A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion

Abstract

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3-106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.