ABSTRACT

Introduction ........................................................................................................ 697

Experimental Procedures.................................................................................. 698

Reagents....................................................................................................... 698

Sugar Crystallinity ..................................................................................... 698

X-ray Diffraction......................................................................................... 698

Glassy Preservation ................................................................................... 699

Enzyme Assay ............................................................................................ 699

Results and Discussion ..................................................................................... 700

Crystallinity Changes as a Function of RH ........................................... 700

LDH Preservation ...................................................................................... 701

Conclusion .......................................................................................................... 701

References ........................................................................................................... 701

Freeze-drying is the conventional way to stabilize labile biological products,

such as proteins and enzymes (Franks et al., 1991). Freeze-dried products

usually contain sugars, polyols, or amino acids as excipients. For example,

trehalose and other glass-forming solutes have been used as excipients for

enzyme EcoRI stabilization (Rossi et al., 1992). Excipients with the ability to

turn into the glassy state during the freeze-drying process help preserve the

physical and chemical integrity of biological products (Fox, 1995). Solute

crystallization from amorphous mixtures is considered to be of importance,

usually undesirable, for many applications of concentrated aqueous

carbohydrate systems; examples include food processing and preservation

of biological activity in freeze-dried pharmaceutical preparations. Recently,

however, it has been proposed that sugar crystallization may, in the case of

the formation of a stable crystalline hydrate, be of benefit in the enhancement

of chemical and physical stability in humid environments. Thus, if a sugar

can crystallize in the form of a hydrate in real time, then depending on the

hydrate stoichiometry, its crystallization from the amorphous state will

prevent the dilution of the remaining amorphous solution phase. If the

residual (noncrystalline) solution phase then becomes sufficiently concen-

trated, its glass-transition temperature will be raised (Kajiwara and Franks,

1997). In this study, raffinose and trehalose, which at ambient conditions

exist as a crystalline pentahydrate and dehydrate, respectively, were used as

amorphous excipients and its ability to stabilize LDH in various humid

conditions was tested.