High performance size-exclusion chromatography (HP-SEC)


High performance size-exclusion chromatography (HP-SEC) is a well-established, robust and highly versatile technique for the characterization and quantification of protein monomers, fragments and aggregates.


In HP-SEC, the sample is injected onto a column that is constantly flushed with mobile phase. The chromatography column itself contains fine and porous beads, which are composed of dextran, agarose, silica or polyacrylamide (the stationary phase). The beads allow small species to migrate into their pores, increasing their retention inside the column, while larger species migrate with the mobile phase without entering the bead pores. Thus, larger species reach the column end faster than smaller species. After separation, the various species are detected using following detectors:

  • Ultraviolet (UV) absorption
  • Fluorescence
  • Refractive index (RI)
  • Multi-angle laser light scattering (MALLS)
  • Charged aerosol detection (CAD)

HP-SEC allows the sizing, quantification and molecular weight determination of fragments, monomers and aggregates. The size range of HP-SEC is defined by the pore size of the column and the method set-up (e.g., mobile phase, flow settings and column dimensions).

Orthogonal techniques to HP-SEC:

  • Asymmetric flow field flow fractionation (AF4) offers an increased upper size limit, allowing for the analysis of larger aggregates and particle. Method development, however, may be more elaborate.
  • Analytical Ultracentrifugation (AUC) characterizes samples in their native buffer while mitigating dilution effects. As a matrix-free separation technique, AUC is very suitable for the verification of HP-SEC results. The modern instruments operated by Coriolis can even reach the throughput of HP-SEC sequences.

Compared to these orthogonal techniques, HP-SEC generally offers a higher resolution and lower limit of detection. Verification of HP-SEC data with orthogonal techniques, such as AUC and AF4, is often recommended and can be performed by Coriolis during method development or on existing methods.

HP-SEC is commonly employed in all stages of research and development of biopharmaceuticals. It is highly valuable during formulation development, stability studies and forced degradation studies, but is most frequently used for batch release testing. Coriolis offers HP-SEC also in full GMP compliance.

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