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 degrees C/60% RH climate chamber, before placing all vials in climate chambers (25 degrees C/60% RH and 40 degrees 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 degrees 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.

Fig. 1

Plasmid DNA map of pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) including selected restriction sites

Fig. 2

Purity of pDERMATT lyophilised with four different disaccharides (filled circles represent sucrose; filled nabla represents trehalose; circles represent maltose; triangles represent lactose) fully dried (a,b) and partly wet (c,d) after storage at 25°C/60% RH (a,c) and 40°C/75% RH (b,d)

Fig. 3

Representative chromatograms of pDERMATT in sucrose (PW) during storage at 40°C/75% RH; at t = 0 (a), t=1 month (b) and t = 6 months (c). 1 µg injection, detection at 260 nm

Fig. 4

Decrease in Ln(AUC) of luciferase expression (photons/s) in human ex vivo skin after tattoo administration (2 mg/ml pVAX:Luc, needle depth 1.5 mm, 100 Hz, 20 s) when formulated in disaccharides vs dissolved in water. pDNA dissolved in water without any excipients gave a significant better expression (Ln(AUC) = 15.6) than when disaccharides are added to the formulation (all p values <0.01). Of the tested disaccharides only maltose gave a significant better expression than trehalose (p value 0.001) and lactose (p value 0.01). Maltose compared to sucrose gave a p value of 0.104