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J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jun 1;1054:80-92. doi: 10.1016/j.jchromb.2017.04.009. Epub 2017 Apr 4.

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures.

Author information

1
Université de Lyon, Université Claude Bernard Lyon 1, Faculté de Pharmacie - ISPB, EA 4446 Bioactive Molecules and Medicinal Chemistry, SFR Santé Lyon-Est CNRS UMS3453 -INSERM US7, 8 avenue Rockefeller, F-69373, Lyon Cedex 8, France; Reaction and Coupling Chemistry Laboratory, MTech, Sanofi Pasteur, 31/33 quai Armand Barbès, 69250 Neuville-sur-Saône, France.
2
Structural Analysis Laboratory, MTech, Sanofi Pasteur, 31/33 quai Armand Barbès, 69250 Neuville-sur-Saône, France.
3
Reaction and Coupling Chemistry Laboratory, MTech, Sanofi Pasteur, 31/33 quai Armand Barbès, 69250 Neuville-sur-Saône, France.
4
Analytical Research and Development Department, Sanofi Pasteur, Campus Mérieux,1541 Avenue Marcel Mérieux, 69280 Marcy L'Etoile, France.
5
Université de Lyon, Université Claude Bernard Lyon 1, Faculté de Pharmacie - ISPB, EA 4446 Bioactive Molecules and Medicinal Chemistry, SFR Santé Lyon-Est CNRS UMS3453 -INSERM US7, 8 avenue Rockefeller, F-69373, Lyon Cedex 8, France. Electronic address: marc.le-borgne@univ-lyon1.fr.

Abstract

Aldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde.

KEYWORDS:

Amino acid; Analytical tools; Formaldehyde; Tetanus toxin; Tetanus toxoid; Vaccine

PMID:
28448854
DOI:
10.1016/j.jchromb.2017.04.009
[Indexed for MEDLINE]

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