title: LC-MS/MS quantification of next-generation biotherapeutics: a case study for an IgE binding Nanobody in cynomolgus monkey plasma.
abstract: BACKGROUND
  Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented.
  
  
  RESULTS
  Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated.
  
  
  CONCLUSIONS
  A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.
authors.affiliation: R.I.C., President Kennedypark 26, 8500 Kortrijk, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Sandra
authors.forename: Kjell
authors.initials: K
authors.lastname: Mortier
authors.forename: Lucie
authors.initials: L
authors.lastname: Jorge
authors.forename: Luis C
authors.initials: LC
authors.lastname: Perez
authors.forename: Pat
authors.initials: P
authors.lastname: Sandra
authors.forename: Sofie
authors.initials: S
authors.lastname: Priem
authors.forename: Sofie
authors.initials: S
authors.lastname: Poelmans
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Bouche
mesh_terms: D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007073:Immunoglobulin E / Q000097:blood* / Q000737:chemistry / Q000276:immunology*; D008252:Macaca fascicularis; D061905:Single-Domain Antibodies / Q000097:blood* / Q000737:chemistry / Q000276:immunology*; D053719:Tandem Mass Spectrometry
chemical_list: D061905:Single-Domain Antibodies; D007073:Immunoglobulin E