Characterization of side reactions during the annealing of small interfering RNAs

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class o...

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Bibliographic Details
Published in:Analytical biochemistry 2011-07, Vol.414 (1), p.47-57
Main Authors: Seiffert, Stephan, Debelak, Harald, Hadwiger, Philipp, Jahn-Hofmann, Kerstin, Roehl, Ingo, Vornlocher, Hans-Peter, Noll, Bernhard
Format: Article
Language:English
Subjects:
annealing
cations
Chromatography, Gel
Chromatography, High Pressure Liquid
differential scanning calorimetry
drugs
Duplex stability
gel chromatography
gene expression
Hot Temperature
Ion-pair reversed-phase HPLC
manufacturing
Mass Spectrometry
Nucleic Acid Conformation
nucleic acids
Oligonucleotide
oligonucleotides
Oligonucleotides - chemistry
product quality
reversed-phase high performance liquid chromatography
Ribose - analogs & derivatives
RNA
RNA, Small Interfering - chemistry
siRNA
Size-exclusion chromatography
small interfering RNA
spectroscopy
temperature
therapeutics
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Summary:Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2′- O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions—such as 2′,3′-isomerization, strand scission, and HF elimination—depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2011.02.040