BR112016022025B1 - METHOD FOR PRODUCTION OF DOUBLE CHAIN BONT/A SOLUBLE PROTEIN, COMPOSITION, LIQUID PHARMACEUTICAL COMPOSITION AND USE - Google Patents

Abstract

MANUFACTURE OF RECOMBINANT NEUROTOXINS FROM CLOSTRIDIUM BOTULINUM The invention provides methods for producing soluble double-chain BoNT/A protein.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to methods for producing recombinant neurotoxins from Clostridium botulinum (C. botulinum) serotype A (BoNT/A).

BACKGROUND OF THE INVENTION

[0002] Botulinum neurotoxin (BoNT) is produced from the bacteria C. Botulinum, in the form of a large protein complex, consisting of BoNT itself, complexed with a number of accessory proteins. Currently, there are at least seven different classes of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, CI, D, E, F and G, all of which share similar structures and modes of action. A possible eighth serotype, serotype H, has recently been reported, however, the sequence is not yet published. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antiserum, with this classification being made by serotype correlated with percent sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on percent amino acid sequence identity.

[0003] BoNTs are the most potent toxins known, with mean lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. BoNTs are adsorbed in the gastrointestinal tract and after entering the general circulation, they bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of the neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave synaptobrevin/vesicle-associated protein (VAMP) membrane; BoNT/C, BoNT/A and BoNT/E cleave the 25 kDa synaptosome-associated protein (SNAP-25); and BoNT/C cleaves syntaxin.

[0004] In nature, clostridial neurotoxins are synthesized as a single-chain polypeptide, which is post-translationally modified by a proteolytic cleavage event, so as to form two polypeptide chains joined by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine residues that provide the disulfide interchain bond. It is this double-stranded form that is the active form of the toxin. The two chains are called the heavy chain (H-chain), which have a molecular mass of approximately 100 kDa, and the light chain (L-chain or LC), which have a molecular mass of approximately 50 kDa. The H-chain comprises a C-terminal targeting component (HC domain) and an N-terminal translocation component (HN domain). The cleavage site is located between the L-chain and the translocation components, in an exposed coil region (see Table 1). Following binding of the HC domain to its target neuron and internalization of the bound toxin into the cell via an endosome, the HN domain promotes translocation of the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non-cytotoxic protease).

[0005] Non-cytotoxic proteases act by proteolytic cleavage of intracellular transport proteins known as SNARE proteins (eg, SNAP-25, VAMP, or Syntaxin) - see, Gerald K. (2002) 'Cell and Molecular Biology' ( 4th Edition), John Wiley & Sons, Inc. The abbreviation SNARE is derived from the term 'Soluble NSF Attachment Receptor', where NSF stands for N-ethylmaleimide Sensitive Factor. SNARE proteins are integrated into intracellular vesicular fusion, thereby, to the secretion of molecules through the vesicular transport of a cell. The function of the protease is a zinc-dependent endopeptidase activity, and it exhibits a high substrate specificity for SNARE proteins. Consequently, once released to a desired target cell, the protease non-cytotoxic is capable of inhibiting cellular secretion from the target cell Clostridial neurotoxin L-chain proteases are non-cytotoxic proteases that cleave SNARE proteins.

[0006] Botulinum neurotoxins are well known for their ability to cause flaccid muscle paralysis. The so-called muscle-relaxing properties have led to botulinum neurotoxins (such as BoNT/A) being employed in a variety of medical and cosmetic procedures, including the treatment of glabellar lines or hyperkinetic facial lines, headache, spasm hemifacial, overactive bladder, hyperhidrosis, nasal labral lines, cervical dystonia, blepharospasm, and spasticity.

[0007] Traditionally, the production of BoNT was performed by culturing the bacteria of C. botulinum, followed by isolation and purification of the botulinum neurotoxin complex. However, BoNT production through this mode is inefficient and provides low protein yields. Furthermore, C. botulinum bacteria are spore-forming bacteria and therefore require specific culture equipment and facilities, which are not required for the culture of bacteria such as, for example, Escherichia coli (E. coli). The increasing use of BoNTs has led to the development of alternative methods for the production and purification of BoNTs. Specifically, methods for producing BoNTs using E. coli have been developed.

[0008] The purification of CNTs from the fermentation solution (using C. botulinum or E. coli) is a particular challenge, since the neurotoxins are contained there as a mixture of unprocessed, partially processed and fully processed polypeptides, present , on the whole, similar biochemical and physical properties. Partially processed neurotoxins are typically generated if endoproteolytic activity has hydrolyzed the peptide bond between the light chain and the coil, while the peptide bond between the coil and the N-terminus of the heavy chain is still intact. Furthermore, partially processed neurotoxin can also be created if endoproteolytic activity has released the coiled peptide from the heavy chain, at the time the peptide bond between the coiled peptide and the C-terminus of the heavy chain has not yet been hydrolyzed. Depending on fermentation conditions and the type of neurotoxin, fully processed polypeptide, which is free of coiled peptide, can be significantly contaminated with 5 to 90% of partially processed or unprocessed polypeptide. In some cases, the neurotoxin is mostly unprocessed, and prior to therapeutic use, it needs to be treated with an endopeptidase in order to become biologically active.

[0009] The prior art describes several attempts to treat clostridial neurotoxins with heterologous proteases, in order to reduce the amount of unprocessed or partially processed precursor protein. The most widely used protease for activating clostridial neurotoxins is trypsin, which, while useful for activating clostridial neurotoxins of serotypes B (BoNT/B) and E (BoNT/E), apparently produces side products, presumably by proteolytic action near the C-terminus of the heavy subunit of BoNT/A and thus appears to disrupt the binding of the toxin to its cellular receptor. More specific cleavage products are theoretically expected from endogenous proteases isolated from native host such as C. botulinum, producer of BoNT/A.

[0010] The present inventors have previously identified several proteases, such as the endogenous proteases of C. botulinum and exogenous proteases, including the protease endoproteinase Lys-C (Lys-C), (which is commercially available and which can be isolated from the bacterium Lysobacter enzymogenes). However, although cleavage of recombinant BoNT/A by this endoproteinase more precisely focuses on native cleavage, the use of Lys-C provides new practical considerations. Specifically, after cleavage of recombinant BoNT/A, Lys-C can remain active in the reaction mixture for days. Therefore, means and methods to reduce the amount of Lys-C in the final product and thereby improve the quality of neurotoxin preparations are highly desirable but not yet available.

[0011] Thus, a technical problem that underlies the present invention can be seen as the provision of means and methods to improve the manufacture of polypeptides from neurotoxins, in order to meet the aforementioned needs. Specifically, there is a need in the art for improved methods for producing recombinant BoNTs, in particular double-stranded activated recombinant BoNT/A.

[0012] The technical problem is solved by the modalities described herein, which are characterized in the appended claims and presented below.

SUMMARY OF THE INVENTION

[0013] Production of recombinant BoNT/A subserotypes can be achieved by expression in Escherichia coli as a single polypeptide, referred to herein as single-chain BoNT/A proteins. After isolation of this single-chain protein by means of fast protein liquid chromatography (FPLC), the single-chain protein must be cleaved to form the active toxin, which is a double-chain protein, linked together by a single disulfide bond. .

[0014] The present inventors have previously discovered that single-chain recombinant BoNT/A subserotypes can be cleaved using the endoproteinase Lys-C (Lys-C) protease to produce double-chain BoNT/A active protein neurotoxin , i.e., a heterodimer of the light and heavy chains of BoNT/A. The Lys-C cleavage site was determined to be identical to that of the endogenous protein by N-terminal sequencing and mass spectrometry. Therefore, the production of active double-chain BoNT/A protein using Lys-C is advantageous in that it results in the production of an authentic double-chain active BoNT/A, namely, a double-chain BoNT/A that is substantially identical to pure active double-stranded BoNT/A. In contrast, trypsin, another common protease used to produce double-stranded active BoNT proteins, promotes cleavage of at least one additional site within the single-chain BoNT/A sequence.

[0015] This additional cleavage reduces the potency of the active double-stranded BoNT/A protein produced.

[0016] Once a single-chain BoNT/A protein has been cleaved by Lys-C, the active double-stranded BoNT/A protein requires purification to remove any remaining host cell proteins and Lys-C protease, the in order to produce the final pure product.

[0017] In particular, from the Lys-C endoproteinase cleavage tests, the inventors found that Lys-C promotes the cleavage of recombinant BoNT/A1 (rBoNT/Al) at very low concentration levels and that it remains active for a period of several days. Consequently, it is important to develop a method that can purify this protease out of the activated toxin.

[0018] The present inventors have developed an efficient method to remove Lys-C activating protease and host cell proteins from BoNT/A after activation using hydrophobic interaction chromatography (HIC). The present inventors have surprisingly discovered that the HIC method not only maximizes the amount of double-stranded active BoNT/A recovered in purification, but also significantly improves Lys-C resolution, i.e., the removal of Lys-C protease. The advantageous properties of the HIC method are counterintuitive when considering the biophysical characteristics of Lys-C and rBoNT/A; based on the isoelectric point (pI) and net charge of BoNT/A and Lys-C, it is assumed that an ion exchange chromatography (IEX) method can resolve the two proteins, rather than the HIC method.

[0019] The present invention solves one or more of the aforementioned problems by providing certain methods as specified in the appended claims.

[0020] Accordingly, the present invention provides a method for producing soluble double-chain BoNT/A protein, said method comprising: a) providing a soluble single-chain BoNT/A protein; b) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution; and c) separating the soluble BoNT/A protein from Lys-C by contacting the solution containing the soluble BoNT/A and Lys-C protein with a hydrophobic surface, where the soluble BoNT/A protein preferably binds to the hydrophobic surface.

[0021] Soluble single-chain BoNT/A protein can be produced in a host cell by expressing a nucleic acid encoding said single-chain BoNT/A protein in an expression system, wherein, optionally, the expression system is a bacterial expression system, preferably wherein the bacterial expression system is an E. coli expression system, and the host cell is an E. coli cell. Said single chain soluble BoNT/A protein may be expressed in the cytoplasm of said host cell, or in a cell-free system. Soluble single-chain BoNT/A protein can be expressed at a level of at least 5 mg/L of culture. Said method may comprise lysing the host cell to provide a host cell homogenate containing said single chain soluble BoNT/A protein (preferably, the single chain soluble BoNT/A protein is present in a concentration of at least 5 mg/L of host cell homogenate).

[0022] The hydrophobic surface used in the method of the present invention may be an inert matrix, on which a linker consisting of aryl or alkyl groups is attached, wherein, optionally, the linker is selected from the group consisting of butyl, phenyl and or octyl. In one embodiment, a high performance hydrophobic surface is used.

[0023] The invention also provides an active double-chain BoNT/A protein, which can be obtained according to the method of the invention.

[0024] The invention further provides a composition comprising an active double-chain BoNT/A protein in accordance with the present invention, wherein said composition is substantially free of Lys-C endoproteinase. Preferably, said composition contains less than 400 pg Lys-C endoproteinase (Lys-C) per 100 ng BoNT/A protein, or less than 300 pg Lys-C per 100 ng BoNT/A protein, or less than 200 pg of Lys-C per 100 ng of BoNT/A protein, or less than 100 pg of Lys-C per 100 ng of BoNT/A protein, or less than 50 pg of Lys-C per 100 ng of BoNT/A protein. Such compositions are suitable for use in therapeutic and cosmetic treatments.

[0025] The invention also provides a liquid pharmaceutical composition, comprising: an active double-chain BoNT/A protein according to the invention; a non-protein stabilizing agent which is a surfactant; and water; wherein said liquid pharmaceutical composition does not comprise a protein stabilizing agent; and wherein said liquid pharmaceutical composition is substantially free of endoproteinase Lys-C (Lys-C). Preferably, said liquid pharmaceutical composition contains less than 400 pg Lys-C per 100 ng BoNT/A protein, or less than 300 pg Lys-C per 100 ng BoNT/A protein, or less than 200 pg of Lys-C per 100 ng BoNT/A protein, or less than 100 pg Lys-C per 100 ng BoNT/A protein, or less than 50 pg Lys-C per 100 ng BoNT/A protein A. Said liquid pharmaceutical composition may further comprise sodium chloride, a buffer, to maintain the pH between 5.5 and 7.5, and a disaccharide; and wherein the water is sterile water.

[0026] The invention also provides an active double-chain BoNT/A protein, a composition thereof or a liquid pharmaceutical composition according to the invention, for use in therapy.

[0027] The invention further provides the use of an active double-chain BoNT/A protein, a composition thereof or a liquid pharmaceutical composition according to the invention, in the manufacture of a medicament.

[0028] The invention also provides a method of treatment comprising administering an active double-chain BoNT/A protein, a composition thereof or a liquid pharmaceutical composition according to the invention, to a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION Botulinum Toxin Serotype A (BoNT/A)

[0029] The term “BoNT' means botulinum neurotoxin and refers to the neurotoxin that can be obtained from the bacteria C. botulinum, such as, for example, BoNT of serotypes A, B, C1, D, E, F or G Also included within the terms 'CNT' and 'BoNT' is recombinant and modified neurotoxin comprising one or more modifications, including chemical modification or genetic modification. The term "genetic modification" means the deletion, substitution or addition of one or more nucleic acid residues resulting from the deletion, substitution or addition of one or more amino acid bases, or the deletion, substitution or addition of said one or more amino acid residues .

[0030] Botulinum neurotoxin serotype A (BoNT/A) is divided into at least eight subserotypes (also known as subtypes), BoNT/AI to B0NT/A8, which share at least 84% to 98% sequence identity of amino acids; BoNT/A proteins within a given subtype share a greater percentage of amino acid sequence identity. Table 1 below shows the precursor, the pure double-stranded neurotoxin of the BoNT/A subtypes and identifies the exposed coil (activation loop) comprising the amino acid sequence cleaved by Lys-C.Table 1 :

[0031] With reference to Table 1, the amino acid positions (residues) indicated for the LC, HN, HCN and Hcc regions correspond to the amino acid positions for said regions in a full-length BoNT/A protein sequence . The Uniprot Accession Numbers shown in Table 1 are examples of the different subserotypes of BoNT/A. Thus, in one embodiment, the amino acid positions of the LC, HN, HCN and Hcc regions indicated in Table 1 correspond to the amino acid positions in the full-length BoNT/A protein sequences identified by the Accession Number.

[0032] By using the methods of the present invention, it is now possible to obtain double-stranded BoNT/A compositions with significantly less contamination by an unprocessed or partially processed BoNT/A, since these contaminants are efficiently processed within of double-chain BoNT/A. The methods of the present invention also allow for effective removal of the Lys-C enzyme used to process the single-stranded BoNT/A protein into the active duplex. In one aspect, the double-chain BoNT/A is a pure double-chain neurotoxin, wherein the C-terminus of the light chain and the N-terminus of the heavy chain are identical to the corresponding isolated fully processed double-chain BoNT/A of the wild-type clostridia species.

[0033] According to the present invention, an active double-chain BoNT/A, including a recombinant double-chain BoNT/A can be produced in the form of a single-chain polypeptide, which is cleaved to form the active form of protein double chain.

[0034] The present methods can be used to produce the duplex of any subtype of BoNT/A, or a homologue or derivative thereof, including, for example, the polypeptide of SEQ ID NO: 1 and derivatives thereof. The term "derivative", as used in connection with this and other aspects of the invention, comprises amino acid mutations such as addition, substitution, deletion or truncation of one or more amino acid residues.

[0035] The single-chain BoNT/A protein may comprise a polypeptide sequence as shown in GenBank, nos: CBZ04958.1,YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782718.1, ZP_02616437.1, ZP_ 02614241 .1, YP_001392361.1, YP_001255575.1. The single chain BoNT/A protein may comprise a polypeptide sequence as shown in:- subserotype A1: UniParc Accession No. UPI0000ED909E (UniProt Access No. P10845, Version 159), UniParc Access No. UPI0000EF85BD (UniProt Accession No. S8B1U4, Version 3, or UniProt Accession No. A2I2R4, version 41), UniParc Accession No. UPI0001A954C8 (UniProt Accession No. C6K838, Version 11), UniParc Accession No. UPI0000001386 (UniProt Accession No. A5HZZ9, Version 57), UniParc Accession No. UPI000003409D (UniProt Accession No. A2I2U2, Version 36), UniParc Accession No. UPI00016529B7 (UniProt Accession No. B1A2D5, Version 21), UniParc Accession No. UPI00027EE164 (UniProt Accession No. J7FGZ9, Version 6); - subserotype A2: Accession UniParc No. UPI0000EF84BD (UniProt Accession No. A2I2R5, Version 28), UniParc Accession No. UPI0001C0B376 (UniProt Accession No. D2KCK3, Version 15), UniParc Accession No. UPI0001C32E84 (UniProt Accession No. D3IV23, Version 10), UniParc Accession No. UPI0001F3B30D (UniProt Accession No. E5F1I1, Version 11), UniParc Accession No. UPI000016EA88 (UniProt Accession No. Q45894, Version 116 or UniProt Accession No. Q58GH1, Version 51), UniParc Accession No. UPI000067C53E (UniProt Accession No. Q2PPK6, Version 33), UniParc Accession No. UPI000290BEB 1 (UniProt Accession No. K4GGE0, Version 6); - subserotype A3: Accession UniParc No. UPI00005B712C (UniProt Access No. Q3LRX9, Version 38), UniParc Access No. Q3LRX9, Version 38 UPI00016DBC11 (UniProt Accession No. B1L2G5, Version 38 or UniProt Accession No. D3IV24, Version 11), UniParc Accession No. UPI000290C3D0 (UniProt Accession No. K4G3L3, Version 7); - subserotype A4: Accession UniParc No. UPI00005B712D (UniProt Access No. Q3LRX8, Version 35), UniParc Access No. Q3LRX8, Version 35 UPI00019DB885 (UniProt Access No. C3KS13, Version 29); - subserotype A5: Accession UniParc No. UPI0001AE7D6A (UniProt Accession No. C7BEA8, Version 14), UniParc Accession No. C7BEA8, Version 14 UPI000198BDAE (UniProt Accession No. E8ZMW0, Version 18 or UniProt Accession No. C1IPK2, Version 20); or - subserotype A6: Accession UniParc No. UPI0001B7D251 (UniProt Accession No. C9WWY7, Version 13). - subserotype A7: Accession UniParc No. UPI000290B995 (UniProt Accession No. K4LN57). - subserotype A8: Accession UniParc No. UPI000559A469 (UniProt Accession No. A0A0A7PDB7); Access UniParc No. UPI0003C9D2A1 (UniProt Accession No. V5N8R1).

[0036] The single-chain BoNT/A protein may comprise a polypeptide sequence, which is a homolog or a derivative having at least 50% sequence identity to one of the above-mentioned BoNT/A polypeptide sequences.

[0037] In one aspect, the polypeptide chain of said single chain BoNT/A protein comprises a sequence selected from any one of SEQ ID NOs: 2 to 7 or 13 or 14. In a more particular aspect, the chain of said single-chain BoNT/A protein polypeptides comprising a sequence selected from any one of SEQ ID NOs: 2 to 7 or 13 or 14, and wherein the second polypeptide is C-terminally cleaved at a basic amino acid residue within of said sequence of any one of SEQ ID NOs: 2 to 7 or 13 or 14. Said sequences represent amino acid sequences of known single-chain BoNT/A protein substrates according to the present invention.

[0038] Single-chain BoNT/A proteins are cleaved at the C-terminus at a basic amino acid residue contained in the sequence (compare Table 1, columns LC and HN). In a preferred aspect, said single chain BoNT/A protein comprises a sequence selected from SEQ ID NO: 2 to 7 and 13-14 (e.g. serotype BoNT/A1, SEQ ID NO: 2).

[0039] The single-chain BoNT/A protein may comprise a derivative of any of SEQ ID NOs: 2 to 7 or 13 or 14, or of SEQ ID NO: 1, or of one of the polypeptide sequences corresponding to the accession numbers identified herein, wherein said derivative has one or more point mutations and/or one or more additional amino acid residues. In another aspect, said derivative has up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, or up to 15 point mutations. By using an activity assay to determine protease activity as described herein, one skilled in the art will be able to determine whether a given derivative is processed by Lys-C.

[0040] The derivative may contain a point mutation that exchanges a basic amino acid residue for a non-basic amino acid residue. The derivative may show at least 50% sequence identity with any one of SEQ ID NOs: 2 to 7 or 13 or 14, SEQ ID NO: 1, one of the polypeptide sequences corresponding to the accession numbers identified herein. Said derivative or polypeptide comprising the derivative may be a Lys-C substrate and be proteolytically cleavable by Lys-C. A typical example is a derivative of SEQ ID NO: 1, comprising, for example, one or more point mutations in the light chain or heavy chain.

[0041] Said single chain BoNT/A protein may comprise: (a) a polypeptide sequence having at least 30% sequence identity, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, with the sequence of SEQ ID NO: 1 (BoNT/A of ATCC 3502, Genbank acc. AAA23262), or with one of the polypeptide sequences corresponding to the accession numbers identified herein. The single-chain BoNT/A protein can comprise the polypeptide sequence of SEQ ID NO: 15, or a polypeptide sequence having at least 30% sequence identity, at least 50%, at least 60%, at least 70 %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more with the sequence of SEQ ID NO: 15.

[0042] The term "sequence identity" as used herein, refers to determining the identity between a reference amino acid sequence and a query sequence, wherein the sequences are aligned such that the highest order equivalence is obtained and that can be calculated using published techniques or methods coded in computer programs, such as, for example, BLASTP, BLASTN, FASTA (Altschul 1990, J. Mol. Biol., 215:403). Percent identity values can be calculated over the entire amino acid sequence or over a region of the amino acid sequence. Thus, for example, for single-chain BoNT/A protein, sequence identity can be calculated over a sequence length of up to 50 amino acid (aa) residues, up to 100 aa residues, up to 150 aa residues, up to 250 aa residues, up to 300 aa residues, up to 350 aa residues, up to 400 aa residues, up to 450 aa residues, up to 500 aa residues, up to 550 aa residues, up to 600 aa residues, up to 650 aa residue, up to 700 aa residue, up to 750 aa residue, up to 800 aa residue, up to 850 aa residue, up to 900 aa residue, up to 950 aa residue, up to 1000 aa residue, up to 1050 aa residue aa, up to 1100 aa residues, up to 1150 aa residues, up to 1200 aa residues, up to 1250 aa residues, or up to more residues, or up to the entire length of the single-chain BoNT/A protein sequence. Sequence identity can be calculated on at least 50 aa residues, at least 100 aa residues, at least 150 aa residues, or at least 250 aa residues.

[0043] A number of programs based on a variety of algorithms are available to those skilled in the art, for comparing different sequences. In this context, the Needleman and Wunsch or Smith and Waterman algorithms provide particularly reliable results. To perform the sequence alignments and calculate the sequence identity values presented here, the commercially available program DNASTAR Lasergene MegAlign, Version 7.1.0, based on the Clustal W algorithm, was used on the entire sequence region with the following adjustments: parameters alignment in pair mode; loss of spacing: 10.00; loss of spacing extension: 0.10; protein weight matrix, Gonnet 250; which, unless otherwise specified, should always be used as standard fits for sequence alignments.

[0044] The term at least 30% means at least 30%, at least 40%, at least 50%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%. The sequence identity of said single chain BoNT/A protein sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 can be determined based on amino acid position 420 to 466 of SEQ ID NO : 1, or said sequence identity may be determined on the basis of any one of SEQ ID NOs: 2 to 7 or 13 or 14. In other words, a single chain BoNT/A protein may comprise a sequence of polypeptides which has at least 30% sequence identity with the polypeptide sequence found between amino acid positions 420 to 466 of the single-chain BoNT/A protein, or at least 30% sequence identity with the polypeptide sequence of any of the single-chain BoNT/A protein, including any of the polypeptide sequences corresponding to the accession numbers identified herein. A polypeptide according to this definition is, for example, the polypeptide obtainable from C. botulinum, C. tetani or C. sporogenes. Said single-chain BoNT/A protein may be, for example, a naturally occurring neurotoxin, or a derivative thereof, comprising one or more amino acid mutations, such as addition, substitution, deletion or truncation of one or more amino acid residues. Included herein are, for example, derivatives missing from, for example, the HC domain of the parent neurotoxin or parts thereof, or those derived from other amino acid residues which replace the HC domain of the neurotoxin, as well as derivatives with an additional light chain or other protein cargo molecule fused at the N-terminus to the BoNT light chain.

[0045] The single-chain BoNT/A protein may contain additional amino acid residues at the N- or -C-terminus, or at an internal position. Additional amino acid residues may be flanked by one or more protease cleavage sites. The additional amino acid sequence can function as a detectable label and/or allow binding to a solid support. An example is a His tag or a GST tag. Another example is the amino acid sequence VPPTPGSAWSHPQFEK (SEQ ID NO: 12) containing the Streptag, preferably added to the C-terminus.

[0046] In another aspect, the biological activity of said single-chain BoNT/A protein can be modulated by proteolytic cleavage. It is well known to a person skilled in the art that the function of various polypeptides can be modulated by proteolytic processing. “Modulated”, as used herein, means augmented or diminished, activated or deactivated. Thus, for example, the biological activity of several clostridial neurotoxins is enhanced or triggered by proteolytic processing of a single-chain neurotoxin into a double-chain neurotoxin, where the double-chain neurotoxin is composed of a light chain and a heavy chain of polypeptides, which are covalently linked through a disulfide bond. The biological activity of the neurotoxin encompasses at least three separate activities: the first activity is a “proteolytic activity” that resides in the light chain of the neurotoxin and that is responsible for the hydrolysis of the peptide bond of one or more polypeptides involved in the regulation of cell membrane fusion . A second activity is a "translocation activity", which resides at the N-terminal end of the heavy chain of the processed neurotoxin, and which is involved in the transport of the light chain across the liposomal membrane and into the cytoplasm. A third activity is a "receptor binding activity", which resides at the C-terminal end of the heavy chain of the processed neurotoxin and which is involved in the binding and uptake of the neurotoxin into a target cell. In a preferred aspect, the term biological activity as used herein means proteolytic activity. In a more preferred aspect, the term means increased proteolytic activity.

[0047] The biological activity of clostridial neurotoxin can be measured by means of various tests, all of which are known to a person skilled in the art. These tests allow the determination of one or more of the aforementioned activities. Thus, for example, the mouse Ld50 assay or the ex-vivo mouse phrenic nerve (MPN) hemidiaphragm assay, as described by Pearce et al., 1994 (Pearce L.B, Borodic G.E, First E.R, MacCallum R.D ( 1994), Toxicol. Appl. Pharmacol., 128:69-77) and by Habermann et al, 1980 (Habermann E, Dreyer F, Bigalke H. (1980), Naunyn Schmiedebergs Arch Pharmacol., 311:33-40) allow determine the toxic effect of a given neurotoxin preparation on a living organism or of an isolated neuromuscular preparation. For establishing the toxic effect in an LD50 assay, the neurotoxin must be biologically active in each of the said three activities mentioned above. In addition, several other assays are available, which allow, for example, to determine whether a neurotoxin or the light chain of the neurotoxin is proteolytically active. These assays are, for example, based on contacting BoNT/A with SNAP-25. Alternatively, a peptide representing the SNAP-25 cleavage site can be used, where the peptide can be labeled to facilitate detection. In a preferred aspect, the biological activity is determined through the use of the aforementioned MPN assay.

[0048] A nucleic acid molecule encoding said single-chain BoNT/A protein can be used in accordance with the present invention. Said nucleic acid molecule optionally may comprise regulatory elements. The term "regulatory elements" as used herein refers to regulatory elements of gene expression, including transcription and translation, and includes elements such as tata box, promoter, enhancer, ribosome binding site, Shine- Dalgarno, IRES region, polyadenylation signal, terminal capping structure and the like. Said regulatory elements may comprise one or more heterologous regulatory elements or one or more homologous regulatory elements. A "homologous regulatory element" is a regulatory element of a wild-type cell, from which the nucleic acid molecule is derived, which is involved in regulating gene expression of the nucleic acid molecule or polypeptide in said cell of the wild type. wild type. A "heterologous regulatory element" is a regulatory element that is not involved in regulating gene expression of the nucleic acid molecule or the polypeptide in said wild-type cell. Regulatory elements for inducible expression, such as inducible promoters, can also be used.

[0049] The nucleic acid molecule may be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA, and/or modified nucleic acid molecules. The nucleic acid molecule can be circular, linear, integrated within a genome, or episomal. Furthermore, coding concatemers for fusion proteins comprising three, four, five, six, seven, eight, nine or ten polypeptides are encompassed. Furthermore, the nucleic acid molecule may contain signal sequences encoding sequences for intracellular transport, such as signals for transport within an intracellular compartment, or for transport across the cell membrane.

[0050] According to the invention, a nucleic acid molecule encoding BoNT/A can be designed to advantageously provide high levels of expression in host cells, particularly bacterial host cells, preferably E. coli cells. Methods for designing nucleic acid molecules to increase protein expression in host cells, particularly bacterial host cells, preferably E. coli cells, are known in the art, and include decreasing the frequency (number of occurrences) of "slow codons" in encoding the nucleic acid sequence.

[0051] In one aspect, a single chain BoNT/A protein is produced using an expression vector comprising a nucleic acid molecule encoding said single chain BoNT/A protein. A vector may be suitable for in vitro and/or in vivo expression of said single chain BoNT/A protein. The vector may be a vector for transient and/or stable gene expression. Furthermore, the vector may comprise regulatory elements and/or selection markers. Said vector may be of viral origin, of phage origin, or of bacterial origin. For example, said expression vector may be a pET-26b(+) vector.

[0052] A nucleic acid molecule or an expression vector encoding a single chain BoNT/A protein can be comprised in a host cell, which comprises the nucleic acid molecule or vector of the present invention. The term "host cell", as used herein, encompasses prokaryotic and/or eukaryotic cells suitable for converting said nucleic acid molecule or said vector and, in particular, single-chain BoNT/A protein. Said host cell may be a host cell which does not express the single chain BoNT/A protein or a homologue thereof. The term "homologue" as used herein refers to a polypeptide comprising a sequence of polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity with a BoNT/A sequence, e.g., the BoNT/A sequence of SEQ ID NO : 1. However, host cells, such as wild-type cells, which express single-chain BoNT/A protein or a homologue thereof are also included. Thus, for example, the host cell can be selected from C. botulinum, C. butyricum, C. baratii and C. tetani, for example, C. botulinum serotypes A, B or F. The host cell can be the strain of Hall (ATCC 3502) of C. botulinum; the ATCC 19397 strain, also known as NCTC 4587 and NCTC 7272 of BoNT/A-producing C. botulinum; the BoNT/A-producing C. botulinum strain NCTC 2916; the Kyoto-F or Mauritius/NCTC 9837 strain of C. botulinum producing BoNT/A2; the Loch Maree/NCTC 2012 strain A254 of C. botulinum producing BoNT/A3; the BoNT/A4 and B-producing C. botulinum strain CDC657; the BoNT/A5 and B3'-producing C. botulinum strain H04402065; the Okra/NCTC 7273 strain of C. botulinum producing BoNT/B1; the BoNT/B and F-producing C. botulinum strain CDC4013/NCTC 12265; or the Langeland/NCTC 10281 strain of C. botulinum producing BoNT/F1. Said host cell may be of the species Clostridium sporogenes, Clostridium perfringens, Clostridium acetobutylicum, B. cereus, B. thuringiensis, B. mycoidis, B. thermoproteolyticus, B. anthracis, B. megaterium, B. subtilis, E.coli, or a yeast cell. Preferably, the host cell is an E. coli host cell, specifically, an E. coli BL21(DE3) or BLR(DE3) cell.

[0053] In one aspect, the single-chain BoNT/A protein is modified within the host cell (i.e., glycosylated, phosphorylated, processed by proteases, etc.). Modification also includes the addition of non-protein cofactors, including metal ions. The host cell may comprise a single chain BoNT/A protein expression inducer. Such a pressure inducer can be a nucleic acid molecule or a polypeptide, or a chemical entity, including a small chemical entity, having the effect of increasing the amount of single-chain BoNT/A protein in cell cultures or lysates thereof. The expression inducer can, for example, increase transcription or translation of a single-stranded BoNT/A protein-encoding nucleic acid molecule. The inducer can be expressed, for example, by recombinant means known to a person skilled in the art. Alternatively, the inducer can be isolated from a cell, for example a clostridial cell.

[0054] A single chain BoNT/A protein can be produced by a method comprising introducing an expression vector, as described herein, for expressing said single chain BoNT/A protein in said host cell. A single chain BoNT/A protein can be produced by providing a host cell comprising a nucleic acid encoding said single chain BoNT/A protein, and expressing said single chain BoNT/A protein in said host cell . Typically, the host cell is a bacterial cell, preferably an E. coli cell. The E. coli host cell can be an E. coli BL21 (DE3) or BLR(DE3) cell.

[0055] Preferably, the single-chain BoNT/A protein is converted in a cell. The cell can be a prokaryotic cell or a eukaryotic cell. In one aspect, the cell is selected from E. coli, B. subtilis or yeast, where the E. coli cell is a highly preferred host cell. Also encompassed by the present invention is the translation of the single-chain BoNT/A protein in a wild-type cell, i.e., a cell isolated from nature, such as any known isolate of Clostridium botulinum, Clostridium butyricum, Clostridium baratii, and Clostridium tetani. In accordance with the present invention, any suitable host cell as described herein can be used.

[0056] Several standard means and methods are available to one skilled in the art for bringing a nucleic acid molecule or a vector into a host cell, and expressing the single-chain BoNT/A protein as a recombinant protein in a cell. Furthermore, one skilled in the art is aware of several standard techniques for extracting proteins and polypeptides from cells or cell lysates (e.g., Recombinant DNA Principles and Methodologies, J. Green, Marcel Dekker Inc., 1998; The Condensed Protocols: From Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2000). Any of these means and methods can be used in the processes of the present invention.

[0057] Typical methods of extracting proteins from a host cell or host cell lysate include centrifugation (clearing) of the cell lysate, ammonium sulfate precipitation of proteins, protein resuspension, centrifugation of suspended proteins, chromatography ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and the like. According to the present invention, various combinations of these steps in different orders can be useful for the purification of a single chain BoNT/A protein.

[0058] As described in more detail below, single-chain BoNT/A protein can be contacted with Lys-C to produce an active double-chain BoNT/A protein. The present inventors have just developed an advantageous method for purifying double-chain BoNT/A protein from reaction mixture of BoNT/A and Lys-C.

Lys-C endoproteinase

[0059] As described herein, a single-chain BoNT/A protein is cleaved to produce the active double-chain form of the BoNT/A protein using endoproteinase Lys-C (Lys-C). The term Lys-C refers to a 33 kDa Lys-C serine endoproteinase, from Lysobacter enzymogenes (Lysyl endopeptidase, LeK, Genbank acc. Q7M135), which specifically promotes cleavage of C-terminal peptide bonds at lysine, or a homologue thereof, having at least 50% sequence identity. In one embodiment, said homolog having at least 50% sequence identity with Lys-C retains the functionality (i.e., proteolytic activity) of Lys-C.

[0060] According to the present invention, the Lys-C enzyme is a proteolytically active polypeptide that may comprise or consist of a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8. In In one aspect, the Lys-C enzyme used in accordance with the present invention is a proteolytically active polypeptide consisting of a polypeptide sequence as shown in SEQ ID NO: 8.

[0061] Typically, Lys-C homologues are able to hydrolyze a single-chain botulinum neurotoxin (e.g., serotype A (BoNT/A), to produce double-chain botulinum neurotoxin (e.g., serotype A (BoNT/A). /A). covered by said term the homologues of said protease, having at least 60% sequence identity.

[0062] The term "proteolytically active polypeptide", as used herein, refers to the catalytic function of the polypeptide and means that the polypeptide is capable of hydrolyzing a peptide bond. In one aspect, "proteolytically active polypeptide" refers to a polypeptide that is capable of hydrolyzing a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7.

[0063] The term "proteolytically inactive polypeptide", as used herein, refers to the catalytic function of the polypeptide and means that the polypeptide is unable to hydrolyze a peptide bond.

[0064] A person skilled in the art can determine whether a Lys-C polypeptide according to the sequence definition mentioned herein is a polypeptide for use according to the present invention, by testing the proteolytic activity of said polypeptide. An assay or test system for determining proteolytic activity comprises contacting a Lys-C polypeptide, which comprises a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 8, with a substrate of test.

[0065] A test substrate typically is a polypeptide that is known to be cleaved by Lys-C. Preferably, the test substrate is a clostridial neurotoxin (CNT), such as BoNT, or a fragment thereof. The test substrate can be, for example, an uncleaved/unprocessed BoNT, referred to herein as single-chain BoNT (scBoNT), and can be, for example, serotypes A, B, CI, D, E, F or G (eg scBoNT/A, scBoNT/B etc.), or the test substrate can be tetanus neurotoxin (TNT). Alternatively, the test substrate may be a fragment of a clostridial neurotoxin, said fragment comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7. The fragment may be a polypeptide of 50 or more amino acid residues, or a peptide of up to 49 amino acid residues. As used throughout this description, the term "polypeptide" refers to molecules having 50 or more amino acid residues, while the term "peptide" refers to molecules having 2 to 49 amino acid residues.

[0066] The test substrate may be a soluble neurotoxin fragment, called an LHN, comprising the light chain polypeptide, the exposed coil peptide region and N-terminal half of the heavy chain polypeptide, and the translocation domain HN. The test substrate can be or comprise a peptide selected from any one of SEQ ID NOs: 2 to 8 (see Table 1). The test substrate can be a chimeric neurotoxin comprising amino acid residues derived from two or more serotypes.

[0067] An assay to determine the proteolytic activity of a Lys-C enzyme, homologue or derivative thereof typically comprises a step of determining the degree of conversion of the test substrate into its cleavage product or products. The observation of one or more cleavage product/products generated upon contacting the polypeptide with the test substrate, or the observation of an increase in the amount of cleavage product/products is indicative of proteolytic activity of the polypeptide. Said determination step may involve comparing the substrate with the cleavage product/products. Said comparison may involve determining the amount of substrate and/or the amount of one or more cleavage product/products, and may also involve calculating the ratio of substrate and cleavage product/products. Furthermore, the assay for determining proteolytic activity may comprise a step of comparing a test sample with a reference sample, where the reference sample typically comprises the steps of: (a) a polypeptide comprising a sequence of polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 8, and which is known to be proteolytically active; and (b) a test substrate known to be cleaved by the polypeptide of step (a).

[0068] The assay for determining proteolytic activity may comprise separating the substrate (e.g., single-chain BoNT/A protein) from the cleavage product/products (e.g., active double-chain BoNT/A) through electrophoresis or through column chromatography and, optionally, through spectrometric analysis. Thus, it may be convenient to label the test substrate with one or more labels in order to more easily detect the decrease in the test substrate and/or the increase in the product(s). The term "tag" as used herein means a detectable marker and includes, for example, a radioactive tag, an antibody and/or a fluorescent tag. The amount of test substrate and/or cleavage product can be determined, for example, by autoradiography or spectrometry methods, including methods based on resonance energy transfer between at least two labels. Alternatively, immunological methods such as Western blot or ELISA can be used for detection.

[0069] In a preferred aspect, a polypeptide is proteolytically active if more than 20%, preferably more than 95% of the test substrate is converted to the cleavage products, such as the light chain and the heavy chain, in 120 minutes at 37°C using a buffer selected from 100 mM Tris-HCl, pH 8.0, or PBS (50 mM Na2HPO4, 150 mM NaCl, pH 7.4). The same conditions apply in case the test substrate is not a full-length BoNT/A, but instead is, for example, a fragment of full-length BoNT/A or a derivative of BoNT/A. In that case, it is clear that the cleavage products will be different. However, the person skilled in the art can quantify the corresponding cleavage products.

[0070] Typically, 100 ng of proteolytically active Lys-C polypeptide and a 1:100 molar ratio to substrate are used in the assay.

[0071] A sample can be taken at intervals in order to follow the catalytic activity over time.

[0072] The assay can be modified, for example, by using multiple amounts of the proteolytically active Lys-V polypeptide.

[0073] SEQ ID NO: 9 shows the polypeptide sequence of a proteolytically inactive polypeptide derived from an ATCC 3502 strain of Clostridium botulinum, GenBank accession No: CAL82988.1, having an amino acid length of 581 residues. SEQ ID NO: 8 shows a proteolytically active derivative of SEQ ID NO: 9, omitting amino acid residues 1 to 248 of SEQ ID NO: 9.

[0074] The term "polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8", refers to a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 8. Furthermore, the term refers to a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8. Said polypeptide may have additional amino acids, for example, in an internal position or in an N-terminal or C-terminal position, with respect to the sequence shown in SEQ ID NO: 8, or in an internal position or an N-terminal or C-terminal position, with with respect to an amino acid sequence that is at least 50% identical to the sequence of SEQ ID NO: 8, where a methionine may be present at the N-terminus of the polypeptide. In addition, the term refers to a polypeptide lacking one or more amino acid residues, for example, at an internal position or at the N-terminal or C-terminal position of a sequence that is at least 50% identical to the sequence of SEQ ID NO: 8. The term "sequence identity" and means for calculating sequence identity are defined herein in relation to BoNT/A, and the same definitions and means also apply for the discussion of Lys-C.

[0075] Typically, the sequence identity of the Lys-C enzyme is determined over the entire length of SEQ ID NO: 8 or 9, ie, over the length of 333aa or 581aa, respectively. The term "at least 50% sequence identity" as used herein means at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity.

[0076] The proteolytically active Lys-C polypeptide may have the same number of amino acids as the reference polypeptide sequence, as shown in SEQ ID NO: 8. Alternatively, the polypeptide may have additional amino acid residues or fewer residues of amino acids. For example, the proteolytically active polypeptide of the present invention may consist of or comprise a truncation mutant of SEQ ID NO: 8 or 9, or a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 8 or 9 The truncation mutant of SEQ ID NO: 9 can, for example, omit one or more amino acid residues at the N-terminus with respect to amino acid position 249. A truncation mutant can be an N-terminal or C-terminal truncation mutant that is proteolytically active. The truncation mutant of SEQ ID NO: 9 can omit amino acid positions 1 to 248 of SEQ ID NO: 9. Alternatively, the truncation mutant of SEQ ID NO: 9 can be a C-terminal truncation mutant. The truncation mutant can omit up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 150 or up to 170 consecutive amino acid residues. The proteolytically active polypeptide can have an amino acid length of at least 200 amino acid (aa) residues, at least 250aa residues, at least 300aa residues, or at least 333aa residues. Alternatively, the proteolytically active polypeptide can have up to 333aa residues, up to 350aa residues, up to 573aa residues, up to 581aa residues, up to 592aa residues, up to 600aa residues, or up to 617aa residues.

[0077] The proteolytically active polypeptide may include a polypeptide comprising additional N-terminal or C-terminal amino acid residues, and/or at an internal position of the polypeptide chain of SEQ ID NO: 8, or one of a sequence of polypeptides having at least 50% sequence identity with the sequence of SEQ ID NO: 8. Such additional amino acid residues may comprise up to 5, up to 10 or even up to 200, 300 or 400 consecutive amino acid residues. The additional amino acid residues may function as inhibitors of proteolytic activity. These additional amino acid residues can be removed by a protease. Alternatively, additional residues that inhibit the proteolytic activity of the polypeptide are excluded. Additional amino acid residues may be flanked by one or more protease cleavage sites. In another aspect, the additional amino acid sequence functions as a detectable marker and/or allows attachment to a solid support.

[0078] In another aspect, the polypeptide chain of SEQ ID NO: 8 or one of a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8 is modified by exchanging one or more amino acid residues. The term "exchange", as used herein, means replacing one amino acid with a different amino acid. For example, up to 1 amino acid (AA), 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 15aa, 20aa, or up to 50aa, may be substituted for the polypeptide sequence. The exchanges may involve conservative or non-conservative amino acid exchanges, with the aim, for example, of increasing or decreasing substrate binding or the proteolytic activity of the polypeptide.

[0079] Typically, the proteolytically active polypeptide encompasses a polypeptide that is capable of hydrolyzing a substrate (eg, a single-chain BoNT/A protein) to two or more native cleavage product(s). The polypeptide of the present invention can hydrolyze the substrate into two or more cleavage products, which are identical or different from the native cleavage products. Preferably, the cleavage products are identical to the native cleavage products.

[0080] The term "native cleavage products" or "native products", as used herein, refers to products that are identical in amino acid sequence when compared to products generated from the same substrate in wild-type cell cultures, from which the substrate originates. In a preferred embodiment, the cleavage product is the double chain neurotoxin of a botulinum neurotoxin or tetanus neurotoxin. In a more preferred embodiment, the double-chain neurotoxin is a neurotoxin isolated from C. botulinum, serotype A, B, CI, D, E, F or G. In yet another aspect, said double-chain neurotoxin is a neurotoxin native double-chain BoNT/A.

[0081] It should be understood that the definitions and explanations presented with respect to the terms described above and below, apply mutatis mutandis to all aspects described herein, unless otherwise indicated.

BoNT Production Methods

[0082] The present invention relates to the use of Lys-C in a method for proteolytically processing a polypeptide, specifically, a single-chain BoNT/A protein, and means for purifying the active BoNT/A double chain produced from Lys-C using hydrophobic interaction chromatography (HIC). In one aspect, the present invention relates to a method for manufacturing an active double-chain BoNT/A, comprising the step of contacting: (a) Lys-C with (b) a single-chain BoNT/A protein, said single-chain BoNT/A protein being susceptible to proteolysis by Lys-C, wherein said contacting results in proteolytic processing of said single-chain BoNT/A protein into at least two cleavage products, preferably including BoNT /A active double strand, and purification of said active double strand using HIC.

[0083] The method of the invention can be used to manufacture proteolytically processed clostridial neurotoxin (CNT) or botulinum neurotoxin (BoNT), specifically, proteolytically processed BoNT/A, i.e., double-stranded active BoNT/A, as described herein. By using the method of the present invention, it is now possible to obtain active double-stranded BoNT/A compositions with significantly less contamination caused by unprocessed or partially processed BoNT/A, since these contaminants are efficiently processed into the BoNT/A double chain. The methods of the present invention also allow an effective removal of the Lys-C enzyme used in the processing of the single-chain BoNT/A protein within the active duplex, i.e., an improved purification of the active double-chain BoNT/A.

[0084] Therefore, the present invention relates to the purification (resolution) of double-chain BoNT/A from Lys-C, comprising separation by hydrophobic interaction chromatography. Accordingly, the invention provides a method for producing a double-chain BoNT/A protein, comprising providing a single-chain BoNT/A protein, with following contacting said BoNT/A protein with Lys-C endoproteinase ( Lys-C) in solution, and separating the soluble BoNT/A protein from Lys-C by contacting the solution containing the BoNT/A protein and Lys-C with a hydrophobic surface, on which the BoNT/A protein preferably , binds to the hydrophobic surface. Typically, contacting the single-chain BoNT/A protein with Lys-C results in cleavage of the single-chain BoNT/A protein into a double-chain form of soluble BoNT/A protein. Preferably, the single-chain BoNT/A protein cleavage product by Lys-C is identical to the native BoNT/A protein cleavage product. Typically, both single-chain and double-chain forms of BoNT/A are soluble.

[0085] One or more fractions collected from a column chromatography can be concentrated, for example, by precipitation or ultrafiltration.

[0086] In one embodiment, where the invention provides a method (as described above) for producing a soluble double-chain BoNT/A protein, the soluble single-chain BoNT/A protein is provided by a method as described above, to produce the single-chain soluble BoNT/A protein in a host cell, preferably a bacterial host cell, more preferably an E. coli host cell.

[0087] Any suitable expression system can be used to produce the single-chain soluble BoNT/A protein. An expression system according to the present invention can be an in vivo expression system or an in vitro (cell-free) expression system. Examples of suitable in vivo and in vitro expression systems are known in the art. As defined herein, an expression system may comprise a suitable host cell and/or a suitable expression vector for use in said host cell. Thus, for example, a suitable expression system may comprise a bacterial host cell and/or a vector expression tubes, suitable for expressing the single chain soluble BoNT/A protein in said bacterial host cell.

[0088] The single-chain BoNT/A protein can be produced in any host cell as described herein, such as a bacterial cell. Typically, an E. coli host cell is sweaty. Single-chain BoNT/A protein can be produced by a method comprising expressing a nucleic acid sequence in a host cell (e.g., an E. coli cell) expression system. Such expression may involve introducing a nucleic acid molecule encoding said single-stranded BoNT/A protein into said host cell, and converting said nucleic acid molecule to produce single-stranded BoNT/A protein. Methods and techniques used for expressing heterologous proteins in expression systems, including E. coli expression systems, are well known in the art.

[0089] Typically, said single-chain soluble BoNT/A protein is expressed in the cytoplasm of said E. coli host cell.

[0090] Soluble single-chain BoNT/A protein can be expressed at a level (concentration) of at least 5 mg/L (e.g., at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 25, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL or more). Typically, the level of single chain BoNT/A protein expression refers to the concentration of single chain BoNT/A protein in the cell culture. In one embodiment, said expression level refers to a crude host cell homogenate (e.g., bacterial host cell), i.e., a crude host cell homogenate (e.g., bacterial host cell), used to express the single chain BoNT/A protein. Thus, in one embodiment, the expression level of the single-chain BoNT/A protein in the crude host cell homogenate, for example, in the crude bacterial host cell homogenate, is at least 5 mg/L (as defined herein). .

[0091] The method for producing a single-chain BoNT/A protein as described above may comprise lysing a host cell, preferably a bacterial host cell, more preferably an E. coli host cell, to provide a host cell homogenate, more preferably a bacterial host cell homogenate, most preferably an E. coli host cell homogenate containing said single chain soluble BoNT/A protein. Methods and techniques used to lyse host cells, such as bacterial cells, particularly E. coli host cells, are known in the art. Examples include the ultrasonication procedure or the use of a French press device.

[0092] Purification of the single-chain BoNT/A protein expressed by C. botulinum or E. coli can be done, for example, as substantially described in the prior art (DasGupta 1984, Toxicon, 22, 415; Sathyamoorthy 1985, J. Biol. Chemistry 260, 10461). In particular, the purification of the neurotoxin may contain one or more precipitation and extraction steps, one or more concentration steps, and also different chromatographic steps. Chain recombinant BoNT/A and methods for its purification are described in the state of the art (Rummel et al., 2004, Mol. Microbiol., 51:631-43).

[0093] The bacterial host cell used to produce the single-chain BoNT/A protein, or a derivative thereof, may be C. botulinum or E. coli. For fermentation, the process described by DasGupta B.R. et al., in the publication Toxicon, vol. 22, No.3, pages 414 to 424, 1984, may be used. Thus, 0.5% of yeast extract and 0.6% of autoclaved yeast paste are added to 2% N-Z-amine, of a type A medium, and the pH of 7.2 will be adjusted with the aid of NaOH 4M, with the medium being prepared in such a way, to be then autoclaved. Autoclaved glucose can be added separately to this medium (20% weight by volume) to reach a final glucose concentration of 0.5% in said medium. The incubation can take place, for example, at a temperature of 37°C, without agitation, whereby the fermentation is discontinued, for example, after 96 hours. Batch fermentation, semi-batch fermentation, repeated batch fermentation or continuous fermentation methods can be used.

[0094] After the occurrence of fermentation and separation of the fermentation medium from the cells, the fermentation medium can be subjected to a first precipitation step in order to remove the large proteins. The precipitation preferably is an acid precipitation. The reaction conditions for such acid precipitation are known to those skilled in the art. Typically, 1.5 M H 2 SO 4 can be used to acidify the supernatant to a pH of 3.5. Centrifugation normally takes place for 20 minutes at 2400x g at a temperature of 4°C. The pellet received from the centrifugation step can be washed with water, preferably repeatedly. Then, the pellet can be extracted with a citric acid-trisodium citrate 0.1 M buffer, pH 5.5, for a period of, for example, one hour. Then, an additional centrifugation step can be performed, for example, at 9800x g, for 20 minutes, at a temperature of 4°C. The pellet thus obtained, optionally, can be extracted again, as previously described. The extraction supernatant, and both supernatants if the extraction is repeated, can then be subjected to protamine sulfate precipitation. Precipitation may continue overnight, for example at a temperature of 8°C. Afterwards, the precipitate can be centrifuged, for example, for 20 minutes at 4°C and 12,000x g. The centrifugation supernatant can be subjected to a precipitation step, for example ammonium sulphate precipitation, whereby other larger proteins can be removed. After the ammonium sulfate precipitation step, another centrifugation step can be added and then the pellet thus obtained can be redissolved and, optionally, subjected to dialysis. The extract, which is preferably dialyzed and centrifuged again, can be subjected to a succession of chromatography steps, with the aim of purifying the neurotoxin. Each of the chromatography steps serves to remove contaminants such as protamine sulfate, remnant DNA, parts of smaller proteins and medium-sized proteins, as well as the hemagglutinins of the botulinum neurotoxin protein complex. For this purpose, one or more chromatography steps can be used in a preferred embodiment. Optionally, the eluate, for example, from the last chromatography step can be filtered to remove microorganisms. Optionally, the eluate can be diluted before filtration and suitable adjuvants can be added. During later steps, another sterile filtration can be performed after adding the adjuvants. In one aspect, filtration is performed in filtration vessels, which may then be subjected to a freeze-drying step.

[0095] The single-chain BoNT/A protein can be contacted with Lys-C after it is isolated from the host cell or from the host cell lysate, and then subjected to the method according to the present invention.

[0096] When the single-chain BoNT/A protein according to the invention is contacted with Lys-C, the proteolytic action of Lys-C cleaves the single-chain protein at a site between the component-chain protease L and the translocation component, in order to produce a double-chain protein, where the two chains are linked by a disulfide bridge. Thus, for example, the two chains formed after cleavage of single-chain BoNT/A, i.e., A2 and A4-A6, at the activation site, constitute a first chain of amino acid residues 1-438 and a second chain of amino acid residues 449-1296 (except in A6, where the second strand has amino acid residues 449-1297), with residues 439447 removed by the cleavage event. Thus, Lys-C can be used to activate the single-chain polypeptide by converting it to the active double-chain form. Thus, advantageously, the use of Lys-C means that it is not necessary to construct an exogenous (non-native) cleavage site within a BoNT/A, also enabling the production of double-stranded native BoNT/A.

[0097] In one embodiment, reference to the Lys-C enzyme includes Lys-C-like enzymes and alternative enzymes, which cleave at the same Lys-C protease cleavage site, as described herein.

[0098] The term "contacting", as used herein, refers to arranging at least two different compounds in physical proximity, in order to allow physical and/or chemical interaction of said compounds. According to the method of the present invention, said two different compounds include a single chain BoNT/A protein and Lys-C, contained in a solution. The contact is performed under certain conditions and for a time that is sufficient to allow the interaction of the single-chain BoNT/A protein and Lys-C.

[0099] The term "being susceptible to proteolysis" refers to a characteristic or requirement of the single-chain BoNT/A protein, being used herein to mean that said single-chain BoNT/A protein is proteolytically cleavable by Lys-C. In other words, "being susceptible to proteolysis" means that the single-chain BoNT/A protein comprises a protease recognition and cleavage site, which allows it to function as a Lys-C substrate. As described herein, the single-chain BoNT/A protein is a Lys-C substrate, being proteolytically processed into two or more cleavage products (preferably, just two peptides, the L-chain or fragment thereof, and H or fragment thereof, joined by a disulfide bond). Using the assay described above, a person skilled in the art can test whether a given single-chain BoNT/A protein is a first polypeptide substrate and thus a "second polypeptide" as defined by the present invention. The term "at least two cleavage products" includes, for example, up to two, three, four, five and even six cleavage products.

[0100] The present method can be used, for example, for preparing a pharmaceutical composition comprising a double-chain BoNT/A or for generating fragments of polypeptides used in mass spectrometry processes. Lys-C and the single-chain BoNT/A protein can be contacted at several steps in the double-chain BoNT/A manufacturing process. For example, the step of contacting Lys-C with the single-chain BoNT/A protein can be carried out within a cell, by expressing Lys-C and the single-chain BoNT/A protein in said cell.

[0101] Alternatively, said contacting step is carried out on a cell lysate or on a purified cell lysate. This includes adding Lys-C to the lysate or purified lysate. Lys-C can be added in several steps during the purification of single-chain BoNT/A protein from cell lysate. For example, Lys-C can be added before or after protein precipitation steps, chromatography ion exchange, hydrophobic interaction chromatography and/or size exclusion chromatography.

[0102] The contact step requires incubation under certain conditions and for a sufficient time for Lys-C to cleave the single-chain BoNT/A protein. Exemplary conditions may comprise adding a buffer selected from the group consisting of 100 mM Tris-HCl, pH 8.0, or PBS (50 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.4). Preferred buffer conditions include 100 mM Tris-HCl, pH 8.0. The term "sufficient time for cleavage" can be determined using the assay described hereinabove. In one aspect, said "sufficient time for cleavage" depends on the degree of cleavage that a proteolytically processed polypeptide should have, or a composition comprising the same should have. In one aspect, the method comprises a step of incubating the Lys-C and single-chain BoNT/A protein for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another aspect, Lys-C and single-chain BoNT/A protein are incubated for up to 30 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or up to 600 minutes. In another aspect, the method comprises a step of incubating Lys-C and single-chain BoNT/A protein at either 4°C or 37°C. In another aspect, the method comprises an incubation step for up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours or up to 16 hours.

[0103] In one embodiment, in which the invention provides a method (as described above) for producing a soluble double-chain BoNT/A protein, the method comprises separating the soluble BoNT/A protein from Lys-C by contacting the solution containing soluble BoNT/A and Lys-C protein with a hydrophobic surface, wherein the soluble BoNT/Lys-C protein preferentially binds to the hydrophobic surface.

[0104] The present inventors have found that high yields of activated double-chain BoNT/A protein can be obtained by using a hydrophobic purification process to separate the activated double-chain polypeptide from Lys-C. Surprisingly, this process provides superior purification over standard purification using ion exchange chromatography, which the present inventors have found to be less effective for separating activated double-stranded polypeptide from Lys-C. Furthermore, the process advantageously provides an activated double-stranded BoNT/A protein free of activation protease, thereby suitable for use in therapy as part of a general purification process.

[0105] As described herein, the production of recombinant active BoNT/A requires a proteolytic step that promotes cleavage of the molecule to the active double-stranded form. This cleavage can be obtained through an in vitro activation step, using the Lys-C endoproteinase. After the activation step it is important to remove Lys-C from the final product, which also prevents any further non-specific BoNT/A cleavage.

[0106] As shown in Table 2, the calculated isoelectric points (pI) of Lys-C and BoNT/A are 6.70 and 6.05, respectively, which indicates that the separation of the two proteins can be obtained by a process Ion Exchange Chromatography (IEX), which exploits the charge difference between the two molecules. A net protein charge is affected by the pH of its surrounding environment and will become more positively or negatively charged depending on the gain or loss of protons. The isoelectric point (pi) is the pH value at which a molecule carries no electrical charge and therefore will not interact with a charged IEX medium. This means that if a protein is at a pH above its isoelectric point then it will carry a net negative charge and will bind to a positively charged medium such as an anion exchanger. Similarly, if the pH of the buffer is below the isoelectric point (pI), then the protein will carry a net positive charge and will not bind to an anion exchanger.

[0107] Furthermore, as illustrated in Table 2, BoNT/A and Lys-C have similar average hydropathicities, but large differences in charge at pHs 4.5 and 8.0. Based on this principle, it can be expected that ion exchange chromatography (IEX) can be used to resolve (separate) BoNT/A and Lys-C. IEX chromatography is a simple and inexpensive chromatographic method, as it does not require the protein introduced into the column to be in a highly saline buffer, which can lead to loss of protein by precipitation.

[0108] The present inventors tested a variety of ion exchange columns, using strong and weak functional groups attached to cross-linked agarose beads, at pH 8.0. When compared to the BoNT/A elution, it was unexpectedly found that Lys-C was eluted at a similar ionic strength (Table 3; Figures 7 to 10), which indicates that Lys-C did not separate as predicted, and that could be present in the final purified BoNT/A product, with an additional possibility of further BoNT/A decomposition.

[0109] The present inventors solved the above problem. In greater detail, surprisingly, the inventors found that optimal separation of Lys-C-BoNT/A is achieved using a hydrophobic separation surface (e.g., using hydrophobic interaction chromatography (HIC). chromatography), which separates proteins according to differences in their surface hydrophobicities, through the use of a reversible interaction between these proteins and the hydrophobic surface of an HIC matrix/resin).

[0110] Typically, the active double-chain BoNT/A preferentially binds to the hydrophobic surface of the HIC resin/matrix (these terms are used interchangeably herein), compared to the binding of Lys-C to the hydrophobic surface . "Preferential" binding can be defined as an increase or improvement in binding of double-stranded active BoNT/A to the hydrophobic surface compared to binding of Lys-C to the hydrophobic surface. For example, double-stranded active BoNT/A can bind to the hydrophobic surface at least twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, 15 times , 20 times, 25 times, 30 times, 40 times, 50 times, 100 times or more the affinity of Lys-C to the hydrophobic surface. In a preferred embodiment, double-stranded active BoNT/A binds to the hydrophobic surface with at least five times, or at least ten times the affinity of Lys-C to the hydrophobic surface.

[0111] In one embodiment, the hydrophobic surface is an inert matrix/resin to which a linker comprising or consisting of aryl or alkyl groups is affixed.

[0112] The term "aryl" refers to aromatic groups, for example, phenyl, naphthyl, thienyl and indolyl.

[0113] The term "alkyl" refers to aliphatic groups, including straight-chain groups, branched-chain groups, cyclic groups, and combinations thereof. An alkyl group can have from 1 to 12 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g. n-propyl, isopropyl), butyl (e.g. n-butyl, isobutyl, sec-butyl, t-butyl), pentyl , hexyl, heptyl and octyl.

[0114] In one embodiment, the hydrophobic surface comprises one or more binders selected from the group consisting of butyl, phenyl, or octyl binders.

[0115] In one embodiment, the hydrophobic surface comprises butyl binders. In one embodiment, the hydrophobic surface comprises phenyl linkers. In one embodiment, the hydrophobic surface comprises octyl ligands.

[0116] The hydrophobic surface may contain any suitable inert matrix/resin, with a suitable hydrophobic binder attached. Thus, for example, the inert matrix/resin may be selected from silica, cross-linked dextran, cross-linked polyacrylamide or cross-linked agarose, and the like. Also, particularly included are polypeptides, glass, polystyrene, polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon, amylases, natural and modified celluloses, polyacrylamide, gabbro and magnetite. The inert matrix is, for example, a polysaccharide matrix selected from the group consisting of sepharose, sephadex, agarose, sephacel, microcellulose, and alginate beads. In another aspect, said matrix/resin may consist of glass beads and/or polypeptide matrices.

[0117] The inert matrix/resin may have any suitable configuration or structural arrangement. Thus, for example, the matrix/resin can be spherical, as in the case of a bead, or cylindrical, as in the case of an internal surface of a test tube, or an external surface of a stick. Alternatively, the matrix can be irregular or flat, such as a test sheet or strip.

[0118] The present inventors have discovered that particularly preferred results for the separation of Lys-C from BoNT/A are obtained by hydrophobic interaction chromatography (HIC), using chromatography matrices/resins containing alkyl or aryl groups, e.g. butyl, phenyl and octyl, coupled to an inert matrix/resin, such as cross-linked agarose or polystyrene beads (Table 4; Figures 1 to 3).

[0119] The use of hydrophobic interaction chromatography (HIC) provides an improved resolution (separation) of Lys-C from double-chain active BoNT/A compared to the use of ion exchange chromatography (IEX).

[0120] According to the present invention, "fast flow" HIC chromatographic resins/matrices can be used. A “fast flow” resin/matrix can be defined as a resin/matrix that has a more uniform average particle size compared to a standard HIC resin/matrix, in addition to using smaller particles. Specifically, a “fast flow” resin/matrix will have a narrower range of smaller particle sizes. Particle size can be quantified in any suitable way known in the art, for example in terms of average particle diameter. Typically, a "fast flow" resin/matrix will comprise particles (eg beads) of an average diameter of less than 100 µm, less than 95 µm, less than 90 µm, or less. In a preferred embodiment, the average diameter of a "fast flow" resin/matrix is less than 90 µm. Typically, the average particle diffusion of a "fast flow" resin/matrix will be from about 20 to about 180 µm, from about 30 to about 170 µm, from about 40 to about 170 µm , from about 40 to about 165 µm. In a preferred embodiment, the average particle diffusion of a "fast flow" resin/matrix is from about 44 to about 165 µm.

[0121] In a preferred embodiment, high performance HIC resins are used, such as High Performance Phenyl resins (PhHP) or High Performance Butyl resins (BuHP). These high-performance HIC matrices/resins typically provide improved Lys-C resolution of double-chain active BoNT/A compared to the ion exchange chromatography (IEX) technique, even when matrices/resins are used. high performance IEX resins such as High Performance Quaternary Amine (QHP) matrix/resin. As discussed herein, the main difference between high performance resins/matrices and other resins/matrices is that the average particle size (again, said size can be quantified by any appropriate means known in the art, e.g. average particle diameter) it is smaller (34 μm vs. 90 μm) and smoother (24-44 μm vs. 44-165 μm), which results in improved resolution.

[0122] A high-performance HIC matrix/resin typically has a more uniform average particle size than a standard HIC resin/matrix, or even a "Fast Flow" resin/matrix as defined herein, and will use smaller particles. Specifically, a high performance HIC resin/matrix will have a narrower range of smaller particle sizes than a standard HIC resin/matrix, or even a “Fast Flow” resin/matrix as defined herein.

[0123] Thus, the present invention provides the use of high performance HIC resins/matrices. Typically, a high performance HIC resin/matrix has an average particle diameter of less than 70 µm, less than 60 µm, less than 50 µm, less than 40 µm, less than 30 µm, or less. In a preferred embodiment, a high performance HIC resin/matrix has an average particle diameter of less than 40 µm, more preferably less than 35 µm, for example an average particle diameter of 34 µm.

[0124] Typically, the average particle diffusion of a high performance HIC resin/matrix will be from about 10 to about 60 µm, from about 10 to about 50 µm, from about 20 to about 50 µm, from about 20 to about 44 µm. In a preferred embodiment, the average particle diffusion of a high performance resin/matrix is from about 22 to about 44 µm.

[0125] Typically, the hydrophobic purification process to separate activated double-stranded BoNT/A protein from Lys-C reduces the concentration of Lys-C by at least two-fold, at least 5-fold, at least 10-fold, at least 10-fold. at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times. In a preferred embodiment, the hydrophobic purification process to separate the activated double-stranded BoNT/A protein from Lys-C reduces the concentration of Lys-C by at least 10-fold.

[0126] The ability of hydrophobic purification to separate activated double-chain BoNT/A protein from Lys-C can also be quantified in terms of the percentage of Lys-C contained in the starting solution comprising activated double-chain BoNT/A and the remaining Lys-C after the hydrophobic purification step. Typically less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20 %, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10 %, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or percentages Less than 0% of the Lys-C remains in the double-stranded BoNT/A product after the hydrophobic purification step. Preferably less than 50%, more preferably less than 25%, even more preferably less than 10% of the Lys-C remains in the double-stranded BoNT/A product after the hydrophobic purification step.

[0127] In a related aspect, the invention provides an active double-chain BoNT/A protein obtainable by a method (as described above) for producing soluble double-chain BoNT/A protein.

[0128] In one aspect, the invention provides a composition comprising an active double-chain BoNT/A protein (as described above), wherein said composition is substantially free of Lys-C.

[0129] Thus, advantageously, the composition is substantially free of Lys-C protease (used to activate the single-chain polypeptide, upon conversion thereof to the active double-chain form), thereby preventing unwanted non-specific cleavage of the BoNT/A protein.

[0130] When the composition (as described above) is substantially free of Lys-C, the composition typically contains less than 400 picograms (pg) of Lys-C per 100 ng of BoNT/A protein; for example less than 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 5, 4, 3, 2, 1 pg or less of Lys-C per 100 ng of BoNT/A protein. In one embodiment, the composition (as described above) contains less than 400 pg Lys-C per 100 ng BoNT/A protein, less than 350 pg Lys-C per 100 ng BoNT/A protein, less than 300 pg Lys-C per 100 ng BoNT/A protein, less than 250 pg Lys-C per 100 ng BoNT/A protein, less than 200 pg Lys-C per 100 ng BoNT/A protein A, less than 150 pg of Lys-C per 100 ng of BoNT/A protein, less than 100 pg of Lys-C per 100 ng of BoNT/A protein, less than 50 pg of Lys-C per 100 ng of BoNT/A protein, less than 40 pg Lys-C per 100 ng BoNT/A protein, less than 30 pg Lys-C per 100 ng BoNT/A protein, less than 20 pg Lys-C per 100 ng BoNT/A protein, less than 15 pg Lys-C per 100 ng BoNT/A protein, less than 14 pg Lys-C per 100 ng BoNT/A protein, less than 13 pg of Lys-C per 100 ng BoNT/A protein, less than 12 pg Lys-C per 100 ng BoNT/A protein, less than 11 pg Lys-C per 100 ng BoNT/A protein, less than 10 pg Lys-C per 100 ng BoNT/A protein, less than 9 pg Lys-C per 100 ng BoNT/A protein, less than 8 pg Lys-C per 100 ng protein BoNT/A, less than 7 pg Lys-C per 100 ng BoNT/A protein, less than 6 pg Lys-C per 100 ng BoNT/A protein, less than 5 pg Lys-C per 100 ng of BoNT/A protein, or even lower values. In a preferred embodiment, the composition (as described above) contains less than 100 pg Lys-C per 100 ng BoNT/A protein, or less than 50 pg Lys-C per 100 ng BoNT/A protein, less less than 20 pg Lys-C per 100 ng BoNT/A protein, less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg Lys-C per 100 ng BoNT/A protein BoNT/A,

[0131] Methods for determining the concentration of Lys-C in a composition are known in the art. By way of example, the concentration of Lys-C in a composition of the invention can be determined using a sandwich ELISA (Enzyme-Linked Immunosorbent Assay) procedure or a colorimetric assay, as described herein.

Pharmaceutical Compositions and Therapeutic Indications

[0132] The present invention also relates to a composition that can be obtained by the method presented here, for the manufacture of a double-chain BoNT/A. In one aspect, said composition comprises a mixture of processed BoNT/A (double chain) and unprocessed BoNT/A (single chain), wherein said mixture may contain less than 5%, 4%, 3%, 2% , or less than 1% unprocessed BoNT/A (single chain). In one aspect of said composition, references to BoNT/A include a derivative thereof as defined herein. The composition may be, for example, a liquid composition or a solid composition, and may contain one or more carriers, adjuvants and/or excipients.

[0133] In another aspect, the present invention is also correlated to a method for manufacturing a drug, that is, a pharmaceutical composition, comprising the steps of the aforementioned method and the subsequent step of formulating purified double-chain BoNT/A as medicine. Typically, said medicament comprises a blend of processed BoNT/A (double chain) and unprocessed BoNT/A (single chain), wherein said blend comprises less than 20%, less than 15%, less than 10%, less 5% or less of unprocessed (double-chain) BoNT/A. In a preferred embodiment, the blend contains less than 5% unprocessed (single chain) BoNT/A, such as less than 4%, less than 3%, less than 2%, or less than 1% BoNT/A unprocessed (single string).

[0134] The present invention also relates to various medical and aesthetic uses (cosmetic application) of the compounds and compositions disclosed herein. Accordingly, the present invention relates to an active double-chain BoNT/A or a composition comprising an active double-chain BoNT/A, according to the invention, for use as a medicine or in a pharmaceutical composition. The present invention also relates to the use of an active double-chain BoNT/A, or a composition comprising the active double-chain BoNT/A, according to the invention, in the manufacture of a medicament. The present invention also relates to the use of an active double-chain BoNT/A, or a composition comprising the active double-chain BoNT/A, according to the invention, for use in a method of treating the body of a human or an animal, through therapy. The present invention also relates to a treatment method, comprising administering the active double-chain BoNT/A or a composition comprising an active double-chain BoNT/A, according to the invention, to a patient in need thereof.

[0135] The term "composition", as used herein, refers to any composition formulated in solid, liquid, aerosol (or gaseous) form, and the like. Said composition comprises, for example, a therapeutically active compound according to the invention, optionally together with suitable auxiliary compounds, such as diluents or vehicles, or additional ingredients. In one embodiment, the therapeutically active compound is double-chain active BoNT/A, according to the present invention. Compositions, particularly pharmaceutical compositions, according to the invention are typically substantially free of Lys-C, as defined above.

[0136] Thus, the present invention provides a solid or liquid pharmaceutical composition, comprising: (a) an active double-chain BoNT/A protein as described above; and (b) a stabilizing agent.

[0137] In one embodiment, the composition (as described above) is substantially free of Lys-C. For example, the composition (as described above) may contain less than 400 pg Lys-C per 100 ng BoNT/A protein, less than 300 pg Lys-C per 100 ng BoNT/A protein, less than 200 pg Lys-C per 100 ng BoNT/A protein, less than 100 pg Lys-C per 100 ng BoNT/A protein, less than 50 pg Lys-C per 100 ng BoNT/A protein A, less than 20 pg Lys-C per 100 ng BoNT/A protein, less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg Lys-C per 100 ng of BoNT/A protein.

[0138] Stabilizing agents that can be used in compositions according to the present invention include protein stabilizers such as albumin, specifically human serum albumin (HSA), and non-protein stabilizers.

[0139] Non-protein stabilizing agents that can be used in the compositions according to the invention include surfactants, specifically, nonionic surfactants. Examples of nonionic surfactants include polysorbates, such as polysorbate 20 or polysorbate 80, and block copolymers, such as poloxamers (ie, polyethylene and propylene glycol copolymers).

[0140] In a specific embodiment, the composition does not comprise a protein as a stabilizing agent.

[0141] According to a specific embodiment of the invention, the pharmaceutical composition is a liquid pharmaceutical composition, comprising: (a) an active double-chain BoNT/A protein, as described above; (b) a non-protein stabilizing agent , which is a surfactant; and (c) water;

[0142] wherein said liquid pharmaceutical composition does not comprise a protein stabilizing agent; It is

[0143] wherein said liquid pharmaceutical composition is substantially free of Lys-C, where "substantially free of Lys-C" is as defined herein (e.g., said liquid pharmaceutical composition contains less than 400 pg of Lys-C per 100 ng of BoNT/A protein, less than 300 pg of Lys-C per 100 ng of BoNT/A protein, less than 200 pg of Lys-C per 100 ng of BoNT/A protein, less than 100 pg of Lys-C per 100 ng BoNT/A protein, less than 50 pg Lys-C per 100 ng BoNT/A protein, less than 20 pg Lys-C per 100 ng BoNT/A protein, less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg Lys-C per 100 ng BoNT/A protein).

[0144] In one embodiment, the active double-chain BoNT/A protein is present in the composition (as described above) at a concentration of 1-1000 ng/ml, or more. Thus, the active double-chain BoNT/A protein is present in the composition (as described above) at a concentration of about 1-500 ng/ml, e.g. about 1, 5, 10, 15, 20, 25 ,30,35,40,45,50,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950 or 1000 ng/ mL, or higher values. In a preferred embodiment, the active double-stranded BoNT/A protein is present at a concentration of about 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml. mL or 700 ng/mL.

[0145] In one embodiment, the surfactant (as described above) is a polysorbate, such as a polysorbate having an average degree of polymerization ranging from 20 to 100 monomer units, and may, for example, be polysorbate 80. In the preferred embodiment, the polysorbate is a vegetable derivative. The surfactant concentration is preferably less than 1% vol/vol, for example from about 0.005% to 0.02% vol/vol, in the case of polysorbate 80.

[0146] The pharmaceutical composition according to the invention may also comprise a crystalline agent.

[0147] The term crystalline agent means an agent that, inter alia, maintains a mechanically strong pie structure for a lyophilized botulinum neurotoxin complex (type A, B, CI, D, E, F or G) or a botulinum neurotoxin of high purity (type A, B, CI, D, E, F or G). When included in solid formulations, crystalline agents also have a filling effect. Crystalline agents suitably include sodium chloride. The crystalline agent concentration may be, for example, from 0.1 to 0.5M, preferably from 0.1 to 0.4M, suitably about 0.15 to 0.3M.

[0148] The pharmaceutical composition according to the invention may also comprise a buffer, to maintain a pH level comprised between 5.5 and 7.5, or between 6.0 and 7.0. The buffer can be any buffer capable of maintaining a suitable pH. For example, the buffer for compositions according to the invention may be chosen from the group consisting of succinate, disodium phosphate/citric acid, and an amino acid such as histidine. The buffer concentration can be, for example, from 1 to 50 mM, preferably from 5 to 20 mM, most preferably about 10 mM.

[0149] The pharmaceutical compositions according to the invention may also comprise a disaccharide.

[0150] The disaccharide used in the compositions according to the invention can be chosen from the group consisting of sucrose, trehalose, mannitol and lactose. In a specific embodiment, the disaccharide is sucrose. The concentration of the disaccharide can be, for example, from 5 to 50 mM, preferably from 5 to 25 mM, more preferably from 10 to 20 mM, and most preferably from about 11.7 mM.

[0151] In a specific embodiment, the pharmaceutical composition is a liquid pharmaceutical composition, comprising: (a) an active double-chain BoNT/A protein, as described above; (b) a non-protein stabilizing agent, which is a surfactant; (c) sodium chloride; (d) a buffer to maintain the pH between 5.5 and 7.5; (e) a disaccharide; and (f) sterilized water;

[0152] wherein said liquid pharmaceutical composition does not comprise a protein stabilizing agent; It is

[0153] wherein said liquid pharmaceutical composition is substantially free of Lys-C, where "substantially free of Lys-C" is as defined herein (e.g., said liquid pharmaceutical composition contains less than 400 pg of Lys-C per 100 ng of BoNT/A protein, less than 300 pg of Lys-C per 100 ng of BoNT/A protein, less than 200 pg of Lys-C per 100 ng of BoNT/A protein, less than 100 pg of Lys-C per 100 ng BoNT/A protein, less than 50 pg Lys-C per 100 ng BoNT/A protein, less than 20 pg Lys-C per 100 ng BoNT/A protein, less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg Lys-C per 100 ng BoNT/A protein).

[0154] According to a specific embodiment, the pharmaceutical composition according to the invention that is in liquid form is sealed in a vial or in a ready-to-use device, such as a syringe, without any liquid/gas interface, being stable for at least three months or at least six months at 23 to 27°C and for at least twelve months at 2-8°C. Examples of pharmaceutical compositions according to the invention will be described in the Examples.

[0155] Monthly decomposition rates for pharmaceutical compositions or formulations of the invention can be below 5% per month for 12 weeks, which means that double-chain BoNT protease function remains stable at 25°C for at least 12 weeks.

[0156] The pharmaceutical compositions of the present invention can be stored in vacuum-dried, lyophilized form, in containers under vacuum pressure, or as stable liquids. Prior to lyophilization, the active double-chain BoNT/A protein can be combined with pharmaceutically acceptable excipients, stabilizers and/or vehicles such as albumin. The lyophilized material can be reconstituted with saline or water to create a solution or composition containing double-chain BoNT/A active protein to be administered to a patient.

[0157] In this context, it should be distinguished for the present invention, the difference between the auxiliary compounds, that is, the compounds that do not contribute to the effects elicited by the compound of the present invention after applying the composition for its desired purpose, and additional ingredients, i.e., compounds that contribute an aftereffect or that modulate the effect of the compound of the present invention. Suitable diluents and/or carriers depend on the purpose for which the composition is to be used, as well as the other ingredients. A person skilled in the art can determine such suitable diluents and/or vehicles without any difficulties.

[0158] The vehicle(s) must be acceptable, in the sense of being compatible with the other ingredients of the formulation, and not being harmful to the receiver(s) of the same(s). The pharmaceutical carrier employed can include a solid, a gel or a liquid. Examples of solid carriers include lactose, magnesia, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Examples of liquid carriers include phosphate buffered saline, syrup, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or diluent may include time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, used alone or together with a wax. Said suitable carriers include those mentioned above and others well known in the art, see, for example, Remington's Pharmaceutical Sciences publication, Mack Publishing Company, Easton, Pennsylvania.

[0159] The diluent(s) is/are selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution; in addition, the pharmaceutical composition or formulation may also include other non-toxic, non-therapeutic, non-immunogenic carriers, adjuvants or stabilizers, and the like.

[0160] In one aspect, a pharmaceutical composition as used herein, comprises the biologically active neurotoxin obtained by the method described in the present invention (i.e. double chain active BoNT/A) and, optionally, one or more pharmaceutically acceptable carriers. The active neurotoxin can be present in liquid or lyophilized form. In one aspect, said compound may be present together with glycerol, protein stabilizers (e.g., human serum albumin (HSA)), or non-protein stabilizers, such as polyvinylpyrrolidone or hyaluronic acid. In one aspect, the pharmaceutical composition is administered topically. Conventionally, drug administration is by the intramuscular or subcutaneous route (typically, close to the sebaceous glands). However, depending on the nature and mode of action of a compound, the pharmaceutical composition can be administered by other routes as well. The double-chain neurotoxin polypeptide is the active ingredient of the composition and, in one aspect, is administered in conventional dosage forms, prepared by combining the drug with standard pharmaceutical carriers in accordance with conventional procedures. These procedures may involve mixing, granulating and compressing, or dissolving the ingredients, if appropriate, into the desired preparation. It should be noted that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient which is to be combined, the route of administration and other well known variables.

[0161] A therapeutically effective dose refers to an amount of the compound, the neurotoxin, to be used in the pharmaceutical composition according to the present invention, which prevents, improves or treats the symptoms accompanying a disease or diseased condition referred to in the present description. The therapeutic efficacy and toxicity of the compound can be determined using standard pharmaceutical procedures, in cell cultures or in animals used in experiments, for example, ED50 (the therapeutically effective dose in 50% of the population), and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as a ratio, LD50/ED50.

[0162] The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any given patient are dependent on a number of factors, including the patient's size, body surface area, age, the specific compound being administered, gender, time and route of administration. , general health status, and other medications that are being administered concurrently. Progress can be monitored through periodic assessment. The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat, ameliorate or prevent a disease or diseased condition indicated in the present description. However, said pharmaceutical compositions can be administered more than once.

[0163] As described herein, the active double-chain BoNT/A proteins according to the invention, and compositions and liquid pharmaceutical compositions thereof, can be used in therapy. Appropriate therapies may include cosmetic treatments and medical treatment methods.

[0164] In a further aspect of the invention, the above-mentioned composition is a medicine or a cosmetic composition. In one aspect, said medicament comprising the biologically active neurotoxin (i.e. the double chain active BoNT/A) can be used for prevention and/or treatment of at least one of the following diseases and disorders: voluntary muscle strength, focal dystonia , including cervical dystonia, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic correction of wrinkles; in a further aspect, also, blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, eyelid apraxia, cervical opening dystonia, antecollis, retrocollis, laterocollis, torticollis, dystonia pharyngeal dystonia, laryngeal dystonia, adductor-type spasmodic dystonia, abductor-type spasmodic dystonia, spasmodic dyspnoea, limb dystonia, arm dystonia, task-specific dystonia, writer's cramp, musician's cramp, golfer's cramp, leg dystonia, adduction thigh, knee flexion by thigh abduction, knee strain, ankle sprain, ankle strain, equinovarus (clubfoot), foot strain dystonia, striated toe, toe flexion, toe strain foot, axial dystonia, Pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalized dystonia, Lubag dystonia, dystonia in corticobasal degeneration, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden Spatz disease, dopa-induced dyskinesia/dopa-induced dystonia, tardive dyskinesia/tardive dystonia, paroxysmal dyskinesia, dyskinesias/dystonias, induced palatal myoclonus with kinesiogenic or non-kinesiogenic action, myoclonic myokymia, rigidity, cramp benign muscularis, hereditary chin tremor, paradoxical jaw muscle activity, hemimasticatory spasms, branchial hypertrophic myopathy, maseteric hypertrophy, anterior tibial hypertrophy, nystagmus, gaze palsy with supranuclear oscillopsia, epilepsy, partial or continuous, spasmodic torticollis operation planning, abductor vocal cord paralysis, recalcitrant mutational dysphoria, upper esophageal sphincter dysfunction, vocal fold granuloma, Gilles de la Tourette's stuttering syndrome, middle ear myoclonus, protective laryngeal closure, post-laryngectomy, speech impairment, protective ptosis , sphincter entropion Odii dysfunction, pseudoachalasia, nonachalsia, esophageal motor disorders, vaginismus, postoperative immobilization tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, use scrotum cosmetic, facial asymmetries of frowning, dimples in the lower lip elevator, rigid person syndrome, tetanic hyperplasia of the prostate, adiposities, treatment of children with strabismus due to cerebral palsy, with concomitant paralytic mixture, after detachment surgery retina, after cataract surgery, in strabismus from myositic aphacia, myopathic strabismus, vertical dissociated shunt as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey's syndrome, Crocodile Tears syndrome, hyperhidrosis, palmoplantar axillary rhinorrhea, relative hypersalivation in strokes, in Parkinson's disease, in spastic conditions of amyotrophic lateral sclerosis, in autoimmune processes of encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic's syndrome, viral infections, bacterial infections, parasitic infections, infections caused by fungi, in hemispheric infarction caused by postapoplectic syndrome of hereditary spastic paraparesis, brainstem infarction, spinal cord infarction, migraine, in central nervous system trauma, hemispheric lesions, brainstem lesions, spinal cord injury spinal, in central nervous system haemorrhage, intracerebral haemorrhage, subarachnoid haemorrhage, subdural haemorrhage, intraspinal haemorrhage, in neoplasms, hemispheric tumours, brainstem tumours, spinal cord tumours, in cases of snoring (patent document WO 2000/033863) . For more details and symptoms, see, for example, the article by Jost, 2007, Drugs 67(5), 669 or the article by Dressier, 2000, Botulinum Toxin Therapy, Thieme Verlag, Stuttgart, N. York.

[0165] In another aspect of the invention, the composition is a cosmetic composition that can be formulated as described for the pharmaceutical composition above. For a cosmetic composition, it is likewise intended that the compound of the present invention, in one aspect, be used in a substantially pure form. Cosmetic compositions, in a further aspect, must be applied intramuscularly. In yet another aspect of the invention, cosmetic compositions comprising the neurotoxin can be formulated as an anti-wrinkle solution.

[0166] All references cited in the present description are hereby incorporated by reference, with respect to the entire content of the disclosure and the content of the disclosure specifically mentioned in the present description. Legends for SEQ ID NOs - SEQ ID NO: 1: BoNT/A from ATCC 3502, Genbank acc. "AAA23262"; - SEQ ID NO: 2: BoNT/A1 Activation Coil; - SEQ ID NO: 3: BoNT/A2/A6 Activation Coil; - SEQ ID NO: 4: BoNT/A3 Activation Coil; - SEQ ID NO: 5: BoNT/A3 Activation Coil; - SEQ ID NO: 6: BoNT/A4 Activation Coil; - SEQ ID NO: 7: BoNT/A5 Activation Coil; - SEQ ID NO: 8: Proteolytically active polypeptide derived from a strain of Clostridium botulinum, ATCC 3502, GenBank Accession No: "CAL82988.1", omitting 248 N-terminal amino acid residues; - SEQ ID NO: 9: Proteolytically inactive polypeptide derived from a strain of Clostridium botulinum, ATCC 3502, GenBank Accession No: "CAL82988.1"; - SEQ ID NO: 10: Nucleic acid sequence encoding SEQ ID NO: 8; - SEQ ID NO: 11: Nucleic acid sequence encoding SEQ ID NO: 9; - SEQ ID NO: 12: "Streptag" amino acid sequence; - SEQ ID NO: 13: BoNT/A7 Activation Coil; - SEQ ID NO: 14: BoNT/A8 Activation Coil; - SEQ ID NO: 15: Amino acid sequence derived from the subserotype A1 of botulinum neurotoxin, single chain, recombinant and cationic (mrBoNT/A1).

List of Figures

[0167] Figure 1: Elution profiles of High Performance Phenyl (PhHP) column, in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0168] Figure 2: Column elution profiles of Phenyl Fast Flow, High Substitution (PhFF-Hi), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line ).

[0169] Figure 3: Elution profiles of High Performance Butyl column (BuHP), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0170] Figure 4: Elution profiles of High Performance Sulfopropyl column (SPHP), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0171] Figure 5: Elution profiles of Sulfopropyl Fast Flow (SPFF) column, in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0172] Figure 6: Elution profiles from the Fast Flow Carboxymethyl column (CMFF), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0173] Figure 7: High Performance Quaternary Amine (QHP) column elution profiles, in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0174] Figure 8: Column elution profiles of Quaternary Amine Fast Flow (QFF), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0175] Figure 9: Diethylaminopropyl column elution profiles (DEAP, "ANX"), in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0176] Figure 10: Diethylaminoethyl (DEAE) column elution profiles, in which the separation of Lys-C (A405 - bars) from BoNT/A was verified (A280 - dotted line).

[0177] Figure 11: Removal of Lys-C from rBoNT/A1 by Hydrophobic Interaction Chromatography (HIC) from a High Performance Phenyl (HP) column. Fractions taken from the HIC PhHP polishing step were analyzed by SDS-PAGE (top). Recombinant BoNT/A1 had a molecular weight of ~149 kiloDaltons (kDa) and molecular weight markers (Benchmark) are labeled in kDa. Samples from the same fractions were also tested in a colorimetric Lys-C assay (bottom), where substrate cleavage releases a yellow chromophore (black box).

[0178] Figure 12: Removal of Lys-C from rBoNT/A2(0) by Hydrophobic Interaction Chromatography (HIC) from a High Performance Phenyl (HP) column. Fractions taken from the HIC Phenyl HP polishing step were analyzed by SDS-PAGE (top). Recombinant BoNT/A2(0) had a molecular weight of ~149 kDa and molecular weight markers (Benchmark) are labeled in kDa. Samples from the same fractions were also tested in a colorimetric Lys-C assay (bottom), where substrate cleavage releases a yellow chromophore (black box).

[0179] Figure 13: Removal of Lys-C from rBoNT/A5(0) by Hydrophobic Interaction Chromatography (HIC) from a High Performance Phenyl (HP) column. Fractions taken from the HIC Phenyl HP polishing step were analyzed by SDS-PAGE (top). Recombinant BoNT/A5(0) had a molecular weight of ~149 kDa and the molecular weight markers (Benchmark) are labeled in kDa. Samples from the same fractions were also tested in a colorimetric Lys-C assay (bottom), where substrate cleavage releases a yellow chromophore (black box).

[0180] Figure 14: Removal of Lys-C from rBoNT/A6(0) by Hydrophobic Interaction Chromatography (HIC) from a High Performance Phenyl (HP) column. Fractions taken from the HIC Phenyl HP polishing step were analyzed by SDS-PAGE (top). Recombinant BoNT/A6(0) had a molecular weight of ~149 kDa and molecular weight markers (Benchmark) are labeled in kDa. Samples from the same fractions were also tested in a colorimetric Lys-C assay (bottom), where substrate cleavage releases a yellow chromophore (black box).

[0181] Figure 15: Removal of Lys-C from rBoNT/A1 by Hydrophobic Interaction Chromatography (HIC) from a High Performance Butyl (HP) column. Fractions taken from the HIC Butyl HP polishing step were analyzed by SDS-PAGE (top). Recombinant BoNT/A1 had a molecular weight of ~149 kDa and molecular weight markers (Benchmark) are labeled in kDa. Samples from the same fractions were also tested in a colorimetric Lys-C assay (bottom), where substrate cleavage releases a yellow chromophore (black box).

EXAMPLES Example 1 - Host culture and expression of soluble rBoNT/A protein

[0182] A single colony of BoNT/A transformed into BLR cells (DE3) is used to inoculate a 250 mL conical flask containing 100 mL of Terrific Broth medium (mTB), supplemented with 0.2% glucosamine and 30 μg /ml of kanamycin. This method could be equally applicable in case of using a Microbank granule/bead, or glycerol raw material (10-100 µL) for vial inoculation.

[0183] The flask is incubated for 16 hours at a temperature of 37°C with agitation at 250 rpm. Then, 10 mL of this starter culture is used to inoculate 2 L conical flasks, each flask containing 1 L, supplemented with 0.2% glucosamine and 30 μg/mL kanamycin. Cells were cultured at 37°C for 2 hours, with agitation at 250 rpm, until an OD600 of 0.5 was reached. At this point, the temperature of the culture is lowered to 16°C. After 1 hour, cells are induced to express BoNT/A by addition of 1 mM IPTG for 20 hours. Next, the cells are collected by means of centrifugation, during 20 minutes at a temperature of 4°C, weighed, and then stored at a temperature of -20°C.

Example 2 - Host BoNT/A protein extraction and expression level analysis

[0184] Pastes of rBoNT/A expression cells are thawed at room temperature and resuspended by pipetting in 3 mL of Tris-NaCl resuspension buffer per gram of cell supplemented with 10 μL of benzonase. Cells are lysed by sonication, at 100W - 10x 30s (in operation) + 45 s (no operation). The lysate is centrifuged at 4000x g for 1 hour at 4°C to obtain soluble BoNT/A in the supernatant.

Bradford Assay for Determining the Total Protein Concentration of Prepared Lysates

[0185] A sample (50 μL) of diluted BoNT/A lysate or standard BSA is added to 1 mL disposable crucibles. Next, 450 µL of Coomassie Bradford Assay reagent is added to each crucible and allowed to incubate at room temperature for 10 minutes before reading the A600. The values obtained for the BSA standards are used to determine the amount of protein in the lysate samples.

Western Blot Semiquantitative Analysis

[0186] A commercial sample of BoNT/A protein purchased from Metabiologics is used to produce SDS-PAGE standards. SDS-PAGE samples of the lysate samples from the expressed cell cultures are then prepared to a known total protein concentration. These samples are placed in a polyacrylamide gel and processed at 220V for 50 minutes. Protein bands are electroblotted onto a nitrocellulose membrane in methanol-free staining buffer at 0.4 mA energy for 1 hour. Membranes are blocked for 1 hour with 0.5% BSA in PBS-0.1% Tween 20 and then probed with an antibody against BoNT/A for 1 hour. Blots are further probed with a secondary HRP-conjugated antibody developed with SuperSignal DuraWest substrate and imaged using a Syngene Imaging instrument.

Example 3 - Activation of Botulinum Neurotoxin A (BoNT/A) through Lys-C

[0187] A single-chain recombinant BoNT/A1 (0.5 mg/mL), dissolved in buffer (Tris/HCl, 50 mM, pH 8.0, 125 mM NaCl) was proteolytically activated through Lys-C ( enzyme:substrate ratio from 1:500 to 1:2500), at 37°C or 4°C, for a period of 2-20 hours, before the reaction is inhibited with 0.4 mM AEBSF, i.e. , (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, a specific serine protease inhibitor. This produces the mature double-chain form of BoNT/A1, where the heavy chain is linked to the light chain by a single disulfide bond (data not shown).

[0188] The cleavage site was determined to be identical to that of the endogenous protein by N-terminal sequencing and mass spectrometry, confirming Lys-C as the activating enzyme of choice (data not shown).

[0189] Lys-C endoproteinase cleavage tests demonstrated that Lys-C cleaved rBoNT/Al at very low concentrations and remained active for a certain period of days (data not shown).

Example 4 - Purification of target BoNT/A protein, free of activation protease

[0190] Using the primary protein sequence of BoNT/A, the properties of BoNT/A and Lys-C were investigated (see Table 2). The predicted properties suggested that Lys-C and BoNT/A have a similar mean hydropathicity (GRAVY value - Grand Average of Hydropathicity), but a large difference in charge at pH 4.5 and 8.0 (see Table 2) .Table 2: Predicted Properties of Lys-C and BoNT/A *GRAVY = average hydropathicity per residue of a molecule (a positive value indicates a hydrophobic molecule).

[0191] Based on this information alone, it was predicted that ion exchange chromatography (IEX) can separate Lys-C from BoNT/A. Therefore, various chromatographic means of purification including IEX chromatography were investigated (see below).

Example 5 - Selection of Fast Protein Liquid Chromatography (FPLC) Columns for Separating Lys-C from BoNT/A Purification by FPLC

[0192] After activation of BoNT/A by Lys-C, a series of FPLC columns were tested for polishing and removal of Lys-C: - three hydrophobic interaction chromatography (HIC) columns: High Performance Phenyl (PhHP ), High Substitution Fast Flow Phenyl (PhFF-Hi), and Butyl HP (BuHP) were tested with Tris and pH 8; - three cation exchange chromatography (CEC) columns: High Performance Sulfopropyl (SPHP), Fast Flow Sulfopropyl (SPFF), and Fast Flow Carboxymethyl (CMFF) were tested with sodium acetate and pH 4.5; and - four anion exchange chromatography (AEC) columns: High Performance Quaternary Amine (QHP), Fast Flow Quaternary Amine (QFF), Diethylaminopropyl (DEAP, “ANX”), and Diethylaminoethyl (DEAE) were tested with Tris and pH 8.

[0193] As soon as a sample was introduced into a column, the column was washed with buffer to remove any non-specifically bound molecules, before applying a linear elution gradient of increasing or decreasing salt concentration (HIC and CEC/AEC, respectively ).

[0194] The reaction conditions and purification steps vary between the different types of columns (see Table 3, below). Table 3 - Selection of conditions for all columns tested for Lys-C separation

[0195] Figures 1 to 10 show the elution profiles (A280) of the BoNT/A1 protein, from different chromatographic columns (dotted lines). The different reaction conditions and purification steps used for the different columns explain the different scales used.

[0196] The fractions collected during the elution gradient were analyzed using a colorimetric assay to determine the activity of Lys-C.

Lys-C Activity Colorimetric Assay

[0197] This assay involved the cleavage of a colorless substrate to produce a yellow chromophore that can be detected photometrically by absorption in light at 405 nm (A405). Therefore, this assay provided a simple method to determine whether Lys-C was present in each fraction.

[0198] Each elution fraction was analyzed using said Lys-C activity colorimetric assay, and the A405 nm was measured.

[0199] The amount (measured in terms of A405) of Lys-C in given elution fractions is shown in the form of bars in Figures 1 to 10.

Results

[0200] By comparing the data from A405 and A280 (in the graphs of figures 1 to 10) it was possible to deduce which column(s) provided/provided the best resolution (separation) of Lys-C from BoNT/A.

[0201] The different alkyl/aryl groups (Bu and Ph) of the three HIC columns used provided different ligands, to which different proteins could interact through the hydrophobic effect. This interaction is further influenced by the density (degree of substitution (Hi/Lo) and bead size (FF/HP) of these hydrophobic groups. For CEC columns, the pH of the sample is adjusted to a value below that of the isoelectric point ( pI) of the target protein, in order to obtain an overall net positive charge, the sample being thus able to bind to the column. The different ligands present in each type of column provide different charge densities and the interaction with different proteins is also influenced by ligand density. These variables similarly apply to AEC columns, where different chemical groups exhibit different charge densities. In this case, the pH of the sample is adjusted to a value above that of the isoelectric point (pI) of the target protein , so as to obtain an overall negative net charge.

[0202] The data from the graphs in figures 1 to 10 are summarized qualitatively in Table 4. Table 4 - Summary of Qualitative Resolution Data of Lys-C/BoNT *Apparent resolution of Lys-C relative to rBoNT/A1: √√√ = Satisfactory; √√ = OK √ = Weak a: High Performance Phenyl (PhHP), High Substitution Fast Flow Phenyl (PhFF-Hi), HP Butyl (BuHP) b: High Performance Sulfopropyl (SPHP), Fast Flow Sulfopropyl ( SPFF), Fast Flow Carboxymethyl (CMFF), High Performance Quaternary Amine (QHP), Fast Flow Quaternary Amine (QFF), Diethylaminopropyl (DEAP, "ANX"), and Diethylaminoethyl (DEAE).

Percent Lys-C recovery and purification after elution from each column type

[0203] The total Lys-C signal in each fraction was normalized to the mean A405 value from the last 5 fractions of the chromatographic step. From there, the percentage of Lys-C present in the peak fractions of the protein was calculated, to indicate the degree of separation of Lys-C from the protein, based on the elution fractions (Table 5, % of Lys-C in the fractions of protein peak (normalized)).

[0204] With respect to the target protein, it is assumed that the entire BoNT/A molecule is eluted under the main peak (ie 100% recovery); therefore, the degree of purification can be expressed as the percentage of total protein introduced, which is not recovered in the main peak (Table 5, % Purification).

[0205] A satisfactory separation of Lys-C from BoNT/A would be that visualized with a low value for the percentage of Lys-C in the protein peak fractions. From there, it appears that CEC is not able to separate Lys-C from BoNT/A1. The high performance AEC column, ie the QHP column, showed some ability to separate Lys-C from BoNT/A. However, it was significantly less effective than the two high performance HIC columns. Therefore, the results demonstrate that when comparing similar items, (ie, standard performance vs. standard performance and high performance vs. high performance), HIC columns showed improved BoNT/A1 Lys-C resolution, with respect to to the CEC or AEC columns.

[0206] The two most promising candidates include the column hydrophobic interaction chromatography (HIC), PhHP and BuHP. Interestingly, these columns utilize high performance beads/beads.

[0207] The main difference between high performance, medium performance and other performances is related to the smaller average particle size (34 μm vs. 90 μm) and also a more uniform size (24-44 μm vs. 44-165 μm). This is consistent with the reported improvements in performance with analytical columns, which use smaller-sized beads/beads (average sizes between 3-30 µm) (GE Healthcare Handbooks, 11-0004-21 & 11-0012-69 and files from data 18-1172-87 AE & 18-1172-88 AD). Table 5 - Summary Relating to Column Performance

[0208] The HIC PhHP column was chosen as the final polishing step to separate Lys-C from BoNT/A (see Example 6, below).

Example 6 - Activation and final purification of recombinant botulinum neurotoxin subserotype A1 (rBoNT/Al)

[0209] Single-chain rBoNT/A1 was purified by fast protein liquid chromatography (FPLC), using hydrophobic interaction chromatography on sepharose, with high-performance butyl (Butyl HP HIC) for capture, followed by an intermediate purification step , using anion exchange chromatography on sepharose, with high performance quaternary ammonium (QHP AEC). This molecule was then incubated with 0.4 μg/mL of Lys-C, at a temperature of 37°C, for 2 hours, to produce the active duplex.

[0210] Lys-C was separated from rBoNT/A1 using high-performance phenyl hydrophobic interaction chromatography on sepharose (PhHP HIC). This involved adjusting the reaction mixture with a high salt Tris buffer prior to introduction onto the PhHP column, followed by washing away the high salt and subsequent elution of the protein with a linear gradient in a high salt Tris buffer. low salt content. The elution fractions were analyzed by means of electrophoresis on a denaturing gel (SDS PAGE - Figure 11, at the top) and by a colorimetric assay of Lys-C activity (Figure 11, at the bottom). Lys-C was shown to elute first in the F16-F21 gradient (as highlighted in the black box), before the activated BoNT molecule (F21-F29). Thus, this shows a satisfactory separation of Lys-C from rBoNT/A1. The peak intensity of the fractions appears to be similar to 30 ng/mL of Lys-C, which suggests that any residual Lys-C present in the rBoNT/A1 fractions would be less than this concentration.

Example 7 - Activation and final purification of recombinant botulinum neurotoxin subserotype A2, endopeptidase negative (rBoNT/A2(0))

[0211] Single-chain rBoNT/A2(0) was purified by fast protein liquid chromatography (FPLC), using hydrophobic interaction chromatography on sepharose, with high-performance butyl (Butyl HP HIC) for capture, followed by a step intermediate purification using high performance quaternary ammonium sepharose anion exchange chromatography (QHP AEC). This molecule was then incubated with 4 μg/mL of Lys-C, at a temperature of 37°C, for 2 hours, to produce the active duplex.

[0212] Lys-C was separated from activated rBoNT/A2(0) using high performance phenyl hydrophobic interaction chromatography on sepharose (PhHP HIC). This involved adjusting the reaction mixture with a high salt Tris buffer prior to introduction onto the Phenyl HP column, followed by washing away the high salt and subsequent elution of the protein with a linear gradient in a Tris buffer. low salt. The elution fractions were analyzed by electrophoresis on a denaturing gel (SDS PAGE) and by a colorimetric Lys-C activity assay (Figure 12). This showed that Lys-C eluted first in the gradient (F5-F11), just before the activated BoNT molecule (F12-F15). Thus, this column shows a satisfactory separation of Lys-C from rBoNT/A2(0).

Example 8 - Activation and final purification of recombinant botulinum neurotoxin subserotype A5, endopeptidase negative (rBoNT/A5(0))

[0213] Single-chain rBoNT/A5(0) was purified by fast protein liquid chromatography (FPLC), using hydrophobic interaction chromatography on sepharose, with high-performance butyl (Butyl HP HIC) for capture, followed by a step intermediate purification using high performance quaternary ammonium sepharose anion exchange chromatography (QHP AEC). This molecule was then incubated with 2.5 μg/mL of Lys-C, at a temperature of 37°C, for 2 hours, to produce the active duplex. Lys-C was separated from activated rBoNT/A5(0) using high performance phenyl hydrophobic interaction chromatography on sepharose (Phenyl HP HIC). This involved adjusting the reaction mixture with a high salt Tris buffer prior to introduction onto the Phenyl HP column, followed by washing away the high salt and subsequent elution of the protein with a linear gradient in a Tris buffer. low salt. The elution fractions were analyzed by electrophoresis on a denaturing gel (SDS PAGE) and by a colorimetric assay of Lys-C activity (Figure 13) - this showed that Lys-C eluted first in the gradient (F10-F39 ), before the activated BoNT molecule (F51-F65). Thus, this column shows a satisfactory separation of Lys-C from rBoNT/A5(0).

Example 9 - Activation and final purification of recombinant botulinum neurotoxin subserotype A6, endopeptidase negative (rBoNT/A6(0))

[0214] Single-chain rBoNT/A6(0) was first purified by precipitation of sodium sulfate and further solubilization in sodium acetate before capture with fast protein liquid chromatography (FPLC) using cation exchange chromatography on sepharose , with high-performance sulfopropyl (SPHP CEC), followed by buffer exchange for Tris buffer, at pH 8. This molecule was then incubated with 0.3 μg/mL of Lys-C, at a temperature of 37°C, for 2 hours, to produce the active duplex. Lys-C was separated from activated rBoNT/A6(0) using high performance phenyl hydrophobic interaction chromatography on sepharose (Phenyl HP HIC). This involved adjusting the reaction mixture with a high salt Tris buffer prior to introduction onto the Phenyl HP column, followed by washing away the high salt and subsequent elution of the protein with a linear gradient in a Tris buffer. low salt. The elution fractions were analyzed by electrophoresis on a denaturing gel (SDS PAGE) and by a colorimetric assay of Lys-C activity (Figure 14) - this showed that Lys-C eluted first in the gradient (F6-F13 ), before the activated BoNT molecule (F16-F27). Thus, excellent separation of Lys-C from rBoNT/A6(0) is shown.

Example 10 - Purification of a derivative of subserotype A1 of recombinant, cationic, single-chain botulinum neurotoxin (mrBoNT/A1)

[0215] The single-chain mrBoNT/A1 (SEQ ID NO: 15) was purified by fast protein liquid chromatography (FPLC) using cation exchange chromatography on sepharose, with high performance sulfopropyl (SPHP CEX) for capture. An intermediate purification step was employed, using high-performance butyl hydrophobic interaction chromatography on sepharose (BuHP HIC), to further remove host cell proteins. The BuHP elution fractions were analyzed by SDS-PAGE and those containing pooled single chain mrBoNT/A1 were purified to the single chain mrBoNT/A1 form.

Example 11 - Activation and final purification of a cationic recombinant botulinum neurotoxin subserotype A1 derivative (mrBoNT/A1)

[0216] The single chain mrBoNT/A1 (SEQ ID NO: 15) was incubated with 0.4 μg/mL Lys-C at 37°C for 2 hours to produce the active double chain. Lys-C was separated from activated mrBoNT/A1 using high-performance butyl hydrophobic interaction chromatography on sepharose (BuHP HIC). This involved adjusting the reaction mixture with a high salt Tris buffer prior to introduction onto the BuHP column, followed by washing away the high salt and subsequent elution of the protein with a linear gradient in a high salt Tris buffer. low salt content. The elution fractions were analyzed by SDS PAGE (Figure 15, top) and by a colorimetric Lys-C activity assay (Figure 15, bottom). Lys-C eluted in the column flow and, previously, in the elution gradient (F1-6, highlighted in the black box, Figure 15, at the bottom), before the activated BoNT molecule (F10-F12). Thus, a satisfactory separation of Lys-C from mrBoNT/A1 is shown.

Example 12 - Formulation comprising active double-chain BoNT/A substantially free of Lys-C

[0217] The following six liquid compositions comprising the double-chain active BoNT/A are prepared (see Table 6, below). Table 6 - Examples of BoNT/A Formulations

[0218] All six compositions are stored at a temperature of 25°C for 12 weeks. The stability of double-stranded BoNT/A protease function is checked during this period using a cell-free endopeptidase assay.

Example 13 - High Sensitivity Lys-C Activity Assay

[0219] A quantitative determination of the final concentration of Lys-C in the purified BoNT/A samples was made by measuring the cleavage of a fluorescent peptide substrate. The fluorescent substrate (Tos-GPK-7-amino-4-trifluoroacetate salt, also called Tos-GPK-AMC) was reconstituted in DMSO, providing a starting material with a concentration of 20 mg/mL (32.7 mM) . This raw material was later diluted with 10 mM water, and fractions were prepared and stored at -20°C. On the day of the assay, an adequate amount of substrate was taken and further diluted with 25 mM Tris, pH 8, 125 mM NaCl, providing a 0.5 mM Lys-C processing solution; then, Lys-C standards (known concentrations) were prepared by diluting the processing solution in an assay buffer (25 mM Tris, 125 mM NaCl, 0.1 mg/mL BSA, pH 8). Standards, starting materials (assay buffer only) and test samples (lots of purified rBoNT/A) were distributed onto black OptiPlate® plates, in triplicate (180 μL per well), and neutralized at 37°C for 10 -15 minutes. Next, 20 μL of substrate was added and the plate was immediately transferred to a plate reader device, where a kinetic reading was performed. The plate was read using excitation/emission filters - 360/40 and 485/20 - at a temperature of 37°C, every 20 minutes, for 5 hours. Data were plotted as time, in minutes (X axis), against absorbance/fluorescence (Y axis). Decreasing values for each Lys-C concentration were used to generate a standard curve for Lys-C concentration. Purified rBoNT/A samples were treated in the same manner and the Lys-C concentration in each rBoNT/A sample was interpolated from the standard curve. Based on the analysis of four independent batches of rBoNT/A, the average concentration of Lys-C in the final material was measured, obtaining 7.3 ± 1.5 pg of Lys-C per 100ng/BoNT/A.

Claims (18)

1. Method for producing double-chain soluble BoNT/A protein, characterized in that it comprises: a) providing a single-chain soluble BoNT/A protein; b) contacting said BoNT/A protein with endoproteinase Lys-C (Lys-C) in solution; and c) separating the soluble BoNT/A protein from Lys-C by contacting the solution containing the soluble BoNT/A and Lys-C protein with a hydrophobic surface, whereby the soluble BoNT/A protein binds to the hydrophobic surface . 2. Method, according to claim 1, characterized in that the single-chain soluble BoNT/A protein is produced: (i) in a host cell, through the expression of a nucleic acid encoding said BoNT protein single-stranded /A in an expression system; or (ii) in an in vitro expression system. 3. Method according to claim 2, characterized in that the host cell is a bacterial host cell and the expression system is a bacterial expression system. 4. Method according to claim 3, characterized in that the bacterial expression system is an E. coli expression system and the host cell is an E. coli cell. 5. Method according to any one of claims 2 to 4, characterized in that said single-chain soluble BoNT/A protein is expressed in the cytoplasm of said host cell. 6. Method according to any one of claims 2 to 5, characterized in that said single-chain soluble BoNT/A protein is expressed at a level of at least 5 mg/L. 7. Method according to any one of claims 2 to 6, characterized in that it comprises lysing the bacterial host cell or the E. coli host cell to provide a bacterial host cell or E. coli host cell homogenate. coli containing said single-chain soluble BoNT/A protein. 8. Method according to any one of claims 1 to 7, characterized in that the hydrophobic surface is an inert matrix, on which a ligand consisting of aryl groups or alkyl groups is fixed. 9. Method according to claim 8, characterized in that the linker is selected from the group consisting of butyl, phenyl or octyl linkers. 10. Method according to claim 9, characterized in that a high-performance hydrophobic surface is used. 11. Composition, characterized in that it comprises an active double-chain BoNT/A protein, which can be obtained by the method defined in any one of claims 1 to 10, in which said active BoNT/A protein of double chain is separated from Lys-C by means of hydrophobic interaction chromatography, and wherein said composition comprises (i) less than 2% single-chain BoNT/A, and (ii) Lys-C, wherein Lys -C is present at a concentration of less than 400 pg of Lys-C per 100 ng of BoNT/A protein. 12. Composition according to claim 11, characterized in that said composition comprises less than 1% of single-chain BoNT/A. 13. Composition according to any one of claims 11 or 12, characterized in that said composition contains endoproteinase Lys-C (Lys-C) in a concentration of less than 300 pg of Lys-C per 100 ng of protein of BoNT/A, or less than 200 pg of Lys-C per 100 ng of BoNT/A protein, or less than 100 pg of Lys-C per 100 ng of BoNT/A protein, or less than 50 pg of Lys -C per 100 ng BoNT/A protein, or less than 20 pg Lys-C per 100 ng BoNT/A protein, or less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg of Lys-C per 100 ng of BoNT/A protein. 14. Liquid pharmaceutical composition, characterized in that it comprises: (i) an active double-chain BoNT/A protein, which can be obtained by the method defined in any one of claims 1 to 10, in which said active protein of double-chain BoNT/A is separated from Lys-C by hydrophobic interaction chromatography; (ii) a non-protein stabilizing agent which is a surfactant; and (iii) water; wherein said liquid pharmaceutical composition: (iv) does not comprise a protein stabilizing agent; (v) ) comprises less than 2% single-chain BoNT/A; and (vi) comprises Lys-C, wherein Lys-C is present at a concentration of less than 400 pg Lys-C per 100 ng BoNT/A protein. 15. Liquid pharmaceutical composition according to claim 14, characterized in that said liquid pharmaceutical composition contains Lys-C endoproteinase (Lys-C) in a concentration of less than 300 pg of Lys-C per 100 ng of protein BoNT/A, or less than 200 pg Lys-C per 100 ng BoNT/A protein, or less than 100 pg Lys-C per 100 ng BoNT/A protein, or less than 50 pg Lys-C per 100 ng BoNT/A protein, or less than 20 pg Lys-C per 100 ng BoNT/A protein, or less than 15 pg Lys-C per 100 ng BoNT/A protein, or less than 10 pg BoNT/A Lys-C per 100 ng of BoNT/A protein. 16. Liquid pharmaceutical composition, according to any one of claims 14 or 15, characterized in that said liquid pharmaceutical composition further comprises: - sodium chloride; - a buffer to maintain the pH between 5.5 and 7.5; and - a disaccharide; wherein the water is sterile water. 17. Composition according to any one of claims 11 to 13, or a liquid pharmaceutical composition according to any one of claims 14 to 16, characterized in that said composition is for use in a method to treat at least one of the following diseases and disorders: glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, bladder overactivity, hyperhidrosis, nasal labral lines, cervical dystonia, blepharospasm, and spasticity. 18. Use of a composition, defined in any one of claims 11 to 13, or a liquid pharmaceutical composition, defined in any one of claims 14 to 16, characterized in that said use is applied in the manufacture of a medicine for treat at least one of the following diseases and disorders: glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, bladder overactivity, hyperhidrosis, nasal labral lines, cervical dystonia, blepharospasm, and spasticity.