Bioanalysis Zone

Minimalistic sample preparation strategies for LC–MS quantification of large molecule biopharmaceuticals: a case study highlighting alpha-1 antitrypsin protein


Background: Large molecule biotherapeutics pose a distinctive bioanalytical challenge for LC–MS assay development, particularly when optimizing sample enrichment steps. Alpha-1 antitrypsin (AAT) is used as an example for highlighting large-molecule assay-development strategies. Results: Two sensitive and selective LC–MS/MS-based quantification assays were developed. Fit-for-purpose assay qualifications for BAL and serum matrices were performed by assessing sensitivity, precision and accuracy, dilution linearity and interferences. Conclusion: Our approach to sample preparation focuses on optimizing the simplest methodology necessary to generate fit-for-purpose bioanalytical assays. To measure AAT protein levels in preclinical species with selectivity and increased assay sensitivity, a minimalistic sample preparation strategy was adopted that included either traditional direct digestion or a more complicated immunoprecipitation enrichment process.

Over the last decade, biologics as novel chemical entities are playing larger roles in discovery pipelines. ligand-binding assay platforms have for years been successfully utilized for protein quantification with LC–MS/MS emerging as a complimentary technique for quantifying large molecule constructs. This technique of surrogate peptide quantification has substantial precedence in the pharmaceutical industry [1–6]. In general, assay challenges associated to LC–MS are typically not MS-based issues but more often related to sample preparation.

Click here to view the full article.


Leave A Comment

Please wait...

Bioanalysis Zone New Investigator Award

Why not start this year by doing something good by nominating an early career scientist for the Bioanalysis Zone New Investigator Award. Complete the details below to register your interest!