There are several orthogonal chromatographic techniques available for the detailed characterization of biopharmaceuticals, offering a high selectivity for separating variants based on hydrophobicity, charge and size.
Reversed Phase-HPLC (RP-HPLC), where components are separated based on their hydrophilicity/hydrophobicity, benefits from its high resolving power and its ability to be linked directly to mass spectrometry. The use of Ultra High Pressure Liquid Chromatography (UPLC) systems has also become more routine.
Ion Exchange chromatography (IEX), is a widely used technique where separation is achieved through charge differences. Among different IEX techniques available, cation exchange chromatography (CEX) is most used for protein characterization.
Size Exclusion Chromatography (SEC) separates biomolecules according to their hydrodynamic radius, i.e. based on the size of the components. A main advantage is the mild mobile phase conditions which cause minimal impact on the conformational structure of the protein. Various detectors may be utilized in SEC, including refractive Index (RI), ultraviolet (UV) and multi-angle laser-light scattering (MALS).
Hydrophilic interaction chromatography (HILIC) provides separation of analytes based on their polarity utilizing a hydrophilic stationary phase with reverse-phase type eluents. Hydrophobic Interaction Chromatography (HIC) separates proteins according to reversible hydrophobic interactions between immobilized hydrophobic ligands on the column and non-polar regions of the protein.