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3D-liquid chromatography as a complex mixture characterization tool for knowledge-based downstream process

Alexander T Hanke1, Eleni Tsintavi1, Maria Del Pilar Ramirez Vazquez1

  • 1Dept. of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.

Biotechnology Progress
|June 16, 2016
PubMed
Summary
This summary is machine-generated.

This study introduces a three-dimensional liquid chromatography method for characterizing therapeutic enzymes and impurities. It efficiently identifies optimal separation techniques and determines crucial adsorption isotherm parameters for process design.

Keywords:
feedstock characterizationhost cell proteinsmulti-dimensional chromatographyprocess development

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Area of Science:

  • Biotechnology
  • Chemical Engineering
  • Chromatography

Background:

  • Developing chromatographic separation processes requires understanding physicochemical properties of products and impurities.
  • Characterization techniques typically measure molecular properties (charge, hydrophobicity, size) or adsorption isotherms.

Purpose of the Study:

  • To demonstrate a three-dimensional liquid chromatography (3D-LC) approach for analyzing therapeutic enzymes in clarified cell homogenates.
  • To enable efficient impurity removal by assigning determined parameters to pseudo-components.
  • To determine adsorption isotherm parameters for mechanistic process design.

Main Methods:

  • Application of a 3D-LC system to a clarified cell homogenate containing a therapeutic enzyme.
  • Each separation dimension measures a distinct molecular property relevant to chromatography.
  • Peak matching across dimensions and against a reference chromatogram for component assignment.
  • High-throughput screening using multiple linear gradients in the second dimension for isotherm parameter regression.

Main Results:

  • Successfully applied 3D-LC to characterize components in a complex biological mixture.
  • Enabled assignment of physicochemical parameters to pseudo-components, guiding impurity removal strategies.
  • Determined adsorption isotherm parameters with an average standard error of 8% using gradient separations.

Conclusions:

  • 3D-LC is an effective tool for comprehensive characterization in bioprocess development.
  • The method facilitates the selection of optimal chromatographic techniques for impurity removal.
  • Accurate isotherm parameters obtained support the development of mechanistic process models.