Multiplexed small molecule impurity monitoring in antibody-based therapeutics by mixed-mode chromatography paired...

With advanced genetic engineering technologies and better understanding of disease biology, antibody-based therapeutics are emerging as promising new generation biopharmaceuticals. These novel antibody formats are carefully designed to possess desired features such as enhanced selectivity. However, their high level of structural complexity with multiple components often leads to long development and complex multi-step manufacturing processes, through which a variety of potential small molecule impurities can be introduced. In this work, an in-process assay was developed in which mixed-mode chromatography coupled with charged aerosol detection was utilized for multiplexed detection of nine reagents commonly used in development and manufacturing of antibody-based therapeutics: isopropyl β-d-1-thiogalactopyranoside, methionine sulfoximine, ampicillin, guanidine, dehydroascorbic acid, glutathione, tris(2-carboxyethyl)phosphine, N-acetyl cysteine, and arginine. This method utilized a mixed-mode column with ion-exchange properties operated in the hydrophilic interaction chromatography mode. Various parameters were systematically optimized and under optimal conditions, the method demonstrated excellent specificity, sensitivity, linearity, precision, accuracy, and was successfully applied to determine residual impurities in multiple samples from antibody-derived molecules.