Anouk Donners

33 Tutorial on LC-MS/MS methods quantifying mAbs instrument related factors such as injection, ionization and fragmentation. Depending on the IS used, different levels of corrections can be obtained (Table 2). Also in ELISA there have been attempts to incorporate IS in the assays [89]. However, the correction ability here is limited to dilution corrections only. In LC-MS/MS, SIL proteins can correct for the entire sample pretreatment and analysis because of their matching amino acid sequence and conformational folding and are considered the gold standard in quantitative proteomics [21, 90]. Unfortunately, these SIL variants of therapeutic proteins are often very expensive and only a limited number are commercially available. As an alternative, Nouri-Nigjeh and colleagues have shown that hybrid calibration, which use the therapeutic mAb of interest as ‘calibrator’ in combination with a SIL peptide or extended-SIL peptide as IS, can obtain accurate and precise results in whole sample digestion methods [90]. This observation was also supported by Prasad and Unadkat stating that SIL peptides can be used when maximum trypsin digestion is ensured [91]. The largest source of variability in this type of work-up originates from ionization suppression due to sample complexity. In contrast to the calibrator protein, flanking SIL peptides and extended peptides are easily dissolved in the sample matrix and due to the lack of structural folding and S-S bonds, provide easier access to the cleavage site. Therefore, the correction for digestion efficiency is expected to be very low using this approach. Furthermore, experiments performed in house using a regular SIL peptide and a SIL extended peptide showed that SIL peptide performed better than SIL extended peptide since the SIL extended peptide produced additional variability during digestion that was not correlated to variability found in calibrator protein digestion. Dimethyl labeling was used by Ji and coworkers and was found to be a cheap way of generating multiple labeled peptides from the protein calibrator [55]. However, reaction conditions need to be carefully optimized to obtain maximum labeling efficiency. This principle has not gained a lot of ground since cheap SIL labeled peptides with high purity can easily be obtained. Analogue proteins are also frequently used to correct for sample purification and digestion (Table 1). However, the peptides generated from these analogue proteins are not identical to the signature peptides of the target mAb. Therefore, differences in charge and or hydrophobicity could lead to suboptimal correction for clean-up and enrichment steps. Moreover, differences in protein folding, solubility and disulfide bond location between the calibrator protein and the analogue may only result in moderate correction for digestion if preceding protein reduction, alkylation and denaturation was suboptimal. Matrix effect correction for ionization relies on the elution order of the signature peptide and the IS. So, depending on the elution similarities between the signature peptide and the analogue peptide, varying levels of corrections can be achieved. This is also true for fragmentation correction, here similarities in amino acid sequences between the signature peptide and the analogue determine the levels of correction. Nevertheless, Li and colleagues have shown that when a selective purification is used, analogue proteins can perform better than SIL peptide or a SIL flanking peptide [56]. Here, sample recovery was the major contributor to the method error and therefore, by including an analogue protein that can experience the same losses as the calibrator protein, correction was achieved. Furthermore, variability in LC-MS/MS analysis is 2