Toxins

Matrix effects (ME) during LC-ESI-MS analysis are a commonly acknowledged issue for a variety of matrices and analytes. Although sample preparation techniques are steadily evolving to reduce ME, the complexity and variability of the urine matrix remain a challenge, especially for multi-analyte methods. To investigate the extent of ME implications on method performance and quantification, we used stable isotope-labelled standards (SIL-IS) of 11 mycotoxins to evaluate the magnitude and variability of ME in urine samples from two cohorts: Bangladeshi adult women (n = 50) and Swedish children of both sexes (n = 340). Significant ME differences were observed between the two cohorts for eight of the 11 mycotoxins. Additionally, intra-cohort ME variability turned out to be very high with interquartile ranges (IQR) above 15% for 14 out of 22 analyte-cohort combinations. Maximum IQR values were observed for sterigmatocystin in the Bangladeshi cohort (318%), strongly impacting quantitative results obtained with matrix(-matched) calibration. Further experiments on a small German cohort of four subjects, each providing four to five urine samples, revealed high variability of ME within each individual. Factors influencing ME were investigated, showing little to no impact of sex and a moderate impact of age for some analytes in the Swedish cohort. Nonetheless, especially the more polar analytes, showing stronger signal suppression, demonstrated clear correlation of ME with density and creatinine concentration of the urine samples. As a result, urine samples with very high or low density or creatinine values require careful handling in regard to sensitivity or quantification errors when matrix(-matched) calibration without SIL-IS is applied.

DaxibotulinumtoxinA for injection (DAXI) is a botulinum neurotoxin (BoNT) drug product comprising the 150 kDa pure BoNT/A1 as the drug substance formulated with a proprietary stabilizing excipient, RTP004. We hypothesized that RTP004 facilitates localization of BoNT/A1 to the neuronal membrane, resulting in increased BoNT internalization and cleavage of the synaptosomal-associated protein of 25 kDa (SNAP-25) within synaptic terminals. We characterized the interaction between RTP004 and BoNT/A1 using in silico and in vitro techniques. In vitro analyses revealed that negative charges on the BoNT/A1 surface were located on the light chain (LC, the catalytic domain) and the C-terminus of the heavy chain (H_C, the receptor-binding domain), potentially providing sites for interaction with the positively charged RTP004 peptide. RTP004 bound to BoNT/A1, but not to human serum albumin (HSA), in both static and dynamic conditions. RTP004, not HSA, enhanced binding of BoNT to artificial membranes and RTP004 dissociated from BoNT under conditions that mimicked physiological conditions of the synaptic vesicle. RTP004 also increased binding of BoNT to the synaptosomal cell membrane and enhanced cleavage of SNAP-25 in a dose-dependent manner. These findings demonstrate that RTP004, not the excipient HSA common in other BoNT/A1 drug products, enhances binding of BoNT to the cell surface, facilitates internalization of BoNT into the cell, and increases SNAP-25 cleavage.

Tetanus toxin evaluation has traditionally relied on mouse LD₅₀ bioassays, which require extensive animal use and time, necessitating development of alternative methods in accordance with 3R principles (Replacement, Reduction, and Refinement). We developed and validated a sandwich enzyme-linked immunosorbent assay (ELISA) as an alternative to animal testing for evaluating tetanus toxin biological activity using 18 environmental and clinical isolates of Clostridium tetani, complemented by an immunochromatographic (IC) assay for rapid screening. The ELISA demonstrated excellent analytical performance with a lower limit of quantification of 2.4 ng/mL (equivalent to 85.4 LD₅₀/mL), favorable linearity (R² = 0.999), precision (CV < 1.7–8.2%), and specificity (<1% cross-reactivity with C. septicum, C. novyi, and C. perfringens). Correlation analysis between ELISA relative potency and observed minimum lethal dose values revealed a robust positive correlation (r = 0.974). Both parallel line assay and single-point quantification methods showed strong correlations with mouse bioactivity measurements (r = 0.998). The IC assay successfully detected all isolates within 15 min. The measurement range of 2.4–45.6 ng/mL effectively covered diverse toxin-production capabilities spanning a 600-fold concentration range. This validated ELISA and IC assay combination provides a reliable, rapid alternative to animal experimentation for tetanus toxin evaluation.