The reported use of TDA for protein characterization is rare. So far, TDA has mainly been employed for nanoparticles ( 11), polymers ( 12– 14) and small molecules ( 15, 16). A plug of solute is injected into a moving solvent stream in an open tubular column which disperses by a combination of radial diffusion and cross-sectional velocity. The method, sometimes termed Taylor-Aris dispersion, was first described by Taylor in 1953 ( 9) and developed further by Aris in 1956 ( 10). It is a fast, absolute method based on the dispersion of a solute plug through a uniform cylindrical tube under laminar Poiseuille flow. Taylor dispersion analysis (TDA) is a comparatively unutilized method to determine the diffusion coefficients and hydrodynamic radii of molecules. In particular, methods that facilitate rapid analysis and screening and possess high accuracy and low sample consumption are highly desirable. Thus, the opportunity for new analytical tools to address such issues exists. (2010) ( 8), NTA is not suitable to size peptides or protein monomers, because they are too small for the size range of ca. Although having several advantages over DLS, as described in detail by Filipe et al. For example, nanoparticle tracking analysis (NTA) is a novel imaging-based technique which measures the diffusion coefficient of individual particles by a microscope and camera ( 8). Many new methods of analysis are being applied to solve the challenge of rapidly sizing biopharmaceutical systems using small quantities. This can be an advantage, e.g., for the sensitive detection of small quantities of aggregates, or a disadvantage when aiming to size small peptides in general or monomeric protein in the presence of larger aggregates. The fact that the intensity of the scattered light is proportional to the sixth power of the radius results in a bias of DLS to larger sizes ( 6). DLS is a popular technique because it is user friendly, is widely available in pharmaceutical labs, and it enables sensitive aggregate detection. The hydrodynamic radius is then derived from the diffusion coefficient by the Stokes Einstein equation ( 2, 6, 7). The best known technique in the field of therapeutic proteins is dynamic light scattering (DLS), which determines the diffusion coefficient of molecules from the intensity fluctuations of scattered light on particles moving according to the Brownian motion ( 6). A second important measurement principle is gaining size information by measuring the diffusion coefficient of the molecules in solution. The combination of HP-SEC and FFF with multi-angle laser light scattering detection ( 4, 5) and analytical ultracentrifugation allows for the determination of both the molar mass and size without relying on molecular weight standards. Size information of peptides and proteins can be gained by separation-based methods, including analytical ultracentrifugation, size-exclusion chromatography (HP-SEC), asymmetrical flow field fractionation (FFF) and SDS-PAGE ( 1– 3). The size analysis of peptides and proteins in solution, and their associated degradation products (mainly aggregates), is a central aspect of analytical characterization during pharmaceutical development.

Dynamic light scattering size range