Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays

• Published in: Toxins Vol. 11; no. 1; p. 35
• Main Authors: Xavier, Bruna, Perobelli, Rafaela Ferreira, Walter, Maurício Elesbão, da Silva, Francielle Santos, Dalmora, Sérgio Luiz
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 12.01.2019; MDPI
• Subjects: Animals; Bioassays; Biocompatibility; Biological activity; Biological Assay; Biological products; Biopharmaceuticals; Biotechnology; botulinum neurotoxin type A; Botulinum toxin; Botulinum Toxins, Type A - analysis; Botulinum Toxins, Type A - toxicity; Cell culture; Cell Line; Cell Survival - drug effects; Chromatography; Chromatography, Gel; Chromatography, Liquid; Chromatography, Reverse-Phase; Cytotoxicity; Female; Humans; LD50 mouse bioassay; Lethal Dose 50; Liquid chromatography; Methods; Mice; Neurotoxins; Pharmaceuticals; Potassium; Proteins; Reproducibility of Results; reversed-phase chromatography; Serotypes; Size exclusion chromatography; Sodium; Standard deviation; Toxicity; Toxins; T−47D cell culture; botulinum neurotoxin type A; reversed-phase chromatography; LD50 mouse bioassay; T−47D cell culture; size-exclusion chromatography
• DOI: 10.3390/toxins11010035
• ISSN: 2072-6651

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD mouse bioassay, the T-47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity ( < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.