Due to the recent clinical success of mRNA technology in the management of the covid-19 pandemic, there has been an increase in new innovations for the production and analysis of this type of therapeutic. mRNA is produced in a cell-free process called in vitro transcription using a polymerase-assisted enzymatic transcription of a target area in selected plasmid DNA. After completed transcription the residual plasmid DNA becomes an unwanted impurity and must be removed. For this purpose, different approaches can be used, such as enzymatic digestion with deoxyribonucleases, chromatographic separation and precipitation techniques. For therapeutics, the current regulatory requirements are that there is less than 10 ng of residual DNA per dose, which can range between 30 and 100 µg of mRNA.
Possible residual plasmid DNA in mRNA samples must be monitored with a suitable analytical method. The current method for quantifying residual DNA uses a quantitative polymerase chain reaction (qPCR). Using qPCR can be quite expensive, requires knowledge of the plasmid sequence and is relatively difficult to interpret. Considering the presented problem, the central theme of this master′s thesis is the development of a HPLC method that is simpler, sufficiently selective, accurate, precise, and sensitive enough to meet regulatory requirements (10 ng of residual plasmid DNA). The results were compared side-by-side with the above-mentioned qPCR method and with agarose gel electrophoresis, that can also be used as a method of choice for checking the presence and integrity of different nucleic acids. In addition to the development of this new HPLC analytical method, we also optimized sample preparation to use the smallest possible amount of RNaseA enzyme (0,4 ng/µg mRNA) and the shortest possible incubation time (0,5 h).
The obtained results indicate successful development of the new HPLC method, as the content of residual plasmid DNA in the analyzed mRNA samples is comparable between all three selected methods.
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