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