Pharm Res (2011) 28:2302–2310 DOI 10.1007/s11095-011-0460-3 RESEARCH PAPER Taylor Dispersion Analysis Compared to Dynamic Light Scattering for the Size Analysis of Therapeutic Peptides and Proteins and Their Aggregates Andrea Hawe & Wendy L. Hulse & Wim Jiskoot & Robert T. Forbes Received: 1 February 2011 / Accepted: 26 April 2011 / Published online: 11 May 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com ABSTRACT Purpose To evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS). Methods The hydrodynamic radius of oxytocin, bovine serum albumin, various monoclonal antibodies (type IgG) and etanercept at concentrations between 0.05 and 50 mg/ml was determined by TDA and DLS. IgGs and etanercept were stressed (elevated temperatures) and analyzed by TDA, DLS and HP-SEC. Results TDA and DLS were comparable in sizing non- stressed peptides and proteins in a concentration range of about 0.5 to 50 mg/ml. TDA performed well even at lower concentrations, where DLS tends to provide theoretically high values of the Z-average radius. However, because of differ- ences in the detection physics, DLS was more weighted towards the detection of aggregates in stressed formulations than TDA. Advantageously, TDA was also able to size the small peptide oxytocin, which was not feasible by DLS. Conclusion TDA allows the accurate determination of the hydrodynamic radius of peptides and proteins over a wide concentration range, with little interference from excipients present in the sample. It is marginally less sensitive than DLS in detecting size increase for stressed protein samples. Andrea Hawe and Wendy L. Hulse share first author. A. Hawe (*) : W. Jiskoot Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research Leiden University P.O. Box 9502, 2300 RA, Leiden The Netherlands e-mail: ahawe@chem.leidenuniv.nl W. L. Hulse : R. T. Forbes PI Research Focus Group, School of Pharmacy University of Bradford Bradford BD7 1DP UK KEY WORDS dynamic light scattering . hydrodynamic size . protein aggregation . protein characterization . Taylor dispersion analysis INTRODUCTION 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. Size information of peptides and proteins can be gained by separation-based methods, including analytical ultracentrifugation, size-exclusion chro- matography (HP-SEC), asymmetrical flow field fraction- ation (FFF) and SDS-PAGE (1–3). 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. A second important measurement principle is gaining size information by measuring the diffusion coefficient of the molecules in solution. 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). The hydrodynamic radius is then derived from the diffusion coefficient by the Stokes Einstein equation (2,6,7). DLS is a popular technique because it is user friendly, is widely available in pharmaceutical labs, and it enables sensitive aggregate detection. 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). This can be an advantage, e.g., for the sensitive detection of small quantities of aggregates, or a disadvantage when aiming to