Liquid chromatography/mass spectrometry (LC/MS) method is becoming an important approach for therapeutic antibody assays as an alternative to the ligand-binding assay (LBA) method. The LC/MS method has some advantages over the LBA method, such as a wider dynamic range and short developing time. However, the development of the LC/MS method is often challenging because of complicated sample preparation processes involving affinity purification, denaturation, reduction and methylation, enzymatic digestion, and peptide purification. In addition, it is difficult to select a sensitive and specific surrogate peptide that allows the determination of the lower limit of quantitation of the analytical target. Another issue remains in the bioanalytical method validation (BMV) of the LC/MS method for large molecules. The BMV guideline on the LC/MS method for small molecules and that on the LBA method are helpful while developing a bioanalytical method for large molecules using LC/MS; however, these guidelines lack inherent characteristics related to bioanalysis of large molecules by the LC/MS method. In this review, we describe points to be considered regarding selection of surrogate peptides and optimization of the sample preparation processes in the LC/MS method for therapeutic antibody assays. Furthermore, we propose criteria for BMV of the LC/MS method. We expect that this review will aid in the development of sensitive, specific, and robust bioanalytical LC/MS methods for therapeutic antibodies.
