Size Exclusion Chromatography (SEC) Technique

Size exclusion chromatography (SEC), is a widely used chromatographic separation tool based on the size (hydrodynamic volume) of large molecules and bio-macromolecular complexes, including but not limited to recombinant proteins, enzymes and antibodies, nucleic acids, polysaccharides, etc. In general, there are two basic types of SEC: one is gel permeation chromatography (GPC) and the other is gel filtration chromatography (GFC). The former technique uses hydrophobic column packing materials and non-aqueous mobile phases (organic solvents) to provide the molecular weight distribution results. On the contrary, the latter uses hydrophilic filler materials and aqueous mobile phases to accomplish the separation.

How Does It Work?

Fig. 1 Schematic illustration of the theory behind SEC.

SEC resin is made of a porous matrix filled with micron-scale polymer beads which lack reactivity and adsorptive properties. The pore sizes of these beads are used to estimate the dimensions of macromolecules. When samples with various sizes flow into the column, molecules larger than the pores elute faster because they are unable to penetrate into the pores, whereas smaller molecules flow more slowly because they would enter the pores. Consequently, larger molecules elute from the column faster while smaller molecules slower, so samples can be sorted isocratically and effectively by their sizes.

Unlike other kinds of chromatography, SEC doesn't need the binding reaction between sample components and chromatographic medium to proceed the separation, so the sample buffer components don't affect the separation directly. What's more, buffer, temperature, pH value and other conditions can be varied to suit different kinds of samples or for further purification requirements. SEC can be performed directly before or behind other chromatography.

Gel Permeation Chromatography (GPC)

GPC usually uses polymer-based chromatography columns. The most commonly used filler is a copolymer of polystyrene and divinylbenzene.

In GPC analysis, the solvent selected must be able to dissolve the sample. The most commonly used solvent is tetrahydrofuran (THF). In addition, other commonly used mobile phases include chloroform, dimethylformamide (DMF), dichlorobenzene, methyl acetate, tetrachloroethane, etc.

Gel Filtration Chromatography (GFC)

The chromatography columns used in GFC method often include two types: silica-based chromatography columns and polymer-based chromatography columns. Normally, the calibration curve of silica gel column is smoother, thus the separation performance is better. However, because that the structure of silica gel will be destroyed if the pores are too large, so that this kind of columns is not suitable for separation of substances with too large molecular weight. On the other hand, the structure and the chemical property of the polymer are both more stable than those of silica gel, so the polymer-based chromatography columns are always used to separate substances with high molecular weight

What Can SEC Be Used For?

• It is well recognized that SEC is an ideal technique for separation of PEGylated species from unreacted protein and other components. PEGylated proteins/peptides have increased hydrodynamic size due to the neutral PEG chains compared to the native protein/peptides. Therefore, native and mono-PEGylated proteins will be separated faster.
• In addition, it can also be used to predict the protein size based on radius of a PEG molecule having the same molecular weight as the total conjugated PEG and the native protein radius.
• Moreover, SEC is suitable for bio-macromolecules that are sensitive to changes of buffer conditions such as metal ion concentration, pH value, etc. These samples can be collected in any SEC buffer with the presents of cofactors, detergents, essential metal ions, denaturants, etc. 

Strengths & Weaknesses of SEC

References

1. Ramos-de-la-Peña A M, et al. Progress and challenges in PEGylated proteins downstream processing: a review of the last 8 years. International Journal of Peptide Research and Therapeutics, 2020, 26(1): 333-348.
2. Santos J H P M, Torres-Obreque K M, et al. Protein PEGylation for the design of biobetters: from reaction to purification processes. Brazilian Journal of Pharmaceutical Sciences, 2018, 54(SPE).