Protective mechanism of stabilizing excipients against dehydration in the freeze-drying of proteins.
OBJECTIVE
To investigate the influence of type and amount of excipient on the preservation of the native structure and the biologic activity of freeze-dried lysozyme and catalase.


METHODS
The secondary structure of protein in the dried form and in aqueous solution was obtained using second derivative infrared spectroscopy and circular dichroism spectra respectively whilst the activity was determined using bioassay.


RESULTS
Small molecular excipients (glycerol, sorbitol, 1,6-anhydroglucose, sucrose, and trehalose) were found to stabilize the activity and/or the native structure of freeze-dried lysozyme and catalase, despite the processing temperatures being above Tg' of excipent-protein mixtures. The preservation of catalase activity required excipient to be present at a lower excipient to enzyme mass ratio than that necessary to preserve native structure in the dried form. Combining dextran with sucrose synergistically protected the native structure of catalase but preserved the activity in an additive manner.


CONCLUSIONS
The results indicate that the stabilization of catalase and lysozyme by excipients during dehydration was mainly due to water substitution rather than the formation of glass; the latter appearing not to be a prerequisite during freeze-drying.
Liao
Yong-Hong
YH
MedPharm, Department of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN England, United Kingdom.
Brown
Marc B
MB
Quader
Abdul
A
Martin
Gary P
GP
D003890:Desiccation / Q000379:methods*; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods; D011506:Proteins / Q000737:chemistry*
D005079:Excipients; D011506:Proteins