Mass Spectrometry Protocol for the Absolute Quantification of a Monoclonal Antibody in Serum with Immunopurification

: We present here an analytical protocol for the sensitive, specific, and accurate absolute quantification of cetuximab, a human:murine chimeric monoclonal antibody, using mass spectrometry. Extraction from human serum is performed with micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of cetuximab. This specific immunocapture step allows sample purification and, in parallel, assessment of the antibody's biological potency. The eluted mAb is digested with trypsin and specific peptides from light and heavy chains are monitored by liquid chromatography coupled with tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode. The limit of quantification of the assay was 20 ng/mL in serum.