Abstract
In vitro transcription (IVT) reaction is an RNA polymerase-catalyzed production of messenger RNA (mRNA) from DNA template, and the unit operation with highest cost of goods in the mRNA drug substance production process. To decrease the cost of mRNA production, reagents should be optimally utilized. Due to the catalytic, multicomponent nature of the IVT reaction, optimization is a multi-factorial problem, ideally suited to design-of-experiment approach for optimization and identification of design space. We derived a data-driven model of the IVT reaction and explored factors that drive process yield (in g/L), including impact of nucleoside triphosphate (NTP) concentration and Mg:NTP ratio on reaction yield and how to optimize the main cost drivers RNA polymerase and DNA template, while minimizing dsRNA formation, a critical quality attribute in mRNA products. We report a methodological approach to derive an optimum reaction design, with which cost efficiency of the reaction was improved by 44%. We demonstrate the validity of the model on mRNA construct of different lengths. Finally, we maximized the yield of the IVT reaction to 24.9 ± 1.5 g/L in batch, thus doubling the highest ever reported IVT yield.
Published Version
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