Improve Ion Exchange Methods with Faster Method Development and Lower Dispersion


Tuesday, September 21, 2021 at 11am EDT | 8am PDT | 4pm BST | 5pm CEST Want faster method development? Better ion exchange methods? Lower dispersion? Learn the latest techniques and innovations to optimize your applications and workflows.

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Event Overview:

Protein biopharmaceuticals such as recombinant monoclonal antibodies are currently in widespread use for the treatment of various life-threatening diseases including cancer and autoimmune diseases 1,2,3. In contrast to small molecule drugs, mAbs are large (150 kDa) and heterogeneous. As a result of the biosynthetic process and subsequent manufacturing and storage, hundreds of different variants may coexist, differing in aspects such as N-glycosylation, N- and C-terminal processing, deamidation, oxidation, amino acid sequence, and disulfide bridges. Due to the structural complexity of mAbs, a variety of tests are required to monitor critical quality attributes (CQAs) throughout the development and manufacturing process, hence, unraveling this complexity represents an analytical challenge. Charge variant analysis is a demanding application for applied liquid chromatography systems due to the use of highly corrosive buffer salts in combination with very shallow gradients for optimal separation.4 Reproducible gradient formation and delivery is essential for successful ion exchange chromatography. Gradient delay volume, UHPLC dispersion volume, pump-mixing behavior, and flow cell geometry can all affect gradient delivery and speed. Choosing the correct pumping system and minimizing delay volume and extra column volume lead to faster method development of ion exchange methods. 

New advances in UHPLC technology resulted in bio-specific LC systems for the analysis of large molecules, and these systems are made in all varieties, including binary and quaternary blending pumps. Binary pumps employ high-pressure mixing of two mobile phases allowing very shallow gradients. Alternatively, quaternary, or flexible pumps, will mix up to four buffers in a low-pressure mixing scenario, enabling buffer blending for faster method development. What makes one system preferred to another? Can a lower delay volume produce better chromatography?

Join Agilent Technologies LC applications scientist Patrick Cronan for an in-depth comparison of high-pressure UHPLC systems ranging from 600 to 1300 Bar with varying delay volumes and explore the effects on ion exchange chromatography.

  1. Sandra, K.; Vandenheede, I.; Sandra, P. Modern Chromatographic and Mass Spectrometric Techniques for Protein Biopharmaceutical Characterization. J. Chromatogr. A 2014, 1335, 81–103.
  2. Fekete, S.; et al. Chromatographic, Electrophoretic and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. 2016, 88, 480–507.
  3. Walsh, G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 2018, 36, 1136–1145.
  4. Schneider S., Agilent Technologies, How Shallow Can You Go? Refining charge variant analysis of mAbs with the Agilent 1290 Infinity II Bio LC System, Application Note 2020.

Key Learning Objectives:

1. Learn how pump-mixing behavior and delay volume affect chromatographic performance

2. Understand how changes in LC tubing volume and material affect peak shape and resolution

3. Learn to pick the correct pumping system to speed method development for your applications and workflows

Who Should Attend:

Analytical Chemists, Large molecule chemists, Biologists, LC and LCMS analysts, Bio Processing technicians


Patrick Cronan
LC Applications Scientist
Agilent Technologies

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