Table 4. Results of the Unstressed and Ethylene Oxide-Stressed BsMAb Samples with the mD-UPLC-MS/MS System^a.

^aFor the characterization of susceptible amino acids for the ethylene oxide adduct formation, stressed and unstressed samples were analyzed with the mD-UPLC-MS/MS system incorporating a ¹D CEX. The stressed sample was incubated for 7 days at 30 °C with 0.01% ethylene oxide, while the unstressed sample was incubated under the same conditions without ethylene oxide. The main and basic peaks of each sample were analyzed in triplicates, and the relative abundance was calculated with the area under the curve (AUC) values of the extracted ion chromatograms (XIC). The relative abundance of the modified peptide was calculated based on the XIC-AUC of the modified peptide divided by the total peak area of the unmodified and modified peptide. The arithmetic mean (n = 3) of the relative abundance is listed together with the standard deviation (SD). For the analysis, the combined enzyme digestion setup (trypsin, LysC) of the mD-UPLC-MS/MS system was used. The data analysis was accomplished with the PMI-Byos (Byonic) software version 4.0–53 (Protein Metrics Inc.) and two separate workflows for trypsin and LysC. For the peptide identification, MS/MS spectra were used. The precursor mass tolerance was set to 10 ppm, and a miss cleavage rate of one was permitted.