Conclusions
Our data clearly demonstrate that the free cysteine (Cys252) on the p40 subunit of recombinant IL-12 is also present in its cysteinylated and homocysteinylated forms at a considerable rate. Our observations, although based on results obtained on an IL-12-derived fusion protein, are consistent with the current literature.
Methods
Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra-high-performance liquid chromatography-MS analysis was performed on a Waters quadrupole time-of-flight Xevo G2-XS mass spectrometer acquiring data in MSE mode. MS data were processed using Expressionist MS Refiner 13.5 (Genedata).
Results
Scouting experiments were performed using two batches of drug substance. An in-depth study of the LC tandem mass spectrometry profiles revealed the presence of additional species related to "free" Cys252; this cysteine residue was also detected in its S-cysteinylated and S-homocysteinylated forms. This result is consistent with that reported in literature so far. The relative abundance of overall "cysteinylated" species resulted in the range between 46% and 36%, which has also been confirmed using orthogonal techniques such as Ellman's assay. Conclusions: Our data clearly demonstrate that the free cysteine (Cys252) on the p40 subunit of recombinant IL-12 is also present in its cysteinylated and homocysteinylated forms at a considerable rate. Our observations, although based on results obtained on an IL-12-derived fusion protein, are consistent with the current literature.