LC-MS/MS Characterization of SOBERANA 02, a Receptor Binding Domain-Tetanus Toxoid Conjugate Vaccine Against SARS-CoV-2

SOBERANA 02 is a safe and effective anti-SARS-CoV-2 conjugate vaccine, produced using the maleimide-thiol chemistry. In this vaccine, Cys⁵³⁸ in the recombinant receptor binding domain (RBD) of SARS-CoV-2 is linked through a N-thiosuccinimidopropionyl linker to lysine residues of tetanus toxoid (TT) preparation. LC-MS/MS analysis revealed the complex protein composition of TT. The fifteen most abundant proteins account for approximately 78% of the total protein mass. The detoxified tetanus neurotoxin (d-TeNT) was the most abundant protein (30%–56%) regardless of the quantification method used. LC-MS/MS analysis of d-TeNT activation reaction with BMPS (3-maleimidopropionic acid N-hydroxysuccinimide ester) showed that 102 (95%) of the 107 lysine residues incorporated a maleimide group. Of them, only 22 Lys residues (20%) were cross-linked to the RBD C-terminal tryptic peptide (⁵³⁸CVNF⁵⁴¹-HHHHHH), probably due to steric hindrance. Affinity chromatography prior to LC-MS/MS analysis was critical to identify the conjugation sites. Linear peptides carrying a conjugated lysine residue and type 2 peptides with stabilized linker forms (hydrolyzed and transcyclized), allowed the identification of twelve and eighteen conjugation sites, respectively. The RBD was also conjugated, but to a lesser extent, to ten other low-abundance carrier proteins. This study represents the first report of a conjugation site assignment in a TT-based conjugate vaccine.