Lyophilization-induced protein denaturation in phosphate buffer systems: Monomeric and tetrameric beta-galactosidase

During freezing in phosphate buffers, selective precipitation of a less sol uble buffer component and subsequent pH shifts may induce protein denaturat ion. Previous reports indicate significantly more inactivation and secondar y structural perturbation of monomeric and tetrameric beta -galactosidase ( beta -gal) during freeze-thawing in sodium phosphate (NaP) buffer as compar ed with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during free zing in the NaP buffer.(1) In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta -gal in eit her NaP or YP buffer resulted in significant, secondary structural perturba tions, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze- drying, indicating that most dehydration-induced unfolding was reversible o n reconstitution of the freeze-dried protein. In contrast, the tetrameric p rotein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried sampl es relative to that measured after freeze-thawing. To ensure optimal protei n stability during freeze-drying, the protein must be protected from both f reezing and dehydration stresses. Although poly(ethylene glycol) and dextra n are preferentially excluded solutes and should confer protection during f reezing, they were unable to prevent lyophilization-induced denaturation. I n addition, Tween did not foster maintenance of native protein during freez e-drying. However, sucrose, which hydrogen bonds to dried protein in the pl ace of lost water, greatly reduced freezing- and drying-induced denaturatio n, as observed by the high retention of native protein in the dried state a s well as the complete recovery of active P-gal on reconstitution. These re sults indicate that addition of an effective stabilizer, such as sucrose, m ay minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.