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

Abstract

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 LD50 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 (p < 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.

Rapid Detection of Botulinum Neurotoxins—A Review

“…One of these such systems is surface plasmon resonance, this has seen development from initial uses [121,122] to more recent and emerging advancements [81,123]. Other such techniques that were first explored several years ago are time-resolved fluorescence assay [124] and liquid chromatography [125]; these have more recently been explored, providing a relatively low level of detection and short detection time [40,126]. The problem with these techniques as detection platforms, is that they often require expensive instrumentation or have simply not been tested across a wide enough set of parameters.…”

“…Due to the fact that the toxin is a biological product which intrinsically leads to variability between production batches, each set has to be tested for safety, potency and stability before it can be used on humans [39]. The testing must be conducted at multiple steps in the manufacturing timeline, this is a mandatory requirement to be able to meet regulatory protocols, leading to marketing consent being approved [40].…”