Characterization of disulfide bridges pattern of recombinant human interleukin 12 fusion protein p40 subunit and identification and quantification of cysteinylated free cysteine 252

RATIONALE: We show evidence of cysteinylation on Cys252 of recombinant human p40 subunit of interleukin 12 (IL‐12). This was reported in 1996. However, no paper detailing this concept has been published yet. Our paper reports the quantification of Cys252 cysteinylation as well as the full disulfide bridges assignment by nonreducing peptide mapping using mass spectrometry (MS) detection. 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 MS(E) 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.