigma, G5767), Sodium thioglycolate (Sigma, T0632), Pure water (ultrapure water or water quality equal to or higher than ultrapure water).

[0052] 1-2. Bacterial culture

[0053] Thaw a vial containing the botulinum strain stored in a cryogenic freezer in a 37°C incubator for 30 minutes. Homogenize the botulinum strain 3 to 5 times in a BSC, inoculate 0.1 ml of the homogenized botulinum strain into the culture medium, and then culture it in an anaerobic incubator at 34 ± 1°C. After culture, measure the OD using a spectrophotometer. 600 value.

[0054] 1-3. Toxin level measurement

[0055] The toxin level was determined by the test method of Botulinum neurotoxin type A DuoSet ELISA (R&D Systems) controlled according to the manufacturer's instructions. 100 μL of capture antibody (prepared by dilution in PBS) was inoculated into each well of a 96-well microplate and the coating lasted for 16 hours or more, and each well was washed with wash buffer for a total of three times. Each well was blocked with reagent diluent and then rinsed with wash buffer for a total of 3 washes.

[0056] After the culture is completed, the culture solution is sterilized using a 0.2 μm injection filter, diluted, and 100 microliters of the culture solution is added to each well and allowed to react for 2 hours, and then each well is washed with a wash buffer, and repeated 3 times in total. 100 microliters of the detection antibody dilution is added to each well and allowed to react for 2 hours, and then each well is washed with a wash buffer, and repeated 3 times in total. 100 microliters of streptomycin-HRP dilution is added to each well and allowed to react for 20 minutes, and each well is washed with a wash buffer, and repeated 3 times in total. 100 microliters of substrate solution is added to each well and allowed to react for 20 minutes, 50 microliters of reaction stop solution is added to each well, and shaken appropriately to terminate the enzyme reaction. Use an ELISA reader to measure the 450nm absorbance in each well.

[0057] Example 2. Selection of peptone to replace TPYG medium

[0058] TPYG medium containing casein peptone, soy peptone, yeast extract, glucose and sodium thioglycolate is a conventional medium frequently used for producing botulinum toxin, and attempts have been made to replace the conventional medium with PYG medium including peptone, yeast extract, glucose and sodium thioglycolate.

[0059] As for peptone, various candidates including plant peptone were selected, and mainly a plant-derived medium having a similar toxin production amount as that of the TYPG medium was selected from soybean-derived medium and other plant-derived medium.

[0060] 2-1. Comparative Example: Preparation of TPYG Medium

[0061] 2 g of plant peptone, 1 g of tryptophan, 1 g of yeast extract and 0.1 g of sodium thioglycolate were weighed using a weighing dish, 50 ml of pure water was added to a 250 ml beaker, the weighed medium components were added to the beaker, and stirred until the components were completely dissolved. The pH was adjusted to 7.2 with 1N sodium hydroxide, the medium in the beaker was transferred to a 100 ml mass cylinder, purified water was added to the mass cylinder to adjust the final liquid level to 95 ml, and the prepared medium and magnetic bar were transferred to a 100 ml glass bottle and covered with a lid.

[0062] 1 g of glucose was weighed using a weighing dish, and the weighed glucose was placed in a 15 ml conical tube containing about 3 ml of pure water and vortexed to fully dissolve the glucose, and then pure water was added to the conical tube to adjust the final solution volume to 5 ml.

[0063] The lid of the glass bottle was slightly opened, and the glass bottle was wrapped with aluminum foil and sterilized in an autoclave at 121° C. for 20 minutes. The sterilized medium and glucose were fully cooled in a BSC, and then the glucose was transferred to the medium and mixed.

[0064] 2-2. Experimental Example: Preparation of PYG medium

[0065] The 12 medium components shown in Table 1 were weighed and dissolved in 50 ml of pure water, the pH was adjusted to 7.2 using 1N sodium hydroxide, pure water was added to adjust to the final liquid level as shown in Table 1, the prepared medium and magnetic bar were transferred to a 100 ml glass bottle, and the lid was closed.

[0066] Table 1

[0067]

[0068] 13 g of glucose was weighed using a weighing dish, and the weighed glucose was dissolved in 65 ml of pure water. The prepared glucose was transferred to a 100 ml glass bottle and covered with a lid.

[0069] The prepared culture medium and glucose were sterilized at 121° C. for 20 minutes, the sterilized culture medium and glucose were sufficiently cooled in a BSC, and 5 ml of the glucose solution was transferred to each culture medium and mixed.

[0070] 2-3. Selection of peptone

[0071] The botulinum strain was cultured in the culture medium prepared in Example 2-1 or Example 2-2 in the same manner as in Example 1-2, and the OD was measured after the culture was completed. 600 The toxin production level was determined using the same ELISA method as in Example 1-3.

[0072] Table 2

[0073]

[0074]

[0075] Results: OD after culture completion 600 The values ​​and toxin production levels are shown in Table 2. Compared with TPYG medium, the three media (PYG1, PYG2 and PYG5) showed similar toxin levels and maximum strain solubility, so OD 600The value was less than 1. These results were verified again by repeated tests. As can be seen from Table 3, PYG1, PYG2 and PYG5 also showed similar toxin production levels as TPYG in repeated tests. Among them, PYG2 medium that does not include animal components and major allergens was used to obtain the best medium composition.

[0076] Table 3

[0077]

[0078]

[0079] Example 3: Optimization of culture medium composition

[0080] To find the optimal concentrations of potato peptone, yeast extract, and glucose determined by the media screening assay, concentration range tests were performed by DoE, and the optimal composition was determined based on the results of factorial, central, and axial experiments.

[0081] 3-1. Factor Experiment

[0082] A factorial experiment was conducted using a medium composition including components in 8 concentration ranges as shown in Table 4. Using the components contained in Table 4, a medium was prepared in the same manner as in Example 2-2, and the botulinum strain was cultured in the prepared medium in the same manner as in Example 1-2, and the OD was measured at the end of the culture. 600 Values, and the toxin production levels were determined by the same ELISA as in Examples 1-3.

[0083] Table 4

[0084]

[0085] Therefore, OD was determined as shown in Table 5. 600 On this basis, a normal distribution analysis including first-order and second-order interactions but not including third-order interactions showed that among the first-order and second-order interactions of peptone, yeast, and glucose, except for the peptone*glucose interaction, the remaining factors had significant effects on the toxin production level ( Figure 1 ), the Pareto chart analysis also shows the same results as the normal distribution analysis ( Figure 2 ).

[0086] Table 5

[0087]

[0088] 3-2. Center point experiment

[0089] 24.5 g of potato peptone E210, 8.75 g of yeast extract, and 0.7 g of sodium thioglycolate of the culture medium for the center point experiment were weighed using a weighing dish, 550 ml of pure water was placed in a 1-liter beaker, and the weighed ingredients were stirred in the beaker until they were completely dissolved. The pH was adjusted to 7.2 using 1N sodium hydroxide, and the culture medium in the beaker was transferred to a 1-liter mass cylinder, and pure water was added to the mass cylinder to adjust the final liquid level to 656.25 ml, and then the final liquid was transferred to a 1-liter beaker and stirred for about 5 minutes. 93.75 ml of the culture medium was transferred to each of a total of 6 100-ml glass bottles, a magnetic bar was injected into the glass bottle, and the lid was covered, and each culture medium was designated as any one of the center points 1 to 6.

[0090] 8 g of glucose was weighed using a weighing dish, and the weighed glucose was completely dissolved in 40 ml of pure water.

[0091] The prepared culture medium and glucose were sterilized at 121°C for 20 minutes, the sterilized culture medium and glucose were fully cooled in a BSC, and 6.25 ml of the glucose solution was transferred to each culture medium and mixed.

[0092] In the medium thus prepared, the botulinum strain was cultured in the same manner as in Example 1-2, and the OD was measured after the culture was completed. 600 The toxin production levels were determined by ELISA in the same manner as in Examples 1-3.

[0093] Table 6

[0094]

[0095]

[0096] The center point experiment of one concentration was repeated 6 times, and the results are shown in Table 6. When judging the presence or absence of the curve based on the results of the center point experiment, the P value is 0.001, and the P value is less than 0.05, which is the criterion for judging significance. This means that the curve exists, and additional tests are performed for surface analysis.

[0097] Table 7

[0098]

[0099] 3-3. Axial point experiment

[0100] As shown in Table 8, the six medium components for the axial point experiment were weighed and dissolved in 50 ml of pure water, the pH was adjusted to 7.2 using 1N sodium hydroxide, and pure water was added to adjust the final liquid level as shown in Table 8. Each prepared medium and magnetic bar were transferred to a 100 ml glass bottle and capped.

[0101] Table 8

[0102]

[0103] 8 g of glucose was weighed using a weighing dish and the weighed glucose was completely dissolved in 40 ml of pure water. The prepared culture medium and glucose were sterilized in an autoclave at 121° C. for 20 minutes, the sterilized culture medium and glucose were fully cooled in a BSC, and then the glucose solution was added to each culture medium in the amount shown in Table 8.

[0104] In the medium thus prepared, the botulinum strain was cultured in the same manner as in Example 1-2, and the OD was measured at the end of the culture. 600 The values ​​were calculated and the toxin production levels were determined by ELSA in the same manner as in Example 1-3. Thus, the axial point values ​​were determined as shown in Table 9.

[0105] Table 9

[0106]

[0107]

[0108] 3-4. Optimal concentration analysis via surface analysis

[0109] Surface analysis was performed based on the results of factor experiments, center point experiments, and axial point experiments. In the model suitability analysis, the normal probability plot, histogram, residual value to fitted value relationship plot, and residual value to class relationship plot were analyzed. Among them, the normal probability plot was determined as a straight line, the histogram appeared in the form of normal distribution, and the residual value to fitted value relationship plot and residual value to class relationship plot had no pattern. On this basis, it was determined that the selected model was appropriate ( Figure 3 ).

[0110] The results were analyzed using surface analysis to determine the composition of the medium. Figure 4 As shown, based on Figure 5 The optimal ratio was calculated based on the results shown, and the optimal concentrations obtained from the optimal ratio are summarized in Table 10.

[0111] Table 10

[0112] Ingredient name Potato Peptone Yeast Extract glucose Optimum concentration (%) 3％ 1％ 1.5％

[0113] 3-5. Determine the optimal composition ratio

[0114] The optimal concentration is set according to the toxin production level by surface analysis, and the ultimate goal is to make the composition have the highest toxin production level and OD after culture. 600 Therefore, by comparing the glucose concentration (which is considered to have an influence on the toxin production level and OD 600 The value (with the greatest impact) was changed to 0.75, 1, 1, 1.25, 1.5, and 1.5% to obtain the best medium composition, and the results are shown in Tables 11 and Figure 6 The optimal medium composition was determined to have 3% potato peptone, 1% yeast extract and 1% glucose.

[0115] Table 11

[0116]

[0117] Example 4: Strain culture experiment based on the optimal culture composition

[0118] The vials containing the botulinum strain stored in the cryogenic freezer were thawed in an incubator at 37°C for 30 minutes. The botulinum strain was homogenized 3 to 5 times in a BSC, and 1 ml of the homogenized botulinum strain was inoculated into 100 ml of PYG medium in a 2-liter cell bag and cultured in an incubator (WAVE25) at 34±1°C. 100 ml of the strain culture was inoculated into 5L of PYG medium between 10 and 24 hours before the strain autolyzed. Then, the absorbance at a wavelength of 600nm and the toxin level were measured over time. Only the culture solution was recovered from each sample using a 0.2 micron antibacterial filter, and then the toxin level was measured in the same manner as in Example 1-3.

[0119] When the above medium composition is used for culturing, the results of measuring the changes in OD value and toxin production level over time show that the medium composition according to the present invention can increase the OD value of the strain culture to 600 The value reaches a maximum value in about 14 to 15 hours, so the culture time can be significantly shortened compared with the conventional botulinum culture medium composition, and the maximum toxin production level can be obtained after about 28 hours of culture at 35°C and about 37 hours of culture at 33°C.

[0120] Example 5: Comparison of culture effects between the best culture composition and other culture compositions

[0121] When using soy peptone and the range outside the optimal composition ratio obtained in Example 3, the culture effect is determined. For this purpose, each culture medium is prepared according to the composition ratio shown in Table 12, and the culture effect of each culture medium is determined. Therefore, it can be seen from Table 13 that the experimental group has advantages in toxin production level or cell culture time compared to the comparative example. Specifically, when using the potato peptone in Comparative Example 1, the time required to reach the maximum cell amount is significantly shortened, but it is found that under the corresponding composition ratio, the maximum toxin production is much lower than the experimental group. On the other hand, when using the soy peptone in Comparative Example 2, it is found that the time required to reach the maximum cell amount is much more than the experimental group.

[0122] Table 12

[0123] glucose Yeast Extract Peptone Experimental Group 1 1 3 (Potato) Comparative Example 1 1 0.1 1 (potato) Comparative Example 2 1 1 3(Soybean)

[0124] Table 13

[0125]

[0126] Industry availability

[0127] According to the present invention, when the botulinum toxin prepared by the present invention is applied to the human body, the botulinum toxin produced by excluding animal-derived components and major allergens can prevent transmissible spongiform encephalopathy caused by the accumulation of abnormal prion protein, as well as adverse reactions caused by allergic reactions (harmful substances, angioedema, atopic dermatitis, anaphylactic shock). In addition, when potato peptone is used to exclude animal-derived components and major allergens, the strain producing botulinum toxin can be induced in a short time to reach the maximum growth amount, so in the process of botulinum toxin production, the effect of shortening production time and reducing production costs can be achieved.

[0128] Although the specific configuration of the present invention has been described in detail, it will be appreciated by those skilled in the art that the content described in the present invention is to provide the present invention to illustrate preferred embodiments and should not be interpreted as limiting the scope of the present invention. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing botulinum toxin, comprising: (a) by using a Clostridium botulinum ) to produce botulinum toxin, wherein the culture medium composition comprises: 3% (w / v) potato peptone, 1% (w / v) yeast extract and 1% (w / v) glucose, and the botulinum toxin is type A botulinum toxin; and (b) recovering the botulinum toxin.

2. The method according to claim 1, wherein the medium composition used for culturing does not include animal-derived products and allergens.

3. The method according to claim 1, wherein the botulinum toxin is recovered within 40 hours after the start of culturing in step (b).

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