Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying.
The stabilizing effect of mannitol during the freeze-drying of proteins was studied using L-lactate dehydrogenase (LDH, rabbit muscle), beta-galactosidase (Escherichia coli) and L-asparaginase (Erwinia chrysanthemi) as model proteins. Crystallization of mannitol was studied by powder X-ray diffraction and differential scanning calorimetry (DSC), in relation to the stabilizing effect. All the enzymes were protected concentration-dependently by amorphous mannitol, but the stabilizing effect was decreased with an increase in mannitol crystallinity. The heat-treatment of frozen solutions above crystallization temperature prior to drying enhanced mannitol crystallization and LDH inactivation. The importance of maintaining excipients in an amorphous state during freeze-drying, previously reported for Aspergillus oryzae beta-galactosidase (K. Izutsu et al., Pharm. Res., 10, 1233 (1993)), was confirmed using three different enzymes.
Izutsu
K
K
National Institute of Health Sciences, Tokyo, Japan.
Yoshioka
S
S
Terao
T
T
D000818:Animals; D003460:Crystallization; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry*; D005612:Freeze Drying; D008353:Mannitol / Q000737:chemistry*; D011817:Rabbits
D004798:Enzymes; D008353:Mannitol