Sequential immunoaffinity-LC/MS assay for quantitation of a therapeutic protein in monkey plasma

Immunocapture-LC/MS has recently been used for quantitating therapeutic proteins/peptides and biomarkers in various matrices. The advantages of LC/MS quantitation include high speci­ficity and selectivity, wide dynamic range, and less susceptibility to interference from endogenous matrix components. We present a highly sensitive sequential immunoaffinity-LC/MS assay for quantitation of a biotherapeutic protein (39 kD) in monkey plasma. The first immunocapture utilized a biotinylat­ed mouse anti-drug antibody to capture the drug in plasma. After tryptic digestion, a unique peptide from the drug was then captured by the sec­ond immunocapture using a mouse anti-peptide antibody for further sample purification. Samples analysis was performed on a microLC-triple quadrupole mass spectrometry system (MS/MS). Both immunocapture procedures were carried out in 96-well plates using a magnetic beads handler. The LLOQ of the assay is 50 pg/mL, which was approximately 100x more sensitive than a corresponding single immunocapture-LC/MS assay either using the anti-drug or anti-peptide antibody.