Taylor Dispersion Analysis for Peptide and Protein Characterization This becomes also obvious when plotting the DLS results (rzave or rmain peak) against the rh (TDA), as shown in Fig. 2. For this comparison of TDA with DLS, additional measurements of rituximab, a model IgG, as well as etanercept and adalimumab diluted in buffer, were includ- ed. With the exception of the small peptide oxytocin findings and practically all formulations at 0.05 mg/ml, rzave (DLS) and rh (TDA) offered comparable results (Fig. 2a,b). The rmain peak (DLS) and rh (TDA) were in good agreement for all analyzed conditions (Fig. 2c). Influence of Excipients on the Analysis The influence of excipients on protein characterization is an important issue, in particular as most therapeutic proteins are applied in complex formulations. DLS eval- uates the scattering of not just the solute of interest in solution. As the above results confirm, with decrease in concentration and decrease in size of the substance of interest, DLS data can become more and more dominated by the scattering interference from excipients present in the formulations. Sugars and surfactants are often used as stabilizing excipients in therapeutic protein formulations. From previous studies for sugar solutions, e.g. sucrose, DLS typically measures two peaks corresponding to radii of about 0.5 nm and 100–200 nm (29). The reason for the size of 100–200 nm is not yet fully understood. To elucidate if 2307 TDA is also influenced by sugars, the etanercept formula- tion, which contains 1% (w/v) sucrose, was analyzed at etanercept concentrations between 0.05 and 50 mg/ml (Fig. 3a). For the protein-free placebo, DLS resulted in an rzave of 288 nm, a main peak at about 200 nm and a second peak <1 nm. In TDA, only a rh of 0.5 nm was measured for the sucrose-containing placebo, whereas the larger size of 200 nm was not detected. Sucrose also disturbed the DLS measurements at the low etanercept concentration of 0.05 mg/ml, where a too high rzave was measured. At higher protein concentrations above 0.5 mg/ml, the protein signal dominates, and the presence of sucrose no longer affects the rzave. Surfactants are a second class of excipients that can disturb DLS measurements, when used above the critical micelle concentration. This is also the case for the adalimumab placebo, which contains 0.1% polysorbate 80. Here, a rzave of about 12 nm was determined by DLS, corresponding to the polysorbate micelles (Fig. 3b). No rh could be resolved for the formulation placebo by TDA, as the formulation buffer was also used as a running buffer for the system. The experiments showed that excipients like sugars and surfactants can disturb the size analysis of proteins by DLS, particularly for formulations with low protein concentra- tions. TDA has more selectivity, on the other hand, and its results were not affected to the same extent by the presence of excipients. Fig. 4 Results for DLS (rZave, rmain peak) and TDA (rh) for non-stressed and aggregated formulations of etanercept (a), adalimumab (b), rituximab (c) and the model IgG (d). *marks rh from TDA, # Zaverage values from DLS, which are significantly higher for the stressed than for the non-stressed formulation (p<0.05). The results are shown as average ± standard deviation of n=3 (DLS) and n=5 (TDA) individual measurements.