Method for producing botulinum toxin

Abstract

The present disclosure relates generally to the field of producing botulinum toxin. More specifically, the present disclosure relates to a method of producing botulinum toxin in a medium that is free or substantially free of animal products. The present disclosure also relates to a medium that is free or substantially free of animal products for producing botulinum toxin.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 951,549, filed December 20, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the field of producing botulinum toxins. More specifically, the present disclosure relates to methods of producing botulinum toxins in media that are free or substantially free of animal products. The present disclosure also relates to media for producing botulinum toxins that are free or substantially free of animal products. [Background technology]

[0003] The following discussion of the background of the present technology is provided merely to aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Seven generally immunologically distinct botulinum neurotoxins have been characterized (botulinum neurotoxin serotypes A, B, C, D, E, F, and G), each distinguished by neutralization by type-specific antibodies. As an example, Botox® is the trademark for the botulinum toxin type A purified neurotoxin complex commercially available from Allergan, Inc. (Irvine, California). Botox is a popular injectable cosmetic treatment for the temporary reduction of fine wrinkles. One unit (U) of botulinum toxin produces an LD of 1000 when injected intraperitoneally into female Swiss Webster mice weighing 18–20 grams each. 50The botulinum toxin serotype is defined as the LD value (Degree of Death) of botulinum toxin (DDT). In other words, one unit of botulinum toxin is the amount of botulinum toxin that kills 50% of a group of female Swiss Webster mice. Seven immunologically distinct botulinum neurotoxins have generally been characterized (botulinum neurotoxin serotypes A, B, C, D, E, F, and G), each distinguished by neutralization by type-specific antibodies. The different botulinum toxin serotypes vary in the animal species they affect and the severity and duration of the paralysis they induce. For example, in rats, botulinum toxin type A is more than 500 times more potent than botulinum toxin type B, as measured by the rate of paralysis produced in rats. Additionally, botulinum toxin type B has been determined to be nontoxic to primates at a dose of 480 U / kg, which is the LD value of botulinum toxin type A in primates. 50 It is about 12 times higher than the conventional method. Botulinum toxin is also known to be useful for treating various diseases. For example, see Patent Document 1 (migraine); Patent Document 2 (headache); Patent Document 3 (headache due to drug overuse); and Patent Document 4 (neuropsychiatric disorders), all of which are incorporated herein by reference.

[0005] Botulinum toxins are traditionally obtained by a culture and fermentation process using one or more animal-derived products (broth medium and blood fractions or blood-derived excipients). Administration of pharmaceutical compositions containing biologically active ingredients derived from animal-derived products to patients can potentially expose the patient to various pathogens and infectious agents. For example, prions may be present in pharmaceutical compositions. Prions are proteinaceous infectious particles hypothesized to arise as abnormal conformational isoforms from the same nucleic acid sequence that makes up the normal protein. Furthermore, infectivity is hypothesized to result from a "recruitment response" of normal isoform proteins to prion protein isoforms at the post-translational level. Apparently, normal endogenous cellular proteins are induced to misfold into the pathogenic prion conformation.

[0006] There is a need to develop a process for producing botulinum toxin in a medium that is free or substantially free of animal-derived products, which minimizes the risks and problems associated with undesirable animal-derived contaminants. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 5,714,468 (issued February 3, 1998) [Patent Document 2] U.S. Patent Application Publication No. 2005 / 019132 (Serial No. 11 / 039,506, filed January 18, 2005) [Patent Document 3] U.S. Patent Application Publication No. 2005 / 0191320 (Serial No. 10 / 789,180, filed February 26, 2004) [Patent Document 4] U.S. Patent No. 7,811,587 (issued October 12, 2010) Summary of the Invention [Means for solving the problem]

[0008] Provided herein are methods for producing a botulinum toxin, the methods comprising the steps of: (a) providing a working cell bank (WCB) containing Clostridium botulinum, (b) adding the working cell bank to a first vessel containing a vegetable toxin production medium (VTPM) and culturing the Clostridium botulinum in the VTPM under conditions that allow the Clostridium botulinum to grow and produce a pre-culture, (c) adding the pre-culture to a second vessel containing VTPM and culturing the Clostridium botulinum under conditions that allow the production of a botulinum toxin, and (d) recovering the botulinum toxin, wherein the VTPM is substantially free of animal-derived products or is free of animal-derived products and comprises plant-derived proteins.

[0009] In some embodiments, the botulinum toxin is botulinum neurotoxin type A (BoNT / A).

[0010] In some embodiments, the vessel used in steps (b) and (c) is a fermentation bag.

[0011] In some embodiments, the conditions in steps (b) and (c) include an anaerobic environment. In some embodiments, the anaerobic environment has a dissolved oxygen (DO) concentration of less than 2%. In some embodiments, the anaerobic environment has a dissolved oxygen (DO) concentration of less than 1%. In some embodiments, the anaerobic environment has a dissolved oxygen (DO) concentration of less than 0.5%.

[0012] In some embodiments, the conditions in step (b) include a temperature of between about 35°C and about 39°C, between about 36°C and about 38°C (or any range therebetween). In some embodiments, the conditions in step (b) include a temperature of about 35.0°C, about 35.5°C, about 36.0°C, about 36.5°C, about 37.0°C, about 37.5°C, about 38.0°C, about 38.5°C, or about 39.0°C. In some embodiments, the conditions in step (b) include a temperature of about 37±1°C. In some embodiments, the conditions in step (b) include a temperature of about 37±0.5°C. In some embodiments, the conditions in step (b) include a temperature of about 37±0.2°C.

[0013] In some embodiments, the conditions in step (c) include a temperature of between about 30°C and about 37°C, between about 31°C and about 36°C, between about 32°C and about 35°C, or between about 32°C and about 34°C (or any range therebetween). In some embodiments, the conditions in step (c) include a temperature of about 33±1°C. In some embodiments, the conditions in step (c) include a temperature of about 33±0.5°C. In some embodiments, the conditions in step (c) include a temperature of about 33±0.2°C.

[0014] In some embodiments, the volume ratio of the WCB to the VTPM in step (b) is about 2.0% or less, about 1.9% or less, about 1.8% or less, about 1.7% or less, about 1.6% or less, about 1.5% or less, about 1.4% or less, about 1.3% or less, about 1.2% or less, about 1.1% or less, about 1.0% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, about 0.09% or less, about 0.08% or less, about 0.07% or less, about 0.06% or less, about 0.05% or less, about 0.04% or less, about 0.03% or less, about 0.02%, or about 0.01% or less (or a range therebetween).

[0015] In some embodiments, the volume ratio of the WCB to the VTPM in step (b) is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 100%, about 100%. %, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

[0016] In some embodiments, the volume ratio of the pre-culture to the VTPM in step (c) is between about 1:2 and about 1:50, between about 1:3 and about 1:45, between about 1:4 and about 1:40, between about 1:5 and about 1:35, between about 1:6 and about 1:30, between about 1:7 and about 1:25, between about 1:8 and about 1:20, or between about 1:8 and about 1:10 (or any range therebetween). In some embodiments, the volume ratio of the pre-culture to the VTPM in step (c) is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, or about 1:50.

[0017] In some embodiments, OD 600 Step (b) is carried out until OD reaches a value in the range of about 0.1 to about 1.0, about 0.1 to about 0.05, or about 0.2 to about 0.4. 600 Step (b) above is carried out until the β-dimer ratio reaches about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0.

[0018] In some embodiments, step (b) is carried out for about 10 hours to about 30 hours, about 15 hours to about 25 hours, or about 17 hours to about 21 hours (or any range therebetween). In some embodiments, step (b) is carried out for about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours. In some embodiments, step (b) is carried out for about 19±2 hours. In some embodiments, step (b) is carried out for about 19±1 hour. In some embodiments, step (b) is carried out for about 19±0.5 hours. In some embodiments, step (b) is carried out for about 19±0.2 hours. In some embodiments, step (b) is carried out for about 19 hours.

[0019] In some embodiments, step (c) is carried out for between about 60 and about 80 hours, about 65 and about 75 hours, or about 67 and about 71 hours (or any range therebetween). In some embodiments, the fermentation process lasts for about 60 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, or about 80 hours. In some embodiments, the fermentation process lasts for about 69±2 hours, about 69±1 hour, about 69±0.5 hours, or about 69±0.2 hours. In one embodiment, step (c) is carried out for about 69 hours.

[0020] In some embodiments, microbiological purity with respect to microorganisms other than C. botulinum is tested in the preculture after step (b) and before step (c).

[0021] In some embodiments, microbiological purity with respect to microorganisms other than C. botulinum is tested in the culture after step (c) and before step (d).

[0022] In some embodiments, the plant-derived protein is wheat peptone. In some embodiments, the concentration of wheat peptone in the VTPM is between about 10 grams per liter and about 30 grams per liter, e.g., about 20 grams per liter. In some embodiments, the concentration of wheat peptone in the VTPM is about 20 grams per liter. In some embodiments, the VTPM comprises wheat peptone, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, and medical antifoam C emulsion.

[0023] In some embodiments, the VTPM comprises wheat peptone, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, medical antifoam C emulsion, distilled water, NaOH, and HCl. In one particular embodiment, the VTPM comprises about 20 grams per liter of wheat peptone, about 20 grams per liter of yeast extract, about 5 grams per liter of D-(+)-glucose, about 0.20 grams per liter of L-cysteine ​​hydrochloride monohydrate, and about 0.24 grams per liter of medical antifoam C emulsion. In one particular embodiment, the pH of the VTPM is between about 6.7 and about 7.2.

[0024] In another aspect, provided herein are compositions comprising Clostridium botulinum and a medium for producing a botulinum toxin, the medium being free or substantially free of animal-derived products and comprising one or more plant-derived proteins. In some embodiments, the one or more plant-derived proteins are wheat peptone, broad bean peptone, potato peptone, pea peptone, rice peptone, or soy peptone, or a combination thereof. In some embodiments, the plant-derived protein is wheat peptone.

[0025] In some embodiments, the concentration of wheat peptone in the VTPM is about 20 grams per liter. In some embodiments, the VTPM comprises wheat peptone, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, and medical antifoam C emulsion.

[0026] In some embodiments, the VTPM comprises wheat peptone, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, medical antifoam C emulsion, distilled water, NaOH, and HCl. In one particular embodiment, the VTPM comprises about 20 grams per liter of wheat peptone, about 20 grams per liter of yeast extract, about 5 grams per liter of D-(+)-glucose, about 0.20 grams per liter of L-cysteine ​​hydrochloride monohydrate, and about 0.24 grams per liter of medical antifoam C emulsion. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows the fermentation process. 400 μl of working cell bank (WCB) is added to 500 mL of growth medium in a 2 L fermentation bag. The bag is purged with filtered nitrogen before inoculation. Fermentation is carried out at 37°C until the OD600 reaches 0.2-0.4. 4500 mL of vegetable toxin production medium (VTPM) is then added to the 5 L culture bag containing 500 mL of preculture. The bag is purged with filtered nitrogen before inoculation. Fermentation is carried out for 69°C for 2 hours at 33°C. [Figure 2]Figure 2 shows the optical density curve at 600 nm for the main culture, based on samples extracted and analyzed from several fermentations carried out as described in Example 2 below. [Figure 3] Figure 3 shows the pH curve for the main culture, which is based on samples extracted and analyzed from several fermentation samples carried out according to Example 2 below. [Figure 4] 4 shows Western blot analysis of BoNT / A heavy chain variants in the main culture performed according to Example 2. Samples were extracted at different time points during fermentation and run alongside reference samples (showing only band 2 or both bands 1 and 2). [Figure 5] 5 shows tables containing cutaways of Western blot analysis of BoNT / A heavy chain variants in samples harvested from main cultures performed at different temperatures. These tables also show the BoNT / A concentrations of the same samples as determined by ELISA. [Figure 6] FIG. 6 shows the toxin content of main cultures harvested at 69 hours grown in wheat peptone-based VTPM or soy peptone-based VTPM. [Figure 7] FIG. 7 shows the toxin concentrations obtained from 69 hour harvests of main cultures grown in VTPM based on potato, broad bean, or wheat-derived peptone. DETAILED DESCRIPTION OF THE INVENTION

[0028] Embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. It is understood that the present technology is not limited to particular methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0029] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and the relevant art, and it is further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Such terms, although not expressly defined below, should be interpreted according to their ordinary meaning.

[0030] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure also is described in terms of any individual element or subgroup of elements of the Markush group.

[0031] As will be understood by those skilled in the art, for any and all purposes, and in view of providing a particularly written description, all ranges disclosed herein also encompass any and all subranges and combinations of such subranges. Any stated range is sufficiently descriptive, and it will be readily recognized that the same range can be divided into at least two, three, four, five, ten, etc. equal parts. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. Additionally, as will be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," and "less than," is inclusive of the recited number and refers to a range that can be successively divided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual element. Thus, for example, a group having 1 to 3 cells refers to a group having 1 cell, a group having 2 cells, or a group having 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1 cell, a group having 2 cells, a group having 3 cells, a group having 4 cells, a group having 5 cells, etc.

[0032] Unless the context indicates otherwise, it is specifically intended that the various features of the technology described herein may be used in any combination. Moreover, the present disclosure also contemplates that, in some embodiments, any feature or combination of features specified herein may be excluded or omitted. For illustration, if the specification states that a composite includes components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, alone or in any combination, may be omitted or discarded.

[0033] Unless expressly stated otherwise, all specific embodiments, features, and terms are intended to include the recited embodiment, feature, or term, and biological equivalents thereof.

[0034] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

[0035] definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless expressly stated to refer to the singular only.

[0036] It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about" or "approximately." The term "about" or "approximately" means that the numbers encompassed are not limited to the exact numbers set forth herein, but are intended to refer to numbers that are substantially near the recited number without departing from the scope of the present invention. As used herein, "about" or "approximately" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of this term is not clear to those of ordinary skill in the art given the context in which it is used, "about" or "approximately" will mean up to plus or minus 15%, 10%, 5%, 1%, or 0.1% of the particular term (e.g., "about 10" should be understood as 10 and a range of 8.5 to 11.5).

[0037] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0038] As used herein, "animal product-free," "essentially animal product-free," or "substantially animal product-free" encompasses "animal protein-free," "essentially animal protein-free," or "substantially animal protein-free," respectively, and refers to the absence, essential absence, or substantial absence of blood-derived, blood pool, and other animal-derived products or compounds. "Animal" refers to mammals (such as humans), birds, reptiles, fish, insects, arachnids, and other animal species. "Animal" excludes microorganisms, such as bacteria. Thus, animal product-free or substantially animal product-free media or processes within the scope of the present disclosure may include botulinum toxin or Clostridium botulinum bacteria. For example, an animal product-free or substantially animal product-free process refers to a process that is substantially, essentially, or completely free of animal-derived proteins, such as immunoglobulins, meat digests, meat by-products, and milk or dairy products or dairy digests. Thus, an example of an animal product-free process is a process that excludes meat and dairy products or meat or dairy products (eg, a bacterial culture process or a bacterial fermentation process).

[0039] As used herein, "botulinum toxin" refers to a botulinum toxin (or its light or heavy chain) recombinantly produced by a non-clostridial species, as well as neurotoxins produced by Clostridium botulinum. As used herein, the term "botulinum toxin" encompasses botulinum toxin serotypes A, B, C, D, E, F, and G. As used herein, botulinum toxin includes both botulinum toxin complexes (i.e., 300, 600, and 900 kDa complexes) and pure botulinum toxin (i.e., approximately 150 kDa). "Purified botulinum toxin" is defined as a botulinum toxin that has been isolated or substantially isolated from other proteins, including proteins that form the botulinum toxin complex. Pure botulinum toxin may be greater than 95% pure, and preferably greater than 99% pure. Botulinum C2 and C3 cytotoxins are not neurotoxins and are therefore excluded from the scope of this disclosure. As used herein, "botulinum toxin" also includes "modified botulinum toxins."

[0040] "Modified botulinum toxin" refers to a botulinum toxin in which at least one of its amino acids has been deleted, modified, or substituted compared to the original botulinum toxin. In addition, the modified botulinum toxin can be a recombinantly produced neurotoxin or a derivative or fragment of a recombinantly produced neurotoxin. The modified botulinum toxin retains at least one biological activity of the original botulinum toxin, such as the ability to bind to a botulinum toxin receptor or the ability to inhibit the release of neurotransmitters from nerves. One example of a modified botulinum toxin is a botulinum toxin having a light chain derived from one botulinum toxin serotype (e.g., serotype A) and a heavy chain derived from a different botulinum toxin serotype (e.g., serotype B). Another example of a modified botulinum toxin is a botulinum toxin that binds to a neurotransmitter, such as substance P.

[0041] As used herein, "culture medium" or "fermentation medium" means any medium for culturing bacteria, either a growth medium to create a seed culture used to inoculate a production medium, or a production medium in which bacteria are grown to produce toxins. One example of a fermentation medium according to the present disclosure is Vegetable Toxin Production Medium (VTPM).

[0042] As used herein, "clostridial neurotoxins" refers to neurotoxins produced by or native to clostridial bacteria (e.g., Clostridium botulinum, Clostridium butyricum, or Clostridium baratii) and recombinantly produced clostridial neurotoxins produced by non-clostridial species. Clostridial toxins produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii, and Clostridium butyricum are the most widely used in therapeutic and cosmetic treatments of humans and other mammals. C. botulinum strains produce seven antigenically distinct botulinum toxins (BoNTs). These toxins have been identified during outbreak botulism investigations in humans (BoNT / A, 7B, FE, and / F), animals (BoNT / C and / D), or isolated from soil (BoNT / G). BoNTs share approximately 35% amino acid identity with each other and the same functional domain organization and overall structural design. As recognized by those skilled in the art, each clostridial toxin may have subspecies that differ somewhat in their amino acid sequences and the nucleic acids encoding these proteins. For example, there are currently five BoNT / A subspecies: BoNT / A1, BoNT / A2, BoNT / A3, BoNT / A4, and BoNT / A5. These particular subspecies exhibit approximately 89% amino acid identity when compared with other BoNT / A subspecies. All seven BoNT serotypes have similar structures and pharmacological properties, but each also exhibits distinct bacteriological properties. In contrast, tetanus toxin (TeNT) is produced by a homogenous group of Clostridium tetani bacteria. Two other clostridial species, C. baratii and C. butyricum, also produce toxins similar to BoNT / F and BoNT / E, BaNT and BuNT, respectively.

[0043] Clostridial toxins are released by Clostridial bacteria as complexes containing the approximately 150 kDa clostridial toxin together with associated non-toxin proteins (NAPs). The identified NAPs include proteins with hemagglutinating activity, such as the approximately 17 kDa hemagglutinin (HA-17), the approximately 33 kDa hemagglutinin (HA-33), and the approximately 70 kDa hemagglutinin (HA-70), as well as the nontoxic nonhemagglutinin (NTNH), an approximately 130 kDa protein (see, e.g., Eric A. Johnson and Marite Bradshaw, Clostridial botulinum and its Neurotoxins: A Metabolic and Cellular Perspective, 39 Toxicon 1703-1722 (2001); and Stephanie Raffestin et al., Organization and Regulation of the Neurotoxin Genes in Clostridium botulinum and Clostridium tetani, 10 Anaerobe 93-100 (2004)). Thus, the botulinum toxin type A complex can be produced by Clostridium bacteria as a 900 kDa, 500 kDa, and 300 kDa form. Botulinum toxin types B and C are apparently produced only as the 500 kDa complex. Botulinum toxin type D is produced as a 300 kDa complex and a 500 kDa complex. Finally, botulinum toxin types E and F are produced only as a complex of approximately 300 kDa. The difference in molecular weight between these complexes is due to different ratios of NAPs. The toxin complex is important for the intoxication process because it appears to protect the toxin from adverse environmental conditions, provide resistance to protease digestion, and facilitate internalization and activation of the toxin.

[0044] Each clostridial toxin is translated as a single-chain polypeptide and then cleaved by proteolytic cleavage within a disulfide loop by naturally occurring proteases. This cleavage occurs within a distinct two-chain loop region formed between two cysteine ​​residues that form a disulfide bridge. This post-translational processing results in a two-chain molecule containing an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC), linked by a single disulfide bond and noncovalent interactions between the two chains. The naturally occurring protease used to convert the single-chain molecule to a two-chain molecule is currently unknown. For some serotypes, e.g., BoNT / A, naturally occurring proteases are endogenously produced by the bacterial serotype, and cleavage occurs intracellularly before the toxin is released into the environment. However, for other serotypes, e.g., BoNT / E, bacterial strains do not appear to produce endogenous proteases capable of converting the single-chain form of the toxin to the two-chain form. In these situations, the toxin is released from the cell as a single-chain toxin, which is then converted to a two-chain form by naturally occurring proteases found in the environment.

[0045] As used herein, "free" or "completely free" means that the substance cannot be detected or its presence cannot be confirmed within the detection range of the equipment or process used.

[0046] As used herein, "essentially free" means that only trace amounts of the substance can be detected. In this disclosure, "essentially free" means that the amount of the substance is less than 0.1% by weight of the total composition, preferably less than 0.01% by weight, and most preferably less than 0.001% by weight.

[0047] As used herein, "substantially free" means that the amount of the material is less than 5% by weight of the total composition, preferably less than 2% by weight, and most preferably less than 1% by weight.

[0048] As used herein, "culture medium" or "fermentation medium" means any medium for culturing bacteria, whether it is a growth medium to create a seed culture used to inoculate a production medium, or a production medium in which bacteria grow to produce toxins.

[0049] As used herein, "working cell bank" or "WCB" refers to an essentially homogeneous population of cells derived from a single master cell bank (MCB). WCBs are generally required for therapeutic development and manufacturing. A WCB is generated from a single vial of MCB that has been grown for several passages and cryopreserved. In other words, cells for a WCB are expanded from an MCB. In practice, when a cell line is to be used over many manufacturing cycles, a two-tiered cell deposit system consisting of a master cell bank (MCB) and a working cell bank (WCB) is widely recommended.

[0050] As used herein, "vegetable toxin production medium" or "VTPM" means a cell culture medium that includes one or more components derived from one or more vegetables (e.g., wheat, soybean, fava bean, potato, pea, etc.). The one or more components derived from one or more vegetables include, but are not limited to, vegetable digests, peptones, or extracts.

[0051] As used herein, "peptone" means a hydrolyzed proteinaceous material formed by enzymatic or acid digestion.

[0052] As used herein, "vegetable extract" means an aqueous extract of any vegetable that contains amino acids and low molecular weight peptides, carbohydrates, vitamins, and other growth factors.

[0053] As used herein, "plant peptone" refers to a plant-derived proteinaceous material hydrolyzed by the use of microbial or vegetable enzymes or by acid hydrolysis. The protein substrate for forming the peptone may be any proteinaceous material of vegetable origin or a protein concentrate isolated from, for example, rice, wheat, or soy flour. The term "yeast peptone" refers to a yeast cell-derived proteinaceous material hydrolyzed by autolysis, the use of microbial or vegetable enzymes, or acid hydrolysis. The plant peptone of the present disclosure refers to the partial digestion product of plant-derived proteins in the form of a mixture containing not only a single amino acid molecule but also peptides consisting of several or several dozen amino acids and complete protein molecules. Preferably, the plant peptone of the present disclosure is soybean peptone, wheat peptone, broad bean peptone, potato peptone, pea peptone, defatted soybean peptone, or lupin bean peptone, most preferably pea peptone and wheat peptone.

[0054] As used herein, "OD 600 " means optical density measured at a wavelength of 600 nm. Those of ordinary skill in the art will understand that OD 600 We recognize that measuring OD is a common method for estimating cell (including bacterial) concentrations in a liquid. 600 Methods for determining OD are described, for example, in S.A. Janke, et al., Microbiological Turbidity Using Standard Photometers, 6 BIOSPEKTRUM 501-02 (1999); K. Harnack, et al. Turbidity Measurements (OD 600 ) with Absorption Spectrometers, 6 BIOSPEKTRUM 503-04 (1999).

[0055] Vegetable Toxin Production Medium (VTPM) Vegetable extracts can be used in media for the growth of pathogenic bacteria and their toxin production. Vegetable extracts are aqueous extracts of plants that contain amino acids and low-molecular-weight peptides, relatively high concentrations of carbohydrates, vitamins, and other growth factors. According to the present disclosure, plant-derived proteins, such as peptones derived from plants including potato, wheat, rice, wheat and rice blends, cotton, or pea, can replace animal-derived products to support the growth of Clostridium botulinum. Peptones that may be used for the purposes of the disclosed VTPM include, but are not limited to, wheat peptone CAS#94350-06-8, wheat peptone E1, wheat peptone E260, pea peptone CAS#100209-45-8, pea peptone A482, pea peptone A2501, potato peptone CAS#100209-45-8, potato peptone E210, potato peptone L8, potato peptone A2401, rice peptone 19560, cotton peptone 200, soy peptone CAS#91079-46-8, soy peptone A3SC, soy peptone A2SC, and other plant or vegetable peptones.

[0056] In some embodiments, the peptone is wheat peptone. In certain embodiments, the concentration of wheat peptone in the fermentation medium is between 5 and 50 g / L of fermentation medium, preferably between 10 and 40 g / L, preferably between 15 and 30 g / L, preferably between 15 and 25 g / L, and more preferably about 20 g / L. According to embodiments, the concentration of wheat peptone in the fermentation medium is about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L.

[0057] According to the present disclosure, the fermentation medium comprises a yeast extract, which is generally obtained by salt-free autolysis of primary yeast cells followed by extensive purification, resulting in a yeast extract free of undesirable components such as spores and DNA.

[0058] According to the present disclosure, the fermentation medium further comprises a yeast extract. In some embodiments, the concentration of the yeast extract in the fermentation medium is between 5 and 50 g / L of fermentation medium, preferably between 10 and 40 g / L, preferably between 15 and 30 g / L, preferably between 15 and 25 g / L, and more preferably about 20 g / L. In some embodiments, the concentration of the yeast extract in the fermentation medium is about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L.

[0059] A variety of carbon sources, including glucose and glycerol, have been used to grow C. botulinum. When using a carbon-containing nitrogen source (C. botulinum can utilize carbon from amino acids), the addition of a separate carbon source is not necessary, although the presence of an additional carbon source results in higher growth rates during fermentation.

[0060] According to the present disclosure, the fermentation medium includes D-(+)-glucose. In some embodiments, the concentration of D-(+)-glucose in the fermentation medium is between 0.5 and 20 g / L of fermentation medium, preferably between 1.0 and 10 g / L, preferably between 2.5 and 7.5 g / L, preferably between 3.5 and 6.5 g / L, and more preferably about 5 g / L. In some embodiments, the concentration of D-(+)-glucose in the fermentation medium is about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1.0 g / L, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, about 5.0 g / L, about 5.5 g / L, about 6.0 g / L, or about 7.0 g / L. about 6.5 g / L, about 7.0 g / L, about 7.5 g / L, about 8.0 g / L, about 8.5 g / L, about 9.0 g / L, about 9.5 g / L, about 10.0 g / L, about 11.0 g / L, about 12.0 g / L, about 13.0 g / L, about 14.0 g / L, about 15.0 g / L, about 16.0 g / L, about 17.0 g / L, about 18.0 g / L, about 19.0 g / L, or about 20.0 g / L.

[0061] According to the present disclosure, the fermentation medium also includes L-cysteine ​​hydrochloride monohydrate. In some embodiments, the concentration of L-cysteine ​​hydrochloride monohydrate in the fermentation medium is between 0.05 and 5 g / L of fermentation medium, preferably between 0.1 and 5 g / L, preferably between 0.1 and 2.5 g / L, preferably between 0.15 and 1.5 g / L, and more preferably about 0.2 g / L. In some embodiments, the concentration of L-cysteine ​​hydrochloride monohydrate in the fermentation medium is about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0.10 g / L, about 0.15 g / L, about 0.20 g / L, about 0.25 g / L, about 0.30 g / L, about 0.35 g / L, about 0.40 g / L, about 0.45 g / L, or about 0. 50 g / L, about 0.55 g / L, about 0.60 g / L, about 0.70 g / L, about 0.75 g / L, about 0.80 g / L, about 0.85 g / L, about 0.90 g / L, about 0.95 g / L, about 1.0 g / L, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, or about 5.0 g / L.

[0062] According to the present disclosure, the fermentation medium may include Medical Antifoam C Emulsion (Dow Corning®). In some embodiments, the concentration of Medical Antifoam C Emulsion in the fermentation medium is between about 0.05 g / L and about 0.50 g / L, between about 0.10 g / L and about 0.40 g / L, between about 0.20 g / L and about 0.30 g / L, or between about 0.22 g / L and about 0.26 g / L (or any range therebetween). In some embodiments, the concentration of the medical antifoam C emulsion in the fermentation medium is about 0.05 g / L, about 0.10 g / L, about 0.12 g / L, about 0.14 g / L, about 0.16 g / L, about 0.18 g / L, about 0.20 g / L, about 0.22 g / L, about 0.24 g / L, about 0.26 g / L, about 0.28 g / L, about 0.30 g / L, about 0.32 g / L, about 0.34 g / L, about 0.36 g / L, about 0.38 g / L, about 0.40 g / L, about 0.45 g / L, or about 0.50 g / L, or any value therebetween. In one embodiment, the concentration of the medical antifoam C emulsion in the fermentation medium is about 0.24 g / L.

[0063] According to the present disclosure, the pH of the VTPM is between 5 and 8, preferably between 6 and 7.8, for example, about 6.1, 6.3, 6.5, 6.7, 6.9, 7.0, 7.1, 7.3, 7.5, and 7.7.

[0064] Culture conditions According to the present disclosure, the first step in producing botulinum toxin is pre-culturing Clostridium botulinum from a working cell bank (WCB). In one specific embodiment, the WCB is generated by first isolating a unique C. botulinum type A1 strain from a soil sample. The strain is further cultured to form spores, which are then frozen in multiple (e.g., 100) 0.5 mL aliquots as a master cell bank (MCB). Individual aliquots of the MCB are cultured to form spores, which are then frozen in multiple (e.g., 500) 0.5 mL aliquots as a WCB.

[0065] In a specific embodiment, the WCB is thawed and added to a fermentation bag containing vegetable toxin production medium (VTPM). In a specific embodiment, the fermentation bag is a sterile, flexible, single-use fermentation bag containing ports and / or tubing for medium feeding, inoculation, sample collection, and gas introduction and exhaust. In a specific embodiment, the fermentation bag is equipped with a 0.2 μm gas filter in the gas inlet and / or tubing to ensure a sterile environment. In a preferred embodiment, the VTPM is preheated to approximately 37±1°C and purged with filtered nitrogen gas to create an anaerobic environment.

[0066] An anaerobic environment is defined as an environment with a dissolved oxygen (DO) of less than 2%. In some embodiments, the dissolved oxygen (DO) may be less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01%. In one particular embodiment, the dissolved oxygen may be about 0%. In one particular embodiment, the WCB is thawed at room temperature for 5 minutes and then vortexed three times for 5 seconds each time before adding 400 μl of the WCB to a fermentation bag containing 500 mL of VTPM.

[0067] OD 600 The fermentation process is continued until the OD reaches an acceptable value, for example, between about 0.1 and about 1.0, between about 0.1 and about 0.5, or preferably between about 0.2 and about 0.4 (or any range therebetween), to create a preculture. 600 reaches a value of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0.

[0068] In a specific embodiment, microbiological purity testing for microorganisms other than C. botulinum is performed as a pre-culture in an in-process control. In a specific embodiment, this testing is performed to detect possible contamination of bacterial cultures during fermentation. Only Clostridium botulinum must be present and detected. This testing is performed in different media and environmental conditions to screen for the possible presence of any contaminating bacteria or fungi in the C. botulinum culture. In a specific embodiment, to detect anaerobic bacteria, 10 μL of the culture is streaked onto a sheep blood agar plate incubated at 30-35°C under anaerobic conditions. In a specific embodiment, to detect aerobic bacteria, 1 mL of the culture is mixed with TSA and incubated at 30-35°C. In a specific embodiment, to detect yeast and mold, 1 mL of the sample is mixed with SAB and incubated at 20-25°C. There should be no growth on the TSA plate or the SAB plate. Additionally, all colonies growing on sheep blood plates must have the same (clostridial) morphology. Colonies from the blood plates must then be Gram stained to show Gram-positive rod-shaped bacteria and spores. Viable counts of C. botulinum are analyzed for in-process monitoring.

[0069] The next step in producing botulinum toxin is the main culture. In one particular embodiment, the preculture is added to a fermentation bag containing 4500 ml of VTPM, preheated to 33±1°C, and purged with filtered nitrogen gas to create an anaerobic environment. An anaerobic environment is defined as an environment with dissolved oxygen (DO) less than 2%. In some embodiments, the dissolved oxygen (DO) is less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% (or In one particular embodiment, the dissolved oxygen may be about 0%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0%.

[0070] In some embodiments, the fermentation process continues for about 60 hours to about 80 hours, about 65 hours to about 75 hours, or about 67 hours to about 71 hours (or any range therebetween). In some embodiments, the fermentation process continues for about 60 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, or about 80 hours. In some embodiments, the fermentation process continues for about 69±2 hours, about 69±1 hour, about 69±0.5 hours, or about 69±0.2 hours. In one embodiment, step (c) is carried out for about 69 hours.

[0071] In one particular embodiment, testing for microbiological purity for microorganisms other than C. botulinum is performed as a pre-culture in an in-process control, and viable counts of C. botulinum are analyzed for in-process monitoring. [Example]

[0072] Example 1: Preculture A 2-L fermentation bag was filled with 500 mL of vegetable toxin production medium (VTPM) (Table 1), preheated to 37°C, and purged with filtered nitrogen gas to create an anaerobic environment. In-process control of dissolved oxygen (DO) was performed until DO was below 2%. One vial of working cell bank (WCB) was thawed at room temperature for 5 minutes, vortexed three times for 5 seconds each, and then 400 μl of WCB was added by pipette under Grade A air supply to the fermentation bag. The fermentation bag was then placed in the bioreactor.

[0073] For the working cell bank (WCB), a proprietary cell bank was used. Briefly, botulinum toxin type A1 was isolated from a soil sample. For this WCB, the toxin operon is 100% identical to that of strain Hall, ATCC 3502, a representative group I (proteolytic) botulinum toxin-producing bacterium.

[0074] Temperature: 37±1℃, stirring angle: 12°, vibration frequency: 12 min -1 The oxygen concentration (DO) and pH were monitored in real time. The fermentation was continued until OD 600 until the OD reached a value in the range of 0.2–0.4 after approximately 19 h. 600 In-process control was carried out.

[0075] At the end of the pre-incubation step, microbiological purity testing was performed for microorganisms other than C. botulinum as a pre-incubation in-process control. Viable counts of C. botulinum were analyzed for in-process monitoring.

[0076] [Table 1]

[0077] Example 2: Main culture and fermentation Under nitrogen gas flow, 4500 ml of VTPM was filled into a 10 L fermentation bag, and the stirring angle was 12° and the vibration frequency was 12 min -1 The bioreactor was preheated to 33±1°C. The preculture from the above step was added by siphoning into the fermentation bag. The bag was purged with nitrogen gas to create an anaerobic environment, and in-process control of dissolved oxygen (DO) was performed until the DO stabilized at less than 2%.

[0078] Fermentation continued for 69±2 hours from inoculation of the main culture. In-process control of the harvested culture is performed by testing for microbiological purity for organisms other than C. botulinum. Viable counts of C. botulinum are analyzed for in-process monitoring.

[0079] Briefly, microbiological purity testing is performed to detect possible contamination of bacterial cultures during fermentation. Only Clostridium botulinum should be present and detectable. This test is performed using different media and environmental conditions to screen for the possible presence of any contaminating bacteria or fungi in the C. botulinum culture. To detect anaerobic bacteria, 10 μL of the culture is streaked onto a sheep blood agar plate incubated at 30–35°C under anaerobic conditions. To detect aerobic bacteria, 1 mL of the culture is mixed with TSA and incubated at 30–35°C. To detect yeast and mold, 1 mL of the culture is mixed with SAB and incubated at 20–25°C. There should be no growth on the TSA and SAB plates. Additionally, all colonies growing on the sheep blood plate should have the same (clostridial) morphology. Colonies from the blood plate are then Gram stained and should show Gram-positive rod-shaped bacteria and spores.

[0080] [Table 2]

[0081] The main culture was sampled and analyzed for optical density at 600 nm (OD 600 The growth of C. botulinum is monitored by measuring the OD . Figure 2 shows the optical density curve (absorbance at 600 nm) for the main culture of C. botulinum in VTPM performed according to Example 2. The absorbance at 600 nm over time provides the growth curve of the main culture. The OD was measured over the first 15 hours. 600 Rapid growth is observed, with the OD increasing to approximately 7. After this, the OD increases due to bacterial lysis and the release of toxin molecules. 600 The OD decreased equally rapidly to approximately 1. For the remaining time, from approximately 40 hours to the 69 ± 2 hour harvest, the OD 600 has remained relatively stable and shows a slight increase.

[0082] Figure 3 shows the results of in-process pH monitoring during fermentation of the main culture. The pH of the main culture fluctuated from an initial pH of 7 to 1000 (OD ) per approximately 15 hours. 600 The typical pattern is that the pH drops to approximately 5.7 at 69 hours (i.e., the time of harvest) and then increases slowly to 6.3 at 69 hours (i.e., the time of harvest).

[0083] The yield of toxin in the main culture at harvest can be measured by a BONT / A-specific ELISA. The average concentration produced in the main culture grown according to Example 2 is 4.9 μg / mL, with a standard deviation of 0.75.

[0084] The ELISA protocol is based on the USP <1103> This is an indirect sandwich ELISA based on the principles and general methods described in "Immunological Test Methods—Enzyme-linked Immunosorbent Assay." This ELISA method is based on the immunological binding and detection of BoNT / A using two different BoNT / A-specific polyclonal antibodies.

[0085] A commercially available BoNT / A toxin-based protein standard serial dilution is prepared by diluting BoNT / A in PBS-Tween solution (0.05% Tween-20) to a concentration range of 3–28 ng / mL. Triplicate samples of the protein standard dilution range in PBS-Tween are added to microplate wells coated with polyclonal anti-BoNT / A antibodies. Incubation allows for antibody identification and binding of BoNT / A antigen to the wells. Each incubation is followed by an automated wash step with PBS-Tween solution.

[0086] Primary detection is achieved by the binding of another polyclonal anti-BoNT / A antibody, forming a sandwich complex. A secondary antibody conjugated to horseradish peroxidase (HRP) is then added, which binds to the primary antibody and detects BoNT / A within the sandwich complex. 3,3',5,5'-tetramethylbenzidine (TMB) substrate is then added to the sample wells. HRP converts the TMB substrate to a blue reaction product. A stop solution is added to halt the conversion of TMB and initiate the yellow color conversion of the remaining TMB. The absorbance of each microplate well is measured at 450 nm using a plate reader, and the measured absorbance is directly proportional to the amount of BoNT / A in the well. Sample absorbance values ​​are calculated by comparison with a standard curve based on absorbance values ​​obtained from standard dilutions of BoNT / A. Results are reported as mean values ​​(units: μg / mL).

[0087] During the fermentation of the main culture, the toxin becomes detectable in the medium after approximately 15 hours. When the toxin content in reduced fermentation samples of the main culture is analyzed by Western blot analysis using a polyclonal anti-BoNT / A antibody, different toxin heavy chain variants are detected. Figure 4 shows an example of such a Western blot analysis, confirming the formation of heavy chain band 1 and heavy chain band 2 during the main fermentation (samples from 20 to 77 hours).

[0088] Early in the fermentation, three major bands are visible, representing the 160 kDa uncleaved protoxin polypeptide, the approximately 100 kDa heavy chain band 1, and the heavy chain band 2, which runs just below fully mature BoNT / A band 1. During fermentation, the uncleaved protoxin polypeptide and heavy chain band 1 gradually disappear and are converted to the mature heavy chain band 2 isoform. At harvest (69 ± 2 hours), only the mature heavy chain band 2 isoform is present in the main culture.

[0089] The maturation of BoNT / A protein to the band 2 heavy chain isoform is regulated by fermentation time, but fermentation temperature is another important factor. Figure 5 shows a table containing cutouts (from Western blot analysis) of BoNT / A heavy chain variants at harvest times for main cultures grown at different temperatures. The table also provides the BoNT / A concentrations (as determined by ELISA) for the same samples. At temperatures of 30°C or lower, maturation is not fully complete by 69 hours of fermentation in the main culture. At fermentation temperatures of 35°C or higher, maturation to the band 2 heavy chain isoform is complete by 69 hours, but the BoNT / A concentration in the medium is low. These data suggest that the optimal temperature for the main culture is approximately 33°C. At this temperature, fully mature BoNT / A can be produced with a high toxin yield.

[0090] Example 3: Comparison with other plant-derived peptones Apart from wheat peptone, VTPM media for the growth of C. botulinum and the production of botulinum toxin can be based on other plant peptones (eg, soybean, potato, or faba bean peptone).

[0091] Figure 6 shows the amount of botulinum toxin obtained in main cultures of C. botulinum grown at 30°C in the same total amount of soy peptone-based VTPM (solid bars) or wheat peptone-based VTPM (shaded bars). The data show that wheat peptone not only results in a somewhat higher toxin yield, but also in a more robust and consistent process.

[0092] Figure 7 shows the toxin concentrations obtained in main cultures of C. botulinum grown at 30°C in VTPM based on potato, broad bean, or wheat peptone. All three VTPM media achieve toxin concentrations above 1 μg / mL at harvest. However, it can be seen that the wheat peptone yield achieves substantially higher amounts of toxin (approximately 4 μg / mL) than the potato and broad bean peptone-based VTPMs.

Claims

1. (a) providing a working cell bank (WCB) containing Clostridium botulinum; (b) adding the working cell bank to a first vessel containing a cell culture medium including wheat peptone and culturing the Clostridium botulinum in the cell culture medium under conditions that allow the Clostridium botulinum to grow and produce a preculture; (c) adding the pre-culture to a second vessel containing the cell culture medium and culturing the Clostridium botulinum under conditions conducive to the production of botulinum toxin; and (d) recovering the botulinum toxin; The cell culture medium is free of animal protein or contains less than 0.1% by weight of animal protein in the total cell culture medium; The cell culture medium contains wheat peptone at a concentration of 15 to 30 g / L. A method for producing botulinum toxin.

2. wherein step (c) comprises culturing the Clostridium botulinum at a temperature of 33±1°C, 33±0.5°C, or 33±0.2°C; The method of claim 1.

3. 3. The method of claim 1 or 2, wherein step (c) is carried out for 69±2 hours.

4. The method according to any one of claims 1 to 3, wherein the botulinum toxin is botulinum neurotoxin type A (BoNT / A).

5. the container used in steps (b) and (c) is a fermentation bag, and / or The conditions of steps (b) and (c) include an anaerobic environment. The method according to any one of claims 1 to 4.

6. 6. The method of claim 5, wherein the anaerobic environment has a dissolved oxygen (DO) concentration of less than 2%.

7. 7. The method of claim 1, wherein the conditions in step (b) include a temperature between 35°C and 39°C.

8. 8. The method of claim 1, wherein the conditions in step (c) include a temperature between 30°C and 37°C.

9. In the step (b), the volume ratio of the WCB to the cell culture medium is 2.0% or less. the volume ratio of the pre-culture to the cell culture medium in step (c) is between 1:2 and 1:50; O.D. ６００ step (b) is carried out until the value of ρ reaches the range of 0.1 to 1.0; and / or Step (b) is carried out for a period of between 10 and 30 hours. The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 2 and 4 to 9, wherein step (c) is carried out for a period of between 60 and 80 hours.

11. C. Microbiological purity with respect to microorganisms other than botulinum is tested in the preculture after step (b) and before step (c), and / or C. Microbiological purity with respect to microorganisms other than botulinum is tested in the culture after step (c) and before step (d). The method according to any one of claims 1 to 10.

12. 12. The method according to any one of claims 1 to 11, wherein the concentration of wheat peptone in the cell culture medium is between 15 g / L and 25 g / L.

13. 13. The method of any one of claims 1 to 12, wherein the cell culture medium comprises wheat peptone, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, and medical antifoam C emulsion.

14. The cell culture medium is wheat peptone between 15g / L and 25g / L; between 5 g / L and 50 g / L of yeast extract; D-(+)-glucose between 0.05 g / L and 20 g / L; Between 0.05 g / L and 0.50 g / L of L-cysteine ​​hydrochloride monohydrate, and Medicinal antifoam C emulsion between 0.05 g / L and 0.50 g / L, The method according to any one of claims 1 to 13.

15. 15. The method of any one of claims 1 to 14, wherein the pH of the cell culture medium is between 6.7 and 7.

2.

16. A method for producing a botulinum toxin comprising the steps of: The medium does not contain animal protein or the amount of animal protein is less than 0.1% by weight of the total medium; The medium comprises wheat peptone at a concentration of 15 to 30 g / L, yeast extract, D-(+)-glucose, L-cysteine ​​hydrochloride monohydrate, and medical antifoam C emulsion.

17. The medium is wheat peptone between 15g / L and 25g / L; between 5 g / L and 50 g / L of yeast extract; D-(+)-glucose between 1 g / L and 20 g / L; Between 0.05 g / L and 0.50 g / L of L-cysteine ​​hydrochloride monohydrate, and Medicinal antifoam C emulsion between 0.05 g / L and 0.50 g / L, 17. The composition of claim 16.

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