Type A botulinum neurotoxin (BoNT/A), a 150 kDa protein, is comprised of a 50 kDa light chain and a 100 kDa heavy chain, covalently linked by an inter-chain disulfide bond 1,2. It is generally produced in association with a group of neurotoxin-associated proteins (NAPs), comprising a non-toxic non-hemagglutinating subunit (NTNHA) and hemagglutinin subunits (HAs) HA-17, HA-33, and HA-70, yielding the final molecular weight of ~900 kDa 3. The majority of botulinum neurotoxin products available on the market are in the form of purified neurotoxin complexes, except for Xeomin®, which includes a 150 kDa neurotoxin without the complexing proteins 4. Meanwhile, as reported earlier, NAP can enhance the structural stability and activity of the neurotoxin (e.g., BoNT/A) 5− 8. Its stabilizing effect can even maintain its oral toxicity 9− 12. Nevertheless, the pharmacological effect is dependent on the neurotoxin dissociated from the complex 2,13,14.
Botulinum neurotoxin products such as Botox®, Dysport® and Xeomin® are marketed as vacuum- or freeze-dried powder set at pH around 7, whereas Myobloc® is a solution for injection set at pH around 5.6 15,16. Botox®, Dysport® and Xeomin® are commercially available botulinum toxin type A, and Myobloc® is type B, suggesting the conformational and colloidal stability of each toxin could differ with its types. Supportively, the conformational stability of therapeutic proteins and antibodies are optimal in very narrow pH range and their unique balances of protein-protein interactions induce different colloidal stability 17− 20. Besides, earlier studies suggested that the pH and ionic strength can be major driving forces for the dissociation of neurotoxin from its complex 13,14,21. Neurotoxins can readily dissociate from the complex within a minute, especially when exposed to alkaline pH, suggesting the existence of a free form (i.e., dissociated) of botulinum neurotoxin as reconstituted 22,23. At acidic pH, the protective activity of NAPs on the neurotoxin indicates they maintain their associated state with the neurotoxin 7,24,25.
On the other hand, all products mentioned contain human serum albumin (HSA) in the formulation to protect BoNT/A from nonspecific binding 26. It stabilizes the neurotoxic proteins during and after manufacturing, suppressing the aggregation of neurotoxic proteins 27. However, the inclusion of HSA introduces the risk of viral and other pathogens transmission since it is derived from humans along with the difficulty in maintaining a uniform quality 28. To counter this risk, recombinant HSA could be an alternative. However, challenges still exist in the analytical method development, as the selective analysis and quantification of the active proteins and their degradation products or aggregates are often difficult in presence of the secondary protein in the form of HSA. For the reasons, the market demands HSA-free formulations of botulinum toxins. Consequently, the stability of the toxins and the mechanism of its dissociation should be investigated thoroughly.
In fact, the existence of the neurotoxin as a complex with NAPs poses challenges, as each of the proteins in the complex portrays individual behavior in different solution conditions. Given their complexity, different methods intended to investigate their similar attributes are often necessary to provide independent confirmation of the protein properties. Enzyme-linked immunoassay (ELISA) has been widely used as a highly sensitive quantification tool for neurotoxins 29–32. However, the process is limited to detection and quantification of neurotoxin regardless of its associated or dissociated state as the neurotoxins are exposed to reagents at pH 7.2 to 7.4 during the analysis.
For size exclusion chromatography (SEC), there is no relevant report of its use in quantifying and discriminating associated and dissociated neurotoxin. Generally, this analysis has been conventionally used for the quantification of proteins in various states as monomers, oligomers, aggregates, and even fragments. The major benefit is the use of elution conditions that allow the characterization of proteins with the least impact in their local environment and conformational structure 33. In this study, the state of neurotoxin was investigated mainly using SEC to evaluate the dissociated BoNT/A from NAPs at different pHs, incubation times, and storage temperatures. Moreover, the impact of polysorbate 20 is investigated since it has been used as a stabilizer in marketed formulations and can be an excipient as a substitute for HSA 28,34,35. Additionally, multiangle light scattering (MALS) and ELISA were utilized further to interpret the SEC chromatograms. Lastly, dynamic light scattering (DLS) was used to access the size distribution and zeta potential of the complexes at different pHs.