Taylor Dispersion Analysis for Peptide and Protein Characterization size small peptides in general or monomeric protein in the presence of larger aggregates. Many new methods of analysis are being applied to solve the challenge of rapidly sizing biopharmaceutical systems using small quantities. 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). Although having several advantages over DLS, as described in detail by Filipe et al. (2010) (8), NTA is not suitable to size peptides or protein monomers, because they are too small for the size range of ca. 30 to 1,000 nm covered by NTA. Thus, the opportunity for new analytical tools to address such issues exists. In particular, methods that facilitate rapid analysis and screening and possess high accuracy and low sample consumption are highly desirable. Taylor dispersion analysis (TDA) is a comparatively unutilized method to determine the diffusion coefficients and hydrodynamic radii of molecules. It is a fast, absolute method based on the dispersion of a solute plug through a uniform cylindrical tube under laminar Poiseuille flow. The method, sometimes termed Taylor-Aris dispersion, was first described by Taylor in 1953 (9) and developed further by Aris in 1956 (10). 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. So far, TDA has mainly been employed for nanoparticles (11), polymers (12–14) and small molecules (15,16). The reported use of TDA for protein characterization is rare. In 1994, Bello et al. sized the proteins ovalbumin and hemoglobin, but the emphasis of their work was to report the major advance of the use of small bore glass capillaries, radii≤50 μm, which enabled the reduction of analysis time and sample consump- tion (17). More recently published, another rare example of a peer-reviewed article involving a protein, is a study on the interaction of the drug propranolol with serum proteins (18). Applications of TDA for the characterization of therapeutic proteins in complex formulations with typically used exci- pients have so far not been described in the literature. In this paper, we present our evaluation of TDA for the determination of the hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formu- lations compared to DLS. The newly commercially available instrument for protein sizing (TDA200) utilizes pixilated UV area imaging to improve the quality of data collection, allowing TDA the potential to routinely measure the hydrodynamic radius of therapeutic proteins and peptides. The detector monitors broadening of a band of a therapeutic protein or small molecule solution injected into a stream of buffer solution and driven through a fused- silica capillary using a capillary electrophoresis type autosampler. The band is imaged at two windows, the first on entry to and the second on exit from a loop in the capillary. Band broadening due to Taylor dispersion is calculated from absorbance versus time data using the peak centre times at the first and second window, t1 and t2, respectively, and the corresponding standard deviations, τ1 and τ2 (band broadening), using Eq. 1: 4kbT t22 Rh ¼ phr2ðt2 t12 t1Þ 2303 1 ð Þ where Rh is hydrodynamic radius, kb is the Boltzman constant, T is the temperature, η is the viscosity, and r is the radius of capillary. Given the interest in the pathways to protein aggregation and analysis thereof (19), the need for analytical methods for aggregates and subvisible particles and for evaluating their performance is high (20). Our comparative evaluation is timely since new analytical tools suggested to be used for protein aggregation analysis still lack comparison to more established methods (19). Another recent commentary strongly supports the need for further work to assess the performance and limitations of methods to assess protein aggregates (21). The authors suggested that potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatog- raphy necessitate an essential need to use orthogonal analytical methods to assure protein product quality. Within the context of methods to assess protein aggregation, the aim of our study was to evaluate the performance of Taylor dispersion analysis (TDA) as a novel method to determine the hydrodynamic radius of thera- peutic proteins and peptides and their aggregates. We report the most extensive evaluation so far of TDA pertinent to the area of therapeutic proteins. Notably, oxytocin, bovine serum albumin, several monoclonal anti- bodies and etanercept are used as models, including stressed and un-stressed formulated products. The results obtained by TDA were compared to those of dynamic light scattering (DLS), which has been so far the standard technique for those applications in the pharmaceutical field. The influence of protein concentration, the presence of excipients, e.g. sugars, and protein aggregation on the measurement results were factors to be investigated using not only model proteins, but also formulated commercial proteins. Capabilities, advantages and limitations of Taylor dispersion analysis compared to DLS were identified and are discussed within the manuscript. Of particular interest was the probing of the relative ability of TDA to indicate formulated product change after thermal stress. MATERIALS AND METHODS Materials The commercial protein products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg were obtained from