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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., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA., (© 2022 Sartorius BIA Separations, D.O.O. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

In recent years considerable attention has been paid to the treatment of postoperative pain, with regard to the favorable effect of adequate analgesia on patient outcome. Multimodal analgesia (e.g., opioids and nonsteroidal anti-inflammatory drugs [NSAIDs] or local anesthetics) is recommended for effective postoperative pain relief. There are few data on the use of NSAIDs in postoperative pain treatment after abdominal surgery. We conducted a randomized, double-blind, placebo-controlled study to assess the analgesic efficacy and safety of ketoprofen after major abdominal surgery. One and nine hours postoperatively patients received 100 mg of ketoprofen i.v. (n = 21) or placebo (n = 22) in addition to a pain-treatment protocol consisting of continuous infusion of tramadol 200 mg and metamizol 5 g over 24 hours with additional 25 mg i.v. tramadol in case of inadequate analgesia. Pain was assessed by numeric rating scale at rest and at deep breath 3, 6, 12, and 24 hours postoperatively and the total dose of tramadol used in the first 24 hours was recorded. Patients in the ketoprofen group had significantly lower pain scores both at rest and at deep breath, at 3 (p < 0.01), 6, and 12 hours (p < 0.05) postoperatively. The 24-hour use of tramadol was significantly lower in the ketoprofen group (p < 0.01), with less nausea and vomiting. There were no bleeding complications or other adverse events related to ketoprofen therapy. The study showed the value of short-term use of ketoprofen to improve the quality of analgesia after major abdominal surgery without significant adverse effects.