Design of a cell-based in vitro assay for the pharmaceutical potency testing of botulinum neurotoxin type-A
Design of a cell-based in vitro assay for the pharmaceutical potency testing of botulinum neurotoxin type-A
Botulinum Neurotoxin type-A (BoNT-A), from the bacterium Clostridium Botulinum is extensively used in the pharmaceutical and medical industries for the treatment of limb dystonia and various spastic movement disorders. Determining the batch potency before release for clinical and therapeutic use is thus fundamental and is currently calculated using the mouse LD50 potency bioassay. Pursuing in vitro alternatives is vital for the replacement, refinement and reduction of animals in research (NC3R’s), and if an alternative is available, it should prioritise the animal method. Human induced pluripotent stem cells and neuroblastoma cell lines are sensitive to BoNT-A and this project screens cell lines for toxin sensitivity with the intention of selecting the most sensitive candidate for use in a GMP-compliant routine laboratory assay for the testing of potency. BoNT-A proteolytically degrades SNAP-25₂₀₆, to yield SNAP-25₁₉₇, thus inhibiting neurotransmitter release and the detection of this cleaved protein determines toxin activity. This project has successfully discovered a cell line for use in an in vitro assay demonstrating that the cells are viable for use after 7 days of growth and incubation in toxin for 48-hours provides the most optimum results. Several cell lines were screened, differentiation methods trialled and iCell Neurons, which do not require an extensive or variable differentiation protocol have been selected as the most sensitive. The method in which the SNAP-25 cleavage as a result of toxin activity is detected proved to be challenging, with the three methods originally selected as potentially sensitive rendering inconclusive or unsuitable results. Western Blot was a viable tool in the selection of the cell line, but this variable method is not a suitable GMP-compliant method for the reliable testing of such a potent and dangerous drug product.
University of Southampton
Davies (Long), Shellie
387d01d3-c935-4eea-bc5d-bb53d513f329
1 February 2018
Davies (Long), Shellie
387d01d3-c935-4eea-bc5d-bb53d513f329
Willaime-Morawek, Sandrine
24a2981f-aa9e-4bf6-ad12-2ccf6b49f1c0
Davies (Long), Shellie
(2018)
Design of a cell-based in vitro assay for the pharmaceutical potency testing of botulinum neurotoxin type-A.
University of Southampton, Doctoral Thesis, 328pp.
Record type:
Thesis
(Doctoral)
Abstract
Botulinum Neurotoxin type-A (BoNT-A), from the bacterium Clostridium Botulinum is extensively used in the pharmaceutical and medical industries for the treatment of limb dystonia and various spastic movement disorders. Determining the batch potency before release for clinical and therapeutic use is thus fundamental and is currently calculated using the mouse LD50 potency bioassay. Pursuing in vitro alternatives is vital for the replacement, refinement and reduction of animals in research (NC3R’s), and if an alternative is available, it should prioritise the animal method. Human induced pluripotent stem cells and neuroblastoma cell lines are sensitive to BoNT-A and this project screens cell lines for toxin sensitivity with the intention of selecting the most sensitive candidate for use in a GMP-compliant routine laboratory assay for the testing of potency. BoNT-A proteolytically degrades SNAP-25₂₀₆, to yield SNAP-25₁₉₇, thus inhibiting neurotransmitter release and the detection of this cleaved protein determines toxin activity. This project has successfully discovered a cell line for use in an in vitro assay demonstrating that the cells are viable for use after 7 days of growth and incubation in toxin for 48-hours provides the most optimum results. Several cell lines were screened, differentiation methods trialled and iCell Neurons, which do not require an extensive or variable differentiation protocol have been selected as the most sensitive. The method in which the SNAP-25 cleavage as a result of toxin activity is detected proved to be challenging, with the three methods originally selected as potentially sensitive rendering inconclusive or unsuitable results. Western Blot was a viable tool in the selection of the cell line, but this variable method is not a suitable GMP-compliant method for the reliable testing of such a potent and dangerous drug product.
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S Long (Davies) Completed MPhil Thesis with Corrections
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Published date: 1 February 2018
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Local EPrints ID: 457142
URI: http://eprints.soton.ac.uk/id/eprint/457142
PURE UUID: c39855d0-a527-4aa3-9cfe-6316efe6f749
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Date deposited: 24 May 2022 17:01
Last modified: 17 Mar 2024 07:20
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Author:
Shellie Davies (Long)
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