Size-Exclusion Chromatography (SEC) Optimization Guide


Table of Contents


ACQUITY UPLC Instrumentation Considerations for Successful SEC Analysis

Importance of Developing a Robust UPLC-based SEC Method

      a. Choosing the Appropriate ACQUITY UPLC Protein BEH SEC Column

      b. Method Development

Considerations When SEC Separated Components Tail and/or Resolution Decreases

      a. Connecting an ACQUITY UPLC Protein BEH SEC Column to an ACQUITY UPLC System

      b. LC System Dispersion Effects on a UPLC SEC Separation

      c. Determining LC System Dispersion Volume

Maximizing ACQUITY UPLC Proetin BEH SEC Column Life

      a. Mobile Phase Preparation and Use

      b. Avoiding Microbial Contamination of the Solvent Delivery System

      c. Cleaning ACQUITY UPLC Systems

      d. Minimizing Sample Particulate Contamination of Protein BEH SEC Columns

      e. Preventing Sample Formulation Constituent Contamination of Protein BEH SEC Columns

Summary and References



Size-exclusion chromatography (SEC) is a technique commonly used throughout the development and commercialization of biotherapeutic proteins. Traditional HPLC-based SEC methods involve the use of columns packed with >3 µm particles containing an appropriate pore size distribution to allow for the separation of proteins of different hydrodynamic volumes (i.e., Stokes Radii). Ideally, the proteins are separated based solely on their relative size in solution. To achieve this, there should be no secondary interactions (e.g., hydrophobic or ionic) between the proteins and the SEC particles. 

In 2010, Waters was the first company to introduce the use of SEC columns containing <3 µm particles (patent pending) as an effective way to increase the resolution of separated components and to decrease analysis times. This technology, as implemented on an appropriately designed UPLC System, enabled the separation and quantitation of monoclonal antibody (mAb) high molecular weight species (>300,000 Da), mAb monomer (150,000 Da), and lower molecular weight species (e.g., <100,000 Da) in <8 minutes (Figure 1). It is the objective of this document to detail the instrument, method, and column-use considerations that are important to obtaining robust UPLC-based SEC separations of proteins with maximum column lifetimes.

Figure 1. ACQUITY UPLC BEH SEC separation of a biotherapeutic monoclonal antibody (mAb).

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