The effect of lyophilization on plasmid DNA activity.

The effect of lyophilization of plasmid DNA's ability to express an encoded protein was studied. Plasmid DNA, pRL-CMV expressing Renilla luciferase, was purified and stored in Tris-ethylenedi-aminetetraacetic acid (EDTA) buffer. Aliquots of the plasmid were lyophilized using analytical equipment, both alone and in the presence of carbohydrate. Samples were rehydrated and subject to functional and structural analyses. Analytical techniques included transfection efficiency in COS-1 cells, agarose gel electrophoresis, dimethylethylenediamine (DMED) assay for abasic sites, circular dichroism measurement, and UV spectroscopy. The lyophilization of pRL-CMV plasmid DNA resulted in a statistically significant loss of transfection efficiency (p < 0.05). Mono- and disaccharides could completely restore transfection efficiency. Agarose gel electrophoresis and the DMED assay demonstrated no change in gross plasmid structure or increase in abasic sites during lyophilization, respectively. Changes in DNA form, as measured by a change in ellipsisity, were observed on lyophilization. However, these changes were transient and were not shown to be responsible for loss of transfection efficiency. A hyperchromic effect was observed at 260 nm after lyophilization and could be reversed by the presence of carbohydrates. Lyophilization causes a decrease in plasmid DNA activity as measured by an in vitro transfection assay. Carbohydrates can ameliorate this decreased activity, which may be due to structural changes seen during the lyophilization process.

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.

Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.

Deterioration of lyophilized pharmaceutical proteins.

The successful use of proteins in pharmaceutical and other commercial applications requires close examination of their relative fragility. Because of the resultant enhanced stability, proteins are often formulated in the solid state, even though dehydration tends to alter their structure. Even in the solid form, however, proteins may become inactivated due to various deleterious processes, e.g., aggregation. This review focuses on such mechanisms, with an emphasis on case studies conducted in our laboratory. Proteins which have both disulfide bonds and free thiols may aggregate via thiol-disulfide exchange, and this process may be facilitated by lyophilization-induced structural perturbations. For proteins possessing disulfides but not free thiols, aggregation also may occur when native disulfides are beta-eliminated, thus giving rise to thiol species which can catalyze disulfide scrambling. Other deleterious processes have also been uncovered, including a formaldehyde-mediated aggregation of formalinized vaccines. It is illustrated how knowledge of such deterioration pathways makes possible the rational development of stable solid protein formulations.

Degradation of macromolecules during preservation of lyophilized pathological tissues.

Methods of preserving biological specimens are becoming more important due to recent advances in molecular biological analysis. Storing samples in a freezer, however, is still the most commonly used method of preserving pathological specimens. We investigated the feasibility of using freeze-dried tissues stored at room temperature as an alternative method of preserving tissue samples for molecular analysis at the DNA, RNA, and protein levels. The freeze-dried tissues were transferred to a sealed vial filled with nitrogen gas and kept for months at room temperature. DNA and protein were stably preserved for at least 24 weeks. RNA, however, showed slight degradation after 10 weeks of storage. Controlling moisture and temperature during long-term storage was found to be important, as it significantly affected the stability of these cellular molecules in tissues. Shelf-stable preservation eliminates the need for storage in a freezer and allows convenient shipping of samples to distant places. These findings should provide practical basis for the development of a convenient and economical way of preserving pathological specimens for a variety of analyses in the field of molecular biology.

Electron microprobe study of the effect of long-term vacuum storage of freeze-dried cryosections on distribution of elements in cells.

The distribution of elements in yeast cells was measured on freshly prepared, freeze-dried cryosections and compared with the distribution obtained on the same sections after storage for 20 months in a vacuum below 2.6 kPa. The average concentration of phosphorus remained unchanged but was equalized throughout the cells, i.e. it migrated from vacuoles into the cytoplasm and mitochondria. Zinc remained preferentially localized in the vacuoles, but the ratio between vacuolar and cytoplasmic zinc concentrations decreased about three-fold. Cytoplasmic and mitochondrial concentrations of potassium remained unchanged while partial release from the vacuoles and subsequently from the cells was observed. This resulted in a homogeneous distribution of potassium in the cells after 20 months and some of the vacuolar potassium appeared in spectra of formvar film measured several micrometres from the cells. A large increase in the sodium (from 160 to 360% more than in fresh sections), magnesium (from 110 to 200% more) and sulphur (from 70 to 350% more) contents was observed in all cellular compartments (except for vacuoles, where only a 20% increase in the magnesium content was observed), while chlorine was almost completely released from the cells. The limitations of the use of long-term vacuum-stored cryosections for electron microprobe analysis of cells are discussed.