Method for purifying botulinum toxin

Abstract

The present technology relates to a commercial-scale method for purifying a botulinum toxin composition obtained from cell culture. The purification method according to the present disclosure is based on a series of filtration and chromatographic separation steps to obtain a highly purified botulinum toxin composition comprising botulinum toxin protein molecules (approximately 150 kDa) in a solution that is free, essentially free, or substantially free of botulinum toxin complexes and animal products, without precipitating or lyophilizing the botulinum toxin protein molecules. The purification method according to the present disclosure can obtain highly pure, highly active free botulinum toxin protein molecules (approximately 150 kDa) in solution without the use of precipitation, centrifugation, or lyophilization steps, and without the need for reconstitution by the end user.

Description

Technical Field

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

[0002] The present technology generally relates to the field of purifying neurotoxin protein molecules. In particular, the present technology relates to a method for purifying botulinum toxin. The botulinum toxin purified therefrom is suitable for therapeutic use, particularly for administration to a patient to achieve a desired therapeutic or cosmetic effect.

Background Art

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

[0004] Generally, seven immunologically distinct botulinum neurotoxins (botulinum neurotoxin serotypes A, B, C, D, E, F, G) are characterized, each being distinguished by neutralization with type-specific antibodies. As an example, Botox® is a trademark for a purified botulinum toxin type A neurotoxin complex commercially available from Allergan, Inc. (Irvine, California). Botox® is popular as an injectable cosmetic treatment for temporarily reducing the appearance of fine wrinkles.

[0005] Conventionally, botulinum toxin containing type A toxin is produced from the fermentation of C. botulinum. However, in addition to the botulinum toxin molecule, a culture solution containing complete bacteria, lysed bacteria, nutrients in the medium, and fermentation by-products may be generated. When the C. botulinum culture solution is filtered to remove complete cell components and / or lysed cell components and, optionally, other fermentation medium residues, a clarified culture is obtained. The clarified culture solution contains botulinum toxin molecules and various impurities, and a purified botulinum toxin (e.g., BoNT / A1) suitable for removing these impurities to obtain a botulinum toxin pharmaceutical composition can be obtained.

[0006] In existing commercial-scale processes for obtaining a pharmaceutically suitable botulinum toxin composition, typically, multiple precipitation steps are used to separate impurity residues from the fermentation process from the toxin complex. For example, cold alcohol fractionation (e.g., the Cohn method) or precipitation is used to remove plasma proteins. However, precipitation techniques for purifying botulinum toxin have problems such as low resolution, low yield, difficulty in operation, difficulty in control and / or verification, and lack of scalability. Also, drying the botulinum toxin (e.g., by lyophilization, precipitation, etc.) substantially reduces its toxicity. This is because the inactivated toxin may form a toxoid and confer immunity to the botulinum toxin in patients, which is a clinical problem.

[0007] Nevertheless, currently approved botulinum toxin products in the United States (e.g., BOTOX COSMETIC®, DYSPORT®, XEOMIN®, and JEUVEAU®) are stored in a lyophilized or freeze-dried state for reasons of stability. Such formulations need to be reconstituted in sterile saline by a physician before administration to a patient. This reconstitution process is associated with taking up the physician's time, the risk of making dilution errors, and the risk of contamination. The suppliers of botulinum toxin also have to train physicians to ensure that the reconstitution process is carried out properly.

[0008] Therefore, there is a need for a controllable and scalable high-yield method for purifying botulinum toxin from a fermentation medium to obtain a highly pure, highly active and pharmaceutically suitable botulinum toxin composition that does not contain, is essentially free of, or substantially free of animal-derived products and does not require reconstitution before administration to a patient. SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure relates to a method for purifying botulinum toxin. The method includes purifying the toxin from a solution containing the toxin, and the process does not include precipitation, centrifugation, or lyophilization.

[0010] In some embodiments, the botulinum toxin is serotype A. In some embodiments, the resulting purified botulinum toxin does not contain, is essentially free of, or substantially free of botulinum toxin complex. In some embodiments, the resulting purified botulinum toxin does not contain, is essentially free of, or substantially free of animal-derived products containing human albumin.

[0011] In some embodiments, the purification includes a filtration step, preferably a tangential flow filtration step. In some embodiments, the filtration step uses a hollow fiber filter. In some embodiments, the purification includes contacting a first chromatography column with a solution containing the toxin to obtain a toxin-containing fraction. In some embodiments, the first chromatography column includes an anion exchange chromatography column. In some embodiments, the anion exchange chromatography column includes Q Sepharose. In some embodiments, the purification further includes recovering the toxin-containing fraction, and the toxin-containing fraction does not adsorb to a first stationary phase.

[0012] In some embodiments, the purification further comprises contacting the toxin-containing fraction with a second chromatography column. In some embodiments, the second chromatography column comprises a cation exchange chromatography column. In some embodiments, the cation exchange chromatography column comprises SP Sepharose. In some embodiments, the purification further comprises eluting the botulinum toxin from the second chromatography column to obtain a first toxin-containing eluate.

[0013] In some embodiments, the purification further comprises filtering the first toxin-containing eluate to obtain a toxin-containing retention solution. In some embodiments, filtering the first toxin-containing eluate includes exchanging the buffer. In some embodiments, filtering the first toxin-containing eluate separates botulinum toxin molecules from non-toxin proteins to obtain free toxin molecules.

[0014] In some embodiments, the purification further comprises contacting the toxin-containing retention solution with a third chromatography column. In some embodiments, the third chromatography column comprises a second anion exchange chromatography column. In some embodiments, the second anion exchange chromatography column comprises Q Sepharose. In some embodiments, the purification further comprises eluting the botulinum toxin from the third chromatography column to obtain a second toxin-containing eluate.

[0015] In some embodiments, the purification further comprises contacting a fourth chromatography column with the second toxin-containing eluate. In some embodiments, the toxin-containing eluate is directly injected into the fourth chromatography column. In some embodiments, the third chromatography column and the fourth chromatography column are interconnected.

[0016] In some embodiments, the fourth chromatography column includes a size exclusion chromatography column. In some embodiments, the size exclusion chromatography column includes a gel filtration chromatography column. In some embodiments, the gel filtration chromatography column includes Superdex 200. In some embodiments, the purification further includes eluting the botulinum toxin from the fourth chromatography column to obtain a purified botulinum toxin.

[0017] In other aspects, a method for purifying the toxin from a solution containing botulinum toxin includes: (a) filtering the solution containing the toxin; (b) contacting the filtered solution containing the toxin from (a) with a first chromatography column, which is an ion exchange chromatography column; (c) collecting a toxin-containing fraction that passes through the first chromatography column without adsorbing to the stationary phase; (d) contacting the toxin-containing fraction with a second chromatography column, which is 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 retention solution; (g) contacting the toxin-containing retention solution from the filtration (f) with a third chromatography column, which is an ion exchange column; (h) eluting the botulinum toxin from the third chromatography column to obtain a second toxin-containing eluate; (i) contacting the second toxin-containing eluate with a fourth chromatography column, which is a size exclusion chromatography column; and (j) eluting the botulinum toxin from the fourth chromatography column to thereby obtain a purified botulinum toxin, and the process does not include precipitation, centrifugation, or lyophilization of the botulinum toxin.

[0018] In some embodiments, the botulinum toxin includes botulinum neurotoxin serotype A. In some embodiments, the obtained purified botulinum toxin does not contain, essentially does not contain, or substantially does not contain a botulinum toxin complex. In some embodiments, the obtained purified botulinum toxin does not contain, essentially does not contain, or substantially does not contain an animal-derived product containing human albumin.

[0019] In some embodiments, the first chromatography column includes an anion exchange chromatography column. In some embodiments, the first chromatography column includes Q Sepharose.

[0020] In some embodiments, the second chromatography column includes a cation exchange chromatography column. In some embodiments, the second chromatography column includes SP Sepharose.

[0021] In some embodiments, the first chromatography column includes an anion exchange chromatography column, and the second chromatography column includes a cation exchange chromatography column. In some embodiments, the first chromatography column includes Q Sepharose, and the second chromatography column includes SP Sepharose.

[0022] In some embodiments, the third chromatography column includes an anion exchange chromatography column. In some embodiments, the third chromatography column includes Q Sepharose.

[0023] In some embodiments, the fourth chromatography column includes a gel filtration column. In some embodiments, the fourth chromatography column includes Superdex 200.

[0024] In some embodiments, 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 includes Q Sepharose, and the fourth chromatography column includes Superdex 200. In some embodiments, the second toxin-containing eluate is directly injected into the fourth chromatography column. In some embodiments, the third chromatography column and the fourth chromatography column are interconnected.

[0025] In some embodiments, 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 one embodiment, the first chromatography column includes Q Sepharose, the second chromatography column includes SP Sepharose, the third chromatography column includes Q Sepharose, and the fourth chromatography column includes Superdex 200.

[0026] In some embodiments, the first, second, third, and fourth chromatography columns are a single-use chromatography system.

[0027] In some embodiments, the filtration (f) dissociates the botulinum toxin protein molecules from non-toxin proteins to obtain free toxin molecules. In some embodiments, the filtration (f) includes buffer exchange.

[0028] In some embodiments, the solution containing botulinum toxin does not contain, is essentially free of, or is substantially free of animal-derived products. In some embodiments, the purification includes contacting the botulinum toxin with a filtered buffer solution having a reduced bioburden.

[0029] In another aspect, the present disclosure relates to a purified botulinum toxin obtained by purifying the toxin from a solution containing the toxin. Here, the process does not include precipitation, centrifugation, or lyophilization. In some embodiments, the purified botulinum toxin is of serotype A. In some embodiments, the purified botulinum toxin does not contain, is essentially free of, or is substantially free of toxin complexes. In some embodiments, the purified botulinum toxin does not contain, is essentially free of, or is substantially free of animal-derived products containing human albumin.

[0030] In another aspect, the present disclosure relates to a composition comprising a purified botulinum toxin in a buffer solution containing phosphate. In some embodiments, the buffer solution further contains acetate. In some embodiments, the buffer solution further contains at least one source of chloride ions. In some embodiments, the at least one source of chloride ions includes sodium chloride. In some embodiments, the buffer solution further contains at least one surfactant. In some embodiments, the surfactant is polysorbate 20.

[0031] In some embodiments of the composition according to the present disclosure, the botulinum toxin is botulinum neurotoxin serotype A. In some embodiments, the composition does not contain, is essentially free of, or is substantially free of botulinum toxin complexes. In some embodiments, the composition does not contain, is essentially free of, or is substantially free of animal-derived products. In some embodiments, the composition does not contain, is essentially free of, or is substantially free of human albumin. In some embodiments, the pH of the composition is between about 6.6 and 6.9.

[0032] The following detailed description is exemplary and explanatory and should not be construed as limiting.

Brief Description of the Drawings

[0033]

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[0034] Embodiments according to the present disclosure are described in sufficient detail below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. It is to be understood, of course, that the technology is not limited to the particular methods, reagents, compounds, compositions, or biological systems that may vary. The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0035] Unless otherwise defined, all terms (including technical and scientific terms) used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined as in a commonly used dictionary shall be interpreted to have a meaning that coincides with the meaning in the context of the present application and the relevant art, and it should further be understood that they shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein. Such terms are not explicitly defined below, but shall be interpreted according to their general meaning.

[0036] Also, when a feature or aspect of the present disclosure is described in a Markush group, those skilled in the art will recognize that the present disclosure is also described with respect to any individual element or sub-group of elements of the Markush group.

[0037] As will be understood by those skilled in the art, for all purposes, and particularly from the perspective of providing a specifically described explanation, all ranges disclosed herein also include any and all sub-ranges and combinations of those sub-ranges. It can be readily recognized that any specified range is sufficiently descriptive and can be easily divided into at least two equal parts, three equal parts, five equal parts, ten equal parts, etc. of the same range. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, and an upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to", "at least", "more than", "less than", etc. refers to a range that includes the recited number and can be sequentially divided into sub-ranges as described above. Finally, as will be understood by those skilled in the art, one range includes each of the individual elements. Thus, for example, a group having 1 to 3 cells refers to a group having 1 cell, a group having 2 cells, or a group having 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1 cell, a group having 2 cells, a group having 3 cells, a group having 4 cells, a group having 5 cells, etc.

[0038] Unless otherwise indicated by context, it is specifically intended that the various features of the invention described herein can be used in any combination. Further, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a certain composite includes components A, B, and C, it is specifically intended that any one of A, B, or C, or combinations thereof, can be omitted or disclaimed, either singly or in any combination.

[0039] Unless otherwise expressly indicated, all specific embodiments, features, and terms are intended to include the recited embodiments, features, or terms, and their biological equivalents.

[0040] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, unless they are inconsistent with the explicit teachings herein. Definitions

[0041] As used herein, the singular forms "a", "an", and "the" include both singular and plural forms unless it is clearly stated otherwise.

[0042] Also, as used herein, "and / or" is interpreted to include not only any and all possible combinations of one or more of the associated listed items, but also the absence of a combination when interpreted as an alternative ("or").

[0043] Even when not expressly stated, all numerical designations are preceded by the terms "about" or "approximately". The terms "about" or "approximately" are intended to mean that the numbers included are not limited to the exact numbers specified in this specification, but rather are numbers that are near the substantially recited numbers without departing from the scope of the present invention. As used herein, "about" or "approximately" is understood by those skilled in the art and varies to some extent in the context in which it is used. If the use of this term is not clear to those skilled in the art considering the context in which it is used, "about" or "approximately" means up to plus or minus 10%, 5%, 1%, or 0.1% of the particular term at most.

[0044] As used herein, "not containing" or "not containing at all" means that the substance cannot be detected within the detection range of the device or process used, or its presence cannot be confirmed.

[0045] As used herein, "essentially not containing" means that only a very small amount of the substance can be detected. In the present disclosure, "essentially not containing" means that the amount of the substance is less than 0.1% by weight of the whole composition, preferably less than 0.01% by weight, and most preferably less than 0.001% by weight.

[0046] As used herein, "substantially not containing" means that the amount of the substance is less than 5% by weight of the whole composition, preferably less than 2% by weight, and most preferably less than 1% by weight.

[0047] As used herein, "botulinum toxin" means, in addition to the neurotoxin produced by Clostridium botulinum, botulinum toxin (or its light chain or heavy chain) recombinantly produced by non-Clostridium species. As used herein, "botulinum toxin" includes types A, B, C, D, E, F, and G. "Botulinum toxin" also includes "modified botulinum toxin".

[0048] As used herein, "botulinum toxin complex" or "toxin complex" encompasses complexes released by Clostridium bacteria that contain botulinum toxin protein molecules (about 150 kDa in all serotypes) and one or more associated non-toxin proteins. These complexes (e.g., having molecular weights of about 300 kDa, 500 kDa, or 900 kDa) are thought to contain non-toxin hemagglutinin proteins ("NTH proteins") and non-toxin non-hemagglutinin proteins ("NTNH proteins"). Thus, a botulinum toxin complex may contain a botulinum toxin molecule (the neurotoxic component) and one or more NTH proteins and / or NTNH proteins. These two types of non-toxin proteins can stabilize the toxin molecule against denaturation and protect the toxin molecule from digestive acids when the toxin is ingested. Also, larger (300 kDa or greater) botulinum toxin complexes can disperse more slowly from the intramuscular injection site compared to botulinum toxin proteins.

[0049] As an example of a toxin complex, botulinum toxin type A complex can be obtained by Clostridium bacteria in 900 kDa form, 500 kDa form, and 300 kDa form. Botulinum toxin types B and C1 are obtained as 500 kDa complexes. Botulinum toxin type D is obtained as 300 kDa and 500 kDa complexes. Finally, botulinum toxin types E and F are obtained as complexes of about 300 kDa.

[0050] Referring to botulinum neurotoxin type A1, when the pH is above about 7, it is known that the non-toxin protein dissociates from the botulinum toxin protein molecule (about 150 kDa). Therefore, these toxin complexes can be dissociated into botulinum toxin protein and hemagglutinin protein by performing a separation step such as column chromatography in a suitable buffer with a pH of about 7 to 8. However, it is known that the botulinum toxin protein is unstable when removing the NTH hemagglutinin protein and / or the NTNH hemagglutinin protein. When the pH and temperature increase, or as a result of the surface expanding or contracting or drying (for example, during lyophilization or precipitation), the toxin loses its toxicity. Furthermore, in the absence of a stabilizer, the toxin loses its specific activity during dilution (for example, during culture, fermentation, and purification).

[0051] As used herein, "modified botulinum toxin" means a botulinum toxin in which at least one amino acid has been deleted, modified, or substituted compared to the native botulinum toxin. Also, the modified botulinum toxin can be a recombinantly produced neurotoxin, or a derivative or fragment of a recombinantly produced neurotoxin. The modified botulinum toxin retains at least one biological activity of the native botulinum toxin, for example, the ability to bind to the botulinum toxin receptor, or the ability to inhibit the release of neurotransmitters from nerves. An example of a modified botulinum toxin is a botulinum toxin having a light chain derived from one botulinum toxin serotype (for example, serotype A) and a heavy chain derived from a different botulinum toxin serotype (for example, serotype B). Thus, the modified botulinum toxin may contain a light chain and a heavy chain derived 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 a botulinum toxin that binds to neurotransmitters.

[0052] As used herein, "purified botulinum toxin", "pure toxin", "free botulinum toxin", "free toxin", or "botulinum toxin protein" is a botulinum toxin isolated or substantially isolated from other proteins including the NTH protein and / or NTNH protein that form the botulinum toxin complex. The purity of the purified botulinum toxin may be greater than 95%, preferably greater than 99%.

[0053] As used herein, "medium" or "fermentation medium" means any medium for culturing bacteria, whether a growth medium for preparing a seed culture used to inoculate a production medium or a production medium in which the bacteria grow to produce toxin.

[0054] As used herein, "animal product free" ("APF"), "essentially animal product free", or "substantially animal product free" each encompasses "animal protein free", "essentially animal protein free", or "substantially animal protein free", and means the absence, essential absence, or substantial absence of blood-derived, blood pool, and other animal-derived products or compounds. In this context, "free", "essentially free", and "substantially free" correspond to the definitions given above. "Animal" means mammals (such as humans), birds, reptiles, fish, insects, spiders, and other animal species. "Animal" excludes microorganisms such as bacteria. Thus, an APF medium or process, or a substantially APF medium or process, within the scope of the present invention can include botulinum toxin or Clostridium botulinum bacteria. For example, an APF process or a substantially APF process means a process that does not contain, or substantially does not contain, for example, immunoglobulins, human albumin, meat digests, meat by-products, and milk or dairy products, or milk digests. Thus, an example of an APF process is a process excluding meat and dairy products or meat or dairy products (e.g., a bacterial culture process or a bacterial fermentation process).

[0055] As used herein, "bioburden" means bacteria living on a non-sterilized surface, inside an apparatus, or in a solution. For example, embodiments of the present technology include filtering a buffer solution to reduce "bioburden", i.e., bacteria living in the buffer solution or bacteria transferred from a surface in contact with the solution (e.g., the surface of a glassware) to the solution.

[0056] As used herein, "tangential flow filtration" and "TFF" refer to a form of filtration useful for clarifying, concentrating, or purifying biological materials (e.g., proteins). In TFF, a solution or suspension containing macromolecules or biological materials may be pumped tangentially along the surface of a membrane. When pressure is applied, a portion of the solution can be pushed out through the pores of the membrane. In this specification, this solution is referred to as the "permeate" (or "filtrate"). Macromolecules, biological materials, and particles that are too large to pass through the pores of the membrane can be retained on the upstream side. In this specification, this solution is referred to as the "retentate". Compared to conventional filtration methods, the retained material does not accumulate on the surface of the membrane. Instead, it can be flowed along the membrane surface by 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.

[0057] As used herein, "permeate" refers to a solution, suspension, or their components that cross a filter or membrane (e.g., a diafiltration membrane, a tangential flow filtration membrane, an ultrafiltration membrane, a microfiltration membrane, or a hollow fiber filter) by passing through the pores of the filter or membrane, and also refers to a solution that has already crossed or passed through the filter or membrane. Generally, solvent molecules and solute molecules smaller than the pore size of the filter or membrane cross the filter or membrane, while molecules larger than the pore size do not cross the filter or membrane.

[0058] As used herein, "toxin-containing permeate" refers to a permeate that contains botulinum toxin molecules when the pore size of the filter is larger than the botulinum toxin molecules to such an extent that it crosses the filter.

[0059] As used herein, "retentate" refers to a solution, suspension, or their components that do not cross the filter or membrane. For example, in the case of tangential flow filtration, the retentate is a solution or suspension component or a part thereof that does not cross the filter or membrane but flows tangentially along it. Generally, molecules larger than the pore size of the filter or membrane do not cross the filter or membrane.

[0060] As used herein, "toxin-containing retentate" refers to a retentate that contains botulinum toxin molecules when the pore size of the filter is smaller than the botulinum toxin molecules to such an extent that it cannot cross the filter.

[0061] As used herein, "membrane differential pressure" or "TMP" refers to the differential gradient of the pressure applied along the length of the filtration membrane to cause a fluid and filterable solutes to pass through or cross the filter or membrane.

[0062] As used herein, "diafiltration" refers to a special type of filtration in which the retention fluid is diluted with a solvent and refiltered to reduce the concentration of soluble permeate components. Diafiltration may or may not increase the concentration of retention components including proteins (e.g., BoNT / A). For example, in continuous diafiltration, the solvent is continuously added to the retention fluid at the same rate at which the permeate is produced. In this case, the volume of the retention fluid and the concentration of the retained components do not change during the process. On the other hand, in discontinuous diafiltration or continuous dilution diafiltration, the solvent is added to the retention fluid side after the filtration step. If the volume of the solvent added to the retention fluid side is less than the volume of the produced permeate, the concentration of the retained components will be higher than the concentration 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-library / non-gated / 02.0629_Buffer_Exchange_STR.pdf (last accessed December 9, 2019).

[0063] As used herein, "diafiltration volume" or "DV" refers to the total volume exchanged during the process of diafiltration. One DV is equal to the volume of the retention fluid at the start of diafiltration. For example, if the volume of the original solution is 1 liter and a volume of permeate approximately equal to 1 liter is obtained by the diafiltration process and the 1L volume of the retention fluid is maintained or restored (e.g., using a buffer solution), the original solution or suspension has been filtered or washed with one DV. Multiple DVs can be exchanged by continuous diafiltration. For example, if the volume of the original retention fluid is 1 liter and a volume of permeate approximately equal to 5 liters is obtained by the diafiltration process, the original solution or suspension has been filtered or washed with five DVs.

[0064] As used herein, "microfiltration" typically refers to filtration of the like using membranes having pore sizes in the range of about 0.1 μm to about 10 μm and greater. See, e.g., Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998).

[0065] As used herein, "ultrafiltration" typically refers to filtration of the like using membranes having pore sizes in the range of about 0.1 μm to about 0.01 μm and less. Alternatively, the nominal membrane pore size expressed in molecular weight may be, for example, about 30 kDa or less to about 750 kDa or less, preferably 50 kDa or less, or 30 kDa or less. This may refer to any technique of passing a solution or suspension through a semipermeable membrane that allows the solvent and small solute molecules to pass through but retains macromolecules. Ultrafiltration may be used to concentrate macromolecules (e.g., proteins such as BoNT / A) in a solution or suspension. See, e.g., Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998).

[0066] As used herein, "chromatography" or "chromatographic separation" refers to a physical separation method in which the components to be separated (e.g., proteins) are partitioned between two phases, a stationary phase and a mobile phase. The molecules to be separated are dissolved in the mobile phase and move through the stationary phase (e.g., porous gel, charged polymer beads, etc.). Separation is possible because different molecules in the sample exhibit different affinities for the stationary phase and similar molecules are separated. Molecules with a high affinity for the stationary phase tend to move more slowly through the stationary phase compared to those with a low affinity. When applied to proteins (e.g., botulinum toxin), proteins can be separated by chromatographic separation based on many different properties. For example, in gel filtration chromatography, proteins in the mobile phase are separated based on their particle size. This is because proteins of different particle sizes move through the porous stationary phase, and small proteins are trapped in the stationary phase and move more slowly. In ion exchange chromatography, proteins are separated based on their charge, and an interaction due to Coulomb force with the stationary phase is obtained.

[0067] As used herein, "chromatography column" or simply "column" refers to a component that contains a chromatography matrix (e.g., stationary phase or solid phase) and is configured such that a mobile phase (e.g., fluid sample or buffer) can pass through it and pass through the stationary phase retained in the column. Non-limiting examples of such columns include those commercially available from G.E. Healthcare. See Chromatography Products: Chromatography columns, systems, resins, and buffer management solutions, G.E. HEALTHCARE, https: / / www.gelifesciences.com / en / us / shop / chromatography (last accessed December 9, 2019).

[0068] As used herein, "fraction" refers to a portion of the mobile phase that is recovered as it exits the column. The components of a "fraction" differ based on the time at which they are recovered. A "fraction" that moves quickly is recovered at an earlier time and contains a relatively high concentration of molecules that move more quickly through the stationary phase. A "fraction" that moves slowly is recovered at a later time and contains a relatively high concentration of molecules that move more slowly through the stationary phase.

[0069] As used herein, "toxin-containing fraction" means a fraction in which botulinum toxin molecules (e.g., BoNT / A molecules) in the mobile phase are recovered from a chromatography column as they exit the column.

[0070] As used herein, "ion exchange chromatography" or "IEX" refers to a separation technique by chromatography that separates molecules based on the polarity and magnitude of their charge (e.g., +2, +1, neutral, -1, -2). IEX retains analyte molecules (e.g., proteins) on the stationary phase based on the degree to which they interact with the stationary phase via Coulombic forces. The surface of the stationary phase presents ionic functional groups that interact with analyte (e.g., botulinum toxin) ions having an opposite charge. To achieve electrical neutralization, these static charges interact with exchangeable counterions in the mobile phase. Analyte molecules compete with these exchangeable counterions for binding. Analyte molecules are retained or "eluted" based on their charge. First, molecules that are not bound or are weakly bound to the stationary phase are flushed away. See, e.g., Ion Exchange Chromatography: Principles and Methods, G.E. HEALTHCARE (2016), https: / / cdn.gelifesciences.com / dmm3bwsv3 / AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13101 (last visited Dec. 9, 2019).

[0071] As used herein, "anion exchange chromatography" or "AIEX" means ion exchange chromatography in which anionic analyte molecules (e.g., proteins) are retained on a cationic stationary phase. See, e.g., Ion Exchange Chromatography: Principles and Methods, G.E. HEALTHCARE (2016), https: / / cdn.gelifesciences.com / dmm3bwsv3 / AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13101 (last visited Dec. 9, 2019).

[0072] As used herein, "cation exchange chromatography" or "CIEX" refers to ion exchange chromatography in which cationic analyte molecules (e.g., proteins) are retained on an anionic stationary phase. See, e.g., Ion Exchange Chromatography: Principles and Methods, G.E. HEALTHCARE (2016), https: / / cdn.gelifesciences.com / dmm3bwsv3 / AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13101 (last visited Dec. 9, 2019).

[0073] As used herein, "elute" means to desorb molecules that were bound to the stationary phase by changing the solution conditions within the chromatography column. The concentration of exchangeable counterions can be increased, or the pH can be changed to affect the binding affinity of the analyte. Molecules that have lost their affinity for the stationary phase and entered the mobile phase "elute" from the column.

[0074] As used herein, "eluate" or "washing solution" refers to an agent, typically a solution, that is used to alter the adsorption of an analyte (e.g., a botulinum toxin molecule) to a stationary phase and / or to remove unbound material from the stationary phase. The elution characteristics of an eluate can depend on, among other factors, for example, pH, ionic strength, and detergent strength.

[0075] As used herein, "eluate" refers to a solution (e.g., a washing solution or buffer solution) that contains unbound material (including "eluted" or desorbed analyte molecules, e.g., botulinum toxin molecules) that has migrated through the stationary phase and exited the column in a chromatographic separation.

[0076] As used herein, "toxin-containing eluate" refers to the mobile phase containing eluted botulinum toxin molecules that have exited a column used for chromatographic separation.

[0077] As used herein, "gel filtration chromatography" or "gel filtration" means size exclusion chromatography that can be used to fractionate molecules (e.g., proteins, protein complexes, polysaccharides, nucleic acids, small molecules, etc.) in a sample into fractions having specific ranges of particle sizes. Alternatively, "gel filtration" can remove all molecules larger than a specific cut-off particle size from a sample. In a gel filtration chromatography column, the stationary phase contains a porous matrix (e.g., beads), and the mobile phase is a solution (e.g., a buffer solution) that flows around the matrix. The matrix may have a defined range of pore sizes known as the "fractionation range". Molecules and complexes that are too large to enter the pores remain in the mobile phase and move through the column with the buffer solution. Small molecules and complexes that can move through the pores enter the stationary phase and move through the gel filtration column via a longer path (i.e., through the pores rather than around the beads). Molecules that can enter the stationary phase are fractionated by particle size. Smaller molecules move through the pores and move more slowly than larger molecules that cannot easily enter the pores. Thus, larger molecules are eluted more quickly. Therefore, sample components that exceed the fractionation range are eluted before components within the fractionation range. Generally, see, e.g., Size Exclusion Chromatography: Principles and Methods, G.E. HEALTHCARE (2018), https: / / cdn.gelifesciences.com / dmm3bwsv3 / AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=11639 (last accessed December 9, 2019).

[0078] As used herein to refer to components of a chromatography system, "single-use" refers to components that are configured to be replaced or discarded after each use and are not intended to be reused in that system. Fermentation medium

[0079] 1, a method 100 for purifying a botulinum toxin can include step 104 of obtaining a solution (e.g., a fermentation medium) containing a botulinum toxin (e.g., BoNT / A). In some embodiments, the solution can be a fermentation medium, preferably a supernatant from a fermentation medium containing intact C. botulinum cells, lysed bacteria, medium nutrients (e.g., vegetable peptones), and fermentation by-products. In some embodiments, the fermentation medium can be substantially free, essentially free, or free of animal products, such as the fermentation medium described in co-pending U.S. Provisional Patent Application No. 62 / 951,549 (i.e., an "APF" fermentation medium).

[0080] The botulinum toxin may be isolated and purified from the fermentation medium using protein purification methods well known to those skilled in the art of protein purification. See generally, e.g., 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).

[0081] As described herein, the disclosed purification methods may include purifying a botulinum toxin complex (e.g., a 900 kDa complex) that is more stable than the 150 kDa botulinum toxin protein molecule, and then separating and purifying the toxin protein molecule from non-toxin proteins (i.e., NTH protein and / or NTNH protein) to obtain a purified botulinum toxin (about 150 kDa) product without precipitation, centrifugation, or lyophilization steps. The product toxin solution may be free, essentially free, or substantially free of toxin complex and / or animal products. Furthermore, since no precipitation, centrifugation, or lyophilization steps are required, the botulinum toxin can be recovered in solution. In contrast, a powder must be reconstituted by the end user prior to administration to a patient. Filtration Filtration 1

[0082] Referring further to FIG. 1, the method may include a first filtration (“Filtration 1”) 106. The first filtration may include filtering a medium or culture solution to remove intact bacteria or lysed bacteria, spores (e.g., C. botulinum spores), and debris, to obtain a toxin-containing permeate 107. The toxin-containing permeate 107 contains botulinum toxin and various impurities, and may be processed to obtain a concentrated botulinum toxin (e.g., BoNT / A).

[0083] In certain embodiments, the first filtration 106 may include removing intact C. botulinum cells or lysed C. botulinum cells (or components thereof) from the fermentation medium using any suitable filtration technique (e.g., diafiltration, tangential flow microfiltration, tangential flow ultrafiltration, hollow fiber filtration, etc.). Filtration techniques 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 / pall / laboratory / literature-library / non-gated / id-34212.pdf (last accessed Dec. 9, 2019); Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998). In one embodiment, the first filtration 106 includes tangential flow microfiltration.

[0084] Depending on the embodiment, the first filtration step 106 may use a filter (e.g., a hollow fiber filter, a tangential flow filtration membrane, etc.). Here, at least the solvent and the botulinum toxin molecule or toxin complex pass through the filter to obtain a toxin-containing permeate 107. If there are intact cells or lysed cells, they do not pass through the filter and are retained in the retentate. Depending on the embodiment, the filter includes pores having a diameter between about 0.1 μm and 10 μm (e.g., about 0.2 μm). In one embodiment, the filter is a hollow fiber filter.

[0085] Depending on the embodiment, the first filtration step 106 may further include concentrating the toxin-containing permeate 107 by any suitable method (e.g., diafiltration) to recover additional botulinum toxin molecules from the retentate and / or the toxin-containing permeate 107. Filtration 2

[0086] Continuing to refer to FIG. 1, the method may further include a second filtration (“filtration 2”) 108 to remove fermentation broth residues (e.g., small peptides, carbohydrates, etc.) from the toxin-containing permeate 107. This step may include any suitable filtration technique (e.g., diafiltration, tangential flow microfiltration, tangential flow ultrafiltration, hollow fiber filtration, etc.). In one embodiment, the second filtration 108 for removing fermentation broth residues includes ultrafiltration using a tangential flow filter (e.g., a hollow fiber filter) having a pore size that allows the fermentation broth residues to pass through but retains the botulinum toxin molecule and / or toxin complex. For example, depending on the embodiment, the pore size of the filter may be 150 kDa or less, 100 kDa or less, or 50 kDa or less. In this case, the botulinum toxin molecule or botulinum toxin complex remains in the retentate obtained from the second filtration 108, and a clarified culture 110 is obtained that contains the botulinum toxin molecule or botulinum toxin complex but does not contain, essentially does not contain, or substantially does not contain intact C. botulinum cells or lysed C. botulinum cells (or components thereof) and fermentation broth residues (e.g., small peptides, carbohydrates, etc.).

[0087] Depending on the embodiment, the clarified culture 110 may be recovered and further purified in subsequent processing steps without precipitating the botulinum toxin molecule or toxin complex from the solution at any time during the purification process. As an advantage, this increases the overall process yield, preserves the toxin activity, and eliminates the need for the end user to reconstitute the lyophilized drug product. Column chromatography Separation by the first chromatography

[0088] Continuing to refer to FIG. 1, some embodiments of 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 complex from the nucleic acids (e.g., DNA and RNA) present in the clarified culture 110. The first chromatographic separation 112 may include any suitable chromatographic separation technique (e.g., ion exchange chromatography including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). Depending on the embodiment, the first chromatographic separation may include anion exchange chromatography (AIEX). In one embodiment, the first chromatographic separation includes AIEX on Q Sepharose.

[0089] The first chromatographic separation 112 may include contacting the first chromatographic column with the clarified culture 110 containing the botulinum toxin or toxin complex. The mobile phase (including the clarified culture 110) may flow through the first stationary phase to separate the botulinum toxin from other remaining impurities (e.g., nucleic acids). The first stationary phase may include any suitable chromatographic matrix (e.g., an agarose bead-based medium such as Q Sepharose FF (GE Healthcare)).

[0090] In certain embodiments, the clarified culture 110 may be adjusted for column chromatography (e.g., by dilution in a buffer solution or buffer exchange). In a particular embodiment, the clarified culture 110 may be prepared in a phosphate buffer having a pH of about 7.5 or less, preferably about 7 or less, more preferably about 6.1 in a phosphate buffer. In some embodiments, the clarified culture 110 may be adjusted to 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.

[0091] In certain embodiments, the separation by the first chromatography 112 may further include washing the botulinum toxin or toxin complex through the stationary phase using a suitable buffer solution (e.g., a phosphate buffer having a pH of 6.1). The purpose of this step is to separate the botulinum toxin complex from other proteins and remove nucleic acids (RNA and DNA), while washing the botulinum toxin complex through the column as much as possible. In some embodiments, the salt concentration of the buffer solution is selected such that the amount of botulinum toxin or toxin complex flowing through the column is maximized and the amount of other proteins flowing through the column is minimized.

[0092] In some embodiments, this buffer solution contains sodium chloride (NaCl) at a concentration of about 15 mM or more, about 20 mM or more, about 30 mM or more, about 40 mM or more, about 50 mM or more, about 60 mM or more, about 70 mM or more, about 80 mM or more, about 90 mM or more, about 100 mM or more, about 150 mM or more, about 200 mM or more, about 250 mM or more, about 300 mM or more, about 350 mM or more, about 400 mM or more, about 450 mM or more, or about 500 mM or more (and ranges therebetween). In some embodiments, this 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 one embodiment, this buffer solution contains NaCl at a concentration of about 150 mM.

[0093] In a particular embodiment, the first chromatography column may operate in a flow-through fractionation mode, and the botulinum toxin and / or toxin complex may pass through the column without adsorbing to the stationary phase. In this configuration, it is not required to elute the botulinum toxin or toxin complex from the column. Instead, the botulinum toxin or toxin complex may exit the column contained in the toxin-containing fraction 114, and the toxin-containing fraction 114 may be recovered and further purified in subsequent processing steps. In some embodiments, the recovery of the toxin-containing fraction 114 is monitored by detecting the absorbance of the flow-through fraction at 280 nm ( 280 "A 280 "), and the toxin-containing fraction 114 is recovered while the peak of A Separation by the second chromatography

[0094] Some embodiments of method 100 may further include a separation step 116 by a second chromatography that purifies the toxin-containing fraction 114 to remove bulky impurities (e.g., other proteins) to obtain a first toxin-containing eluate 118. The separation 116 by the second chromatography may include any suitable chromatographic separation technique (e.g., ion exchange chromatography including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). In a particular embodiment, the separation 116 by the second chromatography includes cation exchange chromatography (CIEX).

[0095] The separation 116 by the second chromatography may include contacting the botulinum toxin or toxin complex with a second chromatography column, and the mobile phase containing the toxin-containing fraction 114 moves through the second stationary phase. The second stationary phase may include any suitable chromatographic matrix (e.g., an agarose bead-based medium such as SP Sepharose FF (GE Healthcare), etc.). In a plurality of embodiments, the botulinum toxin molecule or toxin complex may bind to the second stationary phase. In a plurality of embodiments, the botulinum toxin or toxin complex may bind to the second stationary phase, and the A 280 of the solution for washing through the column is monitored. In a plurality of embodiments, until A 280 returns to the baseline value (which indicates that all of the toxin-containing eluate 118 has passed through the column, but the botulinum toxin and toxin complex remain bound to the second stationary phase), the column is washed with a suitable buffer solution (e.g., 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5).

[0096] In some embodiments, the separation by the second chromatography 116 may further include washing the second chromatography column with a washing buffer to remove any weakly bound proteins, while leaving the botulinum toxin and toxin complex bound to the second stationary phase. This washing step may use any suitable buffer solution (e.g., 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5, 210 mM NaCl). In some embodiments, this washing 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. In some embodiments, this washing 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.

[0097] In some embodiments, the separation by the second chromatography 116 may further include conditioning the toxin-containing fraction 114 for column chromatography (e.g., by dilution or buffer exchange) prior to contacting the toxin-containing fraction 114 with the second chromatography column. For example, in some embodiments, the toxin-containing fraction 114 may be conditioned in an acetic acid - acetate buffer having a pH between about 3 and about 7, between about 3.5 and about 6, or between about 4 and about 5, preferably about 4.5. In some embodiments, the toxin-containing fraction is conditioned in a buffer having a pH 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.

[0098] In some embodiments, the separation by the second chromatography 116 may further include eluting the botulinum toxin or toxin complex 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 promote dissociation of the toxin (or toxin complex) from the stationary phase (e.g., by changing the pH or ionic strength). This elution buffer may be any suitable buffer solution for promoting dissociation of the toxin or toxin complex from the second stationary phase (e.g., 50 mM sodium acetate, 0.2% polysorbate 20, pH 4.5). For example, such a buffer solution may include an acetic acid - acetate buffer with a pH between about 3 and about 7, between about 4 and about 6, between about 4 and about 5, or about 4.5. In some embodiments, the toxin-containing fraction is eluted from the second chromatography column using a buffer with a pH 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.

[0099] In some embodiments, this elution buffer may contain salt at a concentration sufficient to promote dissociation of the botulinum toxin or toxin complex from the second stationary phase. In multiple embodiments, this elution buffer contains NaCl at a concentration of 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, this 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 mM, about 390 mM, or about 400 mM, or any value between these.

[0100] In multiple embodiments, botulinum toxin molecules or toxin complexes are eluted from the column to obtain a first toxin-containing eluate 118. This first toxin-containing eluate may be recovered and further purified in subsequent processing steps. Depending on the embodiment, during elution from the second chromatography column, to detect the presence of botulinum toxin molecules or toxin complexes eluting from the column, A 280 is measured. The peak of A 280 is collected as one fraction to obtain the first toxin-containing eluate 118. Filtration 3

[0101] Some embodiments of the method further include a third filtration step 120 that is performed after the second chromatography-based separation 116 and before the third chromatography-based separation 124. In one embodiment, the third filtration 120 dissociates the NTH protein and / or NTNH protein from the botulinum toxin complex. In multiple embodiments, the third filtration 120 includes a buffer exchange. The buffer exchange may increase the pH of the first toxin-containing eluate 118 (e.g., from about 4.5 to about 8.0) and includes a buffer exchange. In multiple embodiments, the buffer exchange may reduce the salt concentration in the toxin-containing eluate 118 (e.g., from about 270 mM to about 50 mM). In multiple embodiments, the third filtration 120 may concentrate the toxin-containing eluate 118 (e.g., by reducing the volume from about 300 mL to about 50 - 60 mL).

[0102] The third filtration 120 may include any suitable filtration technique (e.g., diafiltration, tangential flow microfiltration, tangential flow ultrafiltration, hollow fiber filtration, etc.). In a particular embodiment, the third filtration 120 may include ultrafiltration using a tangential flow filter (e.g., a hollow fiber filter) having a pore size suitable for retaining dissociated botulinum toxin molecules (150 kDa) and the NTH protein and / or the NTNH protein. Depending on the embodiment, the filter may have a pore size of about 150 kDa or less, about 140 kDa or less, about 130 kDa or less, about 120 kDa or less, about 110 kDa or less, about 100 kDa or less, about 90 kDa or less, about 80 kDa or less, about 70 kDa or less, about 60 kDa or less, about 50 kDa or less, about 40 kDa or less, about 30 kDa, or about 20 kDa or less (or a range therebetween). For example, the tangential flow 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 any value therebetween. In one embodiment, the filter has a pore size of about 30 kDa.

[0103] Depending on the embodiment, the toxin-containing eluate 118 may be concentrated by ultrafiltration and then washed using diafiltration against a buffer solution to dissociate the NTH protein and / or the NTNH protein from the botulinum toxin complex. In a particular embodiment, the botulinum toxin protein molecules and the NTH protein and / or the NTNH protein may remain in the retention liquid 121 (i.e., the toxin-containing retention liquid), while other filtration residues and chromatography media residues are separated and recovered in the permeate.

[0104] The buffer solution used for the third filtration 120 may be any suitable buffer solution (e.g., Tris-HCl buffer) for dissociating the NTH protein and / or NTNH protein in the toxin complex from the botulinum toxin molecule. In some embodiments, the buffer solution used for the third filtration 120 may be the same buffer solution (e.g., Tris-HCl buffer with a pH above 7 as described below) used to condition the third chromatography column.

[0105] In some embodiments, the buffer solution used for the third filtration 120 has a pH suitable for dissociating the NTH protein and / or NTNH protein from the botulinum toxin protein molecule. In some embodiments, the pH of the buffer solution is at least about 7, preferably between about 7 and about 10, preferably between about 7 and about 9, preferably between about 7.5 and about 8.5, preferably about 8.0. In some embodiments, the pH of the buffer solution for the third filtration 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 about 10.0. In one embodiment, the pH of the buffer solution for the third filtration 120 is about 8.0.

[0106] In some embodiments, the toxin-containing retention solution 121 obtained from the third filtration 120 may be recovered and further purified in a subsequent processing step without precipitating the botulinum toxin molecule or toxin complex from the solution at any point during the purification process. As an advantage, this increases the overall process yield and eliminates the need to reconstitute the lyophilized drug product at any point during the purification process or by the end user. Separation by the third chromatography

[0107] Some embodiments of method 100 further include a third chromatographic separation 124 that separates and removes the NTH protein and / or NTNH protein dissociated from the toxin complex in the third filtration 120 while retaining the botulinum toxin protein molecule. The resulting second toxin-containing eluate 126 may contain free botulinum toxin protein molecules (about 150 kDa), may not contain, essentially not contain, or substantially not contain the toxin complex. The third chromatographic separation 124 may include any suitable chromatographic separation technique (e.g., ion exchange chromatography including anion exchange chromatography or cation exchange chromatography, gel filtration chromatography, high performance liquid chromatography, affinity chromatography, etc.). In a particular embodiment, the third chromatographic separation 124 may include anion exchange chromatography (AIEX).

[0108] The third chromatographic separation 124 may include contacting the botulinum toxin or toxin complex in the toxin-containing retention fluid 121 obtained from the third filtration 120 with a third chromatographic column. Here, the toxin-containing retention fluid 121 (and the mobile phase containing free botulinum toxin protein molecules therein) moves through the third stationary phase. The third stationary phase may include any suitable chromatographic matrix (e.g., an agarose bead-based medium such as Q Sepharose FF (GE Healthcare), etc.).

[0109] Depending on the embodiment, the separation 124 by the third chromatography may further include adjusting the toxin-containing retention solution 121 obtained from the third filtration 120 for column chromatography (e.g., by dilution, buffer exchange, filtration, or a combination thereof). For example, the toxin-containing retention solution 121 may be diluted in a buffer solution such as a Tris-HCl buffer with a pH greater than 7, preferably between 7 and 10, preferably between about 7 and about 9, preferably between about 7.5 and about 8.5, or preferably about 8.0. Depending on the embodiment, 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.

[0110] Depending on the embodiment, the botulinum toxin protein molecules, toxin complexes, and NTH protein and / or NTNH protein in the toxin-containing retention solution 121 may adsorb to the third stationary phase. Depending on the embodiment, the toxin-containing retention solution 121 is packed into the third chromatography column and washed with a washing buffer solution to remove non-toxin impurities that are not bound to the third stationary phase. The washing buffer may be any suitable buffer solution (e.g., 20 mM Tris / HCl, 50 mM NaCl, 0.2% polysorbate 20, pH 8.0), and A 280 of the solution passing through the column may be monitored until it reaches the baseline value (which means that all unbound material has flowed through the third chromatography column). 280 of may be monitored.

[0111] The third chromatographic separation 124 may further include eluting the botulinum toxin protein bound to the third stationary phase to obtain a second toxin-containing eluate 126. The elution may include washing the column with one or more buffer solutions using any suitable protein-compatible buffer solution to desorb the botulinum toxin protein molecules from the stationary phase (e.g., by changing the pH, ionic strength, etc.). For example, a Tris-HCl buffer solution with a pH greater than 7, preferably between 7 and 10, preferably between about 7 and about 9, preferably between about 7.5 and about 8.5, or preferably about 8.0 may be used to elute the botulinum toxin molecules from the third stationary phase. 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.

[0112] Depending on the embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column may contain a salt (e.g., NaCl). According to an embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column contains NaCl at a concentration suitable for promoting the desorption of the botulinum toxin molecules from the third stationary phase. In a plurality of embodiments, this elution buffer may contain NaCl at a concentration between about 25 mM and about 250 mM, preferably between about 50 mM and about 200 mM, preferably between about 100 mM and about 150 mM, preferably about 120 mM. According to an embodiment, the elution buffer used to elute the 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 one embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column contains about 120 mM or about 150 mM of NaCl.

[0113] Depending on the embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column further contains a surfactant (e.g., polysorbate 20). Depending on the embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column contains a surfactant at a concentration of about 0.05 vol% to about 1.0 vol%, preferably about 0.10 vol% to about 0.5 vol%, preferably about 0.15 vol% to about 0.25 vol%, preferably about 0.20 vol%. Depending on the embodiment, the buffer solution used to elute the 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 one embodiment, the buffer solution used to elute the botulinum toxin molecules from the third chromatography column contains polysorbate 20 at a concentration of about 0.20 vol%.

[0114] In certain embodiments, without precipitating or lyophilizing the botulinum toxin protein, the second toxin-containing fraction 126 is eluted from the column in subsequent processing steps and recovered while being further purified. In one embodiment, the second toxin-containing fraction 126 is directly injected into a fourth chromatography column for finishing (i.e., removing high molecular weight contaminants including aggregates of the target protein, low molecular weight contaminants including fragments of the target protein, and other impurities that may not have been removed in the previous purification steps). Separation by the fourth chromatography

[0115] Some embodiments of method 100 further include a finishing step of removing aggregates and / or protein impurities 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 technique (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 one embodiment, the fourth chromatographic separation 128 may include gel filtration chromatography.

[0116] 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 chromatographic separation 128 may use a Superdex 200 chromatography solvent (GE Healthcare).

[0117] The fourth chromatographic separation 128 may further include adjusting the second toxin-containing eluate 126 for column chromatography (e.g., by dilution, buffer exchange, filtration, or a combination thereof). Alternatively, in one embodiment, the second toxin-containing eluate 126 obtained from the third chromatographic separation 124 may be directly injected into the fourth chromatographic column without further adjustment. In this configuration, the third and fourth chromatographic columns may be interconnected so that the second toxin-containing eluate 126 can be directly loaded.

[0118] The fourth chromatographic separation 128 may further include washing the botulinum toxin molecules through the stationary phase using a washing solution or buffer to obtain the third toxin-containing eluate 130. This buffer solution may be any suitable protein-compatible buffer. In certain embodiments, this buffer solution may be an acetic acid - acetate buffer solution having a pH of less than about 7, preferably between about 5 and about 7, preferably between about 6 and about 7, preferably between about 6.6 and about 6.9. In some embodiments, the pH of this buffer solution may be about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0119] In some embodiments, the buffer solution used to wash the botulinum toxin molecules through the stationary phase in the fourth chromatographic column further contains a salt (e.g., NaCl). In some embodiments, this washing buffer solution contains NaCl at a concentration of about 100 mM to about 1 M, preferably about 200 mM to about 600 mM, more preferably about 300 mM to about 500 mM, and most preferably about 400 mM. In some embodiments, this buffer solution contains NaCl at 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 1 M. In one embodiment, the buffer solution used to wash the botulinum toxin molecules through the stationary phase in the fourth chromatographic column contains about 350 mM or about 370 mM of NaCl.

[0120] Depending on the embodiment, the buffer solution used to wash the botulinum toxin molecules through the stationary phase of the fourth chromatography column further contains a surfactant (for example, polysorbate 20). Depending on the embodiment, this buffer solution contains the surfactant at a concentration of about 0.05 vol% to about 1.0 vol%, preferably about 0.10 vol% to about 0.5 vol%, preferably about 0.15 vol% to about 0.25 vol%, preferably about 0.20 vol%. Depending on the embodiment, this buffer solution contains the 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 one embodiment, the buffer solution used to wash the botulinum toxin molecules through the stationary phase of the fourth chromatography column contains polysorbate 20 at a concentration of about 0.20 vol%.

[0121] Depending on the embodiment, the presence of free botulinum toxin molecules in the solution passing through the column is monitored by A 280 In one embodiment, the peak of A 280 (indicating the presence of free botulinum toxin molecules) is collected as one fraction to obtain the third toxin-containing eluate 130.

[0122] Depending on the embodiment, without precipitating or lyophilizing the botulinum toxin protein, the third toxin-containing eluate 130 may be eluted from the fourth chromatography column in subsequent processing steps and recovered while being diluted or further purified. Depending on the embodiment, the third toxin-containing eluate 130 may be stored at about 2 to 8 °C until post-purification processing. Depending on the embodiment, the third toxin-containing eluate 130 may be diluted, filtered, and dispensed to obtain a product toxin solution 134. Dilution, filtration, and distribution

[0123] In some embodiments, the method 100 further includes diluting, filtering, and dispensing 132 the third toxin-containing eluate 130 to obtain a product toxin solution 134. In some embodiments, the third toxin-containing eluate 130 may be diluted to a final concentration using any suitable buffer solution. In some embodiments, the diluent buffer solution may have substantially the same composition as the wash buffer used for the fourth chromatographic separation 128. For example, the diluent buffer solution may include an acetic acid-sodium acetate buffer having a pH of less than about 7, preferably between about 5 and about 7, preferably between about 6 and about 7, preferably between about 6.6 and about 6.9. In some embodiments, the pH of this buffer solution is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0124] The method 100 may further include filtering the third toxin-containing eluate 130 (or a diluted form thereof) to reduce bioburden and obtain a product toxin solution 134. Filtering the third toxin-containing eluate 130 may include any suitable filtration technique (e.g., diafiltration, tangential flow microfiltration, tangential flow ultrafiltration, hollow fiber filtration, etc.). In some embodiments, the filter may have a pore size between 0.1 μm and 10 μm (e.g., about 0.2 μm).

[0125] The product toxin solution 134 may be dispensed into a container (e.g., a cryovial) and transported and stored under conditions that preserve the efficacy of the botulinum toxin protein therein. For example, the product toxin solution 134 may be dispensed into a pre-cooled storage container (e.g., a vial housed in a pre-cooled aluminum block) and transported and / or stored.

[0126] Depending on the embodiment, distributing the product toxin solution 134 may include pumping the product toxin solution from a sterilization container (e.g., a single-use sterilization bag) into a primary storage container (e.g., a cryovial). Depending on the embodiment, for example, by storing, moving, and freezing the primary storage container within a pre-cooled aluminum block, the primary storage container may be pre-cooled and maintained at an appropriate temperature (e.g., 0 °C or lower). Thereby, the product toxin solution 134 is maintained at a temperature such that the botulinum toxin molecules are stable and retain their neurotoxicity. Also, this may eliminate the need to lyophilize the product toxin solution 134. The product toxin solution 134 may further be stored at a temperature of about 0 °C or lower, preferably about -70 °C or lower. Process yield

[0127] The overall yield of the process and the yield of each step may be calculated to evaluate the purification process. The yield may be calculated from the concentration of the toxin obtained for each fraction. The yield of each step (i.e., the "step yield") may be calculated by the following formula.

Number

[0128] The overall yield may be calculated using the following formula.

Number

[0129] In some embodiments, 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 any range or value therebetween. In some embodiments, 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%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, or any range or value therebetween.

[0130] In one embodiment, the purity of the drug substance may be determined from SDS-PAGE and / or HPLC-SEC. In one embodiment, the drug substance obtained from the process according to the present disclosure has a purity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (and ranges therebetween). In one embodiment, the purity of the drug substance may be 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 any range or value therebetween.

Example

[0131] Example 1: Chromatography method using four columns for purifying botulinum toxin As an example, an embodiment of the method may be performed according to the following description. Here, botulinum toxin A1 (about 150 kDa) is recovered from a fermentation medium that contains C. botulinum cells, botulinum toxin A1 protein molecules, and toxin complexes, and is substantially free, essentially free, or free of animal-derived products. Such an APF fermentation medium is described in U.S. Patent Co-Pending Provisional Application No. 62 / 951,549. The botulinum toxin herein is filtered and purified according to a process described in more detail below to obtain a product toxin solution that contains botulinum toxin protein molecules and is free, essentially free, or substantially free of toxin complexes without using a precipitation step or a lyophilization step. Preparation of buffer

[0132] As shown in Table 1, a plurality of different buffer solutions may be used in this embodiment of the method. In one embodiment, the buffer (to reduce bioburden) is passed through a 0.2 μm filter and placed in a single-use sterilized bag. The used filter is removed and the bag containing the buffer is stored at room temperature until use.

[0133] [Table 1]

[0134] In Table 1, the first chromatography column was equilibrated using Buffer 1 and 2. The clarified culture for column chromatography was prepared using Buffer 3. The botulinum toxin complex was washed through the first column in the flow-through fraction mode using Buffer 4. The second chromatography column was equilibrated using Buffer 5 and 6. The toxin-containing fraction obtained from the separation by the first chromatography was adjusted for the separation by the second chromatography using Buffer 7. Bulky impurities (e.g., proteins) were washed through the second chromatography column using Buffer 8. The bound botulinum toxin complex was eluted from the second chromatography column using Buffer 9. The NTH protein and / or NTNH protein was separated from the botulinum toxin protein molecule using Buffer 10 (i.e., the first toxin-containing eluate obtained from the separation by the second chromatography was prepared for the separation by the third chromatography). The third chromatography column was equilibrated using Buffer 11 and 12. Impurities were flushed out from the third chromatography column using Buffer 13. The bound botulinum toxin protein molecule was eluted from the third chromatography column using Buffer 14. The fourth chromatography column was equilibrated using Buffer 15. The botulinum toxin molecule was washed through the fourth chromatography column using Buffer 16. The toxin-containing eluate obtained from the separation by the fourth chromatography was diluted to its final concentration before being dispensed into the primary storage container to obtain the product toxin solution. Filtration

[0135] The C. botulinum fermentation medium was obtained by the APF preparation method described in U.S. Patent Co-Pending Provisional Application No. 62 / 951,549. After direct recovery, the culture (about 5 L) was diluted with about 280 mL of buffer solution (1 M sodium acetate, 4 M NaCl, pH 5.5) immediately before filtration to adjust the pH. The diluted solution was filtered by microfiltration (''Filtration 1'') using a 0.2 μm hollow fiber filter by tangential flow filtration to separate spores, intact C. botulinum cells, and lysed C. botulinum cells to obtain a clarified culture. The clarified culture was sampled for in-process control for the absence of C. botulinum. Then, the clarified culture was purified by removing fermentation medium residues (e.g., proteins, carbohydrates, etc.) by tangential flow ultrafiltration (''Filtration 2'') using a 50 kDa hollow fiber filter. Next, the clarified culture was buffer-exchanged in sodium phosphate buffer (pH about 6.1) to condition the clarified culture for column chromatography. This filtration process and other suitable protein filtration processes are known in the art. See, e.g., Munir Cheryan, Ultrafiltration and Microfiltration Handbook (2d ed. 1998). Purification

[0136] A four-column chromatography process has been developed to purify the BoNT / A1 protein molecule (about 150 kDa) without using a precipitation, lyophilization, or centrifugation step. This process results in a product toxin solution that is free of, essentially free of, or substantially free of botulinum toxin complex and / or animal-derived products. This increases the yield and eliminates errors by end-users associated with reconstitution procedures (which are known to reduce the effectiveness of botulinum toxin drug compositions).

[0137] This process protects the toxin using the botulinum toxin complex through a plurality of initial steps while removing proteases and other bulky contaminating proteins. The first chromatographic step is anion exchange chromatography, which removes most of the nucleic acids in the medium during recovery, as otherwise the nucleic acids would interfere with downstream chromatographic steps, thereby inhibiting the denaturation of botulinum toxin molecules during the purification process. This process then separates the botulinum toxin protein molecule from the NTH protein and / or NTNH protein to which it is associated, yielding a product toxin solution that contains, essentially contains, or substantially contains pure botulinum toxin molecules (about 150 kDa) and does not contain the botulinum toxin complex.

[0138] Also, all chromatographic steps used in this process are designed to be performed on single-use chromatography columns that are discarded after one purification procedure, thereby eliminating the costly and time-consuming column regeneration process faced by multi-use chromatography systems. The entire process is carried out using a closed system with single-use bags, tubes, filters, and chromatography columns. Further, preferably, since the process fluid is maintained within the bag, tube, filter, or column, the handling steps in an open system are avoided, protecting the product toxin solution from contamination and safeguarding the operator from exposure to the toxin.

[0139] Table 2 shows an overview of the four-column chromatographic purification method of BoNT / A1 according to the present disclosure. This process includes the following steps. (1) The clarified culture was directly loaded onto a Q Sepharose FF column (packed with 2.5 L in a column with a diameter of 80 mm and a height of 500 mm) in flow-through fractionation mode to separate the botulinum toxin complex from the nucleic acids using sodium phosphate buffer (pH 6.1). In this step, the botulinum toxin complex is not bound to the stationary phase; instead, it flows through the column and is included in the toxin-containing fraction. (2) The toxin-containing fraction from a Q Sepharose FF column (a column with a diameter of 50 mm and a height of 50 mm filled with 100 mL) was adjusted in sodium acetate buffer (pH 4.5) and passed through an SP Sepharose FF column using sodium acetate buffer (pH 4.5). In this step, the botulinum toxin complex was adsorbed to the stationary phase, while bulky impurities (e.g., other proteins) were washed through the column. The toxin complex bound to the column was eluted using sodium acetate buffer (pH 4.5) containing NaCl and recovered in the first toxin-containing eluate. (3) The first toxin-containing eluate was filtered to remove fermentation broth residues and adjusted in a suitable buffer solution for buffer exchange (Tris-HCl buffer, pH 8.0) by tangential flow ultrafiltration ( "filtration 3") using a 30 kDa filter to dissociate botulinum toxin molecules from NTH protein and / or NTNH protein in the toxin complex. Then, the retentate was passed through a Q Sepharose FF column (a column with a diameter of 5 mm and a height of 100 mm filled with 2 mL) to separate free botulinum toxin protein from NTH protein and / or NTNH protein. In this step, the botulinum toxin molecules were adsorbed to the stationary phase, while NTH protein and / or NTNH protein (and other bulky impurities) were washed through the column to some extent (however, most adsorbed more strongly to the stationary phase than the botulinum toxin molecules). The botulinum toxin protein was recovered in the second toxin-containing eluate using Tris-HCl buffer (pH 8.0) containing NaCl. (4) For finishing (i.e., removal of aggregates), the second toxin-containing eluate was directly injected into a Superdex 200 gel filtration column (a column with a diameter of 25 mm and a height of 600 mm filled with 320 mL). In this step, the column was washed with a buffer containing 50 mM sodium acetate, 370 mM NaCl, 0.2% polysorbate 20, 13 mM Na2HPO4, and 17 mM NaOH (pH 6.6 - 6.9), and the purified botulinum toxin protein was recovered in the third toxin-containing eluate.

[0140]

Table 2

[0141] Each of the above column chromatography steps may include on-site cleaning procedures and other column preparation steps prior to performing separation by chromatography. Column preparation and operating procedures are well known in the art. Generally, for example, see 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. DISORD. 20 (2000); Wang Y-c, DERM. LAS FACIAL COSMET. SURG. 58 (2002); Johnson et al., 32 PROTEIN EXPR. & PURIF. 1 (2003); US2003 / 0008367A1. Dilution / Filtration / Distribution

[0142] The purified botulinum toxin protein was diluted in a suitable buffer solution (e.g., 50 mM sodium acetate, 0.2% polysorbate 20, 370 mM NaCl, 13 mM sodium phosphate, 17 mM NaOH) to a final concentration and gently mixed on a platform rocker for about 30 minutes. The dilution was filtered using a 0.2 μm filter, placed in a single-use sterile bag, and dispensed into 1.8 mL cryovials in 0.4 mL aliquots using a dispensing pump and needle. The samples were then rapidly frozen using an aluminum block pre-cooled and stored at -70°C or below. Hereinafter, the final solution prepared for storage according to the above method is referred to as the "drug substance" (or "DS").

[0143] Three different drug substance lots: #16852, #17043, and #19139 were prepared according to the procedure outlined above. Subsequently, the DS lots were tested for appearance, potency, specific activity, and total protein concentration as described in the following examples.

[0144] Example 2: Appearance Test

[0145] Since opalescence in solution may indicate protein aggregation or precipitation, the transparency and color were tested to confirm that the drug substance is clear and colorless. The method is a visual method based on Ph.Eur. 2.2.1, entitled "Clarity and Degree of Opalescence of Liquids" and Ph.Eur. 2.2.2, entitled "Degree of Coloration of Liquids", but is modified to use the vial and volume of the drug substance instead of the container and volume specified in the pharmacopoeial method. Water is used as the reference solution.

[0146] By this method, all the drug substance lots prepared according to Example 1 were clear and colorless solutions. This indicates the absence of detectable aggregation or precipitation of botulinum toxin protein molecules. Example 3: Potency

[0147] Mouse LD 50 The potency of the purified drug substance obtained from Example 1 was determined using an assay. This is an absolute assay that quantitatively measures the potency in the sample being tested. Using gelatin-phosphate buffer as the diluent, 11 dosing groups were established around the target LD 50 value. Dosing groups with potencies of 3.0 to 0.4 units per dose were arranged at approximately logarithmic intervals of 0.0899. The number of deaths was recorded 72 hours after injection, and the LD 50 was calculated using the Spearman-Karber method. The calculation by the Spearman-Karber method is used as the arithmetic mean to determine the midpoint (LD 50 ) of the logarithmic dilution curve for death. The potency of the purified drug substance is LD50 It is expressed as units / mL. See, for example, M.A. Ramakrishnan, Determination of 50% Endpoint Titer Using a Simple Formula, 5 WORLD J. VIROL. 85 - 86 (2016); and G. Kaerber, 162 PATHOL. U PHARMAKOL. 480 - 83 (131); C. Spearman, The Method of “Right and Wrong Cases” (Constant Stimuli) Without Gauss’s Formula, 2 BR. J. PSYCHOL. 227 - 42 (1908).

[0148] Purified drug substance lots #16852, #17043, and #19139 each showed an efficacy value of 11×10 6 LD 50 units / mL, 23×10 6 LD 50 units / mL, and 19×10 6 LD 50 units / mL. These efficacy values, in combination with the volume of the DS produced, confirmed that the above - mentioned process yield is sufficient to produce high - dose drug products from DS lots that are desirable for the commercial manufacture of the DS. Example 4: Total Protein Concentration

[0149] The total protein concentration of the drug substance prepared according to Example 1 was determined according to the bicinchoninic acid (BCA) method. This is because this method has a sensitivity high enough to determine the protein concentration of a typical drug substance lot prepared according to Example 1. See Ph.Eur. 2.5.33 method 4, and USP <507> method II.

[0150] Determine the total protein concentration using a micro BCA protein assay kit, and calculate the protein concentration from the absorbance measured using a standard curve prepared with bovine serum albumin. The results are reported as the average value by measuring two prepared samples of the same.

[0151] For purified drug substance lots #16852, #17043, and #19139, the total protein concentrations were determined to be 59.4 μg / mL, 107.0 μg / mL, and 91.4 μg / mL, respectively. These protein concentration values obtained from the above DS lots are high enough to ensure that the analytical methods for release and characterization are accurate and precise. Example 5: Specific activity

[0152] For the drug substance manufactured according to Example 1, LD 50 Calculate the specific activity from the efficacy value (units / mL) of the drug substance obtained in the assay (Example 3) and the total protein concentration (mg / mL) of the drug substance (Example 4). The specific activity is calculated according to the following formula.

Equation

[0153] For purified drug substance lots #16852, #17043, and #19139, the specific activities were determined to be 1.9×10 8 U / mg, 2.2×10 8 U / mg, and 2.1×10 8 U / mg, respectively. This confirmed that the DS has high purity. This indicates that the above process is suitable for the commercial production of highly purified and sufficiently active complex-free botulinum toxin. Example 6: Protein-related impurities

[0154] 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, titled "Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) - Uniform Percentage Gels". In this method, SDS-PAGE is used in combination with colloidal Coomassie blue staining. The sample is diluted to prepare a standard curve, and non-reduced and reduced samples are analyzed. By correlating the band intensity of the impurity band with the standard curve, the impurities are quantified by densitometry. The results of the impurities are expressed as a percentage of the total protein amount loaded on the gel.

[0155] Figure 2 shows the purity analysis results obtained using SDS-PAGE and colloidal Coomassie blue staining for DS Lot #17043 prepared according to Example 1. From left to right, lanes 1-5 are non-reduced samples of the standard curve (1.2 - 4.0 μg / mL) from Lot #17043, 6-7 are the same samples 1 and 2 (140 μg / mL) of non-reduced Lot #17043, 8 is the molecular weight marker, and 9-10 are the same samples 1 and 2 (140 μg / mL) of reduced Lot #17043. All three drug substance lots prepared according to Example 1 had protein-related impurities of less than 6.0%. In particular, no detectable impurities were seen in Lot #16852. Example 7: Residual Nucleic Acids

[0156] To detect RNA and / or DNA in the drug substance prepared according to Example 1, a limit test for residual nucleic acids was performed. The method used a commercially available RiboGreen RNA Quantification Kit. RiboGreen binds to nucleic acids and generates a fluorescent signal proportional to the amount of nucleic acids in the sample. Since DNA generates a stronger signal than RNA when bound to RiboGreen, by using a standard sample containing RNA, the nucleic acid content of a sample containing DNA can be estimated, and the reported nucleic acid value can be taken as the maximum amount of nucleic acids in the sample.

[0157] The purified drug substance lots #16852, #17043, and #19139 were compared to the reference standard curve with the lowest concentration standard sample with RNA at 0.15 μg / mL. All three lots showed weaker fluorescence for the samples than the standard sample. This corresponds to residual nucleic acid being below 0.15 μg / mL (data not shown). Example 8: Protein Characterization

[0158] The method used to determine the protein characteristics is based on the principles described in Ph.Eur. 2.2.31 Electrophoresis, title “Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) – Uniform Percentage Gels”. The purified drug substance lots #16852, #17043, and #19139 were analyzed on 4–12% Bis-Tris gels to separate the component proteins and stained using silver staining (SilverQuest staining kit). Figure 3 shows the results of SDS-PAGE of the reduced drug substance samples (lanes 6–9) and non-reduced drug substance samples (lanes 2–5) compared to the molecular weight markers (lanes 1 and 10). Lanes 3 and 7 correspond to lot #16852, lanes 4 and 8 correspond to lot #17043, and lanes 5 and 9 correspond to lot #19139. (Lanes 2 and 6 show the results of a drug substance lot (#1014997) prepared according to a chromatographic process with four columns using somewhat different parameters of the process (e.g., a buffer with a slightly higher pH in the first chromatographic separation).)

[0159] Purified drug substance lots #1014997, #16852, #17043, and #19139 exhibit equivalent protein characteristics showing a strong band near 150 kDa (lanes 2, 3, 4, and 5, respectively). This indicates that the primary protein component is free BoNT / A. The reduced samples in lanes 6, 7, 8, and 9 show two major protein components near 100 kDa and near 150 kDa. These correspond to the heavy and light chains of BoNT / A, respectively. Example 9: Molecular Weight Distribution of Drug Substances

[0160] The molecular weight size distribution of drug substance components was monitored using size exclusion chromatography (SEC). This method analyzes the main components in a sample containing the product toxin and any high molecular weight and low molecular weight species (HMW and LMW, respectively) that may be associated with either the process or the product. A UPLC® SEC column is used for separation. The mobile phase is Buffer 17 (Table 1 above), with 0.4 M L-arginine added and the pH between 6.6 and 6.9. L-arginine is added to Buffer 17 to minimize secondary interactions between the separated proteins and the wet parts of the column matrix, filters, and other chromatography system components. Using a flow rate of 0.25 mL / min, two identical samples (from one vial of drug substance) are analyzed without pretreatment or dilution. Detection is at 280 nm. The resulting chromatograms are integrated, and the average area percentage (area %) obtained for the main components is reported for each drug substance lot.

[0161] Figure 4 shows the molecular weight distribution of DS lot #17043 prepared according to Example 1. The major peak corresponds to the free product toxin molecule. All of the purified drug substance lots #16852, #17043, and #19139 showed at least 96% of the main component (BoNT / A). From this data, it was confirmed that the product toxin solution prepared according to the present disclosure contains high-purity botulinum toxin. Example 10: Process Yield

[0162] The process yield of BoNT / A is determined as an indicator showing the robustness of each step in the purification process. To calculate the process yield, the weight of each fraction is measured to determine the volume. To determine the concentration of BoNT / A in each fraction, BoNT / A-specific ELISA is used.

[0163] The ELISA protocol is an indirect sandwich ELISA based on the principles and general methods described in USP <1103> “Immunological Test Methods―Enzyme-linked Immunosorbent Assay”. The ELISA method is based on immunological binding using two different types of BoNT / A-specific polyclonal antibodies and the detection of BoNT / A.

[0164] A protein standard serial dilution solution based on commercially available BoNT / A toxin is prepared by diluting BoNT / A in a PBS-Tween solution (0.05% Tween-20) to a concentration range of 3 to 28 ng / mL. Three identical samples diluted within the range of the protein standard diluent with PBS-Tween are added to microplate wells coated with polyclonal anti-BoNT / A antibody. The antibody is recognized by incubation, and binding of the BoNT / A antigen to the well is obtained. After each incubation, an automatic washing step using PBS-Tween solution is performed.

[0165] The primary detection is performed by the binding of another type of polyclonal anti-BoNT / A antibody, forming a sandwich complex. Then, a secondary antibody conjugated with horseradish peroxidase (HRP) is added. This binds to the primary antibody, enabling the detection of BoNT / A within the sandwich complex. Next, a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate is added to the sample well. HRP converts the TMB substrate, generating a blue reaction product. A stop solution is added to halt the conversion of TMB and initiate the color conversion of the remaining TMB to yellow. The absorbance of each microplate well is detected at 450 nm using a plate reader, and the measured absorbance is made proportional to the amount of BoNT / A in the well. The absorbance value of the sample is calculated by comparing it with a standard curve obtained from the absorbance values of the BoNT / A standard dilutions. The results are reported as an average value (unit: μg / mL). Subsequently, the process yield is calculated according to Equation 1 above.

[0166] Figure 5 shows the average values of the process yields at each step of the purification process for lots #16852, #17043, and #19139. The bar labeled "DS" represents the combined yield spanning the last two chromatography steps. This is because they are interconnected and no sampling is performed between these two steps. This data shows that the yields of these steps vary between 100% and 50%. Example 11: Cumulative Process Yield

[0167] The cumulative process yield of each individual step is calculated by the following formula.

Equation

[0168] Figure 6 shows the average cumulative step yields for each step of the purification process for purified drug substance lots #16852, #17043, and #19139. The bar labeled "DS" represents the combined yield spanning the last two chromatography steps. This is because they are interconnected and no sampling occurs between these two steps. Example 12: Overall Yield

[0169] The overall yield of BoNT / A in the product toxin solution (or "DS") compared to the culture at harvest ("KS") is determined as an indicator of the robustness of the entire purification process. To calculate the process yield, the weights of the KS and DS fractions are measured and the volumes are determined. The concentrations of BoNT / A in KS and DS are determined using BoNT / A-specific ELISA. The overall yield is calculated according to Equation 2 above.

[0170] For purified drug substance lots #16852, #17043, and #19139, the overall yield was determined to be between 13% and 29% (data not shown). Example 13: Purity Improvement Factor (across steps)

[0171] The purity improvement factor of BoNT / A in each individual step is determined as an indicator of the efficiency of each step (and the entire purification process) for removing unwanted protein components (related to the process and the product). To calculate the purity improvement factor for each step, each fraction is analyzed using BoNT / A-specific ELISA for toxin concentration and the micro BCA method (Example 4) for total protein concentration. The improvement in purity for each step is calculated according to the following equation.

Equation

[0172] Here, ToxC represents the toxin concentration, TotPC represents the total protein concentration, the subscript "fraction" represents the toxin concentration or total protein concentration obtained from the current processing step, and the subscript "previous fraction" represents the toxin concentration or total protein concentration obtained from the previous processing step.

[0173] Figure 7 shows the average values of the purity improvement factors across processes for purified drug substance lots #16852, #17043, and #19139. The bars labeled "DS" show the combined purity improvement factors across the last two chromatography processes. This is because these processes are interconnected and no sampling is done between these two processes. From this data, it is clear that the second chromatography column is the process that most contributes to the purification of the toxin. However, all processes (except filtration 1 which removes whole cells or lysed cells and cell components) contribute to the overall purification of the toxin relative to the total protein concentration. Example 14: Cumulative Purity Improvement Factor (across processes)

[0174] The cumulative purity improvement factor for each process is calculated as follows.

Equation

[0175] Figure 8 shows the average cumulative purity improvement factors across processes for purified drug substance lots #16852, #17043, and #19139. The bars labeled "DS" show the combined cumulative purity improvement factors across the last two chromatography processes. This is because they are interconnected and no sampling is done between these two processes.

Claims

1. A method for purifying botulinum toxin from a solution containing botulinum toxin, comprising: (a) filtering the solution containing the toxin; (b) contacting the filtered solution containing the toxin from (a) with a first chromatography column, which is an ion exchange chromatography column; (c) recovering a toxin-containing fraction that passes through the first chromatography column without adsorbing to the stationary phase; (d) contacting the toxin-containing fraction with a second chromatography column, which is 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 retention solution; (g) contacting the toxin-containing retention solution from the filtration (f) with a third chromatography column, which is an ion exchange column; (h) eluting the botulinum toxin from the third chromatography column to obtain a second toxin-containing eluate; (i) contacting the second toxin-containing eluate with a fourth chromatography column, which is a size exclusion chromatography column; and (j) eluting the botulinum toxin from the fourth chromatography column to thereby obtain a purified botulinum toxin, wherein the process does not include precipitation, centrifugation, or lyophilization of the botulinum toxin.

2. (i) the botulinum toxin includes botulinum neurotoxin serotype A; (ii) the obtained purified botulinum toxin does not contain a botulinum toxin complex, or the botulinum toxin complex is present in an amount less than 1% by weight based on the whole composition; (iii) the obtained purified botulinum toxin does not contain an animal-derived product, or the animal-derived product is present in an amount less than 1% by weight based on the whole composition; and / or (iv) the purified botulinum toxin does not contain human albumin, or human albumin is present in an amount less than 1% by weight based on the whole composition. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the first chromatography column includes an anion exchange chromatography column.

4. The method according to any one of claims 1 to 3, wherein the second chromatography column includes a cation exchange chromatography column.

5. The first chromatographic column includes an anion exchange chromatographic column, and the second chromatographic column includes a cation exchange chromatographic column. The method according to any one of claims 1 to 4.

6. The third chromatographic column includes an anion exchange chromatographic column. The method according to any one of claims 1 to 5.

7. The fourth chromatographic column includes a gel filtration column. The method according to any one of claims 1 to 6.

8. The third chromatographic column includes an anion exchange chromatographic column, and the fourth chromatographic column includes a gel filtration chromatographic column. The method according to any one of claims 1 to 7.

9. The second toxin-containing eluate is directly injected into the fourth chromatographic column, or The third chromatographic column and the fourth chromatographic column are interconnected. The method according to any one of claims 1 to 8.

10. The first chromatographic column is an anion exchange chromatographic column, the second chromatographic column is a cation exchange chromatographic column, the third chromatographic column is a second anion exchange chromatographic column, and the fourth chromatographic column is a gel filtration chromatographic column. The method according to any one of claims 1 to 9.

11. The first, second, third, and fourth chromatographic columns are a single-use chromatographic system. The method according to any one of claims 1 to 10.

12. The filtration (f) dissociates the botulinum toxin protein molecules from the non-toxin proteins to obtain free toxin molecules. The method according to any one of claims 1 to 11.

13. The filtration (f) includes buffer exchange. The method according to any one of claims 1 to 12.

14. The solution containing botulinum toxin is a fermentation medium that does not contain animal-derived products, or the solution containing botulinum toxin is a fermentation medium that contains less than 1% by weight of animal-derived products based on the whole composition. The method according to any one of claims 1 to 13.

15. The method according to any one of claims 1 to 14, further comprising contacting the botulinum toxin with a buffer solution that has been filtered to reduce the bioburden.

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