Determination of degradation for sulfo-SIAB, SM(PEG)₂, and sulfo-GMBS by RPLC-UV analysis

Bi-functional N-Hydroxysuccinimide (NHS) linkers are widely used in the conjugation processes linking an immunogen with a carrier protein capable of boosting immunity. A potential vaccine candidate against HIV-1, called fusion peptide (FP), is covalently linked to the recombinant tetanus toxoid heavy-chain fragment C (rTTHC) via this type of linker. A reversed-phase liquid chromatography (RPLC-UV) method was used to monitor the linker's degradation kinetics in various buffers, mimicking the steps in the conjugation process. The kinetics of the reactivities of the linkers are revealed in this study and can provide a good guidance to help effective conjugation process before these linkers are completely hydrolyze to the inactive degradants. Three cross-linkers degradation pathways were evaluated: Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB), PEGylated SMCC (SM(PEG)₂), and N-γ-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS). We have reported kinetics for Sulfo-SIAB.