Abstract
Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.
Highlights
Botulinum neurotoxin (BoNT) is one of the six category A biothreat agents and development of safe and effective antitoxins is a priority to protect from potential bioterror use [1,2]
Differential scanning calorimetry (DSC) thermograms indicated that each antibody in anti-BoNT 9ABE unfolded independently, suggesting that the nine monoclonal antibody (mAb) did not significantly interact. anti-BoNT 9ABE melted over a range encompassing the Tm with the lowest and highest unfolding temperatures and the melting curves of the mixture closely resemble the sum of the melting of the nine individual mAbs (Fig 1A and 1B)
Results from Reverse phase-HPLC (RP-HPLC) and WCX-HPLC were consistent with results of Size exclusion- high-pressure liquid chromatography (SE-HPLC), DSC and Dynamic light scattering (DLS)
Summary
Botulinum neurotoxin (BoNT) is one of the six category A biothreat agents and development of safe and effective antitoxins is a priority to protect from potential bioterror use [1,2]. The sequence diversity leading to multiple serotypes and subserotypes presents technical. Nine-antibody co-formulated mixture with ambient temperature stability manuscript. The funder provided support in the form of salaries for authors MTT, SFJ, ML, DL, CO but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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