Methods of increasing botulinum toxin productivity

Abstract

The present invention relates to a method for culturing a Botuulum Toxin producing strain for increasing the productivity of Botuulum Toxin, and a method for efficiently preparing Botuulum Toxin by culturing a strain using the method. According to the present invention, the production rate of botulinum toxin is increased by 10 times or more by the culture method, and thus commercialized production rate can be achieved.

Description

Technical Field

[0001] The present invention relates to a method for increasing the productivity of botulinum toxin, and more particularly, to a method for culturing a botulinum toxin producing strain for increasing the productivity of botulinum toxin and a method for efficiently preparing botulinum toxin by culturing the strain using the method. Background Art

[0002] Botulinum toxin is a neurotoxin protein produced by bacteria such as Clostridium butyricum, Clostridium baratii and Clostridium botulinum. Botulinum toxin blocks neuromuscular transmission and causes neuroparalytic diseases in humans and animals. Seven different types of botulinum toxins have been identified: A, B, C1, D, E, F, G and H. Each type can be distinguished by type-specific antibodies and differs from one another in the severity of the paralysis they cause and the species of animals they affect.

[0003] Botulinum toxins consist of four groups, from group I to group IV. Group I and group II cause botulism in humans, and group III causes botulism in animals. In contrast, it is well known that group IV does not cause botulism. Group I of botulinum toxins contains proteolytic A, B, and F toxins, and group II of botulinum toxins contains non-proteolytic B, E, and F toxins. In addition, groups I and II can decompose gelatin, and group II can ferment various carbohydrates such as sucrose and mannose.

[0004] The molecular weight of the botulinum toxin protein molecule is about 150 kDa, and it is conjugated with a light chain of about 50 kDa and a heavy chain of about 100 kDa. However, the botulinum toxin released by Clostridium bacteria is a complex formed by the 150 kDa toxin and one or more non-toxin proteins and released. For example, botulinum toxin is released in the form of complexes of 900 kDa, 500 kDa, and 300 kDa.

[0005] Although botulinum toxin is extremely lethal to humans, it is being developed in recent years for the treatment of a variety of conditions, including neuromuscular diseases characterized by overactivity of skeletal muscles. Botulinum toxin A is a trademark of botulinum toxin A commercially developed by Allergan, Inc., which is used to treat blepharospasm, strabismus, cervical dystonia and improvement of glabellar (facial) wrinkles, and other serotypes are being studied and developed for appropriate use in clinical applications.

[0006] Botulinum toxin for clinical use is usually separated from cell culture, and in the past, botulinum toxin was mainly separated by culture, fermentation and purification processes using animal-derived products. However, in the case of using animal-derived products to produce botulinum toxin, when it is administered to patients, there is a risk that various pathogens or infectious substances of animal origin may be administered to patients together. For example, the botulinum toxin composition produced may contain prions together. Prions are disease infection factors of completely different types from bacteria or viruses, molds, parasites, etc. When animals including humans are infected, the brain will produce holes like a sponge, causing nerve cell death, thereby losing the corresponding brain function. Prions can produce abnormal conformational isomers (abnormalcoformational isoforms) from the same nucleic acid sequence that constitutes normal proteins, and their infectivity exists during the "recruitment reaction" during which normal isomers in the post-translational stage become prion protein isomers. It induces normal endogenous cell proteins to misfold (misfold) into pathogenic prion conformations. Creutzfeldt-Jakob disease is a rare neurodegenerative disease of human transmissible spongiform encephalopathies, the infectious agent of which is an abnormal isomer of prion protein. Individuals with Creutzfeldt-Jakob disease can deteriorate from a healthy state to akinetic mutism within 6 months. Therefore, when administering a pharmaceutical composition comprising a biological agent such as botulinum toxin obtained using an animal-derived product, there is a risk of developing a prion-mediated disease such as Creutzfeldt-Jakob disease.

[0007] In order to eliminate the above risks, attempts have been made to exclude animal-derived components from the culture medium in the process of producing botulinum toxin. Representatively, Allergan has proposed a method of fermenting in a culture medium containing soybeans as a plant-derived protein to replace animal-derived components (Korean Patent Publication No. 10-2006-0102330), but there is a problem that it takes a long time to culture the strain in order to produce a sufficient amount of botulinum toxin.

[0008] Food allergy refers to an abnormal reaction caused by an immune response after ingesting food. Generally, allergic reactions are not only symptoms such as urticaria, angioedema, atopic dermatitis, etc. that cause a lot of inconvenience in daily life, but can also be life-threatening in severe cases. The eight main causes announced by the FDA include milk, eggs, fish, shellfish, nuts, peanuts, wheat, and soybeans, accounting for 85-90% of the total. Therefore, it is necessary to replace the existing culture medium with animal-derived ingredients and exclude possible allergens, while increasing the productivity of botulinum toxin by effectively culturing botulinum toxin-producing strains. Furthermore, for the commercial use of botulinum toxin, it is necessary to increase the toxin productivity through a simple and economical process.

[0009] Therefore, the present inventors have made intensive efforts to develop a method for culturing a botulinum toxin-producing strain for increasing the productivity of botulinum toxin. As a result, they have confirmed that the productivity of botulinum toxin can be effectively improved by adjusting pH and using an appropriate medium composition during the cultivation of the botulinum toxin-producing strain, thereby completing the present invention. Summary of the invention

[0010] Technical issues

[0011] The object of the present invention is to provide a method for increasing the productivity of botulinum toxin.

[0012] Technical Solution

[0013] In order to achieve the above object, the present invention provides a method for culturing a botulinum toxin producing strain, the method comprising:

[0014] Step (a), culturing a botulinum toxin-producing strain in a seed culture medium; and

[0015] Step (b), culturing the botulinum toxin producing strain after completing seed culture in a main culture medium at pH 5.0-6.0.

[0016] In the present invention, in the step (a), the culture may be carried out under an anaerobic environment.

[0017] In the present invention, in step (a), the seed culture medium may contain 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

[0018] In the present invention, in the step (a), the seed culture medium may be selected from N 2 , CO 2 and H2 A mixed gas of two or more gases in the composed group is used for gas replacement.

[0019] In the present invention, in the step (a), the culture may be carried out at a temperature of 28° C. to 32° C. for 18 to 30 hours.

[0020] In the present invention, in the step (b), the culture may be carried out under an anaerobic environment.

[0021] In the present invention, in step (b), the main culture medium may contain 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

[0022] In the present invention, in the step (b), the main culture medium may be prepared by titrating the seed culture medium to a pH of 6.8 to 7.2.

[0023] In the present invention, in step (b), the main culture medium may be selected from N 2 , CO 2 and H 2 A mixed gas of two or more gases in the composed group is used for gas replacement.

[0024] The present invention is characterized in that, in the step (b), the culture can be carried out at a temperature of 28° C. to 32° C. for 24 to 144 hours.

[0025] In the present invention, the botulinum toxin may be at least one selected from the group consisting of type E botulinum toxin, non-proteolytic type B botulinum toxin, and non-proteolytic type F botulinum toxin.

[0026] The present invention also provides a culture medium composition for culturing a strain producing one or more botulinum toxins selected from the group consisting of botulinum toxin type E, non-proteolytic botulinum toxin type B and non-proteolytic botulinum toxin type F, comprising 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

[0027] The present invention also provides a method for preparing botulinum toxin, the method comprising:

[0028] Step (a), producing botulinum toxin by culturing a botulinum toxin-producing strain using the method; and

[0029] Step (b), recovering the produced botulinum toxin.

[0030] Effects of the Invention

[0031] When a botulinum toxin producing strain is cultured using the method of the present invention, the growth rate of the strain is significantly increased, and a high yield of botulinum toxin can be obtained. Commercial productivity can be achieved by increasing the productivity of botulinum toxin by more than 10 times. In addition, the present invention has the advantage that the culture medium composition of the present invention does not contain animal-derived products and major allergen substances, and therefore has excellent stability. Furthermore, the advantage of botulinum toxin type E is that it takes effect on the day of injection and lasts for up to one month, so it can be used to prepare biopharmaceuticals, tissue sealing and facial fixation and lifting threads, lipolysis injections, local analgesics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shown are changes in OD600 values ​​of Clostridium botulinum strains over culture time.

[0033] Figure 2 The figure shows the change in pH of the culture solution over time.

[0034] Figure 3 The amount of botulinum toxin is shown over the culture time.

[0035] Figure 4 The amount of toxin according to the composition of the culture medium is shown.

[0036] Figure 5 The amount of toxin is shown as a function of pH adjustment.

[0037] Figure 6 The amount of toxin is shown as a function of pH adjustment. DETAILED DESCRIPTION

[0038] Hereinafter, the present invention will be described in detail.

[0039] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used in this specification is well known and commonly used in the art.

[0040] In the present invention, the term "botulinum toxin" refers to a neurotoxin produced by Clostridium botulinum, and a botulinum toxin (or its light chain or heavy chain) prepared by recombination of a non-Clostridium species. As used in this specification, botulinum toxin also includes a botulinum toxin complex (i.e., a complex of 300, 600 and 900 kDa) and a pure botulinum toxin (i.e., about 150 kDa). The "pure botulinum toxin" is defined as a botulinum toxin separated or substantially separated from other proteins including proteins that form a botulinum toxin complex. The purity of pure botulinum toxin may be 95% or more, preferably 99% or more. Clostridium botulinum C2 and C3 cytotoxins are not neurotoxins and therefore do not fall within the scope of the present invention.

[0041] In the present invention, the botulinum toxin-producing strain may be Clostridium botulinum or a variant thereof, but is not limited thereto. It is obvious to a person skilled in the art that any strain capable of producing botulinum toxin may be used.

[0042] Therefore, in one aspect, the present invention relates to a method for culturing a botulinum toxin producing strain, the method comprising:

[0043] Step (a), culturing a botulinum toxin-producing strain in a seed culture medium; and

[0044] Step (b), culturing the botulinum toxin-producing strain after seed culture in a main culture medium at pH 5.0-6.0, preferably pH 5.2-5.8, more preferably pH 5.3-5.7.

[0045] In the present invention, in the step (a), the culture may be carried out under an anaerobic environment, but is not limited thereto.

[0046] In the present invention, in the step (a), the seed culture medium may contain potato peptone, yeast extract (yeast extract) and glucose (glucose) as effective ingredients, but is not limited thereto.

[0047] In the present invention, the concentration of potato peptone can be 3.0-4.0% (w / v), preferably 3.3-3.8% (w / v), more preferably 3.5% (w / v), the concentration of yeast extract can be 0.5-2.5w / v%, preferably 1.0-1.5w / v%, more preferably 1.2w / v%, and the concentration of glucose can be 0.5-2.5w / v%, preferably 1.0-1.5w / v%, more preferably 1.2w / v%, but is not limited thereto.

[0048] In the present invention, in step (a), the seed culture medium may be selected from N 2 , CO 2 and H 2 The gas replacement may be performed by a mixed gas of two or more gases in the group, but the present invention is not limited thereto.

[0049] In the present invention, in the step (b), the culture may be carried out at a temperature of 28° C. to 32° C. for 24 to 144 hours, preferably 48 to 96 hours, but is not limited thereto.

[0050] In the present invention, in order to maintain the pH in the range of 5.0 to 6.0, preferably 5.2 to 5.8, and more preferably 5.3 to 5.7, the pH is adjusted after 4 hours of main culture to avoid deviating from the pH range, and culture is carried out for 24 to 144 hours, preferably 48 to 96 hours, but not limited thereto. In more detail, titration is performed when the pH begins to deviate from the pH range, and then titrated until the pH no longer changes. The pH titration can be performed with an alkaline substance, preferably sodium hydroxide, but not limited thereto.

[0051] In the present invention, when the strain is subjected to main culture, the pH decreases, reaches pH 5.3 after 4 to 8 hours of main culture, and the pH change of the culture solution slows down after 13 to 17 hours of main culture, and can be maintained at about pH 5.5, but is not limited thereto. In more detail, the pH adjustment can be started after 4 to 8 hours of main culture, and the pH adjustment can be ended when the pH change of the culture solution slows down after 13 to 17 hours of main culture, but is not limited thereto.

[0052] In the present invention, the botulinum toxin may be group II botulinum toxin, but is not limited thereto.

[0053] The botulinum toxin II group may be one or more selected from the group consisting of botulinum toxin type E, non-proteolytic botulinum toxin type B, and non-proteolytic botulinum toxin type F, but is not limited thereto.

[0054] In the present invention, the botulinum toxin productivity of the strain can be increased by more than 5 times, preferably more than 9.5 times, by the method, but not limited thereto.

[0055] In still another aspect, the present invention relates to a culture medium composition for culturing a strain producing one or more botulinum toxins selected from the group consisting of botulinum toxin type E, non-proteolytic botulinum toxin type B and non-proteolytic botulinum toxin type F, comprising 3.0-4.0% (w / v), preferably 3.3-3.8% (w / v), more preferably 3.5% (w / v) potato peptone, 0.5-2.5 w / v%, preferably 1.0-1.5 w / v%, more preferably 1.2 w / v% yeast extract and 0.5-2.5 w / v%, preferably 1.0-1.5 w / v%, more preferably 1.2 w / v% glucose as active ingredients.

[0056] In another aspect, the present invention relates to a method for preparing a botulinum toxin, the method comprising:

[0057] Step (a), producing botulinum toxin by culturing a botulinum toxin-producing strain using the method; and

[0058] Step (b), recovering the produced botulinum toxin.

[0059] In the present invention, "free of animal-derived ingredients" means "substantially free of animal-derived ingredients" or "substantially free of animal protein", which means free of or substantially free of blood-derived, blood pooled and other animal-derived products or compounds. "Animal" refers to mammals (such as humans, etc.), birds, reptiles, fish, insects, spiders or other animal species. "Animal" does not include microorganisms such as bacteria. Therefore, a culture medium or method that does not contain animal products or a culture medium or method that does not substantially contain animal products within the scope of the present invention may contain botulinum toxin or botulinum toxin. For example, a method that does not contain animal products or a method that does not substantially contain animal products refers to a method that does not substantially contain or essentially contain or completely contain animal-derived proteins such as immunoglobulins, meat digests, meat by-products, and milk or dairy products or digests. Therefore, examples of methods that do not contain animal products include methods that exclude meat and dairy products or by-products of meat or dairy products (such as bacterial culture or bacterial fermentation methods, etc.).

[0060] The present invention provides a culture medium comprising at least reduced levels of animal or dairy byproducts, preferably substantially free of animal or dairy products. "Animal or dairy byproducts" refers to compounds or combinations of compounds produced in vivo or in vitro by animal (except bacteria) cells or by animal (except bacteria) cells. Preferred non-animal sources of culture medium components such as proteins, amino acids and nitrogen include plants, microorganisms (such as yeast, etc.) and synthetic compounds.

[0061] The culture medium of the present invention includes a culture medium for fermenting Clostridium botulinum in small or large amounts, a culture medium for growing and culturing Clostridium botulinum for inoculation into a seed culture medium and a fermentation medium (main culture medium), but is not limited thereto.

[0062] As a particularly preferred embodiment of the present invention, the culture medium for the growth of Clostridium botulinum and the production of botulinum toxin may contain potato-derived components, preferably potato peptone, instead of animal-derived components.

[0063] The present invention provides a method for growing Clostridium botulinum, which maximizes the production of botulinum toxin in the shortest time by using a culture medium that is substantially free of animal-derived components. Clostridium botulinum can be grown using a culture medium composition in which the animal-derived components are replaced by potato peptone.

[0064] In a preferred embodiment of the present invention, the growth of Clostridium botulinum is carried out in two steps (seed growth and fermentation). Preferably, both steps are carried out under anaerobic conditions. The seed growth (seed culture) step is generally used to "scale up" the amount of microorganisms in the stored culture. The purpose of the seed growth (seed culture) step is to increase the amount of microorganisms that can be used for fermentation. In addition, the seed growth (seed culture) step revitalizes the microorganisms in the stored culture that are in a relatively low metabolic state and grows into an actively growing culture. In addition, the volume and content of the growable microorganisms used for inoculation into the fermentation medium can be more accurately controlled in the actively growing culture compared to the stored culture. Therefore, the growth of the seed culture used for inoculation into the fermentation medium (main culture medium) is preferred. In addition, several consecutive steps including growth in the seed culture medium can be used to amplify the amount of Clostridium botulinum used for inoculation into the fermentation medium (main culture medium).

[0065] In the fermentation (main culture) step, part or all of the seed culture medium containing Clostridium botulinum derived from the seed growth can be used to inoculate the fermentation medium (main culture). Preferably, about 1-10% of the seed culture medium with Clostridium botulinum from the seed growth (seed culture) step is used to inoculate the fermentation medium (main culture). Fermentation (main culture) is used to produce the maximum amount of microorganisms in a large-scale anaerobic environment.

[0066] The botulinum toxin contained in the culture solution of the strain cultured by the above method can be separated and purified by a protein purification method known to those skilled in the art in the field of protein purification technology.

[0067] The growth of Clostridium botulinum can be carried out in more than one step. Preferably, the growth is carried out in two steps. In the seed growth (seed culture) as the first step, Clostridium botulinum is suspended in the medium composition of the present invention and cultured in an anaerobic environment at 30±2°C for 18-30 hours. Preferably, the seed growth (seed culture) is carried out for about 24 hours. Preferably, before the final growth in the seed culture medium is inoculated into the fermentation medium (main culture medium), the growth in the seed culture medium in any step does not cause cell lysis.

[0068] Then, in order to further grow Clostridium botulinum and recover botulinum toxin, fermentation (main culture) as the second step is performed by inoculating part or all of the medium used for seed growth (seed culture) into the medium composition of the present invention. After inoculation, culture is carried out for 48 to 96 hours under an anaerobic environment at 30±2°C, 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 of the present invention, cell lysis and a decrease in OD value occur after the maximum growth is shown between about 14 and 18 hours, and thus, in the fermentation step, the botulinum toxin is recovered within 96 hours, more preferably within 72 hours, after the start of culturing Clostridium botulinum.

[0069] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0070] Example

[0071] Example 1

[0072] Experimental Materials

[0073] The experimental materials used in the present invention are as follows: purified water (ultrapure water or water with equivalent quality), potato peptone E210 (Organotechnie, 19425), yeast extract (BD, 212750), glucose (Merck, 1.37048.5000), sodium hydroxide pellets (Merck, 1.06482.5000), 5 mL plastic syringe Luer lock (Plasticsyringes Luer-Lock 5 mL) (Henke-ject, 4050-X00V0), 50 mL plastic syringe Luer lock (Plasticsyringes Luer-Lock 50 mL) (Henke-ject, 4850003000), 0.2 μm bottle top filter (0.2 μm bottle top filter). filter) (Merck, S2GPT05RE), Cellulose based depth filter (Pall, NP6PDH41), Cellulose acetate 0.2 μm filter (Pall, CSS92DPRRK)

[0074] Example 2

[0075] Method for preparing culture medium for culturing botulinum toxin producing strain

[0076] 500 mL of PYG medium (3.5% potato peptone, 1.2% yeast extract, 1.2% glucose, pH 7.0 ± 0.2) was used as the seed medium. 5 L of the PYG medium was titrated to pH 7.0 ± 0.2 using 5N sodium hydroxide to prepare the main medium. The main medium was sterilized using a 2 μm filter, and then placed in a cell bag while paying attention to contamination and fixed in an incubator, and the mixture was incubated with a mixed gas (N 2 :CO 2 :H 2 =8:1:1) and use after gas replacement at 0.2 lpm for more than 4 hours.

[0077] Example 3

[0078] Cultivation method of botulinum toxin producing strain

[0079] Example 3-1. Preparation of strains

[0080] Take out one vial of the Research Cell Bank (RCB) stored in a deep freezer. Confirm that the RCB is Clostridium botulinum type E NCTC8550. Thaw in a BSC for 10 minutes.

[0081] Example 3-2. Seed culture

[0082] On a biosafety bench, thawed RCB was inoculated into the seed culture medium using a luer syringe while being careful of contamination. The inoculated seed culture cell bag was placed in an anaerobic chamber replaced with mixed gas at 30°C and cultured for 24 hours. After 24 hours of culture, the seed culture was terminated when the OD600 absorbance reached between 2 and 8.

[0083] Example 3-3. Main culture

[0084] On a biosafety bench, 100 mL of the culture solution from the completed seed culture was inoculated into the main culture medium using a luer syringe while being careful about contamination. After inoculation, the culture was cultured for 72-96 hours under the conditions shown in Table 1.

[0085] Table 1

[0086] Culture conditions for primary culture

[0087] condition Setpoint temperature 30±2℃ Gas pressure 0.2±0.1lpm Gas composition <![CDATA[N 2 :CO 2 :H 2 =8:1:1]]>

[0088] After 4 hours of cultivation, start sampling the culture medium and measure the OD600 value. When the absorbance of the culture medium is above 1, dilute it to 1 / 10 for measurement. When the pH drops below 5.3, connect 1N NaOH to the cell bag and inject it at a rate of 470-620μL / min for 9 hours. Confirm that the pH remains at 5.5±0.2 until the end of the culture. Sample the culture medium and perform ELISA every 24 hours of cultivation. After the cultivation is completed, use a depth filter and a 0.2μm filter to sterilize and store at 4°C.

[0089] Example 4

[0090] Enzyme-Linked Immunosorbent Assay (ELISA) was used to determine the botulinum toxin

[0091] Prepare by diluting 5 μg / mL of Antibody A buffer: Antibody A stock solution in ELISA Plate-Coating Buffer, inoculate 100 μL in each well of 96-well microplates and coat for 16 hours, then wash each well with wash buffer for a total of 3 times. Block each well with Blocking buffer (Super block), and wash each well with wash buffer for a total of 3 times.

[0092] The culture solution sampled during the culture process was diluted, and then 100 μL was added to each well and reacted for 2 hours, and then each well was washed with wash buffer for a total of 3 times. 100 μL of detection antibody dilution was added to each well and reacted for 2 hours, and then each well was washed with wash buffer for a total of 3 times. 100 μL of anti-IgG antibody dilution was added to each well and reacted for 1 hour, and then each well was washed with wash buffer for a total of 5 times. 100 μL of TMB was added to each well and reacted for 20 minutes, and then 50 μL of stop solution was added to each well and shaken appropriately to stop the enzyme reaction. The absorbance at 450 nM was measured in each well using an ELISA reader.

[0093] Example 5

[0094] Comparison of botulinum toxin production according to culture medium composition

[0095] The inventors cultured a botulinum toxin type E producing strain in the culture medium composition disclosed in the prior art patent (Korean Patent Publication No. 10-2020-0114722) (potato peptone: 3%, yeast extract: 1%, glucose: 1%) and the culture medium composition proposed in the present invention (potato peptone: 3.5%, yeast extract: 1.2%, glucose: 1.2%), and compared the amount of botulinum toxin produced.

[0096] As a result, it was confirmed that the toxin amount increased by about 18% ( 0.05 wt %) when the strain was cultured in the medium composition of the present invention compared to the case where the strain was cultured in the medium composition of the prior art patent. Figure 4 ). That is, it was confirmed that when the type E botulinum toxin producing strain was cultured in a medium containing 3.5% potato peptone, 1.2% yeast extraction, and 1.2% glucose, it had excellent toxin production. Thus, it was confirmed that the medium composition for culturing the type E botulinum toxin producing strain is different from that for the type A botulinum toxin producing strain.

[0097] Example 6

[0098] Comparison of botulinum toxin production according to pH titration

[0099] After culturing in the same manner as before, the pH titration conditions of the culture solution were set as shown in Table 2, and the culture was performed while adjusting the pH so as not to deviate from the set range during the culturing process.

[0100] Table 2

[0101] pH titration conditions during culture

[0102] Experimental Group condition Control group pH titration not performed (existing batches) pH 6.0 test group During the culture, the pH was adjusted to 6.0 ± 0.2 pH5.5 test group During the culture, the pH was adjusted to 5.5 ± 0.2

[0103] The culture results confirmed that the control group (Control) as an existing batch did not undergo pH titration, so the pH at the final recovery was 4.85, and the pH6.0 test group and the pH5.5 test group both ended at a pH within the adjustment range. Specifically, at the final recovery time point, it was confirmed that the pH6.0 test group was pH5.8-6.2, and the pH5.5 test group was pH5.3-5.7.

[0104] Table 3

[0105] pH titration results during culture

[0106]

[0107] When comparing the three batches cultured under the conditions of Table 2, the maximum OD value and autolysis percentage of the strain in the pH 6.0 test group titrated to pH 6.0 were the highest, and the control group without pH titration was the lowest. This result proves that the growth of the strain requires appropriate pH adjustment.

[0108] Furthermore, when comparing the toxin amounts of the three batches in Table 3, it was confirmed that the toxin amount of the pH 5.5 test group titrated to pH 5.5 ± 0.2 was the highest at 18.10 mg / L, thus having excellent productivity. Therefore, the experiment was conducted after setting the titration condition to pH 5.5 ± 0.2.

[0109] Example 7

[0110] Comparison of botulinum toxin production over culture time

[0111] When the toxin levels in the culture medium were measured in 24-hour increments, the toxin levels were similar after 48 hours ( Figure 3 ). Since it was confirmed that the toxin amounts were similar after 48 hours, the culture time was set to 48-96 hours.

[0112] Example 8

[0113] Comparison of botulinum toxin production according to culture conditions

[0114] The culture condition parameters were set by DOE experimental design. The results of culturing and measuring the toxin amount under the culture conditions are shown in Table 4.

[0115] Table 4

[0116] Toxin quantity determination results according to DOE experimental design

[0117]

[0118]

[0119] The results of the toxin amount in Table 4 were analyzed by a statistical program (Minitab 18), and it was confirmed that the factor affecting the toxin amount under the set conditions was the pH titration value (Table 5). Under the set conditions, the influence of the culture temperature, gas pressure, and gas type on the toxin amount was not significant.

[0120] The P value was confirmed to be 0.016 under the condition of pH 5.5±0.3. In other words, it was confirmed that the P value of pH was less than 0.05, there was no confounding factor between the parameters, and there was a curvature effect.

[0121] Table 5

[0122] Screening results of factors affecting toxin levels

[0123]

[0124] Specifically, the toxin amount comparison results in Table 4 confirmed that the toxin amount in the center batch titrated to pH 5.5 was the highest. It was confirmed that during the culture process, the toxin amount differed significantly depending on whether or not pH titration was performed and the conditions ( Figure 5 ). In addition, it was confirmed that during the culture process, the pH 5.5±0.2 condition showed a productivity similar to that of the pH 5.5 culture condition ( Figure 6 Therefore, the titration was set to pH 5.5 ± 0.2 ( Figure 6 ).

[0125] Regarding the culture temperature, it was confirmed that there was no significant effect on the toxin amount within the range of 28-32°C, so the culture temperature was set to 30±2°C (Table 5).

[0126] As for the gas pressure, it was confirmed that there was no significant effect on the toxin amount within the range of 100-300 lpm, so the gas pressure was set to 200±100 lpm (Table 5).

[0127] Regarding the type of gas, it was confirmed that both the mixed gas and nitrogen had no significant effect on the toxin amount, so it was determined that both gases could be used (Table 5). However, the results of confirming the toxin amount (Table 4) confirmed that under the central conditions, the average toxin amount (26.34, 16.35 mg / L) of the DOE 3 and DOE 9 batches cultured with mixed gas was about 38% higher than that of the DOE 6 and DOE12 batches cultured with nitrogen. Therefore, when culturing strains, both mixed gas and nitrogen can be used, but it is recommended to use mixed gas.

Claims

1. A method for culturing a botulinum toxin producing strain, characterized in that: The steps include: Step (a), culturing a botulinum toxin-producing strain in a seed culture medium; and Step (b), culturing the botulinum toxin producing strain after completing seed culture in a main culture medium at pH 5.0-6.

0.

2. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (a), the culture is carried out under an anaerobic environment.

3. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (a), the seed culture medium contains 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

4. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (a), the seed culture medium is gas-substituted by a mixed gas comprising two or more gases selected from the group consisting of N2, CO2 and H2.

5. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (a), the culture is carried out at a temperature of 28° C. to 32° C. for 18 to 30 hours.

6. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (b), the culture is carried out under an anaerobic environment.

7. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (b), the main culture medium contains 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

8. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (b), the main culture medium is prepared by titrating the seed culture medium to a pH of 6.8 to 7.

2.

9. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (b), the main culture medium is gas-substituted by a mixed gas comprising two or more gases selected from the group consisting of N 2 , CO 2 and H 2 .

10. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: In the step (b), the culture is carried out at a temperature of 28°C to 32°C for 24 to 144 hours.

11. The method for culturing a botulinum toxin-producing strain according to claim 1, characterized in that: The botulinum toxin is at least one selected from the group consisting of botulinum toxin type E, non-proteolytic botulinum toxin type B, and non-proteolytic botulinum toxin type F.

12. A culture medium composition for culturing a strain producing one or more botulinum toxins selected from the group consisting of botulinum toxin type E, non-proteolytic botulinum toxin type B, and non-proteolytic botulinum toxin type F, characterized in that: The invention comprises 3.0-4.0% (w / v) potato peptone, 0.5-2.5 w / v% yeast extract and 0.5-2.5 w / v% glucose as effective ingredients.

13. A method for preparing botulinum toxin, characterized in that: The steps include: Step (a), producing botulinum toxin by culturing a botulinum toxin-producing strain using the culturing method for a botulinum toxin-producing strain according to any one of claims 1 to 11; as well as Step (b), recovering the produced botulinum toxin.

Images