High performance size-exclusion chromatography (HP-SEC)
High performance size-exclusion chromatography (HP-SEC), also known as gel-filtration chromatography, 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 ultraviolet (UV) absorption, fluorescence-, refractive index (RI)-detectors or multi-angle laser light scattering (MALLS) detectors. 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).
Compared to orthogonal techniques (e.g., AF4 and AUC), HP-SEC generally offers a higher resolution and lower limit of detection. Also, method development is often straightforward and measurement times are short.
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. It is a common technique used for batch release testing and at Coriolis, it can be offered in full GMP compliance.
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