Browsing: quantification

Large molecule quantification by LC–MS PPD IMage
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Monoclonal antibody (mAb) based biotherapeutics have emerged as a dominant class of approved medicines during the past decade in both market share and variety of target diseases. Ligand binding assays (LBAs), such as enzyme-linked immunosorbent assays (ELISA), have been the gold standard for the bioanalysis of mAbs in support of non-clinical and clinical trials. While conventional LBAs provide highly sensitive and specific detection for the quantitation of mAbs, combining an LBA with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection offers additional orthogonal dimensions of selectivity/specificity, extending the assay utility to a multiplexed format otherwise not achievable by LBA alone.

Large molecule quantification by LC–MS LGC_Glu-c_an_orthogonal_and_alternative_enzyme_for_protein_quantitation_SS
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To date, almost all protein LC-MS/MS based bioanalysis has involved the use of trypsin to cleave biopharmaceuticals into manageable peptides for quantitation. In some cases, where trypsin does not generate useful peptide sequences, a different cleavage agent is required. Glu-C is an enzyme that is frequently used for protein characterisation, and cuts proteins on the C-terminal side of the aspartic and glutamic acid residues. These two molecules are acidic amino acids, whereas the ubiquitous trypsin cleaves at the basic residues (lysine and arginine), therefore making this an orthogonal cleavage enzyme.

Large molecule quantification by LC–MS
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In this application note, Yun Alelyunas (Waters Corporation) describes an LC-HRMS method for high sensitivity bioanalysis of monoclonal antibodies by direct mass measurement at the intact level for whole molecule quantification. This emerging approach can serve as an alternative or complement to traditional methods such as ligand binding assay for protein quantification. Methodology and guidance for evaluating and optimizing data processing parameters are discussed in detail.

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