Merz Pharma Gmbh & Co Kgaa

Karl-Heinz Eisele

Harold Victor Taylor

Cornelia Brunn

Michael Pfeil

Josef Friedrich

Kai Harting

Harold V Taylor

Gerd Mander

Gerd J Mander

Klaus Fink

Claudia Jatzke

Cornelia Brünn

Cornelia Bruenn

Harold Taylor

Thomas Wilk

Kerstin Hölscher

Cornelia BRÜNN

Tim STÖVEKEN

Swen Grein

The present invention pertains to a method for standardizing the sensitivity of induced pluripotent stem cell (iPS)-derived neurons to a neurotoxin polypeptide, comprising the steps of: a) cultivating different batches of induced pluripotent stem cell-derived neurons in a cell culture medium comprising GT1b for at least 3 hours; b) contacting the different batches of induced pluripotent stem cell-derived neurons of step a) with a neurotoxin polypeptide; c) cultivating the different batches of induced pluripotent stem cell-derived neurons of step b) for at least 24 hours in the presence of GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity, thereby standardizing the sensitivity of the induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide. The invention further relates to a method for the generation of induced pluripotent stem cell-derived neurons having a standardized sensitivity to a neurotoxin polypeptide, comprising the steps of: a) providing different batches of induced pluripotent stem cell-derived neurons; b) cultivating the different batches of induced pluripotent stem cell-derived neurons of step a) in a cell culture medium comprising GT1b for at least 3 hours, thereby standardizing the sensitivity of the induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide. In addition, encompassed by the present invention is a method for determining the biological activity of a neurotoxin polypeptide, comprising the steps of: a) cultivating induced pluripotent stem cell-derived neurons in a cell culture medium comprising GT1b for at least 3 hours; b) contacting the induced pluripotent stem cell-derived neurons of step a) with a neurotoxin polypeptide; c) cultivating the induced pluripotent stem cell-derived neurons of step b) for at least 24 hours in the presence of GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and d) determining the biological activity of the neurotoxin polypeptide in said cells. Finally, the invention relates to the use of GT1b for a) standardizing the sensitivity of different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide; or b) reducing the variability of the sensitivity of different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide.

Gangliosides for standardizing and increasing the sensitivity of cells to botulinum neurotoxins in in vitro test systems

The present invention pertains to a method for the generation of neurotoxin-sensitive, neuronal differentiated cells comprising the steps of: a) cultivating tumor cells which are able to differentiate into neuronal cells in a culture medium under conditions and for a time which primes said tumor cells for neuronal differentiation; and b) cultivating the tumor cells primed for neuronal differentiation of a) in a differentiation medium having an osmolality of 100 to 270 mOsm/kg, and comprising (i) B27 supplement and/or (ii) N2 supplement, for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronal differentiated cells. The invention further relates to neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention. In addition, the invention encompasses a method for determining the activity of a neurotoxin polypeptide comprising the steps of: a) contacting the neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention with a neurotoxin polypeptide; b) cultivating the neurotoxin-sensitive, neuronal differentiated cells of step a) for 3 to 74 hours or 72 hours under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and c) determining the activity of the neurotoxin polypeptide in the said cells after cultivation according to step b). Finally, the invention provides for a medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

Cellular test systems for the determination of the biological activities of neurotoxin polypeptides

The present invention provides a method for enhancing the specific uptake of a neurotoxin polypeptide into cells, the method comprising: incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least one of the following steps: (i) K-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition, thereby enhancing the specific uptake of the neurotoxin polypeptide into said cells. In addition, the invention pertains to a method for directly determining the biological activity of a neurotoxin polypeptide in cells, comprising: a) incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, the incubation comprising at least one of the following steps: (i) K-mediated depolarization of the cells, (ii) a reduced neurotoxin polypeptide exposition time and/or (iii) agitation of the cells during neurotoxin polypeptide exposition; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d); and f) calculating the amount of substrate cleaved by said neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said neurotoxin polypeptide in said cells.

Methods for enhancing the specific uptake of botulinum neurotoxins into cells

The present invention pertains to a method for determining the biological activity of a neurotoxin, the method comprising the steps of: (a) expressing a fusion protein comprising (i) an anchor protein, (ii) a reporter protein and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, in neurotoxin-sensitive cells; (b) incubating the neurotoxin-sensitive cells of (a) with a neurotoxin and cultivating the cells under conditions which allow the neurotoxin to exert its biological activity; (c) permeabilizing the neurotoxin-sensitive cells of (b) under conditions which allow the release of the reporter protein but not the release of the anchor protein from the permeabilized neurotoxin-sensitive cells; and (d) quantifying the activity of the reporter protein released from the cells, thereby determining the biological activity of the neurotoxin. In addition, the invention relates to a fusion protein comprising (i) an anchor protein, (ii) a reporter protein, and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells. Further encompassed by the present invention is a kit comprising the fusion protein of the invention. Finally, the invention pertains to the use of a fusion protein of the invention for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells.

Methods for the determination of the biological activities of neurotoxin polypeptides

The present invention relates to an antibody which specifically binds to unprocessed and/or partially processed neurotoxin polypeptide or an antibody which specifically binds an epitope consisting of a peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 16 and to methods for the manufacture of such antibodies. Moreover, the present invention relates to a composition comprising processed neurotoxin polypeptide free of unprocessed or partially processed neurotoxin polypeptide and a method for manufacturing said neurotoxin polypeptide based on the antibodies of the invention. The present invention also relates to the use of the aforementioned antibody for separating processed neurotoxin polypeptides from unprocessed or partially processed neurotoxin polypeptides or for determining unprocessed or partially processed neurotoxin polypeptides. The present invention relates to a method for the manufacture of a medicament.

Antibody that specifically binds partially processed or unprocessed neurotoxin polypeptides

Means and methods for manufacturing highly pure neurotoxin

The present invention is concerned with modified neurotoxins. Specifically, it relates to a modified biologically active neurotoxin polypeptide comprising at least one poly-Glycine domain. Also contemplated is a polynucleotide encoding the modified neurotoxin polypeptide having a poly-Glycine domain fused to the N-terminus, to the C-terminus or to both of the heavy and/or light chain of the mature neurotoxin polypeptide, vector comprising it and a host cell comprising such a polynucleotide or a vector. Moreover, envisaged are the aforementioned compounds for use as a medicament for treating various diseases.

Modified neurotoxins with poly-glycine and uses thereof

Novel methods for determining the unknown biological activity of a clostridial neurotoxin in a sample with respect to the known biological activity of a clostridial neurotoxin in a reference sample, comprising the step of comparing the biological activity of a clostridial neurotoxin preparation with the biological activity of a standard preparation of a reference clostridial neurotoxin in certain in vitro systems.

In vitro assay for quantifying clostridial neurotoxin activity

The present invention is concerned with tools for the quality control and safety during manufacture of neurotoxins. In particular, it relates to a method for the determination of the amount of partially processed and/or unprocessedneurotoxin A polypeptide (BoNT/A) in a solution comprising processed and partially processed and/or unprocessed BoNT/A comprising the steps of contacting a sample of the solution with a capture antibody which specifically binds to the partially processed and unprocessed BoNT/A under conditions which allow for binding of the antibody to the partially processed and unprocessed BoNT/A, whereby a complex is formed, and determining the amount of the formed complex, whereby the amount of the complex is indicative for the amount of the partially processed and/or unprocessed BoNT/A in the solution. Moreover, the present invention contemplates a device and a kit for carrying out the method.

Growing community of inventors

Frankfurt am Main, Germany

Karl-Heinz Eisele