EA045619B1 - METHOD FOR PURIFYING BOTULINUM TOXIN - Google Patents

Description

Cross references to related applications

This application claims the priority and effect of an invention under 35 U.S.C. § 119(e) under U.S. Provisional Patent Application Serial No.

62/951,828, filed December 20, 2019, the contents of which are incorporated herein by reference in their entirety.

Technical field

The present invention generally relates to the field of purification of neurotoxin protein molecules. In particular, the present invention relates to a method for purifying botulinum toxin (botulinum toxin). Botulinum toxin purified by this method is suitable for use in therapy, and in particular for administration to a patient in order to achieve the desired therapeutic or aesthetic effect.

Prior Art

The following description of the prior art for the proposed technology is provided solely as an aid to understanding the proposed technology and is not intended to be used to describe the proposed technology or incorporated into the proposed technology.

Seven essentially immunologically distinct botulinum neurotoxins have been described - botulinum neurotoxin serotypes A, B, C, D, E, F and G, which are distinguished by neutralization by type-specific antibodies. As an example, BOTOX® is a trademark of the purified botulinum toxin type A neurotoxin complex commercially available from Allergan, Inc. (Irvine, California). BOTOX® is a popular injectable cosmetic procedure that temporarily visually reduces fine lines and wrinkles.

Botulinum toxins, including type A toxins, are typically produced from fermentation of C. botulinum, which can produce a culture solution containing whole bacteria, lysed bacteria, culture medium nutrients, and fermentation byproducts other than botulinum toxin molecules. Filtration of C. botulinum culture solutions to remove whole and/or lysed cellular components and optionally other residues of the fermentation medium produces a clarified culture. The clarified culture solution contains botulinum toxin molecules and various impurities that can be removed to obtain a concentrated, purified botulinum toxin (eg, BoNT/A1) suitable for inclusion in a pharmaceutical botulinum toxin composition.

Existing industrial processes for preparing pharmaceutically useful botulinum toxin compositions typically use multiple precipitation steps to separate the toxin complex from residual impurities from the fermentation process. For example, cold alcohol fractionation (eg, Cohn method) or precipitation is used to remove plasma proteins. Unfortunately, precipitation methods for the purification of botulinum toxin have disadvantages such as low resolution, low yield, operational difficulties, difficulties in control and/or validation, and lack of scalability. In addition, drying the botulinum toxin (eg, by lyophilization, precipitation, etc.) significantly reduces its toxicity. This is a clinical concern because the inactivated toxin can form a toxoid and immunize patients against botulinum toxin.

However, botulinum toxin products currently approved for use in the United States (e.g., BOTOX COSMETIC®, DYSPORT®, XEOMIN®, and JEUVEAU®) are stored in lyophilized or freeze-dried form for stability reasons. Such compositions require reconstitution by a physician in a sterile saline solution before administration to a patient. This reconstitution stage involves loss of physician time, risk of dilution error, and risk of contamination. The botulinum toxin supplier must also train physicians to ensure that the recovery phase is adequately performed.

Therefore, controlled, scalable, high-throughput methods for purifying botulinum toxins from fermentation media are needed to produce highly pure, highly active, pharmaceutically useful botulinum toxin compositions in a form that contains no, substantially no or virtually no animal products, and which does not require reconstitution before administration to patients.

The essence of the invention

In one aspect, the present invention relates to a method for purifying botulinum toxin, comprising purifying the toxin from a solution containing the toxin, wherein the method does not involve precipitation, centrifugation or lyophilization.

In some embodiments, the toxin is serotype A. In some embodiments, the resulting purified toxin contains no, substantially no, or substantially no botulinum toxin complexes. In some embodiments of the present invention, purified botulinum toxin is not

- 1 045619 contains, essentially does not contain or practically does not contain products of animal origin, including human albumin.

In some embodiments of the present invention, the purification includes a filtration step, preferably a tangential flow filtration step. In some embodiments of the present invention, a hollow fiber filter is used during the filtration step. In some embodiments of the present invention, purification includes contacting a first chromatography column with a solution containing a toxin to obtain a toxin-containing fraction. In some embodiments of the present invention, the first chromatography column includes an anion exchange chromatography column. In some embodiments of the present invention, the anion exchange chromatography column contains Q Sepharose. In some embodiments of the present invention, purification further includes collecting a toxin-containing fraction, wherein the toxin-containing fraction is not adsorbed to the first stationary phase.

In some embodiments of the present invention, purification further includes contacting a second chromatography column with the toxin-containing fraction. In some embodiments of the present invention, the second chromatography column includes a cation exchange chromatography column. In some embodiments of the present invention, the cation exchange chromatography column contains SP Sepharose. In some embodiments of the present invention, the purification further includes eluting the botulinum toxin from the second chromatography column to obtain a first toxin-containing eluate.

In some embodiments of the present invention, the purification further includes filtering the first toxin-containing eluate to obtain a toxin-containing retentate. In some embodiments of the present invention, filtration of the first toxin-containing eluate involves exchanging a buffer. In some embodiments of the present invention, filtration of the first toxin-containing eluate separates botulinum toxin molecules from non-toxin proteins to obtain free toxin molecules.

In some embodiments of the present invention, the purification further includes contacting a third chromatography column with the toxin-containing retentate. In some embodiments of the present invention, the third chromatography column includes a second anion exchange chromatography column. In some embodiments of the present invention, the second anion exchange chromatography column contains Q Sepharose. In some embodiments of the present invention, the purification further comprises eluting the botulinum toxin from the third chromatography column to produce a second toxin-containing eluate.

In some embodiments of the present invention, the purification further includes contacting a fourth chromatography column with a second toxin-containing eluate. In some embodiments of the present invention, the toxin-containing eluate is directly injected onto a fourth chromatography column. In some embodiments of the present invention, the third chromatography column and the fourth chromatography column are connected to each other.

In some embodiments of the present invention, the fourth chromatography column includes a size exclusion chromatography column. In some embodiments of the present invention, the size exclusion chromatography column includes a gel filtration chromatography column. In some embodiments of the present invention, the gel filtration chromatography column contains Superdex 200. In some embodiments of the present invention, the purification further comprises eluting the botulinum toxin from the fourth chromatography column to obtain purified botulinum toxin.

In another aspect of the present invention, a method for purifying botulinum toxin from a solution containing the toxin comprises: (a) filtering the solution containing the toxin; (b) contacting the first chromatography column with the filtered solution containing the toxin from step (a), wherein the first chromatography column is an ion exchange chromatography column; (c) collecting the toxin-containing fraction, wherein the toxin-containing fraction flows through the first chromatography column without adsorption to the stationary phase; (d) contacting a second chromatography column with the toxin-containing fraction, the second chromatography column being an ion exchange chromatography column; (f) eluting the botulinum toxin from the second chromatography column to obtain a first toxin-containing eluate; (f) filtering the first toxin-containing eluate to obtain a toxin-containing retentate; (g) contacting a third chromatography column with the toxin-containing retentate from the filtration step (f), wherein the third chromatography column is an ion exchange column; (h) eluting the botulinum toxin from the third chromatography column to obtain a second toxin-containing eluate; (i) contacting a fourth chromatography column with a second toxin-containing eluate, the fourth chromatography column being a size exclusion chromatography column; and (j) eluting the botulinum toxin from the fourth chromatography column to obtain purified botulinum toxin, wherein the method does not involve precipitation, centrifugation or lyophilization of the botulinum toxin.

In some embodiments, the botulinum toxin is botulinum neurotoxin serotype A. In some embodiments, the resulting purified botulinum toxin contains no, substantially no, or substantially no botulinum toxin complexes. In some embodiments of the present invention, the resulting purified botulinum toxin does not contain, is substantially free of, or substantially free of animal products, including human albumin.

In some embodiments of the present invention, the first chromatography column includes an anion exchange chromatography column. In some embodiments of the present invention, the first chromatography column contains Q Sepharose.

In some embodiments of the present invention, the second chromatography column includes a cation exchange chromatography column. In some embodiments of the present invention, the second chromatography column contains SP Sepharose.

In some embodiments of the present invention, the first chromatography column includes an anion exchange chromatography column and the second chromatography column includes a cation exchange chromatography column. In some embodiments of the present invention, the first chromatography column contains Q Sepharose and the second chromatography column contains SP Sepharose.

In some embodiments of the present invention, the third chromatography column includes an anion exchange chromatography column. In some embodiments of the present invention, the third chromatography column contains Q Sepharose.

In some embodiments of the present invention, the fourth chromatography column includes a gel filtration column. In some embodiments of the present invention, the fourth chromatography column contains Superdex 200.

In some embodiments of the present invention, the third chromatography column includes an anion exchange chromatography column, and the fourth chromatography column includes a gel filtration chromatography column. In some embodiments, the third chromatography column contains Q Sepharose and the fourth chromatography column contains Superdex 200. In some embodiments, the second toxin containing eluate is directly injected onto the fourth chromatography column. In some embodiments of the present invention, the third chromatography column and the fourth chromatography column are connected to each other.

In some embodiments of the present invention, the first chromatography column is an anion exchange chromatography column, the second chromatography column is a cation exchange chromatography column, the third chromatography column is a second anion exchange chromatography column, and the fourth chromatography column is a gel filtration chromatography column. In an embodiment of the present invention, the first chromatography column contains Q Sepharose, the second chromatography column contains SP Sepharose, the third chromatography column contains Q Sepharose, and the fourth chromatography column contains Superdex 200.

In some embodiments of the present invention, the first, second, third, and fourth chromatography columns are disposable chromatography systems.

In some embodiments of the present invention, filtration (f) separates botulinum toxin protein molecules from non-toxin proteins to produce free toxin molecules. In some embodiments of the present invention, filtering (f) includes buffer exchange.

In some embodiments of the present invention, the botulinum toxin-containing solution contains no, substantially no, or substantially no animal products. In some embodiments of the present invention, purification includes contacting the botulinum toxin with a buffer solution, wherein the buffer solution is filtered to reduce microbiological load.

In another aspect, the present invention relates to purified botulinum toxin obtained by purifying the toxin from a solution containing the toxin, the method not involving precipitation, centrifugation or lyophilization. In some embodiments, the purified botulinum toxin is serotype A. In some embodiments, the purified botulinum toxin contains no, substantially no, or substantially no toxin complexes. In some embodiments of the present invention, the purified botulinum toxin contains no, substantially no, or substantially no animal products, including human albumin.

In another aspect, the present invention relates to a composition containing purified botulinum toxin in a buffer solution containing phosphate. In some embodiments of the present invention, the buffer solution also contains acetate. In some embodiments of the present invention, the buffer solution also contains at least one source of chloride ions. In some embodiments of the present invention, the at least one source of chloride ions is sodium chloride. In some embodiments of the present invention, the buffer solution also contains at least one surfactant. In some embodiments of the present invention, the surfactant is polysorbate 20.

In some embodiments of the composition of the present invention, the botulinum toxin is botulinum neurotoxin serotype A. In some embodiments of the present invention, the composition does not contain, substantially does not contain, or substantially does not contain botulinum toxin complexes. In some embodiments of the present invention, the composition contains no, substantially no, or substantially no animal products. In some embodiments of the present invention, the composition contains no, substantially no, or substantially no human albumin. In some embodiments of the present invention, the composition has a pH value ranging from about 6.6 to about 6.9.

The following detailed description is illustrative and explanatory and should not be construed as limiting.

Brief description of graphic materials

Fig. 1 is a flow diagram of an embodiment of a method for purifying a botulinum toxin composition from a fermentation medium according to the present invention.

Fig. 2 shows the results of a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis using colloidal Coomassie staining of the toxin productive solution prepared according to the present invention.

Fig. 3 shows the results of SDS-PAGE analysis of three different productive toxin solutions prepared according to the present invention.

Fig. 4 shows molecular weight distribution results obtained by size exclusion chromatography (SEC) for a productive toxin solution prepared in accordance with the present invention.

Fig. 5 shows the average process yield for successive purification steps for three different productive toxin solutions prepared according to the present invention.

Fig. 6 shows the average cumulative process yield from successive purification steps for three different productive toxin solutions prepared by the present invention.

Fig. 7 shows the average purity improvement across successive purification steps for three different productive toxin solutions produced by the present invention.

Fig. 8 shows the average accumulated purity improvement across successive purification steps for three different productive toxin solutions produced by the present invention.

Intelligence. confirming the possibility of implementing the invention

Hereinafter, embodiments of the present invention will be described in more detail. However, aspects of the present invention may be embodied in various forms and should not be considered limited to the embodiments set forth herein. Rather, these embodiments are presented in order that the description of the present invention will be detailed and complete and will fully disclose the scope of the present invention to those skilled in the art. It should be understood that the proposed technology is not limited to specific methods, reagents, compounds, compositions or biological systems, which, of course, may vary. The terminology used in this description is intended solely to describe specific embodiments of the present invention, and is not limiting.

Unless otherwise indicated, all terms (including technical and scientific terms) used in this work have the standard meaning used by specialists in the field of technology to which the present invention relates. It should also be understood that terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of the application and the relevant art, and should not be interpreted in an idealized or overly formal sense unless when such a definition is explicitly given in this work. Such terms are to be interpreted according to their standard meaning, unless expressly stated otherwise.

Moreover, where features or aspects of the present invention are described in terms of the Markush group, those skilled in the art will appreciate that the present invention is also described in terms of any individual element or subgroup of elements of the Markush group.

As will be appreciated by one skilled in the art, for any purpose, in particular for

- 4 045619 providing a written description, all ranges specified in this work also include all possible sub-ranges and combinations of sub-ranges. Any specified range can easily be considered sufficiently descriptive and allows the range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, any range discussed in this work can easily be broken down into a lower third, a middle third and an upper third, etc. It will also be appreciated by one skilled in the art that all expressions such as up to, at least more than, less than, and the like include the number indicated and refer to ranges that can be further broken down into subranges as discussed higher. Finally, as one skilled in the art will appreciate, the range includes each specific element. Accordingly, for example, the term group containing from 1 cell to 3 cells refers to groups containing 1 cell, 2 cells or 3 cells. Similarly, the term group containing from 1 cell to 5 cells refers to groups containing 1 cell, 2 cells, 3 cells, 4 cells or 5 cells, etc.

Unless the context otherwise requires, this specifically means that the various features of the invention described herein can be used in any combination. Moreover, the present invention also provides that in some embodiments, any feature or combination of features described herein can be omitted or removed. By way of illustration, if the specification states that a complex contains components A, B, and C, then this specifically means that any component A, B, or C, or combination thereof, may be omitted and discarded individually or in any combination.

Unless otherwise expressly stated, all embodiments, features and terms described include both the stated embodiment, feature or term and their biological equivalents.

All patents, patent applications, provisional patent applications and publications referenced or cited in this work are incorporated herein by reference in their entirety, including all graphics and tables, to the extent not inappropriate. the explicitly expressed ideas of this description.

Definitions

When used in the context of the present invention, singular forms denote both the singular and the plural, unless explicitly stated to denote only the singular.

When used in the context of the present invention, the term and/or refers to any possible combinations and covers any possible combinations of one or more of the respective specified elements, as well as the absence of combinations when interpreted in the alternative form (or).

Even if not explicitly stated, all numerical values are preceded by the term approximately or roughly. The terms approximately or approximately mean that the implied number is not limited to the exact number specified in this work, but refers to numbers located essentially around the specified number if they are within the scope of the present invention. When used in the context of the present invention, the terms approximately or approximately will be understandable to those skilled in the art and will vary to a certain extent depending on the context in which they are used. If there are applications of terms that are not obvious to those skilled in the art, then depending on the context in which they are used, the terms approximately or approximately will mean up to plus or minus 10%, 5%, 1% or 0.1% of a particular numbers.

When used in the context of the present invention, the terms does not contain or does not contain at all means that within the detection range of the instrument or method used, the substance cannot be detected or its presence cannot be confirmed.

When used in the context of the present invention, the term essentially free means that only trace amounts of a substance can be detected. In the present invention, substantially free means that the substance is present in a concentration of less than 0.1%, preferably less than 0.01%, and most preferably less than 0.001% by weight of the total composition.

When used in the context of the present invention, the term substantially free means that the substance is present in a concentration of less than 5%, preferably less than 2%, and most preferably less than 1% by weight of the total composition.

As used in the context of the present invention, the term botulinum toxin means a neurotoxin produced by Closthdium botulinum and botulinum toxin (either its light chain or its heavy chain) produced recombinantly using non-clostridial species. The term botulinum toxin, when used in the context of the present invention, covers serotypes A, B, C, D, E, F and G of botulinum toxin. The term botulinum toxin also covers the term modified botulinum toxin.

- 5 045619

When used in the context of the present invention, the terms botulinum toxin complex or toxin complex cover a complex secreted by clostridial bacteria and containing a botulinum toxin protein molecule (approximately 150 kDa in all serotypes) together with one or more associated non-toxin proteins. Complexes (e.g., with molecular weights of about 300 kDa, 500 kDa, or 900 kDa) are expected to contain non-toxin hemagglutinin protein (NTH protein) and non-toxin hemagglutinin protein (NTNH protein). ; from English: non-toxin non-hemagglutinin protein). Accordingly, the botulinum toxin complex may contain a botulinum toxin molecule (neurotoxin component) and one or more NTH and/or NTNH proteins. These two types of non-toxin proteins can stabilize the toxin molecule against denaturation and protect against digestive tract acids if the toxin is ingested. In addition, larger (300 kDa or larger) botulinum toxin complexes may diffuse more slowly from intramuscular injection sites compared to botulinum toxin protein.

As an example of a toxin complex, botulinum toxin type A complex can be produced by clostridial bacteria in forms with molecular weights of 900 kDa, 500 kDa and 300 kDa. Botulinum toxins types B and C1 are produced in the form of a complex with a molecular weight of 500 kDa. Botulinum toxin type D is produced in the form of complexes with molecular weights of 300 kDa and 500 kDa. Finally, botulinum toxins types E and F are produced in the form of complexes with a molecular weight of approximately 300 kDa.

With regard to botulinum toxin type A1, it is known that at pH values greater than about 7, non-toxin proteins are separated from the botulinum toxin protein molecule (approximately 150 kDa). Accordingly, toxin complexes can be separated into botulinum toxin protein and hemagglutinin proteins by subjecting the complex to a separation method, such as column chromatography, in a suitable buffer solution at a pH value ranging from about 7 to about 8. However, botulinum toxin protein is known to be unstable after removal of NTH and/or NTNH of the hemagglutinin protein (or proteins), and the toxin loses its toxicity with increasing pH and temperature or as a result of surface stretching or drying (for example, during lyophilization or precipitation). In addition, the toxin loses its specific activity upon dilution (eg, dilution during cultivation, fermentation, and purification) unless a stabilizing agent is present.

When used in the context of the present invention, the term modified botulinum toxin means a botulinum toxin in which at least one of its amino acids has been removed, modified or replaced compared to the native botulinum toxin. In addition, the modified botulinum toxin may be a recombinantly produced neurotoxin or a derivative or fragment of a recombinantly produced neurotoxin. The modified botulinum toxin retains at least one biological activity of the native botulinum toxin, for example, the ability to bind to the 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 contains a light chain from one botulinum toxin serotype (eg, serotype A) and a heavy chain from another botulinum toxin serotype (eg, serotype B). Accordingly, modified botulinum toxins may contain light and heavy chains from two different serotypes selected from any of serotypes A, B, C, D, E, F or G. Another example of a modified botulinum toxin is botulinum toxin coupled to a neurotransmitter.

When used in the context of the present invention, the terms purified botulinum toxin, pure toxin, free botulinum toxin, free toxin or botulinum toxin protein are defined as botulinum toxin that is separated or substantially separated from other proteins, including NTH and/or NTNH proteins, which form botulinum toxin. toxin complex. The purified botulinum toxin may have a purity greater than 95%, and preferably greater than 99%.

When used in the context of the present invention, the terms medium or fermentation medium mean any medium for cultivating bacteria, that is, either a medium for their growth to produce a seed culture used to inoculate a production medium, or a production medium in which bacteria multiply and produce a toxin.

When used in the context of the present invention, the terms animal product free (APF), substantially animal product free, or substantially animal product free include, respectively, the terms animal protein free, or substantially free of animal protein, or substantially free of animal protein, and means the absence, substantially the absence or practical absence of blood derivatives, blood depots and other products or compounds of animal origin. In this context, the terms does not contain, substantially does not contain and substantially does not contain correspond to the definitions, with

- 6 045619 given above. The term animal means a mammal (eg human), bird, reptile, fish, insect, spider or other species of animal. The term animal does not include microorganisms such as bacteria. Accordingly, an animal product-free (APF) environment or method, or a substantially animal product-free environment or method within the scope of the present invention may include botulinum toxin or the bacterium Clostridium botulinum. For example, a process that does not contain an animal product, or a process that is substantially free of an animal product, means a process that contains little or no animal proteins, such as immunoglobulins, human albumin, hydrolyzed meat, meat by-products, and milk or dairy products. products or hydrolysates. Accordingly, an example of an animal product-free (APF) process is a process (such as a bacterial culture or bacterial fermentation process) that does not use meat or dairy products or meat or dairy by-products.

When used in the context of the present invention, the term microbiological load means bacteria living on the surface, inside a device or in a solution that has not been sterilized. For example, embodiments of the present invention include filtering buffer solutions to reduce the microbiological load, that is, bacteria living in the buffer solution or bacteria that are transferred into the solution from surfaces (eg, glassware surfaces) that have been in contact with the solution.

When used in the context of the present invention, the term tangential flow filtration or TFF; from English: tangential flow filtration refers to a filtration mode that is used to clarify, concentrate and purify biological materials (for example, proteins). During TFF, a solution or suspension containing macromolecules or biological materials can be pumped tangentially along the surface of the membrane. The applied pressure can force some of the solution through the pores in the membrane.

This solution is referred to in this work as permeate (or filtrate). Macromolecules, biological materials and particles that are too large to penetrate the membrane pores may be retained on the inlet side. This solution is referred to in this work as retentate. Unlike conventional filtration methods, retained materials do not accumulate on the surface of the membrane. Instead, they can move along the face of the membrane with the tangential flow of the fluid. See, for example, L. Schwartz and K. Seeley, Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications, Pall Life Sciences (2002), https://laboratory.pall.com/content/dam/pall/laboratory /literature-library/non-gated/id-34212.pdf.

As used in the context of the present invention, the term permeate refers to a solution, suspension, or component thereof that passes a filter or membrane (e.g., diafiltration membrane, tangential flow filtration membrane, ultrafiltration membrane, microfiltration membrane, or hollow fiber filter) by permeating the pores of the filter or membrane, as well as to a solution that has already passed through the filter or membrane or has passed through the filter or membrane. In general, solvent molecules and solute molecules that are smaller than the pore size of the filter or membrane will overcome the filter or membrane, whereas molecules that are larger than the pore size will not overcome the filter or membrane.

When used in the context of the present invention, the term toxin-containing permeate refers to a permeate that contains botulinum toxin molecules, that is, if the pore size of the filter is larger than the botulinum toxin molecules, the botulinum toxin molecules will overcome the filter.

When used in the context of the present invention, the term retentate refers to a solution, suspension, or components thereof that does not penetrate the filter or membrane. For example, in the case of tangential flow filtration, the retentate is a component or part of a solution or suspension that flows tangentially along the filter or membrane, but does not cross the filter or membrane. In general, molecules that are larger than the pore size of the filter or membrane will not penetrate the filter or membrane.

When used in the context of the present invention, the term toxin-containing retentate refers to a retentate that contains botulinum toxin molecules, that is, if the pore size of the filter is smaller than the botulinum toxin molecules, the botulinum toxin molecules cannot penetrate the filter.

When used in the context of the present invention, the term transmembrane pressure or TMP refers to the differential pressure gradient that is applied along the length of the filtration membrane to cause the flow of fluid and filtered solutes through the filter or membrane or across the filter or membranes.

As used in the context of the present invention, the term diafiltration refers to a specialized class of filtration in which the retentate is diluted with a solvent and refiltered to reduce the concentration of the soluble components of the permeate. Diafiltration may or may not result in increased concentrations of retained components, including proteins.

- 7 045619 (for example, BoNT/A). For example, in continuous diafiltration, solvent is continuously added to the retentate at the same rate at which the permeate is formed. In this case, the volume of the retentate and the concentration of the retained components do not change during the process. On the other hand, in intermittent or serial dilution diafiltration, the filtration step is followed by the addition of solvent to the retentate side; if the volume of solvent added to the retentate side is less than the volume of permeate formed, then the retained components will have a higher concentration than in the original solution. Diafiltration can be used to change the pH, ionic strength, salt composition, or other properties of a solution or suspension of macromolecules (e.g., proteins such as BoNT/A). See, for example, L. Schwartz, Diafiltration: A Fast, Efficient, Method for Desalting, or Buffer Exchange of Biological Samples, Pall Life Sciences (2003), https://laboratory.pall.com/content/dam/pall /laboratory/literature-librarv/non-qated/02.0629 Buffer Exchange STR.pdf (last visited Dec. 9, 2019).

When used in the context of the present invention, the term diafiltration volume or DM' (from English: diafiltration volume) refers to the total volume replaced during the diafiltration process. One DV is equal to the volume of retentate at the beginning of diafiltration. For example, if the initial solution volume is one liter, the diafiltration process creates a permeate volume approximately equal to one liter, and the retentate volume is maintained at 1 L or supplemented to 1 L (for example, using a buffer solution), then the original solution or initial suspension is filtered or washed with one DV. Continuous diafiltration allows for the exchange of multiple DVs. For example, if the initial retentate volume is one liter and the diafiltration process produces a permeate volume of approximately five liters, then the feed solution or feed suspension is filtered or washed with five DVs.

As used in the context of the present invention, the term microfiltration refers to a class of filtration that typically employs membrane pore sizes ranging from about 0.1 μm to about 10 μm or greater. See, for example, Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998).

As used in the context of the present invention, the term ultrafiltration refers to a class of filtration that typically employs membrane pore sizes ranging from about 0.1 μm to about 0.01 μm or less. Alternatively, nominal membrane pore sizes can be expressed in terms of molecular weight, for example, from about 30 kDa or less to about 750 kDa or less, preferably 50 kDa or less or 30 kDa or less. The term can refer to any method in which a solution or suspension is applied to a semi-permeable membrane that retains macromolecules but allows solvent and small solute molecules to pass through. Ultrafiltration can be used to concentrate macromolecules (eg proteins such as BoNT/A) into a solution or suspension. See, for example, Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998).

As used in the context of the present invention, the terms chromatography or chromatographic separation refer to a physical separation method in which the components (eg, proteins) to be separated are partitioned between two phases: a stationary phase and a mobile phase. The molecules to be separated are dissolved in a mobile phase, which moves through a stationary phase (eg, porous gel, charged polymer beads, etc.). Separation is possible because different molecules in the sample will exhibit different affinities for the stationary phase, causing similar molecules to separate. Molecules with a higher affinity for the stationary phase will tend to move more slowly through the stationary phase than molecules with a weaker affinity. When applied to proteins (eg botulinum toxins), chromatographic separation can separate proteins based on many different properties. For example, in gel filtration chromatography, proteins present in the mobile phase are separated based on their size, since proteins of different sizes move through a porous stationary phase in which smaller proteins are trapped and their movement is slowed. In ion exchange chromatography, proteins are separated based on their charge and the resulting Coulombic interactions with the stationary phase.

As used in the context of the present invention, the term chromatography column, or simply column, refers to a component containing a chromatography matrix (e.g., a stationary phase or solid phase) and arranged so that a mobile phase, such as a sample fluid or buffer, can pass through the column, in doing so, it passes through the stationary phase held in the column. Non-limiting examples of such columns include those commercially available from G.E. Healthcare. See, for example, Chromatography Products: Chromatography columns, systems, resins, and buffer management solutions, G.E. Healthcare, https://www.gelifesciences.com/en/us/shop/chromatography (last visited Dec. 9, 2019).

When used in the context of the present invention, the term fraction refers to the portion of the mobile phase that is collected at the outlet of the column. The components contained in a fraction will vary depending on the time during which they are collected. The faster moving fractions collected during the initial periods of time will contain relatively higher concentrations of those molecules that move more quickly through the stationary phase; slower-moving fractions collected at later times will contain relatively high concentrations of molecules that move more slowly through the stationary phase.

When used in the context of the present invention, the term toxin-containing fraction means the fraction collected from the chromatography column at the time when botulinum toxin molecules (eg, BoNT/A molecules) leave the column in the mobile phase.

As used in the context of the present invention, the term ion exchange chromatography, or IEX, refers to a chromatographic separation technique in which molecules are separated based on their polarity and the magnitude of their charges (e.g., +2, +1, neutral, -1, -2, etc.). In IEX, analyte molecules (e.g., proteins) are retained on the stationary phase based on the magnitude of their Coulombic interactions with the stationary phase. The surface of the stationary phase exhibits ionic functional groups that interact with analyte ions (eg botulinum toxin) of opposite sign. To achieve electrical neutrality, these stationary charges interact with exchangeable counterions in the mobile phase. The analyte molecules compete with these exchangeable counterions for binding. Analyte molecules are retained or eluted depending on their charge. Initially, molecules that do not bind or bind weakly to the stationary phase are washed out first. See, for example, Ion Exchange Chromatography: Principles and Methods, G.E. Healthcare (2016), https://cdn.gelifesciences.com/dmm3bwsv3/AssetStrearn.aspx?rnediaformatid=10061&destinationid=10016&as setid=13101 (last visited Dec. 9, 2019).

As used in the context of the present invention, the term anion exchange chromatography, or AIEX, refers to a type of ion exchange chromatography in which anionic analyte molecules (eg, proteins) are retained on a cationic stationary phase. See generally, for example, Ion Exchange Chromatography: Principles and Methods, G.E. Healthcare (2016), https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&ass etid=13101 (last visited Dec. 9, 2019).

As used in the context of the present invention, the term cation exchange chromatography, or CIEX, refers to a type of ion exchange chromatography in which cationic analyte molecules (eg, proteins) are retained on an anionic stationary phase. See generally, for example, Ion Exchange Chromatography: Principles and Methods, G.E. Healthcare (2016), https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&ass etid=13101 (last visited Dec. 9, 2019).

When used in the context of the present invention, the term elution refers to the desorption of molecules attached to the stationary phase by changing the solution conditions in the chromatography column. The concentrations of exchangeable counterions can be increased or the pH can be changed to influence the binding affinity of the analyte. Molecules that lose affinity for the stationary phase and enter the mobile phase are eluted from the column.

As used in the context of the present invention, the terms eluent or wash solution refer to an agent, typically a solution, that is used to modify the adsorption of an analyte (eg, botulinum toxin molecule) onto a stationary phase and/or to remove unbound materials from the stationary phase. The elution characteristics of the eluent may depend, for example, on pH, ionic strength and detergent strength, among other factors.

When used in the context of the present invention, the term eluate refers to a solution (eg, a wash solution or a buffer solution) containing unbound materials (including eluted or desorbed analyte molecules, such as botulinum toxin molecules) that moves through the stationary phase and exits the column during chromatographic separation.

When used in the context of the present invention, the term toxin-containing eluate refers to the mobile phase containing eluted botulinum toxin molecules that exits the column during chromatographic separation.

When used in the context of the present invention, the terms gel filtration chromatography or gel filtration mean a type of size exclusion chromatography that can be used to either fractionate molecules (e.g., proteins, protein complexes, polysaccharides, nucleic acids, small molecules, etc.) contained in a sample, into fractions, each of which has a certain size range. Alternatively, gel filtration can remove all molecules from a sample that are larger than a certain cutoff size. In a gel filtration chromatography column, the stationary phase contains a porous matrix (eg, beads) and the mobile phase is a solution (eg, a buffer solution) that flows around the matrix. The matrix may have a pore size within a certain range, which is known as the fractionation range. Molecules and complexes that are too large to penetrate

- 9 045619 into the pores, remain in the mobile phase and move through the column with a buffer solution. Smaller molecules and complexes that can enter the pores penetrate the stationary phase and travel through the gel filtration column along a longer path (that is, through the pores rather than around the beads). Molecules that can enter the stationary phase are fractionated by size. Smaller molecules will migrate through the pores and will therefore be slowed down more than larger molecules that cannot easily enter the pores. Therefore, larger molecules elute faster. Accordingly, sample components exceeding the fractionation range will elute before components within the fractionation range. See generally, for example, Size Exclusion Chromatography: Principles and Methods, G.E. Healthcare (2018), https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&ass etid=11639 (last visited Dec. 9, 2019).

When used in relation to chromatography system components, the term disposable refers to components that are configured to be replaced or destroyed after each use and that are not intended to be reused in the system.

Fermentation medium.

According to FIG. 1, a method 100 for purifying botulinum toxin may include preparing 104 a solution (eg, fermentation medium) containing botulinum toxin (eg, BoNT/A). In some embodiments, the solution may be a fermentation medium, preferably a supernatant from a fermentation medium containing whole C. botulinum cells, lysed bacteria, culture medium nutrients (eg, plant peptones), and fermentation by-products. In some embodiments of the present invention, the fermentation medium may be substantially free, substantially free, or free of animal products (i.e., be an APF fermentation medium), such as the fermentation medium described in co-filed provisional U.S. patent application Ser. No. 62/951,549.

Botulinum toxin can be isolated and purified from the fermentation medium using protein purification methods known to those skilled in the art of protein purification. See generally, for example, Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998); Ozutsumi et al., 49 Appl. Envtl. Microbiol. 939 (1985); GE Healthcare, Strategies for Protein Purification Handbook (2010).

The purification methods of the present invention described herein may involve purifying a botulinum toxin complex (e.g., a 900 kDa molecular weight complex) that is more stable than the 150 kDa botulinum toxin protein molecule, followed by separating and further purifying the toxin protein molecule from non-toxin proteins (eg, NTH and/or NTNH proteins) to obtain a purified botulinum toxin product (approximately 150 kDa) without the use of precipitation, centrifugation or lyophilization steps. The productive toxin solution may be free, substantially free, or substantially free of toxin complexes and/or animal products. Additionally, since no precipitation, lyophilization or centrifugation steps are required, botulinum toxin can be obtained in solution, as opposed to powder, which must be reconstituted by the end user before administration to the patient.

Filtration. Filtration 1.

According to FIG. 1, the method may include a first filtration (Filtration 1) 106, which includes filtering the culture medium or solution to remove whole or lysed bacteria, spores (for example, C. botulinum spores) and debris to produce a toxin-containing permeate 107. The toxin-containing permeate 107 contains botulinum toxin and various impurities and can be processed to produce concentrated botulinum toxin (eg BoNT/A).

In specific embodiments of the present invention, the first filtration 106 comprises removing whole or lysed C. botulinum cells (or components thereof) from the fermentation medium using a suitable filtration method (e.g., diafiltration, cross-flow microfiltration, cross-flow ultrafiltration, hollow fiber filtration, etc. .P.). Filtration methods for purifying biomolecules such as proteins are well known in the art. See, for example, L. Schwartz and K. Seeley, Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications, Pall Life Sciences (2002), https://laboratory. pall.com/content/dam/pal l/laboratory/literature-libraiy/non-gated/id-34212.pdf (last visited Dec. 9, 2019); Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998). In an embodiment of the present invention, the first filtration 106 includes tangential flow microfiltration.

In some embodiments of the present invention, a filter (e.g., a hollow fiber filter, a tangential flow filtration membrane, etc.) may be used in the first filtration step 106, wherein at least solvent and botulinum toxin molecules or toxin complexes pass through the filter and produce a toxin-containing permeate 107, whereas whole or lysed cells, if present, do not pass through the filter and are retained in the retentate. In some embodiments of the present invention, the filter contains pores with a diameter ranging from about 0.1 μm to about 10 μm (eg, about 0.2 μm).

In an embodiment of the present invention, the filter is a hollow fiber filter.

In some embodiments of the present invention, the first filtration step 106 may further comprise concentrating the toxin-containing permeate 107 and separating additional botulinum toxin molecules from the retentate and/or toxin-containing permeate 107 using any suitable method (eg, diafiltration).

Filtration 2.

According to FIG. 1, the method may further include a second filtration (Filtration 2) 108 to remove residual fermentation media (e.g., small peptides, carbohydrates, etc.) from the toxin-containing permeate 107. This step may include any suitable filtration method (e.g., diafiltration, microfiltration tangential flow, tangential flow ultrafiltration, hollow fiber filtration, etc.). In an embodiment of the present invention, the second filtration 108 to remove fermentation media residues includes ultrafiltration using a tangential filter (eg, a hollow fiber filter) with a pore size that allows fermentation media residues to pass through the filter but retains botulinum toxin molecules and/or toxin complexes. For example, in some embodiments of the present invention, the filter may have a pore size of 150 kDa or less, 100 kDa or less, or 50 kDa or less. In this case, botulinum toxin molecules or botulinum toxin complexes remain in the retentate from the second filtration 108 and a clarified culture 110 is formed that contains botulinum toxin molecules or botulinum toxin complexes, but does not contain, substantially does not contain, or substantially does not contain whole or lysed C cells botulinum (or their components) and fermentation medium residues (for example, small peptides, carbohydrates, etc.).

In some embodiments of the present invention, the clarified culture 110 can be collected and further purified during subsequent processing steps without precipitating botulinum toxin molecules or toxin complexes from solution at any time during the purification process. Advantageously, it increases the overall yield of the process, maintains toxin activity, and eliminates the need for reconstitution of the lyophilized drug product by the end user.

Column chromatography. First chromatographic separation.

According to FIG. 1, some embodiments of the method 100 further include purifying the clarified culture 110 using a first chromatographic separation 112 to obtain a first toxin-containing fraction 114. The purpose of this step is to separate the botulinum toxin complexes from the nucleic acids (e.g., DNA and RNA) that are present in the clarified culture 110. The first chromatographic separation 112 may include any suitable chromatographic separation method (eg, ion exchange chromatography, including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, and the like). In some embodiments of the present invention, the first chromatographic separation may include anion exchange chromatography (AIEX). In an embodiment of the present invention, the first chromatographic separation involves AIEX on Q Sepharose.

The first chromatographic separation 112 may include contacting the first chromatography column with a clarified culture 110 that contains botulinum toxins or toxin complexes. The mobile phase (containing the clarified culture 110) can flow through the first stationary phase to separate the botulinum toxin from other residual impurities (eg, nucleic acids). The first stationary phase may contain any suitable chromatography matrix (eg, an agarose bead medium such as Q Sepharose FF (available from GE Healthcare).

In specific embodiments of the present invention, the clarified culture 110 may be conditioned for column chromatography (eg, by dilution in a buffer solution or by buffer exchange). In a specific embodiment of the present invention, the clarified culture 110 can be conditioned in phosphate buffer at a pH of less than or equal to about 7.5, preferably at a pH of less than or equal to about 7, more preferably at a pH of about 6.1. In some embodiments of the present invention, the clarified culture 110 may be conditioned in phosphate buffer at a pH of about 7.5, about 7.4, about 7.3, about 7.2, about 7.1, about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6 or about 5.5.

In specific embodiments of the present invention, the first chromatographic separation 112 may further include washing the botulinum toxin or toxin complexes through a stationary phase using a suitable buffer solution (eg, phosphate buffer pH 6.1). The purpose of this stage is to wash as much as possible

- 11 045619 botulinum toxin complexes through a column with separation of botulinum toxin complexes from other proteins and removal of nucleic acids (RNA and DNA). In some embodiments of the present invention, the concentration of salts in this buffer solution can be selected to maximize the amount of botulinum toxin or toxin complexes flowing through the column and minimizing the amount of other proteins flowing through the column.

In some embodiments of the present invention, the buffer solution contains sodium chloride (NaCl) at a concentration of not less than about 15 mM, not less than about 20 mM, not less than about 30 mM, not less than about 40 mM, not less than about 50 mM, not less than about 60 mM, not less than about 70 mM, not less than about 80 mM, not less than about 90 mM, not less than about 100 mM, not less than about 150 mM, not less than about 200 mM, not less than about 250 mM, not less than about 300 mM, not less than about 350 mM, not less than about 400 mM, not less than about 450 mM, or not less than about 500 mM (or an intermediate value). In some embodiments, the buffer solution contains NaCl at a concentration of about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM. In an embodiment of the present invention, the buffer solution contains NaCl at a concentration of about 150 mM.

In a specific embodiment of the present invention, the first chromatography column can be operated in a flow-through mode, and botulinum toxins and/or toxin complexes can pass through the column without adsorption to the stationary phase. This configuration does not require elution of botulinum toxin or toxin complexes. Instead, the botulinum toxin or toxin complexes may exit the column in a toxin-containing fraction 114, which can be collected and further purified during subsequent processing steps. In some embodiments of the present invention, collection of the toxin-containing fraction 114 is monitored by determining the optical fluence at a wavelength of 280 nm (A ₂₈₀ ; from English: absorbance), with the toxin-containing fraction 114 being collected at the time of the appearance of the A ₂₈₀ peak.

Second chromatographic separation.

Some embodiments of the method 100 further include a second chromatographic separation step 116, during which the toxin-containing fraction 114 can be purified to remove major impurities (e.g., other proteins) and provide a first toxin-containing eluate 118. The second chromatographic separation 116 may include any suitable chromatographic separation method ( for example, ion exchange chromatography, including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). In a specific embodiment, the second chromatographic separation 116 includes cation exchange chromatography (CIEX).

The second chromatographic separation 116 may include contacting botulinum toxins or toxin complexes with a second chromatography column, with a mobile phase containing the toxin-containing fraction 114 moving through the second stationary phase. The second stationary phase may contain any suitable chromatographic matrix (eg, agarose bead based medium such as SP Sepharose FF (available from GE Healthcare)). In embodiments of the present invention, botulinum toxin molecules or toxin complexes may bind to the second stationary phase. In embodiments of the present invention, botulinum toxins or toxin complexes may bind to the second stationary phase while monitoring the A ₂₈₀ solution washing the column. In embodiments of the present invention, the column is washed with a suitable buffer solution (eg, a solution containing 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5) until the A ₂₈₀ returns to its original value, indicating that all of the toxin-containing eluate 118 passed through the column, although botulinum toxins and toxin complexes may remain associated with the second stationary phase.

In some embodiments of the present invention, the second chromatography separation 116 may also include further washing the second chromatography column with wash buffer to remove loosely bound proteins while the botulinum toxins and toxin complexes remain tightly bound to the second stationary phase. Any suitable buffer solution can be used for this washing step (eg, a solution containing 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5, 210 mM NaCl). In some embodiments, the wash buffer may contain NaCl at a concentration of 250 mM or less, 240 mM or less, 230 mM or less, 220 mM or less, 210 mM or less, 200 mM or less, 190 mM or less , 180 mM or less, 170 mM or less, 160 mM or less, 150 mM or less, 140 mM or less, 130 mM or less, 120 mM or less, 110 mM or less, or 100 mM or less

- 12 045619 less, or in even lower concentrations. In some embodiments of the present invention, this wash buffer contains NaCl at a concentration of about 100 mM, about

110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about

220 mM, about 230 mM, about 240 mM, or about 250 mM.

In some embodiments of the present invention, the second chromatographic separation 116 may also include conditioning the toxin-containing fraction 114 for column chromatography (eg, through dilution or buffer exchange) before contacting the toxin-containing fraction 114 with the second chromatography column. For example, in some embodiments of the present invention, toxin-containing fraction 114 can be conditioned by dilution in an acetic acid buffer at a pH ranging from about 3 to about 7, about 3.5 to about 6, or about 4 to about 5. preferably at a pH value of approximately 4.5. In some embodiments of the present invention, the toxin-containing fraction 114 is conditioned in a buffer having a pH value of about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4 ,1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5 ,1, about 5.2, about 5.3, about 5.4 or about 5.5.

In some embodiments of the present invention, the second chromatographic separation 116 may further include eluting botulinum toxin or toxin complexes from the column to obtain a first toxin-containing eluate 118. The elution may include washing the column with one or more buffer solutions that facilitate detachment of the toxin (or toxin complex) from the stationary phase (for example, by changing pH or ionic strength). This elution buffer can be any buffer solution suitable for promoting dissociation of the toxin or toxin complex from the second stationary phase (eg, a solution containing 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5). For example, such a buffer solution may comprise an acetate buffer with a pH ranging from about 3 to about 7, from about 4 to about 5, or a pH of about 4.5. In some embodiments of the present invention, the toxin-containing fraction is eluted from the second chromatography column using a buffer having a pH value of about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, 0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, 0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5.

In some embodiments of the present invention, this elution buffer may contain a salt in a concentration sufficient to stimulate the detachment of botulinum toxins or toxin complexes from the second stationary phase. In embodiments of the present invention, the elution buffer contains NaCl at a concentration equal to 230 mM or more, 240 mM or more, 250 mM or more, 260 mM or more, 270 mM or more, 280 mM or more, 290 mM or more, 300 mM or more, 350 mM or more, or 400 mM or more. In some embodiments of the present invention, the elution buffer contains NaCl at a concentration of about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM , about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 M, about 390 mM or about 400 mM, or any value in between.

In embodiments of the present invention, botulinum toxin molecules or toxin complexes are eluted from the column to produce a first toxin-containing eluate 118, which can be collected and further purified in subsequent processing steps. In some embodiments of the present invention, A ₂₈₀ is measured during elution from a second chromatography column to detect the presence of botulinum toxin molecules or toxin complexes eluted from the column, and the A ₂₈₀ peak is collected as a separate fraction to yield a first toxin-containing eluate 118.

Filtration 3.

Some embodiments of the method further include a third filtration step 120 performed after the second chromatographic separation 116 and before the third chromatographic separation 124. In an embodiment of the present invention, during the third filtration 120, NTH and/or NTNH proteins are separated from the botulinum toxin complexes. In embodiments of the present invention, the third filtration 120 includes a buffer exchange, which can increase the pH of the first toxin-containing eluate 118 (eg, from about 4.5 to about 8.0). In embodiments of the present invention, buffer exchange may reduce the salt concentration in toxin-containing eluate 118 (eg, from about 270 mM to about 50 mM). In embodiments of the present invention, the third filtration 120 can concentrate the toxin-containing eluate 118 (eg, by reducing its volume from about 300 ml to about 50-60 ml).

- 13 045619

The third filtration 120 may include any suitable filtration method (eg, diafiltration, cross-flow microfiltration, cross-flow ultrafiltration, hollow fiber filtration, etc.). In a specific embodiment of the present invention, the third filtration 120 may include ultrafiltration using a tangential filter (eg, a hollow fiber filter) with a pore size suitable for retaining detached botulinum toxin molecules (150 kDa) and NTH and/or NTNH proteins. In some embodiments, the filter may have a pore size of no more than about 150 kDa, no more than about 140 kDa, no more than about 130 kDa, no more than about 120 kDa, no more than about 110 kDa, no more than about 100 kDa, no more than about 90 kDa, not exceeding about 80 kDa, not exceeding about 70 kDa, not exceeding about 60 kDa, not exceeding about 50 kDa, not exceeding about 40 kDa, not exceeding about 30 kDa, or not exceeding about 20 kDa (or intermediate) ). For example, the tangential filter may have a pore size of about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, or about 150 kDa, or having any intermediate value. In an embodiment of the present invention, the filter has a pore size of approximately 30 kDa.

In some embodiments of the present invention, the toxin-containing eluate 118 can be concentrated by ultrafiltration, then washed using diafiltration against a buffer solution to separate the NTH and/or NTNH proteins from the botulinum toxin complexes. In a particular embodiment of the present invention, the botulinum toxin protein molecules and the NTH and/or NTNH proteins may remain in the retentate 121 (ie, the toxin-containing retentate) while other residues from the filtration and chromatography media are separated into the permeate.

The buffer solution used for third filtration 120 may be any buffer solution (eg, Tris-HCl buffer) suitable for detaching the NTH and/or NTNH proteins contained in the toxin complexes from the botulinum toxin molecules. In some embodiments of the present invention, the buffer solution used for the third filtration 120 may be the same buffer that was used to condition the third chromatography column (eg, a Tris-HCl buffer with a pH greater than 7, as discussed below).

In some embodiments of the present invention, the buffer solution used for third filtration 120 has a pH suitable for detaching NTH and/or NTNH proteins from botulinum toxin protein molecules. In some embodiments of the present invention, the pH of the buffer solution is at least about 7, preferably in the range of about 7 to about 10, preferably in the range of about 7 to about 9, preferably in the range of about 7.5 to about 8.5, preferably equal to about 8.0. In some embodiments of the present invention, the pH of third filtration buffer 120 may be about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.5 or approximately 10.0. In an embodiment of the present invention, the pH of the third filtration buffer solution 120 is approximately 8.0.

In some embodiments of the present invention, the toxin-containing retentate 121 from the third filtration 120 can be collected and further purified in subsequent processing steps without precipitating botulinum toxin molecules or toxin complexes from solution at any point in the purification process. This advantageously increases overall process yields and eliminates the need to reconstitute the lyophilized drug product at any point in the purification process or by the end user.

Third chromatographic separation.

Some embodiments of the method 100 further include a third chromatographic separation 124 to separate and remove NTH and/or NTNH proteins dissociated from the toxin complexes during the preceding third filtration 120, while retaining the botulinum toxin protein molecules. The resulting second toxin-containing eluate 126 may contain free botulinum toxin protein molecules (approximately 150 kDa) and may contain no, substantially no, or substantially no toxin complexes. The third chromatographic separation 124 may include any suitable chromatographic separation method (eg, ion exchange chromatography, including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). In a specific embodiment, the third chromatographic separation 124 may include anion exchange chromatography (AIEX).

- 14 045619

The third chromatographic separation 124 may include contacting the botulinum toxin or toxin complexes contained in the toxin-containing retentate 121 from the third filtration 120 with a third chromatography column, wherein the mobile phase containing the toxin-containing retentate 121 (and the free botulinum toxin protein molecules therein) moves through the third stationary phase. The third stationary phase may contain any suitable chromatographic matrix (eg, an agarose bead-based medium such as Q Sepharose FF (available from GE Healthcare)).

In some embodiments of the present invention, the third chromatographic separation 124 may also include conditioning the toxin-containing retentate 121 from the third column chromatography filtration 120 (eg, through dilution, buffer exchange, filtration, or a combination thereof). For example, toxin-containing retentate 121 can be diluted in a buffer solution, such as Tris-HCl buffer, at a pH greater than 7, preferably in the range of 7 to 10, preferably from about 7 to about 9, preferably from about 7, 5 to about 8.5, or preferably at a pH value of about 8.0. In some embodiments, the pH of the buffer solution is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.5, or about 10.0.

In some embodiments of the present invention, botulinum toxin protein molecules, toxin complexes, and NTH and/or NTNH proteins contained in toxin-containing retentate 121 can be adsorbed to the third stationary phase. In some embodiments of the present invention, the toxin-containing retentate 121 is loaded onto a third chromatography column and washed with a wash buffer solution to remove non-toxin impurities that remain unassociated with the third stationary phase. The wash buffer can be any suitable buffer solution (eg, a solution containing 20 mM Tris/HCl, 50 mM NaCl, 0.2% polysorbate 20, pH 8.0), and the A ₂₈₀ of the solution passing through the column can be monitored until until A ₂₈₀ reaches the initial value, indicating that all unbound material has flowed through the third chromatography column.

The third chromatographic separation 124 may further include eluting the botulinum toxin proteins associated with the third stationary phase to produce a second toxin-containing eluate 126. The elution may include washing the column with one or more buffer solutions to desorb the botulinum toxin protein molecules from the stationary phase (e.g., by changing the pH , ionic strength, etc.) using any suitable protein-compatible buffer solution. For example, botulinum toxin molecules can be eluted from the third stationary phase using a Tris-HCl buffer solution at a pH greater than 7, preferably ranging from 7 to 10, preferably from about 7 to about 9, preferably from about 7.5 to about 8.5 or preferably at a pH of about 8.0. In some embodiments, the pH of the buffer solution is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4 or about 8.5.

In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column may contain a salt (eg, NaCl). In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains NaCl at a concentration suitable to promote desorption of botulinum toxin molecules from the third stationary phase. In embodiments of the present invention, this elution buffer may contain NaCl at a concentration ranging from about 25 mM to about 250 mM, preferably from about 50 mM to about 200 mM, preferably from about 100 mM to about 150 mM, preferably - equal to approximately 120 mM. In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains NaCl at a concentration of about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM. In an embodiment of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains NaCl at a concentration of about 120 mM or about 150 mM.

In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column is supplemented

- 15 045619 contains a surfactant (eg polysorbate 20). In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains a surfactant at a concentration ranging from about 0.05% by volume to about 1.0% by volume, preferably - from about 0.10% by volume to about 0.5% by volume, preferably from about 0.15% by volume to about 0.25% by volume, preferably at a concentration of about 0.20% by volume. In some embodiments of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains a surfactant at a concentration of about 0.05 vol.%, about 0.10 vol.%, about 0.15 vol. .%, about 0.20 vol.%, about 0.25 vol.%, about 0.30 vol.%, about 0.35 vol.%, about 0.40 vol.%, about 0.45 vol.% , about 0.50 vol.%, about 0.55 vol.%, about 0.60 vol.%, about 0.65 vol.%, about 0.70 vol.%, about 0.75 vol.%, about 0.80 vol.%, about 0.85 vol.%, about 0.90 vol.%, about 0.95 vol.%, or about 1.0 vol.%. In an embodiment of the present invention, the buffer solution used to elute botulinum toxin molecules from the third chromatography column contains polysorbate 20 at a concentration of about 0.20% by volume.

In specific embodiments of the present invention, the second toxin-containing fraction 126 is collected as it elutes from the column and further purified during subsequent process steps without precipitation or lyophilization of the botulinum toxin proteins. In an embodiment of the present invention, the second toxin-containing fraction 126 is directly injected onto a fourth chromatography column for final deep purification (i.e., removal of high molecular weight impurities, including aggregates of the target protein, and low molecular weight impurities, including fragments of the target protein, and other impurities that may not have been removed during time of previous purification stages).

Fourth chromatographic separation.

Some embodiments of the method 100 further include a final purification step to remove aggregates and/or protein contaminants from the second toxin-containing fraction 126 using a fourth chromatographic separation 128 to obtain a third toxin-containing eluate 130. The fourth chromatographic separation 128 may include any suitable chromatographic separation method (e.g. , ion exchange chromatography, including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). For example, in an embodiment of the present invention, the fourth chromatographic separation 128 may include gel filtration chromatography.

The fourth chromatographic separation 128 may use any suitable protein-compatible chromatographic matrix capable of separating pure botulinum toxin protein molecules from aggregated botulinum toxin and other protein impurities. For example, the fourth chromatography separation 128 may use Superdex 200 chromatography media (manufactured by GE Healthcare).

The fourth chromatographic separation 128 may also include conditioning the second toxin-containing eluate 126 for column chromatography (eg, through dilution, buffer exchange, filtration, or a combination thereof). Alternatively, in a particular embodiment of the present invention, the second toxin-containing eluate 126 from the third chromatography separation 124 can be directly injected onto the fourth chromatography column without additional conditioning. In this configuration, the third and fourth chromatography columns can be coupled to each other to provide direct loading of the second toxin-containing eluate 126.

The fourth chromatographic separation 128 may also include washing botulinum toxin molecules through a stationary phase using a wash solution or buffer to produce a third toxin-containing eluate 130. The buffer solution may be any suitable protein-compatible buffer solution. In a specific embodiment of the present invention, the buffer solution may be an acetate buffer solution having a pH of less than about 7, preferably ranging from about 5 to about 7, preferably from about 6 to about 7, preferably from about 6.6 to approximately 6.9. In some embodiments, the pH of the buffer solution may be about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

In some embodiments of the present invention, the buffer solution used to wash the botulinum toxin molecules through the stationary phase in the fourth chromatography column further contains a salt (eg, NaCl). In some embodiments of the present invention, the wash buffer solution contains NaCl at a concentration ranging from about 100 mM to about 1 M, preferably from about 200 mM to about 600 mM, more preferably from about 300 mM to about 500 mM, most preferably equal to about 400 mM. In some embodiments of the present invention, this buffer

- 16 045619 This solution contains NaCl in a concentration of about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM , about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, or about 1M. In an embodiment of the present invention, the buffer solution used to wash the botulinum toxin molecules through the stationary phase in the fourth chromatography column contains about 350 mM NaCl or about 370 mM NaCl.

In some embodiments of the present invention, the buffer solution used to wash the botulinum toxin molecules through the stationary phase in the fourth chromatography column further contains a surfactant (eg, polysorbate 20). In some embodiments of the present invention, the buffer solution contains a surfactant at a concentration ranging from about 0.05% by volume to about 1.0% by volume, preferably from about 0.10% by volume to about 0% by volume. .5% by volume, preferably from about 0.15% by volume to about 0.25% by volume, preferably at a concentration of about 0.20% by volume. In some embodiments of the present invention, the buffer solution contains a surfactant at a concentration of about 0.05% vol., about 0.10% vol., about 0.15% vol., about 0.20% vol., about 0.25 vol.%, about 0.30 vol.%, about 0.35 vol.%, about

0.40 vol.%, about 0.45 vol.%, about 0.50 vol.%, about 0.55 vol.%, about 0.60 vol.%, about

0.65 vol.%, about 0.70 vol.%, about 0.75 vol.%, about 0.80 vol.%, about 0.85 vol.%, about

0.90 vol.%, about 0.95 vol.%, or about 1.0 vol.%. In an embodiment of the present invention, the buffer solution used to wash botulinum toxin molecules through the stationary phase of the fourth chromatography column contains polysorbate 20 at a concentration of about 0.20% by volume.

In some embodiments of the present invention, the presence of free botulinum toxin molecules in the solution passing through the column is monitored by measuring _A280 . In an embodiment of the present invention, peak A ₂₈₀ (indicative of the presence of free botulinum toxin molecules) is collected as a separate fraction to obtain a third toxin-containing eluate 130.

In some embodiments of the present invention, the third toxin-containing eluate 130 can be collected as it elutes from the fourth chromatography column and either diluted or further purified in subsequent process steps without precipitating or lyophilizing the botulinum toxin proteins. In some embodiments of the present invention, the third toxin-containing eluate 130 can be stored at a temperature ranging from about 2°C to about 8°C until processed after purification. In some embodiments of the present invention, the third toxin-containing eluate 130 can be diluted, filtered and dosed to produce a productive toxin solution 134.

Dilution, filtration and dosing.

In some embodiments of the present invention, the method 100 further includes diluting, filtering and dosing 132 a third toxin-containing eluate 130 to produce a toxin productive solution 134. In some embodiments of the present invention, the third toxin-containing eluate 130 can be diluted to a final concentration using any suitable buffer solution. In some embodiments of the present invention, the diluent buffer solution may be approximately identical in composition to the wash buffer used in the fourth chromatographic separation 128. For example, the diluent buffer solution may comprise an acetic acetate buffer with a pH below about 7, preferably with a pH in the range of about 5 to about 7, preferably from about 6 to about 7, preferably from about 6.6 to about 6.9. In some embodiments, the pH of the buffer solution is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

The method 100 may further include filtering the third toxin-containing eluate 130 (or a diluted form thereof) to reduce the microbiological load and obtain a productive toxin solution 134. Filtration of the third toxin-containing eluate 130 may involve any suitable filtration method (eg, diafiltration, tangential flow microfiltration, hollow fiber filtration, etc.). In some embodiments of the present invention, the filter may have a pore size ranging from 0.1 μm to 10 μm (eg, about 0.2 μm).

The productive toxin solution 134 can be dispensed into containers (eg, cryogenic vials) that can be transported and stored under conditions that maintain the activity of the botulinum toxin proteins contained in the vials. For example, a productive solution of toxin 134

- 17 045619 can be dispensed, transported and/or stored in pre-chilled storage containers (eg vials contained in pre-cooled aluminum blocks).

In some embodiments of the present invention, dispensing the productive toxin solution 134 may involve pumping the productive toxin solution from a sterile container (eg, a sterile disposable bag) into primary storage containers (eg, cryogenic vials). In some embodiments of the present invention, the primary storage containers can be pre-cooled and stored at a suitable temperature (eg, 0° C. or below), for example, by storing, transporting and freezing the primary storage containers in pre-cooled aluminum blocks. In this case, the productive toxin solution 134 is stored at a temperature at which the botulinum toxin molecules are stable and retain neurotoxicity. This may also eliminate the need to lyophilize the toxin production solution 134. Productive toxin solution 134 can also be stored at a temperature of about 0°C or lower, preferably at a temperature of about -70°C or lower.

Output method.

To evaluate a purification process, the overall yield of the process and the yield of each step can be calculated. The yield can be calculated based on the resulting concentration of toxin in each fraction. The output of each process step (i.e., Process Stage Output) can be calculated according to the following formula:

_ - (^fractions) (^fractions) . __

Process stage yield (%) = -------------------------- x 100 (^previous fraction) (^previous fraction) (Equation 1) where C is the toxin concentration, V is the volume, the fraction subscript denotes the toxin concentration or volume from the current treatment step, and the previous fraction subscript denotes the toxin concentration or volume from the previous treatment step. For example, if a process step produced 1.0 μg/ml toxin in a volume equal to 1000 ml, and a previous process step produced 0.5 μg/ml toxin in a volume equal to 4000 ml, then the yield of the method step would be 50%. ([1.0 µg/mlx1000 ml]/[0.5 µg/mlx4000 ml])x 100=50%).

The total yield can be calculated using the following formula:

Total yield (%) = ΒαςΙΒαςΙ _x oo

(Equation 2) where C is the concentration, V is the volume, the subscript DS denotes the drug (i.e., the productive solution of the toxin), and the subscript KS denotes the culture at the time of collection from which cells have been removed by filtration through a pore-size filter equal to 0.2 µm. For example, if the DS contains 50 μg/ml toxin in a volume equal to 100 ml, and the culture at the time of harvest contains 5 μg/ml toxin in a volume equal to 5000 ml, then the overall yield will be 20%. ([50 µg/mlx100 ml]/[5 µg/mlx5000 ml])x 100=20%).

In some embodiments of the present invention, the overall yield is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35% , at least about 40%, or lies in any intermediate range or has any intermediate value. In some embodiments of the present invention, the overall yield is about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32% , about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40%, or anything in between.

In an embodiment of the present invention, the purity of the drug substance can be determined by the SDS-PAGE method and/or by HPLC-SEC. In an embodiment of the present invention, the drug substance produced by the method of the present invention has a purity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least approximately 99% (or somewhere in between). In an embodiment of the present invention, the drug substance may have a purity of about 96.0%, about 96.5%, about 97.0%, about 97.5%, about 98.0%, about 98.5%, about 99.0%, about 99.5%, or about 100.0% (or anything in between).

Description of embodiments of the invention

Example 1. Four-column chromatographic method for the purification of botulinum toxin.

- 18 045619

As an example, an embodiment of the method can be carried out as described below, wherein botulinum toxin A1 (approximately 150 kDa) is isolated from a fermentation medium containing essentially no, substantially no or no animal products containing C. botulinum cells and protein molecules of botulinum toxin A1 and toxin complexes. This APF fermentation medium is described in co-filed provisional US patent application Serial No. 62/951,549. The botulinum toxin contained in this medium is filtered and purified according to the process steps described in more detail below to obtain a productive toxin solution containing botulinum toxin protein molecules and containing no, substantially no or substantially no toxin complexes, without the use of precipitation or lyophilization steps.

Preparation of a buffer solution.

As shown in table. 1, many different buffer solutions can be used in this embodiment of the method. In an embodiment of the present invention, buffer solutions are passed through 0.2 micron filters (to reduce microbiological load) into sterile disposable bags. Used filters are removed and bags containing buffer solutions are stored at room temperature until use.

Table 1

Buffer solutions used in the BoNT/A1 purification method

Buffer solutions used in the BoNT/AI purification method Column chromatography Used buffer solutions First chromatographic separation of Q Sepharose FF 1. 20 mM Na phosphate, 1 M NaCI, pH 6.1 2. 20 mM Na phosphate, 150 mM NaCI, pH 6.1 3. 20 mM Na phosphate, 150 mM NaCl, pH 6.1 4. 20 mM Na phosphate, 150 mM NaCI, pH 6.1 Second chromatographic separation SP Sepharose FF 5. 50 mM Na acetate, 1 M NaCI, 0.2% polysorbate 20, pH 4.5 6. 50 mM Na acetate, 0.2% polysorbate 20, pH 4.5 7. 100 mM Na acetate, 0.4% polysorbate 20, pH 4.5 8. 50 mM Na acetate, 210 mM NaCI, 0.2% polysorbate 20, pH 4.5 9. 50 mM Na acetate, 270 mM NaCI, 0.2% polysorbate 20, pH 4.5 Third chromatographic separation Q Sepharose FF 10. 20 mM Tris-HCI, * 50 mM NaCI, pH 8.0 11. 20 mM Tris-HCI, 1 M NaCI, 0.2% polysorbate 20, pH 8.0 12. 20 mM Tris-HCI, 50 mM NaCI, 0.2% polysorbate 20, pH 8.0 13. 20 mM Tris-HCI, 50 mM NaCI, 0.2% polysorbate 20, pH 8.0 14. 20 mM Tris-HCI, 120 mM NaCI, 0.2% polysorbate 20, pH 8.0 Fourth chromatographic separation Superdex 200 15. 50 mM Na acetate, 370 mM NaCI, 0.2% polysorbate 20, 13 mM Na ₂ HPO ₄ , 17 mM NaOH, pH 6.6-6.9 16. 50 mM Na acetate, 370 mM NaCI, 0. 2% polysorbate 20, 13 mM Na ₂ HPO ₄ , 17 mM NaOH, pH 6.6-6.9 Post-cleaning Final dilution 17. 50 mM Na acetate, 370 mM NaCI, 0.2% polysorbate 20, 13 mM Na ₂ HPO ₄ , 17 mM NaOH, pH 6.6-6.9 *Abbreviations: Tris-HCI = tris(hydroxymethyl)aminomethane hydrochloride

Indicated in the table. 1 buffer solutions 1 and 2 were used to equilibrate the first chromatography column; buffer solution 3 was used to condition the clarified culture for column chromatography; buffer solution 4 was used to wash botulinum toxin complexes through the first column in flow mode; buffer solutions 5 and 6 were used to equilibrate the second chromatography column; buffer solution 7 was used to condition the toxin-containing fraction from the first chromatographic separation for the second chromatographic separation; buffer solution 8 was used to wash bulk impurities (eg proteins) through a second chromatography column; buffer solution 9 was used to elute bound botulinum toxin complexes from the second chromatography column; buffer solution 10 was used to separate NTH and/or NTNH proteins from botulinum toxin protein molecules (ie, to condition the first toxin-containing eluate from the second chromatographic separation for the third chromatographic separation); buffer solutions 11 and 12 were used to equilibrate the third chromatography column; buffer solution 13 was used to wash out impurities from the third chromatography column; buffer solution 14 was used to elute bound botulinum toxin protein molecules from the third chromatography column; buffer solution 15 was used to equilibrate the fourth chromatography column; buffer solution 16 was used to wash botulinum toxin molecules through the fourth chromatography column; and buffer solution 17 was used to dilute the toxin-containing eluate from

- 19 045619 fourth chromatographic separation to final concentration before dispensing it into primary storage containers to obtain a productive toxin solution.

Filtration.

The fermentation medium for C. botulinum was obtained from the APF manufacturing process described in the co-filed provisional US patent application Serial No. 62/951,549. Immediately after harvest, the culture (approximately 5 L) was diluted using approximately 280 ml of buffer solution (1 M sodium acetate, 4 M HCl, pH 5.5) to adjust the pH immediately before filtration. The diluted solution was filtered by microfiltration (Filtration 1 using a hollow fiber filter with a pore size of 0.2 μm for tangential flow filtration to separate spores as well as whole and lysed C. botulinum cells and a clarified culture was obtained. A sample was taken from the clarified culture to monitor the process for the absence of C. botulinum The clarified culture was then purified by removing residual fermentation media (e.g. proteins, carbohydrates, etc.) by tangential flow ultrafiltration (Filtration 2) using a 50 pore size hollow fiber filter kDa. The clarified culture was then subjected to a buffer exchange in sodium phosphate buffer (pH of about 6.1) to condition the clarified culture for column chromatography. This and other suitable methods for filtering proteins are known in the art. See, for example, publication Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998).

Cleaning.

A four-column chromatography method was developed for the purification of BoNT/A1 protein molecules (approximately 150 kDa) without the use of precipitation, lyophilization or centrifugation steps. This method provides a productive toxin solution that contains no, substantially no, or substantially no botulinum toxin complexes and/or animal products. This improves yields and eliminates end-user errors associated with reconstitution procedures that are known to reduce the potency of botulinum toxin drug formulations.

The advantage of this method is that botulinum toxin complexes are retained during the initial stages to protect the toxin while proteases and other bulky protein contaminants are removed. The first chromatographic step, which is anion exchange chromatography, removes most of the nucleic acids present in the culture medium at harvest that would otherwise interfere with subsequent chromatographic steps. This inhibits the denaturation of botulinum toxin molecules during the purification process. The method then separates botulinum toxin protein molecules from their associated NTH and/or NTNH proteins to produce a productive toxin solution that contains pure botulinum toxin molecules (approximately 150 kDa) and contains no, substantially no, or substantially no botulinum toxin complexes .

In addition, all chromatographic steps used in this method are designed so that they can be carried out on disposable chromatography columns, which can be destroyed after a single purification procedure. This eliminates the costly and time-consuming column regeneration steps associated with reusable chromatography systems. The entire process is carried out in a closed system using disposable bags, tubes, filters and chromatography columns. In addition, open processing steps are eliminated because the fluid being processed is preferably permanently contained within bags, tubes, filters or columns, thereby protecting the productive toxin solution from contamination and the operator from exposure to the toxin.

In table 2 summarizes the four-column chromatographic purification method of BoNT/A1 according to the present invention. The method includes the following stages:

(1) The clarified culture was directly injected onto a column containing Q Sepharose FF (2.5 L packed into a column with a diameter of 80 mm and a height of 500 mm), in flow mode using sodium phosphate buffer (pH 6.1) to separate botulinum toxin complexes from nucleic acids. At this stage, the botulinum toxin complexes did not attach to the stationary phase, but instead flowed through the column as part of the toxin-containing fraction.

(2) The toxin-containing fraction from the Q Sepharose FF column (100 ml packed into a 50 mm diameter x 50 mm height column) was conditioned in sodium acetate buffer solution (pH 4.5) and passed through an SP Sepharose FF column using sodium acetate buffer (pH 4.5). At this stage, botulinum toxin complexes were adsorbed onto the stationary phase, while bulk impurities (eg, other proteins) were washed through the column. Toxin complexes bound to the column were eluted using sodium acetate buffer (pH 4.5) containing NaCl and collected as part of the first toxin-containing eluate.

(3) The first toxin-containing eluate was filtered to remove residual fermentation medium and conditioned in a suitable buffer solution by tangential flow ultrafiltration (Filtration 3) for buffer exchange (Tris-HCl buffer, pH 8.0) using a pore size filter of 30 kDa, to detach botulinum toxin molecules from NTH and/or NTNH

- 20 045619 proteins in toxin complexes. The retentate was then passed through a Q Sepharose FF column (2 mL packed into a 5 mm diameter x 100 mm height column) to separate free botulinum toxin proteins from NTH and/or NTNH proteins. At this stage, botulinum toxin molecules were adsorbed to the stationary phase, while NTH and/or NTNH proteins (and other bulk impurities) were washed through the column to some extent (but were predominantly adsorbed to the stationary phase, even more strongly than botulinum toxin molecules). Botulinum toxin proteins were eluted using Tris-HCl buffer solution (pH 8.0) containing NaCl and collected as part of a second toxin-containing eluate.

(4) The second toxin-containing eluate was directly injected onto a Superdex 200 gel filtration column (320 mL packed into a 25 mm diameter x 600 mm height column) for final deep purification (ie, aggregate removal). At this stage, the column was washed with a buffer solution containing 50 mM Na acetate, 370 mM NaCl, 0.2% polysorbate 20, 13 mM Na ₂ HPO ₄ and 17 mM NaOH (pH 6.6-6.9), and purified botulinum proteins The toxin was collected as part of the third toxin-containing eluate.

table 2

Summary of column chromatography steps in the BoNT/A1 purification method

Summary of Column Chromatography Steps Column Way Stationary phase Target 1 AIEX Q Sepharose FF Toxin in effluent, nucleic acids bind to column 2 CIEX SP Sepharose FF Separation of bulky impurities (for example, proteins) 3 AIEX Q Sepharose FF Purification of protein complexes (i.e. NTH and NTNH) 4 Gel filtration Superdex 200 Ultimate deep cleaning; removal of aggregates; buffer replacement

Each of the column chromatography steps described above may include clean-in-place procedures and other column preparation steps prior to chromatographic separations. Procedures for preparing and operating columns are known in the art. See, for example, Ozutsumi et al., 49 Appl. Envtl. Microbiol. 939 (1985); GE Healthcare, Strategies for Protein Purification Handbook (2010); Schmidt et al., 156 Anal. Biochem. 213 (1986); Simpson et al., 165 Methods Enzymol. 76 (1988); Zhou et al., 34 Biochem. 15175 (1995); Kannan et al., 15 Mov. Discord. 20 (2000); Wang Y-c, Dermatol. Las Facial Cosmet. Surg. 58 (2002); Johnson et al., 32 Protein Expr. & Purif. 1 (2003); US 2003/0008367 A1.

Dilution/filtration/dosing.

Purified botulinum toxin proteins were diluted to final concentration in a suitable buffer solution (eg, containing 50 mM sodium acetate, 0.2% polysorbate 20, 370 mM NaCl, 13 mM sodium phosphate, 17 mM NaOH) and mixed gently on a platform shaker for approximately 30 minutes. The diluted product was filtered using a 0.2 µm filter into a sterile disposable bag and dispensed in 0.4 ml aliquots into 1.8 ml cryogenic vials using a dispensing pump and needle. The samples were then quickly frozen using pre-cooled aluminum blocks stored at temperatures below -70°C. The final solutions prepared for storage according to the method are designated drug substance (or DS) below.

Three different batches of drug substance were prepared according to the steps described above: No. 16852, No. 17043, and No. 19139. The DS batches were then tested for appearance, potency, specific activity, and total protein concentration, as discussed in the following examples.

Example 2: Appearance test.

Transparency and color were examined to confirm that the drug substance is clear and colorless, since opalescence in solution may indicate protein aggregation or precipitation. The method is a visual method based on European Pharmacopoeia (Ph. Eur) 2.2.1, entitled Transparency and level of opalescence of liquids, and European Pharmacopoeia 2.2.2, entitled Degree of color of liquids, however with the modification that a bottle was used and the volume of the drug substance, rather than the container and volume specified in the methods according to the Pharmacopoeia. Water was used as a reference solution.

According to this method, all batches of the drug substance prepared according to Example 1 were clear and colorless solutions, indicating the absence of detectable aggregation or precipitation of botulinum toxin protein molecules.

Example 3. Activity.

The activity of the purified drug substance obtained in example 1 was determined by

- 21 045619 vom LD50 analysis for mice. This is an absolute assay that quantitatively measures the activity of the sample tested. Gelatin phosphate buffer was used as a diluent and 11 dose groups were established, centered around the target LD50 value. Dose groups with activities ranging from 3.0 to 0.4 dose units were distributed evenly at intervals of approximately 0.0899 log units. The number of deaths 72 hours after injection was recorded and LD50 was calculated using the Spearman-Kerber method. Spearman-Kerber calculation was used as a mathematical tool to determine the midpoint (LD50) of the logarithmic dilution curve for death. The activity of the purified drug substance is expressed in LD ₅₀ units/ml. See, for example, the publication by M.A. Ramakrishnan, Determination of 50% Endpoint Titer Using a Simple Formula, 5 World J. Virol. 85-86 (2016); see also G. Karber, 162 Pathol. u Pharmakol. 480-83 (131); With Spearman, The Method of Right and Wrong Cases (Constant Stimuli) Without Gauss's Formula, 2 Br. J. Psychol. 227-42 (1908).

Purified drug lot lots No. 16852, No. 17043 and No. 19139 showed potency values of 11 x 10 ⁶ LD ₅₀ units / ml, 23 x 10 6 LD ₅₀ units / ml and 19 x 10 6 LD ₅₀ units / ml, respectively. These activity values, coupled with the volume of DS obtained, confirm that the process yield is sufficient to produce a large number of drug product doses from a DS batch, which is desirable for commercial DS production.

Example 4: Total Protein Concentration.

The total protein concentration of the drug substance prepared in Example 1 was determined by the bicinchoninic acid method because it is sensitive enough to determine the protein concentration in a standard batch of drug substance prepared in Example 1. See Ph. Eur. 2.5.33 method 4; see also USP (United States Pharmacopoeia) <507> method II.

The Micro BCA Protein Assay Kit is used to determine the total protein concentration, and the bovine serum albumin standard curve is used to calculate the protein concentration from the measured absorbance. The result is expressed as the average of two measurements in paired samples.

In batches of purified drug substance No. 16852, No. 17043 and No. 19139, the total protein concentration was determined to be 59.4 μg/ml, 107.0 μg/ml and 91.4 μg/ml, respectively. These protein concentration values obtained for DS batches are high enough to ensure that the analytical extraction and characterization methods are accurate and reliable.

Example 5. Specific activity.

For the drug substance prepared according to Example 1, the specific activity is calculated based on the activity value of the drug substance in units/ml obtained by the LD ₅₀ determination method (Example 3) and the total protein concentration (Example 4) of the drug substance in mg/ml. Specific activity is calculated according to the formula:

/Unit\ Activity (^)

Specific activity I - 1 = -------------------------\mg/ Total protein concentration (—) (Equation 3)

In batches of purified drug substance No. 16852, No. 17043 and No. 19139, the specific activity was determined to be 1.9 x 10 ⁸ U/mg, 2.2 x ^{10 8} U/mg and 2.1 x 10 ⁸ U/mg, respectively. This confirms the high purity of DS, indicating that the process is suitable for the commercial production of highly purified and fully active uncomplexed botulinum toxin.

Example 6: Protein-related impurities.

The method used to determine protein-related impurities in the drug substance prepared according to Example 1 is based on the principles described in Ph. Eur. 2.2.31 Electrophoresis, called Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) - Uniform Percentage Gels. This method uses SDS-PAGE in combination with colloidal Coomassie blue staining. The standard curve is prepared by diluting the sample; analyze unreduced and reduced samples. Impurities are quantified using densitometry by the ratio of the intensity of the impurity band to a standard curve. Results for impurities are expressed as a percentage of the total amount of protein loaded on the gel.

Fig. 2 shows the purity analysis of DS Lot No. 17043 prepared according to Example 1 using SDS-PAGE in combination with colloidal Coomassie Blue staining. Lanes from left to right are as follows: 1-5: unreduced standard curve samples (1.2-4.0 μg/ml) from lot #17043; 6-7: unreduced parallel samples 1 and 2 from lot No. 17043 (140 μg/ml); 8: molecular weight marker; and 9-10: reconstituted parallel samples 1 and 2 from lot #17043 (140 μg/ml). In all three batches of the drug substance,

- 22 045619 prepared according to example 1, protein-related impurities amounted to less than 6.0%, and batch no.

16852, in particular, showed no detectable impurities.

Example 7: Residual Nucleic Acids.

An analysis for the maximum permissible content of residual nucleic acids was performed to detect RNA and/or DNA in the medicinal substance obtained according to example 1. The method uses a commercially available kit for the quantitative determination of RNA RiboGreen RNA quantitation Kit. RiboGreen binds nucleic acids and generates a fluorescent signal proportional to the amount of nucleic acid in the sample. Because DNA generates a more intense signal than RNA when bound to RiboGreen, the nucleic acid content of DNA-containing samples will be overestimated when using standards containing RNA, so the reported values for nucleic acids correspond to the maximum amounts of nucleic acid in the samples.

Purified drug lot lots #16852, #17043, and #19139 were compared to a reference standard curve with a minimum standard containing 0.15 μg/mL RNA. All three batches showed sample fluorescence lower than the standard, corresponding to <0.15 μg/ml residual nucleic acids (data not shown).

Example 8: Protein profile.

The method used for protein profiling is based on the principles described in Ph. Eur. 2.2.31 Electrophoresis called Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE) - Uniform Percentage Gels. Purified drug batches #16852, #17043 and #19139 were run on a 4-12% Bis-Tris gel to separate proteins present and stained using silver staining (SilverQuest staining Kit). Fig. 3 shows SDS-PAGE results for reduced (lanes 6-9) and unreduced (lanes 2-5) drug samples compared to molecular weight markers (1 and 10). Lanes 3 and 7 correspond to lot #16852; Lanes 4 and 8 correspond to batch no. 17043; and lanes 5 and 9 correspond to lot No. 19139. (Lane 2 and 6 show results for a batch of drug substance (no. 1014997) prepared according to the four-column chromatography method using slightly different process parameters (for example, slightly higher pH of the buffer solutions in the first chromatography separation)).

Purified drug lot lots #1014997, #16852, #17043, and #19139 showed comparable protein profiles with a strong band at approximately 150 kDa (lanes 2, 3, 4, and 5, respectively), indicating that the major protein component is free BoNT/A . The reduced samples in lanes 6, 7, 8, and 9 showed two major protein components with molecular weights of approximately 100 kDa and 150 kDa, corresponding to the heavy chain and light chain of BoNT/A, respectively.

Example 9. Molecular weight distribution of the drug substance.

The molecular weight distribution of drug components was monitored using size exclusion chromatography (SEC). The method analyzes the major components in a sample containing the resulting toxin and high and low molecular weight molecules (HMW and LMW, respectively), which may be process impurities or product-related impurities. A UPLC® SEC size exclusion column is used for separation. The mobile phase is buffer solution 17 (see Table 1 above) supplemented with 0.4 M L-arginine, pH ranging from 6.6 to 6.9. L-arginine is added to buffer solution 17 to minimize secondary interactions between the separated proteins and the column matrix, filters, and other wetted parts of the chromatography system. Using a flow rate of 0.25 mL/min, paired samples (from the same drug vial) were analyzed without pretreatment or dilution. Detection is carried out at a wavelength of 280 nm. The resulting chromatograms are integrated and for each batch of the drug substance the resulting average area in percentage (area, %) for the main component is recorded.

Fig. 4 shows the molecular weight distribution for batch DS No. 17043 prepared according to Example 1. The main peak corresponds to the free molecules of the resulting toxin. All batches of purified drug substance #16852, #17043 and #19139 showed at least 96% of the main component (BoNT/A). These data confirm the high level of purity of botulinum toxin in the productive toxin solutions obtained according to the present invention.

Example 10. Output of the method stage.

The yield of the BoNT/A process step is determined as an indicator of the stability of each purification process step. To calculate the yield of the process step, each fraction was weighed to determine the volume. A BoNT/A-specific enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of BoNT/A in each fraction.

The ELISA protocol is an indirect sandwich ELISA assay based on the principles and general method described in United States Pharmacopoeia <1103>, Immunological Test

- 23 045619

Methods - Enzyme-linked Immunosorbent Assay. The ELISA method is based on immunological binding and detection of BoNT/A using two different types of BoNT/A-specific polyclonal antibodies.

A series of standard protein dilutions based on commercial BoNT/A toxin were prepared by diluting BoNT/A in phosphate-buffered saline (PBS) supplemented with Tween (0.05% Tween-20) to a concentration range of 3 ng/ml to 28 ng/ml. The sample, diluted with PBS-Tween to a range of standard protein dilutions, was added in triplicate to microplate wells coated with polyclonal anti-BoNT/A antibody. Incubation results in antibody recognition and binding of the BoNT/A antigen to the well. After each incubation, an automated washing step was carried out using a PBS-Tween solution.

Primary detection is performed through the binding of another type of polyclonal antibody to BoNT/A, resulting in the formation of a sandwich complex. A secondary antibody conjugated to horseradish peroxidase (HRP) is then added. Its binding to the primary antibody allows detection of BoNT/A within the sandwich complex. Substrate 3,3',5,5'-tetramethylbenzidine (TMB) is then added to the sample wells. HRP converts the TMB substrate to produce a blue reaction product. A stop solution is then added to stop the conversion of TMB and initiate the color conversion of the residual TMB to yellow. The optical density in each microplate well is determined at a wavelength of 450 nm using a plate reader spectrophotometer, and the measured optical density is directly proportional to the amount of BoNT/A in the well. Sample absorbance values are calculated by comparison with a standard curve based on absorbance values for standard BoNT/A dilutions. Results are presented as mean values in µg/ml. The yield of the process step is then calculated according to Equation 1 above.

Fig. 5 shows the average process step yields for each purification process step for Lots No. 16852, No. 17043, and No. 19139. The bar labeled DS shows the combined yield of the last two chromatographic steps, as they are related and no samples were taken between them. The data shows that the yield of the process steps varies from about 100% to about 50%.

Example 11. Accumulated output of a method step.

The accumulated output of a process step for each specific process step is calculated using the formula:

JAccumulated yield of the method stage (%) = χ YuO (Equation 4) where C is the toxin concentration; V - volume; the fraction subscript denotes the concentration or volume for the current processing step, and the subscript KS denotes the culture at the time of collection from which cells have been removed by filtration through a 0.2 µm filter. The accumulated yield of the method step for the culture at the time of harvest (KS) is taken as 100%.

Fig. 6 shows the average accumulated process step yield for each purification process step for purified drug batches #16852, #17043, and #19139. The bar labeled DS shows the cumulative yield of the last two chromatographic steps, as they are interrelated and no sampling was made between them. samples.

Example 12. General output.

The total yield of BoNT/A in the toxin production solution (or DS) compared to the culture at the time of collection (KS) is determined as an indicator of the robustness of the overall purification method. To calculate the yield of the KS and DS method, the fractions were weighed to determine their volumes. The concentration of BoNT/A in KS and DS was determined using BoNT/A-specific enzyme-linked immunosorbent assay (ELISA). The total yield was calculated according to Equation 2 above.

For batches of purified drug substance No. 16852, No. 17043 and No. 19139, overall yields were determined to range from about 13% to about 29% (data not shown).

Example 13. Purity improvement indicator (at stages).

The purity improvement rate of BoNT/A at each specific process step is determined as an indicator of the effectiveness of each step (and the entire purification process) in removing undesirable protein components (process impurities or product-related impurities). To calculate the purity improvement rate during the step, each fraction was analyzed for toxin concentration using a BoNT/A-specific ELISA and for total protein concentration using the Micro BCA method (Example 4). The increase in purity at the stage was calculated according to the formula:

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Claims (26)

(ToxSfrasch^) (ToSRSfractions) Indicator of increase in purity at the stage (times) = (Go%C of _{the previous} fraction) (ToxPCprevious, fractions) (Equation 5) where ToxC is the concentration of the toxin, TotPC is the concentration of total protein, the fraction subscript denotes the concentration of the toxin or the concentration of total protein at the current stage treatment, and the subscript of the previous fraction denotes the toxin concentration or total protein concentration in the previous treatment step. Fig. 7 shows the average purity improvement across stages for purified drug lot lots #16852, #17043, and #19139. The bar labeled DS shows the total purity improvement during the last two chromatographic steps, as they are related and there was no processing between them. sampling. It is clear from the data that the second chromatography column is the step that makes the greatest contribution to the purification of the toxin. However, all steps (except Filtration 1, which removes whole or lysed cells and cellular components) contribute to overall toxin purification relative to total protein concentration. Example 14. Accumulated purity improvement rate (at stages). The accumulated purity improvement was calculated for each specific process step as follows: (T'ohSfr _aK ts _II ). „ , _λ (^°^Сfractions) Accumulated indicator of increase in purity at the stage (times) = ——γ.—g— U ^0X ^KS) (ToxPC _KS ) (Equation 6) where ToxC is the toxin concentration, TotPC is the total protein concentration, the fraction subscript denotes the toxin concentration or total protein concentration at the current stage of processing, and the KS subscript denotes the toxin concentration or total protein concentration protein in culture at the time of collection, from which cells have been removed by filtration through a 0.2 μm pore size filter. It is accepted that at the first technological stage of the purification method (fermentation during collection), the purity increase index is equal to 1. Fig. Figure 8 shows the average cumulative purity improvement across steps for purified drug substance batches #16852, #17043, and #19139. The bar labeled DS shows the total accumulated purity improvement for the last two chromatographic steps since they are interrelated and there is no interaction between them. samples were collected. CLAIM 1. A method for purifying botulinum toxin from a solution containing the toxin, comprising: (a) filtering the solution containing the toxin; (b) contacting the first chromatography column with the filtered solution containing the toxin from step (a), wherein the first chromatography column is an ion exchange chromatography column; (c) collecting the toxin-containing fraction, wherein the toxin-containing fraction flows through the first chromatography column without adsorption to the stationary phase; (d) contacting a second chromatography column with the toxin-containing fraction, the second chromatography column being an ion exchange chromatography column; (e) eluting the botulinum toxin from the second chromatography column to obtain a first toxin-containing eluate; (f) filtering the first toxin-containing eluate to obtain a toxin-containing retentate; (g) contacting a third chromatography column with the toxin-containing retentate from the filtration step (f), wherein the third chromatography column is an ion exchange column; (h) eluting the botulinum toxin from the third chromatography column to obtain a second toxin-containing eluate; (i) contacting a fourth chromatography column with a second toxin-containing eluate, the fourth chromatography column being a size exclusion chromatography column; (j) eluting the botulinum toxin from the fourth chromatography column to obtain purified botulinum toxin, wherein the method does not involve precipitation, centrifugation or lyophilization of the botulinum toxin. - 25 045619 2. The method according to claim 1, characterized in that the botulinum toxin contains botulinum neurotoxin serotype A. 3. The method according to claim 1 or 2, characterized in that the resulting purified botulinum toxin does not contain botulinum toxin complexes or the amount of botulinum toxin complexes is less than 5 wt.%. 4. Method according to any one of claims 1-3, characterized in that the resulting purified botulinum toxin does not contain products of animal origin or the amount of products of animal origin is less than 5 wt.%. 5. Method according to any one of claims 1 to 4, characterized in that the purified botulinum toxin does not contain human albumin or the amount of human albumin is less than 5 wt.%. 6. Method according to any one of claims 1 to 5, characterized in that the first chromatography column is an anion exchange chromatography column. 7. Method according to claim 6, characterized in that the first chromatographic column contains Q Sepharose. 8. Method according to any one of claims 1 to 7, characterized in that the second chromatography column is a cation exchange chromatography column. 9. Method according to claim 8, characterized in that the second chromatographic column contains SP Sepharose. 10. Method according to any one of claims 1 to 9, characterized in that the first chromatography column is an anion exchange chromatography column and the second chromatography column is a cation exchange chromatography column. 11. The method according to claim 10, characterized in that the first chromatography column contains Q Sepharose, and the second chromatography column contains SP Sepharose. 12. Method according to any one of claims 1 to 11, characterized in that the third chromatography column is an anion exchange chromatography column. 13. The method according to claim 12, characterized in that the third chromatographic column contains Q Sepharose. 14. The method according to any one of claims 1 to 13, characterized in that the fourth chromatographic column is a gel filtration column; 15. The method according to claim 14, characterized in that the fourth chromatographic column contains Superdex 200. 16. The method according to any one of claims 1 to 15, characterized in that the third chromatography column is an anion exchange chromatography column, and the fourth chromatography column is a gel filtration chromatography column; 17. The method according to claim 16, characterized in that the third chromatography column contains Q Sepharose, and the fourth chromatography column contains Superdex 200. 18. Method according to any one of claims 1 to 17, characterized in that the second toxin-containing eluate is injected directly onto the fourth chromatographic column. 19. Method according to any one of claims 1 to 18, characterized in that the third chromatography column and the fourth chromatography column are connected to each other. 20. The method according to any one of claims 1 to 19, characterized in that the first chromatography column is an anion exchange chromatography column, the second chromatography column is a cation exchange chromatography column, the third chromatography column is a second anion exchange chromatography column, and the fourth chromatography column is a gel filtration chromatography column. 21. The method of claim 20, wherein the first chromatography column contains Q Sepharose, the second chromatography column contains SP Sepharose, the third chromatography column contains Q Sepharose and the fourth chromatography column contains Superdex 200. 22. The method according to any one of claims 1 to 21, characterized in that the first, second, third and fourth chromatographic columns are disposable chromatographic systems. 23. Method according to any one of claims 1 to 22, characterized in that filtration (f) separates botulinum toxin protein molecules from non-toxin proteins to obtain free toxin molecules. 24. Method according to any one of claims 1 to 23, characterized in that filtering (f) includes buffer replacement. 25. Method according to any one of claims 1 to 24, characterized in that the solution containing botulinum toxin is a fermentation medium that does not contain animal products or a fermentation medium in which the amount of animal products is less than 5 wt.%. 26. The method according to any one of claims 1 to 25, characterized in that it further includes bringing the botulinum toxin into contact with a buffer solution, wherein the buffer solution is filtered to reduce the microbiological load. -