Characterisation of Anti-HCP antibodies by 2D differential Gel Electrophoresis

By
  • Arnaud Delobel
  • Alix Verret
  • Nicolas Trinchero
  • Géry Van Vyncht
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Host-cell proteins (HCP) are critical process-related impurities in biotherapeutics, with the potential to impact both product safety and efficacy. While ELISA remains the industry gold standard for HCP quantification due to its sensitivity and throughput, its performance strongly depends on the quality and coverage of the anti-HCP antibodies used. Understanding the limitations of these antibodies is therefore essential for reliable HCP monitoring. This application note presents an analytical approach based on 2D Differential Gel Electrophoresis (2D-DIGE) to characterise anti-HCP antibodies and evaluate their coverage.

The full technical details and methodology are available in the complete application note for download.

ANALYTICAL METHOD HIGHLIGHT

  • 2D-DIGE enables simultaneous separation and comparison of total HCP proteins and those recognised by anti-HCP antibodies using differential fluorescent labelling (Cy3/Cy5).
  • The method allows quantification of antibody coverage through the comparison of total protein spots, recognised spots, and matched spots.
  • Overlay of fluorescence images eliminates the complexity of visual comparison seen in traditional Western Blot approaches.
  • Application to a commercial ELISA kit demonstrated a coverage of 82% for anti-HCP antibodies.

VALUE FOR YOUT HCP MONITORING PROGRAMME

A robust understanding of anti-HCP antibody coverage is essential to ensure that ELISA methods are fit for purpose in biopharmaceutical development and routine quality control. The 2D-DIGE approach provides a structured and reliable way to generate this insight.

  • Supports evaluation of anti-HCP antibody coverage required for ELISA method suitability
  • Improves reliability compared to traditional gel comparison methods
  • Helps assess whether commercial or process-specific ELISA methods are adequate
  • Enables comparison of HCP profiles between manufacturing processes
  • Assists in determining the applicability of platform ELISA methods across products
  • Contributes to informed decisions on method optimisation and analytical strategy
  • Support for early-stage analytical strategies alongside orthogonal methods (e.g. LC/MS)

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