FOURIER-TRANSFORM INFRARED SPECTROSCOPIC INVESTIGATION OF PROTEIN STABILITY IN THE LYOPHILIZED FORM

Upon the removal of water, proteins undergo a major, reversible rearra ngement of their secondary structure, as revealed by FTIR spectroscopy . We have found herein that for recombinant human albumin (rHA) the ex tent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (prot ein concentration, pH, and the presence of excipients such as dextran or NaCl) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rK A's solid-state stability against moisture-induced aggregation. In all cases, the alpha-helix content of rHA drops from 58% in solution to 2 5-35% in the dehydrated state, the beta-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and c onfirmed that it too undergoes a significant alteration of the seconda ry structure upon lyophilization. The extent of this structural reorga nization has been found to be insensitive to the pH of the aqueous sol ution prior to lyophilization from pH 1.9 to 5.1, even though the ther mal transition temperature (T-m) in aqueous solution over this range v aries by 30 degrees C.