Practical Application of Novel Test Methods to Evaluate the Potency of Botulinum Toxin: A Comparison Analysis among Widely Used Products in Korea

Abstract

The safe and effective dosing of botulinum neurotoxins (BoNTs) requires accurate and reliable methods to measure their potency. Several novel methods have been introduced over the past decade; however, only few studies have compared the potency of BoNT products with that of the LD50 and other alternative assays. Therefore, the objective of this study was to comparatively evaluate widely used BoNT products using various test methods. Four types of BoNTs (prabotulinumtoxin A, onabotulinumtoxin A, neubotulinumtoxin A, and letibotulinumtoxin A) were used in this study. The estimated potency was assessed using the LD50 assay, and the total BoNT type A protein levels were measured using the enzyme-linked immunosorbent assay (ELISA). The in vitro efficacy of the BoNTs was determined using fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays. The results showed differences in the total amount of BoNT protein and the cleavage activity of SNAP-25 within all types of BoNTs. The SPR study seemed to be useful for evaluating the potency by specifically measuring intact 19S neurotoxin, and these results provide new insights for assessing different BoNT products.

Keywords: LD50; botulinum toxin; endopeptidase; potency; rapid detection.

Figure 1

Graph summarizing the total amounts of BoNT/A protein present in each of the 100 unit vials indicated. Each dot represents one 100 unit vial of the products. n = 21 for Prabotulinumtoxin A (PraBoNT/A; Batch numbers used, 991451, 991452, 991453, 995158, 995159, and 995160), n = 15 for Onabotulinumtoxin A (OnaBoNT/A; Batch numbers used, C3312 C2F, C3603 C2, C3718 C3, C4049 C3, and C3718 C3F), n = 13 for Neubotulinumtoxin A (NeuBoNT/A; Batch numbers used, FAA1559, FAA15110, FAA16081, FAA16107, FAA16108, and FAA16090), and n = 13 for Letibotulinumtoxin A (LetiBoNT/A; HUA15030, HUA15058, HUA15162, HUA15195, HGA17021, and HGA17003). The bars represent average ± standard deviation (SD). **** p-value < 0.0001; ns = non-significant.

Figure 2

(A) Image depicting the principle of FRET-based analysis used in this study. (B) FRET-based BoNT/A activity assay. The y-axis value, OD 526 nm (reflecting the uncleaved SNAP- 25)/OD 470 nm (reflecting the cleaved SNAP-25), is inversely correlated to toxin activity. Prabotulinumtoxin A (PraBoNT/A), green; Onabotulinumtoxin A (OnaBoNT/A), purple; Neubotulinumtoxin A (NeuBoNT/A), red; Letibotulinumtoxin A (LetiBoNT/A), blue; Standard (the mean of OnaBoNT/A). n = 12 for Prabotulinumtoxin A, n = 9 for Onabotulinumtoxin A, Neubotulinumtoxin A, and Letibotulinumtoxin A.

Figure 3

SPR-based BoNT/A efficacy measurements. (A) Results of the kinetic assay. Graph summarizing K_D values reflecting the toxin activity. Each dot represents a 100-unit vial of the products. n = 36 for prabotulinumtoxin A (PraBoNT/A; Batch numbers used, 991451, 991452, 991453, 995158, 995159, and 995160), n = 30 for onabotulinumtoxin A (OnaBoNT/A; Batch numbers used, C3312 C2F, C3603 C2, C3718 C3, C4049 C3, and C3718 C3F), n = 36 for neubotulinumtoxin A (NeuBoNT/A; Batch numbers used, FAA1559, FAA15110, FAA16081, FAA16107, FAA16108, and FAA16090), n = 36 for Letibotulinumtoxin A (LetiBoNT/A; Batch numbers used, HUA15030, HUA15058, HUA15162, HUA15195, HGA17021, and HGA17003). The bars represent average ± standard deviation (SD). ns, not significant; **** p < 0.0001. Unpaired two-tailed t-tests. (B) Representative SPR sensograms from the anti-SNAP-25 mAb binding to the SNAP-25 AA 1–197 fragment (0.35–70 μg/mL) over a range of high (green) and low (red) concentrations: 0.35, 0.7, 1.4, 3.5, 7, 14, 35, and 70 μg/mL were tested. The sensograms were normalized to fit a 1:1 binding model using the Biacore T200 evaluation software (GE Healthcare Life Sciences). Prabotulinumtoxin A, PraBoNT/A; onabotulinumtoxin A, OnaBoNT/A; neubotulinumtoxin A, neuBoNT/A; and letibotulinumtoxin A, LetiBoNT/A.