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 conjugate vaccine against SARS-CoV-2, produced using the maleimide-thiol chemistry. In this vaccine, the Cys ⁵³⁸ in the recombinant receptor binding domain (RBD) of SARS-CoV-2 is linked, through a thiosuccinimide linker, to lysine residues of tetanus toxoid (TT) preparation. LC-MS/MS analysis revealed that TT is a complex mixture of proteins, similar to other TTs where the detoxified tetanus neurotoxin (d-TeNT) has been shown to be the most abundant protein (30-56%), regardless the quantification method used. The fifteen most abundant proteins account for approximately 78% of the total proteins. LC-MS/MS analysis of the activation process showed that 102 out of 107 lysine residues in the d-TeNT incorporated a maleimide group. In contrast, when tryptic peptides isolated by Ni ²⁺ -NTA affinity chromatography, were analyzed by LC-MS/MS, only 22 Lys residues in the d-TeNT were cross-linked to the RBD C -terminal tryptic peptide ( ⁵³⁸ CVNF ⁵⁴¹ -HHHHHH), probably due to steric hindrance. Twelve and eighteen conjugation sites were assigned based on the identification of linear peptides carrying a conjugated lysine residues (Δm = +1454.58 Da or Δm = +1472.59 Da) and cross-linked peptides with stabilized linker forms, respectively. Eight conjugation sites were coincidently assigned by both strategies. The assignment of the conjugation sites was manually validated by observed regularities (z≥3+, XIC, immonium ions, specific linker fragment ions) not considered by the identification software (PEAKS, Kojak and pLink2). The RBD was also conjugated, but to a lesser extent, to ten other low abundance carrier proteins. To our knowledge, this work is the first report of conjugation site assignment in a TT-based conjugate vaccine.