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Related Experiment Video

Updated: Feb 6, 2026

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
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Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream

Bhumit A Patel1, Adrian Gospodarek1, Michael Larkin2

  • 1a Biologics Process Development and Clinical Manufacturing , Merck & Co., Inc ., Kenilworth , NJ , USA.

Mabs
|August 22, 2018
PubMed
Summary

This study introduces two process analytical technology (PAT) methods combining hydrophobic interaction chromatography (HIC) with multi-angle light scattering (MALS) for real-time monitoring of protein aggregate removal during purification. These novel HIC-MALS systems enable precise control and potential real-time release of biotherapeutics.

Keywords:
MALSPATaggregationchromatographyprocess control

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Area of Science:

  • Biopharmaceutical Manufacturing
  • Analytical Chemistry
  • Process Engineering

Background:

  • Protein therapeutics, such as monoclonal antibodies, require complex purification processes to remove aggregates.
  • Current aggregate removal methods can be challenging and often require post-purification analysis.
  • Process Analytical Technology (PAT) offers opportunities for real-time monitoring and control in biomanufacturing.

Purpose of the Study:

  • To evaluate two PAT applications coupling preparative hydrophobic interaction chromatography (HIC) with multi-angle light scattering (MALS) for aggregate monitoring.
  • To assess the capability of HIC-MALS systems for real-time control of aggregate levels during purification.
  • To demonstrate the potential for real-time release of biotherapeutics using these integrated systems.

Main Methods:

  • Coupling a preparative HIC unit with a multi-angle light scattering (MALS) detector.
  • Implementing an in-line MALS system for real-time start/stop fractionation triggers based on weight-average molar mass (Mw).
  • Utilizing an on-line ultra-high performance size-exclusion liquid chromatography (UHPLC) system with a µMALS detector for aggregate analysis.

Main Results:

  • The in-line HIC-MALS system provided real-time Mw measurements and controlled aggregate levels.
  • The on-line UHPLC-µMALS method confirmed aggregate levels, showing 1.5% dimer, consistent with the in-line Mw increase of ~2750 Da.
  • Both HIC-MALS applications demonstrated effective aggregate monitoring and control capabilities.

Conclusions:

  • Novel HIC-MALS systems are powerful tools for real-time aggregate monitoring during biologics purification.
  • These integrated systems facilitate precise control over aggregate removal, improving process efficiency.
  • The demonstrated technology supports the future implementation of real-time release strategies for biotherapeutics.