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Published on: November 9, 2019
Continuous Purification of mRNA Produced by In Vitro Transcription Using High Performance Countercurrent Membrane
Ziqiao Wang1, Amin Javidanbardan1, Ali Behboudi1
1Robert V. Waltemeyer Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, USA.
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Messenger ribonucleic acid (mRNA) therapeutics produced by in vitro transcription must be purified to remove residual enzymes and free nucleotides. This study examines the use of high-performance countercurrent membrane purification (HPCMP) for the purification of mRNA therapeutics based on differences in the rate of diffusion across semipermeable polyethersulfone hollow fiber membranes. Experiments were performed using two model mRNA constructs using cytosine triphosphates (CTPs) and Proteinase K as model impurities. HPCMP achieved 99.7% removal of CTPs using a residence time of 60 min with no measurable loss of mRNA. Proteinase K removal by HPCMP was reduced in the presence of mRNA due to complexation between the positively charged protein and the negatively charged mRNA, an effect that was independently confirmed by dynamic light scattering and stirred cell diafiltration experiments. The HPCMP process could be operated continuously for more than 24 h without membrane fouling with stable mRNA yield (> 97%) and Proteinase K removal (> 94%). These results clearly demonstrate the feasibility of using HPCMP for continuous purification of mRNA therapeutics.
