. 2022 Apr 6;12(4):216.

doi: 10.3390/bios12040216.

Near-Infrared Transflectance Spectroscopy Discriminates Solutions Containing Two Commercial Formulations of Botulinum Toxin Type A Diluted at Recommended Volumes for Clinical Reconstitution

Antonio Currà^{1

2}, Riccardo Gasbarrone³, Giuseppe Bonifazi^{2

4}, Silvia Serranti^{2

4}, Francesco Fattapposta⁵, Carlo Trompetto^{6

7}, Lucio Marinelli^{6

8}, Paolo Missori⁹, Eugenio Lendaro¹⁰

Affiliations

¹ Academic Neurology Unit, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04019 Terracina, Italy.
² Research Center for Biophotonics, Sapienza University of Rome, 04100 Latina, Italy.
³ Ce.R.S.I.Te.S.-Research and Service Center for Sustainable Technological Innovation, Sapienza University of Rome, 04100 Latina, Italy.
⁴ Department of Chemical Engineering, Materials & Environment, Sapienza University of Rome, 00184 Rome, Italy.
⁵ Neurology Unit, Policlinico Umberto I, Department of Human Neurosciences, Sapienza University of Rome, 00185 Rome, Italy.
⁶ Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16132 Genova, Italy.
⁷ Department of Neuroscience, Division of Neurorehabilitation, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy.
⁸ Department of Neuroscience, Division of Clinical Neurophysiology, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy.
⁹ Neurosurgery Unit, Policlinico Umberto I, Department of Human Neurosciences, Sapienza University of Rome, 00185 Rome, Italy.
¹⁰ Pathology Unit I.C.O.T., Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, 04100 Latina, Italy.

PMID: 35448275
PMCID: PMC9032888
DOI: 10.3390/bios12040216

Near-Infrared Transflectance Spectroscopy Discriminates Solutions Containing Two Commercial Formulations of Botulinum Toxin Type A Diluted at Recommended Volumes for Clinical Reconstitution

Antonio Currà et al. Biosensors (Basel). 2022.

. 2022 Apr 6;12(4):216.

doi: 10.3390/bios12040216.

Authors

Affiliations

¹ Academic Neurology Unit, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04019 Terracina, Italy.
² Research Center for Biophotonics, Sapienza University of Rome, 04100 Latina, Italy.
³ Ce.R.S.I.Te.S.-Research and Service Center for Sustainable Technological Innovation, Sapienza University of Rome, 04100 Latina, Italy.
⁴ Department of Chemical Engineering, Materials & Environment, Sapienza University of Rome, 00184 Rome, Italy.
⁵ Neurology Unit, Policlinico Umberto I, Department of Human Neurosciences, Sapienza University of Rome, 00185 Rome, Italy.
⁶ Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16132 Genova, Italy.
⁷ Department of Neuroscience, Division of Neurorehabilitation, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy.
⁸ Department of Neuroscience, Division of Clinical Neurophysiology, IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy.
⁹ Neurosurgery Unit, Policlinico Umberto I, Department of Human Neurosciences, Sapienza University of Rome, 00185 Rome, Italy.
¹⁰ Pathology Unit I.C.O.T., Department of Medical-Surgical Sciences and Bio-Technologies, Sapienza University of Rome, 04100 Latina, Italy.

PMID: 35448275
PMCID: PMC9032888
DOI: 10.3390/bios12040216

Abstract

Botulinum neurotoxin type A (BoNT-A) is the active substance in pharmaceutical preparations widely used worldwide for the highly effective treatment of various disorders. Among the three commercial formulations of BoNT-A currently available in Italy for neurological indications, abobotulinum A toxin (Dysport^®, Ipsen SpA, Milano, Italy) and incobotulinum A toxin (Xeomin^®, Merz Pharma Italia srl, Milano, Italy) differ in the content of neurotoxin, non-toxic protein, and excipients. Clinical applications of BoNT-A adopt extremely diluted solutions (10^-6 mg/mL) for injection in the target body district. Near-infrared spectroscopy (NIRS) and chemometrics allow rapid, non-invasive, and non-destructive methods for qualitative and quantitative analysis. No data are available to date on the chemometric analysis of the spectral fingerprints acquired from the diluted commercial formulations of BoNT-A. In this proof-of-concept study, we tested whether NIRS can categorize solutions of incobotulinum A toxin (lacking non-toxic proteins) and abobotulinum A toxin (containing non-toxic proteins). Distinct excipients in the two formulations were also analyzed. We acquired transmittance spectra in the visible and short-wave infrared regions (350-2500 nm) by an ASD FieldSpec 4™ Standard-Res Spectrophotoradiometer, using a submerged dip probe designed to read spectra in transflectance mode from liquid samples. After preliminary spectra pre-processing, principal component analysis was applied to characterize the spectral features of the two BoNT-A solutions and those of the various excipients diluted according to clinical standards. Partial least squares-discriminant analysis was used to implement a classification model able to discriminate the BoNT-A solutions and excipients. NIRS distinguished solutions containing distinct BoNT-A commercial formulations (abobotulinum A toxin vs. incobotulinum A toxin) diluted at recommended volumes for clinical reconstitution, distinct proteins (HSA vs. incobotulinum A toxin), very diluted solutions of simple sugars (lactose vs. sucrose), and saline or water. Predictive models of botulinum toxin formulations were also performed with the highest precision and accuracy.

Keywords: NIR spectroscopy; botulinum neurotoxin type A; chemometrics; partial least squares-discriminant analysis; transflectance spectroscopy.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.

Figures

**Figure 1**
Schematic representations of the utilized spectrophotometer (**left**) and the experimental set-up (**right**).

**Figure 2**
Average raw (a) and pre-processed (b) transmittance spectra for solutions of incobotulinum A toxin (inco100alb1sac46), abobotulinum A toxin (abo400alb01lat2), HSA (alb1), sucrose (sacc46), lactose (lact1), saline (NaC09), and water (AD). PC1−PC2 score plot (c) and loading plot of PC1 and PC2 (d) for incobotulinum A toxin (inco100alb1sac46), equiactive abobotulinum A toxin (abo400alb01lat2), HSA (alb1), sucrose (sacc46), lactose (lact1), saline (NaC09), and water (AD).

**Figure 3**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46) and abobotulinum A toxin (abo100alb0025lat05), and their excipients (EccInco, EccAbo). PC1−PC2 score plot (c) and loading plot of PC1 and PC2 (d) for incobotulinum A toxin (inco100alb1sac46), abobotulinum A toxin (abo100alb0025lat05), and their excipients (EccInco, EccAbo).

**Figure 4**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46) and equiactive abobotulinum A toxin (abo400alb01lat2), and their excipients (EccInco, EccAbo). PC1−PC2 score plot (c) and loading plot of PC1 and PC2 (d) for incobotulinum A toxin (inco100alb1sac46), equiactive abobotulinum A toxin (abo400alb01lat2), and their excipients (EccInco, EccAbo).

**Figure 5**
Average raw (a) and pre-processed (b) transmittance spectra for abobotulinum A toxin at standard clinical dilutions (abo100alb0025lat05), and its excipients (EccAbo). PC1−PC2 score plot (c) and loading plot of PC1 (d) for abobotulinum A toxin at standard clinical dilutions (abo100alb0025lat05), and its excipients (EccAbo).

**Figure 6**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46), and its excipients (EccInco). PC1−PC2 score plot (c) and loading plot of PC1 (d) for incobotulinum A toxin (inco100alb1sac46), and its excipients (EccInco).

**Figure 7**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46), and equiactive abobotulinum A toxin (abo400alb01lat2). PC1−PC2 score plot (c) and loading plot of PC1 (d) for incobotulinum A toxin (inco100alb1sac46), and equiactive Abobotulinum A toxin (abo400alb01lat2).

**Figure 8**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46) and abobotulinum A toxin (abo100alb0025lat05). PC1−PC2 score plot (c) and loading plot of PC1 (d) for incobotulinum A toxin (inco100alb1sac46), abobotulinum A toxin (abo100alb0025lat05).

**Figure 9**
Average raw (a) and pre-processed (b) transmittance spectra for incobotulinum A toxin (inco100alb1sac46), and HSA (alb1). Scores on PC1 vs. PC2 (c) and loading plot of PC1 (d) for incobotulinum A toxin (inco100alb1sac46), and HSA (alb1).

**Figure 10**
Average raw (a) and pre-processed (b) transmittance spectra for sucrose (sacc46), and lactose (lact1). PC1−PC2 score plot (c) and loading plot of PC1 (d) for sucrose (sacc46), and lactose (lact1).

**Figure 11**
Average raw (a) and pre-processed (b) transmittance spectra for saline (NaC09), and water (AD). PC1−PC2 score plot (c) and loading plot of PC1 (d) for saline (NaC09), and water (AD).

**Figure 12**
Positions of the discrimination boundary as determined by PLS−DA model (utilized wavelength range: 800−2400 nm) for the two classes: abobotulinum A toxin (a) and incobotulinum A toxin (b).

**Figure 13**
VIP scores determined from PLS−DA model for the two classes of abobotulinum A toxin (VIP scores for Y1) and incobotulinum A toxin (VIP scores for Y2).

**Figure 14**
Positions of the discrimination boundary as determined by PLS−DA model (utilized wavelength ranges: 811–991 nm, 1018–1111 nm, 1352–1414 nm, 1527–1722 nm) for the two classes: abobotulinum A toxin (a) and incobotulinum A toxin (b).

**Figure 15**
Molecular structure of BoNT-A complex reconstructed by fitting the three-dimensional structures of the BoNT-A and neurotoxin-binding protein (NTNHA) and HA70-HA-17-HA33 complex modules into the electron microscopy image. (Adapted with permission from Ref. [25]. Copyright 2013 Lee et al.).

See this image and copyright information in PMC

Cited by

Photonic Sensors in Chemical and Biological Applications.
Balevičius Z. Balevičius Z. Biosensors (Basel). 2022 Nov 15;12(11):1021. doi: 10.3390/bios12111021. Biosensors (Basel). 2022. PMID: 36421139 Free PMC article.

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Near-Infrared Transflectance Spectroscopy Discriminates Solutions Containing Two Commercial Formulations of Botulinum Toxin Type A Diluted at Recommended Volumes for Clinical Reconstitution

Affiliations

Near-Infrared Transflectance Spectroscopy Discriminates Solutions Containing Two Commercial Formulations of Botulinum Toxin Type A Diluted at Recommended Volumes for Clinical Reconstitution

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Conflict of interest statement

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