Evaluation of botulinum toxin type A by bioassays and a validated reversed-phase liquid chromatography method

Abstract

The biological potency of botulinum toxin type A (BoNTA) in biotechnology-derived products was assessed by the mouse LD50 bioassay (MBA) and the T47D cell culture assay.

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

“…The validated SEC method was applied to quantitate the BoNTA in the pharmaceutical preparations, and the results were compared to those obtained from the in vivo and in vitro bioassays and from the RPC method previously studied [28], giving mean differences of an estimated content of 0.71% higher, 0.36% lower, and 0.87% higher, respectively, with non-significant differences ( p > 0.05) (Table 3). Moreover, a significant correlation was demonstrated for the SEC method with the in vivo mouse lethality assay (Pearson’s correlation coefficient r = 0.9984).…”

“…Thus, the bioactivity was tested by the in vivo bioassay, and no toxic effects or mortality of the mice were detected. In addition, the bioactivity of the related proteins detected using the RPC method, although previously assessed after 4 h of exposure to UV light [28], was tested again so as to evaluate the effects after 8 h, which demonstrated little decrease from 98.70% (197.40 U mL −1 ) to 95.20% (190.40 U mL −1 ) (Table 4, 4 f ). The biological activity was generated by the interaction of the protein with its target receptor, and, as observed, the effects of the aggregated forms evaluated by the in vitro assay showed differences related to those of the LD 50 mouse bioassay data.…”

“…The RPC method was accomplished on a Zorbax 300 SB C 18 column (5 µm, 300 Å, 150 mm × 4.6 mm) from Agilent (Santa Clara, CA, USA), maintained at 45 °C, following the procedure previously described [28]. The mobile phase consisted of a 50 mM L −1 sodium phosphate solution at pH 2.8, and acetonitrile (80:20, v / v ), at a flow rate of 0.3 mL min −1 , with detection using PDA set at 214 nm.…”

“…This research aimed to develop and validate a specific SEC method in order to evaluate the content of BoNTA in biotechnology-derived medicines; assess the bioactivity of their high-molecular-weight (HMW) forms; and evaluate the correlation of the results with the LD 50 bioassay, the T-47D assay, and the RPC method. In this context, the current work is an extension of the previously published literature [28], contributing to assuring the quality and clinical efficacy of this biotherapeutic.…”

“…Size-exclusion chromatography (SEC) has become the most widely applied technique for the analysis of native-proteins and for their aggregates, which can be generated during fermentation, purification, and processing, and have the potential to elicit an immunoresponse and may affect the biological activity [26,27]. Reversed-phase chromatography (RPC) methods were applied to assess the related proteins as well as the main peak in biopharmaceutical formulations, combined with mass spectrometry, for the detection of soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) cleavage products generated from formulated BoNTA [28,29].…”