REPLACING SUCCINATE WITH GLYCOLATE BUFFER IMPROVES THE STABILITY OF LYOPHILIZED INTERFERON-GAMMA

Citation
Xm. Lam et al., REPLACING SUCCINATE WITH GLYCOLATE BUFFER IMPROVES THE STABILITY OF LYOPHILIZED INTERFERON-GAMMA, International journal of pharmaceutics, 142(1), 1996, pp. 85-95
Citations number
29
Categorie Soggetti
Pharmacology & Pharmacy
ISSN journal
03785173
Volume
142
Issue
1
Year of publication
1996
Pages
85 - 95
Database
ISI
SICI code
0378-5173(1996)142:1<85:RSWGBI>2.0.ZU;2-W
Abstract
Lyophilization is commonly used to dry protein pharmaceuticals to enha nce their shelf life. During the freezing step of this process, signif icant events (e.g. pH shifting) can occur in the uncrystallized, liqui d portion which influence the stability of the product. Herein, we pre sent evidence of such an effect and the impact on the quality of recom binant human interferon-gamma (IFN-gamma) lyophilized from mannitol-co ntaining succinate buffer at pH 5. In the frozen matrix, we hypothesiz e 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) spectr oscopy. The latter indicated that the succinate buffer lyophilized fro m 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-gamma exhibited a marked bioactivity los s during aqueous incubation at pH 3 compared with pH 5. This loss corr elated with (reversible) unfolding of the IFN-gamma molecule at low pH , as determined by both FT-IR spectroscopy and circular dichroism. We also examined the stability of IFN-gamma following lyophilization from pH 5 in two different buffer systems, succinate and glycolate. The la tter, which appeared to minimize the freeze-induced pH shifting, exhib ited superior solid-state stability upon 4-week incubation at 25 degre es C. Both samples had a similar cake structure (based on X-ray diffra ction and differential scanning calorimetry) and had the same residual moisture content. The data suggest that the difference in stability w as a consequence of the freeze-induced pH shifting in the succinate bu ffer system, resulting in a more perturbed (solid-state) structure for IFN-gamma. This is consistent with our FT-IR spectroscopic analysis o f the lyophilized protein.