37 Tutorial on LC-MS/MS methods quantifying mAbs effective and fast way to remove unwanted proteins and was successfully employed for dinutuximab and infliximab quantification in human serum with LC-MS/MS [66, 85]. The major advantage of AS versus the widely used MeOH pellet digestion method (Table 1), is that the protein pellet retains its tertiary conformation allowing for fast re-solvation for reduction and alkylation [95]. Each of the above-mentioned methods has its merits and challenges. The targeted assay tends to be more time consuming and requires ligands or antibodies which can be scarce and expensive. However, the targeted assay provides lower detection limits because of clean extracts and measures the ‘active and free’ mAb fraction which likely associates with loss of treatment efficacy [72]. In contrast, generic sample purification methods are easy and fast to perform and measure total mAb fraction. Importantly, total and free fraction results can show poor correlation, especially when ADA are present in the sample [21, 96]. Therefore, correlation studies must be performed when switching from one assay to another. Nevertheless, there are numerous reports of good agreements between free fraction measured by ELISA and total fraction measured by LC-MS/MS [48, 56, 60, 78, 97], even in subsets consisting of ADA positive samples, as was demonstrated by Willrich and colleagues in the quantification of infliximab in human serum [66]. Table 3. Estimated and theoretical levels of purifications of human plasma proteins using different sample preparation strategies. Theoretical data for protein A and G purification obtained from Thermo Scientific [93]. Plasma Components Concentration in plasma [g/L] Targeted Purification Protein A Protein G AS Precipitation MeOH Pellet digestion Albumin (60 kDa) 45 ++ ++ ++ ++ + IgG (150 kDa) 10 ++ − − − − Fibrinogen (340 kDA) 2.5 ++ ++ ++ − − Transferrin (80 kDa) 2.5 ++ ++ ++ ++ + IgA (320 kDa) 2 ++ + ++ − − Alpha-1 Anti-Trypsin (54 kDa) 1.5 ++ ++ ++ ++ + Phospholipids (<1 kDa) 1 ++ ++ ++ ++ ++ IgM (900 kDa) 1 ++ + ++ − − IgD (180 kDa) 0.02 ++ ++ ++ − − IgE (200 kDa) 0.0002 ++ ++ ++ − − Notes: ++: efficiently eliminated; +: moderately eliminated; −: not eliminated. Abbreviations: AS: ammonium sulfate; MEOH: methanol. 2
RkJQdWJsaXNoZXIy MTk4NDMw