«2 United States Patent Donovan ‘USOO7189541B2 (10) Patent No. (4s) Date of Patent: US 7,189,541 B2 Mar. 13, 2007 (54) BOTULINUM TOXIN PRODUCTION METHOD, (75) Inventor: Stephen Donovan, Capistrano Beach, cA WS) (73) Assignee: Allergan, Ine, Irvine, CA (US) (*) Notice: Subject to any disclaimer, the term of this patent is extended oF adjusted under 35 USC. 154) by 0 days. (21) Appl. No. 11296015 (22) Filed: Dee. 7, 2008 ws) Prior Publication Data US 200610228777 Al Oct. 12, 2006 Related U.S. Application Data (62) Division of upplicaton No. 10'672,876, led on Sep. 25, 2008, now Pat. No, 7,148,081 (1) Incl. C12 2108 (200601) RN 100 (200601) CRN 120 (200601) (52) US.c ASS/TLAA; 435/252.7; 435/283.6: “435/842; 424/236.1: 4247239. (58) Fleld of Classitication Search 435711, “435/252.7,253.6,842;, 424843, 236.1, 230.1 ‘See application file for complete sear history. 66) References Cited USS. PATENT DOCUMENTS. 6.558926 BI 5/2005, Demmin ea. 2008118598 AL 62003 Hunt sea FOREIGN PATENT DOCUMENTS woovantts 101998 WOoK's1296 ——S/1998 Wo 0103997 Az 7/2000, Wo 0103997 AS 7/2000, WO O1-%6655 102000 woouse72 “22001 (OTHER PUBLICATIONS ‘Naumann, Me, Botulinum toxin type A inthe treatment of| focal allay and palmar Aypehiross wad bee hypehidotic c ‘These toxin types may be produced by selection from the appropriate physiologic group of Clostridium botulinum ‘organisms. The organisms designated as Group Late usually referred to as proteolytic and produce botulinum toxins of types A, B and F. The organisms designated as Group Il are saccharolytie and produce borulimim toxins of types B, F and F. The organisms designated as Group Ill produce only botulinum toxin types C and D and are distinguished from ‘organisms of Groups Land I by the production of significant amounts of propionic acid. Group IV organisms produce ‘only neurotoxin of type G. It's known to obtain a tetanus toxin using specifi media substantially fiee of animal products. See ext US. Pat. No. 6,558,926. But sotably, even the “animal product free” media disclosed by this patent uses Bacto-peptone, @ meat digest). Significantly, production of tetamis toxin. by ‘loscidium tani vs. produetion ofa botulinum wxin by & clostridium botulinum bacterium entails different growth, medi and fermentation parameters and considerations. See ‘eg. Johnson, E.A., et al, Clostridium bonulinum and is neurotoxins: metabolic and cellular perspective, Toxicon 39 (2001), 1703-1722. 8 ‘Wit is nooded therefore are media and processes which are free or substantially live of animal produets, sueh as ‘animal derived proteins, for obtaining or producing bologi- cally active botulinum toxin. SUMMARY, ‘The present invention meet this noed and provides media and processes which are free or substanially free of animal products, sch as animal derived proteins, for obtaining oF producing a biologically active Botulinum toxia, The Botu- Tinum toxin obtained can be used to make hotadinum toxin active ingredient phannaceutical compositions. Delinitions As used herein, the words or terms set forth below have the following definitions. “About” means that the item, parameter o term so qualified encompasses a rage of plus oF mints ten percent above and below the value ofthe stated item, parameter or term, “Administration”, or “to administer” means the step of aiving (ie. administering) a pharmaceutical composition to A subject. The pharmaceutical compositions disclosed herein ‘are “locally administered” bye, intramuscular (im), intr ermal, subcutaneous administration, intathecal admini ‘ration, intracranial, intrperitoneal Gp.) administration, Poorly igen _Femesaion Lipase awwid (nono) + Capmsnes topical (transdermal) and implantation (Le. of a slow-release device such as polymeric implant or miniosmotie pump) utes of administration, “Animal product free” of “substantially animal product fee” encompasses, respectively, “animal protein fee" or “substantially animal protein ree” and means the absence or substantial absence of blood derived, blood pooled and other animal derived products or compounds. “Animal” means a ‘mammal (such 3s human), bird, reptile is, inset, spider fr other animal species. “Animal” excludes microorgan- sms, such as bacteria, Thus, an animal produet free medinza or process or substantially animal product free medium or pracess within the scope of my invention can inchide a botulinum toxin oF a Clostridial botulimum bacterium. For ‘example, an animal product Free process or substantially animal prodet free process means process which i either substantially tree or essentially free or entirely fee of ‘animal derived proteins, such as immunoglobulins, mest gest, meat by products and milk or dairy products or tigests, Thus, an example of an animal product free process is a process (such as a bacterial culturing or bacterial {ermentation process) which excludes meat and dairy prod- vets or meat or dairy by produets.US 7,189,541 B2 9 “Rotuliaum toxin” means a neurotoxin produced by Clostridium botulinum, as well asa botulinum toxin (r the Fight chain or the heavy ehain thereof) made recombinantly by a non-Closeidial species. The phrase “botulinum toxin", ‘2 used herein, encompasses the borulinun toxin serotypes A,B,C, D,A, P and G. Botulinum toxin, as used herein, also ‘encompasses both a botulinum toxin complex (ie. the 300, {600 and 900 kDa complexes) as well asthe purified bon fing toxin (1. about 150 kDa) “Partie botulinum toxin js defined asa botulinum toxin that is isolated, or substan- Wally bolted, from other proeins, including proteins that orm a dotulinum xia complex. purilied borudimen oxi may be greater than 95% pure, and preferably is greater than 59% pure, The botulinum C, and C, eytotoxins, not being neurotoxins, are excluded from the scope of the present ‘invention. Tostridial neurotoxin” means a neurotoxin produced from, or native to, a Closiridial bacterium, such as Closiridium botulinum, Clostridium butyricum oF Clostridium beratt 8 well s a Clostridial neurotoxin made recombinantly by a non-Clostridial species. iatrely free” (Le. “consisting of” terminology) means that within the detection range ofthe instrument or process being used, the substance cannot he detected or its presence ‘cannot he contirmed. ssentally fre” (or “consisting essentially of”) means that only trace amounts ofthe substance can be detected “tmmobilizing” means a step that prevents subject fom, raving one or more body pars. Ifa sufficient number of body parts are immobilized, the subject will accordingly be ‘immobilized. Thus, “immobilizing” encompasses the immo- bilization ofa body part, such asa limb, and/or the complete immobilization of a subject. “Modified Botulinin toxin” means «botulinum toxin tat has hd at least one ofits amino acids deleted, modified, or replaced, as compared to a native botulinum toxin. Ad tionally, the modified Botulinum toxin ean be a recom! nantly produced neurovoxin, oe derivative or fragment of 1 recombinantly made neurotoxin. A modified ulinun toxin resins at least one biological activity of the native botulinum toxin, such as, the ability to bind to & botulinum toxin receptor, of the ability t0 inhibit neurotransmitter release from a neuron, One example of a modified botulinum toxin is a Botulinum toxin that has aight chain fom one ‘olinum toxin serotype (such as serotype A), and a heavy chain from a different boruinwn toxin serotype (Such a8 serotype B). Another example ofa modified horalimum toxin 's.a botulinum toxin coupled to a neurotransmiter, such as substance P, Patient” means a human or non-human subject receiving medical or veterinary care. Accordingly, as disclosed herein the compositions may be used in treating any animal, such ‘9s mammal “Pharmaceutical composition” means a formulation in Which an active ingredient can be a Aotulimum toxin. The sword “formulation” means that there is atleast one ad tional ingredient inthe pharmaceutical eomposition besides a nevrotoxin active ingredient. A pharmaceutical composi tion is therefore a formulation which is suitable Tor diag nostic or therapeutic administration (i. by intramuscular oF subcutaneous injection or by insertion of a depot or implant) to a subject, such as a human patient. The phamceutical ‘composition can be: in a lyophilized or vacuum dried ‘condition; & solution formed after reconstitution of the lyophilized or vacuum dried pharmaceutical composition swith saline or water, or asa solution which does nat require reconstitution, The active ingredient can be one of the 0 o 10 Botulinum toxin serotypes A, B, Cy, D, E, F or G or a Botulinum toxin, all of which can be made natively by CClosridial bacteria, As stated, a pharmaceutieal composition can be liguid or solid, for example vacin-dried. The ‘constituent ingredients of a pharmaceutical composition ean be inched ina single composition (that sal the consti ent ingredients, except for any required reconstitution fui fare present atthe time of initial compounding of the phar :maceutical composition) or as a two-component system, for ccximple a vactumedried composition reconstituted with a ‘ilvent such as saline which diluent contains an ingredient ‘ot present inthe inital compounding of the pharmaceutical ‘composition. A two-component system provides the benefit ‘of allowing incorporation of ingredients Which are_not sulicently compatible for long-term shelf storage with the first component of te two component system. For example the reconstitution vehicle of diluent may include a preset vative which provides sullicint protection against microbial srowth forthe use period, for example one-week of relig- feraed storage, but isnot present during the wo-year freezer Storage period during which time it might degrade the toxin. Other ingredients, which may not be compatible with a CClostridial toxin of other ingredients for long periods of time, may be incorporated inthis manner; that, alded in second vehicle (ie. in the reconstitution fluid) at the ‘approximate time of use. Methods for formulating a Bote Tnum toxin setive ingredient pharmaceutical composition ‘are disclosed in U.S. patent publication 2003 0118598 A. “Substantially fre” means present ata level of less than ‘one percent by weight ofthe pharmaceutical composition “Therapeutic formulation” means a formulation ean be used to iret and thereby alleviate disorder or a disease stich asa disorder ora disease characterined by hyperactivity Ge. spasticity) ofa peripheral muscle, ‘The present invention provides media which comprise at least reduced levels of animal or day byproducts and are preferably substantially free of animal or daity byproducts “Animal or dairy byproduets;” means any compound or combination of compounds which was paodiced in oF by an animal (excluding a bacterial) cell, whether in vivo or in vitro, Preferred non-animal sources of media ingredients such as proteins, amino acids; and nitrogen, include vege tables, microbes (such as yeast) and synthetic compounds. ‘My invention also provides methods for obtaining boc linum toxin using at Teast one medium that is substatilly {re of animal or dairy byproducts, For example, the bot ‘imum toxin can be obtained by eulturing Clostridium botu- ‘num in a fermentation medium which is substantially free of animal products. ‘My invention also encompasses, a botulinum toxin joblained by culturing Clostridium botulinum in a fermen ‘ion medium substantially fee of animal produets and which comprises vegetable derived products. Additionally, 1 bonu- Tinum toxin ean be obtained by culturing Clostridium botu- Tinum in a fermentation medium which is substantially free ‘of animal products and which comprises some soy-based products. Tn another prefered embodiment, botulinum toxin can be obtained by culturing Clostridivm Botulinum in a fer ‘mentation medium substantially free of animal products and containing hydrolyzed soy, 28 a substitute for animal-de- rived products, Preferably, growth na fermentation medium proceeds until at least cell lysis occurs, The source of CClosiridium botulinum uses for inoculation of the fenen- tation medium may be obtained from a seed medium con- taining Clostridium botulinum. Preferably, Clostridium Botulinum grown ina seed medium and used 2s an inoculaUS 7,189,541 B2 un {ora fermentation medium hs not undergone cell lysis. The source of Clastridium botulinum used for inoculation of the seed medium may be obtained from a Iyophilized culture. Clostridium botulinum may be lyophilized as @ eulture in ‘animal milk oF soy milk. Preferably the Closiridivm bot Tinun is lyophilized as a culture in soy mle The present invention also provides a composition com= prising a medium substantially fre of animal-derived prod- leis for culturing Clostridiwn botulinum. none embodiment, the composition comprises 2 medium substantially fee of animal- media and processes which ate free or substantially free of fan animal product or an animal byproduct useful for culture and fermentation of an omganism (such as a Clostridium botulinum bacterium) capable of producing biologically active botulinum toxin The botulinum toxin obtained can be Used for making botulinum toxin active ingredient pharma- ceutical compositions. Thus, growih media are disclosed herein which ave significantly reduced levels of meat oF dairy by-produets and preferred media embodiments are substantially free of such animal products The present invention encompasses my surprising finding that antimalsbased products are not required in media for srowth of Clostridium botulinum, and particularly that veg ‘etable-based products can replace animal-based products ‘ypically employed in such media for the growih of Clostridium botulinum. ‘Media that are in curent use for growih and fermentation ‘of bacteria usually comprise one or more animal derived ingredien’s. In accordance with my invention, preferred media for growth of Clostridium botulinum contain anima rived ingredients which comprise no more than about five to about ten pereent of the total weight of the media. Mare preferably, media within the scope of my invention comprise ho more than about one to less than about Five percent ofthe total weight of the media of anima-derived products, Most preferably, all modia and cultures used for the growth of lostriium botulinum forthe production of botulinum oxi re completely free of animal detived products. These medi Jnclude bu are not limited to media for smal and large scale formentation of Clostridium boninum, media for growth of ‘cultures of Clostridium borulimum used to inoculate the seed (first) media and fermentation (second) media, aswell sand media used for ong-term storage of cultures of Clostridivn boulinum (eg. stock cultures) In cerin preferred embodiments of my invention, the media forthe growth of Clostridium botulinum and prode- tion of borulnunn toxin ean eomprise soy based procs to replace animal derived products. Alternately, instead of @ soy based product there ean be used debittered seed of ins campestis. [tis known the protein content of L ‘campestris sce is very similar to that of soyhean. Prefer: ably, these media include soybean or of L. campestris, 0 o 12 derived produets that are hydrolyzed and that are soluble water. However, insoluble soy or of L. campestris products ‘ean also be used inthe present invention to replace anil products. Common animal derived products which ean be substituted by soy or of. campestris products include beet ‘bar infusion (BHI), animal derived peptone products, such as, Bacto-peplone, hydrolyzed easeins, and dry by-prod- vets such as animal milk. Preferably media containing soy-based or of L. campes- cis based products fr the growth of Clostridium botulinum are similar to commonly used growth: media containing ‘animal derived products except tht Substantially all imal- Serived products are replaced with vegetablederived prod. vets. For example, soy based fermentation media can com- prise a soy based product, a source of earboa such as shucose, salts such as NaCl and KCl, phosphate-containing ‘ingredients such a8 Na,HPOs, KH,PO,, dlvalent cations suel as roa and magnesium, iron powder, and amino acids such as Leeysteine and L-ytosine. Media used t0 grow cultures of Clostridium Botulinum for inoculation (i. the sood or first medium) of the fermentation (Sscond) media preferably contain atleast o soy based product, a source of Salt such as NaCl, and a carbon source sich as glicose ‘The present invention provides a method forthe growth of | Clostridium botulinum that maximizes the production of a Botulinum toxin using media that ae substantially free of animal-derived products. Growth for produetion of Closiridium botulinum aod botadinum toxin can take place by fermentation in media containing soy by-products that replace ingredients derived fom animal by-products. The Jnoctlaat for the fermentation medium can he derived from 1 smaller scaled growth medium (a seed medium). Depend- jing on the size and volume of the fermentation step, the of successive growths in seed media to inerease the biomass of the culture ean vay. To grow a suitable amount of Clostridium botulinum for inoculating the fermentation ‘medium, one step or multiple steps involving growth in a seed! medium can be performed. For a method of growing Clostridium botulinum tat is foe of snimal derived prod ucts, itis preferable that growth of Clostridium Boralimum originates from a culture stored in aon animal derived media, The stored culture, preferably lyophilized, is pro- duced by growth in media containing proteins derived from soy and lacking animal by-products. Growth of Clostridium Botulinum in Termentation medium can take place by inoculation directly from a stored, lyophilized culture, Tn a preferred embodiment of the present invention, growth of Closiridium orulimum proceeds in to phases: Sood growth and fermentation. Both of these phases are carved out in anaerobic environments. The seed growth phase is generally used to “scale-up” the quantity of the ‘microorganism from a stored culture. The purpose of the seed growth phase) i to inrease the quantity of the miero- ‘organism available for fermentation, In addition, the sesd growth phase allows relatively dormant microbes in stored tnltures to rejuvenate and grow into atively growing cul- tures. Furthermore, the volume and quantity of Viable miero- ‘onanism used to inoculate the fermentation culture can be controlled more accurately from an actively growing culture than froma stored culture. Thus, growth ofa seed euture for inoculation of the fementation medium is preferred. In addition, ny number of consecutive steps involving growth in seed media 10 scale-up the quantity of Clostridium botulinum for inoceltion of the fermentation medium can De used, [eis noted that growth of Clostridium boraimum in the fermentation phase ean procecd dirty from the stoned culture by direct inoculationUS 7,189,541 B2 13 In the fermentation phase, a portion of seed medium oF allof' seed medium containing Clostridium Boralinum fom, the seed prowth is used t0 inoculate a fermentation medium. Preferably approximately 2-4% of a seed medium having Clostridium botulinum from the seod growth phase is used to inoculate the fermentation medium. Fermentation is used to produce the maximum amoint of microbe in large-scale anserobie environment (Ljungdahl et al, Marval of indus trial microbiology and biotechnology (1086), edited by Demain et al, American Society for Microbiology, Wash- ington, D.C. page. 84), ‘A botulinum toxin can be isolated and purified using methods of protein purification well known 10 those of ‘ordinary skill in the protein puriication art (Coligan etal Curent Protocols in Protein Science, Wiley & Sons: Ozat- sumi etal. Appl. Environ. Microbiol. 49; 939-943:1988. Por production of Botulinum toxin, cultures of Clostridium botulinum 30 can be grown in a seed medium {or inoculation ofthe fermentation medium. The mumber of successive steps involving growth in a seed medium can vary depending on the seale ofthe production of botulinn toxin in the fermentaion phase. However, as previously «discussed, growth in the fermentation phase may proceed directly from inoculation from a stored culture. Animal based seed media generally are comprised of BEI, bacto- peptone, NaCl, and glucose for growth of Clostridivn ‘orulinum. As previously discussed, alternative seed media ‘ay he prepared in accordance with the present invention ia Which animal-based components are substituted Wit snimal-bosed components, For example but without limit tion, soy-based products ean substitute for BET and bacto- pepione in the seed medium for growth of Clostridium botulinum and produetion of Botulinum Toxin. Preferably, the soy-based product is soluble in water and comprises hydrolyzed soy, although cultures of Clostridium botulinum ‘can grow in media containing insoluble soy. However, levels, ‘of growth and subsequent loxin production are greater in reiia derived from soluble soy products, ‘Any source of say-hased products may be used i nooo dance with the present invention. Preferably, the soy is, hydrolyzed soy. Sources of hydrolyzed soy are available froma variety of commercial vendors, These include but are not limited 10 Hy-Soy (Quest Intemational), Soy peptone (Gibeo) Bac-soytone (Difco), AMISOY (Quest), NZ. soy, (Quest), NZ soy BL4, NZ.soy BLT, SESOM (DMV Inter national Nutritionals, Fraser, N.Y), and SESOMK (DMV). Most preferably. the source of hydrolyzed soy is Ti SESOMK. Other potential sources of hydrolyzed say are knows, Concentrations of Hy-Soy in the seed medium in accor ‘dance with the present invention range between 25-200 ai. Preferably, the concentration of Hy-Soy inthe seed medium ranges between 50-150 giL. Most preferably the eoncentra- tion of Hy-Soy in the seed medium is approximately 100 ‘lL. In addition, the concentration of NaCl ranges between, 0.120 gL. Preferably the concentration of NaCl ranges, between 0.2-1.0 g/L. Most preferably, the concentration of NaCl in the seed medium is approximately 05 wl. The ‘concentration of glucose ranges between 0.1 y/L and 5.0L. Preferably, the concentration of glueose ranges between (05-20. Most preferably the concentration of glucose in the seed medium ts approximately 1.0 gL. Tt i also pre- fered but ot necessary forthe present invention that the hucose is sterilized by autoclaving together with the other ‘componcnts of the seed medim. The preferred pll level of the seed medium prior to growth ranges between 7.5-85. 0 o 14 ‘Most preferably, the pH of the seed medium prior to grow ‘of Clostridium botulinum is approximately 8. Growth of Clostridium botulinum in the seed media ‘may proceed in one or more stages. Preferably, growth inthe ‘ood medinm proceeds in two stages. In stage one, acultare ff Clostridium botulinum is suspended ina quantity of soedmedium and incubated at 3431° C. for 24-48 hours in fan anaerobic environment. Prefersbly, growth in stage one proceeds for approximately 48 hours. In stage two, «portion for all of the stage one medium containing. Clostridium ‘orulinum is used 0 inoculate a stage two seed medium for further growth. After inoculation, the stage two medium is ‘incubated at 3421° C. for approximately 1-4 days also in an ‘anaerobic environment. Preferably, rowih in the stage wo seed medium proceeds for approximately 3 days. It is also preferable that growth in seed media in any stage does not result in cel ysis before inoculation of fermentation media wih the inal grow in seed media, Standard fermentation media containing animal by-prod vets for the growth af Clostridium Botulinum can be based ‘ona recipe of Mueller and Miller (MM J. Bacteriol. 67.271 1984). The ingredients in MM media containing animal by-prochets include BIIL and NZ-CaseTT. NZ-CaseTT is a commercially available source of peptides and amino acids ‘whieh are derived from the enzymatic digestion of casein 4 group of proteins fourd in animal milk, The prese invention demonstrates that non-animal based products may be substituted for BHI and NZ-CaseTT ia fermentation ‘media, Por example but withowt limitation, soy-based prod- vets can replace the anial-based components of MM media ted for fermentation of Clostridium botulinum. Preferably, the soy-based products are Water-soluble and derived from bytrolyzed soy. although as previously discussed, insoluble soy products can also be used to practice the present Any souree of soy-based products may be used in aeeor. {dance withthe present invention. Preferably, the hydrolyzed soy is obtained from Quest International (Sheffield) under the tmdename, Hy-Soy of fom DMV Intemational Natei- sionals (Fraser, NY.) under the tradename, SESOMK. Soluble soy products ca he also obtained from a variety of sources including but not limited to Soy peptone (Cibeo) Bac-soytone (Difeo), AMISOY (Quest), NZ soy (Quest), NZ soy BL, NZ soy BLT, and SESOMK (DMV International Nutetionals, Fraser, N.Y). In another prefered embodiment ofthe present inveation, ‘the medium wsed for fermentation of Clostridium Botulinum is ftee of animal by-products and comprises hydrolyzed soy, hicose, NaCl, Na,HPO,, MgSO,7H,0, KH,PO,, L-cys- teine, L-tyrosine, and powdered ion. As disclosed for the ood medium, hydrolyzed soy can replace animal by-prod- vets in fermeniation medium. These animal by-products include BHI and NZ-CaseTT (enzymatically digested cass). ‘The concentration of Hy-Soy in the fermentation medium {or prodiction of dotulinun toxin preferably ranges between approximately 10-100 gi. Preferably, the concentration of Hy-Soy ranges between approximately 20-60 g/L. Most preferably, the concentration of Hy-Soy in the fermentation ‘medium is approximately 35 /L. For msximal production of botulinum toxin, particularly preferred concentrations of fcompontents in the fermentation medium are approximately Sls alueose: 80 PU NACL,0.5 gL NashPOr: 175 mp KH;P0,,50 mg/L MgSO,7H,0; 125 mg/l. L-oysteine; and 125 mg/L Ltyrosine, The amount of powdered iron used ean range from 50 mg/L to 2000 mg/L. Preferably the amonnt of powdered ira ranges beween approximately, 100 mg/LUS 7,189,541 B2 15, nd 1000 mg/L. Most preferably, the amount of powdered iron used in fermentation media ranges between spproxi- mately 200 mpl. and 600 mg/L. For optimal levels of toxin production, the initial pH (before autoclaving) of the soy-based fermentation media ranges profermbly between approximately 5.510 7.1. Prel- ‘erably the initial pH of the fermentation medium is benseen, approximately 6.0 to 6.2.x described forthe seed medium, the components of the fermentation medium, including lucose and iron, are prelerably autoclaved together for serlzation, Preferably, a portion of the second stage seed medium used for growth of Clostridium borin is used to inoeu- Tate the fermentation medium, Fermentation occurs in an anaerobic chamber at approximately 421° C. for approxi mately 7 to 9 days, Growth is monitored by measuring the ‘optical density (O.D,) of the medium. Fermentation prefer ably is stopped afer cell lysis has proceeded for at least 48 hours a determined by growth measurement (optical den- sity). As cell lyse, he OD. ofthe medium will decrease. Tn a preferred embodiment of the present invention, cultures of Clostridium Botulinum used Tor long-term stor. age of Clostridivm botulinum and inoculation of the seed medium are grown and lyophilized in soy-milk prior t0 storage at 4° C, Cultures of Clostridium botulinum ia animal milk lyophilized for store ean also be use for the pro- ‘duction of otulinum toxin. However to maintain media that are substantially fee of animal by-products throughout the production of Botulinum toxin itis preferred thatthe initial culture of Clostridium botulinum be preserved in soy milk fan not animal mil, EXAMPLES The following examples set forth specific. methods ‘encompassed by the present invention and are not intended to limit the scope of the invention. Clostridium botulinum ‘cultures ean be obtained from several sources, including Lis, Laboratories, Camphell, Calif. All experimeats and media ‘ean be prepared with double-distilled water. Ia all the Examples below “Clostridium botufian’™ moans the Hall A (ATCC designation number 3502) strain of Clostridivn botulinum type A. Example 1 Preparation of an Animal Product Free Seed ‘Medium for Clostridium Bouulinum A control seed mesium can be prepared using the fllow= ing ingredients for each one 1 Iter af medium: NaCl (5 2). Bacto-peptone (10 g), glucose (10 g), BEI (to liter), pl18.1 (adjusted with 5 N NaOH). ‘Atest (animal product foe) sood medium can be prepared using the following ingredients for each one 1 Titer of medium: NaCl (5 g), Soy-peptone (10 g), glucose (10 2). Hy-Soy (35 ptr to make up 1 liter of media uid), pH 8.1 (oajusted with 5 N NaOH). Example 2 Culturing Clostridium Borutinum in an Animal Product Free Seed Mediu Alyophilized culture ofthe Clostridium botulinum can be suspended in I ml of each of the control and test sced medium of Example I, divided (each seed media) into two 0 o 16 tubes of which each ean contain 10 ml of the respective seed ‘media, and then incubated at 34? C. for sbout 24-48 hours. ‘One mil of culture ean be then used 10 inoculate a 125 ml Delong Relleo Culture Flask contsining. 40 ml of (the respective) seed media, The inoculated culture can be inew- bated at 33° C. 21° C. for 24 hours in a Coy Anaerobic Chamber (Coy Laboratory Products Inc, Grass Lake, Mich). Example 3 Preparation of an Animal Product Free Fermentation Media for Clostridium Botulinum A bassl fermentation medium can be prepared sng the following ingredients foreach liters of medium glacose (18 4) NaC (10g), Nall,PO, (1g), KHPO, (0350 2) ‘MgS0,7H,0 (0.1 ), cysteine HC (0.280 a, osine-HC1 (0.280, powderet ion (13), ZC (0.280) and MaCl, 04) ‘A contol fermentation medium can be prepared sing the following ingredients for each two lites of medium pre pared: BHI (300m this erresponds to about 48.5 grams of fry weight beef nea infsion), NZ-CaseTT G0 g). and ‘sal medium (io 2 ier), pl 68. “The basal fementation medium can be prepared stand its adjusted to pl 68. The boc! Bear infusion (BUI) BEL can then be prepared and i's pH adjusted t0 O8 with $ N NaOH, The BHI can then be added to the basal medium, Next the NZ-CaveT Tea be prepared. The NZ-Case TT is then ald tothe to basal medium to which the bee! heart infusion has already boon aed, and dissolved by adtion OHI. The pH eas then be ajusted to 68 wilh SN NaOH ‘This medion ean then be separated ino 8m portions into cach of sixteen 10 nm est tubes, following by autoclaving for 25 minutes at 120°C ‘Ates fermentation medium (anim product few) can be pecpured by substituting a stato source forthe BEL the control fermentation modium. Suitable test wt medium sirogen sources ‘include: Hy.Soy (Quest), AMI-Say (Quest), NZ-Soy (Quest), NZ-Soy BLA (Ques), NZ-Soy BET (Ques), Sheftone D_(Shecld), SESOM (DMV), SES0 (DMV), SE_°%)MK (DMV), Soy Peptone (Gibco), BactoSoyton (Dito), Nutsisoy 2207 (ADM), Bakes Nutsoy (ADM) Nutisoy fou, Soybean tea, Bacto-Yeast Extuet Difco) Yeast Extoct (Gib) HiyrVest 412 (Ques), Hy-Yest 441 (Ques), Hy-Yext 4 (Ques), Hy-est 485 (Quest) Bacto-Malt Exit (Dito) Com Step, and Proo (Traer) The tes ermentation-medium can be prepared asset forth above fora control fensentation mesa exeept tht BEL is excluded and the relevant nitrogen soure can be fest adjusted to pH 638 with 3 N HCTor with § N NaOH. The ‘media canbe allocated ton 8 ml portions to sixteen 100 mm test bes, followed by autoclaving for 20-30 axinutes at 120°C Example 4 Grovsth of Clostridium Botulinum in a Asien Product Free Fermentation Medium A 40 4 portion of the test seed medinm cult (animal product free) can be used tp inoculate each 8 ml contol or {est fermentation medium aliquot in an 8 ml 16«100 mm test tube, The culties ean then be incubated at 331° C. for 24 hours Tubes cain thea be incubated in an anaerobic chamberUS 7,189,541 B2 17 to allow for growth of the bacterium. Fach medium assay ‘can be performed in triplicate (Le. cam involve three inde pendent inoculations of the same medium), and ean also ‘nchude a non-inoculated contol which can be used as the blank forthe spectrophotometer). Growth (as determined by ‘optical density, OD) can be measured every 24 hours with Tumer Speetrophotometer (Model 330) st 660 nm. Cultivae tion should he stopped after cell ysis has lasted for abot 48 hours and fotulinum toxin production can then be mieasure. ‘Adiitional experiments can be earied out with « Hy-Soy Jermentation medium containing the following ingredients {or each $00 mi of the medium: Hy-Soy (17.5 g), glucose G.75 py; NaCl 25g); ‘NasHPO, (0.25 g), MgSO,7H,0 (00025 g), KH,PO, (0.0875 g), L-cysteine (0.0825 2), L-ty- rosine (0.0625 g), powdered iron (0.25 2), pH 63 Example 5 Determination of Ratulinum Toxin Production by (Clostridium Bovulnun Growa in ata Anita Product Free Fermentation Medium ‘The cultured cells of Example 4 can be centrifuge, and the pif of the supernatant then detemnined. The levels of botulinum toxin in a given sample can be measured by ‘ding 2 standard anttoxin and measuring the elapsed time before flocculation. Both KF (the time required for floceu- Jatin to ecu, in minutes) and LE (the limit of Roceulation: ‘equivalent to 1 international unit of standard antivoxin, as ‘established by flocculation) ean be detemnined. 4 ml of Termentation broth can be taken rom each fermentation tube fora given culture, and can be combined together so thst 12 rl (otal ean be mixed ia a 15 ml centrifuge tube, The tubes ‘canbe centrifuged at 5000 rpm (3400 g) for 30 min at 4° C. 1 mialiquots of supematant can be added to tubes containing (0.1-0.6 ml of standaed botulinum toxin antiserum, and the tubes ean be carefully shaken to mix their contents, The tubes can then be placed in a water bath at 4521° C. and the initial time can be recorded, The tubes can be checked frequently, andthe time at which flocculation began can be recorded as KE, The concentration of toxin in the tube ia ‘which flocculation ean be first initiated ean be designated LFF. The concentration of toxin in the the in which Alooctlation ean be initiated! second can be designated L1P. Parallel fermentation, growth and toxin production assays ‘ean be eatied out for both of: (2) the contol seed medium (used to inoculate the contol fermentation medium) and the ‘control fermentation medium, to and; (2) the (animal prod uct five), test seed medium (used 10 inoculate the test formentation medium) and the (animal product free) tst Jermentation medium, Signifieanty, it ean be determined thatthe fermentation of Clostridium hotulinwn in media free ‘of animal products and inoculated from cultures also free of imal products (with soy-bose products replacing the ani- mal products) cat result in a Lf OF approximately 50 or ‘more. Minimally, Lf, equals approximately 10, Prefer IY the Lay is at feast 20, Most preferably the Lig iS treater than 30, “Additionally. it can be determined that variows soy prod= vets support Clostridium botulinum growth in fermentati media lacking BHI, Thus soluble soy preparations can replace BHI for growth of Clostridium Botulinum. The best ‘concentration cu he 12.5 or 25 g/L. Hy-Soy (Shelield) cas, ive the highest growth, Iasoluble soy preparations ean be Jess effective TFunhermore, results can be obtained to show that Quest Hy-Soy, DMV SESOMK, 0 o 18 soy products in terms of their sbilty to place BHI for Closiridium botulinum growth, The results can reveal that the soy products (such as Quest Hy-Soy, DMV SESOMK, ane Quest NZ-Soy} that may be optimal for growth can also be ellotive at replacing BI for toxin production, The best soy product for toxin production can be Quest Ily-Soy at 22.75 ail. Higher concentrations of this produet may py «duce beter growth but not improve toxin production. Simi Jar results can, its proposed, be obtained with SESOMK, for 4 higher concentration may generate inereased growth, but oot ineresse toxin production. NZ-Soy, on the ‘ther hand, may give higher growth and higher toxin pro- duction a its higher concentration, Finally, i ean be determined that soy products can effer: tively replace BHT as well as the NZ-CaseTT. Removal of NZ-CaseTT from soy-hased media can rece growth of shout 2-4 fold. The best soy precact for growth both in the presence and the absence of NZ-CaseTI can be SESOMK. HY-Soy can replace both BHI and NZ-CaseTT for toxin production. However, a longer fermentation eyele of 1 or 2 ays may be necessary. HY-Soy could replace both BHT and NZ-CaseTT in media for toxin production. However, it can be determined that yeast extracts can be inhibitory 10 toxin production, It can be determined that HY-Soy at 22.75 g/l may completely replace both BIL and HY-CaseTT foros production. Unlike the effect on growth where 56.88 a) HY-Soy can be best, 4.13 gil HY'Soy ean be best for the toxin production phase ‘Thus, I have surprisingly determined if Hy-Soy ot [Hy- Soysy-Yest] can replace BHT and Baeto-peptone in media for seed growth of Clostridium bonulimon. In addition, experiments can be designed to determine the optimam fons of components in sd media to produce the levels of Botulinum toxin produetion by the clostridium botulinum, “Toxin production by Clostridium ‘oralinum grow in sed medium and fermentation medium that is Iroc of BHT and NZ-CaseTT eun reach or excood levels attained in media containing BHT and NZ-CaseTT. 1 can be determined that the optimum concentrations of Hy-Soy or [Hy-SoysHy-Yest] for growth in the seed ‘medium. Experiments can confirm that Hy-Soy ean replace BHI and Bacto-peptone as the nitrogen source in seed ‘medium for growth of Clostridium botulinum and for pro= duction of Botuliauss Toxin inthe subsequent fermentation phase. Also, Hy-Soy a nitrogen source in the seed medium, as compared to Hy-Soy plus Hy-Yest, can produce higher levels of Botulinum Toxin in the subsequent fermentation sep. The concentrations of Hy-Soy in seed medium that pradace the best levels of toxin range from approximately 625 pil to 100 p11 Additional experiments designed to determine the op ‘mum concentrations of Hy-Soy in the seed medium for the ‘maximum production of Botulisum toxin by Clostridium botulinum by fermentation. Thus, 30 50 g, 75 and 100 ‘20f Hy-Soy in the see! medium can all rested in prodve- ‘ion of botulinum toxin by fermentation of Clostridium Botulinum aoxl this is comparable or exceeds levels of botulinun: toxin made in seed medium containing BHI and ucto-peptone asa nitrogen source. ean be found that a concentration af 100 i. Hy-Soy in the seed medium resulted in the highest levels of toxin production inthe subsequent fermentation stp. In addition, the data indicate that scedslep-1 of Hy-Soy seed mesinm produced greater growth after 48 hours than after 24 hoursUS 7,189,541 B2 19 ‘Various publications, patents andlor references have bee fed herein, the conteuls of which, in their emiretes, are Jncomporated herein by reference ‘Although the present invention has been deseribed in detail with regard to certain prefered methods, other ‘embodiments, versions, and modifications within the scope ‘of the present invention are possible. For example, a wide variety of animal prod lrce processes are within the seupe of the present inveation Accordingly. the spirit and scope ofthe following claims should not be Timited to the descriptions of the preferred ‘embodiments set forth above. Telaim: 1. A method for production of a botulinum toxin, the method comprising the steps of (@) providing a first medium that is no more that 1% by’ ‘Weight of an animal based product and that comprises 4 protein product of soybea (©) culturing @ Clostridium botulinum bacterium in the Tirst medium under condition at permit growth of the Clostridium botulinum bacterium: (©) providing a seoond medim that is no more than 1% bby weight of an animal based product and that com prises a protein product of soybean: (@) ingeulatng the Second medium wit the frst mediom; (e) culturing the Clostridium Botulinum bacterium in the ‘second medium under conditions that allow production ofa botulinum toxin: and () recovering the botulinum toxin 2. The method of claim 1, wherein in the step of providing first medium, the ist mesiam comprises a bydrolyzed soy, ‘and wherein inthe step of inoculating a second medium, the second medium comprises « hydrolyzed so. '3. The method of claims 1, wherein in the step of culturing a Closridium botulinum in. a first mesium, the conditions ‘comprise a temperature of about 34 degrees Celsius, and furer comprise no decrease in cell density during cul ing, ‘wherein Inthe step of inoculating a sccond medium with 4 first medium, 2 to 4 percent of the first medium is tused to inoculate the second medium, and wherein in the step of culturing the bacterium in the second ‘medium, the conditions that allow growth comprise 2 temperature of approximately 34 degrees Celsius and further comprise culturing until cell density of the cnlture decreases due 0 eel ysis. 20 4. The method of claim 1, wherein dhe botulinum toxin is selected from the group consisting of Botulinum toxins types A,B,C, D.E, Fand G, '. The method of claim 1, wherein the botulinum toxin is Donulinum toxin type A. 6. The method of claim 1, wherein the botulinum toxin is further purified. 7. A method for prxucton of a Borulinam toxin type A the method comprising the steps of () providing a frst medium that is no more that 19% by ‘weight of an animal bas proc and that comprises 2 protein product of soybean; (b) culturing a Clostridium Botulinum type A bacterium in the fist medium under conditions thst permit growth of the Clostridium botulinum bacterium, (©) providing a second medium that is no more than 1% ‘by weight of an animal based product and that com> prises protein product of saybean: (6) inoculating the second medium with the frst medinm: () culturing the Clostridium Borulimem bacterium ia the second medium under condition tit allow production (fa Botulinum toxin type Az ad (0 recovering the botulieum toxin type A. 8. The method of elsim 7, wherein the protein product of soybean is a hydrolyzed soy. 9. A method for production of a bonulinun toxin type A, the method compesing the steps of (a) providing a frst medium that iso more that 1% by Weight of an animal based product and that comprises a hydrolyzed soy (b) culturing a Clastridiua botulinum type A bacterium in ‘the first medium under conditions that permit growth of | the Clostridium botulinum bacterin: (©) providing a second medium that is no more than 1% Dy weight of an animal based product and that com- prises a hydrolyzed soy: (@) inoculating the second medium withthe frst medium: (culturing a the Clostridium botulinum bacteria in the second medium under conditions that allow production (of a Botulinum wxin ype As; and (0 recovering the botulinum toxin type A.