2304 local hospitals (Amsterdam and Leiden, The Netherlands). A humanized IgG type 1 was used as a model protein (molar mass 150 kDa, isoelectric point ~8–9). Oxytocin and BSA were purchased from Sigma (Sigma Aldrich, Zwijndrecht, The Netherlands). All other substances used for preparation of buffers and mobile phase were standard analytical grades. Enbrel®50 mg (50 mg/ml etanercept, 10 mg/ml sucrose, 5.8 mg/ml NaCl, 5.3 mg/ml arginine*HCl and 3.9 mg/ml Na2HPO4*2H20 pH 6.3) and Humira®40 mg (50 mg/ml adalimumab, 2 mg/ml NaCl, 1.52 mg/ml Na2HPO4*2H20, 0.3 mg/ml sodium citrate, 1.3 mg/ml citric acid monohydrate, 12.0 mg/ml mannitol and 1.0 mg/ml polysorbate 80 pH 5.2) were analyzed at different concentrations between 50 and 0.05 mg/ml. The formulations were diluted either with only buffer (3.9 mg/ ml Na2HPO4*2H20 pH 6.3 for Enbrel®50 mg/ml and 1.52 mg/ml Na2HPO4*2H20, 0.3 mg/ml sodium citrate, 1.3 mg/ml citric acid monohydrate, pH 5.2 for Humira®40 mg) or the placebo buffer including all excipients. Mabthera®100 mg (10 mg/ml rituximab, 9 mg/ml NaCl, 7.3 mg/ml sodium citrate dehydrate and 0.7 mg/ml polysorbate 80, pH 6.5) was used at a concentration of 10 mg/ml and the humanized IgG1 (1.0 mg/ml IgG, 100 mM phosphate, pH 7.2) at 1 mg/ ml. Oxytocin (50 mM phosphate, pH 4.5) was used in concentrations between 40 and 0.05 mg/ml and BSA (PBS, pH 7.2) between 50 and 0.05 mg/ml. To induce aggregation, the commercial preparations and the humanized IgG1 were subjected to 10 min thermal stress using 1.5-ml reaction caps and a thermomixer (Eppendorf, Germany). Enbrel®50 mg was stressed at 65 and 70°C, Humira®40 mg and Mabthera®100 mg at 60 and 65°C and the model IgG at 75 and 80°C. Taylor Dispersion Analysis TDA was performed on a TDA200 HT nano-sizing sytem (Paraytec Ltd., York, UK). Samples (56 nl) were injected into fused silica capillary under a continuous flow of buffer (2 mm/s) using a CE injection system (PrinCE, Prince technologies B.V., Netherlands). The corresponding for- mulation buffer was used as running buffer. The detector head is placed inside the CE at a controlled temperature of 25°C. The total capillary length, with ID:OD dimensions of 75:360 μm, was 143.3 cm with length to first window being 46 cm and length between windows 48.5 cm. The capillary was cleaned between samples using a sodium hydroxide wash (1 M). UV detection wavelength used was 214 nm. Dynamic Light Scattering Dynamic light scattering (DLS) was performed on a Zetasizer Nano (Malvern, Herrenberg Germany). Five hundred μl of Hawe, Hulse, Jiskoot and Forbes the formulations were analyzed in plastic cuvettes at 25°C using the automatic mode for identifying the best number of subruns and measurement time (n = 3). The z-average radius (zaverage) and polydispersity index (PDI) were calculated from the correlation function using the Dispersion Technology Software version 6.01 (Malvern, Herrenberg, Germany). Size Exclusion Chromatography (HP-SEC) The non-stressed and stressed samples of Enbrel®50 mg, Humira®40 mg, Mabthera®100 mg, and IgG were analyzed by HP-SEC, using a TSKgel4000SWXL column (Tosoh Biosep, Stuttgart, Germany) on an isocratic HPLC system with a Waters 515 pump, a Waters 717 plus autosampler, a Waters 474 fluorescence detector (Waters, Milford Massachusetts, USA) and a Shimadzu SPD UV/ Vis detector (Shimadzu, Tokyo, Japan) at a flow rate of 0.5 ml/min. For the standard IgG formulations of 1.0 mg/ ml, 50 μl sample was injected. The higher concentrated products were diluted to a concentration of 5 mg/ml using the particular formulation buffer, and 10 μl were injected. The mobile phase was composed of 50 mM sodium phosphate, 150 mM arginine and 0.025% NaN3, pH 6.5. To quantify aggregation in the IgG formulations, UV absorption at 280 nm was used. Statistical Analysis Statistical analysis was performed by a one-way ANOVA with a post-test (Tukey) using α = 0.05 (95% confidence interval) using GraphPad Prism (Version 5.02). Within the statistical analysis, the data (radii from TDA and DLS) for the stressed formulations were compared to the data of the non-stressed formulation as a control. RESULTS AND DISCUSSION Analysis of Peptides and Proteins Varying in Size and Concentration No previous work has explored the use of TDA in its new format to determine the hydrodynamic radius of an extensive range of proteins and peptides differing in size (Table I) over a wide concentration range and in complex formulations containing various excipients. Oxytocin, BSA, etanercept and adalimumab formulated at concentrations between 0.05 and 50 mg/ml were analyzed by TDA and DLS, with the resulting average hydrodynamic radius rh (TDA), as well as the z-average radius rzave (DLS) and average main peak radius rmain peak (DLS) shown in Fig. 1. In comparing the relative strengths and weaknesses of the