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A Useful Method Development Strategy for Reversed-Phase LC Separations of Large Protein Variants in Reversed-Phase



  October 3, 2018

  8:00 a.m. PDT / 11:00 a.m. EDT / 16:00 BST / 17:00 CEST




The number of biotherapeutic drugs including mAbs and their related products such as antibody-drug conjugates (ADCs) has been growing rapidly, particularly in the last decade. The development and commercialization of these complex entities is a complicated process and requires a significant amount of effort and capability—especially in chromatographic separations. This has created a need for chromatographers to have effective strategies for LC separations of large (>50,000 MW) and complex proteins, such as monoclonal antibodies (mAbs), and their variants.

In this web seminar, we will discuss how to approach your method development strategy by adjusting various parameters of your separation to ensure that you are seeing more of the variants of your complex proteins. These parameters include: pore size, bonded-phase chemistry, mobile phase modifiers, mobile phase additives, and column temperature. Systematic selection and adjustment of these variables leads to improved resolution of highly similar protein variants. An example of this approach is high-resolution intact protein LC–MS analysis of difficult IgG mixtures, such as the complex mixtures of lower abundance free sulfhydryl variants present in therapeutic mAbs.

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Key Learning Objectives

  • Develop a strategy for biotherapeutic method development for large, complex proteins (> 50,000 MW)
  • Learn how to utilize larger pore sizes to improve the efficiency and robustness of your large complex protein separations
  • Review several examples in which several different separation parameters were used to improve protein separations

Who Should Attend

  • Method developers for UHPLC/UPLC and HPLC methods for biomolecules in pharmaceutical, chemical, clinical, environmental, agrichemical, university and governmental laboratories
  • LC chromatographers looking to separate monoclonal antibodies (mAbs) (>50,000 MW)


Ben Libert,
Associate Scientist in R&D,
Advanced Materials Technology, Inc.


Ann Thayer,
Contributing Editor,
C&EN Media Group