Peptide and protein based pharmaceutical formulations present unique analytical challenges. Protein aggregation and polymerization of peptides are not uncommon, and must be detected and quantified. For example, a low-dose peptide may be contained within a gelatin capsule, in which case separation and identification of the peptide becomes the proverbial “needle in the haystack” problem, with the higher molecular weight gelatin proteins swamping out the much lower concentration of the peptide active pharmaceutical ingredient.

Multidimensional separations offer an ideal solution to such difficult chromatographic problems. Ideally, two or more chromatographic modes are employed that are chemically orthogonal to one another. The separation power of such a system can be expressed as the total system peak capacity, n_T, which is the product of the peak capacities of the individual chromatographic systems (see equation).

The combination of the reversed-phase and size exclusion modes of HPLC, while not completely orthogonal, nevertheless offers a powerful combination for such separation problems.

Two applications of multidimensional chromatographic approaches are discussed. Determination of molecular aggregation is a critical requirement for protein based formulations. Aggregation is a broad term to describe processes that range from full covalent bonding, to simple hydrophobic association. While both reversed-phase and SEC are widely used for purity and potency determination of peptides and proteins, it will be shown that both modes are required to fully characterize, for example, BSA. The second application will be the determination of low concentrations of an active peptide, in presence of gelatin from a capsule, during dissolution method development.