2D or Not 2D - Agilent 2D-LC Forum

Summary

Recent Advances in 2D-LC for Biopharmaceutical Analysis - Fundamental Studies and Practical Applications

Characterization of biomolecule (e.g., peptides, proteins, oligonucleotides) mixtures is typically quite challenging due to the heterogeneity of the molecules themselves (e.g., due to extensive glycosylation), and/or the matrices in which they are presented to the analytes (e.g., cell culture supernatant). Increasingly, two-dimensional liquid chromatography (2D-LC) methods are being used to improve the depth of characterization of these mixtures, improve the speed of these analyses, or both. This seminar will provide an overview of the key concepts required for successful 2D-LC method development, and several examples of recently developed 2D-LC separations of biomolecules.

Learning Objectives:

• Learn about innovative ways 2D-LC is being used to characterize molecules ranging from oligonucleotides to proteins
• Learn about the most recent fundamental studies that support development of better 2D-LC methods
• Learn about the trend in the use of multi-dimensional liquid chromatography to automate deep characterization of mAbs and other proteins

Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)

Dwight Stoll is Professor of Chemistry at Gustavus Adolphus College in St. Peter, MN. He has authored or co-authored 74 peer-reviewed publications and four book chapters in separation science, and over 130 conference presentations. His primary research focus is on the development of two-dimensional liquid chromatography (2D-LC) for both targeted and untargeted analyses. Within this area he has made contributions on many aspects of the technique including stationary phase characterization, biopharmaceutical analysis, new 2D-LC methodologies and instrumentation, and fundamental aspects including re-equilibration in gradient elution reversed-phase liquid chromatography and analyte focusing. He has taught several short courses on 2D-LC in recent years at venues including Pittcon and the international HPLC 20XX series. He is the 2011 recipient of LCGC’s Emerging Leader in Chromatography Award, the 2015 recipient of the American Chemical Society Division of Analytical Chemistry Award for Young Investigators in Separation Science, and in 2017 he received the Georges Guiochon Faculty Fellowship.

Two-Dimensional Liquid Chromatography (2D-LC): A Tutorial focusing on Best Practices - Part 1

Protein biologics such as mAbs now represent a significant share of pharmaceutical sales and future growth potential, particularly in an era of increasing patent expirations. LC/MS is a powerful analytical method for characterizing therapeutic proteins. In this presentation, we will show the application of high resolution and high mass accuracy Q-TOF for intact mAb measurement, peptide mapping, and characterization of PTM (Post-Translation Modifications). Fast separation using AdvanceBio peptide mapping columns with a state-of-the-art HPLC system will also be highlighted.

Learning Objectives:

• Learn about different modes of 2D-LC and how these are enabled by recent advances in instrumentation
• Learn about column (stationary phase and dimensions) selection and optimizing usage of available 2D separation space

Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)

Two-Dimensional Liquid Chromatography (2D-LC): A Tutorial focusing on Best Practices - Part 2

Monoclonal antibodies (mAb) are glycoproteins of the immunoglobulin (Ig) family. They can be used as therapeutics for the treatment of several life-threatening conditions such as cancer, inflammatory diseases, complications arising after organ transplantation, infectious, and cardiovascular diseases. Like all other protein therapeutics, mAbs can undergo aggregation during production, storage and transportation. Aggregation of therapeutic proteins is undesirable since it can lead to activity loss, decreased solubility, and enhanced immunogenicity. Usually, run under isocratic conditions, SEC method development is fairly straightforward. However, many analysts do not fully explore how changes in system volume effect sizing chromatography. Discover more about the new 1290 Bio LC and learn how minimizing dispersion volume improves resolution.

Learning Objectives:

• Learn about the mobile phase mismatch problem, and different strategies to minimize its effects
• Learn about important method development fundamentals, including loop filling and interface operation
• Learn about strategies for approaching developing 2D-LC methods

Presenter: Dwight R. Stoll, Ph.D. (Professor of Chemistry, Gustavus Adolphus College)

Ask the Experts

Still have 2D-LC questions? Regardless of whether they are hardware, software or application based, bring your questions and ask our expert panelists!

Experts: Dwight Stoll, Stephan Buckenmair, Ulrich Ederhardinger

Presenter: Dr. Stephan Buckenmaier (Principal Scientist, Agilent Technologies, Inc.)

Stephan has received his PhD from the University of the West of England (Bristol, UK) in 2004, where he investigated “Factors influencing Retention and Peak Shape in HPLC” in the group of Prof. David McCalley. Following a Postdoc at the Proteome Center at the University of Tübingen, where he studied diagnostic proteins and post-translational modifications, he joined Agilent in 2006. Currently, Stephan holds a position as Principal Scientist in Research and Development in the Liquid Phase Separation Division located in Waldbronn, Germany. His primary focus is on the development of 2D-LC workflow solutions and collaborations with external companies and academic institutions.

Presenter: Dr. Ulrich Eberhardinger (Product Manager, Agilent Technologies, Inc.)

Ulrich studied chemistry at the University of Stuttgart and earned his PhD in physical chemistry. He works for Agilent since 2002 and has been responsible for the development and market introduction of various hardware, software products and solutions in liquid chromatography and microfluidics such as the bio-inert LC, 1260 and 1290 Infinity II systems, high-performance pumps, method development solution, instrument control software and many others. As the product manager for 2D-LC Software, he shapes the future of multidimensional chromatography at Agilent.