Replacing succinate with glycolate buffer improves the stability of lyophilized interferon-γ

Abstract

Lyophilization is commonly used to dry protein pharmaceuticals to enhance their shelf life. During the freezing step of this process, significant events (e.g. pH shifting) can occur in the uncrystallized, liquid portion which influence the stability of the product. Herein, we present evidence of such an effect and the impact on the quality of recombinant human interferon-γ (IFN-γ) lyophilized from mannitol-containing succinate buffer at pH 5. In the frozen matrix, we hypothesize that the monosodium form of succinic acid crystallized, as evidenced by electrical resistance data, affecting the buffer system's ability to maintain pH, as probed by Fourier-transform infrared (FT-IR) spectroscopy. The latter indicated that the succinate buffer lyophilized from aqueous solution at pH 5 exhibited an ionization state corresponding to that of some 1–2 pH units lower. In exploring the implications for stability, we found that IFN-γ exhibited a marked bioactivity loss during aqueous incubation at pH 3 compared with pH 5. This loss correlated with (reversible) unfolding of the IFN-γ molecule at low pH, as determined by both FT-IR spectroscopy and circular dichroism. We also examined the stability of IFN-γ following lyophilization from pH 5 in two different buffer systems, succinate and glycolate. The latter, which appeared to minimize the freeze-induced pH shifting, exhibited superior solid-state stability upon 4-week incubation at 25°C. Both samples had a similar cake structure (based on X-ray diffraction and differential scanning calorimetry) and had the same residual moisture content. The data suggest that the difference in stability was a consequence of the freeze-induced pH shifting in the succinate buffer system, resulting in a more perturbed (solid-state) structure for IFN-γ. This is consistent with our FT-IR spectroscopic analysis of the lyophilized protein.