Anouk Donners

29 Tutorial on LC-MS/MS methods quantifying mAbs However, top-down and middle-down methods do have some limitations. Firstly, targeted sample purification with anti-idiotypic antibodies or ligands is necessary due to structural similarities between the therapeutic mAb and endogenous IgG. Secondly, the required HRMS apparatus is expensive compared to the triple quadrupole mass spectrometer. Finally, attaining the required lower limit of quantifications (LLOQ) can be challenging because of wider precursor charge distribution, broadened chromatographic peak shape of large proteins, and mAb glycoform heterogeneity [39-41]. Some additional steps can be implemented to gain higher signals and thus lower LLOQ’s. For example, human immunoglobulin G1 in rat serum was successfully quantified with LC-HRMS after target specific purification with anti-idiotypic antibodies followed by deglycosylation [32]. Here, a remarkable LLOQ of 0.1 mg/mL was achieved through this strategy by utilizing a high sample volume (50 mL) in combination with high volume injection (60 mL) and 1mm diameter analytical column. Top-down quantification can also be performed by HPLC coupled to fluorescence detector (FLD). For example, intact trastuzumab, bevacizumab and infliximab in human serum were successfully quantified by HPLC-FLD after targeted purification [42, 43]. Unlike HRMS methods, FLD methods are not affected by signal dilution caused by charge distributions. However, fluorescents measurements have a low specificity. Most proteins have similar excitation and emission spectra which result in noisy and overlapping chromatographic peaks. Furthermore, because of the lower sensitivity, a higher sample volume (~100 mL) is required which limits its applicability. Middle-down strategies can also be used to reduce precursor charge distribution found in intact analysis. Here, only a portion of the mAb is measured such as the light chains after dithiothreitol (DTT) reduction, or Fab regions after limited Lys-C digestion [44, 45]. In contrast to intact mAb measurement, these regions are smaller and are usually free from glycan chains, leading to fewer precursor ions resulting in an increase in signal intensity of the mAb. As can be seen from Figure 2, peptide level quantification via LC-MS/MS or LC-HRMS instruments have low requirement and are thus frequently employed. This approach has been extensively used for numerous mAbs with great success. However, for some fully human/humanized therapeutic mAbs, quantification via signature peptide can be challenging because of the human polyclonal serum background [45]. In these situations, a targeted purification followed by intact or peptide level quantification might be preferred. Bottom-up quantification Currently, bottom-up quantification of mAbs in biological matrices using signature peptides is the most common approach. This principle offers fast, easy and flexible method development with high detection sensitivity using standard triple quadrupole mass spectrometers. 2