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
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.