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Protein biopharmaceuticals have emerged as important for the treatment of various diseases. Protein biopharmaceuticals are structurally diverse and include monoclonal antibodies (mAbs), antibody drug conjugates (ADCs), fusion proteins, hormones, growth factors, cytokines, therapeutic enzymes, vaccines, and anticoagulants. These molecules have substantially reshaped the pharmaceutical market and today over 350 products have been approved in the United States and the European Union. These biopharmaceutical products account for a total sales value of $200 billion which represents around a quarter of the total pharmaceutical market.

The structural complexity of these molecules presents an enormous analytical challenge for characterization and manufacturing QC.  Unlike small molecule drugs, biopharmaceuticals are large – mAbs have a molecular weight of ~150 kDa – and heterogeneous – due to variability of biosynthetic processes and sensitivity to manufacturing and storage conditions.

An emerging tool to tackle this complexity is multidimensional liquid chromatography (LC). The current lecture will highlight the power of 2D-, 3D- and 4D-LC in hyphenation to mass spectrometry (MS) for the detailed characterization of protein biopharmaceuticals.


Topics include:

  • The power of 2D-LC
    • Resolution
    • Orthogonal/complementary information
    • MS compatibility
  • Introduction to protein biopharmaceuticals
  • Comprehensive LC×LC for peptide mapping and surfactant characterization
  • (Multiple) heart-cutting LC–LC-MS of monoclonal antibodies and antibody drug conjugates
  • 3D- and 4D-LC-MS based multi-attribute analysis of antibodies