Development and validation of a robust RP-HPLC method to quantitate residual 2-mercaptoethylamine in drug product formulations containing amino acid additives.

Abstract

Bispecific antibodies have a wide range of applications in cancer immunotherapy, some of which are manufactured by controlled Fab-arm exchange requiring the reductant 2-mercaptoethylamine (2-MEA). As a process impurity, monitoring the residual 2-MEA in bispecific antibody drug product process development is needed. A novel reversed phase-high performance liquid chromatography (RP-HPLC) method for measurement of residual 2-MEA that uses 7-fluorobenzofurazan-4-sulfonic acid ammonium salt (SBD-F) as a fluorescent-detection tag in drug product formulations containing high concentrations of arginine has been developed. Using a thiol tag for residual 2-MEA eliminates any potential interference from conventional tag binding to amine groups of the formulation arginine, and potentially resulting in overestimation of the amount of impurity in a given sample. The new method has been fully validated for specificity, linearity, accuracy, range, limit of quantitation, limit of detection, and robustness. This method therefore has potential to aid in detecting residual 2-MEA content for any process that utilizes 2-MEA for bispecific antibody manufacturing.