Anouk Donners

32 Chapter 2 reduction [88]. Nevertheless, care must be taken when these peptides are used since in vivo degradation of the therapeutic mAb via the above mentioned pathways can lead to an underestimation of circulating mAb as was demonstrated by Bults and colleagues in their research into deamidation of trastuzumab in human plasma [67]. After a list of potential signature peptide candidates is composed, a tryptic digest of the stock standard is preformed and run on LC-MS/MS to identify the various eluting peptides. An example of this is illustrated in Figure 4 where a number of infliximab peptides were identified using a shallow gradient (5-50% acetonitrile 0.1% formic acid in 20 min). It is important to note that due to the slow scan speed of the triple quadrupole mass spectrometer a high stock standards concentration (~100 mg/mL) should be digested. Figure 4. Signature peptides identification of infliximab tryptic digest with a triple quadrupole operating in full scan MS (300-1500 m/z) (A), GLEWVAEIR precursor elucidation though comparison with theoretical mass (B) and finally, conformation of the precursor sequence through fragmentation (20 eV) of [Mþ2H]2þ and product ion scan (150-1500 m/z) (C). Internal standard selection Methods that include an IS are able to correct for various factors causing variability during sample analysis, such as component losses during sample preparation as well as