SURFACE DENATURATION AT SOLID-VOID INTERFACE - A POSSIBLE PATHWAY BY WHICH OPALESCENT PARTICULATES FORM DURING THE STORAGE OF LYOPHILIZED TISSUE-TYPE PLASMINOGEN-ACTIVATOR AT HIGH-TEMPERATURES

During protein lyophilization, it is common practice to complete the f reezing step as fast as possible in order to avoid protein denaturatio n, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that y ielded more opalescent particulates upon long term storage at 50 degre es C, under a 150 mTorr nitrogen seal gas environment. Fast cooling al so resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22 - 1.78 m(2)/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass trans ition temperatures (103 - 105 degrees C). However, during storage the rate of opalescent particulate formation in the lyophilized product (a s determined by UV optical density measurement in the 360 to 340 nm ra nge for the reconstituted solution) was proportional to the cake surfa ce area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denat ures during storage at elevated temperatures. Irreversible denaturatio n at the ice-liquid interface during freezing in lyophilization is unl ikely to occur, since repeated freezing/thawing did not show any adver se effect on the protein. Infrared spectroscopic analysis could not de termine whether protein, upon lyophilization, at the solid-void interf ace would still be in a native form.