Evaluation of Degradation Pathways for Plasmid DNA in Pharmaceutical Formulations via Accelerated Stability Studies

Evans, Robert K.; Xu, Zheng; Bohannon, Kathryn E.; Wang, Bei; Bruner, Mark W.; Volkin, David B.

doi:10.1002/(sici)1520-6017(200001)89:1<76::aid-jps8>3.0.co;2-u

Cited by 115 publications

(114 citation statements)

References 20 publications

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“…6 Outside the cell, they can be generated by mechanisms generally involving metallic ions. 7,8 Oxidation could also result from attacks by ozone, an atmospheric pollutant that rapidly reacts with RNA either in solution or in the solid state. 9 Degradation can also occur through the activity of some metallic complexes catalyzing the hydrolytic cleavage of the phosphodiester bond or by contaminating nucleases.…”

Section: Introductionmentioning

confidence: 99%

An efficient method for long-term room temperature storage of RNA

Fabre

Colotte²,

Luis³

et al. 2013

Eur J Hum Genet

113

110

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RNA is a tool used in many fields, from molecular and cellular biology to medicine and nanotechnology. For most of these uses, the integrity of RNA is required and must be maintained during storage. Even though freezing is currently the storage method of choice, the increasing number of samples to be stored and the costly use of a cold chain have highlighted the need for room temperature preservation methods. Here, we report a new room temperature technology that consists in drying RNA samples in the presence of a stabilizer in stainless steel minicapsules. These air-and water-tight capsules isolate RNA from the atmosphere and maintain an anhydrous and anoxic environment. Through the evaluation of RNA integrity over time at room temperature or 90 1C, we identified atmospheric humidity as a major deleterious factor. The degradation rate dependence in temperature fitted an Arrhenius model, with an activation energy of 28.5 kcal/mol and an extrapolated room temperature degradation rate of 3.2 10 À13 /nt/s (95% confidence interval: 2.3-4.2/nt/s). In these conditions, it is expected that an RNA molecule will be subjected to 0.7-1.3 cut every 1000 nucleotides per century. In addition, we showed that stored RNA is compatible for further analyses, such as reverse transcription-quantitative PCR. No significant change in the C q values was observed over a simulated period of several decades. At last, our data are consistent with a sequence-independent degradation rate of RNA in the solid state.

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Section: Introductionmentioning

confidence: 99%

An efficient method for long-term room temperature storage of RNA

Fabre

Colotte²,

Luis³

et al. 2013

Eur J Hum Genet

113

110

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show abstract

“…Degradation of nucleic acids features in the storage of specimens, for example, cerebrospinal fluid and whole blood, and other samples, including various pathogens, and in forensic applications (1,3,6,9,10,13,16,18,21). In a pharmaceutical context, the stability of plasmids during storage has been investigated (7,11,15). All these studies have investigated storage by freezing based on rapid freeze and thaw cycles included in the experiments, but long-term storage has been neglected.…”

mentioning

confidence: 99%

Impact of Long-Term Storage on Stability of Standard DNA for Nucleic Acid-Based Methods

et al. 2010

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Real-time PCR is dependent upon a calibration function for quantification. While long-term storage of standards saves cost and time, solutions of DNA are prone to degradation. We present here the benchmark treatment for preservation of DNA standards, involving storage in 50% glycerol-double-distilled water, whereby a deviation of 0.2 threshold cycle (C T ) values resulted after 100 days of storage.

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“…Some processes have been known to nick or degrade plasmid DNA. 4 Among them, magnetic fields, 5,6 ultrasonic waves, 7 heating, 8,9 high shearing forces, 10,11 and pH 12,13 are known to rapidly degrade DNA. Standard characterization of DNA degradation has been done using the gel electrophoresis method, where degraded DNA and intact DNA are separated by their different motilities in the gel.…”

Section: Introductionmentioning

confidence: 99%

Characterization of DNA degradation using direct current conductivity and dynamic dielectric relaxation techniques

Sheu¹,

Sheu

2006

AAPS PharmSciTech

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The purpose of this study was to evaluate DNA degradation upon thermal heating using dielectric relaxation and direct current (DC) conductivity methods. Herring sperm DNA, human growth hormone (HgH) plasmid DNA, and secreted alkaline phosphatase (SEAP) plasmid DNA were used as the examples. DNA was heated at 80°C for 1 hour. The dielectric relaxation spectra as a function of the applied field frequency were measured for HgH DNA at 0.5 hours and at 1 hour. The frequency range covered was from 10 kHz to 100 kHz. The DC conductivity measurements were made for all 3 kinds of DNA at 4 time points: 0 hours, 0.5 hours, 0.75 hours, and 1 hour. At each time point the DC conductivity was measured for each sample as a function of concentration via water dilution. The results show that the dielectric relaxation method is less sensitive in characterizing heat-driven DNA degradation. Conversely, DC conductivity is very sensitive. The semiquantitative dependence of the conductivity upon heating suggests that DNA degradation involves more than plasmid DNA nicking. Double strand and single strand breaks may also occur. In addition, herring sperm DNA, HgH DNA, and SEAP DNA, though similar in their DC conductivity functional forms upon dilution, exhibit significant differences in their responses to sustained heating.

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Evaluation of Degradation Pathways for Plasmid DNA in Pharmaceutical Formulations via Accelerated Stability Studies

Cited by 115 publications

References 20 publications

An efficient method for long-term room temperature storage of RNA

An efficient method for long-term room temperature storage of RNA

Impact of Long-Term Storage on Stability of Standard DNA for Nucleic Acid-Based Methods

Characterization of DNA degradation using direct current conductivity and dynamic dielectric relaxation techniques

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