Anouk Donners

27 Tutorial on LC-MS/MS methods quantifying mAbs Figure 1. A) Direct ELISA, B) Indirect ELISA and C) Sandwich ELISA. Top-down, middle-down and bottom-up quantitative proteomics Quantification with LC-MS offers various advantages over LBA and over the years various LC-MS strategies have been explored and reported. Three main strategies are discussed in detail namely topdown, middle-down and bottom-up proteomics. The flow chart in Figure 2 depicts the decision making process for the preferred strategy for quantification based on factors, such as the intended assay specifications in terms of sensitivity and selectivity and the applied instrumentation and materials. For therapeutic drug monitoring of trough levels, in most cases a LLOQ of 1 mg/L would suffice [38]. This would allow for the quantification of the peptide using LC-MS/MS or intact after selective purification using various instruments such as LC-HRMS, LBA or LC-FLD. However, for pharmacokinetic applications more sensitive assays are regularly required (e.g., 100 µg/L or less) to characterize the terminal elimination phase. Therefore, in most cases only LC-MS/MS using bottom-up proteomics to quantify the signature peptide or LBA after selective purification with anti-idiotypic antibodies would be suitable (Figure 2). 2