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

Abstract

Background: Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used for therapeutic and aesthetic applications. Objective: 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. Methods: 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. Results: 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. Conclusion: 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.