Two-Dimensional Liquid Chromatography After 40 Years: State-of-the-Art, and Future Prospects

In this morning session, experts from both academic and industrial laboratories with substantial experience in two-dimensional liquid chromatography (2D-LC) will describe ways that 2D-LC is impacting their workflows for small- and large-molecule analysis, as well as developments in instrumentation, and optimization, and data analysis for 2D-LC. The invited session, presided over by Dwight Stoll of Gustavus Adolphus College, will be held in Room 179A.

Kelly Zhang of Genentech opens the proceedings with a discussion on the implementation of 2D-LC in pharmaceutical research and development. Case studies of many aspects of drug research and development for a wide variety of drug modalities will be examined, as well as a strategy for implementing 2D-LC that goes beyond traditional peak capacity and orthogonality to expand its power to improve analytical efficiency.

Bob Pirok of the University of Amsterdam follows with an examination of tools to facilitate the proliferation of 2D-LC. In this presentation, recent notable developments in the field of comprehensive 2D-LC (LC×LC) are discussed, and the challenges associated with data analysis and method development will be addressed. Pirok will then demonstrate his group’s newly developed peak-tracking algorithm for 2D-LC data, showing its usefulness within the context of both rapid automated data analysis and method development. Finally, the application of the algorithm on real cases of data will be demonstrated, including a LC×LC separation of antibody digests.

Next, Alexandre Goyon of Genentech discusses the implementation of a multidimensional LC–MS setup for a faster and more effective characterization of biotherapeutic products. This work examines how multidimensional LC–MS workflows can be effectively implemented for the characterization of mAb variants by combining the analysis at the reduced intact and peptide levels within the same system.

After a short recess, the program continues with Stoll presenting recent advances in analytical scale peptide separations by 2D-LC. In this presentation, Stoll will discuss results from recent work performed by his group to understand and break through the existing obstacles to higher performing analytical 2D-LC separations of peptides, showing that peak capacities exceeding 10,000 are now possible for online 2D-LC separations in four hours. On the other end of the spectrum, sub-10-second second dimension separations enable high quality 2D-LC separations in less than 20 minutes. 

The session concludes with Zachary Dunn of Merck & Company examining rapid 2D protein-A size exclusion chromatography (ProA-SEC) of monoclonal antibodies (mAb) for titer and aggregation measurements. This work focuses on the development of a 2D protein-A size exclusion chromatography method using entirely commercially available instrumentation. Each individual separation and the transfer of material between dimensions were optimized to develop a method that measures titer and aggregation of a target antibody from HCCF in under 5 minutes. Dunn and his associates determined the effects of each parameter of the method on mAb recovery and stability, as well as speed, robustness, resolution, and accuracy of %HMW in the second dimension.