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Related Concept Videos

Size-Exclusion Chromatography01:08

Size-Exclusion Chromatography

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In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
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Related Experiment Video

Updated: Mar 6, 2026

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
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Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

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Robust quantitation of basic-protein higher-order aggregates using size-exclusion chromatography.

David Gervais1, Andrew Downer1, Darryl King1

  • 1Porton Biopharma Limited, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom.

Journal of Pharmaceutical and Biomedical Analysis
|March 16, 2017
PubMed
Summary
This summary is machine-generated.

Detecting higher-order aggregates (HOA) in basic proteins like Erwinia chrysanthemil-asparaginase (ErA) using size-exclusion chromatography (SEC) is challenging. Coated-silica SEC columns improve HOA detection accuracy and reproducibility.

Keywords:
Higher-order aggregatesNon-specific interactionsSize-exclusion chromatographyl-Asparaginase

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Area of Science:

  • Biopharmaceutical analysis
  • Protein aggregation studies
  • Chromatographic method development

Background:

  • Higher-order aggregates (HOA) detection via size-exclusion chromatography (SEC) can be variable for basic proteins.
  • Erwinia chrysanthemil-asparaginase (ErA), a basic protein (pI 8.6), showed inconsistent HOA detection using standard diol-silica SEC columns.

Purpose of the Study:

  • To investigate the variability in detecting higher-order aggregates (HOA) of a basic protein using different size-exclusion chromatography (SEC) columns.
  • To improve the accuracy and reproducibility of HOA detection for alkaline-pI proteins in SEC analysis.

Main Methods:

  • Analysis of Erwinia chrysanthemil-asparaginase (ErA) tetrameric preparations using diol-silica SEC columns.
  • Comparison of SEC results with orthogonal analytical ultracentrifugation (AUC) measurements.
  • Evaluation of coated-silica SEC columns for improved HOA detection and reproducibility.

Main Results:

  • Diol-silica SEC columns exhibited highly variable recovery and detection of HOA for ErA, with poor agreement with AUC.
  • Only HOA, not the main peak or octamer species, was selectively affected by silanol interactions.
  • Coated-silica SEC columns significantly improved HOA resolution, reproducibility, and agreement with AUC for the basic ErA protein.

Conclusions:

  • Standard SEC columns with exposed silanols can interfere with higher-order aggregate detection in basic proteins.
  • Coated-silica SEC columns offer a more reliable method for assessing HOA in alkaline-pI biopharmaceuticals.
  • Thorough SEC method development and column selection are crucial for accurate aggregate analysis of basic proteins.