Comparison of analytical methods to detect instability of etanercept during thermal stress testing

Several analytical methods were used to detect instability in three batches of etanercept showing significant differences in sensitivity in the order of: Bis-Ans fluorescence > HP-SEC > DLS > Far-UV CD spectroscopy > ELISA > Second derivative UV spectroscopy. The aim was comparing the capability of a set of analytical methods to detect physical instability (focus on aggregation and structural changes) of etanercept during thermal stress testing as early as possible. Pre-filled syringes of Enbrel® 50 mg from three batches were thermally stressed for one week at 50 °C. Samples were taken at days 0, 1, 2, 3, 4 and 7, and analyzed with high-performance liquid size exclusion chromatography (HP-SEC), SDS–PAGE gel electrophoresis, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA). Thermal stress resulted in the formation of small soluble aggregates (HP-SEC, DLS) which were in part covalent (SDS–PAGE), and conformationally changed (Bis-ANS, CD, UV). No significant increase in subvisible particles was detected by light obscuration. An apparent increase in TNF-α binding to etancercept in the stressed formulations was found by ELISA. The three batches were comparable when unstressed, but showed slight differences in aggregation tendency. Bis-ANS fluorescence was most sensitive with respect to early-stage detection of heat-induced instability of etanercept (significant changes already at day 1), followed by HP-SEC (day 2) and DLS (day 3). This points towards a degradation mechanism involving exposure of hydrophobic patches due to partial unfolding followed by aggregation.