Antibody drugs are effective therapeutic agents and provide treatment for many types of diseases such as cancer and rheumatoid arthritis. Because many antibody drugs are developed and approved, quantification technologies of antibodies are required for drug development and individualized therapy. We recently reported a high reproducible and robust therapeutic drug monitoring method for antibody drugs. This method was developed to be applicable to all type of antibody drugs and to provide accurate quantification values. The method is named nano-surface and molecular-orientation limited (nSMOL) proteolysis. nSMOL limits the access of protease to immunoglobulin G molecules and aims to selectively proteolyze on Fab region of substrate. However, the bioanalysis of Tocilizumab using nSMOL has not been validated. We newly discovered that acidified reduction pretreatment of Tocilizumab promotes digestive efficiency by nSMOL proteolysis. Exposure of Tocilizumab to Tris(2-carboxyethyl)phosphine hydrochloride before nSMOL proteolysis significantly improved the recovery of peptides. Under this condition, the quantification range of Tocilizumab in human serum was from 0.781 to 200 μg/mL. The quantification values of quality control samples fulfilled all guideline criteria for bioanalytical validation. The signature peptide with the highest quantitative sensitivity was on H-chain VTMLR. From these results, it is expected that the pretreatment with TCEP will broaden the application of antibody drugs quantification by nSMOL proteolysis.
Keywords: Acidified reduction; Bioanalysis; LC–MS/MS; Method validation; Tocilizumab; nSMOL.
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