30 Chapter 2 Table 1. Published methods for peptide level quantification of therapeutic mAbs with LC-MS/MS. Sample type Internal standard used Rodent Monkey Human Analogue protein SIL peptide SIL protein Extended SIL peptide Dimethyl labeled [33, 47-53] [21, 54-64] [34, 65-85] [47, 63, 76, 79-81, 83] [34, 49, 53, 54, 57, 60, 64, 67-71, 73, 75, 78, 85] [21, 33, 48, 51, 52, 56, 59, 62, 72, 74, 77, 82] [58, 61, 65, 73] [55] Sample purification None (whole digest) Albumin depletion Pellet Digestion Protein A Protein G Anti-human FC antibody Anti-Idiotypic antibody Ligand [21, 47, 55, 60, 73, 78] [33, 54] [49-52, 57, 58, 61, 63, 64, 66, 67, 75, 85] [45, 53, 68-71, 80, 84] [65, 76, 81] [48, 56, 59, 60, 62] [34, 65] [72, 74, 77, 79, 82, 83] Digestion by denaturation Digestion by reduction & alkylation Guanidine Urea Surfactant TFE None TCEP and IAA DTT and IAA DTT [47] [21, 33, 48, 54-56, 59, 74, 82] [34, 50, 60, 62, 73, 78, 85] [66] [49, 51, 57, 64, 67, 71, 75, 82, 83] [33, 48, 60, 65, 77] [21, 34, 47, 50, 52-56, 58, 59, 61-63, 66, 73, 76, 78, 80, 81, 85] [72, 79] Post digest SPE cleanup Mass spectrometer LLOQ method performance RP Ion exchange Triple Quad Q-Trap HRMS ≤0.5mg/L ≤1 mg/L >1mg/L [33, 47, 48, 75, 78] [47, 52, 60, 61, 63, 73] [21, 34, 47, 50, 51, 53, 55, 56, 58-62, 64-68, 71-76, 79-81, 85] [33, 48, 49, 54, 57, 63, 78, 82] [52, 77, 83] [34, 47, 48, 50, 53, 56, 59, 62, 65, 67, 71, 77, 84, 86] [49, 52, 55, 57, 65, 68, 74, 75, 81-83, 85] [21, 33, 51, 54, 58, 60, 63, 64, 66, 73, 76, 78, 80] Abbreviations: SIL: stable isotopically labeled; TFE: trifluoroethanol; TCEP: tris(2-carboxyethyl)phosphine; IAA: iodoacetamide; DTT: dithiothreitol; SPE: solid phase extraction; RP: reversed phase.
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