Quantitative analysis of residual aurintricarboxylic acid in biotherapeutic process streams using liquid chromatography triple quadrupole mass spectrometry.

Aurintricarboxylic acid (ATA) is an excipient that can be added to the therapeutic protein manufacturing process as a component of the Chinese hamster ovary (CHO) cell culture media. ATA inhibits cell apoptosis and promotes cell growth in both serum-free and protein-free media. The addition of ATA is beneficial to the manufacturing process at the cell growth stage, however, residual ATA not consumed by cells can have toxicological effects on patients and its removal is required during downstream processing. To ensure manufacturing control and patient safety, the determination of residual ATA during downstream processing of biotherapeutics is required. The aim of this study was to develop and validate a liquid chromatography triple quadrupole mass spectrometry method for monitoring process clearance of ATA during downstream processing. Chromatographic separation of ATA was achieved using a 50 × 3 mm, 3 µm Imtakt Cadenza HS-C18 column and a triple quadrupole mass spectrometer operated in selected reaction monitoring mode was used for sensitive and specific detection. Linearity was assessed over a range of 62.5 ng/mL to 2000 ng/mL. Accuracy and precision were within 20% of the theoretical spike levels across the three concentration levels evaluated utilising two different antibodies, an IgG1 and an IgG4. Analyte specificity and selectivity were deemed acceptable based on no extraneous peaks detected. The method was successfully applied to monitoring process clearance of ATA in a tangential flow filtration (TFF) unit operation for downstream processing of monoclonal antibodies.