Taylor Dispersion Analysis for Peptide and Protein Characterization Table 1 Peptides and Proteins Included into the Study Molar mass [kDa] 1.007 66.8 ~150 ~150 Literature values hydrodynamic radius [nm] ~0.8 nm (30)a 3.3–4.3 nm (23) 3.7 nm (31) 5–6 nm (24–27) 7.1 nm (22) Oxytocin BSA Monoclonal antibodies: model IgG1, adalimumab, rituximab Etanercept a radius estimated by Stokes-Einstein from diffusion coefficient of D= 4.34*10−6 cm2 s−1 as described in (30) two methods, it is clear that TDA was able to provide consistent hydrodynamic radii for the smaller sized BSA and oxytocin at their lowest concentrations of 0.05 mg/ml. Both DLS and TDA were successful in sizing the larger sized antibody adalimumab at a low concentration of 0.05 mg/ml. For etanercept under the same conditions, neither technique was ideal for sizing purposes. Considering each of the systems in more detail, for oxytocin the rh ranged from 0.9 to 1.1 nm when measured by TDA over all concentrations (Fig. 1a), which is slightly higher than the estimated hydrodynamic radius of about 0.8 nm (22). DLS proved to be less suitable for the analysis 2305 of oxytocin. Because of the small size of oxytocin and particularly at low concentrations, DLS analysis was influenced by the non-protein material in the formulation, e.g., small amounts of dust. Therefore, too high rzave values were measured (6.9–130 nm), in particular at the lower peptide concentrations. However, within the size distribu- tion by intensity plots from DLS (data not shown), a peak at about 0.9 to 1 nm representing oxytocin could be identified for the concentration range of 1 to 40 mg/ml. In the concentration range from 0.5 to 10 mg/ml, TDA and DLS resulted in similar results for the hydrodynamic radius of BSA of 3.8 to 4.3 nm (Fig. 1b), which is in good agreement with the theoretical size of 3.3 to 4.3 nm (23). At 50 mg/ml, TDA resulted in a higher rh of 4.6 nm, whereas rzave and rmain peak in DLS were found at 3.4 nm. At the lowest concentration of 0.05 mg/ml, rzave and rmain peak in DLS produced inconsis- tently higher radii values than theory (8.4 nm and 5.3 nm). For etanercept, a hydrodynamic radius between 7.0 and 8.3 nm was measured by TDA and DLS in the concentra- tion range from 0.5 to 50 mg/ml (Fig. 1c), which is in agreement with the theoretical value of 7.1 nm (22). For the formulation of etanercept at 0.05 mg/ml, TDA obtained a value rh of 4.9 nm, much lower than theoretically expected. For this sample and conditions, it would appear the TDA was operating at the limit of its detection. The rzave (DLS) was unexpectedly high (41.6 nm) for 0.05 mg/ml etaner- Fig. 1 Radius from DLS (Zaverage radius, radius main peak) and Taylor dispersion analysis for oxy- tocin (a), BSA (b), etanercept (c) and adalimumab (d). The theo- retical hydrodynamic radii (com- pare Table 1) are shown as light grey areas. The results are shown as average ± standard deviation of n=3 (DLS) and n=5 (TDA) individual measurements.