Highly Specific and Sensitive Measurements of Human and Monkey Interleukin 21 Using Sequential Protein and Tryptic Peptide Immunoaffinity LC-MS/MS

A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides using antipeptide antibodies, and quantification using nanoflow LC-MS/MS. This assay was developed and qualified in human and cynomolgus monkey serum and tissues with a lower limit of quantitation of 0.78 pg/mL based on the intact cytokine. Both intra- and interbatch precision and accuracy, as well as stability and recovery, were found to be acceptable. IL-21 was not detected in serum from normal healthy volunteers or from autoimmune disease patients. However, IL-21 levels were quantified in cynomolgus monkey spleen and colon tissue and normal and inflammatory bowel disease (IBD) human colon tissue as well as hyperplasia human tonsils.