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KMID : 1146920220520040453
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Journal of Pharmaceutical Investigation
2022 Volume.52 No. 4 p.453 ~ p.463
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Dissociation mechanics and stability of type A botulinum neurotoxin complex by means of biophysical evaluation
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Hada Shavron
Lee Jae-Chul
Lee Eun-Chae
Ji Sun-Kyong
Nam Jeong-Sun
Yun Bum-Jin
Na Dong-Hee
Kim Nam-Ah
Jeong Seong-Hoon
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Abstract
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Purpose: Biophysical characterisation of type A botulinum neurotoxin (BoNT/A) could be challenging since it exists in association with neurotoxin associated proteins (NAPs) as large protein complexes. The objective of this study was to elucidate the dissociation mechanics of BoNT/A complex along with its thermodynamic stability through a combination of analytical tools.
Methods: Size exclusion chromatography (SEC) was mainly used to study the behavior of BoNT/A complex at various pH. In addition, multi-angled light scattering (MALS), enzyme linked immunosorbent assay (ELISA), and dynamic light scattering (DLS) were utilized to validate its chromatographic results.
Results: The dissociation of BoNT/A from its complex was found to be strongly dependent on its pH with higher dissociation towards alkaline pH which was further accelerated with time and temperature. In addition, dissociated BoNT/A at pH 7.4 showed lower thermal stability compared to the associated state even in the presence of polysorbate, as revealed by the SEC chromatogram and aggregation onset temperature. Moreover, the partial reversibility of the dissociated BoNT/A complex after titration of pH to 6.0, suggested vulnerability of BoNT/A towards formation of irreversible aggregates as it dissociates, signifying lower stability profile of the neurotoxin after dissociation.
Conclusion: Overall, BoNT/A was more stable when associated with NAPs at pH 6.0 compared to its dissociated state at pH 7.4. Moreover, the conventional analytical used could be utilized to relatively quantify its amount in different formulations.
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KEYWORD
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Botulinum neurotoxin A complex, Protein aggregation, Biophysical characterization, Size-exclusion chromatography (SEC), Formulation, Neurotoxin stability
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