Validation of the T–47D Cell Culture Bioassay for the Potency Assessment of Botulinum Neurotoxin Type A

Abstract

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used for therapeutic and aesthetic applications. An alternative in vitro cell culture bioassay based on the induction of apoptosis on T−47D breast cancer cells, after exposure to BoNTA, was developed and validated. The T-47D cells (ATCC HTB−133) were seeded at a density of 3 × 105 cells mL−1, and the bioassay was performed with doses of BoNTA, between 3 and 81 U mL−1. The responses were assessed using 10 µL of Alamar Blue®. The absorbances were read at 570 and 600 nm. The results were compared with those of the in vivo LD50 mouse bioassay, showing a non-significant 1.08% higher, mean difference of the estimated potencies (p>0.05). Besides, the biopharmaceutics is analyzed by the size exclusion and reversed-phase liquid chromatography methods, showing a significant correlation with values 1.15% higher and 0.85% lower, respectively, related to the cell culture bioassay. It is concluded that the validated T−47D cell culture assay represents an advancement toward the establishment of an alternative approach for the potency assessment, in the context of the 3 Rs. Besides, the employment of chromatographic methods in conjunction with the bioassays contributes to assessing the quality attributes of the biopharmaceutical formulations of BoNTA.