By Alireza Aghayee, Yamin Htet, Stephan M. Koza, Lindsay Morrison, Henry Shion, Ying Qing Yu; Waters Corporation
Monoclonal antibodies (mAbs) remain one of the fast-growing classes of biopharmaceuticals and are significantly improving the quality of life for patients all around the world. Discovery and development of successful mAb therapeutics requires sophisticated analytical technologies that can rapidly measure the critical product attributes that have profound impact on the safety and efficacy of these drugs. Therefore, high-throughput analytical platforms for the monitoring of proteins produced at the clone selection stage and during process development have been increasingly in demand.
Due to their production in host cell cultures, large sizes, and heterogenous structures monitoring mAb production raises many analytical challenges. Selection of an appropriate clone typically requires parallel incubation of tens to hundreds of transfected lines, requiring many samples to be analyzed. The optimization of cell culture conditions typically also requires parallel cultures with analytics needed for each culture at least once per experiment. This requires high-throughput methods with fast sample preparation and robust analytical instrumentation, along with facile and straightforward data interpretation. As an integral part of the method, automated sample preparation protocols can increase the sample throughput and improve the efficiency of analytical workflows in biopharmaceutical development.
Here, we present a fully automated workflow for sample preparation and LC-MS analysis of mAbs obtained directly from complex samples such as spent cell culture media including host cell protein. The method includes a mAb purification step using Protein A followed by FabRICATOR® (IdeS) digestion and subsequent DTT reduction to yield mAb subunits suitable for high-throughput LC-MS analysis using a Waters™ BioAccord™ LC-MS System.