METHOD FOR PURIFICATION AND ACTIVATION OF BOTULINUM NEUROTOXIN

Abstract

Methods for the isolation and purification of a botulinum neurotoxin protein (BONT) or a polypeptide comprising a BONT receptor-binding domain from a solution are disclosed here. The method comprises contacting the solution containing the protein or polypeptide with a matrix bound to it by a non-toxic non-hemagglutinin (NTNHA) under appropriate conditions for binding, washing the matrix to remove unbound materials, and eluting the protein or polypeptide with a solution that dissociates the bound protein from the NTNHA. Appropriate conditions for binding are a pH less than 7.5 (e.g., 6). Appropriate conditions for dissociation are a pH greater than or equal to 7.5 (e.g., 8). Specific compositions for the methods are also described.

Description

1 / 74 Descriptive Report of the Invention Patent for: "METHOD FOR PURIFICATION AND ACTIVATION OF BOTULINUM NEUROTOXIN" Cross-referencing to related patent applications.

[0001] This patent application claims the benefit of U.S. Provisional Patent Application 62 / 336,958, filed May 16, 2016, the contents of which are incorporated herein by reference in full. SEQUENCE LISTING

[0002] This specification refers to a Sequence Listing (submitted electronically as a .txt file named “0342941-0584_SL.TXT” on May 16, 2017). The .txt file was generated on May 16, 2017 and has a size of 96,153 bytes. All the contents of the Sequence Listing are incorporated herein by reference. FIELD OF THE INVENTION

[0003] The present invention relates to the field of therapeutic use of neurotoxins. FUNDAMENTALS OF THE INVENTION

[0004] Botulinum neurotoxins (BoNTs) are the best-known toxic substances for humans. Seven serotypes of BoNTs (A to G) have been identified; with many subtypes within each serotype. BoNTs are proteins of ~150 kDa produced by different strains of the bacterium Clostridium botulinum (Montal 2010). These toxins cause botulism. Petition 870180151897, dated 11 / 14 / 2018, page 24 / 137 2 / 74 in animals, a serious neurological disease manifested as severe flaccid paralysis and possible death. The molecular basis of this toxicity lies in the ability of BoNTs to bind to and enter motor neurons and release their enzymatic domain into the cytosol, which cleaves the cellular machinery responsible for synaptic vesicle fusion at neuromuscular junctions (NMJs) and inhibits neurotransmission by blocking the release of acetylcholine.

[0005] The neuroinhibitory function of BoNTs has been explored as a treatment strategy for many muscular disorders ranging from strabismus to the control of multiple dystonias (Masuyer et al. 2014), not to mention the sharp increase in cosmetic uses of BoNTs (A) to induce flaccid paralysis in facial muscles to smooth wrinkles. The BoNTs market is close to US$2 billion and is still growing at a rapid pace.

[0006] Currently, there are several changes in the production of BoNTs. BoNTs need to be produced in bacteria and isolated from bacterial lysates. Current therapeutic BoNTs are still produced and isolated using older methodologies similar to those that originated more than 50 years ago when the first batch of BoNT / A prepared in the laboratory was described (Bonventre & Kempe 1959; Pickett 2014). These methods typically involve lengthy incubation / fermentation of bacterial strains. Petition 870180151897, dated 11 / 14 / 2018, page 25 / 137 3 / 74 natural sources that produce these toxins (spore-forming clostridium strains) and many subsequent laborious chromatography steps. In addition to genetic engineering and containment challenges, these processes can also compromise the final field strength, efficacy, and reproducibility of BoNT preparations.

[0007] The expression of recombinant BoNTs from common host systems used for protein production in industry, such as E. coli and insect cells, has been explored recently. An affinity marker, such as His-6 (SEQ ID NO: 1) or GST, is typically fused to BoNTs to facilitate purification via affinity purification. While isolating recombinant BoNTs with affinity markers simplifies purification steps, it introduces new problems. The marker may adversely affect the biological activity of the toxin and / or have undesirable antigenicity. Consequently, the marker has to be removed after purification, which involves additional enzymatic treatment and purification steps. Furthermore, there are often additional residues attached to the toxin by the cleaved marker, creating a non-native N- or C-terminus that can affect activity or promote immunological consequences in a patient.

[0008] Isolating the natural forms of BoNTs is much preferred, but it remains a laborious and time-consuming process. Petition 870180151897, dated 11 / 14 / 2018, page 26 / 137 4 / 74

[0009] Purified BoNTs still need to be activated by limited proteolysis before use. Recombinant BoNTs are typically activated post-purification by incubation with an endoproteinase, such as trypsin. Such activation can cause non-specific degradation and requires an additional purification step to remove the endoproteinase from activation, both of which compromise toxin activity and yield. SUMMARY OF THE INVENTION

[0010] As will become evident to those skilled in the art upon reading the present description, the present invention encompasses the recognition of a problem with the compositions and methods for the production, purification, and / or activation of botulinum neurotoxins (BoNTs) or portions or fragments thereof. Among other things, the present invention identifies the challenges in providing materials and procedures that facilitate the production, purification, and / or activation of BoNTs with the desired characteristics (e.g., relatively uncompromised biological activity; limited introduction of unwanted antigenicity; limited contaminants, such as endoproteinases and / or unwanted degradation products; and high quality, potency, and / or reproducibility of the desired BoNT), while reducing the limitations of previous approaches (by Petition 870180151897, dated 11 / 14 / 2018, page 27 / 137 5 / 74 example, limited production efficiency, time-consuming and / or laborious steps and / or adverse conditions).

[0011] One aspect of the invention relates to a molecule comprising a non-toxic non-hemagglutinin polypeptide (NTNHA) covalently linked to a heterologous affinity moiety. In one embodiment, the NTNHA and the affinity moiety are expressed as a fusion protein. In one embodiment of the compositions described herein, the affinity moiety is located at a selected position of the group consisting of the N-terminal of the NTNHA amino acid sequence, the C-terminal of the NTNHA amino acid sequence, and internal to the NTNHA amino acid sequence. In one embodiment of the compositions described herein, the affinity moiety effectively binds to a binding target under conditions of about pH 6 to about pH 8.In one embodiment of the compositions described herein, the affinity fraction is selected from the group consisting of glutathione-Stransferase (GST), C-myc marker, chitin-binding domain, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, Stag, Strep-tag II, FLA, protein A, protein G, histidine affinity marker (HAT), Poly-His, and maltose-binding protein (MBP). In one embodiment of the compositions described herein, the NTNHA is of serotype A, B, C1, D, E, F, or G. In one embodiment of the compositions described herein, the NTNHA is of... Petition 870180151897, dated 11 / 14 / 2018, p. 28 / 137 6 / 7 4 serotype B. In one embodiment of the compositions described herein, the molecule is in a complex with a compatible botulinum neurotoxin (BoNT) or with a polypeptide comprising a receptor-binding domain thereof. In one embodiment of the compositions described herein, the BoNT or the polypeptide comprises a modified receptor-binding domain of clostridial botulinum serotype B (B-Hc). In one embodiment of the compositions described herein, the molecule is additionally linked to a binding target via the affinity moiety. In one embodiment of the compositions described herein, the binding target is stably linked to a matrix.

[0012] Another aspect of the invention relates to an aqueous solution comprising one of the molecules described herein.

[0013] Another aspect of the invention relates to a nucleic acid that encodes one of the functional NTNHA fusion protein and affinity fraction described herein.

[0014] Another aspect of the invention relates to an expression vector comprising the nucleic acid encoding one of the functional NTNHA fusion proteins and the affinity fraction described herein.

[0015] Another aspect of the invention relates to a host cell comprising and expressing nucleic acid encoding one of the functional NTNHA fusion proteins and the affinity fraction described herein. In one embodiment, the host cell further expresses a botulinum neurotoxin. Petition 870180151897, dated 11 / 14 / 2018, p. 29 / 137 7 / 7 4 (BoNT) compatible. In one embodiment of the host cells described herein, the BoNT comprises a modified receptor-binding domain of the botulinum clostridium serotype B (BHc). In one embodiment of the host cells described herein, the host cell is prokaryotic or eukaryotic. In one embodiment of the host cells described here, the host cell is a bacterial cell, a yeast cell, a mammalian cell, an insect cell, a plant cell, or an amphibian cell.

[0016] Another aspect of the invention relates to a method of purifying Botulinum Neurotoxin (BoNT) comprising contacting the BoNT with a compatible non-toxic non-hemagglutinin (NTNHA) under appropriate conditions for the NTNHA to bind to the BoNT, thereby forming an NTNHA-BoNT complex. In one embodiment, the BoNT is in solution and the NTNHA is bound to a matrix, while the solution is contacted with the matrix to bring the BoNT into contact with the NTNHA. In one embodiment of the methods described herein, the method further comprises washing the matrix to remove unbound materials and eluting the BoNT from the matrix by contacting the matrix with an aqueous solution that dissociates the BoNT from the NTNHA-BoNT complex. In an alternative embodiment of the methods described herein, after contact of the BoNT solution with the NTNHA matrix, the method further comprises washing Petition 870180151897, dated 11 / 14 / 2018, p. 30 / 137 8 / 74 the matrix to remove unbound materials, contact the matrix with a protease under conditions that preserve the NTNHA-BoNT complex and are appropriate for cleavage of BoNT into the NTNHA-BoNT complex, wash the matrix to remove the protease and unbound materials, and elute the BoNT from the matrix by contacting the matrix with an aqueous solution that dissociates the BoNT from the NTNHA-BoNT complex. In one embodiment of the methods described herein, NTNHA is covalently linked to an affinity fraction, the matrix is ​​linked to a binding target of the affinity fraction, and NTNHA is non-covalently linked to the matrix through interactions of the affinity fraction and the binding target. In another embodiment of the methods described herein, NTNHA is covalently linked to the matrix. In another embodiment of the methods described herein, BoNT comprises a modified receptor-binding domain of clostridial botulinum serotype B (B-Hc).In one embodiment of the methods described herein, the aqueous solution that dissociates BoNT from the NTNHA-BoNT complex has a pH > 7.5. In one embodiment of the methods described herein, the solution comprising BoNT is a clean cell extract from cells expressing BoNT. In one embodiment of the methods described herein, the clean cell extract further comprises 1 mM phenylmethylsulfonyl fluoride (PMSF). In one embodiment of the methods described herein, the appropriate conditions for binding comprise placing BoNT in a buffer context. Petition 870180151897, dated 11 / 14 / 2018, p. 31 / 137 9 / 74 binding buffer that has physiological ionic strength and pH < 7.5. In one embodiment of the methods described herein, washing is done with a washing buffer that has physiological ionic strength and a pH < 7.5. In one embodiment of the methods described herein, the binding buffer and / or washing buffer is between 100 to 200 mM KCl or NaCl. In one embodiment of the methods described herein, the binding buffer and / or washing buffer has a pH of about 6. In one embodiment of the methods described herein, the binding buffer and / or washing buffer comprises 50 mM MES, 150 mM NaCl, pH 6. In one embodiment of the methods described herein, the aqueous solution that dissociates BoNT from the NTNHA-BoNT complex is an elution buffer of about 50 mM Tris, 150 mM NaCl. In one embodiment of the methods described herein, the aqueous solution is an elution buffer of about pH 8.In one embodiment of the methods described herein, the affinity fraction is selected from the group consisting of glutathione-S-transferase (GST), C-myc marker, chitin-binding domain, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, histidine affinity marker (HAT), Poly-His, and maltose-binding protein (MBP).

[0017] In one embodiment of the methods described herein, the affinity fraction is GST and the binding target is glutathione. Petition 870180151897, dated 11 / 14 / 2018, p. 32 / 137 10 / 74

[0018] In one embodiment of the methods described herein, NTNHA is present at a molar ratio between about 1:1 and about 10:1 for BoNT, for example, about 2:1, 3:1, 4:1 or 5:1 for BoNT. In one embodiment of the methods described herein, BoNT and NTNHA are co-expressed in the same host cell, for example, E. coli. In one embodiment of the methods described herein, BoNT and NTNHA are expressed in different host cells. In one embodiment of the methods described herein, BoNT is produced recombinantly in a heterologous host cell, such as E. coli. In one embodiment of the methods described herein, BoNT is produced in its native clostridial cell. In one embodiment of the methods described herein, NTNHA is produced recombinantly in a heterologous host cell, such as E. coli. In one embodiment of the methods described here, NTNHA is produced in its native clostridial cell.

[0019] In one embodiment of the methods described herein, the protease is selected from trypsin, pepsin, Lys-C endoproteinase, Lys-N endoproteinase, arginyl endopeptidase, plasmin, omptin, and a clostridial protease as described in EP2524963. In a preferred embodiment, the protease is trypsin or Lys-C endoproteinase. In one embodiment, the protease is a protease that cleaves a non-native (i.e., exogenous) cleavage site of BoNT. In such Petition 870180151897, dated 11 / 14 / 2018, page 33 / 137 11 / 74 clostridial toxins, the cleavage site of the native protease (also known as the activation site) is modified or replaced by a protease cleavage site that is not active for the clostridial toxin. Non-native proteases that may be employed include Enterokinase (DDDDKj (SEQ ID NO: 2)), Factor Xa (IEGRMSEQ ID NO: 3) / IDGRj(SEQ ID NO: 4)), TEV (Tobacco Etching Virus) (ENLYFQ^G (SEQ ID NO: 5)), Thrombin (LVPRjGS (SEQ ID NO: 6)) and PreScission (LEVLFQ1GP (SEQ ID NO: 7)).

[0020] In one embodiment of the methods described herein, the protease is added at a molar ratio of about 1:2 to about 1:1,000 for NTNHA, preferably from about 1:5 to about 1:100 for NTNHA, for example, about 1:10, 1:20, 1:30, 1:40 or 1:50. In one embodiment of the methods described herein, the protease is added at a molar ratio of about 1:2 to about 1:1,000 for BoNT, preferably from about 1:5 to about 1:100 for BoNT, for example, about 1:10, 1:20, 1:30, 1:40 or 1:50. The appropriate conditions for the specific protease used will be determined by those skilled in the art. The exposure time of the protease will also vary with the protease, the concentration used, and the temperature. In one embodiment of the methods described herein, the protease is brought into contact with the matrix at a temperature of about 2°C to about 40°C, preferably from about 4°C to about 37°C, for example, Petition 870180151897, dated 11 / 14 / 2018, p. 34 / 137 12 / 74 4°C, 16°C, 20°C or 37°C. In one embodiment of the methods described herein, the protease is placed in contact with the matrix at room temperature (approximately 20 to 22°C). In another embodiment of the methods described herein, the protease is placed in contact with the matrix for about 10 minutes to about 18 hours, preferably from about 30 minutes to about 5 hours, for example, about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours or 5 hours. In one embodiment of the methods described herein, the protease is brought into contact with the matrix at a pH of about 5.5 to about 8.5, preferably about 6 to 8, for example, at a pH of about 6, 7 or 8. In another embodiment, the protease is selected from the proteases: trypsin and Lys-C endoproteinase and is brought into contact with the matrix at room temperature for about 30 minutes to 2 hours at a pH between 6 and 7.

[0021] In one embodiment of the methods described herein, the protease is added at a molar ratio of about 1:10 to the NTNHA. In one embodiment of the methods described herein, the protease is placed in contact with the matrix at room temperature. In another embodiment of the methods described herein, the protease is placed in contact with the matrix for about 30 minutes to 12 hours.

[0022] Another aspect of the invention relates to a method of purifying Botulinum Neurotoxin (BoNT) comprising placing a clean cell extract comprising BoNT in Petition 870180151897, dated 11 / 14 / 2018, p. 35 / 137 13 / 74 contact with a glutathione-coated matrix that has a non-toxic non-hemagglutinin (NTNHA) compatible fused to it, glutathione-S-transferase, in a binding buffer with a pH of about 6 to form an NTNHA-BoNT complex, washing the matrix with a washing buffer with a pH of about 6 to remove unbound materials, contacting the matrix with a protease in a buffer with a pH of about 6 to cleave the BoNT into the NTNHA-BoNT complex, washing the matrix with a washing buffer with a pH of about 6 to remove the protease and unbound materials, and eluting the BoNT from the matrix by contacting the matrix with an elution buffer having a pH > 7.5 to dissociate the BoNT from the NTNHA-BoNT complex. In one embodiment of the methods described herein, the BoNT comprises a modified receptor-binding domain of clostridial botulinum serotype B (B-Hc).In one embodiment of the methods described herein, the binding buffer and / or washing buffer comprises 50 mM MES, 150 mM NaCl. In another embodiment of the methods described herein, the binding buffer further comprises 1 mM phenylmethylsulfonyl fluoride (PMSF). In another embodiment of the methods described herein, the elution buffer comprises 50 mM Tris, 150 mM NaCl and has a pH of about 8. In another embodiment of the methods described herein, the glutathione-coated matrix is ​​a crude polymer of agarose linked to glutathione. In another embodiment of the methods described herein... Petition 870180151897, dated 11 / 14 / 2018, pp. 36 / 137 14 / 74 The glutathione-coated matrix is ​​a column. In one embodiment of the methods described herein, the glutathione-coated matrix has approximately 5 mg / mL of bound NTNHA. In one embodiment of the methods described herein, the protease is trypsin or Lys-C endoproteinase.

[0023] Another aspect of the invention relates to a method of purifying a polypeptide comprising a receptor-binding domain (Hc polypeptide) of botulinum neurotoxin, comprising the steps of contacting a solution comprising the Hc polypeptide with a matrix having compatible non-toxic non-hemagglutinin (NTNHA) bound to it, under conditions appropriate for the binding of the NTNHA to the Hc-polypeptide to form an NTNHA-Hc polypeptide complex, washing the matrix to remove unbound materials, and eluting the Hc polypeptide from the matrix by contacting the matrix with an aqueous solution that dissociates the Hc polypeptide from the NTNHA-Hc polypeptide complex. In one embodiment of the methods described herein, the receptor-binding domain of the Hc polypeptide is a modified receptor-binding domain of the clostridial botulinum serotype B (BHc). In another embodiment of the methods described herein, the Hc polypeptide is a botulinum neurotoxin (BoNT) polypeptide.In one embodiment of the methods described herein, the Hc polypeptide is a chimeric polypeptide of botulinum neurotoxin (BoNT). Petition 870180151897, dated 11 / 14 / 2018, pp. 37 / 137 15 / 74

[0024] Another aspect of the invention relates to the use of a molecule described herein in a method for, or purification of, a botulinum neurotoxin (BoNT) polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] This patent file or patent application contains at least one color drawing. Copies of this patent or patent application with the color drawing(s) are provided by the Office upon request and payment of the required fee.

[0026] FIGURE 1A and FIGURE 1B illustrate one embodiment of a purification principle or protocol for BoNTs as described herein. FIGURE 1A) Schematic illustration of the pH-dependent bimolecular complexation of BoNTs and NTNHA. LC: light chain, HN: translocation domain, HCN, HCC: N-terminal and C-terminal segments of the receptor-binding domain, respectively. NTNHA has the same domain content as BoNTs; it is presented as a fused GST (Glutathione-S-transferase) protein immobilized on Glutathione-Agarose resin. FIGURE 1B) A flowchart describing a comprehensive purification, activation, and elution protocol for BoNTs using NTNHA from the natural pair binding.

[0027] FIGURE 2A and FIGURE 2B are images of the fractionated proteins from the gel. The experimental results indicate the successful purification of BoNT / B using NTNHA / B as a Petition 870180151897, dated 11 / 14 / 2018, pp. 38 / 137 16 / 74 model complex for BoNTs. FIGURE 2A) A monoclonal antibody against BoNT / B is used to monitor the presence of BoNT / B throughout each purification step described in FIGURE 1B (except that the samples here were not treated with trypsin). FIGURE 2B) An SDS-PAGE gel of the selected samples stained with Coomassie shows the purity of the purified BoNT / B as described in panel A. A main band (~150 kDa) corresponding to BoNT / B is observed in the elution fraction.

[0028] FIGURE 3A and FIGURE 3B are images of two sets of fractionated proteins from the gel. Experimental results indicate that BoNT / B is efficiently activated into NTNHA / B → BoNT / B complexes. FIGURE 3A) Representative immunoblot of activation of NTNHA-bound BoNT / B by trypsin, which separates BoNT / B into two fragments (100 kDa and 50 kDa, respectively). The two BoNT / B fragments remain linked to each other by a single disulfide bond. They separate from each other when DTT is added to reduce the disulfide bond. FIGURE 3B) Coomassie-stained elution fraction shows the toxin bands corresponding to the cleaved toxin fragments (at 100 and 50 kDa, respectively). The 150 kDa band is the full-length toxin portion that remains to be cleaved. Petition 870180151897, dated 11 / 14 / 2018, pp. 39 / 137 17 / 74

[0029] FIGURE 4 is an image of the fractionated proteins from the gel. The experimental results establish a successful purification of the chimeric BoNT / A1B toxin using NTNHA / B. A polyclonal antibody against BoNT / A was used to track the purification steps of a chimeric BoNT / A1B toxin, which is made up of the light chain and translocation domain of BoNT / A1, with the receptor-binding domain of BoNT / B. The full-size BoNT / A1B (the 150 kDa band in the elution fraction) was successfully purified and eluted using NTNHA / B. It was observed that the prominent band at 100 kDa is a degradation product of this chimeric toxin, likely cleaved by endogenous proteases in E. coli.

[0030] Figures 5A to 5I (SEQ ID NO.s 22-30) are a list of the amino acid sequences of the various NTNHA serotypes and variants thereof.

[0031] Figures 6A-6C illustrate one embodiment of a purification principle or protocol for BoNTs, as described herein. FIGURE 6A) Schematic illustration of a pH-dependent bimolecular complexation of BoNTs and NTNHA. LC: light chain, HN: translocation domain, HCN, HCC: N-terminal and C-terminal segments of the receptor-binding domain, respectively. NTNHA has the same domain content as BoNTs and is presented as a fused GST (Glutathione-S-transferase) protein that can be immobilized on Glutathione-Agarose resin. The interaction Petition 870180151897, dated 11 / 14 / 2018, p. 40 / 137 The interaction between BoNT and NTNHA under slightly acidic conditions (e.g., ~pH 6) can be interrupted by manipulating buffer conditions to a neutral to alkaline pH. FIGURE 6B) BoNT isolation and activation protocol. A flowchart describing a strategy for the purification, activation, and elution of labeled and unlabeled BoNTs from crude lysates using NTNHA. FIGURE 6C) SDS-PAGE analysis of a typical isolation of an inactive BoNT (BoNT / B(ry)) from E. coli lysate cleared using GST-NTNHA / B immobilized on crude agarose and glutathione polymers. The ligation and washing steps were performed at pH 6 and elution by changing the buffer to pH 8.

[0032] FIGURE 7A and FIGURE 7B show BoNT / B isolated using immobilized and pure NTNHA and binding to its canonical neuronal receptor. FIGURE 7A) SDS-PAGE analysis (left) shows three elution fractions that are pooled and concentrated (lane 5). A monoclonal antibody against BoNT / B to detect the toxin at all stages (WB, right). The eluted fractions contain unactivated BoNT / B as the main band at ~150 kDa which corresponds to a single-chain BoNT / B(ry) toxin. FIGURE 7B) Binding detected by anisotropy: the full-length eluted toxin shows similar affinity to a FITC-labeled fragment of its canonical synaptic vesicle receptor Synaptotagmin 1 (Syt 1) as its recombinant HC domain; BoNT / B HC does not bind to Syt. Error bars represent the mean + SEM of 3 samples. Petition 870180151897, dated 11 / 14 / 2018, page 41 / 137 19 / 74

[0033] FIGURES 8A to 8C show that the complexed BoNT is efficiently activated while still protected from nonspecific cleavage. FIGURE 8A) Trypsin-mediated activation (cleavage) of BoNT / B (ry) is visualized on an 8% SDS-PAGE. The course of cleavage of the single-chain toxin (SC) results in two fragments: Heavy chain (HC) and Light chain (LC) linked by a single disulfide bond. FIGURE 8B) WB analysis shows that the activation of BoNT / B, although complexed with NTNHA / B, protects it from nonspecific trypsination while allowing efficient scavenging and removal by the endoproteinase. FIGURE 8C) Lys-C endoproteinase can also be used as a specific activator to produce the active toxins and single-chain toxin using this method.

[0034] FIGURE 9 shows the isolation of the chimeric toxin BoNT / A1B1 using NTNHA / B. A polyclonal antibody against BoNT / A is used to screen the purification of a chimeric toxin made of BoNT / A (LC(ry), Hn) fused to the Hc domain of BoNT / B. The eluted fractions contain the non-activated BoNT / A1B1 protein at ~150 kDa. The prominent band at ~70 kDa is likely a fragment of NTNHA / B that is recognized by the polyclonal antibody. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0035] Botulinum neurotoxins (BoNTs) are highly potent protein toxins produced by spore-bearing Clostridium botulinum. In the last two decades, Petition 870180151897, dated 11 / 14 / 2018, p. 42 / 137 In 20 / 74 it was discovered that these deadly agents are useful in treating numerous neuromuscular disorders and in aesthetic applications by blocking the release of neurotransmitters in the injected muscles. Now established as therapeutic agents, BoNTs are widely produced on a large scale by various manufacturers worldwide. Available data suggest that manufacturing procedures rely on decades-old methodologies that utilize spore-forming strains, and toxin isolation is achieved through laborious and inefficient mass purification steps. An improved method for the direct purification and activation of therapeutic BoNTs is needed.

[0036] Botulinum neurotoxins (BoNTs) are the most common toxic substances for humans. Seven serotypes of BoNT proteins (A to G) have been identified as ~150 kDa products of different strains of the bacterium Clostridium botulinum (Montal 2010). These toxins cause botulism in animals, a severe neuromuscular disease manifested as severe flaccid paralysis. The molecular basis of this toxicity lies in the ability of the toxins to bind potently to receptors on motor neurons at the neuromuscular junction (NMJ), internalize by endocytosis, and cross the endosomal membrane to release their enzymatic chain into the cytosol. The released protease then cleaves the cellular machinery (SNARE proteins) responsible for fusion. Petition 870180151897, dated 11 / 14 / 2018, page 43 / 137 21 / 74 of the synaptic vesicle in the NMJ, thus inhibiting neurotransmission by blocking the release of acetylcholine (Blasi et al. 1993; Borden Lacy et al. 1998; Rossetto et al. 2014).

[0037] Botulinum neurotoxins (BoNTs) can also be used as tools to locally control muscle activity, especially uncontrolled activity or abnormalities due to muscle elasticity (Masuyer et al. 2014). This neuroinhibitory function of BoNTs has been explored as a treatment strategy for many muscle disorders, including strabismus and the control of multiple dystonias and lower urinary tract dysfunctions (LUTD) (Jankovic & Brin 1991; Truong & Jost 2006; Visco et al. 2012; Jiang et al. 2015). As a therapeutic and / or cosmetic agent, BoNTs can be used to paralyze facial muscles in order to smooth wrinkles (Hexsel et al. 2011). Additional applications of the toxins aim to alleviate depression and provide prophylactic treatment for migraines (Finzi & Rosenthal 2014; Jackson et al. 2012). The clinical uses of the toxin have garnered much public interest (Sifferlin 2017).

[0038] Botulinum neurotoxins (BoNTs) can be isolated from a spore-forming Clostridium strain growth and subsequently purified to a final product (Pickett 2014). Available data on the processes of Petition 870180151897, dated 11 / 14 / 2018, p. 44 / 137 22 / 74 Production and isolation of BoNTs suggest that producers use cultivation and growth conditions methodologies on native strains similar to those that originated decades ago (Pickett & Perrow 2009; Snipe & Sommer 1928; Duff, Wright, et al. 1957; Duff, Klerer, et al. 1957; Bonventre & Kempe 1959; Schantz & Johnson 1992; Pickett 2014). These methods are limited by the efficiency with which the native Clostridium strain can produce the toxin and typically involve prolonged fermentation periods of the natural toxin source (spore-producing Clostridium strains) after laborious toxin isolation procedures, often under adverse conditions, involving multiple acid / alcohol precipitations, crystallizations, and / or multiple chromatographic steps (DasGupta & Boroff 1967; Tse et al. 1982; Schantz & Johnson 1992; Malizio et al.).

[0039] The production of recombinantly specific BoNTs is possible with the inclusion of an affinity marker (e.g., His6X or GST fusion) to aid in toxin purification using affinity chromatography. However, such approaches have disadvantages, for example, in the use of BoNTs as therapeutic biologics. For instance, affinity markers may adversely affect the biological activity of the toxin and / or have undesirable antigenicity. The removal of a marker after purification also requires further steps. Petition 870180151897, dated 11 / 14 / 2018, page 45 / 137 23 / 74 additional enzymatic and purification steps, simultaneously producing native N- or C-terminal ends in the final product. Additionally, recombinant BoNTs have to be activated post-purification by an endoproteinase to obtain functional and potent dichain toxins. This proteolytic step leads to non-specific degradations that require additional purification steps to remove endoproteinases and / or degradation products. Besides the genetic engineering and containment challenges for toxin production from spore-forming strains and subsequent purifications (Malizio et al. 2000; Pickett 2014), these recombinant approaches can compromise most properties in the final product, ranging from quality and potency to efficient reproducibility. A novel strategy for safely and efficiently isolating active therapeutic BoNTs would be beneficial for large-scale production and easy isolation of BoNTs.

[0040] Studies on the biochemical properties and cellular mechanisms by which clostridial neurotoxins gain entry into the neuronal cytosol have provided some understanding of the structural, molecular, and mechanistic functions of clostridial neurotoxins (Blasi et al. 1993; Borden Lacy et al. 1998; Dong et al. 2006; Rossetto et al. 2014). Foodborne botulism requires the passage of intact toxins and other Petition 870180151897, dated 11 / 14 / 2018, page 46 / 137 24 / 74 products of the bacteria are expelled through the host's gastrointestinal tract. The molecular and structural basis of this ability to evade degradation remained a mystery until recently, when larger complexes called "progenitor toxin complexes" (PTCs) were characterized as constituting the total toxic agent encountered by a specific organism. In addition to the proteolytically active toxin, these multiprotein complexes are typically composed of a non-toxic non-hemagglutinin protein (NTNHA) with a specific serotype and three hemagglutinin proteins (HAs) (Lee et al. 2014). Previously considered to aid in toxin functions (Schantz & Johnson 1992), PTCs are now known to physically shield and protect BoNTs from the harsh gastrointestinal environment to safely reach their destinations: first at the epithelial barriers and subsequently at NMJs where they can be internalized into the cytosol via synaptic vesicle recycling mechanisms.In a structural study (Gu et al. 2012), Gu and collaborators indicated in the atomic report detail a minimally effective PTC (m-PTC) in a non-covalent complex of BoNT / A:NTNHA / A. The cocrystal structure of the toxin:NTNHA complex indicated pH-dependent complex formation. BoNT / A and NTNHA / A were reported to be able to form a rigid complex with nanomolar-level affinity under slightly acidic conditions (~pH 6). Petition 870180151897, dated 11 / 14 / 2018, page 47 / 137 25 / 74 However, it was stated that such complex formation does not occur at neutral to alkaline pH.

[0041] Compositions and methods are described herein with respect to the purification of BoNT using the natural affinity of the BoNT molecule for the non-toxic non-hemagglutinin protein (NTNHA). BoNT naturally forms a dimer complex with the NTNHA chaperone protein and is protected from protease and acid degradation in the gastrointestinal tract. The binding is reversible and pH-dependent, binding at pH < 7 and dissociation at pH > 7.4. The NTNHA protein is added to a mixture containing BoNT at a pH that promotes binding. The BoNT:NTNHA complex is isolated from the other components of the mixture by immobilizing NTNHA on the complex. After washing, BoNT is then released from the complex by increasing the pH to promote dissociation. As this method does not depend on a modification of the BoNT affinity, unlabeled forms of the toxin can be purified.

[0042] The purification methods described here also make it possible to activate BoNT even in the BoNT:NTNHA complex. After activation, BoNT can be released from the complex, thus generating a purified and activated form of the toxin.

[0043] Aspects of the invention relate to a method of purifying a BoNT. Typically, the BoNT is, in the context of an aqueous solution containing contaminating components, such Petition 870180151897, dated 11 / 14 / 2018, p. 48 / 137 26 / 74 as a cell extract. The method comprises combining the solution in the NTNHA molecule under appropriate conditions for the binding of BoNT to the NTNHA. Practically, this may involve combining the NTNHA molecule with an aqueous solution (e.g., cell extract or a cleared cell extract). The BoNT can be isolated by virtue of the NTNHA molecule. Generally, this is accomplished by immobilizing the NTNHA in a matrix. Unbound materials are removed from the complex, for example, by washing the matrix (e.g., using a quantity of washing buffer of 3 to 4 volumes of the matrix). After washing, the BoNT is released from the complex, for example, by eluting the NTNHA-bound matrix, to yield a purified polypeptide.

[0044] BoNT can be activated before the release of the complex by digestion with a protease. This can be accomplished by placing the bound complex in the matrix in contact with a protease under conditions appropriate for the cleavage of BoNT that would not otherwise break down the complex (e.g., maintaining the necessary pH). The protease is removed along with other unbound materials by washing the matrix (e.g., with a washing buffer). The activated and purified BoNT can then be eluted by placing the matrix in contact with an aqueous solution that dissociates the BoNT from the NTNHA complex (e.g., with an elution buffer). In Petition 870180151897, dated 11 / 14 / 2018, page 49 / 137 27 / 74 In some modalities, activation of the polypeptide is neither necessary nor desired.

[0045] The NTNHA used in the method must be compatible with BoNT. The term compatible, when used in reference to NTNHA and BoNT, refers to molecules that are capable of forming a rigid and stable complex with each other. In one embodiment, BoNT and NTNHA are components of the same naturally occurring BoNT serotype protein complex. This occurs when the sequences encoding BoNT and NTNHA are from the same operon. Since the term serotype is used here to describe the NTNHA molecule, being of a serotype refers to an NTNHA molecule derived from an operon encoding a specific BoNT serotype. Compatible can also refer to a BoNT or chimeric polypeptide that has a region (e.g., the Hc region) that is compatible with NTNHA. In one embodiment, the NTNHA and the Hc region of BoNT are both derived from the same naturally occurring BoNT serotype complex.

[0046] NTNHA immobilization in the matrix can occur before or after BoNT binding. In one embodiment, NTNHA is bound to a matrix and a solution comprising BoNT is added to the matrix to bring the Hc polypeptide into contact with NTNHA and promote complex formation. In another embodiment, NTNHA and BoNT are in a complex before NTNHA binds to the matrix. Petition 870180151897, dated 11 / 14 / 2018, p. 50 / 137 28 / 74

[0047] In one embodiment, an affinity fraction is introduced into the NTNHA protein (e.g., by expression as a fusion protein) and the labeled protein is used to bind and isolate BoNT under conditions that promote BoNT:NTNHA binding. The BoNT:NTNHA complex is isolated by affinity purification of NTNHA in the complex.

[0048] Binding buffers, incubation buffers, washing buffers, and protease digestion buffers promote appropriate conditions for the formation and preservation of the Hc-NTNHA complex. This includes, but is not limited to, having a pH that promotes complex formation. Typically, this will be at a pH less than 7.5, for example, less than 6. In one embodiment, the buffer pH is 2 to 8. In one embodiment, the buffer pH is 5 to 7. In one embodiment, the pH is about 5, about 6, or about 7. The binding buffers, incubation buffers, and washing buffers may all be highly similar or the same. The buffers may also contain additional components other than those specified herein. In one embodiment, the buffer also contains a stabilizing agent for the BoNT polypeptide (e.g., serum albumin, polysaccharide, trehalose, or surfactant). The pH of the buffers can be optimized for various components within the specified ranges.Those knowledgeable will realize that the pH of the buffer must preserve the overall structure of the protein. Petition 870180151897, dated 11 / 14 / 2018, page 51 / 137 29 / 74 avoiding a pH that approaches the pI of the protein, which can cause the protein to precipitate.

[0049] Buffers preferably have physiological ionic strength (e.g., in the range of 100 to 200 mM KCl or NaCl). A variety of salts are available to create the required ionic strength. Salt concentrations that are too high can disrupt the interactions due to polar / ionic interference. In one embodiment, the salt concentration is 400 mM or less. Low salt concentration conditions are also expected to work sufficiently. In one embodiment, the salt concentration is 150 mM. In one embodiment, the buffer comprises 50 mM MES, 150 mM NaCl, and has a pH of 6. In one embodiment, the buffer in which binding occurs (binding buffer) further comprises one or more protease inhibitors (e.g., phenylmethylsulfonyl fluoride (PMSF)). In one embodiment, the binding buffer comprises PMSF at a concentration of about 0.1 to 1 mM. In one embodiment, the PMSF is about 1 mM.

[0050] Washing can be carried out, for example, using a washing buffer. A typical amount for washing is 3 to 4 volumes of the matrix.

[0051] The BoNT molecule contains several domains and binds to the NTNHA molecule via its receptor-binding domain (otherwise referred to as the Hc domain). As such, the methods described herein are applicable. Petition 870180151897, dated 11 / 14 / 2018, p. 52 / 137 30 / 74 purification of any polypeptide comprising a receptor-binding domain (Hc polypeptide) of botulinum neurotoxin (e.g., full-size BoNT or fragment thereof comprising the Hc polypeptide or a chimeric polypeptide comprising the Hc domain).

[0052] In one embodiment of the methods described herein, NTNHA is present at a molar ratio between about 1:1 and about 10:1 for the BoNT or for its receptor-binding domain, for example, about 2:1, 3:1, 4:1 or 5:1 for the BoNT or for its receptor-binding domain.

[0053] Activation of bound BoNT or a fragment thereof is achieved by placing the BoNT:NTNHA complex (e.g., when matrix-bound) in contact with an appropriate protease. In one embodiment, the protease cleaves a protein after a lysine residue. In one embodiment, the protease is, without limitation, trypsin, pepsin, Lys-C endoprotease, Lys-N endoproteinase, arginyl endopeptidase, plasmin, omptin, or clostridial protease as described in EP2524963. Preferred conditions result in substantial degradation of NTNHA, any associated affinity fractions, or its binding target. Appropriate conditions for cleavage include the appropriate protease concentration and appropriate conditions for the Petition 870180151897, dated 11 / 14 / 2018, page 53 / 137 31 / 74 protease activity (e.g., temperature, incubation time, buffer components, etc.). Such conditions can be obtained by using an appropriate protease digestion buffer. The amount of protease used can be determined by the amount of NTNHA molecule or the amount of BoNT molecule. In one embodiment, the protease is present at a molar ratio of about 1:2 to about 1:1,000 to the NTNHA molecule. In another embodiment, the protease is present at a molar ratio of about 1:5 to about 1:100 to the NTNHA molecule, for example, about 1:10, 1:20, 1:30, 1:40, or 1:50. In one embodiment of the methods described herein, the protease is added at a molar ratio of about 1:2 to about 1:1,000 for BoNT (e.g., about 1:5 to about 1:100 for BoNT) or about 1:10, 1:20, 1:30, 1:40, or 1:50.

[0054] The appropriate conditions for the specific protease used are determined by those skilled in the art. The exposure time of the protease also varies with the protease, the concentration used, and the temperature. In one embodiment, the protease is exposed at a temperature between 2°C and 40°C, preferably between 4°C and 37°C (e.g., 4°C, 16°C, 20°C, or 37°C). In another embodiment, the protease is exposed at room temperature (approximately 20 to 22°C). Petition 870180151897, dated 11 / 14 / 2018, p. 54 / 137 32 / 74

[0055] In one embodiment, the protease is exposed for about 10 minutes to about 18 hours, preferably between 30 minutes and 5 hours (e.g., about 30 minutes, 1 hour, 2, 3, 4, or 5 hours). In one embodiment, the protease is exposed for about 4 hours. In one embodiment, the protease is Lys-C endoprotease and the incubation time is about 30 minutes.

[0056] In one embodiment, the protease is brought into contact with the matrix at a pH of about 5.5 to about 8.5. In another embodiment, the protease is brought into contact with the matrix at a pH of about 6 to about 8 (e.g., about 6, 7, or 8).

[0057] In one embodiment, the protease is selected from trypsin proteases and Lys-C endoproteinase and is placed in contact with the matrix at room temperature for about 30 minutes to 2 hours at a pH between 6 and 7,

[0058] Elution of BoNT from the BoNT-NTNHA complex is performed using an aqueous solution with a pH that promotes the dissociation of the complex (referred to herein as an elution buffer). Preferably, the elution buffer disrupts the BoNT-NTNHA complex by being of the appropriate pH, while otherwise substantially preserving the integrity of the Hc polypeptide and substantially preserving the immobilization of NTNHA (e.g., preserving the binding of NTNHA to a matrix). The elution buffer preferably also has strength Petition 870180151897, dated 11 / 14 / 2018, page 55 / 137 33 / 74 physiological ionic. A variety of buffers are available and appropriate for use (e.g., Tris, MOPS, HEPES, phosphate buffer, etc.). In one embodiment, the elution buffer is the same as the binding and / or washing buffer, differing only in pH. In one embodiment, the elution buffer is approximately 50 mM Tris, 150 mM NaCl with an appropriate pH discussed herein (e.g., pH 8).

[0059] The elution buffer used (for example, those described herein) may be pH from about 7 to about 11. In one embodiment, the pH is 7.5 or higher. In another embodiment, the pH is about 8. The elution buffer may contain additional components besides those specified herein. The pH of the elution buffer may be optimized for the various components therein.

[0060] Typically, BoNT is purified from a cell extract. In one embodiment, the cell extract is a cleared cell extract. The term “cleared cell extract” refers to an extract that is substantially free of all particulate matter, such as when removed by centrifugation and / or filtration.

[0061] BoNT and NTNHA can be co-expressed in the same host cell, for example, E. coli. The method can utilize the NTNHA expressed in it along with BoNT. Alternatively, BoNT and NTNHA can be expressed in different host cells. The respective cell extracts can be Petition 870180151897, dated 11 / 14 / 2018, page 56 / 137 34 / 74 are used to produce / isolate the respective proteins. BoNT can be produced recombinantly in a heterologous host cell, such as E. coli, or in its native clostridial cell. NTNHA can be produced recombinantly in a heterologous host cell, such as E. coli, or in its native clostridial cell.

[0062] Purification or purified, as used herein, refers to a BoNT or fragment thereof that is substantially pure with respect to other components of a preparation (e.g., other polypeptides). It may refer to a BoNT or fragment that is at least about 50%, 60%, 70% or 75%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% pure with respect to other components. In reformulated form, the terms substantially pure or essentially purified, with respect to a BoNT or fragment, refer to a preparation containing less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1% or less than 1%, of one or more other components (e.g., other polypeptides or cellular components).

[0063] Other aspects of the invention relate to the components used in the methods described herein. One aspect of the invention relates to the NTNHA polypeptide used to bind Petition 870180151897, dated 11 / 14 / 2018, p. 57 / 137 35 / 74 for BoNT. The NTNHA polypeptide can be full-chain NTNHA or a functional fragment thereof. A functional fragment of NTNHA is considered to retain the binding to the Hc domain of the compatible BoNT and protect the BoNT from degradation, while also allowing activation. The NTNHA polypeptide may further comprise additional heterologous amino acids. As the term is used here, heterologous refers to a molecule of a different origin. For example, a heterologous affinity moiety differs from any internal affinity moieties naturally present in the NTNHA molecule.

[0064] Heterologous sequences can be covalently linked to NTNHA (e.g., by expression as a fusion protein or by post-translational modification of the NTNHA molecule). In one embodiment, the additional heterologous amino acid sequences are a heterologous affinity fraction.

[0065] Heterologous amino acid sequences may be present at the N-terminus, C-terminus, or internally. Such sequences, when present, must be designed to preserve the interaction of NTNHA with the Hc domain of BoNT. In one embodiment, the heterologous sequence is an affinity fraction, and there is no intermediate sequence between the affinity fraction and the NTNHA sequence. In one embodiment, the Petition 870180151897, dated 11 / 14 / 2018, p. 58 / 137 36 / 74 heterologous amino acids are located at the N-terminal of NTNHA.

[0066] In one embodiment, the heterologous amino acids do not have a functional protein cleavage site, such as those normally used to remove an affinity marker from a fusion protein. In one embodiment, the invention excludes an NTNHA polypeptide comprising an N-terminus fused myc marker, for example, NTNHA-A1 (Gu et al., Science 335: 977-981 (2012)).

[0067] In one aspect of the invention, the NTNHA polypeptide is stably linked to a matrix. Stable linkage refers to linkage that is not disrupted by the conditions of the various buffers described herein. Linkage to the matrix can be via covalent or non-covalent interactions. In one embodiment, linkage to the matrix is ​​via the interaction of a heterologous affinity moiety in the NTNHA polypeptide with a corresponding linkage moiety in the matrix (e.g., a GST affinity moiety in NTNHA with glutathione present in the matrix).

[0068] In one embodiment, the NTNHA polypeptide in the various forms described herein (e.g., linked to an affinity moiety and / or stably bound to a matrix) is further in a complex with a compatible BoNT or a polypeptide comprising a receptor-binding domain (Hc) thereof. In one embodiment, BoNT or Hc is a native protein. Petition 870180151897, dated 11 / 14 / 2018, p. 59 / 137 37 / 74 In one embodiment, the BoNT or Hc is a genetically modified receptor-binding domain (e.g., with increased binding to a specific receptor).

[0069] In one embodiment, the NTNHA polypeptide comprising the affinity moiety is further linked to a binding target via affinity moiety linkage. The binding target may be further stably linked to a matrix.

[0070] Another aspect of the invention relates to an aqueous solution containing the NTNHA polypeptide described herein. The NTNHA polypeptide in the solution may be in any of the ways described herein, such as linked to an affinity moiety, stably linked to a matrix and / or linked to a binding target by means of an affinity moiety, any of which may be further linked to a compatible BoNT.

[0071] The nucleic acid sequences encoding NTNHA and the affinity fraction fusion protein described herein are also encompassed by the invention. The nucleic acid sequences encoding the proteins can be optimized for E. coli expression. In one embodiment, the nucleic acid sequences are in the context of a vector (e.g., an expression vector). The vectors must be compatible with the host cells in which the nucleic acids are to be propagated and / or expressed. NTNHA Petition 870180151897, dated 11 / 14 / 2018, pp. 60 / 137 38 / 74

[0072] NTNHA is a 140 kDa protein synthesized by Clostridium botulinum. NTNHA genes occur in operons that encode a BoNT protein with a particular serotype. BoNT and NTNHA produced from the same opeon are components of the same naturally occurring BoNT protein complex and form a rigid and stable complex with each other. NTNHA binds to BoNT with a Kd of approximately 30.8 nM, in a 1:1 stoichiometry (Shenyan et al., Science 335: 977-981 (2012)). Preferably, NTNHA is derived from the same Clostridium botulinum strain that produces the serotype (and subtype) of the BoNT or Hc fragment being purified (A, A1, A2, A3, A4A, A4-B, types B, C, C1, D, E, F, or G). Some overlap in binding between serotypes can be expected.The amino acid sequences of the different NTNHA proteins are available from experts, as are the coding sequences of nucleic acids, such as NTNHA proteins derived from operons encoding the BoNT serotypes: A1 (YP_001253341.1), A2 (WP_012704905), B (WP_003404192.1), C1 (YP_398515.1), D (BAA75083.1) and (WP_003409842), F (YP_001390122.1) and G (CAA61228.1). In one embodiment, the invention excludes the use of the NTNHA / A molecule (NTNHA / A1) and the coding nucleic acids. BoNT

[0073] The different serotypes of botulinum neurotoxins are known in the art (A to G) and also Petition 870180151897, dated 11 / 14 / 2018, p. 61 / 137 39 / 74 There are many subtypes (A1, A2, A3, A4-A, A4-B). The methods described here can be used to purify native BoNT (produced by clostridial bacteria) or a recombinant protein. Recombinant BoNT can be produced in any other type of host, such as other prokaryotic cells, eukaryotic cells, tissues, or organisms.

[0074] Muted variants of BoNT (e.g., resulting from amino acid substitutions, insertions, or deletions) can also be isolated. In one embodiment, the variant has greater toxicity (e.g., having greater binding to cellular receptors). Such mutated variants may comprise a “modified receptor-binding domain” or “modified Hc.” A modified Hc, as the term is used herein, has one or more non-naturally occurring substitution mutations that enhance the binding of the C. botulinum neurotoxin molecule to which it is embedded to a C. botulinum neurotoxin receptor located on the surface of a target cell. Such a molecule is typically generated through genetic recombination technology. The modified Hc has a binding activity for the C. botulinum neurotoxin receptor that is stronger than its wild-type homolog.Examples of modified receptor-binding domains are described in US Patent Application 2015 / 166972, the contents of which are incorporated herein by reference. The invention is also useful for... Petition 870180151897, dated 11 / 14 / 2018, page 62 / 137 40 / 74 isolate any molecule that possesses or retains the biological activity of botulinum toxin, such as a fusion (or chimeric) protein, truncated protein, protein fragment, or a mutated variant of botulinum toxin, such as a protein that has one or more amino acids added, deleted, or substituted.

[0075] In one embodiment, the BoNT isolated by the methods described herein has toxic activity. The activity of BoNT can be determined by measuring the proteolytic activity on the appropriate substrate. Botulinum toxin types A and E cleave the SNAP-25 protein, botulinum toxin types B, D, F, and G cleave the vesicle-associated membrane protein (VAMP, called synaptobrevin). Botulinum toxin type C1 cleaves both SNAP25 and also the syntaxin protein. The assays that can be used to determine this activity are known in the art as described in WO 95 / 33850, the contents of which are incorporated herein by reference. Affinity fractions

[0076] NTNHA can be linked to an affinity fraction. The affinity fraction specifically binds to a binding target under the conditions of the methods described herein (e.g., from about pH 6 to about pH 8). A variety of affinity fractions are known in the art and available for use in the invention. An affinity fraction can be a Petition 870180151897, dated 11 / 14 / 2018, pp. 63 / 137 41 / 74 member of a specific binding pair, such as an epitope that is specifically recognized by an antibody. When an epitope is used as the affinity fraction, the antibody is used as the binding target. Many affinity fraction:antibody combinations like this are known in the art and commercially available. Examples include, without limitation, c-myc (Roth et al, (1991) J. Cell Biol. 115: 587596), myc (EQKLISEEDL (SEQ ID NO: 8); Evan GI, et al. (1985) Mol. Cell Biol. 5: 3610-3616; Munro S. and Pelham HRB, (1987) Cell 48:899-907; Borjigin J. and Nathans J., (1994) 269:1471514727; Smith DJ, (1997) BioTechniques 23:116-120) FLAG.RTM. (US Patents 4,703,004; 4,851,341 and 5,011,912), HA, derived from influenza hemagglutinin protein (Wilson IA, et al., (1984) Cell, 37:767; Field J. et al. Mol. Cell Biol. (1988) 8:2159-2165; Xu Y, et al. (2000) Mol Cell Biol. 20:2138-2146), IRS (RYIRS (SEQ ID NO: 9); Liang TC et al. (1996) 329:208-214; Luo W et al.Biophys. 329:215-220), AU1 and AU5 ((DTYRYI (SEQ ID NO: 10) and TDFLYK (SEQ ID NO: 11)); Lim PS et al. (1990) J. Infect. Dis. 162:1263-1269; Goldstein DJ et al. (1983; 1983; Koralnik IJ et al (1993) J. Virol 67:2360-2366), glu-glu (an epitope with 9 amino acids of polyoma virus medium T antigen (EEEEYMPME (SEQ ID NO: 12)); Grussenmeyer, T. et al. (1985) PNAS. USA 82:7952-7954; Rubinfeld. B. et al. (1991) Cell 65:1033-1042), KT3 (an 11 amino acid epitope of Petition 870180151897, of 14 / 11 / 2018, p. 64 / 137 42 / 74 SV40 large T antigen (KPPTPPPEPET (SEQ ID NO: 13)); MacArthur H. and Walter G. (1984) J, Virol. 52:483-491; Martin GA et al. (1990) 63:843-849; Di Paolo G et al. (1997) 272:5175-5182), T7 (an 11-amino acid leader peptide of the T7 main capsid protein (MASMTGGQQMG (SEQ ID NO: 14))), S-TAG, HSV (an 11-amino acid peptide of herpes simplex virus glycoprotein D (QPELAPEDPEDC (SEQ ID NO: 15))), VSV-G (an 11-amino acid epitope of the vesicular stomatitis virus carboxy terminal glycoprotein, (YTDIEMNRLGK (SEQ ID NO: 16)); Kreis T. (1986) EMBO J. 5:931941; Turner JR et al (1996) 271:7738-7744), Anti-Xpress (8-amino acid epitope, (DLYDDDK (SEQ ID NO: 17))) and VS (14-amino acid epitope of paramoxivirus SV5, (GKPIPNPLLGLDST (SEQ ID NO: 18))).

[0077] Another epitope commonly used as an affinity fraction is FLAG.RTM. This sequence typically consists of DYKDDDDK (SEQ ID NO: 19), but any combination of 3 to 6 aspartic or glutamic acid residues is also considered a FLAG.RTM sequence. The FLAG.RTM affinity marker has been effectively used in several expression systems for the purification of recombinant fusion proteins (Brizzard et al. (1994) BioTechniques 16:730-735; Lee et al. (1994) Nature 372:739-746; Xu et al. (1993) Development 117:1223-1237; Dent et al. (1995) Mol. Cell Biol. Petition 870180151897, dated 11 / 14 / 2018, pp. 65 / 137 43 / 74 15:4125-4135; Ritchie et al. (1999) BioChem Journal 338:30510.).

[0078] There are also many affinity fractions that are not epitope-based and these can also be used in the invention. GST (Glutathione-S-transferase) is an affinity fraction foreseen for use in the present invention (US Patents 5,654,176; 6,303,128 and 6,013,462). The polyhistidine affinity fraction is a non-natural consecutive sequence of histidine amino acid residues including any corresponding peptides described in US Patents 5,284,933 and 5,310,663. Typically, such sequences comprise four to ten histidine residues (SEQ ID NO: 20).

[0079] In one embodiment, the affinity fraction is glutathione-S-transferase (GST), C-myc marker, chitin-binding domain, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, histidine affinity marker (HAT), Poly-His, or maltose-binding protein (MBP). In one embodiment, the affinity fraction is not GST, C-myc marker, chitin-binding domain, SBP, cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, HAT, Poly-His, or MBP. In one embodiment, the affinity fraction is AviTag™, V5, Myc, T7, Petition 870180151897, dated 11 / 14 / 2018, pp. 66 / 137 44 / 74 FLAG, HSV, VSV-G, polyHis (typically His6(SEQ ID NO: 1)), biotin, or STREP (WSHPQFEK (SEQ ID NO: 21)). In one embodiment, the affinity fraction is not AviTag™, V5, Myc, T7, FLAG, HSV, VSV-G, polyHis, biotin, or STREP.

[0080] Linking pair members that interact with or are found naturally in the mammalian (human) body, such as antibodies that naturally bind to NTNHA or molecules recognized by transporters in the liver and / or kidneys, are excluded from the compositions described herein. Binding targets for affinity fractions

[0081] Binding targets are used to immobilize the NTNHA polypeptide by binding the affinity fraction. The binding target is typically specific for a given affinity fraction. Binding targets are matrix-linked in such a way that their binding affinity for the affinity fraction is preserved. For example, the binding target for an epitope marker is an antibody that specifically binds to the epitope marker. The binding target for GST is glutathione. The binding target for biotin is avidin or streptavidin. The binding target for STREP is Strep-tactin. The binding target for polyHis is divalent nickel and cobalt ions. The binding target for G protein is the Fc portion of IgG. The binding target for A protein is the Fc portion of immunoglobulin from various species. Petition 870180151897, dated 11 / 14 / 2018, pp. 67 / 137 45 / 74 Headquarters

[0082] Various inert substances normally used to immobilize a molecule by means of physical bonding can be used as the matrix in the invention. The matrix, otherwise referred to as a substrate, can be made of a wide variety of materials and can have a variety of shapes. The materials include, without limitation, metal, metal alloy, polymer, plastic, paper, glass, fabric, packaging material, biological material such as cells, tissues, hydrogels, proteins, peptides, nucleic acids and any combinations thereof. The shapes that the matrix can have include, without limitation, crude polymers (including polymer microbeads, magnetic microbeads and the like), filters, fibers, splints, mesh, tubes, hollow fibers, scaffolds, plates, channels and any combination thereof.Other examples of known substrate matrices in the art include, among others, nucleic acid scaffolds, protein scaffolds, lipid scaffolds, dendrimers, microparticles or microbeads, nanotubes, and microtiter plates. In one embodiment, the matrix components are in the form of a column.

[0083] In one embodiment, the NTNHA polypeptide is linked to the matrix by coupling an affinity moiety present in NTNHA to a binding target present on the matrix surface. Several affinity moieties and binding targets are Petition 870180151897, dated 11 / 14 / 2018, pp. 68 / 137 46 / 74 are available for use, examples of which are discussed herein. In one embodiment, the matrix is ​​coated with glutathione as the binding target (e.g., crude agarose polymer linked to glutathione). In another embodiment, the glutathione-coated matrix is ​​in the form of a column.

[0084] In one embodiment, the NTNHA polypeptide is conjugated directly to a matrix surface via a covalent or non-covalent interaction. This can occur via the N-terminus, the C-terminus, or internally within the molecule. It may also be useful to include a ligand in the NTNHA polypeptide in order to facilitate binding to the substrate.

[0085] Substrate conjugation can be performed using a variety of methods in the technique. Examples of covalent bonding include, but are not limited to, silane coupling (Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol. 134 (1976)) and the use of the NHS reaction or a conjugating agent. Non-covalent bonding can be based on ionic interactions, van der Waals interactions, dipole-dipole interactions, hydrogen bonds, electrostatic interactions, and / or shape recognition interactions. Without limitation, conjugation can include either a stable or unstable bond or a conjugating agent. Exemplary conjugations include, among others, covalent bonding, amide bonding, and additions to multiple bonds. Petition 870180151897, dated 11 / 14 / 2018, pp. 69 / 137 47 / 7 4 carbon-carbon, Huisgen cycloaddition of azide alkyne, Diels-Alder reaction, disulfite linkage, ester linkage, Michael additions, silane linkage, urethane, nucleophilic ring-opening reactions: epoxides, non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolar cycloaddition, temperature-sensitive linking or conjugating agent, radiation-sensitive (IR, near-IR, UV), pH-sensitive linking or conjugating agent, non-covalent linkages (e.g., ionic complex formation, hydrogen bonding, pi-pi interactions, cyclodextrin / inflexibly host-guest interaction) and the like. As used herein, the term conjugating agent means an organic moiety that connects two parts of a compound.The ligands typically comprise a direct linkage or an atom, such as oxygen or sulfur, a unit, such as NR1, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms, wherein one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, NH, C(O)N(R1)2, C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1 is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0086] A variety of conjugation chemistries are available to conjugate two molecules and can be used Petition 870180151897, dated 11 / 14 / 2018, pp. 70 / 137 48 / 74 to link the NTNHA polypeptide to a matrix. Exemplary coupling molecules and / or functional groups for conjugating at least one specific engineered microbe molecule to a substrate include, but are not limited to, a polyethylene glycol (PEG, NH2-PEGX-COOH which may have a PEG spacer arm of various sizes X, wherein 1 < X < 100, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K and the like), maleimide conjugating agent, PASylation, HESylation, bis(sulfosuccinimidyl) suberate conjugating agent, DNA conjugating agent, peptide conjugating agent, silane conjugating agent, polysaccharide conjugating agent, hydrolyzable conjugating agent and any combinations thereof.

[0087] The amount of NTNHA bound to the matrix can be determined and optimized by those skilled in the art. In one embodiment, the matrix has about 20 mg / mL of NTNHA polypeptide. In another embodiment, the matrix has about 5 mg / mL of polypeptide or about 2 mg / mL of polypeptide. Proteases

[0088] Any protease that cleaves BoNT can be used with the methods described herein. Such proteases include, without limitation, trypsin, pepsin, Lys-C endoproteinase, Lys-N endoproteinase, arginyl endopeptidase, plasmin, omptin, and a clostridial protease as described in EP2524963. In one embodiment, the protease is trypsin or Petition 870180151897, dated 11 / 14 / 2018, p. 71 / 137 49 / 74 endoproteinase Lys-C. In one embodiment, the protease is a protease that cleaves a non-native (i.e., exogenous) cleavage site of BoNT. In such clostridial toxins, the native protease cleavage site (also known as the activation site) is modified or replaced by a protease cleavage site that is not active for the clostridial toxin. Non-native proteases that may be employed include Enterokinase (DDDDKj(SEQ ID NO: 2)), Factor Xa (IEGRMSEQ ID NO: 3) / IDGRj(SEQ ID NO: 4)), TEV (Tobacco Engraving Virus) (ENLYFQjG (SEQ ID NO: 5)), Thrombin (LVPRjGS (SEQ ID NO: 6)), and PreScission (LEVLFQjGP (SEQ ID NO: 7)), (oj denotes the cleavage site). Nucleic acid vectors

[0089] Another aspect of the invention relates to a nucleic acid vector comprising the nucleic acid molecule encoding the NTNHA polypeptide described herein. The vector may be a vector solely for the propagation of a nucleic acid sequence in an organism or cell, or it may also be for the expression of the nucleic acid sequence as a polypeptide in the organism or cell.

[0090] In one embodiment, the vector is an expression vector. An expression vector such as this is referred to herein as an expression construct and comprises a nucleic acid molecule described herein operationally linked to the expression vector useful for expressing the nucleic acid molecule. Petition 870180151897, dated 11 / 14 / 2018, pp. 72 / 137 50 / 74 in a cell or cell-free extract. A wide variety of expression vectors can be employed to express a nucleic acid molecule encoding an NTNHA polypeptide described herein, including, without limitation, a viral expression vector (e.g., retrovirus, vaccinia virus, adeno-associated virus, herpesvirus, or bovine papillomavirus), a prokaryotic expression vector, a eukaryotic expression vector, such as, for example, a yeast expression vector, an insect expression vector, a mammalian expression vector, and a cell-free extract expression vector. In one embodiment, the expression vector is a baculovirus expression vector. Suitable expression vectors include, without limitation, the Okayama-Berg cDNA pcDV1 expression vector (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (Invitrogen) or baculovirus-derived vectors.Virus-derived expression vectors can be used for the distribution of the nucleic acids of the invention into a specific cell population. Numerous expression vectors for the production of affinity fraction fusions, such as those described herein, are available in the art. The selection, labeling, and use of an appropriate expression vector are routine procedures performed by those skilled in the art. Host cells Petition 870180151897, dated 11 / 14 / 2018, pp. 73 / 137 51 / 74

[0091] Another aspect of the invention relates to a cell in which one or more of the molecules described herein (e.g., the NTNHA polypeptide and / or the BoNT polypeptide) is propagated and / or expressed. Such a cell is referred to as a host cell. Host cells may be genetically modified to express the molecules described herein, such as by transfection with a vector encoding the proteins, and / or may express one or more of the molecules (e.g., BoNT) naturally. In one embodiment, the host cell comprises a nucleic acid encoding the NTNHA polypeptide (e.g., in the context of a vector). In another embodiment, the host cell expresses the nucleic acid (e.g., from an expression vector). In some embodiments, the cells used according to the present invention include prokaryotic cells and eukaryotic cells.Non-limiting examples of prokaryotic cells are Escherichia coli cells, Clostridium botulinum cells, Clostridium tetani cells, Clostridium berattii cells, Clostridium butyricum cells, or Clostridium perfringens cells. Non-limiting examples of eukaryotic cells are insect cells, yeast cells, amphibian cells, mammalian cells, and plant cells. Non-limiting examples of insect cells are Spodoptera frugiperda cells, Aedes albopictus cells, and Trichoplusia nii cells. Petition 870180151897, dated 11 / 14 / 2018, pp. 74 / 137 52 / 74 cells of Stigmene acrea, Bombyx mori cells, and Drosophila melanogaster cells. Non-limiting examples of yeast cells are Saccharomyces cerevisiae cells, Schizosaccharomyces pombe cells, Pichia pastoris cells, Hansenula polymorpha cells, Kluyveromyces lactis cells, and Yarrowia lipolitica cells.

[0092] Unless otherwise defined, scientific and technical terms used in conjunction with this patent application shall have the meanings that are commonly understood by those skilled in the art. Additionally, unless otherwise required by the context, singular terms shall include plurals and plural terms shall include singulars.

[0093] It should be understood that this invention is not limited to the particular methodology, protocols and reagents described herein and, as such, may vary. The terminology used herein is intended to describe particular embodiments only and should not limit the scope of the present invention, which is defined only by the claims.

[0094] Except in operational examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all cases by the term “about.” The term “about,” when used Petition 870180151897, dated 11 / 14 / 2018, pp. 75 / 137 53 / 74 to describe the present invention, together with the percentages, may mean ±1% or ±5% or ±10%.

[0095] In one aspect, the present invention relates to the compositions, methods and respective component(s) thereof described herein as essential to the invention, still open to the inclusion of non-specific elements, essential or not (“which comprises”). In some embodiments, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel feature(s) of the invention (“which essentially consists of”). This also applies to the steps in a described method, as well as to the compositions and components thereof. In other embodiments, the inventions, compositions, methods and respective components thereof described herein shall be exclusive of any element not considered an essential element for the component, composition or method (“which consists of”).

[0096] All patents, patent applications and publications identified are hereby expressly incorporated by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications, which are to be used in conjunction with the present invention. These publications are provided for description only prior to the filing date of this application. Petition 870180151897, dated 11 / 14 / 2018, pp. 76 / 137 54 / 74 patent. Nothing in relation to it should be construed as an admission that the inventors are not authorized to antedate such description by virtue of prior invention or for any other reason. All statements regarding the date or representation regarding the contents of these documents are based on information available to the applicants and do not constitute any admission regarding the accuracy of the dates or contents of these documents.

[0097] The invention is further illustrated by the following examples, which should not be considered limiting yet. EXAMPLES Example 1 A groundbreaking method for purifying and activating BoNTs.

[0098] Here, a novel method is proposed for purifying and activating unlabeled and natural forms of BoNTs through simple affinity purification steps. This method is based on a unique characteristic of BoNTs: these toxins naturally form a dimer complex with their chaperone protein, known as NTNHA. The biological purpose of this dimer is to protect the toxins from proteases and the harsh acidic environment in the gastrointestinal (GI) tract. The interactions between BoNTs and NTNHA are pH-dependent: they bind at pH <7 and dissociate from each other at pH >7.4. Thus, the introduction of an affinity marker into NTNHA can be used to isolate the natural forms of BoNTs. Petition 870180151897, dated 11 / 14 / 2018, pp. 77 / 137 55 / 74 BoNTs in solutions with pH < 7. The bound BoNTs can then be released simply by increasing the pH of the solution to pH > 7.4. In other words, instead of placing an affinity marker on the BoNTs, their bonding pair can be labeled. This allows the production of natural forms of BoNTs through the convenient affinity purification method.

[0099] In addition to purification, BoNTs need to be activated by limited proteolysis. Recombinant BoNTs are usually activated post-purification with an endoproteinase (such as trypsin). This method has several disadvantages: 1) there is a chance of non-specific cleavage by the endoproteinase, which compromises the activity and yield of the toxin; 2) the endoproteinase needs to be removed after the reaction is complete, requiring an additional separation step that compromises the yield and activity of the toxins.

[0100] The activation site in BoNTs is still exposed on the surface of the BoNT-NTNHA complex, while other susceptible BoNT sites are frequently produced in the complex. This provides an opportunity to treat the toxins with the endoproteinase while the toxin is still in the complex with NTNHA. This approach addresses both problems of previous methods: 1) NTNHA protects the toxins from non-specific cleavage by the endoproteinase; 2) the endoproteinase can be easily removed in a single step. Petition 870180151897, dated 11 / 14 / 2018, pp. 78 / 137 56 / 74 washing step along with all other non-toxin proteins that do not bind to NTNHA. Results

[0101] Each naturally occurring BoNT has its own naturally occurring NTNHA pair. BoNT / B and NTNHA / B were used as prototypes to establish the viability of this approach. Briefly, NTNHA / B was expressed as a fusion protein with the commonly used GST (Glutathione-S-transferase) marker. GST-NTNHA / B was purified, immobilized on crude Glutathione polymers, and subsequently equilibrated with toxin-binding buffer (pH = 6). This resin was then added to E. coli cell lysate containing recombinantly expressed BoNT / B and incubated for 1 hour at 4°C to allow complex formation under pH 6 conditions. Subsequently, the crude polymer-bound complex was washed with binding buffer to remove non-specific contaminants and unbound proteins. The BoNT / B-linked compound was either eluted from the crude polymers using a pH 8 elution buffer or subjected to trypsin treatment to be activated.

[0102] The principle and purification steps are schematically illustrated in FIGURE 1A and FIGURE 1B. The results indicated that BoNT / B can be efficiently purified from crude bacterial lysates using this method (FIGURE 2A), with high yield and purity of Petition 870180151897, dated 11 / 14 / 2018, pp. 79 / 137 57 / 74 final protein (FIGURE 2B). A fraction of the resin containing the NTNHA / B:BoNT / B complex was subjected to trypsin-mediated cleavage. The results, shown in FIGURE 3A and FIGURE 3B, indicate that BoNT can be efficiently activated in a few hours in the crude polymers in the complex and can be subsequently eluted from the crude polymers to produce the native and active toxin.

[0103] If NTNHA, which is specific to a serotype, can be used to purify chimeric toxins containing a segment of that toxin, particularly the receptor-binding domain, this has been explored. The receptor-binding domain mediates most interactions between NTNHA and BoNT. The results, shown in FIGURE 4, indicate the successful use of NTNHA / B to purify a hybrid toxin (BoNT / A1B) containing the receptor-binding domain of BoNT / B.

[0104] These experimental results serve as proof of concept for a method that can be used for the widespread purification of therapeutic toxins: BoNT / A (with NTNHA / A) and BoNT / B (with NTNHA / B), purifying other serotypes of BoNTs (with NTNHAs), purifying recombinant BoNTs containing mutations, purifying chimeric BoNTs (with NTNHAs that bind to the receptor-binding domain or specifically designed chimeric protein NTNHAs). The advantages of this method are 1) the ability to purify BoNTs with Petition 870180151897, dated 11 / 14 / 2018, pp. 80 / 137 58 / 74 N- and C-terminal naturals that are recombinantly expressed through convenient affinity purification, 2) mild buffer conditions (pH 6 to 8) minimize any potential damage to toxins, 3) toxins with pH-dependent liquefaction and highly pure elution yields conveniently reduce the need for further purification, 4) NTNHA protection reduces non-specific cleavage by protease activation during the activation step, and 5) protease activation prior to toxin elution eliminates the need to separately remove the protease. Materials and methods

[0105] Protein expression and purification. NTNHA / B was expressed in E. coli as a glutathione S-transferase fusion protein (GST-NTNHA / B) with the GST being fused to the N-terminal of the NTNHA / B protein; BoNT / B was expressed in E. coli with a C-terminal Hisg marker (SEQ ID NO: 1). Bacterial cultures (1 L) were grown at 37 degrees and protein expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) (250 μM) when the optical density of the culture at 600 nm (ODeoo) reached -0.6 AU. The cultures were then transferred to a shaker incubator at 20 degrees for overnight expression (~16 hours). The bacteria were collected by centrifugation at Petition 870180151897, dated 11 / 14 / 2018, p. 81 / 137 59 / 74 5,500 xg and the resulting precipitates were frozen until purification.

[0106] BoNT / B precipitates were thawed and solubilized in binding buffer (50 mM MES, 150 mM NaCl, pH 6) with 5 mL / gram of dry bacterial precipitate; NTNHA / B precipitates were thawed and solubilized in a different binding buffer (50 mM Tris, 150 mM NaCl, pH 8). 1 mM phenylmethylsulfonyl fluoride (PMSF) was added prior to lysis by sonication on ice (Branson Sonifier 250) for 15 min (3 x 5 min, 50% energy). The crude lysate was then cleared by centrifugation (30,000 x g, 15 min) and the supernatant was filtered using 0.45 µm syringe filters (Nalgene).

[0107] Purification of GST-NTNHA / B. 600 pL of crude Glutathione-Agarose Pierce polymers (50% flow paste; Thermo) equilibrated with binding buffer were added to ~20 mL of the GST-NTNHA / B supernatant and batch-bound for 1 hour at 4 degrees. The crude polymers were recovered by centrifugation (700 x g) and washed twice with 3 volumes of binding buffer resin bed (50 mM Tris, 150 mM NaCl, pH 8). The estimated concentration of purified GST-NTNHA / B was ~0.6 mg / mL (BCA assay and SDSPAGE analysis).

[0108] pH-dependent complexation; protease activation; and elution of purified BoNT. Crude polymers Petition 870180151897, dated 11 / 14 / 2018, p. 82 / 137 60 / 74 agarose-containing GST-NTNHA / B were added to ~5 mL of clarified BoNT / B from E. coli lysate for batch binding for 2 hours at 4 degrees in a conical rocking tube. The crude polymers were collected by (700 x g) and washed twice with 3X the volumes of the resin bed of the binding buffer (MES 50 mM, NaCl 150 mM, pH 6).

[0109] Trypsin or Lys-C endoproteinase (Sigma-Aldrich) was added at a molar ratio of 1:10 at pH 6 (in the crude polymers) to activate the NTNHA-bound toxin in a final volume of 500 μL. The reaction was advanced on a turntable at room temperature and monitored for 4 hours by sampling small aliquots for subsequent analysis. The resin was washed twice with binding buffer to remove proteases and unbound impurities. The purified and activated BoNT was eluted with two volumes of high pH buffer resin (50 mM Tris, 150 mM NaCl, pH 8).

[0110] SDS-PAGE and WB analysis. 10 pL of all samples (with or without the DTT reducing agent) were applied to 9% SDS-PAGE gel. After separation, the gel was stained with Coomassie stain or subjected to standard immunoblot analysis. A human monoclonal antibody was used to detect BoNT / B and a rabbit polyclonal antibody was used to detect the chimeric toxin BoNT / A1B. Example 2 Easy and direct isolation of recombinant BoNTs Petition 870180151897, dated 11 / 14 / 2018, pp. 83 / 137 61 / 74 crude bacterial lysates

[0111] The association between BoNT and NTNHA is facilitated by numerous pH sensors on both molecules that form specific surface recognition (Gu et al. 2012). This interconnected complex protects the active toxin from the harsh acidic environment through which it must pass to reach its cellular target.

[0112] The present Example confirms the feasibility of isolating a recombinant full-size BoNT (inactive BoNT / B, hereinafter referred to as BoNT / B{ry}) that is expressed in E. coli, as described herein. The complex pair that facilitates toxin isolation is a GST-labeled compatible serotype of its recombinant complex pair, NTNHA / B. The GST-labeled NTNHA-B molecule and BoNT / B{RY} were expressed separately in E. coli hosts, and protein production was achieved using standard autoinduction methods (Studier 2005). For the isolation of GST-NTNHA-B, a one-step batch purification with crude agarose-glutathione polymers was performed as described in the methods. Immobilized GST-NTNHA-B was stable for short- to medium-term storage at 4 degrees for approximately one week, although longer storage periods may possibly lead to spontaneous notching as previously reported (Sagane et al. 2002; Gu et al. 2012).This reagent was subsequently used for... Petition 870180151897, dated 11 / 14 / 2018, page 84 / 137 62 / 74 isolate the chimeric BoNT / Bry and BoNT / A1{ry}B1 in a simple workflow (FIGURE 6B), where crude agarose polymers were baited to remove recombinant toxins from crude lysates under favorable conditions (e.g., pH 6.0, 150 mM NaCl). SDS-PAGE analysis of the relevant fractions from the purification scheme is shown in FIGURE 6C. The regenerated GST-NTNHA / B after elution can be rapidly used in another purification cycle to isolate the most compatible toxins from fresh or alternative extracts. The eluted full-length toxin is selectively released from the complex by buffer exchange in the crude polymers and can be visualized by SDSPAGE or western blot (WB) analysis (FIGURE 7A). Furthermore, such mild conditions for isolating full-size (FL) toxins are more likely to preserve the protease's activity and functional roles in binding to its cellular targets.As the canonical neuronal receptor for BoNT / B, a labeled peptide derived from synaptotagmin has been shown to interact with the isolated full-length toxin in an in-vitro fluorescent anisotropy binding assay (FIGURE 7B). The complexed toxin is efficiently activated by exogenous proteases.

[0113] As dichain toxins (AB), BoNTs are expressed as a single polypeptide chain that undergoes activation to generate a functional molecule linked by a Petition 870180151897, dated 11 / 14 / 2018, page 85 / 137 63 / 74 disulfide bridge between the heavy and light chains. “Notching” by exogenous or endogenous proteases that cleave the polypeptide chain between two conserved cysteines that maintain a covalent link to LC and HC can improve potency and may be necessary for maximum potency (FIGURE 6A). The present Example documents that the addition of such proteases (e.g., specifically an exogenous protease) can be incorporated into the workflow of the purification protocol as described herein and, in some embodiments, can help maximize the recovery of active toxins. For example, the complexed GST-NTNHA / B:BoNT / B{ry} can be cleaved by catalytic amounts of trypsin or Lys-C endoproteinase under mild conditions at room temperature, and the notched toxin can be selectively released into a higher pH buffer.Figures 8A to 8C show activation durations of complexed single-chain BoNT / / B{ry} to release the ~50kDa protease domain (LC) and the ~100 kDa HC in samples containing dithiothreitol (DTT). Lower pH binding conditions and lower protease activity (Kasserra & Laidler 1969; Jekel et al. 1983) play a protective role in non-specific / excessive degradation of the toxin and / or NTNHA (FIGURE 8B). The purity and extent of toxin notching can be visualized both on SDS-PAGE gel and detected by WB analysis (FIGURE 8A and FIGURE 8C). Petition 870180151897, dated 11 / 14 / 2018, pp. 86 / 137 64 / 74 Chimeric recombinant toxins can be isolated using a common NTNHA serotype.

[0114] The present Example confirms that a chimeric recombinant botulinum neurotoxin can be purified using a purification protocol based on the complex described herein using the various targets that can serve as therapeutic backbones for future biological products. The receptor-binding domain of BoNTs mediates the most polar contacts with NTNHA (Gu et al. 2012). The present Example confirms that the recombinant botulinum neurotoxin can be purified by forming the complex with NTNHA. A chimeric recombinant protein (BoNT / A1{RY}B1) constructed from inactive BoNT / A LC, BoNT / UM HN, and BoNT / B HC was used as a validation of the concept. Using the same previous recombinant GSTNTNHA / B, complexation and enrichment of the chimeric toxin in the crude NTNHA polymers can be detected despite the low levels of toxin expression (FIGURE 9).The lysate cleared for the chimeric toxin contained degradation products and large impurities that often prevent efficient complexation with immobilized NTNHA. Therefore, the BoNT / A1{RY}B1 lysate was passed once and eluted from a Ni-NTA resin before exposure to NTNHA immobilized on agarose resin. Subsequent activation profiles were obtained accordingly. Petition 870180151897, dated 11 / 14 / 2018, p. 87 / 137 65 / 74 chimeric toxin may be similar to that of BoNT / B{ry}, possibly with varying efficiencies. Discussion

[0115] This example demonstrates the isolation of recombinantly expressed BoNTs using an NTNHA / B as a non-covalent parent complex pair. Both BoNT / B{ry} and NTNHA / B were separately overexpressed in E. coli hosts. NTNHA / B was expressed as a fusion protein with a GST marker attached to its N-terminus as an affinity moiety for solid agarose-glutathione resin. BoNT / B{ry} (and chimeric BoNT / A1{ry}B1) was expressed as the wild-type sequence, except for inactivation mutations and a C-terminal His6X marker. Bacterial lysis in a low pH buffer released these toxins into a lysate that was incubated with crude agarose polymers carrying GST-NTNHA / B.After the complex is formed, the solid media are washed extensively to remove impurities; after which the toxins can be either eluted by high pH buffer exchange or activated by means of an additional step in which an exogenous endoprotease is applied to the resin-bound complex.

[0116] As confirmed by the findings documented in this Example, the present description provides a solution for efficiently isolating therapeutic and active BoNTs. Petition 870180151897, dated 11 / 14 / 2018, pp. 88 / 137 66 / 7 4 from various sources under mild conditions. The improved methodology for isolating, activating, and eluting purified BoNTs could be immensely useful, such as in the large-scale production of therapeutic BoNTs. Potential benefits include the following: 1) efficient isolation of recombinant BoNTs from crude lysates under mild conditions, unlike current practices (Malizio et al. 2000; Donovan 2007); 2) high-purity and activated toxins can be produced using a single purification scheme, as it allows for extensive washing away of contaminants and avoids multiple chromatography steps; 3) immobilized NTNHA can adequately protect against non-specific toxin cleavage in the activation step, which can be rapidly incorporated into the purification protocol (as opposed to common post-purification activation).This can increase the final yields and homogeneity of the final activated toxin; and 4) the immobilized GST-NTNHA can be used for multiple sequential purifications, since it is regenerated at the end of each cycle with little loss; and 5) this methodology can be expanded to the isolation of chimeric therapeutic toxins with compatible receptor-binding domains. Materials and methods Protein expression and purification Petition 870180151897, dated 11 / 14 / 2018, pp. 89 / 137 67 / 74

[0117] NTNHA / B was expressed as a glutathione-S-transferase fusion protein (GST-NTNHA / B) in a pGEX vector; BoNT / B{ry} and BoNT / A1{ry}B1 were expressed with a C-terminal marker (His6x) in a pET32-a vector in E. coli (BL21DE3). Cell cultures (typically 300 mL) were grown in autoinduction media (Formedium™, UK) in 2 L flasks deflected at 37 °C with vigorous shaking (>250 RPM). When the cultures reached OD of ~0.6, the cell cultures were transferred to a shaker incubator at 20 °C for overnight expression (~16 hours). Cells were collected by centrifugation at 5,500 xg and the resulting precipitates were frozen at -20 °C until purification.

[0118] BoNT / B{ry} cell precipitates were thawed and solubilized in binding buffer (50 mM MES, 150 mM NaCl, pH 6) at 5 mL / gram of dry cell precipitate. GST-NTNHA / B cells were thawed and solubilized in TBS binding buffer (50 mM Tris, 150 mM NaCl, pH 8). Phenylmethylsulfonyl fluoride (PMSF) was added to the solubilized cells at a final concentration of 0.1 mM before lysis by sonication on ice (Branson Sonifier 250) for 15 min (3 x 5 min); 30% energy. The crude lysates were then cleared by centrifugation (30,000 x g, 15 min) and the supernatant was filtered using 0.45 µm syringe filters (Nalgene). Petition 870180151897, dated 11 / 14 / 2018, pp. 90 / 137 68 / 74 GST-NTNHA / B purification

[0119] 600 pL of crude Glutathione-Agarose Pierce polymers (50% flowable paste; Thermo) were equilibrated with binding buffer and added to ~20 mL of GST-NTNHA / B supernatant and batch-bound for 1 hour at 4 °C on a gentle balancing platform. The crude polymers were recovered by centrifugation (700 x g) and washed twice with 3X the volumes of the binding buffer resin bed (1X TBS). The estimated concentration of purified GST-NTNHA / B was typically ~0.5 mg / mL (BCA assay and SDS-PAGE analysis). Binding, activation and elution of purified BoNTs

[0120] Crude agarose polymers carrying GSTNTNHA / B were added to 10 to 25 mL of BoNT / B{ry} or clarified BoNT / A1{ry}B1 lysates (in MES, pH 6) buffer and batch-bound for 2 hours at 4°C in a 50 mL conical tube on a rocking platform. The crude polymers were collected by (700 x g) and washed twice with 3X volumes of binding buffer resin bed (MES, pH 6). If no activation is desired, the purified bound toxins can be eluted at this stage as described below.

[0121] Trypsin or Lys-C endoproteinase (SigmaAldrich) was added at a molar ratio of 1:10 endoproteinase:GST-NTNHA / B at pH 6 (in crude polymers) Petition 870180151897, dated 11 / 14 / 2018, pp. 91 / 137 69 / 74 to activate the bound toxin in a final volume of 500 to 1,000 µL. The reaction continued on a tumbling platform at room temperature and was monitored (either for 2 to 4 hours as in FIGURE 8A and FIGURE 8C; or overnight at 4 °C as in FIGURE 8B) by sampling small aliquots for subsequent analysis. The resin was washed twice with binding buffer to remove proteases and impurities. The purified and activated BoNT / B{RY} was eluted in two-volume fractions of high pH buffer resin (TBS: 50 mM Tris, 150 mM NaCl, pH 8). SDS-PAGE and WB analysis

[0122] 10 pL of all samples (with or without the reducing agents DTT or βME) were applied to 8 to 12% SDSPAGE gel. After separation, the gels were stained with Coomassie stain or subjected to the standard Western blotting procedure. A rabbit monoclonal antibody (1:5,000) was used to detect BoNT / B{ry} and a rabbit polyclonal antibody (1:2,000) created against BoNT / A was used to detect BoNT / A1{ry}B1. Fluorescence anisotropy

[0123] The peptide derived (AA 33-53) from human synaptotagmin 1 (Syt 1) was synthesized with an N-terminal FITC tag (GenScript, Piscataway NJ) and used as a receptor in the 50–100 nM binding experiment. The eluted full-length toxins were concentrated in units of Petition 870180151897, dated 11 / 14 / 2018, pp. 92-137 70 / 74 filtration Vivaspin 6 (10K MWCO, GE). Binding experiments (50 µL) were measured on a 96-well black plate (Corning) using a filter-based plate reader (485 / 520 nm excitation / emission). BoNT / aea and BoNT / B Hc were separately expressed and purified and served as the negative and positive controls, respectively. References Blasi, J. et al., 1993. Botulinum neurotoxin A selectively eliminates the synaptic protein SNAP-25. Nature, 365(6442), pp. 160 to 163. Bonventre, PF & Kempe, LL, 1959. Physiology of toxin production by Clostridium botulinum types A and B. III. Effect of pH and temperature during incubation on growth, autolysis, and toxin production. Applied Microbiology, 7, pp. 374-377. Borden Lacy, D. et al., 1998. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nature structural & molecular biology, 5(10), pp. 898-902. DasGupta, B.R. & Boroff, D.A., 1967. Chromatographic isolation of hemagglutinin-free neurotoxin from crystalline toxin of Clostridium botulinum type A. Biochimica et biophysica acta, 147(3), pp.603 a 605. Dong, M. et al., 2006. SV2 is the protein receptor for botulinum neurotoxin A. Science, 312(5773), pp.592 a 596. Petição 870180151897, de 14 / 11 / 2018, pág. 93 / 137 71 / 74 Donovan, S., 2007. Botulinum toxin production method. US Patent. Available a: https: / / www.google.com / patents / US7189541 [Acessado em 10 de março de 2017]. Duff, J.T., Wright, G.G., et al., 1957. Studies on immunity to toxins of Clostridium botulinum. I. A simplified procedure for isolation of type A toxin. Journal of bacteriology, 73(1), pp.42 a 47. Duff, J.T., Klerer, J., et al., 1957. Studies on immunity to toxins of Clostridium botulinum. II. Production and purification of type B toxin for toxoid. Journal of bacteriology, 73(5), pp.597 a 601. Finzi, E. & Rosenthal, N.E., 2014. Treatment of depression with onabotulinumtoxinA: a randomized, doubleblind, placebo controlled trial. Journal of psychiatric research, 52, pp.1 a 6. Gu, S. et al., 2012. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science, 335(6071), pp.977 a 981. Hexsel, C. et al., 2011. Botulinum toxin type A for aging face and aesthetic uses. Dermatologic therapy, 24(1), pp.54 a 61. Jackson, J.L., Kuriyama, A. & Hayashino, Y., 2012. Botulinum toxin A for prophylactic treatment of migraine and tension headaches in adults: a meta-analysis. JAMA: the Petição 870180151897, de 14 / 11 / 2018, pág. 94 / 137 72 / 74 journal of the American Medical Association, 307(16), pp.1.736 a 1.745. Jankovic, J. & Brin, M.F., 1991. Therapeutic uses of botulinum toxin. The New England journal of medicine, 324(17), pp.1.186 a 1.194. Jekel, P.A., Weijer, W.J. & Beintema, J.J., 1983. Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Analytical biochemistry, 134(2), pp.347 a 354. Jiang, Y.-H., Liao, C.-H. & Kuo, H.-C., 2015. Current and potential urological applications of botulinum toxin A. Nature reviews. Urology, 12(9), pp.519 a 533. Kasserra, H.P. & Laidler, K.J., 1969. pH Effects in trypsin catalysis. Canadian journal of chemistry, 47(21), pp.4.021 a 4.029. Lee, K. et al., 2014. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science, 344(6190), pp.1.405 a 1.410. Malizio, C.J., Goodnough, M.C. & Johnson, E.A., 2000. Purification of Clostridium botulinum type A neurotoxin. Methods in molecular biology , 145, pp.27 a 39. Masuyer, G. et al., 2014. Engineered botulinum neurotoxins as new therapeutics. Annual review of pharmacology and toxicology, 54, pp.27 a 51. Petição 870180151897, de 14 / 11 / 2018, pág. 95 / 137 73 / 74 Montal, M., 2010. Botulinum neurotoxin: a marvel of protein design. Annual review of biochemistry, 79, pp.591 a 617. Pickett, A., 2014. Botulinum Toxin as a Clinical Product: Manufacture and Pharmacology. In Clinical Applications of Botulinum Neurotoxin. Current Topics in Neurotoxicity. Springer New York, pp. 7 a 49. Pickett, A. & Perrow, K., 2009. Composition and Molecular Size of Clostridium botulinum Type A ToxinHemagglutinin Complex. The protein journal, 28(5), pp.248 a 249. Rossetto, O., Pirazzini, M. & Montecucco, C., 2014. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nature reviews. Microbiology, 12(8), pp.535 a 549. Sagane, Y. et al., 2002. Spontaneous Nicking in the Nontoxic-Nonhemagglutinin Component of the Clostridium botulinum Toxin Complex. Biochemical and biophysical research communications, 292(2), pp.434 a 440. Schantz, E.J. & Johnson, E.A., 1992. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiological reviews, 56(1), pp.80 a 99. Sifferlin, A., 2017. Botox: The Drug That’s Treating Everything. Time. Available at: Petição 870180151897, de 14 / 11 / 2018, pág. 96 / 137 74 / 74 http: / / time.com / 4623409 / botox-drug-treating-everything / [Acessado em 10 demarço de 2017]. Snipe, P.T. & Sommer, H., 1928. Studies on botulinus toxin 3. Acid precipitation of botulinus toxin. The Journal of infectious diseases, 43(2), pp.152 a 160. Studier, F.W., 2005. Protein production by autoinduction in high-density shaking cultures. Protein expression and purification, 41(1), pp.207 a 234. Truong, D.D. & Jost, W.H., 2006. Botulinum toxin: clinical use. Parkinsonism & related disorders, 12(6), pp.331 a 355. Tse, C.K. et al., 1982. Preparation and characterisation of homogeneous neurotoxin type A from Clostridium botulinum. Its inhibitory action on neuronal release of acetylcholine in the absence and presence of betabungarotoxin. European journal of biochemistry / FEBS, 122(3), pp.493 a 500. Visco, A.G. et al., 2012. Anticholinergic therapy vs. onabotulinumtoxina for urgency urinary incontinence. The New England journal of medicine, 367(19), pp.1.803 a 1.813. Petição 870180151897, de 14 / 11 / 2018, pág. 97 / 137

Claims

1 / 9 CLAIMS 1. Method for purifying botulinum neurotoxin (BoNT) or a polypeptide comprising a receptor-binding domain (Hc polypeptide) of BoNT, characterized in that it comprises the steps of: a) forming an NTNHA-BoNT complex or NTNHA-Hc polypeptide complex by placing BoNT or the BoNT polypeptide (Hc) in contact with a molecule comprising a non-toxic non-hemagglutinin polypeptide (NTNHA) covalently linked to a heterologous affinity moiety; wherein the BoNT or the BoNT polypeptide (Hc) is in solution, and the molecule is bound to a matrix, wherein the solution is placed in contact with the matrix to thus place the BoNT or the BoNT polypeptide (Hc) in contact with the NTNHA.

2. Method according to claim 1, characterized in that it further comprises: b) washing the matrix to thereby remove unbound materials; and c) eluting the BoNT or the BoNT polypeptide (Hc) from the matrix by contacting the matrix with an aqueous solution that dissociates the BoNT or the BoNT polypeptide (Hc) from the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex; or b) washing the matrix to thereby remove unbound materials; Petition 870260050700, dated 05 / 27 / 2026, p.10 / 28 2 / 9 c) Contact the matrix with a protease under conditions that preserve the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex and are appropriate for cleaving the BoNT or the BoNT polypeptide (Hc) into the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex; d) Wash the matrix to remove the protease and unbound materials; ee) Elute the cleaved BoNT or the cleaved BoNT polypeptide (Hc) from the matrix by contacting the matrix with an aqueous solution that dissociates the cleaved BoNT or the cleaved BoNT polypeptide (Hc) from the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex.

3. A method according to any one of claims 1 or 2, characterized in that the matrix is ​​linked to an affinity fraction binding target, and the molecule is non-covalently linked to the matrix through affinity fraction-binding target interactions.

4. A method according to any one of claims 2 or 3, characterized in that the aqueous solution has a pH > 7.

5.

5. Method, according to any one of claims 1 to 4, characterized in that: i) the solution comprising BoNT or BoNT polypeptide (Hc) is a clean cell extract from cells expressing BoNT or BoNT polypeptide (Hc); or Petition 870260050700, dated 05 / 27 / 2026, page 11 / 28 3 / 9 ii) the solution comprising BoNT or BoNT polypeptide (Hc) is a clean cell extract from cells expressing BoNT or BoNT polypeptide (Hc) and the clean cell extract further comprises 1 mM phenylmethylsulfonyl fluoride (PMSF).

6. Method, according to any one of claims 1 to 5, characterized in that the appropriate conditions for binding comprise bringing BoNT or the BoNT polypeptide (Hc) into contact in the context of a binding buffer having a physiological ionic strength and a pH < 7.

5.

7. A method according to any one of claims 1 to 6, characterized in that the washing is done with a washing buffer that is of physiological ionic strength with a pH < 7.

5.

8. Method, according to any one of claims 6 or 7, characterized in that: i) the binding buffer and / or washing buffer is between 100 and 200 mM KCl or NaCl; ii) the binding buffer and / or washing buffer has a pH of 6; iii) the binding buffer and / or washing buffer comprises 50 mM MES, 150 mM NaCl, pH 6; iv) the aqueous solution is an elution buffer of 50 mM Tris, 150 mM NaCl; and / or Petition 870260050700, dated 05 / 27 / 2026, page 12 / 28 4 / 9 v) the aqueous solution is an elution buffer of pH 8.

9. A method according to any one of claims 3 to 8, characterized in that: i) the affinity fraction is selected from the group consisting of glutathione-S-transferase (GST), C-myc marker, chitin-binding domain, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, histidine affinity marker (HAT), Poly-His and maltose-binding protein (MBP); and / or ii) the affinity fraction is GST and the binding target is glutathione.

10. Method, according to any one of claims 2 to 9, characterized in that: i) the protease is trypsin or Lys-C endoproteinase; ii) the protease is added at a molar ratio of 1:2 to 1:1,000 to NTNHA; iii) the protease is placed in contact with the matrix at room temperature; and / or iv) the protease is placed in contact with the matrix for 10 minutes to 18 hours.

11. Method according to claim 1, characterized in that: the molecule is bound to a glutathione-coated matrix and the affinity fraction is glutathione-S transferase; and the (a) contact step comprises placing a clean cell extract comprising BoNT or BoNT polypeptide (Hc) in contact with the matrix in a binding buffer at a pH of 6 to thereby form the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex; and further wherein the method comprises the steps of (b) washing the matrix with a washing buffer at a pH of 6 to thereby remove unbound materials; (ec) elute the BoNT or the BoNT polypeptide (Hc) from the matrix by contacting the matrix with an elution buffer that has a pH > 7.5 to dissociate the BoNT or the BoNT polypeptide (Hc) from the NTNHA-BoNT complex or the NTNHA-polypeptide Hc complex.

12. Method according to claim 1, characterized in that it comprises: the molecule is linked to a glutathione-coated matrix and the affinity fraction is glutathione-Stransferase; and the (a) contact step comprises placing a clean cell extract comprising BoNT or BoNT polypeptide (Hc) in contact with the matrix in a binding buffer at a pH of 6 to thereby form an NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex; and further wherein the method comprises the steps of: (b) washing the matrix with a washing buffer at a pH of 6 to thereby remove unbound materials; Petition 870260050700, dated 27 / 05 / 2026, p.14 / 28 6 / 9 c) place the matrix in contact with a protease in a buffer with a pH of 6 to cleave the BoNT or the BoNT polypeptide (Hc) into the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex; d) wash the matrix with a washing buffer with a pH of 6 to remove the protease and unbound materials; ee) elute the BoNT or the BoNT polypeptide (Hc) from the matrix by placing the matrix in contact with an elution buffer that has a pH > 7.5 to dissociate the BoNT or the BoNT polypeptide (Hc) from the NTNHA-BoNT complex or the NTNHA-Hc polypeptide complex.

13. Method, according to any one of claims 11 or 12, characterized in that: i) the binding buffer and / or the washing buffer comprises 50 mM MES, 150 mM NaCl; ii) the binding buffer and / or washing buffer comprises 50 mM MES, 150 mM NaCl and the binding buffer further comprises 1 mM phenylmethylsulfonyl fluoride (PMSF); iii) the elution buffer comprises 50 mM Tris, 150 mM NaCl, and has a pH of 8; iv) the glutathione-coated matrix is ​​glutathione-linked agarose crude polymer (beads) or the glutathione-coated matrix is ​​a column; Petition 870260050700, dated 05 / 27 / 2026, p. 15 / 28 7 / 9 v) the glutathione-coated matrix has 5 mg / mL of bound NTNHA; and / or vi) the protease is trypsin or Lys-C endoproteinase.

14. Method according to claim 1, characterized in that the Hc polypeptide is a botulinum neurotoxin (BoNT) polypeptide or the Hc polypeptide is a chimeric botulinum neurotoxin (BoNT) polypeptide.

15. Method, according to any one of claims 1 to 14, characterized in that the BoNT or the Hc polypeptide comprises a modified receptor-binding domain of the botulinum clostridial serotype B (BHc).

16. Use of a molecule characterized in that the use is in a method for purifying a botulinum neurotoxin (BoNT) polypeptide or a polypeptide comprising a receptor-binding domain (Hc polypeptide) of BoNT, as defined in claim 1, wherein the molecule is defined in that it comprises a non-toxic non-hemagglutinin polypeptide (NTNHA) covalently linked to a heterologous affinity moiety for use in the isolation and purification of a botulinum neurotoxin (BoNT) protein, or a polypeptide comprising a receptor-binding domain (Hc polypeptide) of BoNT, from a solution, wherein: Petition 870260050700, dated 05 / 27 / 2026, page 16 / 28 8 / 9 the molecule is further linked to a binding target through the affinity moiety; and the NTNHA polypeptide is in a complex with a compatible botulinum neurotoxin (BoNT) or a polypeptide comprising a receptor-binding domain (Hc polypeptide) thereof.

17. Use according to claim 16, characterized in that the complex remains intact in solution at pH 6 and disintegrates in solution at pH 8.

18. Use according to claim 16, characterized in that the compatible BoNT, or a polypeptide comprising a receptor-binding domain (Hc polypeptide) of BoNT, can be activated after exposure to a protease, while remaining in complex with the NTNHA polypeptide.

19. Use according to claim 16, characterized in that NTNHA and the affinity fraction are expressed as a fusion protein.

20. Use according to any one of claims 16 to 19, characterized in that: i) the affinity fraction is located at a selected position of the group consisting of the N-terminal end of the NTNHA amino acid sequence, the C-terminal end of the NTNHA amino acid sequence and internally to the NTNHA amino acid sequence; Petition 870260050700, dated 05 / 27 / 2026, p. 17 / 28 9 / 9 ii) the affinity fraction effectively binds to a binding target under pH 6 to pH 8 conditions; and / or iii) the affinity fraction is selected from the group consisting of glutathione-S-transferase (GST), C-myc Tag, chitin-binding domain, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S-tag, Strep-tag II, FLA, protein A, protein G, histidine affinity marker (HAT), poly-His and maltose-binding protein (MBP).

21. Use, according to any one of claims 16 to 20, characterized in that: i) the NTNHA is of serotype B, A, C1, D, E, F or G; and / or ii) the NTNHA is of serotype B.

22. Use, according to any one of claims 16 to 21, characterized in that the binding target is stably bound to the matrix. Petition 870260050700, dated 05 / 27 / 2026, p. 18 / 28

Images