Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation

Abstract

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25°C/60% RH climate chamber, before placing all vials in climate chambers (25°C/60% RH and 40°C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40°C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.

Stabilization of Plasmid DNA and Lipid-Based Therapeutics as Dehydrated Formulations

“…The interest in preserving DNA as dried preparations has increased vastly in response to the growing demand from a variety of fields, including pharmaceutical sciences [56,82,91,113,121,122], forensics [123], molecular diagnostics [124], and biorepository management [125]) for storing DNA samples or tumor specimen banks [114,126] for prolonged periods of time, sometimes in large numbers, in regions where sophisticated storage equipment may not be available. Although the requirements are quite different in each of these fields, the need for strategies that provide highly stable storage conditions (e.g., room temperature storage) seems to be a common factor.…”

“…However, DNA must resist the freezing and drying stresses encountered during processing before stability can be addressed. In this respect, it has been demonstrated that the addition of disaccharides, such as sucrose and trehalose, are beneficial to prevent loss of SC DNA during lyophilization [55,56,82,91,96,113,115,121,178,179].…”

Preservation of primer and probes on “ready-to-use” 96-well microtiter plates: A step forward towards enhancing throughput and harmonization of real-time PCR applications in food and feed control

“…For TRH (D-(þ)-trehalose dehydrate, Fluka Analytical, Buchs, Switzerland) final concentrations of 0.07% TRH (w/v) e corresponding to a ratio of 20/1 TRH/oligo nucleic acid according to Quaak, Haanen, Beijnen, and Nuijen (2010) e and 4.17% TRH (w/v) were used. For gelatine (GEL; Perfect Block Gelatine, MoBiTec GmbH, Göttingen, Germany), final concentrations of 0.1% GEL (w/v) and 0.17% GEL (w/v) and for polyethylene glycol (PEG 8000; Fluka Analytical Biochemica, Buchs, Switzerland) final concentrations of 0.5% PEG (w/v), 1.5% PEG (w/v) and 8% PEG (w/v) were applied.…”

Accelerated Stability Studies for Moisture-Induced Aggregation of Tetanus Toxoid

“…In general, solid proteins are the preferred mode of storage, since in solution dosage forms, proteins and even plasmid DNA are relatively unstable (1,2). A minimum amount of water is required for maintaining the three-dimensionally folded native conformation of a protein in the solid state.…”