ABSTRACT
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.
Subject(s)
DNA/chemistry , DNA/metabolism , Freeze Drying/adverse effects , Luciferases/genetics , Plasmids/metabolism , Animals , COS Cells , Chemistry Techniques, Analytical/methods , Circular Dichroism , Edetic Acid/chemistry , Monosaccharides/chemistry , Polysaccharides/chemistry , Time Factors , TransfectionABSTRACT
The objective of this study was to evaluate the feasibility of foam fractionation for removal of contaminating endotoxin from biotechnology products, including plasmid DNA and recombinant proteins. After foam fractionation, alone and with bovine serum albumin and Triton X-100 as pro-foaming agents, FITC-labeled endotoxin remains in the bulk solution. These studies suggest that foam fractionation will be ineffective in the purification of plasmid DNA solutions, which are not surface-active and remain in the bulk solution with endotoxin. These data support the use of foam fractionation as an effective method for separating surface-active recombinant proteins, which will concentrate in the foam, away from endotoxin.
Subject(s)
DNA, Recombinant/isolation & purification , Endotoxins/isolation & purification , Recombinant Proteins/isolation & purification , Biotechnology , Fluorescein-5-isothiocyanate , Fluorescent Dyes , PlasmidsABSTRACT
The objective of this study was to characterize the interaction of endotoxin with cationic liposomes used in nonviral gene delivery. Endotoxin-cationic liposome interaction was characterized using fluorescent anisotropy, and the Limulus amebocyte lysate (LAL) assay. Cellular toxicity of endotoxin-cationic liposome complex was examined using a dimethylthiazol diphenyltetrazolium bromide (MTT) assay. The effect of endotoxin on the lipid-DNA complex and subsequent transfection into COS-1 cells was also examined. A competitive interaction occurred between fluoroscein isothiocyanate (FITC)-labeled endotoxin and plasmid DNA for binding dioleoyl glycero trimethylammonium propane:dioleoyl glycero phosphoethanolamine (DOTAP:DOPE) liposomes using fluorescent anisotropy techniques. The LAL assay demonstrated no change in endotoxin activity upon interaction with liposomes. No loss of COS cell viability was detected via the MTT assay during a 5-hr exposure to endotoxin. Transient transfection studies indicate that increasing levels of endotoxin lowered activity more than 90% at 50,000 endotoxin units (EU)/ml. Endotoxin and cationic liposomes interact mainly by an electrostatic attraction. Endotoxin contamination can potentially impact transfection efficiency via competition with plasmid DNA for cationic liposome binding by increasing transfection variability at 50 EU/ml, a concentration of endotoxin contamination that can occur with small-scale plasmid preparations used for in vitro cell transfections, but would not be expected with typical GLP or GMP preparations used in clinical studies.
Subject(s)
Endotoxins/chemistry , Genetic Therapy , Liposomes/chemistry , Transfection , Animals , COS Cells , Fluorescence Polarization , Lipids/chemistry , Plasmids , Static ElectricityABSTRACT
The ability of a plasmid DNA formulation to code for a functional protein was assayed as a marker for plasmid DNA stability using a cotransfection method to measure transcription efficiency. This method shows increased sensitivity and reproducibility over single plasmid transfection methods. Method validation, by measuring DNA degradation rates, demonstrates that buffer choice may be of some importance in the pharmaceutical formulation of plasmid DNA. Degradation rates dependant on citrate buffer concentration were observed. This cotransfection method has proven superior to standard agarose gel electrophoresis in quantifying subtle pRL-CMV plasmid DNA damage and could be used to help predict stability of a final plasmid DNA dosage form.
