28 Chapter 2 Figure 2. General recommendation for therapeutic mAb quantification depending on instruments and materials available and on assay requirements; Quantification on the basis of peptide (bottom-up) using LC-MS/MS or LC-HRMS and intact on the basis of middle-down and top-down strategies with LC-HRMS or intact on the basis of LBA, HPLC-FLD. Bottom-up quantitative proteomics is preceded by denaturation and enzymatic digestion of the therapeutic mAb which releases numerous peptides of different chain lengths. Peptides that are unique for the mAb (signature peptides) are selected for measurement. These peptides are easily and efficiently separated using a standard reverse-phase HPLC system and thereafter quantified on a standard triple quadrupole mass spectrometer. In general, the chromatographic peak shape of the peptides is more symmetrical compared to the peak shape of proteins due to fewer secondary interactions on the stationary phase. Top-down and middle-down quantitative proteomics is based on the measurement of intact or semi-intact proteins. Large proteins such as mAbs can shift to high charge states during electrospray ionization yielding a mass to charge (m/z) ratio within the working range ∼1800 − 4000 of a high resolution mass spectrometers (HRMS) such as Orbitrap or Time-Of-Flight (TOF). These methods do not require protein unfolding and enzymatic digestion which can be challenging and time-consuming to optimize.
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