EA040938B1 - PRODUCTION OF RECOMBINANT CLOSTRIDIUM BOTULINUM NEUROTOXINS - Google Patents

Description

Field of invention

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

State of the art invention

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex consisting of BoNT itself complexed with a number of accessory proteins. There are currently at least seven different classes of botulinum neurotoxin, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, and G, which all have similar structures and mechanisms of action. A possible ninth serotype, H, has recently been reported, but the sequence has not yet been published. Different serotypes of BoNT can be distinguished based on inactivation by a specific neutralizing antiserum, such serotype classification being correlated with percent sequence identity at the amino acid level. The BoNT proteins of a given serotype are further subdivided into different subtypes based on percent amino acid sequence identity.

BoNTs are the most potent toxins known, with median lethal dose (LD ₅₀ ) values in mice ranging from 0.5 to 5 ng/kg depending on serotype. BoNTs are absorbed in the gastrointestinal tract and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve endings and prevent the release of the neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C, BoNT/A and BoNT/E cleave the 25 kDa synaptosomal associated protein (SNAP-25); a BoNT/C splits the syntax.

In nature, clostridial neurotoxins are synthesized as a single-chain polypeptide, which is post-translationally modified through the phenomenon of proteolytic cleavage to form two polypeptide chains connected to each other via a disulfide bridge. The cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine residues that provide the interchain disulfide bridge. It is this bicep form that is the active form of the toxin. The two chains are called the heavy chain (H-chain), which has a molecular weight of approximately 100 kDa, and the light chain (L-chain, or LC), which has a molecular weight of approximately 50 kDa. The H chain contains a C-terminal guide component (HC domain) and an N-terminal translocation component (H _N domain). The cleavage site is located between the L chain and the translocation components in the area of the exposed loop (see Table 1). After binding the H _C domain to its target neuron and internalizing the bound toxin into the cell via the endosome, the H _N domain translocates the L chain across the endosomal membrane into the cytosol, and the L chain provides a protease function (also known as a non-cytotoxic protease).

Non-cytotoxic proteases act through proteolytically cleaving 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 comes from the term soluble NSF binding receptor, where NSF stands for N-ethylmaleimide sensitive factor. SNARE proteins are an integral part of intracellular vesicle fusion and thus the secretion of molecules through the transport of vesicles out of the cell. The protease function is a zinc dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Accordingly, upon delivery to the desired target cell, the non-cytotoxic protease is immediately capable of inhibiting cellular secretion from the target cell. Clostridial neurotoxin L-chain proteases are non-cytotoxic proteases that cleave SNARE proteins.

Botulinum neurotoxins are well known for their ability to induce flaccid muscular paralysis. These muscle relaxant properties have led to botulinum neurotoxins (such as BoNT/A) being used in a number of medical and cosmetic procedures, including the treatment of brow wrinkles or hyperkinetic facial wrinkles, headache, hemifacial spasm, overactive bladder, hyperhidrosis, nasolabial wrinkles, cervical dystonia, blepharospasm and spasticity.

Traditionally, BoNT has been obtained by culture of C. botulinum bacteria, followed by isolation and purification of the botulinum neurotoxin complex. However, obtaining BoNT by this method is inefficient and provides a low protein yield. In addition, C. botulinum are spore-forming bacteria and therefore require specialized culture equipment and facilities that are not required for culturing bacteria such as Escherichia coli (E. coli). The increasing use of BoNT has led to the development of alternative methods for the preparation and purification of BoNT. In particular, methods have been developed to produce BoNT using E. coli.

Purification of CNT from a fermentation solution (whether using C. botulinum or E. coli) is a particular challenge because it contains neurotoxins as a mixture of raw, partially processed, and fully processed polypeptides, all of which have very similar biochemical profiles. and physical properties. Typically, partially processed neurotoxins are obtained if the endoproteolytic activity hydrolyzes the peptide bond between the light chain and the loop, while the peptide bond between the loop and the N-terminus of the heavy chain is not yet affected. Moreover, a partially processed neurotoxin can also be obtained if endoproteolytic activity has released the loop peptide from the heavy chain, while the peptide bond between the loop peptide and the C-terminus of the light chain has not yet been hydrolyzed. Depending on the fermentation conditions and the type of neurotoxin, a fully processed polypeptide that lacks a loop peptide can be significantly contaminated with 5 to 90% partially processed or unprocessed polypeptide. In some cases, the neurotoxin is mostly untreated and needs to be treated with endopeptidase before therapeutic use in order to become biologically active.

The prior art describes various attempts to treat clostridial neurotoxins with heterologous proteases to reduce the amount of unprocessed or partially processed precursor protein. The protease trypsin, most widely used to activate clostridial neurotoxins, being useful for activating clostridial neurotoxin serotypes B (BoNT/B) and E (BoNT/E), appears to produce secondary products, presumably through proteolytic action near the C-terminus of severe subunits of BoNT/A and thus, apparently, destroys the toxin that binds to its cellular receptor. More specific cleavage products are theoretically expected from endogenous proteases isolated from a natural host such as C. botulinum producing BoNT/A.

The present inventors have previously identified various proteases such as endogenous proteases from C. botulinum and exogenous proteases including the protease endoproteinase Lys-C (Lys-C) (which is commercially available and can be isolated from Lysobacter enzymogenes). However, although the cleavage of recombinant BoNT/A by this endoproteinase more closely mirrors natural cleavage, the use of Lys-C gives rise to new practical considerations. In particular, after cleavage of recombinant BoNT/A, Lys-C can remain active in the reaction mixture for days. Therefore, means and methods for reducing the amount of Lys-C in the final product and thereby improving the quality of neurotoxin preparations are highly desirable but still not available.

Thus, the technical problem underlying the present invention can be seen as providing means and methods for improving the production of neurotoxin polypeptides by meeting the aforementioned needs. Specifically, there is a need in the art for improved methods for producing recombinant BoNT, in particular activated biceps recombinant BoNT/A.

The technical problem is solved using the embodiments described in the claims and in this document below.

The essence of the invention

Generation of recombinant BoNT/A subserotypes can be achieved by expression in Escherichia coli as a single polypeptide, herein referred to as BoNT/A single chain proteins. As a result of isolating this single chain protein by rapid protein liquid chromatography (FPLC), the single chain protein must be cleaved to form an active toxin, which is a double chain protein linked together by a single disulfide bond.

The present inventors have previously discovered that recombinant single chain BoNT/A subserotypes can be cleaved using the protease endoproteinase Lys-C (Lys-C) to produce active bicep protein neurotoxin BoNT/A, i.e. heterodimer of light and heavy chains BoNT/A. By N-terminal sequencing and mass spectrometry, the Lys-C cleavage site was determined to be identical to the endogenous protein. Thus, production of an active bicep BoNT/A protein using Lys-C is preferred as it results in an authentic active bicep BoNT/A, namely a bicep BoNT/A that is virtually identical to naturally occurring active bicep BoNT/A. In contrast, trypsin, another common protease used to generate active BoNT double chain proteins, cleaves at least one additional site within the single stranded BoNT/A sequence. This additional cleavage reduces the activity of the produced active BoNT/A bicep protein.

After Lys-C cleaves the BoNT/A single strand protein, purification of the active BoNT/A bicep protein is required to remove any remaining host cell proteins and the Lys-C protease in order to obtain a finished pure product.

In particular, from Lys-C endoproteinase cleavage studies, the inventors found that Lys-C cleaves recombinant BoNT/A1 (rBoNT/A1) at very low concentrations and remains active for days. Therefore, it is important to develop a method that could purify a given protease from an activated toxin.

The authors of the present invention have developed an effective method for removing activated

- 2 040938 Lys-C protease and host cell proteins from BoNT/A after activation by the use of hydrophobic interaction chromatography (HIC). The present inventors surprisingly found that HIC not only maximizes the amount of active bicep BoNT/A recovered from purification, but also significantly improves Lys-C clearance, i.e. removal of Lys-C protease. The preferred properties of HIC are unexpected given 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 was predicted that not HIC but ion exchange chromatography (IEX) would cleave the two proteins.

The present invention solves one or more of the above problems by providing methods in accordance with the claims.

Accordingly, the present invention provides a method for producing a soluble BoNT/A bicep protein, said method comprising:

a) providing a soluble single chain BoNT/A protein;

b) contacting said BoNT/A protein with Lys-C endoproteinase (Lys-C) in solution, and

c) separating the soluble BoNT/A protein from Lys-C by contacting a solution containing the soluble BoNT/A protein and Lys-C with a hydrophobic surface, wherein the soluble BoNT/A protein preferentially binds to the hydrophobic surface.

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

The hydrophobic surface used in the method of the present invention may be an inert matrix to which a ligand consisting of aryl or alkyl groups is attached, optionally the ligand is selected from the group consisting of butyl, phenyl or octyl ligands. In one embodiment, a high performance hydrophobic surface is used.

The invention also provides an active BoNT/A bicep protein obtainable by the method of the invention.

The invention further provides a composition comprising an active BoNT/A bicep protein of the invention; wherein said composition is substantially free of Lys-C endoproteinase. Preferably said composition contains less than 400 pg of Lys-C endoproteinase (Lys-C) per 100 ng of BoNT/A protein, or less than 300 pg of Lys-C per 100 ng of BoNT/A protein, or less than 200 pg of Lys-C per 100 ng of protein BoNT/A, 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. These compositions are suitable for use in therapeutic and cosmetic treatments.

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

The invention also provides an active BoNT/A bicep protein, composition or liquid pharmaceutical composition of the invention for use in therapy.

The invention further provides the use of the active BoNT/A bicep protein, composition or liquid pharmaceutical composition of the invention in the preparation of a medicament.

The invention also provides a method of treatment comprising administering the active BoNT/A bicep protein, composition or liquid pharmaceutical composition of the invention to a patient in need thereof.

Detailed description of the invention

Botulinum toxin serotype A (BoNT/A).

The term BoNT stands for botulinum neurotoxin and refers to a neurotoxin derived from

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C. Botulinum, such as BoNT serotype A, B, Sc D, E, F, or G. Also encompassed by the term CNT and BoNT is a recombinant and modified neurotoxin containing 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 in 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.

The BoNT/A serotype is subdivided into at least eight subserotypes (also known as subtypes), from BoNT/A1 to BoNT/A8, which share at least 84%, up to 98%, overall amino acid sequence identity; BoNT/A proteins within a given subtype have a higher overall percentage of amino acid sequence identity. Tab. 1 (below) shows the precursor, natural bicep neurotoxin of the BoNT/A subtypes, and identifies the exposed loop (activation loop) comprising the amino acid sequence cleaved with Lys-C.

Table 1

EXPOSED toxin IMPACT (ACTIVATION) LC H _N HcN Hec A LOOP BoNT/Al (Р10845) SEQ ID NO: 2 M1-K438 A449-N872 I873-S1092 N1093-L1296 BoNT/A2 (A2I2R5) SEQ ID NO: 3 M1-K438 A449-N872 I873-S1092 N1093-L1296 BoNT/A2 (A2I2R5) SEQ ID NO: 4 M1-K434 A445-N868 I869-S1088 N1089-L1292 BoNT/AZ (B1L2G5) SEQ ID NO: 3 M1-K438 A449-N872 I873-S1092 N1093-L1296 BoNT/AZ (B1L2G5) SEQ ID NO: 4 M1-K434 A445-N868 I869-S1088 N1089-L1292 BoNT/AZ (Q3LRX9) SEQ ID NO: 5 M1-K434 A445-N868 I869-S1088 N1089-L1292 BONT/A4 (Q3LRX8) SEQ ID NO: 6 M1-K438 A449-N872 I873-S1092 N1093-L1296 BONT/A5 (C7BEA8) SEQ ID NO: 7 M1-K438 A449-N872 I873-S1092 N1093-L1296 BoNT/Ab (C9WWY7) SEQ ID NO: 2 M1-K438 A449-N872 I873-S1093 N1094-L1297 BONT/A7 (K4LN57) SEQIE 13 NO: M1-K438 A449-N872 I873-S1092 N1093-L1296 B0NT/A8 (A0A0A7PDB7) SEQIE 14 NO: M1-K438 A449-N872 I873-S1093 N1094-Q1297

Referring to Table. 1, the amino acid positions (residues) indicated for the LC, H _N , H _CN and H _cc regions correspond to the amino acid positions for the indicated regions in the full-length BoNT/A protein sequence. UniProt account numbers given in Table. 1 are examples of subserotypes of various BoNT/A. Thus, in one embodiment, the amino acid positions of the LC, H _N , H _CN and H _CC regions shown in Table. 1 correspond to amino acid positions in the full-length BoNT/A protein sequences identified by the Accession Number.

Using the methods of the present invention, it is now possible to obtain biceps BoNT/A compositions with significantly less contamination with untreated or partially treated BoNT/A, since these contaminants are efficiently processed into biceps.

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BoNT/A. The methods of the present invention also allow the efficient removal of the Lys-C enzyme used to convert the single stranded BoNT/A protein into an active bicep. In one aspect, the BoNT/A bicep is a naturally occurring bicep neurotoxin, wherein the C-terminus of the light chain and the N-terminus of the heavy chain are identical to the corresponding fully processed bicep BoNT/A isolated from wild-type Clostridium.

In accordance with the present invention, an active BoNT/A bicep, including a recombinant BoNT/A bicep, can be prepared as a single chain polypeptide that is cleaved to form the active bicep of the protein.

The presented methods can be used to obtain the bicep of any subtype of BoNT/A or its homologue or derivative, including, for example, the polypeptide of SEQ ID NO: 1 and its derivatives. The term derivative, as used in this and other aspects of the invention, includes amino acid mutations, such as the addition, substitution, deletion, or processing of one or more amino acid residues.

Одноцепочечный белок BoNT/A может содержать полипептидную последовательность, как показано в GenBank NO: 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. A single chain BoNT/A protein may contain a polypeptide sequence as shown in subserotype A1: UniParc Supplement No. UPI0000ED909E (UniProt Supplement No. P10845, Version 159), UniParc Supplement No. UPI0000EF85BD (UniProt Supplement No. S8B1U4, Version 3, or UniProt Supplement No. A2I2R4, version 41), UniParc Supplement No. UPI0001A954C8 (UniProt Supplement No. C6K838, Rev. 11), UniParc Supplement No. UPI0000001386 (UniProt Supplement No. A5HZZ9, Rev. 57), UniParc Supplement No. UPI000003409D (UniProt Supplement No. A2I2U2, Rev. 36), UniParc Supplement No. A2I2U2, Rev. 36, UniParc Supplement No. UPI0007 (6 UPI0001 UniProt Supplement No. B1A2D5, Rev. 21), UniParc Supplement No. UPI00027EE164 (UniProt Supplement No. J7FGZ9, Rev. 6);

subserotype A2: UniParc Supplement No. UPI0000EF84BD (UniProt Supplement No. A2I2R5, Rev. 28), UniParc Supplement No. UPI0001C0B376 (UniProt Supplement No. D2KCK3, Rev. 15), UniParc Supplement No. UPI0001C32E84 (UniProt Supplement No. D3IV23, Rev. 10), UniParc Supplement No. D3IV23, Rev. 10 UniProt Supplement No. E5F1I1, Rev. 11), UniParc Supplement No. UPI000016EA88 (UniProt Supplement No. Q45894, Rev. 116 or UniProt Supplement No. Q58GH1, Rev. 51), UniParc Supplement No. UPI000067C53E (UniProt Supplement No. Q2PPK6, Rev. 33), UniParc Supplement No. UPI000290BEB1 ( UniProt Supplement No. K4GGE0, Version 6);

subserotype A3: UniParc Supplement No. UPI00005B712C (UniProt Supplement No. Q3LRX9, Rev. 38), UniParc Supplement No. UPI00016DBC11 (UniProt Supplement No. B1L2G5, Rev. 38 or UniProt Supplement No. D3IV24, Rev. 11), UniParc Supplement No. UPI000290C3D0 (UniProt Supplement No. K4G3L3 7);

subserotype A4: UniParc Supplement No. UPI00005B712D (UniProt Supplement No. Q3LRX8, Rev. 35), UniParc Supplement No. UPI00019DB885 (UniProt Supplement No. C3KS13, Rev. 29);

subserotype A5: UniParc Supplement No. UPI0001AE7D6A (UniProt Supplement No. C7BEA8, Rev. 14), UniParc Supplement No. UPI000198BDAE (UniProt Supplement No. E8ZMW0, Rev. 18 or UniProt Supplement No. C1IPK2, Rev. 20);

Subserotype A6: UniParc Supplement No. UPI0001B7D251 (UniProt Supplement No. C9WWY7, Version 13), Subserotype A7: UniParc Supplement No. UPI000290B995 (UniProt Supplement No. K4LN57), Subserotype A8: UniParc Supplement No. UPI000559A469 (UniProt Supplement No. A0A0A7PDB7); UniParc Supplement No. UPI0003C9D2A1 (UniProt Supplement No. V5N8R1).

A single stranded BoNT/A protein may comprise a polypeptide sequence that is homologue or derivative having at least 50% sequence identity with one of the above BoNT/A polypeptide sequences.

In one aspect, the polypeptide chain of said BoNT/A single chain protein comprises a sequence selected from any one of SEQ ID NOs: 2 to 7, or 13, or 14. In a more specific aspect, the polypeptide chain of said BoNT/A single chain protein comprises a sequence selected from from any one of SEQ ID NO: 2 to 7, or 13, or 14, and wherein the second polypeptide is a cleaved C-terminus to the main amino acid residue within the specified sequence of any one of SEQ ID NO: 2 to 7, or 13 or 14. These sequences represent the amino acid sequences of known substrates of the BoNT/A single chain protein of the present invention.

Single-stranded BoNT/A proteins are cleaved from the C-terminus to the basic amino acid residue contained in the sequence, compare columns LC and H _N table. 1. In a preferred aspect, said BoNT/A single chain protein comprises a sequence selected from SEQ ID NOs: 2 to 7 and 13-14 (eg BoNT/A1 serotype, SEQ ID NO: 2).

The single chain BoNT/A protein may contain a derivative of any one of SEQ ID NO: 2 to 7, or 13, or 14, or SEQ ID NO: 1, or one of the polypeptide sequences corresponding to the accession numbers identified herein. document, 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, up to 15 point mutations. By using an activity assay to determine protease activity as described herein, the skilled artisan can determine if a given derivative has been treated with Lys-C.

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

Said BoNT/A single chain 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 SEQ ID NO: 1 (BoNT/ A ATCC 3502, Genbank add. AAA23262), or one of the polypeptide sequences corresponding to the accession numbers identified herein. A single stranded BoNT/A protein may comprise a 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.

The term sequence identity, as used herein, refers to determining the identity between a reference amino acid sequence and a search sequence, with the sequences aligned so that a highest order match is obtained, which 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. For example, for a single stranded BoNT/A protein, sequence identity can be calculated from sequence lengths of up to 50 amino acid (aa) residues, up to 100 aa, up to 150 aa, up to 250 aa, 300 aa, 350 aa, 400 aa, 450 аа, 500 аа, 550 аа, 600 аа, 650 аа, 700 аа, 750 аа, 800 аа, 850 аа, 900 аа, 950 аа, 1000 аа, 1050 аа, 1100 аа, 11 50 аа, 12. or more residues, up to the sequence of the full-length single-chain BoNT/A protein. Sequence identity can be calculated from at least 50 aa residues, at least 100 aa, at least 150 aa, or at least 250 aa residues.

To compare different sequences, a number of programs based on various algorithms are available to a skilled worker. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To perform sequence alignment and calculate the sequence identity values listed herein, the commercially available DNASTAR Lasergene MegAlign program version 7.1.0, based on the Clustal W algorithm, was applied over the entire sequence region with the following settings: pairwise alignment parameters: gap penalty: 10.00 , gap length penalty: 0.10, Gonnet 250 amino acid comparison matrix, which, unless otherwise noted, will always be used as standard parameters for sequence alignment.

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 at least 97%, at least 98%, at least 99% or more, up to 100%. The sequence identity of said single chain BoNT/A protein having at least 50% sequence identity with the sequence of SEQ ID NO: 1 can be determined based on amino acid positions 420 to 466 of SEQ ID NO: 1, or said sequence identity can be determined based on on any one of SEQ ID NOs: 2 to 7, or 13, or 14. In other words, a single chain BoNT/A protein may contain a polypeptide sequence that has, for example, at least 30% sequence identity with the polypeptide sequence found between amino acid positions 420 to 466 of the single-stranded BoNT/A protein, or at least 30% sequence identity with the polypeptide sequence of any one of the single-stranded BoNT/A protein, including any one of the polypeptide sequences

- 6 040938 sequences corresponding to the account numbers identified in this document. In accordance with this definition, the polypeptide, for example, is obtained 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 the addition, substitution, deletion or processing of one or more amino acid residues. For example, included are derivatives lacking, for example, the _HC domain of the natural neurotoxin or portions thereof, or derivatives with other amino acid residues replacing the _HC domain of the neurotoxin, as well as derivatives with an additional light chain or other protein-like cargo molecule fused at the N-terminus. with light chain BoNT.

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

In another aspect, the biological activity of said BoNT/A single chain protein can be modulated by proteolytic cleavage. It is well known to the skilled artisan that the function of many polypeptides can be modulated by proteolytic treatment.

Modulated, as used herein, means up or down, activated or inactivated. For example, the biological activity of many clostridial neurotoxins is enhanced or triggered by the proteolytic conversion of a single chain neurotoxin to a bicep neurotoxin, the bicep neurotoxin being composed of light and heavy polypeptide chains that are covalently linked via a disulfide bridge. The biological activity of a neurotoxin encompasses at least three separate actions: the first action is the proteolytic activity inherent in the light chain of the neurotoxin and is responsible for the hydrolysis of the peptide bond of one or more polypeptides involved in the regulation of cell membrane fusion. The second action is the translocation activity inherent in the N-terminal end of the heavy chain of the treated neurotoxin and is involved in the transport of the light chain across the lysosomal membrane into the cytoplasm. The third action is the receptor-binding activity attributable to the C-terminal end of the heavy chain of the treated neurotoxin and is involved in the binding and uptake of the neurotoxin into the target cell. In a preferred aspect, the term biological activity, as used herein, refers to proteolytic activity. In a more preferred aspect, the term refers to increased proteolytic activity.

The biological activity of clostridial neurotoxin can be measured by various assays, all of which are known to those skilled in the art. These assays make it possible to determine one or more of the activities mentioned above. For example, mouse LD ₅₀ assay or ex vivo dome of murine phrenic nerve (MPN) diaphragm assay as described by Pearce et al. 69-77) and Habermann et al., 1980 (Habermann E., Dreyer F., Bigalke H. (1980), Naunyn Schmiedebergs Arch Pharmacol. 311:33-40) allows you to determine the toxic effect of the drug of this neurotoxin on a living organism or isolated neuromuscular drug. To establish a toxic effect in the LD ₅₀ assay, the neurotoxin must be biologically active in each of the three actions mentioned above. Moreover, various other assays are available to determine, for example, whether a neurotoxin or neurotoxin light chain is proteolytically active. These assays are based, for example, on bringing BoNT/A into contact with SNAP-25. Alternatively, a SNAP-25 cleavage site peptide may be used, whereby the peptide may be labeled for easy identification. In a preferred aspect, biological activity is determined by using the MPN assay described above herein.

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

The nucleic acid molecule can be, for example, heterogeneous nuclear RNA, messenger RNA, RNA, DNA, peptidic nucleic acid, locked nucleic acid and/or modified nucleic acid molecules. The nucleic acid molecule can be circular, linear, integrated into the genome, or episomal. Also included are concatemers encoding fusion proteins containing three, four, five, six, seven, eight, nine, or ten polypeptides. Moreover, the nucleic acid molecule may contain sequences encoding signal sequences for intracellular transport, such as signals for transport into the intracellular space or for transport across the cell membrane.

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

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

A nucleic acid molecule or an expression vector encoding a single-stranded BoNT/A protein may be contained in a host cell containing the nucleic acid molecule or vector of the present invention. The term host cell, as used herein, embraces prokaryotic and/or eukaryotic cells suitable for translating said nucleic acid molecule or said vector, and in particular the single stranded BoNT/A protein. Said host cell may be a host cell that does not express the BoNT/A single chain protein or homologue thereof. The term homologue as used herein refers to a polypeptide containing a polypeptide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with a BoNT/A sequence, e.g. a BoNT/A sequence of SEQ ID NO: 1. However, also included are host cells, such as wild-type cells expressing single chain BoNT/A protein or a homologue thereof. For example, the host cell may be selected from C. botulinum. C. butyricum, C. baratii and C. tetani, for example C. botulinum serotype A, B or F. The host cell may be a Hall strain (ATCC 3502) of C. botulinum; BoNT/A producing C. botulinum strain ATCC 19397, also known as NCTC 4587 and NCTC 7272; BoNT/A producing C. botulinum strain NCTC 2916; BoNT/A2 producing C. botulinum strain Kyoto-F or Mauritius/NCTC 9837; BoNT/A3 producing strain A254 Loch Maree/NCTC 2012 C. botulinum; BoNT/A4 and B producing C. botulinum strain CDC657; BoNT/A5 and B3' producing C. botulinum strain H04402 065; BoNT/B1 producing C. botulinum strain Okra/NCTC 7273; BoNT/B and F producing C. botulinum strain CDC4013/NCTC 12265; or BoNT/F1 producing C. botulinum strain Lange land/NCTC 10281. Said host cell may be 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, in particular an E. coli BL21 (DE3) or BLR(DE3) cell.

In one aspect, the BoNT/A single chain protein is modified within the host cell (ie, glycosylated, phosphorylated, protease-treated, etc.). The modification also includes the addition of non-proteinaceous cofactors, including metal ions. The host cell may include an inducer of BoNT/A single chain protein expression. This expression inducer can be a nucleic acid or polypeptide molecule or a chemical building block, including a small chemical building block, which has the effect of increasing the amount of single-stranded BoNT/A protein in host cell cultures or lysates thereof. An expression inducer may, for example, increase transcription or translation of a nucleic acid molecule encoding a single stranded BoNT/A protein. The inducer may, for example, be expressed by recombinant means,

- 8 040938 known to a person skilled in the art. Alternatively, the inducer may be isolated from a cell, such as a clostridial cell.

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

Preferably, the BoNT/A single chain protein is translated in the cell. The cell may be a prokaryotic or eukaryotic cell. In one aspect, the cell is selected from E. coli, B. subtilis, or yeast; E. coli is a highly preferred host cell. The present invention also encompasses the translation of a single stranded BoNT/A protein in a wild-type cell, i.e. a naturally isolated cell such as any known isolate of Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium tetani. Any suitable host cell can be used in accordance with the present invention as described herein.

Various standard means and methods are available to one skilled in the art for introducing a nucleic acid molecule or vector into a host cell and expressing the BoNT/A single chain protein as a recombinant protein in the cell. Moreover, one skilled in the art is aware of many 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 SpriHr Harbor Laboratory, 2000). Any of these means and methods can be used in the methods of the present invention.

Conventional methods for recovering proteins from host cells or a host cell lysate include centrifugation (separation of suspended particles) of the cell lysate, precipitation of proteins with ammonium sulfate, resuspension of proteins, centrifugation of resuspended proteins, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and the like. Some combinations of these steps in different order may be suitable in accordance with the present invention for the purification of a BoNT/A single chain protein.

As described in more detail below, the BoNT/A single chain protein can be contacted with Lys-C to produce an active BoNT/A bicep. The inventors have now developed a preferred method for purifying the BoNT/A bicep from the reaction mixture of BoNT/A and Lys-C.

Endoproteinase Lys-C.

As described herein, the BoNT/A single chain protein is cleaved to form the active double chain form of the BoNT/A protein using Lys-C endoproteinase (Lys-C). The term Lys-C refers to the 33 kDa serine endoproteinase Lys-C from Lysobacter enzymogenes (lysyl endopeptidase, LeK, Genbank add. Q7M135), which specifically cleaves peptide bonds at the C-terminus to a lysine or homologue having at least 50% sequence identity. In one embodiment, its homolog having at least 50% sequence identity to Lys-C retains the functionality (ie, proteolytic activity) of Lys-C.

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

Typically, Lys-C homologues are able to hydrolyze single chain botulinum neurotoxin (eg serotype A (BoNT/A)) to produce biceps botulinum neurotoxin (eg serotype A (BoNT/A)). In one embodiment, the term Lys-C also encompasses functionally equivalent proteases, such as, for example, proteases that recognize the same cleavage sequence as Lys-C and hydrolyze on the carboxyl side of Lys. The term also encompasses homologues of said protease having at least 60% sequence identity.

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, a 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.

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

- 9 040938 peptide bond.

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

The test substrate is typically a polypeptide 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, uncleaved/raw BoNT, referred to herein as single-stranded BoNT (scBoNT), and can be, for example, serotype A, B, C1, D, E, F, or G (e.g., scBoNT /A, scBoNT/B, etc.) or the test substrate may be tetanus neurotoxin (TNT). Alternatively, the test substrate may be a fragment of a clostridial neurotoxin, wherein said fragment contains 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. leftovers. As used throughout this specification, the term polypeptide refers to molecules with 50 or more amino acid residues, while the term peptide refers to molecules with 2-49 amino acid residues.

The test substrate can be a soluble fragment of a neurotoxin called LH _N , containing a light chain polypeptide, a loop-exposed peptide region, and an N-terminal half of the heavy chain polypeptide, the H _N translocation domain. The test substrate may be or contain a peptide selected from any one of SEQ ID NOs: 2 to 8 (see Table 1). The test substrate may be a chimeric neurotoxin containing amino acid residues derived from two or more serotypes.

An assay for determining the proteolytic activity of a Lys-C enzyme, homologue or derivative thereof, typically includes the step of determining the degree of conversion of the test substrate to its cleavage product(s). Observation of one or more cleavage product(s) formed after contacting the polypeptide with a test substrate, or observation of an increase in the amount of cleavage product(s), is indicative of proteolytic activity of the polypeptide. Said determination step may include comparison of substrate and cleavage product(s). Said comparison may include determining the amount of substrate and/or the amount of one or more cleavage products, and may also include counting the ratio of substrate to cleavage product(s). In addition, the assay for determining proteolytic activity may include the step of comparing a test sample with a reference sample, wherein the reference sample typically contains (a) a polypeptide that contains a polypeptide sequence 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 (a).

Assay for determining proteolytic activity may include separation of the substrate (eg, BoNT/A single strand protein) and cleavage product(s) (eg, active BoNT/A bicep) by electrophoresis or by column chromatography and optionally spectrometric analysis. It may be convenient to label the test substrate with one or more labels in order to more easily determine the reduction in test substrate and/or the increase in product(s). The term label, as used herein, refers to a detectable marker and includes, for example, a radioactive label, an antibody, and/or a fluorescent label. The amount of test substrate and/or cleavage product can be determined, for example, by autoradiography or spectrometry methods, including methods based on resonant energy transfer between at least two labels. Alternatively, immunological methods such as Western blotting or ELISA can be used for detection.

In a preferred aspect, the polypeptide is proteolytically active if more than 20%, preferably more than 95% of the test substrate is converted to cleavage products such as light chain and heavy chain in 120 minutes at 37°C using a buffer solution selected from 100 mM Tris-HCl, pH 8.0, or PBS (50 mM Na ₂ HPO ₄ , 150 mM NaCl, pH 7.4). The same conditions apply if the test substrate is not full-length BoNT/A but instead, for example, a fragment of full-length BoNT/A or a derivative of BoNT/A. Obviously, the cleavage products in this case will be different. However, the skilled artisan can quantify the corresponding cleavage products.

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

The sample can be taken at intervals to track catalytic activity over time.

- 10 040938 me.

The assay can be modified, for example, by using multiples of the proteolytically active Lys-C polypeptide.

SEQ ID NO: 9 shows the polypeptide sequence of a proteolytically inactive polypeptide derived from Clostridium botulinum strain ATCC 3502, GenBank Supplement 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 missing from 1 to 248 amino acid residues of SEQ ID NO: 9.

The term polypeptide that contains a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8 refers to a polypeptide that has at least 50% sequence identity with the sequence of SEQ ID NO: 8. In addition, the term refers to a polypeptide , which contains a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8. The specified polypeptide may have additional amino acids, for example, in an internal position or at the N- or C-terminus, to the sequence shown in SEQ ID NO: 8, or internally or at the N- or C-terminus to an amino acid sequence that is at least 50% identical to the sequence of SEQ ID NO: 8, while methionine may be present at the N-terminus of the polypeptide. In addition, the term refers to a polypeptide with one or more amino acid residues missing, for example, at an internal position or at the N- or C-terminus of the sequence shown in SEQ ID NO: 8, or at an internal position or at the N- or C-terminus of a sequence that is at least 50% sequence identical with SEQ ID NO: 8. The term sequence identity and means for calculating sequence identity are defined herein with respect to BoNT/A, and the same definitions and means are also used when referring to Lys-C.

Typically, the sequence identity of the Lys-C enzyme is determined over the entire length of SEQ ID NO: 8 or 9, i.e. along the length, which is 333 aa or 581 aa, 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 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity.

The proteolytically active Lys-C polypeptide may have the same number of amino acids as the sequence of the reference polypeptide as shown in SEQ ID NO: 8. Alternatively, the polypeptide may have additional amino acid residues or fewer amino acid residues. For example, a proteolytically active polypeptide of the present invention may consist of or contain a processing 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. A processing mutant of SEQ ID NO: 9 may for example, one or more amino acid residues are missing from the N-terminus to amino acid position 249. The processing mutant can be an N- or C-terminal processing mutant and/or an internal processing mutant that is proteolytically active.

The processing mutant of SEQ ID NO: 9 may lack amino acid positions 1 to 248 of SEQ ID NO: 9. Alternatively, the processing mutant of SEQ ID NO: 9 may be a C-terminal processing mutant. A processing mutant may lack 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 may have an amino acid length of at least 200 amino acid (aa) residues, at least 250 aa residues, at least 300 aa residues, or at least 333 aa residues. Alternatively, a proteolytically active polypeptide can have up to 333 aa residues, up to 350 aa residues, up to 573 aa residues, up to 581 aa residues, up to 592 aa residues, up to 600 aa residues, or up to 617 aa residues.

A proteolytically active polypeptide may include a polypeptide comprising additional amino acid residues at the N- or C-terminus and/or in the internal position of the polypeptide chain of SEQ ID NO: 8 or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 8 These additional amino acid residues may include up to 5, up to 10, or even up to 200, 300, or up to 400 consecutive amino acid residues.

Additional amino acid residues may function as an inhibitor of proteolytic activity. These additional amino acid residues can be removed by the protease. Alternatively, additional residues that inhibit the proteolytic activity of the polypeptide are excluded. Additional amino acid residues may be surrounded by one or more protease cleavage sites. In another aspect, the additional amino acid sequence functions as a detectable label and/or allows binding to a solid support.

In another aspect, the polypeptide chain of SEQ ID NO: 8 or a polypeptide sequence having

- 11 040938 having at least 50% sequence identity with the sequence of SEQ ID NO: 8, modified by the exchange of one or more amino acid residues. The term exchange, as used herein, refers to the replacement of an amino acid with another amino acid. For example, up to 1 amino acid (aa), 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 15 aa, 20 aa, or up to 50 aa. The exchanges may involve conservative or non-conservative amino acid substitutions aimed, for example, at increasing or decreasing the binding substrate or the proteolytic activity of the polypeptide.

Typically, a proteolytically active polypeptide includes a polypeptide that is capable of hydrolyzing a substrate (eg, single chain BoNT/A protein) into two or more natural cleavage product(s). The polypeptide of the present invention can hydrolyze the substrate into two or more cleavage products that are the same as or different from the natural cleavage products. Preferably the cleavage products are the same as the natural cleavage products.

The term natural cleavage products or natural products, as used herein, refers to products that are identical in amino acid sequence when compared to products derived from the same substrate in the wild-type cell cultures from which the substrate is derived. In a preferred embodiment, the cleavage product is a biceps neurotoxin of botulinum neurotoxin or tetanus neurotoxin. In a more preferred embodiment, the bicep neurotoxin is a neurotoxin isolated from C. botulinum serotype A, B, C1, D, E, F, or G. In another aspect, said bicep neurotoxin is a natural BoNT/A bicep neurotoxin.

It should be understood that the definitions and explanations of terms made above and below, unless otherwise indicated, apply mutatis mutandis for all aspects described in this description.

Ways to get BoNT.

The present invention relates to the use of Lys-C in a method for the proteolytic processing of a polypeptide, specifically the single-stranded BoNT/A protein, and to a means for purifying the active bicep of BoNT/A derived from Lys-C using hydrophobic interaction chromatography (HIC). In one aspect, the present invention relates to a method for producing an active BoNT/A bicep, comprising the step of bringing into contact: (a) Lys-C with (b) a single-stranded BoNT/A protein, wherein said single-stranded BoNT/A protein is proteolyzed 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 an active BoNT/A bicep, and purification of said active bicep using HIC.

The process of the invention can be used to produce proteolytically treated clostridial neurotoxin (CNT) or botulinum neurotoxin (BoNT), specifically proteolytically treated with BoNT/A, ie. active bicep of BoNT/A as described in this document. Using the method of the present invention, it is now possible to obtain compositions of the active BoNT/A bicep with significantly less contamination with untreated or partially treated BoNT/A, since these contaminants are efficiently processed into the BoNT/A bicep. The methods of the present invention also allow the efficient removal of the Lys-C enzyme used to convert the single stranded BoNT/A protein into an active bicep, i.e. improved cleaning of the BoNT/A active bicep.

The present invention thus relates to the purification (cleavage) of the BoNT/A bicep from Lys-C, including separation by hydrophobic interaction chromatography. Accordingly, the invention provides a method for producing a BoNT/A biceps protein, including providing a BoNT/A single strand protein, contacting said BoNT/A protein with a LysC endoproteinase in solution, and separating the BoNT/A protein from Lys-C by contacting a solution containing protein BoNT/A and Lys-C, with a hydrophobic surface, while the BoNT/A protein preferentially 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 soluble bicep form. Preferably, the cleavage product of the BoNT/A single chain protein by Lys-C is the same as the natural cleavage product of BoNT/A. Typically, both the single chain and biceps forms of BoNT/A are soluble.

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

In one embodiment, in which the invention provides a method (as described above) for producing a soluble BoNT/A bicep protein, a soluble BoNT/A single chain protein is provided via a method as described above to obtain a soluble BoNT/A single chain protein in a host cell, preferably a bacterial host cell, most preferably an E. coli host cell.

Any suitable tool can be used to produce a soluble single-stranded BoNT/A protein.

- 12 040938 expression system. In accordance with the present invention, the expression system may be an in vivo or 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 include a suitable host cell and/or a suitable expression vector for use in said host cell. For example, a suitable expression system may include a bacterial host cell and/or an expression vector suitable for expressing a soluble BoNT/A single chain protein in said bacterial host cell.

The BoNT/A single chain protein can be produced in any suitable host cell as described herein, such as a bacterial cell. Typically, an Escherichia coli (E. col) host cell is used. A single stranded BoNT/A protein can be produced by a method involving expression of the nucleic acid sequence in the expression system of a host cell (eg, E. coli cells). Such expression may include introducing a nucleic acid molecule encoding said single stranded BoNT/A protein into said host cell and translating said nucleic acid molecule to produce a single stranded BoNT/A protein. Methods and techniques used to express heterologous proteins in expression systems, including E. coli expression systems, are well known in the art.

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

The soluble single chain BoNT/A protein may 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/l, 1 mg/ml, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100 mg/ml or more). Generally, the level of expression of a single chain BoNT/A protein refers to the level of single chain BoNT/A in cell culture. In one embodiment, said expression level refers to an untreated host cell homogenate (e.g., bacterial host cell), i.e. untreated host cell homogenate (eg, bacterial host cells) used to express the BoNT/A single chain protein. Thus, in one embodiment, the level of expression of the BoNT/A single chain protein in the untreated host cell homogenate, eg, untreated bacterial host cell homogenate, is at least 5 mg/L (as defined herein).

The method for producing a soluble single chain BoNT/A protein as described above may include lysing a host cell, preferably a bacterial host cell, most 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 soluble BoNT/A single chain protein. Methods and techniques used to lyse host cells, such as bacterial cells, in particular E. coli host cells, are known in the art. Examples include sonication or the use of a French press.

Purification of C. botulinum or E. coli expressing single chain BoNT/A protein can be done, for example, as actually described in the prior art (DasGupta 1984, Toxicon 22, 415; Sathyamoorthy 1985, J. Biol. Chemistry 260, 10461). In particular, purification of a neurotoxin may include one or more precipitation and extraction steps, one or more concentration steps, and additional separate chromatographic steps. Recombinant single chain BoNT/A and its purification are described in the prior art (Rummel et al., 2004, Mol. Microbiol. 51:631-43).

The bacterial host cell used to produce the BoNT/A single chain protein or a derivative thereof may be C. botulinum or E. coli. For fermentation, you can use the process described by DasGupta B.R. et al., in Toxicon, vol. 22, no. 3, p. 414-424, 1984. Therefore, 0.5% yeast extract and 0.6% autoclaved yeast paste are added to 2% N-Z-amine type A medium, and a pH of 7.2 will be reached with 4M NaOH, and the medium prepared in this way Thus, it will then be autoclaved. Separately autoclaved glucose (20% w/v) can be added to this medium to achieve a final glucose concentration of 0.5% in the medium. The incubation can take place, for example, at 37° C. without shaking, with the fermentation being interrupted, for example, after 96 hours. Batch fermentation, semi-batch fermentation, repeated batch fermentation or continuous fermentation can be used.

After the actual fermentation and separation of the fermentation medium from the cells, the fermentation medium can be subjected to a first precipitation in order to remove large proteins. The precipitation is preferably an acid precipitation. The reaction conditions for a given acid precipitation are known to those skilled in the art. Typically, 1.5M H2SO4 can be used to acidify the supernatant to pH 3.5. Centrifugation usually occurs within 20 min at 2400xg at 4°C. The precipitate obtained by centrifugation can be washed with water, preferably again. Subsequently, the precipitate can be extracted with a buffer solution of 0.1 M citric acid-trisodium citrate, pH 5.5, for example, within an hour. Into the distance

- 13 040938 Alternatively, an additional centrifugation step can be performed, for example at 9800xg for 20 minutes at 4°C. The precipitate thus obtained can then optionally be re-extracted 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 8°C. Subsequently, the precipitate can be centrifuged, for example, for 20 min at 4°C and at 1200xg. The centrifuge supernatant can be subjected to precipitation, such as ammonium sulfate precipitation, whereby other larger proteins can be removed. After the ammonium sulfate precipitation step, another centrifugation step can be added and the precipitate thus obtained can then be redissolved and optionally dialyzed. The extract, which is preferably dialyzed and re-centrifuged, can be subjected to successive chromatographic steps to purify the neurotoxin. Each of the chromatographic steps serves to remove contaminants such as protamine sulfate, residual DNA, portions of smaller and medium sized proteins, and the hemagglutinins of the botulinum neurotoxin protein complex. To this end, in a preferred embodiment, one or more chromatography steps can be applied. Optionally, the eluate, for example from the last step of the chromatography, can be filtered to remove microorganisms. Optionally, the eluate may be diluted prior to filtration and suitable excipients may be added. During additional steps after the addition of excipients, another sterilizing filtration may be performed. In one aspect, filtration is carried out in reaction vessels, which can then be subjected to a lyophilization step.

The BoNT/A single chain protein can be contacted with Lys-C after it has been isolated from a host cell or host cell lysate and then subjected to the method of the present invention.

When the BoNT/A single chain protein of the invention is contacted with Lys-C, the proteolytic action of Lys-C cleaves the single chain protein at the site between the L chain of the protease component and the translocation component to produce a double chain protein in which the two chains are linked by a disulfide bridge. For example, the two strands formed after cleavage of single strand BoNT/A1, A2 and A4-A6 at the activation site are the first strand amino acid residues 1-438 and the second strand amino acid residues 449-1296 (except for A6, in which the second strand has amino acid residues 449 -1297), while residues 439-447 are removed by a cleavage phenomenon. The two strands formed after cleavage of single strand BoNT/A3 at the activation site are the first strand of amino acid residues 1-434 and the second strand of amino acid residues 445-1292, with residues 435-444 being removed by the cleavage phenomenon. Thus, Lys-C can be used to activate a single chain polypeptide by converting it to its active bicep form. Advantageously, therefore, the use of Lys-C means that there is no need to insert an exogenous (non-natural) cleavage site into BoNT/A, and also makes it possible to obtain a natural BoNT/A bicep.

In one embodiment, reference to Lys-C encompasses Lys-C-like and variant enzymes that cleave at the same protease cleavage site as Lys-C, as described herein.

The term bringing into contact, as used herein, refers to bringing at least two different compounds into physical proximity so as to allow physical and/or chemical interaction of said compounds. According to the method of the present invention, these two different compounds are single chain protein BoNT/A and Lys-C which are included in the solution. Bringing into contact is carried out under conditions and for a time sufficient to allow the interaction of single-stranded protein BoNT/A and Lys-C.

The term proteolyzed refers to a feature or criterion of a BoNT/A single chain protein and is used herein to mean that said BoNT/A single chain protein is proteolytically cleaved by Lys-C. In other words, the term proteolyzed means that the BoNT/A single chain protein includes a protease recognition and cleavage site allowing it to function as a Lys-C substrate. As described herein, the BoNT/A single chain protein is a substrate of Lys-C and is proteolytically processed into two or more cleavage products (preferably two peptides, the L chain or fragment thereof and the H chain or fragment thereof, connected to each other through disulfide bridge). Using the assay described herein above, the skilled artisan can test whether a given BoNT/A single chain protein is a substrate for a first polypeptide, 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 up to six cleavage products.

This method can be used, for example, to prepare a pharmaceutical composition containing the BoNT/A bicep or to create polypeptide fragments used in the method

- 14 040938 mass spectrometry. Lys-C and the single-stranded BoNT/A protein can be contacted at various stages in the process of obtaining the BoNT/A bicep. For example, the step of bringing into contact Lys-C and the BoNT/A single chain protein can occur within the cell, for example, by expressing Lys-C and the BoNT/A single chain protein in said cell.

Alternatively, said contacting step occurs in a cell lysate or in a purified cell lysate. It involves adding Lys-C to a lysate or a purified lysate. Lys-C can be added at various stages during purification of the BoNT/A single chain protein from the cell lysate. For example, Lys-C can be added before or after: protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography and/or size exclusion chromatography.

The contacting step requires incubation under conditions and for a time sufficient for Lys-C to cleave the BoNT/A single chain protein. Illustrative conditions may include the addition of a buffer solution 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 time sufficient for splitting can be determined using the analysis described in this document above. In one aspect, the specified time sufficient for cleavage depends on the degree of cleavage that the proteolytically treated polypeptide or composition containing it should have. In one aspect, the method includes the step of incubating Lys-C and the BoNT/A single chain protein for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another aspect, Lys-C and the BoNT/A single chain 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 includes the step of incubating Lys-C and BoNT/A single chain protein at 4°C or 37°C. In another aspect, the method includes the step of incubating for up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours, or up to 16 hours.

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

The present inventors have found that a high yield of activated bicep protein BoNT/A can be obtained by applying a hydrophobic purification process to separate the activated bicep polypeptide from Lys-C. Surprisingly, this process provides superior purification compared to standard purification using ion exchange chromatography, which the present inventors have found to be less effective in separating the activated bicep polypeptide from Lys-C. In addition, the process advantageously provides an activated BoNT/A bicep protein that does not contain an activated protease and is thus suitable for use in therapy as part of an overall purification process.

As described herein, making active recombinant BoNT/A requires a proteolytic step that cleaves the molecule to its active bicep form. This cleavage can be achieved by an in vitro activation step using Lys-C endoproteinase. After the activation step, it is important to remove Lys-C from the final product, which also prevents any additional non-specific cleavage of BoNT/A.

As shown in Table. 2 below, the calculated isoelectric points (pI) of Lys-C and BoNT/A are 6.70 and 6.05, respectively, indicating that separation of the two proteins could be achieved by ion exchange (IEX) chromatography using the charge difference between the two molecules. . The net charge of a protein is influenced by the pH of its environment and will be more positively or negatively charged depending on whether it gains or loses protons, pI is the pH value at which the molecule carries no electrical charge and therefore will not interact with the charged environment IEX . This means that if a protein is at a pH above its pI, 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 solution is below pI, then the protein will carry a net positive charge and will not bind to the anion exchanger.

Moreover, as illustrated in Table. 2, BoNT/A and Lys-C have similar average hydropathicity but greater charge difference at pH 4.5 and 8.0. Based on this principle, one would expect that ion exchange chromatography (IEX) can be used to cleave (separate) BoNT/A and Lys-C. IEX is a simple and inexpensive chromatography method, so that it does not require the protein loaded onto the column to be in a high saline buffer solution, which would result in loss of the protein through precipitation.

The present inventors tested a variety of anion exchange columns using both strong and weak functionality attached to cross-linked agarose beads at pH 8. When compared with BoNT/A elution, it was found that, surprisingly, Lys-C eluted with similar ionic strength (Table 3; FIGS. 7-10) showing that Lys-C was not separated as predicted and would be present in the final purified BoNT/A product with the additional possibility of further dehydration of BoNT/A.

- 15 040938

The present inventors have solved the above problem. In more detail, the inventors unexpectedly determined that optimal separation of Lys-C-BoNT/A is achieved by using a hydrophobic interface (for example, by hydrophobic interaction chromatography (HIC), which separates proteins according to the difference in hydrophobicity of their surfaces by using a reversible interactions between these proteins and the hydrophobic surface of the HIC matrix/resin).

In general, the active bicep of BoNT/A preferentially binds to the hydrophobic surface of the HIC resin/matrix (these terms are used interchangeably herein) compared to Lys-C to bind to the hydrophobic surface. Preferential binding can be defined as increased or improved binding of the active BoNT/A bicep to a hydrophobic surface compared to Lys-C binding to a hydrophobic surface. For example, the active BoNT/A bicep can bind to a hydrophobic surface at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold. fold, 40-fold, 50-fold, 100-fold or more affinity of Lys-C to the hydrophobic surface. In a preferred embodiment, the active BoNT/A bicep binds to the hydrophobic surface with at least five-fold, or at least ten-fold, Lys-C affinity for the hydrophobic surface.

In one embodiment, the hydrophobic surface is an inert matrix/resin to which is attached a ligand comprising or consisting of aryl or alkyl groups.

The term aryl refers to aromatic groups such as phenyl, naphthyl, thienyl and indolyl.

The term alkyl refers to aliphatic groups, including straight chain, branched chain, cyclic groups, and combinations thereof. The alkyl group may 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.

In one embodiment, the hydrophobic surface contains one or more ligands selected from the group consisting of butyl, phenyl, or octyl ligands.

In one embodiment, the hydrophobic surface contains butyl ligands. In one embodiment, the hydrophobic surface contains phenyl ligands. In one embodiment, the hydrophobic surface contains octyl ligands.

The hydrophobic surface may contain any suitable inert matrix/resin with an appropriately attached hydrophobic ligand. 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, individual polypeptides contain glass, polystyrene, polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbro and magnetite. The inert matrix, for example, is a polysaccharide matrix selected from the group consisting of sepharose, sephadex, agarose, ceftsel, microcellulose, and alginate beads. In another aspect, said inert matrix/resin may be composed of glass beads and/or polypeptide matrices.

The inert matrix/resin may have any suitable structural configuration or arrangement. For example, the matrix/resin may be spherical, as in a bead, or cylindrical, as on the inside of a test tube, or on the outside of a rod. Alternatively, the matrix may be irregular or flat, such as a sheet or test strip.

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

The use of HIC provides improved separation of Lys-C from the active bicep of BoNT/A compared to ion exchange chromatography (IEX).

In accordance with the present invention, fast flow resins/matrices can be used for HIC chromatography. A fast flow resin/matrix can be defined as a resin/matrix with a more uniform average particle size than a standard HIC resin/matrix and will use smaller particles. Specifically, the Fast flow resin/matrix will have a narrower range of smaller particle sizes. Particle size can be quantified by any appropriate method known in the art, such as in terms of average particle diameter. Typically, Fast flow resin/matrix will contain particles (eg, beads) with an average diameter of less than 100 microns, less than 95 microns, less than 90 microns, or less. In a preferred embodiment, the average fast flow resin/matrix diameter is less than 90 microns. Typically, the distribution of medium fast flow resin/matrix particles will be from about 20 to about 180 microns, from about

- 16 040938 to about 170 microns, from about 40 to about 170 microns, from about 40 to about 165 microns. In a preferred embodiment, the distribution of medium fast flow resin/matrix particles is from about 44 to about 165 microns.

In a preferred embodiment, high performance HIC resins are used, such as phenyl high performance (PhHP) or butyl high performance (BuHP) resins. Such high performance HIC matrices/resins generally provide improved separation of Lys-C from the active BoNT/A bicep compared to ion exchange chromatography (IEX), even when high performance IEX matrices/resins such as quaternary amine high performance (QHP) matrices are used. /resin. As discussed herein, the main difference between high performance resins/matrices and others is that the average particle size (again can be quantified by any appropriate method known in the art, e.g. average particle diameter) is smaller (34 µm vs. 90 µm) and more uniform (24-44 µm vs. 44-165 µm), resulting in improved separation.

Generally, a high performance HIC resin/matrix 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.

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

Typically, the average particle distribution of the high performance HIC resin/matrix will be about 10 to about 60 microns, about 10 to about 50 microns, about 20 to about 50 microns, about 20 to about 44 microns. In a preferred embodiment, the high performance HIC resin/matrix medium particle distribution is from about 22 to about 44 microns.

Typically, a hydrophobic purification process to separate the activated BoNT/A bicep protein from Lys-C reduces the concentration of Lys-C at least two-fold, at least 5-fold, at least 10-fold, at least 15-fold , 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, a hydrophobic purification process to separate the activated BoNT/A bicep protein from Lys-C reduces the concentration of Lys-C by at least 10-fold.

The ability of hydrophobic purification to separate the activated BoNT/A bicep from Lys-C can also be quantified in terms of the percentage of Lys-C contained in the initial solution containing the activated BoNT/A bicep and Lys-C that remains after the hydrophobic purification step. As a rule, when obtaining a BoNT/A bicep, after the hydrophobic purification step, less than 80, 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 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 less, including 0% Lys-C. Preferably less than 50%, more preferably less than 25%, even more preferably less than 10% Lys-C is left after the hydrophobic purification step in the preparation of the BoNT/A bicep.

In a related aspect, the invention provides an active BoNT/A bicep protein obtainable by a method (as described above) for producing a soluble BoNT/A bicep protein.

In one aspect, the invention provides a composition comprising an active BoNT/A bicep protein (as described above), said composition being substantially free of Lys-C.

Thus, the composition is advantageously substantially free of the Lys-C protease (used to activate the single chain polypeptide by converting it to its active bicep form), thus preventing unwanted non-specific cleavage of the BoNT/A protein.

However, the composition (as described above) is substantially free of Lys-C, with the composition typically containing less than 400 pg of Lys-C per 100 ng of BoNT/A protein; e.g. less than 400, 350, 300, 250, 200, 150, 100, 90, 8θ, 70, 60, 50, 40, 30, 20, 10, 9, 8, 5, 4, 3, 2, 1 pg or less than Lys-C per 100 ng of BoNT/A protein. In one embodiment, the composition (as described above) contains less than 400 pg of Lys-C per 100 ng of BoNT/A protein, less than 350 pg of Lys-C per 100 ng of BoNT/A protein, less than 300 pg of Lys-C per 100 ng of BoNT protein /A, less than 250 pg of Lys-C per 100 ng of BoNT protein/A, less than 200 pg of Lys-C per 100 ng of BoNT protein/A, less than 150 pg of Lys-C per 100 ng of BoNT protein/A, 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 of Lys-C per 100 ng of BoNT/A protein, less than 30 pg of Lys-C per 100 ng of 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 Lys-C per 100 ng of BoNT/A protein, less than 12 pg of Lys-C per 100

- 17 040938 ng of BoNT/A protein, less than 11 pg of Lys-C per 100 ng of BoNT/A protein, less than 10 pg of Lys-C per 100 ng of BoNT/A protein, less than 9 pg of Lys-C per 100 ng of BoNT/A protein , less than 8 pg of Lys-C per 100 ng of BoNT/A protein, less than 7 pg of Lys-C per 100 ng of BoNT/A protein, less than 6 pg of Lys-C per 100 ng of BoNT/A protein, less than 5 pg of Lys-C per 100 ng of BoNT/A protein or less. In a preferred embodiment, the composition (as described above) contains 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, less than 20 pg of Lys-C per 100 ng of BoNT protein /A, less than 15 pg of Lys-C per 100 ng of BoNT/A protein or less than 10 pg of LysC per 100 ng of BoNT/A protein.

Methods for determining the concentration of Lys-C in a composition are known in the art. As an example, the concentration of Lys-C in the composition of the invention can be determined using a sandwich ELISA (enzyme-linked immunosorbent assay) or calorimetric analysis, as described in this document.

Pharmaceutical compositions and clinical indications.

The present invention also relates to a composition obtainable by the process of the present invention for the production of a BoNT/A bicep. In one aspect, said composition contains a mixture of treated (biceps) and untreated (single chain) BoNT/A, wherein said mixture may contain less than 5, 4, 3, 2, or less than 1% of untreated (single chain) BoNT/A. In the aspect of the specified composition, references to BoNT/A cover its derivative, as defined in this document. The composition may be, for example, a liquid or solid composition and may contain one or more carriers, excipients and/or excipients.

In another aspect, the present invention also relates to a method for producing a medicinal product, i. a pharmaceutical composition comprising the steps of the method mentioned above and an additional step of preparing the finished form of the purified BoNT/A bicep as a drug. Typically, said drug contains a mixture of processed (biceps) and unprocessed (single chain) BoNT/A, wherein said mixture contains less than 20%, less than 15%, less than 10%, less than 5%, or less than untreated (single strand) BoNT/A . In a preferred embodiment, the mixture contains less than 5% raw (single chain) BoNT/A, such as less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% raw (single chain) BoNT/A.

The present invention also relates to various medical and aesthetic (cosmetic) uses for the compounds and compositions disclosed herein. Accordingly, the present invention relates to an active BoNT/A bicep or a composition comprising an active BoNT/A bicep according to the present invention, for use as a drug or in a pharmaceutical composition. The present invention also relates to the use of an active BoNT/A bicep or a composition containing an active BoNT/A bicep of the present invention in the manufacture of medicaments. The present invention also relates to an active BoNT/A bicep or a composition comprising an active BoNT/A bicep of the present invention, for use in a method of treating a human or animal body through therapy. The present invention also relates to a method of treatment comprising administering an active BoNT/A bicep or a composition containing an active BoNT/A bicep of the present invention to a patient in need thereof.

The term composition, as used herein, refers to any composition that is formulated into a solid, liquid, aerosol (or gaseous) form, and the like. Said composition contains, for example, a therapeutically active compound of the invention, optionally together with suitable auxiliary compounds such as diluents or carriers or additional ingredients. In one aspect, the therapeutically active compound is the active BoNT/A bicep of the present invention. Compositions, in particular pharmaceutical compositions of the invention, as a rule, essentially do not contain Lys-C, as defined in this document.

The present invention thus provides a solid or liquid pharmaceutical composition comprising:

(a) an active BoNT/A bicep protein as described above, and (b) a stabilizing agent.

In one embodiment, the composition (as described above) is substantially free of Lys-C. For example, a composition (as described above) may contain 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 A, less than 100 pg 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 of BoNT/A protein or less than 10 pg of Lys-C per 100 ng of BoNT/A protein.

Stabilizing agents that can be used in the composition according to the invention include protein stabilizers such as albumin, in particular human serum albumin (HSA), and non-protein stabilizers.

Non-protein stabilizing agents that can be used in the composition according to the invention include surfactants, in particular non-ionic surfactants. Examples of non-ionic surfactants include polysorbates such as polysorbate 20 or polysorbate 80, and block copolymers such as poloxamers (ie copolymers of polyethylene and propylene glycol).

In a separate embodiment, the composition does not contain protein as a stabilizing agent.

In accordance with a separate embodiment of the invention, the pharmaceutical composition is a liquid pharmaceutical composition containing:

(a) active BoNT/A bicep protein as described above;

(b) a non-protein stabilizing agent, which is a surfactant; and (c) water;

wherein said liquid pharmaceutical composition does not contain a protein stabilizing agent; and wherein said liquid pharmaceutical composition is substantially free of Lys-C while being substantially free of Lys-C has the meaning 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 of 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 protein BoNT/A).

In one embodiment, the active BoNT/A bicep protein is present in the composition (as described above) at a concentration of 1-1000 ng/ml or more. Thus, the active BoNT/A bicep protein may be 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 more. In a preferred embodiment, the active BoNT/A bicep 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, or 700 ng/mL .

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 a preferred embodiment, the polysorbate is of plant origin . The concentration of surfactant is preferably less than 1% v/v, for example, in the case of polysorbate 80 from about 0.005 to 0.02% v/v.

The pharmaceutical composition according to the invention may also contain a crystalline agent.

By crystalline agent is meant an agent which, inter alia, retains the mechanically strong compressed structure of a lyophilized botulinum neurotoxin complex (type A, B, C1, D, E, F or G) or high purity botulinum neurotoxin (type A, B, C1, D, E , F or G). When incorporated into solid formulations, crystalline agents also have a bulking effect. In particular, the crystalline agents contain sodium chloride. The concentration of the crystalline agent may be, for example, from 0.1 to 0.5M, preferably from 0.1 to 0.4M, especially from about 0.15 to 0.3M.

The pharmaceutical composition according to the invention may also contain a buffer solution to maintain the pH between 5.5 and 7.5 or between 6.0 and 7.0. The buffer solution can be any buffer solution capable of maintaining an adequate pH. For example, the buffer solution for the composition according to the invention can be selected from the group consisting of succinate, disodium phosphate/citric acid and an amino acid such as histidine. The concentration of the buffer solution may be, for example, from 1 to 50 mm, preferably from 5 to 20 mm, preferably about 10 mm.

The pharmaceutical composition according to the invention may also contain a disaccharide.

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

In a separate embodiment, the pharmaceutical composition is a liquid pharmaceutical composition containing:

(a) active BoNT/A bicep protein as described above;

(b) a non-protein stabilizing agent, which is a surfactant;

(c) sodium chloride, (d) buffer solution to maintain pH between 5.5 and 7.5;

(e) a disaccharide and

- 19 040938 (f) sterile water, wherein said liquid pharmaceutical composition does not contain a protein stabilizing agent; and wherein said liquid pharmaceutical composition is substantially free of Lys-C while being substantially free of Lys-C has the meaning 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 of 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 protein BoNT/A).

According to a particular embodiment, the pharmaceutical composition of the invention in liquid form is stoppered in a vial or ready-to-use device such as a syringe without a liquid/gas interface and is stable for at least three months or at least at least six months at 23-27°C and for at least twelve months at 2-8°C. Illustrative pharmaceutical compositions of the invention are described in the examples.

Monthly disintegration rates of pharmaceutical compositions or formulations of the invention can be below 5% per month for 12 weeks, which means that the protease function of the BoNT biceps of the compositions or formulations remains stable at 25° C. for at least 12 weeks.

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

In this context, for the present invention, a distinction should be made between auxiliary compounds, i.e. compounds that do not contribute to the results produced by the compound of the present invention when the composition is used for its intended purpose, and additional ingredients, i.e. compounds that contribute to the additional effect or modulated 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 and the other ingredients. A person skilled in the art can determine such suitable diluents and/or carriers without additional difficulty.

The carrier(s) must be acceptable in the sense that it must be compatible with the other ingredients of the formulation and harmless to its recipient. The pharmaceutical carrier used may be a solid, gel or liquid. Illustrative solid carriers are lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum, magnesium stearate, stearic acid, and the like. Illustrative examples of liquid carriers are phosphate buffered saline, molasses, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or diluent may contain a time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate alone or with wax. Said suitable carriers include those mentioned above and others well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

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

In one aspect, the pharmaceutical composition as used herein contains a biologically active neurotoxin obtained by the method of the present invention (ie active BoNT/A biceps), optionally one or more pharmaceutically acceptable carriers. The active neurotoxin may be present in liquid or lyophilized form. In an aspect, said compound may be present together with glycerol, proteinaceous stabilizers (eg, human serum albumin (HSA)) or non-proteinaceous stabilizers such as polyvinylpyrrolidone or hyaluronic acid. In one aspect, the pharmaceutical composition is administered topically. The commonly used drug administration is intramuscular or subcutaneous (usually near the sebaceous glands) administration. However, depending on the nature and mechanism of action of the compound, the pharmaceutical composition may also be administered in other ways. The bichain neurotoxin polypeptide is the active ingredient of the composition, and in one aspect is administered in conventional dosage forms prepared by combining the drug substance with standard pharmaceutical carriers in accordance with conventional procedure. These procedures may include mixing, granulating and compressing or dissolving the ingredients, as appropriate for the desired formulation. It will be appreciated that the form and nature of the pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient with which it is to be combined, by administration and other well known variables.

A therapeutically effective dose refers to the amount of a compound, a neurotoxin, to be used in a pharmaceutical composition of the present invention that prevents, alleviates or treats the symptoms accompanying the disease or condition referred to herein. The therapeutic efficacy and toxicity of a compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example _ED50 (therapeutically effective dose in 50% of the population) and LD ₅₀ (dose lethal to 50% of the population). The dose ratio between therapeutic and toxic results is the therapeutic index and can be expressed as the ratio, LD ₅₀ /ED ₅₀ .

The dosage regimen will be determined by the hospital staff doctor and other clinical factors. As is well known in the medical field, dosages for any patient depend on a variety of factors, including patient size, body surface area, age, particular compound to be administered, sex, time and route of administration, general health and other concomitantly administered drugs, success can be track through periodic evaluations. The pharmaceutical compositions and formulations referred to herein are administered at least once for the purpose of treating or ameliorating or preventing the disease or condition listed herein. However, these pharmaceutical compositions can be administered more than once.

As described herein, active BoNT/A bicep proteins of the invention and compositions and liquid pharmaceutical compositions thereof can be used for treatment. Suitable treatments may include cosmetic treatments and medical treatments.

In a further aspect of the invention, the composition mentioned above is a medicinal or cosmetic composition. In one aspect, said drug containing a biologically active neurotoxin (i.e. active BoNT/A bicep) can be used to prevent and/or treat at least one of the following diseases and disorders: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, hyperhidrosis and correction of cosmetic wrinkles, in an additional aspect also blepharospasm, oromandibular dystonia of the open jaw type, occlusion type, bruxism, Meigs syndrome, lingual dystonia, apraxia century, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, dystonia of the extremities, dystonia of the upper extremities, dystonia when performed with specific tasks, writing spasm, musician's spasm, golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee flexion, knee extension, ankle flexion, ankle leg extension, foot equinovarus deformity, foot dystonia deformity , striatal toe, toe flexion, toe extension, axial dystonia, Leaning Tower syndrome, belly dancer's dystonia, segmental dystonia, hemidystonia, generalized dystonia, Lubag syndrome dystonia, corticobasal degeneration dystonia, Lubag syndrome dystonia, residual 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, residual dyskinesia/residual dystonia, paroxysmal dyskinesia/dystonia, kinesiogenic, non-kinesiogenic, action-induced viem, palatine myoclonus, myokymic myoclonus, rigidity, benign muscle cramps, hereditary trembling of the chin, paradoxical activity of the masticatory muscles, spasms of the masticatory muscles of one half of the face, hypertrophic branchial myopathy, hypertrophy of the masseter muscle, hypertrophy of the anterior tibial muscle, nystagmus, oscillopsia, supranuclear gaze palsy , permanent partial epilepsy, planned surgery for spastic torticollis, abducens glottis paralysis, persistent mutational dysphonia, upper pharyngeal sphincter dysfunction, vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective glottic closure, post-laryngectomy speech disorder, protective ptosis , eyelid volvulus, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal dysmotility, vaginismus, postoperative tremor with immobility, bladder dysfunction, detrusor-sphincter dyssynergia, urinary sphincter spasm blistering, hemifacial spasm, reinnervation dyskinesias, cosmetic use for crow's feet, facial asymmetry due to frown, chin pits, stiff man syndrome, tetanus, prostate hyperplasia, obesity treatment, cerebral palsy, strabismus, mixed, paralytic,

- 21 040938 concomitant, after surgery for retinal detachment, after cataract surgery, for aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation, as an addition to strabismus surgery, convergent strabismus, divergent strabismus, achalasia, anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, axillary, palmar, plantar hyperhidrosis, rhinorrhea, relative hypersalivation in stroke, in Parkinson's disease, in spastic conditions in amyotrophic lateral sclerosis, in autoimmune encephalitis and myelitis processes, multiple sclerosis, transverse myelitis, Devic's disease, viral infections bacterial infections parasitic infections, fungal infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brainstem infarction, spinal cord infarction, migraine, with trauma to the central nervous system, hemispheric lesions, spinal cord injury a, brain stem injury, central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, spinal cord hemorrhage, neoplasia, hemispheric tumors, brain stem tumors, spinal cord tumors, snoring (WO 2000/033863). For details and symptoms see, for example, Jost 2007, Drugs 67(5), 669 or Dressier 2000 in Botulinum Toxin Therapy, Thieme Verlag, Stuttgart, New York.

In another aspect of the invention, the composition is a cosmetic composition which can be formulated as described for a pharmaceutical composition above. Moreover, for a cosmetic composition, it is contemplated that the compound of the present invention is in use aspect in a substantially pure form. In a further aspect, the cosmetic compositions are to be administered intramuscularly. In another further aspect of the invention, cosmetic compositions containing the neurotoxin may be formulated as an anti-wrinkle solution.

All references listed in this specification are hereby incorporated by reference with respect to the contents of their full disclosure and the contents of the disclosure specifically mentioned in this specification.

Key to SEQ ID NO.

SEQ ID NO: 1 BoNT/A ATCC 3502, Genbank add. AAA23262.

SEQ ID NO: 2 BoNT/A1 activation loop.

SEQ ID NO: 3 BoNT/A2/A6 activation loop.

SEQ ID NO: 4 BoNT/A3 activation loop.

SEQ ID NO: 5 BoNT/A3 activation loop.

SEQ ID NO: 6 BoNT/A4 activation loop.

SEQ ID NO: 7 BoNT/A5 activation loop.

SEQ ID NO: 8 Proteolytically active polypeptide derived from Clostridium botulinum strain ATCC 3502, GenBank Supplement No. CAL82988.1, missing 248 N-terminal amino acid residues.

SEQ ID NO: 9 Proteolytically inactive polypeptide derived from Clostridium botulinum strain ATCC 3502, GenBank Supplement 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 loop.

SEQ ID NO: 14 BoNT/A8 activation loop.

SEQ ID NO: 15 Amino acid sequence of single chain, recombinant, cationic derivative of botulinum neurotoxin subserotype A1 (mrBoNT/A1).

List of figures

Fig. 1 shows elution profiles from a phenyl high efficiency (PhHP) column, on which the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 2 shows elution profiles from a highly displaced Fast Flow phenyl column (PhFF-Hi) that assessed the separation of Lys-C (A ₄₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line).

Fig. 3 shows elution profiles from a butyl high efficiency (BuHP) column on which the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 4 shows elution profiles from a sulfopropyl high efficiency (SPHP) column on which the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 5 shows elution profiles from a sulfopropyl Fast Flow (SPFF) column that assessed the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line).

Fig. 6 shows elution profiles from a carboxymethyl Fast Flow (CMFF) column that assessed the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line).

Fig. 7 shows elution profiles from a quaternary amine high efficiency (QHP) column on which the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 8 shows elution profiles from a quaternary amine Fast Flow (QFF) column that assessed the separation of Lys-C (A4o ₅ - bars) from BoNT/A (A ₂₈₀ - dotted line).

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Fig. 9 shows elution profiles from a diethylaminopropyl (DEAP, ANX) column on which the separation of Lys-C (A4 ₀₅ - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 10 shows elution profiles from a diethylaminoethyl (DEAE) column on which the separation of Lys-C (A405 - bars) from BoNT/A (A ₂₈₀ - dotted line) was evaluated.

Fig. 11 - removal of Lys-C from rBoNT/A1 by phenyl HP HIC. Fractions taken from the PhHP HIC purification step were analyzed by SDS-PAGE (top). Recombinant BoNT/A1 has a molecular weight of ~149 kDa and molecular weight markers (Benchmark) were labeled in kDa. Samples of the same fractions were also tested in a Lys-C calorimetric assay (bottom), where cleavage of the substrate releases a yellow chromophore (in black box).

Fig. 12 - removal of Lys-C from rBoNT/A2(0) by phenyl HP HIC. Fractions taken from the phenyl HP HIC purification step were analyzed by SDS-PAGE (top). Recombinant BoNT/A1 has a molecular weight of ~149 kDa and molecular weight markers (Benchmark) were labeled in kDa. Samples of the same fractions were also tested in a Lys-C calorimetric assay (bottom), where cleavage of the substrate releases a yellow chromophore (in black box).

Fig. 13 - removal of Lys-C from rBoNT/A5(0) by phenyl HP HIC. Fractions taken from the phenyl HP HIC purification step were analyzed by SDS-PAGE (top). Recombinant BoNT/A5(0) has a molecular weight of ~149 kDa and molecular weight markers (Benchmark) were labeled in kDa. Samples of the same fractions were also tested in a Lys-C calorimetric assay (bottom), where cleavage of the substrate releases a yellow chromophore (in black box).

Fig. 14 - removal of Lys-C from rBoNT/A6(0) by phenyl HP HIC. Fractions taken from the phenyl HP HIC purification step were analyzed by SDS-PAGE (top). Recombinant BoNT/A6(0) has a molecular weight of ~149 kDa and molecular weight markers (Benchmark) were labeled in kDa. Samples of the same fractions were also tested in a Lys-C calorimetric assay (bottom), where cleavage of the substrate releases a yellow chromophore (in black box).

Fig. 15 - removal of Lys-C from mrBoNT/A1 by butyl HP HIC. Fractions taken from the butyl HP HIC purification step were analyzed by SDS-PAGE (top). Recombinant mrBoNT/A1 has a molecular weight of ~149 kDa and molecular weight markers (Benchmark) were labeled in kDa. Samples of the same fractions were also tested in a Lys-C calorimetric assay (bottom), where cleavage of the substrate releases a yellow chromophore (in black box).

Examples

Example 1 Cultivation of the host and expression of soluble rBoNT/A protein.

A single colony of BLR (DE3) transformable BoNT/A is used to inoculate a 250 ml conical flask containing 100 ml Terrific Broth (mTB) modified medium supplemented with 0.2% glucosamine and 30 μg/ml kanamycin.

This method will be equally applicable when using a Microbank bead or glycerol material (10-100 µl) to inoculate the flask.

The flask is incubated for 16 hours at 37° C. with shaking at 250 rpm. 10 ml of this starter culture is used to inoculate 2 L conical flasks each containing 1 L supplemented with 0.2% glucosamine and 30 μg/ml kanamycin. Cells are grown at 37° C. for 2 hours at 225 rpm until an OD ₆₀₀ of 0.5 is reached. At this point, the cultivation temperature is lowered to 16°C. After 1 hour, cells are induced to express BoNT/A by adding 1 mM IPTG for 20 hours. Cells are harvested by centrifugation for 20 minutes at 4°C, weighed and then stored at -20°C.

Example 2 Extraction of the BoNT/A protein from the host and analysis of the expression level.

The rBoNT/A expressing cell pastes are thawed at room temperature and resuspended by pipetting 3 ml per gram of cells with Tris-NaCl resuspension buffer supplemented with 10 μl benzonase. Cells are lysed by sonication at 100 W - 10 x 30 seconds on+45 seconds off. The lysate is centrifuged at 4000xg for 1 hour at 4°C to obtain soluble rBoNT/A in the supernatant.

Bradford analysis to determine the total protein concentration of the obtained lysates.

A sample (50 µl) of each of the diluted rBoNT/A lysate or BSA standard is added to 1 ml disposable cuvettes. 450 µl Coomassie reagent for Bradford assay is added to each cuvette and allowed to incubate at room temperature for 10 minutes before reading A6 ₀₀ . The values obtained for the BSA standards are used to determine the amount of protein in the lysate samples.

Semiquantitative analysis Western blotting.

A commercial BoNT/A protein sample sold by Metabiologics is used to create SDS-PAGE standards. SDS-PAGE samples with known total protein concentration are then obtained from the lysate samples from the expression cell cultures. These samples were applied to a polyacrylamide gel and treated at 200 V for 50 minutes. Protein bands are electrophoresed onto a nitrocellulose membrane in methanol-free blotting buffer at 0.4 mA for 1 h.

- 23 040938

The membranes are blocked for 1 h with 0.5% BSA in PBS-0.1% Tween 20 and then examined with an antibody to BoNT/A for 1 h. The spots are further examined with HRP conjugated secondary antibodies formed in the substrate SuperSignal DuraWest and rendered using a Syngene display device.

Example 3 Activation of botulinum neurotoxin A (BoNT/A) by Lys-C.

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

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

Lys-C endoproteinase cleavage tests demonstrated that Lys-C cleaved rBoNT/A1 at very low concentrations and remained active for days (data not shown).

Example 4 Purification of a BoNT/A target protein lacking an activating protease.

Using the primary sequence of the BoNT/A protein, the properties of BoNT/A and Lys-C were examined (see Table 2).

Predicted properties suggesting that both Lys-C and BoNT/A have similar average hydropathicity (GRAVY value) but large charge difference at pH 4.5 and 8 (see Table 2).

table 2

Predicted Properties of Lys-C and BoNT/A

Property (calculated) Lys-C BoNT/A R! 6.70 6.05 % Charged Residue (DEKR) 13 25 General Medium Hydropathic (GRAVY)* -0.30 -0.37 Charge at pH 8.0 -5 -12 Charge at pH 4.5 + 13 +72

(* GRAVY=medium hydropathic per molecule residue (positive value indicates hydrophobic molecule)).

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

Example 5 Screening of Fast Protein Liquid Chromatography (FPLC) Columns to Separate Lys-C from BoNT/A.

FPLC cleaning.

After BoNT/A1 activation with Lys-C, a number of FPLC columns were tested for purification and removal of Lys-C: three hydrophobic interaction chromatography (HIC) columns: high performance phenyl (PhHP), highly substituted phenyl Fast Flow (PhFF-Hi), and butyl HP (BuHP) was tested with Tris pH 8; three cation exchange chromatography (CEC) columns: Sulfopropyl High Performance (SPHP), Sulfopropyl Fast Flow (SPFF), and Carboxylmethyl Fast Flow (CMFF) were tested with sodium acetate pH 4.5; and four anion exchange chromatography (AEC) columns: quaternary amine high performance (QHP), quaternary amine Fast Flow (QFF), diethylaminopropyl (DEAP, ANX), and diethylaminoethyl (DEAE)) were tested with Tris pH 8.

After the sample was loaded onto the column, the column was washed with a buffer solution to remove any non-specifically bound molecules before eluting with a linear gradient of increasing or decreasing salt concentration (HIC and CEC/AEC, respectively).

Reaction conditions and purification runs vary between different types of columns (see Table 3 below).

- 24 040938

Table 3

Screening conditions for all columns tested for Lys-C separation

Column Downloaded BoNT/Al (mg) Loaded Lys-C (mcg) buffer solution Elution gradient PhHP 1.9 1.5 High Salt, Tris pH 8 1-0 M, 15 CV BuHP 1.9 1.5 High Salt, Tris pH 8 1-0 M, 15 CV PhFF Hi 1.5 1.2 High Salt, Tris pH 8 1-0 M, 15 CV SPHP 3.4 3.0 Low salt, NaOAc pH 4.5 0-0.5 M, 30 CV SPFF 3.4 3.0 Low salt, NaOAc pH 4.5 0-0.5 M, 30 CV CMFF 3.4 3.0 Low salt, NaOAc pH 4.5 0-0.5 M, 30 CV QHP 2.7 2.0 Low salt, Tris pH 8 0-0.5 M, 30 CV QFF 2.7 2.0 Low salt, Tris pH 8 0-0.5 M, 30 CV ANX 2.7 2.0 Low salt, Tris pH 8 0-0.5 M, 30 CV DEAE 2.7 2.0 Low salt, Tris pH 8 0-0.5 M, 30 CV

Fig. 1-10 show the elution profiles (A ₂₈₀ ) of the BoNT/A1 protein from various chromatographic columns (dashed lines). The different reaction conditions and purification runs used for different columns explain the different scales used.

To evaluate Lys-C activity, fractions collected during the elution gradient were analyzed by calorimetric analysis.

Calorimetric analysis of Lys-C activity.

This assay involved cleavage of a colorless substrate to produce a yellow chromophore that can be detected photometrically by absorbing 405 nm light (A ₄₀₅ ). Thus, this assay provided an easy way to determine whether Lys-C was present in each fraction.

Each elution fraction was analyzed using the indicated Lys-C activity calorimetric assay and A ₄₀₅ nm was measured.

The amount (measured in terms of A ₄₀₅ ) of Lys-C in each elution fraction is shown as bars in FIG. 1-10.

Results.

By comparing data A ₄₀₅ and A ₂₈₀ (in the graphs of Fig. 1-10) it was possible to conclude which column/columns provide the best separation of Lys-C from BoNT/A.

The different alkyl/aryl groups (Bu and Ph) from the three HIC columns used provide different ligands that different proteins can interact with through a 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 below the pH of the target protein pI so that it receives all of the net positive charge and is 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 affected by the density of the ligand. These variables apply similarly to AEC columns, where different chemical groups represent different charge densities. In this case, the pH of the sample is adjusted above the pH of the target protein pI so that it receives all of the net negative charge.

The graphical data from FIG. 1-10 are qualitatively summarized in Table. 4.

- 25 040938

Table 4

The Essence of Qualitative Lys-C/BoNT Data Splitting

Chromatography Hydrophobic interaction ³ Ion exchange ^b cationic anionic

Branch of Lys-C* PhHP /// BuHP /// PhFF Hi y/ y/ SPHP s SPFF CMFF y/ QHP y/ y/ QFF if ANX y/ DEAE

* apparent separation of Lys-C relative to rBoNT/A1:

///=good, =OK,

S=low, ^a Phenyl High Potency (PhHP), Highly Substituted Phenyl Fast Flow (PhFF-Hi), Butyl HP (BuHP), ^b Sulfopropyl High Potency (SPHP), Sulfopropyl Fast Flow (SPFF), Carboxylmethyl Fast Flow (CMFF), Quaternary Amino High Performance (QHP), Quaternary Amino Fast Flow (QFF), Diethylaminopropyl (DEAP, ANX) and Diethylaminoethyl (DEAE).

Percentage of Lys-C recovery and purification after elution from each type of column.

The total Lys-C signal in each fraction was normalized to the average A ₄₀₅ value from the last 5 fractions of the chromatographic step. From this, the percentage of Lys-C present in peak protein fractions was calculated to show the degree of separation of Lys-C from protein based on elution fractions (Table 5, % LysC in peak protein fractions (normalized)).

With regard to the target protein, it is assumed that all BoNT/A molecules elute under the main peak (ie 100% recovery); therefore, the degree of purification can be expressed as the percentage of total loaded protein that is not recovered in the main peak (Table 5, % purification).

A good separation of Lys-C from BoNT/A would be a separation that showed a low percentage of LysC in the peak protein fraction. Hence, it appears that CEC is unable to separate Lys-C from BoNT/A1. The high performance AEC column, QHP, showed some ability to separate Lys-C from BoNT/A1. However, it was significantly less efficient than two high performance HIC columns. Therefore, the results show that in a comparable comparison (ie, standard efficiency vs. standard efficiency, and high efficiency vs. high efficiency), HIC columns showed improved separation of Lys-C from BopT/A1 than either CEC or AEC columns.

The two most promising candidates include HIC - PhHP and BuHP. Interestingly, both of these columns use high performance beads.

The main difference between high performance media and others is that the average particle size is smaller (34 µm vs. 90 µm) and more uniform (24-44 µm vs. 44-165 µm). This is consistent with claimed performance improvements with analytical columns using smaller beads (mean sizes between 3-30 µm) (GE Healthcare handbooks 11-0004-21 & 11-0012-69 and data files 18-1172-87 AE & 18-1172-88 A.D.).

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Table 5

Summary of speaker performance _____________

Column % LysC in peak protein fractions (normalized) % Cleaning PhHP 5 eleven WNR 7 eleven PhFF Hi 43 22 SPHP 85 20 SPFF 82 eleven CMFF 81 10 QHP 26 9 QFF 51 6 ANX 70 7 DEAE 74 9

PhHP HIC was chosen as the final purification 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/A1).

Single strand rBoNT/A1 was purified by rapid protein liquid chromatography (FPLC) using high performance butyl sepharose hydrophobic interaction chromatography (butyl HP HIC) to capture followed by an intermediate purification step using high performance quaternary ammonium sepharose anion exchange chromatography (Q HP AEC). This molecule was then incubated with 0.4 μg/ml Lys-C at 37° C. for 2 hours to obtain an active bicep.

Lys-C was separated from activated rBoNT/A1 using high performance phenyl sepharose hydrophobic interaction chromatography (PhHP HIC). It involved adjusting the reaction mixture with a high salt Tris buffer prior to loading onto a PhHP column, followed by a high salt wash followed by linear gradient elution of the protein in a low salt Tris buffer. Fraction elution was analyzed by denaturing gel electrophoresis (SDS PAGE - FIG. 11 top) and calorimetric analysis of Lys-C activity (FIG. 11 bottom). Early elution of Lys-C in a gradient (F16-F21, outlined in black box) was demonstrated prior to activation of the BoNT molecule (F21-F29). Thus, this shows a good separation of Lys-C from rBoNT/A1. The maximum efficiency of the fractions appears to be similar to 30 ng/ml Lys-C, suggesting that any residual Lys-C present in the rBoNT/A1 fractions would be less than this concentration.

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

Single strand rBoNT/A2(0) was purified by rapid protein liquid chromatography (FPLC) using high performance butyl sepharose hydrophobic interaction chromatography (butyl HP HIC) to capture followed by an intermediate purification step using high performance quaternary ammonium sepharose anion exchange chromatography (Q HP AEC ). This molecule was then incubated with 4 μg/ml Lys-C at 37° C. for 2 hours to obtain an active bicep.

Lys-C was separated from activated rBoNT/A2(0) using high performance phenyl sepharose hydrophobic interaction chromatography (PhHP HIC). It involved adjusting the reaction mixture with a high salt Tris buffer before loading onto a phenyl HP column followed by a high salt wash followed by linear gradient elution of the protein in a low salt Tris buffer. Fraction elution was analyzed by denaturing gel electrophoresis (SDS PAGE) and calorimetric analysis of Lys-C activity (Fig. 12), it showed that Lys-C eluted early in the gradient (F5-F11) just before the activated BoNT molecule (F12- F15). Thus, this column shows good separation of Lys-C from rBoNT/A2(0).

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

Single strand rBoNT/A5(0) was purified by rapid protein liquid chromatography (FPLC) using high performance butyl sepharose hydrophobic interaction chromatography (butyl HP HIC) to capture followed by an intermediate purification step using high performance quaternary ammonium sepharose anion exchange chromatography (Q

- 27 040938

HP AEC). This molecule was then incubated with 2.5 μg/ml Lys-C at 37° C. for 2 hours to obtain an active bicep. Lys-C was separated from activated rBoNT/A5(0) using high performance phenyl sepharose hydrophobic interaction chromatography (phenyl HP HIC). It involved adjusting the reaction mixture with high salt Tris buffer before loading onto a phenyl HP column, followed by high salt washing and subsequent elution of the protein in a linear gradient in low salt Tris buffer. Fraction elution was analyzed by denaturing gel electrophoresis (SDS PAGE) and calorimetric analysis of Lys-C activity (Fig. 13), it showed that Lys-C eluted early in the gradient (F10-F39) before the activated BoNT molecule (F51-F65 ). Thus, this column shows a good separation of Lys-C from rBoNT/A5(0).

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

Single strand rBoNT/A6(0) was first purified by sodium sulfate precipitation and resolubilization into sodium acetate before being captured by FPLC using high performance sulfopropyl sepharose cation exchange chromatography (SP HP CEC) followed by buffer exchange with Tris buffer at pH 8. This molecule was then incubated with 0.3 μg/ml Lys-C at 37° C. for 2 hours to obtain an active bicep. Lys-C was separated from activated rBoNT/A6(0) using high performance phenyl sepharose hydrophobic interaction chromatography (phenyl HP HIC). It involved adjusting the reaction mixture with a high salt Tris buffer before loading onto a phenyl HP column, followed by a high salt wash and subsequent elution of the protein in a linear gradient into a low salt Tris buffer. Fraction elution was analyzed by denaturing gel electrophoresis (SDS PAGE) and calorimetric analysis of Lys-C activity (Fig. 14), it showed that Lys-C eluted early in the gradient (F6-F13) before the activated BoNT molecule (F16-F27 ). This shows excellent separation of Lys-C from rBoNT/A6(0).

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

Single strand mrBoNT/A1 (SEQ ID NO: 15) was purified by rapid protein liquid chromatography (FPLC) using high performance sulfopropyl sepharose cation exchange chromatography (SPHP CEX) to capture. An intermediate purification step using High Performance Butyl Sepharose Hydrophobic Interaction Chromatography (BuHP HIC) was used to further remove host cell proteins. BuHP elution fractions were analyzed by SDS-PAGE, and fractions containing single chain mrBoNT/A1 were collected as purified, single chain mrBoNT/A1.

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

Single strand mrBoNT/A1 (SEQ ID NO: 15) was incubated with 0.4 μg/ml Lys-C at 37°C for 2 hours to obtain an active bicep. Lys-C was separated from activated mrBoNT/A1 using High Performance Butyl Sepharose Hydrophobic Interaction Chromatography (BuHP HIC). It involved adjusting the reaction mixture with a high salt Bis-Tris buffer before loading onto a BuHP column, followed by a high salt wash and subsequent elution of the protein in a linear gradient into a low salt Bis-Tris buffer. Fraction elution was analyzed by SDS-PAGE (FIG. 15 top) and calorimetric analysis of Lys-C activity (FIG. 15 bottom). Lys-C was eluted early and through the column in an elution gradient (F1-6, highlighted in black box, bottom FIG. 15) in front of the activated BoNT molecule (F10-F12). Thus, this shows good separation of Lys-C from mrBoNT/A1.

Example 12 The BoNT/A active bicep formulation is PO substantially free of Lys-C.

Prepared the following six liquid compositions containing the active bicep BoNT/A (table. 6).

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Claims (21)

Table 6 Illustrative BoNT/A Finishes 1 2 3 4 5 6 Polysorbate 80 0.10 mg/ml 0.10 mg/ml 0.10 mg/ml 0.10 mg/ml - - Poloxamer - - - - 0.04 mg/ml 0.04 mg/ml sucrose 4.0 mg/ml - 4.0 mg/ml - 4.0 mg/ml - Mannitol - 4.0 mg/ml - 4.0 mg/ml - 4.0 mg/ml Sodium chloride 8.76 mg/ml 8.76 mg/ml 8.76 mg/ml 8.76 mg/ml 8.76 mg/ml 8.76 mg/ml pH 6, 5 6.5 6.5 6.5 6, 5 6.5 buffer solution L-Histidine/ Hydrochloric acid L-Histidine/ Hydrochloric acid Disodium Phosphate / Anhydrous Citric Acid Disodium Phosphate/ Anhydrous Lemon acids L-Histidine/ Hydrochloric acid L-Histidine/ Hydrochloric acid Bichain BoNT/A 20 ng/ml 20 ng/ml 20 ng/ml 20 ng/ml 20 ng/ml 20 ng/ml MilliQ water how much is needed up to 1 ml how much is needed up to 1 ml how much is needed up to 1 ml how much is needed up to 1 ml how much is needed up to 1 ml how much is needed up to 1 ml All six compositions were stored at 25°C for 12 weeks. The stability of the bicep protease function of BoNT/A was assessed during this period using a cell-free endopeptidase assay. Example 13 High sensitivity Lys-C activity assay. Quantification of the final concentration of Lys-C in the purified BoNT/A samples was performed by cleavage of the fluorescent peptide substrate. The fluorescent substrate (TosGPK-7-amino-4-trifluoroacetate salt, also referred to as Tos-GPK-AMC) was reconstituted in DMSO to provide a stock of 20 mg/mL (32.7 mM). It was further diluted to 10 mM with water and aliquots prepared and stored at -20°C. On the day of analysis, the appropriate amount of substrate was taken and further diluted with 25 mM Tris pH 8, 125 mM NaCl to provide 0.5 mM Lys-C working solution; Lys-C standards (of known concentrations) were then prepared by diluting the working solution in assay buffer (25 mM Tris, 125 mM NaCl, 0.1 mg/ml BSA, pH 8). Standards, blanks (assay buffer only) and test samples (batch of purified rBoNT/A) were dispensed onto black OptiPlates, in triplicate (180 µl per well) and equilibrated to 37°C over 10-15 min. Next, 20 μl of substrate was added and the plate was immediately transferred to a plate reader where kinetic reading was performed. The plate was read using excitation/emission filters 360/40 and 485/20 at 37° C., every 20 min for 5 hours. Data was plotted as time in minutes (x-axis) versus absorbance/fluorescence (y-axis). The slope values for each Lys-C concentration were used to create a standard curve for the Lys-C concentration. Purified rBoNT/A samples were treated in the same manner and the concentration of Lys-C in each rBoNT/A sample was interpolated from the standard curve. Based on the analysis of four independent lots or rBoNT/A, the measured average concentration of Lys-C in the final material was 7.3±1.5 pg of Lys-C per 100 ng/BoNT/A. CLAIM 1. A method for producing an active soluble double-stranded BoNT/A protein, including: a) providing a soluble single chain BoNT/A protein containing an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; b) contacting said soluble BoNT/A single chain protein with Lys-C endoproteinase in solution to produce a soluble BoNT/A double chain protein; And c) separating said soluble BoNT/A double chain protein from Lys-C by contacting a solution containing the soluble BoNT/A double chain protein and Lys-C with a hydrophobic surface to which said soluble BoNT/A double chain protein binds, wherein said soluble the double-stranded BoNT/A protein includes a first chain containing an amino acid sequence that is at least 95% identical to amino acid residues 1-438 of SEQ ID NO: 1; and a second chain containing an amino acid sequence that is at least 95% identical to amino acid residues 449-1296 of SEQ ID NO: 1; where the first and second chains are connected by a disulfide bridge. 2. The method of claim 1, wherein the soluble BoNT/A single chain protein is obtained by expressing a nucleic acid encoding said BoNT/A single chain protein in a host cell or in an in vitro expression system. 3. The method of claim 2, wherein the host cell is a bacterial host cell. 4. The method of claim 3 wherein the host cell is an E. coli cell. 5. A method according to any one of claims 2-4, wherein said soluble BoNT/A single chain protein is expressed in the cytoplasm of said host cell. - 29 040938 6. The method according to any one of claims 2 to 5, wherein said soluble BoNT/A single chain protein is expressed at a level of at least 5 mg/l. 7. The method of any one of claims 3 to 6, comprising lysing the bacterial or E. coli host cell to provide a bacterial or E. coli host cell homogenate containing said soluble BoNT/A single chain protein. 8. The method according to any one of claims 1 to 7, wherein the hydrophobic surface is an inert matrix to which a ligand consisting of aryl or alkyl groups is attached. 9. The method of claim 8 wherein the ligand is a butyl, phenyl or octyl ligand. 10. Muscle relaxant composition containing: (i) active soluble double-stranded BoNT/A protein obtained by the method according to any one of paragraphs.1-9; (ii) less than 2% single-stranded BoNT/A protein; and (iii) Lys-C, where Lys-C is present at a concentration of less than 400 pg of Lys-C per 100 ng of BoNT/A protein; where the active protease function of double-stranded BoNT/A remains stable at 25°C for at least 12 weeks. 11. A composition according to claim 10 containing less than 1% single chain BoNT/A protein. 12. Composition according to claim 10 or 11, containing Lys-C endoproteinase at a concentration of less than 300 pg of Lys-C per 100 ng of BoNT/A protein, or less than 200 pg of Lys-C per 100 ng of 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 of BoNT/A protein, or less than 10 pg of Lys-C per 100 ng of BoNT/A protein. 13. Liquid muscle relaxant pharmaceutical composition containing: a) active soluble BoNT/A double-stranded protein obtained by the method according to any one of claims 1 to 9; b) a non-protein stabilizing agent which is a surfactant; And c) water; wherein said liquid pharmaceutical composition does not contain a protein stabilizing agent; and contains (i) less than 2% single-stranded BoNT/A protein and (ii) and (iii) Lys-C, where Lys-C is present at a concentration of less than 400 pg of Lys-C per 100 ng of BoNT/A protein; where the active protease function of double-stranded BoNT/A remains stable at 25°C for at least 12 weeks. 14. Liquid pharmaceutical composition according to claim 13, containing Lys-C endoproteinase at a concentration of less than 300 pg of Lys-C per 100 ng of BoNT/A protein, or less than 200 pg of Lys-C per 100 ng of 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 of BoNT/A protein, or less than 10 pg of Lys-C per 100 ng of BoNT/A protein. 15. Liquid pharmaceutical composition according to claim 13 or 14, additionally containing sodium chloride, a buffer solution to maintain a pH between 5.5 and 7.5 and a disaccharide; the water being sterile water. 16. Composition according to any one of claims 10-12 for treating a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders , hyperhidrosis, correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia (open-maxillary type, occlusive type), bruxism, Meigs syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, specific task dystonia, writing spasm, musician's spasm, golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee flexion, knee p flexion, flexion of the leg at the ankle joint, extension of the leg at the ankle joint, equinovarus deformity of the foot, deformity due to dystonia of the foot, striatal toe, flexion of the toe, extension of the toe, axial dystonia, Leaning Tower of Pisa syndrome, dystonia of the belly dancer, segmental dystonia, hemidistonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/ dopa-induced dystonia, residual dyskine- - 30 040938 dystonia/residual dystonia, paroxysmal dyskinesia/dystonia, kinesiogenic, non-kinesiogenic, action-induced, palatal myoclonus, myokymic myoclonus, rigidity, benign muscle cramps, hereditary chin trembling, paradoxical activity of masticatory muscles, spasms of masticatory muscles of one side of the face, hypertrophic branchial myopathy, masticatory muscle hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned surgery for spastic torticollis, vocal cord abductor paralysis, persistent mutational dysphonia, upper pharyngeal sphincter dysfunction, vocal cord granuloma, stuttering, Tourette syndrome, middle ear myoclonus, protective glottis closure, post-laryngectomy speech disorder, protective ptosis, eyelid volvulus, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal motility disorder, vaginismus, postoperative onic tremor on immobility, bladder dysfunction, detrusor-sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, crow's feet cosmetic use, facial asymmetry due to frown, chin pit, stiff man syndrome, tetanus, prostatic hyperplasia, obesity treatment , cerebral palsy, strabismus, mixed, paralytic, concomitant, after retinal detachment surgery, after cataract surgery, with aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, convergent strabismus, divergent strabismus, achalasia , anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, axillary, palmar, plantar hyperhidrosis, rhinorrhea, relative hypersalivation in stroke, in Parkinson's disease, in spastic conditions in amyotrophic lateral sclerosis, p in the processes of autoimmune encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic's disease, viral infections, bacterial infections, parasitic infections, fungal infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brainstem infarction, spinal cord infarction, migraine, with trauma of the central nervous system, damage to the hemispheres, damage to the spinal cord, damage to the brain stem, with hemorrhage of the central nervous system, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, hemorrhage in the spinal cord, with neoplasia, tumors of the hemispheres, tumors of the brain stem, tumors of the spinal cord and snoring. 17. Liquid pharmaceutical composition according to any one of claims 13-15 for the treatment of a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal intestinal disorders, hyperhidrosis, correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia (open jaw type, closed jaw type), bruxism, Meigs syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, specific task dystonia, writing spasm, musician's spasm, golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee abduction foot flexion, knee extension, ankle flexion, ankle leg extension, foot equinovarus deformity, foot dystonia deformity, striatal toe, toe flexion, toe extension, axial dystonia, Leaning Tower of Pisa syndrome, belly dancer dystonia , segmental dystonia, hemidistonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual 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, residual dyskinesia/residual dystonia, paroxysmal dyskinesia/dystonia, kinesiogenic, non-kinesiogenic, action-induced, palatal myoclonus, myokymic myoclonus, rigidity, benign muscle cramps, hereditary chin trembling, paradoxical masticatory muscle spasm, masticatory muscle spasms of one half of the face, hypertrophic branchial myopathy, masseter hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned surgery for spastic torticollis, paralysis of the abducens vocal cord muscle, stable mutational dysphonia, dysfunction of the upper pharyngeal sphincter, vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective glottic closure, post-laryngectomy speech disorder, protective ptosis, volvulus of the eyelids, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal dysmotility, vaginismus, postoperative tremor on immobility, bladder dysfunction, detrusor-sphincter dyssynergy, spasm of the bladder sphincter, hemifacial spasm, reinnervation dyskinesias, cosmetic use in crow's feet, facial asymmetry due to frown, chin pits, stiff man syndrome, tetanus, prostate hyperplasia, obesity treatment, cerebral palsy, squint - 31 040938 concomitant, mixed, paralytic, concomitant, after surgery for retinal detachment, after cataract surgery, for aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, convergent strabismus, divergent strabismus, achalasia, anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, axillary, palmar, plantar hyperhidrosis, rhinorrhea, relative hypersalivation in stroke, Parkinson's disease, spastic conditions in amyotrophic lateral sclerosis, autoimmune encephalitis and myelitis processes, multiple sclerosis, transverse myelitis, Devic's disease, viral infections, bacterial infections, parasitic infections, fungal infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brainstem infarction, spinal cord infarction, migraine, with trauma of the central nervous system, lesions hemispheres, spinal cord injury, brain stem injury, central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, spinal cord hemorrhage, neoplasia, hemispheric tumors, brain stem tumors, spinal cord tumors and snoring. 18. The use of a composition according to any one of claims 10-12, or a liquid pharmaceutical composition according to any one of claims 13-15 in the manufacture of a medicament for the treatment of a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, hyperhidrosis, correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia (open-maxillary type, occlusive type), bruxism, Meigs' syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, dystonia when performing specific tasks, writing spasm, musician's cramps, c golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee flexion, knee extension, ankle flexion, ankle leg extension, foot equinovarus deformity, foot dystonia deformity, striatal toe, toe flexion, toe extension, axial dystonia, Leaning Tower syndrome, belly dancer's dystonia, segmental dystonia, hemidystonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual dystonia, dystonia in spinocerebellar ataxia, dystonia in disease Parkinson's disease, Huntington's dystonia, Hallervorden-Spatz's dystonia, dopa-induced dyskinesias/dopa-induced dystonia, residual dyskinesias/residual dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic, non-kinesiogenic, action-induced, palatine myoclonus, myokymic myoclonus, rigidity awn, benign muscle cramps, hereditary chin trembling, paradoxical activity of the masticatory muscles, spasms of the masticatory muscles of one half of the face, hypertrophic branchial myopathy, hypertrophy of the masseter muscle, hypertrophy of the anterior tibial muscle, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned surgery for spastic torticollis, abducens vocal cord paralysis, persistent mutational dysphonia, upper pharyngeal sphincter dysfunction, vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective glottic closure, post-laryngectomy speech disorder, protective ptosis, torsion of the eyelids, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal dysmotility, vaginismus, postoperative tremor on immobility, bladder dysfunction, detrusor-sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesia ii, cosmetic use for crow's feet, facial asymmetry due to frown, chin pit, stiff man syndrome, tetanus, prostatic hyperplasia, obesity treatment, cerebral palsy, strabismus, mixed, paralytic, concomitant, after retinal detachment surgery, after cataract surgery , with aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, convergent strabismus, divergent strabismus, achalasia, anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, hyperhidrosis axillary, palmar, plantar, rhinorrhea , relative hypersalivation in stroke, in Parkinson's disease, in spastic conditions in amyotrophic lateral sclerosis, in the processes of autoimmune encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic's disease, viral infections, bacterial infections, parasitic infections, fungus high infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brain stem infarction, spinal cord infarction, migraine, with trauma to the central nervous system, lesions of the hemispheres, spinal cord injury, damage to the brain stem, with hemorrhage of the central nervous system, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, spinal cord hemorrhage, neoplasia, hemispheric tumors, brainstem tumors, spinal cord tumors and snoring. - 32 040938 19. The use of a liquid pharmaceutical composition according to any one of claims 13-15 in the manufacture of a medicament for the treatment of a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, hyperhidrosis, cosmetic wrinkle correction, blepharospasm, oromandibular dystonia (open jaw type, closed jaw type), bruxism, Meigs syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, specific task dystonia, writing spasm, musician's spasm, golfer's spasm, lower limb dystonia limbs, hip adduction, hip abduction, knee flexion, knee extension, ankle flexion, ankle leg extension, foot equinovarus deformity, foot dystonia deformity, striatal toe, toe flexion, toe extension, axial dystonia, leaning tower syndrome, belly dancer's dystonia, segmental dystonia, hemidystonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, residual dyskinesias/residual dystonias, paroxysmal dyskinesias/dystonias, kinesiogenic, non-kinesiogenic, action-induced, palatal myoclonus, myokymic myoclonus, rigidity, benign muscle vasospasms horns, hereditary tremor of the chin, paradoxical activity of the masticatory muscles, spasms of the masticatory muscles of one half of the face, hypertrophic branchial myopathy, hypertrophy of the masticatory muscle, hypertrophy of the anterior tibial muscle, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned surgery for spastic torticollis, paralysis abducens glottis, persistent mutational dysphonia, upper pharyngeal sphincter dysfunction, vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective glottic closure, post-laryngectomy speech disorder, protective ptosis, torsion of the eyelids, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasia esophageal dysmotility, vaginismus, postoperative tremor on immobility, bladder dysfunction, detrusor sphincter dyssynergy, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use in goose legs, facial asymmetry due to frown, chin pits, stiff man syndrome, tetanus, prostate hyperplasia, obesity treatment, cerebral palsy, strabismus, mixed, paralytic, concomitant, after retinal detachment surgery, after cataract surgery, with aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation as an adjunct to strabismus surgery, convergent strabismus, divergent strabismus, achalasia, anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, hyperhidrosis axillary, palmar, plantar, rhinorrhea, relative hypersalivation in stroke , with Parkinson's disease, with spastic conditions with amyotrophic lateral sclerosis, with processes of autoimmune encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic's disease, viral infections, bacterial infections, parasitic infections, fungal infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brainstem infarction, spinal cord infarction, migraine, in case of trauma of the central nervous system, hemispheric lesions, spinal cord injury, brainstem injury, in case of central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage , hemorrhage in the spinal cord, with neoplasia, tumors of the hemispheres, tumors of the brain stem, tumors of the spinal cord and snoring. 20. A method of treating a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, hyperhidrosis, correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia (open-maxillary type, closed-maxillary type), bruxism, Meigs syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, specific task dystonia, writer's spasm, musician's cramp, golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee flexion, knee extension, goal flexion foot extension, leg extension at the ankle joint, equinovarus deformity of the foot, deformity due to foot dystonia, striatal toe, flexion of the toe, extension of the toe, axial dystonia, Leaning Tower of Pisa syndrome, belly dancer's dystonia, segmental dystonia, hemidistonia, generalized dysplasia - 33 040938 tonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa- induced dystonia, residual dyskinesia/residual dystonia, paroxysmal dyskinesia/dystonia, kinesiogenic, non-kinesiogenic, action-induced, palatal myoclonus, myokymic myoclonus, rigidity, benign muscle cramps, hereditary chin trembling, paradoxical activity of masticatory muscles, spasms of masticatory muscles of one side of the face, hypertrophic branchial myopathy, masseter muscle hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned surgery for spastic torticollis, abducens vocal cord paralysis, sustained m utational dysphonia, dysfunction of the upper pharyngeal sphincter, vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective closure of the glottis, post-laryngectomy speech disorder, protective ptosis, volvulus of the eyelids, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal dysmotility, vaginismus, postoperative tremor on immobility, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, crow's feet cosmetic use, facial asymmetry due to frown, chin pit, stiff man syndrome, tetanus, prostate hyperplasia, obesity treatment, pediatric cerebral paralysis, strabismus, mixed, paralytic, concomitant, after surgery for retinal detachment, after cataract surgery, with aphakia, myositis strabismus, myopathic strabismus, dissociated vertical deviation, as an addition to surgery strabismus, convergent strabismus, divergent strabismus, achalasia, anal fissures, hyperfunction of exocrine glands, Frey's syndrome, crocodile tears syndrome, axillary, palmar, plantar hyperhidrosis, rhinorrhea, relative hypersalivation in stroke, in Parkinson's disease, in spastic conditions in amyotrophic lateral sclerosis , in the processes of autoimmune encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic's disease, viral infections, bacterial infections, parasitic infections, fungal infections, with hereditary spastic paraparesis, post-apoplectic syndrome, hemispheric infarction, brainstem infarction, spinal cord infarction, migraine, in case of trauma of the central nervous system, damage to the hemispheres, damage to the spinal cord, damage to the brain stem, with hemorrhage of the central nervous system, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, hemorrhage in the spinal cord, with neoplasia, tumors of the floor brain stem tumors, spinal cord tumors and snoring, wherein said method comprises administering a composition according to any one of claims 10-12 to a patient in need thereof. 21. A method of treating a disease or disorder selected from the following: striated muscle resistance, focal dystonia, including cervical, cranial dystonia and benign idiopathic blepharospasm, hemifacial spasm and focal spasticity, gastrointestinal disorders, hyperhidrosis, correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia (open-maxillary type, closed-maxillary type), bruxism, Meigs syndrome, lingual dystonia, eyelid apraxia, open cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, adductor spastic dysphonia, abductor spastic dysphonia, spastic dyspnea, limb dystonia, upper limb dystonia, specific task dystonia, writer's spasm, musician's cramp, golfer's spasm, lower limb dystonia, hip adduction, hip abduction, knee flexion, knee extension, goal flexion foot extension, ankle leg extension, foot equinovarus deformity, foot dystonia deformity, striatal toe, toe flexion, toe extension, axial dystonia, Leaning Tower of Pisa syndrome, belly dancer's dystonia, segmental dystonia, hemidystonia, generalized dystonia, dystonia in Lubag syndrome, dystonia in corticobasal degeneration, dystonia in Lubag syndrome, residual dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, residual dyskinesias /residual dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic, non-kinesiogenic, action-induced, palatine myoclonus, myokymic myoclonus, rigidity, benign muscle cramps, hereditary chin trembling, paradoxical masticatory muscle activity, masticatory muscle spasms SC of one half of the face, hypertrophic branchial myopathy, masseter hypertrophy, hypertrophy of the anterior tibial muscle, nystagmus, oscillopsia, supranuclear gaze palsy, partial permanent epilepsy, planned operation for spastic torticollis, paralysis of the abducens vocal cord muscle, stable mutational dysphonia, dysfunction of the upper pharyngeal sphincter , vocal cord granuloma, stuttering, Tourette's syndrome, middle ear myoclonus, protective glottic closure, post-laryngectomy speech disorder, protective ptosis, eyelid volvulus, sphincter of Oddi dysfunction, pseudoachalasia, non-achalasic esophageal dysmotility, -