Immunoaffinity LC-MS/MS to Quantify a PEGylated anti-Factor D Fab Biotherapeutic in Cynomolgus Monkey Serum

Bioanalysis. 2019 Dec;11(23):2161-2173. doi: 10.4155/bio-2019-0082. Epub 2019 Nov 8.


Aim: To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Materials & methods: Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. Results & conclusion: The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.

Keywords: LC–MS/MS; PEGylated Fab; age-related macular degeneration; anti-Factor D; biotherapeutics; geographic atrophy; immunoaffinity; quantification; surrogate peptide.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / administration & dosage
  • Antibodies, Monoclonal / blood*
  • Antibodies, Monoclonal / immunology
  • Chromatography, Affinity
  • Chromatography, Liquid
  • Complement Factor D / administration & dosage
  • Complement Factor D / analysis*
  • Complement Factor D / immunology
  • Intravitreal Injections
  • Macaca fascicularis / blood*
  • Polyethylene Glycols / chemistry*
  • Tandem Mass Spectrometry


  • Antibodies, Monoclonal
  • Polyethylene Glycols
  • Complement Factor D