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.
Highlights
Type-A toxin is one of the seven different serotypes (A to G) currently known to be produced by Clostridium botulinum, and it is the first to receive attention clinically [1]
Botulinum neurotoxin type-A (BoNTA) is a polypeptide that contains 1296 amino acids and has a molecular mass of 150 kDa; it is cleaved by proteases to generate the active di-chain form of the toxin, which is linked by a Toxins 2019, 11, 35; doi:10.3390/toxins11010035
This research aimed to develop and validate a specific size-exclusion chromatography (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 LD50 bioassay, the T-47D assay, Toxins 2019, 11, x FOR PEER REVIEW
Summary
Bruna Xavier 1 , Rafaela Ferreira Perobelli 1 , Maurício Elesbão Walter 1 , Francielle Santos da Silva 1 and Sérgio Luiz Dalmora 2, *. Industrial Pharmacy Department, Federal University of Santa Maria, Santa Maria 97105-900, Brazil. Received: 22 November 2018; Accepted: 10 January 2019; Published: 12 January 2019
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