process and system for obtaining botulinum neurotoxin

专利摘要:
PROCESS AND SYSTEM FOR OBTAINING BOTULINIC NEUROTOXIN.The present invention relates to fast, chromatographic processes and systems, free of animal protein, for obtaining high-performance, high-potency botulinum neurotoxin for research, therapeutic and cosmetic use.
公开号:BR112012000784A2
申请号:R112012000784-0
申请日:2010-07-12
公开日:2020-08-11
发明作者:Jennifer L. Ton；Hemant A. Patel；Ronald C. Bates；Wajdie M. Ahmad
申请人:Allergan, Inc.；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for "PROCESS" SYSTEM FOR OBTAINING BOTULINIC NEUROTOXIN ".
CROSS REFERENCE This application claims the benefit of US Patent Application Serial Number 12 / 502,181, filed on July 13, 2009, the full disclosure of which is incorporated in this document by this specific reference. : BACKGROUND The present invention relates to systems and processes for obtaining a Clostridial neurotoxin, methods for producing a pharmaceutical composition thereof, and therapeutic and cosmetic uses of the pharmaceutical composition thus produced. In particular, the present invention relates to a rapid chromatographic process and system, free of animal protein, to obtain a biologically active botulinum neurotoxin of high potency, high purity and high yield.
A pharmaceutical composition suitable for administration to a human or animal for a therapeutic, diagnostic, research or cosmetic purpose comprises an active ingredient and one or more excipients, buffers, carriers, stabilizers, tonicity adjusters, preservatives and / or bulky agents. The active ingredient in a pharmaceutical composition can be a biological, such as a botulinum neurotoxin. Known methods (such as the Schantz method) for obtaining a botulinum neurotoxin useful as the active ingredient in a pharmaceutical composition are multi-week culture, fermentation and purification processes that use animal-derived proteins, such as meat broth and casein, used in culture and fermentation media, and purification enzymes derived from animals. The administration to a patient of a pharmaceutical composition prepared through the use of products of animal origin can cause risk of administration of pathogens or an infectious agent, such as a prion. In addition, known methods free of animal protein to obtain a botulinum toxin are time-consuming processes (that is, they take more than a week to complete) with numerous steps upstream (culture and fermentation) and downstream (purification), and it still results in ”obtaining a botulinum neurotoxin with detectable impurities.
Botulinum Toxin The genus Clostridium has more than one hundred and twenty-seven species, grouped by morphology and function.
The gram-positive anaerobic bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, the botulinum toxin (synonymously "toxin"), which causes a neuropa: ralitic disease in humans and animals known as botulism.
Symptoms of: botulinum toxin intoxication can progress from difficulty walking, - swallowing, swallowing, and speech to paralysis of the respiratory muscles and death.
One unit of botulinum toxin is defined as LDso when intraperitoneal injection in female Swiss Webster mice weighing about 18 to 20 grams each.
One unit of botulinum toxin is the amount of botulinum toxin that leads to the death of 50% of a group of Swiss Webster female mice.
Seven botulinum neurotoxins, generally immunologically distinct, were characterized, these being botulinum neurotoxins serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies.
The different serotypes of botulinum toxin vary in the animal species in which they affect and in the severity and duration of the paralysis they cause.
Botulinum toxins apparently bind with high affinity to motor cholinergic neurons and move within the neuron and block the presynaptic release of acetylcholine.
Botulinum toxins have been used in clinical settings to treat, for example, neuromuscular disorders characterized by hyperactive skeletal muscles.
Botulinum toxin type A has been proven by the Food and Drug Administration (FDA) in the USA for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over twelve years of age, cervical dystonia, line wrinkles glabular (facial) and for the treatment of hyperhidrosis.
The FDA has also approved a botulinum toxin type B for the treatment of cervical dystonia.
Although all botulinum toxin serotypes apparently inhibit the release of the acetylcholine neurotransmitter at the neuromuscular junction, they do so by affecting different neurosecretory proteins and / or by cleaving these proteins at different sites. Botulinum toxin type A is a zinc endopeptidase that specifically hydrolyzes a peptide bond of the intracellular protein, associated with a vesicle (VAMP, also called synaptobrevine) 25 kilo Daltons protein (kKDa) associated with synaptosoma (SNAP-25). Botulinum toxin type E also cleaves SNAP-25, but it does. different amino acid sequences within this protein, as compared to botulinum toxin type A. Botulinum toxins types B, D, F and G act .— 10 in VWVAMP with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C has been shown to cleave both syntaxin and SNAP-25. These differences in the mechanism of action can affect the relative potency and / or duration of action of the various botulinum toxin serotypes.
The molecular weight of the active botulinum toxin protein molecule (also known as "pure toxin" or as the "neurotoxic component") from a botulinum toxin complex, for all seven known botulinum toxin serotypes, is about 150 kDa. Interestingly, botulinum toxins are released by the Clostridial bacteria as complexes comprising the 150 kDa neurotoxic component together with one or more associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by the Clostridial bacteria as forms of 900 kDa, 500 kDa and 300 kDa (approximate molecular weights). Botulinum toxins types B and C are apparently produced as only a 500 kDa complex. Botulinum toxin type D is produced with both 300 kDa and 500 kDa complexes. Finally, botulinum toxins types E and F are produced only as complexes of approximately 300 kDa. The complexes (ie, molecular weight greater than about 150 kDa) contain hemagglutinin (HA) proteins and a non-toxin protein - non-hemagglutinin (NTNH). Thus, a botulinum toxin complex can comprise a botulinum toxin molecule (the neurotoxic component) and one or more HA proteins and / or NTNH proteins. These two types of
non-toxins (which together with the botulinum toxin molecule can comprise the relevant neurotoxin complex) can act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when the toxin is ingested. In addition, it is possible that larger complexes (greater than about 150 kDa molecular weight) of botulinum toxin may result in a slower rate of diffusion of the botulinum toxin out of an intramuscular injection site of one. botulinum toxin complex. The success of botulinum toxin type A in treating a variety of clinical conditions has led to an interest in other so-10 types of botulinum toxin. Thus, at least botulinum toxins types A, B E and F have been used clinically in humans. In addition, a formulation of the neurotoxic component (ie without the associated non-toxin proteins) is sold in Europe under the trade name XEOMIN (Merz Pharmaceuticals, Frankfurt, Germany).
Botulinum toxin type A is known to be soluble in aqueous solutions at pH 4 to 6.8. At pH above about 7, non-toxin stabilizing proteins dissociate from neurotoxin, resulting in a gradual loss of toxicity, particularly as the pH and temperature increase (Schantz EJ, et al Preparation and characterization of botulinum-numtoxintype A for human treatment ( in particular pages 44-45), chapter 3 of Jankovic, J., et al, Therapy with Botulinum toxin, Marcel Dekker, Inc, 1994).
As with enzymes, the biological activities of botulinum toxins (which are intracellular peptidases) are generally dependent, at least in part, on their three-dimensional conformations. Diluting the toxin in milligram quantities to a solution containing nanograms per milliliter presents difficulties, such as, for example, a tendency for toxin to stick to surfaces and thus reduce the amount of toxin available. Since the toxin can be used for months or years after a pharmaceutical composition containing the toxin has been formulated, the toxin is stabilized with a stabilizing agent such as albumin, sucrose, trehalose and / or gelatin.
A commercially available pharmaceutical composition containing * botulinum toxin is sold under the brand name BOTOXO (purified botulinum toxin type A neurotoxin complex) commercially available from Allergan, Inc., of Irvine, California. Each bottle of 100 units of BOTOXQO consists of about 5 ng of purified botulinum toxin type A complex, 0.5 mg of human serum albumin, and 0.9 mg of sodium chloride, in a vacuum-dried form. reconstituted with normal sterile saline without a condom (0.9% sodium chloride injection). Other pharmaceutical compositions containing commercially available botulinum toxin .-— 10 vehicles include Dysport & (Clostridium botulinum toxin type A complex and hemagglutinin with human serum albumin and lactose in the pharmaceutical composition of botulinum toxin), available from Ipsen Limited, Berkshire , UK as a powder to be reconstituted with 0.9% sodium chloride before use), and MyoBloc "" (an injectable solution comprising botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride at about pH of 5.6, available from Solstice Neurosciences of San Diego, California The neurotoxic component (the 150 kDa toxin molecule) and botulinum toxin complexes (300 kDa to 900 kDa) can be obtained from, for example, List Biological Laboratories , Inc., Campbell, California; Center for Applied Microbiology and Research, Porton Down, UK; Wako (Osaka, Japan), as well as Sigma Chemicals of St Louis, Missouri. Methods free of animal protein and / or chromat ographs for obtaining a botulinum neurotoxin are described in US patents 7,445,914;
7,452,697; 7,354,740; 7,160,699; 7,148,041, and; 7,189,541. Also of interest are patent applications: US 11 / 609,449 entitled "Media for Clostridium Bacterium", filed on December 12, 2006; 12 / 098,896 entitled "Animal Product Free Media and Processes for Obtaining a Botulinum Toxin", filed on April 7, 2008; 11 / 932,689 entitled "Chromatographic Method and System for Purifying a Botulinum Toxin", deposited on October 31, 2007; 11 / 932,789 entitled "Chromatographic Method and System for Purifying a Botulinum Toxin" deposited on October 31, 2007, and; 12 / 234,537, entitled "Animal Product Free Media And Processes
For Obtaining A Botulinum toxin ", deposited on September 19, 2008. í Botulinum toxin for use in a pharmaceutical composition can be obtained by anaerobic fermentation of Clostridium botulinum using the well-known Schantz process (see, for example, Schantz EJ, etal, Properties and use of botulinum toxin and other microbial neurotoxins in medicine, Microbiol Rev 1992 Mar; 56 (1): 80-99; Schantz EJ, et al., Preparation and characterization of botulinum toxin type A for human treatment,. chapter 3 in Jankovic J, ed. Neurological Disease and Therapy. Therapy with botulinum toxin (1994), New York, Marcel Dekker; 1994, pages 41-49, and; - 10 Schantz Eu, etal, Use of crystalline type A botulinum toxin in medical re- search, in: Lewis GE Jr, ed. Biomedical Aspects of Botulism (1981) New York, Academic Press, pages 143-50) .The Schantz process for obtaining a botulinum toxin makes use of animal products, for example, as reagents and as part of the culture medium and fermentation.
A number of steps are required to produce a Clostridial toxin pharmaceutical composition suitable for administration to a human or animal for a therapeutic, diagnostic, research or cosmetic purpose. These steps may include obtaining a purified Clostridial toxin and then formulating the purified Clostridial toxin. A first step may be to platelet and grow colonies of Clostridial bacteria, typically on blood agar plates, in an environment conducive to the growth of anaerobic bacteria, as in a warm anaerobic atmosphere. This step allows to obtain Clostridial colonies with morphology and other desirable characteristics. In a second stage, selected colonies of —Clostridial can be fermented in a first appropriate medium and, if desired, in a second fermentation medium. After a certain fermentation period, the Clostridial bacteria are typically smooth and release the Clostridial toxin in the medium. Third, the medium can be purified in order to obtain a bulk toxin. Typically, the purification of the medium to obtain bulk toxin is carried out using, among other reagents, enzymes of animal origin, such as DNase and RNase, which are used to degrade and facilitate the removal of nucleic acids. The resulting bulk toxin can be a highly purified toxin with a particular specific activity. After stabilization in an appropriate buffer, the bulk toxin can be formulated with one or more excipients to produce a pharmaceutical composition of Clostridial toxin suitable for administration to a human. The pharmaceutical composition of Clostridial toxin can comprise Clostridial toxin as an active pharmaceutical ingredient (API). The pharmaceutical composition can also include one or more excipients, buffers, carriers, stabilizers, preservatives and / or bulky agents.
The Clostridium toxin fermentation step can result - 10 in a fermentation medium solution containing complete Clostridium bacteria, lysed bacteria, culture medium nutrients and fermentation by-products. The filtration of this culture solution in order to remove coarse elements, such as whole and lysed bacteria, provides a harvested / clarified medium. The clarified medium comprises a Clostridial toxin and various impurities and is processed to obtain a concentrated Clostridial toxin, which is called bulk toxin. The fermentation and purification processes to obtain Clostridial toxin in bulk using one or more products of animal origin (such as digested milk and casein, DNase and RNase) are known. An example of this non-animal product ("NAPF") process for obtaining a botulinum toxin complex is the Schantz process and modifications thereof. The Schantz process (from initial plating, cell culture to fermentation and toxin purification) makes use of a number of products derived from animal sources, for example, Bacto Cooked Meat medium - culture flask, plated Columbia Blood Agar for colony growth and selection, and casein in the fermentation medium. In addition, the Schantz purification process of bulk toxin makes use of DNase and RNase from bovine sources to hydrolyze nucleic acids present in the toxin containing fermentation medium. Concerns have been expressed about a potential for viral and transmissible spongiform encephalopathy (TSE), such as bovine spongiform encephalopathy (BSE), contamination when animal products are used in a process to obtain an API and / or in a process to produce ( formulate) a pharmaceutical composition * using said API.
A fermentation process for obtaining a tetanus toxoid that uses reduced amounts of animal products (referred to as fermentation processes free of animal products or "APF"; APF includes free of animal protein) is known, see, for example , US patent 6,558,926. An APF fermentation process to obtain: a Clostridial toxin, has the potential advantage of reducing the (already very low) possibility of contamination of the subsequent toxin in bulk with viruses, .- 10 prions or other undesirable elements that can then accompany the ingredient active pharmaceutical agent, a Clostridial toxin, as it is formulated in a pharmaceutical composition for administration to humans. Chromatography, such as column chromatography, for example, can be used to separate a particular protein (such as a botulinum neurotoxin) from a mixture of proteins, nucleic acids, cell fragments, etc. in a process known as fractionation or purification. The protein mixture typically passes through a plastic or glass column containing, for example, a solid medium, usually porous (usually referred to as beads or resin). Different proteins and other compounds pass through the matrix at different speeds based on their specific chemical characteristics and the way in which these characteristics cause them to interact with the chromatographic medium used.
The choice of medium determines the type of chemical characteristic on which the protein fractionation is based. There are four basic types of column chromatography; ion exchange, gel filtration, affinity and hydrophobic interaction. Ion exchange chromatography achieves fractionation based on the electrostatic charge of the surface using a column packed with small beads carrying a positive or negative charge. In gel filtration chromatography, proteins are fractionated based on their sizes. In affinity chromatography, proteins are separated based on their ability to bind to specific chemical groups (ligands) attached to the beads in the column matrix. The ligands can be biologically specific for a target protein. Hydrophobic interaction chromatography obtains fractionation based on the hydrophobicity of the surface. Column chromatography to purify (fractionate) a Clostridial toxin is well known. See, for example, the following publications:
1. Ozutsumi K ,, et al, Rapid, simplified method for production and purification of tetanus toxin, App & Environ Micro, Apr. 1985, p 939-943, vol à 49, no. 4. (1985) reveals the use of high pressure liquid chromatography (H-PLC) for filtration in ge! to purify tetanus toxin.
- 10 2. Schmidt JJ, et al., Purification of type E botulinum neurotoxin by high-performance ion Exchange chromatography, Anal Biochem 1986 Jul; 156 (1): 213-219 reveals the use of size exclusion chromatography or chromatography ion exchange to purify botulinum toxin type E. The use of protamine sulfate instead of ribonuclease (RNase) is still described.
3. Simpson L.L., et al., Isolation and characterization of the botulinum toxins Simpson LL; Schmidt JJ; Middlebrook JL, In: Harsman S, ed. Methods in Enzymology. Vol. 165, Microbial Toxins: Tools in Enzymology San Diego, CA: Academic Press; vol 165: pages 76-85 (1988) reveals the purification of botulinum neurotoxins using gravity flow chromatography, HPLC, capture steps using an affinity resin, size exclusion chromatography, and ion exchange chromatography (anion and cation), including the use of two different ion exchange columns. Several Sephadex, Sephacel, Trisacryl, S and Q columns are described.
4. Zhou L., et al., Expression and purification of the light chain of botulinum toxin A: A single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain Biochemistry 1995; 34 (46): 15175- 81 (1995) reveals the use of amylose affinity column to purify botulinum neurotoxin from light chain fusion proteins.
5. Kannan K ,, et al., Methods development for the biochemical assessment of Neurobloc (botulinum toxin type B), Mov Disord 2000; 15 (Supp! 2): 20 (2000) reveals the use of size exclusion chromatography for evaluate a botulinum toxin type B.
] 6. Wang Yc, The preparation and quality of botulinum toxin type A for injection (BTXA) and its clinical use, Dermatol Las Faci Cosm Surg 2002; 58 (2002) reveals ion exchange chromatography to purify a toxinabotulin type A. This reference reveals a combination of precipitation and chromatography techniques.
7. Johnson SK,., Et al., Scale-up of the fermentation and purification- S tion of the recombination heavy chain fragment C of botulinum toxin serotype F, expressed in Pichia pastoris, Protein Expr and Purif 2003; 32: 1- 9 (2003) - 10 reveal the use of ion exchange columns and hydrophobic interaction to purify a recombinant heavy chain fragment of a botulinum toxin type Fr.
8. Published patent application U.S. 2003 0008367 A1 (Oguma) discloses the use of ion exchange and lactose columns to purify a botulinum toxin.
The purification methods summarized above refer to the small scale purification of the neurotoxic component of a botulinum toxin complex (i.e., the approximately 150 kDa neurotoxic molecule), or a specific neurotoxic component, as opposed to the purification of the entire complex. of 900 kDa botulinum toxin.
In addition, existing processes, including commercial-scale processes, to obtain an appropriate botulinum toxin to prepare a pharmaceutical botulinum toxin composition typically include a series of precipitation steps to separate the toxin complex from impurities that accompany the botulinum toxin from the fermentation process. Notably, precipitation techniques are widely used in the biopharmaceutical industry to purify a botulinum toxin. For example, fractionation in cold alcohol (Cohn's method) or precipitation is used to remove plasma proteins. Unfortunately, precipitation techniques to purify a botulinum toxin have the disadvantage of low resolution, low productivity, difficulty in operation, difficulty in controlling and / or validating and / or difficulty in scaling up or down. The Patent Application
US serial number 11 / 452,570, published on October 12, 2006, reviews “steps like centrifugation, acid precipitation, ethanol precipitation, acidification steps, and ammonium sulfate precipitation used in various processes free from animal protein and NAPF processes (for a detailed discussion, see Published Patent Application US 2006/0228780, hereby incorporated by reference in its entirety). Some distinctions between an animal protein - free process and a protein - free process. animal protein to obtain a botulinum neurotoxin are shown in these.
TO Therefore, rapid systems and processes are required, on a relatively small scale, but of high yield to obtain highly potent, highly potent botulinum neurotoxin, which can be used for research purposes and / or to produce a pharmaceutical composition.
SUMMARY The present invention achieves this need and provides high-purity, highly potent botulinum neurotoxins obtained by fast, small-scale, commercially useful, high-performance chromatographic systems and processes free of animal protein. The resulting botulinum neurotoxin is useful for producing a pharmaceutical composition. The Clostridial toxin obtained by the practice of our invention is preferably a botulinum neurotoxin complex and more preferably a type A botulinum neurotoxin complex of about 900 kDa or the neurotoxic component 150 kDa thereof. Our invention does not require NAPF reagents, such as DNase and RNase. Definitions The following words and terms used here have the following definitions. "About" means that the item, parameter or term so qualified includes a range of plus or minus ten percent above and below the —valordoitem, parameter or term mentioned. "Administration," or "to administer" means the step of conferring (that is, administering) a pharmaceutical composition or active ingredient to a subject. The pharmaceutical compositions described herein are "locally administered" by, for example, intramuscular (im), intradermal, subcutaneous administration, intrathecal, intracranial, intraperitoneal (ip) topical (transdermal) administration and implant administration routes (i.e. a slow-release device such as a polymeric implant or an osmoptic mini-pump).
"Animal-free" or "substantially animal-free" includes, respectively, "animal-protein-free" or "substantially animal-free" and means the substantial absence or absence of derivatives. blood, pooled blood and other products or compounds of animal origin. "Animal" means a mammal (such as a human), bird, reptile, fish, insect, spider or other animal species. "Animal" excludes microorganisms, such as bacteria. Thus, an APF medium or process or a substantially APF medium or process within the scope of the present invention may include a botulinum toxin or a Closteridial botulinum bacterium. For example, an APF process or a substantially APF process means a process that is substantially free or essentially free of proteins derived from animals, such as immunoglobulins, digested meat, meat and milk by-products, or dairy or dairy products. managed.
"Botulinum toxin" or "botulinum neurotoxin" means a neurotoxin produced by Clostridium botulinum, as well as modified, recombinant, hybrid and chimeric toxins. A recombinant botulinum toxin can have the light chain and / or the heavy chain therein recombinantly by a non-Clostridial species. "Botulinum toxin," as used here, includes botulinum toxin from serotypes A, B, C, D / E, F and G. "Botulinum toxin," as used here, still includes both botulinum toxin complex (ie, the 300, 600 and 900 kDa complexes) as well as pure botulinum toxin (ie, the 150 kDa neurotoxic molecule), all of which are useful in the practice of the present invention. "Purified botulinum toxin" means a pure botulinum toxin or a botulinum toxin complex that is isolated, or substantially isolated, from other proteins and impurities that can accompany botulinum toxin as it is obtained from a culture or fermentation process.
Thus, the purified botulinum toxin can have at least 90%, preferably more than 95%, and more preferably more than 99% of the non-botulinum toxin proteins and impurities removed.
Botulinum cytotoxins C2 and C3, not being neurotoxins, are excluded from the scope of the present invention. "Clostridial neurotoxin" means a neurotoxin produced from, or native to, a Clostridial bacterium, such as Clostridium botulinum, Clostridium-a butyricum or Clostridium beratti, hem as a Clostridial neurotoxin — -10 made by a non-Clostridial species. "Completely exempt" (terminology "consists of") means that within the detection range of the instrument or process being used, the substance cannot be detected or its presence cannot be confirmed. "Essentially exempt" (or "essentially consists of") means that only trace amounts of the substance can be detected. "Modified botulinum toxin" means a botulinum toxin that has at least one of its amino acids deleted, modified or replaced, as compared to a native botulinum toxin.
In addition, the modified botulinum toxin can be a recombinantly produced neurotoxin, or a recombinantly made neurotoxin derivative or fragment.
A modified botulinum toxin retains at least one biological activity of the native botulinum toxin, such as the ability to bind to a botulinum toxin receptor, or the ability to inhibit the release of a neurotransmitter from a neuron.
An example of a modified botulinum toxin is a botulinum toxin that has a light chain of a botulinum toxin serotype (like serotype A), and a heavy chain of a botulinum toxin of a different serotype (like serotype B). Another example of a modified botulinum toxin is a botulinum toxin coupled to a neurotransmitter, such as substance P. "Pharmaceutical composition" means a formulation in which an active ingredient may be a botulinum toxin.
The word "formulation" means
means that there is at least one additional ingredient (such as, but * not limited to, albumin [such as human serum albumin or recombinant human albumin] and / or sodium chloride) in the pharmaceutical composition in addition to botulinum neurotoxin as an active ingredient. A pharmaceutical composition is, therefore, a formulation that is suitable for diagnosis, therapy, or cosmetic administration (for example, by intramuscular or subcutaneous injection or by inserting a depot or implant) to a subject, such as a patient. human. The pharmaceutical composition may be: in a lyophilized or vacuum-dried condition, a solution formed .- 10 after reconstitution of the lyophilized or vacuum-dried pharmaceutical composition with saline or water, for example, or; as a solution that does not require reconstitution. The active ingredient can be one of the botulinum toxin A, B, C1, D, E, F or G serotypes or a botulinum toxin, all of which can be made natively by Clostridial bacteria. As stated, a pharmaceutical composition can be liquid or solid, for example, vacuum dried. Exemplary methods for formulating a pharmaceutical composition containing botulinum toxin as an active ingredient are described in U.S. patent application 20030118598, filed November 5, 2002, hereby incorporated by reference in its entirety.
"Substantially free" means present at a level of less than one percent of a culture medium, fermentation medium, pharmaceutical composition or other material in which the percentage weight of a substance (such as an animal product, animal protein or product or animal-derived protein) is evaluated.
"Therapeutic formulation" means a formulation that can be used to treat and thereby alleviate a disorder or disease and / or associated symptoms thereof, such as a disorder or disease characterized by hyperactivity (eg, spasticity) of a peripheral muscle or gland, (for example, sweat gland).
"Therapeutically effective amount" means the level, amount or concentration of an agent (for example, as a botulinum toxin or pharmaceutical composition comprising botulinum toxin) required
necessary to treat a disease, disorder or condition without causing significant * negative effects or adverse side effects. "Treating", "treating", or "treatment" means relief or reduction (which includes some reduction, a significant reduction, an almost total reduction, and a total reduction), resolution or prevention (temporarily or permanently) of a disease , disorder or condition, such as a deformity in the buttocks, in order to achieve a therapeutic result or. desired cosmetic, such as healing of damaged or injured tissue, or altering, modifying, reinforcing, improving, softening and / or embellishing »» 10 an existing or perceived disease, disorder or condition.
A treatment effect, such as a relief effect from the administration of a botulinum neurotoxin may not appear clinically for 1 to 7 days after the administration of the botulinum neurotoxin to a patient for example and may have an effect duration of around 1 month to about 1 year or any range of time between them, for example, depending on the condition and particular case being treated.
The percentages are based on weight by volume unless stated otherwise.
APF means free of animal product / protein.
CV stands for column volume.
DF stands for diafiltration.
ELISA means enzyme-linked immunosorbent assay.
IAPF, as in "IAPF system" or "IAPF process", means "improved animal protein-free" system or process. An IAPF system or process includes the use of two chromatographic media or three chromatographic media to purify a botulinum toxin or neurotoxin component, as specifically detailed here.
Chromatographic medium includes chromatography resins, as known in the art.
Lots of botulinum neurotoxin obtained using two chromatographic media are here designated as IAPF.
FAPF, as in "FAPF system" or "FAPF process" ", means" improved animal protein-free "system or process. Thus, FAPF is an IAPF process, and a FAPF system or process means that three IS chromatographic media are used to purify a botulinum toxin or neurotoxin component. Lots of botulinum neurotoxin obtained by using three chromatographic media are referred to here as FAPF. NAPF means not free from animal protein.
SDS-PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis.
. SEC-HPLC stands for high performance liquid chromatography by size exclusion. - 10 UF means ultrafiltration.
In one embodiment of the invention, a substantially APF chromatographic process for obtaining a biologically active botulinum neurotoxin is provided, the process comprising the following steps of (a) providing a substantially APF fermentation medium; (b) ferment Clostridium botulinum bacteria in the fermentation medium, and; (c) recovering the biologically active botulinum neurotoxin from the fermentation medium by contacting the fermentation medium with an anion exchange chromatography medium followed by contacting an eluent from the anion exchange chromatography medium with a chromatography medium from cationic exchange, in order to obtain the biologically active botulinum neurotoxin from the substantially APF chromatographic process. In particular embodiments, the process provides a botulinum neurotoxin which comprises less than one part per million (ppm) of residual nucleic acid which is one nanogram or less of the residual nucleic acid for each milligram of the botulinum neurotoxin obtained.
In yet another aspect, the process is carried out in a week or less.
In one example, medium containing a 3: 1: 1 ratio means a culture / botulinum toxin fermentation medium containing 3% Hy-Soy, 1% HyYeast, and 1% glucose. HySoy (Quest, product No. 5X59022) is a source of peptides prepared by enzymatic soy hydrolysis. HyYeast (Hy-Yest, Quest product no. 5210102 or 5210313 is a baker's yeast extract. In another example, the medium containing a 5: 1: 1 ratio means
a culture / fermentation medium of botulinum toxin containing 5% Hy- É Soy, 1% HyYeast, and 1% glucose.
Another modality provides a substantially APF chromatographic process to obtain a biologically active botulinum neurotoxin complex A, the process comprising the following sequential steps of culturing Clostridium botulinum bacteria in a substantially APF culture medium; ferment the bacterium Clostridium botulinum from the medium and culture in about 2 L to about 75 L of a substantially APF fermentation medium, more preferably in about 2 L to about - 10 S50L from a substantially APF fermentation medium, even more preferably - mainly in about 2 L to about 30 L of a substantially APF fermentation medium (particular embodiments have at least one of the culture media and the fermentation medium including a vegetable protein and / or a vegetable protein derivative, for example a hydrolyzed vegetable protein), harvesting the fermentation medium by removing cell fragments present in the fermentation medium using filtration or centrifugation; concentration of the fermentation medium collected by filtration, such as ultrafiltration (UF) for example; dilute the concentrated fermentation medium by adding a buffer. After dilution with the buffer, a first contact step is carried out in which the harvested diluted fermentation medium is contacted with an anion exchange medium so that the biologically active botulinum neurotoxin becomes captured by the anion exchange medium; followed by elution of the botulinum neurotoxin captured from the anion exchange medium to obtain a first eluent containing the botulinum toxin; carry out a second stage of contact with a cation exchange medium to remove impurities from the first eluent, so as to obtain a second eluent containing the botulinum toxin; followed by processing the second eluent by diafiltration (DF); and filtering the second processed eluent, thereby obtaining biologically active botulinum neurotoxin type A complex using a substantially APF chromatographic process. The type A botulinum neurotoxin complex obtained can have a potency of about 2.0 x 107 units / mg to about 6.0 x 10 ”units / mg of type A botulinum neurotoxin complex. botulinum neurotoxin type A containing a potency of between about 2.4 x 10 ”units / mg to about 5.9 x 10th units / mg, for example, can be obtained. In a particular embodiment, the process uses a fermentation medium comprising no more than about 5% w / v of a vegetable-derived protein product, no more than about 2% w / v of an extract of yeast and no more than about 2% w / v glucose, and where the S PH level of the fermentation medium is about 6.5 to about pH 8.0, more preferably about pH 6 , 8 at about pH 7.6, at the beginning of the - 10 fermentation stage. In a particular embodiment, the culture step is conducted until the optical density of the culture medium at about 540 nanometers (nm) is between about 0.8 absorbance units (AU) and about 4.5 AU. The culture stage is preferably initiated by the introduction of a Clostridium botulinum APF working cell bank content to the culture medium, where the content of the working cell bank comprises at least about 1 x 10 forming units colony, preferably from about 1 x 10º to about 5 x 107 colony-forming units of Clostridium botulinum per milliliter of the working cell bank, and where the bacterium Clostridium botulinum in the working cell bank has a substantially uniform phology. In yet another modality, the fermentation stage is carried out for about 60 to 80 hours and until an optical density of the fermentation medium of about 890 nm decreases to between about 0.05 AU to about 0.7 AU . In one aspect, the botulinum neurotoxin obtained by the substantially APF chromatographic process comprises less than 1 ppm of residual nucleic acid and the process is carried out in a week or less.
In yet another modality, an APF process using chromatography to obtain a biologically active botulinum neurotoxin is provided, comprising the sequential steps of: (a) adding Clostridium botulinum bacteria from an APF work cell bank to a medium of APF culture; (b) cultivating the bacterium Clostridium botulinum in the culture medium; (c) ferment the Clostridium botulinum bacteria from step (b) in a fermentation medium
APF feeding until the lysis of the Clostridium botulinum cell occurs; (d) * harvesting the fermentation culture to provide harvested fermentation medium; (e) subjecting the harvested fermentation medium to concentration by filtration; (f) diluting the filtered fermentation medium by adding a buffer to obtain a diluted fermentation medium; (g) a first contact stage in which the diluted fermentation medium is contacted with a capture chromatography medium, where the capture chromatography medium is an exchange and anionic medium; (h) a second contact stage where an eluent from the first contact stage is contacted with chromatographic polishing means, where - 10 omeochromatographic polishing is a cation exchange medium, and (i) filter the eluent from the second contact stage, thus obtaining biologically active botulinum neurotoxin by the improved APF process, where the botulinum neurotoxin obtained comprises 1 ppm residual nucleic acid or less than 1 ppm residual nucleic acid and the process is conducted in a week or less.
In one aspect, a substantially animal-free chromatographic system (APF) for obtaining a biologically active botulinum neurotoxin is provided, comprising a substantially APF fermentation medium, Clostridium botulinum bacteria for fermentation in the defermentation medium, a chromatography medium anion exchange agent to recover the biologically active botulinum neurotoxin from the fermentation medium, and a cation exchange chromatography medium to recover another biologically active botulinum neurotoxin from an eluent from the anion exchange chromatography medium, thereby obtaining biologically active botulinum neurotoxin from a substantially APF chromatographic process. In particular configurations, the system may further comprise a first apparatus for anaerobically cultivating the Clostridium botulinum bacterium in a substantially APF culture medium, and may further be comprised of a second apparatus for anaerobically fermenting the Clostridium boululinum bacterium in the medium of substantially APF fermentation, where the bacterium Clostridium botulinum is obtained from the first apparatus. For clarification, the system may include a harvesting device to remove cell debris
fermentation medium obtained from the second apparatus to provide a harvested fermentation medium.
The harvested fermentation medium can be passed through a concentration and dilution apparatus to concentrate and then dilute the harvested fermentation medium. - In a particular example, the system may also include a hydrophobic interaction medium to recover another biologically active purified botulinum neurotoxin from an elution in the cation exchange chromatography medium.
In addition, a device: filtration to reduce the biologically active botulinum neurotoxin biofuel: active obtained can also be part of the system, to reduce the biologically active botulinum neurotoxin .- 10 biofuel obtained by using two or three chromatography media.
In a specific example, an anae- chamber. A robic tube containing a high efficiency particle air filter integrated into the workspace, to grow Clostridium botulinum bacteria in the substantially APF culture medium, can be used.
Exemplary systems can supply botulinum neurotoxin containing a power of at least about 2.0 x 10 ”units / mg of botulinum neurotoxin and the obtained botulinum neurotoxin comprises ng or less than ng nucleic acid residual for each mg of botulinum neurotoxin obtained.
In particular embodiments, the substantially APF fermentation medium is supplied in an amount of about 2 L to about 75 L; and from about 200 ml to about 1 L of substantially APF culture medium is used.
In another aspect of our invention, a substantially APF system using chromatography to obtain a biologically active botulinum neurotoxin is provided, the system comprising a first apparatus for growing Clostridium botulinum bacteria, the first device capable of contacting a culture medium substantially APF; a second device for fermenting the Clostridium botulinum bacterium that was grown in the first device, the second device capable of contacting a substantially APF fermentation medium; a third apparatus for harvesting the fermentation medium; a fourth apparatus for concentrating the harvested fermentation medium and diluting the filtered fermentation medium; a fifth apparatus for conducting a first purification of botulinum neurotoxin from the harvested medium,
where the fifth device comprises an anionic exchange chromatography medium, thus obtaining a first purified botulinum neurotoxin; and a sixth apparatus for conducting a second purification of the botulinum neurotoxin where the sixth apparatus comprises a cation exchange chromatography medium, so as to obtain a second purified botulinum neurotoxin, where the obtained botulinum neurotoxin has a potency of at least about from 2.0 x 107 units / mg of botulinum neurotoxin to about 5.9 x 10º units / mg of botulinum neurotoxin, the botulinum neurotoxin obtained comprises one ng or less than one ng of residual nucleic acid for each - 10 mg of botulinum neurotoxin obtained and the process is carried out in a week or less. In particular modalities, the botulinum neurotoxin obtained may have a potency of at least 4.4 x 10º units / mg of botulinum neurotoxin. In a particular embodiment of the system, the system may further comprise a seventh apparatus for conducting another purification of the botulinum neurotoxin obtained from the sixth apparatus, where the seventh apparatus comprises a hydrophobic interaction medium, thereby obtaining a third purified botulinum neurotoxin. In an additional embodiment, the system may further comprise an eighth apparatus comprising a membrane for filtering the eluent from the seventh apparatus.
Another aspect of our invention includes a substantially APF chromatographic system for obtaining a biologically active botulinum neurotoxin comprising a first apparatus for anaerobic culture of bacteria Clostridium botulinum, the first apparatus capable of containing from about 200 mL to about 1 L a substantially APF culture medium; a second apparatus comprising an anaerobic chamber containing a high efficiency particle air filter integrated within the chamber capable of containing the first apparatus; a third apparatus for anaerobic fermentation of Clostridium botulinum bacteria that was grown in the first apparatus, the third apparatus capable of containing from about 2 L to about 75 L of a substantially APF fermentation medium, preferably from about 2 L to about 30 L of a substantially APF fermentation medium and including at least one disposable probe selected from the group consisting of a oxidation reduction probe, a pH probe and * a turbidity probe; a fourth apparatus for harvesting the fermentation medium; a fifth apparatus for concentrating the harvested fermentation medium and diluting the filtered fermentation medium; a sixth apparatus for conducting a first purification of botulinum neurotoxin obtained from the harvested fermentation medium, the sixth apparatus comprising an anion exchange chromatography medium, thereby obtaining a first purified botulinum neurotoxin; . a seventh apparatus for conducting a second purification of the botulinum neurotoxin, the seventh apparatus comprising a cation exchange chromatography medium, thereby obtaining a second purified botulinum neurotoxin; an eighth apparatus for conducting a third purification of the second o. purified botulinum neurotoxin, the eighth apparatus comprising hydrophobic interaction medium to thereby obtain a third purified botulinum neurotoxin; and a ninth apparatus for filtering the third purified botulinum neurotoxin, the ninth apparatus comprising a filtration membrane, where the obtained botulinum neurotoxin has a potency of about 2.4 x 107 units / mg of botulinum neurotoxin at about 5.9 x 10 units / mg botulinum neurotoxin, the botulinum neurotoxin obtained comprises one ng or less than one ng of residual nucleic acid for each mg of a botulinum neurotoxin obtained and the process is carried out in a week or less. According to these processes, a biologically active botulinum neurotoxin is thus obtained, and in particular examples, the obtained botulinum neurotoxin has a potency of at least about 4.4 x 10 ”units / mg of botulinum neurotoxin.
According to these processes and systems described here, biologically active botulinum neurotoxin is thus obtained. In particular modalities, the biologically active botulinum neurotoxin obtained by the process and systems described here has a molecular weight of about 900 kDa.
Our invention further includes a method for producing a substantially APF pharmaceutical composition in which the active ingredient is a biologically active botulinum neurotoxin, the method comprising the steps of: (a) obtaining a biologically active botulinum neurotoxin by: (i) providing a fermentation medium which is substantially free of an animal product; (ii) fermenting Clostridium botulinum bacteria in the fermentation medium, and; (iii) recovering the biologically active botulinum neurotoxin from the fermentation medium, using an anion exchange chromatography medium followed by the use of a cation exchange chromatography medium, where the recovered botulinum neurotoxin has a potency of at least about. 2.0 x 107 units / mg botulinum neurotoxin, preferably about 2.4 x 10 units / mg botulinum neurotoxin to about 5.9 x 107 units / mg botulinum neurotoxin, in some modalities at least minus about 4.4 x 10 th units / mg of botulinum neurotoxin, the neurotoxin bo-. tulinic comprises one ng or less than one ng of residual nucleic acid for each mg of a botulinum neurotoxin, and steps (i) to (iii) are completed in a week or less, and; (b) manipulating the botanical neurotoxin with at least one appropriate excipient, thereby producing the pharmaceutical composition substantially APF. In a particular modality, the manipulation step comprises the step of drying the botulinum neurotoxin by a process selected from the group of processes consisting of freeze drying and vacuum drying and where the appropriate excipient is selected - from the group consisting of albumin , human serum albumin, recombinant human serum albumin, gelatin, sucrose, trehalose, hydroxyethyl starch, collagen, lactose, sucrose, sodium chloride, polysaccharide, caprylate, polyvinylpyrrolidone and sodium. Thus, an aspect of our invention also provides substantially APF pharmaceutical compositions prepared by manipulating the biologically active botulinum neurotoxin obtained by the processes and systems described herein.
In addition, our invention further includes a method for treating a condition in a patient, the method comprising the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by the methods to produce a substantially pharmaceutical composition. APF in which the active ingredient is a biologically active botulinum neurotoxin obtained by the APF processes (ie, IAPF and FAPF processes) described herein.
Examples of conditions to be treated are selected from the group consisting of a headache, a migraine, tension headache, sinus headache, a cervicogenic headache, a sweating disorder, axillary hyperhidrosis, hi - palmar perhidrosis, plantar hyperhidrosis, Frey syndrome, a line of hyperkinetic skin, a facial wrinkle, glabellar lines, crow's feet, marionette lines, a nasolabial fold, a skin disorder, achalasia, strabismus, anal fissure chronic, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, prostatic enlargement, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremors, myoclonus, gastrointestinal disorders, diabetes, sialoreia, dysplasia and detrimental nergy -sphincter, post-infarction spasticity, wound healing, juvenile cerebral palsy, smooth muscle spasm, restenosis, focal dystonia, epilepsy, cervical dystonia, disorder thyroid disease, hypercalcemia, obsessive compulsive disorder, arthritic pain, Raynaud's syndrome, stretch marks, peritoneal adhesion, vasospasm, rhinorrhea, muscle contracture, an injured muscle, laryngeal dystonia, dystonic disorders and syndrome
me from carpal tunnel, for example.
In one embodiment, a method for treating a condition in a patient, the method comprising the step of administering locally to the patient an effective amount of the substantially APF pharmaceutical composition prepared by the method including the steps of: (a) obtaining a biologically botulinum neurotoxin active by (i) providing a means of fermentation that is substantially free of an animal product; (ii) ferment bacteria Clostridium botulinum in the fermentation medium, and; (iii) recovering the biologically active botulinum neurotoxin from the fermentation medium, using an anion exchange chromatography medium followed by the use of a cation exchange chromatography medium, where the recovered botulinum neurotoxin has a potency of at least about of 2.0 x 10 units / mg botulinum neurotoxin, the botulinum neurotoxin comprises ng or less than one ng of residual nucleic acid for each mg of a botulinum neurotoxin, and steps (i) to (iii) are completed in a week or less, and; (b) manipulating the botulinum neurotoxin with at least one appropriate excipient, thereby producing the substantially APF pharmaceutical composition, whereby administration of the substantially APF pharmaceutical composition treats the condition.
Local administration of therapeutically effective amounts of a pharmaceutical composition, comprising a biologically active botulinum neurotoxin provided by the IAPF process / system / method described here, can be repeated at intervals of about 2 months to about 6 months or at intervals from about 2 months to about 3 months, for example - 10 times. Exemplary useful dosages administered locally to the patient of a therapeutically effective amount of the substantially APF or pharmaceutical composition, prepared according to the present procedure, can have unit quantities of botulinum neurotoxin between about 0.01 units and about 10,000 units. In particular cases, botulinum neurotoxin is administered in an amount of between about 0.01 unit and about 3000 units. In particular examples, the biologically active botulinum neurotoxin that is the active pharmaceutical ingredient in the pharmaceutical composition is botulinum neurotoxin type A or type B, for example.
Our invention includes a substantially APF process, using chromatography, to obtain a biologically active botulinum neurotoxin. The process may comprise the sequential steps of providing a substantially APF fermentation medium, followed by fermenting Clostridium botulinum bacteria in the fermentation medium and recovering the biologically active botulinum neurotoxin from the fermentation medium using an anion exchange chromatography medium followed by use of an anion exchange chromatography medium. cation exchange chromatography to obtain the biologically active botulinum neurotoxin from the substantially APF chromatographic process. The recovery step may also include the use of a hydrophobic interaction medium after using cation exchange chromatography. The biologically active botulinum neurotoxin obtained can be a botulinum neurotoxin complex or a botulinum toxin neurotoxic component isolated from it with a molecular weight of about 150 kDa free from complex protein complexes. APF processes (using 2-column (IAPF), for example, anionic chromatography followed by cationic; or 3-column (FAPF), for example, anionic followed by cationic followed by hydrophobic interaction chromatography) can be used to obtain a neurotoxin biologically active botulinum toxin as type A, B, C1, D, E, F and G botulinum neurotoxins. The botulinum neurotoxin obtained is preferably a type A botulinum neurotoxin complex.
In one aspect of our invention, the amount of fermentation medium used can comprise from about 2 L to about 75 L of the substantially APF fermentation medium, preferably from about: 2 L to about 30 L of the fermentation medium. substantially APF fermentation. As an example, from about 100 mg to about 5 grams, preferably from about 100 mg to about 3 grams, more preferably from about 100 mg to about 1 gram of a biologically active botulinum neurotoxin is obtained from the process. As an example, from about 20 mg to about 100 mg or from about 20 mg to about 80 mg of the biologically active neurotoxin can be obtained per liter of fermentation medium used. The fermentation medium can comprise protein product of plant origin, yeast extract and glucose, for example. As an example, the fermentation medium comprises about 5% w / v or less of a vegetable-derived protein product. In yet another example, the fermentation medium comprises about 2% w / v or less of a yeast extract. In another embodiment, the fermentation medium comprises about 2% w / v or less of glucose. In a particular example, the fermentation medium comprises about 5% w / v or less of a vegetable-derived protein product, about 2% w / v or less of a yeast extract and about 2% w / v or less of glucose, the product protein of plant origin, yeast extract and glucose being in a proportion according to the declared amounts of percentage w / v. In some embodiments, the fermentation step proceeds between about 60 hours to about 80 hours. In one embodiment, a substantially APF process using chromatography to obtain a biologically active botulinum neurotoxin A, the process comprising the following sequential steps is provided where the bacterium Clostridium botulinum is grown in a substantially APF culture medium, then the bacterium Clostridium botulinum is fermented in the culture medium in about 2 L to about 75 L of a substantially APF fermentation medium, more preferably in about 2 L to about 30 L of a substantially APF fermentation medium, on- . of at least one of the substantially APF culture medium and substantially APF fermentation medium include a vegetable protein, followed by - 10 harvesting fermentation medium by removing cell debris present in the fermentation medium and concentration of fermentation medium = harvested by filtration, and dilution of the concentrated fermentation medium by adding a buffer. Once buffered, a first contact step is performed, in which the harvested diluted fermentation medium is contacted with an anion exchange medium so that the biologically active botulinum neurotoxin is associated with the anion exchange medium, then eluting the botulinum neurotoxin captured from the anion exchange medium proceeds to obtain a first eluent, followed by a second contact step in which the first eluent is contacted with a cation exchange medium to remove impurities from a first eluent, thus obtaining a second eluent; which is then processed, as per UF and / or DF; and then filtering the second processed eluent, thus obtaining biologically active botulinum neurotoxin using a substantially APF process that uses chromatography.
As an example, the time to complete the process, from cultivating the bacteria to obtaining the biologically active botulinum neurotoxin can be between about 50 hours to about 150 hours, more preferably about 80 hours to about 120 hours, for example. In particular modalities, the culture medium comprises no more than about 4% w / v of a vegetable-derived protein product, in another, the culture medium comprises no more than about 2% w / v of one yeast extract and yet another, the culture medium comprises no more than about 2% w / v glucose.
The culture medium can comprise a protein product of: vegetable origin, yeast extract and glucose in any proportion according to the amounts mentioned in percentage w / v.
In a specific example, the pH level of the culture medium can be from about pH 6.5 to about pH38.0, preferably about pH 6.8 to about pH 7.6, more preferably 7.3 at the beginning of culture stage.
The culture step can be conducted for between about 8 hours and about 14 hours, about - 10 hours to 12 hours, preferably about 11 hours, in a temperature of about 33ºC to about 37ºC, preferably in about 34.5 - 10 C, in an anaerobic chamber.
In a particular example, the anaerobic chamber may contain a particular high-efficiency filter within the space. where culture is conducted.
The fermentation step can be conducted for between about 60 hours and about 80 hours, preferably about 72 hours at a temperature of about 33ºC to about 37ºC, preferably at 35ºC.
According to one aspect of our invention, the harvesting step can remove at least about 80% of RNA and DNA contained in the fermentation medium and the anion exchange medium can remove all remaining measurable DNA and RNA (below the detection limit ) in the harvested fermentation medium.
In another aspect, the harvesting step can be conducted for between about 1 hour and about 3 hours, preferably about 2.5 hours.
In particular examples, the harvesting stage can be conducted until 75% of the original volume of the fermentation medium has been collected.
In one aspect of an embodiment, the concentration step can be conducted for between about 30 minutes and about 2 hours, preferably about 0.75 hours.
In another aspect, the dilution step dilutes the fermentation medium harvested back to the initial weight of the fermentation medium at the beginning of the harvest stage.
A first contact step can be conducted for between about 4 hours and about 5 hours, for example.
In one example, the first eluate of the anion exchange resin is collected in spectrophotometer readings of about 150 mAU or greater, until the spectrophotometer readings at 280 nm decrease from the peak apex back to about 150 mAU.
The second contact step can be conducted for between 1 hour and about 3 hours, preferably for about 2 hours.
This second eluate can be collected from the cation exchange resin in spectrophotometer readings of about 100 mAU or greater, until the spectrophotometer readings decrease from the peak peak to about 100 mAU, for example.
A processing step of this second eluant by diafiltration concentration can be carried out for between about 1 hour and about 2 hours, preferably for about 1.5 hours.
In a particular modality. lar, the filtration step includes reducing the biocharging by passing the second eluant through a biocharging reduction filter The biocharging reduction filter - 10 can have a pore size of about 0.1 µm to about 0.3 µm, preferably 0.2 µm.
In particular modalities, the process can still and comprise a third contact stage after the second contact stage, contacting the second eluant to a hydrophobic interaction medium to remove more impurities from the second eluant and, thus, to obtain a third eluent ro.
This third contact stage can be conducted for between 1 hour and about 3 hours, preferably for about 2 hours.
This third eluate can be collected from the hydrophobic interaction medium in spectrophotometer readings of about 50 MAU or greater, until the spectrophotometer readings decrease from the peak peak to about 50 mAU, for example.
Where there is a third contact stage, the concentration and diafiltration processing stage is applied to the third eluent and is conducted for between about 2 hours and about 4 hours.
The reduction of the biocharging by passing the eluent, which is concentrated and diafiltered (by a process of 2 or 3 columns used), through a biocharging reduction filter can consequently be carried out.
In particular modalities, the process also comprises a stage of freezing the biologically active botulinum neurotoxin obtained
gives.
In particular embodiments, the substantially APF culture medium comprises a volume of between about 100 ml and about 500 ml Particular culture steps are initiated by introducing between about 100 µl and about 500 µl. of an APF working cell bank medium containing Clostridium botulinum to the culture medium substantially
APF. The culture step can take place in an anaerobic chamber for at least about 8 hours, preferably about 11 hours, at a temperature of around 34.5ºC + 1ºC, for example. In one example, the working cell bank medium may have a viable cell count assay of at least about 1 x 10th colony forming units / mL of the working cell bank medium, for example about 1 x 10 to about 5 x 107 colony-forming units / ml of the cell bank = working medium, and the bacterium Clostridium botulinum in the work cell bank can be selected to have a substantially uniform morphology - 10 m and 1 In one embodiment, the working cell bank medium includes about 20% by volume of glycerol, such as sterile glycerol, for example. The working cell bank medium can be made by (a) growth of Clostridium botulinum bacteria in an APF medium containing about 2% w / v soy peptone, about 1% w / v yeast extract, and about 1% w / v. of glucose in an anaerobic chamber, at a temperature of about 34.5ºC t + 1ºC to an optical density of an aliquot of the medium measured at a wavelength of about 540 nm is about 2.557 1.0 AU, and; add glycerol to obtain a concentration of glycerol in the medium of about 20%, thus obtaining a working cell bank. A way of storing the work cell bank can be prepared by freezing the work cell bank at about -135ºC, for example. The way of storing the work cell bank, for use in an exemplary process according to the present disclosure, can be defrosted at room temperature and used to start the culture stage.
The culture step can be conducted for between 8 hours and about 14 hours, preferably about 11 hours in a temperature of about 33ºC to about 37ºC, preferably in about 34.5ºC, in a anaerobic chamber, such as an anaerobic chamber / cabin containing a high efficiency particulate air filter (HEPA), preferably within the workspace. The fermentation step can be conducted for between about 20 hours and about 80 hours, preferably about 60 hours to about 80 hours, more preferably for about 72 hours at a temperature of around 33ºC at about 37ºC, preferably at 35ºC. The process may also comprise, for example, and before the culture step, a step that allows the oxidative reduction of the substantially APF culture medium by exposing the medium to the atmosphere of an anaerobic chamber. The process may further include, prior to the fermentation step, a step which allows the oxidative reduction of the substantially APF fermentation medium also by exposing the fermentation medium to the atmosphere of an anaerobic chamber. As an example, the step that allows a substantially oxidative reduction of the culture medium - the APF can be conducted for between about 10 hours and about 14 hours in the anaerobic chamber. Similarly, the step that allows oxidative reduction of the substantially APF fermentation medium in the fermenter can be carried out between about 10 hours and about 14 hours before the start of the fermentation step.
In one embodiment, an APF process, including chromatography, to obtain a biologically active botulinum neurotoxin is described, comprising the following additional steps of adding Clostridium botulihum bacteria from an APF working cell bank to an APF culture medium; cultivate the bacterium Clostridium botulinum in the culture medium; ferment the bacterium Clostridium botulinum from a culture step in an APF fermentation medium until lysis of the Clostridium botulinum cell occurs; harvesting the APF fermentation culture to provide a harvested fermentation medium; subjecting the harvested fermentation medium to concentration by filtration; diluting the filtered fermentation medium by adding a buffer to obtain a diluted fermentation medium; a first contact stage in which the diluted fermentation medium is contacted with a capture chromatography medium, where the capture chromatography medium is an anion exchange medium; a second contact stage where an eluent from the first contact stage is contact with a polishing chromatographic medium, where the polishing chromatographic medium is a cation exchange medium, and filtering the eluent from the second contact stage, thus obtaining neurotoxin botulinum Õ biologically active by the improved APF process. In particular modalities, the process can also comprise the stage of conducting a third stage of contact, after the stage of contact and before the stage of filtration, when contacting the eluent of the second stage of contact with a means of hydrophobic interaction. The lysis phase of Clostridium botulinum can occur between about 35 hours and about 70 hours after the beginning of the fermentation step, for example. The fermentation medium can have a volume of between about 2 L and about 75 L, between about 2 L and about 30 L, or between - 10 about 2L and 20L of the fermentation medium, for example. The total of this process can be conducted for between about 50 hours to about 150 hours, more preferably about 80 hours to about 120 hours. The biologically active botulinum neurotoxin thus obtained by this process can have a potency of about 2.4 x 10º to about 5.9 x 10º units / mg of biologically active botulinum neurotoxin, for example. According to one aspect, at the end of fermentation, from about 40 mg to about 85 mg of botulinum neurotoxin per liter of the fermentation medium can be obtained. Subsequent to several processing stages (filtration / chromatography / filtration passages), from about 30 mg to about 60 mg of botulinum neurotoxin per liter of fermentation medium; from about 5 mg to about 25 mg of botulinum neurotoxin per liter of fermentation medium; from about 6 mg to about 20 mg of botulinum neurotoxin per liter of fermentation medium can be obtained.
According to one embodiment, the pH of the fermentation medium can be adjusted to be between about pH 6.0 and about pH 8, preferably between about pH 6.8 and about pH 7.6 at the beginning of the fermentation, more preferably about pH 7.3. As another example, the substantially APF chromatographic process for obtaining a biologically active botulinum neurotoxin is further provided, the process comprising the steps of obtaining a substantially APF fermentation medium containing a botulinum neurotoxin; contacting the medium with an ion exchange chromatography resin to provide a purified eluent containing a botulinum neurotoxin; contact an eluent with a cation exchange chromatography resin to obtain another purified eluent, and filter the purified eluent to obtain a biologically active botulinum neurotoxin purified from a substantially APF chromatographic process. In particular configurations, a column Anion chromatography can be used that contains from about 600 mL to about 800 mL of an ion exchange chromatography resin.
The anionic chromatography column can have a diameter of about 8 cm to about 10 cm and a height of the ion exchange chromatography resin bed in the column of about 9 - 10 cm about 16 cm, for example .
A flow rate of the medium of fermentation by an ion exchange chromatography resin can be fan about 140 cm / hour at about 250 cm / hour, or from about 150 cm / hour at about 160 cm / hour , for example.
In another aspect, about 150 ml to about 300 ml of cation exchange chromatography resin in a chromatography column can be used in the process, where the cation chromatography column has a diameter of about 5 cm at about 8 cm and the height of the resin bed cation exchange chromatography from about 5 cm to about 11 cm, for example.
The process may include at least one diafiltration step and / or a bioburden reduction step.
The bioburden reduction step can use a capsule filter.
Diafiltration of the purified eluant is preferably carried out before the step of reducing the biocharge.
In one example, the step of diafiltering the most purified eluent is preceded or followed by adjusting the concentration of the most purified diafiltered eluent, and passing the most purified eluent through concentration diafiltration adjusted by a biocharging reduction filter.
The process can provide a botulinum neurotoxin obtained containing potency, as determined by an LD50 bioassay in mice, of at least about 2.0 x107 units / mg of botulinum toxin, such as about 2.4 x 10º at about 6.0 x 107 units / mg botulinum neurotoxin.
Exemplary recovery after the process of about 4 mg to about 25 mg of botulinum toxin can be recovered per liter of fermentation medium, for example.
In another embodiment, an essentially APF process for purifying a biologically active botulinum neurotoxin may comprise the steps of obtaining from about 2 L to about 30 L an AFP fermentation medium that includes a botulinum neurotoxin; harvesting the APF fermentation medium step to provide a harvested APF fermentation medium; performing an ion exchange chromatography at the time of harvesting the APF fermentation medium to provide a first eluent; contact the eluent for an ion exchange chromatography with chromato- medium. cation exchange graph to perform cation exchange chromatography to provide a second eluant; and filter the second eluent from the medium of - 10 cation exchange chromatography, thus obtaining a botulinum neurotoxin It is purified, where the purified botulinum neurotoxin obtained has a potency. from about 2.4 x 107 to about 5.9 x 10 ”units / mg of biologically active botulinum neurotoxin and can be obtained in an amount of between about 4 mg to about 25 mg per liter of APF fermentation used Our invention further comprises a method of manipulation to produce a substantially APF pharmaceutical composition in which the active ingredient is a biologically active botulinum neurotoxin, comprising the steps of obtaining a biologically active botulinum neurotoxin by (i) providing a fermentation medium which is substantially free of animal products; (ii) ferment the Clostridium botulinum bacteria in the fermentation medium, and (iii) recover the biologically active botulinum neurotoxin from the fermentation medium, using an anion exchange chromatography medium followed by use of an exchange chromatography medium cationic; and then manipulating the botulinum neurotoxin with at least one suitable excipient to thereby produce a substantially APF pharmaceutical composition.
In one example, the method includes the step of drying the manipulated botulinum neurotoxin and at least one excipient suitable to obtain a stable form for shipping or storage, by lyophilization or vacuum drying, in which the active ingredient is a biologically botulinum neurotoxin. active, where the fermentation medium comprises a protein product obtained from a vegetable.
The vegetable form from which the protein product can be obtained can be soybean seed, corn or unsweetened malt It is from Lupinus campestris, or hydrolyzed products thereof. The botulinum neurotoxin obtained can have a potency between about 2.0 x 10 ”units / mg of botulinum neurotoxin to about 6.0 x 10º units / mg of botulinum neurotoxin. Botulinum neurotoxin is selected from the group consisting of botulinum neurotoxins types A, B, C1, D, E, F and G, preferably botulinum neurotoxin type A. In particular cases, the neurotoxin S botulinum is obtained as a neurotoxic component of toxin botulinum with a molecular weight of about 150 kDa free of complex protein - 10 complex of the botulinum toxin complex. In particular embodiments, the appropriate excipient is selected from the group consisting of albumin, human serum albumin, recombinant human serum albumin, gelatin, sucrose, trehalose, hydroxyethyl starch, collagen, lactose, sucrose, amino acid, sodium chloride , potassium chloride, polysaccharide, caprylate, polyvinyl-pyrrolidone and potassium citrate. Obtaining the biologically active botulinum neurotoxin may also comprise the stage of using a hydrophobic interaction medium followed by the use of the cation exchange medium. In particular examples, vacuum drying takes place at a temperature of about 20ºC to about 25ºC. In some embodiments, vacuum drying occurs at a pressure of about 70 mmHg to about 90 mmHg, for example. The vacuum drying time can be from about 4 hours to about 5 hours, for example.
Particular aspects of the present disclosure are directed to providing a pharmaceutical composition, which may, for example, comprise a biologically active botulinum neurotoxin complex and an excipient selected from the group consisting of albumin, human serum albumin, recombinant human serum albumin, gelatin , sucrose, trehalose, hydroxyethyl starch, collagen, lactose and sucrose, where the pharmaceutical composition is essentially free of nucleic acid. In particular examples, a pharmaceutical composition is provided and which comprises a biologically active botulinum neurotoxin where the botulinum neurotoxin obtained has a potency between about 2.0x
107 to about 6.0 x 10 th units / mg of biologically active botulinum neurotoxin and at least one excipient, where the composition comprises less than about 12 ppm nucleic acid, preferably less than 1 ppm nucleic acid per mg of botulinum neurotoxin complex
nica
In a particular embodiment, a substantially APF chromatographic process for obtaining a biologically botulinum neurotoxin. active comprises the following additional steps of: cultivating the bacterium Clostrididium botulinum in a culture medium substantially APF for between - 10 about 10 hours and about 12 hours, or until the biomass measurement of the culture medium has an optical density , at a wavelength: of about 540 nanometers (nm), between about 0.8 AU and about 4.5 AU; ferment the Clostridium botulinum bacteria from the culture medium in a substantially APF fermentation medium for about 65 hours to about 75 hours or until the biomass measurement is obtained at the end of the fermentation by measuring the optical density of the fermentation medium using - when an online biomass probe at a wavelength of about 890 nm is between about 0.05 AU and about 0.7 AU; harvest the fermentation medium for about 2.5 hours, whereby cell fragments in the fermentation medium are removed and the weight of the fermentation medium is reduced to about three quarters of its initial weight at the beginning of the co - you; concentrate the fermentation medium harvested by filtration in tannin flow to about a quarter of its initial volume at the beginning of the harvest stage; dilute the concentrated fermentation medium by adding a buffer, where the concentration and dilution steps take place between about 0.5 hour to about 2 hours, whereby during concentration the fermentation medium is reduced to about a quarter of its initial weight at the beginning of the harvest stage, and is then diluted, by adding the buffer, back to its original initial weight at the beginning of the harvest stage; contact the diluted fermentation medium with a capture chromatography medium to capture the biologically active botulinum neurotoxin for a period of about 4 hours to about 5 hours; contact the eluent of the chromatography
capture graph with a first polishing chromatographic medium to conduct a first polishing run to remove impurities from it for a period of time from about 1.5 hours to about 2.5 hours; conducting a second polishing run by passing the eluent from the polishing chromatographic medium through a hydrophobic interaction medium for a period of time from about 1.5 hours to about 2.5 hours; process the eluent of the hydrophobic interaction medium by diafiltration, for a period of time from about 1 hour to about 4 hours; and filter the processed eluent through a biocharge reduction filter, for about 0.5 hour, thus obtaining - 10 biologically active botulinum neurotoxin.
In another aspect, a substantially chromatographic and APF system for obtaining a biologically active botulinum neurotoxin is described, the system comprising: a first apparatus for growing Clostridium botulinum bacteria, the first apparatus capable of containing a substantially APF culture medium; a second apparatus for fermenting Clostrididium botulinum bacteria which was grown in the first apparatus, the second apparatus capable of containing a substantially APF fermentation medium; a third apparatus for harvesting the fermentation medium; a fourth apparatus for conducting concentration and dilution of the medium harvested from the third apparatus; the fourth apparatus comprising tangential flow filtration (TFF); a fifth apparatus for conducting a first purification of a botulinum neurotoxin from the concentrated and diluted medium, the fifth apparatus comprising an anion exchange chromatography medium, thereby obtaining a first purified botulinum neurotoxin; and a sixth device for conducting a second purification of the first purified botulinum neurotoxin, the sixth device comprising medium cation exchange chromatography, and thus obtaining a second purified botulinum neurotoxin. In a particular embodiment, the system can also comprise a seventh device to conduct another purification, purifying the second purified botulinum neurotoxin obtained from the sixth device, where the seventh device comprises a means of hydrophobic interaction, thus obtaining a third purified botulinum neurotoxin. The system can also be comprised of an eighth apparatus containing a filtration membrane to filter the eluent from the sixth or seventh apparatus.
In yet another embodiment, a chromatography column with a diameter of between about 8 cm and about 15 cm contains the anion exchange chromatography medium and the anion exchange chromatography medium can have a column height of between about 8 cm and about 15 in, for example. In yet another example, the fourth device in the system: it comprises a chromatography column that is operated at a flow rate of between about 125 cm / hour and about 200 cm / hour, and the volume - 10 napodeterum volume column between about 500 ml and about 1 L. In one aspect, the fifth apparatus can have a column volume of about 50: ml and about 500 ml, and a bed height of about 8 cm and about 15 in, for example. In some examples, the chromatography column of the fifth apparatus has a column diameter of about 2 cm and about 10 hundred, for example. The chromatography column of the fifth device can have a flow rate of between about 100 cm and about 200 cm / hour. The seventh apparatus in the system may comprise a filtration membrane. In another embodiment, the system may further comprise a ninth apparatus, the ninth apparatus comprising an anaerobic chamber to provide an anaerobic atmosphere where the first apparatus for culturing Clostridium botulinum bacteria is contained therein. This ninth device preferably includes an integrated high efficiency particulate air filter (HEPA) located inside the chamber / workstation. The second device in the system (for fermentation) can include at least one probe for detecting oxidation-reduction potential, or pH, or optical density. In a particular example, at least one disposable probe is selected from the group consisting of a reduction-oxidation probe, a pH probe and a turbidity probe. An eighth apparatus in the system may comprise a tangential flow filtration apparatus for concentration and etrocade buffer. In another embodiment, the system may comprise a tenth apparatus that includes a biocharge reduction apparatus to reduce biocharge. In one example, the biocharge reduction apparatus comprising
of a filter containing a pore size of between about 0.1 µm and 0.3 µm, preferably 0.2 µm.
The system may also include an eleventh apparatus for use after obtaining the second purified botulinum neurotoxin, for storage of the purified botulinum neurotoxin.
In one example, this storage device provides a storage temperature between about -25ºC to about -80ºC. In another aspect, a biologically active botulinum toxin is that provided by an APF process containing the following steps of providing a substantially APF fermentation medium; ferment a bacterium - 10 Clostridium botulinum in the fermentation medium; recovering the biologically active botanical neurotoxin from the fermentation medium using an anion exchange chromatography medium followed by the use of a cation exchange chromatography medium, where the biologically active botulinum toxin obtained has a potency between about 2 , 0 x 10 th units / mg botulinum neurotoxin at about 6.0 x 10 th units / mg botulinum neurotoxin.
In one embodiment, the process further comprises the step of further purifying botulinum neurotoxin using a hydrophobic interaction medium followed by the use of the cation exchange medium.
According to another aspect, a method for treating a condition in a patient is provided, using a pharmaceutical composition comprising the botulinum neurotoxin obtained according to the methods described herein.
A condition can include an illness, discomfort, illness, or deformity or aesthetic appearance.
In one example, the method of treating a condition in a patient comprises the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a botulinum neurotoxin and at least one appropriate excipient, where the botulinum toxin has a potency of about of 1 unit> about .02 picograms to treat the patient's condition.
In a particular example, the botulinum neurotoxin to treat these conditions can be obtained by a process of growing the bacterium Clostridium botulinum in a substantially APF culture medium; obtaining a substantially APF fermentation medium containing the botuli- neurotoxin; single; contacting the medium with an anion exchange chromatography medium to provide a purified eluent containing the botulinum neurotoxin; contact an eluent with a cation exchange chromatography medium to thereby obtain another purified eluent, and filter the purified eluent to thereby obtain the biologically active botulinum neurotoxin purified from a chromatographic process
substantially APF. . In one embodiment a substantially APF chromatographic system for obtaining a biologically active botulinum neurotoxin is included, the system comprising a first apparatus for anae-: robic culture of Clostridium botulinum bacteria, the first apparatus capable of containing and from 200 ml to about 1 L of a substantially APF culture medium; a second device for anaerobic fermentation of Clostridium botulinum bacteria that was grown in the first device, the second device capable of containing from about 5 L to about 75 L, or from about 2 L to about 75 L, or about from 2 L to about 30 L of a substantially APF fermentation medium and include at least one disposable probe selected from the group consisting of oxidation reduction probe, pH probe and turbidity probe; a ninth apparatus to provide an anaerobic atmosphere capable of containing the first apparatus, the ninth apparatus comprising an anaerobic chamber containing a high efficiency particle air filter integrated within the chamber, where said chamber may contain the first apparatus for anaerobic culture of Clostridium botulinum bacterium; a third apparatus for harvesting the fermentation medium; a fourth apparatus for conducting the concentration and dilution of the collected medium, a fifth apparatus for conducting a first purification of botulinum neurotoxin obtained from the fourth apparatus, the fifth apparatus comprising an anion exchange chromatography medium, thus obtaining a first purified botulinum neurotoxin ; a sixth apparatus for conducting the second purification of the first purified botulinum neurotoxin, the sixth apparatus comprising a cation exchange chromatography medium, thereby obtaining a second purified botulinum neurotoxin; a seventh apparatus conducting a third purification of the second purified botulinum neurotoxin, the seventh apparatus comprising a hydrophobic interaction medium, thereby obtaining a third purified botulinum neurotoxin; and an eighth apparatus for concentration and buffer exchange of the third purified botulinum neurotoxin, the eighth apparatus comprising a TFF membrane. In particular examples, the fermentation medium comprises no more than about 5% w / v of a protein product derived from vegetables, no more than about 2% w / v of a yeast extract and no more than about 2% w / v glucose, and where the pH level of the fermentation medium - 10 tion is about pH6.8 to about 7.6, preferably about pH 7.3 Å at the beginning of a fermentation step of 72 hours, for example. In an e. modality, the method can also comprise the step of contacting the purified eluent with a hydrophobic interaction medium to obtain another purified eluent containing the botulinum neurotoxin. In a particular example, the method of treating the conditions may be to use a botulinum neurotoxin that is obtained as a botulinum toxin neurotoxic component with a molecular weight of about 150 kDa free of complexing proteins from a botulinum toxin complex. Exemplary stages of administration can be selected from the group of administration routes for intramuscular, intradermal, subcutaneous, intraglandular, intrathecal, rectal, oral and transdermal administration, and a botulinum neurotoxin is selected from the group consisting of botulinum toxin type A, B , C1, D, E, F or G. Preferably, the botulinum neurotoxin is type A botulinum neurotoxin. In some instances, the system can facilitate a process by which a biologically active botulinum neurotoxin complex can be obtained for use as part of the pharmaceutical composition which comprises less than about 12 ng of nucleic acid per mg of botulinum neurotoxin complex, preferably below 1 ng of nucleic acid per mg of botulinum neurotoxin complex, more preferably containing no measurable nucleic acid (for example below the detection limit). DRAWINGS
Figure 1A is a flowchart showing the main steps in the NAPF process in Example 1. Figure 1B is a flowchart showing the main steps in the IAPF process in Example 2, where the capture and polishing chromatography steps can use 2 columns (exchange anionic followed by cationic exchange) or 3 columns (FAPF) (anionic exchange followed by cationic exchange followed by a hydrophobic interaction column).
DESCRIPTION . Our invention is based on the discovery that a high potency, high purity and biologically active Clostridial neurotoxin, such as a botulinum neurotoxin, can be obtained by using a simple, fast and economical APF chromatographic system and process. Significantly, the use of our system and process can result in a purified botulinum neurotoxin comprising 1 ng (or less than 1 ng) of nucleic acid impurities (RNA and DNA) per 1 mg of a purified botulinum neurotoxin obtained, although no enzyme derived from animals, such as RNase and DNase, is used to purify the fermented botulinum neurotoxin.For example, the use of our system and process can result in a purified botulinum neurotoxin comprising less than about 0.6 ng of nucleic acid impurities (RNA and DNA) per milligram of purified botulinum neurotoxin obtained.The botulinum neurotoxin obtained can be a type A botulinum toxin complex, as a 300 kDa, 500 kDa or 900 kDa complex (approximate molecular weights) or mixtures thereof. The botulinum neurotoxin obtained may also be a type of neurotoxic component of botulinum toxin (ie, without complex proteins) with a molecular weight of about 150 kDa. The botulinum neurotoxin can be any of serotypes A, B, C, D, E, F or G or mixtures thereof. In addition, improved systems and processes can be practiced in conjunction with a recombinant, chimeric or modified hybrid botulinum toxin (light chain, heavy chain, or both chains together). An important aspect of our invention is the use of an anion exchange chromatography (capture) medium followed by the use of cation exchange chromatography (polishing) medium to purify botanical neurotoxin.
of an APF fermentation medium in which the bacterium Clostridium botulinum has been fermented.
We demonstrate that the use of anion exchange followed by the use of cation exchange chromatography medium provides an effective and fast method for obtaining high purity and high yield botulinum neurotoxin.
Previously, the use of ion exchange chromatography was believed to have a detrimental effect on botulinum neurotoxin gel band patterns, thus discouraging the use of ion exchange chromatography for the purification of botulinum neurotoxin.
See, for example, US Patent 7,452,697 in column 55, lines 53 to 57. "The other important aspect of our invention is that this results in high purity botulinum neurotoxin (ie O 1 ng nucleic acid / x mg of botulinum neurotoxin obtained), as set out above.
Another important aspect of our invention is that while the Schantz process requires several weeks (that is, typically about 18 to about 22 days) to grow, ferment and purify botulinum neurotoxin, a system and process within the scope of our invention allows all stages of cultivation, fermentation and purification to be completed in a week or less.
In a preferred embodiment of our invention, all stages of culture, fermentation and purification can be completed in six days or less.
In a more preferred mode of our invention, all stages of culture, fermentation and purification can be completed in about four days or less (for example in about 80 to about 144 hours or within a time / range among these). We invented this faster modality of our invention by developing a process or nine steps (and the system for achieving the process) and finding that each of the eight or nine steps in a particular modality can be completed within the time periods set out here below: about 8 hours to about 14 hours for culture; about 60 hours to about 80 hours for fermentation; about 2.5 hours to harvest; about 2 hours to about 4 hours for concentration and dilution;
about 4 hours to about 6 hours for an ion exchange chromatography (this includes time to elute the captured botulinum toxin); about 2 hours for cation exchange chromatography; about 2 hours for an optional third chromatography step (ie hydrophobic interaction chromatography; about 2 hours to about 4 hours for concentration and diafiltration, and about 1/2 hour for another filtration Thus, the total time required to complete our 8 or 9 steps, the most preferred modality - 10 our invention is about 75 hours to about 150 hours.: Our invention is more efficient and saves time. aspect, our new process uses pre-selected and verified cell lines, and thus ends the steps of the Shantz process of the previous technique of plating and growing cells, selecting and harvesting colonies and expanding cell lines from the collected colonies (prior to the cell culture and fermentation steps) that were necessary to cultivate and then inoculate the fermentation medium In one aspect, our invention begins immediately with the culture of pre-selected cells for inoculation of a APF culture medium, thus saving time and process steps.
Through experimentation, two-column ("IAPF") and three-column ("FAPF" / "FIAPF") chromatography systems and processes were developed to purify a botulinum neurotoxin present in the fermentation medium, the fermentation medium resulting from an APF fermentation of Clostridium botulinum bacteria. Significantly, while an APF fermentation process can reduce or eliminate animal products (such as casein and broth) as nutrients in the medium used to grow and ferment Clostridial bacteria, known APF fermentation processes are typically followed by one or more purification steps that make use of products of animal origin, such as the enzymes DNase and RNase. Our systems and processes for purifying botulinum neurotoxin present in an APF fermentation medium do not use enzymes.
but of animal origin.
Our invention may include loading a harvested fermentation medium (for example clarified by filtration) onto an anion exchange column such as a POROSO & 50HQ ion exchange chromatography resin from Applied Biosystems. In one aspect, a strong anion exchange medium can be used, containing a polystyrene / divinylbenzene based matrix and particle diameter of about 50 µm and dynamic capacity (BSA ds mg / ml) of about 60 to 70. The column anion exchange technology captures the Clostridial neurotoxin (as a botulinum toxin complex) and reduces levels of - 10 impurity. An anion exchange column has been shown to provide an efficient capture of a botulinum toxin complex from the fermented medium with retention of the biological activity of the botulinum toxin complex, while also separating many impurities present with the botulinum toxin in the fermentation medium. An appropriate buffer is used to elute captured (bound) Clostridial elutaneurotoxin from the anion exchange column. In a two-column embodiment of our invention, the eluent (containing the botulinum neurotoxin) from the anion exchange column is loaded over a cation exchange column to further purify the botulinum neurotoxin from impurities. The cation exchange column can be a POROSQE 20HS cation exchange resin from Applied Biosystems. In one aspect, a strong cation exchange medium can be used, containing a polystyrene / divinylbenzene base matrix and particle diameter of about 20 µm and dynamic binding capacity (lysozyme mg / ml) of about> 75. In a three-column modality (FAPF) of our invention, the eluent from the cation exchange column is loaded onto a hydrophobic interaction column such as GE Healthcare's Phenyl Sepharose resin to further purify the botulinum neurotoxin. In one aspect, a matrix of highly bonded agarose beads with a particle size of about 34 µm, which has been derivatized with phenyl groups and have a dynamic binding capacity (chymotrypsinogen mg / ml) of about 45, can be used . After the two-column or three-column process, the eluent from the last column used can be further processed to obtain a highly purified bulk botulinum toxin complex. Processing steps Í after chromatography may include the concentration of buffer exchange for ultrafiltration and diafiltration, sterile filtration and preparation of a solution of purified botulinum toxin complex instead of a suspension (prior technique), preferably in potassium citrate, and in one example, at a concentration of 10mM potassium citrate at a pH of about 6.5.
It is In certain preferred embodiments, the medium for growth (anaerobic culture and anaerobic fermentation) of Clostridium botulinum and - 10 production of botulinum toxin may comprise soy-based products * to replace animal products so that the medium is subs - substantially or totally free from products derived from animals. The culture step increases the amount of microorganism for subsequent fermentation. The culture allows inactive, previously frozen bacteria to rejuvenate in active growth cultures. In addition, the volume and quantity of viable microorganisms used to inoculate the fermentation medium can be controlled with greater precision from a growing culture than it can be from a stored Clostridium botulinum cell bank. and not spreading. Thus, a sample of a work cell bank in APF medium is thawed and placed in the selected APF culture medium. In order to obtain an appropriate level of bacterial growth, the culture medium is used to inoculate the fermentation medium. As an example, from about 1% to about 5%, or an amount in between, of the growth medium containing Clostridium botulinum of the growth phase is used to inoculate the fermentation medium. Fermentation is conducted to produce the maximum amount of microbial cells in a large-scale anaerobic environment (Ljungdahl! Et al., Manual of industrial microbiology and biotechnology (1986), edited by Demain et al, American Society for Microbiology , Washington, DC page 84). Alternatively, the growth of Clostridium botulinum in the fermentation medium can proceed by adding a sample of the working cell bank directly to the fermentation medium.
In the prior art, the growth of Clostridium botulinum in the culture medium typically proceeds in two stages, a first stage of cell plating, cell colony growth, selection and growth, followed by a second stage of inoculation of the culture medium. ture (typically a two-stage step-up culture) and inoculation of the fermentation medium and production of botulinum toxin.
Preferably, growth in the culture medium at any stage does not result in cell lysis prior to inoculation of the fermentation medium with the final growth in culture medium.
Thus, before our invention, it took about four - 10 days to grow the Clostridium botulinum bacterium before the fermentation step begins.
According to our invention we are able to complete e. the entire culture in just 8 to 14 hours because there is no need for the steps previously used for cell plating, subsequent waiting time for colony growth on blood agar plates, selection of colonies for growth in volumes small cultures (for example, 8 to 9 mL) which then provide an inoculum for the culture medium.
According to one aspect of our invention, pre-selected cells are directly used to inoculate the culture medium which is then used to inoculate the full-scale fermentation medium from which botulinum atoxin is eventually purified, thereby eliminating plaque , colony formation, selection and step up steps previously used to grow cells that could inoculate a culture medium that is then used by itself to inoculate the fermentation medium.
Animal-based culture medium (non-APF or "NAPF") usually includes brain and heart infusion medium (BHI), baptopeptone, NaCl, and glucose.
Culture media within the scope of our invention are APF culture media.
For example, a soy based product can be used in place of BHI and bacto-peptone in the culture and fermentation medium.
Preferably, the soy-based product is soluble in water and comprises hydrolyzed soy, although Clostridium botulinum can grow in medium containing insoluble soy.
Any source of soy-based products can be used in accordance with the present invention.
Preferably,
the soy is hydrolyzed soy and the hydrolyzation was carried out using non-animal enzymes. Sources of hydrolyzed or soluble soy include Hy-Soy (International Quest), soy peptone (Gibco) Bac-soytone (Difco), AMISOY (Quest), NZ soy (Quest), NZ BLA soy, NZ BL7 soy, SESOM ( DMV International Nutritionals), eSESOMK (DMV).
EXAMPLES The following examples establish particular modalities for our invention and are not intended to limit the scope of our invention. Unless otherwise stated in the examples "toxin" or "botulinum toxin" means a type A botulinum toxin complex with a: molecular weight of about 900 kDa. Systems and methods described here to purify a botulinum toxin type A complex with a molecular weight of about 900 kDa, already have applicability for the purification of about 150 kDa, about 300 kDa, about 500 kDa as well as other molecular weight toxins, complexes, botulinum toxin serotypes and neurotoxic botulinum toxin component. Example 1 Non-APF (Schantz) Processing for Obtaining a Botulinum Toxin This example establishes the Schantz process of the previous technique for obtaining botulinum neurotoxin. The process is a non-APF process using animal-derived medium and reagents (that is, meat agar blood plates for culture, casein in the fermentation medium and use of RNase and DNase enzymes to purify botulinum neurotoxin). Figure 1A is a flow chart showing the main stages of the Schantz process. The Schantz process has about 16 to 20 main stages, for scale production work it uses a 115 L fermentor and takes about 3 weeks to complete. The Schantz process is started by thawing a non-APF master cell bank flask of Clostridium botulinum (MCB) to room temperature followed by four cultivation steps. First, to select colonies with an appropriate morphology, aliquots of the defrosted MCB vial were streaked onto Columbia plates
Pre-reduced Blood Agar (CBA) and incubated anaerobically for 30 to 48 hours at 34ºC + 1ºC. Second, the selected colonies were inoculated in 9 mL test tubes containing a casein growth medium for 6 to 12 hours at 34 ° C. The contents of the 9 mL tube with the fastest growing medium and the highest density (growth selection step) were then cultured by two-step anaerobic incubations (the third and fourth stages of cultivation), being an incubation of 12 to 30 hours at 34ºC in a 600 ml seed cultivation bottle at 1 L, followed by 10 cultivation in a 15 L to 25 L seed fermenter containing O a casein growth medium for 6 to 16 hours at 35ºC. These two adjustment cultures were carried out in a nutrient medium containing 2% casein hydrolyzate (a casein digest [milk protein]), 1% yeast extract and 1% glucose (dextrose) in water at pH 7.3.
Adjustment cultures were followed by another 60- 96 hour incubation at 35ºC in a commercial scale production fermenter (ie 115 L) in a medium containing casein under a controlled anaerobic atmosphere. The growth of the bacteria is usually complete after 24 to 36 hours, and during the fermentation stage conducted for about 65 to about 72 hours where most cells undergo lysis and release the botulinum neurotoxin. It is believed that the toxin is released by cell lysis and activated by proteases present in the medium. A filtrate of the culture medium can be prepared using a single layer deep filter to remove coarse impurities (i.e., whole and broken cells) thus obtaining a clear solution referenced as a clarified culture. The collection of botulinum neurotoxin from clarified culture was achieved by decreasing the pH of the clarified culture to pH 3.5 with 3M sulfuric acid to precipitate the crude toxin at 20ºC (precipitation by acidification). The crude botulinum neurotoxin was then concentrated (to achieve a reduction in volume) by ultra-microfiltration (microfiltration) (referred to as MF or UF) followed by diafiltration (DF). A 0.1 µm filter was used for the microfiltration step. The harvested raw or crude toxin was then transferred to a
digestion and stabilized by the addition of the protease inhibitor: benzamidine hydrochloride.
DNase and RNase were added to the digested nucleic acids (hydrolysis). Hydrolyzed nucleic acids and low molecular weight impurities were then removed by other stages of UF and DF.
The toxin was then extracted with phosphate buffer pH 6.0 and the cell fragments removed by clarification.
Then three sequential precipitation steps (precipitations with cold ethanol, hydrochloric acid and ammonium sulfate) were carried out.
The purified botulinum neurotoxin complex (bulk toxin) was stored as a suspension in a sodium phosphate buffer of - 10 dio / ammonium sulphate at 2 ° C to 8 ° C.
The completion of this Schantz process (not APF) from Example 1, - including the harvest and purification steps, takes about two to three weeks.
The resulting bulk botulinum neurotoxin was a high quality suspension of a 900 kDa botulinum toxin type A complex prepared from the Clostridium botulinum Hall A strain with a specific potency of D2 X 10 ”U / mg, an Azso / Az78 less than 0.6 and a distinct band pattern in gel electrophoresis, and suitable for use in the formulation of a botulinum toxin pharmaceutical composition.
Botulinum neurotoxin can also be obtained from a non-chromatographic APF process, as set out in Example 7 of US patent 7,452,697, the complete non-chromatographic APF process, (starting in culture until the end of all purification and processing steps) le - taking about two to three weeks to complete.
Alternatively, botulinum neurotoxin can also be obtained from a chromatographic process — APF, as set out in Example 16 of US patent 7,452,697, the APF chromatographic process (starting in culture until the end of all purification steps and processing) taking a week or more to complete.
Example 2 Two and Three Column APF Chromatographic Systems and Processes for Obtaining a Botulinum Neurotoxin Rapid APF systems and processes based on cation exchange chromatography have been developed to obtain a high-performance, high-purity botulinum neurotoxin.
The process of this Example 2 had only 8-10 main steps, for production purposes (ie, to obtain gram quantities of the final botulinum neurotoxin) used a 20 L fermentation bottle and took only 4-7 days, preferably about from 4 to about 6 days, to complete all stages from the beginning of the culture to the conclusion of the final purification and storage of the toxin.
The devices used in the systems described here are discussed below.
Both the two chromatographic process and the three chromatographic process were developed and are established here.
The two-half process: an ion exchange chromatography followed by cation exchange chromatography.
The three media process used ion exchange chromatography followed by cation exchange chromatography followed by hydrophobic interaction chromatography.
HIC removed other impurities such as a 49 kDa impurity (which was a glucose phosphate isomerase from the host cell, as discussed below). Preparation of a work cell bank A new cell bank of Clostridium botulinum has been developed (to be used to start the culture stage) without the use of Columbia Blood Agar plates, which removed the need for selection of colony before cultivation and also eliminated the need to conduct the Shantz process step up in tube cultivation and steps to multiply the seed (cultivation). For this purpose, a stabilized Schantz master cell bank (MCB) was used to create an APF research cell bank (RCB) from which a new APF master cell bank (MCB) and a subsequent work cell bank (WCB ) were generated.
A research cell bank (RCB) was prepared from a colony of MCB Schantz (NAPF). To remove the animal-derived protein from the MCB flask, the cells were washed twice in APF medium containing 2% w / v SPTII (Pepto- na Soy type II), 1% w / v yeast extract, and 1% w / v glucose.
The cells were plated in APF medium under rigid anaerobic conditions using a controlled modular atmosphere anaerobic chamber (MACS) system. An isolated colony was further expanded and stored in APF medium containing about 20% glycerol below -135ºC.
The APF-MCB was prepared under GMP conditions by expanding the oxygen-free RCBemmeioAPF (200 mL, reduced for a minimum of 12 hours in an anaerobic chamber) and grown in a MACS anaerobic chamber at 34.5ºC + 1ºC (stirred at 60 rpm) until the ODs4 of the culture reached 2.5] 1.0 AU. Sterile glycerol was added to the resulting culture to a final concentration of about 20%, after which the mixture was transferred from the 1 ml / microleaker (weak APF-MCB). The bottles went then! frozen quickly in liquid nitrogen, and then stored - below -135ºC. An APF-WCB was prepared under GMP conditions expanded as above. The resulting APF cell banks have been characterized for identity, purity, viability and genetic stability.
Upstream steps (Culture and Fermentation) Our Example 2 process had two general stages; an upstream and a downstream internship. The upstream stage includes expansion of an initial cell line (growth and reproduction of Clostridium botulinum bacteria in a substantially APF culture medium), fermentation, harvesting (removal of cell fragments) to provide a harvested and clarified culture that it is then concentrated and diluted. Thus, in this example, the nine steps of our two-column process are cultivation, fermentation, harvesting, filtration, concentration, capture chromatography (anionic), polishing chromatography (cationic), buffer exchange, reduction of biocharger and bottle filling .
The upstream stage included the use of a culture medium in a 1 L bottle containing 400 mL of reduced seed APF culture medium (in an anaerobic chamber) (2% w / v SPTII, 1% w / v extract of yeast, (adjusted to pH 7.3 with 1N sodium hydroxide and / or 1N hydrochloric acid before autoclaving)) 1% w / sterile glucose added after autoclaving the culture medium). The culture medium (seed) was inoculated with 400 pull of thawed Clostridium botulinum WCB. The Incubation
tion / culture occurred at 34.5ºC O 1.0ºC with agitation at 150 rpm in an anaerobic chamber.
When the optical density of the culture medium at 540 nm was 1.8 s O 1.0 AU, the total contents of the 1 L flask (approximately 400 mL) were transferred to a 20 L production fermentor containing APF fermentation medium adjusted with 1 N sodium hydroxide and / or 1 N hydrochloric acid after steam sterilization to pH 7.3, fermentation medium composed of 3.25% w / v SPTII, 1.2% w / v yeast extract, 1 , 5% w / v of sterile glucose (added after sterilization; sterilization, for example at about - 10 122ºC for 0.5 hour). The temperature and agitation were controlled at 35ºC O: 1ºC and 70 rpm, respectively. The nitrogen coating was adjusted to e 12 slpm and the headspace pressure was adjusted to 5 psig to maintain an anaerobic environment for cell growth. The fermentation pH and cell density were monitored by online pH and turbidity probes, respectively. The three phases for production fermentation include exponential, stationary and autolysis growth phases. Cellular autolysis, which releases the active BONT / A complex in the culture medium, has been observed to occur consistently between 35 hours and the end of fermentation. At the end of the fermentation, the culture was cooled to 25ºC for harvest.
Once the fermentation medium was cooled to 25 ° C, the cell fragments were separated from a type A botulinum neurotoxin complex containing the lysate by deep filtration, first by a gradient pre-filter of nominal 5 to 0 retention rating .9 μm to remove cell fragments, and then by a grading rating of 0.8 to 0.2 μm nominal retention to remove DNA (removal up to about 80%). Both filters were rinsed together with 20 L of water for injection (WFI) before use. A minimum of 15 L of the filtrate was required for further processing, and any excess material was decontaminated after sampling in the process was complete. The filtrate was stored at 4C if not immediately processed by ultrafiltration.
Inside a biosafety cabin (BSC) the filtrate from the harvest stage was concentrated from 15 L to 5 + 0.5 L using a hollow fiber,
tangential flow filtration membrane (TFF) from GE Healthcare. The ultrafiltered material was then diluted with 10 mM sodium phosphate buffer pH 6.5 to a final volume of 20 L. This material was purified by using 2 column chromatography (anionic then cationic) or three columns (anionic, cationic and then hydrophobic interaction). The collected, diluted and ultrafiltered material was stored at 4ºC if not immediately processed by purification. In the Schantz process, the culture stage is ended and the fermentation stage begins based on time and visual observation of the growth of the culture. In contrast, in our Example 2 processes, the determination of when to end the culture step is based on the analysis of .. optical density of the culture fluid, which ensures that the culture is in the logarithmic growth phase at the time of initiation of the fermentation stage, and allows the reduction of the duration of the culture stage from about 8 hours to about 14 hours. Our OD parameter for the finished culture stage maximized the health of the cultured cells and stimulated a robust and abundant botulinum toxin resulting from the fermentation stage. The average optical density (at 540 nm) of the culture medium at the conclusion of the culture was 1.8 AU. The average duration of the fermentation stage 72 hours and the average final turbidity - (Asso) of the fermentation medium at the conclusion of the fermentation stage was 0.15 AU. The average amount of botulinum toxin type A complex present (as determined by ELISA) in the 20 L fermentation medium (complete broth) at the end of the fermentation step was about 64 Ug of botulinum toxin type A complex / mL fermentation medium.
The harvesting step used deep filtration to remove cell fragments and nucleic acids, followed by ultrafiltration and dilution to prepare the fermentation medium for the next step in the process. This collection / clarification of cell fragments is fundamentally different from the Schantz collection process, which uses precipitation by acidification followed by microfiltration and diafiltration to concentrate and change buffers in preparation for further processing.
Downstream steps (Purification) The downstream steps included capture of a botulinum neurotoxin in an anion exchange column, column elution and other separation of impurities by polishing in a cation exchange column, and preferably (in the process of three columns), passage of eluent containing the desired botulinum neurotoxin through a third column, preferably a hydrophobic interaction column (for example chromatography), followed by concentration and buffer exchange using tangential flow filtration (TFF), and reduction of biocharge (for example by another filtration using a - 10 0.2 µm filter) to a final neurocomplex of botulinum toxin type A optimized for cold storage, preferably freezing, and eventual manipulation in a pharmaceutical composition of botulinum neurotoxin complex type A. The sequence of the chromatography and filtration stages intended to remove the product and impurities related to the process, to remove agent accidental potentials and to control the concentration of botulinum neurotoxin type A complex and final botulinum neurotoxin type A buffer matrix to provide a more stable drug substance. A more detailed embodiment of the three-column downstream process is as follows. The clarified (diluted) ultrafiltered material (20 L, as described above) passed through a POROSG & 50HQ ion exchange chromatography resin, the captured botulinum neurotoxin was eluted from the anion exchange column and then ran through a POROSO 20HS resin of cation exchange chromatography, the eluent from which flowed through a phenyl Sepharose HP chromatography resin. The HIC column eluent was subjected to tangential flow filtration of 100 kDa, followed by filtration at 0.2 µm. The resulting type A botulinum neurotoxin complex was frozen for storage.
In this Example, we used in the first chromatography step of the downstream process a POROSGO 50HQ ion exchange chromatography resin packaged in a column with an internal diameter of about 8 cm and a column height of about 15 cm. The operation of the entire PO-ROSQE 50HQ column was completed at room temperature, and the flow was in a downward direction. A botulinum neurotoxin type A complex was eluted from the anion column using a pH exchange step where the most negatively charged components as nucleic acids (for example DNAs and RNAs) and other host cell proteins bound to the column anion exchange.
Details of the anion exchange step were: use of the PO-ROSQE 50HQ column using 0.1 N sodium hydroxide for a minimum contact time of 30 minutes (at least about 3 column volumes, at 230 in / hour ). The column was then equilibrated with 50 - 10 mM sodium phosphate buffer, pH6.5 (at least 5 column volumes). Then, the diluted and clarified and ultrafiltered material (i.e., lysed AFP fermentation material) was loaded at 230 cm / hour onto the POROSG & 50HQ anion exchange column, followed by washing with at least about 20 column volumes. of 50 mM sodium phosphate, pH 6.5 at 230 cm / hour until the absorbance at 280 nm of the column effluent decreased to 0.10 AU, followed by elution with 50 mM sodium acetate, pH 4.8 at 230 cm / hour. The grouped product was collected when the absorbance at 280 nm (A> z3o) increases to at least about 0.15 AU and by the maximum peak equal to or less than about 0.2 AU at the trailing edge, in a vessel containing 1 column volume of 50mM sodium acetate, pH 4.8. This elution group was stored at about 2 ° C to about 8 ° C for up to 48 hours. The second chromatography step in the downstream process of this Example 2 used a POROSQE 20HS cation exchange chromatography resin packaged in a column with an internal diameter of 8 cm and a column height of 5 cm. The complete operation of the POROSO 20HS column was completed at room temperature, and the flow was in a downward direction. A botulinum neurotoxin type A complex is associated with the POROSG 20HS column resin. A botulinum neurotoxin type A complex was then eluted from the column using a salt change step. Impurities related to the product were eluted with the wash buffer and decontamination solution.
Details of the cation exchange step were: use of a PO-
: ROSQ 20HS using 0.1 N sodium hydroxide solution for a minimum contact time of 30 minutes (at least about 3 column volumes, at 230 cm / hour). The column was then equilibrated with a 50 nM sodium acetate buffer, pH 4.8 (at least about 5 column volumes). Then, the product grouped in POROSE & 50HQ (collected as described above, fresh or from refrigeration) was loaded onto the PO-ROSQG 20HS column. The column was washed with a 50 nM sodium acetate buffer, pH 4.8 (at least about 3 column volumes) and then washed again with 50 nM sodium acetate buffer, 150 ã - 10 mM sodium chloride , pH48. A botulinum neurotoxin type A complex was eluted from the POROUS 20HS column with 50 nM sodium acetate buffer, 250 mM sodium chloride, pH 4.8 to 200 mLUmin, the eluate was bypassed a bioprocess collection bag (containing 1 column volume of 50 mM NaH3C202, pH 4.8) when Azs5 increases to about) 0.1 AU for a maximum peak until year of trailing edge of the elution peak decreases for a leakage edge value of O 0.1 AU. The product grouped in POROSQO 20HS was stored in the collection bag at room temperature for up to about 6 hours.
In the three column chromatography medium process of this Example2, the eluent from the second column (cation exchange) passed through a HIC column. The HIC column used was a hydrophobic interaction chromatography Fenil Sepharose HP resin packaged with an internal diameter of about 8 cm and a column height of about 5 cm. The entire operation on the Fenil Sepharose HP column was completed at room temperature, and the flow was in a downward direction. A type A botulinum neurotoxin complex was eluted from the column using a decreasing salt change step. Impurities were eluted during loading and with the wash buffer and decontamination solution. Details of the hydrophobic interaction chromatography step were: a Fenil Sepharose HP column was initially sanitized with a 0.1 N sodium hydroxide solution for a minimum contact of 30 minutes (with at least about 3 column volumes in one hydroxide solution
0.1 N sodium hydroxide at 200 cm / hour). The column was then equilibrated with at least about 5 column volumes of 50 nM sodium acetate buffer, 0.4 M ammonium sulfate, pH 4.8. Then the product grouped in the PO-ROSQO 20HS (cation exchange column) (above) was combined 1: 1 with a 50 nM sodium acetate buffer, 0.8 M ammonium sulfate, pH 4.8 and loaded on a Fenil Sepharose HP column. The column was first washed with at least about 3 column volumes of a 50 nM sodium acetate buffer, 0.4 M ammonium sulfate, pH 4.8, and then washed with a 50 mM sodium phosphate buffer , 0.4 M ammonium sulfate, pH 6.5. The botulinum neurotoxin type A complex was eluted from the column with a 10 mM sodium phosphate buffer, 0.14 M ammonium sulfate, pH 6.5. The eluate was des- .—. viad into a bioprocess collection bag when Azgo increased to O 0.05 AU. The eluate was collected until A> so from the trailing edge of the elution peak decreased to a value of O 0.05 AU. The product grouped in Fenil Sepharose HP was stored in the collection bag at room temperature for up to 6 hours.
A tangential flow filtration system was used to concentrate and diafiltrate the product chromatography step grouped in Phenyl Sepharose HP in the drug formulation buffer. Pall & Filtron Minimate cassettes with a 100 kDa molecular weight cutoff membrane were used for the concentration and diafiltration steps. The formulated material was then passed through a Pall Mini Kleenpak & 0.2 µm filter to reduce potential bioburden. As previously stated, the UF / DF step concentrated the product grouped in Phenyl Sepharose HP (eluent from the HIC column) to a concentration of the BONT / A complex in a concentration of 0.7 g / L and diafiltered the concentrated material with a citrate buffer 10 mM potassium, pH 6.5.
Details of the ultrafiltration / diafiltration process used were as follows. The UF / DF unit and the Pall 100 kDa sulfoine polyether membrane were initially washed with a minimum of 5 L of water for injection (WFI) to remove the packaging solution and sanitized with a minimum of 200 mL of a solution 1 N sodium hydroxide under recirculation conditions for a minimum of 10 minutes, preferably at least 30 minutes, to sanitize the UF / DF unit. Then the membrane and the UF / DF system were equilibrated with sufficient volumes of 10 mM potassium citrate formulation buffer, pH 6.5 until the pH of the permeate and retentate was 6.5. After that, the product grouped in Fenil Sepharose HP was loaded into the MinimateO cassette for tangential flow filtration and the HIC elution concentrated to 0.7 g / L. Following the concentration step, the retentate aggregate was diafiltered against a minimum of 5 diafiltration volumes of the drug formulation buffer (10 - 10 mM potassium citrate, pH6.5) at a transmembrane pressure of 7.5 psig (pounds per square inch). The permeate outlet was then closed and the UF / DF system ran for at least 2 minutes and the system was rinsed with 50 mL of 10 mM potassium citrate, pH 6.5 formulation buffer. After rinsing, the concentration of the BOoNT / A complex in the retentate pool was determined by measuring A> 7; g offline and based on the Az78 reading, the concentration of the retentate pool was adjusted to 0.5 g / L with 10 mM potassium citrate buffer, pH 6.5. The pool of the adjusted concentration retentate was then filtered through a 0.2 µm Pall Mini Kleenpak filter to reduce the potential bioload. The grouped retentate of adjusted concentration and filtered stored in a collection bag at 2ºC to 8ºC for up to 2 days.
The final type A purified botulinum neurotoxin complex obtained was packaged in 1 ml NuncO cryoprobes at 700 μl per bottle and stored frozen. The filling operation was carried out in a class 100 biosafety cabin at room temperature.
The downstream process (including the use of 2 or 3 chromatography columns) was completed in just 1 to 3 days and a obtained type A botulinum neurotoxin complex was stored in a potassium citrate buffer, pH 6, 5 at a concentration of 0.5 g / L as a solution. In comparison, the prior technique of the downstream Schantz process (toxin purification) uses multiple filtration, precipitation, extraction and centrifugation steps to purify a type A botulinum neurotoxin complex and requires 1 to 2 weeks to complete the downstream steps. , and the drug substance
material (recovered botulinum neurotoxin) is stored refrigerated as À a suspension of ammonium sulphate in a concentration of approximately 2.7 g / L. The use of chromatography in place of precipitation and reduced processing time resulted in a significantly improved and consistent downstream process, as described here.
According to one aspect, the concentrations of plant-based products, such as soy-based products, can be Peptone: Soy Type Il Hy-Soy & 8 or SESO0MK (a Kosher soy peptone) in culture and fermentation medium. Hy-SoyO in the seed culture medium can vary between - 10 to 10 to 200 g / L. Preferably, the concentration of Hy-Soy & in the medium É seed varies between 15 to 150 g / L. More preferably, the concentration of Hy-Soy8 in the seed medium is approximately between about 20 to 30 g / L or an amount between them. The concentration of glucose in the medium can only vary between 0.1 g / L and 20 g / L. Preferably, the glucose concentration varies between 0.5 to 15 g / L. Most preferably, the glucose concentration in the culture medium is approximately 10 g / L. Amounts of the yeast extract can be about 5 to 20 g / L, more preferably about 10 to 15 g / L or an amount between them. For example, the PH of the culture medium before the growth of Clostridium botulinum can be approximately pH 7.0 to 7.5, or among them, preferably pH V8.
As an example, amounts of Hy-Soy & in the production of the fermentation medium can vary between 10 to 200 g / L. Preferably, the concentration of Hy-Soy € in the fermentation medium varies between 15 to 150 g / L.
Most preferably, the concentration of Hy-Soy & in the fermentation medium is approximately between about 20 to 40 g / L or an amount between them. The concentration of glucose in the fermentation medium can vary between 0.1 g / L and 20 g / L. Preferably, the glucose concentration varies between 0.5 to 15 g / L or an amount between them. Not necessarily, but as above, glucose can be sterilized by autoclaving along with the other components of the fermentation medium. The pH level of the fermentation medium before growth can be pH 7.0 to 7.8, preferably about 7.0 to 7.5 or between these, more preferably pH 7.3. As shown on the right side of FIG. 1, the two-column APF process used in this Example 2 to obtain a biologically active botulinum neurotoxin complex comprised the following steps: (a) cultivating the bacteria, such as Clostridium botulinum bacteria from a WCB APF flask, in a seed bottle / culture, (b) then ferment the Clostridium botulinum bacterium in a fermenter (toxin production fermenter) containing APF fermentation medium to expand the cell line, proceed with fermentation and botulinum toxin production to a - 10 desired phase of cell lysis will be achieved. Then, (c) harvest (for example, clarification by filtration) the APF fermentation medium to obtain one: harvested fermentation medium, (d) proceed with concentration and dilution resulting in a harvested diluted fermentation medium which is (e) passed through a capture column to remove impurities, (f) by contacting the capture column eluent with a polishing column to remove more impurities, and optionally a second polishing column (g) concentration and change of buffer of the polishing column eluent, (h) followed by biocharging reduction filtration and (i) vial filling.
In one example, the fermentation volume is 20 L, the total process time for all stages was only 4 to 6 days, and high-performance botanical neurotoxin was obtained.
Below are more details of a particular modality within the scope of our invention. The fermentation step was carried out in APF medium using a 30 L stainless steel fermenter.
In this example below, a very small volume of fermentation medium was used while still providing a high-yield, high-purity type A botulinum neurotoxin complex. Using the following protocol, only 20 L or less, for example, of APF fermentation medium was required, instead of the previous larger volumes (for example115L) of fermentation medium required to produce commercially useful amounts for obtain a botulinum neurotoxin.
The MACS anaerobic workstation (Don Whitley) with
Air flow provided an oxygen-deficient environment to manipulate anaerobic organisms. The access and exit of the chamber were through a hatch system, comprising internal and external doors. The unit was temperature controlled to maintain the user's environment inside the chamber. A humidistat-controlled condensation plate ensured the effective removal of excessive moisture in the chamber. The chamber was illuminated for operator use and an alarm for: low gas pressure, continuous flow of. gas, and loss of energy conditions. The chamber was equipped with a HEPA filter to reduce the levels of viable and non-viable particles in the anaerobic chamber. The anaerobic conditions were maintained using the system of: atmospheric friction of Catalyst "Anotox" and Palladium Deoxo "D". À: condensed water from the condensation plate was collected and pipetted into a reservoir from which it was removed. As described above, an APF process was used to prepare a WCB APF, containing cell bank bottles stored below -135ºC. A WCB APF cell bank flask was thawed at room temperature for about 15 min before inoculation into the culture medium, followed by a single culture step as described above to establish a "seed" culture. This was carried out in a controlled modular atmospheric system using aseptic techniques from start to finish, to minimize biocharge. The controlled atmospheric modular system was cleaned before proceeding with the inoculation of the seed culture flask complete with contents of the WCB APF flasks. The culture medium was prepared using 1 N hydrochloric acid and 1 N sodium hydroxide (for pH adjustment), D (+) Glucose, Anhydrous (Mallinckrodt Baker, Catnumber 7730,
4.00 g), Soy Peptone Type Il (SPTII) (Marcor, Cat number 1130, 8.00 g), Water for injection (WFI) 400.0 mL and Yeast extract (YE) (BD Cat number 212730, 4.00 g) . The Peptona Soy Type !! and yeast extract solution were prepared by measuring 300 mL of WFI with a 500mL graduated pipette and poured into a seed culture bottle. The seed culture bottle was placed on a shaker and activated. 8.00 g of SPTIl and 4.00 g of yeast extract were added to the seed culture bottle
and mixed until dissolved. If the dissolution was not complete after mixing, the mixture could be heated to a low level. The pH was measured and adjusted to about 7.30 + 0.05. The medium solution was swelled to about 360 mL with WFI. The seed culture bottle was adequately ventilated to allow the transfer of steam and gas. A 10% glucose (w / v) solution was prepared by measuring about 30 mL of WFI in a 100 mL graduated cylinder and placed in a pre-assembled glucose addition bottle, which was placed on a shaker and this has been activated. About 4.00 g of glucose was added to the glucose addition bottle and mixed - 10 until dissolved (low heating was used if necessary for dissolution) and qs (sufficient amount) of glucose solution to 40 ml with and WEFI. The glucose addition was then capped weakly with the cap. Both bottles of glucose and seed culture are autoclaved at 123ºC for 30 minutes for sterilization. After sterilization, both items were removed from the autoclave and left to cool in a cold biosafety cabin. After aseptic cooling, 10% of the glucose solution was transferred in a second seed culture bottle containing the yeast extract and soy peptone Il solution and mixed, thus providing a complete seed culture bottle.
This bottle of complete seed culture was placed in a previously cleaned MACS (where a prepared anaerobic indicator was placed). The cap of the complete seed culture bottle was released. The complete seed culture bottle was then placed on a shaking plate inside the MACS (the plate shaker was activated at about 150 rom) and the complete seed culture bottle was reduced for a minimum of 12 hours by about 34 , 5ºC +/- 1ºC inside the MACS, after which 1 mL of white medium was sampled for measurement of optical density (for determination of biomass at 540 nm). Then, the complete seed culture bottle at MACS (anaerobic) was inoculated. A WCB-APF culture flask was obtained from the frozen cell bank and brought into the MACS. The flask was thawed for about 10 to 15 minutes, after which about 400 uL of the contents of the flasks were placed directly in the middle of the complete seed culture flask. The cap on the fully cultured culture bottle was released completely and the cap was placed on top of the bottle and the stirring rate was adjusted to 150 rpm. After at least about 11 hours of incubation at MACS, the production in fermentation was understood, as described below. The probes (for example, redox probe, pH probe, turbidity probe, for example from Broadley James and Optek) and the configuration sequence of the fermenter, such as a 30 L stainless steel fermenter, were checked and calibrated , and inserted into their respective fermenter - 10 doors and tightened in place. For example, a fermenter may be an ABEC 30 L Fermentation System (VT) consisting of a 30 L volume fermenting vessel, an agitator system, pipe assembly for connecting utilities (CIP, cleaning steam , CDA, Nitrogen, Oxygen, Process Cooling Water, bio-waste, and plant vapor), instrumentation (pH, temperature, pressure, ReDox, optical density, and mass flow), and four peristaltic pumps. The speed of the bottom mounted agitator was controlled using an Allen-Bradley variable frequency transmission (VFD). Semi-automatic and automatic control of the system is handled by an Allen-Bradley ControlLogix PLC with programming. The system was designed to provide closed loop (proportional-integral-derived) PID control of culture temperature, pressure, pH, and redox during fermentation operations. An Allen-Bradley DeviceNet & (a network level of open devices) is used for control and communication with devices and sensors in the unit.
In order to keep sterile, in balance, the modes of operation and collection, agitation, temperature, pressure and nitrogen cover are operated with the following set points.
For keeping sterile and in balance:
For OPERATION mode:: 2 For harvest mode:
: | Fermenter pressure - = fopsig | To prepare the fermentation medium, the necessary material included D (+) Glucose, Anhydrous (Mallinckrodt Baker, Catnumber 7730, 300.0 9), Pepto Soya Type ll (SPTII) (Marcor, Cat number 1130, 650 , 0 g), Water for injection (WFI, 13 L) and Yeast Extract (YE) (BD Cat number 212730, 240.0 g), along with standard scales, a bottle (20 L, for example), glass bottle (5 L), graduated beakers, stirring bars and stirrers.
About L of WFI was added to the bottle along with a stir bar.
The bottle was placed on a shaker and activated, after which about 650.0 g of Type II Soy Peptone were added, along with about 240.00 g of YE.
The fermentation medium was q.s. (sufficient quantity) to 13 L with WFI, and the bottle was capped.
A 10% glucose solution (w / v) was then prepared by adding about 2 L of WFI in a 5L glass bottle (with stir bar inside). Placed on a shaker and with the stirring bar rotating, about 300.00 g of glucose were added to the bottle, and mixed until dissolved.
The glucose solution was q.s. to 3 L with WFI and the bottle closed, thus providing a solution
10% glucose.
The fermentation medium in the bottle was added to the fermenter and pre-steam volume at the location in the fermenter was recorded and the fermentation sequence of the operation was advanced.
At the end of SIP (steam in place) (122ºC, +/- 1ºC), the volume of post-SIP in the fermenter was noted.
A glucose addition assembly, comprising a vessel containing a tube with a 0.2 um in-line filter (PALL Corp.) and peristaltic pump, was connected to the fermenter and the line was subjected to SIP and allowed to cool.
An addition valve port was opened and about 3 L of glucose (sterile filter) was added, and the appropriate amount of WFI (sterile filter) for q.s. of the total volume of the fermenter to 20 L was added to the glucose addition bottle and pumped through the same line of. glucose filter.
The addition valve door has been closed.
The production of the fermentation medium was then adjusted to about pH 7.3 +/- 0.05, with 1 N sodium hydroxide or sterile 1 N hydrochloric acid, using the addition lines SIP, as required.
Then, the parameters for keeping sterile were adjusted and maintained for about 12 hours before inoculation.
The initial concentration of the glucose medium was measured using a metabolite analyzer and the glucose concentration was recorded.
As stated above, at the end of the culture incubation only (about 11 + 1 hours), 1 mL of sample was taken to measure optical density (OD). The OD was measured offline at 540nm using a spectrophotometer and if within the appropriate range, the OD value was recorded and the culture was used for fermentation.
The fermentation turbidity probe was appropriately zeroed.
The seed inoculum bottle, from the anaerobic chamber, was brought into the fermenter and a seed inoculum assembly was transferred (a seed vessel with APF culture medium inside, the vessel containing a culture inoculum transfer line with a sterile Kleenpak '“” connector mount available from PALL Corp. or Millipore replaced the fermentor valve, and the tubing for Pump 1 was fixed) The fermenter pressure was reduced to 2 psig and the entire volume of the seed inoculum bottle was pumped into the fermenter.
At the end of the inoculation, the online absorbance units (AU) of the fermenter were recorded, the fermentor parameters were set to the OPERATION mode and the time was recorded.
The fermentation then proceeded (fermentation operations can be from about 60 hours to about 80 hours, preferably from about 68 hours to about 76 hours, more preferably about 72 hours) while the samples were taken from the fermenter, in 24 and 48 hours, for example, while maintaining aseptic conditions. Tests that were done on at least one sample and carried out during fermentation may include, among others, off-line optical density measurements, glucose measurements, ELISA, SDS-PAGE, Western blot, for example. Ê At the end of fermentation (at the end of fermentation the volume of the broth is about 18 to 19 L, for example), a sample can be taken (for testing, for example, off-line optical density measurements, glucose measurements, ELISA, SDS-PAGE, western blot and DNA / RNA quantification).
At the end of the fermentation, the optical density online, EFT (time of fermentation elapsed), and time of the end of fermentation were recorded, as well as agitation in rpm, temperature inºC, pressure in psig and nitrogen cover in slpm and redox in mV. Then, the production fermentation broth was harvested, that is, the production fermentation broth is clarified by which, for example, about 15 L of filtrate is collected. The fermentation parameters were adjusted for harvest and the filter assembly for clarification was prepared (CUNO, 3M filtration) which includes a pre-filter, depth filter and at least one pressure gauge. The pre-filter and depth filter were washed with about 20 L of water for injection. After washing, the filtration assembly was connected to the fermenter collection / drain port. The temperature of the fermenter was reduced to about 25ºC, after which the clarification of the fermentation broth begins (record the initial clarification time, initial OD online, initial pH, initial temperature and initial volume of the fermenter). The pressure in the fermenter was increased at a rate of about 1 psi (pound per square inch) approximately every 10 minutes during filtration, until a pressure of about 6 psi was reached, at which the pressure
was maintained until the end of the harvest. This filter removes approximately 80% of the RNA / DNA in the APF fermentation medium (the remainder is essentially reduced during the subsequent chromatography steps, as discussed below), thus ending the dependence / use of RNase and / or It is used to remove said components from the fermentation broth. Process parameters, such as inlet pressure in the pre-filter, inlet pressure in the depth filter, fermenter pressure, agitation and volume of the filtrate were monitored for every 2 L of collected filtrate, at the end of which the time of the end of the clarification and the volume of the collected filtrate were recorded - 10 of them. After completion of the harvest stage, the systems were decontaminated and cleaned.
fa The filtrate bottle was brought in BSC for sampling, of which about <10 mL of filtrate was sampled for offline DO measurements and other analysis (eg ELISA, SDS-PAGE, DNA / RNA and westerblot) . The filtrate was then subjected to ultrafiltration / dilution. A tangential flow filter unit (TFF) mount was maintained. The TFF unit was rinsed for about 90 minutes with WFI at a preferential speed of about 2 L per minute and then the TFF unit was sanitized by running 0.1 N sodium hydroxide (re-circulated) through it and for about 60 minutes, after which 1 L of 10 mM sodium phosphate buffer, pH 6.5 flowed through it, followed by a rinse with WFI for about 30 minutes. The filtrate from the harvest stage (about 15 L) was then passed through TFF (this is carried out in a biosafety cabin), concentrating the filtrate around 5L +/- 0.5L (the concentration stage proceeds at about 2L per minute and at a trans-membrane pressure of about 5 psig). A sample of the permeate can be taken and subjected to ELISA, dsDNA, SDS-PAGE and western blot tests, for example. Once concentrated to about 5 L +/- 0.5 L, the retentate pool was then diluted to about 20 L with about 15L of sterile 10 mM sodium phosphate buffer, pH 6.5, by TFF, in about a speed of 2 L per minute. A sample can then be taken again and subjected to ELISA, DNA / RNA, SDS-PAGE and western blot tests, for example. The ultrafiltration / dilution material (retentate) was stored at 4ºC.
After use, all systems were decontaminated using either 1N sodium hydroxide or sterilization temperatures (steam) and cleaned.
The following materials, equipment and procedures were used to prepare the solutions, buffers, etc., set out below for use in an exemplary process, which is to purify the fermentation medium obtained from the processes of Example 2 in order to obtain a A purified botulinum neurotoxin complex type A. Exemplary buffers used (filtered on a 0.2 micron vacuum filter and their conductivities measured in: mS / cm, for recording) include: 10 mM sodium phosphate, pH 6, 5; 50 mM sodium phosphate, pH 6.5; 50 mM sodium acetate, pH 4.8; 50 MM sodium acetate, 170 mM sodium chloride, pH 4.8; 50 mM sodium acetate, 250 mM sodium chloride, pH 4.8; 50 mM sodium acetate, 1 M sodium chloride, pH 4.8; 50 mM sodium acetate, pH 4.0 and 10 MM citrate, pH 6.5.
The following is an example of operations for purifying and obtaining botulinum neurotoxin type A from the processes of Example 2. All parts that come into contact with the product have been designed and constructed to ensure that they are not reactive and non-absorptive. In addition, all equipment has been designed to allow the use of single-use disposable systems or has been designed and built to facilitate cleaning, cleaning and decontamination according to documented and validated methods. The systems or units are designed to not come into contact with the product while the flow paths are designed to be disposable for single use, including the chromatography columns and all associated tubing. The chromatography components were obtained from AlphaBio and the UF / DF components were obtained from Scilog Inc. The chromatography adjustments used included a peristaltic pump to release the solution with variable speed transmission, block of inlet valves with 5 inputs, block of column valves with an automated 3-valve arrangement, 3-outlet outlet valve block, column effluent monitoring, including pH, conductivity, and UV, peak collection based on UV absorbance, and instrumentation and controls required for complete the purification operations. The control system had both software and hardware designed to control the purification process. The commands and data were entered via an HMI terminal (Human Machine Interface). The operator initiated all functions of the automated process by commands on the HMI and monitored and adjusted the process parameters such as flow rates, pressure, conductivity, pH, UV absorbance and positions of individual valves.
The UF / DF system included a recirculation pump, - 10 diaphragm pump, 2 scales and a tangential flow filter support (TFF). À: recirculation pump interface with 3 disposable pressure sensors and one of the scales (located under the permeate reservoir) to control the flow rate to maintain a defined transmembrane pressure and interrupt, based on the weight of the permeate reservoir . The diafiltration pump had an interface with the second scale (located under the retentate reservoir) to start and stop, based on the constant weight maintenance of the retentate reservoir. After concentrating and diluting the material retained from the harvest stage (collecting the fermentation medium free of animal protein), the material was loaded onto an anion exchange column. The following is the procedure used for packaging and testing the anion exchange column useful in the two column process of Example 2. The prepackaged columns were used for all three chromatographic steps. First, the feed material (APF medium collected that was subjected to ultrafiltration / dilution) was passed through the anion exchange column (Poros 50HQ, from ABI as described above). At least 5 column volumes (CVs) of 50mM sodium phosphate, pH 6.5, were used to balance the anion exchange column (in this example, a capture column).
After equilibration, the loading step was carried out, where the feed material (post-harvest step of harvested fermentation broth, of about 20 L, for example) was loaded onto the anion exchange column at a speed of about 200 cm / hr for example. After passing 0.5 column volume of material loaded through the anion exchange column, the pooled by passing flow (FT) was collected in a container such as a polyethersulfone vessel, while the toxin complex is bound to the column material anion exchange. This was followed by a wash step, where at least about 15 column volumes of the wash buffer (eg 50 MM sodium phosphate at pH 6.5) was passed through the anion exchange column. The washing step was interrupted when the UV, measured at the column outlet, in real time, decreased to less than - 10 or equal to about 80mAU. The volume of the wash buffer and the volume of the bypass / wash was recorded, and a 1 ml sample of the bypass / wash aggregate is taken and tested, for example, for toxin concentration, nucleic acid content , total cellular proteins, SDS-PAGE, qPCR, 2D LC and ELISA.
The next step was the elution step, where the elution buffer (eg 50 MM sodium acetate, pH 4.8) was pumped into the anion exchange column. When the UV reading at the column outlet, in real time, increased to about 150 mAU or more, the eluate collection in a container pre-filled with 1 CV of elution buffer (50 mM sodium acetate, pH4 , 8) started. The eluate cluster collection was interrupted when the UV reading decreased to less than or equal to about 200 mAU (volume collected at this point is between about 1 to about 2 CVs). The chromatography system was then decontaminated and cleaned using 1 N sodium hydroxide.
The eluate pool of the anion exchange column was then prepared for addition over the cation exchange column. The volume of the anion exchange eluate, pH, conductivity and feed temperature were recorded and the eluate pool of the anion exchange column was diluted with 1 CV of 50 MM sodium acetate, pH 4.8.
Following the race for the anion exchange column, the cation exchange chromatography operation was undertaken. The cation exchange column (eg, Poros & 20HS) was equilibrated with a minimum of 5 CVs of equilibration buffer (50 mM sodium acetate, pH 4.8). After equilibration, the diluted eluate pool from the anion exchange column was loaded over the cation exchange column and the total volume loaded was recorded. After passing 0.5 column volume of eluate pool diluted by the cation exchange pool, the pooled flow (FT) was collected. A first wash of the cation exchange column was carried out where about 3 to 5 CVs of 50OmM sodium acetate, pH 4.8, were passed through the column of: cation exchange (volume of the first wash buffer used was recorded ). A second wash was performed, where about 3 CVs of - 10 sodium chloride 170 MM, 50 mM sodium acetate, pH 4.8, were pumped through the column, this eluate being collected in a new container labeled "WASH 2 . Peak ". The collection started when the UV readings increased to more than or equal to 50 mAU. 1 CV was collected and the volume of the second wash buffer was recorded.
The elution of the bulk toxin complex from the cation exchange column was carried out using elution buffer (eg 250 mM sodium chloride in 50 mM sodium acetate, pH 4.8) which was pumped over the exchange column cationic. When the UV elution reading reached at least about 100 MAU, the collection of the eluate started in pre-filled containers with dilution buffers (40 mL of 100 mM potassium phosphate, pH 6.8 and 60 mL of citrate 10 mM potassium, pH 6.5). The eluate collection from the cation exchange column continued until the UV readings decreased to about 100 mAU or less. The total volume of the eluate, after dilution, was recorded. The cation exchange chromatography system was then decontaminated and cleaned.
After eluting the cation exchange column, the eluate was subjected to filtration. A tangential flow filtration system (TFF) was used, using three 100K MWCO membranes (Sartorius AG, Goettingen, Germany) positioned on top of each other. The initial volume of the cation exchange group was noted, as well as the descriptions of the diafiltration / balance and sanitization solutions. For example, the diafiltration solution can be 10 mM potassium citrate, pH 6.5 and the sanitization solution can be hydrophilic.
0.1 N sodium oxide. The installation of the system proceeded with the connection of a reservoir tube containing cationic column eluate (IAPF) or HIC column (FAPF), the eluate containing botulinum toxin was directed by the ultrafiltration pump to the entrance of the tangential flow filtration membrane. A second tube of the permeate outlet from the tangential flow filtration membrane was connected to the ultrafiltration (UF) permeate container. A tube from the retentate outlet of the tangential flow filtration membrane to the retentate reservoir was secured, and a fourth tube of the diafiltration buffer (DF) by the diafiltration pump directed to the retentate reservoir was also ensured. . The É buffer system buffer was washed, as is the membrane, washing the membrane with at least 720 ml of water for injection (WFI) with the retentate directed to the residue, after which the membrane it was washed again with at least about 4200 mL of water for injection with the retentate recirculating to the reservoir. Then, the membrane was sanitized (if necessary) by washing it with at least about 200 mL of IN sodium hydroxide with the retentate directed to the residue, followed by a washing of the membrane with at least about 200 mL of IN NaOH with the retentate recirculating to the reservoir for a minimum of 30 minutes. The equilibrium was then carried out, washing the membrane with an equilibration buffer (10 mM potassium citrate at a pH of 6.5), with the retentate directed to the residue and the permeate pH was within +/- 0.2 pH units of the equilibration buffer (for example, within +/- 0.2 pH 6.5 units).
The concentration of the material (eluate (grouped product) of the cation exchange column) was determined to see if the dilution or concentration (exemplary processing) was appropriate (an example of target concentration may be about 0 , 7 mg / ml). The dilution was achieved using 10 mM potassium citrate, pH 6.5. A target volume was determined, for example, at a product concentration of 0.7 mg / mL (target vol = (initial concentration / initial vol / 0.7 mg / mL).
The product cluster (eluate (consequently processed or not) from the cation exchange column) was loaded onto the membrane and recirculation (with the permeate outlet closed) from the system (TFF system) occurs for at least 2 minutes with no pressure of return, then the permeate valve was slowly opened while adjusting the retentate return pressure valve to a target of about 7 psig transmembrane pressure. For dilution, 10 mM potassium citrate, pH 6.5 is added to the target volume, and transferred to diafiltration without ultrafiltration; for: concentration, ultrafiltration is initiated. For diafiltration: the permeate residue was collected in a new container (target volume of diafiltration is 5X - 10 volume of diafiltration) and the diafiltrate with at least 5 volumes of diafil- It is 10 mM potassium citrate traction, pH 6 , 5. Diafiltration process data were collected at minimum 10 minute intervals (weight of permeate g / vol mL, inlet pressure (psig), retentate pressure (psig), permeate pressure (psig) and pressure transmembrane (psig)). For recirculation and rinsing: with the permeate outlet filter closed, the system was recirculated / conducted for at least 2 minutes with no back pressure and the system was rinsed with at least 20 mL of 10 mM potassium citrate , pH 6.5. The product group includes retentate and mop. A sample can be taken from the product cluster and subjected to verification analysis including, for example, 278hm UV, SDS-Page, LcHPLC tests. SE-HPLC, qPCR, RP-HPLC, Native-Page, AUC, amebocyte lysate Limulus, Western Blot and ELISA. For cleaning after use, the system was washed with 1N sodium hydroxide, recirculated for at least 10 minutes, then the system was washed and stored with 0.1N sodium hydroxide.
Sterile filtration and filling were then conducted for storage and division of the neurotoxin in bulk. The concentration adjustment was carried out to adjust the concentration of the toxin, using 10 mM potassium citrate, pH 6.5, to about 0.5 mg / mL with post-rinsing sample. If the concentration of the toxin was less than about 0.5 mg / mL, then no concentration adjustment is necessary. Using a sterile pipette, 10mL / 0.75mL aliquots in each of the 15 mL / 1.5mL sterile sample tubes were prepared. The product container was gently shaken by hand and the required amount of solution was transferred (containing bulk of the drug substance, that is, botulinum toxin in bulk) in each bottle. The samples were stored in a maximum of 5 days in a refrigerator of 2ºC - 8ºC or 0.75 mL of the group of filtered product was transferred to cryovials. The cryovials are stored at -70ºC +/- 5ºC.
. Example 3 Formulation Method - 10 A pharmaceutical composition suitable for administration to a patient can be prepared by manipulating a botulinum neurotoxin Sao obtained from the process of Example 2 with one or more excipients. An excipient can act to stabilize botulinum toxin during the formulation process and for a subsequent storage period before use. An excipient can still function as a bulking agent and / or to provide a certain tonicity to the pharmaceutical composition. The formulation requires a many-fold dilution of a botulinum neurotoxin obtained from the process of Example 2, mixing with one or more excipients (such as albumin [such as human serum albumin or a recombinant human albumin] and sodium chloride ) to form a toxin composition, and prepare a stable form for storage and transportation of the toxin composition, such as by lyophilization or vacuum drying the composition. Thus, about 1.5 to 1.9 ng of the botulinum toxin type A complex obtained from Example 2 is manipulated with about 0.5 milligrams of recombinant human albumin (Delta Biotechnologies) and about 0.9 milligrams of chloride sodium by mixing these three ingredients together followed by vacuum drying. Vacuum drying can occur from about 20ºC to about 25ºC, at a pressure of about 80 mm Hg, for about 5 hours, during which time the vials in which these components are subjected to lyophilization are sealed and sealed under vacuum, thus obtaining a flask with about 100 units of type A botulinum neurotoxin complex A. The resulting freeze-dried (sprayed) solid product is, at the time of use,
replaced with normal saline (0.9%) and used to treat patients with various indications, such as cervical dystonia and hyperhidrosis. Freeze-drying, vacuum or freeze drying prepares a stable form for storing and transporting the manipulated botulinum neurotoxin.
In another example, about 1.5 to 1.9 ng of botulinum toxin type A in bulk is handled with about 0.5 milligrams of human serum albumin (Baxter / Immuno, Octapharma, and Pharmacia & Upjohn) and about : 0.9 milligram of sodium chloride mixing these three ingredients together followed by lyophilization. Exemplary vacuum drying can take place from about 20ºC to 25ºC, at a pressure of about 80 um Hg, for about 5 hours, during which time the bottles where these components are dried at.: Vacuum are closed under vacuum and sealed, thus obtaining a bottle with about 100 units of botulinum toxin. The resulting solid (sprayed) vacuum-dried product is, at the time of use, reconstituted with normal saline (0.9%) and used to treat patients with various indications, such as cervical dystonia and hyperhidrosis. In addition, a pharmaceutical composition of botulinum toxin may contain human serum albumin and / or lactose, for example. In one example, about 1.5 to 1.9 ng of bulk botulinum toxin type A can be manipulated with about 125 micrograms of human serum albumin, and 2.5 milligrams of lactose and vacuum dried, lyophilized or dried by freezing for storage stability, for example.
In yet another example, about 1.5 to 1.9 ng of botulinum neurotoxin obtained by the processes described here can be combined with about 10 mg trehalose and about 0.5 mg serum albumin (as human serum albumin, native or recombinant), and optionally, about 1 milligram of methionine to provide about 100 units of dry product botulinum toxin. This composition can be lyophilized and reconstituted later with, before use, about 1 mL of sterile distilled water or sterile saline without preservative (sodium chloride 0.9% for injection), for example.
In particular examples, pharmaceutical compositions of botulinum toxin may include sucrose, as in an exemplary formulation containing about 1.5 to 1.9 ng of botulinum neurotoxin obtained by the processes described here combined with 20% human serum albumin and sucrose, which also It can also be lyophilized to provide about 100 units of botulinum toxin type A, and then reconstituted with saline without a condom (in a volume of about 0.5 ml to about 8.0 ml for example). In a particular example, 200 units of botulinum neurotoxin can be combined with about 10 mg of sucrose and 2 mg of human serum albumin per ml, and the resulting composition placed in vials and lyophilized, to be
. subsequently reconstituted before use with physiological saline.
In addition, the formulation can also use the neurotoxic component (that is, about 150 kDa of toxin complex component: botulinum type A, free of complexation proteins) of the toxin complex: in botulinum type A obtainable by IAPF processes described here.
In a purification method about 150 kDa of the neurotoxic component of the associated non-toxic proteins (for example HAs, NTNH), type A neurotoxin is purified from the associated non-toxic proteins of the complex by a modification of the method of Tse et al. (1982) (Goodnough, M.
C., 1994, Thesis, UW, Wis.). The botulinum neurotoxin complex obtained by our IAPF process (which uses steps of 2 anionic-cationic columns or 3 anionic-cationic-HIC columns, as discussed above) is recovered from a DEAE-Sephadex A 50 column (Sigma Chemical Co. , St.
Louis, Mo.), pH 5.5, and is precipitated by the addition of 39 g of solid ammonium sulfate / 100 mL.
The precipitated toxin complex is collected by centrifugation, dialyzed against 25 mM sodium phosphate, pH 7.9, and applied to a DEAE-Sephadex AS5O column balanced with the same buffer.
The neurotoxic component is separated from the non-toxic proteins of the complex and eluted from the column with a linear gradient 0 to 0.5 M.
The partially purified neurotoxin component is recovered from the DEAE-Sephadex ASO column at pH 7.9 and dialyzed against 25 mM sodium phosphate, pH 7.0. The dialyzed toxin is applied to SP-Sephadex C50 (Sigma Chemical Co.) in 25 mM sodium phosphate, pH 7.0. The contaminating material does not bind to the column under these conditions.
Pure neurotoxin (a component of about 150 kDa) is eluted with a 0.25 M gradient | 0 O of sodium chloride.
The pure neurotoxin of about 150 kDa can be further purified by metal affinity chromatography, gel filtration or other protein chromatography methods. As above, this pure neurotoxin (the neurotoxic component of a botulinum toxin complex of about 150 kDa) can be lyophilized, vacuum dried or frozen with various excipients (eg serum albumin, sucrose, lactose, sodium chloride , trehalose, etc.) discussed above.
The bulk botulinum neurotoxin complex obtained by our - IAPF process, can be formulated in several ways. Exemplary patents that reveal various formulations of botulinum, such as US Patent.
6,087,327 (reveals a composition of botulinum toxin types A and B formulated with gelatin); US patent 5,512,547 (Johnson et al) entitled "Pharma- - ceutical Composition of Botulinum Neurotoxin and Method of Preparation" issued April 30, 1996 and claims a type A pure botulinum formulation comprising albumin and trehalose, stable storage at 37ºC ; US patent 5,756,468 (Johnson et al) issued May 26, 1998 ("Pharmaceutical Compositions of Botulinum Toxin or Botulinum Neurotoxin and Method of Preparation"), and claims a lyophilized formulation of botulinum toxin formulation comprising a thioalkyl, albumin and trehalose that can be stored between 25ºC and 42ºC; US patent 5,696,077 (Johnson et al) entitled "Pharmaceutical Composition Containing Botulinum B Complex" issued on December 9, 1997 and claims the formation of a lyophilized type B botulin complex, free of sodium chloride comprising a type B complex and a protein excipient; and US patent application publication 2003 0118598 (Hunt) discloses the use of various excipients such as recombinant albumin, collagen or starch to stabilize a botulinum toxin (all of these US patent applications or US patents are incorporated herein by reference in their totalities), all provide examples of various useful formulations / excipients that can be used to formulate the bulk botulinum neurotoxin provided by our lAPF process and provide a pharmaceutical composition. The obtained botulinum toxin complex can be eluted from an ion exchange column in a pH 7 to 8 buffer to dissociate non-toxin complex proteins from the botulinum toxin molecule, thus providing (depending on the type of fermented Clostridium botulinum bacteria) neurotoxic component of botulinum toxin type A with approximately 150 kDa of molecular weight, and a specific potency of 1-2 X 10º LDso U / mg or greater; or purified botulinum toxin type B with approximately 156 kDa of molecular weight and a specific potency of 1-2 X 10º LDso U / mg or greater, or purified botulinum toxin type F with approximately 155 kDa: of molecular weight and a specific potency of 1 -2 X 107 LDso U / mg or greater.
Our invention provides many benefits.
First, the two- or three-column E processes of Example 2 eliminate the use of reagents and media of animal origin (eg casein hydrolyzate and Columbia Blood Agar plates) thus, markedly decreasing theoretical risks exposure of the patient to prion-like agents or other infectious agents.
Second, the two- and three-column chromatographic processes (and associated systems and apparatus) in example 2 are highly reproducible, as evidenced by the excellent batch for consistency batch.
This improvement translates into a more consistent clinical profile in patients who require repeated treatments with compounds containing commercially available botulinum toxin over several years.
Analytical studies of drug substance (botulinum neurotoxin) of the IAPF processes described here (2 and 3 columns) revealed a lower load of protein-like impurities and nucleic acid.
This lesser load of protein impurities translates into a lower risk of immunogenicity (antibody production). In addition, the improved purity of the IPAF process translates into a lower incidence of non-specific symptoms commonly associated with biological drugs (eg, nasopharyngitis, upper respiratory tract symptoms, musculoskeletal symptoms, headache, etc. .). In addition, the improved scale of diminished dimensions of this process decreases the risk of exposure to “BoNT / A exposure in the laboratory and production staff.
Exemplary advantages of a present invention include, for example:
1. Safety is improved since no component or substance of animal origin (eg human or animal) is used in the process, the use of DNase and RNase, plates of Columbia Blood Agar, kasein is eliminated (replaced by example, by: filtration loaded during the clarification / harvesting stage and modern chromatography techniques, by sowing culture medium directly with cells from a working cell bank, that is, cells previously selected and propagated / maintained: in APF medium; and the culture bottle and fermentation medium replaced with Type II Soy Peptone (SPTII) as a soy source).
2. Between about 50 mg to about 200 mg of high quality botulinum toxin 7 type A complex can be obtained per 10 L of: fermentation medium.
3. The bulk purified toxin is obtained from a process that is robust, consistent, scalable, validable, and in accordance with cGMP. Robust means that the process is reproducible even with about + 10% change in one or more of the process parameters. Valuable means that the process reproductively provides consistent yields of purified toxin. Compliance with cGMP means that the process can be easily converted into a manufacturing process that complies with the current Good Manufacturing Practices required by the FDA.
4. The potency of the final purified botulinum toxin complex reaches or exceeds the potency (for example as determined by the MLD50 assay) of purified botulinum toxin complex obtained from the modified S-chantz or Schantz process.
5. Replacement of any precipitation step with chromatographic steps to purify the botulinum toxin complex in bulk, which improves the specificity of the purification process.
6. The new improved process facilitates scaling down resulting in improved handling and achieving an operational success rate of> 95% (for example, reduced volumes of the typical used 110L to 120 L of fermentation medium for about 10 L to about 50 L, up to less than about 2 L to about 30 L of fermentation medium or an amount
between them). The typical current production scale for the drug substance is 115 L of a non-AFP fermentation medium, and, according to one aspect of our invention, it has been reduced to 20 L of fermentation medium. This reduction in scale is possible by optimizing the synthesis and cell release of the BONT / Abem complex as a general yield throughout the purification steps, resulting in a similar amount of the final bulk botulinum toxin (drug substance) as obtained in the previous processes that require, for example, 5X or even more fermentation volumes (for example 115 L). This reduced scale facilitates easier administration of the work fermentation volume and thus minimizes the potential risk of operator exposure to the BoNT / A complex, an important operational and safety advantage.
7. Due to the potentially lethal nature of the BoNT / A complex, closed systems have been implemented throughout the manufacturing process as described here. Unlike the prior art methods, no drug substance material produced in accordance with the aspects of the present invention is exposed to the environment during transfer between operating units; all operations are completely closed.
8. The bulk botulinum toxin manufacturing process described here is simplified in all stages without sacrificing the identity, quality, purity, or potency of the drug substance during production. A number of steps used in a non-APF process has been eliminated in the redesigned IAPF process, thereby reducing the production time by, for example, 21 days to 6 days or less.
9. The storage condition of botulinum toxin in bulk as a frozen solution greatly improves the stability of the drug substance.
Several publications, patents and / or references have been cited here, the contents of which, in their entirety, are incorporated here as a reference. The grouping of alternative elements or modalities of the invention described here should not be construed as limitations. Each group member can be referenced and individually claimed or in combination with other group members or other elements found here. It must be anticipated that one or more members of a group may be included, or deleted from a group for reasons of convenience and patentability. In addition, any combination of the elements described above in all possible variations thereof is encompassed by the invention unless otherwise indicated here or contradicted in another. clearly by context.
Although the present invention has been described in detail with respect to certain preferred methods, other embodiments, versions and modifications within the scope of the present invention are possible. So, the. The spirit and scope of the following claims should not be limited to the particular descriptions of the modalities set out above in this document.

权利要求:
Claims (15)
[1]
1. Substantially APF chromatographic process to obtain a biologically active botulinum neurotoxin type A complex, comprising the following sequential steps: (a) cultivating Clostridium botulinum bacteria in a substantially APF culture medium; (b) fermenting Clostridium botulinum bacteria from the culture medium in about 2 L to about 75 L of a substantially APF fermentation medium, wherein at least one of the culture medium and the fermentation medium includes a protein vegetable; (c) harvesting the fermentation medium by removing cell fragments present in the fermentation medium; (d) concentrating the fermentation medium harvested by filtration; . (e) dilute the concentrated fermentation medium by adding a buffer; (f) a first contact stage in which the diluted harvested fermentation medium is contacted with an anion exchange medium, so that the biologically active botulinum neurotoxin becomes captured with the anion exchange medium; (g) eluting the botulinum neurotoxin captured from the anion exchange medium in order to obtain a first eluent; (h) a second contact step in which the first eluent is contacted with a cation exchange medium to remove impurities from the first eluent, thereby obtaining a second eluent; (1) process the second eluant by diafiltration; and ()) filter the second processed eluent, thereby obtaining a biologically active botulinum neurotoxin type A complex using a substantially APF chromatographic process, in which the obtained botulinum type A neurotoxin complex has a power of 2.0 x 10 a about 6.0x10 'units / mg of type A botulinum neurotoxin complex.
[2]
A process according to claim 1, wherein the fermentation medium comprises no more than about 5% w / v of a product
of vegetable-derived protein, no more than about 2% w / v of a yeast extract and no more than about 2% w / v of glucose, and where the pH level of the fermentation medium is from about pH 6.5 to about pH 8.0 at the beginning of the fermentation step.
[3]
Process according to claim 1, in which the culture step is carried out until the optical density of the culture medium at about 540 nm is between 0.8 AU and about 4.5 AU.
[4]
A process according to claim 1, wherein the fermentation step is carried out for about 60 to 80 hours and until an optical density of the fermentation medium at about 890 nm decreases to between about 0 and 0.05 AU at about 0.7 AU. The
[5]
5. Process according to claim 1, in which the culture step is initiated by introducing a content of work cell bank: APF Clostridium botulinum into the culture medium, in which the work cell bank comprises at least about 1 x 10 to 5 x 107 "colony-forming units of Clostridium botulinum per milliliter of the working cell bank, and in which the bacterium Clostridium botulinum in the working cell bank has a substantially morphology uniform.
[6]
Process according to claim 1, wherein the botulinum neurotoxin obtained comprises one ng or less than one ng of residual nucleic acid for each mg of the botulinum neurotoxin obtained.
[7]
7. Process, according to claim 1, in which the process is carried out in one week or less.
[8]
8. APF chromatographic process to obtain a biologically active botulinum neurotoxin, which comprises the following sequential steps of: (a) adding Clostridium botulinum bacteria from an APF working cell bank to an APF culture medium; (b) cultivating the Clostridium botulinum bacteria in the culture medium; (c) ferment the Clostridium botulinum bacteria from step (b) in APF fermentation medium until the lysis of the Clostridium cell
tulinum; (d) harvesting the fermentation culture to provide a harvested fermentation medium; (e) subjecting the harvested fermentation medium to concentration by filtration; (f) diluting the filtered fermentation medium by adding a buffer to obtain a diluted fermentation medium; (g) a first contact stage in which the diluted fermentation medium is contacted with a capture chromatography, in which the capture chromatography medium is an anion exchange medium; = ”(H) a second contact stage in which an eluent from the first contact stage is contacted with a polishing chromatography medium, in which the polishing chromatography medium is a 3-way exchange medium cation, and (1) filter eluent from the second contact stage, thus ob-. having biologically active botulinum neurotoxin by the perfected APF process, in which the botulinum neurotoxin obtained comprises one ng or less than one ng of residual nucleic acid for each mg of the botulinum neurotoxin obtained and the process is carried out in a week or any less.
[9]
9. Substantially animal product-free process (APF) using chromatography to obtain a biologically active botulinum neurotoxin, comprising the following steps: (a) providing a substantially APF fermentation medium; (b) fermenting Clostridium botulinum bacteria in the fermentation medium; and (c) recovering the biologically active botulinum neurotoxin from the fermentation medium by contacting the fermentation medium with an anion exchange chromatography medium followed by contacting an eluent from the anion exchange chromatography medium with an anion exchange chromatography. cation exchange, thus obtaining the biologically active botulinum neurotoxin from the substantially APF chromatography process.
[10]
A process according to claim 9, wherein the botulinum neurotoxin obtained comprises one part or less of residual nucleic acid per million of the botulinum neurotoxin obtained.
[11]
11. Process according to claim 9, in which the process is carried out in one week or less.
[12]
12. Biologically active botulinum neurotoxin, in which it is obtained by the process of claim 9.
[13]
13. Substantially APF chromatography system to obtain a biologically active botulinum neurotoxin, comprising: a first device for anaerobic culture of bacteria Clostridid botulinum, the first device capable of containing from about 200 mL to about 1 L of medium substantially APF culture; a second apparatus for anaerobic fermentation of bacteria. Clostridium botulinum that were grown in the first apparatus, the second apparatus capable of containing from about 2 L to about 75 L of a substantially APF fermentation medium and including at least one deselectable probe selected from the group consisting of a reduction-oxidation probe, a pH probe and a turbidity probe; . a third apparatus for harvesting the fermentation medium; a fourth apparatus for concentrating the harvested fermentation medium and diluting the filtered fermentation medium; a fifth apparatus for carrying out a first purification of botulinum neurotoxin obtained from the harvested fermentation medium, the fifth apparatus comprising an anion exchange chromatography medium, thereby obtaining a first purified botulinum neurotoxin; a sixth apparatus for performing a second purification of the botulinum neurotoxin, the sixth apparatus comprising a cation exchange chromatography medium, thereby obtaining a second purified botulinum neurotoxin; a seventh apparatus performing a third purification of botulinum neurotoxin, the seventh apparatus comprising a hydrophobic interaction medium, thereby obtaining a third purified botulinum neurotoxin and an eighth apparatus for filtering the third purified botulinum neurotoxin, the eighth apparatus comprising a membrane of purified filtration,
where the botulinum neurotoxin obtained has a potency of about 2.0 x 107 to about 6.0 x 10º units / mg of botulinum neurotoxin, the botulinum neurotoxin obtained comprises about one ng or less than about one ng of residual nucleic acid for each mg of botulinum neurotoxin obtained and the process is carried out in a week or less.
[14]
14. The system according to claim 13, in which it also comprises a controlled system of modular atmosphere that includes an anaerobic chamber capable of containing the first apparatus, in which an anaerobic chamber has a high efficiency particulate air filter. integrated within the anaerobic chamber.
H
[15]
15. Substantially product-free chromatographic system: animal (APF) to obtain a biologically active botulinum neurotoxin, comprising: (a) a substantially APF fermentation medium; (B) . Clostridium botulinum bacteria for fermentation in the fermentation medium; “15 (c) a half anion exchange chromatography to recover biologically active botulinum F neurotoxin from the fermentation medium; and (d) a cation exchange chromatography medium to recover additional biologically active botulinum neurotoxin from an eluent of the anion exchange chromatography medium, thereby obtaining biologically active botulinum neurotoxin from a substantially APF chromatography process.

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法律状态:
2020-08-25| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-16| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-17| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-12-28| B09B| Patent application refused [chapter 9.2 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US12/502,181|US8129139B2|2009-07-13|2009-07-13|Process for obtaining botulinum neurotoxin|

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US12/502,181|2009-07-13|

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PCT/US2010/041745|WO2011008713A1|2009-07-13|2010-07-12|Process and system for obtaining botulinum neurotoxin|

---