Application of Middle-Down Approach in Quantitation and Catabolite Identification of Protein by LC–high-Resolution Mass Spectrometry

Abstract

Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.

Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top‐down and middle‐down mass spectrometry

“…Improvements in stationary phases [49][50][51][52][53][54][55], low-flow chromatography, and alternative front-end separations [56][57][58] have recently opened the way to higher sensitivity studies. For instance, both the qualitative and quantitative top-down and middle-down analysis of biotherapeutics extracted from plasma has been described in the last years [59][60][61][62][63]. However, the lower sensitivity of these protein-centric approaches, compared to the peptide-centric ones, still represents a challenge to their routine use in preclinical/clinical studies.…”