HK1169171B - Means and methods for the determination of the amount of neurotoxin polypeptide and of its catalytic and proteolytic activities - Google Patents

Description

Means and methods for determining the amount of neurotoxin polypeptide and its catalytic and proteolytic activities

Technical Field

The present invention is in the field of tools for ensuring polypeptide production and quality control. In particular, the present invention relates to a method for determining the amount of processed (active) neurotoxin polypeptide in a solution comprising processed neurotoxin polypeptide and partially processed or unprocessed neurotoxin polypeptide. The invention also relates to a device for determining said amount and to a kit suitable for carrying out the method of the invention.

Background

Clostridium botulinum (Clostridium botulinum) and Clostridium tetani (Clostridium tetani) produce potent neurotoxins, namely botulinum toxin (BoNTs) and tetanus toxin (TeNT), respectively. These Clostridial Neurotoxins (CNTs) specifically bind to neuronal cells and disrupt neurotransmitter release. Each toxin is synthesized as an inactive, unprocessed, single-chain protein of approximately 150 kDa. Post-translational processing includes disulfide bond formation, and limited proteolysis (cleavage) by bacterial proteases. The active neurotoxin consists of two chains connected by disulfide bonds, an approximately 50kDa N-terminal light chain and an approximately 100kDa heavy chain. CNTs consist structurally and functionally of 3 domains, a light chain with catalytic function, a heavy chain comprising a translocation domain (half of the N-terminus) and a receptor binding domain (half of the C-terminus), see Krieglstein1990, Eur J Biochem 188, 39; krieglstein 1991, Eur J Biochem 202, 41; krieglstein 1994, J Protein Chem 13, 49. Botulinum toxin is synthesized as a molecular complex comprising a 150kDa neurotoxin protein and related non-toxic proteins. The size of the complex varies according to the strain of clostridium and the unique neurotoxin serotype, varying between 300kDa, greater than 500kDa and 900 kDa. The non-toxic proteins in these complexes have the effect of stabilizing the neurotoxin and protecting it from degradation, see Silberstein 2004, Pain Practice 4, S19-S26.

Clostridium botulinum secretes 7 antigenically distinct botulinum toxin (BoNT) serotypes, designated serotypes a through G. All these serotypes, together with the associated tetanus neurotoxin (TeNT) secreted by clostridium tetani, are Zn2+ endoproteases that block synaptic exocytosis by cleaving SNARE proteins, see Couesnon, 2006, Microbiology, 152, 759. CNTs cause flaccid muscle paralysis, seen in botulinum poisoning and tetanus, see Fischer 2007, PNAS 104, 10447.

Despite its toxic effects, botulinum toxin complexes have been used as therapeutics for a number of diseases. Botulinum toxin serotype a was approved for human use in the united states in 1989 for the treatment of strabismus, blepharospasm, and other diseases. It is commercially available as a botulinum toxin protein A preparation, for example under the trade name BOTOX (Allergan corporation) or DYSPORT (Ipsen corporation). An improved, uncomplexed botulinum toxin A formulation is commercially available under the trade name XEOMIN (Merzpharmaceuticals GmbH). For therapeutic use, the formulation is injected directly into the muscle in need of treatment. At physiological pH, the toxin is released from the protein complex and produces the desired pharmacological effect. The effects of botulinum toxin are only transient, which is why repeated administration of botulinum toxin may be required to maintain a therapeutic effect.

Clostridial neurotoxins attenuate the strength of voluntary muscles and are effective therapies for strabismus, focal dystonia, including cervical dystonia, and benign idiopathic blepharospasm. They have also been shown to alleviate hemifacial spasm and focal spasticity, and in addition, to be effective in other broad indications such as gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle reduction repair, see Jost 2007, Drugs 67, 669.

In the production of clostridial neurotoxins, qualitative and quantitative determination of active neurotoxin polypeptides and quality control thereof are of great importance. Currently available neurotoxin preparations comprise, in addition to the desired active (processed or mature) neurotoxin, also proteolytically unprocessed precursors and/or partially processed neurotoxin polypeptides. The proteolytically unprocessed precursor or partially processed neurotoxin polypeptide and the mature (active, processed) neurotoxin polypeptide differ in sequence by only a few amino acids. Therefore, it is difficult to quantitatively distinguish them according to their chemical and physical properties. On the other hand, in such preparations, the fraction of proteolytically unprocessed precursor and/or partially processed neurotoxin polypeptide in the total protein may still be significant. This depends in part on the biological system used for production and is the result of biosynthesis and reaction conditions in the fermentation process. Thus, the amount of mature, biologically active neurotoxin polypeptide required in a neurotoxin formulation is predefined and currently very difficult to determine.

There is an urgent need for tools and methods for reliable qualitative and quantitative detection systems for mature (active) neurotoxin polypeptides, which are not currently available.

The technical problem underlying the present invention is therefore to be seen as providing a tool and a method that meet the above-mentioned needs. This technical problem is solved by the embodiments described in the claims and in the following.

Disclosure of Invention

The present invention relates to a method for determining the amount of processed (active) neurotoxin polypeptide in a solution comprising processed neurotoxin polypeptide and partially processed and/or unprocessed neurotoxin polypeptide comprising the steps of:

a) contacting a first portion of the above solution with a first capture antibody that specifically binds to the light chain of a mature neurotoxin polypeptide, partially processed and unprocessed neurotoxin polypeptide under conditions that allow binding of the antibody to the mature neurotoxin, partially processed and unprocessed neurotoxin polypeptide, thereby forming a first antibody complex,

b) contacting a first antibody complex with a detection antibody, thereby forming a first detection complex, wherein the detection antibody specifically binds to the heavy chain of the mature neurotoxin, partially processed and unprocessed neurotoxin polypeptide in the antibody complex formed in step a),

c) contacting a second portion of the solution with a second capture antibody that specifically binds to the linker of the partially processed and unprocessed neurotoxin polypeptide under conditions that allow binding of the antibody to the partially processed and unprocessed neurotoxin polypeptide, thereby forming a second antibody complex,

d) contacting the second antibody complex with a detection antibody, thereby forming a second detection complex,

e) determining the amount of the first and second detection complexes formed in steps b) and d).

f) Calculating the amount of mature neurotoxin polypeptide based on the amount of first and second detection complexes determined in step e).

The above-described method may generally comprise additional steps, including a step of formulating a solution or a step relating to further evaluation of the results obtained in step f). Furthermore, steps a) and b) and steps c) and d) can be carried out simultaneously or successively. In the latter case, steps a) and b) may be carried out before or after steps c) and d). In addition, the determination mentioned in step e) can in that case be carried out after both series of steps have been completed, or the determination in step e) can be carried out after steps a) and b) in respect of the first detection complex, while the determination with respect to the second detection complex is carried out after steps c) and d). The method may be partially or completely performed by means of automated techniques. The incubation and measurement steps may be performed by, for example, a robot. Data analysis and interpretation may be performed by computer-implemented algorithms.

The term "neurotoxin polypeptide" as used in the present invention refers to the 7 different serotypes of botulinum neurotoxin (i.e. BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G), and tetanus neurotoxin (TeNT) (see Table 1), as well as variants thereof.

TABLE 1 botulinum neurotoxin and tetanus neurotoxin

The neurotoxins mentioned herein comprise in principle an N-terminal light chain and a C-terminal heavy chain. The neurotoxin is produced as a single chain precursor molecule, referred to herein as a "raw neurotoxin polypeptide". In the unprocessed neurotoxin, the N-terminal light chain and the C-terminal heavy chain sequences are separated by at least one proteolytic cleavage site. These neurotoxins may comprise a linker sequence located between the light chain and heavy chain sequences, wherein the light chain is located N-terminal from the first cleavage site and the heavy chain is located C-terminal from the second cleavage site. In one aspect of the invention, the linker has the amino acid sequence of SEQ ID NOs: 1 to 16. During processing of the neurotoxin, the linker sequence will be cleaved off. These neurotoxins contain two proteolytic cleavage sites, one at the N-terminus and the other at the C-terminus of the linker sequence. During the processing of such neurotoxins, it is possible to generate intermediates which cleave at only one of the cleavage sites, i.e.the linker sequence is not cleaved off but remains on the N-terminal light chain or the C-terminal heavy chain. This intermediate product is referred to herein as a "partially processed neurotoxin polypeptide". Other neurotoxins may contain only one cleavage site. For these neurotoxins, it is understood that the linker sequence cannot be cleaved off. However, unprocessed neurotoxins can be immunologically recognized through intact proteolytic cleavage sites and flanking sequences. These flanking sequences and cleavage sites are also considered a linker in the present invention. Thus, the term "linker" as used herein and above refers to a sequence between the light and heavy chain sequences for neurotoxin polypeptides having two cleavage sites or a cleavage site and flanking sequences for neurotoxin polypeptides having only one cleavage site. The result of the processing is a "processed neurotoxin polypeptide". The processed neurotoxin polypeptide exhibits the biological properties characteristic of a neurotoxin, namely (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of a protein involved in synaptic vesicle membrane fusion. Thus, a processed neurotoxin polypeptide is sometimes referred to herein as an active or mature neurotoxin polypeptide. The biological activity of the neurotoxin polypeptide, in one aspect, results from all of the above-described biological properties. In vivo assays to assess biological activity include mouse LD50 assays as well as ex vivo mouse hemidiaphragm assays as described by Pearce et aL. and Dressier et. (Pearce 1994, Toxicol Appl Pharmacol 128: 69-77 and Dressier 2005, Mov Disord 20: 1617-1619). Biological activity is usually expressed as Mouse Units (MU). As used herein, 1MU is the amount of neurotoxic component that will kill 50% of the mice in a defined population of mice after intraperitoneal injection, i.e., mouse i.p. ld50.

In one aspect of the methods of the invention, the neurotoxin polypeptide is selected from the group consisting of: a) has the sequence shown in SEQ ID NOs: 17 to 24, and b) a neurotoxin polypeptide having an amino acid sequence identical to that shown in any one of SEQ ID NOs: 17 to 24, or a neurotoxin polypeptide having an amino acid sequence which is at least 40% identical to the amino acid sequence of the neurotoxin polypeptide. The above amino acid sequence shows unprocessed neurotoxin polypeptide. The sequence of the corresponding partially processed or processed neurotoxin polypeptide can be deduced from the above sequence by the information of the cleavage sites provided in table 3 below. In another aspect of the invention, the neurotoxin polypeptide has an amino acid sequence identical to SEQ ID NOs: 17 to 24, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical. As used herein, "identical"/"identity" refers to the sequence identity of amino acid sequences, where the sequences are aligned to give the highest level of match. This can be achieved by using published techniques or methods written in computer programs such as BLASTP, BLASTN, FASTA (Altschul 1990, J Mol Biol 215, 403) and the like. In one aspect, the percent identity value is calculated over the entire amino acid sequence. The skilled person can obtain a series of programs based on various algorithms to align different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. Sequence alignments can be performed using the PileUp program (1987, J Mol Evolution 25, 351; Higgins 1989 CABIOS 5, 151) or the modules Gap and BestFit program in the GCG software package (Genetics computer group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch 1970, J Mol Biol 48; 443; Smith and Waterman 1981, Adv Appl Math 2, 482). In one aspect of the invention, the above sequence identity values, expressed as percentages (%), are determined over the entire sequence area using the GAP program, using the following settings: gap Weight (Gap Weight): 50. length Weight (Length Weight): average Match (Average Match): 10.000, Average Mismatch (Average Mismatch): 0.000, these parameters should always be used as standard settings for sequence alignment unless otherwise indicated. It is to be understood that in one aspect of the invention, the above variants should retain at least one biological property of the neurotoxin, and in one aspect, all of the biological properties of the neurotoxin polypeptide described herein. In another aspect, the variants may be neurotoxins having improved or altered biological properties, e.g., they may contain cleavage sites that are improved in recognition by enzymes, or may be improved in receptor binding or any other property mentioned above. It is envisaged that the concept of the present invention relies on the presence of two or more cleavage sites between the light and heavy chains of the neurotoxin polypeptide, and the nature of the cleavage sites and the particular amino acid sequence between them is not critical, as long as it is specific for the partially processed or unprocessed neurotoxin polypeptide. Thus, another aspect is the replacement of protease recognition sites and linker peptides between the heavy and light chains of the neurotoxin polypeptide.

In another aspect, a neurotoxin polypeptide according to the methods of the invention can be a chimeric molecule. The chimeric molecule, in one aspect, can be a single domain substituted. Thus, in another aspect, a portion of the neurotoxin heavy chain is replaced with a portion of the FC domain of the antibody.

The term "amount" as used in the methods of the invention encompasses the absolute amount of a polypeptide, the relative amount or concentration of said polypeptide, as well as any value or parameter related thereto or derivable therefrom.

The term "solution" as used herein refers to any solvent system comprising the mature neurotoxin polypeptide as well as partially processed and/or unprocessed neurotoxin polypeptide precursors thereof. In addition, the solvent system also comprises a solvent. In various aspects of the invention, solvents encompass water, aqueous buffer systems, organic solvents, and ionic liquids. In one aspect of the invention, it is an aqueous solvent system. In addition, the solvent system, in addition to the mature neurotoxin polypeptide and the partially processed or unprocessed precursor neurotoxin polypeptide and the solvent, may comprise other molecules, including other bacterial polypeptides. In one aspect, the solution used in the method of the invention is a bacterial cell culture or a partially purified or purified preparation derived from the bacterial cell culture.

The term "fraction", as used in accordance with the methods of the present invention, refers to a sample or aliquot of a solution. In one aspect of the method of the invention, the first and second parts referred to herein are substantially identical in their volume and content. This can be accomplished, for example, by determining the total protein content in the first and second fractions, wherein a substantially consistent total protein content indicates that the first and second fractions have substantially the same content. However, in another aspect, the portion used as the first or second portion may be a sample of the solution or a dilution of an aliquot. It will be appreciated that depending on the amount of neurotoxin polypeptide to be determined (i.e. partially processed or unprocessed neurotoxin polypeptide or total neurotoxin), dilution may be required to allow optimal qualitative and quantitative determination. How to perform such dilution is well known to those skilled in the art.

The term "contacting" as used in accordance with the methods of the present invention refers to (i) bringing the above-described capture antibody and neurotoxin contained in a solution, or (ii) bringing the antibody complex and detection antibody into physical proximity such that they can physically and/or chemically interact. Suitable conditions to allow specific interactions are well known to the skilled person. The conditions will depend on the antibody and solution to be used in the method of the invention and can be readily adapted by the skilled person. In addition, the time sufficient to allow interaction can also be readily determined by the skilled person. Furthermore, it is understood that between the individual contacting steps described in the process of the invention, a washing step may be carried out to obtain suitable conditions for the contacting. For example, after the formation of the first antibody complex in step a), the remaining solution should be removed before applying the detection antibody to the antibody complex. Furthermore, after the formation of the first detection complex in step b), it may be necessary to remove the remaining (uncomplexed) detection antibody before determining the amount of the first detection complex in step c). Accordingly, this of course applies equally to steps d) to f).

"antibody" as used herein encompasses monoclonal antibodies, polyclonal antibodies, monoclonal antibodies, polyclonal antibodies, monoclonal,A single chain antibody, a chimeric antibody, a bispecific antibody, a synthetic antibody, or a fragment of any of the antibodies. Fragments of the antibodies include Fab, Fv or scFv fragments, or chemically modified derivatives of any of these fragments. Antibodies can be prepared, for example, by the method described in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies may be used initially in1975, Nature 256, 495, Galfr 1981, MethEnzymol 73, 3. The technique involves fusing mouse myeloma cells with spleen cells derived from the immunized mammal. The antibodies may be further modified using techniques well known in the art. For example, surface plasmon resonance used in the biacore (r) system can be used to improve the efficiency of phage antibodies binding to epitopes, see Schier 1996, human antibodies hybrids 7, 97; malmborg 1995, j.immunol Methods 183, 7. Antibodies as used herein also include functional equivalents of antibodies, i.e., agents capable of specifically binding to a desired epitope or portion of a neurotoxin polypeptide. In one aspect, such functional equivalents include receptors or binding proteins mentioned elsewhere in the specification, or domains thereof capable of mediating said specific binding.

According to the method of the invention, the "first capture antibody" specifically binds to an epitope comprised in the light chain of the mature neurotoxin polypeptide as well as comprised in the partially processed and/or unprocessed neurotoxin polypeptide. Specific binding, as used herein, generally means that the antibody does not significantly cross-react with other epitopes on the heavy chain or linker of the neurotoxin polypeptide to be assayed or on other polypeptides. Specific binding referred to herein can be tested by a variety of well-known techniques, including, for example, competitive assays and Western blots. The epitopes used according to the invention relate to antigenic determinants recognized by antibodies.

In another aspect, the first capture antibody can be replaced with a different capture antibody. To this end, at least one capture antibody may specifically bind to an epitope on the light chain of the unprocessed neurotoxin polypeptide, at least one other capture antibody may specifically bind to an epitope on the light chain of the partially processed neurotoxin polypeptide, and at least one other capture antibody may specifically bind to an epitope on the light chain of the processed neurotoxin polypeptide. It will be appreciated that for the purposes of the methods of the invention, these three classes of antibodies are functionally similar to the first capture antibody. Similarly, capture antibodies that specifically bind to light chain epitopes of partially processed and unprocessed neurotoxin polypeptides can be used in combination with capture antibodies that specifically bind to light chain epitopes of processed neurotoxin polypeptides.

In one aspect, the first capture antibody should be immobilized. Immobilization of the antibody may, in principle, in one aspect, be achieved by binding the antibody to a solid support, reversibly or irreversibly, directly or indirectly (via a linker molecule). In one aspect, the first capture antibody is immobilized prior to performing the methods of the invention. In another aspect, the first capture antibody is immobilized after formation of the first antibody complex but before contacting the complex with the detection antibody. Materials for solid supports are well known to those skilled in the art and include, for example, commercially available polysaccharide matrices selected from: sepharose, Sephadex, agarose, Sephace, microfibrous, and alginate beads; polypeptide substrates, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass, plastic and/or silicon wafers and surfaces, nitrocellulose strips, membranes, sheets, duracells, wells and walls of reaction disks, plastic tubes. In one aspect of the invention, the solid support is made of gamma-irradiated polystyrene.

The term "first antibody complex" refers to a complex comprising a first capture antibody that specifically binds to a processed, partially processed, or unprocessed neurotoxin polypeptide. As described above, the formation of the antibody complex is the result of contacting the first capture antibody with a solution containing the processed, partially processed and/or unprocessed neurotoxin polypeptide.

According to the methods of the invention, a "second capture antibody" specifically binds to an epitope of a linker comprising a unprocessed and/or partially processed neurotoxin polypeptide or part thereof. In the absence of a linker sequence, it is contemplated that the second capture antibody specifically binds to an epitope comprising an uncleaved proteolytic cleavage site or portion thereof. In one aspect of the invention, the second capture antibody does not significantly cross-react with the processed neurotoxin polypeptide. In one aspect, the second immobilized capture antibody specifically binds to a polypeptide consisting essentially of SEQ ID NO:1 to 16 (see table 2 or table 3 below) or comprises or is comprised on an epitope of such an amino acid sequence.

TABLE 2 amino acid sequences of cleavage site and flanking sequences of different neurotoxin polypeptides

TABLE 3 amino acid sequence of linker region

Due to the presence of the above epitope, the unprocessed or partially processed neurotoxin polypeptide can be specifically bound by the second capture antibody, thereby forming a second antibody complex. In one aspect, the second capture antibody is immobilized as explained in detail above.

Accordingly, the term "second antibody complex" refers to a complex comprising a second capture antibody that specifically binds to a partially processed or unprocessed neurotoxin polypeptide. However, the second antibody complex should not contain a processed neurotoxin polypeptide.

According to the method of the invention, the "detection antibody" specifically binds to the first and/or second antibody complex. In one aspect, the detection antibodies to the first and second antibody complexes are identical. However, in another aspect, different detection antibodies may be used for the first and second antibody complexes. In one aspect, the detection antibody specifically binds to an epitope of the heavy chain of the processed, partially processed, and unprocessed neurotoxin polypeptide. Either the first antibody complex or the second antibody complex is capable of binding specifically to the detection antibody due to the presence of the same epitope in both complexes, and is thereby detected in this aspect of the invention.

The result of the specific binding of the detection antibody is the formation of a first detection complex or a second detection complex, respectively.

Thus, the term "first detection complex" refers to a complex comprising a first antibody complex and a detection antibody. Likewise, the term "second detection complex" refers to a complex comprising a second antibody complex and a detection antibody.

In one aspect of the method of the invention, the detection antibody contained in the first or second detection complex is coupled to a detectable label, such that the amount of detection antibody bound to the detection complex can be determined. By determining the amount of bound detection antibody, the amount of first or second antibody complex can be determined, since the amount of bound detection antibody in the detection complex correlates with the amount of antibody complex comprised in the detection complex. Labeling may be performed by direct or indirect methods. Direct labeling involves binding of the label directly (covalently or non-covalently) to the first detection antibody. Indirect labeling involves binding (covalently or non-covalently) to a reagent that specifically binds to the detection antibody and carries a detectable label. Such a reagent may be, for example, a secondary (higher) antibody that specifically binds to the detection antibody. In this case, the secondary antibody will be conjugated to a detectable label. It will be appreciated that higher antibodies may also be used for detection of the detection complexes. Higher antibodies are often used to enhance the signal. Suitable higher antibodies may also be includedIncluding the well-known streptavidin-biotin system (Vectorlaboratories, Inc.), the well-known Dako LSAB^TM2 and LSAB^TM+ (labeled streptavidin-biotin), or Dako PAP (Peroxidase Anti-Peroxidase, Peroxidase-Anti-Peroxidase). In another aspect, the label of the first detection antibody is selected from the group consisting of: fluorescent dyes, chemiluminescent molecules, radioactive labels, and enzymes capable of producing a detectable signal. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, texas red (TexasRed), fluorescein, and Alexa dyes (e.g., Alexa 568). Exemplary radiolabels include³⁵S，¹²⁵I，³²P，³³P, and the like. Alternatively, the detectable label conjugated to the first detection antibody may also be an enzyme capable of producing a detectable signal (e.g., by conversion of a substrate). In one aspect, such an enzyme can be a peroxidase (e.g., horseradish peroxidase) or an alkaline phosphatase.

The term "determining an amount" as used herein relates to determining an absolute amount, a relative amount or a concentration in a quantitative or semi-quantitative manner. The measurement may be based on a chemical, physical or biological property of a detectable label conjugated to the first detection antibody. Suitable measurement means for detection are well known to those skilled in the art and depend on the nature of the detectable label as described above. However, it will be appreciated that the amount of detectable label that can be measured is directly related to the amount of detection complex, which in turn is related to the amount of antibody complex and thus the type of neurotoxin to be measured, i.e. to the total (processed, unprocessed and partially processed neurotoxin), or the amount of unprocessed and partially processed neurotoxin. It will be appreciated that the determination of the amount of neurotoxin polypeptide will, in one aspect, also require calibration of the assay method by application of a standard solution having a predetermined amount of neurotoxin polypeptide. How to make such corrections is well known to those skilled in the art.

The term "calculating" as used in accordance with the method of the present invention relates to a mathematical operation by which the amount of processed neurotoxin can be determined from the amount of total neurotoxin (i.e. processed, unprocessed and partially processed neurotoxin) as well as the amount of partially processed and unprocessed neurotoxin. In one aspect of the method of the invention, the calculating comprises subtracting the amount of partially processed and unprocessed neurotoxin from the amount of total neurotoxin.

Advantageously, using the method of the present invention, the amount of processed neurotoxin in a given preparation can be reliably determined. Accordingly, the quality of the neurotoxin preparation can be improved since it can be checked whether the preparation has a constant amount of the desired processed neurotoxin polypeptide.

In principle, the method of the invention may be performed by coupling the first capture antibody to a solid support, such as a reaction vial. Similarly, the second capture antibody can be coupled to another physically separate solid support (e.g., another reaction vial). Subsequently, the two capture antibodies coupled to the solid support can be contacted with the portion of the solution to be determined containing the processed, unprocessed and/or partially processed neurotoxin polypeptide. The solution may be, for example, a purified bacterial cell culture derived from clostridium. It will be appreciated that a first portion of the solution will be contacted with the first capture antibody on the first solid support and a second portion of the solution will be contacted with the second capture antibody on the second solid support. These fractions are usually of equal volume and are normalized for their content (e.g., their total protein content). The contacting is performed for a sufficient time to enable the first and second capture antibodies to specifically bind to their respective antigens. For example, the contacting may be performed at room temperature for about one hour. Subsequently, the first solution portion and the second solution portion are discarded, and the solid support (e.g., reaction vial) is washed once or twice with a buffer under conditions that do not affect the first and second antibody complexes on the solid support that have now formed with the capture antibodies. After performing the washing step, the (first) detection antibody is added to the solid support under conditions that allow specific binding of the detection antibody. Excess detection antibody may be removed by performing an additional washing step using a suitable buffer. Subsequently, the amount of the first and second detection complexes can be determined by measuring the amount of detection antibody that has specifically bound. This may be achieved depending on the nature of the label that detects the antibody, for example by measuring optical density or fluorescence intensity. The measured amount of detectable label can be used to compare to calibration standards to determine the neurotoxin type, i.e., total (processed, unprocessed and partially processed neurotoxin) or the amount of unprocessed and partially processed neurotoxin, in the first or second detection complex. It is understood that the first test complex represents the amount of total neurotoxin, while the second test complex represents only the amount of partially processed and unprocessed neurotoxin polypeptide. Accordingly, in the above-described arrangement, the amount of processed neurotoxin polypeptide can be calculated by subtracting the amount of partially processed or unprocessed neurotoxin polypeptide from the amount of total neurotoxin polypeptide.

It is to be understood that the above definitions and explanations of terms apply, mutatis mutandis, to all aspects of the description that follow, unless otherwise specified.

The present invention also relates to a method for determining the amount of processed (active) neurotoxin polypeptide in a solution comprising processed neurotoxin polypeptide and partially processed and/or unprocessed neurotoxin polypeptide comprising the steps of:

a) contacting a first portion of the solution with a first capture antibody, thereby forming a first antibody complex, wherein the first capture antibody specifically binds to the heavy chain of a mature neurotoxin polypeptide, partially processed and unprocessed neurotoxin polypeptide, under conditions that allow binding of the antibody to the mature neurotoxin, partially processed and unprocessed neurotoxin polypeptide;

b) contacting a detection antibody with a first antibody complex, thereby forming a first detection complex, wherein the detection antibody specifically binds to the light chain of the mature neurotoxin, partially processed and unprocessed neurotoxin polypeptide in the antibody complex formed in step a);

c) contacting a second portion of the solution with a second capture antibody, thereby forming a second antibody complex, wherein the second capture antibody specifically binds to the linker of the partially processed and unprocessed neurotoxin polypeptide, under conditions that allow binding of the antibody to the partially processed and unprocessed neurotoxin polypeptide;

d) contacting the second antibody complex with a detection antibody, thereby forming a second detection complex,

e) determining the amount of the first and second detection complexes formed in steps b) and d).

f) Calculating the amount of mature neurotoxin polypeptide based on the amount of first and second detection complexes determined in step e).

In another aspect of the methods of the invention, the methods further comprise determining the binding activity of the neurotoxin polypeptide.

The term "binding activity" as used in accordance with the methods of the present invention relates to the ability of the processed neurotoxin polypeptide to bind to surface receptor proteins present on e.g. peripheral cholinergic nerve endings. In one aspect, the receptor proteins include BoNT/A-bound SV2, BoNT/B and BoNT/G-bound synaptotagmins I and II, and Gangliosides (GT)_1B) A co-receptor. In one aspect of the method of the invention, the binding activity can be measured ex vivo using a mimetic substrate that replaces the surface protein receptor by mimicking the binding domain of the surface protein receptor. In one aspect, the mimetic substrate is a labeled peptide derived from the receptor protein. In another aspect, suitable markers include those mentioned elsewhere in the specification, particularly biotin.

Accordingly, the present invention also relates to a method for determining the binding activity of a neurotoxin polypeptide comprising the steps of:

a) contacting a portion of the solution containing the neurotoxin polypeptide with the labeled peptide, thereby forming a complex, and

b) determining the complex formed in step a) based on a label, wherein the presence or absence of a complex, or the amount of a complex, is indicative of the binding activity of the neurotoxin polypeptide in said solution.

The complex can be determined based on the nature of the label used to label the peptide. In one aspect, for example, the biotinylated peptide contained by the complex can be determined by using a streptavidin conjugate that is capable of producing a detectable signal. The presence, absence, or intensity of the signal will indicate the binding activity of the neurotoxin polypeptide in solution or its intensity.

In another aspect of the methods of the invention, the methods further comprise detecting the proteolytic activity of the neurotoxin polypeptide.

The term "proteolytic activity" as used in accordance with the methods of the present invention relates to the ability of the processed neurotoxin to proteolytically cleave an N-ethylmaleimide sensitive attachment receptor (SNARE) protein involved in synaptic vesicle membrane fusion. In one aspect, the cleavage is zinc (II) dependent. The proteolytic activity can be measured using a mimetic substrate that replaces the native SNARE protein. In addition, after cleavage, a detectable label, such as a dye, will be released from the mimetic substrate. In one aspect, the mimetic substrate is a compound having the general formula X-p-nitroanilide (nitroanilide), wherein X is arginine or a peptide having the sequence "arginine-Y", wherein Y represents one or more amino acids, in another aspect, the compound is arginine-p-nitroanilide.

Accordingly, the present invention also relates to a method for determining the proteolytic activity of a neurotoxin, comprising the steps of:

a) contacting a portion of the solution containing the neurotoxin polypeptide with a compound having the general formula X-p-nitroanilide, wherein X is arginine or a peptide having the sequence "arginine-Y", wherein Y represents one or more amino acids, and

b) determining the proteolytic activity of the neurotoxin in said solution based on the amount of p-nitroanilide released from step a) in relation to the amount of neurotoxin polypeptide.

In one aspect, Y represents a polypeptide having the sequence set forth in SEQ ID NOs: 25 or 26, or a pharmaceutically acceptable salt thereof.

The processed neurotoxin polypeptide contained in the solution fraction is capable of cleaving and thereby releasing the paranitroanilide from the remaining peptide. Para-nitroaniline is a dye well known in the art. The proteolytic activity of the neurotoxin polypeptide in the solution is determined based on the amount of paranitroanilide released in relation to the amount of neurotoxin polypeptide.

The invention also relates to a device for determining the amount of processed neurotoxin polypeptide in a solution comprising:

a) an arrangement (arrangement) of a first capture antibody, a second capture antibody and a detection antibody, wherein the arrangement is such that steps a) to e) of the above-described method can be performed; and

b) means for calculating the amount of mature neurotoxin polypeptide based on the amount of first and second detection complexes determined using the arrangement in a).

The term "device" as used herein relates to a system comprising at least the above-described arrangements and means, wherein said arrangements and means are operatively connected to each other to allow the performance of said assays. In one aspect, the arrangement may be the above-mentioned solid support with immobilized capture antibody thereon, which may be physically separate vials, allowing separate contacting of the first and second portions of solution. Furthermore, in one aspect, the device may include a component for determining the amount of the detection compound. Depending on the type of detection antibody used, the assembly will include a detector for detecting the signal generated by the antibody. Furthermore, in one aspect, the assembly may further comprise means for calibration, such as a computer-based algorithm, for comparing the measured signal to calibration standards in order to determine the amount of neurotoxin polypeptide contained in the solution or portion thereof. The device may further comprise means for calculating the amount of mature neurotoxin from the amounts of the first and second detection complexes, e.g. a computer based algorithm for performing the calculation.

The invention also relates to a kit suitable for carrying out the above method, said kit comprising:

a) an arrangement of a first capture antibody, a second capture antibody and a detection antibody, wherein the arrangement is such that steps a) to e) of the above method can be performed;

b) means for calculating the amount of mature neurotoxin polypeptide based on the amount of first and second detection complexes determined using the arrangement of a); and

c) instructions for performing the method.

The term "kit" as used herein refers to a collection of the aforementioned means or reagents of the invention, which may or may not be packaged together. The components of the kit may be contained in separate vials (i.e., as a kit of parts) or provided in a single vial. Furthermore, it is to be understood that the kit of the invention is used to carry out the above mentioned methods. In one aspect, it is contemplated that all components are provided in a ready-to-use manner for carrying out the method. In another aspect, the kit includes instructions for performing the method. The instructions may be provided by way of a user manual in paper or electronic form. For example, the manual may contain instructions explaining the results obtained by performing the above-described method using the kit of the invention.

All references cited in this specification are hereby incorporated by reference in their entirety for their disclosure as well as the disclosure specifically mentioned in this specification.

Drawings

Figure 1, binding schematic of at least one (or more) detection antibody.

Figure 2, schematic representation of the specific binding of a second capture antibody to a partially processed or unprocessed precursor neurotoxin polypeptide and subsequent binding of at least one (or more) detection antibody.

FIG. 3 is a schematic diagram of the determination of the binding activity of neurotoxin polypeptides.

FIG. 4 is a schematic diagram of the determination of the proteolytic activity of neurotoxin polypeptides.

Claims (12)

1. A method for determining the amount of processed neurotoxin polypeptide in a solution comprising processed neurotoxin polypeptide and partially processed and/or unprocessed neurotoxin polypeptide comprising the steps of:

a) contacting a first portion of the solution with a first capture antibody, thereby forming a first antibody complex, wherein the first capture antibody specifically binds to a light chain of a processed, partially processed, and unprocessed neurotoxin polypeptide, wherein the contacting is performed under conditions that allow binding of the antibody to the processed, partially processed, and unprocessed neurotoxin polypeptide,

b) contacting a first antibody complex with a detection antibody, thereby forming a first detection complex, wherein the detection antibody specifically binds to the heavy chain of the processed, unprocessed and partially processed neurotoxin polypeptide in the antibody complex formed in step a),

c) contacting a second portion of the solution with a second capture antibody, thereby forming a second antibody complex, wherein the second capture antibody specifically binds to a peptide epitope of the partially processed or unprocessed neurotoxin polypeptide, wherein the peptide epitope has an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 16, wherein the contacting is performed under conditions that allow the antibody to bind to the partially processed or unprocessed neurotoxin polypeptide,

d) contacting the second antibody complex with a detection antibody, thereby forming a second detection complex,

e) determining the amount of the first and second detection complexes formed in steps b) and d),

f) calculating the amount of processed neurotoxin polypeptide based on the amount of first and second detection complexes determined in step e).

2. A method for determining the amount of processed neurotoxin polypeptide in a solution comprising processed neurotoxin polypeptide and partially processed and/or unprocessed neurotoxin polypeptide comprising the steps of:

a) contacting a first portion of the solution with a first capture antibody, thereby forming a first antibody complex, wherein the first capture antibody specifically binds to a processed neurotoxin polypeptide, a heavy chain of a partially processed and unprocessed neurotoxin polypeptide, wherein the contacting is performed under conditions that allow the antibody to bind to the processed neurotoxin, partially processed and unprocessed neurotoxin polypeptide,

b) contacting a first antibody complex with a detection antibody, thereby forming a first detection complex, wherein the detection antibody specifically binds to the light chain of the processed neurotoxin, partially processed and unprocessed neurotoxin polypeptide in the antibody complex formed in step a),

c) contacting a second portion of the solution with a second capture antibody, thereby forming a second antibody complex, wherein the second capture antibody specifically binds to a peptide epitope of the partially processed and unprocessed neurotoxin polypeptide, wherein the peptide epitope has an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 16, wherein the contacting is performed under conditions that allow the antibody to bind to the partially processed and unprocessed neurotoxin polypeptide,

d) contacting the second antibody complex with a detection antibody, thereby forming a second detection complex,

e) determining the amount of the first and second detection complexes formed in steps b) and d),

f) calculating the amount of processed neurotoxin polypeptide based on the amount of first and second detection complexes determined in step e).

3. The method of claim 1 or 2, wherein the first capture antibody is immobilized.

4. The method of claim 1 or 2, wherein the second capture antibody is immobilized.

5. The method of claim 1 or 2, wherein the calculating in step f) comprises subtracting the determined amount of the second detection complex from the determined amount of the first detection complex.

6. The method of claim 1 or 2, wherein the unprocessed neurotoxin polypeptide is selected from the group consisting of neurotoxin polypeptides set forth in any one of SEQ ID NOs 17 to 24.

7. The method of claim 1 or 2, wherein the method further comprises determining the binding activity of the processed neurotoxin polypeptide.

8. The method of claim 7, comprising the steps of:

a) contacting a portion of the solution containing the processed neurotoxin polypeptide with a biotinylated peptide to form a complex, and

b) determining said complex formed in step a) by means of a streptavidin conjugate capable of generating a detectable signal, wherein the presence, absence or amount of complex is indicative of the binding activity of the processed neurotoxin polypeptide in said solution,

wherein the biotinylated peptide is a biotin-labeled peptide derived from a surface receptor protein on a peripheral cholinergic nerve terminal.

9. The method of claim 1 or 2, wherein the method further comprises determining the proteolytic activity of the processed neurotoxin polypeptide.

10. The method of claim 9, comprising the steps of:

a) contacting a portion of the solution containing the neurotoxin polypeptide with a compound having the general formula X-p-nitroanilide, wherein X is arginine or a peptide having the sequence "arginine-Y", wherein Y represents one or more amino acids, and

b) determining the proteolytic activity of the neurotoxin polypeptide in said solution based on the amount of paranitroanilide released in step a) in relation to the amount of the neurotoxin polypeptide.

11. A device for determining the amount of processed neurotoxin polypeptide in a solution comprising:

a) an arrangement of a first capture antibody, a second capture antibody and a detection antibody, wherein the arrangement allows steps a) to e) of the method of any one of claims 1 to 10 to be carried out; and

b) calculating means, wherein said means calculates the amount of neurotoxin polypeptide processed based on the amount of first and second detection complexes determined using the arrangement in a).

12. A kit suitable for performing the method of any one of claims 1 to 10, the kit comprising:

a) an arrangement of a first capture antibody, a second capture antibody and a detection antibody, wherein the arrangement allows steps a) to e) of the method of any one of claims 1 to 10 to be carried out;

b) calculating means, wherein said means calculates the amount of neurotoxin polypeptide processed based on the amount of first and second detection complexes determined using the arrangement in a),

c) instructions for performing the method.