Monolithic Columns for Biomolecule Analysis

During the development and production of therapeutic proteins, characterization of structural variants that can influence safety and efficacy is a critical challenge that must be met, according to the FDA and other regulatory agencies. For instance, microheterogeneity is a major constraint in developing monoclonal antibody therapeutics. These antibody variants can result from glycosylation, oxidation, mutation, phosphorylation, amino terminal modifications, incomplete processing of the c-terminus, and asparagine deamidation. There is a growing need for better chromatographic separation technologies to reliably resolve these variants. The technologies must combine speed and capacity without compromising resolution. Often, multidimensional chromatography is required to achieve desired separation of molecules of interest. This necessitates use of a high-capacity column in the first dimension to support sufficient sample to identify rare proteins of interest. However, high-resolution columns generally lack the necessary capacity to be considered for the first dimension. Speed and resolution are competing factors in separations using porous media. This trade-off is exacerbated in the separation of large biomolecules such as proteins and oligonucleotides. Under these conditions, increased flow rate can lead to significant broadening of peaks due to the mass transfer problem.