If accessory proteins dissociate, why Don't toxins behave the same? multiscale simulation shows divergent in silico profiles despite an identical core neurotoxin.

Botulinum neurotoxin type A formulations all contain the same 150 kDa core protein, yet they behave as if they were different drugs. Clinicians routinely observe differences in onset, spread, duration, and immunogenicity, despite the fact that accessory proteins dissociate quickly after injection. If dissociation were the full story, these products should act identically. They do not. Using a multiscale in silico AesthetiSIM™ platform and a 10,000-patient digital twin cohort. Simulations were performed under physiologic temperature (37 °C) and pH 7.4, with sensitivity analyses evaluating these conditions. Our simulations confirmed that dissociation alone cannot account for divergent clinical profiles. Instead, excipients and microenvironmental factors create distinct pharmacokinetic and immunologic landscapes that persist even when the neurotoxin core is identical. Lactose drove broader diffusion, sucrose stabilized local confinement, sodium chloride altered electrostatic spread, and the peptide RTP004 prolonged residence by binding extracellular proteoglycans. These formulation-specific interactions shaped receptor engagement, clearance, and immunogenicity, overturning the assumption that dissociation explains everything. The findings demand a shift in perspective: botulinum toxins are not defined solely by their neurotoxin, but by the excipient ecosystem in which they are delivered. This mechanistic framework explains why products remain "non-interchangeable" and shows that the paradox of dissociation is not a paradox at all; it is the predictable outcome of pharmacology embedded in formulation. As these outcomes are derived from computational modelling, they should be viewed as predictive and require confirmation through targeted biochemical, biophysical, and cell-based assays.