Quantification of residual hydrophobic fusion peptide with monomer and dimer forms using reversed-phase liquid...

A fusion peptide mimicking a part of the sequence of HIV-1 envelope glycoprotein with an additional cysteine at its C-terminus (FP8: AVGIGAVFC) was conjugated to a carrier protein through a linker for development of an HIV-1 vaccine. Since this fusion peptide is very hydrophobic with poor solubility and can self-dimerize via a disulfide bond, co-existence of monomeric and dimeric forms presented a major challenge for residual unconjugated FP8 quantification. A reversed-phase liquid chromatography (RPLC) with UV detection was developed to monitor residual FP8 using an experimental correction factor of 0.85 for UV peak area measurement between FP8 dimer and monomer. Therefore, both forms of unconjugated residual FP8 can be measured based on a single FP8 monomer reference curve. Overall, this study demonstrated that the current purification process can remove free residual FP8 to a low level, <20 µg/mL, which showed negligible impact (<10%) for the conjugated FP8 ratio measurement using another method, amino acid analysis.