title: Minimalistic sample preparation strategies for LC-MS quantification of large molecule biopharmaceuticals: a case study highlighting alpha-1 antitrypsin protein.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biomarker, Biomeasure & Mass Spectrometry Group, Pfizer Worldwide Research and Development, Andover, MA, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Wright
authors.forename: Dawn
authors.initials: D
authors.lastname: Dufield
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010455:Peptides; D000515:alpha 1-Antitrypsin; D014357:Trypsin