Better Characterization of Biomolecules Using Agilent AdvanceBio Reversed-Phase Columns

Protein biopharmaceuticals are very heterogeneous, so a number of chromatographic techniques may be required to fully characterize an active pharmaceutical ingredient (API). Methods include size exclusion chromatography for the quantitation of dimers and aggregates, and ion-exchange for the identification of charge variants. Both of these techniques use aqueous eluents and nondenaturing conditions. However, as part of the full characterization of a protein, it is also necessary to look at the primary amino acid sequence and any post-translational modifications to the sequence that may have occurred during the purification or formulation steps of manufacture. To perform this type of analysis, denaturing conditions are required, so reversed-phase HPLC is normally the technique of choice.

Reversed-phase (RP) is one of the three key techniques used in biochromatography and is particularly valuable because of its compatibility with LC/MS detection. And small particle improvements, such as those found in Agilent ZORBAX RRHD 300A, 1.8 um columns, make RP an attractive choice for many biopharmaceutical applications. With the introduction of newer phase chemistries, RP seperations can provide alternative selectivities, some of which can have greater sensitivity of proteins. For example, for intact proteins and large fragment separations, Agilent Poroshell 300 offers unique advantages due to its superficially porous particle technology, which behaves more like a larger pore column to deliver significant speed advantages.