2308 Fig. 5 Results for DLS (rZave) and TDA (rh) for etanercept subjected to a 1-week stability study at 50°C. *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. Analysis of Stressed Protein Formulations TDA and DLS have the advantage that they can be used in batch mode over a range of concentration without the need for prior separation of components and without the need for internal standards. The ability to combine stress testing of protein formulations and rapidly detect any aggregation tendency is an important part of the biopharmaceutical development process. In this section of the work, we wished to see if TDA was able to detect change in the average size of stressed protein samples in a similar manner to DLS. Both techniques were benchmarked using HP-SEC to prove the formation of soluble aggregates. One advantage Hawe, Hulse, Jiskoot and Forbes of TDA over DLS would be its lower injection sample size (previously optimized at 60 nl). TDA and DLS were compared for their ability to detect aggregation in heat- stressed formulations of etanercept, adalimumab, rituximab and a model IgG (Fig. 4), as well as in etanercept samples during a one-week accelerated stability study at 50°C (Fig. 5). Heat stress induced the formation of soluble and non- soluble aggregates within the formulations, as shown by HP-SEC (Table II). In general, rzave and rmain peak (DLS) increased to a greater extent than rh (TDA) for the aggregated formulations (Fig. 4). This is mainly obvious for adalimumab stressed at 65°C (Fig. 4b) and the model IgG stressed at 80°C (Fig. 4d), which contained about 60% soluble and non-soluble aggregates as measure by HP-SEC (Table II). The rzave (DLS) increased from 5.7 nm (adali- mumab) and 7.5 nm (IgG) to about 22 nm, whereas the rh (TDA) changed from 4.6 to 6.9 nm (adalimumab) and from 6.5 to 7.5 nm (IgG) after stressing. For formulations that mainly contained small soluble aggregates, the difference between rzave (DLS) and rh (TDA) was less pronounced. Whilst the absolute values of the average radii of the samples reported by TDA and DLS may have differed markedly on occasion, it is noteworthy that TDA was able to identify that all four tested proteins showed significant increase in size after undergoing the more aggressive stress test. After exposure of the four protein formulations to the less aggressive stress conditions, TDA results showed significant increase in size for three of the four formulations, whereas DLS showed all four to be significant. On inspection of the HP-SEC data, this equates to TDA being able to statistically significantly differentiate between a sample of rituximab containing 99.4% monomer and one containing 98.9% monomer but not able to discriminate between a sample of adalimumab containing 99.3% Table II Size Analysis of Protein Samples by HP-SEC Quantified from UV Detection at 280 nm Monomer [%] Dimer [%] HMW [%] Fragments [%] Recovery [%] Soluble aggregates + non-recovered fraction [%] Etanercept, NS Etanerecept, 10 min 65°C Etanerecept, 10 min 70°C Adalimumab, NS Adalimumab, 10 min 60°C Adalimumab, 10 min 65°C IgG, NS IgG, 10 min 75°C IgG, 10 min 80°C Rituximab, NS Rituximab, 10 min 60°C Rituximab, 10 min 65°C 93.0 88.9 81.3 99.3 96.8 58.3 99.1 95.4 45.0 99.4 98.9 97.3 1.3 3.5 6.0 0.4 1.0 1.8 0.0 3.0 8.0 0.6 0.7 1.0 0.0 2.0 7.4 0.0 1.8 39.5 0.0 0.0 44.3 0.0 0.4 1.7 5.6 5.6 5.3 0.3 0.3 0.3 0.9 1.6 2.7 0.0 0.0 0.0 100.0 96.6 95.8 100.0 101.6 69.6 100.0 94.8 87.2 100.0 100.5 98.5 7.0 14.1 22.1 0.7 1.7 59.4 0.9 9.6 60.8 0.6 0.6 4.2