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Biopharmaceuticals offer great new possibilities and potential in treating various life threatening diseases such as cancer and autoimmune diseases. However, their complexity, resulting from their heterogeneity, represents a great challenge to the analytical chemist and a suite of techniques is required for their characterization and analysis. Reversed phase chromatography, the mainstay of traditional small molecule analysis, is employed at multiple biopharmaceutical levels due to its versatility, inherent relative efficiency and ease of hyphenation to a mass spectrometer. However, these separations often rely on finely balanced critical parameters being correctly optimized. We shall interrogate these critical parameters at each level and discuss best practices for implementation and optimization. The considerable increase in glycosylated biopharmaceuticals and the development of biosimilars and biobetters has led to much greater interest in the type and degree of glycosylation. We will review not only the popularity behind glycosylated biopharmaceuticals but where the heterogeneity comes from and how glycans can be effectively liberated, characterized and analyzed using HILIC.


Topics include:

  • Reversed phase (RP) chromatography is commonly used to analyze at the following levels:
    • Intact biopharmaceuticals
    • Biopharma fragments
    • Biopharma degradants/modifications
    • Peptides
    • Amino acids
  • Why is RP so popular and how does it work?
  • How is it employed at each level?
  • How are methods developed and optimized?
  • HILIC is becoming the technique of choice for glycan and polar peptide analysis
    • Why is analyzing glycans so important?
    • How does HILIC work in the analysis of glycans?
    • How are HILIC methods developed, optimized and made robust?