Effects of Annealing on Product Stability and Visible Bubbles of the Freeze-dried Fusion Protein Etanercept Formulations

OBJECTIVE To investigate the effect of annealing steps during freeze-drying on the visible bubbles and their stability during the reconstitution of freeze-drying etanercept products.

METHODS Etanercept products were obtained by freeze-drying at different annealing temperatures. The appearance of the freeze-dried powder was observed visually, the moisture content of the freeze-dried powder was measured by Carl Fischer method, the bubble volume after reconstitution was measured by vernier caliper, the internal microstructure of the freeze-dried powder was observed by scanning electron microscopy(SEM), and the crystallization of the excipients was detected by X-ray diffraction, and accelerated stability experiments were conducted by storing at 40 ℃ for 1, 2, and 3 months respectively. Then the soluble aggregates in different periods was investigated by size exclusion chromatography, and the stability of freeze-dried formulations before and after 3 months storage was observed by Fourier transform infrared spectroscopy.

RESULTS Annealing could reduce the volume of visible bubbles after reconstitution of freeze-dried etanercept products, and the reduction effect was similar with different annealing temperatures. SEM results showed that annealing increased the size of freeze-dried powder micropores, which resulted in larger bubbles during reconstitution. X-ray diffraction results showed that annealing did not affect the crystal type of mannitol. In the accelerated stability test, the results of size exclusion chromatography showed that annealing could slightly reduce the stability of freeze-dried etanercept formulation, and the effect of different annealing temperatures was similar. The infrared spectra showed that the curves in each group overlapped on the characteristic peaks, indicating that the secondary structure of etanercept remained basically unchanged in the accelerated experiment.

CONCLUSION Annealing can reduce the visible bubble volume after reconstitution and slightly reduce the stability of etanercept formulations, and setting the annealing step during lyophilization can effectively reduce the bubble problem after reconstitution.